CH-FW-DEQ-0009273

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PROTOCOL

AN ORAL (GAVAGE) REPRODUCTION/DEVELOPMENTAL
TOXICITY SCREENING STUDY OF H-28548 IN MICE

(U.S. EPA OPPTS 870.3550 and OECD Guideline 421)

Submitted To:

E.I. du Pont de Nemours and Company
Wilmington, Delaware 19898

DuPont Work Request Number: 18405
DuPont Service Code: 1037
DuPont Study Number: 18405-1037

WIL Research Laboratories, LLC
1407 George Road
Ashland, OH 44805-8946

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1 OBJECTIVE:
To provide preliminary information on the potential adverse effects of the test
substance on male and female reproduction within the scope of a screening study.
This will encompass gonadal function, mating behavior, conception, parturition and
lactation of the F0 generation and the development of offspring from conception
through day 40 of postnatal life.
In addition, a toxicokinetic assessment of plasma levels of the test article will be
performed in the F0 females and the F1 pups at culling and on PND 21 and PND 40.
This study is subject to the applicable regulations of the Organisation for Economic
Cooperation and Development (OECD) Guideline for Testing of Chemicals,
Guideline 421, Reproduction/Development Toxicity Screening Test, July 27, 1995,
and the United States Environmental Protection Agency (EPA) Health Effects Test
Guidelines OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test,
July 2000 and will be conducted in accordance with the EPA/TSCA and FIFRA (40
CFR Part 792 and 40 CFR Part 160) and the OECD Principles of Good Laboratory
Practice.
2 PERSONNEL INVOLVED IN THE STUDY:
2.1 Study Representative:
Susan M. Munley, MA
Research Toxicologist
Developmental, Reproductive and Neurobehavioral Toxicology
DuPont Haskell Laboratory for Health and Environmental Sciences
1090 Elkton Rd., PO Box 50
Newark, DE 19714
Tel: [phone]
Email: [e-mail]

2.2 Principal Investigator, Pathology
Greg P. Sykes, VMD, DACVP, DACLAM, DABT
PharmPath, LLC.
105 Phillips Mill Rd.
West Grove, PA, 19390-9165
Tel: [phone]
Cellular Tel: [phone]
Email: [e-mail]

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2.3 WIL Study Director:
Tammye L. Edwards, BS, LAT
Staff Toxicologist, Developmental and Reproductive Toxicology
WIL Research Laboratories, LLC
1407 George Road
Ashland, Ohio 44805
Tel: [phone] ext. 2105
Fax: [phone]
Email: [e-mail]

2.4 WIL Departmental Responsibilities:
Eddie D. Sloter, PhD
Senior Toxicologist, Developmental
and Reproductive Toxicology
Emergency Contact
Tel: [phone]
Fax: [phone]
Email: [e-mail]

Mark D. Nemec, BS, DABT
President and Chief Operating Officer

Donald G. Stump, PhD, DABT
Director, Developmental and
Reproductive Toxicology

George A. Parker, DVM, PhD, DACVP, DABT
Director, Pathology

Melissa J. Beck, PhD
Assistant Director, Neurosciences

Daniel W. Sved, PhD
Director, Metabolism and Analytical Chemistry

Walter R. Miller, BS, DVM
Clinical Veterinarian,
Head of Surgery and Experimental Medicine

Ronald E. Wilson, BS
Director, Informational Systems

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Carol A. Kopp, BS, LAT
Manager, Gross Pathology and
Developmental Toxicology Laboratory

Heather L. Johnson, BS, RQAP-GLP
Manager, Quality Assurance

Bennett J. Varsho, MPH, DABT
Operations Manager, Developmental and
Reproductive Toxicology and the Formulations Laboratory

Carol S. Wally, BA, SRS, RLATG
Group Supervisor, Sample Processing Laboratory

Robert A. Wally, BS, RAC
Manager, Reporting and Regulatory
Technical Services

2.5 Principal Investigator, Plasma Sample Analysis and Report:
Michael Mawn, PhD
Senior Research Chemist
DuPont Stine-Haskell Research Center
1090 Elkton Road
Bldg. S-315 Lab 1333
Newark, DE 19714-0030
Tel: [phone]
Email: [e-mail]

3 STUDY SCHEDULE:
Proposed Experimental Starting
(Animal Receipt) Date: 5 January 2010

Proposed Experimental Start
(First Day of Dosing) Date: 14 January 2010

Proposed Experimental
Completion/Termination Date: 4 June 2010

Proposed Audited Report Date: To be determined10 September 2010

Formatted: Indent: Hanging: 0.12"
Formatted: Heading 2, Indent: Left: 0", First
line: 0", Tab stops: Not at 0.25"
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4 TEST SUBSTANCE DATA:
4.1 Test Substance Shipment:
Test substance and applicable documentation, including a Certificate of
Analysis, will be shipped under Sponsor’s responsibility to:
Formulations Laboratory (WIL-189225; Tammye Edwards)
Attn: Larry Blessing
WIL Research Laboratories, LLC
1407 George Road
Ashland, Ohio 44805-8946

4.2 Identification:
H-28548 or HFPO Dimer Acid Ammonium Salt
4.3 Haskell Test Substance Number:
H-28548

4.4 Lot Number:
E109540-44A
4.5 Expiration/Retest Date:
13 June 2011

4.6 Purity:
84%
4.7 Storage Conditions:
Controlled room temperature and humidity (approximately 18o to 24oC and 20%
to 70% relative humidity)
4.8 Stability:
The analysis was performed by the Sponsor and documented on the Certificate
of Analysis.
4.9 Physical Description:
To be documented by WIL Research Laboratories, LLC.
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4.10 Reserve Samples:
Reserve samples of the test substance will be taken in accordance with WIL
Standard Operating Procedures and stored in the Archives at WIL Research
Laboratories, LLC indefinitely, unless otherwise specified.
4.11 Personnel Safety Data:
See the Material Safety Data Sheet (MSDS) provided by the Sponsor.
4.12 Test Substance Disposition:
With the exception of the reserve sample for each batch of test substance, which
will be archived as described, all neat test substance remaining at completion of
the in-life phase of the study will be kept for subsequent studies.
5 TEST SYSTEM:
5.1 Species:
Mouse
5.2 Strain:
Charles River Crl:CD1(ICR)
5.3 Source:
Males: Charles River Laboratories, Inc., Raleigh, NC
Females: Charles River Laboratories, Inc., Kingston, NY

