CH-FW-DEQ-0009509

DuPont-18405-1017
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Unpublished Work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 Rat

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-18405-1017

WORK REQUEST NUMBER: 18405

SERVICE CODE NUMBER: 1017

SPONSOR: E.I. du Pont de Nemours and Company
Wilmington, Delaware 19898
U.S.A.
Page 1 of 57
CH-FW-DEQ-0009509
H-28548:
Absorption, Distribution, Metabolism, and Elimination in the Rat DuPont-18405-1017
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.

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CH-FW-DEQ-0009510
H-28548:
Absorption, Distribution, Metabolism, and Elimination in the Rat DuPont-18405-1017
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 29, 2010 March 29, 2010 March 29, 2010
May 24, 2010 May 24, 2010 May 24, 2010
‘
Report/Records:
October 04-07,13, 2010 October 13, 2010 October 14, 2010

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

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CH-FW-DEQ-0009511
H-28548:
Absorption, Distribution, Metabolism, and Elimination in the Rat DuPont-18405-1017
CERTIFICATION
We, the undersigned, declare that this report provides an accurate evaluation of data obtained
from this study.

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CH-FW-DEQ-0009512
H-28548:
Absorption, Distribution, Metabolism, and Elimination in the Rat DuPont-18405-1017
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............................................................................................15
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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CH-FW-DEQ-0009513
H-28548:
Absorption, Distribution, Metabolism, and Elimination in the Rat DuPont-18405-1017
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 Rat urine sample fortification QC results for H-28548............................................................25
Table 2 Rat 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 1540x and final concentration of 34700 ng/g...............................................30
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CH-FW-DEQ-0009514
H-28548:
Absorption, Distribution, Metabolism, and Elimination in the Rat DuPont-18405-1017
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 20x, and d) a 12-hour feces study sample from animal 001M, which
had a total 336x dilution factor and final concentration of 2750 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
rat urine (6 hours after administration) – top and control rat urine fortified with H-28458
test substance -bottom..............................................................................................................35
Figure 8 ESI negative mass spectra of H-28548 observed in dosed female rat 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 rat urine
(6 hours after administration)–top; and control rat 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

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CH-FW-DEQ-0009515
H-28548:
Absorption, Distribution, Metabolism, and Elimination in the Rat DuPont-18405-1017
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 23, 2010 / July 1, 2010

