CH-FW-DEQ-0009146

DuPont-18647-1017
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H-28548: Absorption, Distribution, Metabolism, and Elimination in the Mouse

Work Request Number 18647

Service Code 1017

Protocol

Performing Laboratory: E.I. du Pont de Nemours and Company
DuPont Haskell Global Centers for
Health & Environmental Sciences
P.O. Box 50
Newark, Delaware 19714
U.S.A.

Haskell Animal Welfare
Committee Number: BT197-P
CH-FW-DEQ-0009146
H-28548:
Absorption, Distribution, Metabolism, and Elimination in the Mouse DuPont-18647-1017
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TABLE OF CONTENTS
Page
TABLE OF CONTENTS.................................................................................................................2
INTRODUCTION...........................................................................................................................3
OBJECTIVE....................................................................................................................................3
SPONSOR AND CONTACT INFORMATION.............................................................................3
REGULATORY COMPLIANCE AND TEST GUIDELINES ......................................................3
ANIMAL WELFARE ACT COMPLIANCE..................................................................................4
MATERIALS AND METHODS.....................................................................................................4
A. Test Substance .....................................................................................................................4
B. Test System..........................................................................................................................4
C. Animal Husbandry...............................................................................................................5
D. Pretest Period.......................................................................................................................6
E. Assignment to Groups..........................................................................................................6
F. Dose Preparation, Analysis, and Rates................................................................................6
G. In-Life Phase - Tier 1...........................................................................................................7
H. Quantitation of H-28548......................................................................................................8
I. Quantification and Identification of Metabolites.................................................................8
STATISTICAL ANALYSES..........................................................................................................8
CONTROL OF BIAS ......................................................................................................................8
ADDITIONAL TESTING – TIER 2...............................................................................................8
SAFETY AND HOUSEKEEPING.................................................................................................9
RECORDS AND SAMPLE STORAGE.........................................................................................9
PROPOSED STUDY DATES.........................................................................................................9
REFERENCES ................................................................................................................................9
SIGNATURES...............................................................................Error! Bookmark not defined.

CH-FW-DEQ-0009147
H-28548:
Absorption, Distribution, Metabolism, and Elimination in the Mouse DuPont-18647-1017
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INTRODUCTION
H-28548 is currently under investigation by the sponsor. The data from this study will provide
basic information on the absorption, distribution, metabolism, and elimination (ADME) of
H-28548 in the mouse.
OBJECTIVE
The objective of this study is to determine the ADME of H-28548 in the mouse following a
single oral dose of H-28548 in water (Tier 1). Use of a non-radiolabeled test substance for
determining a material balance and metabolite identification in the mouse is justified based on
results from an in vitro metabolism experiment with rat hepatocytes and rat oral and rat/monkey
intravenous dose kinetic studies, which suggests that H-28548 is not metabolized and is
eliminated rapidly.(1,2,3,4)
SPONSOR AND CONTACT INFORMATION
Sponsor: E.I. du Pont de Nemours and Company
Wilmington, Delaware 19898
U.S.A.

Sponsor Contact: Jane Bradd Andersen
302-999-2377
[e-mail]

Testing Facility Contact: William J. Fasano, Sr.
302-451-3301
[e-mail]