5.4 Number on Study:
100 males and 100 females (minimum of 120 males and 120 females purchased;
males and females will be ordered from separate facilities to ensure the
avoidance of sibling mating). Animals not assigned to study will be transferred
to the stock animal colony or will be euthanized by carbon dioxide inhalation
and the carcasses discarded.
The number of animals used on this study is consistent with OPPTS and OECD
guidelines for reproduction/developmental toxicity screening studies.
5.5 Body Weight Range:
A minimum of 20 grams at randomization.

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5.6 Approximate Age:
The approximate age of the males at randomization will be 42-63 days. The
approximate age of the females at randomization will be 70-80 days.42-63 days
old at randomization.
5.7 Identification System:
Each mouse will be uniquely identified by tattoo markings applied to the tail.
Individual cage cards will be affixed to each cage and will display the animal
number, group number, study number, dosage level and sex of the animal.
5.8 Justification for Selection:
This species and strain of animal is recognized as appropriate for reproduction
studies. WIL Research Laboratories, LLC has reproductive historical control
data in the Crl:CD1(ICR) mouse. This animal model has been proven to be
susceptible to the effects of reproductive toxicants.
6 SPECIFIC MAINTENANCE SCHEDULE:
6.1 Animal Housing:
The animals will be housed, 2-3 per cage, for at least 3 days following receipt.
Thereafter, the mice will be housed individually. The females will be housed
individually in solid bottom cages upon arrival. The F0 males and females will be
individually housed in solid bottom cages (plastic maternity cages) containing
ground corncob nesting material (Bed-O’ Cobs®) in an environmentally
controlled room during the quarantine period and throughout the entire study
until euthanasia. All F1 offspring not euthanized at weaning will be housed by
litter in the plastic cages with nesting material until postnatal day (PND) 28. F1
offspring not selected for the maturation phase will be necropsied on PND 21.
On PND 28, F1 offspring will be individually housed in solid bottom cages
(plastic maternity cages) containing ground corncob nesting material (Bed-O’
Cobs®). The cages will be subject to routine cleaning at a frequency consistent
with maintaining good animal health and WIL Standard Operating Procedures.
The facilities at WIL Research Laboratories, LLC are fully accredited by the
Association for Assessment and Accreditation of Laboratory Animal Care
International (AAALAC International).
6.2 Environmental Conditions:
Controls will be set to maintain temperature at 71 ± 5°F (22 ± 3°C) and relative
humidity at 50 ± 20%. Temperature and relative humidity will be monitored
continuously. Data for these two parameters will be scheduled for automatic
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collection on an hourly basis. Fluorescent lighting controlled by light timers
will provide illumination for a 12-hour light/dark photoperiod. The ventilation
rate will be set at a minimum of 10 room air changes per hour, 100% fresh air.
6.3 Drinking Water:
Reverse osmosis-purified water will be available ad libitum. Filters servicing
the automatic watering system are changed regularly according to WIL
Standard Operating Procedures. The municipal water supplying the laboratory
is analyzed according to WIL Standard Operating Procedures on a routine basis
to ensure that contaminants are not present in concentrations that would be
expected to affect the outcome of the study.
6.4 Basal Diet:
PMI Nutrition International, LLC Certified Rodent LabDiet® 5002 will be
offered ad libitum during the study. Periodic analyses of the certified feed are
performed by the manufacturer to ensure that heavy metals and pesticides are
not present at concentrations that would be expected to affect the outcome of
the study. Results of the analyses are provided to WIL Research Laboratories,
LLC by the manufacturer. Feeders will be changed and sanitized once per
week.
6.5 Enrichment:
All animals will be offered NestletsTM for enrichment that will be replaced as
needed.

7 EXPERIMENTAL DESIGN:
7.1 Animal Receipt and Quarantine:
Each animal will be inspected by a qualified technician upon receipt. Mice
judged to be in good health and suitable as test animals will be immediately
placed in quarantine for a minimum of 9 days. All mice will be initially
weighed, permanently identified by tattoo markings applied to the tail and
receive a clinical observation. During the quarantine period, each mouse will be
observed twice daily for changes in general appearance and behavior. Prior to
the start of the in-life phase, those animals judged to be suitable test subjects
will be identified and receive a detailed physical examination.
7.2 Randomization:
At the conclusion of the quarantine period, animals judged to be suitable test
subjects and meeting acceptable body weight requirements, will be assigned at
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random using a computer program. At that time, the animal numbers and
corresponding body weights will be entered into the WIL Toxicology Data
Management System (WTDMS ). A printout containing the animal numbers
and individual group assignments will be generated based on body weight
stratification into a block design. Animals will then be arranged into the groups
according to the printout. The control group and three test item groups will
consist of 20 25 males and 20 25 females each.
Any animal assigned to the study that is found dead, euthanized in extremis or
exhibits abnormal clinical signs, reduced food consumption or body weight
losses prior to the start of dosing may be replaced by an animal of appropriate
age when possible. Replacement animals will be arbitrarily assigned (not
computer randomized) to the study based on comparable body weights (if
possible) with respect to the animal that was replaced.
7.3 Route and Rationale of Test Item Administration:
The route of administration will be oral (gavage). Historically, this route has
been used extensively for studies of this nature. Appropriately sized flexible,
Teflon®-shafted, stainless steel dosing cannulae will be used for the oral
administration by gavage. The dosing cannulae may or may not be ball-tipped as
appropriate for the age of the animal.Appropriately sized flexible,
Teflon -shafted, stainless steel ball-tipped dosing cannulae will be used for the
oral administration by gavage.
7.4 Organization of Test Groups, Dosage Levels and Treatment Regimen:
7.4.1 Organization of Test Groups:
The dose levels proposed for the current study are 0, 0.1, 0.5, and
5 mg/kg/day and are based on previous and ongoing general toxicity
studies in mice. These levels are currently being tested in an ongoing
(in-life dosing phase complete) subchronic toxicity 90-day gavage study
(DuPont-18405-1307). The doses for the 90-day gavage study were
based on results from a previous 28-day gavage study (DuPont-24459)
in which doses of 0, 0.1, 3, and 30 mg/kg/day were tested.
The following table presents the study group arrangement.
Group
Number
Test
Item
Dosage Level
(mg/kg/day)
Dosage
Concentration
(mg/mL)
Dosage
Volume
(mL/kg)
Number of
Animals
Male Female
1 Vehicle Controlb 0 0 10 25 25
2 H-28548 0.1 0.01 10 25 25
3 H-28548 0.5 0.05 10 25 25
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4 H-28548 5 0.5 10 25 25
a Dosage levels will be corrected for the purity of 84%.
b Deionized Water