In-Life Initiated/Completed: March 23, 2010 / March 30, 2010

Notebook Number(s): E-114321-AH, E-98524-GF, E-114321-AL
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CH-FW-DEQ-0009516
H-28548:
Absorption, Distribution, Metabolism, and Elimination in the Rat DuPont-18405-1017
SUMMARY
The absorption, distribution, metabolism, and elimination of H-28548 were investigated in the
Sprague-Dawley rat. H-28548 was administered in water to 5 male and 5 female rats as a single
oral dose at a target dose level of 30 mg H-28548/kg bodyweight (bw) and a dose volume of
4 mL/kg bw. Rats were housed individually in glass 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, rats 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, 96.6% ± 1.43% and 94.6% ± 8.57% of the
administered dose was accounted for in urine (0-12 hours) from male and female rats,
respectively. At the conclusion of the study (168 hours post-dose), the total accumulated amount
of H-28548 detected in urine was 103% ± 2.73% and 99.8% ± 6.41% of the administered dose
for male and female rats, respectively.
Elimination of H-28548 via urine was rapid and accounted for a majority of the administered
dose for both male and female rats; negligible levels of H-28548 detected in feces from male
(1.35% ± 1.05%) and female rats (0.85% ± 0.58%), were likely contamination from urine.
Cagewash, which is composed of dried excreta (urine and feces), accounted for 0.98% ± 0.52%
and 5.03% ± 5.14% of the administered dose for male and female rats, respectively.
Following oral dosing with H-28548 in water and a 168 hour post-dose collection period,
105.3% ± 2.19% and 105.7% ± 1.42% of the administered dose was recovered from male and
female rats, respectively.
Samples of urine evaluated using LC/MS were found to contain only the parent substance,
H-28548. This finding, taken with the complete recovery of the administered dose in urine,
confirms that H-28548 was rapidly absorbed and eliminated unmetabolized following oral
dosing in the rat.
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CH-FW-DEQ-0009517
H-28548:
Absorption, Distribution, Metabolism, and Elimination in the Rat DuPont-18405-1017
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 rat.
OBJECTIVE
The objective of this study was to determine the ADME of H-28548 in the rat 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 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:
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CH-FW-DEQ-0009518
H-28548:
Absorption, Distribution, Metabolism, and Elimination in the Rat DuPont-18405-1017
• 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:CD(SD) rats were obtained from Charles River Laboratories, Inc. (Raleigh,
North Carolina, U.S.A.).
The Sprague-Dawley rat 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 Sprague-Dawley rat 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.
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CH-FW-DEQ-0009519
H-28548:
Absorption, Distribution, Metabolism, and Elimination in the Rat DuPont-18405-1017
• 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 rats were housed in quarantine. The rats 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 178.5 mg) and mixed with deionized water
(20 mL). The dose solution was prepared at a nominal concentration of 7.5 mg H-28548/mL
(adjusted for purity, 84%), with a target dose level of 30 mg/kg body weight (bw) and a dose
volume of 4 mL/kg bw. The dose level was chosen based on the results of the 28-day daily oral
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CH-FW-DEQ-0009520
H-28548:
Absorption, Distribution, Metabolism, and Elimination in the Rat DuPont-18405-1017
dosing study in rats, where the no-observed-adverse-effect level (NOAEL) was 30 and 300
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).
Rats were housed individually in glass metabolism units and fasted for approximately 16 hours
prior to dosing. Food was returned approximately 2 hours post-dose.
Five male and 5 female rats were administered H-28548 at a nominal target of 30 mg
H-28548/kg bw. Two male and 2 female rats were each administered dose vehicle (deionized
water at 4 mL/kg bw) for collection of control excreta and tissue samples. Rats 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), rats 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.
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CH-FW-DEQ-0009521
H-28548:
Absorption, Distribution, Metabolism, and Elimination in the Rat DuPont-18405-1017
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 50-mL conical polypropylene centrifuge tubes were
thawed to room temperature. HPLC grade water was added to the 40-mL mark, and the weight
of water added was recorded to the nearest 0.1 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 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
using 2 grams of control feces, and prepared for analysis using the same procedure.
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CH-FW-DEQ-0009522
H-28548:
Absorption, Distribution, Metabolism, and Elimination in the Rat DuPont-18405-1017
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,
and 250 ng/mL levels.
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
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CH-FW-DEQ-0009523
H-28548:
Absorption, Distribution, Metabolism, and Elimination in the Rat DuPont-18405-1017
Curtain Gas Flow (CUR): 10.0
GS1: 25
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: 0-6, 6-12 and 12-24 hours; females: 0-6 and
6-12 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 1).
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
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CH-FW-DEQ-0009524
H-28548:
Absorption, Distribution, Metabolism, and Elimination in the Rat DuPont-18405-1017
Column Temperature: 25°C
Solvent A: 0.10% Acetic Acid in HPLC grade water
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.
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CH-FW-DEQ-0009525
H-28548:
Absorption, Distribution, Metabolism, and Elimination in the Rat DuPont-18405-1017
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
0.4 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) = 0.0279 g
• Sample Preparation Factor = 1 / 0.0279 = 35.8
• Final LOD for this sample = 0.1 ng/g x 35.8 = 4 ng/g (reported to 1 significant digit)
• Final LOQ for this sample = 2.5 ng/g x 35.8 = 89.5 ng/g (reported to 3 significant digits)
Example LOD & LOQ Calculation: Feces sample from animal 001M, 120 hour time point
• Water Extraction Weight = 25.3 g. Feces weight = 14.21 grams
• Sample Preparation Factor = (25.3(g) + 14.21 (g)) / 14.21 (g) = 2.78
• Final LOD for this sample = 0.4 ng/g x 2.78 = 1 ng/g (reported to 1 significant digit)
• Final LOQ for this sample = 2.5 ng/g x 2.78 = 6.95 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) = 0.2000 g
• Sample Preparation Factor = 1 / 0.2000 = 5.00
- 18 -
CH-FW-DEQ-0009526
H-28548:
Absorption, Distribution, Metabolism, and Elimination in the Rat DuPont-18405-1017
• Final LOD for this sample = 0.1 ng/g x 5.00 = 0.5 ng/g (reported to 1 significant digit)
• Final LOQ for this sample = 2.5 ng/g x 5.00 = 12.5 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 1540x is shown in Figure 2d. The final
concentration for this sample was 34700 ng/g.
4. Chromatographic Results (feces samples)
H-28548 eluted as a well-resolved peak with a retention time of approximately 5.5 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 20x. A 12 hour feces sample
from animal 001M, which had a total dilution factor of 336x is shown in Figure 3d. The final
concentration for this sample was 2750 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
98-99%. The associated coefficient of variation (CV) ranged from 1-2% 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
85-91%. The associated CV ranged from 3-6% 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 6.82 mg
H-28548/mL, which was approximately 91% of the nominal target (7.5 mg H-28548/mL).
At study initiation (day of dosing), males weighed 247.8 g ± 8.15 g and females weighed 181.1 g
± 4.23 g; the calculated dose rate for male (27.4 ± 0.17 mg/kg bw) and female rats (27.2 ± 0.16
mg/kg bw) were within 10% of the nominal target (30 mg/kg bw).
- 19 -
CH-FW-DEQ-0009527
H-28548:
Absorption, Distribution, Metabolism, and Elimination in the Rat DuPont-18405-1017
C. Urine Data
(Table 4, Figure 4, Appendix C)
Following oral administration of H-28548 in water, 96.6% ± 1.43% and 94.6% ± 8.57% of the
administered dose (0-12 hours) was accounted for in urine from male and female rats,
respectively.
At the conclusion of the study (168 hours post-dose), the cumulative amount of H-28548
detected in urine was 103% ± 2.73% and 99.8% ± 6.41% for male and female rats, respectively.
Elimination of H-28548 via urine was rapid and accounted for the administered dose for both
male and female rats.
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 1.35% ± 1.05% and 0.85% ± 0.58%
for male and female rats, respectively.
The negligible 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,
105.3% ± 2.19% and 105.7% ± 1.42% of the administered dose was recovered from male and
female rats, respectively.
Of the total H-28548 recovered, the majority of administered dose was account for in urine from
both males (103.0% ± 2.73%) and females (99.8% ± 6.41%); lesser amounts of H-28548 were
accounted for in feces (male = 1.35% ± 1.05%; female = 0.85% ± 0.58%). Cagewash, which is
composed of dried excreta (urine and feces) accounted for 0.98% ± 0.52% and 5.03% ± 5.14% of
the administered dose for male and female rats, 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%.
- 20 -
CH-FW-DEQ-0009528
H-28548:
Absorption, Distribution, Metabolism, and Elimination in the Rat DuPont-18405-1017
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 rat 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 rat 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 rat 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, 96.6% ± 1.43% and 94.6% ± 8.57% of the
administered dose was accounted for in urine (0-12 hours) from male and female rats,
respectively. At the conclusion of the study (168 hours post-dose), the total accumulated amount
of H-28548 detected in urine was 103% ± 2.73% and 99.8% ± 6.41% of the administered dose
for male and female rats, respectively.
Elimination of H-28548 via urine was rapid and accounted for a majority of the administered
dose for both male and female rats; negligible levels of H-28548 detected in feces from male
(1.35% ± 1.05%) and female rats (0.85% ± 0.58%), were likely contamination from of urine.
Cagewash, which is composed of dried excreta (urine and feces), accounted for 0.98% ± 0.52%
and 5.03% ± 5.14% of the administered dose for male and female rats, respectively.
Following oral dosing with H-28548 in water and a 168 hour post-dose collection period,
105.3% ± 2.19% and 105.7% ± 1.42% of the administered dose was recovered from male and
female rats, respectively.
- 21 -
CH-FW-DEQ-0009529
H-28548:
Absorption, Distribution, Metabolism, and Elimination in the Rat DuPont-18405-1017
Samples of urine evaluated using LC/MS were found to contain only the parent substance,
H-28548. This finding, taken with the complete recovery of the administered dose in urine,
confirms that H-28548 was rapidly absorbed and eliminated unmetabolized following oral
dosing in the rat.
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.
- 22 -
CH-FW-DEQ-0009530
H-28548:
Absorption, Distribution, Metabolism, and Elimination in the Rat DuPont-18405-1017
TABLES
- 23 -
CH-FW-DEQ-0009531
H-28548:
Absorption, Distribution, Metabolism, and Elimination in the Rat DuPont-18405-1017
TABLES
EXPLANATORY NOTES
ABBREVIATIONS:

CV - coefficient of variation
NA - not applicable
QC - quality control
SD - standard deviation
- 24 -
CH-FW-DEQ-0009532
H-28548:
Absorption, Distribution, Metabolism, and Elimination in the Rat DuPont-18405-1017
Table 1
Rat urine sample fortification QC results for H-28548
Rat Urine
Fortification
Sample
Fortification
Concentration
(ng/g)
Average
Recovery
(%)
CV
(%)

Low 400 99 2
Mid 100,000 98 1
High 1,000,000 99 1

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

Low 250 85 6
Mid 1250 85 3
High 62500 91 4

Table 3
Dosing information
Males Females
Mean SD Mean SD

Subject weight (g)247.8 8.15 181.1 4.23

Test substance received (mg)6.79 0.21 4.93 0.10

Dose (mg/kg bw)27.4 0.17 27.2 0.16

- 25 -
CH-FW-DEQ-0009533
H-28548:
Absorption, Distribution, Metabolism, and Elimination in the Rat DuPont-18405-1017
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 68.6 29.4 87.3 11.6
12 96.6 1.43 94.6 8.57
24 101.2 2.69 96.7 8.82
48 102.4 2.91 98.4 7.46
72 102.8 2.76 99.1 6.92
96 102.9 2.75 99.7 6.48
120 103.0 2.74 99.8 6.44
144 103.0 2.73 99.8 6.41
168 103.0 2.73 99.8 6.41

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

0 NA NA NA NA
6 0.74 1.1 NA NA
12 1.06 0.96 0.36 0.19
24 1.24 0.98 0.50 0.35
48 1.27 0.98 0.64 0.36
72 1.28 0.98 0.75 0.45
96 1.32 1.01 0.82 0.55
120 1.33 1.03 0.83 0.56
144 1.34 1.04 0.84 0.57
168 1.35 1.05 0.85 0.58

