EPA 600/R-11/145 | May 2012 | www.epa.gov/ord
United States
Environmental Protection
Agency
Multiple Daily Low-Dose
Bacillus anthracis Ames
Inhalation Exposures in
the Rabbit
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National Homeland Security Research Center
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EPA/600/R-11/145
Multiple Daily Low-Dose Bacillus anthracis Ames
Inhalation Exposures in the Rabbit
United States Environmental Protection Agency
Cincinnati, Ohio 45268
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Disclaimer
The U.S. Environmental Protection Agency (EPA) through its Office of Research and
Development co-funded and managed the research described herein under Interagency
Agreement DW9792208901 with the Defense Technical Information Center, and in turn through
Battelle Chemical, Biological, Radiological, and Nuclear Defense Information Analysis Center
(CBRNIAC) Contract No. SPO-700-00-D-3180 Delivery Order Number 0603 (Task 794). The
Department of Defense's Defense Threat Reduction Agency collaborated with EPA to fund the
research herein under project numbers BA06TAS022 and CBS.PHYSIO.01.10.SW.005.
No official endorsement should be inferred. EPA does not endorse the purchase or sale of any
commercial products or services.
For questions on this report, please contact Dr. Sarah Taft of the U.S. Environmental Protection
Agency, National Homeland Security Research Center, 26 West Martin Luther King Dr., Mail
Stop NG-16, Cincinnati, Ohio 45268. Dr. Taft can also be reached by phone at (513) 569-7037
or email at Taft. Sarah@epa.gov.
11
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Table of Contents
Disclaimer ii
Table of Contents iii
List of Figures v
Appendices vi
Acronyms and Abbreviations vii
Acknowledgments x
Foreword xi
Executive Summary xii
1 Introduction 1
2 Materials and Methods 4
2.1 Test System 4
2.2 Randomization of Animals 4
2.3 Bacillus anthracis Ames Strain Spores 5
2.4 Aerosol Challenge Generation and Monitoring 6
2.5 Tel em etric Monitoring 9
2.6 Clinical Observations and Body Weights 9
2.7 Blood Collection 10
2.8 Protective Antigen ELISA 10
2.9 Bacteremia 11
2.10 TNA/ELISA 13
2.11 Hematology and C-Reactive Protein 14
2.12 Necropsy and Histopathology 15
2.13 Benchmark Dose Analysis and Dosimetric Adjustment 15
3 Results 19
3.1 Aerosol Challenges 19
3.2 Clinical Observations, Body Weights, and Mortality 22
3.3 Telemetric Monitoring 25
3.4 Circulating Levels of Protective Antigen 37
3.5 Bacteremia 37
in
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3.6 TNA/IgGELISA 41
3.7 Hematology and Clinical Chemistry 41
4 Pathology 50
4.1 Benchmark Dose Analysis 59
4.2 Quality Assurance 59
4.3 Archives 62
5 Discussion and Conclusions 63
6 References 70
List of Tables
Table 1. Study Design and Challenge Doses 5
Table 2. Characterization of Bacillus anthracis Spores 5
Table 3. Blood Collection Schedule 10
Table 4. TaqMan® Gene Expression Assay for the B. anthracis rpoB_571 Gene 12
Table 5. Assumptions Used to Generate Human Equivalent Dose and Human Equivalent
Concentration 17
Table 6. Individual and Group Mean Challenge Doses over the 15 Exposure Days 21
Table 7. Challenge Dose Information for the Group 4 Rabbits That Succumbed to Infection 22
Table 8. Abnormality Summaries by Parameter and Group Along with Fisher's Exact Tests
Comparing the Proportion Abnormal in Each Group by Parameter 26
Table 9. Summary of Individual Gross and Microscopic Observations 52
Table 10. Deviations and Impacts on Data Quality and Results 60
Table 11. Technical System Audit (TSAT) and Data Quality Audit (DQA) Dates 61
Table 12. Summary of Study Findings 69
IV
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List of Figures
Figure 1. Mean challenge doses in CPU for each of the 15 exposure days 20
Figure 2. Kaplan-Meier curves representing time to death and survival data for each group 24
Figure 3. Plot of mean baseline-adjusted activity (counts/min) for each group 29
Figure 4. Plot of baseline-adjusted activity (counts/min) values for each rabbit 29
Figure 5. Plot of mean baseline-adjusted heart rate (BPM) for each group 32
Figure 6. Plot of baseline-adjusted heart rate (BPM) for each rabbit 33
Figure 7. Plot of mean baseline-adjusted respiratory period (RP) respiratory rate (in RCPM) for
each group 34
Figure 8. Plot of baseline-adjusted RP respiratory rate (in RCPM) for each animal 35
Figure 9. Plot of mean baseline-adjusted temperature values for each group 39
Figure 10. Baseline-adjusted temperature values for each rabbit 40
Figure 11. Plots of red blood cell counts (1 x 106 cells/jiL) 43
Figure 12. Plots of hemoglobin concentration (g/dL) 44
Figure 13. Plots of white blood cell counts (1 x 103 cells/jiL) 45
Figure 14. Plots of neutrophil counts (1 x 103 cells/|iL) 47
Figure 15. Plots of lymphocyte counts (1 x 103 cells/|iL) 48
Figure 16. Plots of C-reactive protein levels (mg/dL) 49
Figure 17. Animal 38: Lung, alveoli; pyogranulomatous (epithelioid macrophages,
lymphocytes, and neutrophils) inflammatory reaction to a foreign body (arrow).
Hematoxylin and eosin stain. 40X 54
Figure 18. Animal 37: Lung; normal alveoli (control). Hematoxylin and eosin stain. 40X 55
Figure 19. Animal 31: Lung; alveoli contain interstitial suppurative inflammation and anthrax
bacilli (arrows). Alveolar vessels contain anthrax bacilli (arrowhead). Hematoxylin
and eosin stain. 40X 56
Figure 20. Animal 38: Appendix; lymphocytes undergoing excessive apoptosis (arrow) with
macrophage infiltration (arrowheads). Hematoxylin and eosin stain. 10X 57
Figure 21. Animal 38: Lymph node, mediastinal; lymph node congestion and lymphoid
follicles necrosis/depletion. Hematoxylin and eosin stain. 4X 58
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Appendices
Appendix A Study Protocol
Appendix B Study Deviations and Investigation Reports
Appendix C Bordetella Results and Health Monitoring Status Report
Appendix D Randomization Report
Appendix E Aerosol Report
Appendix F Statistical Report - Telemetry
Appendix G Statistical Report - Mortality
Appendix H Statistical Report-Body Weights
Appendix I Blood Draw Times
Appendix J Statistical Report - Hematology and C-Reactive Protein
Appendix K Individual Clinical Observations
Appendix L Individual Body Weights
Appendix M Individual Mortality Results
Appendix N Individual Circulating PA ELISA Results
Appendix O Individual Bacteremia Culture Results
Appendix P Individual Bacteremia qPCR Results
Appendix Q Individual TNA Results
Appendix R Individual Anti-PA IgG ELISA Results
Appendix S Individual Hematology Results
Appendix T Individual C-Reactive Protein Results
Appendix U Pathology Report
Appendix V Benchmark Dose Study Report
VI
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Acronyms and Abbreviations
ADD average daily dose per animal
ANOVA analysis of variance
APS Aerodynamic Particle Sizer
BBRC Battelle Biomedical Research Center
BMD benchmark dose
BMDX benchmark dose at an x level of BMR
BMDLX lower confidence limit of the BMD at anx level of BMR
BMDS benchmark dose software
BMI Battelle Memorial Institute
BMR benchmark response
BMRX benchmark response at anx level
BPM beats per minute
BL3 Biosafety Level 3
CBRNIAC... Chemical, Biological, Radiological, and Nuclear Defense Information Analysis Center
CPU colony forming unit
cm centimeter
CRP C-reactive protein
Ct cycle threshold
dL deciliter
DNA deoxyribonucleic acid
DQA data quality audit
DR deviation report
ED50 effective dose 50%
EDTA ethylenediaminetetraacetic acid
EF edema factor
ELISA enzyme-linked immunosorbent assay
EPA U.S. Environmental Protection Agency
EU endotoxin unit
FD found dead
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FS final-phase sacrifice
F/T freeze/thaw
g gram
GSD geometric standard deviation
HCT hematocrit
HEC human equivalent concentration
FED human equivalent dose
Hg mercury
HGB hemoglobin
FIRP horseradish peroxidase
IgG immunoglobulin G
InD inhaled dose
IR investigation report
kg kilogram
L liter
LD50 median lethal dose
LF lethal factor
LOD limit of detection
m3 cubic meter
jig microgram
|iL microliter
|im micrometer
mg milligram
min minute
mL milliliter
mm millimeter
MMAD mass median aerodynamic diameter
MTT 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide
NF50 neutralization factor 50%
ng nanogram
nM nanomolar
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NHSRC National Homeland Security Research Center
NIAID National Institute of Allergy and Infectious Diseases
Nffl National Institutes of Health
NIC no template control
NZW New Zealand White
OD optical density
PA protective antigen
PBBK physiologically based biokinetic model
PCR polymerase chain reaction
PLT platelet count
QAP quality assurance plan
qPCR quantitative real-time polymerase chain reaction
RBC red blood cell
RCPM respiratory cycles per minute
RDW red cell distribution width
RNA ribonucleic acid
RP respiratory period
rPA recombinant protective antigen
rpoB DNA-directed ribonucleic acid polymerase subunitbeta
SD standard deviation
SST serum separator tube
TAD total aggregate dose
TATV total accumulated tidal volume
TCAD Threat and Consequence Assessment Division
TNA toxin neutralization assay
TNTC too numerous to count
TSA tryptic soy agar
TSAT technical systems audit
VAP vascular access port
WBC white blood cell
4PL four-parameter logistic log
IX
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Acknowledgments
EPA gratefully acknowledges the permission granted by the National Institute of Allergy and
Infectious Diseases (NIAID) and the National Institutes of Health (NIH) to use their PA enzyme-
linked immunosorbent assay (ELISA), and toxin neutralization assay (TNA) reagents, which
were developed by Battelle for use in a previous NIAID study.
The following individuals from Battelle were principal contributors to this study: Jason Comer,
Ph.D.; Andrew Lair, M.B.A.; Michael Taylor, Ph.D.; Stephanie Hines, M.S.; Zachary
Willenberg, M.S.; Karen Tracy, M.B.A.; Gregory Stark, Ph.D.; Kevin Tordoff, Ph.D.; Roy
Barnewall, D.V.M., Ph.D.; and Crystal Briscoe, D.V.M., D.A.V.C.P.
The following researchers conducted reviews of this project and final report: Edwin Barth,
Ph.D., P.E., C.I.H; Eletha Brady-Roberts; Abdel-Razak Kadry, D.V.M., Ph.D., D.A.B.T; Alan
Sasso, Ph.D.; Frank Schaefer III, Ph.D; Harlal Choudhury, D.V.M., Ph.D., D.A.B.T; and Jay
Zhao Ph.D., M.P.H., D.A.B.T.
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Foreword
Following the events of September 11, 2001, EPA's mission was expanded to address critical
needs related to homeland security. Presidential Directives identify EPA as the primary federal
agency responsible for the country's water supplies and for decontamination following a
chemical, biological, and/or radiological attack.
As part of this expanded mission, the National Homeland Security Research Center (NHSRC)
was established to conduct research and deliver products that improve the capability of EPA to
carry out its homeland security responsibilities. One focus area of this research is the
compilation, development, and evaluation of information on the human health effects of
pathogens that might be used by terrorists. Such information is critical to understanding the risks
associated with biological contamination and supporting the development of site-specific cleanup
goals, treatment technologies, and detection limits.
NHSRC has made this publication available to assist the response community in preparing for
and recovering from disasters involving microbial contamination. This information is intended to
move EPA one step closer to achieving its homeland security goals and its overall mission of
protecting human health and the environment while providing sustainable solutions to our
environmental problems.
Jonathan Herrmann, Director
National Homeland Security Research Center
XI
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Executive Summary
The U.S. Environmental Protection Agency (EPA), as one of the lead federal agencies
supporting decontamination activities after a biological incident (U.S. DHS, 2008), has been
systematically evaluating microbial dose-response data and its application for decision making to
support decontamination activities. Site-specific risk-based decision making following a
biological threat agent release poses extremely difficult and unique challenges, especially for a
persistent agent such as Bacillus anthracis. Inhalation exposure risk from B. anthracis spores can
result from aerosolized spores during a terrorist attack, subsequent re-aerosolized spores before
cleanup takes place, or re-aerosolized residual spores after remediation is complete. The
objective of this study was to evaluate physiological responses following 15 inhalation exposures
(5 days a week for 3 weeks) to low doses of B. anthracis Ames spores representative of potential
exposures that might be encountered in a reoccupancy/reuse scenario.
Three groups of seven New Zealand White (NZW) rabbits were implanted with telemetry
transmitters and subsequently aerosol challenged with average daily inhaled doses of 2.91 x 102
to 1.18 x 104 colony forming units (CPU) of B. anthracis Ames spores. Five control rabbits also
implanted with the telemetry transmitters were challenged with irradiated (nonviable) spores as
sham challenge controls. The rabbits were then monitored for changes in nonspecific parameters:
activity levels, body temperature, heart and respiration rates, hematology, and C-reactive protein.
Bacillus anthracis-specific parameters were also measured and included bacteremia and toxemia
as evidenced by the presence of protective antigen (PA, a polypeptide produced by B. anthracis)
in the serum. All rabbits underwent necropsy, with the lungs and any gross lesions examined
microscopically to identify anthrax-specific lesions. The challenge doses and mortality data were
then used to identify a benchmark dose value for rabbits.
All seven rabbits exposed to a mean daily dose of 2.91 x 102 CPU survived to the end of the
study and showed no physiological changes that could be attributed to the exposures. One of the
seven rabbits exposed to a mean daily dose of 1.29 x 103 CPU died 17.9 days after the first
xn
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challenge. Four of the seven rabbits that received a mean daily dose of 1.18 x 104 CPU died
during the study with a mean time to death of 14.8 days.
The rabbits that died on study presented with increased respiration rate, heart rate, body
temperature, toxemia, and bacteremia. One animal in the highest dose group responded in the
same physiological manner as those that died, but subsequently initiated a robust neutrophilic
response, seroconverted (developed a humoral response to PA), and survived to the end of the
study.
The calculated benchmark dose lower 95% confidence limit for 50% mortality (BMDLso) for the
average daily dose was 2.60 x 103 inhaled CPU, and the calculated BMDL for 10% mortality
(BMDLio) was 2.90 x 102 inhaled CPU. The calculated BMDL50 and BMDLio for the total
accumulated doses were, respectively, 4.40 x 104 and 4.90 xlO3 total inhaled CPU.
These data represent the first characterization of multiple low-dose inhalation exposures of B.
anthracis in an animal model.
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1 Introduction
The U.S. Environmental Protection Agency
(EPA), as one of the lead federal agencies
supporting decontamination activities after a
biological incident (U.S. DHS, 2008), has
been systematically evaluating microbial
dose-response data and its application for
decision making to support decontamination
activities. As part of the response to a
biological incident, risk-based approaches to
decontamination are desirable as they
provide a formalized process to evaluate the
hazard posed by the released material, assist
in the identification of clearance goals, and
facilitate assessment of the residual risk
posed by selected management approaches.
There is significant interest in the
development of a risk-based management
approach for Bacillus anthracis incidents
because of its high lethality and prior use in
a domestic terrorism event in 2001.
B. anthracis poses unique challenges in the
site-specific risk assessment process because
of the combination of high persistence and
documented lethality at low inhalation
doses. Inhalation exposure to B. anthracis
spores can result from spores that aerosolize
upon initial contact with the air as well as
subsequent reaerosolization after settling on
surfaces. Given the high lethality of B.
anthracis spores from the inhalation route of
exposure, the evaluation of clearance goals
requires the ability to model the inhalation
hazard posed by the low levels of spores that
may remain on surfaces subsequent to
decontaminati on.
It has been over 10 years since the anthrax
letter attacks of 2001 and the lack of an
acceptable dose-response relationship
continues to challenge the development of
risk-based management approaches.
Although B. anthracis is the most highly
studied of the currently known biothreat
agents (Wilkening, 2006), there are
significant data gaps in the dose-response
assessment of low dose exposures (Gutting
et al., 2008). The primary data gap is the
lack of dose-response data suitable for
modeling multiple dose exposures in the low
dose-region. For use in risk-based decision
making, studies must assess multiple doses
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in a manner that is consistent with the
recurring exposure pattern of receptors who
will reoccupy the locations of the biological
incident.
In view of the lack of historical low-dose
exposure studies and the critical need for
credible science to support risk-based
cleanup decisions, U.S. EPA (2011)
conducted an acute low-exposure study
(hereafter referred to as acute study). This
study aimed to determine physiological
responses following an acute exposure to
low inhaled doses of B. anthracis Ames
strain spores (hereafter referred to as B.
anthracis) in the rabbit model of disease. In
the acute low-exposure study, four groups of
five NZW rabbits were implanted with D70-
PCT telemetry transmitters and subsequently
aerosol challenged with a single average
inhaled dose of 2.86 x 102, 2.06 x 103, 2.45
x 104, or 2.75 x 105 CPU. The rabbits were
then monitored for 21 days post-challenge
for changes in nonspecific parameters:
activity levels, body temperature, heart and
respiration rates, hematology, and serum
chemistry. Bacillus anthracis-specific
parameters were also measured and included
bacteremia and presence of protective
antigen (PA, a polypeptide produced by B.
anthracis) in the serum (toxemia). All
rabbits underwent necropsy, and the lungs
and any gross lesions were examined
microscopically to identify anthrax-specific
lesions.
In the acute study, four of the five rabbits
that received an average single inhaled dose
of 2.75 x 105 CPU succumbed to infection
with the mean time to death of 4.6 days.
Two of the five rabbits that were exposed to
an average inhaled dose of 2.54 x 104 CPU
died 4.1 and 10.9 days post-challenge. All
rabbits that received an average inhaled dose
of 2.06 x 103 CPU or lower survived to the
end of the study. Animals that succumbed to
disease had pathological changes consistent
with inhalational anthrax in the rabbit
model, including pleural effusion and
inflammation and bacilli observed in the
lungs.
All animals that died in the acute study were
bacteremic and 73% were positive for PA in
serum. Increases in respiration, heart rate,
and body temperature were also observed in
rabbits that succumbed to anthrax. In
addition, neutrophilia and increased liver
enzymes in the sera were associated with
disease. Animals that survived to the end of
the study never became bacteremic or
toxemic.
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The data from the acute study suggest that
an inhaled dose of B. anthracis spores at or
above 2.54 x 104 CPU in the rabbit results in
death and elicits measurable physiological
changes. These data also suggest that
inhaled doses of 2.06 x 103 CPU or lower do
not cause death or adverse changes in the
measured physiological responses in the
rabbit model of disease.
While the acute study determined the
physiological effects of a single low dose
exposure, the effects of repeated exposures,
such as would be encountered in a
reoccupancy/reuse scenario, remained
unknown. To fill this knowledge gap, using
the acute study as a guide, the follow-on
study described in this report was performed
to determine physiological changes arising
during and after 3 weeks of exposure to
sublethal doses of B. anthracis spores.
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2 Materials and Methods
2.1 Test System
The protocol for the study, along with the
methods referred to herein, are provided in
Appendix A. All study deviations are
documented in Appendix B. Thirty male
specific pathogen-free NZW rabbits
(Oryctolagus cuniculus) weighing
approximately 2.7 kilograms (kg) were
purchased from Covance (Denver, PA)
(Appendix C identifies the pathogen list for
testing). Twenty-six rabbits were placed on
study and the remaining four served as
replacements. Rabbits were quarantined for
5 days prior to exposure. The study was
performed at the Battelle Biomedical
Research Center (BBRC) located in West
Jefferson, OH. A veterinarian implanted a
Data Sciences International (St. Paul, MN)
model D70-PCT telemetric device and
vascular access ports (VAPs) in each of the
rabbits prior to the start of the study. Nasal
swabs were taken and sent to Charles River
Research Animal Diagnostic Services
(Wilmington, MA) for Bordetella
bronchiseptica testing to determine any
potential correlation with active B.
bronchiseptica infection and response in this
study. All animals were negative for B.
bronchiseptica infection; the results of the
testing are presented in Appendix C. B
bronchiseptica status was not a criterion for
rabbit placement on the study.
2.2 Randomization of Animals
Prior to challenge, the animals were
randomized by weight into one group of five
and three groups of seven rabbits (Table 1).
The rabbits within each group were
randomized for challenge order based on ear
tag numbers. The SAS® software PLAN
procedure (SAS Institute, Inc., Gary, NC)
was used to randomize the animals. The
rabbits were challenged according to
randomization order and challenge dose
group. For example, the rabbits in Group 1
were challenged first and the rabbits in
Group 4 were challenged last. The
randomization report is located in Appendix
D.
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Table 1. Study Design and Challenge Doses
Group
1 (Sham challenge*)
2
3
4
Targeted
Inhaled Spore
Dose (CFU)
10,000*
100
1,000
10,000
Number of Spore
Challenges!
15
15
15
15
Number of Rabbits
5
7
7
7
* Spores were inactivated/killed by irradiation.
fRabbits were challenged once each day for five straight days (Monday through Friday) each week for three
consecutive weeks.
2.3 Bacillus anthracis Ames Strain Spores
B. anthracis Ames strain spores (spore lot
Ames B36) were used on this study. The
spores were characterized and qualified
prior to release for use (Table 2).
Table 2. Characterization of Bacillus anthracis Spores
Characterization
Colony purity: Colony
morphology on blood agar
% Vegetative cells
% Debris
% Spore refractility
Viable spore count
Guinea pig LD50
Endotoxin content
Phenol content
Acceptance Criteria
Pure culture
<5%
<5%
< 5% nonrefractile spore
> lx!09CFU/mL
< 10 spores/dose intradermal
< l.OEU/mL
0.8-1.2%
Results
Pure culture
0%
0.34%
0.72% nonrefractile spore
1.82x 1010CFU/mL
2.49 spores/dose intradermal
0.14EU/mL
0.83%
EU = endotoxin unit
mL = milliliter
The spores were stored at 4°C to 8°C in
1.0% phenol, washed with endotoxin-free
water four times, and stored at 4°C to 8°C
until diluted for aerosolization. Prior to use,
the spores were diluted to the appropriate
concentration in endotoxin-free sterile water
and 0.01% Tween 20. The spores were then
stored in single-use aliquots until time of
use.
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2.4 Aerosol Challenge Generation and
Monitoring
On each of the 15 challenge days, the rabbits
were placed into a plethysmography
chamber, passed into a Class III biosafety
cabinet system, and aerosol challenged with
9 T
targeted inhaled doses of 1.0 x 10 , 1.0 x 10
or 1.0 x 104CFU of B. anthracis spores
(Table 1). The challenge dose was
controlled by the concentration of spores in
the nebulizer and the length or exposure
time. The sham challenge group was
exposed to l.Ox 104gamma-irradiated
spores as described below.
The volume of material loaded onto the
nebulizer was the same for each
concentration, 8 mL loaded. The dose was
controlled by varying the concentration per
mL in the nebulizer which produced the
resulting aerosol concentration. For
example, log increases in the nebulizer
concentration will result in a log increase in
the aerosol concentration (CFU/L) which
results in the log difference in the dose when
the same volume (TATV) of atmosphere is
inhaled.
A modified Microbiological Research
Establishment type three-jet Collison
nebulizer (BGI, Waltham, MA) with a
precious fluid jar was used to generate a
controlled delivery of aerosolized B.
anthracis spores from a liquid suspension.
This nebulizer was designed to generate
aerosols with an approximate aerodynamic
mean diameter of 1 to 2 micrometers (|im).
The nebulizer was characterized for a
pressure that results in approximately 7.5
liters/minute (L/min) flow, which normally
is approximately 28.0 pounds per square
inch, Collison nebulizer dependent.
Aerosol concentration and aerosol particle
size distribution were determined by
analysis of atmospheric samples drawn from
the exposure chamber. The aerosolized
spores were drawn into a plexiglass
exposure chamber with internal dimensions
of approximately 20.5 centimeters (cm) x
20.5 cm x 40 cm (length x width x height).
Atmospheric samples were collected using
an impinger (model 7541; Ace Glass Inc.,
Vineland, NJ) filled with approximately 20
mL of sterile water that sampled at
approximately 6.0 ± 0.3 L/min. The
sampling rate was achieved by maintaining a
vacuum of > 18 inches Hg across the
exhaust connection of the impinger to
maintain the flow from the impinger critical
orifice. The liquid in the impinger was
diluted and enumerated by the spread plate
technique to quantify culturable spore
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counts per mL; concentrations were reported
in terms of CFU/mL. The impinger flow
rates were recorded throughout the exposure
and the mean rate was used in the dose
calculation. Enumeration results, along with
the volume of liquid in the impinger,
sampling rate, and sampling duration, were
used in the calculation of the aerosol
concentration expressed as CFU/L of air.
The aerosol particle size was determined
during each exposure using an Aerodynamic
Particle Sizer® (APS model 3321; TSI Inc.,
Shoreview, MN), which drew an
atmospheric sample from the exposure
chamber at 0.25 L/min with a diluter (1.0
L/min total with 0.75 L/min from the diluter
and 0.25 L/min from the exposure chamber).
Whole-body plethysmography was
performed in real time on each animal
during challenge to measure important
respiratory parameters. These parameters
(tidal volume, total accumulated tidal
volume [TATV], and minute volume) were
calculated from the measured volumetric
displacement of air caused by the movement
of the thoracic cavity of an animal while it
was in a sealed plethysmographic chamber.
The TATV and the aerosol concentration
were used to calculate the inhaled dose.
The rabbits were physically restrained
within a plethysmography restraint device
with the head protruding out of a port that
was sealed with rubber dental dam material
and held in place with two plexiglass
guillotines. The plethysmograph was
connected to a pneumotach (Hans Rudolph,
Inc., Shawnee, KS) that was attached to a
differential pressure transducer (model DP-
45; Validyne Engineering Corp., Northridge,
CA). Pressure differential measurements
from inhalations and exhalations were
transmitted to BioSystem XA version 1.5.7
software (BioSystem XA, Buxco
Electronics, Sharon, CT), which then
calculated and recorded respiratory function.
Prior to animal exposures, the Buxco
software program was calibrated to establish
unit (baseline) and air volume displacements
from 5 to 40 mL to simulate animal
respiration. This calibration was performed
to encompass the respiration volume range
of the animal model to ensure accurate
TATV measurements.
The inhalation exposure system data for
each exposure were documented to ensure
proper system operation and to provide the
needed information to quantify animal
challenge conditions. Impinger sampling
conditions and enumerated concentration
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results provided culturable bioaerosol
challenge concentration, while
plethysmography measurements
documented the total inhaled volume. Total
inhaled dose, as measured in CPU, was
calculated from aerosol concentration and
total inhaled volume. The LDso was
calculated by dividing the total inhaled dose
by the reported inhalation LDso for the
rabbit. The reported LDso value for rabbits is
1.05 x 105 inhaled CFU/animal (Zaucha et
al., 1998).
Impinger samples were enumerated by the
serial dilution (10"1 to 10~3) and plating on
tryptic soy agar [TSA] plates in triplicate.
Diluted samples were mixed in a capped vial
prior to subsequent dilutions. At different
target dilutions, 0.1 mL was spread onto
each of five TSA plates, which were placed
where C = CFU/mL
A = Average CPU per plate
D = Dilution factor
The total inhaled dose (InD) was calculated
from the impinger sample concentration,
sampling parameters, and exposure time
(Equation 2). This equation assumes near
100% impinger sampling efficiency. The
total number of viable CPU captured during
each exposure was the product of the
in a secondary container and incubated.
r\
Impinger samples from the 1.0 x 10 and 1.0
x 103 CPU targeted inhaled doses were
enumerated by spread plating and by growth
on a filter. Briefly, 1.0 mL of the sample
was passed through a sterile 0.45 |im filter
(Nalgene®) analytical test filter funnel
(Catalog no. 145-0045; Fisher Scientific,
Pittsburgh, PA). The filter then was placed
on top of a TSA plate, incubated for 24 to 72
hours at 37°C ± 2°C, and then enumerated.
The impinger samples from the irradiated
spores were plated without diluting (0.1 mL)
to ensure sterility of the samples. After the
incubation period, the plates were
enumerated to determine the number of
colonies on each plate. Impinger sample
concentration was determined using
Equation 1.
(1)
impinger concentration (C) and the impinger
sampler volume (V). The total number of
viable CPU was divided by the amount of
air (S) that was sampled through the
impinger during the exposure time (T). The
aerosol concentration was (C x V) (S x T)"1.
-------
The InD was calculated as the product of
the aerosol concentration and the TATV.
(2)
where InD = Inhaled dose (CPU)
C = Impinger concentration (CFU/mL)
V= Impinger sampler volume (mL)
S = Sampling rate (6 L/min)
T = Exposure time (min)
TATV= Total accumulated tidal volume (L).
Additional details of the aerosol exposure
system and a detailed schematic are found in
Appendix E.
2.5 Telemetric Monitoring
The rabbits were surgically implanted with
telemetry units (model D70-PCT
transmitters) prior to being placed on study.
Each D70-PCT transmitter contained one
pressure lead and one biopotential lead.
Body temperature, electrocardiogram
activity, and cardiovascular function (heart
rate and respiratory pressure) were
monitored for 30 seconds every 15 min for 7
days prechallenge (baseline) and for 39 days
post-first challenge.
Each animal's cage was equipped with a
Data Sciences International telemetry
receiver. The transmitters, receivers,
consolidation matrices, cabling, and
computers using the Dataquest A.R.T.™ data
acquisition and analysis software are all
components of the PhysioTel® telemetry
system. The Dataquest A.R.T. telemetry
software collected the telemetry parameters
mentioned above. The statistical methods
used to analyze the telemetry data are
presented in Appendix F.
2.6 Clinical Observations and Body
Weights
Throughout the study, the rabbits were
observed twice daily for survivability and
clinical signs of illness that could be
attributable to anthrax infection (e.g.,
moribund, respiratory distress, appetite,
activity, and seizures). Animals were
weighed on Study Days 2, 9, 16, 23, 30, and
37. The statistical methods used to analyze
the survival data and body weights are
described in Appendices G and H,
respectively. Individual clinical observations
and body weights are presented in
Appendices K and L, respectively.
-------
2.7 Blood Collection
On Study Days -3, 2, 4, 9, 11, 16, 18, 23, 25,
30, 32, and 37, blood was collected into
ethylene- diaminetetraacetic acid (EDTA;
-1.0 mL) tubes and serum separator tubes
(SSTs; -2.0 to 2.5 mL) (Table 3). Blood
samples also were taken from animals found
dead or prior to euthanasia. On Study Day
39, all surviving rabbits were terminally
bled via cardiac puncture according to Table
3.
Table 3. Blood Collection Schedule
Study Day
Tube
Type
EDTA
(~mL)
SST
(~mL)
Total per
day (~mL)
Day
-3
1.0
2.5
3.5
Day
2
1.0
2.0
3.0
Day
4
1.0
2.5
3.5
Day
9
1.0
2.0
3.0
Day
11
1.0
2.5
3.5
Day
16
1.0
2.0
3.0
Day
18
1.0
2.5
3.5
Day
23
1.0
2.0
3.0
Day
25
1.0
2.5
3.5
Day
30
1.0
2.0
3.0
Day
32
1.0
2.5
3.5
Day
37
1.0
2.0
3.0
Day
39
2.0
10.0
12.0
Serum was collected from blood samples in
SSTs by centrifugation and was stored at
< -70°C until analyzed. Blood in EDTA
tubes was stored at room temperature if used
within 4 hours of collection; blood was
stored at 2°C through 8°C if not analyzed
within 4 hours. Blood was collected from
the VAP throughout the study. If a VAP
failed, the medial auricular artery or the
marginal ear vein was used for blood
collection regardless of the sample time
point. If a blood sample was not collected
from either the VAP or other appropriate
vessel, it was documented in the study file.
Appendix I contains the exact blood
collection times.
2.8 Protective Antigen ELISA
Serum samples were collected and stored in
a freezer set to maintain < -70°C until
evaluation of quantitative circulating PA
levels by enzyme-linked immunosorbent
assay (ELISA). Double affinity purified
polyclonal, monospecific rabbit anti-PA
immunoglobulin G (IgG) "capture antibody"
was produced by Battelle (Columbus, OH).
It was purified from recombinant PA (rPA)-
vaccinated rabbit serum using first a Protein
A column to bind all IgG antibodies, and
then a PA column to specifically isolate
anti-PA IgG antibodies. The "capture
antibody" was used to coat the wells of a 96-
well plate at a concentration of 2
10
-------
micrograms per mL (|ig/mL). The plates
were blocked with skim milk and then
incubated with rabbit serum samples
containing native PA (Catalog No. NR-164,
Lot No. 5051797; BEI Resources, Manassas,
VA), or a reference standard and quality
control samples consisting of rPA spiked
differentially into naive rabbit serum. The
PA was detected by first incubating with
diluted goat PA anti-serum, followed by
incubation with a bovine anti-goat
horseradish peroxidase (HRP)-conjugated
secondary antibody (Santa Cruz
Biotechnology, Inc., Santa Cruz, CA), then a
2,2'-azinobis [3-ethylbenzothiazoline-6-
sulfonic acid]- diammonium salt substrate
and a stop solution (both from Kirkegaard
and Perry Laboratories, Gaithersburg, MD).
The plates were read and the data were
analyzed using a four-parameter logistic-log
(4PL) model to fit the eight-point calibration
curve. The concentrations of PA in unknown
samples were determined by computer
interpolation from the plot of the reference
standard curve data (SoftMax® Pro;
Molecular Devices, Downington, PA). The
assay was qualified using PA spiked into
rabbit serum resulting in a qualified linear
range, slope, and putative limit of detection
(LOD) used for the assays.
2.9 Bacteremia
A portion of each blood sample from the
EDTA collection tubes was tested for
bacteremia by quantitative spread plate
technique and quantitative real-time
polymerase chain reaction (qPCR).
Quantitative counts were achieved by 10-
fold serial dilutions of the blood samples in
Dulbecco's phosphate buffered saline (11.0
grams [g] NaCl, 5.7 gNaH2PO4, 1.3 g
Na2HPO4 dissolved in 1.0 L of distilled
water, pH adjusted to 6.2, and filter
sterilized) from 1.0 x 101 to 1.0 x 109 and
spread plating 100 microliters (|iL) of each
dilution onto TSA in triplicate. The plates
were enumerated after 24-hour incubation at
37°C ± 2°C. In instances when a blood
sample could not be obtained in an EDTA
collection tube for quantitative bacteremia
culture, the pellet from the SST sample was
streaked on an agar plate to obtain
qualitative results. Colonies with
morphology consistent with B. anthracis
("ground glass"-like appearance) were
enumerated to determine the viable bacterial
load in the blood.
To perform qPCR, total nucleic acid was
isolated from 100 jiL rabbit peripheral
whole blood using the fully automated
bioMerieux NucliSENS® easyMag® kit
11
-------
(bioMerieux, Durham, NC). Based on
published sequence data available in
GenBank (accession number AEO16879),
oligonucleotides were designed that would
amplify a small deoxyribonucleic acid
(DNA) fragment within the coding region of
the B. anthracis DNA-directed ribonucleic
acid (RNA) polymerase subunit beta (rpoB)
gene (Table 4). The rpoB gene was selected
because it is a highly conserved
housekeeping gene. Due to its essential role
in cellular metabolism, at least one copy is
expected to be present in all bacteria. The
qPCR assay was designed to be quantitative
and not diagnostic. Therefore, the primers
and probe used may detect other Bacillus
species, and the specificity of the assay was
not determined.
®
Table 4. TaqMan Gene Expression Assay for the B. anthracis rpoB_571 Gene
Name
rpoB_571
Primer/Probe
Forward
Reverse
Probe
Oligonucleotide Sequence (5'-3')
ATTCAAAACAGCGAAACCAA
TCTATTAAGATTTATGCTCCTGAGTCAGA
6FAM-TGGAGTGGTAGAAGGTGA-NFQ
Accession No.
AEO 16879
GenBank accession numbers are available online at http://www.ncbi.nlm.nih. gov/
®
qPCR reactions consisted of IX TaqMan
Universal PCR Master Mix (AmpliTaq
Gold® DNA polymerase, AmpErase® UNG,
dNTPs with dUTP, passive reference, and
optimized buffer components [Applied
Biosystems Inc., Foster City, CA]), IX Gene
Expression Assay mixture 900 nanomolar
(nM) forward primer, 900 nM reverse
primer, and 250 nM probe (dual-labeled
with FAM™ at the 5' end and a
nonfluorescent quencher at the 3' end; Table
4), nuclease-free distilled water, and either 5
|iL of qualified reference standard plasmid
or 5 |iL of isolated nucleic acid in a total
volume of 50 jiL. qPCR was performed
using an ABI PRISM® 7900HT fast
sequence detection system (Applied
Biosystems Inc.) with the following
conditions: 2 min at 50°C, 10 min at 95°C,
followed by 40 cycles of 95°C for 15
seconds and 60°C for 1 min. All reactions
were performed in triplicate, and each run
contained a nucleic acid isolation negative
control (genomic DNA isolation procedure
using nuclease-free distilled water), a
nucleic acid isolation positive control
12
-------
(genomic DNA isolated from a B. anthracis
vegetative culture), and a master mix only
control (no template control [NIC]).
Following acquisition, data were analyzed
using the sequence detection system
software. Final reportable values were
extrapolated from the reference standard
curve as long as a minimum of two test
sample cycle threshold (Ct) values were
within 0.50 of one another.
2.10 TNA/ELISA
To determine if the rabbits elicited an
immune response following challenge,
serum samples were analyzed by an anti-PA
IgG ELISA and high-throughput toxin
neutralization assay (TNA) as described
below. The ELISA was designed to quantify
IgG antibodies against anthrax PA using
purified rPA as the solid-phase immobilized
antigen, and an enzyme-conjugated anti-
gamma chain secondary antibody was used
as the reporter or signal system. The assay
endpoint was reported as the serum mean
concentration of anti-PA-specific IgG
Og/mL).
Microtiter plates were coated with purified
rPA. Unknown test samples, anti-PA IgG
reference standard serum, and positive
control sera were added to the microtiter
plate. The PA-specific antibodies present in
the samples/standards were allowed to bind
to the rPA coated on the plate. After
washing, the bound anti-PA antibodies were
then detected by a species-specific anti-
gamma chain IgG-HRP conjugate followed
by addition of a peroxidase substrate. The
optical density (OD) values for each plate
were then read on a micropiate reader
(ELxSOO; BioTek, Winooski, VT) at a
wavelength of 405 nanometers using a 490
nanometer reference wavelength. The
ELISA has both primary (plate-level) and
secondary (test sample-level) acceptance
criteria. The anti-PA IgG concentration of
each passing test sample on passing plates
was determined by taking the average of the
acceptable concentrations from the eight-
point dilution of the test sample back-
calculated from the standard curve. Results
were reported in |ig/mL of anti-PA IgG for
each unknown test sample.
The TNA was designed to measure and
qualify the functional ability of serum to
neutralize B. anthracis lethal toxin activity
using an in vitro cytotoxicity assay.
Specifically, cell viability was determined
colorimetrically using atetrazolium salt, 3-
[4, 5-dimethylthiazol-2-yl]-2, 5-
diphenyltetrazolium bromide (MTT) as the
reporter or signal system. The serum -
13
-------
mediated neutralization of anthrax lethal
toxin manifested as a suppression of
cytotoxicity, and hence preservation of cell
viability.
Microtiter cell plates were seeded with a
murine monocyte-macrophage cell line
(J774A.1 cells) and allowed to adhere. In
separate microplates (prep plate), a serial
dilution of the test samples and controls
were prepared. Lethal toxin (lethal factor
[LF] + PA) was added to the prep plate and
incubated to allow for lethal toxin
neutralization by neutralizing antibodies.
The contents of the prep plate were then
transferred to the cell plate and incubated to
allow intoxication to proceed. MTT was
then added to the cell plates to allow viable
cells to reduce the MTT dye. The OD values
for each plate were read on the ELxSOO
microplate reader at a wavelength of 570
nanometers using a 690 nanometer reference
wavelength. The TNA Statistical Analysis
System (SAS®; SAS Institute Inc., Gary,
NC) program then fit the seven-point serial
dilutions of the reference serum standard
and test sample serum OD values to a 4PL
function, which was in turn used to calculate
the reportable values (effective dose 50%
[EDso] and neutralization factor 50%
[NFso]). The EDso was the reciprocal of the
dilution of a serum sample that results in
50% neutralization of the lethal toxin. The
NFso is the quotient of the EDso of the test
sample and the EDso of the reference serum
standard. The NF50 was calculated to
determine the neutralization capacity of the
test sample relative to the reference serum
standard on that plate, thus normalizing day-
to-day assay variability.
2.11 Hematology and C-Reactive Protein
Complete hematological analysis was
performed on blood samples collected in
EDTA tubes using the Advia® 120
hematology analyzer (Siemens Healthcare
Diagnostics, Deerfield, IL) according to the
manufacturer's recommendations.
Hematology analysis included the following
parameters:
• White blood cell (WBC) count
• Neutrophil/lymphocyte ratio
• Differential leukocyte (absolute)
count
• Hemoglobin (HGB)
• Hematocrit (HCT)
• Red blood cell (RBC) count
• Mean corpuscular volume
• Mean corpuscular hemoglobin
• Mean corpuscular hemoglobin
concentration
• Red cell distribution width (ROW)
14
-------
• Platelet count (PLT)
• Mean platelet volume.
The values for the normal ranges of these
parameters were identified by the
manufacturer and were derived from mean
values published by Schalm et al. (1975).
The statistical methods used to evaluate the
hematology data are presented in Appendix
J.
After hematological analysis was complete,
plasma was harvested from the residual
sample by centrifugation. The plasma
sample was then assayed for C-reactive
protein (CRP) levels using the Advia® 1200
chemistry analyzer (Siemens Healthcare
Diagnostics, Deerfield, IL) according to the
manufacturer's recommendations. The
statistical methods used to analyze the CRP
data are described in Appendix J.
2.12 Necropsy and Histopathology
Animals that succumbed to challenge or
were found moribund and euthanized
underwent gross necropsy. Surviving
animals were euthanized and necropsied on
Study Day 39. The lungs and gross lesions
from each animal were collected. The
tissues collected for microscopic evaluation
varied from animal to animal and included
skin, cecum, appendix, and mediastinal
lymph node. The collected tissues were
placed in 10% neutral buffered formalin,
processed to approximately 5 jim slides,
stained with hematoxylin and eosin, and
examined histologically by a board-certified
pathologist. All microscopic findings were
graded semi-quantitatively according to the
following scale, with the associated
numerical score used to calculate average
severity grades for each lesion by group:
• Minimal (Grade 1): the least
detectible lesion
• Mild (Grade 2): an easily
discernible lesion
• Moderate (Grade 3): a change
affecting a large area of the
represented tissue
• Marked (Grade 4): a lesion that
approached maximal.
Gross and microscopic diagnoses were
entered into the PATH/TOX SYSTEM®
(Xybion Medical Systems Corporation,
Cedar Knolls, NJ) for data tabulation and
analysis.
2.13 Benchmark Dose Analysis and
Dosimetric Adjustment
A benchmark dose (BMD) analysis was
conducted using the survival data collected
in this study (challenge doses and mortality).
The outputs of the BMD analysis were then
used as the inputs for a dosimetric
15
-------
adjustment to derive human equivalent dose
(HED) and human equivalent concentration
(HEC) values.
Two dose metrics of inhaled dose were
evaluated in the benchmark dose analysis:
the average daily dose per animal (ADD)
and the total aggregate dose per animal
(TAD). For the ADD, daily inhaled doses
were averaged across all exposure and non-
dosing days until the death of the animal or
the exposure duration for those animals that
survived the length of the study. The
exposure duration of the study was 19 days,
which captures the total number of study
days including days to allow for calculation
of an ADD consistent with EPA guidance
for discontinuous exposures (U.S. EPA,
2002). For the TAD, daily inhaled doses
were summed across all exposure days until
the death of the animal or the exposure
duration for those animals that survived the
length of the study.
For the BMD evaluation, the current version
of EPA's benchmark dose software (BMDS
2.1.2 Version 2.1.2.60, Build 06/11/10)
(U.S. EPA, 2010a) was used to fit models to
the dose-response data. Models from the
BMDS dichotomous and dichotomous-
alternative model suites were evaluated in
the analysis: Weibull model, Weibull model
run as exponential (with the power
coefficient fixed as one), probit, loge probit,
logistic, loge logistic, Gamma model,
dichotomous Hill, probit-background
response, loge probit-background response,
logistic-background response, and loge
logistic-background response. Mortality data
were modeled on an individual basis using
each estimated dose (i.e., with n=l at each
dose).
Benchmark dose analysis estimates the
BMD for a specified level of benchmark
response (BMR) observed. The BMR is
defined as the level of change in the
response rate (in this case mortality). For
example, a BMR of 10% would be
equivalent to a 10% response rate of the
endpoint of interest. For this assessment,
BMRs of 0.50, 0.10, and 0.01 were reported
to allow for comparison of different model
estimates at various points in the dose-
response relationship. When used as inputs
in the calculation of BMDs, these BMR
values correspond to estimates of 50%
lethality (i.e., LD50), 10% lethality, and 1%
lethality; the resulting BMDs would be
written BMD50, BMDio, and BMD0i,
respectively. The 95% lower confidence
limit of the calculated BMD is the
benchmark dose limit (BMDL).
16
-------
A dosimetric adjustment was conducted
using the assumptions identified in Table 5
and the ADD BMDLio value calculated
using the best fitting mathematical model
identified during the benchmark dose
analysis. As part of this adjustment,
assumptions were identified for the human
inhalation rate, the rabbit pulmonary
deposition rate, and human pulmonary
deposition rate. With the exception of
generating a particle size distribution-
specific pulmonary deposition rate using the
Regionally Deposited Dose Ratio (RDDR)
Model (U.S. EPA, 1994), the approach to
calculate the HED and HEC followed that
presented in U.S. EPA (201 Ob).
Table 5. Assumptions Used to Generate Human Equivalent Dose and Human Equivalent
Concentration
Parameter
Rabbit Pulmonary
Deposition Rate
Human Inhalation Rate
Human Deposition Rate
Value
0.056
16
0.2
Units
Unitless
m3/day
Unitless
Source
Value calculated with RDDR Model v. 2.3
(U.S. EPA, 1994) with Inputs of:
• MMAD = 0.82 urn and GSD = 1.53
(Data Source: Figure 3, Appendix E,
Aerosol Report),
• Body Weight of 2,850 g (Data Source:
Appendix L, Individual Body Weights,
Arithmetic Average of Body Weight on
Days 2, 9, and 16), and
• Minute Volume of 1 .3 L (Data Source:
Arithmetic Average of Calculated
Minute Volume, Product of Tidal
Volume Inhaled and Sampling Time
from Table 17, Table 24, and Table 30
[i.e., Days 2, 9, and 15]).
3 1 to <5 1 Years of Age, Mean Value (Table 6-1
in U.S. EPA 2009).
Higher End of the Range of Human
Depositional Values for 1 to 2 urn particles
(Figure 6-6, U.S. EPA 2004).
g - gram
GSD - geometric standard deviation
L - liter
MMAD - median aerodynamic diameter
\im - micron
17
-------
The complete methodology used in the
benchmark analysis and dosimetric
adjustment is provided in Appendix V.
18
-------
3 Results
3.1 Aerosol Challenges
To determine the rabbits' physiological
responses to multiple, daily, low-dose
aerosol exposures to B. anthracis spores,
three groups of seven rabbits were exposed
to targeted inhaled doses of 1.0 x 102 to 1.0
x 104 CPU. The individual mean challenge
doses for the 15 challenge days as well as
the group means are listed in Table 6. Figure
1 illustrates the group mean challenge daily
doses over the 15 challenge days. All
challenge days had consistent dosing except
Challenge Day 3 where the challenge dose
of Group 2 was higher than expected. This
was most likely caused by an error in the
dilution of the challenge material. Plate
counts of the impinger samples revealed that
individual mean actual inhaled doses for the
r\
15 days of challenge ranged from 2.32 x 10
(±1.28x lO^CFUto 1.44x 104(±5.99x
103) CPU. The mass median aerodynamic
diameter (MMAD) for challenge material
for each group as determined by an APS is
presented in Table 6 and Appendix E, Figure
3. Details of the aerosol challenge data are
contained in Appendix E.
19
-------
IU
104 -
£ 103-
o
102 -
I
r
n
JJ
[III
I
I
1
1
1 2 3 4 5 6 7 8 9 10 11 12
Challenge Number
^M Group 2 (targeted dose: 1x10 CPU)
I 1 Group 3 (targeted dose: 1 x 103 CPU)
I — • Group 4 (targeted dose: 1 x 104 CPU)
-T- -T-
-
13 14 15
Figure 1. Mean challenge doses in CFU for each of the 15 exposure days.
20
-------
Table 6. Individual and Group Mean Challenge Doses over the 15 Exposure Days
Group
Group
1
Group
2
Group
3
Group
4
Animal
ID
40
7
5
9
37
13
34
25
15
30
28
19
14
11
2
8
12
18
32
6
33
27
31
39
21
38
Daily Mean Inhaled
Dose (CFU/Animal)
Mean
0
0
0
0
0
3.85 x 102
3.17xl02
2.79 x 102
3.17xl02
2.72 x 102
2.34 x 102
2.32 x 102
7.38 xlO2
1.12xl03
1.33 xlO3
1.41 xlO3
1.30 xlO3
1.21 xlO3
1.44 x 103
6.41 x 103
9.75 x 103
1.06 x 104
1.25 x 104
1.44 x 104
1.32 xlO4
1.27 x 104
SD
0
0
0
0
0
7.57 x 102
4.48 x 102
3.54 xlO2
3.27 x 102
2.33 x 102
1.49 x 102
1.28 xlO2
2.99 xlO2
5.01 xlO2
5.95xl02
6.06 x 102
4.90 xlO2
5.47 xlO2
5.92xl02
2.57 x 103
2.58 xlO3
3.51xl03
3.27 xlO3
5.99xl03
4.97 xlO3
3.77 xlO3
Group Mean
Inhaled Dose
(CFU/Animal
SD)
0
2.91 xlO2
(3.88xl02)
1.22 x 103
(5.59 x 102)
1.17xl04
4.64 x 103
Challenge Dose
(LD50)*
Mean
0
0
0
0
0
8.16X10'1
2.13
1.56
9.40 x 10'1
1.88
1.75
1.19
8.82 x 10'1
6.98 x 10'1
1.37X10"1
5.12X10'1
7.62 x 10'1
1.14
2.02
6.55 x 10'1
3.08E+00
1.90E+00
2.69E+00
2.57E+00
1.43E+00
2.49E+00
SD
0
0
0
0
0
7.22 x 10'3
4.27 x 10"3
3.38 x 10"3
S.llxlO'3
2.22 x 10"3
1.42 x 10"3
1.22 x 10"3
2.85 x 10'3
4.77 x 10'3
5.50 x 10"3
5.76 x 10'3
4.67 x 10'3
5.30 x 10"3
1.78 x 10"2
2.44 x 10'2
2.48 x 10'2
3.48 xlO"2
3.13xlO'2
5.70 x 10'2
4.74 x 10"2
3.60 xlO'2
Group
Mean
Challenge
Dose (LDSO)
0
2.77 x 10'3
(3.70X10'3)
.16 x 10
(5.33 x 10'3)
.12 x 10
(4.43 x 10'2)
MMAD
in jim
(GSD)
0.81
(1.53)
0.79
(1.52)
0.82
(1.53)
0.86
(1.49)
Time to
Death
(day)
Survived
Survived
Survived
Survived
Survived
Survived
Survived
Survived
Survived
Survived
Survived
Survived
Survived
Survived
17.9
Survived
Survived
Survived
Survived
10.9
12.7
20.8
14.7
Survived
Survived
Survived
*LD50= 1.05 x 105 CPU (Source: Zauchaetal., 1998)
SD = standard deviation
GSD = geometric standard deviation
21
-------
Body weights were taken periodically over
the course of the study as another indicator
of disease. The body weights of the study
rabbits remained consistent throughout the
study. Individual body weights and
statistical analysis are provided in
Appendices L and H, respectively.
All of the rabbits in Groups 1 and 2 survived
until the end of the study (Figure 2). One of
the seven Group 3 animals (Rabbit 2) died
17.9 days after the first exposure. This
animal received 14 of the 15 challenge doses
and received an accumulated challenge dose
of 1.86 x 104 CPUs over the course of the
study. Four of the seven Group 4 rabbits
succumbed to disease with a mean time to
death of 14.80 ± 4.28 days. Table 7 shows
the number of challenge doses and
accumulated dose for each of the rabbits that
succumbed to disease.
Table 7. Accumulated Challenge Dose Information for the Rabbits That Succumbed to
Infection
3.2 Clinical Observations, Body Weights,
and Mortality
The majority of animals that succumbed to
disease showed clinical signs consistent with
inhalational anthrax in the rabbit model.
Anorexia and lethargy were the most
common observations prior to the animal's
death. One rabbit, Rabbit 33 (Group 4), was
normal up to the time that it was found dead.
Interestingly, Rabbit 38 (Group 4) showed
clinical signs of disease including lethargy,
anorexia, and respiratory abnormalities on
Study Days 22-27 but returned to normal on
Study Day 28 and survived to the end of the
study. A complete list of individual clinical
observations is presented in Appendix K.
1.1 Rabbit
ID
2
6
33
27
31
Group
3
4
4
4
4
Number of
Challenge
Doses
14
9
10
15
11
Accumulated
Dose
1.86xl04
5.77 xlO4
9.75 x 104
1.51 xlO5
1.37 xlO5
Time to Death
(days)
17.9
10.9
12.7
20.8
14.7
22
-------
The overall Fisher's exact test on the
mortality was significant (P = 0.0425);
however, there were no significant pairwise
differences between the groups. An overall
log-rank test was significant (P = 0.0135),
indicating that the survival distribution in at
least one of the groups was significantly
different from those in the other groups.
Prior to adjusting for multiple comparisons,
the time to death in Group 2 was
significantly greater than that in Group 4.
However, this relationship was no longer
significant after adjusting for the multiple
pairwise comparisons.
23
-------
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Group 2 (100 CPU)
Group 3 (1,000 CPU)
Group 4 (10,000 CPU)
10 15 20 25 30
Time to Death (Days)
35 40
Figure 2. Kaplan-Meier curves representing time to death and survival data for each group.
24
-------
A logistic regression model was fitted to the
survival data and indicated a significant
dose-response relationship with increased
inhaled doses being associated with
decreased probabilities of survival, as
evidenced by the significant P-value
associated with the estimated slope
coefficient of-1.30 (P = 0.0288). The
estimated accumulated inhaled dose LDso
was 8.1 x 103 CPU with a 95% Fieller
confidence interval ranging from 2.3 x
103 CPU to 3.6 x 107 CPU. Individual
mortality data are located in Appendix M,
and complete statistical analysis can be
found in Appendix G.
3.3 Telemetric Monitoring
To determine physiological responses to the
various low spore doses in the NZW rabbits,
telemetric devices were implanted in the
animals and body temperature,
electrocardiogram activity, and
cardiovascular function (heart rate and
respiratory pressure) were monitored for 30
seconds every 15 min. Each observation was
then baseline adjusted according to the
associated clock time, and 6-hour averages
were computed for the baseline-adjusted
values using the following intervals:
midnight-06:00 (inclusive), 06:00-12:00
(inclusive), 12:00-18:00 (inclusive), and
18:00-midnight (inclusive). The standard
deviation of each 6-hour average at baseline
was calculated and used to form the upper
and lower limits for indications of
abnormality. The upper limit was defined to
be three standard deviations above zero,
while the lower limit was defined to be three
standard deviations below zero. An animal
was found to be abnormal if two consecutive
baseline-adjusted 6-hour averages were
outside the upper or lower limits following
challenge. The time of onset for abnormality
was defined as the time associated with the
second abnormal value during the first
occurrence of two consecutive abnormal
values following challenge. The end of
abnormality was defined as the time
associated with the last abnormal value
during the last occurrence of two
consecutive abnormal values following
challenge. Therefore, the duration of
abnormality was defined as the difference
between the time associated with the end of
abnormality and the time associated with the
onset of abnormality.
Estimates and exact binomial 95%
confidence intervals for the proportion of
abnormal animals were calculated within
each group, and an overall two-sided
Fisher's exact test was performed to
25
-------
determine if there was a significant
difference between the proportions of
abnormal animals in each group (at the
0.05 significance level). Table 8 contains the
proportion of animals that were abnormal at
any point during the study by group for each
parameter, as well as the mean duration of
abnormality for those groups having
abnormal animals. In addition, Table 8
contains the results of Fisher's exact tests,
comparing the proportion of animals that
were abnormal in each group by parameter.
There were no significant differences
between the groups for any parameter. The
complete statistical analysis of the telemetry
data is located in Appendix F.
Table 8. Abnormality Summaries by Parameter and Group Along with Fisher's Exact
Tests Comparing the Proportion Abnormal in Each Group by Parameter
Parameter
Activity
Heart Rate
Respiratory
Rate
Temperature
Group
1
2
3
4
1
2
3
4
1
2
3
4
1
2
3
4
Number
Abnormal/N
2/5
2/7
3/7
5/7
5/5
5/7
111
5/7
3/5
111
4/7
4/7
3/5
6/7
4/7
6/7
Proportion
Abnormal
(95% Confidence
Interval)
0.40(0.05,0.85)
0.29 (0.04, 0.71)
0.43(0.10,0.82)
0.71 (0.29, 0.96)
1.00(0.48, 1.00)
0.71 (0.29, 0.96)
1.00(0.59, 1.00)
0.71 (0.29, 0.96)
0.60(0.15,0.95)
1.00(0.59, 1.00)
0.57(0.18,0.90)
0.57(0.18,0.90)
0.6(0.15,0.95)
0.86(0.42, 1.00)
0.57(0.18,0.90)
0.86(0.42, 1.00)
Mean Duration of
Abnormality (Days)*
15.51
7.38
11.42
6.00
16.75
19.10
6.82
7.10
30.58
22.11
6.44
7.44
11.17
23.42
10.81
12.08
Fisher's
Group
Effect
P-Value
0.5161
0.2855
0.2096
0.5542
*Means exclude those animals that were never abnormal
26
-------
Figure 3 illustrates the mean activity levels
of the groups after challenge. Figure 4
shows the activity levels for each animal on
study and highlights the variability within
each group. By Study Day 8, all groups had
experienced a significant decrease from
baseline. This significant decrease from
baseline activity continued intermittently in
each group until Study Day 23, but was
more prevalent in Group 4. On Study Day
17 at 6:00-12:00 and on Study Day 18 at
18:00-midnight, the mean decrease from
baseline in Group 4 was significantly
different from the mean change from
baseline activity in Group 1. On Study Day
19 at 12:00-18:00, on Study Day 20 at
midnight-6:00, 6:00-12:00, and 18:00-
midnight, on Study Day 21 at midnight-
6:00, on Study Day 24 at midnight-6:00,
and on Study Day 37 midnight-6:00, the
mean decrease from baseline activity in
Group 4 was significantly different from the
mean change from baseline in Group 2. On
Study Day 37 at midnight-6:00, the mean
decrease from baseline in Group 4 was
significantly greater than that in Group 3.
27
-------
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Group 3 (1,000 CPU)
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10 20 30
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28
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Figure 3. Plot of mean baseline-adjusted activity (counts/min) for each group.
Group 1
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— Animal 39
— Animal 21
— Animal 31
— Animal 38
10 20 30
Days from Midnight of Challenge Day
40
Figure 4. Plot of baseline-adjusted activity (counts/min) values for each rabbit.
29
-------
Figure 5 illustrates the changes in heart rate
in beats per minute (BPM) by group mean
over the course of the study. Figure 6 shows
the heart rate of each rabbit over the course
of the study. By Study Day 1 at 12:00-
18:00, all groups had experienced significant
increases in heart rate from baseline. These
significant increases continued
intermittently for all groups until Study Day
5 at 6:00-12:00. By Study Day 6 at 12:00-
18:00, all groups had experienced a
significant decrease from baseline. The
significant decreases in heart rate continued
intermittently and with increasing frequency
until the end of the study, with more
prevalence in Groups 1 through 3 after
Study Day 24. On Study Day 24 at 6:00-
12:00 and 12:00-18:00, the decrease from
baseline in Group 1 was significantly
different from that in Groups 2, 3, and 4.
Figure 7 illustrates the mean respiratory
rates of the groups after challenge in
respiratory cycles per minute (RCPM).
Figure 8 shows the respiratory rates for each
rabbit over the course of the study. Each
rabbit that succumbed to disease showed
increased respiration rates. Animals that
died are indicated by truncated data lines in
Figure 8. Interestingly, Rabbit 38 showed an
increase in respiration rates from
approximately Day 21 to Day 26, which
corresponds with the time frame in which
the animal was bacteremic, neutrophilic, and
toxemic.
By Study Day 1 at 12:00-18:00, all groups
had experienced a significant increase in
respiration rates from baseline. These
significant increases from baseline
continued intermittently throughout the
study. In Groups 2 and 3, these significant
increases were more prevalent especially
after Study Day 15 through the end of the
study. Group 4 was the only group that
experienced significant decreases in
respiration rates from baseline, which
occurred on Study Day 5 at 6:00-12:00 and
on Study Day 6 at 6:00-12:00. On Study
Day 4 at midnight-6:00 the mean increase
from baseline in Group 1 was significantly
different from the change from baseline in
Groups 2 and 3. In addition, on Study Day 4
at midnight-6:00, Study Day 5 at 6:00-
12:00 and 12:00-18:00, Study Day 6 at
6:00-12:00, and Study Day 10 at midnight-
6:00, the mean decrease from baseline
respiration rate in Group 4 was significantly
different from the change from baseline in
Group 1. On Study Day 2 at 6:00-12:00, the
mean increase from baseline in Group 2 was
significantly different from the change from
30
-------
baseline in Group 3. On Study Day 13 at
midnight-6:00, the mean increase from
baseline in Group 4 was significantly
different from the change from baseline in
Group 2. On Study Day 1 at 18:00-midnight
and on Study Day 5 at 6:00-12:00, the mean
increase from baseline in Group 3 was
significantly different from the change from
baseline in Group 4.
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Group 3 (1,000 CPU)
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10 20 30
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Figure 5. Plot of mean baseline-adjusted heart rate (BPM) for each group.
32
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10 20 30
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Animal 6
Animal 27
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10 20 30
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40
Figure 6. Plot of baseline-adjusted heart rate (BPM) for each rabbit.
33
-------
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Figure 7. Plot of mean baseline-adjusted respiratory period (RP) respiratory rate (in RCPM) for each group.
34
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10 20 30
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Animal 25
Animal 30
Group 4
Animal 6
Animal 27
Animal 33
Animal 39
— Animal 21
— Animal 31
— Animal 38
10 20 30
Days from Midnight of Challenge Day
40
Figure 8. Plot of baseline-adjusted RP respiratory rate (in RCPM) for each animal.
35
-------
Figure 9 shows the mean body temperatures
of the groups after challenge. Figure 10
shows the body temperatures for each rabbit
over the course of the study. All animals that
succumbed to infection, except Rabbit 6
(Group 4), showed an increase in body
temperature. Rabbit 38 also had a febrile
response from Study Day 18 though 24,
which corresponded with the time that the
animal became bacteremic, toxemic, and
neutrophilic, and had an increased
respiration rate. Several animals showed
sporadic decreases in body temperatures
(Figure 10). These drops in temperature
corresponded to blood draw days in which
acepromazine was used as a sedative to
facilitate blood draws from the ear. This
sedative has been shown to decrease body
temperature, and thus the decreases
observed in the study are an artifact of
sedation (Hobbs et al., 1991; Montane et al.,
2003).
For Group 1, there were significant
increases and decreases in body temperature
from baseline beginning on Study Day 1 at
12:00-18:00 and continuing intermittently
until Study Day 9 at 18:00-midnight. For
Group 2, there were significant increases in
body temperature from baseline starting on
Study Day 0 at 18:00-midnight and
continuing with decreasing frequency
through Study Day 29 at midnight-6:00;
relatively soon thereafter, significant
decreases from baseline were observed
beginning on Study Day 30 at 12:00-18:00
and continuing with increasing frequency
through Study Day 38 at 12:00-18:00. For
Group 3, there was a significant increase
from baseline body temperature beginning
on Study Day 1 at 12:00-18:00 and
continuing intermittently through Study Day
38 at 18:00-midnight. For Group 4, there
was a significant increase from baseline
beginning on Study Day 1 at midnight-6:00
and continuing intermittently through Study
Day 20 at 6:00-12:00. On Study Day 27 at
18:00-midnight, Study Day 33 at 18:00-
midnight, and Study Day 34 at 18:00-
midnight, the mean increase from baseline
in Group 3 was significantly different from
the change from baseline in Group 1. On
Study Day 17 at midnight-6:00, Study Day
19 at 18:00-midnight, and Study Day 20 at
6:00-12:00, the mean increase from baseline
in Group 4 was significantly different from
the change from baseline in Group 1. On
Study Day 36 at 12:00-18:00 and Study Day
37 at 12:00-18:00, the observed mean
decrease from baseline in Group 4 was
significantly different from the change from
baseline body temperature in Group 1. On
36
-------
Study Day 27 at 18:00-midnight, Study Day
29 at 18:00-midnight, and Study Day 32 at
12:00-18:00, the mean change from baseline
in Group 2 was significantly different from
the change from baseline in Group 3. On
Study Day 3 at 6:00-12:00, Study Day 10 at
midnight-6:00, Study Day 19 at 18:00-
midnight, and Study Day 20 at 6:00-12:00,
the mean change from baseline in Group 2
was significantly different from the change
from baseline in Group 4. On Study Day 19
at 18:00-midnight, Study Day 27 at 18:00-
midnight, and Study Day 36 at 12:00-18:00,
the mean change from baseline in Group 3
was significantly different from the change
from baseline in Group 4.
Measurements of inspiratory time,
expiratory time, respiration integral, and
peak amplitude were also conducted. See
Appendix F for figures and complete
statistical analysis.
3.4 Circulating Levels of Protective
Antigen
Toxemia was assessed over the course of the
study via a PA ELISA, which measured
circulating levels of PA. All Group 1 and 2
animals were below the LOD (4.9
nanogram/mL [ng/mL]) at all time points
assayed. The Group 3 animal (Rabbit 2) that
was found dead on Study Day 17 had 158.67
ng/mL of PA detected in the terminal blood
sample. Only one of the four rabbits (Rabbit
27) in Group 4 that succumbed to disease
had detectable levels of PA in the terminal
sample (65330.90 ng/mL PA). Two of the
Group 4 survivors had detectable levels of
PA. Rabbit 38 had 7.70 and 6.28 ng/mL PA
on Study Days 18 and 23, respectively.
However, the toxemia resolved by Day 30.
Rabbit 21 had PA levels of 4.97 ng/mL PA
on Day 25; all other blood samples were
below the LOD. Appendix N contains the
PA ELISA results for each rabbit.
3.5 Bacteremia
All animals in Groups 1 and 2 were negative
for B. anthracis bacteremia by culture on all
study days. The terminal sample from the
Group 3 animal (Rabbit 2) that succumbed
to infection showed a bacterial load in the
blood of 3.87 x 105 CFU/mL. The rest of the
animals in this group never became
bacteremic. The terminal samples of three
out of the four rabbits that died in Group 4
were positive for bacteremia. Rabbits 33, 27,
and 31 had terminal bacteremia levels of
4.13 x 105, 2.60 x 103, and 4.00 x 101,
respectively. One of the nonsurvivors of this
group (Rabbit 6) never showed a positive
bacteremia culture and was found dead 10.9
days after the first challenge. Rabbit 38
37
-------
(Group 4) became bacteremic on Study Day
18 (1.80 x 102), which resolved by the next
blood collection time (Study Day 23). The
other two rabbits that survived to the end of
the study (Rabbits 39 and 21) never became
bacteremic. Individual quantitative
bacteremia culture results are located in
Appendix O.
38
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Group 3 (1,000 CPU)
Group 2 (100 CPU)
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10 20 30
Days from Midnight of Challenge Day
40
Figure 9. Plot of mean baseline-adjusted temperature values for each group.
39
-------
smperature (Celsius)
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— Animal 9 — Animal 37
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Group 2
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— Animal 27 — Animal 31
Animal 33 — Animal 38
— Animal 39
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Days from Midnight of Challenge Day
Figure 10. Baseline-adjusted temperature values for each rabbit.
40
-------
Quantitative bacteremia was also assessed
by qPCR targeting the rpoB gene. These
results were consistent with the culture data
for the terminal samples. Rabbits 2 (Group
3) , 27 (Group 4), 31 (Group 4), and 33
(Group 4) had 3.19 x 103, 2.52x 103, 6.42 x
105, and 7.77 x 103 copies of rpoBI\\L of
blood, respectively. Rabbit 6 (Group 4) was
negative for bacteremia by the qPCR
method. Rabbit 38 (Group 4) was positive
for bacteremia by the qPCR method on
Study Days 23 (3.00 copies/|iL) and 30
(5.00 copies/|iL) despite only being positive
by the culture method on Day 18. This is not
surprising as the qPCR-based method does
not distinguish between viable and killed
bacterial cells. Individual qPCR results are
contained in Appendix P.
3.6 TNA/IgG ELISA
Serum samples taken on Study Days -3, 4,
11, 18, 25, 32, and 39 were analyzed via
TNA and anti-PA IgG ELISA to determine
if the rabbits developed a humoral response
to the repeated B. anthracis exposures. Only
Rabbit 38 (Group 4) had detectable levels of
antibodies by either method, no other animal
seroconverted during the study. The TNA
was used to determine the EDso and NFso of
sera able to neutralize lethal toxin. The EDso
values for Rabbit 38 on Study Days 25, 32,
and 39 were 5858, 12789, and 7250. The
NFso on these study days were 12.71, 26.44,
and 14.82. The IgG ELISA results showed
that Rabbit 38 had 1636.02, 2190.85, and
1728.47 ng/mL of circulating anti-PA IgG
on Study Days 25, 32, and 39, respectively.
Individual results for TNA and IgG ELISA
are provided in Appendices Q and R,
respectively.
3.7 Hematology and Clinical Chemistry
To further assess any physiological effects
of low-dose exposure to B. anthracis, whole
blood and plasma were assayed for a variety
of hematological and CRP (refer to Section
2.11 for a complete list of parameters).
Individual animal hematology and CRP
results are presented in Appendices S and T,
respectively, along with values for the
normal ranges of hematology and CRP.
Analysis of variance (ANOVA) models
were fitted separately to each hematology
parameter and CRP to determine the effects
of challenge dose and study day on group
means. Appendix J contains the results of
extensive statistical analyses of the
hematological and CRP results.
3.7.1 Red Blood Cell Parameters
There were significant decreases in RBCs
from baseline in Group 1 on Study Day 4, in
41
-------
Group 2 on Study Day 16, and in Group 4
on Study Day 11 (Figure 11). There were
significant group effects on Study Days 23
and 25. On Study Day 23, the mean decrease
from baseline in Group 4 was significantly
different than the mean increase from
baseline in Group 3 (P = 0.0489, Tukey's
test). On Study Day 25, the mean decrease
from baseline in Group 4 was significantly
different than the mean increases from
baseline in Groups 1(P = 0.0023, Tukey's
test) and 2 (P = 0.0229, Tukey's test). While
the changes were statistically significant,
their biological relevance is limited as all
rabbits remained in or very close to the
normal range of 4.20-6.70 x 106 RBCs/|iL
(Figure 11).
There were also significant changes in the
HGB concentrations in the blood (Figure
12). The decrease from baseline in Group 1
on Study Day 4 was significant (P < 0.05,
ANOVA). There were also significant group
effects on Study Days 23 and 25. On Study
Day 23, the mean decrease from baseline in
Group 4 was significantly different than the
mean increases from baseline in Groups 1,
2, and 3 (P < 0.05, Tukey's test). On Study
Day 25, the mean decrease from baseline in
Group 4 was significantly different than the
mean changes from baseline in Groups 1, 2,
and 3 (P < 0.05, Tukey's test). Like the RBC
counts, the HGB concentrations fell within
or very near the normal range (9.5-14.5
grams per deciliter [g/dL]; Figure 12) and
statistical differences were not likely to be
clinically relevant.
3.7.2 Total and Differential White Blood
Cell Parameters
The mammalian host responds to
extracellular bacterial infection by increased
hematopoiesis and neutrophilia. To
determine if the rabbits responded to the
multiple exposures of B. anthracis, complete
WBC counts and differentials were
performed. Interestingly, there were no
significant shifts as a proportion of baseline
and no significant differences between the
groups on any postchallenge study day.
While there was no significant mean
increase in WBC counts in the groups,
Rabbit 38 (Group 4) did show an increase in
WBCs well above the normal range of 2.90
- 8.10 x 103 WBCs/|iL (Figure 13). In fact,
the WBC count reached 20.33 x 103 cells/|iL
on Study Day 23 but decreased back into the
normal range by the end of the study.
42
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• Rabbit 13
• Rabbit 15
V Rabbit 19
A Rabbit 25
• Rabbit 23
• Rabbit 30
O Rabbit 34
Normal Range
A
5 ~fift 60 d8 sa Bg sg
*
Counts
Red Blood
12-
10 -
•
Gr<
• Rabbit 11
• Rabbit 12
^ Rabbit 14
A Rabbit 18
• Rabbit 2
• Rabbit 32
O Rabbit 8
Normal Range
FITTf"
Dup 3
Tr«fi"ir?i
-3 24 9 11 1618 2325 3032 3739
Study Day
Figure 11. Plots of red blood cell counts (1 x 106 cells/uL).
14 •
•5
£- 10
1
o 3
O 6
T3
8
00 4
41
G
• Rabbit 21
• Rabbit 27
V Rabbit 31
A Rabbit 33
• Rabbit 3$
• Rabbit 39
O Rabbit 6
Normal Range
O
B ifi !!
roup 4
•1 '- Si II
• •
2 4
9 11
16 IS 23 25
Study Day
30 32
37 39
43
-------
rr 25 -
-O
s
S 20 -
•s
!
g 15 -
O
I i°-
I
1 s-
Group
5 Jt si Ji
A
1
• Rabbit 37
• Rabbit 40
V Rabbit 5
A Rabbit 7
• Rabbit 9
Normal Range
S A
A
30 -
5 25-
5
S
-I 20-
1
i
c 'S -
o
1 5-
Group ."
t It if Sf
3
• Rabbit 11
• Rabbit 12
V Rabbit 14
A Rabbit 18
• Rabbit 2
• Rabbit 32
O Rabbit 8
Normal Range
0/5 i* QO
B AM "I
30 -
rr 25-
•o
s
1 20-
n
A
§
c IS -
a
1 10-
o
1 5-
n -
Group
\f
f Si !J_ '•
)4
9 Rabbit 21
• Rabbit 27
V Rabbit 31
A Rabbit 33
• Rabbit 38
• Rabbit 3d
O Rabbit 6
Normal Range
• • lt • I
•- '* -•"
-3 24 9 11 1618 2325 3032 3739
Study Day
-3 24 9 11 16 18 23 25 30 32 37 39
Study Day
Figure 12. Plots of hemoglobin concentration (g/dL).
44
-------
c
I
O
*
O
CO
£
Group 1
15 -
10-
5 •
n .
• Rabbit 37
• Rabbit 40
V Rabbit S
A Rabbit 7
• Rabbit 9
Normal Range
V
5? V y
O A A
: •• .:
A
Y_ V j ^
A A A Q £ g
•1 *
o
I
O
S
I
Group 3
•>o -
15 •
10 -
5 •
•
9
V
A
•
•
O
Ml
•
Rabbit 11
Rabbit 12
Rabbit 14
Rabbit 18
Rabbit 2
Rabbit 32
Rabbit S
Normal Range
"If
>T ll •• 5.
f
•
I
o
o
Group 2
15 •
10 -
5 -
• Rabbil13
• RabbitIS
^ Rabbit 19
A Rabbit 25
• Rabbil 23
• Rabbil 30
<> Rabbit 34
Ncfmal Range
o j£ Jl&_
$ ~^v f~i~
• U Q • •
A
-V_t^_.a —
8° 5g 89
"5
a.
Group 4
Cn9
J" 20
5- '
^
^
§ «•
O
»
o
"8 10 •
|
^: ..
5 5
,
•
V
A
•
•
O
/s
«•
v^'~ir"
A A
Rabbit 2 1
Rabbit 27
Rabbit 31 B
Rabbit 33
Rabbit 36 •
Rabbit 39
Rabbit 6 g
Normal Range
•
• * "
— R-i — i-* g i
-3 24 9 11 16 IS 23 25 30 32 37 39
Study Day
Figure 13. Plots of white blood cell counts (1 x 103 cells/uL).
24 9 11 16 15 23 25 30 32 37 39
Study Day
45
-------
There were no significant shifts in
neutrophil counts as a proportion of baseline
and no significant differences between the
groups on any postchallenge study day
(Figure 14). However, Rabbit 11 (Group 3,
Day 23), Rabbit 6 (Group 4 , Day 4), Rabbit
27 (Group 4, Day 18), Rabbit 21 (Group 4,
Day 32), and Rabbit 38 (Group 4, Days 23-
32) showed levels of circulating neutrophils
above the normal range of 0.8-2.9 x 103
cells/jiL after the first challenge day (Figure
14). The neutrophilia was most pronounced
in Rabbit 38, which was bacteremic on
Study Day 18 and toxemic on Days 18 and
23. This animal was able to clear the
infection and lived to the end of the study.
There were no significant differences in
lymphocyte counts between the groups on
any postchallenge study day (Figure 15).
There was a significant decrease from
baseline in Group 3 on Study Day 39. It is
unlikely that this decrease was related to the
multiple B. anthracis exposures as it did not
occur until the last study day. Several rabbits
experienced lymphophilia (levels above the
normal range of 2.20-5.30 x 103 cells/|iL)
over the course of the study as illustrated in
Figure 15. The effect of the exposures on the
lymphocyte levels remains to be determined
as there was no clear dose-response
relationship in the measurements.
CRP is an indicator of stress and nonspecific
inflammation. It can also be used as a
marker for liver damage. There was a
significant increase in CRP as a proportion
of baseline in Group 4 on Study Day 2
(P > 0.05, ANOVA) (Figure 16). There were
no significant differences between the
groups on any postchallenge study day. The
normal levels range from 0.25 to 0.29
milligram/deciliter (mg/dL) (Murty et al.,
2010; Setorki et al., 2009). However, several
animals had detectable levels of CRP
(> 0.50 mg/dL) that were considered above
the normal range (Figure 16). The increase
in CRP levels did not correspond with
morbidity or mortality and in most cases
could have resulted from the stress of study
activity. Rabbit 38 showed the highest levels
of CRP between Study Days 18 and 25
topping out at 7.42 mg/dL on Study Day 23.
The increase in CRP corresponded with
bacteremia, toxemia, neutrophilia, and
pyrexia indicating that the increase was in
response to the B. anthracis infection.
46
-------
Group 1
Group 2
Neutrophil Count {tcfyiL)
T rj 4- O) 45 O ?
• Rabbit 37
• Rabbit 40
V Rabbit 5
A Rabbit 7
• Rabbit 9
Nofinal Range
•
1 $5 jo y_ *J_ 8* ••
« ^ ^ ^--^-^ -f
Neutrophil Count OOfyiL)
i u t o> » o S
• Rabbit 13
• Rabbit 15
V Rabbit 19
A Rabbit 25
• Rabbi) 28
• Rabbit 30
O Rabbit 34
Normal Range
\ it 88 11 8S fig |.
%5
Group 3 , Group 4
Neutrophil Count (ICrVuL)
3 »J i O) CD O i
• Rabbit It
• Rabbit 12
^ Rabbit 14
A Rabbit 12
• Rabbit 2
• Rabbit 32
O Rabbit 8
Normal Range
•
A A - A 0 •
l__fl|_JJ__M__"--i-84
Neutrophil Count (10%iL)
3 (J *.
-------
Group 1
Group 2
Lymophocyte Count <10J'/nL)
_% — t*
r> (ji o
-------
Group 1
Group 2
C-Reactive Protien (mg/dL)
? to -t o> o
C-Reactive Protein (mg/dL)
3 M *- Q) 00 I
' — — 1 — — 1 — — 1 —
• Rabbit 37
• Rabbit 40
V Rabbit 5
A Rabbit 7
• Rabbit 9
Limit of Detection
I |" *
!• i •
A
u.
Group 3
• Rabbit 11
• Rabbit 12
V Rabbit 14
A Rabbit 18
• Rabbit 2
• Rabbit 32
•0» Rabbit 8
Limit of Detection
0
•
^h -^ A ^h
O OO Om S~O 0~O OW OO
8 •
j"
C-Reactive Protein
3 ro -t^
C-Reactive Protein (mg/dL)
3 K> •t' O> 00
^ Rabbit 13
• Rabbit 15
V Rabbit 19
A Rabbit 25
• Rabbit 2S
• Rabbit 30
O Rabbit 34
Limit of Detection
0 0 0
v n jf A y
i II oo mo oo oo oo
Group 4
• Rabbit 21
• Rabbit 27
y Rabbit 31
A Rabbit 33
• Rabbit 33
• Rabbit 39
O Rabbit 6
Limit of Detection
•
° j| °A '
•
•
•
•
i • •
• s, _•_•
B oo •_• !_• mr mm "•
-3 24 911 16 18 23 25 30 32 37 39 -3 24 9 11 16 1S 23 25 30 32 37 39
Study Day Study Day
Figure 16. Plots of C-reactive protein levels (mg/dL).
49
-------
4 Pathology
Complete necropsies were performed on all
rabbits following spontaneous death or
euthanasia, including rabbits surviving to
study termination on Study Day 39.
Protocol-specified tissues (lungs and gross
lesions) were sampled and preserved in
10% neutral buffered formalin. Standard
sections of these tissues from all rabbits
were processed to slides, stained with
hematoxylin and eosin, and interpreted by a
board-certified veterinary pathologist.
Table 9 summarizes the pathological
findings from each of the rabbits. The
complete pathology report is located in
Appendix U.
Gross lesions consistent with anthrax in
rabbits (Zaucha, etal., 1998) included
discoloration of the lungs, foci in the
appendix, "accumulation" in the cecum,
and/or enlargement of a mediastinal lymph
node; these lesions were identified in
Rabbits 12 (Group 3), 6 (Group 4), 33
(Group 4), and 27 (Group 4). These lesions
correlated microscopically with hemorrhage,
necrosis, edema/fibrin, and suppurative
(largely heterophilic admixed with bacteria
and/or necrotic debris) inflammation. Gross
lesions in the lungs correlated with multiple
foreign body granulomas/pyogranulomas
(Rabbit 38) as shown microscopically in
Figure 17. Multinucleated giant cells as well
as foreign body granulomas/pyogranulomas
were present in the lungs of challenged
rabbits (survivors and non-survivors) but
were not seen in control rabbits in this study.
These multinucleated cells and
granulomas/pyogranulomas surrounded
foreign material (foreign bodies) consistent
with organic debris [e.g. food particles or
hair and debris from vascular access ports
(Taketoh etal., 2009)].
Abdominal skin "lacerations" were
diagnosed grossly in two rabbits (12, Group
3, and 38, Group 4). These lesions correlated
microscopically with necrosuppurative
inflammation but were not associated with
bacteria. While anthrax may have been a
contributing factor, these lesions were more
likely due to trauma. Sections of left apical
50
-------
and right diaphragmatic lung lobes and gross
lesions were examined microscopically for
evidence of anthrax. Figure 18 shows
normal lung tissue from Rabbit 37 (Group
1).
Microscopic findings consistent with
anthrax (Zaucha et al., 1998) were present in
tissues from all rabbits (survivors and non-
survivors) in Groups 2, 3 and 4. Lesions
typical of anthrax in this study included
suppurative inflammation, necrosis,
lymphocyte necrosis/depletion, hemorrhage,
edema, and/or large rod-shaped bacteria
(bacilli) in the lungs (Figure 19), cecum,
appendix (Figure 20), and mediastinal
lymph nodes (Figure 21). Lung lesions
attributed to B. anthracis were primarily
interstitial and consisted of minimal to mild
suppurative interstitial inflammation and
interstitial and/or intravascular bacteria.
51
-------
Table 9. Summary of Individual Gross and Microscopic Observations
Target
Dose
Sham
Challenge
Control
100 CPU
1000 CPU
Animal
Number/
Death Status*
1.2 40/FS
7/FS
5/FS
9/FS
37/FS
13/FS
34/FS
25/FS
15/FS
30/FS
28/FS
19/FS
14/FS
11/FS
2/FD
8/FS
12/FS
306 (18)/FS
307 (32)/FS
Gross
Findings
Skin:
Laceration(s),
hind limb, red,
left hind limb,
40 x 20 mm
Microscopic Findings
Lung: Unremarkable.
Lung: Unremarkable.
Lung: Perivascular eosinophils, minimal.
Lung: Perivascular eosinophils, minimal.
Lung: Unremarkable.
Lung: Perivascular eosinophils, minimal.
Lung: Foreign body, mild.
Lung: Multinucleated giant cells, mild.
Lung: Unremarkable.
Lung: Perivascular eosinophils, minimal.
Lung: Unremarkable.
Lung: Perivascular eosinophils, mild.
Lung: Unremarkable.
Lung: Perivascular eosinophils, minimal.
Lung: Perivascular eosinophils, minimal.
Lung: Hemorrhage, minimal.
Lung: Inflammation, suppurative, minimal.
Lung: Bacteria, minimal.
Unremarkable.
Lung: Foreign body, minimal.
Lung: Multinucleated giant cells, mild.
Skin: Inflammation, necrosuppurative, marked.
Lung: Unremarkable.
Lung: Perivascular eosinophils, minimal.
52
-------
Table 9. Continued.
Target
Dose
Animal
Number/ Death
Status3
Gross Findings
Microscopic Findings
6/FD
33/FD
27FD
10,000
CPU
31/FD
39/FS
21/FS
38/FS
Cecum: Accumulation
(gas). Samples of
cecum, colon, jejunum,
and appendix were
collected to confirm
lesion.
Cecum: Edema, mild.
Cecum: Edema, hemorrhage and necrosis.
Cecum: Hemorrhage, moderate.
Cecum: Necrosis, moderate.
Lung: Perivascular eosinophils, minimal.
Lymph node,
Mediastinal: Enlarged,
dark, 3x.
Lung: Bacteria, mild.
Lung: Hemorrhage, minimal.
Lung: Inflammation, suppurative, mild.
Lung: Perivascular eosinophils, minimal.
Lymph node, mediastinal: Bacteria, marked.
Lymph node, mediastinal: Edema, fibrin, mild.
Lymph node, mediastinal: Hemorrhage, minimal.
Lymph node, mediastinal: Necrosis/depletion,
lymphoid, marked.
Appendix: Foci,
multiple, red, up to 2 x
2 mm.
Appendix: Hemorrhage, mild.
Appendix: Necrosis/depletion, lymphoid,
moderate.
Appendix: Infiltration cellular, macrophages,
moderate.
Appendix: Note: hemorrhage and necrosis.
Lung: Bacteria, minimal.
Lung: Inflammation, suppurative, minimal.
Lung: Perivascular eosinophils, minimal.
Lung: Bacteria, mild.
Lung: Inflammation, suppurative, minimal.
Lung: Foreign body, minimal.
Lung: Multinucleated giant cells, minimal.
Lung: Unremarkable.
Lung: Discoloration(s),
apical lobe, pale, firm.
Skin: Laceration(s),
abdominal, red,
20 x 15 mm.
Lung: Foreign body, moderate.
Lung: Granuloma/pyogranuloma, moderate.
Lung: Perivascular eosinophils, minimal.
Skin: Inflammation, necrosuppurative, moderate.
Skin: Thrombosis, artery, mild.
*FD = found dead, FS = final-phase sacrifice
mm = millimeters
53
-------
i*w %8«fw ^tfM- Ttw*&
t>*« -SPft&J •***'
-f-^ V ^ :^:- ^>£^»:
Figure 17. Animal 38 (Group 4): Lung, alveoli; pyogranulomatous (epithelioid macrophages, lymphocytes, and neutrophils)
inflammatory reaction to a foreign body (arrow). Hematoxylin and eosin stain. 40X.
54
-------
"T" i.-i % * *
Figure 18. Animal 37 (Group 1): Lung; normal alveoli (control). Hematoxylin and eosin stain. 40X.
55
-------
,» >
'*. "•-
» »
% *».
Figure 19. Animal 31 (Group 4): Lung; alveoli contain interstitial suppurative inflammation and anthrax bacilli (arrows).
Alveolar vessels contain anthrax bacilli (arrowhead). Hematoxylin and eosin stain. 40X.
56
-------
•:--
' ._.
®$i*g$^::^-3&
v*ffit^;?»4*'jjp»£ ;•,«*• •'
%£^-^^%£|^
r£~**^-. -Tt f^-4. ^»\!'O£'*"^r.^r"?i fTHi'^L .il.1^1^ .VjL*^ii^J .:
Figure 20. Animal 38 (Group 4): Appendix; lymphocytes undergoing excessive apoptosis (arrow) with macrophage infiltration
(arrowheads). Hematoxylin and eosin stain. 10X.
57
-------
Figure 21. Animal 38 (Group 4): Lymph node, mediastinal; lymph node congestion and lymphoid follicles necrosis/depletion.
Hematoxylin and eosin stain. 4X.
58
-------
4.1 Benchmark Dose Analysis
The following models exhibited acceptable
fits as measured by p-values and scaled
residuals at BMDLs of interest for the
average daily dose data: Dichotomous-Hill,
Loge Logistic, and Weibull (run as
Exponential). Using existing EPA guidance
(US EPA, 2008a), the loge logistic model
was identified as the best fitting model to the
data. A BMDL50 of 2.6 x 103 inhaled CPU
and a BMDLio of 2.9 x 102 inhaled CPU
were calculated using the model.
The following models exhibited acceptable
fits as measured by p-values and scaled
residuals at BMDLs of interest for the total
aggregate dose data (i.e., cumulative dose
over the course of the challenges):
Dichotomous-Hill, Loge Logistic, and
Weibull (Run as Exponential). Using
existing EPA guidance (US EPA, 2008a),
the loge logistic model was identified as the
best fitting model to the data. A BMDLso of
4.4 x 104 total inhaled CPU and a BMDLio
of 4.9 x 103 total inhaled CPU were
calculated using the model. Using the
average daily dose BMDLio value from the
loge logistic model, the calculated example
values for the HED and HEC were 1,400
inhaled CPU and 87 CPU/cubic meter (m3),
respectively.
Given the relatively small animal numbers
present in each dose group, the spacing of
the doses, and modeling assumptions; the
calculated BMDLs should be considered to
be highly uncertain. The complete
benchmark dose report, including the
dosimetric adjustment, is provided in
Appendix V.
4.2 Quality Assurance
The procedures set forth in the EPA-
approved Quality Assurance Project Plan
(QAPP); Comer (2010) prepared for this
project were adhered to except in those
instances that are documented as deviations
(see Appendix B). In all, there were 15 study
deviation reports (DR) and one investigation
report (IR); no facility deviations occurred
during the study. Each of these deviations is
listed in Table 10 along with the impact on
the quality of the data and results reported
herein. Technical systems audits (TSATs)
and data quality audits (DQAs) performed
for this study along with dates performed,
reported, and findings addressed by project
management are listed in Table 11.
59
-------
Table 10. Deviations and Impacts on Data Quality and Results
Deviation
Number
DR-10019
DR- 10063
DR- 10263
DR- 10264
DR-10350
DR-10351
DR-10352
DR-10353
DR- 10444
DR- 10496
DR- 10645
DR- 10646
DR- 10647
DR-11043
Deviation
Type
Study
Study
Study
Study
Study
Study
Study
Study
Study
Study
Study
Study
Study
Study
Impact on Data Quality and Results
Positive. Decreasing the incubation time to a shorter period allows the plates to be
counted at the same time as the non filtered samples.
Minimal. It cannot be determined which incubator the plates were placed in, but the plates
in both incubators were incubated for an appropriate amount of time and the plates for the
animal in question returned satisfactory results.
Minor. The small difference in the calibration values will not cause noticeable differences
in the data Additionally, postchallenge data are compared to the baseline data, which
would offset any differences caused by an incorrect calibration value.
Minor. Three days of baseline telemetry data will be used for comparison to postchallenge
data during statistical analysis. 2-3 days of baseline data are commonly used for analysis
and was the initial requirement of the draft protocol.
None. A sterility check was performed on all samples prior to their removal from the
Biosafety Level 3 laboratory (BL3); all serum aliquots were negative. This information
was recorded on Microbio-463 and was confirmed by the Safety Officer before the
samples were released from the BL3.
Minimal. There is little possibility that the challenge organism could be present at the Day
-3 time point. None of the other animals tested at this time had challenge organism
colonies present. The quantitative bacteremia results for Day -3 will be reported as
negative for Rabbits 33 and 31.
Minimal. Residual samples will be pooled and sent to the client.
None. All of the counts were zero.
Minimal. The dilution of sham challenge control created was 1:50.2 instead of the SOP
required 1:50. Since this resulted in a preparation that is more dilute, this may have had an
effect on the performance of the sham challenge control within the assay.
Minimal. The initial volume given was within the range specified in the protocol.
Minimal. The permanent equipment used or the assay can be verified by other equipment
use dates; however, the transportable equipment like pipettes cannot be verified.
Minimal, a) The refrigerator information was recorded on the equipment form and can be
verified that its use was within the calibration timeframe. b) Since the freeze thaw did not
specify whether the sample was from the PA aliquot or retention, the exact freeze thaw for
the sample could not be verified.
These specifications improve the fidelity and consistency of the results reported by the
assay.
a. Minimal. The date of printing is captured on the records as 8/21/10, and Form No.
ELISA-036 indicates which technician conducted the data analysis.
b. Minimal. Based on the date of printing, data were analyzed on 8/27/10. Furthermore,
all reportable values captured on Microbio-357 can be transcribed and verified; no
reportable data are affected.
c. Minimal. Although the date and operator cannot be confirmed, it has been verified that
the data have been imported into the database.
d. Minimal. Based on the information on the Sample Tracking System, it is most likely
that the freeze/thaw (F/T) cycles should be: BMI032 = F/T 1, BMI012 = F/T 2,
BMI009 = F/T 3, which is within the permissible number of F/T cycles form ELISA
samples.
60
-------
Table 10. Continued.
Deviation
Number
Deviation
Type
Impact on Data Quality and Results
DR-11044
Study
Minimal. There is less than a 0.2% difference between the actual dilution factor and that
specified on ELISA-008. No data are significantly affected.
IR-419
NA
When using either new rPA lot, 17115A2A or 5051797, for coating there were
inconsistencies with the binding of the currently qualified conjugate 05814. This was
seen in preliminary analyses for study 1078 and it caused many failures with study
plates. An investigation was undertaken to determine the most likely cause of the
inconsistencies leading to failures and to determine a correction course of action to take.
It was determined the conjugate 05814 had degraded and was, as a result, inconsistently
binding. Proposed action was to use a different conjugate lot.
BL3 = Biosafety Level 3
Table 11. Technical System Audit (TSAT) and Data Quality Audit (DQA) Dates
Audit Name
Critical Phase: aerosol challenge, Day
2 blood collection, Day 2 weights,
bacteremia, hematology, CRP, IgG
ELISA, TNA, PCR, and PA ELISA
Telemetry Binder
Aerosol Binders 1-3, including the
Aerosol Exposure Report and Final
Data Tables. (Aerosol Exposure
Events Tables audited 10%)
DNA Isolation and Real-Time PCR
Binder
Hematology/CRP Binder, including
summary sheets
a PA IgG ELISA Binders 1 and 2
Micro Binder
Pathology Binder
Vivo Binder
Inhalation Exposure Report
CircPA ELISA
TNA Binders 1-3, results table added
2/11/11
Audit
Type
TSA
DQA
DQA
DQA
DQA
DQA
DQA
DQA
DQA
DQA
DQA
DQA
Inspection
Start Date
10/5/2010
10/7/2010
10/21/2010
10/20/2010
10/29/2010
11/17/2010
12/13/2010
12/13/2010
12/13/2010
12/13/2010
12/14/2010
12/14/2010
Date
Reported
11/8/2010
10/12/2010
10/21/2010
10/26/2010
11/1/2010
11/19/2010
12/14/2010
12/14/2010
12/14/2010
12/14/2010
12/15/2010
12/15/2010
Response
Date
11/17/2010
10/20/2010
11/1/2010
11/1/2010
11/2/2010
11/23/2010
1/28/2011
5/20/2011
1/18/2011
12/15/2010
1/17/2011
1/28/2011
Closure Date
2/24/2011
11/17/2010
12/17/2010
12/17/2010
12/17/2010
12/17/2010
2/24/2011
6/8/2011
2/24/2011
12/17/2010
2/24/2011
2/24/2011
61
-------
4.3 Archives
Records pertaining to the conduct of the
study were documented in Battelle
laboratory record books that were specific to
this study. These records and the final report
will be archived at Battelle.
62
-------
5 Discussion and Conclusions
The previous study determined the
physiological dose response to an acute
inhalational exposure to various low-dose
concentrations of B. anthracis spores (U.S.
EPA, 2011). That study showed a dose
effect on the physiological changes and that
increases in respiration rate, heart rate, body
temperature, and circulating neutrophils
corresponded to decreased survival rates.
Temperature was the most consistent
indicator of disease outcome. All rabbits that
had a febrile response died on study. The
objective of this present study was to build
on the data of the acute low challenge
inhalational dose study and determine the
physiological responses following multiple
exposures to low-dose concentrations of B.
anthracis spores. A summary of the findings
is presented in Table 12.
Rabbits receiving multiple exposures to
irradiated spores served as sham challenge
controls and showed little physiological
response. In fact, the only discernible
reaction was a rise in CRP levels, which
most probably was the result of stress due to
study activity. This was similar to what was
seen in the acute study when rabbits were
exposed to 1.05 x 107 irradiated spores. This
suggests that exposure to avirulent spore
coat material either acutely or in multiple
doses does not prompt a detectable
physiological reaction.
All of the rabbits exposed to 15 doses of a
mean of 2.91 x 102 CPU (Group 2) lived to
the end of the study and showed minimal
physiological changes due to the exposures.