7.4.2 Vehicle Control Item:
Deionized Water
7.4.3 F0 Treatment Regimen:
The test and control items will be administered once daily at
approximately the same time each day as follows:
7.4.3.1 Males:
F0 males will be dosed for a minimum of 70 days prior to mating
and continuing until the day prior to the scheduled euthanasia.
7.4.3.2 Females:
F0 females will be dosed for a minimum of 14 days prior to
mating and continuing throughout mating, gestation and lactation
until Lactation Day (LD) 20, inclusively, for females that deliver,
with the exception of the 5 females/group that are selected for
blood collection on LD 21, which will also receive a dose on LD
21.F0 females will be dosed for a minimum of 14 days prior to
mating and continuing throughout mating, gestation and lactation
until Lactation Day (LD) 21 for females that deliver. For females
that do not have positive signs of mating or delivery, dosing will
continue until one day prior to euthanasia.
7.4.3.3 F1 Males and Females:
F1 males and females will be dosed beginning in PND 21 through
PND 40, inclusively.F1 males and females will be dosed
beginning in PND 21 until one day prior to euthanasia.
7.4.4 Adjustment of Dosages:
Individual dosages will be calculated based on the most recent body
weight to provide the proper mg/kg/day dosage.
7.5 Preparation and Analysis of Test Item Formulations:
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7.5.1 Method and Frequency of Preparation:
Based on the physical characteristics of the test substance, appropriate
methods will be used to ensure the best possible formulations of the test
substance in the vehicle. Dosing formulations will be stored refrigerated
(2-8°C) for a maximum of 12 days. The Study Director or designee will
visually inspect the formulations prior to the initiation of dosing. This
visual inspection will be performed to ensure that the formulations are
visibly homogeneous and acceptable for dosing. Any special procedures
required for formulation will be documented according to Good
Laboratory Practices and presented in the final report of this study. Test
substance formulations will be prepared approximately weekly and
divided into aliquots for daily dispensation. The test substance and
vehicle formulations will be stirred continuously during dosing.
7.5.2 Homogeneity, Resuspension Homogeneity, Stability and
Concentration Determination of Test Substance Formulations:
Stability and resuspension homogeneity were established on a previous
study (Haas, Draft; WIL-189216). Test substance formulations were
stable and 12 days of room temperature storage or refrigerated storage
(2-8°C) at concentrations of 0.01 mg/mL and 100 mg/mL and
homogenous following resuspension after 12 days of refrigerated storage
(2-8°C). Stability and resuspension homogeneity will not be conducted
on this study.
Homogeneity and concentration will be conducted on the first
formulations prepared for dosing. Four 1-mL samples will be collected
from the top, middle and bottom of the test substance formulations from
the low and high dose groups and the samples analyzed to assess the
homogeneity of the test substance in the mixtures; the middle strata will
serve as the measure of test substance concentration. Four 1-mL
samples will be taken from the middle on the control and the mid-dose
groups and analyzed for concentration of the test substance.
Concentration will be assessed on Week 4, 8, 12, 16 and 19 formulations
prepared for dosing. Four 1-mL samples will be collected from the
middle of each test substance formulation and the control group and
analyzed for test substance content.
7.5.3 Sample Analysis:
Samples will be transferred to the Analytical Chemistry Department at
WIL Research Laboratories, LLC for analysis. Analyses of test article
formulations will be performed using a method developed and validated
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by WIL Research Laboratories, LLC. Initially, two of each set of four
replicate, 1-mL samples will be analyzed; the remaining two 1-mL
samples will be stored frozen (approximately -20°C) at WIL and will
function as back-up samples. Back-up samples will be analyzed if
requested by the Sponsor or Study Director or may be discarded
following results that are within specifications and approval of the Study
Director.
7.6 F0 Breeding:
After a minimum of 70 days for males and 14 days of exposure for females, of
exposure, one female will be cohabitated with one male mouse of the same
treatment group, avoiding sibling mating, in a plastic cage for mating.
Detection of mating will be confirmed by evidence of sperm in the vaginal
lavagethe appearance of a vaginal copulatory plug. After confirmation of
mating, the female will be returned to an individual plastic cage and the day will
be designated as day 0 of gestation.
A maximum of 14 days will be allowed for mating. After 14 days of mating,
any females who have not shown evidence of breeding will be placed in a
plastic cage containing nesting material.
7.7 F0 Parturition and Lactation and F1 Litters:
The day parturition is initiated will be designated as day 0 of lactation. Any
difficulties at the time of parturition will be recorded. When parturition is
judged to be complete, the sex of each pup will be determined, pups will be
examined for gross malformations and the number of stillbirths and live pups
will be recorded. Any changes or abnormalities in nesting and nursing behavior
will be recorded. The dam and litter will remain together until postnatal
day (PND) 21.
7.8 Identification of F1 Litters:
Upon completion of delivery, all pups will be individually identified by tattoo
markings applied to the digits. To reduce variability among the litters, on PND
4, eight pups of equal sex distribution (if possible) from each litter will be
randomly selected. For litters consisting of fewer than eight pups, adjustments
for litter sizes will not be performed. Following selection, the non-selected
PND 4 pups will be euthanized by an intraperitoneal injection of sodium
pentobarbital and discarded.
7.9 General Observations During the Experimental Period:
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7.9.1 Parental Appearance and Behavior:
Each parental mouse (F0) will be observed twice daily for moribundity
and mortality, once in the morning and once in the afternoon. A detailed
physical examination will be conducted weekly. Mortality and all signs
of overt toxicity will be recorded on the day observed. The observations
shall include, but are not limited to, evaluations for changes in
appearance of the skin and fur, eyes and mucous membranes,
respiratory, circulatory, autonomic and central nervous systems,
somatomotor activity and behavior. During the period of expected
parturition, the dams will be observed twice daily for dystocia,
prolonged labor, delayed labor or other difficulties at parturition. All
animals will also be observed on the day of necropsy and findings will
be recorded.
During the treatment period, each animal will be observed at
approximately 1-2 hours following each dose administration for findings
that are potentially related to treatment of that might change before the
next scheduled observation. Additional post dosing observation periods
may be necessary and will be documented in the study records.
7.9.2 Parental Body Weights:
All animals will have a final body weight recorded on the day of
euthanasia.
7.9.2.1 Males:
Recorded individually on a weekly basis, beginning on the first
day of dose administration, until euthanasia.
7.9.2.2 Females:
For those females with evidence of mating, body weights will be
recorded individually on a weekly basis, beginning on the first
day of dose administration, until evidence of copulation is
observed and on gestation days 0, 4, 7, 11, 14 and 18 and on
lactation days 1, 4, 7, 14 and 21.Recorded individually on a
weekly basis, beginning on the first day of dose administration,
until evidence of copulation is observed and on gestation days 0,
4, 7, 11, 14, 17 and 20 and lactation days 1, 4, 7, 14 and 21.
For females with no evidence of mating, individual body weights
will continue to be recorded on a weekly basis until euthanasia.
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7.9.3 Parental Food Consumption*:
Individual food consumption will not be recorded during the breeding
period because the animals are cohabitated at that time.
7.9.3.1 Males:
Recorded individually on a weekly basis, beginning on the first
day of dose administration, until euthanasia.
7.9.3.2 Females:
Recorded individually on a weekly basis beginning on the first
day of dose administration, until the start of the mating period.
Individual food consumption will be recorded on the day
evidence of copulation is observed (GD 0) and on gestation days
4, 7, 11, 14 and 18 and lactation days 1, 4, 7, 14 and 21.Recorded
individually on a weekly basis beginning on the first day of dose
administration, until the start of the mating period. Individual
food consumption will be recorded on the day evidence of
copulation is observed (GD 0) and on gestation days 4, 7, 11, 14,
17 and 20 and lactation days 1, 4, 7, 14 and 21.
