CH-FW-DEQ-0009452

DuPont-18647-1017
Page 1 of 57
TRADE SECRET
Unpublished Work
Copyright ©2010 E.I. du Pont de Nemours and Company

STUDY TITLE: H-28548: Absorption, Distribution, Metabolism, and
Elimination in the Mouse

TEST GUIDELINES: U.S. EPA Health Effects Test Guidelines
OPPTS 870.7485 (1998)

AUTHOR: William J. Fasano, Sr., B.S.

STUDY COMPLETED ON: November 3, 2010

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.

E.I. du Pont de Nemours and Company
DuPont Experimental Station (CCAS)
Wilmington, Delaware 19803
U.S.A.

LABORATORY PROJECT ID: DuPont-18647-1017

WORK REQUEST NUMBER: 18647

SERVICE CODE NUMBER: 1017

SPONSOR: E.I. du Pont de Nemours and Company
Wilmington, Delaware 19898
U.S.A.
CH-FW-DEQ-0009452
H-28548:
Absorption, Distribution, Metabolism, and Elimination in the Mouse DuPont-18647-1017
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GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT
This study was conducted in compliance with U.S. EPA TSCA (40 CFR part 792) Good
Laboratory Practice Standards, which are compatible with current OECD Good Laboratory
Practices, except for the item documented below. The item listed does not impact the validity of
the study.
1. Qualitative analysis of urine samples for structure confirmation and elucidation was
conducted on a non-GLP Liquid Chromatography/Mass Spectrometry (LC/MS) system.
However, the identity of the parent analyte, the only analyte detected, was confirmed in urine
samples using the test substance H-28548, which had a matching nominal mass-to-charge
(m/z) ratio of approximately 329.

CH-FW-DEQ-0009453
H-28548:
Absorption, Distribution, Metabolism, and Elimination in the Mouse DuPont-18647-1017
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QUALITY ASSURANCE STATEMENT

Work Request Number: 18647
Service Code Number: 1017

Key inspections for the above referenced study were completed by the Quality Assurance Unit of
DuPont Haskell and the findings were submitted on the following dates:

Audit Dates
Date Reported to
Study Director
Date Reported to
Management

Protocol:
March 17, 2010 March, 17, 2010 March, 17, 2010

Conduct:
March 31, 2010 March 31, 2010 March 31, 2010
June 09, 2010 June 09, 2010 June 09, 2010

Report/Records:
October 08, 11-13, 2010 October 13, 2010 October 14, 2010

Sponsor Edits 1:
October 28, 2010 October 28, 2010 October 28, 2010

CH-FW-DEQ-0009454
H-28548:
Absorption, Distribution, Metabolism, and Elimination in the Mouse DuPont-18647-1017
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CERTIFICATION
We, the undersigned, declare that this report provides an accurate evaluation of data obtained
from this study.

CH-FW-DEQ-0009455
H-28548:
Absorption, Distribution, Metabolism, and Elimination in the Mouse DuPont-18647-1017
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TABLE OF CONTENTS
Page
GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT...................................2
QUALITY ASSURANCE STATEMENT...................................................................................3
CERTIFICATION.........................................................................................................................4
TABLE OF CONTENTS ..............................................................................................................5
LIST OF TABLES.........................................................................................................................6
LIST OF FIGURES.......................................................................................................................6
LIST OF APPENDICES...............................................................................................................7
STUDY INFORMATION.............................................................................................................8
SUMMARY....................................................................................................................................9
INTRODUCTION........................................................................................................................10
OBJECTIVE ................................................................................................................................10
ANIMAL WELFARE ACT COMPLIANCE ...........................................................................10
MATERIALS AND METHODS................................................................................................10
A. Test Guidelines..................................................................................................................10
B. Test Substance ...................................................................................................................11
C. Test System........................................................................................................................11
D. Animal Husbandry.............................................................................................................11
1. Housing..................................................................................................................................................11
2. Environmental Conditions......................................................................................................................11
3. Feed and Water ......................................................................................................................................11
4. Animal Health and Environmental Monitoring Program.......................................................................11
E. Pretest Period.....................................................................................................................12
F. Assignment to Groups........................................................................................................12
G. Dose Preparation, Analysis, and Rates..............................................................................12
H. In-Life Phase......................................................................................................................13
1. Material Balance and Tissue Distribution..............................................................................................13
I. Quantitation of H-28548....................................................................................................14
1. Sample Receipt.......................................................................................................................................14
2. Sample Preparation Procedure (dose solution and urine samples).........................................................14
3. Sample Preparation Procedure (cage wash samples) .............................................................................14
4. Sample Preparation Procedure (feces samples)......................................................................................14
5. Stock Solutions and Calibration Standards............................................................................................14
6. Instrument and Conditions.....................................................................................................................15
7. Quantitation............................................................................................................................................16
J. Identification of Metabolites..............................................................................................16
1. Liquid Chromatography/Mass Spectrometry (LC/MS)..........................................................................16
2. Data processing......................................................................................................................................17
STATISTICAL ANALYSES......................................................................................................17
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RESULTS AND DISCUSSION..................................................................................................18
A. Quantitation of H-28548 by LC/MS/MS...........................................................................18
1. Calibration Standard Curve....................................................................................................................18
2. Limit of Detection and Limit of Quantitation ........................................................................................18
3. Chromatographic Results (urine, cage wash, and dose samples)...........................................................19
4. Chromatographic Results (feces samples) .............................................................................................19
5. Fortification QC Sample Results............................................................................................................19
B. Dose Formulation Concentration, Animal Body Weights, Dosing Information...............19
C. Urine Data..........................................................................................................................20
D. Feces Data..........................................................................................................................20
E. Material Balance................................................................................................................20
F. Metabolite Identification....................................................................................................21
CONCLUSIONS..........................................................................................................................21
RECORDS AND SAMPLE STORAGE....................................................................................22
REFERENCES.............................................................................................................................22
TABLES........................................................................................................................................23
FIGURES......................................................................................................................................27
APPENDICES..............................................................................................................................38

LIST OF TABLES
Page
Table 1 Mouse urine sample fortification QC results for H-28548.......................................................25
Table 2 Mouse feces sample fortification QC result for H-28548........................................................25
Table 3 Dosing information..................................................................................................................25
Table 4 Urine, cumulative percent of dose ...........................................................................................26
Table 5 Feces, cumulative percent of dose ...........................................................................................26
Table 6 Material balance, percent of dose ............................................................................................26

LIST OF FIGURES
Page
Figure 1 Calibration curve for H-28548.................................................................................................29
Figure 2 The LC/MS/MS chromatograms for a) lowest calibration standard at 2.5 ng/mL, b) urine
control matrix sample, c) low level 400 ng/g fortification QC sample with preparation
factor 40x, and d) a 24-hour urine study sample from animal 001M, which had a total
dilution factor of 1480x and final concentration of 14,800 ng/g..............................................30
CH-FW-DEQ-0009457
H-28548:
Absorption, Distribution, Metabolism, and Elimination in the Mouse DuPont-18647-1017
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Figure 3 The LC/MS/MS chromatograms for a) lowest calibration standard at 2.5 ng/mL, b) feces
control matrix sample, c) low level 250 ng/g fortification QC sample that had a
preparation factor of 6.14x, and d) a 12-hour feces study sample from animal 001M,
which had a 34x dilution factor and final concentration of 775 ng/g.......................................31
Figure 4 Urine, cumulative percent........................................................................................................32
Figure 5 Feces, cumulative percent........................................................................................................33
Figure 6 Material Balance, percent of dose............................................................................................34
Figure 7 Reconstructed m/z 329 + 659 ion chromatograms characteristic of H-28548-dosed female
mouse urine (6 hours after administration) – top and control mouse urine fortified with
H-28458 test substance -bottom...............................................................................................35
Figure 8 ESI negative mass spectra of H-28548 observed in dosed female mouse urine (6 hours
after administration)–top; and control urine fortified with H-28548 test substance –
bottom......................................................................................................................................36
Figure 9 ESI negative daughter ion mass spectra of H28548 observed in dosed female mouse urine
(6 hours after administration)–top; and control mouse urine fortified with H-28548 test
substance – bottom...................................................................................................................37

LIST OF APPENDICES
Page
Appendix A Certificate of Analysis.............................................................................................................40
Appendix B Dosing Information..................................................................................................................42
Appendix C Urine Data................................................................................................................................44
Appendix D Feces Data................................................................................................................................49
Appendix E Cage Wash Data.......................................................................................................................54
Appendix F Material Balance......................................................................................................................56

CH-FW-DEQ-0009458
H-28548:
Absorption, Distribution, Metabolism, and Elimination in the Mouse DuPont-18647-1017
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STUDY INFORMATION
Substance Tested: • HFPO Dimer Acid Ammonium Salt
• 2,3,3,3-tetrafluoro-2-(heptafluoropropoxy)propionic
acid, ammonium salt
• 62037-80-3 (CAS Number)
• H-28548

Haskell Number: 28548

Composition: Proprietary

Purity: 84%

Physical Characteristics: Clear and colorless liquid

Stability: The test substance appeared to be stable under the
conditions of the study; no evidence of instability was
observed.