None of these rabbits was ever bacteremic
or toxemic. The mean accumulated dose of
inhaled CPU for the group after the 15
challenge days was 4.36 x 103 (± 8.11 x 102)
CPU. This average accumulated dose was
higher than the previous study's acute
inhaled dose of 2.06 x 103 CPU
administered to 5 animals in which no
measurable physiological effect was
observed.
In the present study one rabbit (Rabbit 2)
succumbed to disease in the 1.0 x 103 CPU
targeted inhaled dose group. This animal's
mean dose was 1.33 x 103 (± 5.95 x 102)
CPU and it was exposed to 1.86 x 104 total
63
-------
CPU for 14 of the 15 days of challenge. This
latter total exposure corresponded to a group
of five animals that received a single dose of
2.54 x 104 (± 5.21 x 103) CPU in the acute
study in which two of the five rabbits died at
4 and 11 days post-challenge. Each animal
presented with tachycardia, tachypnea,
increased body temperature, neutrophilia,
and bacteremia. Rabbit 2 in the present
study succumbed to disease 17.9 days after
the first challenge and also presented with
tachycardia, tachypnea, increased body
temperature, neutrophilia, bacteremia, and
toxemia. These latter signs did not appear
until just prior to death. Additionally, only
the terminal blood draw was positive for
bacteremia and toxemia. These results
suggest that either an infection was not
established until the majority of the
challenges had taken place or the infection
was localized and the rabbit did not respond
in a systemic manner.
Four of the seven rabbits that received a
mean inhaled dose of 1.0 x 104 (± 4.64 x
103) CPU died during the study with a mean
time to death of 14.80 ± 4.28 days. These
rabbits received an accumulated challenge
inhaled dose ranging from 5.77 x 104 to 2.16
x 105 CPU. This group of animals responded
to the multiple challenges in the same
manner as rabbits exposed to a single dose
of 2.75 x 105 CPU in the previous acute
study. This exposure resulted in four of the
five challenged animals dying on study and
presenting with tachycardia, tachypnea,
increased body temperature, neutrophilia,
bacteremia, and toxemia. However, only one
of the four rabbits that died became toxemic
in the current study. Nine of the eleven
rabbits that died in the acute study
(regardless of group) were toxemic. The
reason for the lack of detection of
circulating PA (the indicator of toxemia) is
not known. This is the first study that the
authors are aware of that used multiple low-
dose exposures so there are no other
comparable studies with which to relate this
finding. It has been established that
inhalational anthrax is a biphasic disease
with a brief remission of bacteremia and
toxemia (Boyer et al., 2009; Brachman,
1980). It is possible that these animals had
circulating PA during the course of the
infection, but the times of toxemia did not
coincide with the blood collections. Most
rabbits that succumb to anthrax have
detectable PA levels in the late stage blood
samples (U.S. EPA, 2011; Mabry et al.,
2006; Kobiler et al., 2006). Therefore, more
work is required to determine why the
64
-------
animals that succumbed to disease after
multiple exposures were not toxemic.
The data generated in the acute study
suggested an "all-or-none" outcome to
disease. That is, once an infection was
established (marked by bacteremia/toxemia
and physiological changes) the disease
progressed to a fulminant state and resulted
in the death of the rabbit. In the current
study, two rabbits showed signs of infection
but lived to the end of the study. Rabbit 38
(Group 4) presented with all of the clinical
signs mentioned above and was both
bacteremic and toxemic during the study.
However, the neutrophilic response was
more robust than that previously seen and
the animal seroconverted by Day 25. This
suggests that the animal was able to mount a
significant innate response to clear the
infection and establish a humoral response
that would counteract any residual
circulating toxin. Another survivor in this
group, Rabbit 21, presented with a fever,
neutrophilia, and toxemia but never
seroconverted. While the neutrophilia was
substantially less than that of Rabbit 38, the
animal survived to the end of the study. The
infection may not have been as severe as
that of Rabbit 38 as indicated by the lower
neutrophilic response and the fact that
Rabbit 21 never seroconverted.
All rabbits, including study survivors,
underwent complete necropsies, and the
lungs and any gross lesions were examined
microscopically. All rabbits (survivors and
non-survivors) had pathological findings
consistent with inhalational anthrax in this
model (Zaucha et al., 1998). Interestingly,
multinucleated giant cells as well as foreign
body granulomas/pyogranulomas were
present in the lungs of challenged rabbits but
were not seen in control rabbits in this study.
In the acute study, multinucleated giant cells
were noted in both exposed and control
animals. However, the lesions were more
severe in challenged rabbits. These
multinucleated cells and
granulomas/pyogranulomas surrounded
foreign material (foreign bodies) consistent
with organic debris (e.g., food particles or
hair and debris from vascular access ports
[Taketoh et al., 2009]). As with the acute
study, these lesions could be the result of
altered foreign particle clearance by alveolar
macrophages (macrophage dysfunction).
Macrophage dysfunction has been reported
to occur in late sepsis (Pahuja et al., 2008).
Prolonged bacteremia/sepsis attributed to
anthrax could alter foreign particle clearance
65
-------
by alveolar macrophages, and lethal toxin
has been shown to inhibit alveolar
macrophage function (Ribot et al., 2006).
However, foreign bodies were also found in
animals that were not bacteremic and had no
bacteria detected in the lungs during
histopathological analysis. It is possible that
a low-grade bacterial load was present in the
lung and affected the function of alveolar
macrophages in animals that had bacteria
not detected by histopathology. While the
foreign bodies were found only in
challenged animals in the present study in
contrast to the previous acute inhalation
exposure study, the causal effect has not
been established. Future studies may provide
insight on the role of low B. anthracis
challenge doses and failure to clear foreign
bodies from the lungs.
Another pathology finding, perivascular
eosinophils in the lungs, is likely attributable
to vascular access port placement and has
been observed in rodent studies (Taketoh et
al., 2009). The observed necrosis in the skin
is likely due to self-inflicted trauma.
The rabbit has been shown to be an
acceptable model for human inhalational
anthrax (Leffel and Pitt, 2006), and the
findings from animals that died on this study
were consistent with previous observations
(Table 12). Dutch Belted rabbits exposed to
100 LDso of B. anthracis spores also
presented with tachycardia, tachypnea,
pyrexia, leukocytosis, and neutrophilia
(Lawrence et al., 2009). Yee et al. (2010)
also showed that bacteremia and toxemia are
associated with fever and hematological
changes during inhalational anthrax disease
progression in NZW rabbits after exposure
to 150 times the LDso of B. anthracis spores.
Taken together with the data presented in
this report, these findings suggest that
disease progression and observed symptoms
are independent of dose once a lethal
infection has been established.
The challenge doses and mortality data
presented here were used in benchmark dose
analysis. While a dose-response relationship
was derived through the successful fit of
mathematical models to the study data, it
should be noted the reported BMDLs and
the measures derived from the BMDLs (i.e.,
HED and HEC) should be considered to be
highly uncertain.
These dose-response data may also provide
preliminary evidence that a threshold in the
average daily dose may be present below
which lethality is unlikely to occur in a
healthy, adult male rabbit population. This is
evidenced by the survival rates of the two
66
-------
lowest dose groups. Further testing of levels
between these two doses may allow
modeling of this potential threshold value.
When comparing the benchmark dose
analyses of the data from this work and the
previously-performed acute study (Hines et
al., 2011), there are preliminary indications
that a discernible relationship may exist
between the measured endpoint of lethality
and the administered dose, exposure
duration, and number of doses. The basis for
this hypothesis is that the total aggregate
dose BMDLio of 4,900 CPU is
approximately 3.5 times the BMDLio of
1,400 CPU from the acute study (i.e., single
dose), and the total aggregate dose BMDL50
of 44,000 CPU is approximately 3.4 times
the BMDLso of 13,000 CPU from the same
acute study. Given the limited availability of
multiple low dose-response data sets for B.
anthracis exposures, techniques to model
the relationship between dose,
concentration, and exposure duration may
provide useful information to further
characterize the hazard posed by acute and
short-term exposure scenarios to low levels
of B. anthracis contamination.
The data presented in this report suggest that
rabbits exposed to multiple inhalation doses
r\
of a mean of 2.91 x 10 CPU of B. anthracis
do not exhibit a measurable physiological
response. Also, animals that exhibit
responses and seroconvert may recover from
infection.
While the work presented here starts to fill
in the knowledge gaps in low dose B.
anthracis exposures, there are some
limitations that are difficult for overcome in
a laboratory setting. For instance, an
intentional release of spores will be more
likely to use dried spores to increase
dispersal and infectivity. This study used a
wet preparation of spores in a very
controlled environment. The challenge dose
was determined mathematically and
deposition of spores in the lungs was not
determined. Evaluating deposition and
accumulation of spores in the lungs will
require serial euthanasia of animals and
CPU counts of the lungs at different times
post-exposure. Performing plate counts of
the lungs was not in the scope of the
presented work but may be included in
future studies. Future studies with a serial
pathology focus could also assist in
confirming the sites involved in the
initiation of infection.
The objective of this study was limited to
identified physiological responses in the
rabbit model of disease. This study assessed
67
-------
PA in the blood, but did not look for other B.
anthracis-specific biomarkers. Future
studies may look for biomarkers, such as
HtrA and NlpC/P60, as identified by Sela-
Abramovich et al. (2009) or develop
additional biomarkers for evaluation. In
tandem with work to identify biomarkers
suitable for modeling infection and disease,
studies could also be conducted to inform
the identify and measure critical biokinetic
and biodynamic parameters to further
inform development of physiologically
based biokinetic models (PBBK) for anthrax
pathogenesis.
68
-------
Table 12. Summary of Study Findings
Group
1
2
2
4
ID
40
7
5
9
37
13
34
25
15
30
28
19
14
11
2
8
12
18
32
6
33
27
31
39
21
38
Mean Inhaled Dose
(CFU/Animal)
Mean
0
0
0
0
0
3.85 x 102
3.17xl02
2.79 x 102
3.17xl02
2.72 x 102
2.34 x 102
2.32 x 102
7.38 xlO2
1.12xl03
1.35xl03
1.40 x 103
1.30xl03
1.24 x 103
1.89xl03
6.41 x 103
9.75 x 103
1.08 x 104
1.25 x 104
1.44 x 104
1.32xl04
1.27 x 104
SD
0
0
0
0
0
7.57 x 102
4.48 x 102
3.54 xlO2
3.27 x 102
2.33 x 102
1.49 x 102
1.28 x 102
2.99 x 102
5.01 x 102
5.78 x 102
6.04 x 102
4.90 x 102
5.56 x 102
1.87 x 103
2.57 x 103
2.58 x 103
3.65 x 103
3.27 x 103
5.99 x 103
4.97 x 103
3.77 x 103
#of
Exposures
15
15
15
15
15
15
15
15
15
15
15
15
15
15
14
15
15
15
15
9
10
14
11
15
15
15
Sum of
Doses
(CFU/
Animal)
0
0
0
0
0
5.78 x 103
4.76 x 103
4.19 xlO3
4.76 x 103
4.07 x 103
3.51 xlO3
3.48 x 103
l.llxlO4
1.68 x 104
2.02 x 104
2.10 xlO4
1.95 x 104
1.85 x 104
2.83 x 104
5.77 x 104
9.75 x 104
1.51 xlO5
1.37 xlO5
2.16 xlO5
1.98 xlO5
1.91 xlO5
Heart
Rate*
4-^>
4-^>
4-^>
4-^>
4-^>
4-^>
4-^>
4-^>
4-^>
4-^>
4-^>
4-^>
•
•
t
<— >
<— >
<— >
<— >
t
t
t
t
•
4-^>
t
Respiratory
Rate*
4-^>
4-^>
4-^>
4-^>
4-^>
4-^>
4-^>
4-^>
4-^>
4-^>
•
•
<— >
<— >
<— >
<— >
<— >
<— >
<— >
<—>•
t
Body
Temp*
4-^>
4-^>
4-^>
4-^>
4-^>
4-^>
4-^>
4-^>
4-^>
4-^>
4-^>
4-^>
•
•
t
<— >
<— >
<— >
<— >
t
t
t
t
<— >
t
t
White
Blood
Countt
4-^>
I
t
4-^>
I
t
t
t
4-^>
4-^>
4-^>
I
t
n
<— >
t
<— >
t
<— >
t
<— >
t
<— >
t
*-^>
t
Neutrophil
t
4
I
4-^>
4-^>
4-^>
4-^>
4-^>
4-^>
4-^>
4-^>
4-^>
•
<— >
<— >
<— >
<— >
<— >
<— >
<— >
<— >
t
CRP|
t
t
4-^>
t
t
t
t
t
4-^>
4-^>
4-^>
t
<— >
t
<— >
t
t
<— >
<— >
t
t
t
t
t
t
t
Bacteremia
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
Toxemia
-
-
-
-
-
-
-
-
-
-
-
-
-
-
+
-
+
-
-
-
-
-
+
-
+
+
Time to
Death
(day)
Survived
Survived
Survived
Survived
Survived
Survived
Survived
Survived
Survived
Survived
Survived
Survived
Survived
Survived
17.9
Survived
Survived
Survived
Survived
10.9
12.7
20.8
14.7
Survived
Survived
Survived
T= Increases in a parameter
1= Decreases in a parameter
<-> = No change in the parameter
+ = Positive or bacteremia culture or toxemia
-= Negative for bacteremia culture or toxemia
*= Changes based on baseline
t = Changes based on normal ranges
69
-------
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T.R. Kozel, E. Saile, C.K. Marston, A.
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Brachman P.S. 1980. Inhalation anthrax.
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Lawrence W.S., J.M. Hardcastle, D.L.
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Jr., G.B. Hubbard, S. Leppla, J.L. Patterson,
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Murty, D., E. Rajesh, D. Raghava, T.V.
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Pahuja, M., C. Iran, H. Wang, and K. Yin.
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2009. Novel and unique diagnostic
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Taketoh, I, S. Komatsu, K. Adachi, R.
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Application to Risk-Based Decision
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73
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APPENDIX A
STUDY PROTOCOL
A-1
-------
BBRC Protocol 1078
Study Number: 1078-CG920794
Battelle Biomedieal
Research Center
Date: 22 July 2010
Page 1 of 19
Rabbit Multiple Dose Anthrax Telemetry Study
Study No. 1078-CG920794
BBRC Director:
BBRC Associate Director:
Study Director:
Sponsor:
Sponsor Representative:
James A. Blank, Ph.D., D.A.B.T.
Jason M. Mott, D.V.M., Ph.D.
Jason E. Comer, Ph.D.
US Environmental Protection Agency
National Homeland Security Research
Center
Threat and Consequence Assessment
Division
26 West Martin Luther King Drive
Cincinnati, OH 45268
Sarah C. Taft, Ph.D.
Mailing Address:
Battelle Biomedieal
Research Center (BBRC)
505 King Ave, JM-7
Columbus, Ohio 43201-2693
A-2
-------
BBRC Protocol 1078
Study Number: 1078-CG920794
Battelle Biomedical
Research Center
Date: 22 July 2010
Page 2 of 19
Approval Signatures
r, Ph.D. Date
Director
).attachment Date
Statistician far signature
Katherine A.B. Knostman, D.V.M., Ph.D., D.A.C.V.P Date
Pathologist
Glorfa S. Sivko, Ph.D., D.V.M. Date
Technical Reviewer
Jasqft M. Mott, D.V.M., Ph.D. Date
BBftC Associate Director
-7 - 2 2. - / O
StepGen MTMiller, D.V.M.. Date
Study Veterinarian
iSonafd W. Cagle, MLS. ~^^ Date
Senior Safety and HQr<$ffM$$Klffient
for signature
Sarah C. Taft, Ph.D. Date
Sponsor Representative
Reviewed and Registered by:
HaroldWTNitz, RQAP-GLP^^ " Date
Quality Assurance Officer
A-3
-------
JUL-1S-2010 15:51
BATTELLE
614 424 7441 P.02/82
Approval Signatures
BBRC Protocol 1078
Study Number: 1078-CG920794
Battelle Biomedical
Research Center
Date: 22 My 2010
Page2ofl9
Jason E, Comer, Ph.D.
Study Director
Date
Gregory V. Stark, Ph.D.
Statistician
Katherine A.B. Knostman, D.V.M., Ph.D., D.A.C.V.P
Pathologist
Date
Date
Gloria S. Sivko, Ph.D., D.V7M.
Technical Reviewer
Date
Jason M. Mott D.V.M., Ph.D.
BBRC Associate Director
Date
Stephen M. Miller, D.V.M..
Study Veterinarian
Date
Donald W.Cagle, M.S.
Senior Safety and Health Advisor
Date
Sarah C. Taft, Ph.D.
Sponsor Representative
Date
Reviewed and Registered by:
Harold W. Nitz, RQAP-GLP
Quality Assurance Officer
Date
rtrrfd P.02
-------
BBRC Protocol 1078
Study Number: 1078-CG920794
Battelle Biomedical
Research Center
Date: 22 July 2010
Page 2 of 19
Approval Signatures
Jason E. Comer, PhJD. Date
Study Director
Gregory V. Stark, Ph,D. Date
Statistician
Katherine A.B. Knostman, D.V.M., Ph.D., D.A.C.V.P Date
Pathologist
Gloria S. Sivko, Ph,D., D.V,M. Date
Technical Reviewer
Jason M. Mott, D. V.M., Ph.D. Date
BBRC Associate Director
Stephen M, Miller, D.V.M.. Date
Study Veterinarian
Donald W. Cagle, M.S. Date
Senior Safety and Health Advisor
Jw^ c. /^-^ a a Tu Its
Sarah C. Taft, Ph.D. (^ ! Date O
Sponsor Representative
Reviewed and Registered by:
Harold W. Nitz, ROAP-GLP Date
Quality Assurance Officer
A-5
-------
BBRC Protocol 1078
Study Number: 1078-CG920794
Battelle Biomedical
Research Center
Date; 22 July 2010
Page 3 of 19
1.0 INTRODUCTION
Bacillus anthrads, the etiologic agent of anthrax, is a gram-positive, rod-shaped,
aerobic and/or facultative anaerobic, spore-forming bacterium. Each route of human
infection, including gastrointestinal, cutaneous, and inhalation, manifests in different
clinical symptoms, with inhalational anthrax being the most lethal. The incubation
period usually varies from 12 hours to five days depending upon the dose and route of
entry. The onset of disease can be longer following inhalation exposure and some
reports suggest a delayed onset of several weeks in low-dose exposures or following
removal of therapeutic intervention. The initial clinical signs and symptoms of
inhalation anthrax are nonspecific and may include malaise, headache, fever, nausea,
and vomiting. These are followed by a sudden onset of respiratory distress with
dyspnea, stridor, cyanosis, and/or chest pain. The onset of respiratory distress is
followed by shock and eventually death with close to 100% mortality.
Anthrax is considered a serious biological terrorist and military threat due to the high
lethality rates of inhalation exposure and the stability of the B. anthrads spore. The
virulence of B. anthrads spores is predicated upon the production of an anti-
phagocytic capsule and two proteinaceous toxins. Three polypeptides, protective
antigen (PA), lethal factor (LF), and edema factor (EF), interact to form two
interlinked toxins. PA and LF combine to produce anthrax lethal toxin (LT), and PA
and EF combine to produce edema toxin (ET). PA binds to a host cell receptor and is
cleaved by furin-like protease. The activated PA then forms a heptameric complex
which competitively binds three molecules of LF and/or EF. The holotoxin is then
taken up by the cell via receptor-mediated endocytosis. A decrease in endosomal pH
results in a conformational change in the PA molecule resulting in a pore structure for
LF and EF translocation into the cytoplasm. LF is a zinc metalloprotease that inhibits
mitogen activated protein kinase signaling. EF, a calcium-dependent adenylate cyclase,
increases cyclic adenosine monophosphate levels in susceptible cells and results in
altered water hemostasis and the inhibition of phagocytosis. Thus both toxins inhibit
the signaling cascades required for the activation of immune cells.
An unfortunate outbreak of inhalational anthrax in Sverdlovsk, Russia provided the
largest set of clinical specimens to study the pathology of human anthrax. Necropsies
of victims of the outbreak consistently showed pathologic characteristics of
inhalational anthrax including, necrotic hemorrhage of the thoracic lymph nodes,
hemorrhagic mediastinitis, and pleura! effusion. Fifty percent of the cases involved
hemorrhagic meningitis, and 92 % showed signs of gastrointestinal tract involvement
(i.e., submucosal hemorrhagic lesions).
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BBRC Protocol 1078
Study Number: 1078-CG920794
Battelle Biomedical
Research Center
Date: 22 July 2010
Page 4 of 19
Quantitative microscopic findings showed that most of the severe pathologic lesions
occurred in the mediastinum and mediastinal lymph nodes, the sites of initial
replication of the bacterium. The investigators also observed peripheral transudate
surrounding fibrin-rich edema, necrosis of veins and arteries, and apoptotic
lymphocytes.
The 2001 anthrax letter attacks resulted in five fatal cases of inhalational anthrax in the
United States. Prior to hospital admission, common nonspecific symptoms included
fever, malaise, and cough. Chest radiographs of these patients revealed pleural
effusion and lung infiltrates and anthrax infection was confirmed by culture.
The objective of this study is to determine physiological markers of disease following
multiple exposures of varying doses of Bacillus anthracis Ames strain spores.
2.0 LOCATION OF TESTING FACILITIES
This study will be performed by Battelle Memorial Institute, Biomedical Research
Center (BBRC) located at State Route 142, West Jefferson, OH 43162. Telemetry and
vascular access port (VAP) implantation surgery and histopathology will be performed
at Battelle Memorial Institute, 505 King Ave., Columbus, OH 43201.
3.0 STUDY OBJECTIVES
The objective of this study is to determine physiological markers of disease following
multiple exposures to B. anthracis Ames strain spores.
4.0 TEST SYSTEM
Animals: Thirty (30) male pathogen free New Zealand White (NZW) rabbits
(Oryctolagus citniculm) weighing at least 2.5 kg will be ordered from Covance
(Denver, PA) for this study. Rabbit age will not be used as a criterion for placement on
study. Twenty six (26) rabbits will be placed on study with the remaining four serving
as replacements. A Battelle veterinarian will implant Data Sciences International
model D70-PCT telemetric devices and femoral or jugular vascular access ports
(VAPs) into the rabbits prior to the start of the study. The rabbits shall be in good
health, free of malformations, and exhibit no signs of clinical disease. The identity of
each rabbit will be confirmed before and after each procedure (challenge, monitoring,
and bleeds) by ear tags and verified against cage cards. The four extra animals will also
be implanted with telemetric devices and VAPs in case either apparatus fails in a study
rabbit.
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BBRC Protocol 1078
Study Number: 1078-CG920794
Battelle Biomedical
Research Center
Date: 22 July 2010
Page 5 of 19
5.0 STUDY DESIGN
5.1 Randomization of Animals: Prior to challenge, rabbits will be randomized by
body weight into three groups of seven and one control group of five. The rabbits
within each group will also be randomized for challenge order (based on ear tag
numbers) and will be challenged according to randomization order and challenge dose
group. For example, the rabbits in Group 1 will be challenged first and the rabbits in
Group 4 will be challenged last. Prior to challenge, any animal with a malfunctioning
VAP or telemetric device will be replaced with one of the four extra animals.
5.2 Aerosol Challenge Generation and Monitoring: Each rabbit will be exposed to
B. anthracis Ames Strain spores once a day for five straight working days each week
for 3 straight weeks (up to 15 times each). On challenge days, rabbits will be placed
into a plethysmography chamber, passed into a Class III cabinet system, and aerosol
challenged with targeted doses of 100, 1,000, and 10,000 CPUs of 5. anthracis (Ames
strain) spores. The negative control group will be exposed to the equivalent of 10,000
CPUs of gamma-irradiated spores.
The first day of challenges will be designated as Study Day 0.
Aqueous suspensions of B. anthracis (Ames strain) will be aerosolized as per SOP
BBRC. XIII-001. Serial dilutions of impinger samples will be plated onto TSA plates
and enumerated as per SOP BBRC. X-054. Negative control animals with be
challenged in a separate hood line from the groups receiving viable spores to mitigate
cross contamination. Impinger samples containing irradiated spores will be plated neat
in triplicate to confirm that the animals were not exposed to viable spores.
Impinger samples from the groups receiving targeted doses of 100 or 1,000 CPUs will
also be enumerated by a filter method according to SOP BBRC X-199. Briefly, 1 mL of
the sample will be passed through a sterile 0.45 micron filter (Nalgene) and an
Analytical Test Filter Funnel, (Fisher catalog number 145-0045). The filter will then be
placed on top of a TSA plate and incubated for 24-72 hours at 37°C ± 2°C. The
colonies that form on the filter will then be enumerated. Please note that colonies
outside the range of 25-250 colonies/filter may be accepted at the discretion of the
study director. Another aliquot of the sample will be enumerated by spread plating
undiluted sample and a 1:10 dilution of the sample.
The aerosol challenge duration will be based upon an estimated aerosol challenge
concentration and a cumulative minute volume gathered "real" time throughout the
exposure.
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BBRC Protocol 1078
Study Number: 1078-CG92G794
Battelle Biomedical
Research Center
Date: 22 July 2010
Page 6 of 19
jL Study Design and Challenge Doses.
Group
Spore dose
(CPU)
10,000* "
Number of Spore
Challenges ' 1
15
100 1 15
1,000 i 15
10,000 [ 15
# of Rabbits 1
5
7
7
7 '
1 (neg) control*
*Spores arc inactivated/killed by irradiation. Challenges will occur in a separate aerosol system from the
viable spore challenges,
1 Will be challenged once a day for 5 straight working days (Monday thru Friday) each week for three
straight weeks
5.3 Animal Weights: Animals will be weighed on Study Days 2, 9, 16, 23, 30. and 37
(Study Day 0 will be the first day of challenges). Animals will also be weighed prior to
delivery to the BBRC and this weight will be used for randomization in to groups.
5.4 Blood Collection Schedule: On Study Days -3, 2, 4, 9, 1 1, 16, 18, 23, 25, 30, 32,
and 37 blood will be collected into EDTA (-1.0 mL) and SST tubes (-2.0 - 2.5 mL)
(Table 2). Collection time points for each rabbit will be relative to the very first
challenge day (Challenge Day 0) on Week 1. If possible, a blood sample will be taken
from animals found dead or prior to euthanasia and divided between the two types of
tubes. On Study Day 39. all surviving rabbits will be terminally bled via cardiac
puncture according to Table 2,
Blood samples collected into SST tubes will be processed to serum in accordance with
SOP BBRC. V-033. Blood collected in EDTA tubes will be stored at room temperature
if utilized within 4 hours of collection. If not analyzed within 4 hours of collection, the
blood will be stored at 2-8 °C. Sera will be stored at < -70 "C until needed.
Blood samples will be collected from VAPs on Study Days for the entire study unless
directed by the study director or the port malfunctions. If a port fails, the medial
auricular artery, the marginal ear vein, or other appropriate vasculature may be utilized
for blood collection if attainable. Rabbits will be sedated with Acepromazine prior to
collecting blood from the ear. If a blood sample cannot be collected from either the
port or other appropriate vasculature, based on study director discretion, it will be
documented in the study file,
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BBRC Protocol 1078
Study Number: 1078-CG920794
Battelle Biomedical
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Date: 22 July 2010
Page 7 of 19
Table 2, Blood collection schedule
Tube
type
EDTA
HH)_
SST
(~ml)
Total per
day~(ml)
i.
Day
-3
1.0
2.5
3.5
Day
2
1.0
2.0
3.0
Day
4
1.0
2.5
3.5
Day
9
1.0
2.0
3.0
Day
11
1.0
2.5
3.5
Day
16
1.0
2.0
3.0
Study I
Day
18
1.0
2.5
3.5
)ay_
Day
23
1.0
2.0
3.0
Day
25
1.0
2.5
3.5
Day
30
1.0
2.0
3.0
Day
32
1.0
2.5
3.5
Day
37
1.0
2.0
3.0
Day
39
2.0
10.0
12.0*
*End of in-life blood sample, minimum draw volume.
5.5 VAP Maintenance: The VAPs will be maintained weekly until the end of the study
or until the loss of patency. The skin over and ~3 inches around the VAP will be
clipped weekly or as needed. Prior to flushing the port, clean the area three times with
Nolvasan scrub and 70% alcohol. There will be 40 seconds between the last Nolvasan
scrub and before final alcohol wash. Place the Huber needle into the VAP, then with a
3ml syringe pull the block. Pull blood samples, if required. Flush with 2mL of 0.9%
saline and block with 1.0 mL of blocking solution (4.0 mL of 1000 1U of heparin in 16
mL of 50% dextrose.
5.6 Toxemia Assessment and TNA/ELISA: A portion of all the serum collected will
be analyzed for circulating PA via the quantitative PA ELISA according to SOP BBRC.
X-180.
Additionally, to determine if the rabbits elicit an immune response following challenge,
serum samples will be analyzed by ELISA and htp-TNA according to SOPs BBRC. X-
101 and X-143 based upon the schedule outlined in Table 3.
5.7 Bacteremia: A portion of each blood sample from the EDTA collection tubes will
be tested for bacleremia by quantitative spread plate technique (SOP BBRC. X-202),
and quantitative real-time PCR (SOP BBRC. X-146),
5.8 Hematology and C-Reactive Protein: Hematology will be performed on blood
samples collected in EDTA tubes using the Advia Hematology Analyzer according to
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BBRC Protocol 1078
Study Number: 1078-CG920794
Battelle Biomedical
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Date: 22 July 2010
Page 8 of 19
SOP BBRC. VI-066. Hematology evaluation will include but not be limited to the
following parameters:
• White blood cell count (WBC)
• N/L ratio (neutrophil/lymphocyte ratio)
• Differential leukocyte (absolute) count
• Hemoglobin (HOB)
» Hematocrit (HCT)
• Red blood cell count (RBC)
• Mean corpuscular volume (MCV)
• Mean corpuscular hemoglobin (MCH)
• Mean corpuscular hemoglobin concentration (MCHC)
• Red cell distribution width (RDW)
• Platelet count (PLT)
* Mean platelet volume (MPV)
After hematological analysis is complete, plasma will be harvested from the residual
sample according to SOP BBRC. V-033. The plasma sample will then be assayed for
C-reactive protein levels (CRP) according to SOP BBRC, VI-077.
The assay priority list is below:
Whole Blood: Bacteremia via quantitative plating >» Hematology >» C-reactive
protein >» Bacteremia via Quantitative PCR .Although lower in priority, the PCR
aliquot has to be removed before hematology to avoid cross contamination.
Sera: Circulating PA ELISA >» Retention sample (maximum 750 uL if possible)
>» Anti-PA IgG ELISA and TNA.
All assays listed in Table 3 will be performed on terminal samples for rabbits that die
on study with the exception of hematology and CRP. Hematology and CRP will not
be performed on samples collected for rabbits that were found dead.
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BBRC Protocol 1078
Study Number: 1078-CG920794
Battelle Biomedical
Research Center
Date: 22 July 2010
Page 9 of 19
Table 3. Schedule of assays to be performed on each study day.
Assay
Hematology
C-reactive
protein
Quant.
bact. culture
Quant.
bact. PCR
circ. PA
EL1SA
TNA, anti-
PAIgG
ELISA
Study Day
Day
-3
•
*
•
•
•
•
Day
2
•
*
•
•
•
Day
4
•
t
•
•
*
•
Day
9
•
•
•
•
*
Day
11
•
•
•
•
«
*
Day
16
i
•
•
•
•
Day
18
t
•
•
•
*
t
Day
23
•
*
*
•
*
Day
25
•
•
•
•
•
*
Day
30
*
•
*
•
•
Day
32
•
•
•
•
•
•
Dav
37
•
•
•
*
•
Day
39
•
•
«
4
*
*
Terminal
*
•
•
*
5.9 Clinical Observations: Following aerosol challenge, rabbits with be observed
twice daily for clinical signs of illness and survivability from anthrax infection (e.g.,
moribund, respiratory distress, appetite, activity, and seizures).
5.10 Telemetric Monitoring: Rabbits will be surgically implanted with telemetry
units (D70-PCT transmitters. Data Sciences International) prior to being placed on
study (allowing sufficient time to allow recovery from the implantation procedure).
Each D70-PCT transmitter contains one pressure lead and one biopotential lead. Body
temperature, Electrocardiogram (ECG) activity, and cardiovascular function (heart rate
and respiratory pressure) will be monitored at least 30 seconds every 15 minutes for 7
days pre-challenge (baseline) and for 39 days after the first challenge day according to
SOP BBRC.VI-087. ECG data will be collected but will not be analyzed, but will be
available for future analysis if desired. Event markers will not be logged. If the
telemetry implants fail post-challenge, the affected parameters will not be recorded;
however the animal will stay on study to collect other study data (i.e. clinical
observations, biological samples, clinical pathology, etc.).
Each animal's cage will be equipped with a Data Sciences International telemetry
receiver. The transmitters, receivers, consolidation matrixes, cabling, and computers
utilizing the Dataquest A.R.T.™ data acquisition and analysis software are all
components of the PhysioTel® Telemetry System. The Dataquest A.R.T.™ telemetry
software will collect the telemetry parameters above.
5.11 Necropsy and Histopathology: Animals that succumb to challenge, or are found
moribund and are euthanized, will undergo a gross necropsy. Surviving animals will
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BBRC Protocol 1078
Study Number: 1078-CG920794
Battelle Biomedical
Research Center
Date: 22 July 2010
Page 10 of 19
be euthanized and necropsied on Study Day 39. The lungs and any gross lesions from
each rabbit will be collected and examined histopathologically.
6.0 ANIMAL CARE AND HUSBANDRY
6.1 Quarantine
Rabbits will be quarantined for 7 days prior to study initiation and will be visually
inspected and released by the study veterinarian prior to study. Animals will be
observed a minimum of twice per day during the quarantine period.
6.2 Veterinary Care
Discomfort and distress will be limited to that which is unavoidable in the conductance
of scientifically valuable research. Animals that develop non-study related illness or
injury will be evaluated by a Battelle veterinarian for determination of treatment or
disposition. In such cases, and if in the opinion of the Study Director and a Battelle
veterinarian, an animal is in a moribund state, that animal will be euthanized. No
treatment will be given for study related signs with the exception that rabbits meeting
the Criteria for Euthanasia will be euthanized.
6.3 Criteria for Euthanasia
The sequelae leading to death in the subcutaneous and inhalation rabbit model have
been published by Zaucha et al, (1998) and confirmed in our laboratory. Although
there is a trend for decreased survival time with increasing dose, it is minimal.
Fulminating disease appears to be an all-or-none response and no protracted illness has
ever been observed, regardless of the dose. Abnormal clinical observations are not
generally apparent until approximately 24-hours before death, at which time rabbits
become progressively lethargic and weak. Several rabbits have exhibited brief periods
of excitation and hyperactivity within hours or minutes before death. These rabbits had
brain or meningeal lesions at necropsy.
The following criteria have been pre-established for euthanasia: presence of any
seizure (denoting meningitis or encephalitis), respiratory distress, dyspnea, or forced
abdominal respirations, unresponsive to touch or external stimuli, and moribundity.
Rabbits that are judged to be moribund by a trained life sciences technician, Battelle
veterinarian, or by the Study Director will be euthanized.
Rabbits that are euthanized will be sedated with Acepromazine or other approved
anesthetic and then administered a commercially prepared euthanasia solution at the
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BBRC Protocol 1078
Study Number: 1078-CG920794
Battelle Biomedical
Research Center
Date: 22 July 2010
Page 11 of 19
prescribed dose.
6.4 Husbandry
Housing: Rabbits will be housed individually in stainless steel cages on racks equipped
with automatic watering systems,
Lighting: The light/dark cycle will be approximately 12 hours each per day, using
fluorescent lighting.
Temperature: Animal room temperatures will be maintained according to Battelle SOP
No. BBRC IV-008.
Humidity: The relative humidity of the animal rooms will be maintained according to
Battelle SOP No. BBRC IV-008.
Diet: PM1, Inc. Certified Rabbit Chow® will be fed per Battelle SOP No. BBRC, VII-
013. No contaminants that would affect the results of the study are known to be present
in the feed.
Water: Water is supplied from the Battelle water system and will be available
ad libitum during the entire study. Water is analyzed at a minimum once per year.
Analysis is carried out following Battelle SOPs. No contaminants that would affect the
results of the study are known to be present in the water.
Enrichment: To promote and enhance the psychological well being of the rabbits,
enrichment will be as described in Battelle SOP No. BBRC. VII-040.
7.0 ANALYTICAL AND STATISTICAL PLAN
7.1 Sample Size:
Groups of 7 animals are sufficient to detect group effects in a one-way analysis of
variance (ANOVA) model with greater than 80% power, when a 2x standard deviation
difference in group means is present.
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BBRC Protocol 1078
Study Number: 1078-CG920794
Battelle Biomedical
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Date: 22 July 2010
Page 12 of 19
7.2 Statistical Methods:
Survival data from the groups will be compared using a Fisher's exact test. To explore
difference in time to death a log-rank test will be conducted or Cox proportional
hazards model will be fitted.
For circulating PA levels, TNA/ELISA, hematology, CRP, and telemetric data,
descriptive statistics will be produced for each animal at each sample collection time.
As all negative control animals are expected to survive, baseline values for each animal
will be used in the assessment of these endpoints, with each animal serving as its own
control. Mean changes in each parameter will be compared to baseline to evaluate any
change in health status. An ANOVA model will be fitted to determine if parameters
changed significantly from baseline and whether there were significant differences
between groups.
To evaluate the change in health status due to challenge, the post-challenge telemetry
endpoints may be adjusted to the baseline averages calculated for each individual
animal or other appropriate method. Statistical evaluation of dose-response curves may
be made at specified time intervals during the post-challenge period. Alternatively, time
to onset of altered telemetric parameters may be evaluated using Cox proportional
hazard models with dose as an explanatory variable.
7.3 Missing Value Handling:
All animals used in this study will be individually identified and accounted for at the
conclusion of the study. Mortality will be recorded as it occurs to the nearest hour
and/or day. If animals are removed from the study for appropriate reasons, mortality
will be reported as a percentage of the total animals remaining. Similarly, if individual
sample results or other measurements are not obtained for appropriate reasons, all
available results will be included in the analysis.
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BBRC Protocol 1078
Study Number: 1078-CG920794
Battelle Biomedical
Research Center
Date: 22 July 2010
Page 13 of 19
8.0 RECORDS TO BE MAINTAINED
8.1 Animals
Animals surviving the challenge(s) will be euthanized on Study Day 39,
8.2 Specimens
Specimens generated during this study (tissue, histology slides, sera, etc.) will be
shipped to the sponsor, if requested, or disposed of in accordance with SOP BBRC.
VII-011.
8.3 Study Records and Materials
All records applicable to this study will be maintained in compliance with BBRC
procedures.
8.4 Study Reports
Reports generated for this study will be written to provide all appropriate information to
the sponsor. The final report will contain all general information on the study.
8.5 Sponsor Study Audits
The documentation specific to this study will only be made available to representatives
of the sponsor, independent auditors contracted through the Sponsor, or other
designees of the Sponsor.
9.0 BIOSAFETY CONSIDERATIONS
Containment Level: The aerosol exposure system will be contained in a class III
biosafety cabinet within the BL-3 laboratory. Rabbits will be housed in the BL-3 for
up to 39 days post-last challenge, after which all surviving rabbits will be anesthetized,
have a terminal bleed taken, and be euthanized. If rabbits are removed from the study
prematurely, mortality will be reported as a percentage of the total animals remaining.
All animals that die or are euthanized will be double bagged, autoclaved, and
incinerated.
Biohazard Safety: Personnel handling anthrax challenged rabbits will wear appropriate
personal protective equipment (PPE) as described in Battelle SOPs. Additionally, all
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BBRC Protocol 1078
Study Number; 1078-CG920794
Battelle Biomedical
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Date: 22 July 2010
Page 14 of 19
personnel working with anthrax or anthrax-exposed animals have received appropriate
vaccination. Only antibiotic sensitive strains of anthrax will be used on this study.
Agents Used in this Protocol - Bacillus anthracis, Ames strain
Other toxic chemicals to be used include sodium hypochlorite and vaporized hydrogen
peroxide for decontamination requirements.
A Battelle Environment, Safety and Health Officer has been provided the opportunity
to review the procedures required to execute this study.
10.0 REFERENCES
Battelle SOP Number BBRC. IV-002, "Standard Operating Procedure (SOP) for
Monitoring Room Lighting in Animal Rooms."
Battelle SOP Number BBRC. IV-008, "Standard Operating Procedure (SOP) for
Monitoring Temperature and Humidity Conditions Using Automated HVAC Control
and Monitoring Systems."
Battelle SOP Number BBRC. V-029, "Standard Operating Procedure (SOP) for the
Operation and Maintenance of the AB1 Prism® 7900HT Fast Sequence Detection
System."
Battelle SOP Number BBRC.V-033, "Standard Operating Procedure (SOP) for the
Processing of Blood, Fecal or Urine Specimens Prior to Analysis."
Battelle SOP Number BBRC.V-061, "Standard Operating Procedure (SOP) for
Performing the Rapid Protective Antigen Electrochemiluminescence (ECL) Screening
Assay using Serum."
Battelle SOP Number MREF. VI-023, "Standard Operating Procedure (SOP) for the
Use and Monitoring of Cold Storage Units (CSU)."
Battelle SOP Number MREF. VI-027, "Standard Operating Procedure (SOP) for the
Operation, Calibration, and Maintenance of Electronic Balances."
Battelle SOP Number BBRC, VI-029, "Standard Operating Procedure (SOP) for the
Use of the Mettler Toledo Balancelink."
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BBRC Protocol 1078
Study Number: 1078-CG920794
Battelle Biomedical
Research Center
Date: 22 July 2010
Page 15 of 19
Battelle SOP Number BBRC. VI-044, "Standard Operating Procedure (SOP) for the
Operation and Maintenance of the Bio Medic Data System DAS-6007 Handheld
Scanner."
Battelle SOP Number MREF. VI-054, "Standard Operating Procedure (SOP) for the
Operation and Maintenance of the Dickson Model SP100 and SP150 Pro Temperature
Data Loggers."
Battelle SOP Number BBRC.VI-066, "Standard Operating Procedure (SOP) for the
Operation and Maintenance of the Siemens (formerly Bayer) Advia®120 Hematology
Analyzer."
Battelle SOP Number BBRC.VI-077, "Standard Operating Procedure (SOP) for the
Operation and Maintenance of the Siemens (formerly Bayer) Advia® 1200 Chemistry
Analyzer."
Battelle SOP Number MREF. VI-084, "Standard Operating Procedure (SOP) for the
Operation And Maintenance Of Primus General Purpose Steam Sterilizer Model: PSS5-
A-MSSD."
Battelle SOP Number BBRC.VI-087, "Standard Operating Procedure (SOP) for Use
and Maintenance of the Data Sciences International (DSI) Telemetry System."
Battelle SOP Number MREF. VI-101, "Standard Operating Procedure (SOP) for the
General Use, Operation and Maintenance of Microscopes."
Battelle SOP Number BBRC. VII-002, "Standard Operating Procedure (SOP) for Feed
Source, Storage, Handling, and Analysis."
Battelle SOP Number BBRC. VII-006, "Standard Operating Procedure (SOP) for
Animal Euthanasia at the Battelle Biomedical Research Center (BBRC)."
Battelle SOP Number BBRC. VII-010, "Standard Operating Procedure (SOP) for
Clinical Observations of Animals at the Battelle Biomedical Research Center (BBRC)."
Battelle SOP Number BBRC. VI1-011, "Standard Operating Procedure (SOP) for
Receipt, Handling, Shipping, and Disposal of Test Materials, Analytical Samples and
Controlled Substances."
Battelle SOP Number BBRC. VII-013, "Standard Operating Procedure (SOP) for Care
of Rabbits."
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BBRC Protocol 1078
Study Number: 1078-CG920794
Battelle Biomedical
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Date: 22 July 2010
Page 16 of 19
Battelle SOP Number BBRC. VII-020, "Standard Operating Procedure (SOP) for the
Collection of Blood Samples from Animals."
Battelle SOP Number BBRC. VII-026, "Standard Operating Procedure for Receipt,
Quarantine, Monitoring, and Release of Experimental Animals,"
Battelle SOP Number BBRC. VII-040, "Standard Operating Procedure for
Environmental Enhancement/Enrichment Plan to Promote the Psychological Weil-
Being of species other than Non-Human Primates."
Battelle SOP Number BBRC. VII-056, "Standard Operating Procedure (SOP) for the
Slow Intravenous Administration by Infusion and Collection of Blood Samples From
Venous Access Ports in Rabbits."
Battelle SOP Number BBRC. VIII-003, "Standard Operating Procedure (SOP) for
Supplying Water and Monitoring Water Quality of the Manual and Automatic Watering
Systems,"
Battelle SOP Number BBRC, X-038, "Standard Operating Procedure (SOP) for the
Operation and Maintenance of BBRC Infectious Waste Sterilizers."
Battelle SOP Number BBRC. X-054, "Standard Operating Procedure (SOP) for
Enumeration of BL-2 and BL-3 Bacterial Samples via the Spread Plate Technique."
Battelle SOP Number MREF. X-074, "Standard Operating Procedure (SOP) for the
Production of Bacillus Anthracis Spores."
Battelle SOP Number BBRC. X-075, "Standard Operating Procedure (SOP) for the
Characterization and Qualification of Bacillus anthracis Spores."
Battelle SOP Number BBRC. X-096, "Standard Operating Procedure (SOP) for the
Qualitative Analysis of Bacteria in Blood and Tissue."
Battelle SOP Number BBRC. X-101, "Standard Operating Procedure (SOP) for
Enzyme Linked Immunosorbent Assay (ELISA) Detection of Bacillus anthracis PA-
Specifie IgG in Sera."
Battelle SOP Number BBRC. X-143, "Standard Operating Procedure (SOP) for the
High Throughput Toxin Neutralization Assay (htp-TNA) Proper."
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BBRC Protocol 1078
Study Number: 1078-CG920794
Battelle Biomedical
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Date: 22 July 2010
Page 17 of 19
Battelle SOP Number BBRC. X-146 "Standard Operating Procedure (SOP) for
Performing the Absolute Quantitative Real-Time Polymerase Chain Reaction Assay
Using Qualified Reference Standard Materials."
Battelle SOP Number MREF. X-160, "Standard Operating Procedure (SOP) for the
Qualification of Negative Control Sera for Use in the Toxin Neutralization Assay
(TNA) and Related Assays."
Battelle SOP Number BBRC. X-164, "Standard Operating Procedure (SOP) for the
Qualification of Reference Serum for Use in the Toxin Neutralization Assay (TNA) and
Related Assays."
Battelle SOP Number BBRC. X-166, "Standard Operating Procedure (SOP) for the
Qualification of Reference Standard Plasmid for Use in Quantitative Real-Time
Polymerase Chain Reaction (qPCR)."
Battelle SOP Number BBRC. X-180, "Standard Operating Procedure (SOP) for
Enzyme Linked Immunosorbent Assay (ELISA) Detection of Bacillus anthracis
Circulating Protective Antigen in Sera."
Battelle SOP Number BBRC. X-186, "Standard Operating Procedure (SOP) for the
Preparation and Analysis of Phenol Samples in Bacillus anthracis Spore Supernatant."
Battelle SOP Number BBRC. X-199, "Standard Operating Procedure (SOP for the
Enumeration of Bacterial Colony Forming Units via the Membrane Filter Count
Method."
Battelle SOP Number BBRC. X-202, "Standard Operating Procedure (SOP) the
Enumeration of Bacteria via the Spread Plate Technique."
Battelle SOP Number BBRC. XI-006, "Standard Operating Procedure (SOP) for the
Calibration and Maintenance of Temperature/Humidity Measuring Instruments and
Equipment."
Battelle SOP Number MREF. XI-007, "Standard Operating Procedure (SOP) for
Labeling Reagents, Solutions, Test, Control and Reference Article/Substances, and
Specimens,"
Battelle SOP Number MREF. XI-025, "Standard Operating Procedure (SOP) for the
General Preparation of Dilutions."
A-20
-------
BBRC Protocol 1078
Study Number: 1078-CG920794
Battelle Biomedical
Research Center
Date: 22 July 2010
Page 18 of 19
Battelle SOP Number BBRC. XI1I-001, "Standard Operating Procedure (SOP) for the
Aerosol Exposure System to Challenge Non-Human Primates and Rabbits to
Aerosolized Agent."
Battelle SOP Number BBRC. XIH-002, "Standard Operating Procedure (SOP) for the
Maintenance and Decontamination of the Aerosol System."
Battelle SOP Number BBRC. XIII-005, "Standard Operating Procedure (SOP) for the
Cleaning, Maintenance and Use of Collison Nebulizers and Impingers."
Battelle SOP Number MREF. XIII-006, "Standard Operating Procedure (SOP) for the
Verification of Mass Flow Meters, Mass Flow Controllers, Magnehelics and Gauges."
Battelle SOP Number BBRC. XIII-008, "Standard Operating Procedure (SOP) for
Programming the Buxco Biosystem XA Data Acquisition Software for Pulmonary
Analysis during Animal Inhalation Studies."
Battelle SOP Number BBRC. XIII-009, "Standard Operating Procedure (SOP) for the
Calibration and Operation of the Buxco Biosystem, Preamplifier System and Pressure
Transducers."
Battelle SOP Number BBRC. XIII-010, "Standard Operating Procedure (SOP) for the
Cleaning, Decontamination, and Maintenance of NHP and Rabbit Plethysmography
Boxes."
Battelle SOP Number BBRC, XIII-011, "Standard Operating Procedure (SOP) for
Using and Checking the Calibration of the Aerodynamic Particle Sizer 3321."
Battelle SOP Number BBRC. XIH-012, "Standard Operating Procedure (SOP) for
Conducting Spray Factor Testing of Aerosolized Bacillus anthracis Spores Using the
Battelle Aerosol Exposure System."
Battelle SOP Number BBRC. XII1-018, "Standard Operating Procedure (SOP) for the
Calibration and Operation of the Buxco Biosystem XA (Windows Version 2.7.9)."
Battelle SOP Number BBRC. XIII-022, "Standard Operating Procedure (SOP) for the
Operation of the Aerosol Challenge Database."
Inglesby, T.V., Henderson, D.A., Bartlett. J.G., Ascher, M.S., Eitzen, E., Friedlander,
A.M., Hauer, J., McDade, J., Osterholm, M.T., O'Toole, T., Parke, G., Perl, T.M.,
A-21
-------
BBRC Protocol 1078
Study Number: 1078-CG920794
Battelle Biomedical
Research Center
Date: 22 July 2010
Page 19 of 19
Russell, P,K,, and K. Tonat. 1999. Anthrax as a biological weapon: medical and public
health management. Working Group on Civilian Biodefense. JAMA. 281:1735-1745.
Zaucha, GM; Pitt, LM; Estep, J; Ivins, BE; and Friedlander, AM (1998), The
pathology of experimental anthrax in rabbits exposed by inhalation and subcutaneous
inoculation. Arch. Pathol. Lab. Med. 122: 982-992.
A-22
-------
APPENDIX B
STUDY DEVIATIONS AND INVESTIGATION REPORTS
B-1
-------
BATTELLE BIOMEDICAL RESEARCH CENTER
DEVIATION FORM
Deviation No. (Assigned by QAU):-po i Q Q i q
CAQ No. (Assigned by QAU):
Standard or Procedure Deviated:
HI Protocol (Number and Amendment No. if applicable): 1078
D SOP (Number and Revision Number):
D Method (Number and Revision Number):
D GLP (Section):
D Other:
Type of Deviation (check one):
Facility
Study (fill out study info) Study Number: 1078-CG920794
Study Title: Rabbit Multiple Dose Anthrax Telemetry Study
Record Affected (describe Title, Binder name, location, Form no. etc.): Microbiology binder, form
Microbio-455J <
Date of Deviation(s): July 26-Jnty-3frand August ^-August 13
Description of Deviation:
Protocol states that filtered samples will be incubated for 24-72 hours before being read. For the time period
of July 26-Jttry-30 the protocol was deviated because samples were incubated for less than 24 hours, generally
about 16-24 hours ^ 4 ~> ^ */"/'»
Root Cause of Deviation:
Filtered samples were incubated with non-filtered samples and were taken out and counted at the same time.
Colonies were found to be of a size easy to count. Waiting beyond 24 hours would result in colonies too
large to count.
Corrective Action: £ u> UVN f////,o
For the remainder of the aerosol challenge (August /-13) the samples will continue to be counted following
an incubation of 16-72 hours.
Impact of Deviation: Positive: Extending the incubation time to a shorter period allows the plates to be
counted at the same time as the non filtered samples.
If deviation is planned, effective date: &*2/lfr4o 8/13/10 $-$-/0 -to f-i3~/0
Deviation Form Prepared by/Date: RD 8/2/10 s *- /A
Deviation Reviewed and Corrective Action Accepted by/Date (study Director or Responsible individual):
/=-.r /o
Deviation Reviewed and Corrective Action Accepted by/Date (Superyj^-, Supervisor Representative, or Group Manager):
Circle One: ViveTMigix?, Mol Tox^.^gsg^Stffilstry, BDS, MCB, Facility, QA, Study Management, Other
~
Deviation Reviewed and Registered by QAU/Date:
D Category I
H Category II
(See SOP XI-023 for details)
Form No. Facility-OSS-OS (Revised 12/2/2009) Page 1 of 1
B-2
-------
BATTELLE BIOMEDICAL RESEARCH CENTER
DEVIATION FORM
) Deviation No. (Assigned by QAU): ~p^p \r\r\/ •?
CAQ No. (Assigned by QAU):
Standard or Procedure Deviated:
D Protocol (Number and Amendment No. if applicable):
0 SOP (Number and Revision Number): XI-009-03
D Method (Number and Revision Number):
D GLP (Section):
D Other:
Type of Deviation (check one):
D Facility
0 Study (fill out study info) Study Number: 1078-CG920794
Study Title: Rabbit Multiple Dose Anthrax Telemetry Study
Record Affected (describe Title, Binder name, location, Form no. etc.): Microbiology binder, Form
Microbio-380
Date of Deviation(s): 8/2/10
Description of Deviation: An animal ID was not listed as being included in either incubator.
s Root Cause of Deviation: Animal ID 28 was inadvertently left off of the list of samples in both of the
/incubators listed on the paperwork.
Corrective Action: The technician reviewed SOP XI-009 and was reminded to verify that all animal IDs are
accounted for on the incubation paperwork. As/d C. i \, I D
Impact of Deviation: Minimal. It cannot be determined which incubator the plates were placed in, but the
plates in both incubators were incubated for an appropriate amount of time and the plates for the animal in
question returned satisfactory results.
If deviation is planned, effective date: NA
Deviation Form Prepared by/Date: yf^^? n II, I A
Deviation Reviewed and Corrective Action Accepted by/Date (Study Director or Responsible individual):
Deviation Reviewed and Corrective Action Accepted by/Date (Supervisor, Supervisor Representative, or Group Manager):
Circle One: Vivo, Micro, Mol Tox, Aerosol, Chemistry, BDS, MCB, Facility, QA, Study Management, Other
Deviation. Reviewed and
[istered by QAU/Date:
El Category I
D Category II
(See SOP XI-023 for details)
Form No. Facility-OSS-OS (Revised 12/2/2009)
Page 1 of 1
B-3
-------
JBATTEIILE BIO|lEpIC:AL RESEARCH CENTER
Deviation No. (Assigned by QAU): ^ . . ~. ,.,
CAQ No. (Assigned by QAU): k/ft
Standafdi)r*Erbcedure Deviated:
D Protocol (Number and Amendment No. if applicable):
H SOP (Number and Revision Number): BBRC VI-087
D Method (Number and Revision Number):
D GLP (Section):
D Other:
Type of Deviation (check one):
D Facility
m Study (fiU out study info) Study Number: 1078-CG920794
Study Title: Rabbit Multiple Dose Anthrax Telemetry Study
Record Affected (describe Title, Binder name, location, Form no. etc.): Telemetry Data
Date of Deviation(s): 7/22/10
Description of Deviation: BBRC VI-087 states: Refer to Form No. Telemetry-006, or equivalent,
for the transmitter serial number and calibration information.
On 7/22/10 the configuration was created with incorrect calibration values for IDs 30 and 38. The ECG
calibration for ID 30 was entered as 22.6 mV when 22.3 mV should have been entered. Temperature
calibration 3 of ID 38 was entered as 756.3, when 756.6 should have been entered.
Root Cause of Deviation: This was caused by operator error while entering the calibration values from the
calibration cards into the telemetry computer. Data collection was required to start when the animals arrived
to the facility, which did not allow sufficient time to enter and review all configuration values.
Corrective Action: When possible, transmitter calibration values will be entered prior to animal arrival to
allow more time to enter and review calibration values.
Impact of Deviation: Minor. The small difference in the calibration values will not cause noticeable
differences in the data. Additionally, post-challenge data is compared to the baseline data which would offset
any differences caused by an incorrect calibration value.
If deviation is planned, effective date: NA
Deviation Form Prepared by/Date: t/^\
Deviation Reviewed and Corrective Action Accepted by/Date (study Director or Responsible individual):
.&—•a,,"- •— ——•
Deviation Renewed and Corrective Action Accepted by/Date (Supervisor, Supervisor Representative, or Group Manager):
Circle One: Vivo,(Miji<5, Mol Tox, Aerosol, Chemistry, BDS, MCB, Facility, QA, Study Management, Other
Deviation Reviewed and Registered by QAU/Date:
D Category I
13 Category II
(See SOP XI-023 for details)
Form No. Facility-OSS-OS (Revised 12/2/2009) Page 1 of 1
B-4
-------
Deviation No. (Assigned by QATJ):,,
DR.-
CAQ No. (Assigned by QAU):
Standard or Procedure Deviated:
Protocol (Number and Amendment No. if applicable): BBRC Protocol 1078
SOP (Number and Revision Number):
Method (Number and Revision Number):
GLP (Section):
Other:
Type of Deviation (check one):
D Facility
@ Study (fill out study info) Study Number: 1078-CG920794
Study Title: Rabbit Multiple Dose Anthrax Telemetry Study
Record Affected (describe Title, Binder name, location, Form no. etc.): 1078-CG920794 Telemetry Data
Date of Deviation(s):
7/23/10
)
Description of Deviation:
Protocol 1078 states "Body temperature, Electrocardiogram (ECG) activity, and cardiovascular function
(heart rate and respiratory pressure) will be monitored at least 30 seconds every 15 minutes for 7 days pre-
challenge (baseline) and for 39 days after the first challenge day according to SOP BBRC.VI-087."
Parameters were monitored for 3 days pre-challenge.
Root Cause of Deviation:
The draft protocol initially required 3 days of pre-challenge baseline data and was used when preparing the
schedule for the start of data collection. Also, the study animals arrived to the BBRC 6 days pre-challenge, 1
day after baseline collection should have started.
Corrective Action:
Telemetry lead was reminded to verify start of data collection with study director when a final protocol is not
available.
Impact of Deviation:
Minor. Three days of baseline telemetry data will be used for comparison to post-challenge data during
statistical analysis. 2-3 days of baseline data is commonly used for analysis and was the initial requirement of
the draft protocol.
If deviation is planned, effective date: NA
Deviation Form Prepared by/Date:
y
Deviation Reviewed and Corrective Action Accepted by/Date (study DirectororResponsibieindividual):
^-^
-------
BATTEBEE BIOMEDlCAL RESEARCH CENTER
Jj • '' ?* DEVIATION FORM ,
Deviation No. (Assigned by QAU):^ . , q CAQ No. (Assigned by QAU): MA
Deviation Reviewed and Registered by QAU/Date:
(0
D Category I
El Category II
(See SOP XI-023 for details)
Form No. Facility-OSS-OS (Revised 12/2/2009)
Page 2 of 2
B-6
-------
£* 'feh,:,,... .
;-
BATTEIXE BIOMftDICAL RESEARCiljCENTER :--\
'
Deviation No. (Assigned by QAU):
>
» Q^Z £i /\
CAQ No. (Assigned by QAU): Nf A
. . - -
D Protocol (Number and Amendment No. if applicable)
JS'SOP (Number and Revision Number): X-090-06
D Method (Number and Revision Number):
D GLP (Section):
D Other:
- -, ... ,. . . .
D Facility
J3"Study (fiU out study info) Study Number: 1 078-CG920794
Study Title: Rabbit Multiple Dose Anthrax Telemetry Study
Record Affected (describe Title, Binder name, location, Form no. etc.): Form No. BBRC BL3-026 and Microbio-
435
Date of Deviation(s): 8/9/2010, 9/7/10, and 9/13/2010
Description of Deviation:
BBRC SOP X-090 Section V. B. states the following: "It is the responsibility of the Battelle Safety, Health and
Emergency Response Representative, BSTI to provide approval for the release of tested sample(s) from the BSL-3
laboratory via form No. BBRC BL3-026, or equivalent." Thus, the deviation is as follows: On 8/9/2010, 9/7/10, and
9/13/2010 a technician passed serum out of the BL3 prior to receiving signature authority from the Safety Officer on
^Form No. BBRC BL3-026. The safety officer did, however, approve the sterility of the samples on Form No.
Microbio-463 before release. On 8/10/10,9/10/10, and 9/14/10, respectively, a signature was received on Form No.
BBRC BL3-026 to authorize their release.
Root Cause of Deviation: Technician did not get a signature on BBRC-BL3-026 prior to removal of samples out of the
BL3.
Corrective Action: Ensure that the safety officer signs Form No. BBRC-BL3-026 prior to pulling samples out of the
BL3. Forms and/or SOP could be revised to eliminate the redundancy of the safety officer having to sign in two places.
Impact of Deviation: None. A sterility check was performed on all samples prior to their removal from the BL3; all
serum aliquots were negative. This information was recorded on Microbio-463 and was confirmed by the Safety
Officer before the samples were released from the BL3.
If deviation is planned, effective date: NA
Deviation Form Prepared by/Date:
Deviation Reviewed and Corrective Action Accepted by/Date (StudyJMr^etor or Responsible Individual):
/2 T-ro
Deviation Reviewed and CorrecJ^e Action Accepted by/Date (Supervisor, Supervisor Representative, or Group
Manager): Circle One; VivoVMicjx*; Mol Tox, Aerosol, Chemistry, BDS, MCB, Facility, QA, Study
Management, Other
Deviation Reviewed and Registered by QAU/Date:
•n
$5 Category I
D Category H
(See SOP XI-023 for
details)
Form No. Facility-OSS-OS (Revised 12/2/2009) Page 1 of 1
B-7
-------
BATTELLE BJtijGEDICAL RE$P£
Deviation No. (Assigned by QAU):
CAQ No. (Assigned by QAU):
"•'' ':'. ' ;; * l~^''W^|^.'.;^f|i^^
D Protocol (Number and Amendment No. if applicable):
El SOP (Number and Revision Number): XI-009-03
D Method (Number and Revision Number):
D GLP (Section):
D Other:
D Facility
El Study (fill out study info) Study Number: 1078-CG920794
Study Title: Rabbit Multiple Dose Anthrax Telemetry Study
Record Affected (describe Title, Binder name, location, Form no. etc.): Study 1078-CG920794 Microbiology
Binder. Enumerations for Quantitative Bacteremia; Form No. Microbio-472
Date of Deviation(s): 7/24/10
Description of Deviation: According to SOP XI-009 Section VA4: Study raw data and other data entries made in
supporting records must be recorded clearly, accurately, legibly, completely and promptly in indelible black or blue
ink. This includes, but is not limited to observations, calculations, measurements, materials used, test and control
articles, critical reagents/solutions, equipment information (see Reference HI.F. and Section V.A.10. of this SOP), and
experimental and/or operational details to confirm the performance of the operation as specified. The technician did not
\ accurately record the results of the challenge organism for animal IDs 33 and 31 Day -3 El quantitative bacteremia
plates.
Root Cause of Deviation: The technician recorded a challenge organism colony as present on Day -3 El plates for
animal IDs 33 and 31. As the results are written, it states there is one colony of the challenge organism and one
contaminant present on one of the three plates for each animal ID. All three El plates for animal IDs 33 and 31 should
read 0, with a comment on one plate each that contamination was present.
Corrective Action: Upon speaking with the Study Director, the technician has been asked to review SOP XI-009 for
proper documentation and recording procedures. Proper documentation was also discussed between a study
coordinator and the technician. The technician has been reminded that only challenge organism colonies present are
recorded as the result for quantitative bacteremias. Contamination is documented with a footnote.
Impact of Deviation: Minimal. There is little possibility that the challenge organism could be present at the Day -3
time point. None of the other animals tested at this time had challenge organism colonies present. The quantitative
bacteremia results for Day -3 will be reported as negative for animals 33 and 31.
-rn» •7» • -• -w ftr, . . _ -. _ . " ' ——
If deviation is planned, effective date: NA
Deviation Form Prepared by/Date: j<^ \Q I ^C]1j Q (D
Deviation Reviewed and Corrective Action Accepted by/Date (Study Dirgctoi^or Responsible Individual):
°
Deviation Reviewed and Correc^ye Action Accepted by/Date (Supervisor, Supervisor Representative, or Group
Manager): Circle One; Vivo^cr^, Mol Tox, Aerosol^Chemistry, BDS, MCB, Facility, QA, Study
Management, Other C^ ^
Deviation Reviewed and Registered by QAU/Date:
ISp1 Category I
D Category H
(SeeSOPXI-023for
details)
Form No. Facility-OSS-OS (Revised 12/2/2009) Page 1 of 1
f£spons\b\e -kc,Wv\uan \e> no Icm&r &~ S£c.HelVe_
-------
|TELLE BIOMEDICAL RESEARCH CENTER
^Deviation No. (Assigned by QAU): j A ^? pr n
CAQ No. (Assigned by QAU):
$S Protocol (Number and Amendment No. if applicable): 1078 CG920794
D SOP (Number and Revision Number):
D Method (Number and Revision Number):
D GLP (Section):
D Other:
•> * SI^V^Jip; ' ?j?ji^ ' '*':'Type of Deviation%heclteone):
D Facility
JS'Study (fill out study info) Study Number: 1078-CG920794
Study Title; Rabbit Multiple Dose Anthrax Telemetry Study
Record Affected (describe Title, Binder name, location, Form no. etc.): Microbio-463
DateofDeviation(s): 8/13/2010
Description of Deviation:
On 8/13/2010 a technician did not make a retention aliquot for terminal animal 2, however, made an aliquot
for Circulating PA, Anti-IgG ELISA and TNA.
Root Cause of Deviation:
Th& protocol does not specifically state a retention aliquot is required on terminal animals. The protocol
/states, "all assays listed in Table 3 will be performed on terminal samples for rabbits that die on study with
the exception of hematology and CRP." Table 3 does not list retention as an aliquot/assay required on
terminal samples. However, prior to Table 3 the protocol states a priority list for collected sera. This list
states Circulating PA ELISA > Retention Sample > Anti-PA IgG ELISA and TNA. Because table 3 did not
list retention as an aliquot/assay required, the technician only made aliquots for Circulating PA, Anti-IgG
ELISA, and TNA.
Corrective Action:
Notify the study director of protocol ambiguities prior to the start of the study.
Impact of Deviation: Minimal. Residual samples will be pooled and sent to the client.
If deviation is planned, effective date: NA
Deviation Form Prepared by/Date.
Deviation Reviewed and Corrective Action Accepted by/Date (stgayD%t9rj»Sesponsibieindividual):
/2 •i±'.ic>
Deviation Renewed and Corrective Action Accepted by/Date (Supervisor, Supervisor Representative, or Group Manager):
Circle One: Vivo/|Micrj(< Mol Tox, AerosoL Chemistry, BJ1S, MCB, Facility, QA, Study Management, Other
Deviation Reviewed
and Registered
by QAU/Date:
~AVVoJb: 2,
— — — — — o
1
i In
J3" Category I
jEf Category II
(See SOP XI-023 for details)
Form No. Facility-OSS-OS (Revised 12/2/2009) Page 1 of 1
B-9
-------
•BATTEELE BtOMEDICAL RfSEARCp;.CENTE|L--:
v '' Jr DEv&noN jfoRM I • -::-: 11; "
) Deviation No. (Assigned by QAU):
CAQ No. (Assigned by QAU):
• '%fe':.^::'1; '"-. *'^|I|!%H':|V-:^^^
D Protocol (Number and Amendment No. if applicable):
0 SOP (Number and Revision Number): BBRC XI-009-03
D Method (Number and Revision Number):
D GLP (Section):
D Other:
|jTy|e of Delation (check (>ne):
" *"*"';s -'^'t ^-vii-S. " * ^ ' 1"/ "•" ^; -un;., '
,*^!S?F-. %; '^'^
D Facility
0 Study (fill out study info) Study Number: 1078-CG920794
Study Title: Rabbit Multiple Dose Anthrax Telemetry Study
Record Affected (describe Title, Binder name, location, Form no. etc.): Microbiology Binder, Form No.
Microbio-472
DateofDeviation(s): 8/28/10
Description of Deviation: BBRC SOP XI-009-03 states that study raw data and other data entries made in
supporting records must be recorded clearly, accurately, legibly, completely and promptly in indelible black
or blue ink. This includes, but is not limited to observations, calculations, measurements, materials used, test
I and control articles, critical reagents/solutions, equipment information and experimental and/or operational
details to confirm the performance of the operation as specified. On 8/28/10, all of the results were not
documented correctly.
Root Cause of Deviation: The technician did not include footnote 2 in the appropriate results section on the
form, meaning that all counts were zero.
Corrective Action: The technician was reminded to verify that all results are documented correctly before
submitting paperwork. (The responsible technician is no longer a Battelle employee and cannot sign this
form.)
Impact of Deviation: None. All of the counts were zero.
If deviation is planned, effective date: NA
Deviation Form Prepared by/Date:
Deviation Reviewed and Corrective Action Accepted by/Date (StSd^M^ste^r Responsible individual):
^—^^
Deviation ReYfpwed and Corrective Action Accepted by/Date (Supervisor, Supervisor Representative, or Group Manager):
Circle One: Vivc/MicroMoI Tox, Aerosol, ChemistarBDS, MCB, Facilitj^QA,Study Management, Other
Deviation Reviewed and Registered by QAU/Date:
=
53 Category I
D Category II
(See SOP XI-023 for details)
Form No. Facility-OSS-OS (Revised 12/2/2009)
Page 1 Of 1
B-10
-------
|v.;., ^AJ
Deviation No. (Assigned by QAU):-pg_
CAQ No. (Assigned by QAU):
: Standard
D Protocol (Number and Amendment No. if applicable):
0 SOP (Number and Revision Number): XI-009-03
D Method (Number and Revision Number):
D GLP (Section):
D Other:
IPtType'df Deviation (check one)
Facility
0 Study (fill out study info) Study Number: 1078-CG920794
Study Title: Rabbit Multiple Dose Anthrax Telemetry Study
Record Affected (describe Title, Binder name, location, Form no. etc.): S-MP, C-MP, and T-MP
Preparation, form No. Microbio-447
Date of Deviation^): 9/2/10, 9/3/10, 9/8/10
Description of Deviation: Section V.B.S.b of SOP XI-009 states that all quantitative data collected for a
regulatory study must be recorded the same day the work was performed. Any omissions or errors discovered
must be entered into the study record as a deviation. When preparing the negative control (Lot BMI012), a
math error in the dilution calculation was noted. The negative control is to be prepared at a 1:50 dilution,
with 1400uL total volume prepared. This would require 28uL of the neat negative control serum into 1372uL
of diluent. 1378uL of diluent is recorded, yielding a 1:50.2 dilution.
Root Cause of Deviation: The technicians preparing controls did not notice the math error, and therefore did
not correct it at the time of preparation. The correction was made several weeks after the work was
performed. It is likely that the incorrect diluent volume recorded was actually the diluent volume used, but
this cannot be verified with the information given.
Corrective Action: Technicians have been reminded to check all math, verify all dilutions, and back
calculate all calculations prior to performing work on a study. It has been stressed that the accuracy of the
dilutions created can make a difference between passing plates and unnecessary repeating of plates due to an
incorrectly prepared critical reagent. ^fa& U-"M-I"C7
Impact of Deviation: Minimal. The dilution of negative control created was 1:50.2 instead of the SOP
required 1:50. Since this resulted in a preparation that is more dilute, this may have had an effect on the
performance of the negative control within the assay.
If deviation is planned, effective date: NA
Deviation Form Prepared by/Date: /,// ,./,., /
JMT /'//T f/o
Deviation Reviewed and Corrective Action Accepted by/Date
-------
Deviation No. (Assigned by QAU)rr)|7,
CAQ No. (Assigned by QAU):
Standard
D Protocol (Number and Amendment No. if applicable):
H SOP (Number and Revision Number): XI-009-03
D Method (Number and Revision Number):
D GLP (Section):
D Other:
(chejck one):
D Facility
H Study (fill out study info) Study Number: 1078-C920794
Study Title: Rabbit Multiple Dose Anthrax Telemetry Study
Record Affected (describe Title, Binder name, location, Form no. etc.): VIVO Binder, Form No. Animal
Prep-038-00
DateofDeviation(s): 8/4/10
Description of Deviation:
Technician failed to sign that "Prior to Administration, the initial volume to be given was verified against the
Acepromazine range specified in the protocol."
Root Cause of Deviation:
Technician failed to double check that all areas on the form were filled out at the end of study activities.
Corrective Action:
Technician will double check that all areas of forms are properly and completely filled out at the end of each
study activity, technician was also asked to review SOP XI-009-03
Impact of Deviation: Minimal, due to the initial volume given was within the range specified in the protocol.
If deviation is planned, effective date:
Deviation Form Prepared by/Date:
Deviation Reviewed and Corrective Action Accepted by/DatC^studyDirs^ or Responsible individual):
Deviation Reviewed and Corrective Action Accepted by/Date (Supervisor, Supervisor Representative, or Group Manager):
Circle One: Ym&Micro. Mol Tox, Aerosol, Chemistry, BDS, MCB, Facility, QA, Study Management, Other
Deviation Reviewed and Registered by QAU/Date:
10
M Category I
D Category II
(See SOP XI-023 for details)
Form No. Facility-035-05 (Revised 12/2/2009)
Page 1 of 1
B-12
-------
BATTELLE BIO|IEDICAE?fESEARCH CENTER^
Deviation No. (Assigned by QAU):, IQ/UC:
CAQ No. (Assigned by QAU): y^
.Standard'orP:g61cedureDeviated:*^ «ff* -s"^
•ifrft? - -^- <.,.«M jrfM*' ;'f^ ••*-•:.-••. .,.,«
D Protocol (Number and Amendment No. if applicable):
II SOP (Number and Revision Number): XI-009-03
D Method (Number and Revision Number):
D GLP (Section):
D Other:
Type of Deviation (check one):
D Facility
H Study (ffll out study info) Study Number: 1078-CG920794
Study Title: Rabbit Multiple dose Anthrax Telemetry Study
Record Affected (describe Title, Binder name, location, Form no. etc.): 1078 PA ELISA binder- CircPA-
007
DateofDeviation(s): 10/12/10
Description of Deviation:
According to SOP XI-009, all information and equipment must be recorded and verified at the time of use.
The technician did not record the equipment used in 10/12/10.
Root Cause of Deviation:
The technician inadvertently missed printing the equipment form for recording equipment used.
Corrective Action:
The technician was reminded to confirm that all necessary paperwork is printed and to review paperwork
thoroughly for accuracy and completion.
(\-\\-vo
Impact of Deviation: Minimal - The permanent equipment used for the assay can be verified per other
equipment use dates; however, the transportable equipment like pipettes cannot be verified.
If deviation is planned, effective date: NA
Deviation Form Prepared by/Date:
^ J
Deviation Reviewed and Corrective Action Accepted by/Date^srudy K«ct9> or Responsible individual):
Deviation Renewed and Corrective Action Accepted by/Date (Sujfg^lsor, Supervisor Representative, or Group Manager):
Circle One: \i\(^mcro^Aol Tox, AerosoJ^Chemistry, BOS^MCB, Facility, QA, Study Management, Other
Deviation Reviewed and Registered by QAU/Date:
z.i
SI Category I
D Category II
(See SOP XI-023 for details)
Form No. Facility-OSS-OS (Revised 12/2/2009) Page 1 of 1
B-13
-------
BATTELiE BIOMEDICAL RESEARC&C1
Deviation No. (Assigned by QAU):^£> i*/ ,,/
CAQ No. (Assigned by QAU): A/A
Ijfard'dr ProcedureflJeviated:
D Protocol (Number and Amendment No. if applicable):
M SOP (Number and Revision Number): XI-009-03
D Method (Number and Revision Number):
D GLP (Section):
D Other:
Type of Deviation (check one):
Facility
Study (fill out study info) Study Number: 1078-CG920794
Study Title: Rabbit Multiple dose Anthrax Telemetry Study
Record Affected (describe Title, Binder name, location, Form no. etc.): 1078 PA ELISA binder; Freeze
thaw paperwork form MREF TNA-025
Date of Deviation(s): 10/4/10,10/5/10,10/7/10, 10/11/10
Description of Deviation:
According to SOP XI-009, all information and equipment, reagents, and sample IDs must be recorded and
x verified at the time of use. uku-n.
/a) The technician recorded the CSU that the samples were pulled from but did not record the refrigerator were*
they were placed to thaw overnight. ~^ff/1( /
b) Some retention samples were used to supplement the PA aliquot for re-analysis. The freeze thaw was
recorded, but it wasn't specified if it was for the PA aliquot or the retention sample. Therefore, the retention
sample freeze thaw could not be verified.
Root Cause of Deviation:
a) The technician inadvertently missed recording the refrigerator information on the freeze thaw forms.
b) The technician recorded on the process sheets that retention samples were also used but did not
include the comment on the freeze thaw forms as well.
Corrective Action:
The technician was asked to review SOP-XI-009 and review all paperwork thoroughly for accuracy and
completion, y^ \\-\\-\Q
Impact of Deviation: Minimal - a) The refrigerator information was recorded on the equipment form and
can be verified that its use was within the calibration timeframe. b) Since the freeze thaw did not specify
whether the sample was from the PA aliquot or retention, the exact freeze thaw for the sample could not be
verified.
If deviation is planned, effective date: NA
Deviation Form Prepared by/Date:
Deviation Reviewed and Corrective Action Accepted by/Date^t^Diresidi or Responsible individual):
Form No. Facility-035-05 (Revised 12/2/2009) Page 1 of 2
-------
1
BATTELLE BIOMEDICALjRESEARCH CEJfTER
^'DEVIATION FORM'
Deviation No. (Assigned by QAU):
CAQ No. (Assigned by QAU):
Deviation Reviewed and Corrective Action Accepted by/Date (Supervisor, Supervisor Representative, or Group Manager):
Circle One: Viyo/VIicro)Mol Tox, Aerosol, Chemistry, BDS, MCB, Facility, QA, Study Management, Other
Deviation Reviewed and Registered by QAU/Date:
0 Category I
D Category II
(See SOP XI-023 for details)
Form No. Facility-OSS-OS (Revised 12/2/2009)
Page 2 of 2
B-15
-------
BATTELLE BIOMEDICAL RESEARCH CENTER
DEVIATION FORM
Deviation No. (Assigned by QAU): £)(L'
CAQ No. (Assigned by QAU):
Standard or Procedure Deviated:
O Protocol (Number and Amendment No. if applicable):
S SOP (Number and Revision Number): X-180-03
D Method (Number and Revision Number):
D GLP (Section):
D Other:
Type of Deviation (check one):
Facility
Study (fill out study info) Study Number: 1078-CG920794
Study Title: Rabbit Multiple Dose Anthrax Telemetry Study
Record Affected (describe Title, Binder name, location, Form no. etc.): 1078 CircPA binder; All 1078
Softmax Pro Results packets
Dateof Deviation(s): 5/4/2010
Description of Deviation: The current PA ELISA SOP X-180 is vague in its guidance for TS, QC, and NC
censoring. The following provides clarification for censoring until the SOP may be appropriately updated:
Change No. #1 to Page 17, BBRC.X-180-03 SOP Acceptance Criteria, make the following change:
Section V.C.5.c.3.i of SOP X-180-03 states that for censoring the reference standard (RS) the following is to
be done, "Up to two full dilution pairs (or four individual dilution points) between standard dilution 1 — 8 (or
"SI" - "S8") of the RS may be censored or 'masked.' Censor the RS as needed to ensure that the PCs meet
the criteria described below."
Instead (intended to replace the underlined portion above), the RS will be censored only on plates in which
two or more of the QC fail, and only if censoring improves the RS curve as judged by the overall percent
recovery values moving closer to 100% rather than further away from 100% recovery (hence, worsening the
RS curve). The RS should not be censored on plates hi which the QCs fail but the RS is generally well
behaved, and censoring only serves to worsen the percent recoveries. Also, the reference standard will not be
manipulated on a plate in which 2 or 3 of the QCs pass the acceptance criteria.
lIl ll l ll l l I l I l l l I lIlllli+++iI I I I I I I I I I l lII+++++I l I I I i I I l I I l lI l l l I l l I I I IlI l I ll l
Change No. #2 to Page 17, BBRC.X-180-03 SOP Acceptance Criteria, make the following changes:
Section V.C.5.c.3.ii of SOP X-180-03 currently states that for censoring the quality controls (QCs) and the
test samples (TSs) the following is to be done (combining the guidance from both sections),".. .the QCs/TSs
may be censored as needed to obtain an intra-assay %CV of < 30%. If the % CV is >30%, dilution points
may be censored one at a time (starting with the back calculated concentration furthest from the mean) until
the % CV is acceptable. It is appropriate to censor to only one dilution point if necessary."
Instead (for the underlined portion above), the QCs and TSs will be censored as follows (these guidelines are
Form No. Facility-035-05 (Revised 12/2/2009) Page 1 of 3
B-16
-------
BATTELLE BIOMEDICAL RESEARCH CENTER
DEVIATION FORM
Deviation No. (Assigned by QAU):
CAQ No. (Assigned by QAU): A/A-
based on the premise that the asymptotes of the RS curve are less reliable for PA concentration interpolation
compared to the central or "linear" portion of the RS curve, as defined by approximately S3 - S6):
1) If a sample demonstrates "high PA binding activity" as evident from high ODs tracking along the upper
asymptote of the RS, then the most reliable data point(s) from which to interpolate the PA concentration
would be starting at the bottom of the sample titration, since the bottom portion of the sample titration
falls closest to the "linear" portion of the RS curve. Begin censoring using the "top-down" approach
(starting with the first sample dilution at 1:1 and progress in order toward the 1:125 dilution). If during
censoring, 2 points from the sample titration fall hi the "linear" portion of the curve have >30% CV, then
the sample is considered non-parallel and a concentration value should not be reported.
2) If a sample demonstrates "low PA binding activity" as evident from low ODs tracking along the lower
asymptote of the RS, then the most reliable data point(s) from which to interpolate the PA concentration
would be starting at the top of the sample titration, since the top portion of the sample titration falls
closest to the "linear" portion of the RS curve. Begin censoring using the "bottom-up" approach (starting
with the last sample dilution at 1:125 and progress hi order toward the 1:1 dilution). If during censoring 2
points from the sample titration fall in the "linear" portion of the curve have >30% CV, then the sample is
considered non-parallel and a concentration value should not be reported.
3) In the event that 2-4 points from the sample titration fall in the "linear" portion of the curve, utilize the
following censoring logic:
a. If 2 points from the sample titration fall in the "linear" portion of the curve have >30% CV, then the
sample is considered non-parallel and a concentration value should not be reported.
b. If the 3-4 points of the sample titration demonstrates a %CV of >30%, and the 3-4 sample titration
points that fall into the linear range of the RS curve, then censor based on the titration point(s) that is
furthest from the mean. In order to have a reportable value, at least two points must be used. If the
CV of the 2 or 3 remaining values is <30%, then report the mean concentration of the 2 or 3 remaining
values.
c. If the ODs of the 2 remaining dilution points are within S3 - S6 and still have >30% CV, then the
sample is considered non-parallel and a concentration value should not be reported.
4) Occasionally a sample titration demonstrates a "hook" either at the high or low end of the titration curve.
a. "High-hooks" are found in PA samples of varying activity where the sample increases in OD signal
between the 1:1 and 1:5 dilution or the 1:1 and 1:25, and then hi turn decreases hi OD signal between
the 1:5 and 1:25 or between 1:25 and 1:125 signal, hi general, the sample dilution ODs must decrease
as diluted. Only dilutions showing this monotonic, downward trend in OD may be used to determine
a reportable value. These types of high-hooks are caused by an un-explained matrix effect inherent to
the individual sample (usually an individual animal) and it is appropriate to censor the "hook effect"
by deleting the ODs from the non-monotonic dilution(s). Starting with the monotonic ODs, the TS
censoring rules described above apply.
Form No. Facility-OSS-OS (Revised 12/2/2009) Page 2 of 3
B-17
-------
BATTELLE BIOMEDICAL RESEARCH CENTER
DEVIATION FORM
Deviation No. (Assigned by QAU): ^, i o&HI
CAQ No. (Assigned by QAU): A/4
b. "Low-hooks" are found in low-activity PA samples where the sample signal is low (meaning, the
lowest dilution of the sample begins in the linear range of the RS and higher dilutions reach the lower
asymptote) in the 1:1 and usually the 1:5 dilution wells, but then aberrantly jumps in the 1:25 or the
1:125 dilution wells. The high dilution factors then inflate the erroneous signal to cause a large
concentration to be reported. These types of low-hooks are caused by an un-explained noise effect
likely due to a plate effect (improper washing by the automatic plate washer, cross contamination of
wells, etc) and it is appropriate to censor the "hook effect" by deleting the ODs from the affected
dilution (usually the 1:25 or 1:125, but occasionally it is the 1:5). As stated above, the sample dilution
ODs must decrease as diluted. Only dilutions showing this monotonic, downward trend in OD may
be used to determine a reportable value. After censoring the affected well, the TS censoring rules
described above apply. In addition, low hooks may also affect the negative control sample. If a
negative control sample displays OD values after the 1:1 dilution that are non-monotonic and result in
a back-calculated concentration from the RS, they may be censored as per the rules described above.
Change No. #3 to Page 17, BBRC.X-180-03 SOP Acceptance Criteria, make the following addition:
Occasionally a precipitant is observed hi the wells after the addition of ABTS. This cause of the precipitant
(observed to also look like filaments) is not known and is currently under investigation. If the precipitant is
observed in specific wells, it will be noted and the test operator may need to in turn censor the OD values
from those wells.
Root Cause of Deviation: The censoring specifications for the RS described in SOP X-180-03 are somewhat
vague and require clarification to ensure that censoring is performed properly, consistently, and hi the spirit of
good scientific judgment.
Corrective Action: SOP BBRC X-180 has been updated with the guidances outlined hi this deviation.
Impact of Deviation: These specifications improve the fidelity and consistency of the results reported by the
assay.
If deviation is planned, effective date:
Deviation Form Prepared by/Date:
Deviation Reviewed and Corrective Action Accepted oyfE^ate <$&& DirectorroRe^ponsibie individual):
-------
Deviation No. (Assigned by QAU): DR-11043
CAQ No. (Assigned by QAU):
rWbr Proure Deviated:'?%
D Protocol (Number and Amendment No. if applicable):
0 SOP (Number and Revision Number): XI-009-03
D Method (Number and Revision Number):
D GLP (Section):
D Other:
..-.'.'• '' |. "'-'\^ ' • Type of Deviation
-------
CENTER
Deviation No. (Assigned by QAU): DR-11043
CAQ No. (Assigned by QAU): IS/4
Corrective Action:
a. The staff member is no longer employed at this facility - no corrective action can be taken.
b. The staff member has been reminded that the "Data Analyzed By/Date" section is transcribable and
can therefore be pre-typed, while the signature for form completion is raw data that must be signed the
date the work is performed.
c. The staff member was advised to review SOP XI-009 and sign for all work at the time of completion.
d. The technician was advised to record all information promptly at the time work is completed. New
implementation of the sample tracking system is under development to assist in accurately tracking
freeze thaw cycles of sample and critical reagents.
Impact of Deviation:
a. Minimal. The date of printing is captured on the records as 8/21/10, and Form No. ELIS A-036
indicates which technician conducted the data analysis.
b. Minimal. Based on the date of printing, data were analyzed on 8/27/10. Furthermore, all reportable
values captured on Microbio-357 can be transcribed and verified: no reportable data are affected.
c. Minimal. Although the date and operator cannot be confirmed, it has been verified that data have
been imported into the database.
d. Minimal. Based on the information in the Sample Tracking System, it is most likely that the F/T
cycles should be: BMI032 = F/T 1, BMI012 = F/T 2, BMI009 = F/T 3, which is within the permissible
number of F/T cycles for ELISA samples.
If deviation is planned, effective date: n/a
Deviation Form Prepared by/Date:
r J
Deviation Reviewed and Corrective Action Accepted by/Date (study Director or Responsible individual):
Deviation Reviewed and Corrective Action Accepted^y^DateS^u'pervisor, Supervisor Repre,senta/// D Category II
(See SOP XI-023 for details)
Form No. Facility-OSS-OS (Revised 12/2/2009) Page 2 of 2
B-20
-------
ifMTELLE BIOMEDICAL RjESE^CH CENTER
:Si,- . : DELATION jfetlM :\.
Deviation No. (Assigned by QAU): DR-11044
CAQ No. (Assigned by QAU):
Standard
D Protocol (Number and Amendment No. if applicable):
0 SOP (Number and Revision Number): X-101-08
D Method (Number and Revision Number):
D GLP (Section):
D Other:
i':;'=i .- .- ;:-l%;,, '^I^:J. -?' I W;«,.^P^^PeA1|l^<)n (check,<>f&): ".\
D Facility
El Study (fill out study info) Study Number: 1078-CG920794
Study Title: Rabbit Multiple Dose Anthrax Telemetry Study
Record Affected (describe Title, Binder name, location, Form no. etc.): a-PA IgG ELISAIR-419 Data
Binder
a. Form No. Microbio-447, "ELISA Sample (S-MP, C-MP, T-MP) Preparation form," For Lot No.
081310-JNG.
b. Form No. Microbio-447, "ELISA Sample (S-MP, C-MP, T-MP) Preparation form," For Lot No.
081310-GEM.
Date of Deviation(s): 8/13/2010
Description of Deviation: SOP X-101-08 section a. regarding Reference Standard Master Box (S-MP)
preparation states: "Refer to Form No. ELISA-008 for directions on how to dilute a specific lot of species-
specific reference sera. For each S-MP prepare an appropriate volume of 2X concentrated reference standard
using MP diluent according to Form No. ELISA-008 for the specific lot being tested."
Form No. ELISA-008 specifies a starting plate dilution of 1:530 for BMI-009, the reference standard used for
the assays in question. A 2X concentration would therefore require a dilution of 1:265. For the Batch Lot
No. above, the primary dilution of the reference standard was conducted by transferring 4pL of neat sera into
1054uL of diluent - the total volume was listed as 1060(oL even though the sum of the volumes is 1058jiL.
The actual primary dilution prepared was 1:264.5 for a final plate dilution of 1:529.
Root Cause of Deviation: A math error specifying 1054(iL instead of 1056|±L resulted in the slight variation
in dilutional concentrations.
Corrective Action: Staff members were instructed to review all pre-typed information and calculations prior
to performing study tasks. Because there is less than a 0.2% difference between dilution factors of 529 and
530, no additional action will be taken.
Impact of Deviation: Minimal, there is less than a 0.2% difference between the actual dilution factor and that
specified in ELISA-008. No data are significantly affected.
If deviation is planned, effective date: n/a
Deviation Form Prepared by/Date:
Form No. Facility-035-05 (Revised 12/2/2009)
Page 1 of 2
B-21
-------
»;|Syf:. • • : ;
Deviation No. (Assigned
Deviation Reviewed and
Deviation Reviewed and
Circle One: Vivo. Micro. Mol Tox. A
Deviation Reviewed and
BATTELtE;BIOMEDICAL RlSE A&Efi CEftPER
.„. . ^ ;;fi^ATIONroRM;|l:\ . flp- I
byQAU):DR-11044
' '• ''•''-/,'",'',''/, ''fy&}\^'f-^f-i( Tft. 5"\u\tt
Corrective Action Accepted by/DaJertsupervisor, Supervisor Representative, or Group Manager):
erosol, Chemistry, BDS, MCB, Facility, QA, Study Management, Other \ t . * f-f I /
?W *falu
Registered by QAU/Date:
M ^"
El Category I
D Category II
(See SOP XI-023 for details)
Form No. Facility-OSS-OS (Revised 12/2/2009)
Page 2 Of 2
B-22
-------
BATTELLE BIOMEDICAL RESEARCH CENTER
INVESTIGATION REPORT FORM
STUDY # 1078-CG920794 IR# 419
Describe problem and reason for investigation: When using either new rPA lot, 17115A2A or 5051797, for
coating there were inconsistencies with the binding of the currently qualified conjugate 05814. This was seen
in preliminary analyses for study 1078 and it caused many failures with study plates. An investigation was
undertaken to determine the most likely cause of the inconsistencies leading to failures and determine a
correction course of action to take.
Check applicable boxes:
0 Verified instrument/equipment setup and conditions. 0 Verified instrument/equipment calibration.
0 Performed instrument/equipment check. 0 Verified analysis/operation procedures.
0 Verified sample/reagent/standard expiration date, 0 Verified calculations.
storage and preparation. 0 Verified all other operation specific requirements
Q Notified Sponsor (if applicable) 0 Verified operator training
Describe additional investigation:
First, we compared the old qualified conjugate 03068A and the new qualified conjugate 05814 on split plates
coated with 5051797 on one half and 17115A2A on the other half. This was to test which rPA works best.
• This test plate layout was performed on 8/13/10 by GEM (plate 081210-617) & JNG (plate 081210-
618). After looking at the results the study director determined that rPA 17115A2A should be used.
Second, we tested conjugate 03068A and 05814 at their approved dilutions and also tested 05814 at a higher
dilution on plates coated with rPA 17115A2A. This was performed to determine the performance of the
conjugates.
• This testing occurred on plates 081510-621 to -623 performed by KAS. All plates passed and
conjugate seemed to bind well for each lot and both dilutions of 05814. The study director wanted to
further test the effect of more dilutions.
Third, we tested 05814 at several dilutions using 03068A on the RS & QC's. This test was performed to get
further data on the performance of the conjugates and determine if the conjugate 05814 needed to be re-
qualified.
• These tests were performed on plates 081610-648 to -651 by KAS and plates 081910-654 to -657 by
AH. The results showed no significant applicable improvement by increasing the dilution used. It
was noted that ODs were closer to their expected values on plates that were more recently coated.
Fourth, we tested the possibility of plate age being a factor as we saw a better performance on plates with a
shorter incubation period in the testing result from the third experiment.
• The shorter room temperature incubated plates, 082510-666 to -667, were used by GEM. Two other
plates were coated overnight, 082510-668 to -669, and were run by KGM. Results did not show a
significant difference due to plate age.
Lastly, we tested a direct comparison between a plate with 03068A and 05814. This was to decide which
conjugate performs better and then to utilize that for 1078 study work.
• KGM compared conjugates on plates 082610-673 & -672 as did GZ on plates 082610-670 & -671.
Study director decided that conjugate 03068A would be best for use on study 1078 and study work
continued with that conjugate as a result.
Describe cause (if determined) and proposed action to be taken/recommendations:
Form No. MREF QAU-021-02 (Revised 11/01/06) Page 1 of 2 B-23
-------
BATTELLE BIOMEDICAL RESEARCH CENTER
INVESTIGATION REPORT FORM
STUDY # 1078-CG920794 IR# 419
It was detennined the conjugate 05814 had degraded and was as a result, inconsistently binding. Proposed
action was to use a different conjugate lot.
Completed by/date: *~d //-¥-/# _ Reviewed and accepted by/date: Tfc
QAU Assigned IR # by/date J(3/?/l/,||D -u~5fzs|d CAQ # Issued: MA
Describe action taken:
Action taken was to switch to the older qualified conjugate 03 068 A and to begin qualifying a new conjugate.
Describe measures taken to prevent recurrence:
A suggestion was made that when it has been greater than 6 months since reagents have been used that we
run a test plate of those reagents in order to determine that they are still functioning properly prior to starting
study sample analyses.
Completed by/date: ^~^l(-^-JC> _ Reviewed and accepted by/date: T(I 5llb(n
QAU Registration by/date:
Form No. MREF QAU-021-02 (Revised 11/01/06) Page 2 of 2 B-24
-------
APPENDIX C
BORDETELLA RESULTS
C-1
-------
Printed: Wednesday, August 11, 2010 at 9:49
Charles River Research Animal Diagnostic Services
251 Ballardvale Street, Wilmington, MA 01887 USA
Tel: 800-338-9680 Fax: 978-658-7698
Bacteriology Results Report
Sponsor: Battelle Accession #: 2010-035209
M-8 Received: 28 M 2010
OSKingAve Department: Approved by Richard D. Fister, 03 Aug 2010, 08:29*
:olumbus, OH 43201
JSA
ittn: Jason Comer Bill Method: Credit Card
'el: 614-424-5825 Test Specimen: Nasal Rabbit, Lot V100261001261
Bordetella Screen - Respiratory
Sample #:
Code :
B. bronchiseptica
Other
1234567
40 7 5 9 37 13 34
.
-
8 9 10
25 15 30
.
-
Sample #:
Code :
B. bronchiseptica
Other
11 12 13 14 15 16 17
28 19 14 11 2 8 12
.
-
Sample #:
Code:
B. bronchiseptica
Other
21 22 23 24 25 26
33 27 31 39 21 38
.
-
18 19 20
18 32 6
.
-
Remarks: - = Negative/No Growth; 1 = Rare/Few Colonies; 2 = Several Colonies; 3 = Moderate Growth; 4 = Heavy Growth;
NI = Not Interpreted: culture could not be interpreted due to overgrowth of Proteus; NT = Not Tested.
*This report has been electronically signed by laboratory personnel. The name of the individual who approved these results appears in the header of
this service report. All services are performed in accordance with and subject to General Terms and Conditions of Sale found in the Charles River
Laboratories-Research Models and Services catalogue and on the back of invoices.
CR ILIMS Form: FM-703 Rev 2
Page 1 of 1
C-2
-------
APPENDIX D
RANDOMIZATION REPORT
D-1
-------
Balfelle
I. he Business of Innovation
Dale August 9, 2010
TO Jason Comer
From Heather Mayfield
Subject Study No. 1078-CG920794: Animal
Randomization Report and Supporting
Documentation
Project Number £G9207J41107^STATI3104i
Internal Distribution
RA Lordo/SIA Files (Judd)
NA Niemuth
GV Stark
HJ Mayfield
RMO
s:\BBRC\Anthrax\EPA\Study 1078-CG920?94\
Study 1078 Animal Randomization & Sup Doc.doc
Overview
This memorandum describes the animal randomization process and provides the supporting
documentation for BBRC Study No. 1078-CG920794, The SAS® system (version 9.1.3) was
used to transfer, process, and analyze the data.