For females with no evidence of mating, individual food
consumption will continue to be recorded on a weekly basis
following the end of the mating period until euthanasia.
7.9.4 Examination of Offspring:
7.9.4.1 Appearance and Behavior:
All pups will be observed daily for general appearance and
behavior and survival during lactation. A detailed physical
examination will be recorded for each pup on PND 1, 4, 7, 14
and 21. Any abnormalities in nesting and nursing behavior will
be recorded. The pups will be sexed on PND 0, 4, 14 and 21.
7.9.4.2 Body Weights:
Each pup will be weighed on PND 1, 4, 7, 14 and 21.
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7.9.5 Pup Deaths:
7.9.5.1 Pups 0 to 4 Days of Age:
Moribund pups will be euthanized by an intraperitoneal injection
of sodium pentobarbital. Stillborn pups, pups found dead
between birth and PND 4, and any pups that are euthanized in
extremis will be dissected (including the heart and the brain
examined by a mid-coronal slice) by a technique described by
Stuckhardt and Poppe (Stuckhardt and Poppe, 1984). If a
skeletal anomaly is suspected, the pups will be eviscerated,
cleared and stained with Alizarin Red S as described by Dawson
(Dawson, 1926) and examined. Representative specimens with
malformations may be preserved in 10% neutral buffered
formalin at the discretion of the study director.
7.9.5.2 Pups 5 Days of Age to Weaning:
Moribund pups will be euthanized by an intraperitoneal injection
of sodium pentobarbital (prior to PND 11) or by carbon dioxide
inhalation. A gross necropsy will be performed on pups found
dead or euthanized in extremis, and gross lesions will be saved
for possible future histopathological examination in 10% neutral
buffered formalin. If a skeletal anomaly is suspected, the pups
will be eviscerated, cleared and stained with Alizarin Red S as
described by Dawson (Dawson, 1926) and examined.
7.10 Selection of F1 Generation and Termination of PND 21 Nonselected Pups:
One male and one female pup per litter will be selected for the F1 generation on
or prior to PND 21. Only pups not expected to survive due to notable physical
limitations will not be available for selection. A detailed evaluation of each pup
excluded from selection will be recorded.
All PND 21 pups not selected for the F1 generation will be euthanized by carbon
dioxide inhalation. A gross necropsy examination will be performed with an
emphasis on evaluation of developmental morphology and organs of the
reproductive system. Any gross lesions will be saved for possible future
histopathological examination in 10% neutral buffered formalin.
7.11 Euthanasia of F0 Generation:
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7.11.1 Females:
7.11.1.1 Females Which Deliver:
On lactation day 21, all F0 females that delivered will be
euthanized by carbon dioxide inhalation. A gross examination
will be performed and tissues preserved as described in Section
8.1. The number of former implantation sites will be recorded.
Organ weights will be collected and tissues preserved as
described in Section 8.2.
7.11.1.2 Females Which Fail to Deliver:
On post-mating day 23 (females with evidence of mating) or
post-cohabitation day 23 (females without evidence of
copulation), the F0 females which fail to deliver will be
euthanized by carbon dioxide inhalation. On post-mating day 25
(females with evidence of copulation) or post-cohabitation day
25 (females without evidence of copulation), the F0 females
which fail to deliver will be euthanized by carbon dioxide
inhalation. A gross necropsy examination will be performed and
tissues will be preserved as described in Section 8.1. Organ
weights will be collected as described in Section 8.2 with the
exception of any ammonium sulfide stained uterus, which will be
discarded. Uteri which appear nongravid by macroscopic
examination will be opened and placed in a 10% ammonium
sulfide solution (Salewski, 1964) for detection of early
implantation loss.
7.11.1.3 Females with Total Litter Loss:
Females with total litter loss will be euthanized by carbon
dioxide inhalation on the same day. The number of former
implantation sites will be recorded and the number of corpora
lutea (if litter loss occurs on or before PND 4) will be recorded.
A gross necropsy examination will be performed and tissues
preserved as described in Section 8.1. Organ weights will be
collected as described in Section 8.2.
7.11.1.4 F0 Deaths and Animals Euthanized in Extremis:
Females not surviving until the scheduled euthanasia will have a
gross necropsy examination performed and tissues preserved as
described in Section 8.1. Animals not expected to survive to the
next observation period (moribund) will be euthanized by carbon
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dioxide inhalation and have a gross necropsy examination
performed and tissues preserved as described in Section 8.1.
Organ weights will not be collected from found dead or
euthanized in extremis females. The number and location of
implantation sites or scars will be recorded for females dying or
euthanized during gestation and lactation. The number of
corpora lutea will be recorded for females dying or euthanized
during gestation and up to and including lactation day 4. Uteri
which appear nongravid by macroscopic examination will be
opened and placed in a 10% ammonium sulfide solution
(Salewski, 1964) for detection of early implantation loss.
Viable fetuses will be euthanized by an intrathoracic injection of
sodium pentobarbital. Recognizable fetuses will be examined
externally for gross abnormalities. Representative specimens
with malformations may be preserved in 10% neutral-buffered
formalin, at the discretion of the study director. For females
found dead or euthanized in extremis during lactation, all pups
will be examined externally and subjected to a necropsy
examination according to Section 7.9.5.
7.11.2 Males:
Following completion of the mating period, all F0 males will be
euthanized by carbon dioxide inhalation and subjected to a gross
necropsy and tissue preservation as described in Section 8.1. Organ
weights will be collected as described in Section 8.2.
Males not surviving until the scheduled euthanasia will be subjected to a
gross necropsy and tissue preservation as described in Section 8.1. Any
males not expected to survive to the next observation period (moribund)
will be euthanized by carbon dioxide inhalation and also necropsied and
have tissues preserved as described in Section 8.1. Organ weights will
not be collected.
7.12 F1 Generation General Observations During The Experimental Period:
7.12.1 F1 Clinical Observations:
Following weaning and selection, the mice will be observed twice daily
for moribundity and mortality, once in the morning and once in the
afternoon. Clinical observations will be recorded dailyA detailed
physical examinations will be conducted weekly. Mortality and all signs
of overt toxicity will be recorded on the day observed. The observations
shall include, but are not limited to, evaluation for changes in
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appearance of the skin and fur, eyes, mucous membranes, respiratory,
circulatory, autonomic and central nervous system function,
somatomotor activity and behavior patterns. All animals will also be
observed on the day of necropsy and any findings will be recorded.
During the treatment period, each animal will be observed at
approximately 1-2 hours following each dose administration for findings
that are potentially related to treatment of that might change before the
next scheduled observation. Additional post dosing observation periods
may be necessary and will be documented in the study records.
7.12.2 F1 Body Weights and Food Consumption:
F1 males and females will be have a body weight recorded
approximately weekly, beginning with the start of test diet substance
administration until euthanasia (PND 21, 28, 35 and 40). All animals
will have a final body weight recorded on the day of euthanasia.
F1 males and females will have food consumption recorded individually
on an approximately weekly basis beginning on PND 28 until euthanasia
(PND 28, 35 and 40). Food consumption will not be collected from
PND 21 to PND 28 during group housing for the F1 males and females.
7.13 F1 Postweaning Developmental Landmarks:
Offspring selected for the F1 generation will be evaluated for attainment of the
following landmarks of sexual maturity:
7.13.1 Balanopreputial Separation:
Each male pup will be observed for balanopreputial separation
beginning on PND 25 as described by Korenbrot et al. (Korenbrot
1977). Examination of the males will continue daily until
balanopreputial separation is present. The body weight of each male
will be recorded on the day of attainment of balanopreputial separation.
7.13.2 Vaginal Patency:
Each female pup will be observed for vaginal patency beginning on
PND 21 (only those selected for the F1 generation) as described by
Adams et al. (Adams 1985). Examination of the females will continue
daily until vaginal patency is present. The body weight of each female
will be recorded on the day of attainment of vaginal patency.