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

Urine 103.0 2.73 99.8 6.41
Feces 1.35 1.05 0.85 0.58
Cage Wash 0.98 0.52 5.03 5.14
Total 105.3 2.19 105.7 1.42

- 26 -
CH-FW-DEQ-0009534
H-28548:
Absorption, Distribution, Metabolism, and Elimination in the Rat DuPont-18405-1017
FIGURES
- 27 -
CH-FW-DEQ-0009535
H-28548:
Absorption, Distribution, Metabolism, and Elimination in the Rat DuPont-18405-1017
FIGURES
EXPLANATORY NOTES
ABBREVIATIONS:

QC - quality control
cps - counts per second
m/z - mass-to-charge ratio
min - minute
- 28 -
CH-FW-DEQ-0009536
H-28548:
Absorption, Distribution, Metabolism, and Elimination in the Rat DuPont-18405-1017
Figure 1
Calibration curve for H-28548

- 29 -
CH-FW-DEQ-0009537
H-28548:
Absorption, Distribution, Metabolism, and Elimination in the Rat DuPont-18405-1017
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 1540x and
final concentration of 34700 ng/g
a)

b)

c)

d)

- 30 -
CH-FW-DEQ-0009538
H-28548:
Absorption, Distribution, Metabolism, and Elimination in the Rat DuPont-18405-1017
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
20x, and d) a 12-hour feces study sample from animal 001M, which had a total 336x dilution
factor and final concentration of 2750 ng/g
a)

b)

c)

d)

- 31 -
CH-FW-DEQ-0009539
H-28548:
Absorption, Distribution, Metabolism, and Elimination in the Rat DuPont-18405-1017
Figure 4
Urine, cumulative percent
0
10
20
30
40
50
60
70
80
90
100
110
0 24 48 72 96 120 144 168 192
hours post dosecumulative percent of dose in urinemales
females

- 32 -
CH-FW-DEQ-0009540
H-28548:
Absorption, Distribution, Metabolism, and Elimination in the Rat DuPont-18405-1017
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

- 33 -
CH-FW-DEQ-0009541
H-28548:
Absorption, Distribution, Metabolism, and Elimination in the Rat DuPont-18405-1017
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

- 34 -
CH-FW-DEQ-0009542
H-28548:
Absorption, Distribution, Metabolism, and Elimination in the Rat DuPont-18405-1017
Figure 7
Reconstructed m/z 329 + 659 ion chromatograms characteristic of H-28548-dosed female rat
urine (6 hours after administration) – top and control rat urine fortified with H-28458 test
substance -bottom

- 35 -
CH-FW-DEQ-0009543
H-28548:
Absorption, Distribution, Metabolism, and Elimination in the Rat DuPont-18405-1017
Figure 8
ESI negative mass spectra of H-28548 observed in dosed female rat urine (6 hours after
administration)–top; and control urine fortified with H-28548 test substance – bottom

- 36 -
CH-FW-DEQ-0009544
H-28548:
Absorption, Distribution, Metabolism, and Elimination in the Rat DuPont-18405-1017
Figure 9
ESI negative daughter ion mass spectra of H28548 observed in dosed female rat urine (6 hours
after administration)–top; and control rat urine fortified with H-28548 test substance – bottom

- 37 -
CH-FW-DEQ-0009545
H-28548:
Absorption, Distribution, Metabolism, and Elimination in the Rat DuPont-18405-1017
APPENDICES
- 38 -
CH-FW-DEQ-0009546
H-28548:
Absorption, Distribution, Metabolism, and Elimination in the Rat DuPont-18405-1017
APPENDICES
EXPLANATORY NOTES
ABBREVIATIONS:

F - female
h - hours
LOQ - limit of quantification
M - male
NA - not applicable
ND - not detected
SD - standard deviation
- 39 -
CH-FW-DEQ-0009547
H-28548:
Absorption, Distribution, Metabolism, and Elimination in the Rat DuPont-18405-1017
Appendix A
Certificate of Analysis
- 40 -
CH-FW-DEQ-0009548
H-28548:
Absorption, Distribution, Metabolism, and Elimination in the Rat DuPont-18405-1017

- 41 -
CH-FW-DEQ-0009549
H-28548:
Absorption, Distribution, Metabolism, and Elimination in the Rat DuPont-18405-1017
Appendix B
Dosing Information
- 42 -
CH-FW-DEQ-0009550
H-28548:
Absorption, Distribution, Metabolism, and Elimination in the Rat DuPont-18405-1017

Dosing Information

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

001M 247.8 6.78 27.4
002M 241.0 6.56 27.2
003M 255.0 6.97 27.3
004M 256.7 7.01 27.3
005M 238.4 6.60 27.7
Mean 247.8 6.79 27.4
SD 8.15 0.21 0.17