Sponsor Approval: found on the Work Authorization Form

REGULATORY COMPLIANCE AND TEST GUIDELINES
This study will be conducted in compliance with the following good laboratory practice(s),
which are compatible with current OECD Good Laboratory Practices:
• U.S. EPA TSCA (40 CFR part 792) Good Laboratory Practice Standards
This study will be conducted in compliance with the following test guideline:
• U.S. EPA, OPPTS 870.7485. Metabolism and Pharmacokinetics, Health Effects Test
Guidelines (1998)
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Absorption, Distribution, Metabolism, and Elimination in the Mouse DuPont-18647-1017
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ANIMAL WELFARE ACT COMPLIANCE
This study will comply with all applicable sections of the Final Rules of the Animal Welfare Act
regulations (9 CFR) and the Guidelines from the Guide for the Care and Use of Laboratory
Animals (NRC 1996). The sponsor should make particular note of the following:
• The signature of the sponsor and/or the study director ensures that the study described in this
protocol does not unnecessarily duplicate previous experiments, and is in compliance with
the DuPont Policy on Animal Testing.
• Whenever possible, procedures used in this study have been designed to implement a
reduction, replacement, and/or refinement in the use of animals in an effort 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.
• DuPont Haskell policy is that animals experiencing severe pain or distress that cannot be
relieved will be painlessly euthanized, as deemed appropriate by the veterinary staff and
study director or appropriate designee. The sponsor will be advised by the study director of
all circumstances that 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 and the recommendations of the American Veterinary Medical Association
(AVMA), 2007 Guidelines on Euthanasia.
• This protocol has been reviewed by the Haskell Animal Welfare Committee and complies
with acceptable standards for animal welfare and humane care.
• DuPont Haskell is accredited by the Association for the Assessment and Accreditation of
Laboratory Animal Care (AAALAC) International.
MATERIALS AND METHODS
A. Test Substance
The test substance (CAS registry number 62037-80-3) will be supplied by the sponsor and
assigned Haskell number 28548.
B. Test System
Male and female Crl:CD1(ICR) mice will be obtained from Charles River Laboratories, Inc.
(Raleigh, North Carolina, U.S.A.).
The Crl:CD1(ICR) mouse was chosen for this study because of the extensive experience with
this strain and its suitability with respect to longevity, sensitivity, and low incidence of
spontaneous diseases. Furthermore, the Crl:CD1(ICR) mouse has been used previously for
toxicokinetic and toxicity testing of this chemical.
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H-28548:
Absorption, Distribution, Metabolism, and Elimination in the Mouse DuPont-18647-1017
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At the time of dosing, mice should be sexually mature, and the weight variation should not
exceed ±20% of the mean weight by dose group. Each animal will be assigned a unique
identification number to be used throughout the study. The last 3 digits of the animal
identification number will be marked on the tail of each animal in indelible ink.
C. Animal Husbandry
1. Housing
During the pretest period, animals may be housed in solid bottom caging with bedding. Animals
will be moved to metabolism units or lined solid bottom caging for the in-life phase of the study.
2. Environmental Conditions
Animal rooms will be maintained at a temperature of 18-26ºC (64-79ºF) and a relative humidity
of 30-70%. Animal rooms will be artificially illuminated (fluorescent light) on an approximate
12 hour light/dark cycle. Unless judged by the study director or the laboratory veterinarian to
have significantly affected the results of the study, the relative humidity and temperature ranges
in the housing rooms will be recorded but will not be included in the final report.
3. Feed and Water
All animals will be provided tap water ad libitum and fed PMI® Nutrition International, LLC
Certified Rodent LabDiet® 5002 ad libitum. When housed in metabolism units, feed will be
supplied as ground chow.
4. Animal Health and Environmental Monitoring Program
As specified in the DuPont Haskell animal health and environmental monitoring program, the
following procedures are performed periodically to ensure that contaminant levels are below
those that would be expected to impact the scientific integrity of the study:
• Water samples are analyzed for total bacterial counts, and the presence of coliforms, lead,
and other contaminants.
• Samples from freshly washed cages and cage racks are analyzed to ensure adequate
sanitation by the cagewashers.