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7.14 Euthanasia of F1 Generation:
7.14.1 Scheduled Necropsy
On PND 40, all F1 animals will be euthanized by carbon dioxide
inhalation. A gross necropsy examination will be performed with an
emphasis on evaluation of developmental morphology and organs of the
reproductive system. Any gross lesions will be saved for possible future
histopathological examination in 10% neutral buffered formalin.
7.14.2 Unscheduled Deaths or Animals Euthanized in Extremis
Any F1 animals not surviving until the scheduled euthanasia or not
expected to survive to the next observation period (euthanized by carbon
dioxide inhalation) will be necropsied. A gross necropsy examination
will be performed with an emphasis on evaluation of developmental
morphology and organs of the reproductive system. Any gross lesions
will be saved for possible future histopathological examination in 10%
neutral buffered formalin.

7.15 Plasma Sample Collection and Analysis:
7.15.1 Interval:
Blood samples will be collected at 2 hours post dose administration on
LD 21 at necropsy from 5 randomly selected F0 females per group that
delivered. A blood sample will be collected from all females that failed
to deliver on post-mating day 23 at the time of the scheduled necropsy
(not timed).
In addition, all control females that delivered but were not selected for
blood collection as indicated above, will have blood samples taken on
LD 21 at the time of scheduled necropsy (not timed) to provide control
animal plasma for method development work to be conducted by the
Sponsor. These control samples will be processed and shipped as
described for the study samples.
Formatted: Heading 2, Indent: Left: 0"
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Blood samples will also be collected from the F1 culled pups on PND 4
from 10 randomly chosen litters in each group following culling and
data collection.
On PND 21, blood samples will be collected from 5 randomly selected
F1 males and females in each group at the time of the scheduled
necropsy (not timed) that are not selected for the F1 generation.
On PND 40, blood samples will be collected at 2 hours dose
administration at necropsy from 5 randomly selected F1 males and
females in each group.
7.15.2 Route of Collection:
Blood samples will be collected via the vena cava following euthanasia
by carbon dioxide inhalation from the F0 females and the F1 PND 21 and
PND 40 animals.
Blood samples will be collected via decapitation from the PND 4 pups
and pooled by litter.
7.15.3 Target Blood Volume:
For the F0 females and the F1 PND 21 and PND 40 animals, 1.0 mL or
as much as possible, will be collected into pre-chilled, uniquely-labeled
tubes. For the PND 4 pups, blood will be pooled by litter from all the
culled pups in each litter to obtain as much blood as possible.
7.15.4 Anticoagulant:
K3EDTA

