SERA
United States
Environmental Protection
Agency
EPA 600/R-11/075 | September 2011 j www.epa.gov/ord
Acute Low Dose
Bacillus anthracis Ames
Inhalation Exposures in
the Rabbit
§f M
Office of Research and Development
National Homeland Security Research Center
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Acute Low Dose Bacillus anthracis Ames
Inhalation Exposures in the Rabbit
United States Environmental Protection Agency
National Homeland Security Research Center
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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 DW97922089 with the Defense Technical Information Center, and in turn through
Battelle Chemical, Biological, Radiological, and Nuclear Defense Information Analysis Center
(CBRNIAC) Contract No. SP0-700-00-D-3180, Delivery Order Number 0396 (Task 503) and
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.PHYSI0.01.10.SW.005.
This report has been reviewed by the Agency but does not necessarily reflect the Agency's
views. 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.
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Foreword
Following the events of September 11, 2001, the 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 in
carrying 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 to prepare for and
recover 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
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Executive Summary
Credible dose-response relationships are needed to more accurately assess the risk posed by
exposure to low level Bacillus anthracis contamination during or following a release. The
objective of this study was to evaluate physiological responses following an acute exposure to
low doses of B. anthracis Ames spores.
Groups of five New Zealand White (NZW) rabbits were implanted with D70-PCT telemetery
transmitters and subsequently aerosol challenged with average inhaled doses of 286 to 2.75 x 105
colony forming units (CFU). Control rabbits were challenged with irradiated (non-viable) spores
as negative controls or with an average inhaled dose of 8.27 x 106 CFU as positive controls. The
rabbits were then monitored for changes in non-specific 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.
All of the five positive control animals died on study; the mean time to death was 3.47 days.
Four of the five rabbits that received an average inhaled dose of 2.75 x 105 CFU 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 CFU died 4.1 and 10.9 days post-challenge. All rabbits that
received an average inhaled dose of 2.06 x 10 CFU 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 on study were bacteremic, and 73% were positive for PA in serum.
Increases in respiration, heart rate, and body temperature were observed in rabbits that
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succumbed to anthrax. Neutrophila and increased liver enzymes were also associated with
disease. Animals that survived to the end of the study never became bacteremic or toxemic.
These data suggest in the rabbit that an inhaled dose of B. anthracis spores at or above 2.54 x 104
CFU results in death and elicits measureable physiological changes. These data also suggest that
in the rabbit model of disease, inhaled doses of 2.06 x 10 CFU or lower do not cause death or
adverse changes in the measured physiological responses.
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Acknowledgments
The 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.; Michael Etheridge; Gregory Stark, Ph.D.; Kevin Tordoff, Ph.D.; Roy
Barnewall, D.V.M., Ph.D.; and Crystal Briscoe, D.V.M.
The following researchers conducted reviews of this project and final report. From the EPA
Office of Research and Development: Edwin Barth, Ph.D., P.E., C.I.H; Eletha Brady-Roberts;
Dennis Lye, Ph.D.; Abdel-Razak Kadry, D.V.M., Ph.D., D.A.B.T; Frank Schaefer III, Ph.D.; and
Eric Villegas, Ph.D. From the U.S. Naval Surface Warfare Center, Dahlgren Division: Bradford
Gutting, Ph.D.
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Contents
Disclaimer iii
Foreword iv
Executive Summary vi
Acknowledgments viii
Acronyms and Abbreviations xiii
1 Introduction 1
2 Materials and Methods 5
2.1 Test System 5
2.2 Randomization of Animals 5
2.3 Bacillus anthracis Ames Strain Spores 6
2.4 Aerosol Challenge Generation and Monitoring 7
2.5 Stability of Aerosolized Spores 11
2.6 Tel em etric Monitoring 12
2.7 Clinical Observations and Body Weights 12
2.8 Blood Collection 12
2.9 Protective Antigen ELISA 13
2.10 Bacteremia 14
2.11 T\ A/HI.IS A 16
2.12 Hematology and Clinical Chemistry 17
2.13 Necropsy and Histopathology 18
3 Results 19
3.1 Aerosol Challenges 19
3.2 Stability of Aerosolized Spores 22
3.3 Clinical Observations, Body Weights, and Mortality 23
3.4 Tel em etric Monitoring 25
3.5 Circulating Levels of Protective Antigen 36
3.6 Bacteremia 37
3.7 TNA/IgG ELISA 38
3.8 Hematology and Clinical Chemistry 38
3.9 Pathology 50
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3.10 Quality Assurance 59
3.11 Archives 59
4 Discussion and Conclusions 63
5 References 68
Tables
Table 1. Study Design and Challenge Doses 6
Table 2. Characterization of Bacillus anthracis Spores 7
Table 3. Blood Collection Schedule and Volume 13
Table 4. TaqMan® Gene Expression Assay for the B. anthracis rpoB_571 Gene 15
Table 5. Individual and Group Mean Challenge Doses 21
Table 6. Heat Shock Enumerations 23
Table 7. Abnormality Summaries by Parameter and Group Along with Fisher's Exact
Tests Comparing the Proportion Abnormal in Each Group by Parameter 27
Table 8. Mean Neutrophil Levels 41
Table 9. Mean Levels of C-Reactive Protein over the Course of the Study 45
Table 10. Mean Aspartate Aminotransferase Levels over the Course of the Study 48
Table 11. Pathology of the Rabbit Tissues 52
Table 12. Deviations and Impacts on Data Quality and Results 60
Table 13. Technical System Audit and Data Quality Audit Dates 62
Table 14. Summary Table of Responses Measured by Rabbit 67
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Figures
Figure 1. Kaplan-Meier curves representing time to death and survival data for each group. .25
Figure 2. Plot of mean baseline adjusted activity values for each group 28
Figure 3. Plot of baseline adjusted activity values for each rabbit 29
Figure 4. Plot of mean baseline adjusted heart rate values for each group 30
Figure 5. Plot of baseline adjusted heart rate values for each rabbit 31
Figure 6. Plot of mean baseline adjusted respiratory rate values for each group 32
Figure 7. Plot of mean baseline adjusted respiratory rate values for each rabbit 33
Figure 8. Plot of mean baseline adjusted temperature values for each group 34
Figure 9. Baseline adjusted temperature values for each rabbit 35
Figure 10. Neutrophil levels 42
Figure 11. C-reactive protein levels 46
Figure 12. Aspartate aminotransferase levels 49
Figure 13. Hematoxylin and eosin stain of lung alveoli showing interstitial inflammation,
and intravascular and interstitial B. anthracis bacilli (arrow) for rabbit L23225
(Group 4), 20X 54
Figure 14. Hematoxylin and eosin stain of lymph node showing B. anthracis in the sinuses
(arrow head) for rabbit L23213 (Group 6), 4X 55
Figure 15. Hematoxylin and eosin stain of lymph node showing hemorrhage and lymphocyte
depletion for rabbit L23213 (Group 6), 4X 56
Figure 16. Hematoxylin and eosin stain of thymus from rabbit L23234 (Group 5) showing
lymphocyte depletion (arrow heads), 4X 57
Figure 17. Hematoxylin and eosin stain of a lung section from rabbit L23204 (Group 6)
showing multinucleated giant cell foreign body (arrow), 20X 58
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Appendices
Appendix A Study Protocol
Appendix B Study Deviations
Appendix C Bordetella Results
Appendix D Spray Factor Report
Appendix E Aerosol Report
Appendix F Statistical Analysis of Telemetry Data
Appendix G Blood Draw Times
Appendix H Final Report on Statistical Analysis
ofPA-ELISA, qPCR, and Quantitative Bacteremia Data
Appendix I Statistical Analysis of Hematology Data
Appendix J Clinical Chemistry Statistical Report
Appendix K Heat Shock Results
Appendix L Clinical Observations
Appendix M Body Weights
Appendix N Individual Mortality Results
Appendix O Final Survival Statistical Report
Appendix P Individual Circulating PA ELISA Results
Appendix Q Individual Bacteremia Culture Results
Appendix R Individual Bacteremia qPCR Results
Appendix S Individual TNA Results
Appendix T Individual Anti-PA IgG ELIS A Results
Appendix U Iindividual HematologyResults
Appendix V Individual Clinical Chemistry Results
Appendix W Anatomic Pathology
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Acronyms and Abbreviations
ALT alanine aminotransferase
APS aerodynamic particle sizer
AST asparate aminotransferase
BBRC Battelle Biomedical Research Center
CFU colony forming unit
cm centimeter
CRP C-reactive protein
Ct cycle threshold
dL deciliter
DNA deoxyribonucleic acid
DQA Data Quality Audit
ED50 effective dose 50%
EDTA ethylene diamine tetraacetic acid
EF edema factor
ELISA enzyme-linked immunosorbent assay
EPA U.S. Environmental Protection Agency
EU endotoxin unit
FD found dead
FS final phase sacrifice
g gram
HCT hematocrit
Hg mercury
HGB hemoglobin
HRP horseradish peroxidase
IgG immunoglobulin G
InD inhaled dose
kg kilogram
L liter
LDH lactate dehydrogenase
LD50 median lethal dose
LF lethal factor
Xlll
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LOD limit of detection
LOQ limit of quantitation
|ig microgram
|iL microliter
|im micrometer
mg milligram
min minute
mL milliliter
mm millimeter
MMAD median aerodynamic diameter
MTT 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide
NF50 neutralization factor 50%
ng nanogram
NHSRC National Homeland Security Research Center
nM nanomolar
NTC no template control
NZW New Zealand White
OD optical density
PA protective antigen
PCR polymerase chain reaction
PLT platelet count
QAP Quality Assurance Plan
qPCR quantitative real-time polymerase chain reaction
RDW red cell distribution width
RNA ribonucleic acid
rPA recombinant protective antigen
rpoB DNA-directed ribonucleic acid polymerase subunit beta
SDH sorbitol dehydrogenase
SF spray factor
SST serum separator tube
TATV total accumulated tidal volume
TNA toxin neutralization assay
TNTC too numerous to count
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TSA tryptic soy agar
TSAT Technical System Audit
U units
VAP vascular access port
WBC white blood cell
4PL four parameter logistic log
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1 In
The U.S. Environmental Protection
Agency's (EPA) is responsible for assessing
the health risks associated with the
intentional release of hazardous and toxic
chemical, biological, and radiological threat
agents. EPA is currently developing tools,
technologies, and methodologies to advance
microbial risk assessment to support site-
specific cleanup, treatment, and detection
for risk-based decision making. One of the
greatest challenges faced by EPA is the
assessment of the risk posed by low levels of
biothreat agent contamination, either
immediately following a release or after
decontamination when low levels of residual
agent may remain. Bacillus anthracis, the
causative agent of anthrax, is one current
focus of EPA risk assessment activities.
Bacillus anthracis is a gram-positive, rod-
shaped, aerobic and/or facultative anaerobic,
spore-forming bacterium. Each potential
exposure route—gastrointestinal, cutaneous,
and inhalation—manifests itself in different
clinical symptoms, with inhalational anthrax
being the most lethal. The incubation period
usually varies from one to five days
depending upon the dose and route of
exposure. The onset of disease can be longer
following inhalation exposure; some reports
suggest a delayed onset of several weeks in
low dose exposure or following removal of
therapeutic intervention. The initial clinical
signs and symptoms of inhalational 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 rate (reviewed in Dixon et al.,
1999).
Bacillus anthracis is considered to be one of
the most significant biothreat agents due to
the high lethality rates from inhalation
exposure and the stability and persistence of
the spore in the environment (Inglesby et al.,
1999). The virulence of B. anthracis is
predicated upon the production of an anti-
phagocytic capsule and the anthrax toxins
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(Drysdale et al., 2005; Acenzi et al., 2002).
Three polypeptides, protective antigen (PA),
lethal factor (LF), and edema factor (EF),
interact to form two interlinked toxins of
anthrax lethal toxin and edema toxin. The
anthrax lethal toxin is produced when PA
and LF combine; the edema toxin is
produced when PA and EF combine. PA
binds to a host cell receptor and is cleaved
by a furin-like protease (Klimpel et al.,
1992). The activated PA then forms a
heptameric complex that competitively
binds three molecules of LF and/or EF
(Milne et al., 1994). The holotoxin is then
taken up by the cell via receptor-mediated
endocytosis. A decrease in endosomal pH
produces a conformational change in the PA
molecule, resulting in a pore structure for LF
and EF translocation into the cytoplasm
(Acenzi et al., 2002). LF is a zinc
metalloprotease that inhibits mitogen-
activated protein kinase signaling (Duesbery
et al., 1998; Pellizzari et al., 1999; Vitale et
al., 2000). EF, a calcium-dependent
adenylate cyclase, increases cyclic
adenosine monophosphate levels in
susceptible cells, which results in altered
water homeostasis and the inhibition of
phagocytosis (Leppla 1982; O'Brien et al,
1985). Thus, both toxins inhibit the
signaling cascades required for the
activation of immune cells to combat
disease.