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

001F 180.5 4.94 27.4
002F 184.0 5.01 27.2
003F 177.1 4.83 27.3
004F 177.3 4.84 27.3
005F 186.8 5.04 27.0
Mean 181.1 4.93 27.2
SD 4.23 0.10 0.16
- 43 -
CH-FW-DEQ-0009551
H-28548:
Absorption, Distribution, Metabolism, and Elimination in the Rat DuPont-18405-1017
Appendix C
Urine Data
- 44 -
CH-FW-DEQ-0009552
H-28548:
Absorption, Distribution, Metabolism, and Elimination in the Rat DuPont-18405-1017

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 6782583 Pre-dose 17.314 ND NA NA NA
6 h 4.837 1180000 5707660 84.2 84.2
12 h 2.752 301000 828352 12.2 96.4
24 h 6.358 34700 220623 3.25 99.6
48 h 22.254 1880 41838 0.62 100.2
72 h 27.818 2470 68710 1.01 101.2
96 h 28.961 262 7588 0.11 101.4
120 h 35.357 <89.5 NA NA 101.4
144 h 44.394 <94.3 NA NA 101.4
168 h 30.637 <93.3 NA NA 101.4
101.4

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

002M 6564450 Pre-dose 32.306 ND NA NA NA
6 h 4.228 1250000 5285000 80.5 80.5
12 h 2.688 417000 1120896 17.1 97.6
24 h 5.969 35500 211900 3.23 100.8
48 h 15.124 5270 79703 1.21 102.0
72 h 10.694 1530 16362 0.25 102.3
96 h 15.311 544 8329 0.13 102.4
120 h 44.439 93.7 4164 0.06 102.5
144 h 43.144 <95.5 NA NA 102.5
168 h 37.473 <95.8 NA NA 102.5
102.5

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

003M 6973450 Pre-dose 27.787 ND NA NA NA
6 h 3.323 1810000 6014630 86.3 86.3
12 h 2.077 383000 795491 11.4 97.7
24 h 6.729 55100 370768 5.32 103.0
48 h 17.212 4470 76938 1.10 104.1
72 h 16.394 781 12804 0.18 104.3
96 h 23.082 213 4916 0.07 104.3
120 h 17.137 <96.5 NA NA 104.3
144 h 23.439 <99.3 NA NA 104.3
168 h 15.733 <89.5 NA NA 104.3
104.3
- 45 -
CH-FW-DEQ-0009553
H-28548:
Absorption, Distribution, Metabolism, and Elimination in the Rat DuPont-18405-1017

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 7007533 Pre-dose 38.816 ND NA NA NA
6 h 4.373 1210000 5291330 75.5 75.5
12 h 4.77 275000 1311750 18.7 94.2
24 h 11.983 21800 261229 3.73 98.0
48 h 23.195 3890 90229 1.29 99.2
72 h 25.345 729 18477 0.26 99.5
96 h 22.124 756 16726 0.24 99.7
120 h 21.971 143 3142 0.04 99.8
144 h 21.943 101 2216 0.03 99.8
168 h 16.324 <95.8 NA NA 99.8
99.8

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

005M 6598533 Pre-dose 14.027 ND NA NA NA
6 h 2.27 479000 1087330 16.48 16.48
12 h 4.264 1250000 5330000 80.78 97.3
24 h 5.469 90400 494398 7.49 104.7
48 h 16.676 6630 110562 1.68 106.4
72 h 22.14 691 15299 0.23 106.7
96 h 20.349 663 13491 0.20 106.9
120 h 16.363 <87.5 NA NA 106.9
144 h 22.415 <94.3 NA NA 106.9
168 h 19.651 <93.3 NA NA 106.9
106.9

Timepoint
(hours)
Cumulative
Mean SD

0 h NA NA
6 h 68.6 29.4
12 h 96.6 1.43
24 h 101.2 2.69
48 h 102.4 2.91
72 h 102.8 2.76
96 h 102.9 2.75
120 h 103.0 2.74
144 h 103.0 2.73
168 h 103.0 2.73
- 46 -
CH-FW-DEQ-0009554
H-28548:
Absorption, Distribution, Metabolism, and Elimination in the Rat DuPont-18405-1017

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 4942083 Pre-dose 13.129 ND NA NA NA
6 h 2.268 1600000 3628800 73.4 73.4
12 h 2.657 468000 1243476 25.2 98.6
24 h 6.746 34600 233412 4.72 103.3
48 h 14.826 3400 50408 1.02 104.3
72 h 16.819 1290 21697 0.44 104.8
96 h 19.122 567 10842 0.22 105.0
120 h 11.956 230 2750 0.06 105.0
144 h 26.05 142 3699 0.07 105.1
168 h 19.482 <100 NA NA 105.1
105.1

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

002F 5010250 Pre-dose 14.268 ND NA NA NA
6 h 2.424 1800000 4363200 87.1 87.1
12 h 1.805 54100 97651 1.9 89.0
24 h 7.05 7630 53792 1.07 90.1
48 h 13.206 8310 109742 2.19 92.3
72 h 8.605 3240 27880 0.56 92.9
96 h 19.158 4300 82379 1.64 94.5
120 h 14.669 820 12029 0.24 94.7
144 h 20.706 285 5901 0.12 94.9
168 h 16.431 <105 NA NA 94.9
94.9