Study Initiated/Completed: March 16, 2010 / (see report cover page)

Experimental Start/Termination: March 16, 2010 / June 11, 2010

In-Life Initiated/Completed: March 31, 2010 / April 7, 2010

Notebook Number(s): E-114321-AM, E-114321-AI, E-98524-GE
CH-FW-DEQ-0009459
H-28548:
Absorption, Distribution, Metabolism, and Elimination in the Mouse DuPont-18647-1017
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SUMMARY
The absorption, distribution, metabolism, and elimination of H-28548 were investigated in the
Crl:CD1(ICR) mouse. H-28548 was administered in water to 5 male and 5 female mice as a
single oral dose at a target dose level of 3 mg H-28548/kg bodyweight (bw) and a dose volume
of 10 mL/kg bw. Mice were housed individually in metabolism units and urine and feces were
collected on dry ice predose and postdose at 0-6 hours, 6-12 hours, 12-24 hours, and every 24
hours until 168 hours post-dose. At 168 hours post-dose, mice were asphyxiated by exposure to
carbon dioxide and then sacrificed by exsanguination. H-28548 was quantitated in urine, feces,
and cagewash by liquid chromatograpy tamdem mass spectrometry (LC/MS/MS). Urine
samples were further evaluted by LC/MS to confirm the identity of the parent analyte and
determine if H-28548 was eliminated metabolized or unmetabolized.
Following oral administration of H-28548 in water, 30.8% ± 5.37% and 39.3% ± 5.58% of the
administered dose was accounted for in urine (0-12 hours) from male and female mice,
respectively. At the conclusion of the study (168 hours post-dose), the total accumulated amount
of H-28548 detected in urine was 89.5% ± 6.91% and 91.5% ± 6.04% of the administered dose
for male and female mice, respectively.
Elimination of H-28548 via urine accounted for a majority of the administered dose for both
male and female mice; minor levels of H-28548 detected in feces from male (2.00% ± 1.01%)
and female mice (1.91% ± 0.85%) were likely contamination from urine.
Cagewash, which is composed of dried excreta (urine and feces), accounted for 9.64% ± 3.99%
and 6.25% ± 3.16% of the administered dose for male and female mice, respectively.
Following oral dosing with H-28548 in water and a 168 hour post-dose collection period,
101.2% ± 3.22% and 99.7% ± 2.95% of the administered dose was recovered from male and
female mice, respectively.
Samples of urine evaluated using LC/MS were found to contain only the parent substance,
H-28548. This finding, taken with recovery of the administered dose in urine, confirms that
H-28548 was rapidly absorbed and eliminated unmetabolized following oral dosing in the
mouse.
CH-FW-DEQ-0009460
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Absorption, Distribution, Metabolism, and Elimination in the Mouse DuPont-18647-1017
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INTRODUCTION
The data from this study provides basic information on the absorption, distribution, metabolism,
and elimination (ADME) of H-28548 following oral dosing in the mouse.
OBJECTIVE
The objective of this study was to determine the ADME of H-28548 in the mouse following a
single oral dose of H-28548 in water. 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 and monkey intravenous
dose kinetic studies, which suggests that H-28548 is not metabolized and is eliminated
rapidly.(1,2,3,4)
ANIMAL WELFARE ACT COMPLIANCE
This study complied 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). All studies conducted by or for DuPont Haskell adhere to the following
principles:
• The sponsor and/or the study director ensures that the study described in this report 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.
• DuPont Haskell policy is that animals experiencing severe pain or distress that cannot be
relieved are painlessly euthanized, as deemed appropriate by the veterinary staff and study
director or appropriate designee.
• Methods of euthanasia used during this study were in conformance with the above referenced
regulation and the recommendations of the American Veterinary Medical Association
(AVMA), 2007 Guidelines on Euthanasia.
• DuPont Haskell is accredited by the Association for the Assessment and Accreditation of
Laboratory Animal Care (AAALAC) International.
MATERIALS AND METHODS
A. Test Guidelines
The study design complied with the following test guideline:
CH-FW-DEQ-0009461
H-28548:
Absorption, Distribution, Metabolism, and Elimination in the Mouse DuPont-18647-1017
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• U.S. EPA, OPPTS 870.7485. Metabolism and Pharmacokinetics, Health Effects Test
Guidelines (1998)
B. Test Substance
The test substance (CAS registry number 62037-80-3) was supplied by the sponsor and assigned
Haskell number 28548.
C. Test System
Male and female Crl:CD1(ICR) mice were 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.
Each animal was assigned a unique identification number to be used throughout the study. The
last 3 digits of the animal identification number were marked on the tail of each animal in
indelible ink.
D. Animal Husbandry
1. Housing
During the pretest period, animals were housed individually in solid bottom caging with bedding.
Animals were moved to metabolism units for the in-life phase of the study.
2. Environmental Conditions
Animal rooms were maintained at a temperature of 18-26ºC (64-79ºF) and a relative humidity of
30-70%. Animal rooms were artificially illuminated (fluorescent light) on an approximate
12 hour light/dark cycle.
3. Feed and Water
All animals were provided tap water ad libitum and fed PMI® Nutrition International, LLC
Certified Rodent LabDiet® 5002 ad libitum. When housed in metabolism units, feed was
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.
CH-FW-DEQ-0009462
H-28548:
Absorption, Distribution, Metabolism, and Elimination in the Mouse DuPont-18647-1017
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• 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. Evaluation of these data did not indicate any conditions that
affected the validity of the study.
E. Pretest Period
Upon arrival at DuPont Haskell, all mice were housed in quarantine. The mice were:
• 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 were released from quarantine by the laboratory animal veterinarian or designee
based on body weights and clinical signs.
F. Assignment to Groups
Animals were 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 by sex was less
than 4% of the mean weight.
Each animal was assigned an animal number and a cage identification number. The animal
number and cage identification number were both included on the cage label.
At study start, the animals were at least 8 weeks old.
G. Dose Preparation, Analysis, and Rates
The test substance was prepared for administration by oral gavage. This route was chosen
because it is most commonly used for toxicity studies with H-28548.
H-28548 was weighed into a vial (approximately 35.6 mg) and mixed with deionized water
(100 mL). The dose solution was prepared at a nominal concentration of 0.3 mg H-28548/mL
(adjusted for purity, 84%), 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
CH-FW-DEQ-0009463
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Absorption, Distribution, Metabolism, and Elimination in the Mouse DuPont-18647-1017
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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)
The dosing solution was prepared prior to the day of use and was stored refrigerated at 1-10°C
prior to dosing.
H. In-Life Phase
1. Material Balance and Tissue Distribution
The conduct of this study was designed to comply with the Tier 1 requirements of U.S. EPA,
OPPTS 870.7485 - Metabolism and Pharmacokinetics, Health Effects Test Guidelines (1998).
Mice were housed individually in metabolism units and fasted for approximately 3 hours prior to
dosing. Food was returned approximately 2 hours post-dose.
Five male and 5 female mice were administered H-28548 at a nominal target of 3 mg
H-28548/kg bw. Two male and 2 female mice were each administered dose vehicle (deionized
water at 10 mL/kg bw) for collection of control excreta and tissue samples. Mice were returned
to individual metabolism units following dosing.
Urine and feces were collected on dry ice predose and at 0-6 h, 6-12 h, 12-24 h, and every 24
hours until 168 hours post dose. Evidence supporting a lack of metabolism of H-28548 in rat
hepatocytes and rat oral dose administration studies, precluded 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 were killed by CO2 asphyxiation
followed by exsanguination. The following tissues (Tier 1) were collected:
liver
fat
G.I. tract (and contents)
kidney
spleen
whole blood
residual carcass