7.15.5 Sample Handling and Plasma Preparation:
Samples will be kept on wet ice, protected from light, until centrifugation.
All samples will be centrifuged [approximately 3000 rpm (approximately
2060 x g) for approximately 10 min] at approximately 4oC. Plasma will
be transferred into new, uniquely-labeled polypropylene tubes.

7.15.6 Label Information:
Samples will include study number, dose group, animal number, interval,
sample type and date and time of blood collection.

Formatted: Heading 3, Indent: Left: 0"
Formatted: Heading 3, Indent: Left: 0"
Formatted: Heading 3, Indent: Left: 0", Tab
stops: Not at 1"
Formatted: Heading 3, Indent: Left: 0"
Formatted: Heading 3, Indent: Left: 0"
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7.15.7 Storage:
Plasma samples will be stored frozen at approximately -20°C until
analysis. The time and date the samples were placed in the freezer will be
recorded.

7.15.8 Sample Shipment:
Frozen samples in dry ice, an inventory list and documentation of actual
blood collection times for each animal will be shipped on the first Monday
or Tuesday after the last sample is collected. The recipient will be notified
at least 24 hours in advance of any shipment. Samples will be shipped
overnight to:

Michael Mawn, PhD
Senior Research Chemist
DuPont Stine-Haskell Research Center
1090 Elkton Road
Bldg. S-315 Lab 1334
Newark, DE 19714-0030
Tel: [phone]
Email: [e-mail]

7.15.9 Plasma Analyses and Report:
Plasma samples will be analyzed for the test article content after solvent
protein precipitation with LC/MS/MS analysis. The method of analysis
will be documented in the study records and final report. The Principal
Investigator for the plasma analysis will be responsible for all
bioanalytical delegated-phase activities and will issue a formal
bioanalytical/plasma analyses report from the data generated that will be
included as an appendix in the final report. A Quality Assurance and GLP
compliance statement signed by Sponsor and archival location of the data
will be provided to the WIL Study Director for inclusion in the Final
Report.

8 ANATOMIC PATHOLOGY:
8.1 Macroscopic Examination:
A complete necropsy will be conducted on all F0 parental animals dying
spontaneously, euthanized in extremis (by carbon dioxide inhalation) or at
termination. This will include examination of the external surface, all orifices,
the cranial cavity, the external surface of the brain and the thoracic, abdominal
Formatted: Heading 3, Indent: Left: 0"
Formatted: Heading 3, Indent: Left: 0"
Formatted: Heading 3, Indent: Left: 0"
Formatted: Normal, Indent: Left: 0"
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and pelvic cavities including viscera. For F0 females, the number of former
implantation sites will be recorded.
At the time of necropsy, the following tissues and organs will be collected and
placed in 10% neutral-buffered formalin (except as noted):
Coagulating gland
Kidneys (2)
Liver
Mammary gland (females only)
Ovaries and oviduct (2)
Pituitary

Prostate
Seminal vesicles (2)
Testes with epididymides (2)a
and vas deferens
Uterusb with cervix and vagina
All gross lesionsc
a - Testes and epididymides will be fixed in Bouin's solution. Care will be taken to
ensure separation between the left and right organs.
b - Any uterus stained in 10% ammonium solution for detection of implantation sites
will be discarded and will not be preserved in 10% neutral buffered formalin.
c - Representative sections of corresponding organs from a sufficient number of
controls will be retained for comparison, if possible.
8.2 Organ Weights:
The following organs will be weighed from all F0 parental animals euthanized at
scheduled termination. Organ-to-final-body weight and organ-to-brain weight
ratios will be evaluated.
Brain
Epididymides*
Kidneys
Liver
Ovaries (with oviducts)
Pituitary
Testes*
* - These paired organs will be weighed separately.

8.3 Microscopic Examination:
Microscopic examination of hematoxylin-eosin stained paraffin sections will be
performed on the listed tissues from all F0 parental animals from the control and
high-dose groups and from all parental animals dying spontaneously or
euthanized in extremis and from any animals in the low and mid dose groups
with impaired fertility (males that did not sire a litter or females that did not
deliver a litter). Microscopic examination of hematoxylin-eosin stained paraffin
sections will be performed on the following tissues from all F0 parental animals
from the control and high-dose groups and from all parental animals dying
spontaneously or euthanized in extremis. If a target organ is identified in the
high-dose group, this organ will be examined from all animals in the low and
mid-dose groups (at additional cost):
Cervix Seminal vesicles
Formatted: Indent: Left: 0", Hanging: 1.31"
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Coagulating gland
Epididymides
Ovaries and oviduct
Prostate
Testes
Uterus
Vagina
All gross (internal) lesions

The slides will be prepared by WIL Research Laboratories, LLC and then shipped
to Sponsor at the address and contact below for examination by the Principal
Investigator, Pathology.