An outbreak of inhalational anthrax in
Sverdlovsk, Russia in 1979 provided the
largest set of clinical specimens to study the
pathology of human anthrax (Grinberg et al.,
2001). Autopsies of outbreak victims
consistently showed pathologic
characteristics of inhalational anthrax, such
as necrotic hemorrhage of the thoracic
lymph nodes, hemorrhagic mediastinitis, and
pleural effusion. Fifty percent of the cases
involved hemorrhagic meningitis, and 92%
showed signs of gastrointestinal tract
involvement (i.e., submucosal hemorrhagic
lesions). 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 (Grinberg et al., 2001). 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
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and lung infiltrates, with B. anthracis
infection ultimately confirmed by culture
(Jernigan et al., 2001).
Although almost 10 years have passed since
the anthrax letter attacks of 2001, the lack of
an acceptable dose-response relationship for
B. anthracis continues to challenge the
development of effective risk-based
approaches for these events. Bacillus
anthracis is the most highly studied of the
currently known biothreat agents
(Wilkening, 2006), yet there are significant
data gaps in the dose-response assessment of
low dose exposures (Gutting et al., 2008),
the selection of appropriate animal models
for low dose exposures (Leffel and Pitt,
2006), and necessary data for the
extrapolation from animal to human dose-
response relationships (Coleman et al.,
2008). Collectively, these data gaps limit
progress in the development of a dose-
response relationship suitable for application
for risk-based decision making.
With some exceptions (e.g., Druett et al.,
1953), historical B. anthracis exposure
studies with published dose-response data
often relied on high doses that were
associated with median or higher levels of
lethality. Earlier studies were conducted
with a variety of test species (e.g., guinea
pigs, rabbits, various nonhuman primate
species) and exposure products (e.g.,
differing B. anthracis strains, particle sizes).
There are very few animal exposure studies
that published raw dose-response data with
adequate dose ranges for low dose
extrapolation and sufficiently described
exposure products to allow dosimetric
adjustments.
There has been a significant increase in B.
anthracis challenge studies conducted over
the past 20 years. However, these studies
were typically designed to test vaccine
effectiveness (Fellows et al., 2001), assess
the pathology of infection (Zaucha et al.,
1998), or evaluate physiologic response to
infection (Lawrence et al., 2009). Animals
were challenged with doses that were
multiples of the median lethal doses (LD50)
(e.g., doses that are 200 times or more of the
identified median lethality value (e.g.,
Fellows et al., 2001)). While dose-response
data may be obtained from control group
animals that did not receive the vaccine,
these groups typically show levels of
mortality at or near 100%. As a result, these
data are of limited value to assess dose-
response relationships.
Conducted just over a century after the first
identification of the rabbit as a potential
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animal model for anthrax and published in
1886, the Zaucha et al. (1998) study has
been identified as the definitive work
characterizing the anthrax disease
pathogenesis of rabbits. Zaucha et al. (1998)
described widespread similarities in end-
stage pathology between rabbits and
humans, as well as concordance with
nonhuman primates. Despite differences in
pathology such as a more rapid disease
progression for rabbits than humans
including clinical evidence for regional
lymph node involvement, Zaucha et al.
(1998) concluded that rabbits were a suitable
pathogenesis model for human disease.
While there are a number of studies within
the past 30 years that cite a LD50 for the
New Zealand White (NZW) rabbit
(Oryctolagus cuniculus), the published
values are clustered near the Zaucha et al.
(1998) value of 1.05 x 105 inhaled colony
forming units (CFU)/animal and ranged
from 105 to 1.1 x 105 CFU/animal. It should
be noted that none of the studies present
original dose-response data, and the actual
values are cited as laboratory-derived values
from an unpublished data set, as secondary
data, or as personal communications.
In view of the lack of low dose exposure
studies and the critical need for credible
science to support risk-based cleanup
decisions, this study was conducted 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.
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2 Materials and Methods
2.1 Test System
The protocol for the study, along with the
methods referred to herein, is provided in
Appendix A. All study deviations are
documented in Appendix B. Thirty-five
male pathogen-free NZW rabbits
(Oryctolagus cuniculus) weighing
approximately 3.5 kilograms (kg) were
purchased from Covance (Denver, PA).
Thirty rabbits were placed on study and the
remaining five served as replacements. The
study was performed at the Battelle
Biomedical Research Center (BBRC)
located in West Jefferson, OH. The rabbits
were fitted with vascular access ports (VAP)
at Covance before being shipped to the
study facility. A veterinarian implanted a
Data Sciences International (St. Paul, MN)
model D70-PCT telemetric device in all of
the rabbits prior 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 if there
were any potential correlation with active B.
bronchiseptica infection and responses in
this study. The results of the testing are
presented in Appendix C. Bordetella
bronchiseptica status was not a criterion for
rabbit placement on the study.
2.2 Randomization of Animals
Prior to challenge, the rabbits were
randomized by weight into six groups of
five rabbits per group (Table 1). The rabbits
within each group were randomized for
challenge order (based on ear tag numbers
provided by the supplier). The SAS®
software PLAN procedure (SAS Institute,
Inc., Cary, 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 6 were challenged last.
Prior to challenge, any animals with a
malfunctioning vascular access port (VAP)
or telemetric device were replaced with one
of the five extra animals. Due to patency
issues, all replacement animals with
functional ports were used. Also, animals in
the control groups (Groups 1 and 6) with
working ports were exchanged with animals
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from the experimental groups (Groups 2
through 5) with malfunctioning ports. The
group switching occurred as follows:
L23215 (formerly Group 1) was switched
with L23223 (formerly Group 2); L23227
Table 1. Study Design and Challenge Doses
(formerly Group 6) was switched with
L24213 (formerly Group 4); L23232
(formerly Group 4) was switched with
L23205 (formerly Group 6).
Group
Targeted Inhaled
No. of Spore
No. ofRabbits
Dose (CFU)
Challenges
1 (Negative control)
100 x LD50t
1
5
2
100
1
5
3
1000
1
5
4
10,000
1
5
5
100,000
1
5
6 (High dose positive control)
100 x ldJ'
1
5
Negative controls were challenged with irradiated spores
tLD50 = 1.05 x 105 CFU per Zaucha et al. (1998)
2.3 Bacillus anthracis Ames Strain
Spores
Bacillus anthracis Ames strain spores (spore
lot Ames B35) were used on this study. The
spores were characterized and qualified
prior to release for use (Table 2).
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Table 2. Characterization of Bacillus anthracis Spores
Characte rization
Acceptance Criteria
Results
Colony purity: Colony morphology on
blood agar
Pure culture
Pure culture
% Vegetative cells
<5%
0%
% Debris
<5%
0.5%
% Spore refractility
< 5% Non-refractile spore
0.4% Non-refractile spore
Viable spore count
> 1 xlO9 CFU/mL
8.56 x 109 CFU/mL
Guinea pig LD50
<10 Spores/dose (intradermal)
3.67 Spores/dose (intradermal)
Endotoxin content
< l.OEU/mL
< 0.102 EU/mL
Phenol content
0.8 - 1.2%
0.99%
EU = Endotoxin unit
inL = 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.
2.4 Aerosol Challenge Generation and
Monitoring
Prior to the start of the study, spray factor
(SF) testing was performed on the lower
target inhaled doses of B. anthracis spores to
ensure the challenge system was capable of
achieving the low dose aerosol. It was
confirmed that the targeted doses were
achievable. Appendix D contains the
methods and results of the preliminary SF
study.
The SF was a numeric correlation between
nebulizer concentration and the resulting
generated aerosol concentration. The SF was
calculated by dividing the aerosol
concentration by the starting nebulizer
suspension concentration and was used to
predict aerosol concentration for a given
starting suspension concentration. During
SF testing the overall mean SF was
determined. The formula for determining the
SF is presented in Equation 1.
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(1)
The aerosol system performance
characteristics were evaluated by
aerosolizing four target nebulizer
concentrations of B. anthracis spores
multiple times over a three day period. The
target nebulizer concentrations tested over
the three days were 1.0 x 104, 1.0 x 105, 1.0
x 106, and 1.0 x 107CFU/mL. Specifically,
each concentration was tested a total of nine
times (three times per day over three days).
Each test consisted of a 10-minute (min)
period of aerosol generation and sample
collection. The SF values determined in
testing were used to plan the exposure
durations needed in the actual single
exposure study, the details of which are
discussed in the paragraphs that follow.
On Study Day 0 (defined as Challenge Day),
the rabbits were placed into a
plethysmography chamber, passed into a
Class III biosafety cabinet system, and
aerosol challenged with targeted inhaled
doses of 1.0 x 102, 1.0 x 103, 1.0 x 104, or
1.0 x 105 CFU of B. anthracis spores (Table
1). A high dose control group was
challenged with 100 x LD50 value and the
negative control group was exposed to an
equivalent of 100 x LD50 value of gamma
irradiated spores as described below.
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.
These nebulizers were designed to generate
aerosols having an approximate
aerodynamic mean diameter of 1 to
2 micrometers (|im). Each nebulizer was
characterized for a pressure that results in
approximately 7.5 L/min flow, which
normally is approximately 28.0 pounds per
square inch, Collison nebulizer dependant.
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 in an
impinger (Model 7541; Ace Glass Inc.,
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Vinland, 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 counts per mL;
concentrations were reported in terms of
CFU/mL. 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.,
St. Paul, 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 min 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 data generated for each animal were
used to determine the TATV, which along
with the aerosol concentration, was 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., 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 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
9
-------
of the animal model to ensure accurate
TATV measurements.
The inhalation exposure system data for
each exposure were 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 culturable bioaerosol challenge
concentration, while plethysmography
measurements documented the total inhaled
volume. Total inhaled dose, as measured in
CFU, was calculated from aerosol
concentration and total inhaled volume. The
number of LD50 was calculated by dividing
the total inhaled dose by the reported
inhalation LD50 for the rabbit. The reported
LD50 value for rabbits is 105,000 inhaled
CFU/animal (Zaucha et al., 1998).
Impinger samples were enumerated by the
1 3
spread plate method (10 to 10 ontryptic
soy agar [TSA] plates in triplicate),
where C = CFU/mL
A = Average CFU per plate
D = Dilution factor.
The total inhaled dose (InD) was calculated
from the impinger sample concentration,
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.
Impinger samples from the 1.0 x 10 and 1.0
-3
x 10 CFU 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 Number 145-0045; Fisher
Scientific, Pittsburgh, PA), the filter 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 neat 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 2.
(2)
sampling parameters, and exposure duration
(Equation 3). This equation assumes 100%
10
-------
impinger sampling efficiency. The total
number of viable spores captured during
each exposure was the product of the
impinger concentration (C) and the impinger
sampler volume (V). The total number of
viable spores 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 by the product of
the aerosol concentration and the TATV.
(3)
where InD = Inhaled dose (CFU)
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).
A detailed schematic of the exposure system is located in Appendix E.
2.5 Stability of Aerosolized Spores
To determine the effect of aerosolization on
spore stability, the nebulizer and impinger
samples from the high dose group (Group 6)
were plated after the challenges with and
without heat treatment (30 min at 65°C ±
2°C). This process was repeated twice on
the Group 6 impinger samples because the
first heat treatment was done incorrectly and
the nebulizer samples were not included.
Briefly, the entire volume of impinger
sample (-19 to 20 mL) contained in a 50 mL
conical tube was placed in a 60°C water
bath. However, it was unlikely that the
samples reached the desired temperature for
the necessary length of time because of the
volume of sample and type of tube used.