After collection, these samples were stored at approximately -10C.
Over the course of the experiment, residual feed was collected into a single container and stored
refrigerated at 1-10°C. Cages were rinsed with deionized water, which was collected into a
single container. Cage wash was stored at room temperature and/or refrigerated at 1-10°C.
CH-FW-DEQ-0009464
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I. Quantitation of H-28548
1. Sample Receipt
The dose solution, urine, feces, and cage wash samples were received and stored at
approximately -20C by the analytical laboratory upon receipt and when not in use.
2. Sample Preparation Procedure (dose solution and urine samples)
The frozen samples were thawed to room temperature and mixed briefly before sampling. A
pipette was used to transfer 25 µL of sample into an empty HPLC vial, and the sample weight
was recorded to the nearest 0.0001 gram. The pipette was then used to add 975 µL of HPLC
grade water, and mixed. The initial sample preparation dilution factor = 1/sample weight (g).
Additional sample dilutions were performed with HPLC grade water to ensure that the sample
peak area results were within the calibration curve limits. Quality control fortification samples
were also prepared at low, mid and high levels in control urine, and prepared for analysis using
the same procedure.
3. Sample Preparation Procedure (cage wash samples)
The frozen cage wash samples were thawed to room temperature and mixed briefly before
sampling. A pipette was used to transfer 200 µL of sample into an empty HPLC vial, and the
sample weight was recorded to the nearest 0.0001 gram. The pipette was then used to add
800 µL of HPLC grade water, and mixed. The initial sample preparation factor = 1/sample
weight (g).
4. Sample Preparation Procedure (feces samples)
The frozen feces samples submitted in 15-mL conical polypropylene centrifuge tubes were
thawed to room temperature. HPLC grade water was added to the 13-mL mark, and the weight
of water added was recorded to the nearest 0.01 gram. Five ball bearings (5/32” diameter) were
added to the sample tubes and sealed. The samples were homogenized using a Genogrinder for
5 minutes at 1400 strokes/minute (SPEX CertiPrep Genogrinder 2000, Metuchen, New Jersey
U.S.A.). After homogenization, the samples were placed in a refrigerator for overnight
extraction. After overnight extraction the samples were shaken to mix and centrifuged for
10 minutes at 4150 rpm at 20ºC. Approximately 1.5 mL of supernatant was added to a 1.7 mL
microcentrifuge tube and further centrifuged for 15 minutes at 14,000 rpm and 20 ºC. A syringe
filter (PALL Acrodisc - 25 mm with 0.2 µm Nylon Membrane) was then used to filter
approximately 1 mL supernatant into a HPLC vial for analysis. The preparation factor = (H2O
weight (g) + feces weight (g)) / feces weight (g). Additional sample dilutions were performed
with pooled control feces extract to ensure that the sample peak area results were within the
calibration curve limits. Quality control fortification samples were also prepared at low, mid and
high levels using 2 grams of control feces, and prepared for analysis using the same procedure.
5. Stock Solutions and Calibration Standards
A stock solution of H-28548 was prepared in HPLC grade water. The stock solution was diluted
with HPLC grade water to prepare calibration standards at 0, 2.50, 5.00, 12.5, 25.0, 62.5, 156,
CH-FW-DEQ-0009465
H-28548:
Absorption, Distribution, Metabolism, and Elimination in the Mouse DuPont-18647-1017
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and 250 ng/mL levels. The calibration standards used for the urine, cage wash, and dose solution
sample types were prepared in HPLC grade water. The calibration standards used for the feces
samples were prepared using control feces extract to correct for matrix effects.
6. Instrument and Conditions
The prepared samples were analyzed by LC/MS/MS using the following conditions:
Method 1 Quantitation of H-28548 in urine, feces, and cagewash
HPLC Instrument: Agilent Model 1100

HPLC Parameters:
Column (Urine, dose
solution, and cage wash):
Zorbax SB-C8; 2.1x100 mm with 3.5 micron particle size
Column (Feces) Zorbax SB-C8; 2.1x30 mm with 3.5 micron particle size
Mobile Phase: A: 0.15% acetic acid in HPLC grade water
B: 0.15% acetic acid in acetonitrile

Column Temperature: 35 °C
Injection Volume: 5 µL urine, dose and cage wash samples
2 µL for feces samples

HPLC Gradient
(Urine, dose solution,
Total Time
(min)
Flow Rate
(µL/min)
A
(%)
B
(%)

and cage wash samples) 0.00 400 65.0 35.0
5.00 400 65.0 35.0

HPLC Gradient
(Feces samples)
Total Time
(min)
Flow Rate
(µL/min)
A
(%)
B
(%)

0.00 400 95.0 5.0
2.00 400 95.0 5.0
2.10 400 70.0 30.0
4.50 400 50.0 50.0
6.00 400 5.0 95.0
9.00 400 5.0 95.0
9.10 400 95.0 5.0
11.0 400 95.0 5.0

MS Parameters:
Ion Source: Turbo Spray, Negative Ion
Temperature (TEM): 120ºC
Dwell 250 msec
Curtain Gas Flow (CUR): 10.0
GS1: 25
CH-FW-DEQ-0009466
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Absorption, Distribution, Metabolism, and Elimination in the Mouse DuPont-18647-1017
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GS2: 25
IonSpray (IS) Voltage: -4500
CAD 6.00
EP -10.0
Quadrupole Resolution: Quad. 1: Unit
Quad. 3: Unit
MRM Settings Q1 Mass Q3 Mass DP CE CXP
H-28548 329.0 285.00 -20.0 -6.0 -7.0

7. Quantitation
The samples, calibration standards, and fortification quality control plasma samples were
analyzed by LC/MS/MS. The calibration standard curve was generated by regression analysis
using the chromatographic peak areas of the calibration standard solutions. The peak areas for
the study samples and fortification QC samples were compared to the calibration standard curve
to determine the concentration of the analyte. Any samples with peak areas above the upper
calibration standard were diluted to ensure that the peak areas were within the calibration curve.
J. Identification of Metabolites
Samples of urine were pooled across animals for a given time interval where the mean percent of
the administered dose (by sex) was ≥5% (males and females: 0-6, 6-12, 12-24, 24-48, and 48-72
hours; feces extract samples were not pooled since the total mean percent of dose for each
collection interval (by sex) was <5% of the administered dose.
Samples of pooled urine (25 µL) were diluted to 500 µL with Nanopure water prior to analysis.
Samples of the diluted urine (20 µL) were qualitatively screened by LC/HRMS for metabolites.
Retention time and mass spectral confirmation of the parent was performed by spiking control
urine with approximately 40 ppm (v/v) of the test material (H-28548) and analyzing the spiked
sample using the identical method for the study samples (Method 2).
1. Liquid Chromatography/Mass Spectrometry (LC/MS)
Method 2 Qualitative LC/MS Confirmation and Structural Elucidation of
metabolites in urine
HPLC/MS System: Agilent 1100 HPLC with column thermostat and binary pump,
autosampler, variable wavelength detector (S/N DE63058654 -
Agilent Inc., Little Falls, Delaware, U.S.A.). Thermo-Fisher
OrbiTrap FT-MS (S/N 1016B - Thermo-Fisher Scientific Inc.,
San Jose, California, U.S.A.). The associated computer is loaded
with Thermo-Fisher Xcaliber Software (v 2.0.7)

HPLC Conditions:
Column: Agilent Zorbax SB-C18 column (2.1 x 150 mm) 3.5 µm particle
size
Column Temperature: 25°C
Solvent A: 0.10% Acetic Acid in HPLC grade water
CH-FW-DEQ-0009467
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Absorption, Distribution, Metabolism, and Elimination in the Mouse DuPont-18647-1017
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Solvent B: 0.10% Acetic acid in acetonitrile
Gradient: Time
(min)
A
(%)
B
(%)

0.0 98.0 2.0
20.00 0.0 100.0
25.00 0.0 100.0
25.10 98.0 2.0
30.00 98.0 2.0
Flow Rate: 0.30 mL/min
Run Time: 30.00 min
Injection Volume: 20 µL
UV Wavelength: 190-400 nm

MS Conditions:
Ionization Mode: Electrospray negative ion
Source Voltage: 3.6 kV
Capillary Temperature: 330°C
Tube Lens voltage: 140 V
Source Current: 100 µA
Data Acquisition Function: Full Scan = 120-1000 Da (Profile mode), Mass Resolution =
30,000
Daughter Scans (Da)
Identity Daughters
of
Start Mass End Mass
H-28548 329 90 500
Collision Energy: 25 V daughter ion scan only
Scan Time Full scan 0.95 sec/scan; Daughter ion scan 0.3 sec/scan
Collision Gas and Pressure: Argon at 0.000602 mbar