Carolyn Lloyd
DuPont Haskell Global Centers for Health & Environmental Sciences
Investigative Sciences, S320/531
1090 Elkton Road
Newark, DE 19714-0050
Tel: [phone]
Fax: [phone]
Email: [e-mail]

The examination of the slides will be performed by the Principal Investigator for
Pathology. A final pathology report will be prepared and submitted to WIL
Research for inclusion as an appendix in the main study final report. A Quality
Assurance and GLP compliance statement signed by the performing laboratory
will be provided to the WIL Study Director for inclusion in the Final Report.
The Sponsor is responsible for archiving of raw data associated with the
conduct of the pathological examination.

9 DURATION OF STUDY:
The two generations to be studied (parental animals and first generation offspring)
will be termed F0 and F1, respectively. The conduct of this study will require
approximately 22 weeks for acclimation, mating, gestation and lactation of the F0
generation.
10 STATISTICAL METHODS:
All analyses will be two-tailed for significance levels of 5% and 1%. All means will
be presented with standard deviations. All statistical tests will be performed by a
computer with appropriate programming as referenced below. The litter, rather than
the pup, will be considered as the experimental unit.
10.1 Parental In-Life Data:
Continuous data variables [mean body weights, body weight gains and food
consumption at each interval], pre-coital intervals, gestation length, former
implantation sites, unaccounted-for sites, mean days of attainment of
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developmental landmarks (balanopreputial separation and vaginal patency) and
the body weight on the day of attainment will be subjected to a parametric one-
way analysis of variance (ANOVA) (Snedecor, 1980) to determine intergroup
difference. If the results of the ANOVA are significant (p<0.05), Dunnett's test
(Dunnett, 1964) will be applied to the data to compare the treated groups to the
control group.
Male and female mating, fertility, copulation and conception indices of the
treated groups will be compared to the control group using the Chi-square test
with Yates' correction factor (Hollander, 1999).
10.2 Litter Data:
The mean litter proportions (% per litter) of pup viability during the postnatal
period and sex ratio at birth will be subjected to the Kruskal-Wallis
nonparametric ANOVA test (Kruskal, 1952) to determine intergroup difference.
If the results of the ANOVA are significant (p<0.05), the Dunn’s Test (Dunn,
1964) will be applied to compare the treated groups to the control group. Mean
numbers of pups born, live litter size and litter weights will be subjected to the
parametric ANOVA test (Snedecor, 1980) and Dunnett’s test (Dunnett, 1964) as
described above with the litter representing the experimental unit.
10.3 Histopathology and Organ Weight Data:
Histopathological findings of each treated group will be compared to those of
the control group by the Fisher’s Exact test (Steel, 1980). Organ weights
(absolute and relative to body weights and relative to brain weights) will be
subjected to a parametric ANOVA test (Snedecor, 1980) and Dunnett's test
(1964) as described above.