Certified animal feed is used, guaranteed by the manufacturer to meet specified nutritional
requirements and not to exceed stated maximum concentrations of key contaminants, including
specified heavy metals, aflatoxin, chlorinated hydrocarbons, and organophosphates. The
presence of these contaminants below the maximum concentration stated by the manufacturer
would not be expected to impact the integrity of the study.
The animal health and environmental monitoring program is administered by the attending
laboratory animal veterinarian. Data are maintained separately from study records and may be
included in the final report at the discretion of the study director.
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Absorption, Distribution, Metabolism, and Elimination in the Mouse DuPont-18647-1017
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D. Pretest Period
Upon arrival at DuPont Haskell, all mice will be housed in quarantine. The mice will be:
• quarantined for at least 6 days.
• identified temporarily by cage identification.
• weighed at least 3 times during quarantine and once prior to dosing.
• observed with respect to weight gain and any gross signs of disease or injury.
The animals will be released from quarantine by the laboratory animal veterinarian or designee
based on body weights and clinical signs.
Animals that are accidentally killed or removed from study during the pretest period will be
discarded without necropsy. Animals that are found dead or are sacrificed in extremis during the
pretest period will be given a gross examination to check for the presence of disease. Dependant
upon these findings, further diagnostic procedures may be employed at the discretion of the
study director, a pathologist, or the laboratory animal veterinarian. The results will not be
included in the final report unless considered significant to the evaluation of the study.
E. Assignment to Groups
Animals will be selected for use on study based on adequate body weight gain and freedom from
any clinical signs of disease or injury. The weight variation of selected animals should not
exceed ± 20% of the mean weight.
Each animal will be assigned an animal number and a cage identification number. The animal
number and cage identification number will both be included on the cage label.
At study start, the animals should be 6-8 weeks old.
Animals that have not been assigned to a test group, or which have been removed from study
prior to dosing for out-of-range body weights, will be released for other laboratory purposes or
be sacrificed and discarded without anatomic pathology evaluation.
F. Dose Preparation, Analysis, and Rates
The test substance will be prepared for administration by oral gavage. This route was chosen
because it is most commonly used for toxicity studies with H-28548.
H-28548 will be weighed into a vial and mixed with deionized water (dose vehicle). The dose
solution will be prepared at a nominal concentration of 0.3 mg H-28548/mL (adjusted for purity),
with a target dose level of 3 mg/kg body weight (bw) and a dose volume of 10 mL/kg bw. The
dose level was chosen based on the results of the 28-day daily oral dosing study in mice, where
the no-observed-adverse-effect level (NOAEL) was 0.1 and 3 mg/kg/day for males and females,
respectively.(5)
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Absorption, Distribution, Metabolism, and Elimination in the Mouse DuPont-18647-1017
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The dosing solution/suspension may be prepared on the day of use or prior to the day of use and
stored refrigerated at 1-10°C.
LC/MS/MS will be used to confirm the chemical concentration of H-28548 in the dosing
solution/suspension.
G. In-Life Phase - Tier 1
1. Material Balance and Tissue Distribution
Mice will be housed individually in a metabolism unit and fasted for approximately 3 hours prior
to dosing. Food will be returned approximately 2 hours post-dose.
Five male and 5 female mice will be administered H-28548 at 3 mg/kg bw. Two male and
2 female mice will each be administered dose vehicle (deionized water at 10 mL/kg bw) for
collection of control excreta and tissue samples. Mice will be returned to individual metabolism
unit following dosing.
Urine and feces will be collected on dry ice predose and at 0-6, 6-12, 12-24, and every 24 hours
until 168 hours post dose. Evidence supporting a lack of metabolism of H-28548 in rat
hepatocytes and rat oral kinetic studies, precludes the necessity for a radiolabeled form of
H-28548 and collection of expired air.
At the end of the experiment (168 hours post dose), mice will be killed by CO2 asphyxiation
followed by exsanguination. The following tissues (Tier 1) will be collected:
liver
fat
G.I. tract (and contents)
kidney
spleen
whole blood
residual carcass