Therefore the process was repeated with
modifications: 1.0 mL of impinger and
nebulizer sample from Group 6 was
transferred to a 2.0 mL vial and placed into a
heating block. One mL of tap water was also
placed in a similar vial containing a
thermometer. These samples were incubated
for 30 min at 65°C and enumerated by serial
plating on TSA. The data collected from the
second heat treatment were used to evaluate
the difference between nebulizer and
impinger samples. A two-tailed t-test
(Microsoft Office Excel) was used to
determine if any identified differences were
statistically significant (Appendices F, H, I,
J, and O).
11
-------
2.6 Telemetric Monitoring
The rabbits were surgically implanted with
telemetry units (D70-PCT transmitters, Data
Sciences International) 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 seven days pre-
challenge (baseline) and for 21 days post-
challenge.
Each animal's cage was equipped with a
Data Sciences International telemetry
receiver. The transmitters, receivers,
consolidation matrixes, cabling, and
TM
computers using the Dataquest A.R.T. data
acquisition and analysis software are all
components of the PhysioTel® Telemetry
TM
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. Telemetry surgery
and histopathology were performed at
Battelle Memorial Institute in Columbus,
OH.
2.7 Clinical Observations and Body
Weights
Following challenge, the rabbits were
observed twice daily for survivability and
clinical signs of illness that could be
attributable to B. anthracis infection (e.g.,
moribund, respiratory distress, appetite,
activity, and seizures) except on Study Day
12 when they were only observed in the
morning. Animals were weighed on Study
Days 0 (Challenge Day), 1, 2, 3, 7, 14 and
21.
2.8 Blood Collection
On Study Days -3, 1, 2, 3, 7, and 14 blood
was collected into ethylene diamine
tetraacetic acid (EDTA; -1.5 mL) and serum
separator tubes (SST; -3.5 to 4.5 mL)
(Table 3). Blood was drawn at 24, 48, and
72 hours (± 2 hours) from the median
challenge time of each group. Blood
samples also were taken from animals found
dead or prior to euthanasia. At 21 days post-
challenge, all surviving rabbits were
terminally bled via cardiac puncture
according to Table 3.
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 four hours of collection; blood was
stored at 2°C through 8°C if not analyzed
within four hours. Blood was collected from
12
-------
vascular access ports on Study Days -3, 1,2,
and 3 for both the EDTA tubes and SSTs.
After Day 3, blood collected in the EDTA
tubes was obtained from the medial
auricular artery or the marginal ear vein. If a
port failed, the medial auricular artery or the
marginal ear vein was utilized for blood
Table 3. Blood Collection Schedule and Volume
'Terminal blood sample, minimum draw volume
2.9 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 over 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-
collection regardless of the sample time
point. If a blood sample was not collected
from either the port or other appropriate
vessel, it was documented in the study file.
Appendix G contains the exact blood
collection times.
well plate at a concentration of 2
micrograms per mL (|ig/mL). The plates
were blocked with skim milk and then
incubated with rabbit serum samples
containing native PA (Catalogue 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 Biologicals,
Santa Cruz, CA), then a 2,2'-azinobis [3-
Tube Type
Study Day
-3 1 2 3 7 14 21
EDTA (~mL)
1.5
1.5
1.5
1.5
1.5
1.5
*
1.5
SST (~mL)
4.5
3.5
3.5
3.5
3.5
3.5
*
15
Total/day (~mL)
6
5
5
5
5
5
16.5
13
-------
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 the 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. The statistical
methods used to analyze the PA ELISA data
are given in Appendix H.
2.10 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). The Day
-3 samples were assayed for bacteremia via
streaking an inoculating loop of collected
blood on blood agar.
For qualitative bacteremia culture analysis,
blood samples were streaked over blood
agar plates and read for growth and
morphology consistent with B. anthracis
after a minimum incubation of 48 hours at
37°C ± 2°C. 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 g
NaThPOzi, 1.3 gNa2HPC>4 dissolved in 1.0 L
of distilled water, pH adjusted to 6.2 and
filter sterilized) from 1 x 101 to 1 x 109 and
spread plating 100 microliters (|iL) of each
dilution on to 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 the
morphology constant 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 |iL rabbit peripheral
whole blood using the fully automated
bioMerieux NucliSENS® EasyMAG™ kit
(bioMerieux, Durham, NC).
Based on published sequence data available
in GenBank (Accession Number
AE016879), oligonucleotides were designed
14
-------
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
Primer/Probe
Oligonucleotide Sequence (5'-3')
Accession No/
rpoB_571
Forward
ATT CAAAACAGCGAAACCAA
AE016879
Reverse
T CT ATT AAGATTT AT GCT CCT GAGT CAGA
Probe
6FAM-T GGAGT GGT AGAAGGT GA-NF Q
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 non-
fluorescent 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 |iL. 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 (genomic DNA isolated
from a B. anthracis vegetative culture), and
a master mix only control (no template
control, NTC). Following acquisition, data
were analyzed using the Sequence Detection
System software (Applied Biosystems Inc.).
Final reportable values were extrapolated
15
-------
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. The statistical methods used to
analyze the bacteremia culture and qPCR
data are located in Appendix H.
2.11 TNA/ELISA
To determine if the rabbits elicited an
immune response following challenge,
serum samples were analyzed by ELISA and
high throughput toxin neutralization assay
(TNA) as described below.
The ELISA was designed to quantify IgG
antibodies against B. anthracis anthrax toxin
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 (|ig/mL).
Microtiter plates were coated with purified
rPA. Unknown test samples, anti-PA IgG
reference standard serum, and positive
control sera are 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
(ELx800; 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 |ag/m L 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 (PA+LF)
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-
mediated neutralization of anthrax lethal
toxin manifested as a suppression of
16
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cytotoxicity, and hence preservation of cell
viability.
Microtiter cell plates were seeded with
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 (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 a BioTek microplate reader
(ELx800, BioTeK) at a wavelength of 570
nanometers using a 690 nanometer reference
wavelength. The TNA Statistical Analysis
System 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% [ED50] and neutralization factor
50% [NF50]).
2.12 Hematology and Clinical Chemistry
Complete hematological analysis was
performed on blood samples collected in
EDTA tubes using the Advia 120
Hematology Analyzer (Siemens Heathcare
Diagnostics, Deerfield, IL) according to
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 count
• Mean corpuscular volume
• Mean corpuscular hemoglobin
• Mean corpuscular hemoglobin
concentration
• Red cell distribution width (RDW)
• 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
I.
Clinical chemistry was performed on all
serum samples using Advia 1200 Chemistry
analyzer (Siemens Heathcare Diagnostics)
according to manufacturer's
recommendations. Evaluation included the
following parameters:
17
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• Alanine aminotransferase (ALT)
• Asparate aminotransferase (AST)
• Total bilirubin
• Total protein
• Blood urea nitrogen
• Creatinine
• Calcium
• Sodium
• Potassium
• Chloride
• Phosphorus
• Lactate dehydrogenase (LDH)
• Sorbitol dehydrogenase (SDH)
• C-reactive protein (CRP).
The normal ranges of these parameters were
determined by scientists based on mean
values appearing in Thrall et al. (2004),
Queensberry and Carpenter (1997), and
University of Nebraska Institutional Animal
Care and Use Committee (2009). The
statistical methods used to analyze the
clinical chemistry data are described in
Appendix J.
2.13 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 21. The lungs and gross lesions
from each animal were collected. The
meninges, ependyma, ventricles, frontal
cortex, hippocampus, thalamus, cerebellum,
and brain stem were included when gross
lesions were present in the brain. The brain
and standard sections of all other collected
tissues were placed in 10% neutral buffered
formalin, processed to approximately 5 |im
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.
18
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Results
lower spore concentrations. As the spore
concentration increased in the nebulizer,
more spores were aerosolized and analyzed,
thereby accounting for a larger percent of
the total mass. The result was a shift to a
larger measured particle size or MMAD.
This occurred until a spore concentration
was reached in the aerosol where the
majority of particles counted and sized were
only spores and the MMAD reflected
predominately individual spores. As a result,
the particle size did not shift any further
since the spores were predominately the
largest particle.
3
3.1 Aerosol Challenges
To determine the physiological responses of
low dose aerosol exposure to B. anthracis
spores, four groups of five rabbits were
exposed to targeted inhaled doses of 1 x 10
to 1 x 105 CFU. The individual challenge
doses as well as the group means are listed
in Table 5. Plate counts of the impinger
samples revealed that mean actual inhaled
doses were 2.86 x 102 (± 4.32 x 101) CFU to
2.75 x 105 (± 7.41 x 104) CFU. Group 6
animals were exposed to a targeted inhaled
dose of 79 x LD50 equivalents and served as
a positive control. The mass median
aerodynamic diameter (MMAD) for
challenge material for each group as
determined by an aerodynamic particle sizer
(APS) is presented in Table 5 and Appendix
E, Figure 2. The MMAD values increased
with the challenge dose because the APS
instrument sizes all particles sampled and
counted by the instrument. The APS does
not distinguish between water droplets,
debris particles, spores, etc. As a result, a
greater proportion of small water droplet and
debris were counted relative to spores at
As a negative control, Group 1 rabbits were
exposed to 100 x LD50 equivalents of
gamma irradiated spores. The impinger
plates from animal L23216 (Group 1) were
positive for B. anthracis growth. The sample
was re-plated and was still positive for B.
anthracis; therefore the original results of 3
CFU/100 |iL were reported. The rest of the
impinger samples from Group 1 were sterile
and the irradiated material was confirmed
sterile by the U.S. Centers for Disease
Control and Prevention prior to shipment to
19
-------
the study facility. This suggested that contamination during the plating procedure
growth most likely occurred because of of the individual sample.
20
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Table 5. Individual and Group Mean Challenge Doses
Group
Animal
ID
Inhaled
Dose
(CFU/
Animal)
Mean Inhaled
Dose (SD)
(CFU/Animal)
Challenge
Dose
(LD50)t
Mean
Challenge
Dose (LDS0)
Mass Median
Aerodynamic
Diameter, (jim)
(Geometric
Standard
Deviation)
Time to
Death
(day)
1
L23220
0.00
2.00
(4.58 x 10"1)
0.00
0.00
(0.00)
0.96
(1.33)
Survived
L23216*
1.00 X 101
0.00
Survived
L23218
0.00
0.00
Survived
L23223
0.00
0.00
Survived
L23222
0.00
0.00
Survived
2
L23215
3.22 x 102
2.86 x 10-
(4.32 x 101)
3.00 X 10"3
3.00 x 10"3
(4.10 x 10"4)
0.82
(1.48)
Survived
L23206
2.98 x 102
3.00 x 10"3
Survived
L23210
2.18 x 102
2.00 x 10"3
Survived
L23219
3.21 x 102
3.00 x 10"3
Survived
L23211
2.73 x 102
3.00 x 10"3
Survived
3
L23217
1.48 x 103
2.06 x 103
(3.42 x 10")
1.40 x 10"2
1.9 x 10"
(3.25 x 10"3)
0.92
(1.57)
Survived
L23230
2.02 x 103
1.90 x 10"2
Survived
L23228
2.23 x 103
2.10 x 10"2
Survived
L23227
2.32 x 103
2.20 x 10"2
Survived
L23229
2.24 x 103
2.10 x 10"2
Survived
4
L23235
1.76 x 104
2.54 x 104
(5.21 x 103)
1.68 x 10"1
2.42 x 10"1
(4.96 x lO""'
0.87
(1.59)
11
L23205
2.73 x 104
2.60 x 10"1
Survived
L23225
2.59 x 104
2.47 x 10"1
4
L23231
2.41 x 104
2.29 x 10"1
Survived
L23207
3.19 x 104
3.04 x 10"1
Survived
5
L23201
1.78 x 105
2.75 x 105
(7.41 x 104)
1.69
2.62
(7.06 x 10"1)
1.12
(1.33)
4
L23234
2.96 x 105
2.82
6
L23212
3.29 x 105
3.13
Survived
L23200
2.19 x 105
2.09
3
L23214
3.54 x 105
3.37
6
6
L23204
5.95 x 106
8.27 x 106
(1.69 x 106)
5.67 x 101
7.88 x 101
(1.61 xlO1)
1.12
(1.31)
4
L23203
8.86 x 106
8.44 x 101
5
L23213
7.29 x 106
6.94 x 101
3
L23221
8.88 x 106
8.46 x 101
2
L23232
1.04 x 107
9.88 x 101
4
*
Enumeration plates for this sample were positive for B. anthracis
^50= 1.05 x 105 CFU (Source: Zauclia et al.. 1998)
21
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Details of the aerosol challenge data were
contained in Appendix E. A report
characterizing the spray factors of the low
dose aerosols was provided in Appendix D.