2. Data processing
All chromatograms were screened for differences (chromatographic peaks) in control versus
H28548-dosed urine samples using IntelliExtract™; v. 12.0.1 (ACD, Toronto, Ontario, Canada)
control-sample comparison software.
STATISTICAL ANALYSES
Group data were represented as a mean ± SD.
CH-FW-DEQ-0009468
H-28548:
Absorption, Distribution, Metabolism, and Elimination in the Mouse DuPont-18647-1017
- 18 -
RESULTS AND DISCUSSION
A. Quantitation of H-28548 by LC/MS/MS
(Tables 1-2, Figures 1-3)
1. Calibration Standard Curve
A calibration curve for H-28548 is shown in Figure 1. The curve was generated based on
resulting peak areas of the H-28548 analyte using a quadratic equation, and 1/x weighing.
2. Limit of Detection and Limit of Quantitation
The limit of detection (LOD) and limit of quantitation (LOQ) were determined by comparing the
peak-to-peak noise in chromatograms of control matrix versus the signal of the lowest level
calibration standard. The initial LOD was calculated as 3 times the concentration equivalent of
the mean noise level. The initial LOQ was based on the lowest calibration standard
concentration, which had at least a 10x signal-to-noise ratio. For a sample preparation factor of
1x the initial urine and cage wash sample LOD was 0.1 ng/g and for feces the initial LOD was
0.08 ng/g. For a sample preparation factor of 1x the urine, cage wash, and feces matrices all
have an initial LOQ of 2.5 ng/g. The final LOD and LOQ for each sample was determined by
multiplying the initial values by the sample preparation factor.
Example LOD & LOQ Calculation: Urine sample from animal 001M, 120 hour time point
• 25 µL aliquot sample weight (g) = 0.0294 g
• Sample Preparation Factor = 1 / 0.0294 = 34.0
• Final LOD for this sample = 0.1 ng/g x 34.0 = 3 ng/g (reported to 1 significant digit)
• Final LOQ for this sample = 2.5 ng/g x 34.0 = 85.0 ng/g (reported to 3 significant digits)
Example LOD & LOQ Calculation: Feces sample from animal 001M, 120 hour time point
• Water Extraction Weight = 9.89 g. Feces weight = 2.869 grams
• Sample Preparation Factor = (9.89(g) + 2.869 (g)) / 2.869 (g) = 4.45
• Final LOD for this sample = 0.08 ng/g x 4.45 = 0.4 ng/g (reported to 1 significant digit)
• Final LOQ for this sample = 2.5 ng/g x 4.45 = 11.1 ng/g (reported to 3 significant digits)
Example LOD & LOQ Calculation: Cage wash sample from animal 001M, 168 hour time point
• 200 µL aliquot sample weight (g) = 0.2097 g
• Sample Preparation Factor = 1 / 0.2097 = 4.77
CH-FW-DEQ-0009469
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Absorption, Distribution, Metabolism, and Elimination in the Mouse DuPont-18647-1017
- 19 -
• Final LOD for this sample = 0.1 ng/g x 4.77 = 0.5 ng/g (reported to 1 significant digit)
• Final LOQ for this sample = 2.5 ng/g x 4.77 = 11.9 ng/g (reported to 3 significant digits)
None of the predose urine or feces samples had detectable levels of H-28548.
3. Chromatographic Results (urine, cage wash, and dose samples)
H-28548 eluted as a well-resolved peak with a retention time of approximately 2.4 minutes. An
example chromatogram for the lowest calibration standard at 2.5 ng/mL is shown in Figure 2a.
An example chromatogram of a urine control matrix sample is shown in Figure 2b (H-28548 was
not detected). A low level fortification quality control (QC) sample is shown in Figure 2c, which
was fortified at a level of 400 ng/g, and had a preparation factor of 40x. A 24-hour urine sample
from animal 001M, which had a total dilution factor of 1480x is shown in Figure 2d. The final
concentration for this sample was 14,800 ng/g.
4. Chromatographic Results (feces samples)
H-28548 eluted as a well-resolved peak with a retention time of approximately 5 minutes. An
example chromatogram for the lowest calibration standard at 2.5 ng/mL is shown in Figure 3a.
An example chromatogram of a feces control matrix sample is shown in Figure 3b (H-28548 was
not detected). A low level fortification quality control (QC) sample is shown in Figure 3c, which
was fortified at a level of 250 ng/g, and had a preparation factor of 6.14x. A 12 hour feces
sample from animal 001M, which had a total dilution factor of 34.0x is shown in Figure 3d. The
final concentration for this sample was 775 ng/g.
5. Fortification QC Sample Results
The average QC fortification results for the urine matrix are provided in Table 1. The average
recoveries for the low level, mid level, and high level fortification standards ranged from
101-102%. The associated coefficient of variation (CV) was 1% for each level and demonstrates
acceptable method performance.
The average QC fortification results for the feces matrix are provided in Table 2. The average
recoveries for the low level, mid level, and high level fortification standards ranged from
88-100%. The associated CV ranged from 2-4% and demonstrates acceptable method
performance.
B. Dose Formulation Concentration, Animal Body Weights, Dosing Information
(Table 3, Appendices A-B)
The concentration of H-28548 in the dose solution, as confirmed by LC/MS, was 0.29 mg
H-28548/mL, which was >96% of the nominal target (0.3 mg H-28548/mL).
At study initiation (day of dosing), males weighed 27.0 g ± 0.47 g and females weighed 24.5 g ±
0.52 g; the calculated dose rate for male (2.91 ± 0.03 mg/kg bw) and female mice (2.91 ± 0.06
mg/kg bw) were within 3% of the nominal target (3 mg/kg bw).
CH-FW-DEQ-0009470
H-28548:
Absorption, Distribution, Metabolism, and Elimination in the Mouse DuPont-18647-1017
- 20 -
C. Urine Data
(Table 4, Figure 4, Appendix C)
Following oral administration of H-28548 in water, 30.8% ± 5.37% and 39.3% ± 5.58% of the
administered dose (0-12 hours) was accounted for in urine from male and female mice,
respectively.
At the conclusion of the study (168 hours post-dose), the cumulative amount of H-28548
detected in urine was 89.5% ± 6.91% and 91.5% ± 6.04% for male and female mice,
respectively.
Elimination of H-28548 via urine accounted for the administered dose for both male and female
mice.
D. Feces Data
(Table 5, Figure 5, Appendix D)
Following oral administration of H-28548 in water, the cumulative amount of H-28548 detected
in feces over the entire collection period (0-168 hours) was 2.00% ± 1.01% and 1.91% ± 0.85%
for male and female mice, respectively.
The minor amount of H-28548 detected in feces was likely contamination from of urine. Given
the high levels of H-28548 in urine, and the design of the urine/feces collection system of the
metabolism units, feces likely became contaminated with small amounts of urine when
contacting surfaces in transit to the feces collection vessel.
E. Material Balance
(Table 6, Figure 6, Appendices E-F)
Following oral dosing with H-28548 in water and a 168 hour post-dose collection period,
101.2% ± 3.22% and 99.7% ± 2.95% of the administered dose was recovered from male and
female mice, respectively.
Of the total H-28548 recovered, the majority of administered dose was account for in urine from
both males (89.5% ± 6.91%) and females (91.5% ± 6.04%); lesser amounts of H-28548 were
accounted for in feces (male = 2.00% ± 1.01%; female = 1.91% ± 0.85%). Cagewash, which is
composed of dried excreta (urine and feces) accounted for 9.64% ± 3.99% and 6.25% ± 3.16% of
the administered dose for male and female mice, respectively.
The carcass and residual feed were not analyzed for H-28548 because analysis of urine, feces
and cagewash accounted for the majority of administered dose with an overall recovery of 100%
± 10%.
CH-FW-DEQ-0009471
H-28548:
Absorption, Distribution, Metabolism, and Elimination in the Mouse DuPont-18647-1017
- 21 -
F. Metabolite Identification
(Figures 7-9)
H-28548 was detected in its anionic form by negative ESI mass spectrometry. A representative
reconstructed chromatogram of ions characteristic of H-28548 (parent) for the 6 hour female
dosed mouse urine sample and control urine fortified with the H-28548 test substance is shown
in Figure 7.
The LC/MS mass spectrum of H-28548 in urine shows a significant amount of its proton bound
dimer (m/z 658.943 Da) and sodium bound dimer (m/z 680.923 Da) (Figure 8); the dimer and the
sodium dimer were created in the MS system and were not present in the sample itself. The
molecular anion (m/z 328.968) was observed in both urine from a mouse dosed with H-28548
and the urine fortified with the test substance H-28548, but at a low intensity relative to the
dimer adducts. These dimers are not to be confused with a covalent dimer, such as the HFPO
acid dimer parent, but are charged dimers sometimes formed, in-source, as a result of the
desolvation and ionization processes necessary to be observed by electrospray ionization mass
spectrometry.
The daughter ion mass spectra of the parent ion 328.97 Da for urine from a mouse dosed with
H-28548 and urine fortified with the H-28548 test substance shows the same 2 characteristic
fragment ions at m/z 284.977, the loss of CO2 and 169.989, [C3F7]- (Figure 9).
Subsequent to collection of the LC/MS, all sample data were screened for suspected metabolites
manually and automatically for unexpected metabolites using the IntelliExtractTM control-
comparison data processing tool. In all cases, there was no evidence of metabolism observed in
any of the samples by either method and only the anionic form of the residual parent, H-28548,
was detected.
CONCLUSIONS
Following oral administration of H-28548 in water, 30.8% ± 5.37% and 39.3% ± 5.58% of the
administered dose was accounted for in urine (0-12 hours) from male and female mice,
respectively. At the conclusion of the study (168 hours post-dose), the total accumulated amount
of H-28548 detected in urine was 89.5% ± 6.91% and 91.5% ± 6.04% of the administered dose
for male and female mice, respectively.
Elimination of H-28548 via urine accounted for a majority of the administered dose for both
male and female mice; minor levels of H-28548 detected in feces from male (2.00% ± 1.01%)
and female mice (1.91% ± 0.85%) were likely contamination from of urine.
Cagewash, which is composed of dried excreta (urine and feces), accounted for 9.64% ± 3.99%
and 6.25% ± 3.16% of the administered dose for male and female mice, respectively.
Following oral dosing with H-28548 in water and a 168 hour post-dose collection period,
101.2% ± 3.22% and 99.7% ± 2.95% of the administered dose was recovered from male and
female mice, respectively.
CH-FW-DEQ-0009472
H-28548:
Absorption, Distribution, Metabolism, and Elimination in the Mouse DuPont-18647-1017
- 22 -
Samples of urine evaluated using LC/MS were found to contain only the parent substance,
H-28548. This finding, taken with recovery of the administered dose in urine, confirms that
H-28548 was rapidly absorbed and eliminated unmetabolized following oral dosing in the
mouse.
RECORDS AND SAMPLE STORAGE
Specimens (if applicable), raw data, the protocol, amendments (if any), and the final report will
be retained at DuPont Haskell, Newark, Delaware, Iron Mountain Records Management,
Wilmington, Delaware, or Quality Associates Incorporated, Fulton, Maryland.
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 Rats with a
28-Day Recovery. Unpublished report, DuPont-24447.
CH-FW-DEQ-0009473
H-28548:
Absorption, Distribution, Metabolism, and Elimination in the Mouse DuPont-18647-1017
- 23 -
TABLES
CH-FW-DEQ-0009474
H-28548:
Absorption, Distribution, Metabolism, and Elimination in the Mouse DuPont-18647-1017
- 24 -
TABLES
EXPLANATORY NOTES
ABBREVIATIONS:

CV - coefficient of variation
NA - not applicable
QC - quality control
SD - standard deviation
CH-FW-DEQ-0009475
H-28548:
Absorption, Distribution, Metabolism, and Elimination in the Mouse DuPont-18647-1017
- 25 -
Table 1
Mouse urine sample fortification QC results for H-28548
Mouse Urine
Fortification
Sample
Fortification
Concentration
(ng/g)
Average
Recovery
(%)
CV
(%)

Low 400 102 1
Mid 100,000 100 1
High 1,000,000 101 1

Table 2
Mouse feces sample fortification QC result for H-28548
Mouse Feces
Fortification
Sample
Fortification
Concentration
(ng/g)
Average
Recovery
(%)
CV
(%)

Low 250 88 4
Mid 1250 95 4
High 50,000 100 2

Table 3
Dosing information
Males Females
Mean SD Mean SD

Subject weight (g) 27.0 0.47 24.5 0.52

Test substance received (mg) 0.079 0.001 0.071 0.002

Dose (mg/kg bw) 2.91 0.03 2.91 0.06

CH-FW-DEQ-0009476
H-28548:
Absorption, Distribution, Metabolism, and Elimination in the Mouse DuPont-18647-1017
- 26 -
Table 4
Urine, cumulative percent of dose
Post-Dose Time Point Males Females
(hours) Mean SD Mean SD

Pre-dose NA NA NA NA
6 14.1 5.19 17.2 4.41
12 30.8 5.37 39.3 5.58
24 54.9 6.26 61.4 5.99
48 72.7 8.10 77.9 5.58
72 80.0 7.22 84.3 6.64
96 84.1 7.12 87.7 6.67
120 86.5 7.16 89.5 6.55
144 88.2 7.14 90.7 6.22
168 89.5 6.91 91.5 6.04

Table 5
Feces, cumulative percent of dose
Post-Dose Time Point Males Females
(hours) Mean SD Mean SD

0 NA NA NA NA
6 0.31 0.4 NA NA
12 0.56 0.38 0.70 0.46
24 0.86 0.37 0.89 0.55
48 1.34 0.56 1.40 0.47
72 1.54 0.63 1.63 0.58
96 1.71 0.73 1.70 0.60
120 1.80 0.78 1.80 0.71
144 1.89 0.89 1.89 0.82
168 2.00 1.01 1.91 0.85

Table 6
Material balance, percent of dose
Males Females
Mean SD Mean SD

Urine 89.5 6.91 91.5 6.04
Feces 2.00 1.01 1.91 0.85
Cage Wash 9.64 3.99 6.25 3.16
Total 101.2 3.22 99.7 2.95

CH-FW-DEQ-0009477
H-28548:
Absorption, Distribution, Metabolism, and Elimination in the Mouse DuPont-18647-1017
- 27 -
FIGURES
CH-FW-DEQ-0009478
H-28548:
Absorption, Distribution, Metabolism, and Elimination in the Mouse DuPont-18647-1017
- 28 -
FIGURES
EXPLANATORY NOTES
ABBREVIATIONS:

QC - quality control
cps - counts per second
m/z - mass-to-charge ratio
min - minute
CH-FW-DEQ-0009479
H-28548:
Absorption, Distribution, Metabolism, and Elimination in the Mouse DuPont-18647-1017
- 29 -
Figure 1
Calibration curve for H-28548

CH-FW-DEQ-0009480
H-28548:
Absorption, Distribution, Metabolism, and Elimination in the Mouse DuPont-18647-1017
- 30 -
Figure 2
The LC/MS/MS chromatograms for a) lowest calibration standard at 2.5 ng/mL, b) urine control
matrix sample, c) low level 400 ng/g fortification QC sample with preparation factor 40x, and d)
a 24-hour urine study sample from animal 001M, which had a total dilution factor of 1480x and
final concentration of 14,800 ng/g
a)

b)

c)

d)

CH-FW-DEQ-0009481
H-28548:
Absorption, Distribution, Metabolism, and Elimination in the Mouse DuPont-18647-1017
- 31 -
Figure 3
The LC/MS/MS chromatograms for a) lowest calibration standard at 2.5 ng/mL, b) feces control
matrix sample, c) low level 250 ng/g fortification QC sample that had a preparation factor of
6.14x, and d) a 12-hour feces study sample from animal 001M, which had a 34x dilution factor
and final concentration of 775 ng/g
a)

b)

c)

d)

CH-FW-DEQ-0009482
H-28548:
Absorption, Distribution, Metabolism, and Elimination in the Mouse DuPont-18647-1017
- 32 -
Figure 4
Urine, cumulative percent
0
10
20
30
40
50
60
70
80
90
100
0 24487296120144168192
hours post dosecumulative percent of dose in urinemales
females

CH-FW-DEQ-0009483
H-28548:
Absorption, Distribution, Metabolism, and Elimination in the Mouse DuPont-18647-1017
- 33 -
Figure 5
Feces, cumulative percent
0.0
0.5
1.0
1.5
2.0
2.5
3.0
0 24 48 72 96 120 144 168 192
hours post dosecumulative percent of dose in fecesmales
females

CH-FW-DEQ-0009484
H-28548:
Absorption, Distribution, Metabolism, and Elimination in the Mouse DuPont-18647-1017
- 34 -
Figure 6
Material Balance, percent of dose
0
10
20
30
40
50
60
70
80
90
100
110
urine feces cage wash TotalPercent of doseMales
Females

CH-FW-DEQ-0009485
H-28548:
Absorption, Distribution, Metabolism, and Elimination in the Mouse DuPont-18647-1017
- 35 -
Figure 7
Reconstructed m/z 329 + 659 ion chromatograms characteristic of H-28548-dosed female mouse
urine (6 hours after administration) – top and control mouse urine fortified with H-28458 test
substance -bottom

CH-FW-DEQ-0009486
H-28548:
Absorption, Distribution, Metabolism, and Elimination in the Mouse DuPont-18647-1017
- 36 -
Figure 8
ESI negative mass spectra of H-28548 observed in dosed female mouse urine (6 hours after
administration)–top; and control urine fortified with H-28548 test substance – bottom

CH-FW-DEQ-0009487
H-28548:
Absorption, Distribution, Metabolism, and Elimination in the Mouse DuPont-18647-1017
- 37 -
Figure 9
ESI negative daughter ion mass spectra of H28548 observed in dosed female mouse urine
(6 hours after administration)–top; and control mouse urine fortified with H-28548 test substance
– bottom