11 QUALITY ASSURANCE:
The study will be audited by the WIL Quality Assurance Unit while in progress to
assure compliance with the study protocol and protocol amendments, WIL Standard
Operating Procedures and the appropriate provisions of EPA/TSCA and FIFRA Good
Laboratory Practice Standards published in the Federal Register (40 CFR Part 792
and 40 CFR Part 160) and the OECD Principles of Good Laboratory Practice. The
final report will be audited by the WIL Quality Assurance Unit prior to submission to
the Sponsor Representative to assure that the final report accurately describes the
conduct and the findings of the study.
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The plasma samples analysis and the pathological examination of the slides will be
conducted following the Standard Operating Procedures of the performing laboratory
and in accordance with GLPs.The pathological examination of the slides will be
conducted following the Standard Operating Procedures of the performing laboratory
and in accordance with GLPs. Quality Assurance monitoring of these analyses for
SOP and GLP compliance is the responsibility of the performing laboratory.
Inspection reports will be supplied to the Study Director. Upon completion of the
prescribed activities and submission of the results to the Sponsor and Study Director
the performing laboratory will provide a signed Quality Assurance Statement to the
Sponsor (copy to the Study Director). The results will be included in the final report.
This study will be included on the WIL master list of regulated studies.
12 RECORDS TO BE MAINTAINED:
All original raw data records, as defined by WIL SOPs and the applicable GLPs, will
be stored as described in Section 13 in the Archives at WIL Research Laboratories,
LLC.
The Sponsor will be responsible for the archival of the raw data and records for the
plasma sample analyses and the pathological examination.The Sponsor will be
responsible for the archival of the raw data and records for the pathological
examination.
13 WORK PRODUCT:
The Sponsor will have title to all documentation records, raw data, slides, specimens
and other work product generated during the performance of the study. Any
remaining plasma samples and formulation samples will be discarded after the
issuance of the Final Report. Any remaining formulation samples will be discarded
after the issuance of the Final Report. All work product, including raw paper data,
pertinent electronic storage media and specimens, will be retained for a period of six
months following issuance of the final report in the Archives at WIL Research
Laboratories, LLC. Thereafter, WIL Research Laboratories, LLC will charge a
monthly archiving fee for retention of all work product. All work product will be
stored in compliance with regulatory requirements.
Any work product, including documents, specimens, and samples, that are required
by this protocol, its amendments, or other written instructions of the Sponsor, to be
shipped by WIL Research Laboratories, LLC to another location will be appropriately
packaged and labeled as defined by WIL’s SOPs and delivered to a common carrier
for shipment. WIL Research Laboratories, LLC will not be responsible for shipment
following delivery to the common carrier.
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All work product generated at a performing laboratory will be retained at an
appropriate archive facility as designated by the SOPs of the performing laboratory.
14 REPORTS:
The final report will contain a summary, test item data, methods and procedures,
maternal and pup data WIL Historical Control Data, the analytical chemistry report,
the plasma analysis report, the pathology report and an interpretation and discussion
of the study results. The final report will contain a summary, test item data, methods
and procedures, maternal and pup data WIL Historical Control Data, the analytical
chemistry report, pathology report and an interpretation and discussion of the study
results. The final report will be comprehensive and shall define level(s) inducing
toxic effects as well as no-effect level(s) under the conditions of this investigation.
The report will contain all information necessary to conform with current OPPTS and
OECD specifications.
WIL Research Laboratories, LLC will submit one copy of an audited draft report in a
timely manner upon completion of data collection prior to issuance of the final report.
One revision will be permitted as part of the cost of the study, from which the
Sponsor's reasonable revisions and suggestions will be incorporated into the final
report, as appropriate. Additional changes or revisions may be made, at extra cost. It
is expected that the Sponsor will review the draft report and provide comments to
WIL Research Laboratories, LLC within a two-month time frame following
submission. WIL Research Laboratories, LLC will submit the final report within one
month following receipt of comments. If the Sponsor’s comments and/or
authorization to finalize the report have not been received at WIL Research
Laboratories, LLC within one year following submission of the draft report, WIL
Research Laboratories, LLC may elect to finalize the report following appropriate
written notification to the Sponsor. Two electronic copies (PDF) of the final report
on CD-R will be provided. Requests for paper copies of the final report may result in
additional charges.
15 ANIMAL WELFARE ACT COMPLIANCE:
This study will comply with all applicable sections of the Final Rules of the Animal
Welfare Act (AWA) regulations (9 CFR Parts 1, 2 and 3). The Sponsor should make
particular note of the following:
• The Sponsor Representative's signature on this protocol documents for the Study
Director the Sponsor's assurance that the study described in this protocol does not
unnecessarily duplicate previous experiments.
• Whenever possible, procedures used in this study have been designed to avoid or
minimize discomfort, distress or pain to animals. All methods are described in this
study protocol or in written laboratory Standard Operating Procedures.
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• Animals that experience severe pain or distress that cannot be relieved will be
painlessly euthanized as deemed appropriate by the veterinary staff and Study
Director. The Sponsor will be advised by the Study Director of all circumstances
which could lead to this action in as timely a manner as possible.
• Methods of euthanasia used during this study are in conformance with the
above-referenced regulation.
• The Sponsor/Study Director has considered alternatives to procedures that may
cause more than momentary or slight pain or distress to the animals and has
provided a written narrative description (AWA covered species) of the methods and
sources used to determine that alternatives are not available.
16 PROTOCOL MODIFICATION:
Modification of the protocol may be accomplished during the course of this
investigation. However, no changes will be made in the study design without the
verbal or written permission of the Sponsor. In the event that the Sponsor verbally
requests or approves a change in the protocol, such changes will be made by
appropriate documentation in the form of protocol amendment. All alterations of the
protocol and reasons for the modification(s) will be signed by the Study Director and
the Sponsor Representative.
17 REFERENCES:
Adams, J.; Buelke-Sam, J.; Kimmel, C.A.; Nelson, C.J.; Reiter, L.W.; Sobotka, T.J.;
Tilson, H.A.; Nelson, B.K. Collaborative behavioral teratology study: protocol design
and testing procedure. Neurobehavioral Toxicology and Teratology 1985, 7, 579-586.
Dawson, A.B. A note on the staining of the skeleton of cleared specimens with Alizarin
Red S. Stain Technology 1926, 1, 123-124.
Dunn, O.J. Multiple comparisons using rank sums. Technometrics 1964, 6(3), 241-252.
Dunnett, C.W. New tables for multiple comparisons with a control. Biometrics 1964,
20, 482-491
Haas, M. A 90-Day Oral (Gavage) Study of H-28548 in Rats with a 28-Day Recovery.
WIL-189216, Draft.
Hollander, M.; Wolfe, D.A. Nonparametric Statistical Methods, 2nd ed.; Hollander, M.,
Wolfe, D.A., Eds.; John Wiley and Sons, Inc.: New York, NY, 1999; p 468.
CH-FW-DEQ-0009299
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Korenbrot, C.C.; Huhtaniemi, I.T.; Weiner, R.W. Preputial separation as an external
sign of pubertal development in the male rat. Biology of Reproduction 1977, 17, 298-
303.
Kruskal, W.H.; Wallis, W.A. Use of ranks in one-criterion variance analysis. Journal
of the American Statistical Association 1952, 47, 583-621.
Salewski, E. Färbemethode zum makroskopischen Nachweis von Implantationsstellen
am Uterus der Ratte. [Staining method for a macroscopic test for implantation sites in
the uterus of the rat]. Naunyn - Schmiedebergs Archiv für Experimentelle Pathologie
und Pharmakologie 1964, 247, 367.
Snedecor, G.W.; Cochran, W.G. One Way Classifications; Analysis of Variance. In
Statistical Methods, 7th ed.; The Iowa State University Press: Ames, IA, 1980;
pp 215-237.
Steel, R.G.D.; Torrie, J.H. Principles and Procedures of Statistics, A Biometrical
Approach, 2nd ed.; McGraw-Hill Book Company: New York, NY, 1980; pp 504-506.

Stuckhardt, J.L.; Poppe, S.M. Fresh visceral examination of rat and rabbit fetuses used
in teratogenicity testing. Teratogenesis, Carcinogenesis and Mutagenesis 1984, 4,
181-188.
18 PROTOCOL APPROVAL:
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Sponsor approval received via ________________ on _______________.
Date

E. I. du Pont de Nemours and Company

______________________________________ _____________
Susan M. Munley, MA Date
Sponsor Representative

WIL Research Laboratories, LLC

______________________________________ _____________
Tammye L. Edwards, BS, LAT Date
Study Director

______________________________________ _____________
Donald G. Stump, PhD, DABT Date
Director, Developmental and
Reproductive Toxicology

CH-FW-DEQ-0009301