3.2 Stability of Aerosolized Spores
To determine the stability of spores after the
aerosolization process, both nebulizer and
impinger samples from the Group 6
exposure runs were subjected to heat shock
(65°C for 30 min). Table 6 shows the heat
shock results from individual samples. The
mean pre-and post-heat shock nebulizer
counts were 1.25 x 109 (± 2.02 x 108) CFU
and 1.21 x 109 (± 2.05 x 108) CFU,
respectively. There was no significant
difference between the pre- and post-heat
shock counts for the nebulizer samples (P =
0.55). There was a significant difference
between the pre- and post-heat shock counts
for the impinger samples (P = 0.02). The
mean impinger pre-heat shock counts were
1.49 x 106 (± 4.30 x 105) CFU/mL but
dropped to 1.18 x 106 (± 3.30 x 105)
CFU/mL after the heat shock. This reduction
in viable spores may have been caused by
the spore coat being damaged during the
aerosolization process. Also the impinger
samples were processed twice for heat
treatment which, while unlikely, may have
had an impact on viability. The sample was
processed twice because the first heat-shock
procedure was done incorrectly as described
in the Materials and Methods Section. The
results from the failed heat treatment are
presented in Appendix K.
22
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Table 6. Heat Shock Enumerations
Sample Type
Sample ID
Enumeration
Pre-He at Shock
(CFU/mL)
Enumeration
Post-Heat Shock
(CFU/mL)
Nebulizer
L23204
9.30 x 10s
8.80 x 10s
Nebulizer
L23203
1.48 x 109
1.26 x 109
Nebulizer
L23213
1.22 x 109
1.18 x 109
Nebulizer
L23221
1.33 x 109
1.27 x 109
Nebulizer
L23232
1.27 x 109
1.44 x 109
Impinger
L23204
7.28 x 10s
6.16 x 105
Impinger
L23203
1.72 x 106
1.16 x 106
Impinger
L23213
1.68 x 106
1.34 x 106
Impinger
L23221
1.60 x 106
1.43 x 106
Impinger
L23232
1.73 x 106
1.35 x 106
3.3 Clinical Observations, Body
Weights, and Mortality
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. A
complete list of individual clinical
observations is presented in Appendix L.
The body weights of the study rabbits
remained consistent throughout the course
of the study. Individual body weights are
provided in Appendix M.
All of the rabbits in Groups 1 through 3
survived until the end of the study. Two of
the five Group 4 rabbits died 4.14 days
(L23225) and 10.85 days (L23235) post-
challenge. Rabbits L23225 and L23235 were
dosed with 2.6 x 104 CFU (0.247 x LD50)
and 1.8 x 104 (0.168 x LD50), respectively.
Four of the five Group 5 rabbits died, with a
mean time to death of 4.64 days post-
challenge. These animals received a mean
inhaled dose of 2.8 x 105 (± 7.41 x 104) CFU
or 2.62 x LD50 ± 7.06 x 10"1 equivalents of B.
cmthracis spores. All five of the high-dose
control group (Group 6) animals succumbed
to infection, with a mean time to death of
3.47 ± 1.18 days post-challenge. Appendix
N contains the individual mortality data for
each animal.
An overall Fisher's exact test on the
mortality data was significant (P < 0.0001),
indicating that the proportion of surviving
animals in at least one of the groups was
significantly different than those in the other
groups. Based on the unadjusted Fisher's
23
-------
exact tests, the proportions of surviving
animals in the negative control group
(Group 1) and in the two lowest dose groups
(Groups 2 and 3) were significantly greater
than those in Group 5 and in the high-dose
control group (Group 6). However, after
adjusting for the multiple comparisons, there
were no significant pairwise differences
between the groups since the multiple
comparisons have a more stringent
requirement for significance. The overall
log-rank test was significant (P < 0.0001),
indicating that the survival distribution in at
least one of the groups was significantly
different than those in the other groups.
Based on the unadjusted log-rank tests, the
times to death in the negative control group
(Group 1) and Groups 2 and 3 were
significantly greater than those in Groups 5
and the high dose control group (Group 6).
Furthermore, Group 4 also experienced
significantly greater times to death than the
high dose control group (Group 6).
However, after adjusting for the multiple
pairwise comparisons, only the times to
death in the negative control group
(Group 1) and in the two lowest dose groups
(Groups 2 and 3) were significantly greater
than that in the high dose control group
(Group 6). Figure 1 displays the
Kaplan-Meier curves for each of the
six dose groups. A dose-response
relationship was observed, with increased
target inhaled doses generally being
associated with decreased times to death and
greater mortality.
24
-------
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1,000 CFU (Group 3)
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100,000 CFU (Group 5)
High Dose Control (Group 6)
1
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Time to Death (Days)
21
Figure 1. Kaplan-Meier curves representing time to death and survival data for each
group.
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 -2.21 (P =
0.0147). The estimated LD50 was 5.18 x
104 CFU with a 95% Fieller confidence
interval ranging from 6.14 x 10J CFU to
7.27 x 105 CFU. This study's estimated
LD50 of 5.18 x 104 CFU was about half that
of the commonly cited Zaucha et al. (1998)
LD50 of 1.05 x 105 CFU. A list of the
individual mortality data is located in
Appendix N and complete statistical
analysis can be found in Appendix O.
3.4 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
25
-------
associated clock time, and six-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).
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
determine if there was a significant
difference between the proportions of
abnormal animals in each group (at the
0.05 significance level). Table 7 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. Note that some animals
died prior to becoming abnormal. In
addition, Table 7 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.
26
-------
Table 7. Abnormality Summaries by Parameter and Group Along with Fisher's Exact
Tests Comparing the Proportion Abnormal in Each Group by Parameter
Parameter
Group
No.
Abnormal/
Total
Proportion
Abnormal
(95% Confidence
Interval)
Mean Duration
of Abnormal
(Days)#
Fisher's Group
Effect
(P-value)
1
0/5
0.00 (0.00, 0.52)
NA
2
1/5
0.20 (0.01, 0.72)
0
Activity
3
0/5
0.00 (0.00, 0.52)
NA
0.0558
4
0/5
0.00 (0.00, 0.52)
NA
5
3/5
0.60 (0.15, 0.95)
0
6
0/5
0.00 (0.00, 0.52)
NA
1
4/5
0.80 (0.28, 0.99)
8.69
2
4/5
0.80 (0.28, 0.99)
11.63
Heart rate
3
3/5
0.60 (0.15, 0.95)
0.42
1
4
4/5
0.80 (0.28, 0.99)
8.81
5
4/5
0.80 (0.28, 0.99)
1.31
6
4/5
0.80 (0.28, 0.99)
1.31
1
2/5
0.40 (0.05, 0.85)
0
2
4/5
0.80 (0.28, 0.99)
1.06
Respiratory rate
3
2/5
0.40 (0.05, 0.85)
5.38
0.2832
4
2/5
0.40 (0.05, 0.85)
2.25
5
3/5
0.60 (0.15, 0.95)
2.08
6
5/5
1.00 (0.48, 1.00)
1.5
1
4/5
0.80 (0.28, 0.99)
6.19
2
3/5
0.60 (0.15, 0.95)
4.17
Temperature
3
3/5
0.60 (0.15, 0.95)
1.67
0.8752
4
2/5
0.40 (0.05, 0.85)
3.63
5
4/5
0.80 (0.28, 0.99)
2
6
4/5
0.80 (0.28, 0.99)
2.06
#Means exclude those animals that were never abnormal
NA = There were no abnormal animals for this group
27
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Figure 2. Plot of mean baseline adjusted activity values for each group.
Group 1
Group 2
Group 3
Group 4
Group 5
Group 6
¦ i i i i—
28
A reading was categorized as abnormal if it
fell outside of the criterion of three times the
standard deviation above or three times the
standard deviation below the mean baseline.
Figure 2 illustrates the mean activity levels
of the groups after challenge. Figure 3
shows the activity levels for each animal on
study and highlights the variability within
each group. Differences were observed in
the activity levels of each group. For four
consecutive six-hour intervals beginning on
Study Day 3 at 12:00 - 18:00, there were
significant decreases from baseline in Group
6. On Study Day 3 at 12:00 - 18:00 and
Study Day 4 at 06:00 - 12:00, the mean
decrease from baseline in Group 6 was
significantly different from the mean
increases from baseline in Groups 1 and 3.
28
-------
Baseline Adjusted Activity (Counts/Minute)
Baseline Adjusted Activity (Counts/Minute)
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Figure 8. Plot of mean baseline adjusted temperature values for each group.
Group 1
Group 2
Group 3
Group 4
Group 5
Group 6
28
Figure 8 plots the mean body temperatures
of the groups after challenge. Figure 9
shows the body temperatures for each rabbit
over the course of the study. Like the
respiration rate data, the body temperature
of each rabbit that succumbed to disease
increased just prior to death. Rabbit L23221
(Group 6) showed a decrease in body
temperature starting at midnight of the
challenge day when compared to baseline.
The cause of this drop in body temperature
is not known. The animal may have had an
elevated temperature prior to challenge or
there may have been a problem with the
transponder. However, this animal still
followed the group trend and showed a spike
in body temperature prior to death.
34
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A comparison of group means showed that
for seven consecutive six-hour intervals
beginning on Study Day 2 at 06:00 - 12:00,
there were significant increases in body
temperature from baseline in Group 6.
Excluding Study Day 3 at 12:00 - 18:00, the
mean increases from baseline at these study
times were significantly different from the
mean changes from baseline in the lower
dose groups (Groups 1 through 5). On Study
Day 5 at 06:00 -12:00 and 12:00 - 18:00,
there were significant decreases from
baseline in Group 5 that were significantly
different than the mean changes from
baseline in Groups 1 through 4. The
decrease in the Group 5 body temperature
coincided with the time that the animals
succumbed to disease. The increase in mean
body temperature of Group 4 is attributable
to the two animals that died in this group.
Rabbit L23225 showed an increase in body
temperature from approximately Study Day
2 to Study Day 4. This animal died on Study
Day 4. Rabbit L23235 had an elevated body
temperature ~8 through 11 days post-
challenge and died on Study Day 11 (Figure
9).
Measurements on inspiratory time,
expiratory time, respiration integral, and
peak amplitude were also conducted (see
Appendix F for figures and complete
statistical analysis).
3.5 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 animals in
Groups 1, 2, and 3 had observations less
than the LOD (2.0 nanogram/mL [ng/mL])
at all time points assayed. One Group 4,
three Group 5, and four Group 6 animals
had detectable levels of circulating PA at
some point during the study.
Rabbit L23225 (Group 4) had 51.199 ng/mL
of circulating PA on Study Day 3 and this
increased to 32,964.272 ng/mL on Study
Day 4 when the terminal sample was taken.
Rabbit L23235 (Group 4) never became
positive for circulating PA but died on Study
Day 11. This animal may have become
positive after Study Day 7. However, the
blood collection schedule did not collect a
sample between Days 7 and 11, and a
terminal blood sample was not obtained
from this animal.
Rabbit L23201 (Group 5) had PA levels of
64.857 and 471.046 ng/mL on Study Days 2
and 3, respectively (no terminal sample was
obtained). Rabbits L23214 and L23234
36
-------
(Group 5) had 28.579 (Study 2) and 20.082
ng/mL (Study Day 3) of circulating PA,
respectively. Terminal samples were not
collected for either of these rabbits.
Interestingly, rabbit L23200 (Group 5) never
had detectable levels of PA but died on
Study Day 3. The terminal sample for this
animal was also below the LOD.
Rabbit L23203 (Group 6) had 2.833 and
1.631 ng/mL on Study Days 2 and 3,
respectively. Rabbit L23232 had 2.107
ng/mL of circulating PA on Study Day 3.
Rabbit L23213 (Group 6) had 125.095
ng/mL of circulating PA on Study Day 2 and
a terminal level of 383.515 ng/mL. Rabbit
L23221 (Group 6) was below the LOD on
Study Day 1 and died on Study Day 2 prior
to the blood draw, so no terminal sample
was obtained. The complete statistical report
is presented in Appendix H and the PA
ELISA results for individual animals are
contained in Appendix P.