CH-FW-DEQ-0009488
H-28548:
Absorption, Distribution, Metabolism, and Elimination in the Mouse DuPont-18647-1017
- 38 -
APPENDICES
CH-FW-DEQ-0009489
H-28548:
Absorption, Distribution, Metabolism, and Elimination in the Mouse DuPont-18647-1017
- 39 -
APPENDICES
EXPLANATORY NOTES
ABBREVIATIONS:

F - female
h - hours
LOQ - limit of quantification
M - male
NA - not applicable
ND - not detected
SD - standard deviation
CH-FW-DEQ-0009490
H-28548:
Absorption, Distribution, Metabolism, and Elimination in the Mouse DuPont-18647-1017
- 40 -
Appendix A
Certificate of Analysis
CH-FW-DEQ-0009491
H-28548:
Absorption, Distribution, Metabolism, and Elimination in the Mouse DuPont-18647-1017
- 41 -

CH-FW-DEQ-0009492
H-28548:
Absorption, Distribution, Metabolism, and Elimination in the Mouse DuPont-18647-1017
- 42 -
Appendix B
Dosing Information
CH-FW-DEQ-0009493
H-28548:
Absorption, Distribution, Metabolism, and Elimination in the Mouse DuPont-18647-1017
- 43 -

Dosing Information

Males
Subject
Subject
weight
(g)
Compound
received
(mg)
Dose
rate
(mg/kg)

001M 27.7 0.081 2.91
002M 27.2 0.079 2.90
003M 26.8 0.077 2.89
004M 26.6 0.077 2.90
005M 26.6 0.079 2.95
Mean 27.0 0.079 2.91
SD 0.47 0.001 0.03

Females
Subject
Subject
weight
(g)
Compound
received
(mg)
Dose
rate
(mg/kg)

001F 24.3 0.073 3.01
002F 24.2 0.070 2.88
003F 24.5 0.070 2.85
004F 24.1 0.071 2.94
005F 25.4 0.073 2.89
Mean 24.5 0.071 2.91
SD 0.52 0.002 0.06
CH-FW-DEQ-0009494
H-28548:
Absorption, Distribution, Metabolism, and Elimination in the Mouse DuPont-18647-1017
- 44 -
Appendix C
Urine Data
CH-FW-DEQ-0009495
H-28548:
Absorption, Distribution, Metabolism, and Elimination in the Mouse DuPont-18647-1017
- 45 -

Urine Data - Males

Animal
Number
Total
H-28548
(ng)
Timepoint
(hours)
Sample
weight (g)
Concentration
H-28548
(ng/g)
Total
Amount
(ng H-28548) Percent
Cumulative
(%)

001M 80620 Pre-dose 1.23 ND NA NA NA
6 h 0.467 36000 16812 20.9 20.9
12 h 0.24 31300 7512 9.32 30.2
24 h 1.608 14800 23798 29.5 59.7
48 h 1.672 8060 13476 16.7 76.4
72 h 1.794 2780 4987 6.19 82.6
96 h 1.907 1720 3280 4.07 86.7
120 h 1.795 1070 1921 2.38 89.0
144 h 1.58 722 1141 1.41 90.5
168 h 1.798 537 966 1.20 91.7
91.7

Animal
Number
Total
H-28548
(ng)
Timepoint
(hours)
Sample
weight (g)
Concentration
H-28548
(ng/g)
Total Amount
(ng H-28548) Percent
Cumulative
(%)

002M 78880 Pre-dose 2.155 ND NA NA NA
6 h 0.599 24300 14556 18.5 18.5
12 h 0.311 32900 10232 13.0 31.4
24 h 1.403 12000 16836 21.3 52.8
48 h 2.12 6600 13992 17.7 70.5
72 h 2.489 2720 6770 8.58 79.1
96 h 2.833 1240 3513 4.45 83.5
120 h 2.866 755 2164 2.74 86.3
144 h 2.595 506 1313 1.66 88.0
168 h 3.139 326 1023 1.30 89.2
89.2

Animal
Number
Total
H-28548
(ng)
Timepoint
(hours)
Sample
weight (g)
Concentration
H-28548
(ng/g)
Total Amount
(ng H-28548) Percent
Cumulative
(%)

003M 77430 Pre-dose 1.441 ND NA NA NA
6 h 0.312 29100 9079 11.7 11.7
12 h 0.576 27300 15725 20.3 32.0
24 h 0.95 18200 17290 22.3 54.4
48 h 1.656 11500 19044 24.6 79.0
72 h 1.275 3940 5024 6.49 85.4
96 h 1.375 2460 3383 4.37 89.8
120 h 1.191 1600 1906 2.46 92.3
144 h 1.334 1020 1361 1.76 94.0
168 h 1.469 669 983 1.27 95.3
95.3
CH-FW-DEQ-0009496
H-28548:
Absorption, Distribution, Metabolism, and Elimination in the Mouse DuPont-18647-1017
- 46 -

Urine Data - Males

Animal
Number
Total
H-28548
(ng)
Timepoint
(hours)
Sample
weight (g)
Concentration
H-28548
(ng/g)
Total
Amount
(ng H-28548) Percent
Cumulative
(%)

004M 77140 Pre-dose 1.291 ND NA NA NA
6 h 0.27 26300 7101 9.2 9.21
12 h 0.227 45800 10397 13.5 22.7
24 h 0.897 19900 17850 23.1 45.8
48 h 0.917 11500 10546 13.7 59.5
72 h 1.385 4650 6440 8.35 67.8
96 h 1.315 2470 3248 4.21 72.1
120 h 0.803 2170 1743 2.26 74.3
144 h 1.236 1140 1409 1.83 76.1
168 h 1.43 924 1321 1.71 77.9
77.9

Animal
Number
Total
H-28548
(ng)
Timepoint
(hours)
Sample
weight (g)
Concentration
H-28548
(ng/g)
Total Amount
(ng H-28548) Percent
Cumulative
(%)

005M 78590 Pre-dose 1.621 ND NA NA NA
6 h 0.176 46600 8202 10.4 10.4
12 h 0.366 58500 21411 27.2 37.7
24 h 0.883 21400 18896 24.0 61.7
48 h 1.515 8560 12968 16.5 78.2
72 h 1.432 3620 5184 6.60 84.8
96 h 1.318 2100 2768 3.52 88.3
120 h 1.76 1010 1778 2.26 90.6
144 h 1.995 781 1558 1.98 92.6
168 h 2.318 358 830 1.06 93.6
93.6

Timepoint
(hours)
Cumulative
Mean SD

0 h NA NA
6 h 14.1 5.2
12 h 30.8 5.37
24 h 54.9 6.26
48 h 72.7 8.10
72 h 80.0 7.22
96 h 84.1 7.12
120 h 86.5 7.16
144 h 88.2 7.14
168 h 89.5 6.91
CH-FW-DEQ-0009497
H-28548:
Absorption, Distribution, Metabolism, and Elimination in the Mouse DuPont-18647-1017
- 47 -

Urine Data - Females

Animal
Number
Total
H-28548
(ng)
Timepoint
(hours)
Sample
weight (g)
Concentration
H-28548
(ng/g)
Total
Amount
(ng H-28548) Percent
Cumulative
(%)

001F 73080 Pre-dose 1.165 ND NA NA NA
6 h 0.318 28400 9031 12.4 12.4
12 h 0.305 57500 17538 24.0 36.4
24 h 0.999 19600 19580 26.8 63.1
48 h 2.114 6260 13234 18.11 81.3
72 h 1.355 2780 3767 5.15 86.4
96 h 2.392 1230 2942 4.03 90.4
120 h 2.333 486 1134 1.55 92.0
144 h 2.149 270 580 0.79 92.8
168 h 2.425 183 444 0.61 93.4
93.4

Animal
Number
Total
H-28548
(ng)
Timepoint
(hours)
Sample
weight (g)
Concentration
H-28548
(ng/g)
Total Amount
(ng H-28548) Percent
Cumulative
(%)

002F 69600 Pre-dose 1.164 ND NA NA NA
6 h 0.414 32500 13455 19.3 19.3
12 h 0.29 45800 13282 19.1 38.4
24 h 0.958 15600 14945 21.5 59.9
48 h 1.856 6580 12212 17.5 77.4
72 h 1.442 2490 3591 5.16 82.6
96 h 1.931 1100 2124 3.05 85.6
120 h 2.51 476 1195 1.72 87.4
144 h 2.853 501 1429 2.05 89.4
168 h 1.959 443 868 1.25 90.7
90.7

Animal
Number
Total
H-28548
(ng)
Timepoint
(hours)
Sample
weight (g)
Concentration
H-28548
(ng/g)
Total Amount
(ng H-28548) Percent
Cumulative
(%)