After collection, these samples will be stored at approximately -10C.
Over the course of the experiment, residual feed will be collected (if necessary) into a single
container and stored refrigerated at 1-10°C. Cages will be rinsed with deionized water, which
will be collected into a single container. Cage wash will be stored at room temperature and/or
refrigerated at 1-10°C.
Per the testing guideline, if it is determined that a significant amount of the administered dose
(<90%) is unaccounted for in the excreta (urine and feces, and cagewash, which is primarily
dried excreta, then data on the percent of the total amount of H-28548 in collected tissues as well
as residual carcass will be determined. Analysis of residual feed (if collected) will only occur if
a significant amount of the dose is still unaccounted for following the sequential and step-wise
analysis of excreta (urine and feces), cagewash, tissues residual carcass, and residual feed (if
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Absorption, Distribution, Metabolism, and Elimination in the Mouse DuPont-18647-1017
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collected). Total recovery should approximate 100 ± 10% of the amount of H-28548
administered.
H. Quantitation of H-28548
H-28548 will be quantitated in urine, feces and cagewash using LC/MS/MS, which may involve
direct analysis (urine and cagewash) or solvent extraction (feces), followed by analysis.
H-28548 will be quantitated in collected tissues, carcass, and residual feed (if collected), only if
the mean recovery of the administered dose in urine and feces, and cagewash, which is primarily
dried excreta, is <90%. Details of the methodologies employed will be documented in the study
records and presented in the final report.
I. Quantification and Identification of Metabolites
The profile of H-28548 (and metabolites, if any) will be evaluated in urine and feces samples.
Samples may be pooled across animals for a given time interval. Feces samples will be extracted
using appropriate solvent(s). Urine and fecal extracts may be filtered if necessary prior to
LC/MS analysis.
It is expected that H-28548 will not be metabolized by the mouse. However, any metabolites
that are found to represent greater than 5% of the administered dose will be submitted for
structural identification by mass spectroscopy or other suitable methods. The methodology
relating to identification of H-28548 and metabolites will be documented in the study records
and presented in the final report.
STATISTICAL ANALYSES
Group data will be represented as a mean ± SD. Other statistical evaluations may be performed
at the discretion of the study director and sponsor.
CONTROL OF BIAS
Effective mixing of samples and avoidance of cross-contamination will be used to control bias
during sample collection and analysis.
ADDITIONAL TESTING – TIER 2
Additional studies (Tier 2) may be added by amendment to this protocol if required. Studies at
the Tier 2 level are designed to answer questions about the disposition of test chemicals based on
the existing toxicology data base and/or results of Tier 1 testing. Only those studies that address
a specific concern not resolved at Tier 1 will be conducted following a discussion with the
Agency and a mutual agreed path forward.
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H-28548:
Absorption, Distribution, Metabolism, and Elimination in the Mouse DuPont-18647-1017
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SAFETY AND HOUSEKEEPING
All chemicals used during this study will be handled according to the procedures specified in the
MSDS and disposed of according to the Stine-Haskell Waste Disposal Guidelines and the area
Safety, Health and Environmental (SHE) manual.
RECORDS AND SAMPLE STORAGE
All raw data, the protocol, amendments (if any), and the final report will be retained.
PROPOSED STUDY DATES
Experimental Start: March 2010

Experimental Termination: June 2010

REFERENCES
1. DuPont Haskell (2007). In Vitro Rat Hepatocyte Screen. Unpublished report,
DuPont-23460.
2. DuPont Haskell (2008). Repeated Dose Oral Toxicity 7-Day Gavage Study in Rats.
Unpublished report, DuPont-24009.
3. DuPont Haskell (2007). Biopersistence and Pharmacokinetic Screen in Rats. Unpublished
report, DuPont-24281.
4. DuPont Haskell (2009). Cross-Species Comparison of FRD-902 Plasma Pharmacokinetics in
the Rat and Primate Following Intravenous Dosing. Unpublished report,
DuPont-17751-1579 RV1.
5. DuPont-Haskell (2008). A 28-Day Oral (Gavage) Toxicity Study of H-28397 in Mice with a
28-Day Recovery. Unpublished report, DuPont-24459.
CH-FW-DEQ-0009154
H-28548:
Absorption, Distribution, Metabolism, and Elimination in the Mouse DuPont-18647-1017
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CH-FW-DEQ-0009155