CH-FW-DEQ-0008766

Study Number: WIL-189225
PROTOCOL AMENDMENT 4
Sponsor: E.I. du Pont de Nemours and Company
Title of Study:
An Oral (Gavage) Reproduction/Developmental Toxicity Screening Study of H-28548 in
Mice
Protocol Modifications:
1)1 OBJECTIVE:
The following is added to this section:
In addition, a toxicokinetic assessment of plasma levels of the test article will be
performed in the F0 females and the F1 pups at culling and on PND 21 and PND 40.
2)2.4 WIL Departmental Responsibilities:
The following person is added to this section:
Carol S. Wally, BA, SRS, RLATG
Group Supervisor, Sample Processing Laboratory
3) The following sections are added to the protocol:
2.5 Principal Investigator, Plasma Sample Analysis and Report:
Michael Mawn, PhD
Senior Research Chemist
DuPont Stine-Haskell Research Center
1090 Elkton Road
Bldg. S-315 Lab 1333
Newark, DE 19714-0030
Tel: [phone]
Email:[e-mail]
CH-FW-DEQ-0008766
WIL-189225
Page 2 of 5 Protocol Amendment 4
4) The following sections are added to the protocol:
7.9 Plasma Sample Collection and Analysis:
7.9.1 Interval:
Blood samples will be collected at the time of scheduled necropsy on PND 21
from 5 randomly selected F0 females per group that delivered. A plasma sample
will be collected from all females that failed to deliver on post-mating day 23 at
the time of the scheduled necropsy.
Blood samples will also be collected from the F1 culled pups on PND 4 from 10
randomly chosen litters in each group following culling and data collection.
On PND 21, blood samples will be collected from 5 randomly selected F1 males
and females in each group at the time of the scheduled necropsy that are not
selected for the F1 generation.
On PND 40, blood samples will be collected from 5 randomly selected F1 males
and females in each group at the time of the scheduled necropsy.
7.9.2 Route of Collection:
Blood samples will be collected via the vena cava following euthanasia by carbon
dioxide inhalation from the F0 females and the F1 PND 21 and PND 40 animals.
Blood samples will be collected via decapitation from the PND 4 pups and pooled
by litter.
7.9.3 Target Blood Volume:
For the F0 females and the F1 PND 21 and PND 40 animals, 1.0 mL or as much as
possible, will be collected into pre-chilled, uniquely-labeled tubes. For the PND 4
pups, blood will be pooled by litter from all the culled pups in each litter to obtain
as much blood as possible.
CH-FW-DEQ-0008767
WIL-189225
Page 3 of 5 Protocol Amendment 4
7.9.4 Anticoagulant:
K3EDTA
7.9.5 Sample Handling and Plasma Preparation:
Samples will be kept on wet ice, protected from light, until centrifugation. All
samples will be centrifuged [approximately 3000 rpm (approximately 2060xg) for
approximately 10 min] at approximately 4oC. Plasma will be transferred into
new, uniquely-labeled polypropylene tubes.
7.9.6 Label Information:
Samples will include study number, dose group, animal number, interval, sample
type and date and time of blood collection.
7.9.7 Storage:
Plasma samples will be stored frozen at approximately -20°C until analysis. The
time and date the samples were placed in the freezer will be recorded.
7.9.8 Sample Shipment:
Frozen samples in dry ice, an inventory list and documentation of actual blood
collection times for each animal will be shipped on the first Monday or Tuesday
after the last sample is collected. The recipient will be notified at least 24 hours
in advance of any shipment. Samples will be shipped overnight to:
Michael Mawn, PhD
Senior Research Chemist
DuPont Stine-Haskell Research Center
1090 Elkton Road
Bldg. S-315 Lab 1333
Newark, DE 19714-0030
Tel: [phone]
Email:[e-mail]
7.9.9 Plasma Analyses and Report:
Plasma samples will be analyzed for the test article content using a previously
validated method. The Principal Investigator, Plasma Analysis will be
responsible for all bioanalytical delegated-phase activities and will issue a formal
CH-FW-DEQ-0008768
WIL-189225
Page 4 of 5 Protocol Amendment 4
bioanalytical/plasma analyses report from the data generated that will be included
as an appendix in the final report. A Quality Assurance and GLP compliance
statement signed by Sponsor and archival location of the data will be provided to
the WIL Study Director for inclusion in the Final Report.
5)11 QUALITY ASSURANCE:
The first sentence of the second paragraph is changed to the following:
The plasma samples analysis and the pathological examination of the slides will be
conducted following the Standard Operating Procedures of the performing laboratory
and in accordance with GLPs.
6)12 RECORDS TO BE MAINTAINED:
The second paragraph is changed to the following:
The Sponsor will be responsible for the archival of the raw data and records for the
plasma sample analyses and the pathological examination.
7)13 WORK PRODUCT:
The second sentence of the first paragraph is changed to the following:
Any remaining plasma samples and formulation samples will be discarded after the
issuance of the Final Report.
8)14 REPORTS:
The second sentence of the first paragraph is changed to the following:
The final report will contain a summary, test item data, methods and procedures,
maternal and pup data WIL Historical Control Data, the analytical chemistry report,
the plasma samples report, the pathology report and an interpretation and discussion
of the study results.
CH-FW-DEQ-0008769
WIL-189225
Page 5 of 5 Protocol Amendment 4
Reasons for Protocol Modification:
1-8) Blood collection for plasma sample analyses is added to the protocol at the
Sponsor’s request to characterize the exposure levels of the test substance.
Approval:
Sponsor’s approval was obtained via email on April 12, 2010.
WIL Research Laboratories, LLC
_________________________________________________
Tammye L. Edwards, BS, LAT Date
StudyDirector
_________________________________________________
Donald G. Stump, PhD, DABT Date
Director, Developmental
and Reproductive Toxicology
E. I. du Pont de Nemours and Company
_________________________________________________
Susan M. Munley, MA Date
Sponsor Representative
CH-FW-DEQ-0008770