3.6 Bacteremia
All animals in Groups 1, 2, and 3 were
negative for B. anthracis bacteremia by
culture on all study days. Two out of five
rabbits in Group 4 were bacteremic at some
point during the study, and both succumbed
to disease. Rabbit L23225 (Group 4) had a
bacterial burden of 4.53 x 103 CFU/mL of
blood on Study Day 2 and 8.03 x 105
CFU/mL at the time of death. This animal
was also positive on Study Day 3, but the
bacterial load was below the limit of
quantitation (LOQ) (2.5 x 103 CFU/mL).
Rabbit L23235 (Group 4) had 1.70 x 106
CFU/mL at the time of death; all other
samples for this animal were negative.
Four of the five Group 5 animals became
bacteremic at some time point during the
study. Three of the four animals that died in
this group were bacteremic at the time of
death. Rabbit L23214 (Group 5) was
negative for bacteremia culture at the time
of death (5.8 days post-challenge), but had
8.23 x 103 CFU/mL of blood on Study Day
2.
All of the Group 6 rabbits (5/5) had positive
bacteremia cultures during the study. Only
one animal (L23232) did not have positive
bacteremia culture at the time of death
(Study Day 4), but had 2.77 x 103 CFU/mL
of blood on Study Day 3. A list of individual
bacteremia culture results per animal is
provided in Appendix Q.
Quantitative bacteremia was also assessed
by qPCR targeting the rpoB gene. The two
Group 4 animals that were positive by
bacteremia culture (L23225 and L23235)
37
-------
were also positive for qPCR. Rabbit L23225
was positive on Study Days 1 (21
copies/|iL), 2 (18 copies/|iL) and 3 (109
copies/|iL). The animal died on Study Day 4
and no qPCR sample could be taken. The
Day -3 sample for L23225 was also positive
with a value of 234 copies/|iL, suggesting
the sample was contaminated with B.
anthracis DNA. The sample was repeated,
but still positive with a value of 15
copies/|iL. It is possible that the qPCR assay
detected another Bacillus species as this is a
quantitative method and not used for
identification.
The pPCR data mimicked the culture results
for rabbit L23235 (Group 4); only the
terminal sample was positive (7,394
copies/|iL).
Four out of five Group 5 rabbits were
positive for qPCR at some point during the
study, and all four that died were positive at
the time of death with a mean value of 7.2 x
104(± 1.35 x 105) copies/|iL. Four out of
five rabbits in Group 6 were also positive for
qPCR at some point during the study. Rabbit
L23221 died on Study Day 2 prior to sample
collection, and a qPCR sample could not be
taken on Study Day 1. This animal was
negative for qPCR at the time of death, but
positive by culture (2.58 x 106 CFU/mL). A
complete list of bacteremia and qPCR
results is given in Appendix R. The
complete statistical analysis of the
bacteremia data can be found in Appendix
H.
3.7 TNA/IgG ELISA
Individual TNA and IgG ELISA results are
presented in Appendices S and T,
respectively. All samples were below the
LOQ for both assays. All ELISA samples
had 0 |ig/mL of anti-PA IgG except for one
animal. Rabbit L23229 (Group 3) had levels
of 0.386, 0.502, and 0.404 |ig/mL on Study
Days 7, 14, and 21. While these samples did
produce results, they were well under the
LOQ of 5.0 |ig/mL and even the LOD of 1.0
|ig/mL, The samples were repeated, but the
ELISA plate did not pass acceptance
criteria, therefore only the original data were
reported.
3.8 Hematology and Clinical Chemistry
To further assess any physiological effects
of low dose exposure to B. anthracis, whole
blood and sera were assayed for a variety of
hematological and clinical chemistry
parameters (refer to Section 2.12 for a
complete list of parameters). A list of
individual animal hematology and clinical
chemistry results for all hematology and
clinical chemistry parameters is provided in
38
-------
Appendices U and V, respectively. Values
for the normal range of hematology and
clinical parameters are also provided in
Appendices U and V, respectively.
Appendices I and J contain the results of
extensive statistical analyses of the
hematological and clinical chemistry
parameters, respectively. The statistical
results provided in Appendices I and J were,
as stated in the Appendices, utilized to
perform a group-level analysis of
hematological and clinical chemistry
parameters. However, the interpretation of
the group-level statistics in this study was
hampered by the low number of starting
animals per group and the further decrease
in the group number from animal deaths due
to the treatment effects. Additionally, the
group means could be biased due to the
presence of animals in a group that were a
mixture of non-infected and infected
individuals. In recognition of these
concerns, an analysis was also conducted at
the level of the individual animal comparing
individual results to published normal
ranges of parameter values described in the
literature (see Appendices U and V for an
identification of normal values). In the
discussion below, the results of both the
group and individual-level analyses were
used to formulate findings based on these
data.
The mammalian host responds to
extracellular bacterial infection by increased
hematopoiesis and neutrophilia. To
determine if the rabbits responded to the
various doses of B. anthracis, complete
WBC counts and differentials were
performed. For hematology parameters, the
threshold for an abnormal parameter value
was defined as each individual animal's
baseline (Study Day -3) parameter value
plus or minus two standard deviations. Since
each animal had only one baseline value for
each parameter, the standard deviation
associated with each parameter was
calculated using the baseline values of all
animals. Animals were determined to have
an abnormal parameter value when their
observed value was above the upper
threshold or below the lower threshold for
that respective parameter.
There was a positive relationship between
the challenge dose and circulating
neutrophils, with an increase in the
challenge dose resulting in an increase in the
concentration of circulating neturophils.
Table 8 shows the mean neutrophil levels by
group over the study, and Figure 10 shows
the neutrophil counts by individual rabbit. A
39
-------
decrease in the time to neutrophilia was also
observed with an increase in the challenge
dose. The highest dose group (Group 6) had
the greatest and earliest increase in
neutrophils from baseline levels followed by
Group 5 and then Group 4. However, only a
few rabbits had levels above the normal
range. Groups 1 through 3 did not exhibit a
neutrophilic response following infection,
but two Group 1 rabbits had higher than
normal levels on Day -3.
Group 6 had a significant increase in
neutrophils from baseline to Study Day 2,
with a mean of 4.37 (± 1.53) x 103/|iL
compared to 1.68 (± 0.27) x 103/|iL at
baseline. This shift from baseline for Group
6 was statistically different from all other
groups on this day (P < 0.05; Tukey Test).
While this shift from baseline was
significant, only two animals (L23204 and
L23203) had levels outside of the normal
range of 0.80 to 2.90 x 103/|iL (Schalm et
al., 1975).
40
-------
Table 8. Mean Neutrophil Levels
Neutrophil Counts (103/pL)
Day-3
Day 1
Day 2
Day 3
Day 7
Day 14
Day 21
N
5
5
5
5
5
5
5
Group 1
Mean
2.5
1.34
1.3
1.3
1.5
1.17
0.91
Std Dev
1.36
0.78
0.51
0.67
0.71
0.76
0.19
N
5
5
5
4a
4a
5
4a
Group 2
Average
1.73
1.48
1.4
1.08
1.09
1.34
0.6
Std Dev
0.34
0.21
0.33
0.2
0.18
0.48
0.14
N
5
5
5
5
5
5
5
Group 3
Mean
1.64
1.52
1.46
1.4
1.44
1.45
0.75
Std Dev
0.36
0.37
0.37
0.38
0.36
0.61
0.17
N
5
5
5
5
4
3
3
Group 4
Mean
1.8
1.87
1.99
1.22
2.32
1.4
0.57
Std Dev
0.35
0.66
1.07
0.72
1.25
0.12
0.18
N
5
5
5
3
1
1
1
Group 5
Mean
2.59
1.94
2.17
3.48
1.32
0.82
0.56
Std Dev
1.04
1.23
1.56
2.11
NA
NA
NA
N
3b
4
3
3
--
--
--
Group 6
Mean
1.68
2.07
4.37
1.28
--
--
--
Std Dev
0.27
0.25
1.53
0.69
--
--
--
N = Number of samples analyzed
NA = Not applicable (only one sample)
Std Dev = Standard deviation
-- = No rabbits alive at time point
a Only four out of the five rabbits alive at the time point were assayed
b Only three out of the five rabbits alive at the time point were assayed
41
-------
Group 1
#
L23216
~
L23218
~
L23220
o
L23222
~
L23223
Normal Range
-o--
f
ai
£
-n-
£
w A
I I I
IB
Group 3
6 -
•
L23217
~
L23227
~
L23228
o
L23229
~
L23230
Normal Range
S
D
z
8
6
2 ~ ~
m
8
2
~
S
1
Group 5
•
L23200
L23201
~
L23212
O
L23214
A
L23234
Normal Range
~
o
~
~
1
$
~
o
S
0
1
Group 2
#
L23206
~
L23210
~
123211
0
L23215
~
L23219
Normal Range
_Sil
t
Group 4
•
L23205
~
L23207
~
L23225
o
L23231
~
L23235
Normal Range
ooo
••I
o
f
Group 6
•
L23203
V
L23204
~
L23213
O
L23221
~
L23232
Normal Range
~
~
S T
— I -
Study Days
Study Days
Figure 10. Neutrophil levels.
42
-------
On Study Day 3, the neutrophil level peaked
in Group 5. Two of the three rabbits alive at
this time point in Group 5 had neutrophil
levels of 4.86 and 4.53 x 103/|iL (Figure 10).
These two animals died prior to the next
blood draw. Two Group 4 rabbits showed
abnormally high neutrophil levels (L23225
[3.68 x 103/|iL, Day 2] and L23235 [3.97 x
-3
10 /|iL, Day 7]); both succumbed to
infection. The other animals in this group
were not neutrophilic at any time during the
study.
While the level of circulating neutrophils
was the only parameter that exhibited an
obvious dose response, significant changes
from baseline were observed in PLT, WBC
counts, and the numbers of circulating
neutrophils, lymphocytes, and monocytes.
However, these slight variations in the
measured parameters did not appear to be an
effect of the B. anthracis challenge doses as
there was no direct relationship between
challenge dose and change in any of these
parameters. Figures and complete statistical
analysis of all hematological parameters are
located in Appendix I.
CRP is an indicator of stress and non-
specific inflammation. CRP can also be used
as a marker for liver damage. Table 9 shows
the levels of CRP during the course of the
study, and Figure 11 shows the levels of
CRP by individual animal. The negative
control group (Group 1) had a mean CRP
level of 1.80 mg/deciliter (dL) on Day -3.
This abnormally high level of CRP was
from two animals (L23220 and L23223) that
had levels of 2.98 and 5.26 mg/dL,
respectively (Figure 11). All groups had at
least one animal with detectable levels of
CRP through Study Day 3 (Figure 11).
There were significant increases to the CRP
level in relation to the baseline level in
Groups 2, 3, 5, and 6 at Study Day 1 (P <
0.05), with the increases in Groups 2 and 5
both significantly different than the decrease
in Group 1. As a proportion of baseline, the
levels for Groups 5 and 6 significantly
increased at Study Day 2 (P < 0.05), with
the increase in Group 6 significantly greater
than those in Groups 1, 2, 3, and 4. There
were also significant increases as a
proportion of baseline in the levels for
Groups 5 and 6 at Study Day 3 (P < 0.05).
The increase in the CRP level of Group 6
was significantly different than the CRP
level changes in Groups 1, 2, 3, and 4, while
the increase in Group 5 was significantly
different than the decrease in Group 1.
Levels of CRP peaked on Study Day 3 in
43
-------
Groups 4, 5, and 6; Group 6 demonstrated
the highest level.