Methods
Twenty-six (26) New Zealand White rabbits were available to be assigned to the study. Animals
were randomly assigned to one of four groups (3 groups of 7 and 1 group of 5) by weight
utilizing the SAS* PLAN procedure. Next, animals were assigned a random challenge order
within each study group utilizing random numbers generated by the SAS® RANUNI function.
Data Inputs/Outputs
The Excel spreadsheet 1078 IDS AND WEIGHTS FOR RANDO.XLSX, containing the animal
ID and weight for the animals available to be placed on study, was sent to SIA statisticians on
July 19, 2010, by Jason Comer. The data were read in from the Excel spreadsheet with the SAS®
program ANIMALRAND_1078_CG920794_071910.SAS used for the randomization. The
allocation of animals to groups and challenge order was output by SAS® to create the Excel
spreadsheet RANDOMIZATION_1078_071910.XLS.
Performance Test Methods/Results
A copy of the Excel 1078 IDS AND WEIGHTS FOR RANDO.XLSX and
RANDOMIZATION_1078_071910.XLS, the SAS® program ANIMALRAND_1078_
CG920794_071910.SAS, and the SAS® are The correct transfer of data was
verified by printing out the 1078 IDS AND WEIGHTS FOR RANDO.XLSX
and comparing it to a listing of the in SAS*. The randomization was conducted with the
SAS* PLAN procedure, which not require performance testing.
[signatures on next page]
D-2
-------
Memorandum to: Jason Comer
Subject: Study No. 1078-CG920794: Animal Randomization Report and
Supporting Documentation
August 9, 2010
Page 2
RESPONSIBLE INDIVIDUAL
RESPONSIBLEpMVimJAL
INDEPENDENT REVIEWER
HJMrbhf
Attachments
For Review and Approval
Originator
Concurrence
Approved
Name
Heather Mayfield
Greg Stark
Nancy Niemuth
Initials
L-f/Y^
JiLM4x--^
~A/
Date
£/
-------
Memorandum to: Jason Comer
Subject: Study No, 1078-CG920794: Animal Randomization Report and
Supporting Documentation
August 9, 2010
Page 3
SUPPORTING DOCUMENTATION FOR
Study No. 1078-CG920794
ANIMAL RANDOMIZATION REPORT AND
SUPPORTING DOCUMENTATION
D-4
-------
Study No. 1Q78-C692Q794
Rabbit ID
2
5
6
7
8
9
11
12
13
14
15
18 /
19
21
25
27
28
30
31
32
33
34
37
38
39
40
Body weight (kg)
-------
Study No, 1078-CG920794
Program Name: Ani«talRand_1Q78_CG920794_Q?1910.SAS
Purpose: Randomize animals into treatment groups,
Project: 1Q78-CG920794 , /
Charge Number (s): CG920794-1078STAT //C7/M
Date Created: 06/29/10
Input Data:
Output Data: Listing of randomization scheme,
Other Files Used:
Comments; 26 rabbits will be randomized into three groups of seven and one control group of
five
Jason Comer's 7/19/10 email ". Please note that we lost several animals due to
complications with surgery and only have 26. There will be no extras, "
Rabbits in each groups will be assigned a random challenge order.
Author: Heather Mayfield
%let randloc=C:\Documents and Settings\mayfieldh\My Documents\BBRC\Proc Plan\Anthrax\EPA\1078-
CG920794;
libname randan "Srandloc.";
titlel 'Study Number 1078-CG9207941;
%let stnum=1078;
%let date=071910;
options LS=120 PS=74 pageno=1;
proc printto print="&randloc.\AniroalRandomizaticm_&stnum._Freq_&date..1st" new;
run;
*** read animal data ***;
libname ex1 odbc noprompt="DSN=Excel Files;
DBQ=&randloc.\1078 IDs and Weights for rando.xlsx";
Data animal&stnum._A;
set EX1."Sheet1$A1:B27"N;
run;
data randan.animal_&stnum._&date.;
set animal&stnum._A ;
run;
proc print data=randan.aninalJlstnuB._&date.;
title2'Animal Data';
run;
title2;
proc sort data=randan.animal_SstnuB._&date. out=aninals;
by Body_weight kg_;
run;
data middle other;
set animals;
if 10<_n_
-------
Study No. 1078-CG920794
*0ther has light, medium-light, medium heavy, and heavy weight blocks, ;
data unrand;
do block=l to 4;
do cell=i to 5;
if cell=1 then Group=l;
else group=,;
output;
end;
end;
run;
*********** Randomize to treatment groups********
title2 'Animals Randomized to Groups';
proc plan ;*seed ;
factors blook=4 ordered cell=5;
output data =unrand out = random;
run;
quit;
title2;
proc sort data=random;
by block cell;
run;
data randomother(drop=block cell);
merge other random;*merge w/o by correct here;
run;
data unrand2;
do cell=1 to 6;
if cell=1 then Group=1;
else group=.;
output;
end;
run;
*********** Randomize to treatment groups**********
title2 'Animals Randomized to Groups';
proc plan ;*seed ;
factors cell=6;
output data =unrand2 out = rando«2;
run;
quit;
titlea;
proc sort data=rando«2;
by cell;
run;
randomitiiddle{drop= cell);
Middle random2;*merge w/o by correct here;
run;
groupl group234;
randonother;
if group=1 output groupl;
D-7
-------
Study No. 1078-CG920794
else output group234;
run;
proc sort data=group234;
by Body_weigtit k
run;
data unrandS;
do block=1 to 7;
do cell=1 to 3;
group=cell+1;
output;
end;
end;
run;
*********** Randomize to treatment groups******************;
title2 'Animals Randomized to Groups';
proc plan ;*seed ;
factors block=7 ordered cell=3;
output data =unrand3 out = randoms;
run;
quit;
title2;
proc sort data=random3;
by block cell;
run;
data randomgroup234(drop=block cell);
merge group234 randoms;*merge w/o by correct here;
run;
data animal_randomization&stnum._&date.;
set group! randomgroup234;
chaldet=ranuni(-1);
run;
proc sort data=animal_randomization&stnum._&date,;
by group chaldet;
run;
data randan.animal_randofflization&stnum._&date.(drop=chaldet);
set animal_randomization&stnum._&date.;
retain Challenge_0rder;
by group chaldet;
if first.group then Challenge_0rder=1;
else Challenge_Order=Challenge_Order+1;
run;
titles 'Frequency of Animals In Groups';
proc freq data=randan.animal_randomization&stnum._&date.;
table group group*Challenge_Order;
run;
title2;
proc sort data=randan.animal_randomization&stnum.__&date. out=randbygroup;
D-8
-------
Study No. 1078-CG920794
by group Challenge_0rder;
run;
libname libout odbc nopro«pt="DSN=Excel Files;
DBQ=&randloc. \Randomization_&stnuin._&date..xls"
PRESERVE_COL_NAMES=yes;
data libout."By Group Order'n;
set randbygroup;
by group Challenge_0rder;
run;
libname libout clear;
proc glut data=randbygroup;
class group;
model Body_weight kg_=group;
run;
Proc printto;
run;
D-9
-------
Study Number 1078-CQ920794
Animal Data
13:35 Monday, July 19, 2010 1
Body_
Obs
1
2
3
4
5
8
7
8
9
10
11
12
13
14
15
18
17
18
19
20
21
22
23
24
25
26
Rabbit_
ID
2 »X"
S
8
7
8 W
9
11
12
13
14
15
18 ^
19
21
25 t/"
27
28
30
31
32
33
34
37
38
39
40
weight
2
2
2
2
2
2
2
2
2
2
2
Z
2
2
2.
2.
2.
2.
2.
3,
2.
2.
2.
2.
2.
3.
k8^
,514 \/
,819
,910
,75?
.891»/
.880
.575
,819
.688
.718
.873
.707 £/
.554
•842
.917 /
.695
.808
.757
887
025
532
707
411
859
774
013
t\
I
D-10
-------
Study ttustber 1078-C6920794 13:31 Monday, July 19, 2010
Animals Randomized to Groups
Factor
block
eeil
The PUW
Select
4
S
4 Procedure
Levels
4
5
Order
Ordered
Random
block
1 1 S 2 4 3
2 23451
3 S 3 1 2 4
4 45321
D-11
-------
Study Nunber 1078-C6920794 13:31 Monday, July 19, 2010 3
Animals Randomized to Groups
The PLAN Procedure
Factor Select Levels Order
cell 6 6 RandOfR
cell---
3 8 1 2 4 S
D-12
-------
Study Number 1078-00920794 13:31 Monday, July 19, 2010 4
Animals Randomized to Groups
The PLAN Procedure
Factor Select Levels Order
block 7 "I Ordered
sell 3 3 Random
block
1 312
2 312
3 231
4 213
5 123
8 231
7 231
D-13
-------
Study Number 1078-CG920794
Frequency of Animals in Groups
The FREO Procedure
Cumulative Cumulative
Group Frequency Percent Frequency Percent
13:31 Monday, July 19, 2010
1
2
3
4
S
7
7
7
19.23
26.92
28.92
28.92
6
12
19
26
19.23
46 . 1 5
73.08
100.00
Table of Group by Challenge_0rder
Group Challenge_0rder
Frequency[
Percent
Row Pet
Col Pot
1
2
3
4
Total
1
1
3.85
20.00
25.00
1
3. 85
14.29
25.00
1
3.85
14.29
25.00
1
3.85
14.29
25.00
4
15.38
2
1
3.85
20.00
25.00
1
3.85
14.29
25.00
1
3.85
14.29
2S.OO
1
3.85
14.29
25.00
4
15.38
3
!
3. 85
20.00
25.00
1
3.85
14.29
25.00
1
3.85
14.29
25.00
1
3.85
14.29
25.00
4
15.38
4
1
3.85
20.00
25.00
1
3.85
14.29
25.00
1
3.85
14.29
25.00
1
3.85
14.29
25.00
4
15.38
5
1
3.85
20.00
25.00
1
3.85
14.29
25.00
1
3.85
14.29
25.00
1
3.85
14.29
25.00
4
15.38
6
0
0.00
0.00
0.00
1
3.85
14.29
33.33
1
3.85
14.29
33.33
1
3.85
14.29
33.33
3
11.54
?
0
0.00
0.00
0.00
1
3.85
14.29
33.33
1
3.85
14.29
33.33
1
3.85
14.29
33.33
3
11 .54
Total
5
19.23
7
26.92
7
26.92
7
26.92
26
100.00
D-14
-------
Study Nuniber 1078-CG920794
The SIM Procedure
Class LevttI Information
Class Levels Values
Group 4 1234
13:31 Monday, July 19, 2010 8
Nombgr of Observations Read 26
Number of Observations Used 26
D-15
-------
Study Nuntaer 1078-CG920794
The GLM Procedure
Dependent Variable: Body_weight kg_ Body weight {kg!
13:3! Monday, July 19, 2010 7
Source
Modal
Error
Corrected Total
DF
3
22
as
Sura of
Squares
0.00118497
0.826188S7
0.82735154
Source
Group
Source
Group
Mean Square F Value Pr > F
0.00039499 0.01 0.9977
0.02846212
R-Scjuare Coeff Var Root USE Body_weight kg_ Mean
0.001889 6.145293 0.168707 2,745308
DF Type I SS Mean Square F Value Pr > f
3 0.00118497 0.00038499 0.01 0.9977
OF Type III SS Mean Square F Value Pr -• F
3 0.00118497 0.00039499 0.01 ' 0.997/
*1
D-16
-------
Study No. 1078-CG920794
RabbitJD Body_weight kg_ Group ChallengejDrder
1
2
3
4
5
1
2
3
4
5
6
7
1
2
3
4
5
6
1
1
2
3
4
5
6
JD Body weight
"40
7
5
9
37
13
34
25
15
30
28
19
14
11
2
8
12
18
32
6
33
27
31
39
21
38
_kg_ Group
3.013
2.757
2.619
2.88
2.411
2.656
2.707
2.917
2.873
2.757
2.806
2.554
2.718
2.575
2.514
2.891
2.819
2.707
3.025
2.91
2.532
2.595
2.667
2.774
2.842
2.859
1
1
1
1
1
2
2
2
2
2
2
2
3
3
3
3
3
3
3
4
4
4
4
4
4
4
/
D-17
-------
APPENDIX E
AEROSOL REPORT
1078-CG920794 Aerosol Report E-l
-------
Table of Contents
1. Methods E-3
2. Experimental Setup and Test Conditions E-6
2.1 Aerosol Generation Subsystem E-6
2.2 Delivery Subsystem E-6
2.3 Exposure Chamber E-7
2.4 Sampling/Monitoring E-7
2.5 Plethysmography E-8
3. Inhalation Results E-9
3.1 Impinger Sample Analysis E-9
3.2 Inhaled Dose Calculation E-9
4. Particle Size Results E-10
5. References E-40
List of Tables
Table 1. Aerosol Data Summary Sheet (Day 1) E-14
Table 2. Aerosol Data Summary Sheet (Day 2) E-14
Table 3. Aerosol Data Summary Sheet (Day 3) E-15
Table 4. Aerosol Data Summary Sheet (Day 4) E-15
Table 5. Aerosol Data Summary Sheet (Day 5) E-16
Table 6. Aerosol Data Summary Sheet (Day 6) E-16
Table 7. Aerosol Data Summary Sheet (Day 7) E-17
Table 8. Aerosol Data Summary Sheet (Day 8) E-17
Table 9. Aerosol Data Summary Sheet (Day 9) E-18
Table 10. Aerosol Data Summary Sheet (Day 10) E-18
Table 11. Aerosol Data Summary Sheet (Day 11) E-19
Table 12. Aerosol Data Summary Sheet (Day 12) E-19
Table 13. Aerosol Data Summary Sheet (Day 13) E-20
Table 14. Aerosol Data Summary Sheet (Day 14) E-20
Table 15. Aerosol Data Summary Sheet (Day 15) E-21
Table 16. Aerosol Data Summary Sheet (Day 1) E-24
1078-CG920794 Aerosol Report E-2
-------
Table 17. Aerosol Data Summary Sheet (Day 2) E-25
Table 18. Aerosol Data Summary Sheet (Day 3) E-26
Table 19. Aerosol Data Summary Sheet (Day 4) E-27
Table 20. Aerosol Data Summary Sheet (Day 5) E-28
Table 21. Aerosol Data Summary Sheet (Day 6) E-29
Table 22. Aerosol Data Summary Sheet (Day 7) E-30
Table 23. Aerosol Data Summary Sheet (Day 8) E-31
Table 24. Aerosol Data Summary Sheet (Day 9) E-32
Table 25. Aerosol Data Summary Sheet (Day 10) E-33
Table 26. Aerosol Data Summary Sheet (Day 11) E-34
Table 27. Aerosol Data Summary Sheet (Day 12) E-35
Table 28. Aerosol Data Summary Sheet (Day 13) E-36
Table 29. Aerosol Data Summary Sheet (Day 14) E-37
Table 30. Aerosol Data Summary Sheet (Day 15) E-38
List of Figures
Figure 1. Exposure system diagram E-12
Figure 2. Log probability size distribution plot E-22
Figure 3. Log probability size distribution plot E-39
1078-CG920794 Aerosol Report E-3
-------
List of Acronyms
APS aerodynamic particle sizer
BBRC Battelle Biomedical Research Center
BSC III Class III biological safety cabinet
C impinger concentration
cm centimeter
CPU colony forming units
D dilution factor
d diameter
GSD geometric standard deviation
HEPA high efficiency particulate air
InD total inhaled dose
L liter
LDso Value median lethal dose equivalent
MFC mass flow controller
MFM mass flow meter
min minute
mL milliliter
MMAD mass median aerodynamic diameter
SOP standard operating procedure
T exposure time
TATV total accumulated tidal volume
TSA tryptic soy agar
|j.m micrometer
V impinger sampler volume
1078-CG920794 Aerosol Report E-4
-------
1. Methods
Standard operating procedures (SOPs) were followed during animal aerosol exposure challenges.
Aerosol procedures are described in SOP BBRC Number XIII-001. Procedures for using the
Aerodynamic Particle Sizer® (APS) are described in SOP BBRC Number XIII-011. The
procedures for operating the plethysmography system are described in SOP BBRC Numbers
XIII-008 and XIII-009. A schematic of the exposure system is shown in Figure 1.
1078-CG920794 Aerosol Report E-5
-------
2. Experimental Setup and Test Conditions
The Battelle large animal exposure system can be divided into four subsystems plus an auxiliary
plethysmography subsystem. A description of each subsystem is found below.
2.1 Aerosol Generation Subsystem
For this study both large animal systems were used, one for the non-viable spores and the other
for the viable spores. For all intents and purposes these systems were operated identically with
the same operation ranges for both. Air was supplied to the systems by an in-house air system
filtered through two high efficiency particulate air (HEPA) capsule filters and a carbon filter.
The air was split into dilution airflow of approximately 8.6 L/min and a nebulizer bypass airflow
of 7.5 L/min, maintained by mass flow controllers (MFC). The dilution air was humidified via a
bubbler as needed to maintain humidity within a range of 53% to 83% for the non-viable spore
system and 56 to 80% for the viable spore system. A modified Microbiological Research
Establishment type three-jet Collison nebulizer (BGI, Waltham, MA) with a precious fluid jar
was used to generate a controlled delivery of aerosolized non-viable B. anthracis spores, (spore
lot Ames B36 irradiated prior to challenge days) or viable B. anthracis spores, (spore lot Ames
B36). These nebulizers are designed to generate aerosols having an approximate mean diameter
of 1-2 micrometer (|im). Each nebulizer was characterized for a pressure that results in an
approximately 7.5 L/min flow, which normally is approximately 25 to 36 pounds per square
inch, Collison nebulizer dependant.
2.2 Delivery Subsystem
After the agent aerosol was generated by the Collison nebulizer, it exited the Collison and
traveled down a 3.75 cm diameter, 40 cm long cylinder (mixing tube) that mixed and dried the
aerosol with dilution air. The aerosol then entered the top of the exposure chamber through
another cylinder with a tapered 14 cm long slit on each side. The total airflow entering the
exposure chamber was approximately 16 L/min. The aerosol entered the chamber through these
slits to fill the exposure chamber, washed over the exposure target (muzzle or head), and was
then exhausted out of the exposure chamber through another cylinder at the bottom that
contained slots on two sides, each 19.5 cm in length. The aerosol was pulled through the
chamber using a vacuum pump that maintained a slight negative pressure (from -0.2 to -0.01
1078-CG920794 Aerosol Report E-6
-------
inches of water) within the exposure chamber, as measured using a differential pressure gauge
(magnehelic). The exhaust aerosol was filtered by two HEPA cartridge filters before exiting the
system. Both systems used for this study were built to the same specifications.
2.3 Exposure Chamber
The exposure chamber was a plexiglass box with internal dimensions of approximately 20.5 x
20.5 x 40 cm (Length x Width x Height). A port approximately 15 cm in diameter was located on
one side of the chamber where an animal's head or muzzle entered into the exposure chamber.
Rubber dental dam was stretched across the opening and held in place with an o-ring gasket. The
animal's head or muzzle was pushed through a small hole in the dental dam, producing a seal to
decrease leakage around the opening. Four additional ports are located in the chamber; two ports
for collection of aerosol samples (one for enumeration and one for aerosol particle sizing), one
port to measure temperature and humidity, and one port to measure the differential pressure
within the exposure chamber in relation to the surrounding atmosphere within the Class III
biological safety cabinet (BSC III). Thus, the sampling from the impinger and APS spectrometer
and exposure of the animal all occur from the same chamber. The aerosol system was operated
within a self-contained BSC III.
2.4 Sampling/Monitoring
Aerosol concentration and aerosol particle size distribution were determined by analysis of
atmospheric samples drawn from the exposure chamber. The atmospheric samples were
collected in an impinger (Model 7541, Ace Glass Inc.) filled with approximately 20 mL of sterile
water that sampled at approximately 6.0 ±0.3 L/min. The sampling rate was achieved by
maintaining a vacuum of >18 inches Hg across the exhaust connection of the impinger to
maintain the flow from the impinger critical orifice. The liquid in the impinger was diluted and
enumerated by the spread plate technique to quantify viable spore counts per mL. Concentrations
are reported in terms of colony forming units per mL (CFU/mL). Enumeration results, along with
the volume of liquid in the impinger, sampling rate, and sampling duration, are used in the
calculation of the aerosol concentration expressed as CFU/L of air.
The aerosol particle size was determined during each exposure using an APS spectrometer,
which draws an atmospheric sample from the exposure chamber at 0.25 L/min with a diluter (1.0
1078-CG920794 Aerosol Report E-7
-------
L/min total with 0.75 L/min. from the diluter and 0.25 L/min. from the exposure chamber). An
APS was used because of its advantages over other methods. These advantages include near real-
time data measurements, aerodynamic diameter measurements, ease of instrument operation, and
the generation of electronic data that is easy to process and export to a report.
2.5 Plethysmography
Body plethysmography was performed real-time on each animal during agent challenge to
measure important respiratory parameters. These parameters (tidal volume, total accumulated
Tidal volume, and minute volume) were calculated from the measured volumetric displacement
of air caused by the movement of the thoracic cavity of an animal while it was in a sealed
chamber called a plethysmograph. The data generated for each animal was used to determine the
total accumulated tidal volume (TATV), which along with the aerosol concentration was used in
calculating the inhaled dose. During the rabbit exposure, the anesthetized animal was placed in
dorsal recumbence in a custom-made plexiglass plethysmograph with the head protruding out of
a port that was sealed with rubber dental dam and held into place with two plexiglass guillotines.
The plethysmograph was connected to a pneumotach (Hans Rudolph, Inc., Kansas City, MO)
that was attached to a differential pressure transducer (Model DP-45; Validyne Engineering
Corp., North Ridge, CA). Pressure differential measurements from inhalations and exhalations
were transmitted to Biosystems XA version 1.5.7 software (Biosystems XA, Buxco Electronics,
Sharon, CT) which then calculated and recorded respiratory function. Prior to animal exposures,
the plethysmography was calibrated to establish unit (baseline) and air volume displacements
from 5 to 40 mL to simulate animal respiration. This calibration was performed to encompass the
respiration volume range of the animal model for accurate TATV measurements.
1078-CG920794 Aerosol Report E-8
-------
3. Inhalation Results
The inhalation exposure system data for each exposure was documented on appropriate forms to
ensure proper system operation and to provide the needed information to quantify animal
challenge conditions. Impinger sampling conditions and enumerated concentration results
provided viable bioaerosol challenge concentration while plethysmography measurements
documented the total inhaled volume. Total inhaled dose (CPU) was calculated from aerosol
concentration and total inhaled volume. The number of median lethal dose equivalents (LDso
value) was calculated by dividing the total inhaled dose by the reported inhalation LDso for each
particular species of animal. The reported LDso for rabbits is 105,000 CPU, (Zaucha, et. al.1998).
Tables 1 through 30 show the inhalation results for this study.
3.1 Impinger Sample Analysis
Impinger samples were enumerated by the spread plate method, SOP BBRC X-054 following
serial dilutions to determine viable spore concentration. Diluted samples were mixed in a capped
vial prior to subsequent dilutions. At different target dilutions, 0.1 mL was spread onto each of
five TSA plates, which were placed in a secondary container and incubated at the appropriate
temperature for the appropriate time. After the incubation period, the plates were enumerated to
determine the number of colonies on each plate. Impinger sample concentration was determined
using the equation below:
C = (A-D)/0.1mL (1)
C = CFU/mL
A = average CPU per plate
D = dilution factor
3.2 Inhaled Dose Calculation
The total inhaled dose (InD) was calculated from the impinger sample concentration, sampling
parameters, and exposure duration according to the equation below. The total number of viable
spores captured during each exposure was the product of the impinger concentration and the
impinger volume (C x V). The total number of viable organisms was divided by the amount that
was sampled through the impinger during the exposure time (S x T). The aerosol concentration
1078-CG920794 Aerosol Report E-9
-------
was (C x V) (S x T)"1. The inhaled dose was the product of the aerosol concentration multiplied
by the total accumulated tidal volume:
InD = (C x V) (S x TX^TATV) (2)
InD = Total inhaled dose (CPU)
C = Impinger concentration (CFU/mL)
V = Impinger sampler volume (mL)
S = Sampling rate (6 L/min)
T = Exposure time (min)
TATV = Total accumulated tidal volume (L)
1078-CG920794 Aerosol Report E-10
-------
4. Particle Size Results
The aerodynamic size of aerosol particles primarily dictates aerosol transport characteristics, and
in the case of inhalation studies, the sites of lung deposition. The aerodynamic equivalent
diameter is the diameter of a sphere, with density = 1 g/cm3, that has the same terminal settling
velocity as the aerosol being evaluated. For inhalation exposures, the mass median aerodynamic
diameter (MMAD) of the aerosol is typically reported along with the geometric standard
deviation. Aerosol size distribution plays a critical role in inhalation studies. The biological
effects of inhaled aerosols can be dependent upon the sites and degree of deposition within the
respiratory tract. Further, the size and shape of inhaled aerosols is a critical factor in determining
deposition mechanisms and the extent of penetration into the lung and alveolar regions. As a
general rule, aerosols with aerodynamic particle sizes less than 1-5 mm are desired for inhalation
studies. Above this size, a larger portion of the aerosol is deposited in the upper respiratory tract
(Hinds, 1999). It is important to know the aerosol particle size since large particles containing
bacterial organisms deposited in the upper respiratory tract may not cause disease, or may
require a higher quantity (dosage) to cause disease or may cause only an upper respiratory
disease. Therefore, if the objective is to maximize deep lung deposition, then an aerosol with a
size on the order of 1 to 5 mm or lower, as opposed to larger aerosols is desired.
Figure 2 and Figure 3 show a log - probability plot representing the average of all APS particle
size distributions obtained from exposure testing. The MMAD and geometric standard deviation
(GSD) are also shown.
The MMAD for the log - probability plot (Figure 2 and Figure 3) was determined from
averaging the cumulative median size (50% mass) from the aerosol size distributions obtained
from the APS for all aerosol exposures. The GSD was determined from taking the cumulative
average of the GSD calculated by the APS for each exposure test. The GSD represents one
standard deviation for a normal distribution, and is determined by the following equation:
GSD = d84%/d50% (3)
Where d84% is the particle size diameter (d) at a cumulative % mass of 84% and d50% is the
particle size diameter (d) at a cumulative mass of 50% (Hinds, 1999).
1078-CG920794 Aerosol Report E-l 1
-------
Valve
Valve
Pressure
Regulator
Differential
Pressure—J\
Gauge (7\ o
Pressure <*—
Regulate
Temperature
And Humidity
Monitor
eumotach
i
Plethysmograph
MaxII Preamplifier
Figure 1. Exposure system diagram.
1078-CG920794 Aerosol Report
E-12
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Non-Viable Spore Challenge Data
1078-CG920794 Aerosol Report E-13
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Table 1. Aerosol Data Summary Sheet (Day 1)
Study No. 1078-CG920794 Rabbits 07-26-10 Irradiated Spores
Impinger Avg. Impinger Total Accum.
Rabbit ID Neb Impinger [] Volume Impinger Sample Sample Aerosol [] Tidal Volume Inhaled Dose Ames LD50 Spray Temp RH
(CFU/mL) (CFU/mL) (mL) (CPU) Rate (L/min) Time (min) (CFU/L) inhaled (L) (CFU/animal) Equivalents Factor (°F) (%)
40
7
5
9
37
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
19.00
19.40
19.80
19.40
20.00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
6.3
6.2
6.3
6.3
6.3
7.00
8.67
7.17
7.00
7.00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
17.42
10.02
10.02
12.88
14.12
0
0
0
0
0
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
NC
NC
NC
NC
NC
72.6
NA
72.0
71.9
72.0
60.6
NA
83.0
76.7
63.7
NA = Temp./RH probe malfunction. Readings not obtained.
NC = Not calculable due to zero impinger counts
Table 2. Aerosol Data Summary Sheet (Day 2)
Study No. 1078-CG920794 Rabbits 07-27-10 Irradiated Spores
Impinger Avg. Impinger Total Accum.
Rabbit ID Neb Impinger [] Volume Impinger Sample Sample Aerosol [] Tidal Volume Inhaled Dose Ames LD50 Spray Temp RH
(CFU/mL) (CFU/mL) (mL) (CFU) Rate (L/min) Time (min) (CFU/L) inhaled (L) (CFU/animal) Equivalents Factor (° F) (%)
40
7
5
9
37
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
19.20
19.40
19.20
19.60
19.60
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
6.3
6.3
6.3
6.3
6.3
7.00
7.00
7.00
7.00
7.00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
12.71
10.34
10.56
13.91
13.41
0
0
0
0
0
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
NC
NC
NC
NC
NC
67.0
68.1
68.4
69.2
69.7
69.6
74.4
66.2
68.8
65.1
NC = Not calculable due to zero impinger counts
1078-CG920794 Aerosol Report
E-14
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Table 3. Aerosol Data Summary Sheet (Day 3)
Study No. 1078-CG920794 Rabbits 07-28-10 Irradiated Spores
Impinger Avg. Impinger Total Accum.
Rabbit ID Neb Impinger [] Volume Impinger Sample Sample Aerosol [] Tidal Volume Inhaled Dose Ames LD50 Spray Temp RH
(CFU/mL) (CFU/mL) (ml) (CPU) Rate (L/min) Time (min) (CFU/L) inhaled (L) (CFU/animal) Equivalents Factor (° F) (%)
40
7
5
9
37
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
18.60
18.40
18.80
19.00
17.80
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
6.3
6.2
6.3
6.3
6.3
12.12
15.13
7.95
7.93
15.75
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
10.01
10.02
10.02
10.01
9.99
0
0
0
0
0
0.000
0.000
0.000
0.000
0.000
NC
NC
NC
NC
NC
68.1
69.0
69.8
70.3
70.6
67.9
65.6
65.9
65.6
61.9
NC = Not calculable due to zero impinger counts
Table 4. Aerosol Data Summary Sheet (Day 4)
Study No. 1078-CG920794 Rabbits 07-29-10 Irradiated Spores
Impinger Avg. Impinger Total Accum.
Rabbit ID Neb Impinger [] Volume Impinger Sample Sample Aerosol [] Tidal Volume Inhaled Dose Ames LD50 Spray Temp RH
(CFU/mL) (CFU/mL) (mL) (CFU) Rate (L/min) Time (min) (CFU/L) inhaled (L) (CFU/animal) Equivalents Factor (° F) (%)
40
7
5
9
37
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
19.20
19.40
19.20
19.20
19.40
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
6.3
6.3
6.3
6.3
6.3
7.00
7.17
7.00
7.00
7.00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
17.00
10.02
12.77
12.83
11.19
0
0
0
0
0
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
NC
NC
NC
NC
NC
67.9
68.7
69.2
69.7
70.2
76.9
74.4
70.2
70.5
73.2
NC = Not calculable due to zero impinger counts
1078-CG920794 Aerosol Report
E-15
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Table 5. Aerosol Data Summary Sheet (Day 5)
Study No. 1078-CG920794 Rabbits 07-30-10 Irradiated Spores
Impinger Avg. Impinger Total Accum.
Rabbit ID Neb Impinger [] Volume Impinger Sample Sample Aerosol [] Tidal Volume Inhaled Dose Ames LD50 Spray Temp RH
(CFU/mL) (CFU/mL) (ml) (CPU) Rate (L/min) Time (min) (CFU/L) inhaled (L) (CFU/animal) Equivalents Factor (° F) (%)
40
7
5
9
37
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
18.00
18.40
19.40
19.20
18.40
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
6.2
6.2
6.2
6.2
6.1
14.83
14.82
8.27
7.63
14.28
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
10.02
10.01
10.03
10.03
10.04
0
0
0
0
0
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
NC
NC
NC
NC
NC
67.4
68.3
69.1
69.6
69.9
71.9
70.4
72.7
71.9
67.5
NC = Not calculable due to zero impinger counts
Table 6. Aerosol Data Summary Sheet (Day 6)
Study No. 1078-CG920794 Rabbits 08-02-10 Irradiated Spores
Impinger Avg. Impinger Total Accum.
Rabbit ID Neb Impinger [] Volume Impinger Sample Sample Aerosol [] Tidal Volume Inhaled Dose Ames LD50 Spray Temp RH
(CFU/mL) (CFU/mL) (mL) (CFU) Rate (L/min) Time (min) (CFU/L) inhaled (L) (CFU/animal) Equivalents Factor (° F) (%)
40
7
5
9
37
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
18.50
19.00
19.50
19.25
19.25
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
6.2
6.2
6.3
6.3
6.3
7.72
7.98
8.12
7.00
6.68
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
10.02
10.01
10.01
13.09
17.15
0
0
0
0
0
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
NC
NC
NC
NC
NC
69.7
70.0
70.2
70.7
70.9
52.9
61.0
60.3
63.6
61.1
NC = Not calculable due to zero impinger counts
1078-CG920794 Aerosol Report
E-16
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Table 7. Aerosol Data Summary Sheet (Day 7)
Study No. 1078-CG920794 Rabbits 08-03-10 Irradiated Spores
Rabbit ID
40
7
5
9
37
Neb
(CFU/mL)
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
Impinger [ ]
(CFU/mL)
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
Impinger
Volume
(ml)
18.50
19.25
19.25
19.25
19.00
Impinger
(CPU)
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
Avg. Impinger
Sample
Rate (L/min)
6.3
6.3
6.3
6.2
6.2
Sample
Time (min)
8.22
7.10
7.00
6.85
7.00
Aerosol [ ]
(CFU/L)
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
Total Accum.
Tidal Volume
inhaled (L)
10.00
10.02
11.41
17.01
14.08
Inhaled Dose
(CFU/animal)
0
0
0
0
0
Ames LD50
Equivalents
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
Spray
Factor
NC
NC
NC
NC
NC
Temp
(°F)
66.7
66.7
68.4
69.1
69.3
RH
(%)
70.6
74.4
73.8
78.4
72.8
NC = Not calculable due to zero impinger counts
Table 8. Aerosol Data Summary Sheet (Day 8)
Study No. 1078-CG920794 Rabbits 08-04-10 Irradiated Spores
Impinger Avg. Impinger Total Accum.
Rabbit ID Neb Impinger [] Volume Impinger Sample Sample Aerosol [] Tidal Volume Inhaled Dose Ames LD50 Spray Temp RH
(CFU/mL) (CFU/mL) (ml) (CFU) Rate (L/min) Time (min) (CFU/L) inhaled (L) (CFU/animal) Equivalents Factor (° F) (%)
40
7
5
9
37
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
18.50
18.50
19.00
19.25
18.50
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
6.3
6.3
6.2
6.3
6.3
11.57
14.47
7.95
7.00
11.63
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
10.00
10.00
10.00
11.24
10.00
0
0
0
0
0
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
NC
NC
NC
NC
NC
68.5
69.1
69.7
70.2
70.4
64.1
62.5
63.9
64.7
60.3
NC = Not calculable due to zero impinger counts
1078-CG920794 Aerosol Report
E-17
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Table 9. Aerosol Data Summary Sheet (Day 9)
Study No. 1078-CG920794 Rabbits 08-05-10 Irradiated Spores
Impinger Avg. Impinger Total Accum.
Rabbit ID Neb Impinger [] Volume Impinger Sample Sample Aerosol [] Tidal Volume Inhaled Dose Ames LD50 Spray Temp RH
(CFU/mL) (CFU/mL) (ml) (CPU) Rate (L/min) Time (min) (CFU/L) inhaled (L) (CFU/animal) Equivalents Factor (° F) (%)
40
7
5
9
37
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
19.00
19.50
19.50
19.50
19.50
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
6.2
6.2
6.2
6.2
6.2
7.00
7.00
7.00
7.00
7.15
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
11.24
10.25
12.22
14.24
10.01
0
0
0
0
0
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
NC
NC
NC
NC
NC
67.3
68.3
68.8
69.2
69.4
68.6
73.8
72.7
73.7
70.2
NC = Not calculable due to zero impinger counts
Table 10. Aerosol Data Summary Sheet (Day 10)
Study No. 1078-CG920794 Rabbits 08-06-10 Irradiated Spores
Impinger Avg. Impinger Total Accum.
Rabbit ID Neb Impinger [] Volume Impinger Sample Sample Aerosol [] Tidal Volume Inhaled Dose Ames LD50 Spray Temp RH
(CFU/mL) (CFU/mL) (mL) (CFU) Rate (L/min) Time (min) (CFU/L) inhaled (L) (CFU/animal) Equivalents Factor (° F) (%)
40
7
5
9
37
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
18.60
19.00
19.60
19.60
19.00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
6.3
6.3
6.3
6.3
6.2
10.83
13.47
7.00
7.00
12.77
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
10.01
10.00
10.57
14.88
10.01
0
0
0
0
0
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
NC
NC
NC
NC
NC
67.8
68.5
69.1
69.7
69.7
68.2
66.0
69.3
73.7
62.0
NC = Not calculable due to zero impinger counts
1078-CG920794 Aerosol Report
E-18
-------
Table 11. Aerosol Data Summary Sheet (Day 11)
Study No. 1078-CG920794 Rabbits 08-09-10 Irradiated Spores
Impinger Avg. Impinger Total Accum.
Rabbit ID Neb Impinger [] Volume Impinger Sample Sample Aerosol [] Tidal Volume Inhaled Dose Ames LD50 Spray Temp RH
(CFU/mL) (CFU/mL) (ml) (CPU) Rate (L/min) Time (min) (CFU/L) inhaled (L) (CFU/animal) Equivalents Factor (° F) (%)
40
7
5
9
37
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
18.80
19.60
19.40
19.40
19.40
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
6.1
6.1
6.1
6.1
6.2
7.00
7.00
7.00
7.00
7.00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
11.65
10.87
10.99
10.42
11.64
0
0
0
0
0
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
NC
NC
NC
NC
NC
69.5
69.9
70.1
70.5
70.8
67.9
69.1
69.9
74.3
70.0
NC = Not calculable due to zero impinger counts
Table 12. Aerosol Data Summary Sheet (Day 12)
Study No. 1078-CG920794 Rabbits 08-10-10 Irradiated Spores
Impinger Avg. Impinger Total Accum.
Rabbit ID Neb Impinger [] Volume Impinger Sample Sample Aerosol [] Tidal Volume Inhaled Dose Ames LD50 Spray Temp RH
(CFU/mL) (CFU/mL) (mL) (CFU) Rate (L/min) Time (min) (CFU/L) inhaled (L) (CFU/animal) Equivalents Factor (° F) (%)
40
7
5
9
37
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
19.20
19.60
19.60
19.60
19.60
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
6.2
6.2
6.2
6.2
6.2
7.00
7.00
7.00
7.00
7.12
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
16.43
10.51
10.83
14.41
10.00
0
0
0
0
0
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
NC
NC
NC
NC
NC
67.5
67.9
68.4
68.9
69.3
76.2
73.8
71.0
72.7
72.3
NC = Not calculable due to zero impinger counts
1078-CG920794 Aerosol Report
E-19
-------
Table 13. Aerosol Data Summary Sheet (Day 13)
Study No. 1078-CG920794 Rabbits 08-11-10 Irradiated Spores
Impinger Avg. Impinger Total Accum.
Rabbit ID Neb Impinger [] Volume Impinger Sample Sample Aerosol [] Tidal Volume Inhaled Dose Ames LD50 Spray Temp RH
(CFU/mL) (CFU/mL) (ml) (CPU) Rate (L/min) Time (min) (CFU/L) inhaled (L) (CFU/animal) Equivalents Factor (° F) (%)
40
7
5
9
37
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
18.60
19.20
19.40
19.40
18.00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
6.3
6.3
6.3
6.3
6.3
7.75
12.65
7.00
7.00
16.75
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
10.01
10.03
10.45
13.63
10.01
0
0
0
0
0
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
NC
NC
NC
NC
NC
67.1
67.8
68.4
69.0
69.4
74.5
70.0
71.2
73.7
67.1
NC = Not calculable due to zero impinger counts
Table 14. Aerosol Data Summary Sheet (Day 14)
Study No. 1078-CG920794 Rabbits 08-12-10 Irradiated Spores
Impinger Avg. Impinger Total Accum.
Rabbit ID Neb Impinger [] Volume Impinger Sample Sample Aerosol [] Tidal Volume Inhaled Dose Ames LD50 Spray Temp RH
(CFU/mL) (CFU/mL) (ml) (CFU) Rate (L/min) Time (min) (CFU/L) inhaled (L) (CFU/animal) Equivalents Factor (° F) (%)
40
7
5
9
37
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
18.80
19.40
19.20
19.40
19.60
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
6.3
6.3
6.3
6.3
6.3
7.95
7.00
7.00
7.00
7.00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
10.09
10.16
11.15
14.35
11.34
0
0
0
0
0
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
NC
NC
NC
NC
NC
68.8
69.0
69.2
69.8
69.9
76.6
74.7
71.2
76.8
70.9
NC = Not calculable due to zero impinger counts
1078-CG920794 Aerosol Report
E-20
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Table 15. Aerosol Data Summary Sheet (Day 15)
Study No. 1078-CG920794 Rabbits 08-13-10 Irradiated Spores
Impinger Avg. Impinger Total Accum.
Rabbit ID Neb Impinger [] Volume Impinger Sample Sample Aerosol [] Tidal Volume Inhaled Dose Ames LD50 Spray Temp RH
(CFU/mL) (CFU/mL) (ml) (CPU) Rate (L/min) Time (min) (CFU/L) inhaled (L) (CFU/animal) Equivalents Factor (° F) (%)
40
7
5
9
37
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
18.40
19.20
19.80
19.40
19.00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
6.3
6.2
6.2
6.2
6.1
10.02
10.43
7.00
7.47
11.35
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
10.02
10.02
10.30
14.34
10.01
0
0
0
0
0
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
O.OOE+00
NC
NC
NC
NC
NC
67.0
67.8
68.7
68.9
69.5
68.6
68.6
70.1
70.5
67.3
NC = Not calculable due to zero impinger counts
1078-CG920794 Aerosol Report
E-21
-------
1078-CG920794 Log Probability Size Distribution Plot
Daily Averages Non-Viable
99.99
cc
98 -
84.14 -
50 -
15.87 -
2 -
0.1
0.1
0.5 1 2
Size (jam)
Daily Averages MMAD=0.81nm GSD=1.53
Figure 2. Log probability size distribution plot.
10
1078-CG920794 Aerosol Report
E-22
-------
Viable Spore Challenge Data
1078-CG920794 Aerosol Report E-23
-------
Table 16. Aerosol Data Summary Sheet (Day 1)
Study No. 1078-CG920794 Rabbits 07-26-10 Viable Spores
Impinger Avg. Impinger Total Accum.
Rabbit ID Neb Impinger [] Volume Impinger Sample Sample Aerosol [] Tidal Volume Inhaled Dose Ames LD50 Spray Temp RH
(CFU/mL) (CFU/mL) (mL) (CPU) Rate (L/min) Time (min) (CFU/L) inhaled (L) (CFU/animal) Equivalents Factor (°F) (%)
13
34
25
15
30
28
19
14
11
2
8
12
18
32
6
33
27
31
39
21
38
1.32E+04
1.32E+04
1.32E+04
1.32E+04
1.32E+04
1.32E+04
1.32E+04
1.18E+05
1.18E+05
1.18E+05
1.18E+05
1.18E+05
1.18E+05
1.18E+05
1.15E+06
1.15E+06
1.15E+06
1.15E+06
1.15E+06
1.15E+06
1.15E+06
2.18E+01
1 .98E+01
2.18E+01
2.46E+01
3.50E+01
1 .38E+01
2.26E+01
1 .86E+02
1 .75E+02
3.64E+02
3.08E+02
1 .83E+02
2.52E+02
2.82E+02
2.04E+03
3.02E+03
3.16E+03
2.96E+03
3.06E+03
3.06E+03
3.14E+03
19.80
19.80
19.60
19.60
19.20
19.80
19.60
19.00
19.40
19.60
19.60
19.60
19.60
19.80
19.20
19.60
19.40
19.60
19.40
19.80
19.40
4.32E+02
3.92E+02
4.27E+02
4.82E+02
6.72E+02
2.73E+02
4.43E+02
3.53E+03
3.40E+03
7.13E+03
6.04E+03
3.59E+03
4.94E+03
5.58E+03
3.92E+04
5.92E+04
6.13E+04
5.80E+04
5.94E+04
6.06E+04
6.09E+04
6.1
6.1
6.1
6.1
6.1
6.0
6.1
6.1
6.1
6.1
6.1
6.1
6.1
6.1
6.1
6.1
6.1
6.1
6.1
6.1
6.1
7.00
5.78
8.13
7.87
11.22
7.00
7.00
9.10
7.00
8.75
7.00
7.00
7.00
7.00
9.40
7.25
7.00
7.00
6.53
7.00
7.00
1.01E+01
1.11E+01
8.62E+00
1.00E+01
9.82E+00
6.51 E+00
1.04E+01
6.37E+01
7.95E+01
1.34E+02
1.41E+02
8.40E+01
1.16E+02
1.31E+02
6.83E+02
1.34E+03
1.44E+03
1.36E+03
1.49E+03
1.42E+03
1.43E+03
12.45
17.02
10.01
10.02
10.01
10.80
11.54
10.00
10.25
10.00
13.61
11.03
12.23
10.54
10.01
10.01
12.07
11.41
17.02
14.07
10.42
1.26E+02
1.89E+02
8.62E+01
1.01E+02
9.83E+01
7.03E+01
1.20E+02
6.37E+02
8.15E+02
1.34E+03
1.92E+03
9.27E+02
1.41E+03
1.38E+03
6.84E+03
1.34E+04
1.73E+04
1.55E+04
2.54E+04
2.00E+04
1.49E+04
1 .20E-03
1 .80E-03
8.21 E-04
9.58E-04
9.36E-04
6.69E-04
1.14E-03
6.06E-03
7.76E-03
1 .27E-02
1 .83E-02
8.82E-03
1 .35E-02
1 .31 E-02
6.51 E-02
1 .28E-01
1 .65E-01
1 .48E-01
2.42E-01
1 .90E-01
1 .42E-01
7.66E-07
8.42E-07
6.53E-07
7.61 E-07
7.44E-07
4.93E-07
7.86E-07
5.40E-07
6.74E-07
1.13E-06
1.20E-06
7.12E-07
9.80E-07
1.11E-06
5.94E-07
1.16E-06
1.25E-06
1.18E-06
1.30E-06
1.23E-06
1.24E-06
73.1
73.5
73.6
73.8
73.8
74.5
74.6
74.7
74.6
74.6
74.5
74.5
74.9
74.8
74.6
74.6
74.5
74.6
74.3
75.3
74.5
67.4
65.6
64.1
61.8
64.4
68.6
65.5
62.7
63.5
63.8
69.8
66.5
63.4
61.8
64.9
65.9
64.9
64.3
64.9
74.8
64.5
1078-CG920794 Aerosol Report
E-24
-------
Table 17. Aerosol Data Summary Sheet (Day 2)
Study No. 1078-CG920794 Rabbits 07-27-10 Viable Spores
Impinger Avg. Impinger Total Accum.
Rabbit ID Neb Impinger [] Volume Impinger Sample Sample Aerosol [] Tidal Volume Inhaled Dose Ames LD50 Spray Temp RH
(CFU/mL) (CFU/mL) (mL) (CPU) Rate (L/min) Time (min) (CFU/L) inhaled (L) (CFU/animal) Equivalents Factor (°F) (%)
13
34
25
15
30
28
19
14
11
2
8
12
18
32
6
33
27
31
39
21
38
1.32E+04
1.32E+04
1.32E+04
1.32E+04
1.32E+04
1.32E+04
1.32E+04
1.18E+05
1.18E+05
1.18E+05
1.18E+05
1.18E+05
1.18E+05
1.18E+05
1.15E+06
1.15E+06
1.15E+06
1.15E+06
1.15E+06
1.15E+06
1.15E+06
8.84E+01
4.62E+01
4.28E+01
2.88E+01
3.68E+01
2.90E+01
4.26E+01
1 .76E+02
3.38E+02
4.04E+02
3.42E+02
3.60E+02
1 .81 E+02
2.82E+02
2.80E+03
2.66E+03
2.96E+03
2.42E+03
1 .73E+03
2.03E+03
3.32E+03
18.80
19.20
19.00
19.60
19.20
19.60
18.80
18.80
19.40
19.40
19.60
19.40
19.20
19.60
19.00
19.40
19.00
19.60
19.60
19.80
19.60
1 .66E+03
8.87E+02
8.13E+02
5.64E+02
7.07E+02
5.68E+02
8.01 E+02
3.31 E+03
6.56E+03
7.84E+03
6.70E+03
6.98E+03
3.48E+03
5.53E+03
5.32E+04
5.16E+04
5.62E+04
4.74E+04
3.39E+04
4.02E+04
6.51 E+04
5.9
6.0
6.0
6.0
6.0
6.0
6.0
5.9
6.0
5.9
5.9
5.9
6.0
6.0
6.0
6.0
5.9
6.0
5.9
6.0
6.0
10.00
9.10
8.88
7.00
7.98
7.00
7.00
8.45
8.55
9.50
7.05
7.62
7.00
7.00
8.37
7.55
7.70
7.37
6.12
7.10
7.72
2.82E+01
1.62E+01
1.53E+01
1.34E+01
1.48E+01
1.35E+01
1.91E+01
6.64E+01
1.28E+02
1.40E+02
1.61 E+02
1.55E+02
8.27E+01
1.32E+02
1.06E+03
1.14E+03
1.24E+03
1.07E+03
9.39E+02
9.44E+02
1.40E+03
10.01
10.01
10.01
12.55
10.02
10.65
12.28
10.03
10.02
10.02
10.04
10.14
11.17
10.34
10.00
10.02
10.01
10.01
17.73
10.01
9.99
2.82E+02
1.63E+02
1.53E+02
1.69E+02
1.48E+02
1.44E+02
2.34E+02
6.66E+02
1.28E+03
1.40E+03
1.62E+03
1.58E+03
9.24E+02
1.36E+03
1.06E+04
1.14E+04
1.24E+04
1.07E+04
1.66E+04
9.44E+03
1.40E+04
2.69E-03
1 .55E-03
1 .46E-03
1 .61 E-03
1 .41 E-03
1 .37E-03
2.23E-03
6.34E-03
1 .22E-02
1 .33E-02
1 .54E-02
1 .50E-02
8.80E-03
1 .30E-02
1.01E-01
1 .09E-01
1.18E-01
1 .02E-01
1 .59E-01
8.99E-02
1 .34E-01
2.13E-06
1.23E-06
1.16E-06
1.02E-06
1.12E-06
1.03E-06
1.44E-06
5.62E-07
1.08E-06
1.19E-06
1.37E-06
1.32E-06
7.01 E-07
1.12E-06
9.21 E-07
9.91 E-07
1.08E-06
9.33E-07
8.17E-07
8.20E-07
1.22E-06
71.2
71.8
71.9
72.8
72.8
73.4
73.8
73.8
73.8
74.0
74.1
74.3
74.5
74.6
74.8
74.5
74.7
74.5
74.6
74.7
74.6
65.5
63.9
70.4
74.6
67.9
70.2
74.4
65.8
66.2
66.0
67.2
68.0
69.3
69.6
63.9
62.8
68.2
66.7
69.4
71.3
64.5
1078-CG920794 Aerosol Report
E-25
-------
Table 18. Aerosol Data Summary Sheet (Day 3)
Study No. 1078-CG920794 Rabbits 07-28-10 Viable Spores - Repeats
Impinger Avg. Impinger Total Accum.
Rabbit ID Neb Impinger [] Volume Impinger Sample Sample Aerosol [] Tidal Volume Inhaled Dose Ames LD50 Spray Temp RH
(CFU/mL) (CFU/mL) (mL) (CPU) Rate (L/min) Time (mi n) (CFU/L) inhaled (L) (CFU/animal) Equivalents Factor (°F) (%)
13*
34*
25*
15*
30*
28*
19*
14
11
2
8
12
18
32
6
33
27
31
39
21
38
1.32E+04
1.32E+04
1.32E+04
1.32E+04
1.32E+04
1.32E+04
1.32E+04
1.18E+05
1.18E+05
1.18E+05
1.18E+05
1.18E+05
1.18E+05
1.18E+05
1.15E+06
1.15E+06
1.15E+06
1.15E+06
1.15E+06
1.15E+06
1.15E+06
6.08E+02
4.38E+02
3.86E+02
5.10E+02
3.20E+02
1.17E+02
9.18E+01
2.44E+02
3.64E+02
7.46E+02
3.42E+02
3.02E+02
4.70E+02
3.66E+02
1 .47E+03
2.13E+03
2.50E+03
2.84E+03
3.48E+03
3.64E+03
3.50E+03
19.40
19.80
19.60
19.40
19.60
19.80
19.40
19.60
19.60
19.40
19.80
19.60
19.40
19.80
19.80
19.40
19.60
19.60
19.80
19.60
19.80
1.18E+04
8.67E+03
7.57E+03
9.89E+03
6.27E+03
2.32E+03
1.78E+03
4.78E+03
7.13E+03
1.45E+04
6.77E+03
5.92E+03
9.12E+03
7.25E+03
2.91 E+04
4.13E+04
4.90E+04
5.57E+04
6.89E+04
7.13E+04
6.93E+04
6.0
6.0
6.0
6.0
6.0
6.0
6.0
6.0
6.0
6.0
5.9
5.9
6.0
5.9
5.9
6.0
5.9
5.9
6.0
5.9
6.0
7.00
7.53
8.18
11.33
9.88
7.00
7.00
8.13
7.00
9.83
7.00
7.00
9.05
7.00
7.37
8.50
7.27
7.00
7.00
7.00
7.40
2.81 E+02
1 .92E+02
1 .54E+02
1 .46E+02
1 .06E+02
5.52E+01
4.24E+01
9.80E+01
1 .70E+02
2.45E+02
1 .64E+02
1 .43E+02
1 .68E+02
1 .75E+02
6.69E+02
8.10E+02
1.14E+03
1 .35E+03
1 .64E+03
1 .73E+03
1 .56E+03
11.05
10.01
10.01
10.00
10.00
11.57
11.47
10.01
10.30
10.01
10.51
11.28
10.01
13.22
10.01
10.01
10.01
11.98
14.19
11.71
10.01
3.10E+03
1 .92E+03
1 .54E+03
1 .46E+03
1 .06E+03
6.38E+02
4.86E+02
9.81 E+02
1 .75E+03
2.46E+03
1 .72E+03
1 .62E+03
1 .68E+03
2.32E+03
6.70E+03
8.11E+03
1.14E+04
1 .61 E+04
2.33E+04
2.02E+04
1 .56E+04
2.96E-02
1.83E-02
1.47E-02
1.39E-02
1.01E-02
6.08E-03
4.63E-03
9.35E-03
1.67E-02
2.34E-02
1.64E-02
1.54E-02
1.60E-02
2.21 E-02
6.38E-02
7.72E-02
1.09E-01
1.54E-01
2.22E-01
1.93E-01
1.49E-01
2.13E-05
1.45E-05
1.17E-05
1.10E-05
8.02E-06
4.18E-06
3.21 E-06
8.31 E-07
1.44E-06
2.08E-06
1.39E-06
1.21 E-06
1.42E-06
1.49E-06
5.82E-07
7.05E-07
9.93E-07
1.17E-06
1.43E-06
1.50E-06
1.36E-06
73.8
74.0
74.1
74.7
74.6
75.2
75.1
74.8
74.9
75.1
75.2
75.3
75.2
75.3
75.4
75.2
75.3
75.5
75.7
75.7
75.5
60.9
60.8
66.2
69.8
64.1
68.8
64.8
62.6
68.3
58.8
69.7
62.3
56.4
64.3
63.7
56.5
66.8
64.5
66.0
69.1
64.1
' re-enumerations
1078-CG920794 Aerosol Report
E-26
-------
Table 19. Aerosol Data Summary Sheet (Day 4)
Study No. 1078-CG920794 Rabbits 07-29-10 Viable Spores
Impinger Avg. Impinger Total Accum.
Rabbit ID Neb Impinger [] Volume Impinger Sample Sample Aerosol [] Tidal Volume Inhaled Dose Ames LD50 Spray Temp RH
(CFU/mL) (CFU/mL) (mL) (CPU) Rate (L/min) Time (min) (CFU/L) inhaled (L) (CFU/animal) Equivalents Factor (°F) (%)
13
34
25
15
30
28
19
14
11
2
8
12
18
32
6
33
27
31
39
21
38
1.32E+04
1.32E+04
1.32E+04
1.32E+04
1.32E+04
1.32E+04
1.32E+04
1.18E+05
1.18E+05
1.18E+05
1.18E+05
1.18E+05
1.18E+05
1.18E+05
1.15E+06
1.15E+06
1.15E+06
1.15E+06
1.15E+06
1.15E+06
1.15E+06
5.80E+01
5.14E+01
4.66E+01
4.54E+01
8.94E+01
7.32E+01
1.12E+02
3.10E+02
3.80E+02
5.92E+02
4.88E+02
4.60E+02
8.10E+02
4.36E+02
1 .81 E+03
1 .61 E+03
3.00E+03
2.74E+03
2.94E+03
2.05E+03
2.82E+03
19.60
19.60
19.80
19.40
19.40
19.60
19.40
19.60
19.60
19.60
19.80
19.40
19.40
19.40
19.60
19.80
19.40
19.40
19.80
19.80
19.60
1.14E+03
1 .01 E+03
9.23E+02
8.81 E+02
1 .73E+03
1 .43E+03
2.17E+03
6.08E+03
7.45E+03
1.16E+04
9.66E+03
8.92E+03
1 .57E+04
8.46E+03
3.55E+04
3.19E+04
5.82E+04
5.32E+04
5.82E+04
4.06E+04
5.53E+04
5.8
5.8
5.8
5.8
5.9
5.8
5.8
5.8
5.9
5.9
5.9
5.9
5.9
5.9
5.9
5.9
5.9
5.9
5.9
5.9
5.9
7.53
7.00
7.22
7.00
9.08
7.00
7.43
7.85
7.00
7.58
7.50
7.28
10.15
7.00
7.90
7.00
9.20
7.00
7.00
7.00
7.78
2.60E+01
2.48E+01
2.20E+01
2.17E+01
3.24E+01
3.53E+01
5.04E+01
1.33E+02
1.80E+02
2.59E+02
2.18E+02
2.08E+02
2.62E+02
2.05E+02
7.61 E+02
7.72E+02
1.07E+03
1.29E+03
1.41 E+03
9.83E+02
1.20E+03
10.00
12.21
10.01
13.11
10.01
11.63
10.01
10.01
11.08
10.02
10.01
10.01
10.04
13.46
10.01
11.15
10.01
10.88
13.22
11.04
10.01
2.60E+02
3.03E+02
2.21 E+02
2.84E+02
3.24E+02
4. 11 E+02
5.05E+02
1.34E+03
2.00E+03
2.60E+03
2.19E+03
2.08E+03
2.63E+03
2.76E+03
7.62E+03
8.61 E+03
1.07E+04
1.40E+04
1.86E+04
1.09E+04
1.21E+04
2.48E-03
2.89E-03
2.10E-03
2.71 E-03
3.09E-03
3.91 E-03
4.81 E-03
1 .27E-02
1 .90E-02
2.48E-02
2.08E-02
1 .98E-02
2.51 E-02
2.63E-02
7.26E-02
8.20E-02
1 .02E-01
1 .33E-01
1 .77E-01
1 .03E-01
1.15E-01
1.97E-06
1.88E-06
1.67E-06
1.64E-06
2.45E-06
2.68E-06
3.82E-06
1.13E-06
1.53E-06
2.20E-06
1.85E-06
1.76E-06
2.22E-06
1.74E-06
6.62E-07
6.71 E-07
9.32E-07
1.12E-06
1.23E-06
8.55E-07
1.05E-06
71.7
72.3
72.6
73.3
73.1
73.9
74.3
74.0
74.3
74.2
74.2
74.3
74.5
74.7
74.6
74.6
74.5
74.5
74.5
74.7
74.6
65.6
67.0
63.8
71.0
66.2
70.2
73.1
65.5
68.9
70.3
68.6
66.1
69.9
68.1
66.1
69.1
67.6
65.6
68.1
69.1
64.9
1078-CG920794 Aerosol Report
E-27
-------
Table 20. Aerosol Data Summary Sheet (Day 5)
Study No. 1078-CG920794 Rabbits 07-30-10 Viable Spores
Impinger Avg. Impinger Total Accum.
Rabbit ID Neb Impinger [] Volume Impinger Sample Sample Aerosol [] Tidal Volume Inhaled Dose Ames LD50 Spray Temp RH
(CFU/mL) (CFU/mL) (mL) (CPU) Rate (L/min) Time (min) (CFU/L) inhaled (L) (CFU/animal) Equivalents Factor (°F) (%)
13
34
25
15
30
28
19
14
11
2
8
12
18
32
6
33
27
31
39
21
38
1.32E+04
1.32E+04
1.32E+04
1.32E+04
1.32E+04
1.32E+04
1.32E+04
1.18E+05
1.18E+05
1.18E+05
1.18E+05
1.18E+05
1.18E+05
1.18E+05
1.15E+06
1.15E+06
1.15E+06
1.15E+06
1.15E+06
1.15E+06
1.15E+06
8.38E+01
7.02E+01
4.82E+01
6.12E+01
9.84E+01
8.14E+01
8.08E+01
3.42E+02
2.76E+02
4.26E+02
3.70E+02
4.26E+02
3.08E+02
4.44E+02
1 .42E+03
3.54E+03
2.84E+03
2.24E+03
1 .93E+03
2.58E+03
2.78E+03
19.00
19.40
19.40
19.60
19.40
19.40
19.60
19.40
19.40
19.20
19.40
19.60
19.60
19.40
19.80
19.20
19.40
19.80
19.60
19.80
19.40
1 .59E+03
1 .36E+03
9.35E+02
1 .20E+03
1 .91 E+03
1 .58E+03
1 .58E+03
6.63E+03
5.35E+03
8.18E+03
7.18E+03
8.35E+03
6.04E+03
8.61 E+03
2.81 E+04
6.80E+04
5.51 E+04
4.44E+04
3.78E+04
5. 11 E+04
5.39E+04
6.0
6.0
6.0
6.0
6.0
6.0
6.0
6.0
6.0
6.0
6.0
6.0
6.0
6.0
6.0
6.0
6.0
6.0
6.0
6.0
6.0
7.00
7.00
7.17
7.00
8.60
7.00
7.00
8.07
7.00
9.42
7.00
7.00
7.27
7.93
8.12
9.60
8.32
7.08
7.00
7.00
7.00
3.79E+01
3.24E+01
2.17E+01
2.86E+01
3.70E+01
3.76E+01
3.77E+01
1.37E+02
1.27E+02
1.45E+02
1.71E+02
1.99E+02
1.38E+02
1.81E+02
5.77E+02
1.18E+03
1.10E+03
1.04E+03
9.01 E+02
1.22E+03
1.28E+03
10.91
10.65
10.01
13.62
10.01
11.01
10.52
10.01
11.85
10.01
15.59
10.88
10.01
10.01
10.00
10.00
10.01
12.31
10.24
12.78
11.53
4.14E+02
3.45E+02
2.18E+02
3.89E+02
3.70E+02
4.14E+02
3.97E+02
1.37E+03
1.51 E+03
1.45E+03
2.66E+03
2.16E+03
1.39E+03
1.81 E+03
5.77E+03
1.18E+04
1.10E+04
1.29E+04
9.22E+03
1.55E+04
1.48E+04
3.94E-03
3.29E-03
2.07E-03
3.70E-03
3.53E-03
3.94E-03
3.78E-03
1 .31 E-02
1 .44E-02
1 .38E-02
2.54E-02
2.06E-02
1 .32E-02
1 .73E-02
5.50E-02
1.12E-01
1 .05E-01
1 .22E-01
8.78E-02
1 .48E-01
1.41E-01
2.87E-06
2.46E-06
1.65E-06
2.16E-06
2.80E-06
2.85E-06
2.86E-06
1.16E-06
1.08E-06
1.23E-06
1.45E-06
1.68E-06
1.17E-06
1.53E-06
5.02E-07
1.03E-06
9.60E-07
9.08E-07
7.83E-07
1.06E-06
1.12E-06
72.3
72.4
72.6
73.0
73.0
73.2
73.9
73.6
73.6
73.5
73.8
73.7
75.0
74.2
74.3
73.9
74.4
74.2
74.2
74.4
74.4
67.3
64.1
65.9
73.1
68.0
71.0
76.0
69.4
67.3
63.0
69.2
62.1
64.5
62.6
67.2
60.0
72.3
67.2
67.1
69.6
63.0
1078-CG920794 Aerosol Report
E-28
-------
Table 21. Aerosol Data Summary Sheet (Day 6)
Study No. 1078-CG920794 Rabbits 08-02-10 Viable Spores
Impinger Avg. Impinger Total Accum.
Rabbit ID Neb Impinger [] Volume Impinger Sample Sample Aerosol [] Tidal Volume Inhaled Dose Ames LD50 Spray Temp RH
(CFU/mL) (CFU/mL) (mL) (CPU) Rate (L/min) Time (min) (CFU/L) inhaled (L) (CFU/animal) Equivalents Factor (°F) (%)
13
34
25
15
30
28
19
14
11
2
8
12
18
32
6
33
27
31
39
21
38
1.32E+04
1.32E+04
1.32E+04
1.32E+04
1.32E+04
1.32E+04
1.32E+04
1.18E+05
1.18E+05
1.18E+05
1.18E+05
1.18E+05
1.18E+05
1.18E+05
1.15E+06
1.15E+06
1.15E+06
1.15E+06
1.15E+06
1.15E+06
1.15E+06
6.22E+01
5.88E+01
4.20E+01
4.16E+01
5.42E+01
3.86E+01
3.36E+01
1.16E+02
1 .39E+02
2.28E+02
1 .51 E+02
2.76E+02
1 .51 E+02
3.16E+02
1 .61 E+03
1 .90E+03
3.28E+03
1 .92E+03
3.22E+03
1 .97E+03
3.08E+03
18.75
19.25
19.25
19.25
19.40
19.60
19.60
19.40
19.40
19.40
19.60
19.80
19.40
19.40
19.00
19.20
19.00
19.25
19.25
19.25
19.25
1.17E+03
1.13E+03
8.09E+02
8.01 E+02
1 .05E+03
7.57E+02
6.59E+02
2.25E+03
2.70E+03
4.42E+03
2.96E+03
5.46E+03
2.93E+03
6.13E+03
3.06E+04
3.65E+04
6.23E+04
3.70E+04
6.20E+04
3.79E+04
5.93E+04
5.9
5.9
6.0
6.0
6.0
6.0
6.0
6.0
6.0
6.0
6.0
6.0
6.0
6.0
6.0
6.0
6.0
6.0
6.0
5.9
5.9
7.00
8.12
7.50
7.00
8.20
7.45
7.00
7.70
7.70
7.53
7.00
7.50
7.12
8.12
8.05
8.07
8.78
7.00
8.00
7.53
8.27
2.82E+01
2.36E+01
1.80E+01
1.91E+01
2.14E+01
1.69E+01
1.57E+01
4.87E+01
5.84E+01
9.79E+01
7.05E+01
1.21 E+02
6.86E+01
1.26E+02
6.33E+02
7.53E+02
1.18E+03
8.80E+02
1.29E+03
8.54E+02
1.22E+03
10.07
10.02
10.02
15.66
10.01
10.01
11.48
10.01
10.00
10.00
11.79
10.01
11.25
10.02
10.01
10.01
10.03
10.47
10.02
9.95
10.03
2.84E+02
2.37E+02
1.80E+02
2.99E+02
2.14E+02
1.69E+02
1.80E+02
4.88E+02
5.84E+02
9.79E+02
8.31 E+02
1.22E+03
7.71 E+02
1.26E+03
6.34E+03
7.54E+03
1.19E+04
9.21 E+03
1.29E+04
8.49E+03
1.22E+04
2.71 E-03
2.25E-03
1 .71 E-03
2.84E-03
2.04E-03
1 .61 E-03
1 .71 E-03
4.64E-03
5.56E-03
9.32E-03
7.91 E-03
1.16E-02
7.35E-03
1 .20E-02
6.04E-02
7.18E-02
1.13E-01
8.77E-02
1 .23E-01
8.09E-02
1.16E-01
2.14E-06
1.79E-06
1.36E-06
1.44E-06
1.62E-06
1.28E-06
1.19E-06
4.13E-07
4.95E-07
8.30E-07
5.97E-07
1.03E-06
5.81 E-07
1.07E-06
5.51 E-07
6.55E-07
1.03E-06
7.65E-07
1.12E-06
7.42E-07
1.06E-06
71.6
71.9
72.4
73.5
73.3
74.0
74.4
74.2
74.3
74.6
74.8
74.8
74.9
75.0
75.1
74.3
74.9
75.1
75.1
75.5
75.3
69.0
60.7
62.1
78.6
63.0
69.0
71.8
64.0
66.2
68.3
65.9
65.5
65.0
67.7
62.7
70.3
67.5
64.0
62.4
67.3
63.9
1078-CG920794 Aerosol Report
E-29
-------
Table 22. Aerosol Data Summary Sheet (Day 7)
Study No. 1078-CG920794 Rabbits 08-03-10 Viable Spores
Impinger Avg. Impinger Total Accum.
Rabbit ID Neb Impinger [] Volume Impinger Sample Sample Aerosol [] Tidal Volume Inhaled Dose Ames LD50 Spray Temp RH
(CFU/mL) (CFU/mL) (mL) (CPU) Rate (L/min) Time (min) (CFU/L) inhaled (L) (CFU/animal) Equivalents Factor (°F) (%)
13
34
25
15
30
28
19
14
11
2
8
12
18
32
6
33
27
31
39
21
38
1.32E+04
1.32E+04
1.32E+04
1.32E+04
1.32E+04
1.32E+04
1.32E+04
1.18E+05
1.18E+05
1.18E+05
1.18E+05
1.18E+05
1.18E+05
1.18E+05
1.15E+06
1.15E+06
1.15E+06
1.15E+06
1.15E+06
1.15E+06
1.15E+06
5.66E+01
5.10E+01
4.92E+01
3.82E+01
4.38E+01
3.14E+01
3.28E+01
1 .36E+02
1 .36E+02
2.62E+02
1 .55E+02
1 .38E+02
1 .62E+02
1 .96E+02
1 .85E+02
1 .52E+03
2.13E+03
1 .36E+03
1 .67E+03
1 .63E+03
2.11E+03
18.25
19.25
19.25
19.25
19.50
19.50
19.50
19.50
19.50
19.75
19.75
19.75
19.50
19.25
19.25
19.50
19.50
19.50
19.50
19.50
19.50
1 .03E+03
9.82E+02
9.47E+02
7.35E+02
8.54E+02
6.12E+02
6.40E+02
2.65E+03
2.65E+03
5.17E+03
3.06E+03
2.73E+03
3.16E+03
3.77E+03
3.56E+03
2.96E+04
4.15E+04
2.65E+04
3.26E+04
3.18E+04
4.11E+04
6.0
6.0
6.0
6.0
6.0
6.0
6.0
6.0
6.0
6.0
6.1
6.1
6.1
6.0
6.0
6.0
6.0
6.0
6.0
6.0
6.0
8.03
7.92
7.97
7.00
9.22
7.00
7.00
8.60
8.12
8.08
7.00
7.63
7.93
7.00
8.65
7.00
7.80
7.00
7.00
7.00
7.00
2.14E+01
2.07E+01
1.98E+01
1.75E+01
1.54E+01
1.46E+01
1.52E+01
5.14E+01
5.44E+01
1.07E+02
7.17E+01
5.86E+01
6.53E+01
8.98E+01
6.86E+01
7.06E+02
8.88E+02
6.31 E+02
7.75E+02
7.57E+02
9.80E+02
10.02
10.02
10.01
12.99
10.01
10.60
10.22
10.01
10.01
10.00
11.98
10.01
10.01
11.81
10.01
10.15
10.02
11.11
10.98
11.13
10.30
2.15E+02
2.07E+02
1.98E+02
2.27E+02
1.55E+02
1.55E+02
1.56E+02
5.14E+02
5.45E+02
1.07E+03
8.59E+02
5.86E+02
6.54E+02
1.06E+03
6.87E+02
7.16E+03
8.89E+03
7.02E+03
8.51 E+03
8.42E+03
1.01E+04
2.05E-03
1 .97E-03
1 .89E-03
2.17E-03
1 .47E-03
1 .47E-03
1 .48E-03
4.90E-03
5.19E-03
1 .02E-02
8.18E-03
5.58E-03
6.23E-03
1 .01 E-02
6.54E-03
6.82E-02
8.47E-02
6.68E-02
8. 11 E-02
8.02E-02
9.61 E-02
1.62E-06
1.57E-06
1.50E-06
1.33E-06
1.17E-06
1.10E-06
1.15E-06
4.36E-07
4.61 E-07
9.05E-07
6.08E-07
4.96E-07
5.53E-07
7.61 E-07
5.97E-08
6.14E-07
7.72E-07
5.49E-07
6.74E-07
6.58E-07
8.52E-07
71.2
71.8
72.2
72.9
72.8
73.7
73.9
73.7
73.7
73.8
74.2
74.0
74.1
74.7
74.5
74.3
74.5
74.7
74.8
74.9
74.7
66.7
66.4
64.6
73.7
65.4
73.4
70.2
65.6
64.7
67.5
69.4
66.1
66.8
71.8
64.4
65.1
65.8
64.0
64.9
67.8
65.0
1078-CG920794 Aerosol Report
E-30
-------
Table 23. Aerosol Data Summary Sheet (Day 8)
Study No. 1078-CG920794 Rabbits 08-04-10 Viable Spores
Impinger Avg. Impinger Total Accum.
Rabbit ID Neb Impinger [] Volume Impinger Sample Sample Aerosol [] Tidal Volume Inhaled Dose Ames LD50 Spray Temp RH
(CFU/mL) (CFU/mL) (mL) (CPU) Rate (L/min) Time (min) (CFU/L) inhaled (L) (CFU/animal) Equivalents Factor (°F) (%)
13
34
25
15
30
28
19
14
11
2
8
12
18
32
6
33
27
31
39
21
38
1.32E+04
1.32E+04
1.32E+04
1.32E+04
1.32E+04
1.32E+04
1.32E+04
1.18E+05
1.18E+05
1.18E+05
1.18E+05
1.18E+05
1.18E+05
1.18E+05
1.15E+06
1.15E+06
1.15E+06
1.15E+06
1.15E+06
1.15E+06
1.15E+06
4.38E+01
3.92E+01
5.36E+01
5.62E+01
8.02E+01
4.02E+01
3.84E+01
1 .22E+02
1 .44E+02
1 .47E+02
1 .68E+02
1 .53E+02
1 .20E+02
1 .51 E+02
1 .47E+03
1 .49E+03
1 .77E+03
1 .34E+03
1 .90E+03
1 .80E+03
1 .33E+03
18.75
19.50
19.25
19.50
19.00
19.00
19.00
19.00
19.00
19.50
19.25
19.50
19.25
19.00
19.50
19.50
19.00
19.50
19.50
19.50
19.50
8.21 E+02
7.64E+02
1 .03E+03
1.10E+03
1 .52E+03
7.64E+02
7.30E+02
2.32E+03
2.74E+03
2.87E+03
3.23E+03
2.98E+03
2.31 E+03
2.87E+03
2.87E+04
2.91 E+04
3.36E+04
2.61 E+04
3.71 E+04
3.51 E+04
2.59E+04
5.8
5.8
5.8
5.8
5.8
5.8
5.8
5.8
5.8
5.8
5.8
5.8
5.8
5.8
5.8
5.8
5.8
5.8
5.8
5.8
5.8
7.00
7.00
7.22
7.00
8.90
7.00
7.00
7.13
7.00
7.00
7.00
7.00
7.00
7.00
7.25
7.00
7.42
7.00
7.47
7.00
7.00
2.02E+01
1.88E+01
2.46E+01
2.70E+01
2.95E+01
1.88E+01
1.80E+01
5.61 E+01
6.74E+01
7.06E+01
7.97E+01
7.35E+01
5.69E+01
7.07E+01
6.82E+02
7.16E+02
7.81 E+02
6.44E+02
8.55E+02
8.65E+02
6.39E+02
10.04
11.45
10.01
13.18
10.00
10.70
12.66
10.00
10.20
10.07
11.46
12.14
11.35
12.91
10.01
11.55
10.01
12.35
10.01
12.74
10.38
2.03E+02
2.16E+02
2.47E+02
3.56E+02
2.95E+02
2.01 E+02
2.28E+02
5.61 E+02
6.87E+02
7. 11 E+02
9.13E+02
8.92E+02
6.46E+02
9.12E+02
6.82E+03
8.27E+03
7.82E+03
7.95E+03
8.56E+03
1.10E+04
6.63E+03
1 .93E-03
2.05E-03
2.35E-03
3.39E-03
2.81 E-03
1 .92E-03
2.17E-03
5.34E-03
6.55E-03
6.77E-03
8.69E-03
8.50E-03
6.15E-03
8.69E-03
6.50E-02
7.87E-02
7.45E-02
7.57E-02
8.15E-02
1 .05E-01
6.31 E-02
1.53E-06
1.43E-06
1.87E-06
2.04E-06
2.24E-06
1.43E-06
1.36E-06
4.75E-07
5.71 E-07
5.98E-07
6.75E-07
6.23E-07
4.82E-07
5.99E-07
5.93E-07
6.22E-07
6.80E-07
5.60E-07
7.44E-07
7.52E-07
5.55E-07
74.5
74.4
74.3
74.7
74.4
74.6
75.2
74.8
74.7
74.6
74.7
74.7
74.8
74.9
74.9
74.8
75.0
75.0
75.1
75.3
75.2
64.2
58.0
71.3
75.6
72.7
73.4
79.5
67.7
69.1
65.0
68.1
67.8
66.8
65.5
66.0
64.3
69.0
64.1
70.6
72.4
69.5
1078-CG920794 Aerosol Report
E-31
-------
Table 24. Aerosol Data Summary Sheet (Day 9)
Study No. 1078-CG920794 Rabbits 08-05-10 Viable Spores
Impinger Avg. Impinger Total Accum.
Rabbit ID Neb Impinger [] Volume Impinger Sample Sample Aerosol [] Tidal Volume Inhaled Dose Ames LD50 Spray Temp RH
(CFU/mL) (CFU/mL) (mL) (CPU) Rate (L/min) Time (min) (CFU/L) inhaled (L) (CFU/animal) Equivalents Factor (°F) (%)
13
34
25
15
30
28
19
14
11
2
8
12
18
32
6
33
27
31
39
21
38
1.32E+04
1.32E+04
1.32E+04
1.32E+04
1.32E+04
1.32E+04
1.32E+04
1.18E+05
1.18E+05
1.18E+05
1.18E+05
1.18E+05
1.18E+05
1.18E+05
1.15E+06
1.15E+06
1.15E+06
1.15E+06
1.15E+06
1.15E+06
1.15E+06
3.98E+01
4.94E+01
5.88E+01
4.74E+01
6.04E+01
3.54E+01
3.62E+01
1 .53E+02
2.50E+02
3.26E+02
3.06E+02
2.80E+02
3.18E+02
3.04E+02
1 .56E+03
3.60E+03
3.80E+03
3.08E+03
3.00E+03
2.62E+03
2.98E+03
19.25
19.00
19.25
19.25
19.00
19.50
19.50
19.00
19.25
19.00
19.25
19.25
18.50
19.25
19.25
19.50
19.25
19.50
19.25
19.50
19.50
7.66E+02
9.39E+02
1.13E+03
9.12E+02
1.15E+03
6.90E+02
7.06E+02
2.91 E+03
4.81 E+03
6.19E+03
5.89E+03
5.39E+03
5.88E+03
5.85E+03
3.00E+04
7.02E+04
7.32E+04
6.01 E+04
5.78E+04
5. 11 E+04
5.81 E+04
6.0
6.1
6.0
6.1
6.0
6.0
6.0
6.0
6.0
6.0
6.0
6.0
6.0
6.0
6.0
6.0
6.0
6.0
6.0
6.0
6.0
7.60
8.75
8.33
7.00
9.43
7.27
7.77
7.42
7.00
8.48
7.92
7.00
7.08
7.00
7.98
8.48
8.22
7.00
7.00
7.00
7.25
1.68E+01
1.76E+01
2.26E+01
2.14E+01
2.03E+01
1.58E+01
1.51E+01
6.53E+01
1.15E+02
1.22E+02
1.24E+02
1.28E+02
1.38E+02
1.39E+02
6.27E+02
1.38E+03
1.48E+03
1.43E+03
1.38E+03
1.22E+03
1.34E+03
10.01
10.01
10.68
10.54
10.01
10.00
10.01
10.01
11.72
10.02
10.02
10.54
10.02
11.06
10.02
10.01
10.01
10.79
12.18
12.99
10.33
1.68E+02
1.76E+02
2.42E+02
2.25E+02
2.03E+02
1.58E+02
1.52E+02
6.54E+02
1.34E+03
1.22E+03
1.24E+03
1.35E+03
1.39E+03
1.54E+03
6.28E+03
1.38E+04
1.48E+04
1.54E+04
1.67E+04
1.58E+04
1.38E+04
1 .60E-03
1 .68E-03
2.30E-03
2.15E-03
1 .93E-03
1 .51 E-03
1 .44E-03
6.22E-03
1 .28E-02
1.16E-02
1.18E-02
1 .29E-02
1 .32E-02
1 .47E-02
5.99E-02
1 .32E-01
1.41E-01
1 .47E-01
1 .60E-01
1 .50E-01
1.31E-01
1.27E-06
1.33E-06
1.72E-06
1.62E-06
1.54E-06
1.20E-06
1.15E-06
5.53E-07
9.71 E-07
1.03E-06
1.05E-06
1.09E-06
1.17E-06
1.18E-06
5.45E-07
1.20E-06
1.29E-06
1.24E-06
1.20E-06
1.06E-06
1.16E-06
71.9
72.1
72.5
73.0
73.1
73.8
73.8
73.6
73.9
73.6
73.8
73.4
73.7
74.0
73.9
74.1
74.2
74.2
74.5
74.7
74.6
73.7
67.8
66.5
73.2
70.8
77.1
76.1
73.1
74.9
72.8
73.7
70.7
72.6
73.0
68.9
73.0
73.0
70.9
73.0
72.4
68.5
1078-CG920794 Aerosol Report
E-32
-------
Table 25. Aerosol Data Summary Sheet (Day 10)
Study No. 1078-CG920794 Rabbits 08-06-10 Viable Spores
Impinger Avg. Impinger Total Accum.
Rabbit ID Neb Impinger [] Volume Impinger Sample Sample Aerosol [] Tidal Volume Inhaled Dose Ames LD50 Spray Temp RH
(CFU/mL) (CFU/mL) (mL) (CPU) Rate (L/min) Time (min) (CFU/L) inhaled (L) (CFU/animal) Equivalents Factor (°F) (%)
13
34
25
15
30
28
19
14
11
2
8
12
18
32
33
27
31
39
21
38
1.32E+04
1.32E+04
1.32E+04
1.32E+04
1.32E+04
1.32E+04
1.32E+04
1.18E+05
1.18E+05
1.18E+05
1.18E+05
1.18E+05
1.18E+05
1.18E+05
1.15E+06
1.15E+06
1.15E+06
1.15E+06
1.15E+06
1.15E+06
4.64E+01
5.24E+01
9.66E+01
6.90E+01
7.10E+01
5.88E+01
3.06E+01
1 .73E+02
3.50E+02
4.84E+02
3.98E+02
3.04E+02
4.52E+02
3.30E+02
1 .65E+03
4.34E+03
3.04E+03
5.08E+03
2.76E+03
5.24E+03
19.00
19.60
18.40
19.60
19.25
19.25
19.50
19.25
19.40
19.40
19.60
19.60
19.20
19.80
19.60
19.20
19.60
19.40
19.40
19.20
8.82E+02
1 .03E+03
1 .78E+03
1 .35E+03
1 .37E+03
1.13E+03
5.97E+02
3.33E+03
6.79E+03
9.39E+03
7.80E+03
5.96E+03
8.68E+03
6.53E+03
3.23E+04
8.33E+04
5.96E+04
9.86E+04
5.35E+04
1 .01 E+05
5.8
5.9
5.9
5.9
5.8
5.8
5.8
5.8
5.8
5.8
5.9
5.9
5.9
5.9
5.9
5.9
5.9
5.9
5.9
5.9
8.52
7.35
9.42
7.97
8.80
8.47
7.00
8.23
9.22
10.00
8.17
7.60
9.48
7.00
7.42
9.27
7.00
9.15
7.00
9.15
1.78E+01
2.37E+01
3.20E+01
2.88E+01
2.68E+01
2.30E+01
1.47E+01
6.98E+01
1.27E+02
1.62E+02
1.62E+02
1.33E+02
1.55E+02
1.58E+02
7.39E+02
1.52E+03
1.44E+03
1.83E+03
1.30E+03
1.86E+03
10.52
10.01
10.01
10.00
10.02
10.03
12.81
10.01
10.01
10.01
10.01
10.01
10.01
10.36
10.01
10.00
10.84
10.01
11.14
10.01
1.88E+02
2.37E+02
3.20E+02
2.88E+02
2.68E+02
2.31 E+02
1.88E+02
6.98E+02
1.27E+03
1.62E+03
1.62E+03
1.33E+03
1.55E+03
1.64E+03
7.39E+03
1.52E+04
1.56E+04
1.83E+04
1.44E+04
1.87E+04
1 .79E-03
2.26E-03
3.05E-03
2.74E-03
2.56E-03
2.20E-03
1 .79E-03
6.65E-03
1 .21 E-02
1 .54E-02
1 .54E-02
1 .27E-02
1 .48E-02
1 .56E-02
7.04E-02
1 .45E-01
1 .49E-01
1 .74E-01
1 .38E-01
1 .78E-01
1.35E-06
1.79E-06
2.42E-06
2.18E-06
2.03E-06
1.75E-06
1.11E-06
5.91 E-07
1.08E-06
1.37E-06
1.37E-06
1.13E-06
1.31E-06
1.34E-06
6.42E-07
1.32E-06
1.25E-06
1.59E-06
1.13E-06
1.62E-06
71.5
71.7
71.6
72.3
72.1
72.6
73.0
72.9
72.5
72.8
73.3
73.6
73.7
73.8
73.7
73.8
73.9
73.8
74.2
73.9
72.7
68.7
65.0
76.4
69.2
74.7
76.0
72.6
70.6
69.2
75.0
71.1
66.7
69.6
67.0
72.7
69.2
67.2
72.8
67.8
1078-CG920794 Aerosol Report
E-33
-------
Table 26. Aerosol Data Summary Sheet (Day 11)
Study No. 1078-CG920794 Rabbits 08-09-10 Viable Spores
Impinger Avg. Impinger Total Accum.
Rabbit ID Neb Impinger [] Volume Impinger Sample Sample Aerosol [] Tidal Volume Inhaled Dose Ames LD50 Spray Temp RH
(CFU/mL) (CFU/mL) (mL) (CPU) Rate (L/min) Time (min) (CFU/L) inhaled (L) (CFU/animal) Equivalents Factor (°F) (%)
13
34
25
15
30
28
19
14
11
2
8
12
18
32
27
31
39
21
38
1.32E+04
1.32E+04
1.32E+04
1.32E+04
1.32E+04
1.32E+04
1.32E+04
1.18E+05
1.18E+05
1.18E+05
1.18E+05
1.18E+05
1.18E+05
1.18E+05
1.15E+06
1.15E+06
1.15E+06
1.15E+06
1.15E+06
3.12E+01
3.36E+01
4.66E+01
4.30E+01
6.02E+01
3.36E+01
3.54E+01
1 .30E+02
2.78E+02
1 .56E+02
3.08E+02
2.92E+02
3.10E+02
2.68E+02
1 .87E+03
2.19E+03
2.56E+03
3.96E+03
3.42E+03
19.00
19.80
19.60
19.80
19.40
19.80
19.60
19.60
19.80
19.60
19.60
19.80
19.80
19.40
19.80
20.00
19.80
19.60
19.40
5.93E+02
6.65E+02
9.13E+02
8.51 E+02
1.17E+03
6.65E+02
6.94E+02
2.55E+03
5.50E+03
3.06E+03
6.04E+03
5.78E+03
6.14E+03
5.20E+03
3.70E+04
4.38E+04
5.07E+04
7.76E+04
6.63E+04
5.9
6.0
6.0
5.9
5.9
5.9
6.0
6.0
6.0
5.9
6.0
5.9
6.0
6.0
5.9
6.0
6.0
5.9
5.9
7.00
7.00
8.38
8.52
9.47
7.00
7.00
7.00
7.00
7.00
7.30
7.00
7.20
7.00
8.13
7.00
7.00
7.00
7.12
1.44E+01
1.58E+01
1.82E+01
1.69E+01
2.09E+01
1.61E+01
1.65E+01
6.07E+01
1.31 E+02
7.40E+01
1.38E+02
1.40E+02
1.42E+02
1.24E+02
7.72E+02
1.04E+03
1.21E+03
1.88E+03
1.58E+03
10.40
10.87
10.01
10.00
10.01
10.70
10.91
10.86
13.61
12.87
10.01
10.24
10.00
10.62
10.18
12.20
16.12
10.95
10.01
1.49E+02
1.72E+02
1.82E+02
1.69E+02
2.09E+02
1.72E+02
1.80E+02
6.59E+02
1.78E+03
9.53E+02
1.38E+03
1.43E+03
1.42E+03
1.31E+03
7.86E+03
1.27E+04
1.95E+04
2.06E+04
1.58E+04
1 .42E-03
1 .64E-03
1 .73E-03
1 .61 E-03
1 .99E-03
1 .64E-03
1 .72E-03
6.27E-03
1 .70E-02
9.07E-03
1 .31 E-02
1 .37E-02
1 .35E-02
1 .25E-02
7.48E-02
1.21E-01
1 .85E-01
1 .96E-01
1.51E-01
1.09E-06
1.20E-06
1.38E-06
1.28E-06
1.58E-06
1.22E-06
1.25E-06
5.14E-07
1.11E-06
6.27E-07
1.17E-06
1.19E-06
1.20E-06
1.05E-06
6.71 E-07
9.07E-07
1.05E-06
1.63E-06
1.37E-06
71.8
72.0
72.1
73.0
72.8
73.5
73.9
73.6
73.6
73.7
73.7
73.6
73.6
73.8
73.9
74.0
74.1
74.2
74.1
72.9
70.9
68.7
80.0
71.1
74.5
78.1
68.7
66.7
73.2
70.6
68.1
74.2
67.6
70.1
64.2
70.0
73.3
72.0
1078-CG920794 Aerosol Report
E-34
-------
Table 27. Aerosol Data Summary Sheet (Day 12)
Study No. 1078-CG920794 Rabbits 08-10-10 Viable Spores
Impinger Avg. Impinger Total Accum.
Rabbit ID Neb Impinger [] Volume Impinger Sample Sample Aerosol [] Tidal Volume Inhaled Dose Ames LD50 Spray Temp RH
(CFU/mL) (CFU/mL) (mL) (CPU) Rate (L/min) Time (min) (CFU/L) inhaled (L) (CFU/animal) Equivalents Factor (°F) (%)
13
34
25
15
30
28
19
14
11
2
8
12
18
32
27
39
21
38
1.32E+04
1.32E+04
1.32E+04
1.32E+04
1.32E+04
1.32E+04
1.32E+04
1.18E+05
1.18E+05
1.18E+05
1.18E+05
1.18E+05
1.18E+05
1.18E+05
1.15E+06
1.15E+06
1.15E+06
1.15E+06
1 .92E+01
2.56E+01
3.92E+01
4.54E+01
6.26E+01
5.76E+01
3.92E+01
1 .26E+02
1 .42E+02
3.02E+02
1 .55E+02
3.08E+02
1 .31 E+02
1 .60E+02
1 .53E+03
2.60E+03
1 .70E+03
1 .76E+03
19.00
19.60
19.40
19.60
19.60
19.60
19.40
19.60
19.40
19.40
19.40
19.60
19.60
19.40
19.60
19.60
19.40
19.20
3.65E+02
5.02E+02
7.60E+02
8.90E+02
1 .23E+03
1.13E+03
7.60E+02
2.47E+03
2.75E+03
5.86E+03
3.01 E+03
6.04E+03
2.57E+03
3.10E+03
3.00E+04
5.10E+04
3.30E+04
3.38E+04
5.9
6.0
5.9
5.9
5.9
5.9
5.9
5.9
5.9
5.9
5.9
5.9
5.9
5.9
5.9
5.9
5.9
5.9
7.00
7.00
7.87
7.00
8.22
7.83
7.13
7.53
7.73
8.87
7.00
7.70
7.00
7.85
8.32
8.12
7.28
7.03
8.83E+00
1.19E+01
1.64E+01
2.15E+01
2.53E+01
2.44E+01
1.81E+01
5.56E+01
6.04E+01
1.12E+02
7.28E+01
1.33E+02
6.22E+01
6.70E+01
6. 11 E+02
1.06E+03
7.68E+02
8.15E+02
10.83
11.29
10.01
10.23
10.00
10.01
10.03
10.00
10.01
10.01
10.41
10.02
14.39
10.01
10.00
10.01
10.02
10.02
9.57E+01
1.35E+02
1.64E+02
2.20E+02
2.53E+02
2.45E+02
1.81 E+02
5.56E+02
6.05E+02
1.12E+03
7.58E+02
1.33E+03
8.95E+02
6.71 E+02
6. 11 E+03
1.06E+04
7.69E+03
8.16E+03
9.11E-04
1 .28E-03
1 .56E-03
2.10E-03
2.41 E-03
2.33E-03
1 .73E-03
5.29E-03
5.76E-03
1 .07E-02
7.22E-03
1 .27E-02
8.52E-03
6.39E-03
5.82E-02
1.01E-01
7.33E-02
7.77E-02
6.69E-07
9.05E-07
1.24E-06
1.63E-06
1.92E-06
1.85E-06
1.37E-06
4.71 E-07
5.12E-07
9.49E-07
6.17E-07
1.13E-06
5.27E-07
5.68E-07
5.31 E-07
9.25E-07
6.68E-07
7.08E-07
70.6
70.9
71.6
72.2
71.2
72.9
73.2
72.9
73.0
73.1
73.2
73.3
73.6
73.6
73.4
73.4
73.8
73.8
72.0
71.1
71.3
80.0
68.2
71.3
73.0
65.1
66.1
68.8
69.1
70.4
74.2
69.5
67.7
67.9
74.4
67.4
1078-CG920794 Aerosol Report
E-35
-------
Table 28. Aerosol Data Summary Sheet (Day 13)
Study No. 1078-CG920794 Rabbits 08-11-10 Viable Spores
Impinger Avg. Impinger Total Accum.
Rabbit ID Neb Impinger [] Volume Impinger Sample Sample Aerosol [] Tidal Volume Inhaled Dose Ames LD50 Spray Temp RH
(CFU/mL) (CFU/mL) (mL) (CPU) Rate (L/min) Time (min) (CFU/L) inhaled (L) (CFU/animal) Equivalents Factor (°F) (%)
13
34
25
15
30
28
19
14
11
2
8
12
18
32
27
39
21
38
1.32E+04
1.32E+04
1.32E+04
1.32E+04
1.32E+04
1.32E+04
1.32E+04
1.18E+05
1.18E+05
1.18E+05
1.18E+05
1.18E+05
1.18E+05
1.18E+05
1.15E+06
1.15E+06
1.15E+06
1.15E+06
4.32E+01
5.92E+01
3.04E+01
3.26E+01
7.18E+01
6.58E+01
7.18E+01
2.92E+02
2.70E+02
3.12E+02
3.30E+02
2.98E+02
3.02E+02
4.68E+02
2.52E+03
2.70E+03
2.80E+03
3.56E+03
18.00
18.60
19.60
19.60
19.20
19.40
19.00
19.20
19.60
19.60
19.60
19.40
19.40
19.20
19.40
19.60
19.60
19.60
7.78E+02
1.10E+03
5.96E+02
6.39E+02
1 .38E+03
1 .28E+03
1 .36E+03
5.61 E+03
5.29E+03
6.12E+03
6.47E+03
5.78E+03
5.86E+03
8.99E+03
4.89E+04
5.29E+04
5.49E+04
6.98E+04
5.8
5.8
5.8
5.9
5.9
5.9
5.9
5.9
5.9
5.9
5.9
5.9
5.9
5.8
5.8
5.8
5.8
5.8
14.02
11.82
7.47
7.28
9.78
8.98
10.22
9.95
8.72
8.00
7.17
7.15
8.38
9.93
7.48
7.40
7.00
7.75
9.56E+00
1.61E+01
1.38E+01
1.49E+01
2.39E+01
2.41 E+01
2.26E+01
9.55E+01
1.03E+02
1.30E+02
1.53E+02
1.37E+02
1.18E+02
1.56E+02
1.13E+03
1.23E+03
1.35E+03
1.55E+03
10.01
10.02
10.01
10.01
10.00
10.00
10.00
10.00
10.00
10.01
10.01
10.01
10.03
10.01
10.01
10.02
11.32
10.00
9.57E+01
1.61E+02
1.38E+02
1.49E+02
2.39E+02
2.41 E+02
2.26E+02
9.55E+02
1.03E+03
1.30E+03
1.53E+03
1.37E+03
1.19E+03
1.56E+03
1.13E+04
1.24E+04
1.53E+04
1.55E+04
9.12E-04
1 .53E-03
1 .31 E-03
1 .42E-03
2.28E-03
2.29E-03
2.15E-03
9.10E-03
9.80E-03
1 .24E-02
1 .46E-02
1 .31 E-02
1.13E-02
1 .49E-02
1 .07E-01
1.18E-01
1 .46E-01
1 .48E-01
7.24E-07
1.22E-06
1.04E-06
1.13E-06
1.81E-06
1.83E-06
1.71E-06
8.09E-07
8.72E-07
1.10E-06
1.30E-06
1.16E-06
1.00E-06
1.32E-06
9.80E-07
1.07E-06
1.18E-06
1.35E-06
71.7
72.1
72.6
73.2
72.8
73.7
73.6
73.7
73.4
73.6
73.6
73.9
73.9
74.2
74.3
74.6
74.9
74.7
64.8
63.8
67.8
75.6
66.0
76.1
65.3
69.9
66.7
65.3
62.7
66.4
61.1
62.6
68.6
65.8
70.8
66.9
1078-CG920794 Aerosol Report
E-36
-------
Table 29. Aerosol Data Summary Sheet (Day 14)
Study No. 1078-CG920794 Rabbits 08-12-10 Viable Spores
Impinger Avg. Impinger Total Accum.