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

003F 4826200 Pre-dose 16.215 ND NA NA NA
6 h 2.807 1350000 3789450 78.5 78.5
12 h 2.866 63500 181991 3.8 82.3
24 h 5.164 20400 105346 2.18 84.5
48 h 13.609 14400 195970 4.06 88.5
72 h 15.306 5910 90458 1.87 90.4
96 h 19.344 1360 26308 0.55 91.0
120 h 13.411 247 3313 0.07 91.0
144 h 12.458 184 2292 0.05 91.1
168 h 16.058 <94.3 NA NA 91.1
91.1
- 47 -
CH-FW-DEQ-0009555
H-28548:
Absorption, Distribution, Metabolism, and Elimination in the Rat DuPont-18405-1017

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 4839833 Pre-dose 19.326 ND NA NA NA
6 h 4.46 1070000 4772200 98.6 98.6
12 h 2.937 38200 112193 2.3 100.9
24 h 9.506 8310 78995 1.63 102.6
48 h 23.155 1130 26165 0.54 103.1
72 h 21.058 870 18320 0.38 103.5
96 h 27.669 377 10431 0.22 103.7
120 h 29.855 168 5016 0.10 103.8
144 h 32.112 <95.8 NA NA 103.8
168 h 31.889 <97.0 NA NA 103.8
103.8

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

005F 5037517 Pre-dose 14.453 ND NA NA NA
6 h 3.101 1610000 4992610 99.11 99.11
12 h 3.072 48400 148685 2.95 102.1
24 h 5.328 9410 50136 1.00 103.1
48 h 18.573 2490 46247 0.92 104.0
72 h 17.462 549 9587 0.19 104.2
96 h 18.381 199 3658 0.07 104.2
120 h 18.371 <90.3 NA NA 104.2
144 h 17.949 <96.5 NA NA 104.2
168 h 16.373 <92.3 NA NA 104.2
104.2

Timepoint
(hours)
Cumulative
Mean SD

0 h NA NA
6 h 87.3 11.6
12 h 94.6 8.57
24 h 96.7 8.82
48 h 98.4 7.46
72 h 99.1 6.92
96 h 99.7 6.48
120 h 99.8 6.44
144 h 99.8 6.41
168 h 99.8 6.41
- 48 -
CH-FW-DEQ-0009556
H-28548:
Absorption, Distribution, Metabolism, and Elimination in the Rat DuPont-18405-1017
Appendix D
Feces Data
- 49 -
CH-FW-DEQ-0009557
H-28548:
Absorption, Distribution, Metabolism, and Elimination in the Rat DuPont-18405-1017

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 6782583 0h 1.976 ND NA NA NA
6 h 0.986 8240 8125 0.12 0.12
12 h 4.871 2750 13395 0.20 0.32
24 h 13.463 935 12588 0.19 0.50
48 h 11.986 42.7 512 0.01 0.51
72 h 12.734 46.3 590 0.01 0.52
96 h 12.325 343 4227 0.06 0.58
120 h 14.21 <6.95 NA NA 0.58
144 h 13.565 97 1316 0.02 0.60
168 h 12.641 <7.88 NA NA 0.60
0.60

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

002M 6564450 0h 3.741 ND NA NA NA
6 h 0.192 840000 161280 2.46 2.46
12 h 2.023 4280 8658 0.13 2.59
24 h 4.008 2660 10661 0.16 2.75
48 h 4.893 181 886 0.01 2.76
72 h 7.026 126 885 0.01 2.78
96 h 8.513 790 6725 0.10 2.88
120 h 10.031 321 3220 0.05 2.93
144 h 11.438 289 3306 0.05 2.98
168 h 8.604 108 929 0.01 2.99
2.99

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

003M 6973450 0h 3.057 ND NA NA NA
6 h 0.659 3530 2326 0.03 0.03
12 h 4.431 4240 18787 0.27 0.30
24 h 7.07 1560 11029 0.16 0.46
48 h 10.082 96.7 975 0.01 0.47
72 h 11.949 28.9 345 0.00 0.48
96 h 10.879 108 1175 0.02 0.50
120 h 13.489 34.3 463 0.01 0.50
144 h 12.518 20.6 258 0.00 0.51
168 h 11.799 <8.50 NA NA 0.51
0.51
- 50 -
CH-FW-DEQ-0009558
H-28548:
Absorption, Distribution, Metabolism, and Elimination in the Rat DuPont-18405-1017

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 7007533 0h 6.556 ND NA NA NA
6 h 0.27 285000 76950 1.10 1.10
12 h 4.487 4630 20775 0.30 1.39
24 h 4.822 4050 19529 0.28 1.67
48 h 10.318 467 4819 0.07 1.74
72 h 9.184 79.4 729 0.01 1.75
96 h 10.598 63.7 675 0.01 1.76
120 h 12.049 30 361 0.01 1.77
144 h 10.912 15.5 169 0.00 1.77
168 h 13.156 <7.58 NA NA 1.77
1.77