44
-------
Table 9. Mean Levels of C-Reactive Protein over the Course of the Study
C-Reactive Protein Levels (mg/dL)
Day-3
Day 1
Day 2
Day 3
Day 7
Day 14
Day 21
N
5
5
5
5
5
5
5
Group 1
Mean
1.8
0.9
1.4
0.65
0.79
0.72
0.25
Std Dev
2.27
0.97
1.46
0.66
1.2
1.06
0
N
5
5
5
5
4a
3b
5
Group 2
Average
0.25
1.38
0.52
0.41
0.36
0.25
0.25
Std Dev
0
0.61
0.31
0.35
0.22
0
0
N
5
5
5
5
5
5
5
Group 3
Mean
0.36
0.93
0.4
0.31
0.25
0.37
0.31
Std Dev
0.25
0.75
0.34
0.13
0
0.27
0.13
N
5
5
5
5
4
3
3
Group 4
Mean
0.49
1.06
1.91
2.12
2.06
0.25
0.25
Std Dev
0.55
1
3.39
4.18
3.62
0
0
N
5
5
5
4
1
1
1
Group 5
Mean
0.4
1.78
1.44
2.9
0.25
0.25
0.25
Std Dev
0.34
0.66
1.23
1.77
NA
NA
NA
N
4a
4
3
3
--
--
--
Group 6
Mean
0.33
1.83
6.45
11.22
--
--
--
Std Dev
0.14
0.97
2.03
1.63
--
--
--
N = Number of samples analyzed
NA = Not applicable (only one sample)
Std Dev = Standard deviation
-- = No rabbits alive at time point
a Only four out of the five rabbits alive at the time point were assayed
b Only three out of the five rabbits alive at the time point were assayed
45
-------
_ 12-
_i
XJ
I 10-
C
B o .
o »
£
I I"'
C
- ft
o 8 ¦
d:
o
J 6 •
Group 2
•
L23206
T
L23210
~
L23211
o
L23215
~
L23219
Limit of
Detection
12 •
10 •
8 ¦
CL
0)
•3
Group 3
•
L23217
~
L23227
~
L23228
o
L23229
~
L23230
Limit of
Detection
^ 12"
_j
f 10 *
c
o 8 '
£
-------
The three animals from Group 6 that were
alive on Study Day 3 had a mean CRP level
of 11.22 mg/dL compared to 0.33 mg/dL at
baseline. Groups 4 and 5 had mean levels of
2.12 (±4.18) and 2.90 (± 1.77) mg/dL on
Study Day 3. The increase in Group 4 stems
from the one animal (L23225) that died on
Study Day 4. The rest of the animals in this
group had levels of < 0.5 mg/dL.
AST, ALT, LDH, and SDH were also used
as markers for liver damage. Table 10 shows
AST levels over the course of the study, and
Figure 12 shows the AST levels by
individual rabbit. The normal range for AST
in rabbits is 0.0 to 120.0 units (U)/L (Thrall
et al., 2004; Queensberry and Carpenter,
1997; and University of Nebraska
Institutional Animal Care and Use
Committee, 2009). Only the Group 5 mean
levels on Days 2 and 3 were above the
normal range (Table 10). However, there
was a significant increase in AST as a
proportion of baseline in Groups 5 and 6 on
Study Day 2, and in Group 5 at Study Day 3.
Group 5 showed the greatest increase in
AST levels with a mean level of 565.0 U/L
on Study Day 3. The increase in Group 5 at
Study Day 3 was significantly different (P <
0.0001) than the changes in Groups 1, 2, 3,
4, and 6. There were significant increases as
a proportion of baseline in Groups 2, 3, 4,
and 5 at Study Day 3, and the increase in
Group 4 was significantly different than the
decrease in Group 1.
Among the other liver function parameters,
there were similar results in terms of
significance at Study Days 2, 3, and 7. At
Study Day 2, the increase as a proportion of
baseline in the high dose control group
(Group 6) was significantly different than
the change as a proportion of baseline in the
negative control group (Group 1) for CRP
and SDH. At Study Day 3, the increase as a
proportion of baseline in the Group 5 was
significantly different from the changes as a
proportion of baseline in the lower dose
groups (Groups 1 through 4) for ALT, LDH,
and SDH.
47
-------
Table 10. Mean Aspartate Aminotransferase Levels over the Course of the Study
Aspartate Aminotransferase Levels (U/L)
Day-3
Day 1
Day 2
Day 3
Day 7
Day 14
Day 21
N
5
5
5
5
5
5
5
Group 1
Mean
25.6
19.4
20.3
20.2
22.1
30.5
25
Std Dev
10.6
3.7
4.5
3.4
4.7
13.5
6
N
5
5
5
5
4a
3b
5
Group 2
Average
13.3
15.8
14.9
17.6
18.8
18.1
17.8
Std Dev
2.5
2.6
1.8
2.6
5.1
1.7
2.6
N
5
5
5
5
5
5
5
Group 3
Mean
12.5
14.6
15.9
13.6
16.5
48.9
17.1
Std Dev
2
2.9
3.6
3.9
3.6
56.8
4.8
N
5
5
5
5
4
3
3
Group 4
Mean
14.2
16.2
25.8
20.5
23.3
19.2
20.2
Std Dev
2.9
4.8
26.5
3.4
5.1
4
5.3
N
5
5
5
4
1
1
1
Group 5
Mean
17.2
20
154.3
565
23.7
20.5
21.8
Std Dev
6.8
6.7
306.1
533.6
NA
NA
NA
N
4a
4
3
3
—
--
—
Group 6
Mean
11.7
15.2
69.5
22.4
—
--
—
Std Dev
1.9
1.9
22.2
4.8
--
--
--
N = Number of samples analyzed
NA = Not applicable (only one sample)
Std Dev = Standard deviation
-- = No rabbits alive at time point
a Only four out of the five rabbits alive at the time point were assayed
b Only three out of the five rabbits alive at the time point were assayed
48
-------
1400
1200 -
1000 ¦
800
600 -
400
Group 1
• L23216
~ L23218
~ L23220
O L23222
~ L23223
Normal Range
200
150 •
100 ¦
50 -
0 ¦
8 aft
*
i
i
•o
I
I
9
1400 -
1200 -
1000 -
800 -
600 -
400 -
200
150 -
5 100
|
< 50
0
Group 2
•
L23206
~
L23210
~
L23211
o
L23215
~
L23219
Normal Range
_j>__ ft • i.
ID
3J
1400
1200
1000
800
600
400
Group 3
•
L23217
~
L23227
~
L23228
o
L23229
A
L23230
Normal Range
~
3 ass
*
~
*
6
1400
1200
1000
800
600
400
200
150
ro 100
|
< 50
0
Group 4
# L23205
~ L23207
~ L23225
O L23231
A L23235
Normal Range
a
6 8f ® ^
I
I
Jo
I
I I
I I
I I
IO I
£
<
1200 ¦
1000 -
800 -
600 -
400 •
200/
150 -
Group 5
~
O
•
L23200
~
L23201
~
L23212
o
L23214
~
L23234
Normal Range
7 14
Study Days
I £ ¦ n
Figure 12. Aspartate aminotransferase levels.
Group 6
•
L23203
~
L23204
~
L23213
o
L23221
~
L23232
Normal Range
7 14
Study Days
49
-------
3.10 Pathology
All rabbits were necropsied when they
succumbed to infection or at the end of the
study. Gross lesions in unscheduled death
rabbits (rabbits that succumbed to challenge
with B. anthracis) included discoloration of
the brain (meninges), crusting of the skin,
abdominal and thoracic cavity fluid,
bronchial and mediastinal lymph node
enlargement, small intestinal fluid, and
thymic fluid. These gross lesions were
typical of anthrax in rabbits (Zaucha et al.,
1998) and correlated histologically with
hemorrhage, necrosis, edema, and
suppurative inflammation. Individual rabbit
findings are presented in Table 11 and
Appendix W. Lungs and gross lesions were
examined microscopically for evidence of
anthrax. Microscopic lesions typical of
anthrax (Zaucha et al., 1998) were present in
all rabbits that died on study and most
surviving rabbits challenged with live
spores, and included minimal to moderate
suppurative inflammation [predominately
degenerate and viable heterophils
(polymorphonuclear cells)], necrosis,
hemorrhage, fibrin and/or large rod-shaped
bacteria in the lungs, bronchial and
mediastinal lymph nodes, skin, small
intestine, brain (meninges), and thymus.
Lung lesions consisted of suppurative
inflammation and bacteria generally found
in the pulmonary interstitium, and associated
with alveolar capillaries or larger pulmonary
blood vessels (Figure 13).
Other anthrax-related lesions were found in
lymph nodes, meninges, skin, thymus, and
small intestine. Lymph node findings
included hemorrhage, lymphoid necrosis,
fibrin, and bacteria. Figure 14 and Figure 15
show representative lesions. There were
minimal to mild intravascular bacteria,
suppurative inflammation, and hemorrhage
(with vascular necrosis) primarily in the
meninges of the brain of one rabbit (L23232,
Group 6). The skin of rabbit L23234 (Group
5) had areas of suppurative
inflammation/edema, hemorrhage, necrosis,
and intra- and extravascular bacteria. The
thymuses of rabbits L23234 and L23232 had
mild to moderate atrophy of lymphoid tissue
and evidence of edema. L23234 also had
intravascular bacteria in this tissue (Figure
16). Additionally, there was suppurative
inflammation and intra- and extravascular
bacteria in the small intestine (jejunum) of
one rabbit (L23221, Group 6). Where
suppurative inflammation occurred in tissues
(other than the lung), it was generally
associated with bacteria (bacilli).
50
-------
Multinucleated giant cells "tending to
organize" into microscopic granulomas in
one animal (L23217,Group 3) were present
in the lungs of some rabbits in all groups,
including control (irradiated spore
challenged) rabbits. These multinucleated
giant cells were randomly distributed
throughout the lung in ruptured or intact
airways or less frequently in or near
pulmonary blood vessels or lymphatics. The
multinucleated giant cells often surrounded
or contained birefringent foreign debris
(Figure 17). These cells and debris were also
seen in the lungs of two control animals; but
occurred with greater frequency and severity
in live spore challenged rabbits. Cell
aggregates of this type were not described in
a previous acute anthrax rabbit study
(Zaucha et al., 1998); hence, it is uncertain if
anthrax is contributory to the development
of this lesion. While many pulmonary
macrophages in challenged rabbits contained
foamy or granular cytoplasm and cellular
debris, bacilli were not identifiable in most
cases.