Rabbit ID Neb Impinger [] Volume Impinger Sample Sample Aerosol [] Tidal Volume Inhaled Dose Ames LD50 Spray Temp RH
(CFU/mL) (CFU/mL) (mL) (CPU) Rate (L/min) Time (min) (CFU/L) inhaled (L) (CFU/animal) Equivalents Factor (°F) (%)
13
34
25
15
30
28
19
14
11
2
8
12
18
32
27
39
21
38
1.32E+04
1.32E+04
1.32E+04
1.32E+04
1.32E+04
1.32E+04
1.32E+04
1.18E+05
1.18E+05
1.18E+05
1.18E+05
1.18E+05
1.18E+05
1.18E+05
1.15E+06
1.15E+06
1.15E+06
1.15E+06
1.18E+01
1 .88E+01
2.48E+01
2.10E+01
1 .76E+01
1 .76E+01
1 .78E+01
9.22E+01
1 .06E+02
1 .27E+02
8.62E+01
8.52E+01
1.17E+02
1 .05E+02
8.96E+02
1.12E+03
9.80E+02
1 .32E+03
18.20
19.20
18.80
19.00
18.60
19.20
19.00
18.80
19.00
18.80
19.40
18.80
18.80
18.60
19.00
18.80
18.80
18.80
2.15E+02
3.61 E+02
4.66E+02
3.99E+02
3.27E+02
3.38E+02
3.38E+02
1 .73E+03
2.01 E+03
2.39E+03
1 .67E+03
1 .60E+03
2.20E+03
1 .95E+03
1 .70E+04
2.11E+04
1 .84E+04
2.48E+04
5.9
5.9
6.0
6.0
6.0
6.0
6.1
6.1
6.1
6.1
6.2
6.1
6.1
6.1
6.0
5.9
6.0
6.1
7.00
7.00
7.95
7.00
9.47
8.17
8.10
8.88
7.72
9.47
7.87
8.45
9.32
8.17
7.30
8.30
7.00
8.12
5.20E+00
8.74E+00
9.77E+00
9.50E+00
5.76E+00
6.89E+00
6.84E+00
3.20E+01
4.28E+01
4.13E+01
3.43E+01
3.11E+01
3.87E+01
3.92E+01
3.89E+02
4.30E+02
4.39E+02
5.01 E+02
10.97
10.31
10.01
14.10
10.01
10.01
10.01
10.00
10.00
10.00
10.00
10.00
10.01
10.04
10.01
10.02
10.86
10.01
5.70E+01
9.01 E+01
9.78E+01
1.34E+02
5.77E+01
6.90E+01
6.85E+01
3.20E+02
4.28E+02
4.13E+02
3.43E+02
3. 11 E+02
3.87E+02
3.93E+02
3.89E+03
4.31 E+03
4.76E+03
5.02E+03
5.43E-04
8.58E-04
9.32E-04
1 .28E-03
5.49E-04
6.57E-04
6.53E-04
3.05E-03
4.07E-03
3.94E-03
3.26E-03
2.96E-03
3.69E-03
3.75E-03
3.71 E-02
4.10E-02
4.54E-02
4.78E-02
3.94E-07
6.62E-07
7.40E-07
7.20E-07
4.36E-07
5.22E-07
5.19E-07
2.71 E-07
3.62E-07
3.50E-07
2.90E-07
2.63E-07
3.28E-07
3.32E-07
3.38E-07
3.74E-07
3.81 E-07
4.36E-07
71.0
71.4
71.4
71.8
71.7
72.6
72.8
72.8
72.5
72.8
72.8
72.9
73.0
73.1
73.2
73.3
73.7
73.7
73.1
71.9
71.5
74.0
67.5
75.4
75.0
75.7
67.3
72.7
73.0
72.1
74.7
70.5
70.2
71.1
73.7
70.3
1078-CG920794 Aerosol Report
E-37
-------
Table 30. Aerosol Data Summary Sheet (Day 15)
Study No. 1078-CG920794 Rabbits 08-13-10 Viable Spores
Impinger Avg. Impinger Total Accum.
Rabbit ID Neb Impinger [] Volume Impinger Sample Sample Aerosol [] Tidal Volume Inhaled Dose Ames LD50 Spray Temp RH
(CFU/mL) (CFU/mL) (mL) (CPU) Rate (L/min) Time (min) (CFU/L) inhaled (L) (CFU/animal) Equivalents Factor (°F) (%)
13
34
25
15
30
28
19
14
11
8
12
18
32
27
39
21
38
1.32E+04
1.32E+04
1.32E+04
1.32E+04
1.32E+04
1.32E+04
1.32E+04
1.18E+05
1.18E+05
1.18E+05
1.18E+05
1.18E+05
1.18E+05
1.15E+06
1.15E+06
1.15E+06
1.15E+06
7.48E+01
9.52E+01
5.34E+01
4.04E+01
4.78E+01
4.56E+01
7.38E+01
1 .58E+02
3.02E+02
4.02E+02
3.60E+02
3.44E+02
3.36E+02
2.09E+03
2.28E+03
3.36E+03
2.82E+03
18.00
18.80
19.60
19.40
19.60
19.60
19.00
19.60
19.60
19.80
19.20
19.40
19.80
19.40
19.60
19.80
19.40
1 .35E+03
1 .79E+03
1 .05E+03
7.84E+02
9.37E+02
8.94E+02
1 .40E+03
3.10E+03
5.92E+03
7.96E+03
6.91 E+03
6.67E+03
6.65E+03
4.05E+04
4.47E+04
6.65E+04
5.47E+04
6.0
6.0
6.0
6.0
6.0
6.1
6.1
6.1
6.1
6.0
6.0
6.0
6.0
5.9
6.0
5.9
5.9
15.92
14.33
8.60
7.00
8.72
7.68
13.13
7.62
8.27
8.35
8.40
8.67
7.00
8.28
7.25
7.13
7.00
1.41E+01
2.08E+01
2.03E+01
1.87E+01
1.79E+01
1.91E+01
1.75E+01
6.66E+01
1.17E+02
1.59E+02
1.37E+02
1.28E+02
1.58E+02
8.30E+02
1.03E+03
1.58E+03
1.32E+03
10.01
10.00
10.01
15.65
10.01
10.01
10.00
10.01
10.01
10.00
10.00
10.01
10.12
10.01
10.28
10.01
10.30
1.41E+02
2.08E+02
2.03E+02
2.92E+02
1.79E+02
1.91E+02
1.75E+02
6.67E+02
1.17E+03
1.59E+03
1.37E+03
1.28E+03
1.60E+03
8.31 E+03
1.06E+04
1.58E+04
1.36E+04
1 .34E-03
1 .98E-03
1 .93E-03
2.78E-03
1 .71 E-03
1 .82E-03
1 .67E-03
6.35E-03
1.12E-02
1 .51 E-02
1 .31 E-02
1 .22E-02
1 .53E-02
7.91 E-02
1.01E-01
1.51E-01
1 .30E-01
1.07E-06
1.58E-06
1.54E-06
1.41E-06
1.36E-06
1.45E-06
1.33E-06
5.65E-07
9.94E-07
1.35E-06
1.16E-06
1.09E-06
1.34E-06
7.22E-07
8.93E-07
1.38E-06
1.15E-06
71.0
71.3
71.8
72.2
72.1
72.9
72.6
73.2
72.9
73.1
73.1
73.1
73.4
73.3
73.4
73.5
73.5
65.1
62.5
71.7
76.2
70.9
71.8
60.6
71.7
67.3
69.6
63.4
62.8
68.2
68.8
69.5
73.2
68.1
1078-CG920794 Aerosol Report
E-38
-------
1078-CG920794 Log Probability Size Distribution Plot
Daily Averages Viable
cc
99.99
98 -
84.14 -
50 -
15.87 -
2 -
0.1
0.1
0.5
10
Size (jam)
• Low (1.32e4 cfu/mL) MMAD=0.79|am GSD=1.52
O Mid (1.18e5 cfu/mL) MMAD=0.82|jm GSD=1.53
T High (1.15e6 cfu/mL) MMAD=0.86^m GSD=1.49
Figure 3. Log probability size distribution plot.
1078-CG920794 Aerosol Report
E-39
-------
5. References
1. Hinds, William C. Aerosol Technology: Properties, Behavior, and Measurement of Airborne
Particles. Second Edition 1999. Pages 94-97. Publisher: Wiley-Interscience, John Wiley &
Sons Inc., New York, New York.
2. Zaucha, G.M., Pitt, L.M., Estep, I, Ivins, B.E., and Friedlander, A.M., 1998. The pathology
of experimental rabbits exposed by inhalation and subcutaneous inoculation. Arch. Pathol.
Lab. Med. 122:982-992.
3. Battelle SOP BBRC X-054. "Standard Operating Procedure (SOP) for the Enumeration of
BL-2 and BL-3 Bacterial Samples via the Spread Plate Technique"
4. Battelle SOP BBRC XIII-001. "Standard Operating Procedure (SOP) for the Aerosol
Exposure System to Challenge Non-Human Primates and Rabbits to Aerosolized Agent"
5. Battelle SOP BBRC XIII-008. "Standard Operating Procedure (SOP) for Programming the
Buxco BioSystem XA Data Acquisition Software for Pulmonary Analysis During Animal
Inhalation Studies"
6. Battelle SOP BBRC XIII-009. "Standard Operating Procedure (SOP) for the Calibration and
Operation of the Buxco BioSystem, Preamplifier System and Pressure Transducers"
7. Battelle SOP BBRC XIII-011. "Standard Operating Procedure (SOP) for Using and
Checking the Calibration of the Aerodynamic Particle Sizer 3321"
,- • ,, ,
Report Prepared By: <<••-"' ^*^_———---- - _ i!//jf//()
Eric M. Benson Date
Aerosol Technician Specialist
Reviewed By; t',< >•*• -J :W. (,v,*. .,.,-.• -•.' .\/ \
Roy li7Barnewall, D.V.M., Ph.1).
Aerosol Director
1078-CG920794 Aerosol Report E-40
-------
APPENDIX F
STATISTICAL REPORT - TELEMETRY
1078-CG920794 Statistical Report - Telemetry F-1
-------
Table of Contents
1. Introduction F-7
2. Statistical Methods F-8
3. Results F-10
4. Conclusions F-16
List of Tables
Table 1. Study Design F-7
Table 2. Summary of the ANOVA Results for the Baseline Adjusted Six-Hour
Averages for Activity (Counts/Minutes) F-17
Table 3. Summary of the ANOVA Results for the Baseline Adjusted Six-Hour
Averages for Heart Rate (BPM) F-21
Table 4. Summary of the ANOVA Results for the Baseline Adjusted Six-Hour
Averages for RP Expiratory Time (Seconds) F-26
Table 5. Summary of the ANOVA Results for the Baseline Adjusted Six-Hour
Averages for RP Inspiratory Time (Seconds) F-30
Table 6. Summary of the ANOVA Results for the Baseline Adjusted Six-Hour
Averages for RP Integral (mmHg-seconds) F-34
Table 7. Summary of the ANOVA Results for the Baseline Adjusted Six-Hour
Averages for RP Peak Amplitude (mmHg) F-38
Table 8. Summary of the ANOVA Results for the Baseline Adjusted Six-Hour
Averages for RP Respiratory Rate (RCPM) F-42
Table 9. Summary of the ANOVA Results for the Baseline Adjusted Six-Hour
Averages for Temperature (Celsius) F-46
Table 10. Abnormality Summaries by Parameter and Group Along with Fisher's Exact
Tests Comparing the Proportion Abnormal in Each Group by Parameter F-51
Table 11. Results of Overall Log-Rank Tests Comparing the Time to Abnormality
between Groups by Parameter F-52
Table 12. Results of Overall Log-RankTests Comparing the Duration of Abnormality
between Groups by Parameter F-52
1078-CG920794 Statistical Report - Telemetry
F-2
-------
List of Figures
Figure la. Plot of baseline adjusted Activity (counts/minute) for each animal in Group 1.. F-53
Figure Ib. Plot of baseline adjusted Activity (counts/minute) for each animal in Group 2.. F-53
Figure Ic. Plot of baseline adjusted Activity (counts/minute) for each animal in Group 3.. F-54
Figure Id. Plot of baseline adjusted Activity (counts/minute) for each animal in Group 4.. F-54
Figure 2a. Plot of baseline adjusted Heart Rate (BPM) for each animal in Group 1 F-55
Figure 2b. Plot of baseline adjusted Heart Rate (BPM) for each animal in Group 2 F-55
Figure 2c. Plot of baseline adjusted Heart Rate (BPM) for each animal in Group 3 F-56
Figure 2d. Plot of baseline adjusted Heart Rate (BPM) for each animal in Group 4 F-56
Figure 3a. Plot of baseline adjusted RP Expiratory Time (seconds) for each animal in
Group 1 F-57
Figure 3b. Plot of baseline adjusted RP Expiratory Time (seconds) for each animal in
Group 2 F-57
Figure 3c. Plot of baseline adjusted RP Expiratory Time (seconds) for each animal in
Group 3 F-58
Figure 3d. Plot of baseline adjusted RP Expiratory Time (seconds) for each animal in
Group 4 F-58
Figure 4a. Plot of baseline adjusted RP Inspiratory Time (seconds) for each animal in
Group 1 F-59
Figure 4b. Plot of baseline adjusted RP Inspiratory Time (seconds) for each animal in
Group 2 F-59
Figure 4c. Plot of baseline adjusted RP Inspiratory Time (seconds) for each animal in
Group 3 F-60
Figure 4d. Plot of baseline adjusted RP Inspiratory Time (seconds) for each animal in
Group 4 F-60
Figure 5a. Plot of baseline adjusted RP Integral (mmHg-seconds) for each animal in
Group 1 F-61
Figure 5b. Plot of baseline adjusted RP Integral (mmHg-seconds) for each animal in
Group 2 F-61
Figure 5c. Plot of baseline adjusted RP Integral (mmHg-seconds) for each animal in
Group 3 F-62
Figure 5d. Plot of baseline adjusted RP Integral (mmHg-seconds) for each animal in
Group 4 F-62
Figure 6a. Plot of baseline adjusted RP Peak Amplitude (mmHg) for each animal in
Group 1 F-63
1078-CG920794 Statistical Report - Telemetry F-3
-------
Figure 6b. Plot of baseline adjusted RP Peak Amplitude (mmHg) for each animal in
Group 2 F-63
Figure 6c. Plot of baseline adjusted RP Peak Amplitude (mmHg) for each animal in
Group 3 F-64
Figure 6d. Plot of baseline adjusted RP Peak Amplitude (mmHg) for each animal in
Group 4 F-64
Figure 7a. Plot of baseline adjusted RP Respiratory Rate (RCPM) for each animal in
Group 1 F-65
Figure 7b. Plot of baseline adjusted RP Respiratory Rate (RCPM) for each animal in
Group 2 F-65
Figure 7c. Plot of baseline adjusted RP Respiratory Rate (RCPM) for each animal in
Group 3 F-66
Figure 7d. Plot of baseline adjusted RP Respiratory Rate (RCPM) for each animal in
Group 4 F-66
Figure 8a. Plot of baseline adjusted Temperature (Celsius) for each animal in Group 1 F-67
Figure 8b. Plot of baseline adjusted Temperature (Celsius) for each animal in Group 2 F-67
Figure 8c. Plot of baseline adjusted Temperature (Celsius) for each animal in Group 3 F-68
Figure 8d. Plot of baseline adjusted Temperature (Celsius) for each animal in Group 4 F-68
Figure 9. Plot of mean baseline adjusted Activity (counts/minute) for each group F-69
Figure 10. Plot of mean baseline adjusted Heart Rate (BPM) for each group F-69
Figure 11. Plot of mean baseline adjusted RP Expiratory Time (seconds) for each group... F-70
Figure 12. Plot of mean baseline adjusted RP Inspiratory Time (seconds) for each group.. F-70
Figure 13. Plot of mean baseline adjusted RP Integral (mmHg-seconds) for each group .... F-71
Figure 14. Plot of mean baseline adjusted RP Peak Amplitude (mmHg) for each group F-71
Figure 15. Plot of mean baseline adjusted RP Respiratory Rate (RCPM) for each group.... F-72
Figure 16. Plot of mean baseline adjusted Temperature (Celsius) for each group F-72
Figure 17. Kaplan-Meier curves for time to abnormality based on Activity F-73
Figure 18. Kaplan-Meier curves for time to abnormality based on Heart Rate F-73
Figure 19. Kaplan-Meier curves for time to abnormality based on RP Expiratory Time F-74
Figure 20. Kaplan-Meier curves for time to abnormality based on RP Inspiratory Time .... F-74
Figure 21. Kaplan-Meier curves for time to abnormality based on RP Integral F-75
Figure 22. Kaplan-Meier curves for time to abnormality based on RP Peak Amplitude F-75
Figure 23. Kaplan-Meier curves for time to abnormality based on RP Respiratory Rate .... F-76
Figure 24. Kaplan-Meier curves for time to abnormality based on Temperature F-76
1078-CG920794 Statistical Report - Telemetry F-4
-------
Figure 25. Kaplan-Meier curves for duration of abnormality based on Activity F-77
Figure 26. Kaplan-Meier curves for duration of abnormality based on Heart Rate F-77
Figure 27. Kaplan-Meier curves for duration of abnormality based on RP Expiratory
Time F-78
Figure 28. Kaplan-Meier curves for duration of abnormality based on RP Inspiratory
Time F-78
Figure 29. Kaplan-Meier curves for duration of abnormality based on RP Integral F-79
Figure 30. Kaplan-Meier curves for duration of abnormality based on RP Peak
Amplitude F-79
Figure 31. Kaplan-Meier curves for duration of abnormality based on RP Respiratory
Rate F-80
Figure 32. Kaplan-Meier curves for duration of abnormality based on Temperature F-80
1078-CG920794 Statistical Report - Telemetry F-5
-------
List of Acronyms
ANOVA Analysis of variance
BBRC Battelle Biomedical Research Center
BPM Beats per minute
CPU Colony forming units
ECG Electrocardiogram
N Number of animals
RCPM Respiratory cycles per minute
RP Respiratory period
NZW New Zealand White
1078-CG920794 Statistical Report - Telemetry F-6
-------
1. Introduction
This report summarizes the statistical analysis of telemetry data collected under Battelle
Biomedical Research Center (BBRC) Study No. 1078-CG920794. Twenty-six (26)
pathogen-free New Zealand White (NZW) rabbits were randomly assigned to one of four groups
of animals as shown in Table 1. Beginning on Study Day 0, animals were exposed to
Bacillus anthracis (Ames strain) spores once a day for five straight working days each week for
three straight weeks, at targeted doses shown in Table 1. The control group (Group 1) was
exposed to gamma-irradiated spores.
Table 1. Study Design
Group
1
2
3
4
Number of
Animals per
Group
5
7
7
7
Target Spore Dose
(CPU)
10,000*
100
1,000
10,000
Number of Spore
Challenges
15
CPU Colony forming units.
* These spores were inactivated by radiation.
Telemetry data were collected for activity, respiratory period (RP) expiratory time, RP
inspiratory time, RP integral, RP peak amplitude, RP respiratory rate, and body temperature. The
telemetry data were collected for at least 30 seconds every 15 minutes throughout the study.
Heart rate data was recreated from electrocardiogram (ECG) data; therefore, the collection times
were rounded to the nearest 15-minute clock time. Approximately three days of baseline data
were collected prior to the first challenge for each animal, while the post-challenge data were
collected for surviving animals up to 39 days following the first challenge. All telemetry data
collected after an animal's time of death were excluded from the statistical analysis, as were all
records that had each respiratory parameter recorded as missing and an activity recorded as either
missing or zero.
1078-CG920794 Statistical Report - Telemetry
F-7
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2. Statistical Methods
The analysis described below was performed separately for each animal and for each of the
animal's telemetry parameters. The mean telemetry value was computed for every 15-minute
clock time (00:00, 00:15, ..., 23:45) at baseline. The heart rate data was recreated from ECG
data; therefore, the collection times were rounded to the nearest 15-minute clock time. Each
observation was then baseline adjusted according to the associated clock time, and six-hour
averages were computed for the baseline adjusted values using the following intervals:
midnight-6AM (inclusive), 6AM-noon (inclusive), noon-6PM (inclusive), and 6PM-midnight
(inclusive). The standard deviation of each six-hour average at baseline was calculated and used
to form the upper and lower limits for indications of abnormality. The upper limit was defined to
be three standard deviations above zero, while the lower limit was defined to be three standard
deviations below zero. An animal was found to be abnormal if two consecutive baseline adjusted
six-hour averages were outside the upper or lower limits following challenge. The time of onset
for abnormality was defined as the time associated with the second abnormal value during the
first occurrence of two consecutive abnormal values following challenge. The end of
abnormality was defined as the time associated with the last abnormal value during the last
occurrence of two consecutive abnormal values following challenge. Therefore, the duration of
abnormality was defined as the difference between the time associated with the end of
abnormality and the time associated with the onset of abnormality.
To determine if the baseline adjusted telemetry results were significantly different between the
groups; the following analysis of variance (ANOVA) model was fitted separately at each study
time:
YdlJ= [i + Group,- + 6y
where ¥<#/ is the baseline adjusted six-hour average telemetry value for the }th animal in
Group i (i=l to 4) at study time d, \a is an overall constant, and sij is the random error left
unexplained by the model. Least square mean estimates from the ANOVA models were
calculated and approximate t-tests were performed to determine if, for each group, there was a
significant shift in the telemetry values between baseline and each study time, after adjusting for
the clock time. This tests if the mean baseline adjusted telemetry value is significantly different
from zero. Additionally, Tukey's multiple comparisons procedure was performed to determine
1078-CG920794 Statistical Report - Telemetry F-8
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which pairs of groups had mean baseline adjusted telemetry values that were significantly
different from each other. Under the Tukey procedure, the set of all comparisons within each
parameter and study time combination are made at a joint 0.05 level of significance.
Estimates and exact binomial 95% confidence intervals for the proportion of animals that
became abnormal were calculated within each group. An overall two-sided Fisher's exact test
was performed to determine if there was a significant difference between the proportions of
abnormal animals in each group. For those groups with abnormal animals, the mean duration of
abnormality was also calculated.
For each telemetry parameter, an overall log-rank test was performed to determine if there was a
significant difference between the times to abnormality within each group. Similarly for each
parameter, an overall log-rank test was performed to determine if there was a significant
difference between the duration of abnormality within each group. If the overall log-rank test for
a parameter was significant, then pairwise log-rank tests were performed to evaluate all pairwise
group comparisons. The Bonferroni-Holm adjustment was used to maintain an overall 0.05 level
of significance among the multiple pairwise comparisons made within each telemetry parameter.
All statistical analyses were conducted using Stata (StataCorp LP; College Station, TX;
Version 11.1) and R (Version 2.9.2) software that has been performance tested by Battelle staff.
All results were reported at the 0.05 level of significance.
1078-CG920794 Statistical Report - Telemetry F-9
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3. Results
Since the animals were challenged at various times on Study Day 0 and the clock time is of
interest when analyzing telemetry data, all figures are presented in terms of "days from midnight
of challenge day" instead of "hours post-challenge". Figures la through 8d display the baseline
adjusted six-hour averages for the animals within each group for each of the telemetry
parameters. Figures 9 through 16 display the mean baseline adjusted six-hour averages within
each group for each of the telemetry parameters, respectively.
Tables 2 through 9 contain test results within each group at each study time, testing whether the
mean baseline adjusted value was significantly different from zero (at the 0.05 level) for each
telemetry parameter. In each cell, the estimate of the mean baseline adjusted value is shown for
that parameter, group, and study time. Following the estimate, an up arrow (t) indicates that the
mean baseline adjusted value was significantly greater than zero, while a down arrow (-1)
indicates that it was significantly less than zero. These tables also contain group effect p-values
for each study time, as well as test results from the Tukey's pairwise comparisons procedure that
was used to identify pairs of groups with significantly different mean baseline adjusted values.
Under the Tukey procedure, the set of comparisons within each parameter and study time is
made at a joint 95% confidence level. Each significant difference is shown as the estimated
difference between the mean baseline adjusted values for the pair of groups under consideration,
the direction of the comparison (i.e., which group experienced a larger mean baseline adjusted
value), and the corresponding Tukey-adjusted p-value. P-values less than 0.05 provide evidence
of a significant difference.
The results at some study times were based on smaller sample sizes due to missing data or due to
animal deaths prior to the end of the study. A summary of the results from Tables 2 through 9 is
discussed below for each parameter.
Activity (Table 2, Figures la-Id): By Study Day 8, all groups had experienced a significant
decrease from baseline. This significant decrease from baseline continued intermittently in each
group until Study Day 23, but was more prevalent in Group 4. All significant pairwise group
comparisons involved Group 4. On Study Day 17 at 6AM-Noon and Study Day 18 at 6PM-
Midnight, the mean decrease from baseline in Group 4 was significantly different from the mean
1078-CG920794 Statistical Report - Telemetry F-10
-------
change from baseline in Group 1. On Study Day 19 at Noon-6PM, on Study Day 20 at Midnight-
6AM, 6AM-Noon, and 6PM-Midnight, on Study Day 21 at Midnight-6AM, on Study Day 24 at
Midnight-6AM, and on Study Day Midnight-6AM, the mean decrease from baseline in Group 4
was significantly different from the mean change from baseline in Group 2. On Study Day 37 at
Midnight-6AM, the mean decrease from baseline in Group 4 was significantly greater than that
in Group 3.
Heart Rate (Table 3, Figures 2a-2d): By Study Day 1 at Noon-6PM, all groups had experienced
significant increases from baseline. These significant increases continued intermittently for all
groups until Study Day 5 at 6AM-Noon. By Study Day 6 at Noon-6PM, all groups had
experienced a significant decrease from baseline. The significant decreases continued
intermittently and with increasing frequency until the end of the study, with more prevalence in
Groups 1 through 3 after Study Day 24. On Study Day 24 at 6AM-Noon and Noon-6PM, the
mean decrease from baseline in Group 1 was significantly greater than those in Groups 2
through 4.
RP Expiratory Time (Table 4, Figures 3a-3d): Significant decreases from baseline only occurred
in Groups 2 and 3 beginning on Study Day 0 at 6AM-Noon and continuing intermittently until
Study Day 39 at Midnight-6AM, but with increasing frequency after Study Day 25. On Study
Day 16 at Midnight-6AM, the mean decrease from baseline in Group 2 was significantly greater
than that in Group 3.
RP Inspiratory Time (Table 5, Figures 4a-4d): Significant decreases from baseline occurred in
Groups 1, 2, and 3 without any detectable pattern. In addition, there were significant increases
from baseline at three study times for either Group 2 or 3. On Study Day 1 at Midnight-6AM,
Study Day 2 at Midnight-6AM, and Study Day 5 at Midnight-6AM, the mean decrease from
baseline in Group 1 was significantly different from the mean increase from baseline in Group 2.
On Study Day 19 at 6AM-Noon, the mean decrease from baseline in Group 1 was significantly
different from the increase from baseline in Group 3. On Study Day 1 at Midnight-6AM, Study
Day 14 at Midnight-6AM, and Study Day 25 at 6AM-Noon and 6PM-Midnight, the mean
decrease from baseline in Group 4 was significantly different from the mean change from
baseline in Group 2. On Study Day 25 at 6AM-Noon and 6PM-Midnight, the mean increase
1078-CG920794 Statistical Report - Telemetry F-11
-------
from baseline in Group 3 was significantly different from the mean decrease from baseline in
Group 4.
RP Integral (Table 6, Figures 5a-5d): In Group 3, there were significant increases from baseline
starting on Study Day 5 at 6PM-Midnight and continuing intermittently through Study Day 17 at
6PM-Midnight and less frequently from Study Day 30 at Midnight-6AM through Study Day 38
at Midnight-6AM. In Group 2, there was a significant increase from baseline starting on Study
Day 32 at 6AM-Noon and continuing intermittently through Study Day 39 at 6AM-Noon. In
Group 4, there were significant increases from baseline on Study Day 9 at 6PM-Midnight, Study
Day 10 at Midnight-6AM, and Study Day 13 at Midnight-6AM. On Study Day 5 at
6PM-Midnight and Study Day 6 at Noon-6PM and 6PM-Midnight, the mean increase from
baseline in Group 3 was significantly different from the mean decrease from baseline in Group 2.
On Study Day 14 at Midnight-6AM and 6PM-Midnight and Study Day 15 at Midnight-6AM, the
mean increase from baseline in Group 3 was significantly different from the mean decrease from
baseline in Group 1.
RP Peak Amplitude (Table 7, Figures 6a-6d): In Group 2, there was typically a significant
increase from baseline beginning on Study Day 36 at 6AM-Noon and ending at Study Day 39 at
6AM-Noon. In Group 3, there was a significant increase from baseline starting on Study Day 5 at
Midnight-6AM and continuing consistently through Study Day 17 at Midnight-6AM. On Study
Day 5 at 6PM-Midnight, Study Day 6 at Midnight-6AM, Study Day 11 at Midnight-6AM, Study
Day 11 at 6AM-Noon, Study Day 12 at 6AM-Noon, and Study Day 14 Midnight-6AM through
Study Day 15 6AM-Noon, the mean increase from baseline in Group 3 was significantly
different from the mean decrease from baseline in Group 1. On Study Day 5 at 6PM-Midnight,
Study Day 6 at Midnight-6AM and 6AM-Noon, Study Day 14 at Midnight-6AM and Noon-
6PM, and Study Day 15 at 6AM-Noon the mean increase from baseline in Group 3 was
significantly different from the mean change from baseline in Group 2. On Study Day 5 at 6PM-
Midnight, the mean decrease from baseline in Group 2 was significantly different from the mean
increase from baseline in Group 4. On Study Day 14 at Noon-6PM, the mean increase from
baseline in Group 3 was significantly greater than that in Group 4.
1078-CG920794 Statistical Report - Telemetry F-12
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RP Respiratory Rate (Table 8, Figures 7a-7d): By Study Day 1 at Noon-6PM, all groups had
experienced a significant increase from baseline. These significant increases from baseline
continued intermittently throughout the study. In Groups 2 and 3, these significant increases
were more prevalent especially after Study Day 15 through the end of the study. Group 4 was the
only group that experienced significant decreases from baseline which occurred on Study Day 5
at 6AM-Noon and on Study Day 6 at 6AM-Noon. On Study Day 4 at Midnight-6AM, the mean
increase from baseline in Group 1 was significantly different from the mean changes from
baseline in Groups 2 and 3. Also, on Study Day 4 at Midnight-6AM, Study Day 5 at 6AM-Noon
and Noon-6PM, Study Day 6 at 6AM-Noon, and Study Day 10 at Midnight-6AM, the mean
decrease from baseline in Group 4 was significantly different from the mean increase from
baseline in Group 1. On Study Day 2 at 6AM-Noon, the mean increase from baseline in Group 2
was significantly greater than that in Group 3. On Study Day 13 at Midnight-6AM, the mean
increase from baseline in Group 4 was significantly different from the mean decrease from
baseline in Group 2. On Study Day 1 at 6PM-Midnight and on Study Day 5 at 6AM-Noon, the
mean increase from baseline in Group 3 was significantly different from the mean decrease from
baseline in Group 4.
Temperature (Table 9, Figures 8a-8d): In Group 1, there were significant increases and
decreases from baseline beginning on Study Day 1 at Noon-6PM and continuing intermittently
until Study Day 9 at 6PM-Midnight. In Group 2, there were significant increases from baseline
starting on Study Day 0 at 6PM-Midnight and continuing with decreasing frequency through
Study Day 29 at Midnight-6AM. Also in Group 2, significant decreases from baseline were
observed beginning on Study Day 30 at Noon-6PM and continuing with increasing frequency
through Study Day 38 at Noon-6PM. In Group 3, there was a significant increase from baseline
beginning on Study Day 1 atNoon-6PM and continuing intermittently through Study Day 38 at
6PM-Midnight. In Group 4, there was a significant increase from baseline beginning on Study
Day 1 at Midnight-6AM and continuing intermittently through Study Day 20 at 6AM-Noon. On
Study Day 27 at 6PM-Midnight, Study Day 33 at 6PM-Midnight, and Study Day 34 at 6PM-
Midnight, the mean increase from baseline in Group 3 was significantly different from the mean
decrease from baseline in Group 1. On Study Day 17 at Midnight-6AM, Study Day 19 at 6PM-
Midnight, and Study Day 20 at 6AM-Noon, the mean increase from baseline in Group 4 was
1078-CG920794 Statistical Report - Telemetry F-13
-------
significantly different from the mean change from baseline in Group 1. On Study Day 36 at
Noon-6PM and Study Day 37 at Noon-6PM, the mean decrease from baseline in Group 4 was
significantly different from the mean increase from baseline in Group 1. On Study Day 27 at
6PM-Midnight, Study Day 29 at 6PM-Midnight, and Study Day 32 at Noon-6PM, the mean
increase from baseline in Group 3 was significantly different from the mean change from
baseline in Group 2. On Study Day 3 at 6AM-Noon, Study Day 10 at Midnight-6AM, Study Day
19 at 6PM-Midnight, and Study Day 20 at 6AM-Noon, the mean increase from baseline in
Group 4 was significantly different from the mean change from baseline in Group 2. On Study
Day 19 at 6PM-Midnight, Study Day 27 at 6PM-Midnight, and Study Day 36 at Noon-6PM, the
mean change from baseline in Group 3 was significantly different from the mean change from
baseline in Group 4.
Table 10 contains the proportion of animals that were abnormal at any time during the study by
group for each parameter, as well as the mean duration of abnormality for the groups that had
abnormal animals. Note that some animals died prior to becoming abnormal. In addition,
Table 10 contains the results of Fisher's exact tests comparing the proportion of animals that
were abnormal in each group by parameter. The proportions of animals that became abnormal
were not significantly different between the groups for any of the telemetry parameters.
Table 11 contains the results of the overall log-rank tests for each parameter comparing the times
to abnormality between the groups. The times to abnormality were not significantly different
between the groups for any of the telemetry parameters. Figures 17 through 24 display the
Kaplan-Meier curves associated with time to abnormality for activity, heart rate, RP expiratory
time, RP inspiratory time, RP integral, RP peak amplitude, RP respiratory rate, and temperature,
respectively. The dots displayed throughout the Kaplan-Meier curves indicate that the time to
abnormality for an animal could not be observed beyond the indicated study time. For example,
if an animal were to die prior to experiencing abnormality then the time to abnormality for that
animal would be unobserved and censored at the animal's time of death or if an animal survived
the length of the study then the animal's time to death would be censored at the end of study.
Table 12 contains the results of the overall log-rank tests for each parameter comparing the
duration of abnormality between the groups. The durations of abnormality were not significantly
1078-CG920794 Statistical Report - Telemetry F-14
-------
different between the groups for any of the telemetry parameters. Figures 25 through 32 display
the Kaplan-Meier curves associated with duration of abnormality for activity, heart rate, RP
expiratory time, RP inspiratory time, RP integral, RP peak amplitude, and RP respiratory rate,
and temperature, respectively. The dots displayed throughout the Kaplan-Meier curves indicate
that the duration of abnormality for an animal could not be observed beyond the indicated study
time. For example, if an animal were still abnormal at the time of death or at the end of the study,
then the duration of abnormality for that animal would be unobserved.
1078-CG920794 Statistical Report - Telemetry F-15
-------
4. Conclusions
For activity, most significant shifts from baseline were decreases and all significant pairwise
group comparisons involved the targeted 10,000 CPU dose group (Group 4). On Study Day 17 at
6AM-Noon, on Study Day 19 at Noon-6PM, on Study Day 20 at Midnight-6AM, 6AM-Noon,
and 6PM-Midnight, on Study Day 21 at Midnight-6AM, on Study Day 24 at Midnight-6AM, and
on Study Day 37 at Midnight-6AM, the mean decrease from baseline activity in the targeted
10,000 CPU dose group (Group 4) was significantly different from the mean change from
baseline activity in the targeted 100 CPU dose group (Group 2). Prior to Study Day 6, most
significant shifts from baseline for heart rate were increases, while most after Study Day 6 were
decreases. On Study Day 24 at 6AM-Noon and Noon-6PM, the mean decrease from baseline
heart rate in the control group (Group 1) was significantly greater than those in the challenged
groups (Groups 2 through 4). There were no significant shifts from baseline RP expiratory time
in the control group (Group 1) or the targeted 10,000 CPU dose group (Group 4). There were no
significant shifts from baseline RP inspiratory time in the targeted 10,000 CPU dose group
(Group 4). There were no significant shifts from baseline RP integral in the control group
(Group 1) and all significant pairwise group comparisons involved the targeted 1,000 CPU dose
group (Group 3). There were no significant shifts from baseline RP peak amplitude in the control
group (Group 1) or the targeted 10,000 CPU dose group (Group 4) and all but one significant
pairwise group comparisons involved the targeted 1,000 CPU dose group (Group 3). There were
significant increases from baseline RP respiratory rate in all groups at some time during the
study; however, they were more prevalent in the targeted 100 CPU and 1,000 CPU dose groups
(Groups 2 and 3, repectively). There were significant increases from baseline temperature in all
groups at some time during the study and all significant pairwise group comparisons involved
either the targeted 1,000 CPU or 10,000 CPU dose groups (Groups 3 or 4, respectively). In terms
of the proportion of animals that became abnormal, time to abnormality, and duration of
abnormality, there were no significant differences between the groups for any of the telemetry
parameters.
1078-CG920794 Statistical Report - Telemetry F-16
-------
Table 2. Summary of the ANOVA Results for the Baseline Adjusted Six-Hour Averages
for Activity (Counts/Minute)
Study
Day
0
1
2
3
4
5
6
7
8
9
10
Time
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Mean Baseline Adjusted Value,
by Group
1
1.26
0.51
-0.02
-0.13
0.12
0.18
0.06
-0.09
1.38|
-0.36
0.12
-0.11
-0.24
-0.34
0.03
-0.37
1.20
-0.09
-0.20
-0.40
-0.62
0.17
-0.27
-0.16
-0.69|
-0.06
-0.35
-0.33
-0.25
-0.02
-0.38
-0.23
-0.09
-0.05
-0.43
-0.28
0.81
0.26
0.24
0.07
-0.12
0.12
0.00
2
2.59
0.00
-0.24
-0.14
0.28
-0.20
-0.29
-0.14
0.45
-0.08
-0.39
-0.24
-0.09
-0.40
-0.34
-0.11
0.62
-0.39
-0.48
-0.24
-0.25
-0.12
-0.20
-0.08
-0.27
-0.25
-0.22
0.11
0.00
-0.26
-0.46|
-0.07
0.00
-0.29
-0.50
-0.21
0.71
0.29
-0.40
-0.09
0.11
0.06
-0.19
3
NA
0.45
-0.31
-0.11
-0.06
0.09
0.02
-0.09
0.42
-0.08
-0.05
-0.19
-0.17
0.07
-0.14
-0.08
0.20
-0.22
-0.20
-0.14
-0.25
-0.44
0.25
-0.01
-0.28
-0.27
-0.04
-0.06
-0.64|
-0.11
-0.12
-0.05
-0.33
-0.06
-0.41
-0.22
0.63
0.05
0.10
-0.11
-0.10
-0.22
-0.12
4
NA
0.43
-0.43
0.00
-0.01
0.08
-0.56|
-0.22
0.75|
-0.11
-0.54|
-0.18
-0.42|
0.00
-0.62|
-0.33
0.30
-0.16
-0.55|
-0.15
-0.51|
-0.32
-0.13
-0.10
-0.27
-0.42
-0.55|
-0.36
-0.56|
-0.03
-0.42
-0.04
-0.38
-0.24
-0.59|
-0.52|
0.57
0.44
-0.24
-0.36
0.05
0.11
-0.47
Group
Effect
P-Value
0.4460
0.4940
0.5805
0.7176
0.5958
0.6716
0.0685
0.9197
0.1959
0.9103
0.0622
0.9560
0.5066
0.5777
0.2685
0.4573
0.4668
0.9126
0.5694
0.8237
0.5463
0.3937
0.4168
0.9590
0.3443
0.7470
0.1947
0.4427
0.1750
0.9354
0.5552
0.9380
0.7363
0.8428
0.9500
0.6484
0.9675
0.9087
0.4261
0.6555
0.8385
0.8751
0.7284
Estimated Difference
(Relationship)
Tukey's P-Value*
1078-CG920794 Statistical Report - Telemetry
F-17
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Table 2. (Continued)
Study
Day
11
12
13
14
15
16
17
18
19
20
Time
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Mean Baseline Adjusted Value,
by Group
1
-0.16
-0.25
-0.06
-0.07
-0.32
-0.66|
-0.09
-0.16
-0.07
-0.60
0.06
-0.10
-0.07
-0.05
-0.03
-0.35
-0.12
0.14
-0.44
-0.18
-0.03
-0.12
-0.08
-0.38
-0.29
0.43
-0.28
-0.47
-0.32
0.44
-0.19
-0.10
-0.16
-0.67|
-0.28
-0.28
-0.43
-0.981
-0.41
-0.35
2
-0.04
0.49
-0.26
-0.33
-0.01
-0.39
-0.18
-0.20
-0.01
-0.18
-0.19
-0.10
0.23
0.40
-0.25
-0.32
0.26
0.27
-0.23
-0.15
0.23
0.38
-0.25
-0.34
0.06
0.20
-0.14
-0.25
-0.17
0.31
-0.14
-0.51|
-0.29
-0.25
-0.08
-0.27
-0.04
-0.14
-0.23
-0.24
3
-0.28
0.46
-0.45
-0.08
-0.23
-0.66|
-0.60
-0.02
-0.02
-0.67|
-0.78
-0.12
-0.26
0.11
-0.69
-0.32
-0.27
-0.26
-0.94|
-0.45
-0.28
-0.08
-0.38
-0.52|
-0.27
-0.26
-0.37
-0.49
-0.18
-0.14
-0.20
-0.49
-0.24
-0.67|
-0.80|
-0.38
-0.29
-0.711
-0.52
-0.38
4
-0.01
0.31
0.07
-0.19
-0.17
-0.501
-0.781
-0.56
-0.30
-0.37
-0.92
-0.71
-0.29
0.18
-0.75
-0.761
-0.19
0.08
-0.69
-0.89
-0.43
0.57
-0.91
-1.051
-0.71
-1.111
-0.961
-0.80
-0.73
-0.03
-1.011
-1.261
-0.821
-1.101
-1.401
-0.73
-1.141
-1.301
-1.361
-1.301
Group
Effect
P-Value
0.7960
0.3689
0.8382
0.8054
0.7057
0.6539
0.2988
0.3682
0.7930
0.4480
0.2120
0.3396
0.4274
0.8571
0.4608
0.5255
0.5335
0.5665
0.4288
0.2617
0.3379
0.3295
0.4652
0.1082
0.3538
0.0162*
0.1565
0.5931
0.3686
0.7492
0.0606
0.0205 *
0.2426
0.0784
0.0396 *
0.7050
0.0201 *
0.0171 *
0.1634
0.0196*
Estimated Difference
(Relationship)
Tukey's P-Value*
1. 54 (4<1) 0.0166
1.31 (4<2) 0.0321
1.16 (4<1) 0.0123
1.31 (4<2) 0.0381
1. 10 (4<2) 0.0124
1. 16 (4<2) 0.0172
1.06(4<2)0.0167
1078-CG920794 Statistical Report - Telemetry
F-18
-------
Table 2. (Continued)
Study
Day
21
22
23
24
25
26
27
28
29
30
31
Time
Midnight -6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight -6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight -6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight -6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight -6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight -6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight -6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight -6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight -6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight -6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight -6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Mean Baseline Adjusted Value,
by Group
1
-0.48
-0.75|
-0.51
-0.53
-0.33
-0.92|
-0.22
-0.35
-0.44
-0.24
-0.52
-0.35
-0.51
-0.54
-0.60
-0.55
-0.56
-0.52
-0.66
-0.14
-0.46
-0.94|
-0.07
-0.26
-0.42
-0.79|
-0.48
-0.44
-0.36
-0.49
-0.46
-0.38
-0.61
-1.07|
-0.39
-0.44
-0.61
-0.28
-0.51
-0.48
-0.51
-0.80
-0.44
-0.63
2
-0.07
-0.26
-0.26
-0.39
0.01
-0.44
-0.36
-0.62|
0.14
0.45
-0.22
-0.44
-0.15
-0.20
-0.20
-0.16
-0.14
0.27
-0.15
-0.33
-0.09
-0.33
-0.08
-0.22
0.12
-0.12
-0.10
-0.33
0.16
-0.10
-0.34
-0.35
0.12
-0.22
-0.24
-0.25
0.23
0.51
-0.28
-0.40
-0.08
-0.33
-0.13
-0.18
3
-0.33
-0.66|
-0.90|
-0.47
-0.38
-0.69|
-0.88|
-0.56|
-0.39
-0.25
-0.99|
-0.13
-0.20
-0.57
-0.79
-0.43
-0.21
-0.13
-0.81
-0.19
-0.23
-0.78|
-0.65
-0.28
-0.09
-0.69|
-0.72
-0.05
-0.07
-0.38
-0.74
-0.19
-0.27
-0.80|
-0.79
-0.10
-0.09
-0.19
-0.971
-0.28
-0.23
-0.741
-0.52
-0.29
4
-0.951
-0.61
-1.45
-0.98
-0.64
-0.77
-1.38
-1.181
-1.03
-0.50
-1.17
-1.04
-1.24
-1.25
-1.70
-0.95
-0.97
-0.53
-1.50
-1.01
-0.74
-1.27
-1.37
-0.49
-0.67
-0.61
-1.49
-1.04
-0.92
-0.36
-1.48
-1.07
-0.73
-1.24
-1.53
-0.72
-0.42
-0.53
-1.52
-1.11
-0.89
-1.13
-1.28
-1.26
Group
Effect
P-Value
0.0483 *
0.4936
0.0734
0.3777
0.4265
0.5307
0.1501
0.1421
0.1066
0.0764
0.2569
0.2137
0.0389*
0.1635
0.1377
0.2810
0.2423
0.1632
0.1756
0.1083
0.5828
0.1259
0.1449
0.9716
0.3134
0.1906
0.1929
0.1025
0.1339
0.7820
0.3427
0.1999
0.0677
0.0681
0.2153
0.7038
0.3361
0.2561
0.1909
0.2642
0.3883
0.3839
0.4368
0.1548
Estimated Difference
(Relationship)
Tukey's P-Value*
0.88 (4<2) 0.031 8
1 .09 (4<2) 0.0348
1078-CG920794 Statistical Report - Telemetry
F-19
-------
Table 2. (Continued)
Study
Day
32
33
34
35
36
37
38
39
Time
Midnight -6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight -6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight -6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight -6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight -6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight -6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight -6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight -6AM
6AM - Noon
Mean Baseline Adjusted Value,
by Group
1
-0.36
-0.42
-0.57
-0.62
-0.41
-0.89|
-0.20
-0.47
-0.45
-0.74
-0.63
-0.25
-0.43
-0.51
-0.45
-0.33
-0.35
-0.63
-0.27
-0.44
-0.47
-0.49
-0.67
-0.40
-0.54
-0.821
-0.39
-0.39
-0.36
-0.65
2
-0.02
0.25
-0.18
-0.31
0.01
-0.19
-0.13
-0.13
-0.01
-0.34
-0.30
-0.37
-0.09
-0.19
-0.43
-0.10
0.05
-0.28
-0.39
-0.40
-0.06
-0.08
-0.33
-0.29
-0.08
-0.39
-0.38
-0.37
0.18
0.06
3
-0.26
-0.40
-0.88
-0.46
-0.27
-0.60
-0.72
-0.11
-0.11
-0.70|
-0.87
-0.03
-0.12
-0.54
-0.81
0.12
-0.14
-0.89|
-1.00|
-0.21
-0.17
-0.15
-0.93|
-0.27
-0.18
-0.851
-0.911
-0.05
-0.10
-0.53
4
-0.65
-0.08
-1.29
-1.26
-1.11
-1.29
-1.65
-0.68
-0.71
-1.08
-1.57
-0.83
-0.77
-0.89
-1.49
-1.05
-0.81
-1.16
-1.88
-1.18
-1.21
-0.60
-1.53
-1.09
-0.94
-1.09
-1.47
-0.83
-0.85
-0.13
Group
Effect
P-Value
0.4727
0.4141
0.3110
0.0918
0.1943
0.1484
0.1651
0.7059
0.4405
0.3990
0.2993
0.3355
0.4592
0.5453
0.4024
0.0954
0.2472
0.0963
0.0758
0.1611
0.0197*
0.6323
0.2759
0.0855
0.0941
0.2692
0.2685
0.4049
0.1937
0.4250
Estimated Difference
(Relationship)
Tukey's P-Value*
1.16(4<2)0.0155
1. 04 (4<3) 0.0358
# Cells contain all pairwise comparisons that were significant at the 0.05 level. The format within each
cell is (1) the difference of means, (2) the relationship between the corresponding pair of group
means shown in parentheses [For example, "(1<2)" indicates that the mean baseline adjusted value
in Group 2 was significantly greater than that in Group 1], and (3) the Tukey-adjusted p-value.
NA Data was not available for this group at this study time.
|, 1 "|" indicates that the mean at the study time was significantly greater than that at baseline, while
"1" indicates that the mean at the study time was significantly less than that at baseline (at the
0.05 level).
* Group effect was significant at the 0.05 level.
1078-CG920794 Statistical Report - Telemetry
F-20
-------
Table 3. Summary of the ANOVA Results for the Baseline Adjusted Six-Hour Averages
for Heart Rate (BPM)
Study
Day
0
1
2
3
4
5
6
7
8
9
Time
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Mean Baseline Adjusted Value,
by Group
1
11.69
11.51
5.85
2.39
-6.54
11.16|
18.63|
9.58
-13.61
4.90
19.96|
9.99
-5.54
9.99
16.66
6.91
-8.06
20.22|
24.82|
12.07
11.40
5.22
11.68
10.31
-0.76
-19.64|
-10.48
-3.12
-22.29J,
-0.77
-4.01
-1.48
-16.89J,
-9.11
-10.12
-10.54
-38.06J,
-7.31
26.41
2
22.39
7.89
14.33|
8.29
-6.58
10.22|
17.21|
16.10|
-18.28|
8.49
20.02|
18.66T
-4.42
10.30
11.44
7.84
-12.93
8.97
22.48|
23.90|
19.05|
2.66
15.69
28.23|
1.99
-12.96|
0.55
10.48
-12.04|
-3.97
7.71
16.33
-8.72
-11.06|
-10.99
0.14
-29.37J,
-15.15|
9.36
3
NA
6.14
1.48
-4.38
-11.49|
9.10|
8.28
-3.38
-35.25|
8.99
19.48|
3.23
-11.21
5.58
5.24
-6.91
-22.34|
14.76|
19.97|
1.63
10.43|
-6.71
8.92
0.06
-5.15
-12.58|
-13.16
-17.33
-22.48J,
-0.80
8.03
6.99
-12.40|
-2.91
-14.53
-10.96
-44.99J,
-8.96
24.15|
4
NA
8.86
2.32
4.58
-10.33J,
10.42|
14.03|
5.81
-26.39|
7.11
18.15|
11.26
-8.30
8.69
11.90
5.77
-15.53
6.01
17.64|
15.15
5.68
-0.79
2.26
2.81
-0.27
-16.74|
-16.03
-8.24
-20.13J,
-4.02
-6.29
-2.08
-19.68J,
-2.58
0.98
-2.26
-35.18J,
-19.65|
15.29
Group
Effect
P-Value
0.4463
0.8343
0.3218
0.4670
0.7360
0.9822
0.5074
0.0960
0.1988
0.9534
0.9939
0.1912
0.7519
0.8699
0.7603
0.3249
0.5722
0.3102
0.9076
0.1792
0.0919
0.2043
0.5647
0.1022
0.4592
0.3634
0.4951
0.2276
0.3760
0.9057
0.3325
0.2658
0.3688
0.4336
0.5906
0.7381
0.7464
0.3869
0.5795
Estimated Difference
(Relationship)
Tukey's P-Value*
1078-CG920794 Statistical Report - Telemetry
F-21
-------
Table 3. (Continued)
Study
Day
10
11
12
13
14
15
16
17
18
19
Time
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Mean Baseline Adjusted Value,
by Group
1
14.27
-12.56
-3.13
23.47
22.05
3.17
-2.90
12.83
15.45
-11.43
-18.63
-0.61
4.34
-17.47
-8.14
3.06
2.73
-12.14
-2.16
11.14
16.24
-15.18
-13.53
5.40
7.30
-12.04
-13.85J,
-11.06
-10.22
-22.091
-30.09J,
-23.35
-18.06
-25.10
-10.54
-4.90
-5.85
-26.22J,
-23.69|
-10.83
2
23.74T
-10.18
1.93
13.04
28.32|
2.79
-8.92
3.50
14.79
-16.97|
-29.101
-11.00
-2.44
-27.59|
-27.55|
-18.64|
5.16
-4.06
-4.44
6.67
19.06
-11.12
-13.80
4.33
21.21|
-4.08
-20.52J,
-2.91
7.62
-16.66|
-31.34|
-18.35
-5.78
-18.09
-15.67|
-8.88
-2.82
-23.43J,
-27.95|
-15.52
3
14.54
-18.90J,
-7.00
11.69
17.55
-10.94
-13.67
9.34
13.56
-19.97|
-30.29|
-1.92
6.82
-26.62J,
-23.131
-2.22
4.72
-16.92|
-13.41
4.62
8.96
-23.69|
-22.04J,
-9.22
-3.44
-20.57|
-14.97|
-3.06
1.90
-19.59|
-24.28J,
-5.42
6.68
-18.77
-5.44
-7.48
-9.81
-23.79J,
-27.46|
-13.90
4
10.74
-21.64|
-0.39
15.22
15.84
3.40
-5.48
16.63
24.68|
-8.41
-19.58|
16.22
31.68
-3.23
-23.38|
-6.87
7.59
-14.38
-13.45
6.29
18.58
-5.71
-24.72
-13.94
-3.48
-13.98
-16.48
-4.09
0.35
-23.99|
-35.45J,
-18.19
-12.64
-20.99
-18.51
-12.32
-12.31
-25.80J,
-25.47|
6.43
Group
Effect
P-Value
0.7014
0.4178
0.8770
0.9107
0.7850
0.5551
0.8380
0.8104
0.8218
0.5901
0.5794
0.4173
0.2790
0.1084
0.2634
0.1888
0.9861
0.4534
0.6713
0.9758
0.8736
0.5048
0.6048
0.3558
0.1851
0.1472
0.7082
0.9136
0.5236
0.6790
0.5061
0.7521
0.7285
0.9660
0.3791
0.9525
0.9081
0.9423
0.9359
0.5033
Estimated Difference
(Relationship)
Tukey's P-Value*
1078-CG920794 Statistical Report - Telemetry
F-22
-------
Table 3. (Continued)
Study
Day
20
21
22
23
24
25
26
27
28
Time
Midnight -6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight -6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight -6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight -6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight -6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight -6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight -6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight -6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight -6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Mean Baseline Adjusted Value,
by Group
1
-10.21
-30.69J,
-28.121
-23.87
-20.05
-35.13J,
-37.29|
-34.68J,
-29.88|
-32.78J,
-29.94|
-20.06
-23.92
-28.66|
-16.24
11.09
-5.25
-60.19J,
-95.46|
-67.061
-49.08J,
-35.25|
-12.51
2.28
-1.50
-16.97
-21.42|
-16.05
-21.99
-15.41
-24.75|
-24.76
-25.36
-31.10|
-29.051
-27.08J,
2
-1.05
-29.95J,
-34.41|
-28.06J,
-13.95
-35.04J,
-36.161
-28.78J,
-14.00
-27.74|
-28.29|
-25.42|
-13.34
-25.47|
-25.151
2.39
8.92
-25.111
-36.71|
-36.25|
-23.05J,
-28.85|
-11.23
1.27
5.16
-20.97|
-24.051
-18.74|
-10.16
-19.22|
-18.21|
-13.92
-2.10
-29.53|
-28.001
-13.46
3
-15.40
-30.01 1
-30.97|
-30.60J,
-25.32
-35.45J,
-40.18|
-32.85J,
-32.121
-35.61 1
-30.06|
-34.20J,
-29.46J,
-30.01|
-28.91 1
5.80
3.86
-25.30J,
-36.64|
-34.23|
-30.97J,
-33.63|
-13.85
-4.76
-7.54
-25.111
-26.151
-14.01
-18.59
-21.44|
-24.45|
-5.82
-14.17
-30.48|
-35.70|
-7.12
4
15.45
-7.15
-19.12
-18.99
-7.55
-21.19
-49.97|
-38.45
-35.69
-45.81|
-53.35|
-47.06
-36.81
-36.121
-49.35
-12.61
-14.31
-28.92
-46.36|
-36.98
-25.07
-25.81
-32.99
-3.22
-1.14
-14.07
-22.50
-17.86
-19.11
-16.22
-22.12
-17.74
-21.49
-28.58
-42.92|
-32.26
Group
Effect
P-Value
0.3356
0.1087
0.4607
0.8502
0.7164
0.5978
0.4116
0.9456
0.4073
0.2593
0.2238
0.4128
0.4478
0.5818
0.2923
0.6263
0.3754
0.0020 *
<0.0001 *
0.1475
0.2511
0.8643
0.4163
0.9571
0.7488
0.7008
0.9710
0.9752
0.8226
0.8905
0.8785
0.4884
0.3753
0.9980
0.5428
0.2266
Estimated Difference
(Relationship)
Tukey's P-Value*
-35.07 (1<2) 0.0027
-34.89 (1<3) 0.0039
-31 . 26 (1<4) 0.0340
-58.75 (1<2) <0.0001
-58.82 (1<3) <0.0001
-49.11 (1<4) 0.0024
1078-CG920794 Statistical Report - Telemetry
F-23
-------
Table 3. (Continued)
Study
Day
29
30
31
32
33
34
35
36
37
Time
Midnight -6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight -6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight -6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight -6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight -6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight -6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight -6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight -6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight -6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Mean Baseline Adjusted Value,
by Group
1
-27.86|
-33.71|
-21.32|
-8.87
-16.80
-28.57|
-20.98
-32.50J,
-32.82|
-34.99J,
-45.94|
-42.50|
-37.28J,
-37.111
-23.35J,
-21.63
-27.40
-33.45|
-33.71|
-19.31
-24.45
-34.98|
-28.52J,
-22.11
-25.75
-39.97|
-28.99J,
-11.88
-18.54
-46.67J,
-33.011
-22.73
-25.12
-38.12J,
-19.01
-19.70
2
-6.16
-30.78J,
-27.57|
-8.97
-2.67
-23.03J,
-13.88
-15.81
-15.48
-35.60J,
-38.63|
-31.72|
-21.19|
-30.78|
-14.92
-13.19
-9.99
-34.86|
-32.50J,
-12.72
-1.72
-35.40|
-25.43|
-10.03
-0.88
-34.58|
-30.81 1
-5.26
1.72
-37.43|
-35.97|
-9.05
0.23
-32.48J,
-19.64|
-6.47
3
-12.62
-34.46J,
-31.48|
-2.56
-9.60
-27.85|
-22.081
-19.51|
-26.81|
-37.38J,
-37.87|
-30.75J,
-28.70J,
-33.94|
-14.41
-11.94
-20.74
-33.23|
-31.08J,
-4.86
-12.97
-29.51|
-27.43|
-3.68
-10.41
-38.161
-31.94|
-5.39
-5.84
-42.91|
-36.76|
-10.71
-11.43
-33.24J,
-9.13
-7.78
4
-14.22
-28.61
-32.22
-20.62
-12.07
-27.89
-33.57
-30.42
-33.09
-32.09J,
-35.75|
-40.17
-34.94
-35.72|
-18.08
-27.16
-31.57
-46.36|
-43.76|
-25.63
-27.69
-40.05|
-33.16
-22.43
-28.31
-41.70|
-41.38
-21.84
-30.78
-40.77|
-44.83
-21.21
-25.18
-29.82
-22.23
-13.76
Group
Effect
P-Value
0.3165
0.9067
0.7266
0.7451
0.6872
0.9268
0.4893
0.3678
0.4163
0.9453
0.6937
0.7865
0.6436
0.8689
0.7999
0.6045
0.5081
0.4498
0.6880
0.6669
0.4180
0.7996
0.9470
0.5495
0.2195
0.8987
0.8217
0.7304
0.2296
0.7754
0.8611
0.7220
0.2560
0.9354
0.7427
0.7258
Estimated Difference
(Relationship)
Tukey's P-Value*
1078-CG920794 Statistical Report - Telemetry
F-24
-------
Table 3. (Continued)
Study
Day
38
39
Time
Midnight -6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight -6AM
6AM - Noon
Mean Baseline Adjusted Value,
by Group
1
-31.01|
-41.96|
-36.56|
-24.70
-27.32
-60.17|
2
-5.40
-43.37|
-41.05|
-12.93
-0.45
-48.131
3
-15.20
-43.74|
-39.42|
-17.92
-16.32
-62.30J,
4
-31.23
-36.13
-42.42
-29.89
-36.07
-69.60J,
Group
Effect
P-Value
0.1913
0.9056
0.9782
0.7239
0.1444
0.4570
Estimated Difference
(Relationship)
Tukey's P-Value*
# Cells contain all pairwise comparisons that were significant at the 0.05 level. The format within each
cell is (1) the difference of means, (2) the relationship between the corresponding pair of group
means shown in parentheses [For example, "(1<2)" indicates that the mean baseline adjusted value
in Group 2 was significantly greater than that in Group 1], and (3) the Tukey-adjusted p-value.
NA Data was not available for this group at this study time.
|, J, "|" indicates that the mean at the study time was significantly greater than that at baseline, while
"J," indicates that the mean at the study time was significantly less than that at baseline (at the
0.05 level).
* Group effect was significant at the 0.05 level.
1078-CG920794 Statistical Report - Telemetry
F-25
-------
Table 4. Summary of the ANOVA Results for the Baseline Adjusted Six-Hour Averages
for RP Expiratory Time (Seconds)
Study
Day
0
1
2
3
4
5
6
7
8
9
10
Time
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Mean Baseline Adjusted Value,
by Group
1
-0.07
-0.05
0.04
0.00
0.00
-0.04
-0.02
0.02
0.06
0.04
-0.04
-0.04
0.00
-0.01
-0.06
-0.04
0.04
-0.02
-0.05
-0.02
0.00
-0.04
-0.09
-0.06
0.02
0.02
0.00
-0.02
0.04
-0.02
-0.02
-0.01
0.02
-0.03
-0.02
-0.02
0.09
0.01
-0.10
-0.09
-0.04
-0.11
-0.10
2
-0.23|
-0.06|
-0.02
-0.01
-0.03
-0.05|
-0.01
0.00
-0.05
-0.03
-0.03
0.00
0.00
-0.07
0.00
0.02
-0.03
-0.04
-0.01
-0.01
-0.02
-0.06
-0.02
-0.03
0.03
-0.02
-0.03
-0.02
0.00
-0.03
-0.03
-0.02
0.00
-0.02
0.01
0.01
-0.05
-0.07
-0.03
-0.01
0.03
-0.02
0.01
3
NA
-0.09|
-0.02
-0.01
0.00
-0.03
-0.05
0.02
0.06
0.00
-0.04
0.00
0.02
-0.04
-0.02
0.03
0.05
-0.01
-0.04
0.00
-0.01
-0.03
-0.07
0.01
0.03
-0.04
-0.02
0.04
0.03
-0.07
-0.07
-0.03
0.01
-0.05
0.00
-0.01
0.03
-0.04
-0.12|
-0.04
-0.02
-0.06
-0.11
4
NA
-0.03
0.02
0.01
0.02
-0.03
0.05
0.04
-0.01
-0.01
0.00
0.04
0.04
-0.02
-0.01
0.06
-0.02
0.01
-0.03
-0.01
0.03
0.01
0.00
0.02
0.05
0.01
-0.01
-0.01
0.01
-0.03
-0.02
-0.05
0.00
-0.01
0.01
-0.04
-0.02
-0.04
-0.07
-0.01
-0.02
-0.09
-0.08
Group
Effect
P-Value
0.2303
0.4301
0.1971
0.8226
0.5398
0.8870
0.1079
0.7157
0.0367 *
0.5082
0.7861
0.5450
0.8434
0.6614
0.5643
0.1275
0.2461
0.8102
0.9354
0.9831
0.7613
0.4432
0.3260
0.5559
0.9243
0.4250
0.9640
0.5966
0.8399
0.6721
0.6873
0.7643
0.9881
0.8660
0.9823
0.6680
0.0953
0.6906
0.3885
0.4767
0.5682
0.6847
0.5270
Estimated Difference
(Relationship)
Tukey's P-Value*
1078-CG920794 Statistical Report - Telemetry
F-26
-------
Table 4. (Continued)
Study
Day
11
12
13
14
15
16
17
18
19
20
21
Time
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Mean Baseline Adjusted Value,
by Group
1
-0.04
0.06
0.03
-0.07
-0.07
0.01
-0.04
-0.11
-0.11
-0.01
-0.06
-0.11
-0.12
0.00
-0.04
-0.10
-0.08
-0.03
-0.04
-0.12
-0.09
0.05
-0.01
-0.03
-0.03
0.00
-0.05
-0.09
-0.12
0.03
-0.03
-0.06
-0.05
0.02
-0.03
-0.08
-0.06
0.05
0.01
-0.05
-0.03
0.01
-0.05
-0.06
2
0.00
0.00
0.02
0.07
0.08
0.12
0.08
0.04
0.07
0.14
0.03
0.06
-0.06
-0.06
-0.06
-0.08
-0.11|
-0.06|
-0.06
-0.08
-0.13|
-0.05
-0.01
-0.07
-0.03
-0.03
-0.06
-0.05
-0.03
-0.01
-0.03
-0.05
-0.02
0.00
-0.08
-0.05
-0.08
-0.02
-0.06
-0.05
-0.03
0.01
-0.05
-0.05
3
-0.04
0.02
-0.01
-0.09
-0.05
0.02
-0.03
-0.09
-0.02
0.06
-0.02
-0.07
-0.04
-0.06
-0.06
-0.06
-0.05
-0.04
-0.03
-0.04
0.00
-0.04
0.03
-0.02
-0.06
-0.04
-0.06
-0.07
-0.11|
-0.05
-0.05
-0.04
-0.07
-0.01
-0.09
-0.07
-0.03
0.01
-0.06
-0.07
-0.05
-0.04
-0.08
-0.03
4
-0.08
-0.06
-0.01
-0.09
-0.11
0.00
-0.04
-0.16
-0.21
-0.02
-0.07
-0.12
-0.14
-0.03
-0.03
-0.08
-0.03
-0.01
0.02
-0.04
-0.03
-0.10
-0.04
-0.01
-0.01
-0.01
0.02
-0.03
0.00
-0.06
-0.02
-0.01
0.00
0.02
0.03
-0.07
-0.07
0.02
-0.02
-0.07
-0.08
-0.05
-0.04
-0.09
Group
Effect
P-Value
0.7490
0.4971
0.9792
0.2976
0.3435
0.4570
0.6093
0.2805
0.2110
0.6623
0.8478
0.4752
0.3849
0.5038
0.9432
0.9283
0.5403
0.5476
0.5431
0.5476
0.0366 *
0.2376
0.8787
0.6204
0.9223
0.8942
0.6032
0.8045
0.2417
0.6218
0.9732
0.9266
0.6676
0.9212
0.3969
0.9724
0.9248
0.7217
0.6195
0.9788
0.8881
0.7225
0.9254
0.8662
Estimated Difference
(Relationship)
Tukey's P-Value*
-0.13(2<3)0.0313
1078-CG920794 Statistical Report - Telemetry
F-27
-------
Table 4. (Continued)
Study
Day
22
23
24
25
26
27
28
29
30
31
Time
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Mean Baseline Adjusted Value,
by Group
1
-0.07
-0.04
-0.03
-0.06
-0.05
-0.02
0.02
-0.10
-0.11
-0.02
0.08
-0.03
0.02
-0.01
0.00
-0.09
-0.08
-0.01
-0.04
-0.07
-0.07
-0.03
-0.06
-0.07
-0.06
-0.02
-0.01
-0.06
0.00
0.05
-0.05
-0.08
-0.02
0.03
-0.03
0.01
0.03
0.02
-0.03
0.00
2
-0.05
-0.02
-0.06
-0.03
-0.05
-0.08|
0.00
-0.05
-0.05
-0.02
-0.07
-0.05
-0.07
-0.04
-0.04
-0.09
-0.07
-0.04
-0.13|
-0.06
-0.05
-0.03
-0.12|
-0.07
-0.07
-0.03
-0.08
-0.08
-0.08
-0.03
-0.08|
-0.09
-0.11
-0.07
-0.06
-0.06
-0.05
-0.02
-0.11|
-0.12|
3
-0.01
-0.02
-0.06
-0.09
-0.01
-0.06
-0.02
-0.11
-0.05
-0.02
-0.05
-0.01
0.00
-0.06
-0.06
-0.09
-0.05
-0.01
-0.12|
-0.11
-0.03
-0.07|
-0.09
-0.15|
-0.11
-0.03
-0.08
-0.15|
-0.06
-0.02
-0.07
-0.16|
-0.11
-0.10|
-0.06
-0.10
-0.03
-0.01
-0.11|
-0.10
4
-0.09
-0.08
-0.06
-0.06
-0.06
-0.14
-0.02
-0.09
-0.04
-0.02
-0.01
-0.03
-0.01
-0.08
0.07
-0.07
0.00
0.01
-0.02
-0.03
-0.01
-0.01
-0.03
-0.06
-0.03
0.02
-0.01
-0.03
-0.05
0.05
0.02
-0.03
-0.04
-0.06
0.05
-0.02
0.03
0.04
-0.01
0.00
Group
Effect
P-Value
0.7908
0.6902
0.9532
0.8090
0.9382
0.3185
0.9679
0.8372
0.7805
0.9994
0.1963
0.9221
0.7197
0.7461
0.4628
0.9931
0.7799
0.8455
0.2646
0.7308
0.8745
0.5612
0.6285
0.5994
0.7659
0.8981
0.6086
0.4187
0.8161
0.3640
0.3569
0.5360
0.6069
0.2239
0.2726
0.5025
0.4989
0.7265
0.2634
0.3400
Estimated Difference
(Relationship)
Tukey's P-Value*
1078-CG920794 Statistical Report - Telemetry
F-28
-------
Table 4. (Continued)
Study
Day
32
33
34
35
36
37
38
39
Time
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Mean Baseline Adjusted Value,
by Group
1
0.00
0.04
0.03
-0.03
-0.03
0.00
-0.09
-0.12
-0.02
-0.02
-0.07
-0.11
-0.05
-0.02
-0.09
-0.14
-0.05
0.03
-0.08
-0.10
-0.08
0.00
-0.04
-0.09
-0.06
0.01
-0.08
-0.10
-0.05
0.06
2
-0.07
-0.05
-0.07
-0.08
-0.09
-0.05
-0.12|
-0.11
-0.08
-0.02
-0.13|
-0.13|
-0.08
-0.03
-0.10|
-0.13|
-0.12|
-0.04
-0.08
-0.13|
-0.09|
-0.05
-0.06
-0.10
-0.091
0.00
-0.081
-0.141
-0.111
-0.07
3
-0.05
-0.05
-0.05
-0.11
-0.07
-0.06
-0.121
-0.171
-0.09
-0.04
-0.141
-0.181
-0.10
-0.07
-0.121
-0.181
-0.09
-0.02
-0.10
-0.181
-0.07
-0.04
-0.07
-0.12
-0.05
-0.02
-0.121
-0.161
-0.09
-0.06
4
0.02
-0.02
0.01
0.08
0.07
0.08
-0.01
-0.06
-0.04
-0.01
0.01
-0.10
-0.04
0.02
-0.02
-0.07
-0.09
-0.07
-0.10
-0.19
-0.08
-0.09
0.02
-0.10
-0.04
-0.01
-0.02
-0.14
-0.11
-0.12
Group
Effect
P-Value
0.6238
0.3253
0.4048
0.1941
0.2705
0.1092
0.5301
0.6770
0.8377
0.9443
0.1304
0.8093
0.8315
0.3695
0.5036
0.7201
0.8136
0.6481
0.9879
0.6769
0.9764
0.6534
0.7602
0.9773
0.8254
0.9400
0.4239
0.8677
0.7425
0.0754
Estimated Difference
(Relationship)
Tukey's P-Value*
# Cells contain all pairwise comparisons that were significant at the 0.05 level. The format within each
cell is (1) the difference of means, (2) the relationship between the corresponding pair of group
means shown in parentheses [For example, "(1<2)" indicates that the mean baseline adjusted value
in Group 2 was significantly greater than that in Group 1], and (3) the Tukey-adjusted p-value.
NA Data was not available for this group at this study time.
|, 1 "|" indicates that the mean at the study time was significantly greater than that at baseline, while
"1" indicates that the mean at the study time was significantly less than that at baseline (at the
0.05 level).
* Group effect was significant at the 0.05 level.
1078-CG920794 Statistical Report - Telemetry
F-29
-------
Table 5. Summary of the ANOVA Results for the Baseline Adjusted Six-Hour Averages
for RP Inspiratory Time (Seconds)
Study
Day
0
1
2
3
4
5
6
7
8
9
10
Time
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Mean Baseline Adjusted Value,
by Group
1
-0.14
-0.03
-0.04
-0.04
-0.09|
-0.03
-0.07
-0.08
-0.13|
-0.04
-0.08
-0.04
-0.09|
-0.07
-0.02
-0.07
-0.09
-0.07
-0.08
-0.08
-0.07
-0.09
-0.09
-0.06
-0.06
-0.06
-0.08
-0.05
-0.08
-0.08
-0.09
-0.07
-0.06
-0.04
-0.09
-0.10
-0.08
-0.03
-0.11
-0.05
-0.09
-0.05
-0.14
2
-0.09
-0.02
0.01
0.03|
-0.02
-0.04|
-0.01
0.02
-0.04
-0.01
-0.02
0.02
-0.02
-0.02
-0.03
0.01
-0.04
-0.04
-0.02
0.02
0.01
-0.03
-0.04
0.00
0.00
-0.02
-0.03
0.01
-0.01
-0.03
-0.03
0.02
-0.02
-0.03
-0.03
0.00
-0.04
-0.01
-0.01
0.05
0.00
-0.01
-0.04
3
NA
-0.02
0.02
0.00
-0.01
-0.06|
-0.01
-0.02
-0.04
-0.04
0.01
0.02
-0.01
-0.04
-0.03
-0.02
-0.05
-0.06|
-0.02
-0.02
-0.01
-0.04
-0.01
-0.02
0.00
-0.03
0.00
-0.01
0.00
-0.01
-0.02
-0.01
0.00
-0.03
0.03
0.04
-0.02
-0.01
-0.01
0.03
0.01
-0.02
-0.05
4
NA
-0.01
-0.02
-0.03
-0.02
-0.03
-0.02
0.00
-0.05
-0.01
-0.03
-0.01
-0.01
-0.04
-0.03
-0.02
-0.04
-0.03
-0.02
0.01
0.05
-0.01
-0.01
-0.02
0.04
0.01
-0.04
0.00
0.00
-0.03
-0.04
-0.02
0.01
-0.01
0.02
0.05
0.01
0.00
-0.02
0.07
0.02
0.01
0.01
Group
Effect
P-Value
0.4084
0.9169
0.1500
0.0031 *
0.0630
0.2686
0.4263
0.0312*
0.1400
0.5472
0.1229
0.2823
0.0786
0.6780
0.9902
0.2826
0.6357
0.7331
0.3752
0.0341 *
0.1248
0.3963
0.3215
0.5143
0.3257
0.4677
0.4628
0.7323
0.3132
0.5928
0.5639
0.3222
0.5108
0.8671
0.1288
0.1181
0.1682
0.9030
0.2840
0.2746
0.1313
0.8389
0.2063
Estimated Difference
(Relationship)
Tukey's P-Value*
-0.07 (1<2) 0.0068
0.06 (4<2) 0.0085
-0.1 0(1<2) 0.0224
-0.1 0(1<2) 0.0310
1078-CG920794 Statistical Report - Telemetry
F-30
-------
Table 5. (Continued)
Study
Day
11
12
13
14
15
16
17
18
19
20
21
Time
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Mean Baseline Adjusted Value,
by Group
1
-0.12
-0.10
0.03
-0.09
-0.10
-0.05
-0.02
-0.06
-0.03
0.00
-0.03
-0.08
-0.03
-0.09
-0.06
-0.10
-0.07
-0.07
-0.07
-0.13
-0.06
-0.10
-0.05
-0.11|
-0.08
-0.08
-0.06
-0.03
-0.01
-0.08
-0.05
-0.10
-0.04
-0.06
-0.08
-0.11
-0.10
-0.07
-0.09
-0.07
-0.05
-0.06
-0.03
-0.01
2
0.05
0.00
0.02
-0.01
0.08
0.08
0.05
0.05
0.10
0.1 Of
0.03
0.05
0.03
0.00
0.00
-0.02
0.02
0.01
-0.01
-0.05
0.01
-0.01
-0.03
-0.04
0.02
-0.01
-0.02
-0.02
0.01
-0.02
-0.01
-0.03
0.05
0.03
-0.03
-0.03
0.04
0.02
-0.01
0.00
0.03
0.03
-0.01
-0.02
3
-0.02
-0.07
-0.03
-0.01
-0.01
0.02
-0.03
-0.04
-0.03
0.00
-0.04
-0.03
-0.03
0.00
-0.04
-0.02
-0.02
0.00
-0.01
-0.03
-0.05
-0.03
-0.06|
-0.02
-0.01
0.00
-0.07
-0.03
-0.05
-0.01
-0.03
-0.02
-0.01
0.04
0.02
0.00
-0.02
0.07
-0.01
-0.01
0.01
0.04
-0.03
0.01
4
0.03
-0.07
0.03
-0.03
0.03
0.01
0.04
-0.11
-0.07
0.02
0.01
-0.03
-0.09
-0.07
-0.08
-0.13
-0.07
-0.02
-0.04
-0.09
-0.05
-0.08
-0.04
-0.07
-0.08
-0.03
-0.05
-0.07
-0.03
-0.06
-0.06
-0.10
-0.06
-0.02
-0.05
-0.12
-0.07
-0.06
-0.10
-0.11
-0.08
-0.05
-0.05
-0.10
Group
Effect
P-Value
0.0737
0.3943
0.8508
0.7892
0.0882
0.2401
0.5779
0.2586
0.0679
0.2204
0.6897
0.3016
0.0385*
0.2292
0.4134
0.1307
0.1584
0.0727
0.7181
0.3214
0.3952
0.2175
0.8022
0.2491
0.2390
0.1904
0.7530
0.8462
0.7084
0.3543
0.5249
0.1938
0.0964
0.0249 *
0.2050
0.0932
0.0535
0.0378*
0.1857
0.3121
0.3505
0.2119
0.9162
0.4589
Estimated Difference
(Relationship)
Tukey's P-Value*
0. 1 2 (4<2) 0.0260
-0.10(1<3) 0.0299
1078-CG920794 Statistical Report - Telemetry
F-31
-------
Table 5. (Continued)
Study
Day
22
23
24
25
26
27
28
29
30
31
Time
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Mean Baseline Adjusted Value,
by Group
1
-0.01
-0.01
-0.04
-0.04
-0.04
-0.04
-0.06
-0.06
-0.02
-0.02
0.00
-0.01
-0.01
0.00
0.04
-0.04
0.02
-0.02
-0.09
-0.08
-0.03
-0.06
-0.10
-0.06
-0.05
-0.06
-0.08
-0.08
-0.09
-0.08
-0.09
-0.12
-0.07
-0.12
-0.09
-0.06
-0.07
-0.07
-0.06
-0.06
2
0.04
0.01
-0.01
0.01
0.03
-0.03
-0.04
-0.04
0.05
0.00
-0.02
0.01
0.09|
0.02
-0.01
-0.02
0.03
0.03
-0.06
-0.02
0.02
-0.01
-0.071
-0.04
0.01
-0.02
-0.05
-0.03
0.00
-0.01
-0.06
-0.07
0.02
-0.02
-0.05
-0.03
0.03
-0.02
-0.05
-0.01
3
0.01
0.04
0.02
0.02
0.02
0.03
0.01
0.01
0.00
0.08T
0.00
0.02
0.04
0.03
0.00
0.01
-0.01
0.04
-0.04
0.00
0.00
0.03
-0.04
-0.05
-0.02
0.02
-0.04
-0.05
-0.03
0.03
-0.02
-0.05
-0.02
-0.04
-0.03
-0.02
-0.01
0.04
-0.04
-0.01
4
-0.07
-0.05
-0.09
-0.12
-0.10
-0.11
-0.07
-0.17
-0.10
-0.06
-0.05
-0.11
-0.06
-0.11
-0.05
-0.14
-0.05
-0.05
-0.10
-0.13
-0.07
-0.05
-0.06
-0.14
-0.06
-0.07
-0.04
-0.09
-0.06
-0.02
-0.02
-0.12
-0.05
-0.06
-0.03
-0.08
0.02
-0.01
-0.03
0.01
Group
Effect
P-Value
0.4444
0.5177
0.4248
0.2119
0.1516
0.1277
0.4634
0.0985
0.1742
0.0515
0.8675
0.2122
0.1041
0.0095*
0.5853
0.0237 *
0.2159
0.2756
0.6564
0.1726
0.2388
0.1525
0.6111
0.4807
0.5183
0.3274
0.8391
0.8045
0.3043
0.2763
0.3490
0.4837
0.5654
0.2767
0.5762
0.5923
0.4929
0.3242
0.9264
0.7792
Estimated Difference
(Relationship)
Tukey's P-Value*
0.14 (4<3) 0.0085
0.1 3 (4<2) 0.01 17
0.1 5 (4<3) 0.01 58
0.12 (4<2) 0.0471
1078-CG920794 Statistical Report - Telemetry
F-32
-------
Table 5. (Continued)
Study
Day
32
33
34
35
36
37
38
39
Time
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Mean Baseline Adjusted Value,
by Group
1
-0.04
-0.09
-0.05
-0.06
-0.04
-0.07
-0.07
-0.09
-0.08
-0.05
-0.07
-0.08
-0.03
-0.05
-0.07
-0.11
-0.05
-0.05
-0.07
-0.11
-0.01
-0.08
-0.06
-0.12
-0.02
-0.07
-0.10
-0.08
-0.07
-0.06
2
0.03
-0.02
-0.02
-0.01
0.04
-0.01
-0.061
-0.06
0.03
-0.01
-0.071
-0.081
0.01
0.00
-0.06
-0.07
0.01
0.01
-0.06
-0.091
-0.01
-0.03
-0.04
-0.05
0.01
0.01
-0.06
-0.05
0.01
-0.02
3
-0.01
-0.01
-0.03
0.00
0.00
-0.01
0.01
-0.04
0.02
0.02
-0.01
-0.06
0.00
0.04
-0.03
-0.04
-0.03
0.06
-0.04
-0.05
-0.01
0.00
-0.05
-0.03
0.00
0.03
0.00
-0.03
-0.01
0.01
4
-0.04
-0.01
-0.03
0.01
0.06
0.01
0.00
-0.11
0.03
0.08
-0.04
-0.13
0.03
0.02
0.01
-0.10
-0.05
-0.01
-0.05
-0.14
-0.11
-0.08
-0.03
-0.06
0.07
0.05
-0.02
-0.07
0.01
-0.01
Group
Effect
P-Value
0.5303
0.5198
0.7734
0.4454
0.1151
0.4687
0.0434 *
0.5531
0.0511
0.2730
0.2723
0.6033
0.7210
0.6614
0.4681
0.6187
0.4939
0.2266
0.9188
0.3941
0.3860
0.3076
0.9634
0.4805
0.4444
0.2755
0.2160
0.8748
0.4562
0.4787
Estimated Difference
(Relationship)
Tukey's P-Value*
# Cells contain all pairwise comparisons that were significant at the 0.05 level. The format within each
cell is (1) the difference of means, (2) the relationship between the corresponding pair of group
means shown in parentheses [For example, "(1<2)" indicates that the mean baseline adjusted value
in Group 2 was significantly greater than that in Group 1], and (3) the Tukey-adjusted p-value.
NA Data was not available for this group at this study time.
|, 1 "|" indicates that the mean at the study time was significantly greater than that at baseline, while
"1" indicates that the mean at the study time was significantly less than that at baseline (at the
0.05 level).
* Group effect was significant at the 0.05 level.
1078-CG920794 Statistical Report - Telemetry
F-33
-------
Table 6. Summary of the ANOVA Results for the Baseline Adjusted Six-Hour Averages
for RP Integral (mmHg-seconds)
Study
Day
0
1
2
3
4
5
6
7
8
9
10
Time
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Mean Baseline Adjusted Value, by
Group
1
0.35
-0.66
-1.40
-0.81
-0.14
0.62
-0.30
-1.13
-1.05
0.03
-0.47
-0.72
-0.56
0.32
0.06
-0.52
-1.10
-0.73
-0.62
-0.41
-1.02
-0.25
-0.18
0.06
-0.50
0.13
-0.12
0.00
0.25
0.05
0.03
-0.09
0.13
0.28
-0.39
-0.73
-1.33
-0.11
0.34
-0.31
0.42
0.01
-0.83
2
4.76
1.71
0.65
1.17
1.30
1.78
1.06
-0.07
1.09
0.57
-0.14
-1.81
-1.30
-0.78
-2.86
-8.14
0.21
-0.76
-1.50
-0.65
-0.84
0.92
-1.12
-0.89
-1.50
-0.35
-0.29
0.95
1.59
2.16|
1.45
1.22
1.63
1.87
1.46
0.59
1.65
2.70
1.09
0.18
1.14
0.90
0.50
3
NA
1.90
1.03
0.38
0.93
1.21
0.30
-0.92
-1.12
0.78
1.19
0.37
0.31
1.27
1.15
0.93
0.00
1.44
1.55
1.28
0.88
1.79
2.29|
2.03
1.31
2.14|
2.10|
1.19
1.27
2.44|
2.13|
2.73|
1.65
2.41
1.93
2.93|
1.34
2.97|
3.44|
3.33|
2.16|
2.61
3.56|
4
NA
0.52
-0.63
-0.45
0.17
0.42
-0.10
-0.28
0.17
0.79
0.94
0.60
-0.27
0.21
0.86
0.27
-0.09
-0.19
0.74
1.47
0.13
0.61
1.09
1.38
0.25
0.36
0.73
1.83
1.22
1.25
1.24
1.95
0.97
1.31
0.66
1.28
0.77
2.07
2.51|
2.84|
1.82
1.89
1.96
Group
Effect
P-Value
0.1812
0.4913
0.0747
0.6685
0.8957
0.9487
0.9363
0.9481
0.7318
0.9789
0.6368
0.3831
0.5939
0.4438
0.0900
0.1326
0.7677
0.1732
0.0650
0.2615
0.3145
0.4989
0.0269 *
0.2048
0.1087
0.0197*
0.0222 *
0.7453
0.7779
0.3632
0.5186
0.2415
0.6511
0.6472
0.4848
0.0804
0.2486
0.4536
0.1248
0.0621
0.4953
0.5419
0.1735
Estimated Difference
(Relationship)
Tukey's P-Value*
-3.41 (2<3) 0.0207
-2.49 (2<3) 0.01 60
-2.39 (2<3) 0.0210
1078-CG920794 Statistical Report - Telemetry
F-34
-------
Table 6. (Continued)
Study
Day
11
12
13
14
15
16
17
18
19
20
Time
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Mean Baseline Adjusted Value, by
Group
1
-1.55
-1.58
-0.42
-1.20
-1.58
-1.91
-0.57
-1.19
-0.78
-0.76
-0.53
-0.72
-1.26
-0.93
-0.49
-1.17
-1.52
-0.72
-0.75
-0.31
-1.12
-1.28
0.01
-0.90
-0.97
-1.16
0.00
0.29
0.70
-0.92
-0.85
-1.04
-1.67
-2.16
-1.58
-0.98
-1.27
-2.19
-0.60
-0.66
2
0.32
1.00
2.15
1.37
0.22
0.18
1.84
1.36
1.06
1.06
2.18
1.34
0.73
1.43
0.42
0.51
0.89
1.20
0.65
0.88
1.36
0.87
-0.03
1.02
-0.41
-0.02
1.56
1.84
1.48
1.39
1.96
2.18
2.26
2.51
3.98
3.31
3.31
2.97
4.10
3.56
3
2.95|
2.59|
2.40
3.33|
2.97
1.81
2.52
3.34|
2.86
2.15T
2.12
2.96|
3.15|
2.72|
3.09T
3.03|
3.62|
2.40|
2.15|
2.44
2.50|
1.63
2.25
2.90|
3.22|
1.85
2.29
2.91|
2.89
1.88
3.46
3.19
3.68
2.47
3.65
3.94
3.22
2.56
3.10
3.69
4
3.06
2.35
1.43
2.93
4.30
1.41
1.98
3.43
4.58|
0.74
0.54
0.35
2.28
1.32
0.48
1.00
1.23
0.93
-0.18
0.36
0.99
1.39
0.33
-0.28
0.73
0.60
0.05
1.10
2.15
2.26
0.50
0.52
0.92
0.54
0.96
1.62
2.73
1.85
2.08
2.50
Group
Effect
P-Value
0.0790
0.0860
0.6323
0.1722
0.0886
0.0643
0.6224
0.1156
0.0830
0.1208
0.6394
0.1296
0.0216*
0.1013
0.0690
0.0491 *
0.0568
0.1357
0.1888
0.4784
0.2143
0.3791
0.6026
0.0540
0.0634
0.3028
0.6138
0.5666
0.7281
0.7004
0.6296
0.5116
0.1942
0.3178
0.3605
0.3295
0.3502
0.2120
0.5093
0.4056
Estimated Difference
(Relationship)
Tukey's P-Value*
-4.41 (1<3)0.0186
-4.21 (1<3) 0.0389
-5. 14 (1<3) 0.0399
1078-CG920794 Statistical Report - Telemetry
F-35
-------
Table 6. (Continued)
Study
Day
21
22
23
24
25
26
27
28
29
30
31
Time
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Mean Baseline Adjusted Value, by
Group
1
-0.69
-0.84
-0.30
0.18
0.25
-0.51
0.18
-0.01
-0.20
-0.97
-0.77
-0.39
-0.10
0.10
0.15
0.60
0.58
0.25
0.19
0.28
0.38
-0.26
-0.33
0.22
0.60
-0.61
0.16
0.07
0.40
0.11
0.26
0.03
-0.45
-0.23
-0.02
-0.48
-0.01
-0.25
0.48
0.01
0.10
0.01
0.76
0.20
2
2.12
3.09
4.17
3.25
2.68
3.00
3.66
3.25
2.26
3.21
2.67
2.87
2.39
2.60
3.76
3.07
2.78
2.65
3.82
3.07
2.85
2.82
4.27
3.14
2.85
2.62
3.85
2.89
2.82
3.38
3.52
2.81
2.87
2.94
3.56
2.60
3.29
3.20
3.00
3.36
3.27
3.21
4.25
4.07
3
3.82T
2.59
3.97
3.73
3.37
2.99
3.48
4.13
3.51
3.29
2.56
4.10
3.63
2.47
2.77
3.22
3.59
3.21
3.48
4.20
3.47
3.03
3.99
3.77
2.83
3.04
3.50
4.11
4.29
2.51
3.77
4.20
3.96
2.87
3.69
4.48
4.55T
3.50
3.46
3.76
3.97
2.69
3.84
4.07
4
3.55
2.83
2.15
2.36
3.07
2.73
2.25
2.59
3.00
3.59
2.31
2.61
3.22
2.45
2.32
2.56
2.97
3.44
2.39
2.21
2.39
2.04
1.96
1.73
1.75
1.52
1.41
0.87
1.19
0.69
0.55
0.97
1.35
0.36
-0.45
0.12
1.19
0.43
-0.29
-0.35
0.12
-0.66
-0.64
-0.59
Group
Effect
P-Value
0.2721
0.4047
0.4475
0.5588
0.5870
0.4826
0.6880
0.5423
0.5444
0.2969
0.6400
0.4464
0.5721
0.7901
0.7271
0.7392
0.6950
0.5233
0.6770
0.6360
0.7022
0.6514
0.4875
0.6464
0.8006
0.5241
0.6109
0.5225
0.4981
0.6248
0.6190
0.4428
0.4377
0.5066
0.3226
0.3728
0.3082
0.2926
0.4901
0.4258
0.3607
0.3882
0.4341
0.3092
Estimated Difference
(Relationship)
Tukey's P-Value*
1078-CG920794 Statistical Report - Telemetry
F-36
-------
Table 6. (Continued)
Study
Day
32
33
34
35
36
37
38
39
Time
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Mean Baseline Adjusted Value, by
Group
1
0.41
-0.17
0.24
0.16
0.33
0.10
0.78
0.58
0.17
0.38
0.99
1.03
0.57
0.46
1.11
1.11
0.52
0.02
1.14
0.95
0.60
0.73
0.69
0.89
0.62
-0.15
1.38
0.79
0.98
0.83
2
3.40
3.58T
3.83
3.41
3.55
3.58T
4.72
3.59
3.26
2.96
4.60|
3.74
2.63
3.03
4.21
3.99
3.95
3.45T
4.51|
4.02|
3.02
3.44|
4.24
3.47
3.16
2.65
4.81T
4.01
3.44
3.65|
3
4.34T
2.80
3.80
4.38
4.45|
3.38
3.93
4.96T
4.00
3.39
4.02
4.85
3.99
2.73
4.09
4.73
4.16
2.93
3.83
4.74|
3.99
3.03
3.81
4.44|
4.21 1
3.21
4.26
4.96
4.35
3.21
4
0.02
0.69
-0.06
-0.33
0.32
0.26
-0.10
0.96
1.19
0.65
0.76
1.58
1.94
0.95
1.12
1.47
2.20
1.49
1.08
1.62
1.70
1.23
0.51
2.23
2.14
1.49
2.00
2.48
3.16
2.76
Group
Effect
P-Value
0.3170
0.4108
0.4437
0.2929
0.3524
0.3506
0.4430
0.4372
0.4613
0.5666
0.5153
0.5757
0.6672
0.6838
0.6057
0.5579
0.5583
0.4121
0.4843
0.4391
0.5780
0.5348
0.5250
0.5592
0.5283
0.4359
0.6799
0.5698
0.6710
0.6287
Estimated Difference
(Relationship)
Tukey's P-Value*
# Cells contain all pairwise comparisons that were significant at the 0.05 level. The format within each
cell is (1) the difference of means, (2) the relationship between the corresponding pair of group
means shown in parentheses [For example, "(1<2)" indicates that the mean baseline adjusted value
in Group 2 was significantly greater than that in Group 1], and (3) the Tukey-adjusted p-value.
NA Data was not available for this group at this study time.
|, J, "|" indicates that the mean at the study time was significantly greater than that at baseline, while
"J," indicates that the mean at the study time was significantly less than that at baseline (at the
0.05 level).
* Group effect was significant at the 0.05 level.