003F 69890 Pre-dose 2.575 ND NA NA NA
6 h 0.287 55400 15900 22.7 22.7
12 h 0.41 45000 18450 26.4 49.1
24 h 0.873 17300 15103 21.6 70.8
48 h 1.975 4540 8967 12.8 83.6
72 h 2.041 2580 5266 7.53 91.1
96 h 2.368 736 1743 2.49 93.6
120 h 3.804 334 1271 1.82 95.4
144 h 2.232 231 516 0.74 96.2
168 h 1.971 175 345 0.49 96.7
96.7
CH-FW-DEQ-0009498
H-28548:
Absorption, Distribution, Metabolism, and Elimination in the Mouse DuPont-18647-1017
- 48 -

Urine Data - Females

Animal
Number
Total
H-28548
(ng)
Timepoint
(hours)
Sample
weight (g)
Concentration
H-28548
(ng/g)
Total
Amount
(ng H-28548) Percent
Cumulative
(%)

004F 70760 Pre-dose 0.706 ND NA NA NA
6 h 0.481 27200 13083 18.5 18.5
12 h 0.251 50000 12550 17.7 36.2
24 h 0.642 20800 13354 18.9 55.1
48 h 1.447 6760 9782 13.8 68.9
72 h 1.384 2490 3446 4.87 73.8
96 h 1.433 1600 2293 3.24 77.0
120 h 1.553 909 1412 2.00 79.0
144 h 1.718 603 1036 1.46 80.5
168 h 1.52 484 736 1.04 81.5
81.5

Animal
Number
Total
H-28548
(ng)
Timepoint
(hours)
Sample
weight (g)
Concentration
H-28548
(ng/g)
Total Amount
(ng H-28548) Percent
Cumulative
(%)

005F 73370 Pre-dose 1.152 ND NA NA NA
6 h 0.266 35900 9549 13.0 13.0
12 h 0.483 35500 17147 23.4 36.4
24 h 1.092 14600 15943 21.7 58.1
48 h 2.702 5510 14888 20.3 78.4
72 h 4.6 1490 6854 9.34 87.7
96 h 5.534 548 3033 4.13 91.9
120 h 3.029 396 1199 1.63 93.5
144 h 2.981 254 757 1.03 94.5
168 h 3.908 169 660 0.90 95.4
95.4

Timepoint
(hours)
Cumulative
Mean SD

0 h NA NA
6 h 17.2 4.4
12 h 39.3 5.58
24 h 61.4 5.99
48 h 77.9 5.58
72 h 84.3 6.64
96 h 87.7 6.67
120 h 89.5 6.55
144 h 90.7 6.22
168 h 91.5 6.04
CH-FW-DEQ-0009499
H-28548:
Absorption, Distribution, Metabolism, and Elimination in the Mouse DuPont-18647-1017
- 49 -
Appendix D
Feces Data
CH-FW-DEQ-0009500
H-28548:
Absorption, Distribution, Metabolism, and Elimination in the Mouse DuPont-18647-1017
- 50 -

Feces Data - Males

Animal
Number
Total
H-28548
(ng)
Timepoint
(hours)
Sample
weight (g)
Concentration
H-28548
(ng/g)
Total
Amount
(ng H-28548) Percent
Cumulative
(%)

001M 80620 0h 1.683 ND NA NA NA
6 h 0.307 512 157 0.19 0.19
12 h 0.396 775 307 0.38 0.58
24 h 2.099 95.1 200 0.25 0.82
48 h 2.381 54.7 130 0.16 0.98
72 h 2.805 28.9 81 0.10 1.09
96 h 2.463 13.5 33 0.04 1.13
120 h 2.869 <11.1 NA NA 1.13
144 h 2.447 <13.0 NA NA 1.13
168 h 2.563 <12.5 NA NA 1.13
1.13

Animal
Number
Total
H-28548
(ng)
Timepoint
(hours)
Sample
weight (g)
Concentration
H-28548
(ng/g)
Total Amount
(ng H-28548) Percent
Cumulative
(%)

002M 78880 0h 1.478 ND NA NA NA
6 h 0.367 122 45 0.06 0.06
12 h 0.701 149 104 0.13 0.19
24 h 1.694 88.9 151 0.19 0.38
48 h 3.18 48.4 154 0.20 0.58
72 h 3.207 29.5 95 0.12 0.70
96 h 3.302 29.4 97 0.12 0.82
120 h 3.173 24.6 78 0.10 0.92
144 h 2.993 12.3 37 0.05 0.96
168 h 3.291 19.7 65 0.08 1.05
1.05

Animal
Number
Total
H-28548
(ng)
Timepoint
(hours)
Sample
weight (g)
Concentration
H-28548
(ng/g)
Total Amount
(ng H-28548) Percent
Cumulative
(%)

003M 77430 0h 1.598 ND NA NA NA
6 h 0.626 1320 826 1.07 1.07
12 h 0.553 133 74 0.09 1.16
24 h 1.533 56.4 86 0.11 1.27
48 h 3.188 50.8 162 0.21 1.48
72 h 3.226 110 355 0.46 1.94
96 h 3.354 69.2 232 0.30 2.24
120 h 3.243 33.3 108 0.14 2.38
144 h 3.262 <9.88 NA NA 2.38
168 h 3.563 <8.95 NA NA 2.38
2.38
CH-FW-DEQ-0009501
H-28548:
Absorption, Distribution, Metabolism, and Elimination in the Mouse DuPont-18647-1017
- 51 -

Feces Data - Males

Animal
Number
Total
H-28548
(ng)
Timepoint
(hours)
Sample
weight (g)
Concentration
H-28548
(ng/g)
Total
Amount
(ng H-28548) Percent
Cumulative
(%)

004M 77140 0h 1.716 ND NA NA NA
6 h 0.432 265 114 0.15 0.15
12 h 0.945 122 115 0.15 0.30
24 h 2.18 120 262 0.34 0.64
48 h 3.803 275 1046 1.36 1.99
72 h 3.745 43.5 163 0.21 2.20
96 h 3.744 73.9 277 0.36 2.56
120 h 3.734 30.7 115 0.15 2.71
144 h 3.682 83.5 307 0.40 3.11
168 h 4.069 76.6 312 0.40 3.51
3.51

Animal
Number
Total
H-28548
(ng)
Timepoint
(hours)
Sample
weight (g)
Concentration
H-28548
(ng/g)
Total Amount
(ng H-28548) Percent
Cumulative
(%)

005M 78590 0h 1.971 ND NA NA NA
6 h 0.309 194 60 0.076 0.08
12 h 0.713 554 395 0.50 0.58
24 h 1.718 274 471 0.60 1.18
48 h 3.088 126 389 0.50 1.67
72 h 2.916 20.7 60 0.08 1.75
96 h 2.943 19.6 58 0.073 1.82
120 h 3.199 11.5 37 0.047 1.87
144 h 3.159 <10.2 NA NA 1.87
168 h 3.358 11.1 37 0.047 1.92
1.92

Timepoint
(hours)
Cumulative
Mean SD

0 h NA NA
6 h 0.31 0.43
12 h 0.56 0.38
24 h 0.86 0.37
48 h 1.34 0.56
72 h 1.54 0.63
96 h 1.71 0.73
120 h 1.80 0.78
144 h 1.89 0.89
168 h 2.00 1.01
CH-FW-DEQ-0009502
H-28548:
Absorption, Distribution, Metabolism, and Elimination in the Mouse DuPont-18647-1017
- 52 -

Feces Data - Females

Animal
Number
Total
H-28548
(ng)
Timepoint
(hours)
Sample
weight (g)
Concentration
H-28548
(ng/g)
Total
Amount
(ng H-28548) Percent
Cumulative
(%)

001F 73808 0h 1.685 ND NA NA NA
6 h 0.309 663 205 0.28 0.28
12 h 0.544 97.4 53 0.07 0.35
24 h 1.624 75.6 123 0.17 0.52
48 h 3.620 99.2 359 0.49 1.01
72 h 3.480 19.7 69 0.09 1.11
96 h 3.748 27.9 105 0.14 1.25
120 h 3.428 <10.4 NA NA 1.25
144 h 3.082 <10.5 NA NA 1.25
168 h 3.765 <8.53 NA NA 1.25
1.25

Animal
Number
Total
H-28548
(ng)
Timepoint
(hours)
Sample
weight (g)
Concentration
H-28548
(ng/g)
Total Amount
(ng H-28548) Percent
Cumulative
(%)

002F 69600 0h 1.714 ND NA NA NA
6 h 0.494 1640 810 1.16 1.16
12 h 0.421 107 45 0.06 1.23
24 h 1.454 152 221 0.32 1.55
48 h 3.517 37 130 0.19 1.73
72 h 3.279 90.5 297 0.43 2.16
96 h 3.457 17.9 62 0.09 2.25
120 h 3.576 58.8 210 0.30 2.55
144 h 3.767 48.1 181 0.26 2.81
168 h 3.093 13.6 42 0.06 2.87
2.87