51
-------
Table 11. Pathology of the Rabbit Tissues
Group
Number
Animal
Number/
Death Status
Gross Findings
Microscopic Findings
1
100 ld50
(Irradiated)
101 (L23220)/FS
Lung: Unremarkable
102 (L23216)/FS
Lung: Unremarkable
103 (L23218)/FS
Lung: Multi-nucleated giant cells, minimal
104 (L23223)/FS
Lung: Unremarkable
105 (L23222)/FS
Lung: Multi-nucleated giant cells, minimal
2
100 CFU
201 (L23215)/FS
Lung: Inflammation, suppurative, minimal
Lung: Multi-nucleated giant cells, minimal
202 (L23206)/FS
Lung: Inflammation, suppurative, minimal
203 (L23210)/FS
Lung: Unremarkable
204 (L23219)/FS
Lung: Inflammation, suppurative, minimal
Lung: Multi-nucleated giant cells, minimal
205 (L23211)/FS
Lung: Unremarkable
3
1000 CFU
301 (L23217)/FS
Lung: Inflammation, suppurative, moderate
Lung: Multi-nucleated giant cells, moderate
Lung: Perivascular eosinophils, moderate
302 (L23230)/FS
Lung: Inflammation, suppurative, minimal
303 (L23228)/FS
Lung: Inflammation, suppurative, minimal
Lung: Multi-nucleated giant cells, minimal
304 (L23227)/FS
Lung: Bacteria (bacilli), minimal
305 (L23229)/FS
Lung: Inflammation, suppurative, minimal
4
10,000
LD50
401(L23235)/FD
Lung: Inflammation, suppurative, minimal
402 (L23205)/FS
Lung: Inflammation, nonsuppurative, minimal
Lung: Perivascular eosinophils, minimal
403 (L23225)/FD
Cavity, abdominal: Fluid, red,
-30 mL
Cavity, thoracic: Fluid, red, ~15
mL
Lung: Inflammation, suppurative, mild
Lung: Bacteria (bacilli), moderate
404 (L23231)/FS
Skin: Crust(s), liindlimb, red,
left liind limb,
20 mm x 20 mm
Lung: Inflammation, nonsuppurative, minimal
Skin: Necrosis, moderate
405 (L23207)/FS
Lung: Multi-nucleated giant cells, mild
Lung: Bacteria (bacilli), minimal
Lung: Inflammation, nonsuppurative, minimal
5
100,000
LD50
501 (L23201)/FD
Cavity, thoracic: Fluid, red, ~15
mL
Lung: Inflammation, suppurative, mild
Lung: Multi-nucleated giant cells, mild
Lung: Bacteria (bacilli), mild
502 (L23234)/FD
Cavity, thoracic: Fluid, red,
~10 mL
Skin: Crust(s), liindlimb, dark,
left liindlimb, 30 mm 5 mm
Skin: Crust(s), dark, back, 5
mm 5 mm
Skin: Fluid, abdominal, clear,
ventral abdomen, ~8 mL
Thymus: Fluid, clear, ~8 mL
Lung: Inflammation, suppurative, moderate
Lung: Multi-nucleated giant cells, mild
Lung: Bacteria (bacilli), moderate
Skin: Necrosis, mild
Skin: Bacteria (bacilli), moderate
Skin: Inflammation, suppurative, mild
Skin: Hemorrhage, mild
Thymus: Atrophy, lymphoid, moderate
Thymus: Edema, mild
Thymus: Bacteria, mild
503 (L23212)/FS
Lung: Inflammation, suppurative, minimal
52
-------
Table 11. (continued)
Group
Number
Animal Number/
Death Status
Gross Findings
Microscopic Findings
5
100,000
LD50
(cont'd)
504 (L23200)/FD
Cavity, thoracic: Fluid, red, ~20
inL
Lung: Inflammation suppurative, minimal
Lung: Multi-nucleated giant cells, minimal
505 (L23214)/FD
Lung: Inflammation suppurative, mild
Lung: Multi-nucleated giant cells, mild
Lung: Bacteria (bacilli), mild
601 (L23204)/FD
Cavity, thoracic: Fluid, red, ~15
inL
Lung: Inflammation suppurative, mild
Lung: Multi-nucleated giant cells, mild
Lung: Bacteria (bacilli), mild
602 (L23203)/FD
Lung: Inflammation suppurative, mild
Lung: Multi-nucleated giant cells, mild
Lung: Bacteria (bacilli), mild
6
100 LD50
603 (L23232)/FD
Brain: Discoloration(s),
meninges, diffuse, red, affects
all lobes
Lymph node, bronchial:
Enlarged, dark, 3x
Lymph node, mediastinal:
Enlarged, dark, 3x
Brain: Inflammation, suppurative, moderate
Brain: Hemorrhage, mild
Brain: Bacteria, minimal
Lung: Inflammation suppurative, moderate
Lung: Bacteria (bacilli), minimal
Lymph node, bronchial:
Fibrin, minimal
Hemorrhage, mild
Necrosis, lymphoid, mild
Bacteria (bacilli), minimal
Lymph node, mediastinal:
Fibrin minimal
Histiocytosis, mild
Necrosis, lymphoid, mild
Necrosis, vascular, mild
Bacteria (bacilli), minimal
Thymus: Atrophy, lymphoid, mild
Thymus: Edema, mild
604 (L23221)/FD
Cavity, abdominal: Fluid, red,
~60 mL
Intestine, small: Fluid,
jejunum, green, ~50 mL
Abdomen was distended
Intestine, small:
Inflammation suppurative, mild
Bacteria (bacilli), mild
Lung: Inflammation suppurative, mild
Lung: Bacteria (bacilli), moderate
605 (L23213)/FD
Lymph node, mediastinal:
Enlarged, dark, 3x
Lung: Inflammation suppurative, mild
Lung: Bacteria (bacilli), minimal
Lymph node, mediastinal:
Fibrin moderate
Hemorrhage, moderate
Necrosis, lymphoid, marked
Bacteria (bacilli), marked
FD = Found dead
FS = Final phase sacrifice
53
-------
V,< . , - * >\ AT? i
•,d' < <"* ••}?"' j ^ ,»,>» * ?• | ? * u » %
. ... ^ "A. . c\ - r*\.v
' *» ^ * •' x 11 vj' » < . •*¦-* : \
'• 4 W rm &' I *^C\/ \
* -* v4^ \ s Ws& *> *. V-C A.
'¦*- ir^,f /V
' « I ^ t ¦• ,. • ' » •»»
' ; . - . ^ v »- •v. .v t* • v.ti ««? x*
- *« *1 •< « V i , V* .« • v>- •
*•» •¦. - • «. ' -v . ~ * r I \ f * .»*«. ,» * ^
» «V { »•. • * ~ *>. ' • fr • % * . i fo
V + • ••' : •••>•'• ¦' ¦ *n
\
¦» '
•' •:, . w i >•>« *
j.-' . ¦>_ * * :•
H: -.?• * ~'. -0-
#- • ^4* -i • 1
't.
I
(***
5v , 7 "
J * * t fi
4 • * A
^ *j & . v r
*'4
i. i1
v. vcy v'». ».
•j* w%
-; .V,»
? • * ..
¦* 4 * "
*¦; /\
v ?VT,
1 . r« "
V " . ." S •* ' „ *1 . • -
f. »:^r-Vi i •
e. Jl.* * . « ^ a ff 0
Figure 13. Hematoxylin and eosin stain of lung alveoli showing interstitial inflammation,
and intravascular and interstitial B. anthracis bacilli (arrow) for rabbit L23225 (Group 4),
20X.
54
-------
4
4
Figure 14. Hematoxylin and eosin stain of lymph node showing B. anthracis in the sinuses
(arrow head) for rabbit L23213 (Group 6), 4X.
55
-------
Figure 15. Hematoxylin and eosin stain of lymph node showing hemorrhage and
lymphocyte depletion for rabbit L23213 (Group 6), 4X.
56
-------
4 4
V Tip v ,
Figure 16. Hematoxylin and eosin stain of thymus from rabbit L23234 (Group 5) showing
lymphocyte depletion (arrow heads), 4X.
57
-------
~ fS* *
.
;'5*
* * i5'«v
I* i* r ^ _ - _
\ #• s*- * ;• ••"•-
y>.r .-«*. is- /• »•-' l.
f »•
•*
,-v~, »-
vJjfc w|
Figure 17. Hematoxylin and eosin stain of a lung section from rabbit L23204 (Group 6)
showing multinucleated giant cell foreign body (arrow), 20X.
58
-------
3.11 Quality Assurance
The procedures set forth in the EPA
approved Quality Assurance Plan (QAP)
prepared for this project were adhered to
except in those instances that were
documented as deviations (see Appendix B).
In all there were seventeen study deviations;
no facility deviations occurred during the
study. Each deviation is listed in Table 12,
along with the impact on the quality of the
data and results reported herein. Technical
System Audits (TSAT) and Data Quality
Audits (DQA) performed for this study,
along with dates they were performed,
reported, addressed by the Study Director
and project management, and closed, are
listed in Table 13.
3.12 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.
59
-------
Table 12. Deviations and Impacts on Data Quality and Results
Deviation
Number
Deviation
Type
Impact on Data Quality and Results
7891
Protocol
None. The dye is not necessary to visualize the colonies.
7893
SOP
None. The spores are not viable and do not need to be diluted or plated for
enumeration.
8169
SOP
XI-009 deviation (numerous documentation deviations).
8170
Protocol
a) The protocol directed that the nebulizer and impinger samples from the
high dose group (Group 6) be plated after the challenges with and without
heat treatment (30 min at 65 ± 2°C). Only the impinger samples were heat
shocked and plated on 9/18/09. The heat shock temperature was set at
60°C.
b) Bacteremia testing was performed qualitatively from the blood pellet
obtained in the SST following centrifugation for sample L23214 (Day 3).
c) Bacteremia testing was performed qualitatively from the EDTA tube for
sample L23232 (terminal).
8176
SOP
Study activities initiated before protocol was signed. As per the SOP (XI-
107), "Process control is accomplished by following established procedures,
methods, and study protocols/' The preparation of the spores for challenge
was initiated before the protocol was signed by the study director.
8177
SOP
The challenge material with dilution IDs "1020-3e4", "1020-3e5", and "1020-
3e6"' was not prepared and enumerated at least one day prior to the challenge.
8178
SOP
Per SOP X-202, the acceptable colony range is 25 to 250. Samples with
results falling outside this range were not repeated. Results below this range
will be reported as For sample L23221 (terminal), the results will be
calculated from the obtained results. For dilution ID "E4"', counts of 255, 260,
and Too Numerous To Count (TNTC) were obtained. The plates with the
quantifiable values will be used to calculate a reportable result of 2.58 x 106.
8476
Study
Minimal. The technicians performed the necessary assignments to carry out
the study activities properly but failed to record and/or verify all required
information on the forms for accuracy and completeness at the end of each
task.
8477
Study
Minimal. The animals that had missed afternoon observation on 9/30/09 were
all observed as normal the morning of 9/30/09 and the following morning on
10/1/09. The animals that had a missed AM observation on 10/9/09 were
scheduled for euthanasia that morning and each animal was handled
individually during that procedure so any adverse conditions would have been
noted. The study was not negatively impacted in either instance.
8581
Study
None. The challenge material described above was prepared on the day of the
challenge from the "1020SSF le7" material prepared on 8/27/09, which had
been enumerated prior to the day of challenge. The 3e4, 3e5 and 3e6
challenge material was then enumerated by two analysts the day of challenge.
The enumerated values were accepted by the study director and aerosol
director and spray factor calculations were determined based on these
enumerated values.
60
-------
Deviation
Number
Deviation
Type
Impact on Data Quality and Results
8582
Study
Minimal. The pre-run sample is enumerated as a verification of the aerosol
system and the enumeration values obtained are not utilized in any
calculation. The enumeration values obtained from runs 4, 5, and 6 will be
reported and utilized in the spray factor calculations. For the starting material
from 9/3/09, the challenge enumeration from the previous day (7.86 x 106
CFU/mL) will be utilized in the spray factor calculations.
8584
Study
a) Minimal. The filters used for the procedure cannot be verified. The
impinge samples were filter and reportable results were obtained.
b) Minimal. The equipment and reagents are accurately documented on the
form. All other dates were accurately documented on the form.
c) Minimal. The sample was plated appropriately and a reportable result was
obtained. 8.62 x 105 CFU/mL will be used in the spray factor calculation.
8591
Study
Minimal. The system is still operating at a negative pressure as is necessary
for correct operation. Due to variations in animal respiration, it is difficult to
avoid some readings over -0.2" H20.
8612
Study
None. The reason for taking blood samples from the ear after Day 2 was to
avoid false positive bacteremia results because of a colonized port. All
animals with positive bacteremias on Day 3 succumbed to disease suggesting
the positive results were from systemic infection and not a colonized port.
8713
SOP
Minor documentation errors.
9383
SOP
TNA plates (111809-376 to -378), 111809-379 to -380, 111909-381) failed
primary acceptance criteria but will be passed and the data reported per the
study director.
9410
SOP
1) BBRC SOP VI-077 section V.D. "Raw data must be saved on a daily basis
to a diskette." Raw data was unable to be saved to electronic media due to
instrument malfunction.
2) BBRC SOP VI-077 section V.F. "Parameters for the analytical methods
will then be repeated after a study sample has been completed." Quality
control material was unable to be analyzed at end of sample analysis due to
instrument malfunction.
61
-------
Table 13. Technical System Audit and Data Quality Audit Dates
Audit Name
Audit
Type
Inspection
Start Date
Date
Reported
Response
Date
Closure
Date
Aerosol challenge; Blood collections;
Weights; Hematology, Bacteremia
plating
TSAT
9/18/2009
10/12/2009
2/1/2010
2/26/2010
IgG ELISA, PA ELISA, QPCR, TNA
TSAT
10/22/2009
12/7/2009
2/1/2010
5/25/2010
Quarantine telemetry implant and
housing
TSAT
11/9/2009
11/23/2009
1/21/2010
1/22/2010
Clinical Chemistry/Hematology
DQA
11/20/2009
12/8/2009
12/9/2009
2/25/2010
In vivo Binder
DQA
11/20/2009
12/8/2009
12/9/2009
2/9/2010
Micro and Aerosol Telemetry Binders
B0004642 and 4547
DQA
12/1/2009
12/14/2009
1/7/2010
1/25/2010
DNA Isolation and QPCR
DQA
12/2/2009
12/8/2009
12/11/2009
1/25/2010
Telemetry Binder
DQA
12/2/2009
12/8/2009
1/9/2010
1/25/2010
PA ELISA Binder
DQA
12/11/2009
12/21/2009
12/21/2009
2/25/2010
Spray Factor - Aerosol portion binder
DQA
1/25/2010
1/27/2010
1/27/2010
2/9/2010
Spray Factor - Micro binder
DQA
1/25/2010
1/27/2010
1/27/2010
2/9/2010
ELISA Study Binder
DQA
2/10/2010
2/12/2010
4/6/2010
5/25/2010
TNA Binder B0005163
DQA
2/10/2010
2/12/2010
4/6/2010
5/25/2010
DNA Isolation and Real-time PCR,
Binder 2
DQA
3/19/2010
3/22/2010
3/22/2010
5/25/2010
62
-------
4 Discussion
The anthrax letter attacks of 2001 resulted in
12 cases of cutaneous and 11 cases of
inhalational anthrax. Five of the 11
individuals with the inhalational form of the
disease died (Jernigan et al., 2001). The
processing of these spore-containing letters
through the U.S. mail resulted in the
contamination of numerous government and
private facilities (Canter, 2005). As part of
the remedial process, a standard of
nondetection of culturable spores was used
to confirm that the decontamination of
buildings was successful. Almost ten years
later, the criterion for remediation remains
nondetection (Coleman et al., 2008). One
reason for the continuing lack of a risk-
based standard is the unavailability of
credible dose-response data for B. anthracis
that could be used to estimate the risk posed
by low dose inhalation exposures. This
study was proposed to develop dose-
response data that could be used to begin to
fill the data gap present in the low dose
range.