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

005M 6598533 0h 3.975 ND NA NA NA
6 h 0.861 150 129 0.002 0.00
12 h 1.357 33100 44917 0.68 0.68
24 h 9.349 820 7666 0.12 0.80
48 h 11.721 258 3024 0.05 0.84
72 h 9.353 45.3 424 0.01 0.85
96 h 10.824 27.5 298 0.005 0.86
120 h 11.997 15.4 NA NA 0.86
144 h 10.608 <9.53 NA NA 0.86
168 h 11.942 <8.53 NA NA 0.86
0.86

Timepoint
(hours)
Cumulative
Mean SD

0 h NA NA
6 h 0.74 1.06
12 h 1.06 0.96
24 h 1.24 0.98
48 h 1.27 0.98
72 h 1.28 0.98
96 h 1.32 1.01
120 h 1.33 1.03
144 h 1.34 1.04
168 h 1.35 1.05
- 51 -
CH-FW-DEQ-0009559
H-28548:
Absorption, Distribution, Metabolism, and Elimination in the Rat DuPont-18405-1017

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 4942083 0h 2.131 ND NA NA NA
6 h NA NA NA NA NA
12 h 2.866 4830 13843 0.28 0.28
24 h 2.097 1360 2852 0.06 0.34
48 h 6.556 776 5087 0.10 0.44
72 h 7.927 376 2981 0.06 0.50
96 h 10.519 152 1599 0.03 0.53
120 h 8.874 12.1 107 0.00 0.54
144 h 6.313 50.1 316 0.01 0.54
168 h 9.804 12.8 NA NA 0.54
0.54

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

002F 5010250 0h 2.05 ND NA NA NA
6 h NA NA NA NA NA
12 h 4.308 2750 11847 0.24 0.24
24 h 7.505 475 3565 0.07 0.31
48 h 9.265 2570 23811 0.48 0.78
72 h 8.336 1610 13421 0.27 1.05
96 h 6.43 146 939 0.02 1.07
120 h 7.961 21 167 0.00 1.07
144 h 8.581 29 249 0.00 1.08
168 h 10.028 15 NA NA 1.08
1.08

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

003F 4826200 0h 5.063 ND NA NA NA
6 h NA NA NA NA NA
12 h 5.571 5950 33147 0.69 0.69
24 h 6.342 3300 20929 0.43 1.12
48 h 6.924 590 4085 0.08 1.21
72 h 10.313 934 9632 0.20 1.40
96 h 8.19 1640 13432 0.28 1.68
120 h 9.821 161 1581 0.03 1.72
144 h 6.169 205 1265 0.03 1.74
168 h 8.197 92.6 759 0.02 1.76
1.76
- 52 -
CH-FW-DEQ-0009560
H-28548:
Absorption, Distribution, Metabolism, and Elimination in the Rat DuPont-18405-1017

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 4839833 0h 3.683 ND NA NA NA
6 h NA NA NA NA NA
12 h 5.018 2270 11391 0.24 0.24
24 h 3.763 692 2604 0.05 0.29
48 h 8.256 106 875 0.02 0.31
72 h 9.595 180 1727 0.04 0.34
96 h 8.822 76.2 672 0.01 0.36
120 h 10.684 17.2 184 0.00 0.36
144 h 7.927 43.6 346 0.01 0.37
168 h 9.069 12 109 0.00 0.37
0.37

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

005F 5037517 0h 4.772 ND NA NA NA
6 h NA NA NA NA NA
12 h 5.056 3700 18707 0.37 0.37
24 h 4.32 945 4082 0.08 0.45
48 h 8.301 78.3 650 0.01 0.47
72 h 9.681 48.9 473 0.01 0.47
96 h 8.19 36.7 301 0.01 0.48
120 h 8.962 <11.0 NA NA 0.48
144 h 8.749 <11.3 NA NA 0.48
168 h 7.452 <13.3 NA NA 0.48
0.48

Timepoint
(hours)
Cumulative
Mean SD

0 h NA NA
6 h NA NA
12 h 0.36 0.19
24 h 0.50 0.35
48 h 0.64 0.36
72 h 0.75 0.45
96 h 0.82 0.55
120 h 0.83 0.56
144 h 0.84 0.57
168 h 0.85 0.58
- 53 -
CH-FW-DEQ-0009561
H-28548:
Absorption, Distribution, Metabolism, and Elimination in the Rat DuPont-18405-1017
Appendix E
Cage Wash Data
- 54 -
CH-FW-DEQ-0009562
H-28548:
Absorption, Distribution, Metabolism, and Elimination in the Rat DuPont-18405-1017