1078-CG920794 Statistical Report - Telemetry
F-37
-------
Table 7. Summary of the ANOVA Results for the Baseline Adjusted Six-Hour Averages
for RP Peak Amplitude (mmHg)
Study
Day
0
1
2
3
4
5
6
7
8
9
Time
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Mean Baseline Adjusted Value, by
Group
1
-0.51
-2.14
-1.98
-1.06
-0.07
0.91
-0.64
-1.56
-1.11
0.28
-1.35
-1.23
-0.73
0.24
-0.26
-0.97
-1.24
-1.23
-1.50
-1.02
-1.92
-0.99
-1.27
-0.46
-0.72
0.11
-0.45
-0.31
0.70
-0.30
-0.38
-0.17
0.42
0.39
-0.88
-1.61
-1.34
0.36
-0.43
2
4.98
1.60
1.29
2.67
1.20
2.00
1.64
-0.51
0.21
0.71
-1.43
-3.38
-3.48
-2.80
-5.70
-11.00
0.89
-0.63
-1.95
-0.25
-0.48
1.55
-2.07
-1.88
-1.40
0.22
0.18
1.33
2.88
4.79
3.22
2.71
3.26
3.68
2.74
0.85
2.06
4.44
1.54
3
NA
2.15
1.47
0.42
1.37
1.36
0.37
-0.93
-1.19
0.76
1.43
1.11
0.78
0.89
1.61
1.81
1.27
1.67
1.87
2.59T
1.26
2.62T
3.55|
3.51|
2.36|
3.13|
3.46|
2.57
2.36
3.46
3.09
3.97|
3.17
3.23
3.44
4.09|
3.09
4.91|
4.41 1
4
NA
0.36
-0.14
-0.35
0.82
0.20
-0.35
-0.34
0.26
1.23
0.78
0.62
-0.13
-0.32
0.86
0.60
0.32
-0.06
0.66
1.50
0.60
1.08
1.72
1.70
0.83
0.48
1.16
2.08
1.83
1.75
1.55
1.73
1.50
1.60
1.01
1.06
1.65
3.07
2.93
Group
Effect
P-Value
0.2621
0.5696
0.2381
0.5938
0.9818
0.9733
0.9519
0.9927
0.9673
0.9843
0.5443
0.3362
0.2575
0.3686
0.0922
0.1225
0.8297
0.6034
0.1090
0.1242
0.3344
0.1093
0.0012*
0.0499 *
0.0469 *
0.1278
0.1136
0.8405
0.9072
0.5706
0.7223
0.4692
0.6988
0.7245
0.4224
0.0467 *
0.2485
0.4949
0.3063
Estimated Difference
(Relationship)
Tukey's P-Value*
-4.82 (1<3) 0.01 93
-3.79 (2<4) 0.0424
-5.62 (2<3) 0.001 3
-5.39 (2<3) 0.0410
-3.76 (2<3) 0.0402
-5.70 (1<3) 0.0357
1078-CG920794 Statistical Report - Telemetry
F-38
-------
Table 7. (Continued)
Study
Day
10
11
12
13
14
15
16
17
18
19
Time
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Mean Baseline Adjusted Value, by
Group
1
-0.93
0.62
-0.50
-2.64
-2.94
-2.43
-0.68
-2.94
-3.71
-3.00
-1.27
-2.82
-2.17
-1.38
-1.60
-2.04
-3.09
-1.83
-1.34
-3.08
-3.09
-1.50
-2.09
-1.41
-2.66
-2.03
0.04
-1.52
-1.92
-1.96
-0.47
0.23
0.42
-0.80
-1.66
-2.62
-2.99
-3.57
-3.08
-2.69
2
0.36
2.05
0.90
-0.08
0.03
0.84
3.78
1.93
0.73
1.54
4.16
2.75
1.90
2.85
4.73
2.72
0.17
1.35
0.38
0.42
-0.23
0.90
1.11
0.37
0.66
0.80
0.36
1.08
-0.56
0.25
3.28
3.86
2.77
3.82
4.47
4.09
4.66
5.11
7.23
6.37
3
5.18|
3.43|
4.00|
4.49
4.21
4.23|
3.96
4.73
4.49
3.47|
3.66
4.35
3.77|
3.61 1
2.94
4.08|
4.51|
4.37|
5.07|
4.58|
5.42|
4.28|
3.50|
3.59
3.77
2.96
4.20|
4.57|
4.85|
3.28
3.10
3.71
3.49
2.52
5.73
5.03
5.81
4.69
6.06
5.96
4
3.69
2.86
2.24
1.96
3.22
2.32
1.58
2.38
4.35
1.00
2.73
2.94
3.38
0.82
-0.06
-0.66
1.41
1.06
0.29
0.61
0.39
0.52
-0.37
-0.68
-0.07
0.38
-0.59
-1.47
-0.65
0.19
-0.72
0.99
2.64
2.55
-0.10
-0.43
0.32
0.36
0.87
1.08
Group
Effect
P-Value
0.0450 *
0.5045
0.3129
0.2095
0.0920
0.0264 *
0.4788
0.1839
0.0642
0.0177*
0.4265
0.1779
0.1282
0.1834
0.2648
0.0745
0.0023 *
0.0087*
0.0034 *
0.0233 *
0.0123*
0.0019*
0.1365
0.2837
0.1690
0.1583
0.2158
0.1299
0.0422 *
0.2195
0.5976
0.7957
0.8998
0.7815
0.4846
0.5108
0.2881
0.2830
0.3233
0.3767
Estimated Difference
(Relationship)
Tukey's P-Value*
-6.65 (1<3) 0.01 86
-6.48 (1<3) 0.0099
-7.59 (1<3) 0.001 5
-4.34 (2<3) 0.0352
-6.1 9 (1<3) 0.0054
4.79 (4<3) 0.0423
-6.42 (1<3) 0.0053
-4.70 (2<3) 0.01 71
-7.65 (1<3) 0.01 62
-8.50 (1<3) 0.01 18
-5.78 (1<3) 0.0016
-3.38 (2<3) 0.0307
1078-CG920794 Statistical Report - Telemetry
F-39
-------
Table 7. (Continued)
Study
Day
20
21
22
23
24
25
26
27
28
29
30
Time
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Mean Baseline Adjusted Value, by
Group
1
-2.70
-3.20
-1.05
-1.53
-1.71
-1.32
-0.73
-0.14
-0.02
-0.71
0.06
-0.40
-0.37
-1.54
-0.71
-1.39
-1.03
0.34
1.23
1.25
0.97
0.62
0.82
-0.08
0.35
-0.23
-0.83
-0.40
0.39
-1.16
-0.45
-0.43
0.06
0.28
0.23
-0.45
-0.71
0.02
-0.48
-1.49
-0.40
-0.34
0.23
0.00
2
6.26
5.93
7.39
6.84
4.37
6.01
7.60|
6.50
4.88
5.58
7.16
6.58
4.22
5.73
6.45
4.97
4.29
4.61
6.72
6.36
6.05
5.44
7.80
5.46
5.11
5.17
7.17
6.26
5.25
4.64
6.82
5.50
4.93
5.80
5.84
4.52
4.80
5.23
6.16
4.48
5.41
5.53
6.14
6.18
3
5.20
4.95
5.81
5.81
6.04
5.00
6.46
5.90
5.68
5.59
6.05
6.53
5.81
5.63
4.77
5.89
5.83
4.71
4.65
5.69
6.11
5.68
5.83
6.47
5.59
5.33
5.96
5.92
4.74
4.94
5.26
5.82
6.23
4.27
6.13
6.21
6.06
5.13
6.12
6.19
6.60
5.42
5.67
5.44
4
2.52
2.23
2.50
2.61
3.92
3.59
2.57
1.61
2.26
2.36
2.00
2.66
2.60
3.55
2.62
2.12
3.12
3.00
2.77
2.74
3.33
3.84
3.33
1.67
2.59
2.39
1.87
0.94
0.76
0.76
0.30
-1.30
-1.23
-1.47
-1.93
-1.11
-1.54
-2.17
-3.53
-3.21
-2.02
-3.26
-3.65
-4.02
Group
Effect
P-Value
0.3105
0.2650
0.4183
0.4278
0.3665
0.4171
0.3499
0.5626
0.5481
0.4734
0.5134
0.5755
0.5670
0.3438
0.5544
0.5606
0.5806
0.7817
0.7494
0.7542
0.6979
0.5919
0.6025
0.6359
0.7108
0.6204
0.4023
0.5520
0.6288
0.4606
0.4342
0.5028
0.4191
0.4524
0.3922
0.4424
0.3910
0.3411
0.1946
0.3645
0.2406
0.1864
0.2548
0.3635
Estimated Difference
(Relationship)
Tukey's P-Value*
1078-CG920794 Statistical Report - Telemetry
F-40
-------
Table 7. (Continued)
Study
Day
31
32
33
34
35
36
37
38
39
Time
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Mean Baseline Adjusted Value, by
Group
1
0.10
-0.05
1.25
0.24
0.41
-0.10
0.43
-0.11
0.10
0.17
0.39
-0.11
-0.48
0.57
1.23
0.91
0.36
0.75
1.38
0.96
0.31
0.47
1.37
0.78
0.40
0.90
0.77
0.57
0.28
-0.57
1.54
0.47
0.97
1.42
2
5.71
5.67
7.27
7.54
6.01
6.39
7.35
6.67
6.25
6.50
7.99
6.23
5.56
5.16
7.72
6.19
4.51
5.19
6.78
6.92
6.45
6.21 1
7.72|
6.65
5.03
6.41 1
8.03
6.34
5.56
5.55
8.48T
6.93
5.70
6.34|
3
6.02
5.09
5.86
5.87
6.66
4.84
6.30
6.78
6.91
5.94
6.59
7.35
6.23
5.88
6.35
6.91
6.30
5.00
6.55
6.75
6.47
5.64
6.22
6.78
6.29
5.28
6.23
6.50
6.26
5.67
6.91
7.50
6.75
5.83
4
-3.86
-3.97
-4.29
-3.47
-3.37
-1.90
-3.12
-2.69
-1.81
-1.32
-2.46
-1.20
-0.41
-0.77
-0.67
-0.29
0.69
0.30
0.08
-0.23
0.77
-0.10
-0.57
-0.23
-0.06
-0.77
-0.01
2.08
1.83
1.42
2.15
2.26
3.25
3.58
Group
Effect
P-Value
0.2616
0.2369
0.2295
0.2868
0.2368
0.2761
0.2593
0.2939
0.3354
0.2905
0.2967
0.4290
0.3808
0.5022
0.4276
0.5764
0.6187
0.6329
0.5370
0.5306
0.4756
0.3422
0.3398
0.4209
0.4579
0.3745
0.4492
0.6136
0.5756
0.3914
0.5782
0.5844
0.6659
0.6493
Estimated Difference
(Relationship)
Tukey's P-Value*
# Cells contain all pairwise comparisons that were significant at the 0.05 level. The format within each
cell is (1) the difference of means, (2) the relationship between the corresponding pair of group
means shown in parentheses [For example, "(1<2)" indicates that the mean baseline adjusted value
in Group 2 was significantly greater than that in Group 1], and (3) the Tukey-adjusted p-value.
NA Data was not available for this group at this study time.
|, J, "|" indicates that the mean at the study time was significantly greater than that at baseline, while
"J," indicates that the mean at the study time was significantly less than that at baseline (at the
0.05 level).
* Group effect was significant at the 0.05 level.
1078-CG920794 Statistical Report - Telemetry
F-41
-------
Table 8. Summary of the ANOVA Results for the Baseline Adjusted Six-Hour Averages
for RP Respiratory Rate (RCPM)
Study
Day
0
1
2
3
4
5
6
7
8
9
Time
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Mean Baseline Adjusted Value, by
Group
1
11.90
3.71
-0.76
0.87
5.33|
4.01
2.63
1.43
3.41
-0.25
3.65
3.09
4.74
4.57
2.72
4.61|
1.35
5.02
6.06
4.54
2.38
7.92|
9.43|
4.78
2.64
2.82
3.36
2.42
2.28
6.36
4.09
2.43
1.99
4.64
4.14
4.54
0.73
1.90
10.32|
2
21.83|
4.30|
0.37
-0.14
3.31 f
4.84|
0.81
0.84
6.57|
1.88
2.33
-0.42
1.34
5.08|
0.99
-1.57
5.79|
4.65|
2.09
0.73
1.18
4.83|
3.12
3.02
-1.30
2.55
3.33
1.90
1.66
3.45
3.23
0.70
1.40
2.87
1.80
0.55
6.49|
4.98
2.03
3
NA
6.52|
0.32
0.94
0.73
4.94|
3.07|
0.22
0.47
2.83
1.76
-0.58
0.10
5.32|
3.28|
0.26
0.85
4.79|
3.42
0.76
1.64
4.55|
4.75
0.43
0.04
4.49T
2.66
-0.18
0.20
6.82|
5.51|
2.38
0.72
5.76|
-0.43
0.34
2.01
4.32
8.48|
4
NA
3.36
-0.49
1.00
0.71
3.30|
-1.93
-2.41
4.84|
0.84
0.79
-1.39
-1.70
3.53
0.53
-2.42
4.12
0.78
1.38
0.02
-4.05|
-0.25
-0.15
-0.34
-4.18|
0.33
1.65
0.65
-0.31
3.05
2.73
3.23
0.50
2.11
-1.59
0.09
3.25
3.48
2.81
Group
Effect
P-Value
0.3139
0.3481
0.8891
0.6979
0.0538
0.7367
0.0486 *
0.1226
0.0678
0.5052
0.6709
0.2065
0.1233
0.8615
0.4479
0.0016*
0.2209
0.4114
0.4909
0.4900
0.0221 *
0.0352 *
0.0869
0.3217
0.0396 *
0.1803
0.9083
0.7577
0.6334
0.4097
0.7524
0.6438
0.7934
0.4707
0.3683
0.3597
0.2301
0.8739
0.0592
Estimated Difference
(Relationship)
Tukey's P-Value*
5.00 (4<3) 0.0435
6. 10(3<2) 0.0499
7.03 (4<1) 0.0013
4.35 (3<1) 0.0431
6. 18 (2<1) 0.0059
5.69 (4<3) 0.0402
6.43 (4<1) 0.0470
8. 17 (4<1) 0.0281
6.82 (4<1) 0.0307
1078-CG920794 Statistical Report - Telemetry
F-42
-------
Table 8. (Continued)
Study
Day
10
11
12
13
14
15
16
17
18
19
Time
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Mean Baseline Adjusted Value, by
Group
1
5.78
7.34
12.62
13.15
7.39
2.66
-0.50
7.25
7.57
1.30
3.23
8.04
6.23
0.46
5.39
8.56
6.48
4.86
6.53
10.03
5.77
5.68
6.75
13.62|
7.45
2.54
3.80
6.02
4.15
4.51
7.80
5.64
5.70
2.76
4.82
8.18
4.01
2.01
6.68
10.32|
2
-0.55
0.66
3.91
3.43
0.75
2.78
0.54
1.27
-2.80
-6.24
-2.30
0.17
-1.19
-5.56
1.50
-0.01
3.10
4.21
3.34
5.32
5.05
3.71|
4.10
6.86
7.12|
6.03|
4.15
6.40|
2.72
4.14|
4.44
3.65
1.70
3.79
3.11
4.50
-0.15
0.00
6.59|
4.56
3
0.72
1.94
6.42
10.25|
2.53
4.89
2.75
7.41
3.35
-1.35
4.38
9.25
3.29
-2.62
2.95
7.29
3.27
4.78
8.03|
6.39
3.32
3.78|
3.34
5.12
2.61
6.77|
3.59
3.03
3.42
3.67
8.45|
8.10|
11.06|
8.04
5.30
3.82
3.09
-0.44
4.65
4.96
4
-2.19
1.57
6.12
2.93
3.04
9.22|
0.11
4.74
5.21
3.20
4.56
16.54|
18.43|
2.52
4.64
6.91
14.55
10.39
7.45
12.15
2.92
2.25
1.68
4.98
4.14
10.55
4.73
3.04
3.61
3.17
1.54
4.47
2.08
7.94
4.29
4.73
2.32
-0.52
1.02
9.90
Group
Effect
P-Value
0.0255*
0.1826
0.5096
0.1656
0.4343
0.4464
0.9468
0.5703
0.2382
0.3467
0.6896
0.0812
0.0343 *
0.3283
0.9176
0.3389
0.2354
0.7155
0.7287
0.6306
0.8512
0.7041
0.8449
0.3649
0.4385
0.2870
0.9974
0.6346
0.9811
0.9752
0.2862
0.7956
0.4634
0.7404
0.8893
0.7075
0.5708
0.8192
0.5732
0.3029
Estimated Difference
(Relationship)
Tukey's P-Value*
7.97 (4<1) 0.0180
-1 9.62 (2<4) 0.0236
1078-CG920794 Statistical Report - Telemetry
F-43
-------
Table 8. (Continued)
Study
Day
20
21
22
23
24
25
26
27
28
29
30
Time
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Mean Baseline Adjusted Value, by
Group
1
7.15
0.65
5.35
6.15
3.43
2.50
5.25
3.68
3.41
1.47
5.60
4.62
3.65
6.93
2.82
6.81
5.59
2.71
-3.11
1.20
-0.13
3.35
-2.40
4.85
3.07
1.91
9.45
8.61
5.14
5.81
11.80|
7.06
5.90
5.95
7.11
7.73
3.89
2.22
9.69|
11.13
5.10
8.62
8.79
2.65
2
3.17
0.42
3.96
4.13
1.15
-0.90
3.65
5.13
1.49
0.94
4.32
2.01
2.82
8.2T\
2.53
4.96
2.27
1.31
6.24
2.84
0.45
3.18
2.89
6.43
3.36
0.95
11.75|
5.25
2.99
2.97
10.77|
7.29|
5.31
4.72
8.26|
7.04
6.67
2.87
9.53|
9.74|
6.54
7.50|
6.80|
4.76
3
2.12
-2.05
5.12
5.71
2.16
0.90
7.14
2.95
0.58
0.76
3.80
5.25
0.66
5.53
2.01
6.60
2.15
-1.33
3.40
0.74
-1.02
5.79
3.04
6.86
2.91
0.11
11.15|
7.69
1.73
2.77
8.96|
13.44|
7.53|
2.33
8.68|
12.49|
5.31
1.32
6.67|
13.77|
7.49
11.19|
6.78|
8.00|
4
8.87
4.05
9.17
14.50
14.31
11.44
10.70
17.35
16.81
14.59
16.19
16.86
15.36
21.11|
13.61
17.35
12.96
8.84
7.66
9.64
5.53
14.37
2.79
10.66
4.77
4.45
8.57
9.05
3.80
4.14
6.50
10.05
5.75
5.15
4.73
6.33
5.41
-0.61
2.49
7.21
5.35
9.17
1.14
5.81
Group
Effect
P-Value
0.4799
0.4420
0.7190
0.3985
0.3165
0.3152
0.6919
0.2429
0.2131
0.2522
0.3129
0.2978
0.3130
0.1001
0.4231
0.2741
0.3718
0.4409
0.3641
0.4388
0.5580
0.0837
0.7673
0.7778
0.9730
0.5126
0.9090
0.8984
0.8649
0.7125
0.7072
0.4228
0.9336
0.7503
0.8558
0.5815
0.9629
0.7498
0.1905
0.7287
0.9652
0.6959
0.3584
0.6558
Estimated Difference
(Relationship)
Tukey's P-Value*
1078-CG920794 Statistical Report - Telemetry
F-44
-------
Table 8. (Continued)
Study
Day
31
32
33
34
35
36
37
38
39
Time
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Mean Baseline Adjusted Value, by
Group
1
1.23
3.63
6.89
2.61
2.15
5.80T
3.48
3.88
3.40
5.54
12.651
11.61
5.25
5.20
10.00
11.16
4.08
5.40
13.12|
13.72
5.11
3.79
11.51
12.24
4.27
9.67
9.80
12.45
4.58
5.12
13.751
11.54
6.11
2.17
2
2.85
3.08
9.29|
8.06|
4.32
6.14T
5.98
5.49
4.42
4.96|
11.821
10.45|
4.07
2.17
12.95|
13.43|
5.49
3.07
11.60|
13.77|
8.53T
3.57
10.38|
14.18|
6.79|
6.99|
7.49
9.42|
5.88|
0.53
10.021
12.86|
7.71|
7.71|
3
3.28
-0.02
11.50|
8.29
4.04
6.87T
6.52
7.34|
4.31
5.86|
9.21 1
14.67|
3.57
3.55
11.36|
15.38|
5.86
4.18
11.08|
14.58|
7.43
0.16
9.10|
15.85|
5.31
6.54|
9.22|
10.99|
4.45
2.02
9.32|
13.80|
5.19
5.58|
4
-0.57
-0.64
2.74
0.73
0.70
4.05
3.08
-2.45
-4.28
-2.32
1.57
8.54
0.99
-1.82
2.90
12.02
1.30
-0.15
2.89
9.02
9.42
9.99
12.36
19.07
14.50
13.68
2.58
9.13
0.76
0.55
3.97
10.91
5.11
11.26
Group
Effect
P-Value
0.7923
0.3504
0.3735
0.4830
0.8419
0.7111
0.8271
0.1783
0.1249
0.1122
0.1805
0.7835
0.8494
0.2567
0.2697
0.9096
0.7199
0.5491
0.2178
0.8610
0.8989
0.4376
0.9279
0.7787
0.2801
0.3791
0.6999
0.9306
0.4507
0.5528
0.3516
0.9682
0.8653
0.1662
Estimated Difference
(Relationship)
Tukey's P-Value*
# Cells contain all pairwise comparisons that were significant at the 0.05 level. The format within each
cell is (1) the difference of means, (2) the relationship between the corresponding pair of group
means shown in parentheses [For example, "(1<2)" indicates that the mean baseline adjusted value
in Group 2 was significantly greater than that in Group 1], and (3) the Tukey-adjusted p-value.
NA Data was not available for this group at this study time.
|, J, "|" indicates that the mean at the study time was significantly greater than that at baseline, while
"J," indicates that the mean at the study time was significantly less than that at baseline (at the
0.05 level).
* Group effect was significant at the 0.05 level.
1078-CG920794 Statistical Report - Telemetry
F-45
-------
Table 9. Summary of the ANOVA Results for the Baseline Adjusted Six-Hour Averages
for Temperature (Celsius)
Study
Day
0
1
2
3
4
5
6
7
8
9
Time
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Mean Baseline Adjusted Value, by
Group
1
0.04
0.25
-0.02
0.03
0.01
0.27|
0.06
0.09
-0.61|
0.00
0.22|
0.16|
0.03
0.30
0.16
0.10
-0.53|
0.27|
0.28|
0.17|
0.13
0.27
0.17
0.08
0.05
0.12
0.01
0.03
-0.05
0.23
0.04
0.05
-0.05
0.23
0.02
0.03
-0.61|
0.06
0.34|
2
-0.07
0.07
0.08|
0.06
-0.02
0.11
0.12|
0.11|
-0.06
0.11
0.19|
0.17|
-0.06
0.07
0.12
0.12|
-0.06
0.10
0.19|
0.11|
0.05
-0.01
0.14|
0.13|
-0.01
-0.09
0.06
0.09
-0.07
0.03
0.09
0.11|
-0.05
-0.04
0.02
0.05
-0.12
0.01
0.16
3
NA
0.16
-0.02
0.01
0.01
0.26|
0.06
0.09|
-0.27
0.08
0.18|
0.11|
0.02
0.18
0.10
0.13|
-0.24
0.22|
0.18|
0.13|
0.07
0.12
0.11
0.07
0.05
0.09
0.01
-0.02
-0.02
0.17
0.03
0.08
0.04
0.16
-0.03
0.01
-0.34
0.11
0.28|
4
NA
0.13
0.03
0.11|
0.04
0.22|
0.10|
0.09|
-0.21
0.09
0.23|
0.13|
0.09|
0.22
0.17|
0.14|
-0.19
0.14
0.26|
0.22|
0.16|
0.19
0.15|
0.12|
0.12|
0.09
0.00
0.03
0.00
0.21
0.07
0.14|
0.03
0.16
0.08
0.09
-0.24
0.09
0.26|
Group
Effect
P-Value
0.5990
0.5434
0.0786
0.0908
0.6618
0.4428
0.6318
0.9495
0.1610
0.8955
0.8553
0.6936
0.0574
0.5019
0.7641
0.9155
0.3005
0.3763
0.6149
0.1659
0.3521
0.2582
0.9172
0.7150
0.3470
0.2664
0.8981
0.4432
0.6543
0.4452
0.7558
0.4638
0.4791
0.2057
0.7395
0.6412
0.1969
0.8065
0.4239
Estimated Difference
(Relationship)
Tukey's P-Value*
-0.14 (2<4) 0.0351
1078-CG920794 Statistical Report - Telemetry
F-46
-------
Table 9. (Continued)
Study
Day
10
11
12
13
14
15
16
17
18
19
Time
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Mean Baseline Adjusted Value, by
Group
1
0.12
0.04
0.34
0.18
0.09
-0.53
-0.35
0.04
0.14
-0.01
0.10
0.11
0.10
-0.03
0.10
0.09
0.07
-0.03
0.20
0.15
0.15
-0.02
0.19
0.18
0.15
-0.23
0.09
0.10
0.06
0.01
0.16
0.01
0.03
-0.37
0.15
0.13
0.10
-0.08
0.05
0.13
2
0.17|
-0.10
0.01
0.08
0.15
-0.06
0.02
0.11
0.13
-0.12
-0.16
0.03
0.01
-0.16
-0.20
-0.01
0.14
0.03
0.02
0.14
0.24|
0.01
0.07
0.17|
0.26|
-0.04
-0.29
0.11
0.18|
0.00
-0.02
0.07
0.11
-0.26
-0.12
0.07
0.12
-0.08
-0.11
0.07
3
0.21 1
0.08
0.23
0.20
0.18
0.03
0.26
0.19
0.22
0.04
0.08
0.15
0.14
0.02
0.07
0.16
0.16
0.13
0.19|
0.07
0.17|
0.06
0.13
0.10
0.19|
-0.04
0.15
0.12
0.19|
0.12
0.55|
0.30|
0.31|
-0.04
0.20
0.20
0.24|
0.08
0.17
0.14
4
0.17|
0.12
0.00
0.11
-0.05
-0.34
0.16
0.27
0.58|
0.27
0.12
0.60|
0.61|
0.23
0.01
0.17
0.39
0.30
-0.05
-0.25
0.12
-0.09
-0.07
0.01
0.21
0.08
0.13
0.20
0.44|
0.39
0.33
0.25
0.22
0.10
0.52
0.55|
0.29
-0.02
0.53
1.11|
Group
Effect
P-Value
0.8211
0.0321 *
0.3599
0.8763
0.6568
0.4677
0.3183
0.5869
0.2359
0.3476
0.3749
0.1901
0.1208
0.2299
0.2459
0.3584
0.4600
0.3682
0.0627
0.0892
0.6932
0.5297
0.3906
0.2725
0.6898
0.2623
0.1899
0.9313
0.0563
0.4248
0.3072
0.3649
0.2852
0.1052
0.1263
0.1615
0.3111
0.2477
0.2366
0.0181 *
Estimated Difference
(Relationship)
Tukey's P-Value*
-0.22 (2<4) 0.0297
-0.38 (1<4) 0.0378
-0.97 (3<4) 0.0360
-1.04(2<4)0.0187
-0.98 (1<4) 0.0437
1078-CG920794 Statistical Report - Telemetry
F-47
-------
Table 9. (Continued)
Study
Day
20
21
22
23
24
25
26
27
28
29
Time
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Mean Baseline Adjusted Value, by
Group
1
0.05
-0.17
-0.03
0.05
0.00
-0.08
0.05
-0.05
-0.04
-0.08
0.06
0.04
-0.01
-0.04
0.05
0.18
0.10
-0.08
-0.14
-0.10
-0.05
0.01
0.10
0_23_t
0.14
-0.06
0.10
0.10
0.11
-0.06
0.03
-0.01
0.09
0.00
0.10
0.00
0.03
-0.14
0.04
0.07
2
0.10
-0.11
-0.15
-0.04
0.06
-0.07
-0.11
-0.01
0.06
-0.11
-0.12
-0.05
0.10
-0.03
-0.28
0.16|
0.23|
-0.02
-0.06
0.00
0.04
-0.16
-0.20
0.14|
0.18|
-0.06
-0.03
0.04
0.14|
-0.03
-0.12
0.03
0.13
-0.04
-0.12
-0.01
0.12|
-0.09
-0.13
-0.01
3
0.15
0.02
0.14
0.06
0.12
0.05
0.19
0.05
0.05
0.04
0.17
0.08
0.09
0.10
-0.06
0.18|
0.21 1
0.05
0.13
0.05
0.07
0.04
0.07
0.13
0.18|
0.04
0.17
0.12
0.14
0.08
0.14
0.24|
0.18T
0.08
0.14
0.15|
0.12|
0.03
0.09
0.23|
4
0.77|
0.76|
0.63
0.30
0.32
-0.45
0.28
0.15
0.20
0.11
0.03
0.28
0.50
0.28
-0.16
0.19
0.20
0.05
-0.12
-0.04
0.04
0.00
-0.24
0.00
0.10
-0.06
-0.16
-0.01
0.05
-0.01
-0.16
-0.03
0.05
0.02
-0.21
0.03
0.19
-0.07
-0.18
0.00
Group
Effect
P-Value
0.0458 *
0.0148*
0.0722
0.5870
0.8311
0.8189
0.3525
0.3563
0.1439
0.0483 *
0.1957
0.1446
0.1358
0.1213
0.5372
0.9913
0.6619
0.5484
0.2227
0.1913
0.5211
0.3194
0.1261
0.1445
0.8542
0.4563
0.2066
0.6616
0.7726
0.3985
0.1189
0.0133*
0.5951
0.6234
0.0624
0.1878
0.2550
0.1856
0.1871
0.0396 *
Estimated Difference
(Relationship)
Tukey's P-Value*
-0.87 (2<4) 0.01 92
-0.93 (1<4) 0.01 89
-0.25 (1<3) 0.0275
-0.21 (2<3) 0.0403
0.27 (4<3) 0.0456
-0.24 (2<3) 0.0357
1078-CG920794 Statistical Report - Telemetry
F-48
-------
Table 9. (Continued)
Study
Day
30
31
32
33
34
35
36
37
38
Time
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Midnight - 6AM
6AM - Noon
Noon - 6PM
6PM - Midnight
Mean Baseline Adjusted Value, by
Group
1
0.06
-0.01
0.06
0.02
0.06
-0.07
0.06
-0.10
-0.03
0.03
0.03
0.03
-0.01
-0.04
0.09
-0.05
0.04
-0.08
0.03
-0.03
-0.03
-0.08
0.09
-0.01
0.04
-0.09
0.03
-0.03
0.01
-0.09
0.08
0.10
0.01
-0.13
0.01
-0.03
2
0.11
-0.09
-0.23|
0.05
0.10
-0.14
-0.15
-0.08
0.02
-0.06
-0.26|
0.06
0.15|
-0.06
-0.16
0.04
0.12
-0.10
-0.18
0.09
0.11
-0.11
-0.18
0.05
0.12
-0.18|
-0.23|
0.04
0.08
-0.32|
-0.31|
0.08
0.14
-0.18|
-0.24|
0.03
3
0.18T
0.06
0.01
0.10
0.09
-0.03
0.10
0.05
0.07
0.10
0.13
0.16
0.17|
0.09
0.13
0.21 1
0.14
0.01
0.06
0.25|
0.10
0.01
0.08
0.20T
0.15
-0.09
0.06
0.19T
0.07
-0.07
-0.02
0.15T
0.12
-0.05
0.04
0.18|
4
0.20
0.04
-0.20
0.02
0.06
-0.05
-0.17
-0.10
-0.03
0.11
-0.07
0.06
0.20
-0.01
-0.14
0.00
0.09
-0.06
-0.16
-0.03
0.05
-0.07
-0.13
-0.02
-0.21
-0.37
-0.40
-0.02
-0.10
-0.04
-0.51
0.13
0.13
-0.01
-0.15
0.08
Group
Effect
P-Value
0.4022
0.5075
0.1738
0.7775
0.9760
0.6086
0.1040
0.3340
0.6402
0.2000
0.0356 *
0.6590
0.2074
0.2961
0.0490 *
0.0469 *
0.6841
0.4703
0.1777
0.0182*
0.4614
0.4668
0.0661
0.1109
0.0577
0.1312
0.0086 *
0.1282
0.1417
0.0904
0.0252 *
0.7430
0.5262
0.3728
0.1405
0.1363
Estimated Difference
(Relationship)
Tukey's P-Value*
-0.39 (2<3) 0.0275
-0.26 (1<3) 0.0429
-0.28 (1<3) 0.0222
0.43 (4<1) 0.0378
0.46 (4<3) 0.0201
0.59 (4<1) 0.0454
1078-CG920794 Statistical Report - Telemetry
F-49
-------
Table 9. (Continued)
Study
Day
39
Time
Midnight - 6AM
6AM - Noon
Mean Baseline Adjusted Value, by
Group
1
-0.02
-0.10
2
0.10
-0.14
3
0.12
-0.09
4
0.04
-0.05
Group
Effect
P-Value
0.4091
0.8824
Estimated Difference
(Relationship)
Tukey's P-Value*
# Cells contain all pairwise comparisons that were significant at the 0.05 level. The format within each
cell is (1) the difference of means, (2) the relationship between the corresponding pair of group
means shown in parentheses [For example, "(1<2)" indicates that the mean baseline adjusted value
in Group 2 was significantly greater than that in Group 1], and (3) the Tukey-adjusted p-value.
NA Data was not available for this group at this study time.
|, J, "|" indicates that the mean at the study time was significantly greater than that at baseline, while
"J," indicates that the mean at the study time was significantly less than that at baseline (at the
0.05 level).
* Group effect was significant at the 0.05 level.
1078-CG920794 Statistical Report - Telemetry
F-50
-------
Table 10. Abnormality Summaries by Parameter and Group Along with Fisher's Exact
Tests Comparing the Proportion Abnormal in Each Group by Parameter
Parameter
Activity
Heart Rate
RP
Expiratory
Time
RP
Inspiratory
Time
RP Integral
RP Peak
Amplitude
RP
Respiratory
Rate
Temperature
Group
1
2
3
4
1
2
3
4
1
2
3
4
1
2
3
4
1
2
3
4
1
2
3
4
1
2
3
4
1
2
3
4
Number
Abnormal/N
2/5
2/7
3/7
5/7
5/5
5/7
111
5/7
3/5
6/7
5/7
4/7
2/5
4/7
5/7
3/7
3/5
6/7
5/7
4/7
4/5
6/7
111
5/7
3/5
111
4/7
4/7
3/5
6/7
4/7
6/7
Proportion Abnormal
(95% Confidence Interval)
0.40 (0.05, 0.85)
0.29 (0.04, 0.71)
0.43(0.10,0.82)
0.71 (0.29, 0.96)
1.00(0.48, 1.00)
0.71 (0.29, 0.96)
1.00(0.59, 1.00)
0.71 (0.29, 0.96)
0.60(0.15,0.95)
0.86(0.42, 1.00)
0.71 (0.29, 0.96)
0.57(0.18,0.90)
0.40 (0.05, 0.85)
0.57(0.18,0.90)
0.71 (0.29, 0.96)
0.43(0.10,0.82)
0.60(0.15,0.95)
0.86(0.42, 1.00)
0.71 (0.29, 0.96)
0.57(0.18,0.90)
0.80 (0.28, 0.99)
0.86(0.42, 1.00)
1.00(0.59, 1.00)
0.71 (0.29, 0.96)
0.60(0.15,0.95)
1.00(0.59, 1.00)
0.57(0.18,0.90)
0.57(0.18,0.90)
0.6(0.15,0.95)
0.86(0.42, 1.00)
0.57(0.18,0.90)
0.86(0.42, 1.00)
Mean Duration of
Abnormal (Days)*
15.51
7.38
11.42
6.00
16.75
19.10
6.82
7.10
25.08
21.63
15.60
9.94
11.00
22.19
12.90
6.58
27.17
26.96
12.35
11.38
21.50
28.54
22.46
8.00
30.58
22.11
6.44
7.44
11.17
23.42
10.81
12.08
Fisher's
Group Effect
P-Value
0.5161
0.2855
0.7925
0.8224
0.7925
0.68788
0.2096
0.5542
N Number of animals.
# Means exclude those animals that were never abnormal.
1078-CG920794 Statistical Report - Telemetry
F-51
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Table 11. Results of Overall Log-Rank Tests Comparing the Time to Abnormality between
Groups by Parameter
Parameter
Activity
Heart Rate
RP Expiratory Time
RP Inspiratory Time
RP Integral
RP Peak Amplitude
RP Respiratory Rate
Temperature
Group Effect
P-Value
0.2687
0.5098
0.9260
0.8017
0.7576
0.5836
0.1549
0.6605
Group effect was significant at the 0.05 level.
Table 12. Results of Overall Log-Rank Tests Comparing the Duration of Abnormality
between Groups by Parameter
Parameter
Activity
Heart Rate
RP Expiratory Time
RP Inspiratory Time
RP Integral
RP Peak Amplitude
RP Respiratory Rate
Temperature
Group Effect P-Value
0.1384
0.4335
0.9781
0.5030
0.7944
0.2260
0.0710
0.3558
NA No animals had a duration of abnormality greater than zero days.
* Group effect was significant at the 0.05 level.
1078-CG920794 Statistical Report - Telemetry
F-52
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10-
8-
6-
4-
2-
0-
-2-
-4-
Animal 5
Animal 9
Animal 40
Animal 7
Animal 37
10 20 30
Days from Midnight of Challenge Day
40
Figure la. Plot of baseline adjusted Activity (counts/minute) for each animal in
Group 1.
10-
8-
6-
4-
2-
0-
-2-
-4-
Animal 13
Animal 19
Animal 28
Animal 34
— Animal 15
— Animal 25
— Animal 30
mPW
10 20 30
Days from Midnight of Challenge Day
40
Figure Ib. Plot of baseline adjusted Activity (counts/minute) for each animal in
Group 2.
1078-CG920794 Statistical Report - Telemetry
F-53
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10-
8-
6-
4-
2-
0-
-2-
-4-
Animal 2
Animal 11
Animal 14
Animal 32
Animal 8
Animal 12
Animal 18
10 20 30
Days from Midnight of Challenge Day
40
Figure Ic. Plot of baseline adjusted Activity (counts/minute) for each animal in
Group 3.
10-
8-
6-
4-
2-
0-
-2-
-4-
Animal 6
Animal 27
Animal 33
Animal 39
— Animal 21
— Animal 31
— Animal 38
10 20 30
Days from Midnight of Challenge Day
40
Figure Id. Plot of baseline adjusted Activity (counts/minute) for each animal in
Group 4.
1078-CG920794 Statistical Report - Telemetry
F-54
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175-
150-
125-
100-
75-
50-
25-
0-
-25-
-50-
-75-
-100-
-125-
-150-
Animal 5
Animal 9
Animal 40
Animal 7
Animal 37
0 10 20 30
Days from Midnight of Challenge Day
40
Figure 2a. Plot of baseline adjusted Heart Rate (BPM) for each animal in Group 1.
175-
150-
125-
100-
75-
50-
25-
0-
-25-
-50-
-75-
-100-
-125-
-150-
Animal 13
Animal 19
Animal 28
Animal 34
— Animal 15
— Animal 25
— Animal 30
10 20 30
Days from Midnight of Challenge Day
40
Figure 2b. Plot of baseline adjusted Heart Rate (BPM) for each animal in Group 2.
1078-CG920794 Statistical Report - Telemetry
F-55
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175-
150-
125-
100-
75-
50-
25-
0-
-25-
-50-
-75-
-100-
-125-
-150-
Animal 2
Animal 11
Animal 14
Animal 8
Animal 12
Animal 18
10 20 30
Days from Midnight of Challenge Day
40
Figure 2c. Plot of baseline adjusted Heart Rate (BPM) for each animal in Group 3.
175-
150-
125-
100-
75-
50-
25-
0-
-25-
-50-
-75-
-100-
-125-
-150-
Animal 6
Animal 27
Animal 33
Animal 39
— Animal 21
— Animal 31
— Animal 38
10 20 30
Days from Midnight of Challenge Day
40
Figure 2d. Plot of baseline adjusted Heart Rate (BPM) for each animal in Group 4.
1078-CG920794 Statistical Report - Telemetry
F-56
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1.5-
1-
.5-
0-
-.5-
-1-
-1.5-
Animal 5
Animal 9
Animal 40
Animal 7
Animal 37
vW^
10 20 30 40
Days from Midnight of Challenge Day
Figure 3a. Plot of baseline adjusted RP Expiratory Time (seconds) for each animal in
Group 1.
1.5-
1-
.5-
o-
-.5-
-1-
-1.5-
Animal 13
Animal 19
Animal 28
Animal 34
— Animal 15
— Animal 25
— Animal 30
10 20 30
Days from Midnight of Challenge Day
40
Figure 3b. Plot of baseline adjusted RP Expiratory Time (seconds) for each animal in
Group 2.
1078-CG920794 Statistical Report - Telemetry
F-57
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1.5-
1-
.5-
o-
-.5-
-1-
-1.5-
^''V^'%
— Animal 2 — Animal 8
— Animal 11 — Animal 12
— Animal 14 — Animal 18
— Animal 32
**#%^
0 10 20 30 40
Days from Midnight of Challenge Day
Figure 3c. Plot of I
Group '.
1.5-
1-
.5-
o-
-.5-
-1-
-1.5-
baseline adjusted RP Expiratory Time (seconds) for each animal in
5.
>*^i^^M^
— Animal 6 — Animal 21
— Animal 27 — Animal 31
- Animal 33 — Animal 38
— Animal 39
&w)#tt||tK
10 20 30
Days from Midnight of Challenge Day
40
Figure 3d. Plot of baseline adjusted RP Expiratory Time (seconds) for each animal in
Group 4.
1078-CG920794 Statistical Report - Telemetry
F-58
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.25-
0-
-.25-
-.5-
Animal 5
Animal 9
Animal 40
Animal 7
Animal 37
10 20 30
Days from Midnight of Challenge Day
40
Figure 4a. Plot of baseline adjusted RP Inspiratory Time (seconds) for each animal in
Group 1.
.5-
.25-
0-
-.25-
-.5-
Animal 13
Animal 19
Animal 28
Animal 34
Animal 15
Animal 25
Animal 30
10 20 30
Days from Midnight of Challenge Day
40
Figure 4b. Plot of baseline adjusted RP Inspiratory Time (seconds) for each animal in
Group 2.
1078-CG920794 Statistical Report - Telemetry
F-59
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.5-
.25-
o-
-.25-
-.5-
Animal 2
Animal 11
Animal 14
Animal 32
— Animal 8
— Animal 12
— Animal 18
10 20 30
Days from Midnight of Challenge Day
40
Figure 4c. Plot of baseline adjusted RP Inspiratory Time (seconds) for each animal in
Group 3.
.5-
.25-
o-
-.25-
-.5-
Animal 6
Animal 27
Animal 33
Animal 39
— Animal 21
— Animal 31
— Animal 38
10 20 30
Days from Midnight of Challenge Day
40
Figure 4d. Plot of baseline adjusted RP Inspiratory Time (seconds) for each animal in
Group 4.
1078-CG920794 Statistical Report - Telemetry
F-60
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25-
15-
5-
-5-
-15-
-25-
-35-
Animal 5
Animal 9
Animal 40
Animal 7
Animal 37
10 20 30
Days from Midnight of Challenge Day
40
Figure 5a. Plot of baseline adjusted RP Integral (mmHg-seconds) for each animal in
Group 1.
25-
15-
5-
-5-
-15-
-25-
-35-
Animal 13
Animal 19
Animal 28
Animal 34
— Animal 15
— Animal 25
— Animal 30
10 20 30
Days from Midnight of Challenge Day
40
Figure 5b. Plot of baseline adjusted RP Integral (mmHg-seconds) for each animal in
Group 2.
1078-CG920794 Statistical Report - Telemetry
F-61
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25-
15-
5-
-5-
-15-
-25-
-35-
Animal 2
Animal 11
Animal 14
Animal 32
Animal 8
Animal 12
Animal 18
10 20 30
Days from Midnight of Challenge Day
40
Figure 5c. Plot of baseline adjusted RP Integral (mmHg-seconds) for each animal in
Group 3.
25-
15-
5-
-5-
-15-
-25-
-35-
Animal 6
Animal 27
Animal 33
Animal 39
Animal 21
Animal 31
Animal 38
10 20 30
Days from Midnight of Challenge Day
40
Figure 5d. Plot of baseline adjusted RP Integral (mmHg-seconds) for each animal in
Group 4.
1078-CG920794 Statistical Report - Telemetry
F-62
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50-
40-
30-
20-
10-
0-
-10-
-20-
-30-
-40-
-50-
Animal 5
Animal 9
Animal 40
Animal 7
Animal 37
10 20 30
Days from Midnight of Challenge Day
40
Figure 6a. Plot of baseline adjusted RP Peak Amplitude (mmHg) for each animal in
Group 1.
50-
40-
30-
20-
10-
o-
-10-
-20-
-30-
-40-
-50-
Animal 13
Animal 19
Animal 28
Animal 34
— Animal 15
— Animal 25
— Animal 30
10 20 30
Days from Midnight of Challenge Day
40
Figure 6b. Plot of baseline adjusted RP Peak Amplitude (mmHg) for each animal in
Group 2.
1078-CG920794 Statistical Report - Telemetry
F-63
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50-
40-
30-
20-
10-
0-
-10-
-20-
-30-
-40-
-50-
Animal 2
Animal 11
Animal 14
Animal 32
— Animal 8
— Animal 12
— Animal 18
10 20 30
Days from Midnight of Challenge Day
40
Figure 6c. Plot of baseline adjusted RP Peak Amplitude (mmHg) for each animal in
Group 3.
50-
40-
30-
20-
10-
o-
-10-
-20-
-30-
-40-
-50-
Animal 6
Animal 27
Animal 33
Animal 39
Animal 21
Animal 31
Animal 38
10 20 30
Days from Midnight of Challenge Day
40
Figure 6d. Plot of baseline adjusted RP Peak Amplitude (mmHg) for each animal in
Group 4.
1078-CG920794 Statistical Report - Telemetry
F-64
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60-
50-
40-
30-
20-
10-
0-
-10-
-20-
-30-
-40-
-50-
Animal 5
Animal 9
Animal 40
— Animal 7
— Animal 37
10 20 30
Days from Midnight of Challenge Day
40
Figure 7a. Plot of baseline adjusted RP Respiratory Rate (RCPM) for each animal in
Group 1.
60-
50-
40-
30-
20-
10-
o-
-10-
-20-
-30-
-40-
-50-
Animal 13
Animal 19
Animal 28
Animal 34
— Animal 15
— Animal 25
— Animal 30
10 20 30
Days from Midnight of Challenge Day
40
Figure 7b. Plot of baseline adjusted RP Respiratory Rate (RCPM) for each animal in
Group 2.
1078-CG920794 Statistical Report - Telemetry
F-65
-------
60-
50-
40-
30-
20-
10-
0-
-10-
-20-
-30-
-40-
-50-
Animal 2
Animal 11
Animal 14
Animal 32
— Animal 8
— Animal 12
— Animal 18
10 20 30
Days from Midnight of Challenge Day
40
Figure 7c. Plot of baseline adjusted RP Respiratory Rate (RCPM) for each animal in
Group 3.
60-
50-
40-
30-
20-
10-
0-
-10-
-20-
-30-
-40-
-50-
Animal 6
Animal 27
Animal 33
Animal 39
Animal 21
Animal 31
Animal 38
10 20 30
Days from Midnight of Challenge Day
40
Figure 7d. Plot of baseline adjusted RP Respiratory Rate (RCPM) for each animal in
Group 4.
1078-CG920794 Statistical Report - Telemetry
F-66
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5-
4-
3-
2-
1-
0-
-1-
-2-
-3-
-4-
-5-
I
Animal 5
Animal 9
Animal 40
Animal 7
Animal 37
tyJ&sMWW^
10 20 30
Days from Midnight of Challenge Day
40
Figure 8a. Plot of baseline adjusted Temperature (Celsius) for each animal in Group 1.
5-
4-
3-
2-
1-
o-
-1-
-2-
-3-
-4-
-5-
Animal 13
Animal 19
Animal 28
Animal 34
— Animal 15
— Animal 25
— Animal 30
10 20 30
Days from Midnight of Challenge Day
40
Figure 8b. Plot of baseline adjusted Temperature (Celsius) for each animal in Group 2.
1078-CG920794 Statistical Report - Telemetry
F-67
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Figure 8c.
5-
4-
3-
2-
1-
o-
-1-
-2-
-3-
-4-
-5-
— Animal 2 — Animal 8
— Animal 11 — Animal 12
— Animal 14 — Animal 18
— Animal 32
NA..U AAM^ * * iiAL'/^f4* U U^ M UifA^f *>A>-
•pAVc^Al>$^J*>*^^^
•
F
0 10 20 30 40
Days from Midnight of Challenge Day
Plotc
5-
4-
3-
2-
1-
o-
-1-
-2-
-3-
-4-
-5-
»f baseline adjusted Temperature (Celsius) for each animal in Group 3
(I/I Jldlfe
WMMMMW
PI V^
— Animal 6 — Animal 21
— Animal 27 — Animal 31
- Animal 33 — Animal 38
— Animal 39
A
. bd,Va&JS*iAJtfcjAAMiw u /%*<
Y&YWwyv™^
10 20 30
Days from Midnight of Challenge Day
40
Figure 8d. Plot of baseline adjusted Temperature (Celsius) for each animal in Group 4.
1078-CG920794 Statistical Report - Telemetry
F-68
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3-
2-
1-
0-
-1-
-2-
-3-
Group 1 (Control)
Group 3 (1,000 CPU)
Group 2 (100 CPU)
Group 4 (10,000 CPU)
10 20 30
Days from Midnight of Challenge Day
40
Figure 9. Plot of mean baseline adjusted Activity (counts/minute) for each group.
75-
50-
25-
0-
-25-
-50-
-75-
-100-
-125-
Group 1 (Control)
Group 3 (1,000 CPU)
Group 2 (100 CPU)
Group 4 (10,000 CPU)
10 20 30
Days from Midnight of Challenge Day
40
Figure 10. Plot of mean baseline adjusted Heart Rate (BPM) for each group.
1078-CG920794 Statistical Report - Telemetry
F-69
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.25-
.2-
.15-
.1-
.05-
0-
-.05-
-.1-
-.15-
-.2-
-.25-
Group 1 (Control)
Group 3 (1,000 CPU)
Group 2 (100 CPU)
Group 4 (10,000 CPU)
10 20 30
Days from Midnight of Challenge Day
40
Figure 11. Plot of mean baseline adjusted RP Expiratory Time (seconds) for each group.
.2-
.15-
.1-
.05-
o-
-.05-
-.1-
-.15-
-.2-
Group 1 (Control)
Group 3 (1,000 CPU)
Group 2 (100 CPU)
Group 4 (10,000 CPU)
10 20 30
Days from Midnight of Challenge Day
40
Figure 12. Plot of mean baseline adjusted RP Inspiratory Time (seconds) for each
group.
1078-CG920794 Statistical Report - Telemetry
F-70
-------
10-
8-
6-
4-
2-
0-
-2-
-4-
-6-
-8-
-10-
Group 1 (Control)
Group 3 (1,000 CPU)
Group 2 (100 CPU)
Group 4 (10,000 CPU)
10 20 30
Days from Midnight of Challenge Day
40
Figure 13. Plot of mean baseline adjusted RP Integral (mmHg-seconds) for each group.
15-
10-
5-
o-
-5-
-10-
-15-
Group 1 (Control)
Group 3 (1,000 CPU)
Group 2 (100 CPU)
Group 4 (10,000 CPU)
10 20 30
Days from Midnight of Challenge Day
40
Figure 14. Plot of mean baseline adjusted RP Peak Amplitude (mmHg) for each group.
1078-CG920794 Statistical Report - Telemetry
F-71
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30-
20-
10-
0-
-10-
Group 1 (Control)
Group 3 (1,000 CPU)
Group 2 (100 CPU)
Group 4 (10,000 CPU)
10 20 30
Days from Midnight of Challenge Day
40
Figure 15. Plot of mean baseline adjusted RP Respiratory Rate (RCPM) for each group.
1.5-
1-
.5-
0-
-.5-
-1-
Group 1 (Control)
Group 3 (1,000 CPU)
Group 2 (100 CPU)
Group 4 (10,000 CPU)
10 20 30
Days from Midnight of Challenge Day
40
Figure 16. Plot of mean baseline adjusted Temperature (Celsius) for each group.
1078-CG920794 Statistical Report - Telemetry
F-72
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0.90-
0.80-
0.70-
0.60-
0.50-
0.40-
0.30-
0.20-
0.10
n nn
Group 1 (Contrc
Group 3 (1,000
• A
A
8 12 16 20 24 28 32
Days from Midnight of Challenge Day
36
40
Figure 17. Kaplan-Meier curves for time to abnormality based on Activity.
I.UU
0.90-
0.80-
0.70-
0.50-
0.40-
0.30-
0.20-
0.10-
0.00-
!|
— i
G
G
5
roup 1 (Control)
roup2(100CFU)
roup 3 (1,000 CPU)
roup 4 (10, 000 CPU)
8 12 16 20 24 28 32
Days from Midnight of Challenge Day
36 40
Figure 18. Kaplan-Meier curves for time to abnormality based on Heart Rate.
1078-CG920794 Statistical Report - Telemetry
F-73
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1.00-
0.90-
0.80-
0.70-
0.60-
0.50-
0.40-
0.30-
0.20-
0.10-
0.00-
Group 1 (Control)
Group 3 (1,000 CPU)
Group 2 (100 CPU)
Group 4 (10,000 CPU)
8 12 16 20 24 28 32
Days from Midnight of Challenge Day
36
40
Figure 19. Kaplan-Meier curves for time to abnormality based on RP Expiratory Time.
I.UU
0.90-
0.70-
0.50-
0.40-
0 30-
0.20-
.1 U
n nn-
(jro
(jro
jp i
jp o
•_
• •
8 12 16 20 24 28 32
Days from Midnight of Challenge Day
36 40
Figure 20. Kaplan-Meier curves for time to abnormality based on RP Inspiratory Time.
1078-CG920794 Statistical Report - Telemetry
F-74
-------
1 .UU
0.90-
0.80-
0.70-
0.60-
0.50-
0.40-
0.30-
0.20-
0.10-
0.00-
I L
•
•
(jioup 1 (uontioi) - (jioup z (luu ui U)
(jroup o (1,1)1)1) UrU) (jroup 4 (lU.UUU UrU)
8 12 16 20 24 28 32
Days from Midnight of Challenge Day
36 40
Figure 21. Kaplan-Meier curves for time to abnormality based on RP Integral.
1.00-
0.90-
0.80-
0.70-
0.60-
0.50-
0.40-
0.30-
0.20-
0.10-
0.00-
f
Group 1 (Control)
Group 2 (100 CPU)
L
|
^
.
8 12 16 20 24 28 32
Days from Midnight of Challenge Day
36
40
Figure 22. Kaplan-Meier curves for time to abnormality based on RP Peak Amplitude.
1078-CG920794 Statistical Report - Telemetry
F-75
-------
0.90-
0.80-
0.70-
0.60-
0.50-
0.40-
0.30-
0.20-
010
o.oo-
1
(
roup 1 (Co
roup 3 (1,0
I
^~»
8 12 16 20 24 28 32
Days from Midnight of Challenge Day
36
40
Figure 23. Kaplan-Meier curves for time to abnormality based on RP Respiratory Rate.
1.00-
0.90
0.80-
0.70-
0.50-
OA n
0.30-
0.20-
0.10-
0.00-
— •
I
Group 3 (1,000 CPU)
8 12 16 20 24 28 32
Days from Midnight of Challenge Day
36 40
Figure 24. Kaplan-Meier curves for time to abnormality based on Temperature.
1078-CG920794 Statistical Report - Telemetry
F-76
-------
0.90-
0.80-
0.70-
0.60-
.ou
0.40-
0.30-
0.20-
0.10-
o.oo-
^-. A f l~* JL l\
8 12 16 20 24 28 32
Days from Midnight of Challenge Day
36
40
Figure 25. Kaplan-Meier curves for duration of abnormality based on Activity.
0.90-
0.80-
0.70-
.bU
0.40-
0.30-
0.20-
0.10-
o.oo-
Group 1 (Co
•_
ntrol)
OCFU)
00 CPU)
000 CPU)
8 12 16 20 24 28 32
Days from Midnight of Challenge Day
36
40
Figure 26. Kaplan-Meier curves for duration of abnormality based on Heart Rate.
1078-CG920794 Statistical Report - Telemetry
F-77
-------
1.00-
0.90-
0.80-
0.70-
0.60-
0.50-
0.40-
0.30-
0.20-
0.10-
0.00-
Group 1 (Control)
Group 2 (100 CPU)
Group 3 (1,000 CPU)
Group 4 (10,000 CPU)
8 12 16 20 24 28 32
Days from Midnight of Challenge Day
36
40
Figure 27. Kaplan-Meier Curves for duration of abnormality based on RP Expiratory
Time.
0.90-
0.80-
0.70-
0.60-
.ou
0.40-
0.30-
.ZU
0.10-
o.oo-
j*
— 1
Group 1 (Control)
)
j)
8 12 16 20 24 28 32
Days from Midnight of Challenge Day
36 40
Figure 28. Kaplan-Meier curves for duration of abnormality based on RP Inspiratory
Time.
1078-CG920794 Statistical Report - Telemetry
F-78
-------
0.90-
0.80-
0.70-
0.60-
0.50-
0.40-
0.30-
0.20-
0.10-
o.oo-
n
\
8 12 16 20 24 28 32
Days from Midnight of Challenge Day
36
40
Figure 29. Kaplan-Meier curves for duration to abnormality based on RP Integral.
1.00-
0.90-
0.80-
0.70-
0.60-
.ou
0.40-
0.30-
0.20-
0.10-
o.oo-
^ > i^
Group 3 (1,0
itrol)
CPU)
DO CPU)
DOO CPU)
A
^
• m
8 12 16 20 24 28 32
Days from Midnight of Challenge Day
36 40
Figure 30. Kaplan-Meier curves for duration to abnormality based on RP Amplitude.
1078-CG920794 Statistical Report - Telemetry
F-79
-------
0.90-
0.80-
0.70-
0.60-
0.50-
0.40-
0.30-
0.20-
0.10-
o.oo-
• (•
ntrol)
OCFU)
)00 CPU)
000 CPU)
•
8 12 16 20 24 28 32
Days from Midnight of Challenge Day
36
40
Figure 31. Kaplan-Meier curves for duration of abnormality based on RP Respiratory
Rate.
1.00-
0.90-
0.80-
0.70-
0.60-
0.50-
0.40-
0.30-
0.20-
0.10-
0.00-
Group 1 (Control)
Group 2 (100 CPU)
Group 3 (1,000 CPU)
Group 4 (10,000 CPU)
8 12 16 20 24 28 32
Days from Midnight of Challenge Day
36
40
Figure 32. Kaplan-Meier curves for duration of abnormality based on Temperature.
1078-CG920794 Statistical Report - Telemetry
F-80
-------
APPENDIX G
STATISTICAL REPORT - MORTALITY
1078-CG920794 - Statistical Report - Mortality G-l
-------
Table of Contents
1. Introduction G-4
2. Statistical Methods G-5
3. Results G-6
4. Conclusions G-7
List of Tables
Table 1. Study Design G-4
Table 2. Proportion of Surviving Animals with Exact 95% Confidence Interval by Group. G-7
Table 3. Results of Two-Sided Pairwise Fisher's Exact Tests G-8
Table 4. Results of Pairwise Log-Rank Tests G-8
List of Figures
Figure 1. Estimated logistic regression curve and observed survival or mortality G-8
Figure 2. Kaplan-Meier curves representing time to death and survival data for each group.... G-9
1078-CG920794 - Statistical Report - Mortality
G-2
-------
List of Acronyms
BBRC Battelle Biomedical Research Center
CPU Colony forming unit
LDso Median lethal dose
N Number of animals
1078-CG920794 - Statistical Report - Mortality G-3
-------
1. Introduction
This report summarizes the statistical analysis of survival data collected under Battelle
Biomedical Research Center (BBRC) Study No. 1078-CG920794. Twenty-six (26) pathogen free
New Zealand White rabbits were randomly assigned to one of four groups of animals as shown
in Table 1. Beginning on Study Day 0, animals were exposed to Bacillus anthracis (Ames strain)
spores once a day for five straight working days each week for three straight weeks, at targeted
doses shown in Table 1. The control group (Group 1) was exposed to gamma-irradiated spores.
Table 1. Study Design
Group
1
2
3
4
Number of Animals
per Group
5
7
7
7
Target Spore Dose
(CPU)
10,000*
100
1,000
10,000
Number of Spore
Challenges
15
*These spores were inactivated by radiation.
1078-CG920794 - Statistical Report - Mortality
G-4
-------
2. Statistical Methods
Estimates with exact 95% binomial confidence intervals for the proportion of surviving animals
within each group were calculated. An overall two-sided Fisher's exact test was performed to
determine if the proportions of surviving animals were significantly different between the
groups. If the overall Fisher's exact test was significant, then pairwise two-sided Fisher's exact
tests were performed to determine which pairs of groups were significantly different from each
other. A Bonferroni-Holm adjustment was made to maintain an overall 0.05 level of significance
for the multiple pairwise comparisons.
For each animal, the geometric mean inhaled dose from the 15 spore challenges was calculated
and used in the statistical analysis. Excluding the control group (Group 1), a logistic regression
model was fitted to the survival data as a function of the base -10 log transformed geometric
mean inhaled dose to determine the effect of dose on lethality. The median lethal dose (LD50)
was then estimated from the predicted logistic regression curve, along with 95% Fieller's
confidence intervals.
The time-to-death data were analyzed in combination with the survival data to determine if there
were significant differences between the groups in terms of susceptibility to challenge.
Kaplan-Meier curves were plotted for each group and an overall log-rank test was performed to
determine if the survival distributions within the groups were significantly different from each
other. If the overall log-rank test was significant, then pairwise log-rank tests were performed to
determine which groups were significantly different from each other. Again, the
Bonferroni-Holm adjustment was used to maintain an overall 0.05 level of significance for the
multiple pairwise comparisons.
All statistical analyses were performed using Stata (StataCorp LP; College Station, TX;
version 11.1). All results are reported at the 0.05 level of significance.
1078-CG920794 - Statistical Report - Mortality G-5
-------
3. Results
Table 2 contains the estimated proportion of surviving animals within each group, along with
exact binomial 95% confidence intervals. All animals in Groups 1 and 2 survived the length of
the study. Six of the seven animals in Group 3 survived, and three of the seven animals in
Group 4 survived.
The overall Fisher's exact test was significant (p-value=0.0425). Table 3 contains the unadjusted
and Bonferroni-Holm adjusted p-values from pairwise Fisher's exact tests. When all animals in
both comparison groups survived, the Fisher's exact tests could not be performed; therefore,
p-values of 1.0000 were substituted to indicate that the groups were not significantly different
from each other. Regardless of the adjustment for multiple comparisons, there were no
significant pairwise differences between the groups.
The logistic regression model fitted to the survival data indicated a significant dose response
relationship with increased inhaled doses being associated with decreased probabilities of
survival, as evidenced by the significant p-value associated with the estimated slope coefficient
of-1.30 (p-value=0.0288). The estimated LD50 was 8,094 colony forming units (CFU) with a
95% Fieller confidence interval ranging from 2,276 CFU to 36,135,187 CFU. Figure 1 displays
the fitted logistic regression model overlaid on the observed survival (or mortality) data.
The overall log-rank test was significant (p-value=0.0135), indicating that the survival
distribution in at least one of the groups was significantly different from those in the other
groups. Table 4 contains the unadjusted and Bonferroni-Holm adjusted p-values from pairwise
log-rank tests. When all animals in both comparison groups survived, the log-rank tests could not
be performed; therefore, p-values of 1.0000 were substituted to indicate that the groups were not
significantly different from each other. Prior to adjusting for multiple comparisons, the time to
death in Group 2 was significantly greater than that in Group 4. However, this relationship was
no longer significant after adjusting for the multiple pairwise comparisons. Figure 2 displays the
Kaplan-Meier curves for each of the four dose groups. Since all animals in Groups 1 and 2
survived the length of the study, Group 2 was plotted with a slight offset so that the curves would
be distinguishable. A dose response relationship was observed, with increased target doses
generally being associated with decreased times to death and greater mortality.
1078-CG920794 - Statistical Report - Mortality G-6
-------
4. Conclusions
The proportion of surviving animals decreased for groups that received higher targeted spore
doses. All animals in the control group and the targeted 100 CPU dose group (Groups 1 and 2,
respectively) survived the length of the study. Six of seven animals in the targeted 1,000 CPU
dose group (Group 3) survived, while only three of seven animals in the targeted 10,000 CPU
dose group (Group 4) survived. There were no significant differences in survival rates between
any pair of groups according to a Fisher's exact test.
The results for the logistic regression model fitted to the survival data indicated a significant
dose response relationship with increased inhaled doses being associated with decreased
probabilities of survival. The estimated LDso was 8,094 CPU per challenge day with a 95%
Fieller confidence interval ranging from 2,276 CPU to 36,135,187 CPU.
The overall log-rank test indicated that the survival distribution in at least one of the groups was
significantly different from those in the other groups. Prior to adjusting for multiple
comparisons, the time to death in the targeted 100 CPU dose group (Group 2) was significantly
greater than that in the targeted 10,000 CPU dose group (Group 4). However, this relationship
was no longer significant after adjusting for the multiple pairwise comparisons. A dose response
relationship was observed in the Kaplan-Meier plots, with increased target doses generally being
associated with decreased times to death and greater mortality.
Table 2. Proportion of Surviving Animals with Exact 95% Confidence Interval by Group
Group
1
2
3
4
Number of Surviving
Animals / N
5/5
7/7
6/7
3/7
Proportion Survived
(Exact 95% Confidence Interval)
1.00(0.48, 1.00)
1.00(0.59, 1.00)
0.86(0.42, 1.00)
0.43(0.10,0.82)
N Number of animals.
1078-CG920794 - Statistical Report - Mortality
G-7
-------
Table 3. Results of Two-Sided Pairwise Fisher's Exact Tests
Group
1
2
3
4
Two-Sided Pairwise Fisher's Exact Test P-Values
Unadjusted P-Values
2
1.00003
2222222
3
1.0000
1.0000
Y/////A
4
0.0808
0.0699
0.2657
Bonferroni-Holm Adjusted P-Values
2
1.00003
22222
3
1.0000
1.0000
w^
4
0.3497
0.3497
0.7972
/////////;
aAp-value of 1.0000 was substituted since all animals in both groups survived.
Table 4. Results of Pairwise Log-Rank Tests
Group
1
2
3
4
Pairwise Log-Rank Test P-Values
Unadjusted P-Values
2
1.0000a
Y//////S
'///////.
'//////A
3
0.3980
0.3173
'//////S
//////A
4
0.0526
0.0221*
0.0916
'///////
Bonferroni-Holm Adjusted P-Values
2
1.00003
'//////,
///////,
Y/////A
3
0.6346
0.6346
'/////;
/////A
4
0.2102
0.1103
0.2747
'/////////
aA p-value of 1.0000 was substituted since all animals in both groups survived.
*Comparison was significant at the 0.05 level.
10'
Geometric Mean Inhaled Dose (CPU)
Figure 1. Estimated logistic regression curve and observed survival or mortality.
1078-CG920794 - Statistical Report - Mortality
G-8
-------
I.LT
0.9-
0.8-
0.7-
0.6-
0.5-
0.4-
0.3-
0.2-
0.1-
0.0-
"
\-
Group 1 (Control)
Group 2 (100 CPU)
Group 3 (1,000 CPU)
- Group 4 (10,000 CPU)
10 15 20 25 30
Time to Death (Days)
35 40
All animals in Groups 1 and 2 survived the length of the study.
Figure 2. Kaplan-Meier curves representing time to death and survival data for each
group.
1078-CG920794 - Statistical Report - Mortality
G-9
-------
APPENDIX H
STASTICIAL REPORT - BODY WEIGHTS
1078-CG920794 - Statistical Report Body Weights H-l
-------
Table of Contents
1. Introduction H-4
2. Statistical Methods H-5
3. Results H-6
4. Conclusions H-7
List of Tables
Table 1. Study Design H-4
Table 2. Means with 95% Confidence Intervals for Weight (kilograms) by Group and Study
Day H-8
Table 3. Summary of Test Results for Group Comparisons of Body Weight by Study Day H-8
List of Figures
Figure 1. Group mean weights with 95% confidence intervals over time H-9
1078-CG920794 - Statistical Report Body Weights H-2
-------
List of Acronyms
ANOVA analysis of variance
BBRC Battelle Biomedical Research Center
CPU colony forming unit
N number of animals
1078-CG920794 - Statistical Report Body Weights H-3
-------
1. Introduction
This report summarizes the statistical analysis of body weight data collected under Battelle
Biomedical Research Center (BBRC) Study No. 1078-CG920794. Twenty-six (26)
pathogen-free New Zealand White rabbits were randomly assigned to one of four groups of
animals as shown in Table 1. Beginning on Study Day 0, animals were exposed to
Bacillus anthracis (Ames strain) spores once a day for five straight working days each week for
three straight weeks, at targeted doses shown in Table 1. The control group (Group 1) was
exposed to gamma-irradiated spores. Animals were weighed on Study Days 2, 9, 16, 23, 30
and 37.
Table 1. Study Design
Group
1
2
3
4
Number of Animals
per Group
5
7
7
7
Target Spore Dose
(CPU)
10,000*
100
1,000
10,000
Number of Spore
Challenges
15
Body Weight
Measurements
Study days 2, 9, 16,
23, 30, and 37
CPU Colony forming units.
* These spores were inactivated by radiation.
1078-CG920794 - Statistical Report Body Weights
H-4
-------
2. Statistical Methods
An analysis of variance (ANOVA) model fitted to the body weight data with effects for group,
study day, and the interaction between group and study day was used to assess the model
assumption of normality and to identify potential outliers. Standardized residuals from this
ANOVA model were obtained and a hypothesis test was performed to assess the model
assumption of normality for the untransformed data. Weight was then transformed by taking the
base-10 logarithm of the original values, and the ANOVA model was refitted. A hypothesis test
was again performed to assess the model assumption of normality for the log-transformed data.
If the assumption of normality was more reasonable for the log-transformed data than it was for
the untransformed data, then the log-transformed data were used throughout the analysis.
Deleted studentized residuals, which are the standardized residuals from the model fitted to all
data except the current observation, were computed for each observation. If the absolute value
of the deleted studentized residual was greater than 4, then the observation was considered a
potential outlier. If any potential outliers were identified, then the statistical analysis was
performed both with and without these observations to evaluate their effect on the results.
On each study day, the following ANOVA model was fitted to the body weight data to determine
if there were significant differences between the groups:
Ydtj = M. + group,• + eij (1)
where ¥<#/ is the observed weight for they'th animal in Group /' (/'=!, 2, 3, 4) on Study
Day d(d=2, 9, 16, 23, 30, and 37), u^ is an overall constant, Group t is the effect of Group /',
and Sy is the random error left unexplained by the model. Tukey's multiple comparisons
procedure was also performed on each study day to determine which pairs of groups had mean
body weights that were significantly different from each other.
All statistical analyses were performed using Stata (StataCorp LP; College Station, TX;
Version 11.1). All results are reported at the 0.05 level of significance.
1078-CG920794 - Statistical Report Body Weights H-5
-------
3. Results
The model assumption of normality was not more reasonable when the models were fitted to the
log-transformed data; therefore, all models were fitted to the untransformed values.
Additionally, no potential outliers were identified.
Table 2 contains descriptive statistics (including means with 95% confidence intervals) for the
weights within each group on each study day. All animals in Groups 1 and 2 survived the length
of the study. One of seven animals in Group 3 died prior to Study Day 23. In Group 4, three of
seven animals died prior to Study Day 16 and one additional animal died prior to Study Day 23.
The analyses performed on each study day included only surviving animals and, thus, are based
on smaller sample sizes for these groups on later study days. Figure 1 displays the group mean
weights with 95% confidence intervals for Study Days 2 through 37.
Table 3 contains the results obtained from fitting ANOVA models with a group effect to the
body weight data on each study day. There were no significant differences between the groups
on any study day.
1078-CG920794 - Statistical Report Body Weights H-6
-------
4. Conclusions
All animals in the control group and the targeted 100 colony forming units (CPU) dose group
(Groups 1 and 2, respectively) survived the length of the study. One of seven animals in the
targeted 1,000 CPU dose group (Group 3) died prior to Study Day 23. In the targeted
10,000 CPU dose group (Group 4), three of seven animals died prior to Study Day 16 and one
additional animal died prior to Study Day 23. The analyses performed on each study day include
only surviving animals and, thus, are based on smaller sample sizes for these groups on later
study days. Body weights were not significantly different between the groups on any study day.
1078-CG920794 - Statistical Report Body Weights H-7
-------
Table 2. Means with 95% Confidence Intervals for Weight (kilograms) by Group and
Study Day
Group
1
2
3
4
Study
Day
2
9
16
23
30
37
2
9
16
23
30
37
2
9
16
23
30
37
2
9
16
23
30
37
N
5
5
5
5
5
5
7
7
7
7
7
7
7
7
7
6
6
6
7
7
4
3
3
3
Mean
(95% Confidence Interval)
2.82(2.50,3.13)
2.87 (2.55, 3.20)
2.96 (2.66, 3.26)
2.96 (2.67, 3.26)
3.00 (2.74, 3.25)
3.04 (2.79, 3.29)
2.76 (2.66, 2.87)
2.84 (2.74, 2.94)
2.92 (2.83, 3.01)
2.95 (2.85, 3.04)
2.99(2.91,3.07)
3.02(2.92,3.12)
2.78 (2.59, 2.97)
2.84 (2.66, 3.02)
2.91 (2.70,3.11)
2.98(2.76,3.19)
3.03 (2.83, 3.23)
3.06 (2.85, 3.26)
2.77 (2.66, 2.89)
2.81 (2.71,2.92)
2.94(2.75,3.14)
2.94 (2.47, 3.40)
2.97 (2.43, 3.51)
2.96 (2.39, 3.53)
N Number of animals.
Table 3. Summary of Test Results for Group Comparisons of Body Weight by Study Day
Study Day
2
9
16
23
30
37
Mean Body Weight (Kilograms), by Group
1
2.82
2.87
2.96
2.96
3.00
3.04
2
2.76
2.84
2.92
2.95
2.99
3.02
3
2.78
2.84
2.91
2.98
3.03
3.06
4
2.77
2.81
2.94
2.94
2.97
2.96
Group Effect
P-Value
0.9632
0.9481
0.9606
0.9881
0.9521
0.8865
1078-CG920794 - Statistical Report Body Weights
H-8
-------
3.75-
3.50-
3.25-
3.00-
2.75-
2.50-
2.25-
—A
Group 1 (Control)
— Group 2 (100 CPU)
Group 3 (1 ,000 CPU)
Group 4 (10,000 CPU)
16
23
30
37
Study Day
Figure 1. Group mean weights with 95% confidence intervals over time.
1078-CG920794 - Statistical Report Body Weights
H-9
-------
APPENDIX I
BLOOD DRAW TIMES
1-1
-------
1078-CG920794
Blood Draws
Challenge
Order
1
1
s
;
Aramai ID
t)
1
&
lr
I 3
4
1,
11
J
<3
j
3
n
Sex
V
M
f/
M
f/
M
V
M
M
M
M
ft*
f/
M
h'
IV
M
M
*d
M
!><*
!V
Group
1
1
1
1
1
I
3
3
3
3
Study 0
Date
7/23/2010 '
7/23/2010
7/23/2010
7/23/2010
7/23/2010
7/23/2010
7/23/2010
7/23/2010
7/23/2010
7/23/20IO
7/23/2010
7/23/2010
7/23/2810
7/B/2D10
7/23/20 IS
7/23/2010
7/23/2010
7/23/2S18
7/23/2010
7/23/2018
7/23/20 IS
7/23/2010
?/23/20lfl
7/23/2G10
7/23/2010
7/23/2010
§y(-3J
Time
0948 €
0900
0901
S9S1C
&90S
091$
<3315
0921
0925
HMB
0957
1002
100?
1039 C
1011
1048 C
1019
1024
1027
1031
1035
1SS4
10S8
Study
Datg
//2 20 J
7/,> * 1
7/*8// °
"y */ x
/~ i i"1
A.fe *01u
7/21"1 2r10
'/*.P/2iulJ
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!/£>• I i
7/2H' 10
7/^S/zt,1'
"7.^2 U
7/2S/JD1C
7/28/2 C la
7/2S/2C10
7/2S/20U
7/2S/2010
7/28/20 1U
7/2S/2010
7/2S/2Q10
7/2S/2010
7/28/2^10
Day 2
OS3r C
LS23
Q827
1844 C
1,923
UJ29
J333
0937
G 4S
0^57
100J
1308
U847C
lull
OSb4C
1047 C
l'J27
It30
1034
1039
1042
1055
sed
Study
7/30/2Q1Q
7/30/2010
7/30/2010
7/SS/2010
7/30/2010
7/30/2G10
7/30/2010
7/30/2Q10
7/30/2010
7/30/2010
7/30/2010
7/30/2010
7/30/2010
7/30/2010
7/30/2010
7/30/2010
7/30/2010
7/30/2€10
7/30/2010
7/30/2010
7/30/2010
7/3Q/2010
7/30/2010
7/30/2010
Day 4
Time
OS21 C
0747
0754
0829 C
0903
0846
08S3
Q859
0906
0911
0917
0923
0842 C
0929
0847 C
Q8S2C
0935
0940
OSS7C
0950
0953
0957
1002
1005
Stud
Date
8/4/20 1G
S/4/2Q10
S/4/2010
8/4/2010
8/4/2010
S/4/2010
8/4/2010
S/4/2010
8/4/20 10
S/4/2010
8/4/2010
8/4/2010
8/4/2010
S/4/2010
8/4/2010
8/4/201Q
S/4/2010
8/4/2010
8/4/2010
S/4/2010
8/4/2010
8/4/2010
S/4/2010
S/4/2010
C0ay9
0825C
0830 C
Q800
QSOS
OS38C
OS11
OS57
OSS6
0908
0912
0913
0917
0918
0844 C
0920
ossoc
0857 C
0920
0925
0901 C
0930
0935
0936
0940
0940
Study
8/6/2010
8/6/2010
8/6/2010
8/6/2010
8/6/2010
8/6/2010
8/8/2010
8/6/2010
S/6/2010
8/6/2010
8/6/2010
S/6/2010
8/8/2010
8/6/2010
8/6/2010
8/6/2010
8/6/2010
8/6/2010
S/6/2010
S/6/2010
8/6/2010
8/6/2010
8/6/2010
Day 11
Time
0836 C
0839 C
€S43
QSSQ
0842 C
085S
0928
0929
0934
0934
0939
0939
0945
0900 C
G9SQ
0910 C
0916C
09SO
1004
0919C
095?
1009
1009
Study
Date
8/1 1/20 IS
8/11/2S10
8/11/2010
8/11/2010
8/11/2010
8/11/2010
8/11/2010
8/11/2010
8/11/20 10
8/11/2010
8/11/2010
8/11/2010
a/ii/2010
8/11/2010
.8/11/2010
8/11/2010
8/11/2010
^8/11/2010
8/11/2010
8/11/20 IS
Day 16
Time
084 ^C
USS4'
09^2
093<;
09C"* t
1020 L
WZ*(
09C
ISO
10 ""
10 U
1102*
1047
090 C
105 ">
0913 C
0924 C
1131 C
1SS9
11.0?
1118
Study
f(l ^^
v/l-s * lw
° r<2 i
*• H ^O1
±JJL^ J
t'n - j i
<• r ,
- i ',..'
V 1 ii-K
«M« 11
V !•'. ii
f ia . I
'__^--J~
fc 1^ .
>• 1 ^i!,
' i : ; 1
8/13/21 '
^^-^^
S/13/2C1
^>__-^==B-'
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8/13/2ta
8/1 3/20 1C
Day 18
tt.o t
1 ._
CM*
- n
^-^
i '"L
ji i
T J ft
^~^~
^^
1
1 '-I
Po«* I of 2
-------
107S-CG920794
Blood Draws
Group
Study Day 23
Study Day 2S
Study Day 30
Study Day 32
Study Day 37
Day 39- Terminal
A) Initial sample drawn at 1003 unable to obtain full sample, more blood drawn at 1039 after animal found deat
8} Only able to attain - 0,25 ml on blood draw, drew ~ 0,5 ml at 1140
C| Acepromazirw (0.7 ml) was administered prior to blood colled
-------
APPENDIX J
STATISTICAL REPORT - HEMATOLOGY
AND C-REACTIVE PROTEIN
1078-CG920794 - Hematology and C-Reactive Protein J-l
-------
Table of Contents
1. Introduction J-6
2. Statistical Methods J-8
3. Results J-10
4. Conclusions J-15
Attachment 1 1-1
List of Tables
Table 1. Study Design J-6
Table 2. Potential Hematology and CRP Outliers J-16
TableS. Summary of ANOVA Results for Baseline (Study Day-3) Data J-16
Table 4a. Descriptive Statistics for Red Blood Cell Count (RBC, 106 cells/jiL),
by Group and Study Day J-17
Table 4b. Test Results for Red Blood Cell Count (RBC, 106 cells/jiL) J-18
Table 5a. Descriptive Statistics for Hemoglobin (HGB, g/dL) by Group and Study Day J-19
Table 5b. Test Results for Hemoglobin (HGB, g/dL) J-20
Table 6a. Descriptive Statistics for Hematocrit (HCT, %) by Group and Study Day J-21
Table 6b. Test Results for Hematocrit (HCT, %) J-22
Table 7a. Descriptive Statistics for Mean Corpuscular Volume (MCV, fL)
by Group and Study Day J-23
Table 7b. Test Results for Mean Corpuscular Volume (MCV, fL) J-24
Table 8a. Descriptive Statistics for Mean Corpuscular Hemoglobin (MCH, pg)
by Group and Study Day J-25
Table 8b. Test Results for Mean Corpuscular Hemoglobin (MCH, pg) J-26
Table 9a. Descriptive Statistics for Mean Corpuscular Hemoglobin Concentration
(MCHC, g/dL) by Group and Study Day J-27
Table 9b. Test Results for Mean Corpuscular Hemoglobin Concentration
(MCHC, g/dL) J-28
Table lOa. Descriptive Statistics for Red Cell Distribution Width (ROW, %) by Group
and Study Day J-29
Table lOb. Test Results for Red Cell Distribution Width (ROW, %) J-30
Table 1 la. Descriptive Statistics for Platelet Count (PLT, 103 cells/jiL) by Group and
Study Day J-31
Table lib. Test Results for Platelet Count (PLT, 103 cells/|iL) J-32
1078-CG920794 - Hematology and C-Reactive Protein J-2
-------
Table 12a. Descriptive Statistics for Mean Platelet Volume (MPV, fL) by Group and
Study Day J-33
Table 12b. Test Results for Mean Platelet Volume (MPV, fL) J-34
Table 13a. Descriptive Statistics for White Blood Cell Count (WBC, 103 cells/jiL)
by Group and Study Day J-35
Table 13b. Test Results for White Blood Cell Count (WBC, 103 cells/jiL) J-36
Table 14a. Descriptive Statistics for Neutrophil Count (103 cells/jiL) by Group and
Study Day J-37
Table 14b. Test Results for Neutrophil Count (103 cells/jiL) J-38
Table 15a. Descriptive Statistics for Lymphocyte Count (103 cells/|iL) by Group and
Study Day J-39
Table 15b. Test Results for Lymphocyte Count (103 cells/jiL) J-40
Table 16a. Descriptive Statistics for Neutrophil Count/Lymphocyte Count Ratio
by Group and Study Day J-41
Table 16b. Test Results for Neutrophil Count/Lymphocyte Count Ratio J-42
Table 17a. Descriptive Statistics for Monocyte Count (103 cells/jiL) by Group and
Study Day J-43
Table 17b. Test Results for Monocyte Count (103 cells/jiL) J-44
Table 18a. Descriptive Statistics for Eosinophil Count (103 cells/jiL) by Group and
Study Day J-45
Table 18b. Test Results for Eosinophil Count(103 cells/jiL) J-46
Table 19a. Descriptive Statistics for Basophil Count (103 cells/jiL) by Group and Study
Day J-47
Table 19b. Test Results for Basophil Count (103 cells/jiL) J-48
Table 20a. Descriptive Statistics for C-Reactive Protein (103 cells/jiL) by Group and
Study Day J-49
Table 20b. Test Results for C-Reactive Protein (103 cells/jiL) J-50
Table 1-1. Test Results for Red Cell Distribution Width (ROW, %) with Potential
Outliers Excluded 1-2
Table 1-2. Test Results for Hemoglobin (RGB, g/dL) with Potential Outliers Excluded 1-3
1078-CG920794 - Hematology and C-Reactive Protein J-3
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List of Figures
Figure 1. Plot of Red Blood Cell Count over time J-51
Figure 2. Plot of Hemoglobin overtime J-51
Figure 3. Plot of Hematocrit overtime J-52
Figure 4. Plot of Mean Corpuscular Volume (MCV) overtime J-52
Figure 5. Plot of Mean Corpuscular Hemoglobin (MCH) overtime J-53
Figure 6. Plot of Mean Corpuscular Hemoglobin Concentration (MCHC) overtime J-53
Figure 7. Plot of Red Cell Distribution Width (ROW) overtime J-54
Figure 8. Plot of Platelet Count (PLT) overtime J-54
Figure 9. Plot of Mean Platelet Volume (MPV) overtime J-55
Figure 10. Plot of White Blood Cell Count over time J-55
Figure 11. Plot of Neutrophil Count overtime J-56
Figure 12. Plot of Lymphocyte Count overtime J-56
Figure 13. Plot of Neutrophil Count/Lymphocyte Count Ratio over time J-57
Figure 14. Plot of Monocyte Count overtime J-57
Figure 15. Plot of Eosinophil Count overtime J-58
Figure 16. Plot of Basophil Count overtime J-58
Figure 17. Plot of C-Reactive Protein overtime J-59
1078-CG920794 - Hematology and C-Reactive Protein J-4
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List of Acronyms
ANOVA Analysis of Variance
BBRC Battelle Biomedical Research Center
CPU Colony Forming Units
CRP C-Reactive Protein
dL deciliter
fL femtoliter
g gram
HCT Hematocrit
HGB Hemoglobin
MCH Mean Corpuscular Hemoglobin
MCHC Mean Corpuscular Hemoglobin Concentration
MCV Mean Corpuscular Volume
MPV Mean Platelet Volume
N Number of Animals
N/L Ratio Neutrophil Count/Lymphocyte Count Ratio
PLT Platelet Count
RBC Red Blood Cell Count
ROW Red Cell Distribution Width
WBC White Blood Cell Count
uL microliter
um micrometer
1078-CG920794 - Hematology and C-Reactive Protein J-5
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1. Introduction
This report summarizes the statistical analysis of hematology data collected under Battelle
Biomedical Research Center (BBRC) Study No. 1078-CG920794. Twenty-six (26)
pathogen-free New Zealand White rabbits were randomly assigned to one of four groups of
animals as shown in Table 1. Beginning on Study Day 0, animals were exposed to
Bacillus anthracis (Ames strain) spores once a day for five straight working days each week for
three straight weeks, at targeted doses shown in Table 1. The control group (Group 1) was
exposed to gamma irradiated spores.
Table 1. Study Design
Group
1
2
3
4
Number of Animals
per Group
5
7
7
7
Target Spore Dose (CPU)
10,000*
100
1,000
10,000
Hematology and CRP Blood
Collection Study Days
-3,2,4,9, 11, 16, 18,23,
25, 30, 32, 37, and 39a
Spores are gamma-irradiated (negative control)
Terminal blood draw
Blood samples were collected for hematology and C-reactive protein (CRP) analysis as indicated
in Table 1. Blood collection on Study Day -3 served as a pre-challenge baseline for each animal.
The hematology parameters that were included in this analysis are:
Red Blood Cell Parameters
• Red blood cell count (RBC, 106 cells/uL)
• Hemoglobin (RGB, g/dL)
• Hematocrit (HCT, %)
• Mean corpuscular volume (MCV, fL)
• Mean corpuscular hemoglobin (MCH, pg)
• Mean corpuscular hemoglobin concentration (MCHC, g/dL)
• Red cell distribution width (ROW, %)
Platelet Count and Volume
• Platelet count (PLT, 103 cells/uL)
• Mean platelet volume (MPV, fL)
1078-CG920794 - Hematology and C-Reactive Protein
J-6
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Total and Differential White Blood Cell Parameters
• White blood cell count (WBC, 103 cells/uL)
• Neutrophil count (103 cells/uL)
• Lymphocyte count (103 cells/uL)
• Neutrophil count/lymphocyte count ratio (N/L ratio)
• Monocyte count (103 cells/uL)
• Eosinophil count (103 cells/uL)
• Basophil count (103 cells/uL)
Hematology and CRP analysis was performed at four levels:
1. Descriptive statistics (including arithmetic or geometric means and 95% confidence
intervals) were calculated for each parameter, by group and Study Day.
2. A baseline analysis, using the measurements from Study Day -3, was performed for
each parameter to determine if there were significant differences between the groups
prior to the administration of challenge.
3. Estimates for the mean shift from baseline (the measurement on Study Day -3) were
obtained for each parameter, group, and Study Day. These shifts were evaluated to
determine if they were significantly different from "no shift."
4. The mean shifts from baseline for each parameter and Study Day were compared
between the groups. Those groups having mean shifts that were significantly different
from each other were reported.
1078-CG920794 - Hematology and C-Reactive Protein J-7
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2. Statistical Methods
Analysis of variance (ANOVA) models fitted separately to each hematology parameter and CRP
with effects for group, Study Day, and the interaction between group and Study Day were used
to assess the model assumption of normality and to identify potential outliers. Standardized
residuals from these ANOVA models were obtained and a hypothesis test was performed for
each parameter to assess the model assumption of normality for the untransformed data. Each
parameter was then transformed by taking the base-10 logarithm of the parameter values.
However, prior to taking the base-10 logarithm, parameter values recorded as zero were replaced
with one half of the smallest observed non-zero value associated with the respective parameter.
The ANOVA models were then refitted using the base-10 log-transformed values, and a
hypothesis test was again performed for each parameter to assess the model assumption of
normality for the log-transformed data. If the assumption of normality was more reasonable for
the log-transformed data than it was for the untransformed data, then the log-transformed values
were used throughout the analysis for this parameter. The deleted studentized residuals, which
are the standardized residuals from the model fitted to the data having the current observation
removed, were computed for each observation. If the absolute value of the deleted studentized
residual was greater than 4, then the observation was considered a potential outlier. If any
potential outliers were identified, then the following analyses were performed both with and
without these observations to evaluate their effect on the results.
For each hematology parameter and CRP, the following ANOVA model was fitted to the data at
Study Day -3 to determine if there were significant differences between the groups at baseline:
Yy = n + group, + El/ (1)
where Yy is the observed hematology result for theyth animal in group /' (/'=! to 4) at the
baseline, [j, is an overall constant, and Sy is the random error left unexplained by the model.
Tukey's multiple comparisons procedure was also performed for each parameter to determine
which pairs of groups had baseline means that were significantly different from each other;
however, the results are only presented if significant differences were identified. If the parameter
was log-transformed for analysis, then the same model was used with Ytj replaced by Log(Yy),
the base-10 log-transformed parameter value for theyth animal in group / (/'=! to 4).
1078-CG920794 - Hematology and C-Reactive Protein J-8
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To determine if the mean shifts from baseline were significantly different between the groups,
the following ANOVA model was fitted separately for each hematology parameter and CRP on
each post-challenge Study Day:
Ydy - YblJ = V + group, + By (2)
where ¥<#/ is the observed hematology result for they'th animal in group /' (/'=! to 4) on Study
Day d (d=2, 4, 9, 11, 16, 18, 23, 25, 30, 32, and 39), Y&, is the observed hematology result for
they'th animal in Group / at baseline (Study Day -3), n is an overall constant, and sy- is the
random error left unexplained by the model. If a parameter was log-transformed for the analysis,
then the same model was used with Y<#/ and Y&, replaced with their base-10 log-transformed
counterparts Log(Y^) and Log(Yiy-), respectively. Least square mean estimates from the
ANOVA models were calculated and approximate t-tests were performed to determine if, for
each group, there was a significant shift between baseline and each post-challenge Study Day.
For untransformed data, this tests whether the difference of means is significantly different from
zero. For log-transformed data, this tests whether the ratio of geometric means is significantly
different from one. Additionally, Tukey's multiple comparisons procedure was performed to
determine which pairs of groups had mean shifts from baseline that were significantly different
from each other. Under the Tukey procedure, the set of all comparisons within each parameter
and Study Day combination are made at a joint 95% confidence level.
All statistical analyses were conducted using Stata (Version 11.1) and R software that has been
performance tested by Battelle staff. All results are reported at the 0.05 level of significance.
1078-CG920794 - Hematology and C-Reactive Protein J-9
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3. Results
The model assumption of normality was more reasonable for 9 of 16 hematology parameters and
CRP data when models were fitted to the base-10 log-transformed data. These parameters were:
MCV, MCH, RDW, MPV, WBC, neutrophil count, N/L ratio, monocyte count, and eosinophil
count. Therefore, models were fitted to base-10 log-transformed values in the final analysis for
these parameters. Table 2 contains a list of eleven hematology values that were identified as
potential outliers using the procedure described above. The statistical analyses of the hematology
data were performed both with and without the potential outliers to evaluate their effect on the
results. Attachment I contain the parameters that experienced changes in significance after the
potential outliers were excluded.
Table 3 contains the results of the ANOVA models fitted at baseline (Study Day -3). The group
effect was significant at the baseline for RDW and MPV. Tukey's multiple comparisons
procedure indicated that the baseline mean in Group 2 was significantly greater than those in
Groups 3 and 4 for MPV; however, no significant pairwise differences were identified for RDW.
Significant group effects at baseline are not necessarily detrimental to the analysis since using
the shift from baseline accounts for any differences between the groups at baseline. However, if
the significant differences between the groups at baseline are systematically related to how the
groups were treated, then the significant group effects at other days throughout the study could
be attributed to the differences at baseline. Considering that the random probability of measuring
a significant difference when none truly exists is 0.05, two significant differences out of 17 is not
enough evidence to say there was an a priori difference between the groups.
Descriptive statistics and group comparisons for each parameter are presented in pairs of tables,
where the table numbers 4 through 20 are associated with the parameter of interest. For each
parameter, Table "a" contains the descriptive statistics and Table "b" contains the test results for
comparing the mean shifts from baseline within each group at each post-challenge Study Day.
Tables 4a through 19a contain descriptive statistics (including means with 95% confidence
intervals for untransformed data, or geometric means with 95% confidence intervals for base-10
log-transformed data) for the hematology parameter results within each group on each Study
1078-CG920794 - Hematology and C-Reactive Protein J-10
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Day. Table 20a presents similar information for CRP. The results on some Study Days were
based on smaller sample sizes due to missing data or due to animal deaths prior to the end of the
study. Figures 1 through 16 display means with 95% confidence intervals for untransformed
data, or geometric means with 95% confidence intervals for base-10 log-transformed data, over
the course of the study for each hematology parameter, respectively. Figure 17 displays similar
information for CRP. For plotting purposes, confidence intervals were not displayed for a
particular Study Day on which a group had only two animals.
Tables 4b through 19b contain test results for the mean shift from baseline within each group and
post-challenge Study Day for each hematology parameter, respectively. Table 20b contains
similar information for CRP. In each cell, an estimate of the shift (difference or ratio) from
baseline is shown for that parameter, group, and post-challenge Study Day. Following the shift
estimate, an up arrow (T) indicates a significant increase from baseline, while a down arrow (-1)
indicates a significant decrease from baseline. These tables also contain test results for
significant differences between the group shifts from baseline at each post-challenge Study Day.
The results from Tables 4b through 20b are discussed below in groups of related parameters.
Red Blood Cell Parameters
• RBC (Tables 4a-b, Figure 1): There were significant decreases from baseline in
Group 1 on Study Day 4, in Group 2 on Study Day 16, and in Group 4 on Study
Day 11. There were significant group effects on Study Days 23 and 25. On Study
Day 23, the mean decrease from baseline in Group 4 was significantly different than
the mean increase from baseline in Group 3. On Study Day 25, the mean decrease
from baseline in Group 4 was significantly different than the mean increases from
baseline in Groups 1 and 2.
• HGB (Tables 5a-b, Figure 2): There was a significant decrease from baseline in
Group 1 on Study Day 4. There were significant group effects on Study Days 23
and 25. On Study Day 23, the mean decrease from baseline in Group 4 was
significantly different than the mean increases from baseline in Groups 1, 2, and 3.
On Study Day 25, the mean decrease from baseline in Group 4 was significantly
different than the mean changes from baseline in Groups 1, 2, and 3.
1078-CG920794 - Hematology and C-Reactive Protein J-l 1
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• HCT (Tables 6a-b, Figure 3): There was a significant decrease from baseline in
Group 1 on Study Day 4. There was a significant group effect on Study Day 25. On
Study Day 25, the mean decrease from baseline in Group 4 was significantly different
than the mean increase from baseline in Group 1.
• MCV (Tables 7a-b, Figure 4): There were significant decreases as a proportion of
baseline in Group 1 on Study Day 39, in Group 3 on Study Day 32, and in Group 4 on
Study Days 18, 32, 37, and 39. There were significant group effects on Study Days 32
and 39. On Study Day 32, the mean decrease as a proportion of baseline in Group 4
was significantly different than the mean increase as a proportion of baseline in
Group 1. On Study Day 39, the mean decrease as a proportion of baseline in Group 4
was significantly greater than that in Group 2.
• MCH (Tables 8a-b, Figure 5): There were no significant shifts as a proportion of
baseline and no significant differences between the groups on any post-challenge
Study Day.
• MCHC (Tables 9a-b, Figure 6): There were significant increases from baseline in
Group 2 on Study Days 18 and 23, and in Group 3 on Study Day 18. On Study
Day 25, the mean decrease from baseline in Group 4 was significantly different than
the mean increase from baseline in Group 3.
• RDW (Tables lOa-b, Figure 7): There was a significant increase as a proportion of
baseline in Group 2 on Study Day 18. There was a significant Group effect on Study
Day 18. On Study Day 18, the mean increase as a proportion of baseline in Group 2
was significantly different than the mean decrease as a proportion of baseline in
Group 4. On Study Day 25, the mean decrease as a proportion of baseline in Group 1
was significantly different than the mean increase as a proportion of baseline in
Group 4.
1078-CG920794 - Hematology and C-Reactive Protein J-12
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Platelet Count and Volume
• PLT (Tables lla-b, Figure 8): There were significant decreases from baseline in
Groups 1 and 2 on Study Day 39, in Group 3 on Study Days 4 and 39, and in Group 4
on Study Day 11. There were no significant differences between the groups on any
post-challenge Study Day.
• MPV (Tables 12a-b, Figure 9): There were significant increases as a proportion of
baseline in Group 1 on Study Days 4 and 9, in Group 2 on Study Day 9, in Group 3
on Study Days 9 and 39, and in Group 4 on Study Day 9. There were significant
decreases as a proportion of baseline in Group 1 on Study Day 37, in Group 2 on
Study Days 2, 23, 30, and 37, and in Group 3 on Study Day 37. There were
significant group effects on Study Days 4, 9, and 16; however, no significant pairwise
group comparisons were identified on Study Day 9. On Study Day 4, the mean
increase as a proportion of baseline in Group 1 was significantly different than the
mean changes as a proportion of baseline in Groups 2, 3, and 4. On Study Day 16, the
mean decrease as a proportion of baseline in Group 2 was significantly different than
the mean increase as a proportion of baseline in Group 1.
Total and Differential White Blood Cell Parameters
• WBC (Tables 13a-b, Figure 10): There were no significant shifts as a proportion of
baseline and no significant differences between the groups on any post-challenge
Study Day.
• Neutrophil Count (Tables 14a-b, Figure 11): There were no significant shifts as a
proportion of baseline and no significant differences between the groups on any
post-challenge Study Day.
• Lymphocyte Count (Tables 15a-b, Figure 12): There was a significant decrease
from baseline in Group 3 on Study Day 39. There were no significant differences
between the groups on any post-challenge Study Day.
1078-CG920794 - Hematology and C-Reactive Protein J-13
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N/L Ratio (Tables 16a-b, Figure 13): There was a significant decrease as a
proportion of baseline in Group 1 on Study Day 18. There were no significant
differences between the groups on any post-challenge Study Day.
Monocyte Count (Tables 17a-b, Figure 14): There was a significant decrease as a
proportion of baseline in Group 2 on Study Day 39. On Study Day 9, the mean
decrease as a proportion of baseline in Group 1 was significantly different than the
mean increase as a proportion of baseline in Group 3.
Eosinophil Count (Tables 18a-b, Figure 15): There were no significant shifts as a
proportion of baseline on any post-challenge Study Day. There was a significant
group effect on Study Day 37. On Study Day 37, the mean increase as a proportion of
baseline in Group 1 was significantly different than the mean decrease as a proportion
of baseline in Group 3.