Animal
Number
Total
H-28548
(ng)
Timepoint
(hours)
Sample
weight (g)
Concentration
H-28548
(ng/g)
Total Amount
(ng H-28548) Percent
Cumulative
(%)

003F 69890 0h 1.635 ND NA NA NA
6 h 0.303 182 55 0.08 0.08
12 h 0.5 101 51 0.07 0.15
24 h 1.178 45.9 54 0.08 0.23
48 h 2.807 191 536 0.77 1.00
72 h 3.128 22.2 69 0.10 1.09
96 h 3.01 <10.5 NA NA 1.09
120 h 3.452 10.9 38 0.05 1.15
144 h 2.569 <12.3 NA NA 1.15
168 h 3.105 <10.1 NA NA 1.15
1.15
CH-FW-DEQ-0009503
H-28548:
Absorption, Distribution, Metabolism, and Elimination in the Mouse DuPont-18647-1017
- 53 -

Feces Data - Females

Animal
Number
Total
H-28548
(ng)
Timepoint
(hours)
Sample
weight (g)
Concentration
H-28548
(ng/g)
Total
Amount
(ng H-28548) Percent
Cumulative
(%)

004F 70760 0h 1.604 ND NA NA NA
6 h 0.5 592 296 0.42 0.42
12 h 0.895 524 469 0.66 1.08
24 h 1.283 134 172 0.24 1.32
48 h 3.21 158 507 0.72 2.04
72 h 3.42 57.5 197 0.28 2.32
96 h 3.845 18.3 70 0.10 2.42
120 h 3.698 29.7 110 0.16 2.57
144 h 3.603 32.8 118 0.17 2.74
168 h 3.898 11.7 46 0.06 2.80
2.80

Animal
Number
Total
H-28548
(ng)
Timepoint
(hours)
Sample
weight (g)
Concentration
H-28548
(ng/g)
Total Amount
(ng H-28548) Percent
Cumulative
(%)

005F 73370 0h 2.015 ND NA NA NA
6 h 0.448 536 240 0.33 0.33
12 h 1.051 256 269 0.37 0.69
24 h 1.855 63.2 117 0.16 0.85
48 h 3.817 67.5 258 0.35 1.20
72 h 3.367 60.7 204 0.28 1.48
96 h 3.699 <8.35 NA NA 1.48
120 h 3.217 <9.75 NA NA 1.48
144 h 3.495 <9.20 NA NA 1.48
168 h 3.346 <9.63 NA NA 1.48
1.48

Timepoint
(hours)
Cumulative
Mean SD

0 h NA NA
6 h NA NA
12 h 0.70 0.46
24 h 0.89 0.55
48 h 1.40 0.47
72 h 1.63 0.58
96 h 1.70 0.60
120 h 1.80 0.71
144 h 1.89 0.82
168 h 1.91 0.85
CH-FW-DEQ-0009504
H-28548:
Absorption, Distribution, Metabolism, and Elimination in the Mouse DuPont-18647-1017
- 54 -
Appendix E
Cage Wash Data
CH-FW-DEQ-0009505
H-28548:
Absorption, Distribution, Metabolism, and Elimination in the Mouse DuPont-18647-1017
- 55 -

Cage Wash Data - 168 hours

Animal
Number
Total
H-28548
(ng)
Timepoint
(hours)
Sample
Weight
(g)
Concentration
H-28548
(ng/g)
Total Amount
(ng H-28548) Percent

001M 80620 168 h 274.506 32.3 8867 11.0
002M 78880 168 h 274.514 30.8 8455 10.7
003M 77430 168 h 214.319 24.4 5229 6.75
004M 77140 168 h 207.445 55.7 11555 15.0
005M 78590 168 h 214.205 17.4 3727 4.74

Mean 9.64
SD 3.99

Animal
Number
Total
H-28548
(ng)
Timepoint
(hours)
Sample
Weight
(g)
Concentration
H-28548
(ng/g)
Total Amount
(ng H-28548) Percent

001F 73080 168 h 227.935 12.9 2940 4.02
002F 69600 168 h 230.137 14.4 3314 4.76
003F 69890 168 h 257.292 15.3 3937 5.63
004F 70760 168 h 212.796 39.3 8363 11.8
005F 73370 168 h 244.357 15.1 3690 5.03

Mean 6.25
SD 3.16
CH-FW-DEQ-0009506
H-28548:
Absorption, Distribution, Metabolism, and Elimination in the Mouse DuPont-18647-1017
- 56 -
Appendix F
Material Balance
CH-FW-DEQ-0009507
H-28548:
Absorption, Distribution, Metabolism, and Elimination in the Mouse DuPont-18647-1017
- 57 -

Material Balance

001M 002M 003M 004M 005M Mean SD

urine 6 h 20.9 18.5 11.7 9.21 10.4 14.1 5.19
urine 12 h 9.32 13.0 20.3 13.5 27.2 16.7 7.12
urine 24 h 29.5 21.3 22.3 23.1 24.0 24.1 3.20
urine 48 h 16.7 17.7 24.6 13.7 16.5 17.8 4.06
urine 72 h 6.19 8.58 6.49 8.35 6.60 7.24 1.13
urine 96 h 4.07 4.45 4.37 4.21 3.52 4.12 0.37
urine 120 h 2.38 2.74 2.46 2.26 2.26 2.42 0.20
urine 144 h 1.41 1.66 1.76 1.83 1.98 1.73 0.21
urine 168 h 1.20 1.30 1.27 1.71 1.06 1.31 0.25
Subtotal 91.7 89.2 95.3 77.9 93.6 89.5 6.91

feces 6 h 0.19 0.06 1.07 0.15 0.08 0.31 0.43
feces 12 h 0.38 0.13 0.09 0.15 0.50 0.25 0.18
feces 24 h 0.25 0.19 0.11 0.34 0.60 0.30 0.19
feces 48 h 0.16 0.20 0.21 1.36 0.50 0.48 0.51
feces 72 h 0.10 0.12 0.458 0.21 0.08 0.19 0.16
feces 96 h 0.04 0.12 0.30 0.36 0.073 0.18 0.14
feces 120 h <LOQ 0.10 0.14 0.15 0.047 0.11 0.05
feces 144 h <LOQ 0.05 <LOQ 0.399 <LOQ 0.22 0.25
feces 168 h <LOQ 0.08 <LOQ 0.40 0.05 0.18 0.20
Subtotal 1.13 1.05 2.38 3.51 1.92 2.00 1.01

cage wash 168 h 11.00 10.72 6.75 14.98 4.74 9.64 3.99

Total 103.8 101.0 104.4 96.3 100.3 101.2 3.22

001F 002F 003F 004F 005F Mean SD

urine 6 h 12.4 19.3 22.7 18.5 13.0 17.2 4.41
urine 12 h 24.0 19.1 26.4 17.7 23.4 22.1 3.60
urine 24 h 26.8 21.5 21.6 18.9 21.7 22.1 2.88
urine 48 h 18.11 17.5 12.8 13.8 20.3 16.5 3.11
urine 72 h 5.15 5.16 7.53 4.87 9.34 6.41 1.96
urine 96 h 4.03 3.05 2.49 3.24 4.13 3.39 0.69
urine 120 h 1.55 1.72 1.82 2.00 1.63 1.74 0.17
urine 144 h 0.79 2.05 0.74 1.46 1.03 1.22 0.55
urine 168 h 0.61 1.25 0.49 1.04 0.90 0.86 0.31
Subtotal 93.4 90.7 96.7 81.5 95.4 91.5 6.04

feces 6 h 0.28 1.16 0.08 0.42 0.33 0.45 NA
feces 12 h 0.07 0.06 0.07 0.66 0.37 0.25 0.27
feces 24 h 0.17 0.32 0.08 0.24 0.16 0.19 0.09
feces 48 h 0.49 0.19 0.77 0.72 0.35 0.50 0.24
feces 72 h 0.09 0.43 0.10 0.28 0.28 0.24 0.14
feces 96 h 0.14 0.09 <LOQ 0.10 <LOQ 0.11 0.03
feces 120 h <LOQ 0.302 0.05 0.16 <LOQ 0.17 0.12
feces 144 h <LOQ 0.260 <LOQ 0.17 <LOQ 0.21 0.07
feces 168 h <LOQ 0.060 <LOQ 0.064 <LOQ 0.06 0.00
Subtotal 1.25 2.87 1.15 2.80 1.48 1.91 0.85

cage wash 168 h 4.02 4.76 5.63 11.8 5.03 6.25 3.16

Total 98.7 98.3 103.4 96.2 102.0 99.7 2.95

CH-FW-DEQ-0009508