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 6782583 168 h 691.966 174 120402 1.78
002M 6564450 168 h 838.827 64 53685 0.82
003M 6973450 168 h 757.65 44 33337 0.48
004M 7007533 168 h 802.957 103 82705 1.18
005M 6598533 168 h 778.34 55 42809 0.65

Mean 0.98
SD 0.51

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

001F 4942083 168 h 798.971 125 99871 2.02
002F 5010250 168 h 977.258 397 387971 7.74
003F 4826200 168 h 1249.369 496 619687 12.84
004F 4839833 168 h 784.33 87 68237 1.41
005F 5037517 168 h 793.16 72 57108 1.13

Mean 5.03
SD 5.14
- 55 -
CH-FW-DEQ-0009563
H-28548:
Absorption, Distribution, Metabolism, and Elimination in the Rat DuPont-18405-1017
Appendix F
Material Balance
- 56 -
CH-FW-DEQ-0009564
H-28548:
Absorption, Distribution, Metabolism, and Elimination in the Rat DuPont-18405-1017

Material Balance

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

urine 6 h 84.2 80.5 86.3 75.5 16.5 68.6 29.4
urine 12 h 12.2 17.1 11.4 18.7 80.8 28.0 29.6
urine 24 h 3.25 3.23 5.32 3.73 7.49 4.60 1.83
urine 48 h 0.62 1.21 1.10 1.29 1.68 1.18 0.38
urine 72 h 1.01 0.25 0.18 0.26 0.23 0.39 0.35
urine 96 h 0.11 0.13 0.07 0.24 0.20 0.15 0.07
urine 120 h <LOQ 0.06 <LOQ 0.04 <LOQ 0.05 NA
urine 144 h <LOQ <LOQ <LOQ 0.03 <LOQ 0.03 NA
urine 168 h <LOQ <LOQ <LOQ <LOQ <LOQ NA NA
Subtotal 101.4 102.5 104.3 99.8 106.9 103.0 2.73

feces 6 h 0.12 2.46 0.03 1.10 0.00 0.74 1.06
feces 12 h 0.20 0.13 0.27 0.30 0.68 0.32 0.21
feces 24 h 0.19 0.16 0.16 0.28 0.12 0.18 0.06
feces 48 h 0.01 0.01 0.01 0.07 0.05 0.03 0.03
feces 72 h 0.01 0.01 0.005 0.01 0.01 0.01 0.00
feces 96 h 0.06 0.10 0.02 0.01 0.005 0.04 0.04
feces 120 h <LOQ 0.05 0.01 0.01 0.003 0.02 0.02
feces 144 h 0.02 0.05 0.004 0.002 <LOQ 0.02 0.02
feces 168 h <LOQ 0.01 <LOQ <LOQ <LOQ 0.01 NA
Subtotal 0.60 2.99 0.51 1.77 0.86 1.35 1.05

cage wash 168 h 1.78 0.82 0.48 1.18 0.65 0.98 0.52

Total 103.7 106.3 105.3 102.8 108.4 105.3 2.19

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

urine 6 h 73.43 87.09 78.52 98.60 99.11 87.3 11.6
urine 12 h 25.16 1.95 3.77 2.32 2.95 7.23 10.0
urine 24 h 4.72 1.07 2.18 1.63 1.00 2.12 1.53
urine 48 h 1.02 2.19 4.06 0.54 0.92 1.75 1.43
urine 72 h 0.44 0.56 1.87 0.38 0.19 0.69 0.68
urine 96 h 0.22 1.64 0.55 0.22 0.07 0.54 0.64
urine 120 h 0.06 0.24 0.07 0.10 <LOQ 0.12 0.08
urine 144 h 0.07 0.12 0.05 <LOQ <LOQ 0.08 0.04
urine 168 h <LOQ <LOQ <LOQ <LOQ <LOQ NA NA
Subtotal 105.1 94.9 91.1 103.8 104.2 99.8 6.41

feces 6 h <LOQ <LOQ <LOQ <LOQ <LOQ NA NA
feces 12 h 0.28 0.24 0.69 0.24 0.37 0.36 0.19
feces 24 h 0.06 0.07 0.43 0.05 0.08 0.14 0.16
feces 48 h 0.10 0.48 0.08 0.02 0.01 0.14 0.19
feces 72 h 0.06 0.27 0.20 0.04 0.01 0.11 0.11
feces 96 h 0.03 0.02 0.28 0.01 0.01 0.07 0.12
feces 120 h 0.002 0.003 0.03 0.00 <LOQ 0.01 0.01
feces 144 h <LOQ 0.005 0.03 0.01 <LOQ 0.01 0.01
feces 168 h <LOQ 0.003 0.02 0.002 <LOQ 0.01 0.01
Subtotal 0.54 1.08 1.76 0.37 0.48 0.85 0.58

cage wash 168 h 2.02 7.74 12.8 1.41 1.13 5.03 5.14

Total 107.7 103.7 105.7 105.6 105.8 105.7 1.42
- 57 -
CH-FW-DEQ-0009565