Basophil Count (Tables 19a-b, Figure 16): There was a significant increase from
baseline in Group 2 on Study Day 23. There were no significant differences between
the groups on any post-challenge Study Day.
C-Reactive Protein
• CRP (Tables 20a-b, Figure 17): There was a significant increase as a proportion of
baseline in Group 4 on Study Day 2. There were no significant differences between
the groups on any post-challenge Study Day.
1078-CG920794 - Hematology and C-Reactive Protein J-14
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4. Conclusions
Among the red blood cell parameters, all significant differences between the group mean shifts
from baseline involved the targeted 10,000 CPU dose group (Group 4). On Study Day 25, the
mean change from baseline in the targeted 10,000 CPU dose group (Group 4) was significantly
different than that in at least one of the other groups (Groups 1, 2, and 3) for RBC, HGB, HCT,
MCHC, and RDW.
Among the platelet counts and volume, the mean shifts from baseline were not significantly
different between the groups on any post-challenge Study Day for PLT. For MPV, the mean
increase as a proportion of baseline in the control group (Group 1) was significantly different
than the mean changes as a proportion of baseline in the challenged groups (Groups 2, 3, and 4)
on Study Day 4, and the mean decrease as a proportion of baseline in the targeted 100 CPU dose
group (Group 2) was significantly different than the mean increase as a proportion of baseline in
the control group (Group 1) on Study Day 16.
Among the white blood cell parameters, the mean shifts from baseline were significantly
different between the groups only for monocyte count and eosinophil count. For monocyte count,
the mean decrease as a proportion of baseline in the control group (Group 1) was significantly
different than the mean increase as a proportion of baseline in the targeted 1,000 CPU dose
group (Group 3) on Study Day 9. For eosinophil count, the mean increase as a proportion of
baseline in the control group (Group 1) was significantly different than the mean decrease as a
proportion of baseline in the targeted 1,000 CFU dose group (Group 3) on Study Day 37.
1078-CG920794 - Hematology and C-Reactive Protein J-15
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Table 2. Potential Hematology and CRT Outliers
Parameter
Hemoglobin
Red Cell Distribution
Width1
Platelet Count
White Blood Cell Count1
Neutrophil CountT
C-Reactive ProteinT
Animal
6
38
39
38
39
7
11
40
38
Group
4
4
4
4
4
1
3
1
4
Study
Day
9
25
30
25
25
37
25
4
39
23
23
Parameter
Value
15.2
17.9
16.8
12.3
2044
1408
547
1.60
1.23
0.24
7.42
Deleted Studentized
Residual
4.03
5.09
5.08
-5.09
5.59
4.03
-5.59
-4.30
-4.36
-4.10
4.70
t Distribution was log-normal for this parameter. Parameter values are reported on the original scale,
while the residuals are reported on the log-transformed scale.
Table 3. Summary of ANOVA Results for Baseline (Study Day -3) Data
Parameter
Red Blood Cell Count
Hemoglobin
Hematocrit
Mean Corpuscular Volume1
Mean Corpuscular Hemoglobin1
Mean Corpuscular Hemoglobin Concentration
Red Cell Distribution Width1
Platelet Count
Mean Platelet Volume1
White Blood Cell Count1
Neutrophil CountT
Lymphocyte Count
Neutrophil Count/Lymphocyte Count RatioT
Monocyte Counf
Eosinophil CountT
Basophil Count
C-Reactive ProteinT
Group Effect
P-Value
0.5079
0.6553
0.5376
0.6306
0.7228
0.1048
0.0392*
0.3253
0.0255*
0.3865
0.2256
0.2257
0.1454
0.2461
0.3489
0.5573
0.3295
Estimated Difference
(Relationship)
Tukey's P- Value #
0.04 (3<2) 0.0339
0.04 (4<2) 0.0425
Indicates that values for this parameter were log-transformed for the analysis.
The overall group effect was significant at the 0.05 level.
Cells contain all pairwise comparisons that were significant at the 0.05 level. The format within each
cell is: (1) the ratio of group geometric means at baseline, (2) the relationship between the
corresponding pair of group geometric means at baseline shown in parentheses, and (3) the
Tukey-adjusted p-value.
1078-CG920794 - Hematology and C-Reactive Protein
J-16
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Table 4a. Descriptive Statistics for Red Blood Cell Count (RBC, 106 cells/uL), by Group
and Study Day
Group
1
2
3
Study
Day
-3
2
4
9
11
16
18
23
25
30
32
37
39
-3
2
4
9
11
16
18
23
25
30
32
37
39
-3
2
4
9
11
16
18
23
25
30
32
37
39
N
5
5
4
5
5
4
5
4
3
5
1
5
5
7
7
7
7
7
7
7
6
6
7
3
7
7
7
7
7
6
6
5
6
3
6
3
5
4
6
Mean
(95% Confidence Interval)
5.60 (4.87, 6.33)
5.52(4.90,6.14)
5.12(4.04,6.19)
5.45(4.76,6.14)
5.57(4.91,6.23)
5.54 (4.45, 6.63)
5.46(4.74,6.19)
5.91 (5.22, 6.60)
6.57(5.32, 7.81)
5.61 (4.88, 6.34)
5.45 (-)
5.87(5.11,6.63)
5.02 (3.55, 6.49)
5.99 (5.72, 6.26)
5.98 (5.66, 6.30)
5.83(5.53,6.14)
5.86(5.61,6.11)
5.93 (5.43, 6.43)
5.71 (5.53, 5.89)
5.78 (5.49, 6.07)
6.04(5.58,6.51)
6.06 (5.65, 6.46)
5.96 (5.60, 6.32)
5.76 (4.93, 6.58)
6.11 (5.86,6.36)
5.70 (5.32, 6.08)
5.86(5.54,6.19)
5.73 (5.45, 6.00)
5.59(5.28,5.91)
5.66 (5.40, 5.92)
5.53(5.14,5.92)
6.04 (5.48, 6.59)
5.86 (5.39, 6.34)
6.03(5.56,6.51)
5.85(5.53,6.17)
5.86 (4.69, 7.03)
5.75 (5.22, 6.28)
5.92 (5.39, 6.46)
5.48 (4.56, 6.39)
1078-CG920794 - Hematology and C-Reactive Protein
J-17
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Table 4a. (Continued)
Group
4
Study
Day
-3
2
4
9
11
16
18
23
25
30
32
37
39
N
7
7
4
7
6
4
4
2
2
3
3
3
3
Mean
(95% Confidence Interval)
5.85 (5.40, 6.30)
5.66 (5.27, 6.06)
5.75(5.38,6.11)
5.78 (5.09, 6.47)
5.46 (5.07, 5.84)
5.88 (5.73, 6.04)
5.84 (5.44, 6.24)
5.45(0.00a, 15.04)
5.44(0.00a, 13.57)
5.78(4.55,7.01)
5.45 (4.64, 6.25)
5.82 (4.79, 6.85)
5.90(5.15,6.64)
Confidence interval could not be calculated since only one observation was available for this group
on this Study Day.
a Negative lower confidence limit was set to 0 since negative values are not possible.
Table 4b. Test Results for Red Blood Cell Count (RBC, 106 cells/uL)
Red Blood Cell Count
Study
Day
2
4
9
11
16
18
23
25
30
32
37
39
Mean Shift from Baseline, by Group
1
-0.08
-0.64|
-0.15
-0.03
-0.10
-0.14
0.16
0.63
0.01
0.25
0.27
-0.58
2
-0.01
-0.16
-0.13
-0.06
-0.28|
-0.21
0.05
0.06
-0.03
-0.08
0.12
-0.29
3
-0.14
-0.27
-0.27
-0.29
0.17
-0.07
0.23
-0.08
0.14
-0.20
0.00
-0.46
4
-0.18
-0.36
-0.07
-0.34|
-0.09
-0.13
-0.98
-0.98
-0.32
-0.65
-0.28
-0.20
Group
Effect
P-Value
0.7544
0.2161
0.7885
0.2088
0.1074
0.9154
0.0432*
0.0040*
0.5839
0.2839
0.2678
0.8059
Estimated Difference
(Relationship)
Tukey's P-Value*
1 .21 (4<3) 0.0489
1. 62 (4<1) 0.0023
1 .05 (4<2) 0.0229
T,4
Cells contain all pairwise comparisons that were significant at the 0.05 level. The format within each
cell is: (1) the difference of group mean shifts, (2) the relationship between the corresponding pair of
group mean shifts shown in parentheses, and (3) the Tukey-adjusted p-value.
"|" indicates the mean at the Study Day was significantly greater than that at baseline; "J," indicates
the mean at the Study Day was significantly less than that at baseline (at the 0.05 level).
The overall group effect was significant at the 0.05 level.
1078-CG920794 - Hematology and C-Reactive Protein
J-18
-------
Table 5a. Descriptive Statistics for Hemoglobin (HGB, g/dL) by Group and Study Day
Group
1
2
3
Study
Day
-3
2
4
9
11
16
18
23
25
30
32
37
39
-3
2
4
9
11
16
18
23
25
30
32
37
39
-3
2
4
9
11
16
18
23
25
30
32
37
39
N
5
5
4
5
5
4
5
4
3
5
1
5
5
7
7
7
7
7
7
7
6
6
7
3
7
7
7
7
7
6
6
5
6
3
6
3
5
4
6
Mean
(95% Confidence Interval)
11.6(10.2, 13.0)
11.5(10.4, 12.6)
10.5(8.5, 12.6)
11.4(10.2, 12.5)
11.7(10.3, 13.0)
11.5(9.6, 13.3)
11.3(10.1, 12.6)
12.2(11.1, 13.3)
13.3(10.8, 15.7)
11.6(10.2, 13.1)
11.5(~)
12.0(10.8, 13.3)
10.3(7.4, 13.2)
12.2(11.5, 12.8)
12.2(11.6, 12.9)
11.9(11.2, 12.5)
11.9(11.4, 12.5)
12.1 (11.0, 13.3)
11.7(11.3, 12.2)
11.8(11.1, 12.5)
12.3(11.4, 13.2)
12.2(11.5, 13.0)
12.0(11.4, 12.7)
11.6(10.4, 12.7)
12.3(11.8, 12.8)
11.6(10.9, 12.3)
12.1 (11.7, 12.5)
11.9(11.4, 12.4)
11.6(11.1, 12.1)
11.8(11.5, 12.2)
11.6(10.9, 12.2)
12.6(11.7, 13.5)
12.1 (11.1, 13.1)
12.7(12.5, 12.9)
12.1 (11.4, 12.8)
12.3(9.1, 15.5)
11.8(11.3, 12.3)
12.2(11.3, 13.0)
11.3(9.8, 12.7)
1078-CG920794 - Hematology and C-Reactive Protein
J-19
-------
Table 5a. (Continued)
Group
4
Study
Day
-3
2
4
9
11
16
18
23
25
30
32
37
39
N
7
7
4
7
6
4
4
2
2
3
3
3
3
Mean
(95% Confidence Interval)
12.0(11.1, 12.9)
11.7(10.8, 12.5)
11.8(10.8, 12.7)
11.9(10.5, 13.3)
11.3(10.5, 12.0)
12.2(11.7, 12.6)
11.9(11.6, 12.2)
10.7(0.0a, 31.0)
1 0.7 (0.0a, 28.5)
11.6(8.9, 14.3)
10.9(8.4, 13.4)
11.4(8.6, 14.2)
11.7(9.0, 14.5)
Confidence interval could not be calculated since only one observation was available for this group
on this Study Day.
a Negative lower confidence limit was set to 0 since negative values are not possible.
Table 5b. Test Results for Hemoglobin (HGB, g/dL)
Hemoglobin
Study
Day
2
4
9
11
16
18
23
25
30
32
37
39
Mean Shift from Baseline, by
Group
1
-0.12
-1.351
-0.22
0.06
-0.13
-0.26
0.35
1.03
0.04
0.70
0.44
-1.30
2
0.04
-0.31
-0.24
-0.04
-0.44
-0.39
0.18
0.12
-0.14
-0.10
0.13
-0.56
3
-0.17
-0.49
-0.38
-0.50
0.48
-0.13
0.50
-0.10
0.17
-0.46
-0.05
-0.95
4
-0.33
-0.77
-0.09
-0.67
-0.20
-0.47
-2.40
-2.40
-1.03
-1.70
-1.23
-0.90
Group Effect
P-Value
0.7542
0.1264
0.9395
0.2609
0.1157
0.8869
0.0351*
0.0088*
0.4223
0.2362
0.1090
0.8199
Estimated Difference
(Relationship)
Tukey'sP-Value*
2.75 (4<1) 0.0430
2.58 (4<2) 0.0438
2.90 (4<3) 0.0429
3.43 (4<1) 0.0056
2.52 (4<2) 0.021 8
2.30 (4<3) 0.0370
T,4
Cells contain all pairwise comparisons that were significant at the 0.05 level. The format within each
cell is: (1) the difference of group mean shifts, (2) the relationship between the corresponding pair of
group mean shifts shown in parentheses, and (3) the Tukey-adjusted p-value.
"|" indicates the mean at the Study Day was significantly greater than that at baseline; "J," indicates
the mean at the Study Day was significantly less than that at baseline (at the 0.05 level).
The overall group effect was significant at the 0.05 level.
1078-CG920794 - Hematology and C-Reactive Protein
J-20
-------
Table 6a. Descriptive Statistics for Hematocrit (HCT, %) by Group and Study Day
Group
1
2
3
Study
Day
-3
2
4
9
11
16
18
23
25
30
32
37
39
-3
2
4
9
11
16
18
23
25
30
32
37
39
-3
2
4
9
11
16
18
23
25
30
32
37
39
N
5
5
4
5
5
4
5
4
3
5
1
5
5
7
7
7
7
7
7
7
6
6
7
3
7
7
7
7
7
6
6
5
6
3
6
3
5
4
6
Mean
(95% Confidence Interval)
35.5 (30.5, 40.5)
35.0(31.6,38.5)
32.1 (25.0, 39.2)
34.8 (30.6, 39.0)
35.7(31.2,40.2)
35.4(29.0,41.7)
34.5 (30.7, 38.3)
36.5 (33.0, 40.1)
40.6 (32.6, 48.6)
35.1 (30.9, 39.3)
34.5 (-)
36.3(31.9,40.7)
31.1 (21.7,40.4)
37.8 (36.0, 39.5)
37.7 (35.6, 39.8)
36.9 (34.7, 39.1)
37.3(36.1,38.5)
38.0(34.6,41.4)
36.3 (34.7, 37.8)
36.1 (34.0, 38.2)
37.4 (34.4, 40.4)
37.7 (34.6, 40.8)
37.2 (34.8, 39.5)
34.8(31.3,38.3)
37.8(36.1,39.5)
35.5(33.1,37.9)
37.2 (35.9, 38.6)
36.5 (34.6, 38.4)
35.3 (33.0, 37.5)
36.1 (34.7, 37.5)
36.0 (33.4, 38.5)
38.9 (35.9, 42.0)
36.7 (33.6, 39.8)
38.6 (36.6, 40.6)
36.6(34.1,39.1)
38.5 (26.6, 50.5)
35.8 (34.2, 37.3)
36.8 (33.2, 40.4)
34.0 (29.4, 38.7)
1078-CG920794 - Hematology and C-Reactive Protein
J-21
-------
Table 6a. (Continued)
Group
4
Study Day
-3
2
4
9
11
16
18
23
25
30
32
37
39
N
7
7
4
7
6
4
4
2
2
3
3
3
3
Mean
(95% Confidence Interval)
37.8(34.3,41.4)
36.7(34.1,39.3)
36.9 (34.2, 39.6)
37.7 (33.2, 42.3)
35.8 (32.8, 38.7)
38.5 (37.6, 39.3)
37.5 (35.5, 39.4)
34.2 (0.0a, 101.5)
36.3 (0.0a, 87.1)
37.3 (29.9, 44.8)
34.3 (29.5, 39.2)
36.4 (28.3, 44.5)
37.1 (31.0,43.2)
Confidence interval could not be calculated since only one observation was available for this group
on this Study Day.
a Negative lower confidence limit was set to 0 since negative values are not possible.
Table 6b. Test Results for Hematocrit (HCT, %)
Hematocrit
Study
Day
2
4
9
11
16
18
23
25
30
32
37
39
Mean Shift from Baseline, by
Group
1
-0.48
-4.251
-0.68
0.18
-0.45
-1.02
0.23
2.93
-0.42
2.20
0.82
-4.46
2
-0.10
-0.89
-0.51
0.21
-1.51
-1.70
-0.30
0.00
-0.61
-1.47
0.01
-2.29
3
-0.73
-1.94
-1.55
-1.23
1.50
-0.97
0.67
-1.07
0.80
-2.06
-0.65
-3.63
4
-1.13
-3.05
-0.07
-2.00
-1.13
-2.13
-8.35
-6.25
-3.23
-6.23
-4.20
-3.43
Group Effect
P-Value
0.8645
0.2157
0.8047
0.2519
0.1121
0.8544
0.0579
0.0284*
0.4166
0.1439
0.1505
0.8440
Estimated Difference
(Relationship)
Tukey's P-Value*
9. 18 (4<1) 0.0188
T,4
Cells contain all pairwise comparisons that were significant at the 0.05 level. The format within each
cell is: (1) the difference of group mean shifts, (2) the relationship between the corresponding pair of
group mean shifts shown in parentheses, and (3) the Tukey-adjusted p-value.
"|" indicates the mean at the Study Day was significantly greater than that at baseline; "J," indicates
the mean at the Study Day was significantly less than that at baseline (at the 0.05 level).
The overall group effect was significant at the 0.05 level.
1078-CG920794 - Hematology and C-Reactive Protein
J-22
-------
Table 7a. Descriptive Statistics for Mean Corpuscular Volume (MCV, fL) by Group and
Study Day
Group
1
2
3
Study
Day
-3
2
4
9
11
16
18
23
25
30
32
37
39
-3
2
4
9
11
16
18
23
25
30
32
37
39
-3
2
4
9
11
16
18
23
25
30
32
37
39
N
5
5
4
5
5
4
5
4
3
5
1
5
5
7
7
7
7
7
7
7
6
6
7
3
7
7
7
7
7
6
6
5
6
3
6
3
5
4
6
Geometric Mean
(95% Confidence Interval)
63.4(61.6,65.2)
63.6 (62.2, 64.9)
62.6 (60.3, 65.0)
64.0 (62.7, 65.3)
64.1 (62.4, 65.8)
63.9 (62.0, 65.8)
63.2(61.6,64.9)
62.0 (59.3, 64.7)
61.9(60.2,63.5)
62.7(61.3,64.1)
63.2 (-)
61.9(60.6,63.2)
61.8(60.6,63.1)
63.1 (61.9,64.3)
63.0(61.8,64.3)
63.2(61.4,65.1)
63.6 (62.5, 64.7)
64.0 (62.8, 65.2)
63.5(62.1,65.0)
62.3(61.0,63.6)
61.8(60.5,63.2)
62.2 (60.4, 64.1)
62.2(61.2,63.3)
60.6(57.1,64.2)
61.8(60.7,63.0)
62.3(61.0,63.6)
63.6 (60.9, 66.4)
63.7(61.0,66.6)
63.0(60.1,66.1)
63.8 (60.2, 67.7)
65.0(61.7,68.4)
64.6 (60.7, 68.7)
62.6 (59.7, 65.7)
64.0 (56.6, 72.5)
62.5 (59.0, 66.3)
65.6 (56.9, 75.6)
62.3 (58.3, 66.6)
62.0 (55.9, 68.9)
62.4 (59.6, 65.3)
1078-CG920794 - Hematology and C-Reactive Protein
J-23
-------
Table 7a. (Continued)
Group
4
Study
Day
-3
2
4
9
11
16
18
23
25
30
32
37
39
N
7
7
4
7
6
4
4
2
2
3
3
3
3
Geometric Mean
(95% Confidence Interval)
64.6 (62.4, 66.8)
64.7 (62.9, 66.7)
64.3 (60.6, 68.2)
65.3 (63.8, 66.8)
65.4 (63.7, 67.2)
65.3 (63.6, 67.2)
64.2 (62.8, 65.6)
62.7 (50.7, 77.6)
66.7(60.1, 74.1)
64.7 (62.6, 66.8)
63.0 (60.8, 65.3)
62.5 (59.7, 65.4)
62.9 (60.3, 65.7)
Confidence interval could not be calculated since only one observation was available for this group
on this Study Day.
Table 7b. Test Results for Mean Corpuscular Volume (MCV, fL)
Mean Corpuscular Volume1
Study
Day
2
4
9
11
16
18
23
25
30
32
37
39
Mean Shift as a Proportion
from Baseline, by Group
1
1.00
0.99
1.01
1.01
1.01
1.00
0.98
0.98
0.99
1.02
0.98
0.98|
2
1.00
1.00
1.01
1.01
1.01
0.99
0.98
0.99
0.99
0.97
0.98
0.99
3
1.00
0.99
1.00
1.02
1.01
0.99
0.98
0.98
0.99
0.98|
0.98
0.98
4
1.00
0.98
1.01
1.01
0.99
0.97|
0.95
1.01
0.97
0.95|
0.94|
0.95|
Group Effect
P-Value
0.9591
0.2406
0.8678
0.8177
0.1814
0.1402
0.4505
0.2851
0.5572
0.0123*
0.0868
0.0431*
Estimated Ratio
(Relationship)
Tukey's P-Value*
1.07(4<1)0.0110
1 .04 (4<2) 0.0296
t
#
T,4
Indicates that values for this parameter were log-transformed for the analysis.
Cells contain all pairwise comparisons that were significant at the 0.05 level. The format within each
cell is: (1) the ratio of group mean shifts, (2) the relationship between the corresponding pair of
group mean shifts shown in parentheses, and (3) the Tukey-adjusted p-value.
"t" indicates the geometric mean at the Study Day was significantly greater than that at baseline;
"J," indicates the geometric mean at the Study Day was significantly less than that at baseline (at the
0.05 level).
The overall group effect was significant at the 0.05 level.
1078-CG920794 - Hematology and C-Reactive Protein
J-24
-------
Table 8a. Descriptive Statistics for Mean Corpuscular Hemoglobin (MCH, pg) by Group
and Study Day
Group
1
2
3
Study
Day
-3
2
4
9
11
16
18
23
25
30
32
37
39
-3
2
4
9
11
16
18
23
25
30
32
37
39
-3
2
4
9
11
16
18
23
25
30
32
37
39
N
5
5
4
5
5
4
5
4
3
5
1
5
5
7
7
7
7
7
7
7
6
6
7
3
7
7
7
7
7
6
6
5
6
3
6
3
5
4
6
Geometric Mean
(95% Confidence Interval)
20.7(20.1,21.4)
20.8(20.2,21.3)
20.5(19.7,21.4)
20.9(20.1,21.6)
20.9(20.3,21.5)
20.8(19.9,21.6)
20.8(20.2,21.4)
20.7(20.0,21.4)
20.2(19.0,21.5)
20.8(20.3,21.3)
21.2(~)
20.5(19.8,21.3)
20.7(20.1,21.2)
20.3(19.7,21.0)
20.5(19.8,21.1)
20.3(19.7,21.0)
20.4(19.7,21.0)
20.4 (20.0, 20.9)
20.5(20.0,21.1)
20.4(19.8,21.1)
20.3(19.7,20.9)
20.2(19.8,20.7)
20.2(19.6,20.9)
20.1 (18.4,22.0)
20.2(19.6,20.8)
20.4(19.8,21.0)
20.7(19.8,21.5)
20.8(20.2,21.5)
20.7(20.1,21.4)
20.9(20.1,21.7)
20.9(20.1,21.8)
20.9(19.8,22.0)
20.6(19.7,21.6)
21.1 (19.1,23.2)
20.7(19.9,21.5)
21.0(19.3,22.8)
20.5(19.3,21.9)
20.6(19.0,22.2)
20.6(19.6,21.6)
1078-CG920794 - Hematology and C-Reactive Protein
J-25
-------
Table 8a. (Continued)
Group
4
Study Day
-3
2
4
9
11
16
18
23
25
30
32
37
39
N
7
7
4
7
6
4
4
2
2
3
3
3
3
Geometric Mean
(95% Confidence Interval)
20.4(19.9,21.0)
20.6(20.0,21.2)
20.5(19.3,21.8)
20.6(20.2,21.0)
20.6(20.0,21.3)
20.6(19.4,21.9)
20.4(19.3,21.7)
19.6(16.1,23.8)
19.5(16.6,23.0)
20.0(17.6,22.7)
20.0(18.0,22.3)
19.6(17.5,22.0)
19.9(17.8,22.1)
Confidence interval could not be calculated since only one observation was available for this group
on this Study Day.
Table 8b. Test Results for Mean Corpuscular Hemoglobin (MCH, pg)
Mean Corpuscular Hemoglobin1
Study
Day
2
4
9
11
16
18
23
25
30
32
37
39
Mean Shift as a Proportion
from Baseline, by Group
1
1.00
0.99
1.01
1.01
1.01
1.00
1.00
0.98
1.00
1.02
0.99
1.00
2
1.01
1.00
1.00
1.01
1.01
1.00
1.01
1.00
1.00
1.01
0.99
1.00
3
1.01
1.00
1.01
1.01
1.01
1.00
1.00
1.00
0.98
0.99
1.00
1.00
4
1.01
1.00
1.01
1.00
1.00
0.99
0.96
0.96
0.97
0.97
0.95
0.96
Group Effect
P-Value
0.6843
0.7866
0.5964
0.9808
0.4839
0.3509
0.2630
0.0886
0.1597
0.2488
0.0534
0.0825
Estimated Ratio
(Relationship)
Tukey'sP-Value*
t
#
Indicates that values for this parameter were log-transformed for the analysis.
Cells contain all pairwise comparisons that were significant at the 0.05 level. The format within each
cell is: (1) the ratio of group mean shifts, (2) the relationship between the corresponding pair of
group mean shifts shown in parentheses, and (3) the Tukey-adjusted p-value.
1078-CG920794 - Hematology and C-Reactive Protein
J-26
-------
Table 9a. Descriptive Statistics for Mean Corpuscular Hemoglobin Concentration
(MCHC, g/dL) by Group and Study Day
Group
1
2
3
Study
Day
-3
2
4
9
11
16
18
23
25
30
32
37
39
-3
2
4
9
11
16
18
23
25
30
32
37
39
-3
2
4
9
11
16
18
23
25
30
32
37
39
N
5
5
4
5
5
4
5
4
3
5
1
5
5
7
7
7
7
7
7
7
6
6
7
3
7
7
7
7
7
6
6
5
6
3
6
3
5
4
6
Mean
(95% Confidence Interval)
32.7(31.5,33.8)
32.7 (32.4, 32.9)
32.8(31.7,34.0)
32.6(31.7,33.6)
32.7(31.9,33.5)
32.5(31.9,33.1)
32.9 (32.5, 33.3)
33.4 (32.8, 34.0)
32.7(31.4,34.1)
33.2 (32.3, 34.1)
33.5 (-)
33.1 (32.5, 33.7)
33.4 (32.5, 34.3)
32.2(31.5,32.9)
32.5(31.8,33.1)
32.2(31.6,32.8)
32.0(31.4,32.6)
31.9(31.7,32.2)
32.4(31.7,33.0)
32.7(32.1,33.3)
32.8 (32.3, 33.3)
32.5(31.7,33.2)
32.5(31.8,33.1)
33.3 (32.0, 34.5)
32.6 (32.0, 33.2)
32.8 (32.0, 33.5)
32.5(32.1,32.9)
32.7(32.1,33.2)
32.9(32.1,33.7)
32.7(31.8,33.6)
32.2(31.5,32.9)
32.4(31.9,32.9)
32.9 (32.4, 33.4)
32.9(31.9,34.0)
33.1 (32.3, 33.9)
32.0 (30.2, 33.8)
32.9 (32.6, 33.3)
33.1 (32.1,34.1)
33.0 (32.3, 33.8)
1078-CG920794 - Hematology and C-Reactive Protein
J-27
-------
Table 9a. (Continued)
Group
4
Study
Day
-3
2
4
9
11
16
18
23
25
30
32
37
39
N
7
7
4
7
6
4
4
2
2
3
3
3
3
Mean
(95% Confidence Interval)
31.7(31.1,32.4)
31.8(31.1,32.5)
31.9(31.3,32.5)
31.5(30.8,32.2)
31.5(30.9,32.1)
31.6(30.4,32.8)
31.9(30.6,33.1)
31.2(29.9,32.5)
29.4(21.8,37.0)
31.0(26.2,35.8)
31.8(28.8,34.8)
31.4(28.6,34.2)
31.6(29.2,33.9)
Confidence interval could not be calculated since only one observation was available for this group
on this Study Day.
Table 9b. Test Results for Mean Corpuscular Hemoglobin Concentration (MCHC, g/dL)
Mean Corpuscular Hemoglobin Concentration
Study
Day
2
4
9
11
16
18
23
25
30
32
37
39
Mean Shift from Baseline, by
Group
1
0.00
0.08
-0.06
-0.02
0.10
0.18
0.67
0.23
0.52
0.00
0.44
0.70
2
0.29
0.01
-0.19
-0.26
0.16
0.51|
0.77|
0.42
0.27
1.10
0.41
0.57
3
0.17
0.43
0.30
-0.33
-0.10
0.45|
0.70
0.65
-0.23
0.48
0.50
0.62
4
0.06
0.48
-0.20
-0.13
0.33
0.58
0.45
-1.35
-0.13
0.63
0.27
0.43
Group Effect
P-Value
0.7912
0.5351
0.2699
0.6484
0.6689
0.4966
0.9464
0.0618
0.4258
0.3221
0.9659
0.9672
Estimated Difference
(Relationship)
Tukey'sP-Value*
2.00 (4<3) 0.0438
#
T,4
Cells contain all pairwise comparisons that were significant at the 0.05 level. The format within each
cell is: (1) the difference of group mean shifts, (2) the relationship between the corresponding pair of
group mean shifts shown in parentheses, and (3) the Tukey-adjusted p-value.
"|" indicates the mean at the Study Day was significantly greater than that at baseline; "J," indicates
the mean at the Study Day was significantly less than that at baseline (at the 0.05 level).
1078-CG920794 - Hematology and C-Reactive Protein
J-28
-------
Table lOa. Descriptive Statistics for Red Cell Distribution Width (RDW, %) by Group and
Study Day
Group
1
2
3
Study
Day
-3
2
4
9
11
16
18
23
25
30
32
37
39
-3
2
4
9
11
16
18
23
25
30
32
37
39
-3
2
4
9
11
16
18
23
25
30
32
37
39
N
5
5
4
5
5
4
5
4
3
5
1
5
5
7
7
7
7
7
7
7
6
6
7
3
7
7
7
7
7
6
6
5
6
3
6
3
5
4
6
Geometric Mean
(95% Confidence Interval)
12.7(11.8, 13.8)
12.4(11.7, 13.3)
12.5(12.0, 13.1)
12.7(11.8, 13.7)
12.4(11.8, 13.1)
12.5(11.4, 13.6)
12.4(11.7, 13.1)
12.0(11.2, 12.8)
11.6(10.7, 12.5)
12.2(11.8, 12.5)
12.1 (-)
12.2(11.9, 12.6)
12.3(11.9, 12.6)
11.9(11.5, 12.4)
11.7(11.3, 12.2)
12.0(11.4, 12.6)
12.1 (11.5, 12.7)
12.0(11.6, 12.5)
12.0(11.5, 12.5)
12.5(11.9, 13.1)
11.7(11.2, 12.2)
11.6(11.2, 12.2)
11.8(11.5, 12.1)
12.1 (11.6, 12.6)
11.8(11.5, 12.2)
11.8(11.6, 12.1)
12.0(11.6, 12.5)
11.9(11.4, 12.4)
11.9(11.4, 12.3)
12.0(11.4, 12.6)
12.1 (11.3, 13.0)
12.0(11.2, 13.0)
11.9(11.5, 12.3)
11.6(11.0, 12.3)
11.6(11.2, 12.0)
11.8(10.6, 13.2)
11.8(11.2, 12.5)
11.6(10.9, 12.4)
11.7(11.2, 12.2)
1078-CG920794 - Hematology and C-Reactive Protein
J-29
-------
Table lOa. (Continued)
Group
4
Study
Day
-3
2
4
9
11
16
18
23
25
30
32
37
39
N
7
7
4
7
6
4
4
2
2
3
3
3
3
Geometric Mean
(95% Confidence Interval)
12.7(12.2, 13.2)
12.4(11.8, 12.9)
12.6(11.5, 13.8)
12.7(12.1, 13.3)
12.4(11.6, 13.3)
12.5(11.7, 13.3)
12.1 (11.3, 13.0)
13.6(4.2,43.7)
14.8(1.4, 160.9)
13.5(8.5,21.6)
13.6(9.4, 19.6)
12.9(9.7, 17.2)
12.9(10.0, 16.7)
Confidence interval could not be calculated since only one observation was available for this group
on this Study Day.
Table lOb. Test Results for Red Cell Distribution Width (RDW, %)
Red Cell Distribution Width1
Study
Day
2
4
9
11
16
18
23
25
30
32
37
39
Mean Shift as a Proportion
from Baseline, by Group
1
0.98
0.99
1.00
0.98
1.00
0.97
0.95
0.92
0.96
0.95
0.96
0.96
2
0.98
1.00
1.01
1.01
1.01
1.05|
0.98
0.97
0.99
1.00
0.99
0.99
3
0.99
0.98
1.01
1.00
0.99
1.00
0.98
0.97
1.00
0.99
0.97
0.98
4
0.98
1.00
1.00
0.97
0.98
0.95
1.04
1.14
1.05
1.05
1.00
1.00
Group Effect
P-Value
0.7815
0.7898
0.9052
0.3128
0.7196
0.0204*
0.2958
0.0544
0.4365
0.5851
0.6306
0.6035
Estimated Ratio
(Relationship)
Tukey'sP-Value*
1.10(4<2) 0.0278
1 .24 (1<4) 0.0389
t
#
T, 4
Indicates that values for this parameter were log-transformed for the analysis.
Cells contain all pairwise comparisons that were significant at the 0.05 level. The format within each
cell is: (1) the ratio of group mean shifts, (2) the relationship between the corresponding pair of
group mean shifts shown in parentheses, and (3) the Tukey-adjusted p-value.
"t" indicates the geometric mean at the Study Day was significantly greater than that at baseline;
"J," indicates the geometric mean at the Study Day was significantly less than that at baseline (at the
0.05 level).
The overall group effect was significant at the 0.05 level.
1078-CG920794 - Hematology and C-Reactive Protein
J-30
-------
Table lla. Descriptive Statistics for Platelet Count (PLT, 103 cells/jiL) by Group and
Study Day
Group
1
2
3
Study
Day
-3
2
4
9
11
16
18
23
25
30
32
37
39
-3
2
4
9
11
16
18
23
25
30
32
37
39
-3
2
4
9
11
16
18
23
25
30
32
37
39
N
5
5
4
5
5
4
5
4
3
5
1
5
5
7
7
7
7
7
7
7
6
6
7
3
7
7
7
7
7
6
6
5
6
3
6
3
5
4
6
Mean
(95% Confidence Interval)
627 (387, 868)
634 (386, 882)
364 (Oa, 746)
518(179,858)
597 (325, 869)
446(171,722)
527 (368, 687)
368 (Oa, 805)
503(168, 839)
430(148,712)
447 (-)
446 (306, 585)
236 (Oa, 493)
547(378,716)
520 (370, 670)
548 (468, 627)
598 (500, 695)
576 (478, 674)
492 (437, 548)
471 (359, 583)
483 (365, 601)
386 (268, 503)
408 (332, 485)
400(176,624)
429(347,511)
250(143, 358)
565 (348, 783)
516(413,618)
413(222,605)
586(457, 715)
438 (309, 566)
461 (385, 536)
537 (353, 721)
401 (110,692)
395(166,625)
498 (Oa, 1019)
385 (262, 509)
498 (387, 609)
210(23,396)
1078-CG920794 - Hematology and C-Reactive Protein
J-31
-------
Table lla. (Continued)
Group
4
Study
Day
-3
2
4
9
11
16
18
23
25
30
32
37
39
N
7
7
4
7
6
4
4
2
2
3
3
3
3
Mean
(95% Confidence Interval)
744 (533, 955)
680 (483, 878)
649(238, 1060)
670 (532, 808)
489 (225, 752)
594 (260, 928)
630 (396, 863)
1 020 (Oa, 6750)
1296(0a, 10806)
732 (Oa, 1974)
615(0a, 1448)
765 (Oa, 2149)
716(0a, 1440)
Confidence interval could not be calculated since only one observation was available for this group
on this Study Day.
a Negative lower confidence limit was set to 0 since negative values are not possible.
Table lib. Test Results for Platelet Count (PLT, 103 cells/uL)
Platelet Count
Study
Day
2
4
9
11
16
18
23
25
30
32
37
39
Mean Shift from Baseline,
by Group
1
7.00
-209.25
-109.20
-30.60
-202.00
-100.00
-204.75
-129.67
-197.40
55.00
-181.80
-391 .60|
2
-27.00
0.57
50.57
28.71
-54.57
-76.14
-55.17
-152.17
-138.57
-113.67
-118.00
-296. 71 1
3
-49.71
-152.141
30.00
-188.67
5.40
-18.83
4.67
-160.67
-92.00
-126.00
-184.25
-346.50J,
4
-63.57
-49.75
-74.29
-303.501
-119.25
-83.75
179.50
455.00
-48.33
-164.67
-15.00
-63.67
Group Effect
P-Value
0.8491
0.1120
0.4877
0.1052
0.4353
0.8636
0.3292
0.1110
0.7641
0.7869
0.6654
0.4431
Estimated Difference
(Relationship)
Tukey's P-Value*
T,4
Cells contain all pairwise comparisons that were significant at the 0.05 level. The format within each
cell is (1) the difference of group mean shifts, (2) the relationship between the corresponding pair of
group mean shifts shown in parentheses, and (3) the Tukey-adjusted p-value.
"|" indicates the mean at the Study Day was significantly greater than that at baseline; "J," indicates
the mean at the Study Day was significantly less than that at baseline (at the 0.05 level).
1078-CG920794 - Hematology and C-Reactive Protein
J-32
-------
Table 12a. Descriptive Statistics for Mean Platelet Volume (MPV, fL) by Group and Study
Day
Group
1
2
3
Study
Day
-3
2
4
9
11
16
18
23
25
30
32
37
39
-3
2
4
9
11
16
18
23
25
30
32
37
39
-3
2
4
9
11
16
18
23
25
30
32
37
39
N
5
5
4
5
5
4
5
4
3
5
1
5
5
7
7
7
7
7
7
7
6
6
7
3
7
7
7
7
7
6
6
5
6
3
6
3
5
4
6
Geometric Mean
(95% Confidence Interval)
6.9 (6.2, 7.8)
6.2 (5.8, 6.7)
9.4(5.9, 14.8)
8.6 (8.0, 9.3)
8.0(6.3, 10.2)
7.9(7.1,8.9)
7.8 (7.3, 8.4)
7.4(5.2, 10.4)
6.8 (5.3, 8.8)
6.8 (5.8, 8.0)
6.2 (-)
6.4 (5.6, 7.3)
9.4(6.6, 13.3)
7.4 (7.3, 7.6)
6.6(6.2,7.1)
7.3 (6.8, 7.7)
8.5 (8.3, 8.6)
7.0 (6.6, 7.5)
6.9 (6.2, 7.7)
7.9 (6.6, 9.4)
6.3 (5.9, 6.7)
6.6 (5.9, 7.5)
6.8 (6.4, 7.3)
6.7(4.1, 10.9)
6.3 (5.9, 6.6)
8.5(6.5, 11.1)
6.7(6.3,7.1)
6.4 (5.7, 7.2)
6.9 (6.4, 7.5)
7.6(7.2,8.1)
7.8 (6.4, 9.4)
6.7 (5.8, 7.8)
6.3(5.6,7.1)
6.6 (5.9, 7.4)
6.5 (5.4, 7.9)
6.6 (4.6, 9.4)
6.7(4.9,9.1)
5.8(5.1,6.6)
9.0(6.9, 11.8)
1078-CG920794 - Hematology and C-Reactive Protein
J-33
-------
Table 12a. (Continued)
Group
4
Study
Day
-3
2
4
9
11
16
18
23
25
30
32
37
39
N
7
7
4
7
6
4
4
2
2
3
3
3
3
Geometric Mean
(95% Confidence Interval)
6.7 (6.3, 7.2)
6.6 (6.0, 7.2)
6.5 (6.0, 7.0)
8.3 (7.8, 8.7)
6.9 (6.0, 7.8)
6.4(6.1,6.7)
6.5(6.0,7.1)
7.0(3.7, 13.2)
7.9(1.4,46.0)
6.7 (4.8, 9.2)
7.0(4.1, 12.1)
6.2(5.1,7.5)
6.9(3.5, 13.5)
Confidence interval could not be calculated since only one observation was available for this group
on this Study Day.
Table 12b. Test Results for Mean Platelet Volume (MPV, fL)
t
#
T, 4
Mean Platelet Volume1
Study
Day
2
4
9
11
16
18
23
25
30
32
37
39
Mean Shift as a Proportion from
Baseline, by Group
1
0.90
1.33|
1.24|
1.16
1.15
1.13
1.05
0.97
0.98
0.87
0.92|
1.35
2
0.89|
0.98
1.14|
0.95
0.93
1.06
0.85|
0.90
0.92|
0.91
0.84|
1.14
3
0.96
1.03
1.13|
1.16
0.99
0.93
0.94
0.97
0.94
0.99
0.89|
1.33|
4
0.97
0.96
1.22|
1.01
0.93
0.95
0.97
1.09
0.98
1.03
0.91
1.01
Group Effect
P-Value
0.2070
0.0064*
0.0376*
0.0583
0.0362*
0.1046
0.1236
0.4534
0.4191
0.7925
0.1247
0.3745
Estimated Ratio
(Relationship)
Tu key's P-Value*
1. 37 (2<1) 0.0071
1. 30 (3<1) 0.0258
1. 39 (4<1) 0.0130
1. 23 (2<1) 0.0343
Indicates that values for this parameter were log-transformed for the analysis.
Cells contain all pairwise comparisons that were significant at the 0.05 level. The format within each
cell is: (1) the ratio of group mean shifts, (2) the relationship between the corresponding pair of
group mean shifts shown in parentheses, and (3) the Tukey-adjusted p-value.
"t" indicates the geometric mean at the Study Day was significantly greater than that at baseline;
"J," indicates the geometric mean at the Study Day was significantly less than that at baseline (at the
0.05 level).
The overall group effect was significant at the 0.05 level.
1078-CG920794 - Hematology and C-Reactive Protein
J-34
-------
Table 13a. Descriptive Statistics for White Blood Cell Count (WBC, 103 cells/jiL) by
Group and Study Day
Group
1
2
3
Study
Day
-3
2
4
9
11
16
18
23
25
30
32
37
39
-3
2
4
9
11
16
18
23
25
30
32
37
39
-3
2
4
9
11
16
18
23
25
30
32
37
39
N
5
5
4
5
5
4
5
4
3
5
1
5
5
7
7
7
7
7
7
7
6
6
7
3
7
7
7
7
7
6
6
5
6
3
6
3
5
4
6
Geometric Mean
(95% Confidence Interval)
5.76 (3.92, 8.48)
6.09 (4.80, 7.74)
4.41 (1.46, 13.31)
5.78 (3.88, 8.63)
5.88 (4.63, 7.46)
6.21 (4.55, 8.49)
6.04(4.49,8.13)
5.67(2.84, 11.31)
6.02(3.06, 11.85)
6.06(4.16,8.82)
6.51 (-)
6.60 (5.22, 8.34)
3.21 (1.25,8.22)
6.91 (5.77, 8.27)
7.12(5.74,8.82)
7.26 (6.06, 8.71)
7.35 (6.24, 8.67)
7.20 (6.23, 8.32)
6.93(5.90,8.14)
6.99 (5.74, 8.50)
6.56 (5.65, 7.61)
6.09 (5.02, 7.40)
6.66 (5.86, 7.58)
5.41 (4.88,6.01)
6.38(5.14,7.92)
4.38 (3.02, 6.35)
6.18(5.16,7.40)
5.32 (4.55, 6.23)
5.37 (4.37, 6.62)
5.86 (4.89, 7.02)
6.34(5.16,7.79)
5.85 (4.65, 7.37)
5.87(4.41,7.82)
7.33(4.10, 13.08)
6.01 (4.68, 7.73)
6.93(3.53, 13.59)
5.92(4.81,7.29)
5.63 (4.68, 6.76)
3.63(1.97,6.68)
1078-CG920794 - Hematology and C-Reactive Protein
J-35
-------
Table 13a. (Continued)
Group
4
Study
Day
-3
2
4
9
11
16
18
23
25
30
32
37
39
N
7
7
4
7
6
4
4
2
2
3
3
3
3
Geometric Mean
(95% Confidence Interval)
7.16(5.62,9.13)
7.00(5.38,9.10)
8.23(5.54, 12.22)
6.79 (5.74, 8.03)
6.58(4.21, 10.29)
7.28 (5.35, 9.92)
9.36(8.17, 10.72)
13.49(0.07,2475.61)
13.27(0.26,675.42)
9.12(2.37, 35.15)
8.85(5.51, 14.20)
6.81 (3.81, 12.17)
6.54 (4.77, 8.97)
Confidence interval could not be calculated since only one observation was available for this group
on this Study Day.
Table 13b. Test Results for White Blood Cell Count (WBC, 103 cells/uL)
t
#
White Blood Cell Count1
Study
Day
2
4
9
11
16
18
23
25
30
32
37
39
Mean Shift as a Proportk
Baseline, by Grou
1
1.06
0.84
1.00
1.02
1.06
1.05
1.08
1.09
1.05
1.45
1.14
0.56
2
1.03
1.05
1.06
1.04
1.00
1.01
0.97
0.90
0.96
0.75
0.92
0.63
3
0.86
0.87
0.92
0.98
1.00
0.92
1.24
0.95
0.96
0.98
0.81
0.57
in from
p
4
0.98
0.99
0.95
0.94
0.89
1.14
1.60
1.57
1.08
1.05
0.81
0.78
Group Effect
P-Value
0.2954
0.5770
0.7112
0.8349
0.8006
0.7318
0.4020
0.1273
0.9490
0.1716
0.1815
0.8894
Estimated Ratio
(Relationship)
Tukey'sP-Value*
Indicates that values for this parameter were log-transformed for the analysis.
Cells contain all pairwise comparisons that were significant at the 0.05 level. The format within each
cell is: (1) the ratio of group mean shifts, (2) the relationship between the corresponding pair of
group mean shifts shown in parentheses, and (3) the Tukey-adjusted p-value.
1078-CG920794 - Hematology and C-Reactive Protein
J-36
-------
Table 14a. Descriptive Statistics for Neutrophil Count (103 cells/jiL) by Group and
Study Day
Group
1
2
3
Study
Day
-3
2
4
9
11
16
18
23
25
30
32
37
39
-3
2
4
9
11
16
18
23
25
30
32
37
39
-3
2
4
9
11
16
18
23
25
30
32
37
39
N
5
5
4
5
5
4
5
4
3
5
1
5
5
7
7
7
7
7
7
7
6
6
7
3
7
7
7
7
7
6
6
5
6
3
6
3
5
4
6
Geometric Mean
(95% Confidence Interval)
1.69(0.77,3.74)
1.55(1.03,2.33)
1.14(0.28,4.56)
1.25(0.94, 1.67)
1.36(0.76,2.45)
1.40(1.15, 1.70)
1.16(0.87, 1.55)
1.00(0.22,4.52)
1.33(0.54,3.25)
1.21 (0.72,2.03)
1.39(~)
1.53(1.40, 1.66)
0.52(0.16, 1.66)
1.61 (1.16,2.23)
1.42(1.02, 1.96)
1.55(1.26, 1.91)
1.41 (1.19, 1.69)
1.51 (1.21, 1.88)
1.40(1.19, 1.64)
1.29(1.14, 1.46)
1.57(1.26, 1.97)
1.39(1.14, 1.69)
1.39(1.14, 1.70)
1.25(0.89, 1.74)
1.39(1.10, 1.77)
0.75(0.50, 1.12)
1.61 (1.22,2.14)
1.20(1.04, 1.40)
1.29(0.95, 1.76)
1.49(1.23, 1.81)
1.54(1.24, 1.92)
1.33(1.06, 1.68)
1.49(1.06,2.10)
2.56 (0.85, 7.67)
1.59(1.03,2.45)
1.74(1.16,2.62)
1.68(1.08,2.63)
1.40(1.13, 1.72)
0.88(0.36,2.13)
1078-CG920794 - Hematology and C-Reactive Protein
J-37
-------
Table 14a. (Continued)
Group
4
Study
Day
-3
2
4
9
11
16
18
23
25
30
32
37
39
N
7
7
4
7
6
4
4
2
2
3
3
3
3
Geometric Mean
(95% Confidence Interval)
2.35(1.90,2.92)
2.06(1.60,2.65)
2.41 (0.89, 6.55)
1.99(1.52,2.60)
1.81 (1.13,2.88)
1.41 (0.69,2.89)
2.02(0.99,4.12)
4.04 (0.00, 70945.34)
3.00(0.00, 1133098.47)
2.31 (0.19,27.74)
3.02(0.58, 15.81)
1.31 (1.20, 1.44)
1.39(0.36,5.32)
Confidence interval could not be calculated since only one observation was available for this group
on this Study Day.
Table 14b. Test Results for Neutrophil Count (103 cells/uL)
Neutrophil Count1
Study
Day
2
4
9
11
16
18
23
25
30
32
37
39
Mean Shift as a Proportion
from Baseline, by Group
1
0.92
0.86
0.74
0.81
0.82
0.69
0.75
0.97
0.71
1.19
0.90
0.31
2
0.88
0.96
0.88
0.93
0.87
0.80
0.98
0.87
0.87
0.71
0.86
0.46
3
0.75
0.80
0.86
0.90
0.91
0.86
1.58
0.92
0.83
1.04
0.75
0.51
4
0.87
0.96
0.84
0.75
0.55
0.79
1.78
1.32
0.87
1.13
0.49
0.52
Group Effect
P-Value
0.7568
0.9257
0.8840
0.7220
0.4450
0.8898
0.4806
0.7763
0.9419
0.6774
0.2981
0.8322
Estimated Ratio
(Relationship)
Tu key's P-Value*
t Indicates that values for this parameter were log-transformed for the analysis.
# Cells contain all pairwise comparisons that were significant at the 0.05 level. The format within each
cell is: (1) the ratio of group mean shifts, (2) the relationship between the corresponding pair of
group mean shifts shown in parentheses, and (3) the Tukey-adjusted p-value.
1078-CG920794 - Hematology and C-Reactive Protein
J-38
-------
Table 15a. Descriptive Statistics for Lymphocyte Count (103 cells/uL) by Group and Study
Day
Group
1
2
3
Study
Day
-3
2
4
9
11
16
18
23
25
30
32
37
39
-3
2
4
9
11
16
18
23
25
30
32
37
39
-3
2
4
9
11
16
18
23
25
30
32
37
39
N
5
5
4
5
5
4
5
4
3
5
1
5
5
7
7
7
7
7
7
7
6
6
7
3
7
7
7
7
7
6
6
5
6
3
6
3
5
4
6
Mean
(95% Confidence Interval)
3.50 (2.20, 4.80)
4.02 (2.68, 5.37)
3.37 (0.44, 6.29)
4.35(1.75,6.95)
4.00(2.41,5.60)
4.38 (2.55, 6.21)
4.45(2.51,6.39)
4.32(1.44,7.20)
4.27 (0.76, 7.77)
4.35(2.56,6.13)
4.49 (-)
4.57 (2.74, 6.40)
2.87(0.71, 5.02)
4.78(4.01,5.55)
5.17(4.23,6.11)
5.19(4.01,6.37)
5.45 (4.44, 6.45)
5.17(4.41,5.92)
5.06(3.98,6.14)
5.24(3.91,6.56)
4.39(3.51,5.27)
4.23(3.15, 5.31)
4.74 (3.95, 5.52)
3.57 (2.92, 4.22)
4.50(3.39,5.61)
3.53(2.17,4.90)
4.10(3.42,4.77)
3.73 (3.02, 4.44)
3.65 (2.65, 4.64)
3.83 (2.79, 4.88)
4.37 (3.36, 5.38)
4.15(3.22,5.08)
4.05 (2.66, 5.44)
4.20 (0.75, 7.64)
3.89 (2.84, 4.95)
4.57(1.26,7.88)
3.58(2.61,4.54)
3.69 (3.06, 4.31)
2.52(1.07, 3.98)
1078-CG920794 - Hematology and C-Reactive Protein
J-39
-------
Table 15a. (Continued)
Group
4
Study
Day
-3
2
4
9
11
16
18
23
25
30
32
37
39
N
7
7
4
7
6
4
4
2
2
3
3
3
3
Mean
(95% Confidence Interval)
4.47(3.11,5.83)
4.60 (2.70, 6.50)
4.99(3.19,6.79)
4.36(3.17,5.56)
4.53 (2.60, 6.46)
5.28 (3.94, 6.62)
6.55(5.94,7.17)
7.79 (0.00a, 24.38)
7.78(3.14, 12.41)
5.77(0.02, 11.53)
4.63(1.26,8.01)
4.85(0.52,9.19)
4.42 (2.56, 6.27)
Confidence interval could not be calculated since only one observation was available for this group
on this Study Day.
a Negative lower confidence limit was set to 0 since negative values are not possible.
Table 15b. Test Results for Lymphocyte Count (103 cells/uL)
#
Lymphocyte Count
Study
Day
2
4
9
11
16
18
23
25
30
32
37
39
Mean Shift from Baseline,
by Group
1
0.53
-0.25
0.85
0.51
0.83
0.95
0.70
0.38
0.85
1.68
1.07
-0.63
2
0.39
0.41
0.67
0.39
0.28
0.46
-0.33
-0.49
-0.04
-1.33
-0.28
-1.25
3
-0.37
-0.45
-0.35
0.11
0.24
-0.13
0.37
-0.29
-0.00
-0.37
-0.81
-1.66|
4
0.13
-0.22
-0.11
0.24
0.23
1.51
2.18
2.16
0.67
-0.47
-0.25
-0.69
Group Effect
P-Value
0.2704
0.5563
0.1985
0.8899
0.8293
0.2451
0.3101
0.1161
0.6610
0.1112
0.0704
0.6146
Estimated Difference
(Relationship)
Tu key's P-Value*
Cells contain all pairwise comparisons that were significant at the 0.05 level. The format within each
cell is: (1) the difference of group mean shifts, (2) the relationship between the corresponding pair of
group mean shifts shown in parentheses, and (3) the Tukey-adjusted p-value.
|, J, "|" indicates the mean at the Study Day was significantly greater than that at baseline; "J," indicates
the mean at the Study Day was significantly less than that at baseline (at the 0.05 level).
1078-CG920794 - Hematology and C-Reactive Protein
J-40
-------
Table 16a. Descriptive Statistics for Neutrophil Count/Lymphocyte Count Ratio by Group
and Study Day
Group
1
2
3
Study
Day
-3
2
4
9
11
16
18
23
25
30
32
37
39
-3
2
4
9
11
16
18
23
25
30
32
37
39
-3
2
4
9
11
16
18
23
25
30
32
37
39
N
5
5
4
5
5
4
5
4
3
5
1
5
5
7
7
7
7
7
7
7
6
6
7
3
7
7
7
7
7
6
6
5
6
3
6
3
5
4
6
Geometric Mean
(95% Confidence Interval)
0.498(0.221, 1.124)
0.396 (0.227, 0.691)
0.390 (0.225, 0.677)
0.312(0.181, 0.536)
0.356(0.148,0.855)
0.327 (0.252, 0.423)
0.273(0.172,0.433)
0.245(0.074,0.810)
0.322(0.154, 0.673)
0.289(0.200,0.419)
0.31 0(-)
0.347 (0.237, 0.506)
0.222(0.136, 0.362)
0.341 (0.256, 0.454)
0.279(0.214, 0.362)
0.306 (0.229, 0.409)
0.264(0.210, 0.332)
0.295(0.230, 0.378)
0.281 (0.229, 0.346)
0.255(0.207, 0.314)
0.364 (0.285, 0.465)
0.336(0.275, 0.411)
0.298 (0.235, 0.379)
0.350(0.221, 0.552)
0.318(0.267,0.380)
0.227(0.192, 0.270)
0.399 (0.334, 0.476)
0.329 (0.283, 0.381)
0.368 (0.243, 0.560)
0.402(0.271, 0.597)
0.360 (0.292, 0.443)
0.325(0.271,0.390)
0.386 (0.246, 0.605)
0.631 (0.126,3.159)
0.418(0.280, 0.625)
0.393 (0.270, 0.571)
0.479 (0.272, 0.842)
0.381 (0.333, 0.437)
0.402(0.176, 0.920)
1078-CG920794 - Hematology and C-Reactive Protein
J-41
-------
Table 16a. (Continued)
Group
4
Study
Day
-3
2
4
9
11
16
18
23
25
30
32
37
39
N
7
7
4
7
6
4
4
2
2
3
3
3
3
Geometric Mean
(95% Confidence Interval)
0.558 (0.373, 0.835)
0.492(0.265,0.914)
0.491 (0.168, 1.435)
0.477 (0.292, 0.780)
0.434 (0.275, 0.683)
0.270(0.160,0.454)
0.308(0.142, 0.670)
0.525(0.000, 1078.118)
0.386 (0.000, 265028.326)
0.427 (0.040, 4.586)
0.671 (0.072, 6.265)
0.281 (0.123, 0.642)
0.317(0.060, 1.669)
Confidence interval could not be calculated since only one observation was available for this group
on this Study Day.
Table 16b. Test Results for Neutrophil Count/Lymphocyte Count Ratio
Neutrophil Count/Lymphocyte Count Ratio1
Study
Day
2
4
9
11
16
18
23
25
30
32
37
39
Mean Shift as a Proportic
Baseline, by Grou
1
0.79
1.03
0.63
0.72
0.66
0.551
0.65
0.88
0.58
0.74
0.70
0.45
2
0.82
0.90
0.77
0.87
0.83
0.75
1.06
0.98
0.87
0.95
0.93
0.67
3
0.82
0.92
0.96
0.89
0.86
0.92
1.47
1.00
0.85
1.16
0.91
0.96
an from
p
4
0.88
1.00
0.86
0.73
0.52
0.60
1.28
0.94
0.80
1.25
0.53
0.59
Group Effect
P-Value
0.9518
0.9726
0.3502
0.6277
0.3631
0.2191
0.3969
0.9857
0.4913
0.7897
0.2121
0.3655
Estimated Ratio
(Relationship)
Tukey's P-Value*
t
#
T, 4
Indicates that values for this parameter were log-transformed for the analysis.
Cells contain all pairwise comparisons that were significant at the 0.05 level. The format within each
cell is: (1) the ratio of group mean shifts, (2) the relationship between the corresponding pair of
group mean shifts shown in parentheses, and (3) the Tukey-adjusted p-value.
"t" indicates the geometric mean at the Study Day was significantly greater than that at baseline;
"J," indicates the geometric mean at the Study Day was significantly less than that at baseline (at the
0.05 level).
1078-CG920794 - Hematology and C-Reactive Protein
J-42
-------
Table 17a. Descriptive Statistics for Monocyte Count (103 cells/uL) by Group and Study
Day
Group
1
2
3
Study
Day
-3
2
4
9
11
16
18
23
25
30
32
37
39
-3
2
4
9
11
16
18
23
25
30
32
37
39
-3
2
4
9
11
16
18
23
25
30
32
37
39
N
5
5
4
5
5
4
5
4
3
5
1
5
4
7
7
7
7
7
7
7
6
6
7
3
7
7
7
7
7
6
6
5
6
3
6
3
5
4
6
Geometric Mean
(95% Confidence Interval)
0.09 (0.04, 0.21)
0.09(0.04,0.18)
0.05(0.01,0.26)
0.05 (0.03, 0.09)
0.09(0.05,0.18)
0.08(0.04,0.16)
0.07 (0.05, 0.09)
0.06 (0.02, 0.20)
0.10(0.01,0.71)
0.11 (0.07,0.17)
0.06 (-)
0.08 (0.03, 0.20)
0.04(0.01,0.24)
0.10(0.06,0.15)
0.12(0.08,0.20)
0.10(0.06,0.17)
0.10(0.06,0.16)
0.10(0.07,0.14)
0.12(0.08,0.18)
0.11 (0.07,0.18)
0.10(0.05,0.18)
0.09(0.06,0.13)
0.09(0.07,0.11)
0.07 (0.02, 0.20)
0.10(0.05,0.23)
0.03 (0.02, 0.06)
0.09(0.06,0.13)
0.09(0.06,0.12)
0.08(0.04,0.16)
0.13(0.08,0.22)
0.08(0.05,0.12)
0.07(0.04,0.15)
0.10(0.06,0.16)
0.10(0.02,0.44)
0.12(0.06,0.25)
0.16(0.01,3.26)
0.13(0.07,0.26)
0.12(0.03,0.41)
0.06(0.02,0.16)
1078-CG920794 - Hematology and C-Reactive Protein
J-43
-------
Table 17a. (Continued)
Group
4
Study
Day
-3
2
4
9
11
16
18
23
25
30
32
37
39
N
7
7
4
7
6
4
4
2
2
3
3
3
3
Geometric Mean
(95% Confidence Interval)
0.14(0.11,0.20)
0.14(0.08,0.26)
0.15(0.04,0.60)
0.12(0.08,0.18)
0.12(0.05,0.28)
0.15(0.05,0.42)
0.18(0.06,0.53)
0.48(0.00,3207918.13)
0.50(0.00,514082.32)
0.25 (0.02, 2.57)
0.22 (0.02, 2.82)
0.15(0.04,0.58)
0.11 (0.03,0.38)
Confidence interval could not be calculated since only one observation was available for this group
on this Study Day.
Table lib. Test Results for Monocyte Count (103 cells/jiL)
t
#
T, 4
Monocyte Count1
Study
Day
2
4
9
11
16
18
23
25
30
32
37
39
Mean Shift as a Proportic
Baseline, by Grou
1
0.93
0.64
0.55
0.96
0.80
0.73
0.76
1.04
1.14
1.50
0.83
0.54
2
1.28
1.02
1.05
1.00
1.20
1.18
1.08
0.93
0.91
0.51
1.06
0.31|
3
0.94
0.85
1.36
0.75
0.84
0.98
1.00
1.24
1.34
1.32
1.12
0.61
an from
p
4
0.99
0.90
0.84
0.80
0.94
1.12
3.24
3.40
1.35
1.21
0.84
0.59
Group Effect
P-Value
0.7183
0.7599
0.0537
0.8501
0.6687
0.6203
0.2750
0.2561
0.7953
0.3380
0.8672
0.5866
Estimated Ratio
(Relationship)
Tukey'sP-Value*
2.46 (1<3) 0.0409
Indicates that values for this parameter were log-transformed for the analysis.
Cells contain all pairwise comparisons that were significant at the 0.05 level. The format within each
cell is: (1) the ratio of group mean shifts, (2) the relationship between the corresponding pair of
group mean shifts shown in parentheses, and (3) the Tukey-adjusted p-value.
"t" indicates the geometric mean at the Study Day was significantly greater than that at baseline;
"J," indicates the geometric mean at the Study Day was significantly less than that at baseline (at the
0.05 level).
1078-CG920794 - Hematology and C-Reactive Protein
J-44
-------
Table 18a. Descriptive Statistics for Eosinophil Count (103 cells/uL) by Group and Study
Day
Group
1
2
3
Study
Day
-3
2
4
9
11
16
18
23
25
30
32
37
39
-3
2
4
9
11
16
18
23
25
30
32
37
39
-3
2
4
9
11
16
18
23
25
30
32
37
39
N
5
5
4
5
5
4
5
4
3
5
1
5
5
7
7
7
7
7
7
7
6
6
7
3
7
7
7
7
7
6
6
5
6
3
6
3
5
4
6
Geometric Mean
(95% Confidence Interval)
0.14(0.10,0.18)
0.14(0.08,0.24)
0.11 (0.05,0.23)
0.13(0.09,0.19)
0.14(0.10,0.21)
0.19(0.11,0.33)
0.19(0.14,0.25)
0.16(0.08,0.32)
0.16(0.08,0.34)
0.16(0.13,0.20)
0.21 (-)
0.19(0.18,0.20)
0.14(0.11,0.19)
0.16(0.12,0.21)
0.18(0.12,0.26)
0.17(0.13,0.23)
0.15(0.11,0.19)
0.16(0.13,0.18)
0.15(0.13,0.18)
0.14(0.12,0.18)
0.15(0.11,0.22)
0.17(0.13,0.22)
0.15(0.11,0.19)
0.14(0.12,0.17)
0.15(0.13,0.17)
0.12(0.09,0.16)
0.18(0.14,0.23)
0.15(0.11,0.20)
0.17(0.13,0.22)
0.16(0.12,0.21)
0.17(0.16,0.19)
0.15(0.12,0.19)
0.13(0.09,0.19)
0.15(0.10,0.21)
0.16(0.12,0.21)
0.24(0.10,0.56)
0.16(0.09,0.27)
0.14(0.12,0.16)
0.13(0.09,0.21)
1078-CG920794 - Hematology and C-Reactive Protein
J-45
-------
Table 18a. (Continued)
Group
4
Study
Day
-3
2
4
9
11
16
18
23
25
30
32
37
39
N
7
7
4
7
6
4
4
2
2
3
3
3
3
Geometric Mean
(95% Confidence Interval)
0.14(0.12,0.17)
0.13(0.09,0.20)
0.19(0.14, 0.25)
0.14(0.11,0.18)
0.16(0.11, 0.23)
0.14(0.08,0.23)
0.15(0.08, 0.29)
0.15(0.15,0.15)
0.13(0.00, 3.80)
0.16(0.08,0.33)
0.16(0.11, 0.26)
0.17(0.14,0.21)
0.16(0.03, 0.83)
Confidence interval could not be calculated since only one observation was available for this group
on this Study Day.
Table 18b. Test Results for Eosinophil Count (103 cells/uL)
Eosinophil Count1
Study
Day
2
4
9
11
16
18
23
25
30
32
37
39
Mean Shift as a Proportion from
Baseline, by Group
1
1.03
0.76
0.95
1.03
1.40
1.37
1.09
1.14
1.20
1.24
1.37
1.04
2
1.13
1.08
0.95
1.00
0.96
0.92
1.06
1.15
0.94
0.90
0.96
0.77
3
0.84
0.95
0.95
0.97
0.85
0.78
0.80
0.93
1.22
0.93
0.76
0.79
4
0.94
1.30
0.98
1.15
0.90
0.98
1.12
0.97
1.14
1.16
1.22
1.13
Group Effect
P-Value
0.5673
0.1536
0.9929
0.8155
0.2067
0.0911
0.6402
0.7508
0.6631
0.7732
0.0249*
0.4833
Estimated Ratio
(Relationship)
Tu key's P-Value*
1. 80 (3<1) 0.0227
t Indicates that values for this parameter were log-transformed for the analysis.
# Cells contain all pairwise comparisons that were significant at the 0.05 level. The format within each
cell is: (1) the ratio of group mean shifts, (2) the relationship between the corresponding pair of
group mean shifts shown in parentheses, and (3) the Tukey-adjusted p-value.
* The overall group effect was significant at the 0.05 level.
1078-CG920794 - Hematology and C-Reactive Protein
J-46
-------
Table 19a. Descriptive Statistics for Basophil Count (103 cells/uL) by Group and Study
Day
Group
1
2
3
Study
Day
-3
2
4
9
11
16
18
23
25
30
32
37
39
-3
2
4
9
11
16
18
23
25
30
32
37
39
-3
2
4
9
11
16
18
23
25
30
32
37
39
N
5
5
4
5
5
4
5
4
3
5
1
5
5
7
7
7
7
7
7
7
6
6
7
3
7
7
7
7
7
6
6
5
6
3
6
3
5
4
6
Mean
(95% Confidence Interval)
0.22 (0.03, 0.41)
0.27 (0.05, 0.50)
0.14(0.00a, 0.32)
0.22 (0.06, 0.38)
0.22 (0.07, 0.38)
0.23(0.01,0.44)
0.29(0.12,0.45)
0.22 (0.03, 0.40)
0.22 (0.00a, 0.48)
0.35 (0.05, 0.65)
0.36 (-)
0.30(0.17,0.44)
0.13(0.00a, 0.28)
0.26 (0.09, 0.43)
0.27(0.11,0.43)
0.31 (0.12,0.50)
0.30(0.13,0.46)
0.29(0.15,0.43)
0.26(0.15,0.38)
0.30(0.15,0.45)
0.33(0.12,0.53)
0.28 (0.09, 0.47)
0.32(0.15,0.49)
0.38 (0.00a, 0.79)
0.30(0.15,0.44)
0.18(0.05,0.30)
0.23(0.13,0.32)
0.20(0.10,0.30)
0.20 (0.09, 0.32)
0.27(0.17,0.36)
0.23(0.13,0.33)
0.20 (0.06, 0.33)
0.19(0.10,0.29)
0.24 (0.00a, 0.65)
0.26(0.15,0.37)
0.28(0.01,0.56)
0.30(0.14,0.47)
0.27(0.14,0.40)
0.14(0.05,0.23)
1078-CG920794 - Hematology and C-Reactive Protein
J-47
-------
Table 19a. (Continued)
Group
4
Study
Day
-3
2
4
9
11
16
18
23
25
30
32
37
39
N
7
7
4
7
6
4
4
2
2
3
3
3
3
Mean
(95% Confidence Interval)
0.16(0.08,0.24)
0.19(0.06,0.32)
0.23(0.01,0.44)
0.18(0.09,0.27)
0.22 (0.09, 0.34)
0.26(0.11,0.41)
0.27 (0.09, 0.44)
0.40 (0.00a, 2.05)
0.42 (0.00a, 3.15)
0.40 (0.00a, 0.90)
0.40 (0.04, 0.75)
0.42 (0.00a, 0.90)
0.28 (0.00a, 0.71)
Confidence interval could not be calculated since only one observation was available for this group
on this Study Day.
a Negative lower confidence limit was set to 0 since negative values are not possible.
Table 19b. Test Results for Basophil Count (103 cells/uL)
Basophil Count
Study
Day
2
4
9
11
16
18
23
25
30
32
37
39
Mean Shift from Baseline, by Group
1
0.06
-0.03
0.00
0.00
0.01
0.07
0.05
0.09
0.13
0.08
0.09
-0.08
2
0.01
0.05
0.03
0.03
0.00
0.04
0.10|
0.05
0.06
0.09
0.04
-0.09
3
-0.03
-0.02
0.05
-0.01
0.01
-0.03
0.04
0.04
0.03
0.10
0.01
-0.08
4
0.03
0.04
0.02
0.04
0.06
0.06
0.13
0.15
0.18
0.17
0.19
0.06
Group Effect
P-Value
0.1120
0.3090
0.7242
0.8349
0.7620
0.5612
0.6075
0.6998
0.5113
0.8650
0.2922
0.7349
Estimated Difference
(Relationship)
Tukey'sP-Value*
T,4
Cells contain all pairwise comparisons that were significant at the 0.05 level. The format within each
cell is: (1) the difference of group mean shifts, (2) the relationship between the corresponding pair of
group mean shifts shown in parentheses, and (3) the Tukey-adjusted p-value.
"|" indicates the mean at the Study Day was significantly greater than that at baseline; "J," indicates
the mean at the Study Day was significantly less than that at baseline (at the 0.05 level).
1078-CG920794 - Hematology and C-Reactive Protein
J-48
-------
Table 20a. Descriptive Statistics for C-Reactive Protein (103 cells/uL) by Group and
Study Day
Group
1
2
3
Study
Day
-3
2
4
9
11
16
18
23
25
30
32
37
39
-3
2
4
9
11
16
18
23
25
30
32
37
39
-3
2
4
9
11
16
18
23
25
30
32
37
39
N
5
5
2
4
5
3
5
4
4
4
5
5
4
7
6
7
7
6
5
7
5
6
6
5
6
7
7
7
6
5
6
6
6
2
6
2
6
4
6
Geometric Mean
(95% Confidence Interval)
0.47(0.16, 1.39)
0.63(0.31, 1.30)
1.04(0.01, 105.17)
0.39(0.17,0.87)
0.35(0.14,0.85)
0.33(0.10, 1.07)
0.36(0.13, 1.00)
0.25 (0.25, 0.25)
0.25 (0.25, 0.25)
0.25 (0.25, 0.25)
0.25 (0.25, 0.25)
0.25 (0.25, 0.25)
0.25 (0.25, 0.25)
0.37(0.19,0.69)
0.44 (0.22, 0.88)
0.38 (0.23, 0.62)
0.25 (0.25, 0.25)
0.29 (0.20, 0.44)
0.34(0.14,0.82)
0.25 (0.25, 0.25)
0.25 (0.25, 0.25)
0.25 (0.25, 0.25)
0.25 (0.25, 0.25)
0.30(0.18,0.53)
0.25 (0.25, 0.25)
0.34(0.16,0.73)
0.25 (0.25, 0.25)
0.28(0.21,0.39)
0.30(0.19,0.47)
0.25 (0.25, 0.25)
0.39(0.17,0.90)
0.39(0.18,0.83)
0.29(0.19,0.45)
0.25 (0.25, 0.25)
0.28(0.21,0.38)
0.25 (0.25, 0.25)
0.47(0.16, 1.35)
0.25 (0.25, 0.25)
0.29 (0.20, 0.43)
1078-CG920794 - Hematology and C-Reactive Protein
J-49
-------
Table 20a. (Continued)
Group
4
Study
Day
-3
2
4
9
11
16
18
23
25
30
32
37
39
N
7
7
4
6
5
3
4
3
3
2
3
2
3
Geometric Mean
(95% Confidence Interval)
0.43 (0.22, 0.84)
0.80(0.34, 1.88)
0.46(0.15, 1.42)
0.42(0.18, 1.00)
0.32(0.16,0.61)
0.43(0.04,4.18)
1.03(0.15,7.26)
0.77(0.01, 100.14)
0.88 (0.02, 48.84)
0.69(0.00,281753.42)
0.43 (0.04, 4.24)
1.20(0.00,5246.62)
0.44(0.13, 1.53)
Table 20b. Test Results for C-Reactive Protein (103 cells/uL)
C-Reactive ProteinT
Study
Day
2
4
9
11
16
18
23
25
30
32
37
39
Mean Shift as a Proportion from
Baseline, by Group
1
1.35
2.09
0.70
0.74
0.73
0.77
0.71
0.64
0.64
0.53
0.53
0.46
2
1.12
1.03
0.68
0.75
0.80
0.68
0.59
0.64
0.75
0.71
0.64
0.93
3
1.14
1.19
1.00
1.56
1.54
1.18
1.00
1.12
1.00
1.88
1.00
1.16
4
1.86|
1.04
1.16
0.60
1.35
2.35
1.46
1.66
1.95
0.80
1.55
0.83
Group Effect
P-Value
0.4243
0.7210
0.6774
0.1996
0.4062
0.2266
0.7314
0.3604
0.3995
0.1860
0.3728
0.4306
Estimated Ratio
(Relationship)
Tukey'sP-Value*
t
#
T,4
Indicates that values for this parameter were log-transformed for the analysis.
Cells contain all pairwise comparisons that were significant at the 0.05 level. The format within each
cell is: (1) the ratio of group mean shifts, (2) the relationship between the corresponding pair of
group mean shifts shown in parentheses, and (3) the Tukey-adjusted p-value.
"t" indicates the geometric mean at the Study Day was significantly greater than that at baseline;
"J," indicates the geometric mean at the Study Day was significantly less than that at baseline (at the
0.05 level).
1078-CG920794 - Hematology and C-Reactive Protein
J-50
-------
Jfl
"CD
o
8-
7-
6-
5-
4-
3-
Group 1 (Control)
Group 2 (100 CPU)
Group 3 (1,000 CPU)
Group 4 (10,000 CPU)
-3
11 16 18 23 25 30 32 37 39
Study Day
Figure 1. Plot of Red Blood Cell Count over time.
16-
15-
14-
13-
12-
11-
10-
9-
8-
7-
Group 1 (Control)
Group 2 (100 CPU)
Group 3 (1,000 CPU)
Group 4 (10,000 CPU)
-3 2 4 9 11 16 18 23 25 30 32 37 39
Study Day
Figure 2. Plot of Hemoglobin over time.
1078-CG920794 - Hematology and C-Reactive Protein
J-51
-------
o
o
-t->
CO
E
CD
X
51-
46-
41-
36-
31-
26-
21-
Group 1 (Control)
Group 2 (100 CPU)
Group 3 (1,000 CPU)
Group 4 (10,000 CPU)
-3 2 4 9 11 16 18 23 25 30 32 37 39
Study Day
Figure 3. Plot of Hematocrit over time.
80-
75-
70-
65-
60-
55-
Group 1 (Control)
Group 2 (100 CPU)
Group 3 (1,000 CPU)
Group 4 (10,000 CPU)
-3 2 4 9 11 16 18 23 25 30 32 37 39
Study Day
Figure 4. Plot of Mean Corpuscular Volume (MCV) over time.
1078-CG920794 - Hematology and C-Reactive Protein
J-52
-------
24-
23-
22-
21
20
19-
18-
17-
Group 1 (Control)
Group 2 (100 CPU)
Group 3 (1,000 CPU)
Group 4 (10,000 CPU)
2 4 9 11 16 18 23 25 30 32 37 39
Study Day
Figure 5. Plot of Mean Corpuscular Hemoglobin (MCH) over time.
36-
34-
32-
30-
28-
26-
-3
Group 1 (Control)
Group 2 (100 CPU)
Group 3 (1,000 CPU)
Group 4 (10,000 CPU)
11 16 18 23 25 30 32 37 39
Study Day
Figure 6. Plot of Mean Corpuscular Hemoglobin Concentration (MCHC) over time.
1078-CG920794 - Hematology and C-Reactive Protein
J-53
-------
24-
20-
16-
12-
8-
Group 1 (Control)
Group 2 (100 CPU)
Group 3 (1,000 CPU)
Group 4 (10,000 CPU)
-3 2 4 9 11 16 18 23 25 30 32 37 39
Study Day
Figure 7. Plot of Red Cell Distribution Width (RDW) over time.
"CD
o
c
D
O
O
2400
2000
1600
1200
800-
400-
0-
Group 1 (Control)
Group 2 (100 CPU)
Group 3 (1,000 CPU)
Group 4 (10,000 CPU)
-324 9 11 16 18 23 25 30 32 37 39
Study Day
Negative lower bounds were set to zero.
Figure 8. Plot of Platelet Count (PLT) over time.
1078-CG920794 - Hematology and C-Reactive Protein
J-54
-------
Jfl
"CD
o
15-
12-
9-
6-
3-
Group 1 (Control)
Group 2 (100 CPU)
Group 3 (1,000 CPU)
Group 4 (10,000 CPU)
-3 2 4 9 11 16 18 23 25 30 32 37 39
Study Day
Figure 9. Plot of Mean Platelet Volume (MPV) over time.
100:
"CD
o
10-
1-
Group 1 (Control)
Group 2 (100 CPU)
Group 3 (1,000 CPU)
Group 4 (10,000 CPU)
-324 9 11 16 18 23 25 30 32 37 39
Study Day
Figure 10. Plot of White Blood Cell Count over time.
1078-CG920794 - Hematology and C-Reactive Protein
J-55
-------
100.0-
"CD
o
10.0-
1.0-
0.1-
Group 1 (Control)
Group 2 (100 CPU)
Group 3 (1,000 CPU)
Group 4 (10,000 CPU)
-3 2 4 9 11 16 18 23 25 30 32 37 39
Study Day
Figure 11. Plot of Neutrophil Count over time.
"CD
o
12-
9-
Group 1 (Control)
Group 2 (100 CPU)
Group 3 (1,000 CPU)
Group 4 (10,000 CPU)
-3 2 4 9 11 16 18 23 25 30 32 37 39
Study Day
Figure 12. Plot of Lymphocyte Count over time.
1078-CG920794 - Hematology and C-Reactive Protein
J-56
-------
10.00-
o
O
3
o
c
o
1.00-
0.10-
0.01-
Group 1 (Control)
Group 2 (100 CPU)
Group 3 (1,000 CPU)
Group 4 (10,000 CPU)
-32 4 9 11 16 18 23 25 30 32 37 39
Study Day
Figure 13. Plot of Neutrophil Count/Lymphocyte Count Ratio over time.
"CD
o
Id.OOCh
1.000^
0.010^
0.001-
Group 1 (Control)
Group 2 (100 CPU)
Group 3 (1,000 CPU)
Group 4 (10,000 CPU)
-32 4 9 11 16 18 23 25 30 32 37 39
Study Day
Figure 14. Plot of Monocyte Count over time.
1078-CG920794 - Hematology and C-Reactive Protein
J-57
-------
1.00-
Jfl
"CD
o
0.10-
0.01-
Group 1 (Control)
Group 2 (100 CPU)
Group 3 (1,000 CPU)
Group 4 (10,000 CPU)
-3 2 4 9 11 16 18 23 25 30 32 37 39
Study Day
Figure 15. Plot of Eosinophil Count over time.
"CD
o
1.0-
0.8-
0.6-
0.4-
0.2-
0.0-
Group 1 (Control)
Group 2 (100 CPU)
Group 3 (1,000 CPU)
Group 4 (10,000 CPU)
-3 2 4 9 11 16 18 23 25 30 32 37 39
Study Day
Negative lower bounds were set to zero.
Figure 16. Plot of Basophil Count over time.
1078-CG920794 - Hematology and C-Reactive Protein
J-58
-------
100.000 =
10.000 =
1.000 =
0.100 =
0.010 =
0.001-
Group 1 (Control)
Group 2 (100 CPU)
Group 3 (1,000 CPU)
Group 4 (10,000 CPU)
-3 2 4 9 11 16 18 23 25 30 32 37 39
Study Day
Figure 17. Plot of C-Reactive Protein over time.
1078-CG920794 - Hematology and C-Reactive Protein
J-59
-------
ATTACHMENT I
RESULTS OF ANALYSIS REPEATED
WITH POTENTIAL OUTLIERS EXCLUDED
1078-CG920794 - Hematology and C-Reactive Protein 1-1
-------
ATTACHMENT I: RESULTS OF ANALYSIS REPEATED
WITH POTENTIAL OUTLIERS EXCLUDED
Eleven (11) potential outliers were identified in Table 2 of the report. To determine the effect of
the potential outliers on the statistical analysis, the analysis was performed on the data with these
observations excluded. The results that had a change in significance after excluding the potential
outliers are presented below.
Tables A-l and A-2 contain test results for those parameters that experienced changes in
significance due to the exclusion of the potential outliers, when compared to the corresponding
results shown in Tables 4b through 19b where the potential outliers were not excluded. Table
entries are shown in bold if the significance changed in comparison to the corresponding results
shown in Tables 4b through 19b. With the potential outliers excluded, the following changes in
significance were noted:
• HGB (Table A-l): There was a significant decrease from baseline in group 4 on
Study Day 9.
• RDW (Table A-2): There was a significant decrease as a proportion of baseline in
group 1 on Study Day 25. On Study Day 25, there was no longer a significant
difference between mean changes as a proportion of baseline in Groups 1 and 4.
Table 1-1. Test Results for Hemoglobin (HGB, g/dL) with Potential Outliers Excluded
Hemoglobin
Study
Day
9
Mean Shift from Baseline, by Group
1
-0.22
2
-0.24
3
-0.38
4
-0.58|
Group Effect
P-Value
0.5691
Estimated Difference
(Relationship)
Tukey'sP-Value*
# Cells contain all significant pain/vise group comparisons at the 0.05 level. The format within each cell
is: difference of shifts (relationship between corresponding group mean shifts) Tukey-adjusted
p-value.
|, J, "|" indicates the mean at the Study Day was significantly greater than that at baseline; "J," indicates
the mean at the Study Day was significantly less than that at baseline (at the 0.05 level).
1078-CG920794 - Hematology and C-Reactive Protein
1-2
-------
Table 1-2. Test Results for Red Cell Distribution Width (RDW, %) with Potential
Outliers Excluded
Red Cell Distribution Width1
Study
Day
25
Mean Shift as a Proportion of
Baseline, by Group
1
0.92|
2
0.97
3
0.97
4
NA
Group Effect
P-Value
0.0613
Estimated Ratio
(Relationship)
Tukey's P-Value*
Groups 1 and 4 were no longer
significantly different.
t
#
T,4
NA
Indicates that values for this parameter were log-transformed for the analysis.
Cells contain all significant pain/vise group comparisons at the 0.05 level. The format within each cell
is: ratio of shifts (relationship between corresponding group mean shifts) Tukey-adjusted p-value.
"t" indicates the geometric mean at the Study Day was significantly greater than that at baseline;
"J," indicates the geometric mean at the Study Day was significantly less than that at baseline (at the
0.05 level).
There were no measurements available for this group on this Study Day.
1078-CG920794 - Hematology and C-Reactive Protein
1-3
-------
APPENDIX K
INDIVIDUAL CLINICAL OBSERVATIONS
K-1
-------
1078-CG920794
Naive Observations
Challenge
Order
i
2
3
4
5
i
2
3
4
5
6
7
1
2
3
4
6
7
1
2
3
4
5
6
Animal SO
40
7
5
9
37
13
34
25
15
30
28
19
14
11
2
8
12
32
6
33
27
31
39
21
Sex
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
Group
1
1
1
1
3
3
3
3
3
4
4
4
4
4
4
4
7/20
/2010
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
7/21
N
N
N
N
N
N
NE
N
N
N
N
N
N
N
NE
N
N
N
N
N
N
N
N
N
NE
/2010
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
7/22
AM
N
N
\
N
N
N
NE
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
/2010
PM
N
N
N
N
N
N
N
N
N
N
N
N
N
fs
N
N
N
N
N
N
N
N
N
N
N
7/23
AM
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
/2010
PM
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
7/24
AM
N
M
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
/2010
PM
N
N
N
N
N
N
N ~
N
N
N
N
N
N
N
N
N
N
N
N
N
N
AM
r. i
K
t\
K j
N ' •*
__
N i
- N J
J j
M i
. t
PM
N
N
N
N
N
N
N
N
N
N
M
N I N
N j N
N 1 N
1 N 1 N
N , N
•"""—— * ™™™™™,
N | N
- N _ , N
N N
N N '
N j -i
N 1 N
N 1 'J
N 1 N
_il 1 N
N 1 n
AM • '
. "> ,
'I 1
J 1
'* .
__•< |
1
' j
\ '
I
-T ^
r, i
r.
N [
f: i
'< i
f.. "
h .
N
f>.
N = Normal
NE = Not Eating
* Animals received from LAR, all appear N
a Animals challenged after am observations, refer to Clinical Observations
Printed By: _
QC Review- d f
-------
'Sm,};! amount of stool
'Animal found prostrate, unresponsive to touch, and gasping at 1055, died at 1113 prior
Animal found dead sfter am observations
V»t notified about NE, vet ordered weight to be taken and yogurt to be added to food
-------
Vet notified about NE, vet ordered weight to be taken and yogurt to be added to food
Ved yogurt, appears animal did not eat previous yogurt feeding, but no peitets m f
filmpaired use of right front iimb. N unless condition worwns
-------
FD s found t>ad
0 = Other |requsres
3311
"S*nst! amount of stool
Animal found prostrate, unresponsive to touf h and ea^piAj; 3! 10SS died s! 111'^ • ' t /i ' '
Vet notified about NE, vet ordered weight to be taken and vogurt to be adifcd to food
%d yogyrt, appear animal did not eat previous yogurt feeding, but no pellets n feeder, still dehydrated
-------
N = Normal
NE = tSiot CaEing
Di = Dkrrhea
SS ™ Soft Stool
NS •- fto StooJ
RA ~ Re^ptratofy Aboofmaliiies
L * Lethargk
FD s Found Dead
0 ~ Other (mquirfi'i com rsent)
Animal found prostrate, unresponsive to touch, and gasping at 1055, died at 11U prior to adr
'Animal found dead after am observations
*V« notified about NE, vet ordered weight to be taken and yogurt to be added to food
5Fed yogurt, appears animal did not eat previous yojurt feeding, but no pellets In feeder, still a
Impaired use of right front limb, N unless condition worsens
-------
1078-CG920794
Naive Observations
Challenge
Order
i
2
3
4
5
i
2
3
4
5
6
7
1
2
3
4
6
7
1
2
3
4
5
6
Animal SO
40
7
5
9
37
13
34
25
15
30
28
19
14
11
2
8
12
32
6
33
27
31
39
21
Sex
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
Group
1
1
1
1
3
3
3
3
3
4
4
4
4
4
4
4
7/20
/2010
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
7/21
N
N
N
N
N
N
NE
N
N
N
N
N
N
N
NE
N
N
N
N
N
N
N
N
N
NE
/2010
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
7/22
AM
N
N
\
N
N
N
NE
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
/2010
PM
N
N
N
N
N
N
N
N
N
N
N
N
N
fs
N
N
N
N
N
N
N
N
N
N
N
7/23
AM
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
/2010
PM
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
7/24
AM
N
M
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
/2010
PM
N
N
N
N
N
N
N ~
N
N
N
N
N
N
N
N
N
N
N
N
N
N
AM
r. i
K
t\
K j
N ' •*
__
N i
- N J
J j
M i
. t
PM
N
N
N
N
N
N
N
N
N
N
M
N I N
N j N
N 1 N
1 N 1 N
N , N
•"""—— * ™™™™™,
N | N
- N _ , N
N N
N N '
N j -i
N 1 N
N 1 'J
N 1 N
_il 1 N
N 1 n
AM • '
. "> ,
'I 1
J 1
'* .
__•< |
1
' j
\ '
I
-T ^
r, i
r.
N [
f: i
'< i
f.. "
h .
N
f>.
N = Normal
NE = Not Eating
* Animals received from LAR, all appear N
a Animals challenged after am observations, refer to Clinical Observations
Printed By: _
QC Review- d f
-------
'Sm,};! amount of stool
'Animal found prostrate, unresponsive to touch, and gasping at 1055, died at 1113 prior
Animal found dead sfter am observations
V»t notified about NE, vet ordered weight to be taken and yogurt to be added to food
-------
Vet notified about NE, vet ordered weight to be taken and yogurt to be added to food
Ved yogurt, appears animal did not eat previous yogurt feeding, but no peitets m f
filmpaired use of right front iimb. N unless condition worwns
-------
FD s found t>ad
0 = Other |requsres
3311
"S*nst! amount of stool
Animal found prostrate, unresponsive to touf h and ea^piAj; 3! 10SS died s! 111'^ • ' t /i ' '
Vet notified about NE, vet ordered weight to be taken and vogurt to be adifcd to food
%d yogyrt, appear animal did not eat previous yogurt feeding, but no pellets n feeder, still dehydrated
-------
N = Normai
NE = tSiot CaEing
Di = Dkrrhea
SS ™ Soft Stool
NS •- fto StooJ
RA ~ Re^ptratofy Aboofmaliiies
L * Lethargk
FD s Found Dead
0 ~ Other (mquirfi'i com rsent)
Animal found prostrate, unresponsive to touch, and gasping at 1055, died at 11U prior to adr
'Animal found dead after am observations
*V« notified about NE, vet ordered weight to be taken and yogurt to be added to food
5Fed yogurt, appears animal did not eat previous yojurt feeding, but no pellets In feeder, still a
Impaired use of right front limb, N unless condition worsens
-------
APPENDIX L
INDIVIDUAL BODY WEIGHTS
L-1
-------
107S-CG920794
Body Weights
10/20/2010
Challenge
Order
f
!
i
1 i
1 I
s
i
i
I
Animal ID
j i
i
] i
t
I-
t
1
' 1 -•'-}
Sex Group
iV* i
M f 1
_ i f
V i
M ' ,'
M J
M } - "
M f
i „. i _ „
M '
M T
M 1 '
M j 1
- M i. _
M I i
M , ,
M r <
M 1
M f ^
M
M f 4
M j ,
M |
M I
M * ,
Stud
"" ^'2 10
/ ^ ^ ilO
^' ?l< 40
"•• no
i -< 2 no
/'" i 10
i '/nio
7 *11G
/ , "110
7 ^c .. 110
/'2~ '110
/ .. ^ . i 10
2' 110
°V- UlO
~j '">on
/' - .CIO
~i > 2 10
^-,'2t 10
7 2 ' -10
/ 7" ilO
y Day 2
Weight
3,13
2.89
2.6S
2.46
2.68
2.75
2.89
2,85
2.81
2.55
2 79
2.55
2,55
2.93
2.79
2 72
3,12
2.86
2.63
2.59
2.75
kg)
2.86
2.90
Stud
8/4/2010
8/4/2010
8/4/2010
8/4/2010
8/4/2010
8/4/2010
8/4/2010
8/4/2010
8/4/2010
8/4/2010
8/4/2010
8/4/2010
8/4/2010
8/4/2010
8/4/2010
8/4/2010
8/4/2010
8/4/2010
8/4/2010
8/4/2010
8/4/2010
8/4/2010
8/4/2010
yDay9
3.16
2.94
2,80
2,47
2.75
2.81
2.95
2.91
2.87
2.94
2.66
2.65
2.63
2.96
2.86
3.19
2.77
2.66
2.71
2.77
2.95
2.89
2.95
Stud
8/11/2010
8/11/2010
8/11/2010
8/11/2010
8/11/2010
8/11/2010
8/11/2010
8/11/2010
8/11/2010
8/11/2010
8/11/2010
8/11/2010
8/11/2010
8/11/2010
8/11/2010
8/11/2010
8/11/2010
8/11/2010
8/11/2010
8/11/2010
i Day 16
Weight (kg)
3.22
3,05
2.85
2.60
2.77
2.91
2.98
3.02
2.93
3.00
2.80
2.56
2.79
3,06
2.94
3 27
2.76
299
3 00
3.02
Date
8/18/2010
8/18/2010
8/18/2010
8/18/201C
8/18/2010
8/18/2010
8/18/2010
8/18/2010
8/18/201C
8/18/201C
8/18/2010
8/18/201C
8/18/201C
8/18/2010
8/18/2010
8/18/2010
8/18/2010
3/18/2010
8/18/2010
8/18/2010
Weight {kgj
3 23
2 08
2 90
3.10
2 60
2 80
2 98
2 95
3,07
2.94
3 05
2 33
2.70
3 05
2.95
3 31
3.06
3,03
2.72
Study
Date
8/2S/2010
8/25/2010
8/25/2010
8/25/201C
8/25/2010
8/25/2010
8/2S/2010
8/25/2010
8/25/2010
8/25/2010
8/25/2010
8/25/2010
8/25/2010
8/25/2010
8/25/2010
8/25/2010
F'2 .'2010
8/25/2010
8/25/2010
8/25/2010
Weight (kg)
3.2S
3.00
2 OQ
__JL06__
2.68
2.86
3.03
3.01
3.09
3.06
2,88
3.06
3.09
3.01
? ', i
5 Ji
^—^
^-^
_-^='
3.11
3.08
2.72
Stud
Date
9/1/2010
9/1/2010
9/1/2010
9/1/2010
9/1/2010
9/1/2010
9/1/2010
9/1/2010
9/1/2010
9/1/2010
9/1/2010
9/1/2010
9/l/201o]
9/1/2010
,^H
9/1/2010
_____
* u '01 J1
' 1 . 'ID
^
~f~~^
9/1/2010
9/1/2010
9/1/2010
Weight {kgj
3.30
3.0S
3.01
3,09
2.74
f 2~87~
3.04
3.06
3.18
2.96
3.09
2.91
3.08
2.79
3.11
3 03
; r
/
«~~--~~~^~~J~
3.13
3.05
2.70
Animal Deceased
-------
APPENDIX M
INDIVIVUAL MORTALITY RESULTS
1078-CG920794 - Individual Mortality Results M-1
-------
1078-CG920794
Mortality
12/15/2010
Animal ID
40
7
5
9 ,
37
13
34
25
15
30
28
19
14
11
2
8
12
18
32
6
33
27
31
39
21
38
Group ID
1
1
1
1
l
2
2
2
2
2
2
2
3
3
3
3
3
3
3
4
4
4
4
4
4
4
Challenge Date
&Time
7/26/10 9:32
7/26/10 9:46
7/26/10 10:00
7/26/10 10:11
7/26/10 10:22
7/26/10 10:55
7/26/10 11:11
7/26/10 11:22
7/26/10 11:33
7/26/10 11:48
7/26/10 11:58
7/26/10 12:09
7/26/10 12:25
7/26/10 12:36
7/26/10 12:49
7/26/10 12:59
7/26/10 13:12
7/26/10 13:28
7/26/10 13:39
7/26/10 13:53
7/26/10 14:05
7/26/10 14:16
7/26/10 14:27
7/26/10 14:36
7/26/10 14:47
7/26/10 14:57
Date & Time o
Death
9/3/10 9:09
9/3/109:27
9/3/10 9:36
9/3/109:58
9/3/10 10:17
9/3/10 10:32
9/3/10 10:50
9/3/10 10:57
9/3/10 11:10
9/3/10 11:24
9/3/10 11:33
9/3/10 12:21
9/3/10 12:34
9/3/10 12:12
8/13/10 10:39
9/3/10 12:37
9/3/10 12:51
9/3/10 13:01
9/3/10 13:08
8/6/10 11:13
8/8/10 7:57
8/16/108:26
8/10/10 8:16
9/3/10 13:16
9/3/10 13:24
9/3/10 13:31
Time to
Death (days)
39.0
39,0
39.0
39.0
39.0
39.0
39.0
39.0
39.0
39.0
39.0
39.0
39.0
39.0
17.9
39.0
39.0
39.0
39.0
10.9
12.7
20.8
14.7
38.9
38.9
38.9
Found Dead or
Moribund/Euthanized/
Survived
Survived
Survived
Survived
Survived
Survived
Survived
Survived
Survived
Survived
Survived
Survived
Survived
Survived
Survived
Found Dead
Survived
Survived
Survived
Survived
Found Dead
Found Dead
Found Dead
Found Dead
Survived
Survived
Survived
By:
QC
M-2
-------
APPENDIX N
INDIVIDUAL CIRCULATING PA ELIZA RESULTS
1078-CG920794 - Individual Pa ELIZA Results N-1
-------
9°
o
ffl
m
r^
N
BD- below limit of defection (LLOQ 4.9 ng/mL per QD-186)
results from failing plates and will be accepted per Study Director's discretion. Refer to memo titled Acceptance of failing PA ELISA results
Lineout means no sample was obtained at that timepoint.
1078-CG920794 PA Results Summary (ng/mL)
droup
s
I
I
i
i
"
I ,
">
1
1
^
—
>
*
i
"t
_! "~
>
_
i
l)a\ -3
Day 2
HI) 1 BD
BD
BD
Bl)
BD
BO
BD
BD
Hi)
HI) j
HI)
BD "
Bl)
BD"
BD 1
BD
BD
BD
BD
BD
BD
BD
BD 1
Bt) J
»D_ .r
BD 1
BD
BD
BD
BD
BD
BD
[ BD |
BD
BD
BD
BD
BD
BD
BD
BD
BD
BD
BD
BD
BD
BD
~~BD
BD
BD
BD
Day 4
BD
BD
BD
| -^—
BD
BD
BD
BD
BD
BD
BD
_BDi
BD
BD
BD
BD
BD
BD
BD
BD
BD
BD
__
BD
BD
BD
Day 9
BD
BD
BD
BD
BD
BD
BD
BD
BD
BD
BD
BD
__
BD
BD
BD
BD
BD
BD
___
BD
BD
— __
BD
BD
BD
Day 1 1
BD
BD
BD
BD
BD
BD
BD
BD
BD
BD
BD
BD
BD
BD
BD
BD
BD
BD
BD
BD
BD
BD
BD
BD
BD
Day 16
BD
BD
BD
BD
BD
BD
BD
BD
BD
BD
BD
BD
BD
BD
BD
BD
BD
BD
BD
RD
BD
BD
BD
Day 18
BD
BD
BD
BD
BD
BD
BD
BD
BD
BD
BD
Rn
BD
BD
^*^~~"
BD
BD
BD
RD
RH
BD
BD
7.669
Day 23
BD
BD
BD
BD
BD
BD
BD
BD
BD
BD
BD
o r\
BD
BD
— -— ^"^'
BD
BD
BD
Rn
^^
-^~^-
BD
BD
6.280
Day 25
BD
BD
BD
BD
BD
BD
BD
BD
BD
BD
BD
BD
BD
^---""''""^
BD
BD
BD
^^
*^~^
BD
4.967
BD
Day 30
BD
BD
BD
BD
BD
BD
BD
BD
BD
BD
BD
OU
BD
BD
^-—"""""""'
BD
BD
BD
oD
— ^^
^^-
BD
BD
BD
Day 32
BD
BD
BD
BD
BD
BD
BD
BD
BD
BD
BD
BD
BD
BD
— —-""""~*"~
BD
BD
BD
BD
^^
^5^~
BD
BD
BD
Day 37
BD
BD
BD
BD
BD
BD
BD
BD
BD
BD
BD
BD
BD
BD
^^""~""
BD
BD
BD
BD
-=^Z
-==~^Z
BD
BD
BD
Day 39
BD
BD
BD
BD
BD
Terminal
^-^
^^^
____^
BD ^-^""^l
BD
BD
BD
BD
BD
~___^-^H
r^I-----^
^^^
BD ^~~*~~
— — ™™ .^-- "^
BD ^^^J,
BD L— — - """""""
^^~---""""' !S\66S
BD
BD
BD
^-^1
^~^~
_JD_JU^^
^^^J, BD
^^^
-e£±l_^
BD i
BD
BD
^^^
()*.\\o \<.)*
^
, ")
-------
1078-CG920794 PA Results Comparison for Samples From Failing Plates (ng/mL)
i ,1 •
f
Plate ID
ww 10-001
110') 104)0 1
OiJ'010-002
_
'
^ ~ ~"
•)
H!U JO-tRH
I'M 1104)01
UN 10-004
0'>5
-------
APPENDIX O
INDIVIDUAL BACTEREMIA CULTURE RESULTS
1078-CG920794 - Individual Bacteremia Culture Results O-1
-------
9°
o
O3
Q)
Q)'
O
^m*Y^f |o Group
i 1 1
> - - —4
t ;• t 4-
•
i -1 . .
[—--H—
3
r 3
4. 3
' - i 3
— -r-t— T—
L "4_*'
f • . [_ 4
"1 4
^ ' ! 4
Day -3
_____
0
0
0
0
0
0
0
0
0
0
0, C
0
0, C
0
0
0
Day 2
0
0
0
0
0
0
0, C
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Day 4
0
0
0
0
0
0
0
0
0
0
0
0
o
0
0, C
0
0
0
0
0
Q, C
0
0
_
o
Day 9
0
0
0
0
0
0
0
o
0
1 _
0
0, C
0
0
0, C
0
0
0
0
0
0
0
0
0, C
0 C
1078-CG920794 Quantitative Bacteremia (CFU/mL)*
Day 11
0
0
0
o
0
0
0
_
0
0
0
0
o,c
0
0
0
0
0
0
0, C
Q
Day 16
0
0, C
0
o
0
0
0
0
0
0
0
_
0, C
0
0
0
0
J. .'., (L^SX.
0, C
Day 18
0
0
0
0
0
0
0
0
0, C
0
0, C
0
0
0
0
5W%f?t
0, C
Day 23
0
0
0
0
0
o
0
0
0
0
0
0
0
0
'"**.•?* A*"!1'
te..v**-:ft.'
0
Day 25
0
0
0
0
0, C
0
o
0
0
0
o
0
0
0
0
.^yte*.*.
0
Day 30
Q
Q
0
0
0
0
0
0
0
0
0
0
0
0
0
o
.-s.v^v:
0 |
0
Day 32
0
0
0
0
0
o
0
1 o
0
0
0
0
0
0
o
0
0
•?*.$%?;•.•
o |
o
Day 37
0
0
0
0
0
0
0
o
0
0
o
0
0
0
0
0
o
^M^:
o
0
Day 39
0
0
0
0
0
o
0
0
0 j
0
0
o
0
0
0
0
,V4»"w^|;p"
*3h&&*
0
0
0
Terminal
j^.i^HMII mffl^S^J
Unm'iyn.ir^Nil .t.lfa
V ~'v-' >..''
v ,|Vf;w
im^miniiii^g..!^....^^*..
0
4.13E+OS
2 60E+03
4.00E4-01
* in brackets indicate results derived from mean colony counts outside of the countable range (25-250 colonies)
0 = for fl. Anthracis
C = indicates presence of organism other than 8, anthrads
Printed By/Date:
QC/Tech Review By/Date:
j -2S- 1 1
-------
APPENDIX P
INDIVIDUAL BACTEREMIA QPCR RESULTS
-------
1078-CG920794
PCR Result Table *
r\i
CL
Group \nimal II). Da> -3
— --)•—--
1 9 iog
< 10 log
, ,n J J°°-_
15 J_ Log
19 , 1 OQ
2 25 I log
>() j ~ | o 2 J Dav 4 _!_ Day 9
} log 4 LOQ | LOQ
-log
iog
i i og - LOQ
IOQ
-iog
;) og
1 fog | i < >Q
log
I -i-°Q
1 LOQ
- 1 og
log
i og
L log -iog
-i og T -LOQ
— _-*•)_ — i.
• I OQ
iog
iog
-log
-iog
-I OQ
-i og
Log
IOQ_J Log
I og ,
--I og
IOQ
I OQ
log
iog
-i og
i og
--i og
-Log
-•I og Log
- -4 — — -
iog.
i og
log
-i og
-log
i og
Log
Log
LOQ
LOQ
-Log
LOQ
LOQ
LOQ
-LOQ
Dtt\ >9 Icintin.jl
i OQ j < i og log,,,--'-""""
i og] t og ; -i og i -— """"
-Ig 1^-- "'
^^\ ^~^\ ^>f=-:-
i^ToQ -log^i og"4*^! o()1 ,--"'-'
-I OQ
-log
-'LOQ
- Log
-i og i - 1 OQ 1 - 1 ( >g r -- •""
- log iog i oo , , -
iog - og • ~ogr~-- ""
-10O! LOO -lool ,-•""""
, i _-_( _ 1 •> - "V U^- 1
i og 1 -i og -loo i oo i _---*
^^^"
-i og
^^v
^
^^^l^^'^^^' -i(K)
- i og j - og f i oo I , - """*"
^~^ ^^" ^••^'i h r\ IH
^"^^r'^^^" -M,
Q Tj~---~"**~
*AI! rssuits are in copies/|4L
**See memo dated 8/25/10
MS-Ho sample
s of quantification
1 of 1
Printb i
-------
APPENDIX Q
INDIVIDUAL TNA RESULTS
Q-1
-------
1078-CG920794 TNA RL
Plate ID Information
JA
NA
NA
092810-116
NA
NA
NA
NA
NA
091310-906
AGN
NA
NA
NA
JA
092810-113
091310-905
NA
NA
NA
NA
NA
091310-905
092810-115
NA
NA
NA
NA
AGN
NA
AGN
092810-112
092810-115
a
QDSQ LOD=23
Printed By:
1 of 6
QC/Tech Review by:
\\\! 3-
-------
\iiiin«l ID Intoimaiinn
\ti i nni i i1 i\\ s
amp i »
1 . ' " i - f , 1 '
f- •- , ' -! 2— 5"_"1
L r ". ' ,_ f',L _
I _ "J"LJ . . 1
L - JL z 1 4 . L * '
1- ' 4 " V J '
- ^ -' * -"-i _
, - J_' .: 4^ '^ \..
' -,. ''J ' 4 .'' .T .
*" "" 1 , • 1 U i
J--»_r' «."<• • _
i, 11 1 L'I '
< ,"<, 1 T ,/1
i ~ i r
i
L ' V1 , t _•'•-_
- 1 '_j . "'i ..
- - „ L " * -',""'
' - 4 - n' -, -''-j.' _
> - 1 •_• 1 " ' • .< -
_, 'll» ' . . ''J1 J . .
' -I. '1. ... '" 'J~
J '^1 4- "'.*''
-t ' ; I- ''I*
( _j ' , 0 1
• _! r ° *
1 ' 'A- " _l ' 1 ' _t
' T , , * j r i , •
" » 'i- " 4 ( • '
1 „• ' ^ '' f -1-
« , ^ t , «. i ,(
'- - .. 1'Ai,-- 1
, c _/ '' 1 *'fl j; '' _J
' , I-i. .1 •- l , 1.
L » ' 'i-' |. ' - j_
[ i , I 1, i ,,
'• , ( .' " 1 L •
t 1 ' - '-L ' -
>- 1 -, "J-'T •'_!>.,«
, - 4^ i1 r ""'.<• .
. 1 .,„ j- A -' .. .
1-1 ^'-. . L -!--' -
1 ' ,1, T -t ^ 1
.. '. " ".. ']_: • j. .
w -'- r '^' J C.J.
t 'J I L , %X™*'C '• ' ~"
•' I --... 1 '•»' i/. _
"- J_ j 4 HL>. * j
- "' 4 l 'x, I _'-! ."" .,
} 'L 1__. .' ^n,,,, J
[ '- 1 ' .4 1 ,41 i, 1
Test 1 Test 2
NFSO NFSO
0 0
, 0 0
0 0
P l
^ t
r \
11 ij
II
j(
!
v t J
^ 1
^
l,O
" J
C
J J
u< 0
v
ji i
1
n
"> V.
1 5
0 n
i, l
i. O
v i
ir^"
' 1
I u"T
^ tJ1
r, 7
' i,0
T -Q
v ^}
1 j 3
! T
Std Dev-
NFSO
0.000
0.000
0.000
0.028
0.000
0,000
0.000
o.ooo
0.000
0.000
0.000
0.000
0.000
o.ooo
0.000
0.000
0.000
0.000
0,000
0,000
0.000
0.000
0.000
0.000
0,000
0.000
0.000
0.000
0.000
0.000
o.ooo
0.000
0.000
0.000
0.000
0.000
o.ooo
o.ooo
0.000
0.000
o.ooo
o.ooo
0.000
0.000
0.000
0.000
0.000
0.000
o.ooo
0.000
0.000
o.ooo
0.000
0.013
0.000
0.000
0.000
0.000
0.000
%CV NFSO
0%
0%
0%
173%
0%
0%
0%
0%
0%
0%
0%
0%
0%
0%
0%
0%
0%
0%
0%
0%
0%
0%
0%
0%
0%
0%
0%
0%
0%
0%
0%
0%
0%
0%
0%
0%
0%
0%
0%
0%
0%
0%
0%
0%
0%
0%
0%
0%
0%
0%
0%
0%
0%
173%
0%
0%
0%
0%
0%
Median NFSO
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0,000
0,000
0,000
0,000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0,000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0,000
0.000
0.000
0.000
0.000
0,000
0,000
0.000
0.000
0.000
0.000
0,000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0,000
0,000
0,000
o.ooo
0,000
1078-CG920794 TNA
CO
6
QDSO LOD=23
Printed By:
QC/Tech Review by:
il/v ,MH1
-------
1078-CG02U794 TNA PI -•(
Animal ID Information
\ntnul III 1 HIM Point 1 N \ taiiink Illi
\IHlMt
, - —i J ._ - ^] ,A~!
<.] ?' 1 i\ , . j JA
' n, * 1 1" 1 VN
r " ..-, - i ' I' , , A'.N
1 - j 1 T 'o'1 " j A N
>- - j -; r ;,,-; -'-.N
|_ •« > • ~f ., t t 7' N
' , <• '0, , •' -f ;
i 1 J ^ *v J 1 * 1 , " ™ T A' '<
1" '• J, ' 1 ' ' J_/ NJ
1 ' ' » ' ' ' i O 1 «' N
^ , ' 1 ^ • • I , ' 1 l ".
' , '< 2* ' , ' A< N
, ' 1 ' j * :' _ " * £ ii • ^ ,
i. - ' 1 - —I - ' !'~ -I''*"—
-) -" t ' 1 ( ^ 7 ** N
- 1 4 - 5 - ' .'1 t' 7 '_ . / N
«. ] ^ i * 1 A' iN
.* - ^ _- * - _i . ' _
esl 1
Plate in
091310-913
091310-910
092710-034
092710-036
082310-722
10908 10-893
091310-910
082310-720
092710-035
092710-037
082310-723
082310-725
091310-910
082310-720
0927104334
092710-037
OS23 10-722
082510-757
090810-894
092710-033
082310-721
[j»271 0-035
092710-037
PJ8231 0-724
091310-913
090010-894
^090810-889
- '- . ->' J -• ' i . 1 ^TM2710^36
r^*<< i 2 ! y '* l A \ fiAtmn-??1?
: ' . 1 j . ; , 1 • I ?-
V t L> 1 A.VJ
J" , ] .',> , * \
_ " ." .i- I < ' •'• *' "<
., J 1 _4 ' r 0 _^, '7 **j
_li • " L ' >V ' ' AC N
L- • _"_? 1" , • " 2- i '^
2 - J -i ' - L •"-• 1 A '•
- - _,' J ' *' fl ' NNH
- *• ' -V j • ' ' K\
»u 7 J J ' J H " '"
H5I251Q-7S7
05)0810-893
082310-721
082310-723
082310-725
J35111M1-L
091310-910
^JB^S 10-720
,092710-034
092710^036
082310-722
1 i •,, 1t i',\- A' \1_091310-913
i1 J ' ' ' " • ^_ |M ..T
P -i > i " ~, ' A"7]
--' ! -J 1 '"-'"' 4 kf'T
4' - - -» - ' ' -i ' , - ff<
l ' I1 . j Jl P v 1 ] A A
- „.- t' - r - ' [ ' : -j- J'
• . i j • L' , 1 Ar,»ri
JJ91310-910
090810-889
092710-034
092710-036
082310-722
082510-757
082310-723
> '' " L ', " T U "1 082510-757
"'_,«"" 4 l ^ " A SJ
,' J •" _ -*' ' i * 1 K''IJ
|" > 4. ' -1 1 ""-^
1,1 1 i ' j kN
k .. 'i ; 11, r"i
"/ V4 r,4™[A.N
090810-893
092710-033
082310-720
092710-035
092710-037
082310-723
Plate ID Information
Analyst
JA
AGN
JA
JA
JA
JA
AGN
JA
JA
JA
JA
AGN
JA
JA
JA
JA
JA
JA
JA
JA
JA
JA
JA
JA
JA
JA
AGN
KN
JA
Test 2
Plate ID
092710-044
091310-914
092710-040
09271 0-042
092710-045
091310-914
082510-751
092710-041
092710-043
082510-754
082510-756
091310-914
082510-751
092710-040
092710-043
082510-753
091310-909
092710-039
082510-752
092710-041
092710-043
082510-755
092710-044
091310-904
092710-040
092710-042
082510-760
092710-038
["08251 0-752
JA | 082510-754
JA "'
JA
AGN
JA
JA
JA
JA
NA
AGN
JA
JA
JA
JA
AGN
JA
AGN
JA
JA
JA
JA
JA
JA
082510-756
092710-045
091310-914
082510-751
092710-040
092710-042
082510-753
NA
091310-914
091310-904
092710-040
092710-042
082510-753
082510-780
082510-754
082510-780
092710-045
092710-039
082510-751
092710-041
092710-043
082510-754
Analyst
NA
NA |
KN
KN
AGN
NA
AGN
KN
KN
NA
NA
NA
AGN
KN
KN
NA
NA
AGN
JA
KN
KN
NA
NA
NA
KN
KN
NA
KN
JA
NA
NA
AGN
NA
AGN
KN
KN
NA
NA
NA
NA
KN
KN
NA
NA
NA
NA
AGN
AGN
AGN
KN
KN
NA
Tests
Plate II)
NA
NA
092810-112
092810-114
092810-083
NA
090810-890
092810-113
092810-115
NA
NA
NA
090810-890
092810-112
Ud2o10-115
NA
NA
092810-077
091 31 0-906
092810-113
092810-115
NA
NA
NA
092810-112
092810-114
NA
092810-116
091310-906
NA
NA
092810-083
NA
090810-890
092810-112
092810-114
NA
NA
NA
NA
092810-112
092810-114
NA
NA
NA
NA
092810-083
092810-077
090810-890
092810-113
092810-115
NA
Test 4
NA
NA
NA
NA
NA
NA
JA
NA
NA
NA
NA
NA
JA
NA
NA
NA
NA
KN
AGN
NA
NA
NA
NA
NA
NA
NA
NA
NA
AGN
NA
NA
NA
NA
JA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
KN
JA
NA
NA
NA
Plate ID
NA
NA
NA
NA
NA
NA
091310-905
NA
NA
NA
NA
NA
091310-905
NA
NA
NA
NA
092810-111
091310-912
NA
NA
NA
NA
NA
NA
NA
NA
NA
091310-912
NA
NA
NA
NA
091310-905
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
092810-111
091310-905
NA
NA
NA
F.D50
Test I
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
o
0
0
o
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Test 2
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
o
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
NA
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Test "!
NA t.\ i , ;
NA I NA i I r t
NA i '.A i f
o
o
0
NA
0
0
0
NA
NA
NA
0
0
c
NA
NA
~ t »• - -+ - '
--£--- -: :--. '':: :
r A , j , >
- -j - .. J ^
^" l.o 1 ~ o ' ' - ' 1 j
NA I i | i i, 1
~ "MA" J I"'-,'
NA ]_ i ,
VV 4 - L . . '•'
—. j ,
NA ~* J r ~" ,
N ' ~*~ " I
. ri/i 1
0 t - - - '
0 i T "
" o t -*\" " -, i - * , -
0 1 "\A ' . - ' , - ! '
NA ] NA T ' '" " ! . '
NA 1 N, ~ i -r - -- -j
NA \A , . 1 - ' (.
NA 1 N", ) ' , [ ]
C 1 N'-, . i .' I "- , "
C 1 -.A "* ' - = ' ' -I f -
NA | NA I ' , I i ' l i i
NA \A j
o , ] r ""'""'", ' ,
NA NA t "''',, 1
Nft
C
NA
C
C
C
NA
NA
NA
NA
C
0
NA
J" l ' "i
r,A ' 4 " ' 1 ' " ' '
NA "
_^_ i . - ^ .
NA" * ~ v :j* " r
NA ~* J "" ' "1 i, ,
K , -"'-,•
N/\ j i '
NA 1 ~ " ' " r
NA ! NA i ] '
c T 1 "* „ *
NA | NA * r
NA j '
-------
1078-CG920794 TNA
10
a
QD50 100=23
Printed By:
4 of 6
QC/Tech Review by:
fT\H ^1-11- 1 1
-------
1078-CG920794 TNA
\ninial 10 Information
Plate ID Information
ED50
\nimil II> I nnt I o'nl IN V
Analyst
NA
:
t.
- 4 -
AGN
J
}
4
NA
L_
KN
8440.374079
4508.651414
AGN
12769.078381
15258.337052
7249.765087
092810-111
091310-912
NA
a
QD50 LOD=23
Printed B,
5 of 6
QC/Tech Review by:
-------
\nimal ID Information
j IN\ Sample IDi
NFSO
1 " _*_ _
4—
1-
::
^ j
4 -' J
4 r • '
7 i o7
l< ,4_ I
J" „ _ _
* L
_
_NA_
NA
32,880
J
22,235
_NA_
_NA_
_NA_
NA"
_NA_
_JL_
_£_
NA
JIA_
_NA_
NA
_NA_
NA
14,679
NA
_NA_
NA
_NA_
"~NA
0,000
0,000
Std Dev
NFSO
0.000
0.000
0.000
3.996
0.000
4.571
0,000
0%
Median NFSO
0.000
0.000
0.000
0,000
14.819
0.000
0.000
1078-CG920794 TNA
a
QD50 LOD=23
Printed By:
6 of 6
QG/Tech Review by:
P)U I '1 - i 1 - < !
-------
APPENDIX R
INDIVIDUAL ANTI-PA IGG ELISA RESULTS
R-1
-------
1Q78-CG920794 IgG ELISA
1078-CG920794 IgG ELISA Reportable Values fue/mU
Animal
ID
40
/
5
9
o /
13
34
25
15
30
19
14
11
*L
0
12
18
32
6
33
2. /
31
39
21
38
Group
1*
1*
1*
2
z.
z
£.
£~
2
3
3
3
o
3
o
u
4
4
4
4
4
4
4
Spore Dose
(CPU)
10,000*
10,000*
10,000*
10,000*
10,000*
100
100
100
100
100
100
100
1,000
1,000
1,000
| ^QQQ
1,000
1,000
1,000
10,000
10,000
10,000
10,000
10,000
10,000
10,000
Day -3
-------
APPENDIX S
INDIVIDUAL HEMATOLOGY RESULTS
S-1
-------
Study 1078-CG920794
Hematoiogy
Parameter Group J Animal ID
White Blood Ceil Count 1
(2.9-S.lAlO'M) l 1
1
1
2
2
]
1 2
2
_ j 2
2
37
40
5
7
9
Average
Std Dev
13
15
19
25
28
30
Day
8.35
5.39
7.59
4.48
4.16
5.99
1.88
7.66
6.75
1 7.92
6.16
S.14
6.24
34 ~]~9-26
AverageJ 7,02
j Std Dev
3
" " " . i 1
3
3
3
3
3
4
4
f 4
4
4
4
4
11
12
14
18
2
32
Average
Std Dev
1.37
7.09
4.62
6.28
8.40
5.22
6.07
-3
6.30 L
6.28
1.23
21
27
31
33
38
39
6
Average
Std Dev
8.37
7.54
6.44
4.17
7.81
9.15
8.00
7.35
1.63
Day 2
6.78
5.01
7.49
6.71
4.91
6.18
1.16
8.34
8.11
7.63
8.00
4.97
5.22
8.62
7.27
1.52
6.41
4.66
6.58
4.44
4.73
4.80
6.12
5.39
0.93
5.72
7.65
,7**
1 4.29
6.91
10.63
7.90
. ,47-23
| 1.97
Day 4
QNS
5.27
7.63
1.60
5.89
5.10
2.54
9,7CT
8.27
*
7.03
7.70
5.53
5.81
7.64
7.38
1.43
6.78
3.66
7.27
5.07
5.19
5.00
5.46
5.49
1.20
5.98
QNS
QNS
QNS
7.84
9.12
10.73
8.42
2.01
*
Day 9
4.54
4.73
9.54
6.57
*
*
4.76
6.05
2.17
9.39
7.58
~
7.40
8.00
5.37
6.47
7.95
7.45
1.27
6.32
4.66
7.33
5.01
QNS
5.69
6.59
5.93
1.01
6.46
8.76
5.59
5.28
6.99
7.09
8.01
6.88
—
1.24
Day 11 Day 16
5.45 5 17
5.72 1 QNS
7.46
6.66
4.53
5.96
1.13
7.53
7.90
7.80
—
6.69J__
8.75
5.41
6.76
7.86
7.27
1.081
5.78
QNS
7.65
6.67
4.82
5.65
8.08
6.44
1.26
6.69
10.00
6.43
2.94
7.85
8.20
7.02 ~
2.37
—
*
6.86
5.39
6.31
1.25
8.66
7.00
6.54
8.89
5.64
6.36
6.08
7.02
1.27
6.56
4.95
7.00
QNS
4.64
QNS
6.50
5,93
1.06
5.62
8.60
-
-
8.30
i
7.02
7.39
1.36
*
-
Day 18 ) Day 23 t Day 25 : Day 30 Day 32 Day 37
474 QNS QNS 6.93 . (INS ' 6,?'t
529
8.59
6.89
5.43
6.19
1.56
7.69
7.75
4,97
9.85
6.71
6.24
6.65
7.12
1.53
6.88
4.21
7.93
6.08
4.19
7.02
6.05
1.55
8.62
10.25
—
9.88
8.79
9.39
0.80
* ^ 3 12 \ ', 4.5b 3.84 ' ' QNS 5 bis
' 8.73 | 7.86 i J 8.49 ' QNS 8.97
6.59 ; QNS ' b.72 651 b 85
1 5.74 \ , fa.09 5.38 ' ' QNS ' 5 M '
; 6.05 , 6.17 ' ' 5 1J ; /./8 ' b 9i
i " , i
(
i .. i .
' l "
20.33 ,18.08s ,15.09, 11.01 S '>«)
8.95 9.74 9.80 ' 8 Od ' 8.92
] '
, ~ 1 -
14.64 i 13 91 10 01 B 96 r, <<4
J 8.05 4 ! 5.90 | , 4.98 1 78 ' 1.7J
r\i
th
Bold: > Normal Range
Underline; < Normal Range
— No Value
* Clot Removed
QMS - Sample Volume not Sufficient for analysis
Page 1 of 2"2
-------
Stydy 1078-CG920794
Hematology
Parameter Group
Red Blood Celi Count 1
(4.2-6.7xiOA6/uL) 1
Animal ID
37
40
: I_L 5 _
i
i
r j
i 2
2
2
2
1 2
Day -3
4.99
5-44l
5.91
7 5.20
9 T6.46
Average T 5.60
Std Dev 0.59
13 I 5.96
15 T 6.26
19 fs.58
25 [6.33
30
.....; 2_ 34
Average
Std Dev
3
3
3
1 3
3
3
3
,
4
4
4
4
I 4
4
4
[
11
_ _ „__
14
18
2
_ 32
8
Average
Std Dev
21
27
31
33
5.71
5.85
6.23
5.99
0.29
5.51
Day 2
4.80
5.57
5.81
5.32
6.11
0.50
5.81
6.34
5.83
6.12
5.79
5.85
5.431
5.87
6.47J
5.86J
0.35
5.45
5.59
5.74
""5.18
38 6.51
39 | 6.34
6 1 6.13
Average
Std Dev
5.85
0.49
«^ — ! i j
6.52
5.65
5.63
6.06
5.98
Day 4
QNS
5.33
5.47
4.11
5.55
5.12
0.68
5.67
5.96
0.34
5.67
5.66
5.69 |
5.39
5.54
5.81
6.32
5.73
0.29
5.40
5.58
6.21
5.10
5.95
5.30
6.11
5.66
0.43
5.50
6.43
5.46
5.90
Day 9
4.79
5.55
5.94
* 4.96
6.01
5.45
0.56
5.91
5.83
0.33
5.75
5.30
5.36
5.29
^
5.56
5.73
6.01
5.66
6.31
*
*
Day 11
5.29
5.58
5.66
4.95
| 6.38
1 5.57
J0.53
1
~|~5.74~
5.75
5.54
6.021
5.86]
0.27J
5.48
5.80
5.49
5.56
QNS
5.65 5.52
6.25
5.59
0.34
5.62
QNS
QNS
QNS~
5.88
5.49
5.99
5.75
0.23
I 6.11
5.66
j 0.25
j 5.48
5.42
4.94
5.75
5.79
7.34
5.78
5.93
5.55
7.12
Day 16
4.77 "
QNS
6.00
5.16
6.22
5.54
0.69
I
5.48
5.87
T5.55
5.58
5.78
5.82
5.93
0.54
5.43
QNS
5.78 1_
5.34
5.08
^
5.43
6.13
5.53
| 0.37
[
0.75
i
5.39
5.54
5.57
4.77
5.83
5.65
-
5.46
5.97
5.77
5.80
5.51
5.71
0.19
f"
5.87
6.27 '
6.23
QNS
5.34
QNS !
6.47
6.04"
To-45
i
5.76
5.86
1
— 1 —
*
0.37
5.98
5.94
—
5.89
0.10
Day 18 Day
' 4.63 QNS
5.55 * 5.78
' 6.02 6.11
5.16
5.96
5.46
0.58
5.48
6.19
' 5.53
f 6.08
5.93
584 '
5.41
5.78
0.31
1
5.80
, 5.97
6.17
t 5.24
5.51
6.49
5.86 "
0.45
5.51
5.83
, 5.90
, 6.12 '
j
5.84
0.25 '
[ '
5.37
6.37
' 5.91
0.43
QNS
6.28
5.68
6.83
5.89
588
5.70
6.04
0.44
5.89
6.25
5.96
QNS
QNS
QNS
6.03
0.19
QNS
4.69
6.20
5.4b
1.07
23 Day 25 Day 30 t Day 32 Day 37
QNS , 4. tl QNS 4«0
6 08 b 91 , * ' QNS * 5 38
1 6 54 , 5 75 QNS ' o _V
QNS | 5.4J S 45 5 81
7.08 i < b,26 QNS ' ^ 6 Vi
6.57 j S.bl ' S.4S ' S.i;[-- '
5.90 > 5. S3 ' 5.40 S M
j 5.74 [ 5.69 ' ' QNS b 14
' 5.81 | S.79 606 j S 'H
6.06 5 96 b /6 ti 11
038 0 39 t Q 3^ O..'/
5.84 ' b 83 b ^2 ' S \r>
6.25 i ' QNS i b.20 , Qf.S
S.86 | 6 34 ! 5..SO " Chi
5.46 i b 40 ! QNS " 5 /'j
T ]
5 57 ! f QNS | •> 52 ' b fc-S
' 6.1.' j 1 QNS 6 2J n41
5.85 , ' 5 S6 S./S ' ' 51U
0.30 | 04/ 043 0 U
QNS 5.60 S •;/ , S $<
i i
i
4.80 I S 40 ' S 08 S 40
6 08 , 6 34 ' S 69 . 6. .' 5
1
b44 5/8 S45 ; ,S ,-2
0.91 0 50 0 j2 > '04?
Bold: > Normal Range
Underline: < Normal Range
- No Value
* Clot Removed
QNS - Sample Volume not Sufficient for analysis
CO
th
Page 2 of 22
1C)
-------
Study 1078-CG920794
Hematology
Parameter j Group
Hemoglobin 1
(9.5- 14.5 g/dL) i 1
1
1
1
j - -
i 2
i 2
' 2
2
2
2
2
1
1
Animal ID
37 1
40
5
7
9
Average
Std Dev
15
Day -3 Day 2 Day 4 J Day 9 I Day 11
10.5
__
11.8
10.8~
10.2
11.8
11.7
11.1
13.3 1 12.6
iHT ^^
1.1
| 0.9
12.7 12.3
12.9 1 j 13.0
19 10.8 11.3
28
30
34 ~|
Average
Std Dev
l 3 _!_ U
! ,
! 3
\ 3
- . j ...
i 3
1 3
|
r
12
14
18
2
32
8
Average
Std Dev
12.7
12.0
13.2
J12.0
12.0 [ll.7
12.2
12.2
0.7
12.2
12.1
12.1
| 12.2
^0.7
|
12.5
VLB
12.3~| 12.1
11.9
11.4
12.0
12.7
12.1
0.4
.. t _ 1 ]
i 4 • 21
! 4
< 4
4
4
4
27
31
33
38
39
4 6
1 Average
*
Std Dev
11.1
11.7
1 11.9
[12.5
11.9
0.5
11.7 11.8
11.6 11.6
11.6
10.5
13.7
12.5
HT
12.6
10.5
12.3
10.4
12.4
12.0 11.7
1.0
QNS
11.3
10.9
8.6
11.3
10.5
1.3
12.1
12.2
10.6
12.8
11.4
12.2
11.8
—
0.9
11.9
0.7
12.4
11.0
11.4
10.9
11.8
11.4
12.3
11.6
0.6
12.1
QNS
QNS
QNS
12.1
10.9
12.0
11.8
0.6
i 10.3
11.8
12.0
* 10.5
12.3
11.4
0.9
12.2
12.4
11.0
12.6
12.0
11.4
12.0
11.9
0.6
12.1
* 12.0
11.7
11.5
QNS
11.4
12.2
11.8
0.3
11.7
11.3
11.7
10.1
11.7
11.6
15.2
11.9
1.6
*jTl.3
*
11.9
Tl.4
10.4
(Ts.s
-
11.7
1.1
12.1
12.1
(10.9
14.7
_
_
11.6
12.0
11.6
12.1
12.1
QNS
12.2
11.1
10.8
11.2
12.1
11.6
0.6
11.7
11.6
11.4
9.8
11.8
11.3
11.3
0.7
Dayl
QNS
10.9
—
12.8
11.5
1.2
11.9
12.0
10.9
12.1
12. l"
11.9
11.3
11.7
0.5
__ -
*
z
6 Day
* 9.9
11.7
12.1
10.8
12.2
11.3
1.0
jll.7
12.7
10.7
-1
12.2
12.4
12.1
10.8
' 11.8
0.8
12.9 | 12.8
12.7
13.3
QNS
11.4
QNS
12.8
12.6
18 Day 23 Day 25 Day 30 Day 32 ; Day 3
' QNS , QNS 9.9 ' QNS 10 4 '
* ' 12.2 , ' 12.6 12.5 ' QNS !.'.;.
12.4 i ; 12.8 , , ll.b QNS V $
! 113 QNS ( 11.4 ^ 115 ( 1M
13.0 , 14.4 ' 12.8 . QNS ' 15.2
12.2 ' 133 | ll.fa 11.5 120
1 0. 7 1.0 ' 1 1 ' ,00 10
j QNS ^ QNS ' 12.0 ' QMS l.l S
12.8 • 12.8 , 13.1 • QNS 12.? '
11.1 ' ' 11.4 ' 11 2 * ' 11 3 ' 11 •' '
i 13.6 j , 133 | 12 y | QNS > 13 J
! 124 ' 12 ? 'l20 ' 11 3 ' 12.0
12.2 j 11.9 ' 11.6 QNS 12,4
11.6 , 11.7 , 11. S '' 12.1 i li.S
! 123 ^ j 12.2 ; > 12.0 : 11 b L> i
\ 0.9 ' 0.7 i 07 0.5 0 ^
12.8 12 8 ' 12.6 11 7 12 8
12.1 ] 12.6 ' 12 /' QNS , 12.4 QNS
13.0 12.7 12 3 ' ; 134 ' 116 QNS
10.6
•""•
11.3
12.6
12.1
0.7 0.9
12.5
12.2
-
—
12 2
11.8
-
12.2
0.3
Ti'i
118
120
11.9
•— -
0.2
: QNS _ 11.; ; w 9 ' ' QNS 11 3
! QNS 11 5 ; ' QNS , ,113 ll.b
i QNS 12.1 ' QNS ' 12 0 12.4 '
' 12.7 ' 12 1 i ' 123 11 8 12. /
0.1 O.b , 13 04 lUi
_ QNS QNS ' 11.9 ' 11 7 114
1 " , ' '
i 9 1 93 10 4 ') « 10 1
1? 3 , 12 1 ' 12 5 iH 12 ,'
10 / 10 / lib' 10 9 11.4
1 2.3 2.0 ! 1 1.1 I " 1.0 1.1
Bold: > Normal Range
Underline: < Normal Range
— No Value
* Clot Removed
QNS - Sample Volume not Sufficient for analysis
Page 3 of 22
-------
1Q78-CG920794 IgG ELISA
1078-CG920794 IgG ELISA Reportable Values fue/mU
Animal
ID
40
/
5
9
o /
13
34
25
15
30
19
14
11
*L
0
12
18
32
6
33
2. /
31
39
21
38
Group
1*
1*
1*
2
z.
z
£.
£~
2
3
3
3
o
3
o
u
4
4
4
4
4
4
4
Spore Dose
(CPU)
10,000*
10,000*
10,000*
10,000*
10,000*
100
100
100
100
100
100
100
1,000
1,000
1,000
| ^QQQ
1,000
1,000
1,000
10,000
10,000
10,000
10,000
10,000
10,000
10,000
Day -3
-------
Study 1078-CG920794
Hematology
Parameter j Group
Hemoglobin 1
(9.5- 14.5 g/dL) i 1
1
1
1
j - -
i 2
i 2
' 2
2
2
2
2
1
1
Animal ID
37 1
40
5
7
9
Average
Std Dev
15
Day -3 Day 2 Day 4 J Day 9 I Day 11
10.5
__
11.8
10.8~
10.2
11.8
11.7
11.1
13.3 1 12.6
iHT ^^
1.1
| 0.9
12.7 12.3
12.9 1 j 13.0
19 10.8 11.3
28
30
34 ~|
Average
Std Dev
l 3 _!_ U
! ,
! 3
\ 3
- . j ...
i 3
1 3
|
r
12
14
18
2
32
8
Average
Std Dev
12.7
12.0
13.2
J12.0
12.0 [ll.7
12.2
12.2
0.7
12.2
12.1
12.1
| 12.2
^0.7
|
12.5
VLB
12.3~| 12.1
11.9
11.4
12.0
12.7
12.1
0.4
.. t _ 1 ]
i 4 • 21
! 4
< 4
4
4
4
27
31
33
38
39
4 6
1 Average
*
Std Dev
11.1
11.7
1 11.9
[12.5
11.9
0.5
11.7 11.8
11.6 11.6
11.6
10.5
13.7
12.5
HT
12.6
10.5
12.3
10.4
12.4
12.0 11.7
1.0
QNS
11.3
10.9
8.6
11.3
10.5
1.3
12.1
12.2
10.6
12.8
11.4
12.2
11.8
—
0.9
11.9
0.7
12.4
11.0
11.4
10.9
11.8
11.4
12.3
11.6
0.6
12.1
QNS
QNS
QNS
12.1
10.9
12.0
11.8
0.6
i 10.3
11.8
12.0
* 10.5
12.3
11.4
0.9
12.2
12.4
11.0
12.6
12.0
11.4
12.0
11.9
0.6
12.1
* 12.0
11.7
11.5
QNS
11.4
12.2
11.8
0.3
11.7
11.3
11.7
10.1
11.7
11.6
15.2
11.9
1.6
*jTl.3
*
11.9
Tl.4
10.4
(Ts.s
-
11.7
1.1
12.1
12.1
(10.9
14.7
_
_
11.6
12.0
11.6
12.1
12.1
QNS
12.2
11.1
10.8
11.2
12.1
11.6
0.6
11.7
11.6
11.4
9.8
11.8
11.3
11.3
0.7
Dayl
QNS
10.9
—
12.8
11.5
1.2
11.9
12.0
10.9
12.1
12. l"
11.9
11.3
11.7
0.5
__ -
*
z
6 Day
* 9.9
11.7
12.1
10.8
12.2
11.3
1.0
jll.7
12.7
10.7
-1
12.2
12.4
12.1
10.8
' 11.8
0.8
12.9 | 12.8
12.7
13.3
QNS
11.4
QNS
12.8
12.6
18 Day 23 Day 25 Day 30 Day 32 ; Day 3
' QNS , QNS 9.9 ' QNS 10 4 '
* ' 12.2 , ' 12.6 12.5 ' QNS !.'.;.
12.4 i ; 12.8 , , ll.b QNS V $
! 113 QNS ( 11.4 ^ 115 ( 1M
13.0 , 14.4 ' 12.8 . QNS ' 15.2
12.2 ' 133 | ll.fa 11.5 120
1 0. 7 1.0 ' 1 1 ' ,00 10
j QNS ^ QNS ' 12.0 ' QMS l.l S
12.8 • 12.8 , 13.1 • QNS 12.? '
11.1 ' ' 11.4 ' 11 2 * ' 11 3 ' 11 •' '
i 13.6 j , 133 | 12 y | QNS > 13 J
! 124 ' 12 ? 'l20 ' 11 3 ' 12.0
12.2 j 11.9 ' 11.6 QNS 12,4
11.6 , 11.7 , 11. S '' 12.1 i li.S
! 123 ^ j 12.2 ; > 12.0 : 11 b L> i
\ 0.9 ' 0.7 i 07 0.5 0 ^
12.8 12 8 ' 12.6 11 7 12 8
12.1 ] 12.6 ' 12 /' QNS , 12.4 QNS
13.0 12.7 12 3 ' ; 134 ' 116 QNS
10.6
•""•
11.3
12.6
12.1
0.7 0.9
12.5
12.2
-
—
12 2
11.8
-
12.2
0.3
Ti'i
118
120
11.9
•— -
0.2
: QNS _ 11.; ; w 9 ' ' QNS 11 3
! QNS 11 5 ; ' QNS , ,113 ll.b
i QNS 12.1 ' QNS ' 12 0 12.4 '
' 12.7 ' 12 1 i ' 123 11 8 12. /
0.1 O.b , 13 04 lUi
_ QNS QNS ' 11.9 ' 11 7 114
1 " , ' '
i 9 1 93 10 4 ') « 10 1
1? 3 , 12 1 ' 12 5 iH 12 ,'
10 / 10 / lib' 10 9 11.4
1 2.3 2.0 ! 1 1.1 I " 1.0 1.1
Bold: > Normal Range
Underline: < Normal Range
— No Value
* Clot Removed
QNS - Sample Volume not Sufficient for analysis
Page 3 of 22
-------
Study 1078-CG9Z0794
Hematology
Parameter t Group
Hernatotrit ! 1
(27.2 -45.9 Vu) 1
1
1
1
t
2
' 2
2
r 2
1 2
2
, 3
3
3
3
3
3
4
t 4
f 4
Animal ID
37
Day -3 [ Day 2 Day 4
32.3
40 34.4
5 1 36.6
7 j 32.3
9 J42.0
Average 35.5
^ Std Dev TTo"
f [ ~1
13 38.2
15 39.6"
19 34.6
25 40.1
-fj
36.3
37.7
34 37.9
Average 37.8
Std Dev j 1.9
1
11
12
14
18
2
32
J_
Average
Std Dev
21
27
31
, _* .33.
1 ^
4
4
38
39
6
Average
Std Dev
37.6
37,4
38.8
36.8
34.6
36.6
38.8
37.2
1.4
36.6
36.6
35.8
32.4
44.5
40.6
38.2
37.8
3.8
31.1
35.8
36.0
33.7
38.6
j 35.0
2.8
QNS
34.1
33.4
25.5
35.3
1 32.1
4.5
37.5,
j 40.7
36.1
[ 41.0
JJ35,8_
lisj"
36.8
37.7
2.3
j
t 38.7
35.0
38.4
33.2
35.8
| 36.3
j 38.1
36.5
1 2-°
36.8
36.4
39.6
32.1
39.7
34.0
38.2
36.7
2.8
36.8
37.6
33.5
40.2
34.9
39.2
36.0
36.9
2.3
39.1
31.9
35.6
32.7
35.6
35.2
36.9
35.3
2.4
37.7
QNS
QNS
QNS
38.4
34.5
37.1
36.9
1.7
1
*
*
Day 9
31.3
35.3
37.3
31,5
38.8
34.8
3.4
37.2
38.2
35.6
39.4
36.9
36.0
37.5
37.3
1.3
38.0
35.7
36.9
34.4
QNS
35.1
36.6
36.1
1.3
37.2
35.3
36.8
31.1
—
38.3
38.1
47.4
37.7
4.9
1
*
*
Day
34.8
35.3
35.3
31.6
41.5
35.7
3.6
37.4
37.6
34.6
45.9
36.1
37.9
36.4
38.0
3.7
37.6
QNS
39.2
33.5
33.5
34.6
37.4
36.0
2.4
36.6
37.1
J 36-°
30.3
38.2
36.3
—
35.8
2.8
LI 1 Day 16 Day 1
J 31-1
QNS
37.4
33.1
39.8
35.4
4.0
354
37.5
34.1
37.2
37.4
38.0
34.2
36.3 ,
1.7
39.9
38,6
41.6
QNS
35.0
QNS (
39.6
38.9
2.5
38.5
38.0 l
-
*
39.2
38.1
"38.5
*]_ 30.1
35.0
37.0
fSjf
1 37'6
31
349
' 387
, 335
37.5
37.4
37.6
32.9
1 36.1
2.3
i
38.7
' 36.1
' 40.3
32.6
34.2
38.3
136.7
, 2.9
36.0
37.5
1
37.3
39.0
37.5
1.2 ,
8 t Day 23 Day 25 Day 30 , Day 32 , Day 3
QNS QNS 30.0 QNS -50 n
* | 363 ' 37.8 ' ' 36.4 * QNS ' 36 <
36.4 , 39.9 3V3 , QNS */ -i
34 0 QNS _ 34.t> • , 34.5 ' <5 :-
23 32 ] 33 i 0.0 ' 3'.
QNS ! QNS , 35.7 , QNS ," -i
388 i 41.0 ' 403 QN.s ' JH '
343 34.8 [ " 35.1 * 34 i> ' ib 4
] 42.1 41.7 ' 41.3 QNS , " 41 .'
37.2 373 j | Bb.b '" ' ii 5 370 "
37.4 363 ] t 35.8 _ QNS iS ?
^ 34.6 35.1 i Vvj < 363 . 55 V
37.4 37 7 ' W.: ' 34 & ' ^ S
29 3.0 , .' 5 1.4 , 1 g
' 39.4 39 9 ' 3<-X9 tt 7 ' *•) S '
37.8 37 4 ' QNS i/ 0 , QNS
38 6 38 3 42 S ! % S QNS
QNS 33 b ^3 > i QNS i jiS 4
i \ , ' i
QNS , 34 3 ' QNS ' 3-< ^ ^4 -'
QNS 36.2 , QNS 36.O , ^7 3
38.6 _ 36.6 ' 38 S ' 35 S ' 3b 8
0 8 2.4 ' 4.8 13 .M
i
QNS , QNS ( 3S.8 ^S b 3 -SO
39.5 _ 40.3 40 8 -ft 3 4 Normal Range
Underline: < Normal Range
- No Value
* Clot Removed
QMS - Sample Volume not Sufficient for analysis
Page 4 of 22
IO
-------
Stydy 1078-CG920794
Hematology
Parameter 1 Group ] Animal ID
Moan Corpuitular Volume 1 37
(59.1- 75.4 fl) 1 40~~
1 ' 5
1 ' 7
1 9
Day -3
64.8
63.2J
61-9T
62.1
65.0
Average 63.4
i Std Dev 1.5
1 2 ^ 13 l_64.2
t 2 , 15 63.2
j 2 19 62.1
\ Z 25 63.4
2 28 ~~\ 63.5
2 30 t 64.4
j 2 34 [ 60.8
j Average 63.1
1
Day 2
64.9
64.2
62.0
63.4
63.3
63.6
1.1
64.5
I 64.2
J 61.9
J_62.8
Std Dev "T 1.3
' 3 11
3 12
3 14
3 18
3 2
3 32
3 8
j i Average
1 Std Dev
I t
I 4 1 21
1 4 I 27
; 4 si
' 4 33
i 4 38
4 39
4 6
Average
| Std Dev
68.2
61.2
67.0
63.0
63.8
62.4
59.9
63.6
3.0
1
—
,
67.1
65.5
623J
62.6
68.3
64.T1
62.3
2.4
i 1
63.4
63.7
60.8
63.0
1.3
68.3
61.9
67.4
61.6
Day 4 Day 9
QNS
64.0
60.9
61.9
63.7
62.6
1.5
64.8
63.0
61.0
_
64.5
62.5
60.3
63.8J
3.1
68.2
65.2
63.8
62.9
66.7
64.1
62.5
64.8
2.1
1
^
62.5
64.0
66.4
60.9
63.2
2.0
68.0
60.3
66.4
61.7
63.9
65.4
63.6
*
62.8
63.5
64.6
64.0
1.0
65.1
63.5
62.9
62.4
*
62.3
59.0
63.1
64.1
65.0
62.3
63.6
*
*
Day 11
65.8
63.3
62.4
63.9
... ... -j— ...
J5-1
64.1 I
Day 16 Day 1
65.2 * 65.1 ,
_QNS
62.3
64.2
63.9
63.9
1.4 T | 1.2
| ^
65.2
63.4
62.3
64.4
1.2
69.3
61.6
67.2
___„
____
61.8
QNS
63.6
59.9
63.9
3.2 3.6
67.2
QNS
QNS
QNS
65.4
62.9
61.9
64.4
2.4
n57.9
65.3
64.2
63.0
66.6
65.7
64.5
65.3
1.6
64.8
65.6
62.4
64.0
1.3
69.3
QNS
67.8
62.6
66.0
[64.7
63.9
61.5
] 62.3
63.7
61.0
-Sf—
67.9
— •
66.9
64.6
63.4
65.6
64.4
-
65.5
-
1.7
64.8
65.6
63.0
61.5 '
63.6 !
63.0
63.2 1
1.3
63.5
62.5
1 60.6
1 61.7
63.0
' 64.3
62.1 60.7
63.6 " 62.3^
1.6 1.4
68.1
61.5
66.9
QNS
65.6
QNS
61.1
i *
66.7 !
60.5
65.3
62.3
T
62.1
59.1
64.6 ! 62.7
3.2 1 2.9
66.8
64.8
~~
-
J_
i 65.3
64.4
i
65.6 63.3 ,
64.2
_
65.4
1.1
63.8
64.2
0.9
8 , Day 23 , Day 25 i Day 30 Day 32 Day 3?
; QNS ' QNS 63.8 ' QNS t,2 »
* 62.8 i 62.2 ' 61. / ' * QNS fel.7
59.6 | ! 61.1 , 61 4 QNS ' ' WJ *
63.4 , QNS b3.8 63.2 ' G2.S
62.1 ' | 62.3 ; ' b2.6 QNS ' W.O
62.0 ' 61.9 ; • bJ 7 6} 2 ijl ^
1.7 ' 0.7 1.1 j 00 10
i
QNS ! ! QNS ' 63.b ( ' QNS ( e'9
61.7 ' 64.8 62.7 QNS ' t>> '
60.4 ' 60.1 60.6 * ( 59.6 60.n
61.6 62.1 ' b2.4 QNS fa>.0
63.1 63.2 62.7 ' • 62 2 ' 63 4
63.6 62.8 62 9 QNS ' 62 1
1 60.7 ' ', 60.4 ' 60 9 ^.9 hi} 3
61 9 ' 62.2 fa2 i ' 60 6 ' til B
, 13 [ 1 1.8 , i 1.1 ' 1.4 . 1.-*
6fe.8 l ( 68.3 i 68 S ' b7 0 ' bS 0
60.6 j 59.8 QNS 60.U , QMS
64.8 i ! b5.3 67.0 ' ' t.4.7 ' QNS
QNS ' fal.4 61 5 _ QNS ' bl .'
QNS a : 61.6 * , QNS bl.b 01. }
QNS ] 59.1 QNS 58.9 S8.1
64.1 62.6 • 65.7 62 4 61 .'
3.2 3.5 3.7 34 ( 4;
QNS [ QNS , 64.0 63.S ' 62.8
61.7 ! I b?.3 \ bS.6 63,.-1 6i 1
63.8 ' ' 66.2 ' ' 64.4 b2.0 c<4
62.8 ' 66.8 64. / ' G3.0 6: S
1.5 ' 0.8 O.S ' 09 ' 'it
CO
th
Bold: > Normal Range
Underline: < Normal Range
- No Value
* Clot Removed
QNS - Sample Volume not Sufficient for analysis
Page 5 of 22
-------
Study 1078-CG920794
Hematology
Parameter \ Group i Animal ID
Mean Corpuscu'ar Hemoglobin ' I
(19.7- 24.6 pg) " 1
I
' 3.
37
40
5
7
9
Day -3
21.0
21.4
19.9
20.8
20.6
1 AverageJ 20.7
J ? J
Std Dev J0.6
ty^
13 21.3
j 2 15
1 2 I 19~
2
2
2
25
28
30
t 2 1 34
. . _ i _
Average
Std Dev
Day 2
21.2
20.1
20.8
J20.6
I 20.8
20.5
19,4
20.0
21-°L
20.5
19.6
20.3
0.7
J i
3
3
3
~ " ! 3 "
3
3
i
4
i 4
- - *• ~
4
4 f
4
11 22.2
12 19.8
14 21.3
18
2
32
8
Average
Std Dev
20.4
21.0
20.4
19.6
20.7
0.9
21 1 21.4
27 _J_20.7
31 £20.1
33 [20.2
38 T 21.0
4 | 39
4 J~ 6
i Average
j
Std Dev
19.7
20.1
20.5
0.4
21.2
Day 4 Day 9 Day 11
QNS
21.1
19.9
j 20.8
| 20.4
20.61
19A\
20.2 L_
21.2
20.8
19.9
20.5
0.7
| 22.1
20.5
21.3
20.6
21.2
20.4
19.8
20.8
0.8
21.8
yj 20.8
,
0.6
20.2
20.6
20.7
19.7
20.6
0.5
21.5
21.2
i 20.1
* | 21.2
21.3
20.5
19.3
19.9
20.8
20.7
20.0
] 20.4
0.7
_
21.6
20.7
20.4
20.9
0.6
21.3
20.6
19.3
19.9
20.9
20.7
J 19.9
—
*
21.2
20.6
21.3 f
20.2 f
19.6
20.7
0.7
21.6
QNS
QNS
20.3
20.6 ]
0.7~
QNS
20.6
19.9
—
20.4
0.7
22.1
20.7
*
21.4
21.2
20.2
21.0
20.8
—
—
20.9
0.5
21.0
20.4
Day 16 Day
I 21.3
QNS
1 20.1
[21.1
20.6
—
19.6
20.6
[ 20.8
20.7
20.0
20.4
21.11
20.7|£
QNS T
20.7
19.9
20.9
0.7
21.3
20.0
20.5J
0.8
21.0
20.5
20.3
20.4
0.5
22.2
QNS
20.8
0.5
21.6
20.5
19.7
20.2
21.0
^20.4
J 20.5
_J 20-6
21.0 V'
20.7 [
21.3J
20.7
19.7
20.9
joT
21.7
20.0 I
20.7
20.6
0.4
20.9
20.5
20.4
20.2
*
20.0J
— |
20.6
0.6
—
0.6
21.9
20.2
21.3
QNS
21.4
QNS
19.8
20.9
0.9
~21.6
20.8
—
_
20.3
19.8
—
20.6
0.8
* 21.4
1 21.0
20.2
[21.0
20.4
20.8
LJ 0-5
21.4
20.5
' 19.3
20.0
j 20.9
20.8
20.0
20.4
0.7
22.1
20.3
21.1
1 20.3
* 20.5
19.4
j 20.6
0.9
21.3
20.8
20.1
"19.6
0.8
18 t Day 23 Day 25 Day 30 Day 32 Day 37
QNS ; QNS 21 i ; QNS ! n 2
* 21.0 ! ! 20.7 i i 21 2 ' * QMS ' i 20 /
20.2 ] ! 19.7 i ;d.2 ' QNS 14 /
21.1 QNS ' 21.0 ' ' 21 2 ' 1'O.S
20.5 20.3 ' ' 20 5 ; QNS ' :0 2
j 20.7 20.2 , 20.S ' ' 21.2 ' .'0 ••
0.4 , O.b i 0.4 i 0.0 ' ' 0,*
I ' (
QNS QNS * 21.3 ' , QNS _ " 21 2
_ 20.4 20.3 20.5 ' QNS j .?0 -I
19.5 19.6 ' 19 4 [ * ly 5 14 j
1 19.9 ; • 19.9 ! 19.5 . QNS ' ' 14 f.
21.0 208 ; j ?0.7 ' 20.c> " 20S
. 20.7 , 20.6 ' ?0.4 ( QNS 20 3
20.3 ' j 20.1 1H.8 ' ! 20.0 , .'J D
20.3 20.2 7 ; 20.2 ' 20. 1 , ' JO ' "
j 0.5 : ' 0.4 07 0 7 0.6
21.8 ' 22.0 i 21.6 ; : 2J.9 ( 21 -j
20.2 ! 20.3 ' QNS 20 0 , ' QN\
21.2 21.0 ' ' 21 1 21.J ' ! QNS
QNS , 20.6 i .'0.2 I QNb ' _'U 4
QNS 20.6 I , QNS ' ; 20.4 ' ] 20 i-
_ QNS 19.7 , i QNS ! , 13.2 • ' 19..)
21.1 ; 20.7 , ' 21.0 ', 20.5 ' 2().h
0.8 0.8 ' 0 7 '10 'in
1 | 1 ' -
QNS ' QNS " 21.2 ( ' 21.0 , ' .'U 5
; - ' - ' ..
19.3 19.3 ' 19.2 ! 19.3 IB /
19.9 1 19.8 ' ' 19.7 198 ' 'Wo
19.6 19.6 i 20.0 ", * 20.0 1') u
0.4 ' 0.4 1 u i 0 q 0 ')
Bold: > Normal Range
Underline: < Normal Range
— No Value
* Clot Removed
QNS - Sample Volume not Sufficient for analysis
Page 6 of 22
-------
Study 1078-CG920I94
Hematology
Parameter I Group Animal ID i Day -3 | Day 2 Day 4
Mean Corpuscular Hemoglobin Concentration ' 1 j 37 1 32.4
(31 - 34.9 g/dL) ' 1 40 |_3~3.8
'1^51 32.1
1 1 7 [33.5
1 1 , 9
Average
Std Dev
}
.2 13
; 2 | 15
- 2 19
2 25
2 28
2 30
2 34
Average
Std Dev
!
1 3 11
1 3 12
' 3 14
3 18
t 3 i 2
I 3 1 32
! 3 [_ 8
i Average
1 Std Dev
j
4 | 21
, 4 f 27
1 4 ! 31
[ 4 j~~ 33
| 4 38
4 39
4 6
1 ' Average
1 Std Dev
31.6
32.7
0.9
33.1
32.5
31.3
32.7
Z
31.5
33.0 1
31.8
32.2
32.21
0.7
32.5
32.4
31.8
32.3
32.9
_ — ,
32.8
32.8
32.5
0.4
31.9
31.7
32.4 f
32.3 i_
30.7
30.8
J2-3
31.7
0.7
32.9
32.4
32.8
32.6
32.7
0.2
32.9
32.1
31.3
32.2
33.5
32.6
_____
32.5
0.7
32.3
33.1
31.6
33.4
32.8
32.7
32.8
32.7
0.6
32.0
31.8 |
31.7
32.8
31.1
30.7
32.4
31.8
0.7
QNS
33.0
32.6
33.7
32.0
32.8
0.7
32.8
32.5
*
31.61
31.9
32.5
31.3 j
32.9
32.2
0.6 |
31.9
34.3
*
32.0
33.4
33.3 1
32.4 1
33.2
32.9
i
32.2
QNS
QNS
QNS
31.5
31.7
32.2
31.9
0.4
L DaV9
32.9
33.3
32.0
33.3
31.6
32.6
0.8
#
*
~~
32.7
32.4
I 30.8
31.9
32.6
31.8
31.9
32.0
0.6
31.8
33.6
31.7
33.6
QNS
32.5
33.2
_
—
32.7
0.9
31.4
32.1
31.9
32.3
30.6
30.4
32.0
31.5
0.8
H
Day 11
32.61
33.6
32.3
32.9
31.9
32.7
0.6 J
- _j__j
32.2
32.2
31.5
31.9
32.2
31.6
32.0
31.9 ,
0.3
32.1 j
QNS
31.0
33.0
32.3
32.4
32.3
32.2
0.7
31.9
31.3
31.8
32.2
30.8
31.0
#
-
31.5
0.6
i
Day 16 Day
32.7 L* 32.8
QNS
32.2
32.9
32.2
32.5
0.4
33.4
32.1
32.0
32.4
32.4
31.2
33.0
32.4
0.7
32.2
32.9'
31.9
QNS '
32.6
QNS
32.3 ,
32.4
0.4
32.4
32.1
i
31.0
30,9
31.6 ^
0.8 '
L 33.3
32.8
33.0
32.4
32.9
0.3
33.7
32.8
31.8
32.4
33.1
32.3
32.9
32.7
0.6
. 33.1
33.5
' 32.2
32.6
33.1
32.8
32.9
0.5
32.6
32.3
31.7
30.8
31.9
0.8
18 Day 23 Day 25 Day 30 '' Day 32 Day 37
QNS ' QNS 33.1 QNb ' 33 '
* < 33.5 j ( 33 3 34.4 * QNS « c-
33.9 ' | 32.2 32 4 Qfjb : •».' 3
33.3 QNS 32 9 ' ' 33.5 ' ' 33 0
33.0 I ,327^ ^ 32.7 ' QNS 32 c '
_ 33.4 i 32.7" 33.2 33 S i4 l
0.4 j 0.6 ! 0.7 00 05
1 1 , '
QNS ] QNS ' i 33.S QNb « C,
, 33.0 ! 31.3 32.6 QNS *2 J>
32.3 i 32.7 32.0 ' ' 12 7 ' ^ i
' 32.3 i 32.0 ( il.3 , QNS 3i 7
33.3 32.8 ' 32 9 H.b 32.4
32.6 ! ! 32.8 ' 32.4 QNS ' V b
33.4 ' , 33.2 ' 32 b 33 S 3-i 1 '
32.8 , _ 32.5 ! | 32.S 33.3 32. r-
0.5 07 0,7 ' Ob Ot-
,
32.6 ' 32.? 31.6 }2 7 U :;
33.4 33.9 ' QNS ' 33.3 QNS
32.8 _ 32.1 31.5 : ' 32 8 QN1<
QNS ' , 33.5 ' 32.8 QNS 31 3
QNS , 33.4 ' QNS ' ' 33 1 ' 3"-t f>
QNS ' 33.4 ! QNS ' 32 7 A3.-5
32.9 | ^3.1 ' 32.0 ' 3? 9 " 33 1 '
0.4 0.7 ' 0.7 0.3 ' 0.6
QNS ! QNS | 33.1 ' ' ^2.9 ' iJ '
— i ' — i -
31.3 !" 28 S 29. i 30 b ! AA6
31.1 30.0 i 306 ' 31.9 ' 4U9
! ! '
| -
| 31.2 , 29.4 il.O il.S • 314
0.1 ! O.S ' 1.9 l 1 .' 1 !
op
th
Bold: > Normal Range
Underline: < Normal Range
— No Value
* Clot Removed
QNS - Sample Volume not Sufficient for analysis
Page 7 of 22
-------
Study 1078-CG920794
Hematology
Parameter 1 Group
Red Ceil Distribution Width 1
(10.3 - 14.4 ?i) 1
1
1
1
1 2
1 2
* 2
2
2
2
; 2
; 3
1 3
i 3
3
1 3
1 3
3
-
Animal ID
37
40 n
~~ 5
Day -3 Day 2
13.1
13.9
12.1
7 1 12.8
9 t 11.9J
Average 12,8
Std Dev
13
15
19
25
28
30
34
Average
Std Dev
,_ JL1 _ -
1? .
14
18
2
32
8
Average
Std Dev
1 4
J 4
I 4
4
4
4
0.8
11.7
11.7
12.9
11.8
11.2
12.0
12.3
11.9
.0-5 1.
11.9
11.8
11.9
11.5
12.9
12.0
12.4
12.1
0.5
21 12.5
27
31
12.1
13.5
33 12.5
38 13.2
39
6
Average
Std Dev
12.9
12.0
12.7
0.6
13.0
13.2
11.9
12.4
11.8
12.5
0.6
11.8
11.6
12.2
11.2
11.2
11.7
_
12.6
11.8
0.5
11.3
12.1
11.6
11.5
12.9
12.0
12.1
11.9
0.5
12.0
11.8
12.8
12.1
13.0
13.2j
11.8
12.4
0.6
Day 4
QNS
13.0
12.2
12.5 *
12.4
12.5
0.3
12.2
•
11.9
12.3
11.3
11.5
11.5
13.3
12.0
0.7
11.4
11.8 *
11.6
11.5
—
12.8
11.9
12.1
11.9
0.5
1
12TT
QNS
QNS
QNS
12.9
13.5
11.9
12.6
0.7
Day 9
12.9
13.0
12.1
„
^
*
13.7
11.8
12.7J
0.8
12.5
12.2 )
12.2
n.gj
11.1
11.8
13.0
12.1
0.6
11.9
12.1
11.6,
11.3
QNS
12.2
12.9
12.0
0.6
12.7
12.0
13.0
11.8
13.2
13.6
12.7
12.7
0.6
Day 11
12.6 r~
12.6
12.1
13.1
11.8
12.4
0.5
12.3
11.9
12.5
11.7
11.4
11.7
12.9
12.1
0.5
11.6
QNS
11.5
11.1
13.1
12.5
12.8
12.1
0.8
12.3
11.7
H—
13.2
13.4
-
12.5
0.8
_
*
Day 16 Day
12.6
QNS
12.2
13.4
11.7
12.5
* 12.5
12.3
12.1
13.2
11.7
12.4
6.7 ] 0.6
— H
12.4 12.6
12.5
12.3
11.4 :
11.5
11.5 '
12.6 :
12.0
0.5 "'
11.5
11.8 !
11.4
QNS
13.2
QNS
12.3
12.0
0.7 .
12.1
12.0
_ '
_
12.8 :
13.0
-
12.5
0.5
i
12.7
12.7
12.7
11.7
11.6
13.6
' 12.5
0.7
1 12.1
11.7
* 11.3
11.9
,
12.0
" 12.5
11.9
0.4 "
'11.9
_ 11.8
11.8
12.9
12.1
0.5
18 i Day
QNS
* 12.0
, 11.6
' 12.7
11.6
12.0
05
QNS
11.9
12.1
11.6
11.2
11.1
* 11.7
0.5
11.9
11.5
11.4
' QNS
QNS
f QNS
, 11.6
' 0.3_
QNS
. - 4
14.9
12.4
' 13.7
1.8
Z3 j Day 25 j Day 30 , Day 32 , Day 3
QNS ; _ 12 3 QNS l.'.b
12 0 ' 12.0 * QNS ' i: ?
' 11.3 _ 12.5 , QNS j ' J '
] QNS ' 12. ^ U.I 1? .'
| 11.4 [ , 11.8 ' QNS 11 3
ll.b ' 12.2 U 1 r .,'
04 . 0.3 ' 0.0 ' 0 3
] QNS 12.1 QNS ' L> 0
11.7 120 i QNS ' 1.1 •> '
, 12 2 12. J , * , 1?,3 ' ' 12 •»
11.5 I 11.6 QNS 11 '
11.? i : ll.b ' 12.1 lib
; 11.1 | [ 11.3 ' , QNS J t >
' 12.2 11,8 , 11.9 l> 0
11.7 ' , 11.8 V 1 ' ' 11 A
0.5 03 0.2 H4
, t '
1 12 1 t 12.4 ' , 12 S , • 1J 1
11.3 QNS 1 ' 11.6 QNs
11.4 > lib "llS UN-*
' 11.3 j ' 11.4 , QNS ' 11 3
11 5 , QMS | 11 2 11 '
; 12.0 ' QNS ' ',121' 12 L> ,
11.6 , 11 b 11 8 ' 11 /
1 0.4 ; | O.S [ ,05' ' 0^
QNS ' 12 0 ' i 12 4 11 y
I
i \
1 p
1 17.9 , , 16.8 * 16.1 14.7
1 12.3 ' 12 3 12.5 12 1
> 15.1 , 13.7 ! 13. / ' 1")
4.0 ; ) 1 \ 2,1 lb
I '
q>
fh
Bold: > Normal Range
ynderline: < Normal Range
- No Value
* Clot Removed
QNS - Sample Volume not Sufficient for analysis
Page 8 of 22
-------
Stydy 1078-CG920794
Hematology
Parameter j Group Animal ID Day -3
Platelet Count ' 1 37
(13/-558*ioVuL) 1 40
1 5
1 7
: i 9
I Average
Std Dev
1
j 2 13
846
544
545
__
397|
sioT
627J
194T
1
602
2 a 15 _L.665
2 19
2 25
674
571
2 28 1 176
2 30 451
2 ! 34 690
| Average
I Std Dev
1 i
! 3 11
[ 3 ~| 12
3 J 14
^3 ] 18
3 ' 2
3 ! 32
3 8
Average
Day 2
962
622
445
507
636 1
634 [
200 |
763
624
604
509
331
—
547 I
183
583
200
_
407
779j
622
J5LL
^64)
565
Std Dev 235
1
4 1 21 659
4 27 513
4 ' 31 842
4 33 1058
4 38 986
_
308
501
520
162
^
Day 4
QNS
540
413
12 *
489
364
240
667
646
540
| 550
502
548
433
417
^
714
412
590
496
516
111
641
654
688
_
4 ! 39 ] 695 ]_
46
Average
[ Std Dev
I 1
455
744
228 |
880
1008
532
511
417
548
Day 9
235
326
*
555
532 |
943
518
273
780
686
485
617
86
539
86
389
*
687
330
591
271
413
207
597
QNS
360
680 J
214
QNS
QNS
1000
622
539
508
568
598
105
j Day 11
837
586
280
518
763
597
219
704
_
575
44Y)
438
727
QNS
670
664
586
123
502
377
649
259
697
600
775
931
657
526
670
149
617
459
696
436
579
539
576
106
Day 16
255
QNS
351
550
629
446
173
*
604
529
492
489
454
460
419
492
60
T '
499
QNS
438
626
454
318
291
] 438
123
376 4
673
513
55 |*
763
552
Z_
489
-
521
389
401
QNS
491
QNS
soil
61
350
504
_
826
696
—
594
210
o
Day 18 j Day 23 i Day 25 i Day 30 j Day 32 , Day 37 n r, 3$
618 QNS ' QNS , 674 QNS 585 ' - -
390 30 479 ' 48 ' QNS V>,, ',7'
385
603
641
527
128
568
642
522
295
j 367
404
471
|
121
561
364
413 j
787
699
399
537
175
441
615 '
796
666 t
_
630
147
262 • , 382 319 QNb ' ' -.'37
555 ' i QNS 48? 447 460 I *
625 ' 649 | 577 i QNS ', 500
368 ( ' 503 430 . 447 ' 44b
275 ' : 135 '227 " 0 ' ' 11 '
QNS _ QNS , ' 382 _ QNS 579 ' . .; '
594 21.' ' 526 [ QNS • 4?5 .\->
429 , 304 303 , * 412 431 >->
585 ' _ 529 ' 413 • QNb ' 46o :
445 | ! 424 337 304 fc2
302 399 ' 509 ^ QNS ' 3Ib ', ' '
J 541 446 ' 389 , 483 •*«.' '
483 ! , 386 408 l ' 400 ! 4.1'! J ;
112 | ' 112 83 4 6.1 ]
i
1471 ' ' 2044 { 1305' 1002, 1408 .1:4', " '
569 i 54 / 50fe 438 ' 4(<3 •;: ;
1020 1296' /3? blS * /h= -i
638 ' 1059! 1 500 1 I 335 "-57 >,-,
\ ! | :
Bold: > Normal Range
Underline: < Normal Range
— No Value
* Clot Removed
QNS - Sample Volume not Sufficient for analysis
Page 9 of 22
-------
Study 1078-CG920794
Hematology
Parameter Group [Animal 10
Mean Platelet Volume 1 1 37
(5.2 -7.7 ft) 1 40
1 f 5
1 ' 7
Day -3
6.6 J
7-°J_
7.9
7.1
1 , 9 | 6.2
Average i 7.0
_ ] Std Dev 0.6
2 13
2 15
2 19
1 2 25
2 28
7.8
7.4
7.4
7.3
7.5
.
Day 2
6.5 i
5.9
6.6
6.3
| 5.9
6.2
0.3
1 6-6
2 30 7.5
2 34 t 7.2
Average j 7.4
Std Dev
3 11
3 12
3 14
3 18
3 1 2
• 3 32
0.2
-
6.7
6.8
7.6
6.7
6.5
6.6
3 8 6.2
' , Average 6.7
Std Dev 0.4
4 21 j 6.6
4 27 7.0
4 ' 31
4 1 33
4 i 38
t 4 t 39
1 4 6
|_ i Average
t Std Dev
6.4
6.9
7.0
7.5
6.5
6.8
0.5
6.3
6.6
6.3
7.1
7.5
Day 4 Day 9 Day 11
QNS
7.9
8.6
14.4
7.9
j 9.7
1 3.2
8.3
6.0
6.6J
as"""]
6.1
6.7
7.4
5.8
7.6
L 5J
t 5.9
6.5
0.8
6.2
6.7
6.3
8.0
6.9
6.8
8.3
8.6
9.6
* 8.3
8.4
8.6
0.6
7.4
7.3
7.4
6.8
7.3
0.5
6.5
8.2
7.0
6.5
7.1
6.6
6.0
6.5
6.4
6.6
0.7
6.5
6.9
0.6
6.1
QNS
8.4
8.6
8.4
8.6
8.2
*
*
_
:
8.6
8.4
8.5
0.2
7.8
* L7-3
| 8.1
| 6.9
QNS
7.8
7.8
7.6
0.4
7.0
7.6
11.2
7.8
7.2
1 Day 16
8.0
QNS
8.6
S.Q
, 7.2
8.2 ; 8.0
1.7
6.7
7.0
6.5
7.3
8.1
0.6
T~6.0
-
—
_
7.6
6.1
7.9
7.4
7.0 T 7.4
6.8 j~
7.1 J
0.5
j_6,5
6.4 <
7.0"'
0.8
6.3
IQNS 6.8 [
8.1
QNS
QNS
6.6
6.8
6.4
6.5
_^
8.3
7.5
8.7
8.8
7.8
7.0
6.8
8.3
10.6
8.1
7.9
1.5
7.1
7.3
6.0
7.1
QNS
7.8
QNS
5.7
6.7
0.8
~T ' •-
6.6
6.4
-
8.4 ^ -
6.1 6,1
6.6 6.4 ^
8.7
8.3
0.5
1 ~
6.9
0.9
-
6.4
0.2
j
4 Day
* 7.8
7.7
8.6
7.4
7.6
7.8
0.5
6.8
8.1
6.3
10.2
7.8
10.0
6.7
8.0
1.6
7.3
6.0
7.2
] 6.0
! 5.8
5.7
1 6.3
0.7
6.3
6.7
6.9
6.2
6.5
0.3
18 Day 23 , Day 25 Day 30 ; Day 32 Day 37
' QNS , QNS i b.5 QNS o j
* , 10.0 ' [ 6.2 6.6 '*, QNS ' b .-i
7.5 i 7.b | 8.6 i QNS < / b
' 6.3 j f QNS ; b.4 ' fa ,? i o '.
6.3 |_ ! fa.7 63 ' QNS Vb
7.5 , 6.8 6.9 fa..' b-i
1 1.7 • 0.7 : 1.0 ' 0.0 ' 0 .'
i ' 1 i
i „ i
QNS ! ; QNS i /.O ; QNS 6 1
6.3 i 1 8.2 77' QNS ^ f-, i
' 5.9 | ' 6.7 u.b * ' S Normal Range
Underline: < Normal Range
- No Value
* Clot Removed
QNS - Sample Volume not Sufficient for analysis
Page 10 of 22
-------
Study 1078-CG920794
Hematology
Parameter i 6royp Animal ID Day -3
Neutrophils Percentage 1 37 54.4
(17.2-40"-'.} 1 40
1 5
1 7
, 1 9
j Average
! J Std Dev
1 2 J 13
L 2_J_ 15
. 2 19
: 2 L 25
• 2 28
• 2 30
1 2 1 S4
] Average
Std Dev
30.6
25.1
26.2
19.9
31.2
—
13.5
21.5 j
23.1
[J6.3T-
21.5
18.0
24.1
22.3
—
J?A
5.8
1 3 11 27.8
] 3 12 24.5
! 3 14 30.6
' 3 18 29.1
! 3 2 20.4 1
i A-.a 32 _
i 3 1 8
27.9
i — —
23.8
| f Average 26.3
1 Std Oev 3.5
I
1 4 21 44.5
1 4 [ 27 29.8
4 31 37.6
4 ^ 33 44.9
4 38 33.4
1 4 39 ' 21.5
1 4 6
I Average
1 Std Dev
25.9
33.9~
9.0
Day 2
39.8
25.2
18.4
.__
23.6
24.5
26.3
8.0
14.7
19.2
26.5
19.7
18.5
17.6
25.7
20.3
4.3
121.8
28.7
] 22.4
21.0
23.1
22.9
19.5
"22.8
—
2.9
23.9
24.8
43.0
59.0
30.6
16.3
25.3
31.8
14.5
Day
QNS
4 Day 9
28.5
f23-1 '
19.4
34.5
*
26.4
6.6
13.3
20.3
25.8
23.3
21.9
20.6
27.7
21.8
4.6
22.4
26.2
21.8
j 40.8
29.0
20.6
14.8
25.1
8.2
19.7
QNS
QNS
QNS
35.6
34.2
18.5
15.0
*
*
20.8 1
24.3
22.6
7.3
12.9
17.3
23.6
21.8
20.8
19.2
21.0
19.5
3.5
*
21.5
27.4
24.3
39.6
21.4
QNS
22.3
21.6
26.1
7.0
21.6
28.9
38.7
51.7
28.0
18.2
49.0 28.9
31.4
13JJ
Day 11 Day 16 Day 18 Day
48.0 23.9
22.1 QNS
* 26.9
16.3
16.8 19.4 13.8
23.9 23.2
15.6 23.8
43.1
18.7
25.3 P" 22.6 ( 19.8
13.2 2.1 5.3
1
17.4
18.7
24.7
^28.4
24.7
18.8
16.5
21.3
4.6
23.8
_
30.9J
11.2
QNS
23.1
31.5
26.0
22.5
20.5
24.6
3.8
21.4
27.3
42.6
29.8
29.5
19.4
15.3
23.3
23.3
19.1
> 18,1
' 19.8
23.5
20.3
3.2
21.4
24.9
, 25.6
1 QNS
|23.7
_j QNS
1 18.9
22.9
2.7
17.5
16.4
24.1
16.3
21.1
19.7
15.9
18.7
3.1
20.8
33.7
22.7
40.8
1 22.6
' 18.2
' 26.5
8.8
1 " •'
U13.8
22.0
" I
*
' 25.0
18.7
-
28.3 19.9
8.2 4.8
17.2
~| 31.9
26.8
14.6
22.6
8.1
1 QNS
* IA
' 16.6
! 24.9
29.9
19.8
9.8
QNS
25.4
28.1
23.1
1 25.3
' 18.5
1 24.9
j 24.2
i 3.2
1
48.3
139.1
22.5
] QNS
QNS
QNS
36.6
13.1
' QNS
1
42.8
| 20.9
1
31.9
15.5
23 Day 25 Day 30 Day 32 Da}
_ QNS 25.9 ' QNS ' Jn.i
19.4 15.7 " QNS 24 1
19.S 17.2 QNS It .
QNS ..'1.0 ' 213 ' .!.: '
28.2 213 QNS 2S'.
22 5 ?0.2 21 j tM r
5.0 4.0 00 1 ^
QNS ! 20.b , QNb ' .'•; :
! 203 | 2.] 3 , ' QNS n t
' 25.2 ' >7 i ' * 2fc.S .'t. ,
21.2 , , 17.5 ; QNS ( IS >
21.8 ' 19. b , ?^ 6 ' .'-) ^
22.1 • | Ib.j , QNS 19 ,
27.0 ' 24.3 ! 19 b ' 20.t
22.9 • [ ?!..' _ ?3 2 [ 2lL
i.b ' 3.7 ' 'IS ? t>
' 25.6 ^ 23.3 * 344 ' 2 < '-
17.9 • 1 QNS 41.6 ; QNi
22.5 23.2 20.S ' QNi
_ 35.4 , 29. S QNS .>s ^
31.5 i ' QNS '' 20.S 24 ]
293 ^ QNS ' ^0.7 ' 26 '•
27.0 ' 25 3 .'9.0 ' 2S.,
6.3 ' 3.6 ' 9.1 IS
i
QNS ' 23.7 ' 44.9 < Jl ^
1 i f- r
j '
45.5 48.5 49.0 22,1
11.2 ' 143 ' IS j Jb..'
28.4 ' , 28.8 ' .-i/3 1^ c
24.3 ' 17.7 ' ' 168 t 1
[
Bold: > Normal Range
Underline: < Normal Range
- No Value
* Clot Removed
QNS - Sample Volume not Sufficient for analysis
Page 11 of 22
•i-10
-------
Stydy 1078-CG920794
Hematology
Parameter Group
Lymphocytes Percentage 1
{57.5-81.1".) 1
1
1
' I
2
2
2
2
2
2
2
3
3
3
3
3
3
3
4
4
4
4
4
^
4
Animal ID Day -3 Day 2 Day 4
37
40
36.2
61.0
5 I 70.5
7
9
Average
Std Dev
13
15
19
25
28
30
34
Average
Std Dev
11
12
14
18
2
32
8
Average
Std Dev
21
62.8
72.7
60.6
14.5
67.1
67.3
48.5
55.7
75.0
74.3
71.5
69.8
68.7
6.5
62.0
67.7
63.0
63.6
69.1 L
61.8
71.2
65.5
3.8
48.6
27 64.5
31 55.4
33 48.0
38 1 59.2
39 72.4
6 69.2
1 Average | 59.6
f StdDevJ^.6
—
76.5
67.0
69.1
65.1
h!0.3
76.0
70.2
63.3
75.7
73.1
78.3
64.6
QNS
60.6
73.7
70.5
66.1
7.0
78.5
69.3
62.5
71.6
5.8
67.1
65.0
71.8
67.8
70.8
64.7
74.5
68.8
3.7
66.8
70.0
49.1
37.9
57.8
77.6
69.2
61.2
13.8
73.1
68.7
75.4
62.5
70.0
6.1
68.4
67.1
*
Day 9
55.9
69.6
81.3
71.2
71.5
69.9
9.1
78.5
73.2
66.5
74.5
71.5
77.1
69.2
72.9
4.2
68.9
*
72.2
47.3
65.7
65.5
75.1
65.9
8.9
71.7
QNS
QNS
QNS
55.1
73.0
43.7
60.9
14.1
65.4
68.0
47.0
*
#
—
Day 11 Day 16 j Day 18 i Day
41.3
67.6
76.9
68.9
78.8
66.7
15.0
1
74.3
70.9
67.2
65.5
67.2 '
76.8?
"75.1^
I
QNS
64.5
69.3
63.9
8.5
70.2
65.9
_
52.3
43.6
63.3
75.7
JJ54J
62.2
10.9
71.0
4.5
66.3
QNS
70.9
61.1
68.4 '
69.3
70.5
67.8 (
3.7 ,
69.3
68.4
46.91
64.5
60.4
73.8 l
-
63.9
9.5
64.6
[75.0
69.0
66.9
* 59.3
71.8
' 81.0
• 68.8
72.7
68.9 70.7
4.5 7.8
'76.7
67.4
66.7
, 76.4
1 73.8
75.3
65.3
71.7
5.0
68.9
67.7"
68.6
QNS
71.8
QNS
72.9
r70.0
2.2
1 76.0
73.6
-
*
63.8
JJ74.5
_
, 72.0
5.5
1
i 74.2 l
72.5
615
79.1 "
72.3
74.6
74.6
72.7 (
5.4
71.0
60.0
' 71.1
54.0
, -
67.4
73.2
66.1
17.5
74.1
63.2
63.2
81.0
~
1 70.4
8.8 [
1
QNS
* 84.8
78.1
66.2
60.1
72.3
11.2
QNS
63.6
59.5
715
, 64.5
76.3
1 62.2
66.3
6.3
42.7
552
71.8
, QNS
QNS
QNS
56.6
! 14.6
QNS
44 7
725
, 58.6
19.7
n Day 25 ; Day 30 Day 32 Day 37
' QNS ' 57.8 l QNS t>: I
69.6 i 723 ' * QNS < CiS 1
' 74.6 , 77 (j QNS 78 4 '
QNS , 70.5 69 0 ' /O 1
62.0 6/3 ' QNS ' 59 8
_ 68.7 ' 69.1 69.0 ' ; b/ 1
63' 73 | 0 0 74
s
i QNS | /.'.O QNS , b?..l
68.9 _ 68 i ( QNS ' [ oi A
, 66.4 ' 64.4 » l hi 3 ' 6? 4
[ 72.? 77.4 QNS \ /b 7
; 70.7 70.4 t>7.? ' , t>S 7
\ 72.3 [ 783 QNS 74 J
57.2 61.7 b?.0 ' ' bS ->
j 68 0 ; 70.4 ' 65 8 i 68 7
58 fe.2 i ; 2 52
t>5.6 < 63 2 1 5J 0 b4 .'
i 75.8 , ' QNS ' 52 0 QNS
' 71 8 | 66 1 | ' o8.2 QN'- '
53 1 ' 62.9 QNS b7.4
" ' ! 1- *
_ 59.0 ' QNS _ 69.y ' b? i
Sfa.7 QNS 57 / ' b2 2
63.7 64.1 j bOO ] > ob.,'
1 8 9 ' 1.8 ' 8 6 ' .' 5
! QNS 64 0 ' 44 ! \ b/ >5
"l '
I ~ ' I
i " ' •
~ ~
[ 41 0 i ' 41.0 3a S fil.4
83.6 i 80.2 ' 75.6 ' /<. •<
i
L 62.3 fal.7 , , 53 1 " 68 /
! 30.1 ! i 19.7 19.6 ' 7 *
\ *
-1- -
CO
th
Bold: > Normal Range
.Underline: < Normal Range
— No Value
* Clot Removed
QNS - Sample Volume not Sufficient for analysis
Page 12 of 22
•lO
-------
Study 1078-CG82Q794
Hematology
^r
Parameter 1 Group
Monocytus Percentage 1
(0-6.1^) " 1
1
1
1
Animal ID Day -3
1 37
1 40
5
1
9
Average
Std Dev
2
2
2
_. 2 _
2
I 2
2
_ _ _ _ 3
3
3
3
3
- . - - - . i „...,
3
4
4
4
4
4
; 4
4
13
15
19
25
28
2.7
1.3
1.2
0.9
3.1
1.8
1.0
—
1.8
1.2
2.4
0.8
1.5
30 1.2
34 j 1.5
Average \ 1.5
Std Dev 0.5
1 _ _ _
11 1.8
12 1.0
14 2.4
18 , 1.1
2 1.1
32 1.9
_ J ,
1.4
Average J 1.5
Day 2
i.sj
1.2
3.1
0.9
1.3
1.6
0.9
1.8
2.4
2.1
2.1
—
1.5
1.0
1.9
1.8
0.5
1.9
1.2
2.3
1.7
Std Dev ] 0.5
21
27
31
33 _ .
38
39
6
Average
!
3.4i
£=n
2.4
2.6
1.7
, _„.
1.8
1.7
2.1
Std Dev ,0.7
1.3
1.2
1.9
1.6
0.4
3.1
1.6
Day 4
QNS
1.3
1.0
_
0.9 *
1.8
1.3
0.4
1.2
1.7
4.1
1.1
1.2
0.9
0.9
1.6
1.1
1.0
0.9
1.7
~| 1.0
2.1
1.0 j
3.8
1.2
3.6
2.3 I
1.2
0.8
2.2
5.7
1.9
1.8
[Day 9
1.0
*
1.0 *
1.2
0.7
0.9
1.0
0.2
1.8
1.3
i 2.9
0.7
1.5
0.9
*
-
1.6
QNS
QNS
QNS
1.5
0.9
5.0
2.3
1.9
1.3
,,',
0.7
3.0
1.2
2.6
1.9
QNS
2.5
Day 11
3.0
1.2
2.1
1.0
1.1
1.7
0.9
0.9
2.6
1.2
1.3
1.7
1.5
0.9
1.4
0.6
2.7
2.3
0.7
1.3
1.3
2.7
1.8
1.7
1.3
3.3
1.9
I 0.8
1.5
QNS
1.3
~
Day 16
1.1 '
QNS
1 — -1-
1.4
u>3-
0.4
^
1.0
2.1 (
3.3
1.0
L ' 1.8
1.0
3.1
1.9
1.0
|
1.4
0.9
" 1.6 ,
0.9
0.9 |
1.1
1.7
^
1.2
0.3
1.3
5.3
1.1
2.7
1.9
-
2.2
1.7
A
QNS
0.9~|
QNS
2.1
1.4
0.5
1.8
1.0
i
—
4.2
2.5
2.4
1.4 1
Day 18 J Day
1.2 " j QNS
1.4 * " 0.5
1.1 ' 1.4
0.8
1.1
1.1
0.2
1.4
5.5 '
0.8
1.2
1.7
1.1
2.0
}1'6
1.2
I 1.0
I 1.3
; 2.3
2.7
" -2"° ,
1.8 "•
0.7
1 1.7
4.8
1 1.1
2.3
1.7 j
0.8
1.6
1.1
0.5
QNS
2.2
• 35
1.0
19
1.0
0.9
1 1.8
10
1.8
0.9
1.6
QNS
QNS
' QNS
1.4
0.5
QNS
8.2
1.5
- t —
4.9
4.7
23 ] Day 25 j Day 30 j Day 32 ' Day 37 Dsv 1* ,
QNS ' P.3 , QNS P ? i : ' ",
1.0 ; 1.9 ' QNS : Ob I
1 2.4 ' 12 QNS ' I •>
QNS ' Li 0.9 i> '" '„< 4 -,
\ 2.0 ' 28 \ QNS 24
1.8 1 9 0.9 ' IP 1, •
0.7 07 ' 0.0 10 " 'J
- . j , ,
i QNS | i 2 QNS 2 : • •
1 1.7 ' 13 , QNS 3 3 , <;
1.4 , , 1 8 * 't ? 1 ' b .'
1.5 l.i ' QNS ' 0 -> ~ •>
0.9 10 ' 0 P 0 '>
1.2 13 i QNS 11
2l' 1.4 0 8 L 0 ' - •' !
| 1.5 | l.i 13 21
I 0.4 0.2 0 / 1 ' ',
1.8 5.0 ' 4 2 [ 41 -' ' ;
,10 ' QNS '?4' QNi '
I./ I 34 , 2l' ' QNb ' u>* ',
1 3.4 ' 0.8 [ QNS 1 : .
8.3 , ; 4.9 , ; 4.5 , ' 1 , ' ' ' i
,17 13 \ OH •?.! i -
1
5.0 i.l , 11 • ?.? i
4.7 ' ' 1.8 | .? 0 ' | 0 7
^
Bold: > Normal Range
Underline: < Normal Range
- No Value
* Clot Removed
QNS - Sample Volume not Sufficient for analysis
Page 13 of 22
-------
Study 1Q78-CG920794
Hematology
Parameter Group , Animal ID
Day -3 Day 2
Eosinophib Percentage \ 1 37 1.2
t0.1-7>.) 1 40 n 2.6 1
1 ' 5
: 1 r 7
1 " 9 "
Average
Std Dev
i 2 13
2 15
2 19
: 2 « j
2 28
2 30
, 2 34
Average
Std Dev
3 11
| 3 j 12
3 14
3 18
3 2
3 32
3 8 ,
Average
Std Dev
2.2
3.8
2.8
2.5
0.9
3.1
2.7
"2.5"
1.7
3.2
2.0
1.2
2.3
0.7 ~
3.8
3.2
2.4
2.1
4.3
3.0
1.9
3.0
0.9
1
' 4 21
4 27
4 31
4 ; 33
4 • 38
4 39
4 , 6
Average
Std Dev
2.0
2.6
1.6
3.1
1.9
_
~.-~_
2.6
3.1
0.9
3.3
¥
0.9
___
2.1
4.3
1.4
3.6
1.9
2.6
2.7
1.0
1
3.8
2.1
1.7
3.5
2.9
4.1
2.2
2.9
0.9
3.6
1.8
2.2
1.4
2.3
1.3^ 1.6
2.0
2.1
0.6
1.1
2.0
0.8
Day 4
QNS
3.8
1.2
4.6^
2.2
3.0
1.5
*
—
2.2 r
2.1
3.3
1.3
4.2
2.2
1.9
2.5
1.0
3.1
3.5
1.9
3.6
3.0
5.9
2.2
3.3
1.3
3.8
QNS
QNS
QNS
2.3
2.4
1.4
2.5
1.0
*
Day 9
' 2.9 I*
3.5 *
1.4
2.4
1.8
2.4
0.8
1.6
2.1 |
3.1
1.6
—
3.1
1.4
1.8 1
2.1
0.7
2.2
2.5
2.3
3.6
QNS
4.3
2.2
2.9
0.9
3.1
_
1.8
2.7
1.8
2.0
1.7
1.8
2.1
0.5
Day 11
1.7
3.1
2.5
2.6
2.4
0.5
2.1
1.8
3.1
2.1 r
2.8
2.0
1.5
2.2
0.6
2.8
QNS
2.2
2.5
3.2
3.4
2.5
2.8
0.5
4.1
1.2
2.5
3.5
2.5
1.8
-
2.6
1.1
*
Day 16 Day 18 | Day 23 Day 2S Day 30 Day 32 Day 37
3.5 • * 4.0 i QNS ' QNS l 29 QNS 2 S
QNS
2.2
2.1 <
5.8
3.4
1.7
1.9 !
2.1
3.2
1.6
2.6
2.1
1.9
2.2
0.5
\
2.9
2.5
1.8
QNS ~
2.7
QNS |
2.6
2.5
0.4
3.1
1.3
__
2.1
1.4
- ,
2.0
0.8
2.6
2.3
1 2.4
4.9
3.2
1.2
2.1
'"1.5
4.1
1.2
2.2
2.4
' 2.0
! 2.2
} 0.9
t
2.7
3.1
1.9
1.1
3.4
2.1
2.4
0.8
2.9
1.5
1.4
; 1.0
1.7
* ! 2.9 3.6 ' 3.5 - ' QNS " i <
,21 15 2.3 ', QNS ' 2.0
2.4 ' QNS 2.3 ' 3.2 ' .V
i 4.5 3.7 ' 3.1 QNS i 1
'3.0 29! 2.8 3,2 ? 9
1.1 • 1.2 05 0.0 ' 0 S
i •
QNS i QNS 20, ' UN} , J 2
1.5 1 9 ' 1.7 QNS 1 r
' 3.7 ' 4.0 ' 3.3 * 20 314
| 1.6 2.? i 1.4 QNi 14
3.9 ' ,3.1 33; ? 4 .' y
2.6 ! | 2.8 l ; 1 7 QNS ' 3.0
! 1.9 3.1 , 2.9 ' 23 2 >'•<
j 2.5 2.9 '2.3 25 ,>'»
i 1.1 0.7 ^ ' 0.8 | 0.-! 0 7
1 2.0 ~24 29' ' 2 S ; } -i
! 2.4 2 & f • QNS , '18; QNS
1.8 ; l.b 3.9 ! 42 ' QM^ '
' QNS , 3.3 35' QNS ' .M)
1 QNS 2.9 i QNS ' 2.b " 3.0
QNS | 3.3 , ' QNS ' ?.4 ' ? S
2.1 ! 2.7 | ; 3.4 2.8 ? i
0.3 i 0.7 ' 05 O.o O 4
i '
QNS QNS , 2.4 ,' 1,8 ( ? a
1 "" ! '
, 0.7 I ' 0.9 14 j ' 1.8 ! 3.:
• 1.7 , 1.0 , 1.7 .'0 l-i
1.2 , ' 1.0 1.8 1.9 : c
\ 0.7 • 0.1 !' 0,S 0.1 0.'
1 ' '
in
th
A <
Bold: > Normal Range
Underline: < Normal Range
-- No Value
* Clot Removed
QNS - Sample Volume not Sufficient for analysis
Page 14 of 22
-------
Study 1Q78-CG920794
Hematology
Parameter Group
Ba>ophi!s Percentage 1
(2.2- 9.7 \) 1
1
1
1
12
I 2
2
2
Animal ID , Day -3
37 5.3 i
40
5
To
7 6.2
9
Average
_L§
3.6 "
Std Dev 2.3
13
15
19
25
- - ). 2 28
2
30
I 2' i 34
3
. _ i !__
• - - 1
i 3
Average
Std Dev
6.0
5.6
3.0
Q.9
2.8
1.0
-||-j—
2.1
11 4.5
12 3.6
14 fjl.6
1_«. 3.9_
J 3 2
1 3
| 3
4
4
4
I 4
4
4
4
32
r 8
Average
Std Dev
21
27
31
5.0
5.2
1.6
Day 2
7.7 i
6,1
1.0
5.2
2.2
4.4
2.8
4.6
_
Day 4
QNS
5.8
1.0
4.4
6.0
3.8 |
10 1
2.9
1.1
4.9
3.5
1.9
5.2
2.9
—
3.6
1.5
M
18
2.8
33 1.2
38
~39
3.7
2.7
6 09
Average , 2.1
J I Std Dev
1.0
18
5.9
1.9
6.9
16
3.2
2.3
4.7
6.5
4.3
—
Day 9
6.0
7.4
0.9
* J4.8
_L_^
4.1
11 |
3.8
0.8
6.9
4.0
1 2-4
2.0
3.8]
2.1
2.6
1.7
3.5
06
5.4
3.2
0.7
2.5
1.7
|
5.0
2.3
2.0
7.3
2.8
5.0
5.9
3.7
1.5
3.0
15
6.4
3.9
2.0
*
1.4
5.8
2.2 1
3.7
2.3
3.1
QNS
QNS
QNS
5.4
2.3
0.9
2.9
4.3
3.4
*
Day 11 Day 16 Day 1
5.8
6.1
13
|3.9
_
—
—
18
3.8
2.2
6.9
QNS
14
4.7
1 2.0
3.8
5.3
5.9
3.8
2.6
3.3
0.8
5.8
3.9
19
2.5
7.8
QNS
6.1
3.9
4.7
1.9
3.8
1.9
19
3.5
0.7
4.7
3.0
0.9
5.3
QNS
2.3
2.5
5.1
5.1
3.1
1.9
3.7
16
6.1
T3.8
—
3.9
1.6
3.7 1"
4.4 |
3.5
1.4
3.8
1 2.1
—
2.6 j
15 j
2.6
0.9
4.8
2.9
-
2.9
1.4
1.7
5.4
3.8
2.4
QNS
0.8
QNS
3.5
3.2
1.7
4.8
2.1
1 ~~
*
4.7
2.9
_
3.6
1.3
* 8.5
7.8 ,
1.6
4.7
2.7
5.1
3.0
4.7
|7.3
^ 4.6
u Z5
3.0
1.4
6.4 '
_2.'l
, 4.3
2J.
2.8 ,
T
3.7
4.1
3.1
11 '
J
_ «
] 1.6
3.5
2.1
2.9
1.2
\_ Day 23 [ Day 25 Day 30 | Day 32 , Day 37
QNS ( _ QNS 10.9 QNS b :
* 4.2 ' 65 , b& * QNS " b.'
1-6 i i 1.3 | '15* QNS 1 ^
5.7 i QNS ' , 4.9 S.5 ' H y
3.8 ' ] 4.0 'r 5.4 QNS ' b4
3.8 , i.y ' 5.9 55 4 u
1-7 26 3.4 ! 0.0 1 :
QNS ) ' QNS ' ,43, ' QNS 4 4
7.2 ' ! 7.1 ,641 ' QNS Go
49 ,30 32 ! * S.I ' 3,8
2-6 ' 22 2.2 * QNs 2 2
3.9 , 3.6 ' ( 5 fa , 57 4 s
1.6 | 1.7 ' 1 / I QNS ? 3
10.0 , ' 10.4 i. 5 , 10.3 8 t
5.0 j ' 4.7 i 4.7 ' /O 46
3.1 | , 3.4 ' _> 7 28 J. ,;
5.0 , 4.5 ' 5.D fa fa vs
2.3 ! , 2.4 ' ' QNS ' !.<. " QN'.
1.9 i i 2.4 32 4 S QNS
" QNS j i 4.6 ' 3 i ' QNS < 0
; .. i • ;
QNS ' ,50 QNS _ 54' 4 H
QNS ^ 63 ' QNS i b9 55
31, 4.?! 40 ' 51 4S
1-7 , 1.5 14 20 13
!
QNS ; QNS | 6.8 4 8 5 S
t
26 '3.5 i 4.1 ! " bO 107
3.1 , ' l_l 23 43 -so
1 '
2 9 j ! 2.8 ,44 '44 ' t> S
0.4 _ ' 1 0 2 -5 0.9 i y
l
Bold: > Normal Range
Underline: < Normal Range
- No Value
* Clot Removed
QNS - Sample Volume not Sufficient for analysis
Page 15 of 22
-------
Study 1078-CG920794
Hematology
Parameter 1 Group
Nfutrophib 1
(0.8-2.9xlOVuL) ' 1
' 1
-- " i l
1
j Animal ID
37
40
5
7
i. _9 _
Average
1 Std Dev
j
2
2
13
Day -3
4.54
1.65
130
1.17
0.83
2.02
1.47
1.65
15 1,56
2 19 ' 2.87
2 ( 25 1.32
2
2
, 2
i 3
1 3
1 3
1 3
3
3
3
i _,
, 4
4
4
4
4
; 4
4
—
Day 2
2.70
1.27
1.38
1.58
1.20
1.63
0.62
|~ 1.23
28 I 0.93
30 1 l.SlT
34 ; 2.06 [
Average
Std Dev
11
12
1.70J
0.62
rT.97
1.13
14 J 1.92
18
2
32
8
Average
2.45
1.06
_
1.55
2.02
1.57
0.92
0.92
2.22
Day 4 Day 9 Day 11 Day]
QNS
1.50
1.76
0.31
2.03
1.55
*
1.40
0.76
1.29
_„____.
1.68
1.81
—
1.49
0.51
] 1.40
1.70l
1.50
1.68
Std Dev 0.49
1
21 1 3.72
27
31
1.34
1.47
0.93
1.80
1.21
0.87
1.45
1.37
1.15
1.28
0.27
1.20 |
2.11
1.59
0.36
1.09
1.10
1.19
1.22
j 0.19
2.24 I
2.42]
33 1.87
38 n.ei
39 fi.97
6 2.07
Average 2.41
Std Dev ! 0.63
1"
1.37
1.52
0.96
1.59
1.21
1.32
1.75
1.74
* 2.62 | 1.24
* 1.26
1.26
1.59
QNS
0.71
1.49
0.71
1.31
1.48
1.12
1.24 f
1.67
1.44
~O27~
*
2.07
1.51
1.03
0.81
1.36
0.44
1.90
3.24J
2.53
2.11
1.73
2.00
z
2.13
0.61
.1^,
QNS
1.36
1.28
1.78
1.65
2.49
1.51
1.59
1.28
1.41
0.17
1.33
1.63
1.34
1 *T
1.30
_LL55_
0.44
__
1.98
QNS
1.27
1.42
J 1.52
QNS
QNS
2.79
1.95
1 5.26
2.80
1.77
0.29
1.40
~_
2.53
2.16
2.73
1.96
_J_1.29
2.32
2.06
0.55
1.38
QNS
1.52
1.70
1.02
1.26
1.43
1.41
to.23
1.40
1.77
2.10
1.25
1.27
1.66
1.57
0.33
1.43
2.73
1.23
1.79
QNS
1.10
QNS
fl.23
11.35
— —
2.74
0.88
2.32
] 1.59
-
*
— —
0.27
0.77
1.89
-_
_
2.08
1.31
1.95
"OTij
1.51
0.59
-b
*
__
__
— -
i
Day 18 Day 23 | Day 25
J..27 QNS ' QNS
0.86 * 0 24 • 0.88 !
1.19
1.59
1.02
1.19
0.28
1.35
1.27
J|o
1.60
1.41
1.23
1.05
1.30
0.17
1.43
1.42
1.80
2.48
__
0.95
1.28
1.56
0.53
1.49
3,27
¥.65
1.28
2.17
0.95
..— .,
1.45 1.56
, 1.64 ! QNS
, 1.72 ^ 1.71
1.26 i 1 38
, 0.69 0.44
QNS ' QNS
1.90 _ 1,26
1.74 ; 1 68 ,
1.89 ^ 1.77 '
158 ' ' 1.17
1.08 1.14 |
1.43 i 1.44 !
1 60 ' 1.41 ,
0.31 0.27
4.21^ ' 1.72 j
2.20" 0.71 i
t 1.81 ! 1.79 [
| QNS " 2.41 _
—
i i
' QNS ' i 1.73
QNS ! 1,76 !
' 2.74 j 1.69
i 1.29 ' ' 0.5S
QNS QNS (
'
! 8.71 8.23
, 1.87 [ ' 1.09
' 5.29 . 4 66 ,
1 4.84 'j 5.05
t \
I Day 30 j Day 32 , Day 37
' 1.79 ' QNS ' iol
O.bO * QNS 1 •!/
1.46 < QNS l.i-'j
1 1.41 1 1 39 ! l.V~
, 1 15 ' QNS ' l.bV-
1.28 , 1 j9 ' 1 Si
0.44 ' 0.00 ' 0 it-
{ i i
137 QNS 1 SI
1 87 j QNS ' ' J iJ'3
l.faS * 137 t.Jb
1.3b ' ^ QNS ' 161
1.20 , 1 32 ' in
0 98 QNS 0 ^T
1 49 1.07 _ 1 14 "
1.42 ' i,2S ' 1 4 -!
030 , 0.16 ' ' 0 ,-t/
164 ; ' 2.34 ' ' 1.46
QNS i , 2 33 . QNi
2 10 147 ' QN-i
1.S4 : ( QNS ' l.'jl
QNS ' 0.^9 ' l.is
QMS | 1.70 ' 1 SI
1.76 1/7 14'
0.30 , , 0.58 ( O.I/
1.21 : ' 3.50 ' I.Jo
f >
7.32 i , 5.40 . • I < •
140 ' 146 m
3 31 | t 145 ' 1 31
i.4/ ' 1M/ oOS
Bold: > Normal Range
Underjine: < Normal Range
- No Value
* Clot Removed
QNS - Sample Volume not Sufficient for analysis
Page 16 of 22
/He
-------
Study 1078-CG920794
Hematology
Parameter Group [ Animal ID
Day -3
Lymphocytes 1 37 3.021
(2.2-5.3vioJ/uL) I 40 [_3.29j
1,5
1 1 7
19
Average
Std Dev
5.35
isl]
3.02
3.50
ToT
2 13 5.14
2 I 15 1 4.55
2 1 19 4.41
2 T 25 j 4.62
2 1 28 ' 3.82
. 2 30
2 34
Average
Std Dev
3 11
3 ' 12
3 14
3 18
3 2
3 32
38
Average
I 1 Std Dev
4.46
6.46
4.78
0.84
Day 2
3.29 |
3.22
5.73
4.49
3.39
4.02
1.09
~634~
1 5.69
_l_4-83
4.40
3.12
3.96
5.35
3.60
3.75"
4.49
4.10
0.73
, 1
4 I 21 J~4~07
4 27 j 4.87
: 4 31 i 3.57
4 33 | 2.00
4 38
4 39
4.62
6.62
4 4 6 ^ 5.53
Average
f Std Dev
j r 1
6.05
3.63
4.08
5.57
Day 4
QNS
3.20 i
5.62
1.13
3.52
3.37
1.84
[7.6!
5.17
1.02
4.31
3.03
4.73
3.01
3.35
3.11
4.56
^3.73
0.77
._™._
4.47
1.47
3.82
T35~
3.70
1.63
3.99
8.25
5.73
4.39
5.63
3.80
*
Day 9
2.54 *
3.29 *
7.84
4.68
3.40
U.35
2.10
4.38
4.78
5.19
1.27
4.64
2.46
5.25
2.40
3.41
3.27
4.10
J 3.65
5.47
4.60
2.05
!
1.07
4.29
QNS
QNS
QNS
4.32
*
~
7.38
5.56
4.92
5.96
3.84
Day 11
2.25 1
3.87
5.74
4.58
3.58
4.00
IT1-29
4.98
s.sq!
5.45)
1.09 j
4.36
3.05
4.98
2.36
QNS
TI3-67
Jj4.57
183
6.66}
4.69 t
4.99
1.13
0.99
4.54
5.77
2.93
2.30
5.60
5.60
4.50
5.73
3.64
5.19
5.91
Day 16
3.34
QNS
5.85
4.73
3.61
4.38
Pl.15
5. IT!
0.82J
3.83
QNS
5.43
4.08
i 3.29
JTs.91
3 5-7°
4.37
4.43
5.37 I
5.19 j
4,36
1.29
0.96
4.64
6.84
3.02
1.90
4.74
_
6.64
4.72
4.37
6.79
4.16
4.79
3.97
5.06
1.17
*
Dayl
2.81
3.80
6.96
4.74
3.94 •
4.45 "
1.56
.
5.71
5.62
3.06
7.80
4.85 '
4.65
4.96
5.24
1.43
8 Day 23 Day 2S Day 30 Day 32 Day 37
QNS t QNS 4.00 ' QNS " i <*j
* | 2.64 3.17 , ,?./8 • * QNS 3 /O
' 6.82 5.86 j 6.59 , t QNS ' 7.04
4.36 QNS ^ 4.74 ' 4 41) ( '48!
3.45 l 3.77 3.62 ! QNS l 3.40
432 4.27- I" 4 35 4,49' 4 1* I
1-81 1.41 • 1.44 : 0.00 1.4K
QNS QNS , 4.81 QNS ' 5.22
4.75 4.27 i 5.71 > , QNS ' ' 5 in "
3.68 ; ' 4.43 J 3 9b ' ' ' 3.27 i ^
_ 5.84 6.09 ; ' 6.03 ' QNS ' 6.65 "
*4.03 3.79 ' ^ 43i; ^ 3.75 " 3 t-,
4.46 3.73 ' ; 4.55 QNS Io6
3.58 3.06 ( : 3.78 > 3.b9~ ' i i>0
4.39 | 4.23 t , 4.74 ' , 3.57 ' 4 SO
0.84 1.03 , 0.85 , O.J6 * ' 1 ."n
1 i
4.52
3.35
4.81
QNS
[Tss
*
6.06J
— 1
4.53
1.84
QNS
4.74
4.15
0.75
4.27
6.33
-
-
5.29
5.23
—
5.28
0.84
4.88
2.53 '
1 5.64 '
"
3.28
2.83
5.14
4.05
1.33
6.39
6.47
6.24
7.12
^6.56
0.39
_ 3.72 , " 439 ; 4.45 *.S4 401
3.11 300 QNS ' 291 Qto
5.76 ' 5.70 i ( 5.96 ' 4 91 • ' QNS.
QNS ^ 3.61 , 3.30 ' QNS 4 tU
QNS ' 3.25 QNS • 143 3 .X'
QNS ( ; 3.40 QNS ' *.}ij ] 3 50
4.20 , : 3.89 \ i 4.57 ' 3.58 ' 3 tfl
139 ' . 1.00 ; i W 0/8 U.-5'i
i ! ' ( '
^ QNS ' QNS I 3.28 , l 3.44 ' 4.02 '
, " I
9.10 | 7.41 ' 6.18 ! 4.35 ' "it?
6.49 '. 8.14 " _ 7.86 6.11 6.86
7.80 ^ /.78 ' 5 11 \ 4.63 4 »'-,
, 1-85 ' 0.52 ! ; 2.32 ( l.iO 1 f-.
__J | ]^__
Bold: > Normal Range
Underline: < Normal Range
— No Value
* Clot Removed
QNS - Sample Volume not Sufficient for analysis
Page 17 of 22
lie
-------
Study 1078-CG920794
Hematology
Parameter j Group] Animal ID Day -3
Monocytes i 1 j 37 J 0.23~Q_
(0 - 0.4 xlQ3/uL) 1 ' 40
15
1 7
1 1 ! 9~ ~~
0.07
0.09
Day 2
0.09
0.06
0.23
0.04 I i 0.06
0.13 i 0.07
: Average | 0.11
; Std Dev 0.07
- - ': i - l__ J_
; 2 , 13
; 2 • is'""""
2 19
0.14
0.08
0.19
; 2 25 0,05
1 2 28
2 30
2 34
i Average
Std Dev
3 11
0.08
0.07
0.14
0.11
0.05
0.13
! 3 12 0.05
|3 14 0-15
3 18 , 0.09
{ 3 2
i 3 32
I 3 | 8
Average
] Std Dev
0.06
0.11
0.09
0.10
0.04
4 21 i 0.28
4 27 1 0.10
: 4 31 _Jo".is
4 33
I 4 38
4 39
4 6
Average
j Std Dev
0.11
0.10
0.07
Day 4 Day 9
QNS
0.07
0.07
lo.oi
to. 11
1
JilSj
1 0.19
T 0.16
J0.17
0.07
0.05
0.17
0.14
0.05
0.12
0.06
0.15
—
0.13 j
0.17
0.13
0.15
0.06
I i
0.08
0.06
0.07
*
0.04 F
0.11
0.14
0.29
0.09
0.07
0.05
0.04 *
0.05
Hxi2
0.04
0.04
0.06
Day 11
0.17
* 0.07
6.6TT
o.TT
0.10
0.22
J 0.06
0.07
0.12
0.08
Jo.07
0.06
0.11
0.09
0.04
_____
0.12
0.16
0.03
~oiF
0.05
0.08
0.06
0.11
0.11
0.06
[ 0.19
3T°.06
0^04j
d.iTj
0.31
0.10
1 o.io
0.04
0.26 j
0.12
0.28
0.17
0.08
0.10
QNS
QNS
QNS
0.12
0.08
0.54
^
0.21
0.22
0.19
0.10
QNS
0.14
0.18
0.14
0.05
0.08
0.12
-
0.151
0.10
0.12
0.09
0.26
0.13
0.06
0.15
0.07
0.05
0.10
0.05 |
0.07
0.21
0.08
0.11
0.09
0.10
0.07
0.10
0.05
0.09
QNS
0.10
0.06
—
Day 16
0.06
_QNS
0.15
0.06
0.08
0.09
0.04
0.09
0.15
0.21
0.09
0.10
6.06
*
I
—
0.19 |
0.13
0.06
| 6.09
0.04
0.04^f
0.06
0.14
0.08
0.04
0.09
0.11
QNS
—
0.04^
QNS
0.14
0.08
I 0.04
1 0.10
0.09 ( 0.08
6.34J "r -
0.03
___
0.15
_
0.15
0.11
*
L~
0.35
"017
0.18
0.12
Day 18 Day
0.06 1 QNS
0.08 * 0.02
0.09 [ 0.12
' 0.06 i 0.05
, 0.06 '
0.07 '
0.01
0.11 '
0.17
0.28
0.08
0.08
6.11
0.07
6.13
0.07
0.08
0.04
0.11
0.14
0.11
0.14
0.10 i
_ „_
1- — - —
0.12
0.17
-
0.47
0.10 '
0.22
jxi?:
, 0.09
' 0.07
* 0.04
! QNS
O.lb
0.21
0.08
' 0.12
0.06
0.05
0.11
, 0.06
0.15
0.05
0.13
( QNS
QNS
QNS
0.11
0.05
" QNS
; 1.66
' 0.14
0.90
( 1.07
23 1 Day 25 I Day 30 ! Day 32 Day 37
4 QNS ' [ 0 16 QNS 0 ]7
j ' 0.04 | 0.07 * ' QNS 004
, 0.19 , 0.11 t QNS 'nil
1 QNb DOS 0.06 00-i
, [ 0.12 , 0.15 QNi. 0 1-
! i 0.12 0.11 OOb 0.10
0.08 0.04 0.00 OOb
1 , QNS ' ' 008 ' QNS ' 0.:..
] 0.11 | _ 0.11 QNS , (Ub
0.09 ( 0 11 ' 0 11 ' 0 .',*
0 12 ' ( 0.10 QNS ' 0.08
, 0 05 i \ 0.06 ' 0 05 0.0r-
0 06 ' 0.08 ' QNS ' 0 Ob
0.11 . 0.09 0.05 004
0.09 , 0.09 t " 0.07 0.14
0.03 p 0.0? ' 003 0 11
0.12 0,35 ! 1 0.28 " 0.20
0.04 j ' QNb O.lA QMS *
0.14 030 'f ' 0.15 , i)NS
1 0.23 , , 0.04 ' ' QNS ' 0.1 1
0.08 , ' QNS 1 ' O.Ob U.OJ
! 0.26 ( ; fJNS | ' 0.12 ' 0 it,
1 0 15 , 0.23 i 0.1S ' 0.14
0.09 _ 0.17 O.OS O.Oy
QNS ( O.lb 0.32 ',) 1 i
, 1.50 , 0.73 ' ' 0.49 0 l!l
i 0 17 ( 0.1 i ' 0.07 " 0 ,'t-
0.84 , 034 0..>M ' O.I/
1 0.94 | 034 , 0.21 0 li<
Bold: > Normal Range
Underline: < Normal Range
- No Value
* Clot Removed
QNS - Sample Volume not Sufficient for analysis
Page 18 of 22
(£> '4 IO
-------
Stydy 1078-CG920794
Hematology
Parameter Group
tosmophils 1
(0-0.4xiO'/uL) 1
1
1
1
4 2
2
Animal ID
37
40
5
7
9
Day -3
o.iol
o.iTj^
0.17
0.17
0.12
Average 0,14
Std Dev 0.03
13
0.24
15 0.19
1 2 19 I0'20
2 25 0,11
i
2
2
I 3
3
1 3
3
3
_ L 3
3
28 0.17
Day 2
0.18
0.16
0.07
0.22
0.13
0.15
1 0.06
J 0.22
j 0.17
0.33
0.11
0.18
30 j 0.12n 0.10
34 0.11
Average
Std Dev
11
0.16
0.05
0.27
12 lfl.15
14 1 0.15
18 0.18
2 0.22
32
8
Average
Std Dev
4
4
4
4
4
4
4
21
27
31
33
38
39
6
Average
Std Dev
0.18
0.12
0.18
0.05
0.22
0.19
0.08
0.16
0.19
0.10
0.13
0.15
0.12
0.16
0.14
0.03 |
0.25
0.10
0.11
0.15
0.14
0.20
Day
QNS
0.20
0.09
0.07
0.13
0.12
0.06
0.21
0.18
0.23
0.10
0.23
—
0.13
0.15
0.05
0.21
0.14
0.17
0.06
0.16
0.17
0.09
0.14
0.05
0.13
0.15
0.18
0.05
0.21
0.13
0.14
0.18
4
Day 9
0.13
0.16
0.14
* 0.16
J °-08
0.13
0.03
1
0.15
0.16
0.23
*
_
0.15
0.29
0.12
0.17
0.06
0.22
QNS
QNS
QNS
0.18
0.22
0.15
0.19
0.03
_j_
__
0.12
0.17
0.09
0.15
0.15
0.04
*
F
J
• -
-
Day 11
0.09
0.18
0.19
0.15
0.12
0.15 '
0.04
0.16
0.15
0.21
0.18
0.15
0.14
0.12
0.16
0.03
.
~
-
0.14 • 0.16
0.12
0.17
0.18
QNS
0.24
0.14
0.17
0.04
0.20
0.16
0.15
0.09
0.14
0.12
0.14
0.14
0.03
j
-
QNS
0.17
0.17
0.16
0.19
0.20
-
o.iiff
0.02 I
0.28
0.12
0.16
0.10
0.20
0.15
-
0.17
0.06
*
—
Day 16
0.18
QNS
0.17
0.14
0.31
^0.20
0.08
0.16
0.14
0.21
0.15
0.14
0.14
0.12
0.15
0.03
0.19
0.13
0.13
QNS
0.13
QNS
0.17
0.15
0.03
0.18
0.11
-
-
0.18
0.10
I
LMi
poSf
*
-
"
Day
0.19
0.14
0.20
0.17
0.26
0.19
0.04
0.16
0.11
0.21
0.12
0.15
0.15
0.13
0.15
0.03
0.18
0.13
0.15
0.07
0.14
0.15
0.14
0.04
0.25
0.16
0.14
0.09
0.16
0.07 1
18 u_Day23 Day 25 Day 30 [ Day 32 , Day 37
QNS QNS j O.-'O ' QNS 0 Ib
* _ 0.09 0 16 l 0 H , * QNS 0 jo
0.18 0.1? l 0 Id , QMS ' 0 IK
' 0.16 i QNS i 0 15 ; 0 .'i 0 50
0.26 ' 0.22 0.16 ' QNS (J i'-)
017 017 017 O.'l 0.19
0.07 O.OS 0.03 000 O'U
1 (
QNS '' QNS 0 13 ' t QNS 0.1.'
0.11 ' 0.12 014 QNS 0.13
0.23 0 27 ' _ 0.20 ' * 0.15 0 IS
' 0.13 0.18 ] ' 0 11 _ QNi 0 IJt
0.24 ! 0 17 O.:0 I j 0 14 * o It.
0.15 0 14 _ 0.10 i QNS 0.1H
' 0 11 0.16 0.18 ' 0 1-5 ' O.l1"'
0.16 0 17 ' O.lb 014 0 is
0.06 O.OS 0.04 ' 001 0 02
0.17 O.lb ' 0.21 0 !<•> 0 14
0.13 ' 0.11 QNS , 0.10 QNs
0.14 ' , 0.12 _ 0 35 _ 0.31 ' UMS
QNS 0.23 0 18 ' QMS * 0.1"
1 , * l
QNS _ 016 , QNS 0 U , 0.13
QNS ' 0.20 QNS ' 0.1^ U 14
0.15 Olfa | 0.2S ^ 0.17 ' 0.14
, 0.02 0.05 0.04 • "' O.Oi) OH]
1 ' '
QNS QNS ' ' 0 1? 0.14 ' O.lo
- ' \ " i . ~ „ ' ' ,
( r '
0.15 0.17 , 0.21 0 :0 ' , 0.1'!
0.15 0.10 0.17 0 Ifa 0 i"'
' 0.15 j 0.14 0.17 0 I/ 0 I1" "
0.00 0.05 O.OS OCH ' O'i.
"••"" • - • ™r r- 1 -4-
Bold: > Normal Range
Underline: < Normal Range
- No Value
* Clot Removed
QNS - Sample Volume not Sufficient for analysis
Page 19 of 22
,4
-------
Study 1Q78-CG920794
Hematology
Parameter , Groyp
Baiophib 1
Animal ID I Day -3 Day 2 Day 4
37 0,44 0.52
(0 1 - 0,6 <103M) 1 40 [ 0.22
1
1
5 0.08
7 0,28
9 0.07
Average I 0.22
T Std Dev
2
2
, 2
1 2 n
f 2
t 2 '
13
15
19
25
28
30
2 1 34
| Average
~~
3
3
3
3
3
3
! 4
4
4
4
, 4
4
4
Std Dev
11
12
14
18
2
32
8
Average
Std Dev
21
27
31. .
33
.3*_
39
6
Average
Std Dev
0.15
0.46
0.38
0.24
0.06
0.15
0.06 I
0.48
0.31
—
0.08
0.35
0.11
_
0.27
0,18
0.38
0.49
0.29
0.26
0.18
0.32
0.17
d.ioT^
0.331
0.26]
0.31 1
0.10
0.23
0.10
"^1
0.13 1
0.13 j
0.18
0.05
0.29
0.25
0.08
6.16~
0.09
h
i
0.08
0.15
0.06
0.43
0.27
0.17
0.34
QNS
0.31
0.08
0.07
0.09
0.14
0.12
-
0.14
0.12
0.26
0.09
0.33
0.12
0.20
0.11
0.15
—
0.13
0.26
0.03
0.37
0.34
0.05
—
0.19
0.14
0.46
0.54
0.31
0.08
0.21
0.05
0.53
0.31
0.21
0.34
0,08
0.15
0.37
Day 9 Day 11 Day 16 ! Day
0.27
0.35
0.09
* 0.32
0.07
0.22
0.13
0.47
0.45
0.27
0.12
0.16
0.09
0.51
0.30
0.18
0.27
* 0.16
0.08
0.29
0.12
0.20
0.13
0.18
QNS
QNS
QNS
0.42
0.21
0.10
0.23
0.18
0.39
QNS
0.35
0.26
0.27
*
*
0.32
0,35
0.10
0.26
0.08
0.22
0.12
0.40
0.46
0.26
0.23
Tois
0.05
0.45
0.29
_
0.15
0.31
QNS
0.09
0,25
0.17
0,20
0.04
0.33
0.21
0.07
0.18
0.14 0.10
0.18
0.26
0.08
0.21
0.36 * 0,40
QNS
] °-11
0.32
To.ii
1 0.23
0.13
0.44
0.35
0.21
0.17 I
0.21
0.10
0.37
0,26
0,12
0.35
0.23
0.10
0.26
0.21
0.17
0.03 *
0.38
0.24
—
0.22
0.12
0.35 *
0,19
0.17
QNS
0,04
QNS
0.23'
0.20
0.11
0.27 '
0.18
-
_
0.39
0.20
_
0.26
0.09
0.41
0.14
0,33
0.15
0.29
0.13
0.36
0.57
0.23
0.24
| 0.20
009
j 0.43
1 0.30
0.16
1 0.30
j 0.09
^0.23
0.10
0.15
0.29
t 019
[ 0.09
' 0,37
_ 0.16
\ 0.35
' 0.18
• 0.27
0.11
18 Day 23 Day 25 j Day 30 j Day 32 Day 37
QNS QNS ! 0.76 , ' QNS ' 0 ?')
* 0.13 0.30 i 0.25 * i QNS t) 3.H
0.14 ' 0.10 t ! 013 QNS "()!•*"
0,38 t ( QNS 0.33 , 0 3t> 0.>
( 0.22 ; 0.25 ' < 0.29 : ' QNS , 0 Jb '
0.2? _ 0.22 j 0.35 ' • Q.ib ' 0 30
0.12 ' 0.10 ' 0.24 000 ' 0 11
1 |
' QNS i ' QNS [ ' 0.29 QMS 0,?-.
1 0.54 i 0.44 ; [ 0.54 > QNS OM
0.30 0 20 0.20 i * O..'fe ' 0..J"
0.21 1 ! 0.19 ' 0.17 * QNS ' 0 l^s
0.24 i ' 0.19 ' ' 0,34 \ ' 0.3? ' ' 0 .%4
0.09 ' 009 , 0.10 ' QNS 0 ii
0.57 ' i 0.56 ' '058 0.5? 04?
0.33 I j 0.28 , 0 32 _ ' 0 3S 0 ->0
0.19 ' i 0.18 i " 0.18 ' 0.16 0 lb
0.43 ' i 0.30 0 39 0 45 '0 -io
0.13 r 009 ' , QNS ! 0.11 ' - ON.-)
0.15 ! 1 0.19 | ' 0.29 ' l 033 ' QNS "
QNS i 0 31 ( 0.17 _ QNS ( 0 18
QNS > ' 0.28 j QNS ' 0.25 ' 0 «?i '
QNS 0.38 ' ' QNS 0.38 ' L'.JtJ
0.24 > 0.26 , 028 030 ' 027 '
0 17 0.10 ,011 0.13 00*
* i |
QNS QNS , 0 35 ' 0.37 ' ' 0 JJ
i t
0.53 0.63 , , 0.62 0.55 0.64
0.27 ' 0.20 " ' 0.23 , 0.2 / 0 .V
0.40 , 0.42 I 0 40 0.40 0 4?
0.18 030 '• 0,20 0 14 ' 0 20
Bold: > Normal Range
Underline.: < Normal Range
- No Value
* Clot Removed
QMS - Sample Volume not Sufficient for analysis
•<••*:
Page 20 of 22
-------
Stydy 1078-CG920794
Hematology
Parameter j Group
Neutrophils/Lymphoiytfes Ratio 1
1
! i
l
1 l
I 2
! 2
[ 2
2
2
Animal ID Day -3 Day 2
37
40
1.503
0.502
5 0.355!
7
9
°-416L
0.275
Average [0.610
[ Std Dev J0.506
13
15
19
25
28
2 30
2
34
h
0.321
— ~ — — \—~—
0.3431
0.651
0.286
0.243
0.339
0.319
Average J_0.357
3
3
3
3
3
Std Dev JO. 134
_ 4
0.821J
0.394J
0.241
0.352
0.354
0.432
0.224
0.194
0.272
Day 4
QNS
0.469
0.313
0.274
0.577
J 0.408
] 0.140
0.418 1
0.260J
0.253
0.225
_J0.399
|0.289
0.086
11 |0_.448J
12
14
18
2
3 ] 32
3
; 4::
0.362
0.485
0.458
0.294
0.453
8 0.334
Average (0.405
Std Dev
21
4 1 27
4
4
4
4
4 ]
31
33
38
0.074
0.914
0.460
0.678
0.935
0.565
39 JO.298
6
j Average
i
Std Dev
0.374
0.603
0.252
i
0.325
Day 9
0.610
0.264
0,185
* 0.293
10.338
0.170J
0.293J
0.412
0.320
0.318
0.274
0.441
0.318
0.442]
0.311J
0.309
0.325
JO261
[0.332
0.056
0.338
0.162
0.164
*
—
*
~~
0.237T
0.356
0.292
0.292
0.249
0.090
0.328
0390 *"
0.303
0.862
0.443
0.359
0.355
0.876
1,552
JO529
0.210
0.366
0,607
0.315
0.198
0.406
0.215
0.304
0.270
0.061
0.312]
0,420
0.357
0.275 1
QNS
QNS
i QNS
0.646
0.293
0.468
1.122
0.839
QNS
0.346
0.311
0.431
Day 11
1.164
0.326
0.220
0.347
0.198
0.451
0.404
0.234
0.264
0.367
0.435
0.368
0.245
0.220
0.305
0.084
Day 16
0.371
QNS
0.258
0.336
0.355
—
0.330
0.050
0.200
0.345
*
Day
0.452
0.226
0.171
0.335
0.259
1 0.289
0.348'
0.250
To.245
[0.263
0.360
QNS
0.326
0.515
"To. 380
0.204^
0.308
0.438
j 0.737
1.187
0.442
0.584
0.397
0.240
0.325
0.291
0.366
0.079
0.308)
0.399
0.907
0.463
0.489
0.447
0.543
0.324
0.262
—
0.472
*
0.360
0.287
0.063
0.310
0.367
0.372
^QNS
0.330
QNS
0.259
0.328
0.046
0.180
0.299
0.393!
0.231
1
0.250
1
1
0.281J
0.089T
0.109
i0.236
0.226
18 Day 23
QNS ,
* ' 0.091
' 0.213
,0376
0.499
[0.295;
0.179
j
QNS !
0.400
0.392 ! 0.473;
0.205 0,324"
0.29l' ,0.392J
0.2651 0.242!
0.212 10.399
0.261
0.065
0.293
0.561
0.319
0.756
„_
0.336
0.249
0419
0 198
0233
0505
0425
0180
».
0.336
0.154
^372
' 0.079
i '
i 1.132'
!0.707!
' 0.314,
QNS i
J
1 ^
i QNS .
QNS i
^0718
, 0 409
QNS
i
i " ,
0957
,0288:
0.623;
1 0.473 '
Day 25 Day 30 Day 32 Day 3?
1 QNS '0.448 QNS 0.4? j
0.278! 0216 * QNS ' 0 W
I0.26b' ,0.222' QNS (Ul%
! - t •
QNS O.J9/ '0.310 '0.3''
0.454f 0.318* QNS ' CUt-iS
J03321 0300, 0310 ' 0 3W
•0.105 0.094 1 ! 0000 1 0 (Hi
QNS _ | 0.285 , QNS ' ' 0 34 •"
0.295 ! 'o.3? 7 ' QNS rC 354
0.379 to.424; * 0.419' (04\X
0.291 a 0.226] ' QNS ' 0.24.-
0309 0.278 L 035,'' ,03V
0.306. 0.715' QNS ,026,'
0.471 ' 10394* ' 0.290 ' 0»'
0342 i '0307, 0354 "o 3J:
0.071 0.080 !O.OfeS 'o.Url
i
I
0392 l 0 3t>9 0.663 LM'.-4
0.237, ' QNS ,0.801 ', QNS
0.314] [03!>2i '0299* QNS
0.668 '0.4fa/ QNS :037?
0532 [ QNS 0397 (1 -,s;
0.518' ' QNS , ,0.531< 0.4*1
0.443] 0396 ! 0518* 058;
0.159, ; 0.062' 0.22?' 0.0,54
QNS J .OSb^ 1.01/' 0 JH
l
1.111' 1.184 1.24 1' '03o'
0.134; '01/S1 <(U-!9 '0194
0.622 0577 0.83 3 ' 0 29 j
0.691* 0.535, " 0.52b OU&M
Bold: > Normal Range
Underline: < Normal Range
- No Value
* Clot Removed
QNS - Sample Volume not Sufficient for analysis
Page 21 of 22
ID •
-------
Stydy 1078-CG920794
Hematology
Parameter Group
Neutrophils/Lymphocytes Percentage Ratio 1
i 1
1
1
' 1
f
2
2
2
2
2
2
2
.Animal ID, Day-3j Day 2 | Day 4
37 1 1,503
40 10,502
5 Ja356
7 [0.417
9 [0.274
Average
Std Dev
13
15
19
25
28
0.610
0.506
_|0.821
0.320
0.343
0.652
0.287
0.242
30 0.337
34 0.319
Average JO357
Std Dev 0.134
3
3
3
3
3
3
3
!
11 0.448
12 0.362
14
18
2
32
8
Average
Std Dev
4
4
4
" i 4
! 4
14
..' , 4
21
27
0.486
0.458
0.295
0.451
0.334
0.405
0.073
0.916
0.462
31 1o.679
33 0.935
38
39
6
Average
0.564
0.297
0.374
0.604
Std Dev [0.252
0.393
0.241
0.352
0.355
0.432
0.225
0.193
1 QNS
0.274]_
0.419
0.260
JO.253
JO.225
JO398
0.289
0.086
0.325
0.442
0.312
0.470
0.313
0.275
0.578
0.409
Day
0.612
0.266
*
0.141 1
0.169
0.293
0.413
0.319
0.319
1 0.273
0.443
0.310
0.326J
0.354
0.262
0.333
0.055
0.358
J0354
[0.876
1.557
0.529
0.210
0.366
0.607
0.318
0.091
0.185
0.292
0.340
'•)
*
*
0.339
0.163
0.164
0.236
0.355
0.293
0.291
0.249
0.327
0.390
*
0.302
0.863
0.441
0.315 1
0.197
0.405
0.216
1 0.275
1 QNS
QNS
1 QNS
0.469
0.646
0.293
1,121
0.584
0.303
0.270
0.061
—
Day 11
1.162
0.327
0.218
0.347
0.198
0.450
0.403
0.234
0.264
0.368
0.434
0.368
0.245
0.312
0.419
0.357)
0.843
QNS
0.346
0.312
0.431
0.205
0.308
i 0.439
0.397
0.740
1.186
0.442
0.240
0.447
0.543
0.324
0.220
0.304
0.084
0.359
_
Day 16 Day 1
0.370
QNS
0.259
0.336
0.356
0.330
0.050
0.199
0.346
J3.349
0.250
"O245
0.263
1 0.360
QNS
0.3261
o.sieT
0.380)
0.325
0.291
0.366
0.079
0.309
0.399
0.908
0.462
0.488
0.263
—
0.472
0.231
0.288
* 0.454
' 0.227}
0.170
0.336;
0.257
0.289
0.110
' 0.236
! 0.226
1 0.392 !
0.206
0.2921
1 0.264
0.213
0.261
0.063 i '0.065
0.311
0.368
0.373
QNS
0.330
QNS
0.259
0.328
1 0.047
1
0.182
0.299
*
_
_
0.392
0.251
0.293
0.562 "
0.319
0.756
'0.335
0.249
0.419
0.198
0.232
0.505
1
0.424
0.180
• •• — — "
~ 1
0.281J" 0.335
0.088J 0.154
8 L_Day23 1 Day 25 Day 30 | Day 32 , Day 3?
QNS QNS ! ' 0.448' '' QNS i 041^
* 0.090, '0.2791 0.?!?' ' , QNS ' '(MA.,
0.213; ,0.265' 10222' QNS 0"1-
0.376' QNS' '0298, 0 3G ' 0.216' ; QNS ! 026'
0.400 ' " 0.472' 0394, " 0,2^1- '029s
^0.372' 0.342! ,0307 ,0.354, 'o.Vn
0.079 [ lOO/l' ,0079' 0064, 'OOoJ
' 1 ' ' !
, 1.131J 0390 '0.369! '0662! 'o36fa
0.708! 0.236 ' 'QNS' ' 0.800' QNb
0.313, 0.313, 0.351, ;0.3Ul" QNS
QNS ' ^0.667 0.469 QNS , 0.37.
QNS , ,0.534j * QNS i , 0.298 '0359
! QNS ! j051/' ' QNS ' 0532 043'
_ 0.718 10.443! 1 0.396 [ fOS18, 0.3;-
' 0.409J 0.159- '0,064, 0.22 1 'U0<-
QNS ' QNS , ,0 370' 1016' 0 •!].)
i
i
""
' '
0.957 '1.110 1.133! ,l.'41j 'uSu1^
0.288 ' 0.134 0178 0 .'39* 'dl'i1-'
0.623' 1 0.622 0577, '0832! 0 .'<•',
0.473 0.690 05-?-! 05^i>' 0'i«i-N'
Bold: > Normal Range
UnjJgrljne: < Normal Range
- No Value
* Clot Removed
QNS - Sample Volume not Sufficient for analysis
Page 22 of 22
•410
-------
APPENDIX T
INDIVIDUAL C-REACTIVE PROTEIN RESULTS
T-1
-------
Study 1078-CG920794
C-Reactive Protein (CRP)
Parameter Group Animal ID Day -3
C-Reactive Protein 1 37 1.48
(mg/dL) 1 40 'o.Ss!
1 5 ,<0.sf
1 7 <0.5i
1 9 <0.5
Average ' 0.64
Std Dev ' 0.57
1
l ' 1
2 13 <0 5 '
2 , 15 t<0.5
I 19 0 67 ,
2 ; 25 <0.s'
, 2 \ 28 ,^0.5.
2 '• 30 i<0,5*
, 2 ^ 34 1.36 j
Average 0.47
Std Dev 0.42 1
Day 2
^1.14
*0,89
<0-5L
0.56
0.71
0.7l
Day 4 [ Day 9
-
0.72
-
-
1.49
1.11
0.34^ 0.54
|QNS
QNS
QNS
0.55
<0.5
<0.5
„
0.65
043_
[6.21
<0.5
0.52
0.69
<0.5
-
<0.5
1.23
0.53
0.39
; j
; 3 11 <0.5|
: 3 • 12 <0.5T~"
3 14 <0.5 !
,
3 2 <0-C(~
1 3 32 ^°-5l J
3 _ 8
<0.5
0.61
,
0.00' 'o.Ooi '0.00 0.00 i OOO' ,)!•:
i T
- 1 QNS i - , QNS ! <-0.5 ' - ' QNS <0 b • v
<0.5\ |<0.5, i^-O.S ,"<0.s'
-------
Study 1078-CG920794
C-Reactive Protein (CRP)
Parameter Group Animal ID Day -3
Hemolyiis Index 1 ' 37 - 1 -
1 40 . -- +
1 5 ' - : -
1 ' 7 ' - ! 4-
Day 2 ] Day 4 J Day 9
-
--
--
-
. 1 , 9 - - -
2 13 ~ . -- *,++
2 15 -- ; +
2 19 ; ~ 1 +
2 ; 25 - ; -
2 ' 28 - "! +
2 so ;--";+"
2 34 - 1 4-
3 11 ',..','.
- - _
--
--
-
- i -
. ^
-
-
-
-
QNS
QNS
-_j QNS
-
-
.
+
4-
_=_
-
-
—
-
-
QNS
Day 11
-
-
--
-
—
-
4-
4-
4-
Day 16
—
-
—
—
- + 7 _
QNS
| I
— -
4- — -
4-4-
4-
4.
.._
-
-
—
_
—
3 12 ! - , . _ .. - I -
3 14 I -- i -
3 18 ; - | 4-
3 ~2 - | +
3 32 " - '4-4.
3 8 ~ I -
1 4 21 1 -- I '-"
! - - f i
4 27 1 - i 4-
i 4 31 j - j -
4 33 ' - -
4 38 -- "' -
4 39 -- ' -
I 4 i 6 1 - ! 4-
1 ! 1 1
~
-
" 7 "
+
-
I
-
—
-
..
.. \.
-
-
_
-
-
"
„
4-
+
4-
+
QNS
-
QNS
+
Day 18 Day 23 Dav25 Dav 30 Da« ^? na« *-t ;
—
—
-
_ [ _
_.
—
-
, J
QNS| - - - IQNS
. — .
-_]_ -
-
-
_
-
-
_QNS_
QNS
- i - QNS
- ' ~ f +
- QNS
-
_
—
.. | . ~* „
_ 1
-
-
-
QNS
-
—
-
-
--
-
-
+
QNS
+
4-
QNS
—
--
I
1
irl +
_.
—
-
-
1
IONS"
-
-
-
+
QNS
4-
++
4-
-
__
—
"
.
QNS -
4- - 1 -
—
—
QNS
-
-
- 1
-
•
—
__(
+"
Jl_4
T
4-
.
-
- QNS' - 1 - - - + - - ' .
+ j - QNS - . QNS " +
- - ' - i 4. • .. ! 1
i '
i ' , "
-\ - QNS! -- 'QNS -- + l - (IMS " .
, — - _
4- i -- - - 4- ' -- 4
- ' -- QNS - - , -- 4- ! - + .. +
++ - - 1 „ , . .. . ++ "
_
~ 1
-- , 4- ..! + '. ' (1Nb' , '
1
- H - '
----- + - QNS - 4- - » -
, - , . j . . ,
-- j QNS! - ' - - ' QMS . " +
- QNS -- + - QNb --
+.
- QNS -- i - ; -- QNS i . +
l ,
+ - ' - ' .. " ' , '
_
_
— '
-- ' - I -- - '
[ - i - : - -- - . - - , - + -
, s *
- No Value
- Negative; * Slight; -H- Moderate
*«• Rflanv; +++* Severe
QNS Saiwpte vol. not sufficient ibr analysis
///// tflStmroeM unable to calculate results
<0,S - Value < IOO, use LOO/2 In cakulaUon of Avg. and Std Dev.
Page 2 of 3
-------
Study 1078-CG920794
C-Reactive Protein (CRP)
Parameter Group Animal ID Day -3 . Day 2
iipemia Index 1 37 - ' - i -
1 40 .--'.'--
i s ..;.„-
1 7 ---,-.'.
i 9 ; ~ i -'_„'-
2 13 ' - -I - '•" -
2 15 - ' - ' -
2 19 -- | - , -
2 25 ~ ' . ! _.~
Qi
-
..
—
"I
y 4
QNS
QNS
QNS
Day9
-
—
—
_
-
.
QNS
Day 11
—
—
„_
_
j_ Day 16
-^ -
_
I
_
QNS
QNS^
Day
_
18
_T."
Day 23 1 Day 25 ' Day 30 Day 32 Day 37 Oav
- QNS1 - - ' - - - , - .. ' .
- i - - QNS -- ' QNS
-~ -1 - - _'.;..' '. .. . ' •
—
i i
ill!
111
1
-
-
2 28 - ' . .. 1 .
2 30 ...'...
~__1 "
_ -
~ 1 .-
2 , 34 i -
3 11 --" j - , -
3 ' 12 - ! - ' -
3 14 --'-.-
3 18
3 2 ' -- > - ' -
3 ' 32 '-"''- -
3 , g
•4 21 - ' - ""--""
—
-
i —
r~ - } - QNS - ]_- _^
- 1 -
1 — — i
4 _
1
^
I
i
4 27 ^ _
4 31
. 4 • 33
4 38
4 39 - + - ' -
.
- J2"
QNS -
QNS
QNS
QNS
|
- 1 -
,
^
—
-
._
„
QNS
±
1
QNS -
QNS
-
-
—
—
QNS
-
.
!
- 'QNS - QNSi - ' - - QNS! -
1 t
K; | .. i . i . ' >
QNS
- ^ -i - •
j- ; - - ! ! • , Ul" ' -
\- - - - ( r
" ~T_" - QNS - - - ' - - .
,.
__
- "";:
_
-
QNS - I - - QNS
QNS! -
1 j
| 4 | 6 - ^ - - J -
— — 1 L .
-
QNS
4_
—
_
„_
-
—
__
-
- IQNS; --;.'.. QNS - ' . ' ..
- \ -; ' -- '
- QNS - ! - - i QNS .. ' . .. ' .
— -j— ] — - < „
—
:: .
--
~
—
_r. - -- ; •- --
i -,
- HQ Value
• Negative; + Slight; -H- Moderate
44-4- Many; +*+4 Severe
QNS Sample ysi not sufficient for analysis
///// fnstmment wnabte to calculate rasyJts
-------
APPENDIX U
PATHOLOGY REPORT
1078-CG920794 - Pathology Report U-1
-------
Table of Contents
1.0 Introduction U-5
2.0 Pathology U-7
2.1 Necropsy U-7
3.0 Histopathology U-8
4.0 Conclusions U-23
5.0 References U-24
Appendix 1 1-1
List of Tables
Table 1. Study Design and Challenge Doses U-5
Table 2. Mortality in Rabbits Aerosol Challenged with B. anthracis Spores U-7
Table 3. Incidence Summary of Microscopic Nonneoplastic Graded Observations with
Average Severity - Males, Day 39 U-9
Table 4. Incidence Summary of Microscopic Nonneoplastic Graded Observations with
Average Severity -Males, Unscheduled (Continued) U-12
Table 5. Summary of Individual Gross and Microscopic Observations, Males (Continued).. U-15
1078-CG920794 - Pathology Report U-2
-------
List of Figures
Figure 1-1. Animal# 103 (37): Lung; normal alveoli (Control). H& E Stain. 40X 1-2
Figure 1-2. Animal# 404 (31): Lung; alveoli contain interstitial suppurative inflammation
and anthrax bacilli (arrows). Alveolar vessels contain anthrax bacilli (arrows).
H&E Stain. 40X 1-3
Figure 1-3. Animal# 407 (38): Lung, alveoli; aggregates of inflammatory cells surrounding
a foreign body (arrow). H&E Stain. 10X 1-4
Figure 1-4. Animal# 407 (38): Lung, alveoli; pyogranulomatous (epithelioid macrophages,
lymphocytes, and neutrophils) inflammatory reaction to a foreign body (arrow).
H&E Stain. 40X 1-5
Figure 1-5. Animal# 202 (34): Lung, alveoli; multinucleated giant cells are found within
alveolar spaces (arrows). H&E Stain. 10X 1-6
Figure 1-6. Animal# 202 (34): Lung, alveoli; multinucleated giant cells surrounding a
foreign body (arrow). H&E Stain. 40X 1-7
Figure 1-7. Animal# 407 (38): Lymph node, mediastinal; lymph node congestion and
lymphoid follicles necrosis/depletion. H&E Stain. 4X 1-8
Figure 1-8. Animal# 407 (38): Appendix; lymphocytes undergoing excessive apoptosis
(arrow) with macrophage infiltration (arrowheads). H&E Stain. 1 OX 1-9
1078-CG920794 - Pathology Report U-3
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List of Acronyms
CPU colony forming units
mm millimeter
SOP standard operating procedure
1078-CG920794 - Pathology Report U-4
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1.0 Introduction
The objective of this Study was to determine physiological markers of disease following multiple
exposures to Bacillus anthracis Ames strain spores. This narrative addresses gross and
microscopic findings in selected tissues.
Prior to challenge, New Zealand white male rabbits were randomized into three groups of seven
and one control group of five. Each rabbit was aerosol challenged with targeted doses of B.
anthracis Ames strain spores as outlined in Table 1.
Table 1. Study Design and Challenge Doses
Group
1
(Negative) Control3
2
3
4
Spore Dose
(CFU)
10,000a
100
1000
10,000
Number of Spore
Challenges"
15
15
15
15
Number of
Rabbits
5
7
7
7
Negative controls were challenged with irradiated spores.
Rabbits were challenged once a day
each week for three straight weeks.
b Rabbits were challenged once a day for five straight working days (Monday through Friday)
Complete necropsies were performed on all rabbits following spontaneous death or euthanasia,
including rabbits surviving to study termination on Study Day 39, according to Standard
Operating Procedure (SOP) PATH Xl-001. Protocol-specified tissues (lungs and gross lesions)
were sampled and preserved in 10% neutral buffered formalin. Standard sections of these tissues
from all rabbits were processed to slides, stained with hematoxylin and eosin, and interpreted by
a board-certified veterinary pathologist. All microscopic findings were graded semi-
quantitatively according to the following scale, with the associated numerical score used to
calculate average severity grades for each lesion by group. Minimal (Grade 1) represented the
least detectible lesion; mild (Grade 2) represented an easily discernible lesion; moderate
(Grade 3) represented a change affecting a large area of the represented tissue; and marked
(Grade 4) represented a lesion that approached maximal. The incidence summary of microscopic
observations with weighted average severity is presented in Table 3 (survivors on Day 39) and
1078-CG920794 - Pathology Report U-5
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Table 4 (unscheduled-death rabbits). In all tables, average severity for a given lesion was
calculated as the sum of severity scores in a study group divided by the total number of animals
examined in the group (unweighted).
Gross and microscopic diagnoses were entered into the PATH/TOX SYSTEM (Xybion Medical
Systems Corporation, Cedar Knolls, New Jersey) for data tabulation and analysis.
1078-CG920794 - Pathology Report U-6
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2.0 Pathology
2.1. Necropsy
One rabbit (14%) in Group 3 and four rabbits (57%) in Group 4 died or became moribund and
were euthanized between 10 and 21 days after the initial challenge (see Table 2).
Table 2. Mortality in Rabbits Aerosol Challenged with B. anthracis Spores
Group Legend: 1= Control; 2=100 cfu; 3=1,000 cfu;
Mortality (%)
Number Dead
Number of Animals
Group: la
0
0
5
2
0
0
7
4=10,000 cfu
3
14
1
7
4
57
4
7
a Control animals (Group 1) were challenged with 10,000 cfu irradiated spores.
Gross lesions consistent with anthrax in rabbits (Zaucha, et al, 1998) included discoloration of
the lungs, foci in the appendix, "accumulation" in the cecum, and/or enlargement of a
mediastinal lymph node; and were found in rabbits [305 (12), 401 (6), 402 (33), and 403 (27)].
These lesions correlated microscopically with hemorrhage, necrosis, edema/fibrin, and
suppurative (largely heterophilic admixed with bacteria and/or necrotic debris) inflammation.
Gross lesions in the lungs correlated with multiple foreign body granulomas/pyogranulomas
[rabbit 407 (38)] microscopically and were attributed to anthrax (indirectly).
Hindlimb/abdominal skin "lacerations" were diagnosed grossly in two rabbits [305 (12) and 407
(38)]. These lesions correlated microscopically with necrosuppurative inflammation but were
not associated with bacteria as seen in the single dose rabbits in a related study (Study 1020-
CG920503). While anthrax may have been a contributing factor, these lesions were more likely
due to trauma. Gross and microscopic lesions are summarized in Table 5 and are listed in the
Individual Gross and Microscopic Observations Table 6.
1078-CG920794 - Pathology Report U-7
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3.0 Histopathology
Sections of left apical and right diaphragmatic lung lobes and gross lesions were examined
microscopically for evidence of anthrax. There were no missing tissues.
Microscopic findings consistent with anthrax (Zaucha, etal, 1998) were present in tissues from
all rabbits. Lesions typical of anthrax in this Study included suppurative inflammation, necrosis,
lymphocyte necrosis/depletion, hemorrhage, edema, and/or large rod shaped bacteria (bacilli) in
the lungs, cecum, appendix, and mediastinal lymph nodes. Lung lesions attributed to B.
anthracis were primarily interstitial and consisted of minimal to mild suppurative interstitial
inflammation and interstitial and/or intravascular bacteria.
Multinucleated giant cells as well as foreign body granulomas/pyogranulmas were present in the
lungs of challenged rabbits but were not seen in control rabbits in this Study. In a related single-
dose study (1020-CG9290503), multinucleated giant cells were noted in both exposed and
control animals. However, the lesions were more severe in challenged rabbits. These
multinucleated cells and granulomas/pyogranulomas surrounded foreign material (foreign
bodies) consistent with organic debris [e.g. food particles or hair and debris from vascular access
ports (Taketoh, etal, 2009)]. As with Study 1020-CG920503, these lesions were likely the result
of altered foreign particle clearance by alveolar macrophages (macrophage dysfunction).
Macrophage dysfunction has been reported to occur in late sepsis (Pahuja, et al, 2008).
Prolonged bacteremia/sepsis attributed to anthrax could alter foreign particle clearance by
alveolar macrophages. One lesion, perivascular eosinophils in the lungs, was likely attributed to
vascular access port placement and has been observed in rodent studies (Taketoh, et al, 2009).
Necrosis in the skin was likely due to self-inflicted trauma.
1078-CG920794 - Pathology Report U-8
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Table 3. Incidence Summary of Microscopic Nonneoplastic Graded Observations with Average Severity - Males, Day 39
Tissue/Observation Group:
Appendix Number Examined:
Hemorrhage
Average Severity:
Infiltration Cellular, Macrophages
Average Severity:
Necrosis/Depletion, Lymphoid
Average Severity:
Cecum Number Examined:
Edema
Average Severity:
Hemorrhage
Average Severity:
Necrosis
Average Severity:
Lung Number Examined:
Bacteria
Average Severity:
Foreign Body
Average Severity:
Granuloma/Pyogranulomatous
Average Severity:
Hemorrhage
Average Severity:
1
0
-
-
-
-
-
-
0
-
-
-
-
-
-
5
0
0.0
0
0.0
0
0.0
0
0.0
Number Observed
2
0
-
-
-
-
-
-
0
-
-
-
-
-
-
7
0
0.0
1
0.3
0
0.0
0
0.0
Per Group
3
0
-
-
-
-
-
-
0
-
-
-
-
-
-
6
0
0.0
1
0.2
0
0.0
0
0.0
4
0
-
-
-
-
-
-
0
-
-
-
-
-
-
3
0
0.0
1
1.0
1
1.0
0
0.0
Group Legend: 1=CONTROL; 2=100 CFU; 3=1,000 CFU; 4=10,000 CFU
1078-CG920794 - Pathology Report U-9
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Table 3. Incidence Summary of Microscopic Nonneoplastic Graded Observations with Average Severity - Males, Day 39
(Continued)
Tissue/Observation Group:
Inflammation, Suppurative, Interstitial
Average Severity:
Multinucleated Giant Cells
Average Severity:
Perivascular Eosinophils
Average Severity:
Lymph Node, Mediastinal Number Examined:
Bacteria
Average Severity:
Edema/Fibrin
Average Severity:
Hemorrhage
Average Severity:
Necrosis/Depletion, Lymphoid
Average Severity:
Skin Number Examined:
Inflammation, Necrosuppurative
Average Severity:
Artery Thrombosis
Average Severity
1
0
0.0
0
0.0
2
0.4
0
-
-
-
-
-
-
-
-
0
-
-
-
-
Number Observed
2
0
0.0
1
0.3
3
0.6
0
-
-
-
-
-
-
-
-
0
-
-
-
-
Per Group
3
0
0.0
1
0.3
3
0.5
0
-
-
-
-
-
-
-
-
1
1
4.0
-
-
4
0
0.0
1
0.3
1
0.3
0
-
-
-
-
-
-
-
-
1
1
3.0
1
2.0
Group Legend: 1=CONTROL; 2=100 CFU; 3=1,000 CFU; 4=10,000 CFU
1078-CG920794 - Pathology Report U-10
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Table 4. Incidence Summary of Microscopic Nonneoplastic Graded Observations with Average Severity - Males, Unscheduled
Tissue/Observation Group:
Appendix Number Examined: 0
Hemorrhage
Average Severity:
Infiltration Cellular, Macrophages
Average Severity:
Necrosis/Depletion, Lymphoid
Average Severity:
Cecum Number Examined: 0
Edema
Average Severity:
Hemorrhage
Average Severity:
Necrosis
Average Severity:
Lung Number Examined: 0
Bacteria
Average Severity:
Foreign Body
Average Severity:
Granuloma/Pyogranulomatous
Average Severity:
Hemorrhage
Average Severity:
Number Observed Per Group
2 3
0 0
-
-
-
-
-
-
0 0
-
-
-
-
-
-
0 1
1
1.0
0
0.0
0
0.0
1
1.0
4
1
1
2.0
1
3.0
1
3.0
1
1
2.0
1
3.0
1
3.0
4
3
1.3
0
0.0
0
0.0
1
0.3
Group Legend: 1=CONTROL; 2=100 CFU; 3=1,000 CFU; 4=10,000 CFU
1078-CG920794 - Pathology Report U-11
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Table 4. Incidence Summary of Microscopic Nonneoplastic Graded Observations with Average Severity - Males, Unscheduled
(Continued)
Tissue/Obsei vation Group: 1
Inflammation, Suppurative
Average Severity:
Multinucleated Giant Cells
Average Severity:
Perivascular Eosinophils
Average Severity:
Lymph Node, Mediastinal Number Examined: 0
Bacteria
Average Severity:
Edema/Fibrin
Average Severity:
Hemorrhage
Average Severity:
Necrosi s/D epl eti on/Lymphoi d
Average Severity:
Skin Number Examined: 0
Inflammation, Necrosuppurative
Average Severity:
Thrombosis, Artery
Average Severity:
Number Observed Per Group
2 3
1
1.0
0
0.0
0
0.0
0 0
0 0
4
3
1.0
0
0.0
3
0.8
1
1
4.0
1
2.0
1
1.0
1
4.0
0
Group Legend: 1=CONTROL; 2=100 CFU; 3=1,000 CFU; 4=10,000 CFU
1078-CG920794 - Pathology Report U-12
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Table 5. Summary of Individual Gross and Microscopic Observations, Males
Group
Number
Control
100 CPU
1000 CPU
Animal Number/
Death Status"
101 (40)/FS
102 (7)/FS
103 (5)/FS
104 (9)/FS
105 (37)/FS
201 (13)/FS
202 (34)/FS
203 (25)/FS
204(15)/FS
205 (30)/FS
206 (28)/FS
207 (19)/FS
301 (14)/FS
302(11)/FS
303 (2)/FD
304 (8)/FS
305 (12)/FS
Gross Findings
Skin: Laceration(s), hindlimb, red, left
hindlimb, 40 x 20 mm
Microscopic Findings
Lung: Unremarkable.
Lung: Unremarkable.
Lung: Perivascular eosinophils, minimal.
Lung: Perivascular eosinophils, minimal.
Lung: Unremarkable.
Lung: Perivascular eosinophils, minimal.
Lung: Foreign body, mild.
Lung: Multinucleated giant cells, mild.
Lung: Unremarkable.
Lung: Perivascular eosinophils, minimal.
Lung: Unremarkable.
Lung: Perivascular eosinophils, mild.
Lung: Unremarkable.
Lung: Perivascular eosinophils, minimal.
Lung: Perivascular eosinophils, minimal.
Lung: Hemorrhage, minimal.
Lung: Inflammation, suppurative, minimal.
Lung: Bacteria, minimal.
Unremarkable.
Lung: Foreign body, minimal.
Lung: Multinucleated giant cells, mild.
Skin: Inflammation, necrosuppurative, marked.
aFD = Found Dead, FS = Final Phase Sacrifice
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U-13
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Table 5. Summary of Individual Gross and Microscopic Observations, Males (Continued)
Group
Number
1000 CPU
10,000
CPU
Animal Number/
Death Status"
306(18)/FS
307 (32)/FS
401 (6)/FD
402 (33)/FD
403 (27)/FD
Gross Findings
Cecum: Accumulation (gas). Samples of
cecum, colon, jejunum, and appendix were
collected to confirm lesion.
Lymph Node, Mediastinal: Enlarged, dark,
3x.
Appendix: Foci, multiple, red, up to 2 x 2
mm.
Microscopic Findings
Lung: Unremarkable.
Lung: Perivascular eosinophils, minimal.
Cecum: Edema, mild.
Cecum: Edema, hemorrhage and necrosis.
Cecum: Hemorrhage, moderate.
Cecum: Necrosis, moderate.
Lung: Perivascular eosinophils, minimal.
Lung: Bacteria, mild.
Lung: Hemorrhage, minimal.
Lung: Inflammation, suppurative, mild.
Lung: Perivascular eosinophils, minimal.
Lymph Node, Mediastinal: Bacteria, Marked.
Lymph Node, Mediastinal: Edema, fibrin,
mild.
Lymph Node, Mediastinal: Hemorrhage,
minimal.
Lymph Node, Mediastinal: Necrosis/depletion,
lymphoid, marked.
Appendix: Hemorrhage, mild.
Appendix: Necrosis/depletion, lymphoid,
moderate.
aFD = Found Dead, FS = Final Phase Sacrifice
1078-CG920794 - Pathology Report
U-14
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Table 5. Summary of Individual Gross and Microscopic Observations, Males (Continued)
Group
Number
10,000
CPU
Animal Number/
Death Status"
403 (27)/FD
(Continued)
404(31)/FD
405 (39)
406 (21)
407 (38)
Gross Findings
Lung: Discoloration(s), apical lobe, pale,
firm.
Skin: Laceration(s), abdominal, red,
20 x 15mm.
Microscopic Findings
Appendix: Infiltration cellular, macrophages,
moderate.
Appendix: Note: hemorrhage and necrosis.
Lung: Bacteria, minimal.
Lung: Inflammation, suppurative, minimal.
Lung: Perivascular eosinophils, minimal.
Lung: Bacteria, mild.
Lung: Inflammation, suppurative, minimal.
Lung: Foreign body, minimal.
Lung: Multinucleated giant cells, minimal.
Lung: Unremarkable.
Lung: Foreign body, moderate.
Lung: Granuloma/pyrogranuloma, moderate.
Lung: Perivascular eosinophils, minimal.
Skin: Inflammation, necrosuppurative,
moderate.
Skin: Thrombosis, artery, mild.
aFD = Found Dead, FS = Final Phase Sacrifice
Blank Space = No gross lesions observed on tissue
1078-CG920794 - Pathology Report
U-15
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Table 6. Individual Gross and Microscopic Observations, Males
Animal ID: 101 (40) Group: CONTROL
Day of Death: 39 (Final Phase Sacrifice)
Tissue
Gross Observation(s)
Microscopic Observation(s)
Lung
No gross lesions observed on tissue.
Unremarkable.
Animal ID: 102 (7) Group: CONTROL
Day of Death: 39 (Final Phase Sacrifice)
Tissue
Gross Observation(s)
Microscopic Observation(s)
Lung
No gross lesions observed on tissues.
Unremarkable.
Animal ID: 103 (5) Group: CONTROL
Day of Death: 39 (Final Phase Sacrifice)
Tissue
Gross Observation(s)
Microscopic Observation(s)
Lung
No gross lesions observed on tissue.
Perivascular eosinophils, minimal.
Animal ID: 104 (9) Group: CONTROL
Day of Death: 39 (Final Phase Sacrifice)
Tissue
Gross Observation(s)
Microscopic Observation(s)
Lung
No gross lesions observed on tissue.
Perivascular eosinophils, minimal.
1078-CG920794 - Pathology Report
U-16
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Table 6. Individual Gross and Microscopic Observations, Males (Continued)
Animal ID: 105 (37) Group: CONTROL
Day of Death: 39 (Final Phase Sacrifice)
Tissue
Gross Observation(s)
Microscopic Observation(s)
Lung
No gross observed on tissue.
Unremarkable.
Animal ID: 201 (13) Group: 100 CPU
Day of Death: 39 (Final Phase Sacrifice)
Tissue
Gross Observation(s)
Microscopic Observation(s)
Lung
No gross lesions observed on tissue.
Perivascular eosinophils, minimal.
Animal ID: 202 (34) Group: 100 CPU
Day of Death: 39 (Final Phase Sacrifice)
Tissue
Gross Observation(s)
Microscopic Observation(s)
Lung
No gross lesions observed on tissue.
Foreign body, mild.
Multinucleated giant cells, mild.
Animal ID: 203 (25) Group: 100 CPU
Day of Death: 39 (Final Phase Sacrifice)
Tissue
Gross Observation(s)
Microscopic Observation(s)
Lung
No gross lesions observed on tissue.
Unremarkable.
1078-CG920794 - Pathology Report
U-17
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Table 6. Individual Gross and Microscopic Observations, Males (Continued)
Animal ID: 204 (15) Group: 100 CPU
Day of Death: 39 (Final Phase Sacrifice)
Tissue Gross Observation(s) Microscopic Observation(s)
Lung No gross lesions observed on tissue. Perivascular eosinophils, minimal.
Animal ID: 205 (30) Group: 100 CPU
Day of Death: 39 (Final Phase Sacrifice)
Tissue Gross Observation(s) Microscopic Observation(s)
Lung No gross lesions observed on tissue. Unremarkable.
Animal ID: 206 (28) Group: 100 CPU
Day of Death: 39 (Final Phase Sacrifice)
Tissue Gross Observation(s) Microscopic Observation(s)
Lung No gross lesions observed on tissue. Perivascular eosinophils, mild.
Animal ID: 207 (19) Group: 100 CPU
Day of Death: 39 (Final Phase Sacrifice)
Tissue Gross Observation(s) Microscopic Observation(s)
Lung No gross lesions observed on tissue. Unremarkable.
1078-CG920794 - Pathology Report U-18
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Table 6. Individual Gross and Microscopic Observations, Males (Continued)
Animal ID: 301 (14) Group: 1,000 CPU
Day of Death: 39 (Final Phase Sacrifice)
Tissue
Gross Observation(s)
Microscopic Observation(s)
Lung
No gross lesions observed on tissue.
Perivascular eosinophils, minimal.
Animal ID: 302 (11) Group: 1,000 CPU
Day of Death: 39 (Final Phase Sacrifice)
Tissue
Gross Observation(s)
Microscopic Observation(s)
Lung
No gross lesions observed on tissue.
Perivascular eosinophils, minimal.
Animal ID: 303 (2) Group: 1,000 CPU
Day of Death: 18 (Found Dead)
Tissue
Gross Observation(s)
Microscopic Observation(s)
Lung
No gross lesions observed on tissue.
Hemorrhage, minimal.
Inflammation, suppurative, minimal.
Bacteria, minimal.
Animal ID: 304 (8) Group: 1,000 CPU
Day of Death: 39 (Final Phase Sacrifice)
Tissue
Gross Observation(s)
Microscopic Observation(s)
Lung
No gross lesions observed on tissue.
Unremarkable.
1078-CG920794 - Pathology Report
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Table 6. Individual Gross and Microscopic Observations, Males (Continued)
Animal ID: 305 (12) Group: 1,000 CPU
Day of Death: 39 (Final Phase Sacrifice)
Microscopic Observation(s)
Tissue
Gross Observation(s)
Foreign body, minimal.
Multinucleated giant cells, mild.
Lung
No gross lesions observed on tissue.
Inflammation, necrosuppurative, marked.
Skin
Laceration(s), hindlimb, red, left
hindlimb, 40x20mm.
Animal ID: 306 (18) Group: 1,000 CPU
Day of Death: 39 (Final Phase Sacrifice)
Tissue
Gross Observation(s)
Microscopic Observation(s)
Lung
No gross lesions observed on tissue.
Unremarkable.
Animal ID: 307 (32) Group: 1,000 CPU
Day of Death: 39 (Final Phase Sacrifice)
Tissue
Gross Observation(s)
Microscopic Observation(s)
Lung
No gross lesions observed on tissue.
Perivascular eosinophils, minimal.
Animal ID: 401 (6) Group: 10,000 CPU
Day of Death: 11 (Found Dead)
Tissue
Gross Observation(s)
Microscopic Observation(s)
Cecum
Accumulation (gas). Samples of cecum,
colon, jejunum, and appendix were
collected to confirm lesion.
Edema, mild.
Hemorrhage, moderate.
Necrosis, moderate.
Lung
No gross lesions observed on tissue.
Perivascular eosinophils, minimal.
1078-CG920794 - Pathology Report
U-20
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Table 6. Individual Gross and Microscopic Observations, Males (Continued)
Animal ID: 402 (33) Group: 10,000 CPU
Day of Death: 13 (Found Dead)
Tissue
Gross Observation(s)
Microscopic Observation(s)
Lung
No gross lesions observed on tissue.
Perivascular eosinophils, minimal.
Hemorrhage, minimal.
Inflammation, suppurative, mild.
Bacteria, mild.
Lymph Node, Mediastinal Enlarged, dark, 3x.
Necrosis/depletion, lymphoid, marked.
Bacteria, marked.
Hemorrhage, minimal.
Edema/fibrin, mild.
Animal ID: 403 (27) Group: 10,000 CPU
Day of Death: 21 (Found Dead)
Tissue
Gross Observation(s)
Microscopic Observation(s)
Appendix
Foci, multiple, red, up to 2x2mm.
Hemorrhage, mild.
Necrosis/depletion, lymphoid, moderate.
Infiltration cellular, macrophages, moderate.
Lung
No gross lesions observed on tissue.
Perivascular eosinophils, minimal.
Inflammation, suppurative, minimal.
Bacteria, minimal.
Animal ID: 404 (31) Group: 10,000 CPU
Day of Death: 15 (Found Dead)
Tissue
Gross Observation(s)
Microscopic Observation(s)
Lung
No gross lesions observed on tissue.
Inflammation, suppurative, minimal.
Bacteria, mild.
1078-CG920794 - Pathology Report
U-21
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Table 6. Individual Gross and Microscopic Observations, Males (Continued)
Animal ID: 405 (39) Group: 10,000 CPU
Day of Death: 39 (Final Phase Sacrifice)
Microscopic Observation(s)
Tissue
Gross Observation(s)
--- _.___ .-__ _ \~ s g
No gross lesions observed on tissue. Foreign body, minimal.
Lung
^-, — j -y
Multinucleated giant cells, minimum.
Animal ID: 406 (21) Group: 10,000 CPU
Day of Death: 39 (Final Phase Sacrifice)
Tissue
Gross Observation(s)
Microscopic Observation(s)
Lung
No gross lesions observed on tissue.
Unremarkable.
Animal ID: 407 (38) Group: 10,000 CPU
Day of Death: 39 (Final Phase Sacrifice)
Microscopic Observation(s)
Tissue
Gross Observation(s)
Perivascular eosinophils, minimal.
Granul oma/pyogranul om a, moderate.
Foreign body, moderate.
Lung
Discoloration(s), apical lobe, pale, firm.
Inflammation, necrosuppurative, moderate.
Thrombosis, artery, mild.
Skin
Laceration(s), abdominal, red, 20x15mm.
1078-CG920794 - Pathology Report
U-22
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4.0 Conclusions
In this multiple-dose anthrax Study, lesions typical of anthrax were generally acute and
consisted of suppurative inflammation, hemorrhage, edema, lymphocyte destruction, and/or
intravascular and intralesional bacilli. Multinucleated giant cells (as seen with Study 1020-
CG920503) were also frequently present in the lungs of challenged animals and were
attributed to anthrax septicemia. Additionally, one multiple-dosed rabbit also had well-
formed granulomas/pyogranulomas distributed randomly throughout one lung lobe.
Multinucleated giant cells and/or foreign body granulomas of the severity observed in these
single and multiple dosed rabbits are not typical of anthrax.
1078-CG920794 - Pathology Report U-23
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5.0 References
Zaucha, G.M., Pitt, L.M., Estep, I, Ivins, B.E., and Friedlander, A.M. (1998) The pathology
of experimental anthrax in rabbits exposed by inhalation and subcutaneous inoculation. Arch
PatholLabMed 122(11):982-992.
Taketoh, J., Komatsu, S., Adachi, K., and Asanuma, K. (2009) Application of an indwelling
vascular access port for intravenous administration in a repeated and intermittent dose
toxicity study in rats. Journal of Toxicological Sciences (34):39-52.
Pahuja, M., Iran, C., Wang, H., and Yin, K. (2008) Alveolar macrophage suppression in
sepsis is associated with high Mobility Group Box 1 Transmigration. Shock, (29): 754-760.
1078-CG920794 - Pathology Report U-24
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Battelle Study No. 1078-CG920794
March 21, 2011
Prepared By:
DRAFT
Crystal M. Briscoe, D.V.M. Date
Diplomate, A.C.V.P.
Study Pathologist
Approved By:
Anthony Skowronek, D.V.M., Ph.D. Date
Diplomate, A.C.V.P.
Technical Review
1078-CG920794 - Pathology Report U-25
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APPENDIX I
1078-CG920794 - Pathology Report I-1
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&s
Figure 1-1. Animal# 103 (37): Lung; normal alveoli (Control). H & E Stain. 40X
1078-CG920794 - Pathology Report
1-2
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Figure 1-2. Animal# 404 (31): Lung; alveoli contain interstitial suppurative inflammation
and anthrax bacilli (arrows). Alveolar vessels contain anthrax bacilli (arrows). H&E Stain.
40X
1078-CG920794 - Pathology Report
i-:
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Figure 1-3. Animal# 407 (38): Lung, alveoli; aggregates of inflammatory cells surrounding
a foreign body (arrow). H&E Stain. 10X
1078-CG920794 - Pathology Report
1-4
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Figure 1-4. Animal# 407 (38): Lung, alveoli; pyogranulomatous (epithelioid macrophages,
lymphocytes, and neutrophils) inflammatory reaction to a foreign body (arrow). H&E
Stain. 40X
1078-CG920794 - Pathology Report
1-5
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1078-CG920794 - Pathology Report
1-6
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1078-CG920794 - Pathology Report
1-7
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Figure 1-7. Animal# 407 (38): Lymph node, mediastinal; lymph node congestion and
lymphoid follicles necrosis/depletion. H&E Stain. 4X
1078-CG920794 - Pathology Report
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1078-CG920794 - Pathology Report
1-9
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APPENDIX V
BENCHMARK DOSE STUDY REPORT
1078-CG920794 - Benchmark Dose Study Report V-1
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Table of Contents
1. Introduction V-5
2. Methods V-6
2.1 Calculation of Doses V-6
2.2 Dose-Response Analysis Using Benchmark Dose Software V-7
2.2.1 Benchmark Dose Models for Dose-Response Relationships V-7
2.2.2 ten Berge Models V-9
2.3 Calculation of Human Equivalent Dose and Human Equivalent Concentration V-10
3. Results V-ll
3.1 Average Daily Dose V-ll
3.2 Total Aggregate Dose V-12
3.3 ten Berge V-12
3.4 Human Equivalent Dose and Human Equivalent Concentration V-13
4. Discussion V-15
5. References V-17
List of Tables
Table 1. Raw Data Used in Benchmark Dose Analysis by Individual Animal V-7
Table 2. Assumptions Used to Generate Human Equivalent Dose and Human Equivalent
Concentration V-10
Table 3. Model Parameters, Standard Errors, 95% Confidence Limits, and AIC Values for
the Statistically Significant Mathematical Model Fits for the Average Daily Dose
Data V-ll
Table 4. The BMD and BMDL at Identified BMRs for the Average Daily Dose Data V-12
Table 5. Model Parameters, Standard Errors, 95% Confidence Limits, and AIC Values for
the Statistically Significant Mathematical Model Fits for the Aggregate Dose Data. V-13
Table 6. The BMD and BMDL at Identified BMRs for the Total Aggregate Dose Data V-13
Table 7. Comparison of Single Dose and Multiple Dose Study Results for Rabbit
Inhalation Exposures to Bacillus anthracis (Ames) Spores V-16
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List of Figures
Figure 1. Interspecies Extrapolation Using BMDS Results V-14
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Acronyms and Abbreviations
ADD Average Daily Dose per Animal
AIC Akaike Information Criterion
BMDX Benchmark Dose at anx level of BMR
BMDLX Benchmark Dose Limit at anx level of BMR
BMDS Benchmark Dose Software
BMRx Benchmark Response at anx level
CPU Colony Forming Units
EPA US Environmental Protection Agency
GSD geometric standard deviation
HEC Human Equivalent Concentration
HED Human Equivalent Dose
LDso median lethal dose
MMAD median aerodynamic diameter
RDDR Regionally Deposited Dose Ratio
TAD Total Aggregate Dose per Animal
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1. Introduction
A benchmark dose analysis was conducted using Study 1078 data developed from rabbit
inhalation exposures to Bacillus anthracis spores over a 15-dose series. The outputs of the
benchmark dose analysis were then used as the inputs for an interspecies extrapolation to derive
human equivalent dose (HED) and human equivalent concentration (HEC) values. One potential
use of the calculated HED and HEC values is the development of cleanup goals to evaluate the
hazard posed by B. anthracis releases.
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2. Methods
2.1 Calculation of Doses
Individual rabbit inhaled doses (Colony Forming Units [CFU]/animal) were obtained from the
1078-CG920794 Inhalation Exposure Report (Tables 16 through 30). The nominal doses (i.e.,
100 CFU/animal/day, 1,000 CFU/animal/day, and 10,000 CFU/animal/day) were not used as
inputs in the dose-response analysis. For noninteger1 reported inhaled doses, the integer of the
inhaled dose was used in all dose calculations.
Two dose metrics of inhaled dose were evaluated in the benchmark dose analysis - the average
daily dose per animal (ADD) and the total aggregate dose per animal (TAD). The ADD
(CFU/Animal/Day) was calculated as shown in Equation 1. For the ADD, daily inhaled doses
were averaged across all exposure days until the death of the animal or the exposure duration for
those animals that survived the length of the study. The exposure duration of the study was 19
days, which captures the total number of study days including nondosing days to allow for
calculation of an average daily dose consistent with EPA guidance for discontinuous exposure
assessment (US EPA, 2002).2 The dates when each animal died during the study were obtained
from Appendix N. The TAD (Total CFU/Animal) was calculated as shown Equation 2. For the
TAD, daily inhaled doses were summed across all exposure days until the death of the animal or
the exposure duration for those animals that survived the length of the study. The calculated
ADD and TAD dose values and the presence or absence of the study endpoint (i.e., death or no
death) by individual animal are shown in Table 1.
Calculation of Average Daily Dose (1)
CFU
£ Daily Inhaled Dose (-
Average Daily Dose per Animal (ADD) =
Total Number of Days of Exposure (Days}
1 The value for the air concentration was quantified using the arithmetic average of triplicate plate counts from B.
anthracis spores captured by the measurement impingers. As such, the air concentrations and calculated daily
inhaled doses as reported included noninteger values.
2 Rabbit Number 27 died three days (i.e., August 16th) after the study exposures were completed on August 13th. For
the calculation of that individual ADD, the total number of days of exposure was calculated the same as for all
survivors (i.e., 19 days of exposure, the full period of the study) and the individual rabbit was identified as
exhibiting the lethality endpoint.
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Calculation of Total Aggregate Dose
(2)
Total Aggregate Dose per Animal (TAD} = / Daily Inhaled Dose (•
Z_i
CPU
Animal'
Table 1. Raw Data Used in Benchmark Dose Analysis by Individual Animal
Nominal Dose
Group
(CFU/Animal/Day)
100
1,000
10,000
Rabbit
Number
13
34
25
15
30
28
19
14
11
2
8
12
18
32
6
33
27
31
39
21
38
Average Daily Dose
(Inhaled CFU/Animal)
304
250
220
250
214
184
182
582
883
1,040
1,110
1,030
958
1,140
5,240
7,500
8,360
9,140
11,300
10,400
10,000
Total Aggregate Dose
(Inhaled CFU/Animal)
5,780
4,760
4,190
4,760
4,070
3,510
3,480
11,100
16,800
18,600
21,200
19,600
18,200
21,600
57,700
97,500
159,000
137,000
216,000
198,000
191,000
Death
During Study
Death
Death
Death
Death
Death
2.2 Dose-Response Analysis Using Benchmark Dose Software
2.2.1 Benchmark Dose Models for Dose-Response Relationships
For the benchmark dose evaluation, the current version of the US Environmental Protection
Agency's (EPA) Benchmark Dose Software (BMDS) (BMDS 2.1.2 Version 2.1.2.60, Build
06/11/10) (US EPA 2010a) was used to fit models to the dose-response data. Models from the
BMDS dichotomous and dichotomous-alternative model suites were used for analysis: Weibull
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model, Weibull model run as exponential (with the power coefficient fixed as one), probit, loge
probit, logistic, loge logistic, Gamma model, dichotomous Hill, probit-background response, loge
probit-background response, logistic-background response, and loge logistic-background
response.
The overall goal of benchmark dose analysis is to fit a mathematical function that best describes
the dose-response relationship in the observable low dose region of the data to enable
extrapolation to doses lower than those tested and/or interpolation among the test doses to
identify a given response level. Benchmark dose analysis estimates the dose, termed a
benchmark dose (BMD), for a specified level of benchmark dose response (BMR) observed. The
BMR is defined as the level of change in the response rate. For example, a BMR of 10% would
be equivalent to a 10% response rate of the endpoint of interest. The BMDS allows for the
change in response rate to be calculated as one of added or extra risk; extra risk was selected for
all analyses.
EPA (2008a) recommends a BMR value of 0.10 for use with dichotomous data sets for chemical
hazards when deriving a point of departure value, although users may make data-specific
determinations to select other values. To date, EPA has not developed guidance for the selection
of BMRs when conducting microbial benchmark dose analysis. For this assessment, BMRs of
0.50, 0.10, and 0.01 were reported to allow for comparison of different model estimates at
various points in the dose-response relationship. When used as inputs to the calculation of
BMDs, these BMR values correspond to estimates of 50% lethality (i.e., LD50), 10% lethality,
and 1% lethality; the resulting BMDs would be written BMD50, BMDio, and BMD0i,
respectively. The Benchmark Dose Limit (BMDL) is the 95% lower statistical confidence limit
of the calculated BMD when the 95% lower confidence limit is applied to the estimated slope
parameter value.
The BMDS software places a number of default restrictions on the slope and power values for
specified models. These restrictions operate in the slope parameter for the loge probit and loge
logistic models, where the value of the slope parameter is restricted to be equal or greater than
one, and in the power term for the gamma, Weibull, loge logistic, and loge probit models, where
the value of the power term is restricted to be greater than or equal to one. All default slope and
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power term restrictions were maintained in this analysis which prevents the modeling of supra-
linear response in the low dose region.3
The background parameter was directly specified as zero for those models allowing this selection
(i.e., loge logistic, loge probit, Weibull, and Weibull run as exponential) and the g parameter was
specified as zero for the dichotomous Hill model to ensure model fits did not incorporate a
background incidence of lethality.
Statistically valid model fits and BMD values for a given data set were identified using EPA
guidance (US EPA, 2008a). For each model, two BMDS outputs describing the fit of an
individual model to the data were evaluated: the global goodness of fit as measured by the
model-calculated Chi-square p-value and the scaled residuals calculated for each dose group.
The p-value reflects the overall goodness of fit, and a p-value of greater than 0.1 was used to
identify a statistically valid fit. The scaled residual is the difference between the model estimate
of response for an individual or dose group relative to its measured value. Scaled residuals
closest to the BMD are of most concern for benchmark dose analysis as they indicate the fit of
the model to the data in the dose region of greatest interest.
When comparing the fit of different models with valid statistical fits and equivalent restrictions,
the lowest BMDL was selected when the calculated BMDLs were not within a three-fold range
(US EPA, 2008a). However, if the BMDLs were within a three-fold range, the model with the
lowest calculated value of the Akaike Information Criterion (AIC) was selected (US EPA,
2008a). The AIC value was calculated using the log-likelihood at the maximum likelihood
estimates for the model parameters and the number of model degrees of freedom. The AIC value
is more appropriately used to compare fits across models than the Chi-square p-values because
these p-values cannot be used to compare the fits among different families of models or models
with differing numbers of parameters.
2.2.2 ten Berge Models
The ten Berge model (ten Berge, 1986), also known as Concentration x Time model, is also
available for evaluation using the BMDS. This model is appropriate for "data that identifies
3 Historically, microbial dose-response models (i.e., exponential, beta-Poisson) have exhibited linearity in the low
dose region and are mathematically precluded from displaying supra-linear behavior. Since the use of the power
term default value of one maintains this limit on supra-linear behavior in the low dose region, the power term default
that was originally recommended for use with chemical hazards was maintained.
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concentration (or dose values) and durations of exposure (the time component, typically shorter-
term durations), as well as responses (dichotomous response rates) to estimate a concentration-
time-response relationship" (U.S. EPA, 2008b). The BMDS calculates a Chi-square p-value for
each tested model and a Student t value is produced to determine the statistical significance of
model coefficients (U.S. EPA, 2008b).
2.3 Calculation of Human Equivalent Dose and Human Equivalent Concentration
An interspecies extrapolation was conducted using the assumptions identified in Table 2 and the
BMDLio value calculated using the best fitting mathematical model identified during the
benchmark dose analysis. With the exception of generating a particle size distribution-specific
pulmonary deposition rate using the Regionally Deposited Dose Ratio (RDDR) Model (U.S.
EPA, 1994), the approach to calculate the HED and HEC followed that presented in U.S. EPA
(201 Ob).
Table 2. Assumptions Used to Generate Human Equivalent Dose and Human Equivalent
Concentration
Parameter
Rabbit Pulmonary Deposition Rate
Human Inhalation Rate
Human Deposition Rate
Value
0.056
16
0.2
Units
Unitless
m3/day
Unitless
Source
Calculated with RDDR Model v. 2.3 (U.S. EPA,
1994) with Inputs of MMAD = 0.82 and
GSD=1.53 (Data Source: Figure 3, Aerosol
Exposure Report), Body Weight of 2850 g
(Arithmetic Average of Body Weight on Days, 2, 9,
and 16), and Minute Volume of 1.3 L (Average of
Calculated Minute Volume from Days 2, 9 and 15)
3 1 to <51 yrs, Mean Value, Table 6-1, Exposure
Factors Handbook, EPA 2009
Higher End of the Range of Human Depositional
Values for 1 to 2 uM particles, Figure 6-6 , U.S.
EPA 2004
GSD - geometric standard deviation
MMAD - median aerodynamic diameter
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3. Results
3.1 Average Daily Dose
The following models exhibited acceptable fits as measured by p-values and scaled residuals at
BMDLs of interest: Dichotomous-Hill, Loge Logistic, and Weibull (run as Exponential) (Table
3). The calculated BMDL values were within a three-fold range when compared at BMDLio
values (Table 4). Therefore, the model with the lowest AIC value was selected per existing EPA
benchmark dose guidance (US EPA, 2008a). Accordingly, the loge logistic model was identified
as the best fitting model to the data.
This model calculated a BMDL50 of 2,600 inhaled CPU and a BMDLio of 290 inhaled CPU
(Table 4). Calculated BMDs and BMDLs for identified BMRs are provided in Table 4.
Table 3. Model Parameters, Standard Errors, 95% Confidence Limits, and AIC Values for
the Statistically Significant Mathematical Model Fits for the Average Daily Dose Data
Model
Dichotomous-
Hill
(p=0.80)
Loge Logistic
(P=0.71)
Weibull (Run as
Exponential)
(p=0.48)
Slope
(Standard
Error)
95%
Confidence
Limit
5.88(12.4)
-18.4 to 30.2
!(*)
*
9.47E-5
(4.31E-05)
1.02E-05to
0.000179
Intercept
(Standard
Error)
95%
Confidence
Limit
-41.7(85.9)
-2 10 to 127
-8.83 (*)
*
Parameter Not in
Model
Power
(Standard
Error)
95%
Confidence
Limit
Parameter Not
in Model
Parameter Not
in Model
Power Set to 1
v and g
Parameters
(Standard
Error)
95%
Confidence
Limit
v: 0.568 (0.189)
0.199 to 0.938
g: Parameter
Specified as 0
Parameters Not
in Model
Parameters Not
in Model
AIC
Values
20.9394
18.9504
19.6503
Value of
Scaled
Residual
Closest to
BMD10
-0.299
-0.36
-0.333
* Standard Error not calculated by BMDS due to recognized error in its calculation
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Table 4. The BMD and BMDL at Identified BMRs for the Average Daily Dose Data
Dichotomous-Hill
Loge Logistic
(Best Fitting Model)
Weibull (Run as
Exponential)
BMR = 0.50
BMD50= 1,700
BMDL50= 980
BMD50= 6,800
BMDL50= 2,600
BMD50= 7,300
BMDL50=3,700
BMR = 0.10
BMD10=920
BMDL10=230
BMD10=760
BMDLJ o=290
BMD10= 1,100
BMDLJ o=570
BMR = 0.01
BMDoi = 600
BMDLoi = 19
BMD01 = 68
BMDLoi = 25
BMD0i=110
BMDLoi = 54
3.2 Total Aggregate Dose
The following models exhibited acceptable fits as measured by p-values and scaled residuals at
BMDLs of interest: Dichotomous-Hill, Loge Logistic, and Weibull (Run as Exponential) (Table
5). The calculated BMDL values were within a three-fold range when compared at BMDLio
values (Table 6). Therefore, the model with the lowest AIC value was selected per existing EPA
benchmark dose guidance (US EPA, 2008a). Accordingly, the loge logistic model was identified
as the best fitting model to the data.
This model calculated a BMDL50 of 44,000 total inhaled CPU and a BMDLio of 4,900 total
inhaled CPU (Table 6). Calculated BMDs and BMDLs for identified BMRs are provided in
Table 6.
3.3 ten Berge
The dose-response data sets using the ADD and TAD dose metrics were unable to be
successfully fit to the ten Berge model using the BMDS.
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Table 5. Model Parameters, Standard Errors, 95% Confidence Limits, and AIC Values for
the Statistically Significant Mathematical Model Fits for the Aggregate Dose Data
Model
Dichotomous-
Hill
(p=0.75)
Loge Logistic
(p=0.62)
(Best Fitting
Model)
Weibull (Run as
Exponential)
(p=0.33)
Slope
(Standard
Error)
95%
Confidence
Limit
4.27 (4.56)
-4.66 to 13.2
1C)
*
5.24E-06
(2.37E-06)
5.87E-7to
9.89E-06
Intercept
(Standard
Error)
95%
Confidence
Limit
-42.9(45.0)
-131 to 45.2
-11.7 (*)
*
Parameter Not
in Model
Power
(Standard
Error)
95%
Confidence
Limit
Parameter Not
in Model
Parameter Not
in Model
Power Set to 1
v and g
Parameters
(Standard
Error)
95%
Confidence
Limit
v: 0.563
(0.190)
0.192 to 0.935
g: Parameter
Specified as 0
Parameters Not
in Model
Parameters Not
in Model
AIC
Values
21.3862
20.3447
21.549
Value of Scaled
Residual Closest
to BMD10
-0.352
-0.309
-0.329
* Standard Error not calculated by BMDS due to recognized error in its calculation
Table 6. The BMD and BMDL at Identified BMRs for the Total Aggregate Dose Data
Dichotomous-Hill
Loge Logistic
(Best Fitting Model)
Weibull (Run as
Exponential)
BMR = 0.50
BMD5o = 38,000
BMDL50= 19,000
BMD50= 120,000
BMDL50= 44,000
BMD50= 130,000
BMDL50= 68,000
BMR = 0.10
BMD10= 16,000
BMDL10= 4,500
BMD10= 13,000
BMDL10= 4,900
BMD10= 20,000
BMDL10= 10,000
BMR = 0.01
BMD0i = 9,100
BMDLoi = 380
BMDoi = 1,200
BMDL0i= 450
BMD01 = 1,900
BMDLm = 980
3.4 Human Equivalent Dose and Human Equivalent Concentration
Using the ADD BMDLio value from the loge logistic model, the calculated values for the HED
and HEC were 1,400 inhaled CPU and 87 CFU/m3, respectively. The values for the intermediate
calculations of the interspecies extrapolation are provided in Figure 1.
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Animal
Receptor
Interspecies Extrapolation
Point of Exposure Potential Dose
EnvironmentalAir
Concentration
Human Equivalent
Concentration
Environmental
AirConcentration
87 CFU/m3
Animal
Inhaled Dose
BMDL10 = 4,900
Inhaled CPU
Human Equivalent
Dose
Human
Inhaled Dose
1,400 Inhaled CPU
Deposited Dose
to Alveolar
Region of Animal
Lung
270 Deposited
CPU
Deposited Dose
to Alveolar
Region of
Human Lung
270 Deposited
CPU
Human
Receptor
Figure 1. Interspecies extrapolation using BMDS results.
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4. Discussion
Study 1078 represents the first reported study designed to derive the dose-response relationship
of a multiple dose inhalation exposure to B. anthracis spores in the rabbit animal model. While
the rabbit is recognized as a suitable animal model for B. anthracis disease modeling (Leffel and
Pitt, 2006), few dose-response data sets suitable for analysis are found in the literature. Study
1078 is a continuation of the single dose acute study reported by Comer (2010) that built upon
the single dose study design to include multiple doses while maintaining consistency with the
protocol of the previous study.
The dose-response data for Study 1078 may also provide preliminary evidence that a threshold in
the ADD may be present below which lethality is unlikely to occur in a healthy, adult male rabbit
population. This is evidenced by of the survival rates of the two lowest dose groups. As shown in
Table 1, no lethality was exhibited in the 100 CFU/animal/day nominal dose group and only 14%
lethality was exhibited in the 1,000 CFU/animal/day nominal dose group. Further testing of
levels between these two doses may allow modeling of this threshold value.
When comparing the benchmark dose analyses for data from these two studies as reported in
Hines et al. (2011) for the first study and detailed in this report for Study 1078, there are
preliminary indications that a discernable relationship may be present in the measured endpoint
of lethality with the administered dose, number and timing of the administered doses (Table 7).
The basis for this hypothesis is that the total aggregate dose BMDLio of 4,900 CFU is
approximately 3.5 times the BMDLio of 1,400 CFU from the single dose acute study and the
total aggregate dose BMDL50 of 44,000 CFU is approximately 3.4 times the BMDL50 of 13,000
CFU from the same single dose acute study. This implies that Haber's Law, or the microbial
equivalent of the independent action hypothesis4, may not hold for B. anthracis exposures in
rabbits for the tested dose ranges and timing of doses evaluated in Study 1078 and Comer
(2010). Haber's Law, originally derived during the early 1900's for acute inhalation exposures to
volatile chemicals, describes a dose-response relationship whereby the product of concentration
(or dose, in this case) and time (or, number of doses as was tested here) is the sole determinant of
4 One aspect of the independent hypothesis for microbial effects predicts that the length of time of dose
administration (e.g., number of doses over which total dose administered) should not affect the probability of
response (Rubin, 1987).
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toxicity. The law also assumes that each element, concentration and time, contributes equally to
the toxic effect. However, this law does not hold for acute inhalation exposures to even a small
set of tested volatile chemicals (ten Berge, 1986). The model developed by ten Berge (ten
Berge, 1986) allows for an evaluation of differing exponents (i.e., other than one) on the
concentration x time equation terms. The fit of the study data to the ADD was evaluated using
the BMDS and the ten Berge model was unable to converge with the data and provide reportable
results. However, further analysis of this potential relationship using other software or models is
still desirable to definitively assess the potential for this relationship. For example, preliminary
work is being conducted using dynamic dose-response response modeling as a mechanism to
capture the time dependence of dosing on response (Mayer et al., 2010). Given the limited
availability of dose-response data sets for B. anthracis exposures, techniques to model the
relationship between dose, concentration, number of doses, and exposure duration may provide
useful information to further define the hazard posed by acute and short-term exposure scenarios
to low levels of B. anthracis contamination.
Table 7. Comparison of Single Dose and Multiple Dose Study Results for Rabbit Inhalation
Exposures to Bacillus anthracis (Ames) Spores
BMR = 0.50
BMR = 0.10
Other Reported Measures
or Study Notes
Single Dose Study Results
Hinesetal.,2011
(Dichotomous-Hill Model)
Gutting et al., 2008
Zaucha et al., 1998
(Probit Model)
BMDgo = 52,000
BMDLgo = 13,000
NA
BMDgo = 105,000
BMD10 = 5,700
BMDL10 = 1,400
NA
NA
NA
When Dosed with up to
3,360 Inhaled B. anthracis
CFU, 4 out of 4 Rabbits
Survived the Exposure
NA
Multiple Dose Study Results
Study 1078
(ADD, Daily Dose over
19 Days)
(Loge Logistic Model)
Study 1078
(TAD, Aggregate Dose
over 19 Days)
(Loge Logistic Model)
BMDgo = 6,800
BMDLgo = 2,600
BMDgo = 120,000
BMDLgo = 44,000
BMD10 = 760
BMDL10 = 290
BMD10 = 13,000
BMDL10 = 4,900
NA
NA
1078-CG920794 - Benchmark Dose Study Report
V-16
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U.S. EPA. 20lOa Benchmark Dose Software (BMDS) 2.2.2 Version 2.1.2.60 (Build 6/11/10).
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