The objective of this work was to determine
physiological responses following an acute
and Conclusions
inhalation exposure of B. anthracis in the
NZW rabbit at various low dose
concentrations. NZW rabbits were exposed
to targeted inhaled doses of 1.0 x 10 , 1.0 x
103, 1.0 x 104, and 1.0 x 105 CFU of B.
anthracis. Preliminary SF studies were
conducted to ensure that the lower doses
could be successfully delivered. The actual
doses were approximately 2.06 to 2.86 times
the targeted inhaled dose and remained
relatively consistent from animal to animal
within a group. Based on the actual doses
administered, the LD50 was determined to be
5.18 x 104 CFU, which is about 50% of the
published LD50 of 1.05 x 105 from Zaucha et
al. (1998). The Zaucha et al. (1998) LD50
value falls within the confidence intervals
established for this study of 6.14 x 103 CFU
to 7.27 x 105 CFU. As no confidence
intervals were identified in Zaucha et al.
(1998), it is not possible to determine how
the LD50 presented here compares to the
variability in the Zaucha data. It is also
possible that selection of dose inputs (i.e.,
targeted versus actual dose) that were used
in Zaucha et al. (1998) dose-response
63
-------
analysis may also have affected the value of
their calculated LD50, if similar variability
between the targeted versus actual doses
were present in their study.
As described in this report, Group 6 served
as a positive control group and these rabbits
were challenged with a targeted inhaled dose
of 1.00 x 107 CFU (actual equaled 8.27 x
106 CFU). Each of these five positive
control animals succumbed to infection with
a mean time to death of 3.47 days post-
challenge. All of the animals in this group
were bacteremic prior to death and all
animals except one were shown to be
toxemic at some time in the study. In
addition, the positive control animals
showed increases in respiration rate, heart
rate, body temperature, and circulating
neutrophils prior to death, and had
pathology findings consistent with
inhalational anthrax. Enzymes indicative of
liver damage (AST) were also elevated in
this group; however, there was no gross
evidence of liver damage. These
observations correspond to the findings of
Lawrence et al. (2009), who evaluated the
physiological response of Dutch Belted
n
Rabbits exposed to 1.00 x 10 B. anthracis
spores via nasal instillation. The Dutch
Belted Rabbits also presented with
tachycardia, fever, and neutrophilia prior to
death, which corresponded to the
observations in the positive control animals
(Lawrence et al., 2009). This strain of rabbit
also showed significant increase in ALT
levels. Interestingly, no significant increase
in AST was observed in the Dutch Belted
Rabbits, whereas there was a significant
increase in the NZW rabbits from Groups 5
and 6 in the current study. This may be the
result of differences in the two strains of
rabbit used in the studies.
In Group 5, four out of five of the rabbits
that received 2.75 X 105 CFU showed results
consistent with those observed in the
positive control group and died on study.
The pathology in this group was not as
extensive as that seen in the positive control
group, but was generally consistent with
pathology previously reported (Zaucha et
al., 1998) for inhalational anthrax in the
rabbit model. The only animal survivor in
Group 5 (L23212) received an actual inhaled
dose of 3.29 x 105 CFU. This animal did not
become bacteremic or toxemic and did not
demonstrate increases in heart rate,
respiration, or body temperature. End of
study pathology examination showed
minimal acute inflammation in the lung,
64
-------
which may or may not have been induced by
exposure to the B. anthracis.
Group 4 animals received an average
inhaled dose of 2.54 x 104 CFU and only
two out of five rabbits succumbed to
infection. However, the two animals that
died (L23235 and L23225) were bacteremic
at the time of death. Rabbit L23235 never
become toxemic (i.e., circulating levels of
PA were not found to be elevated)
suggesting that detectable levels of
circulating PA may not be an appropriate
marker for disease. However, both of the
animals that succumbed showed increases in
heart and body temperature prior to death.
All animals that received inhaled doses less
than 2.06 x 103 CFU (i.e., Groups 2 and 3)
survived to the end of the study and were
not observed to be bacteremic or toxemic at
any time. Rabbit L23227 (Group 3) was
noted as having bacteria (Bacilli) in the lung
upon microscopic examination; however, it
cannot be confirmed that these were B.
anthracis. The entire lung was fixed in 10%
buffered formalin, so the tissue could not be
cultured. As the animal was never
bacteremic but bacilli were observed in the
lungs, it was possible, though unlikely, that
a few of the spores germinated in the lungs
of L23227 but never established a systemic
infection. None of the surviving rabbits
seroconverted, suggesting that a B. anthracis
infection was not established.
Nine of the rabbits on study were positive
for Bordetella bronchiseptica prior to
challenge with B. anthracis. All challenge
groups (except Group 6) contained at least
one positive animal. The B. bronchiseptica
infection was not found to affect survival or
any physiological parameters measured.
(Please note two samples were labeled
L23223, both samples were negative and
one of the samples was most probably from
animal L23233.)
During histopathologic examination, it was
also noted that many of the rabbits had
multi-nucleated giant cells in the lungs. This
finding did not correspond with challenge
dose or B.bronchiseptica infection.
However, it is possible that these lesions
were caused by an indirect effect of
prolonged anthrax septicemia on
macrophage function resulting in altered
foreign particle clearance. Macrophage
dysfunction has been described as occurring
late in sepsis (Pahuja et al., 2008). Inhaled
debris or emboli from indwelling vascular
access ports may have also contributed to
the development of multinucleated giant
cells/granulomas. Pulmonary multinucleated
65
-------
giant cells/granulomas, thromboemboli, and
perivascular eosinophils have been
described in animals fitted with indwelling
vascular access ports (Taketoh et al., 2009).
Further research is required to determine if
this pathological finding was caused by
exposure to the B. anthracis spores.
Table 14 showed the physiological changes
that occurred after challenge with various
doses of B. anthracis spores. These data
presented a profile of the physiological
responses of the rabbit model when
challenged with various doses of B.
anthracis spores. The table illustrated that as
the challenge dose increases, so do the
number of parameters affected.
In the present study there was a trend toward
increases in these heart and respiration
parameters in the higher challenge dose
groups relative to the pre-challenge baseline.
Increased heart and respiration rates were
commonly associated with death, but a few
animals showed increases in these
parameters and lived. Temperature was the
most consistent indicator of disease
outcome. All rabbits that had a febrile
response died on study. Increased
neutrophils and increased liver enzymes
have been associated with inhalational
anthrax in the rabbit model (Lawrence et al.,
2009). However, if one were to use
literature-based values to determine
departures from normality, only seven out of
the 11 animals that died would have been
found to exhibit neutrophilia and only three
of the 11 that died would have been found to
exhibit elevated levels of AST. The majority
of rabbits had detectable levels of CRP
regardless of levels of the challenge dose,
indicating that this parameter was not a good
indicator of disease outcome.
While the above parameters show a
physiological response to exposure over a
certain threshold, they are not specific and
may be caused by a myriad of bacteria. The
diagnostic indicators assayed here were
bacteremia and toxemia. These specific
indicators were only detected in animals that
succumbed to disease, suggesting that if the
bacteria were able to escape the lungs and
spread systemically then the result is death.
Overall, the data indicate that challenge
doses of B. anthracis below the level
sufficient to establish systemic infection do
not produce observable physiological
responses; however, doses that trigger a
response result in death.
66
-------
Table 14. Summary Table of Responses Measured by Rabbit
Animal
ID
Dose
Group
Inhaled
Dose
(CFU/
Animal)
Heart
Rate3
Resp.
Rate3
Temp3
Neut.
Levels"
CRP"
AST"
Tox
Bact
Time
to
Death
(days)
L23220
1
0
<—>
u
<—>
u
T
<—>
-
-
NA
L23216
1
1.00 xio1
T
T
<—>
T
<—>
-
-
NA
L23218
1
0
T
<—>
<—>
<—>
T
<—>
-
-
NA
L23223
1
0
<—>
<—>
<—>
<—>
<—>
-
-
NA
L23222
1
0
<—>
<—>
T
T
<—>
-
-
NA
L23215
2
3.22 xlO2
<—>
<—>
1
T
<—>
-
-
NA
L23206
2
2.98 xlO2
<—>
1
<—>
1
T
<—>
-
-
NA
L23210
2
2.18 x 102
<—>
1
1
T
<—>
-
-
NA
L23219
2
3.21 xlO2
<—>
1
1
T
<—>
-
-
NA
L23211
2
2.73 xlO2
T
T
1
T
<—>
-
-
NA
L23217
3
1.48 xlO3
4
1
<—>
<—>
<—>
<—>
-
-
NA
L23230
3
2.02 xlO3
T
1
1
T
<—>
-
-
NA
L23228
3
2.23 xlO3
<—>
<—>
1
T
T
-
-
NA
L23227
3
2.32 xlO3
<—>
<—>
1
<—>
<—>
-
-
NA
L23229
3
2.24 xlO3
<—>
T
<—>
1
T
<—>
-
-
NA
L23235
4
1.76 xlO4
T
T
T
T
T
<—>
-
+
11
L23205
4
2.73 xlO4
<—>
T
<—>
<—>
<—>
-
-
NA
L23225
4
2.59 xlO4
T
<—>
T
U
T
<—>
+
+
4
L23231
4
2.41 xlO4
T
<—>
<—>
1
<—>
<—>
-
-
NA
L23207
4
3.19 x 104
<—>
<—>
1
T
<—>
-
-
NA
L23201
5
1.78 xlO5
U
T
T
T
T
T
+
+
4
L23234
5
2.96 xlO5
T
T
T
T
T
T
+
+
6
L23212
5
3.29 xlO5
<—>
<—>
<—>
1
T
<—>
-
-
NA
L23200
5
2.19 xlO5
4
T
T
1
<—>
<—>
-
+
3
L23214
5
3.54 xlO5
T
T
T
T
T
T
+
+
6
L23204
6
5.95x10°
T
T
T
T
T
<—>
+
+
4
L23203
6
8.86x10°
T
T
T
T
T
<—>
+
+
5
L23213
6
7.29x10°
T
T
T
<—>
T
<—>
+
+
3
L23221
6
8.88x10°
T
T
T
<—>
T
<—>
-
+
2
L23232
6
1.04 xlO7
T
T
T
1
T
<—>
+
+
4
| = Increases in a parameter
I = Decreases in a parameter
= No change in the parameter
+ = Positive or bacteremia culture or toxemia
- = Negative for bacteremia culture or toxemia
a = Changes based on baseline
b = Changes base on normal ranges
67
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APPENDIX A
STUDY PROTOCOL
A-l
-------
BBRC Protocol 1020
Study Number: 1020-CG920503
Battelle Biomedical
Research Center
Date: 15 September 2009
Page 1 of 16
Rabbit Single Dose Anthrax Telemetry Study
Study No. 1020-CG920503
BBRC Director:
BBRC Associate Director:
Study Director:
Sponsor:
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
Sponsor Representative:
Sarah C. Taft, Ph.D.
Mailing Address:
Battelle Biomedical
Research Center (BBRC)
505 King Ave, JM-7
Columbus, Ohio 43201-2693
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BBRC Protocol 1020
Study Number: 1020-CG920503
Battelle Biomedical
Research Center
Date: 15 September 2009
Page 2 of 16
Approval Signatures
JaSon E. Comer, Ph.D.
Study Director
See attachment
for signature
Gregory V. Stark, Ph.D.
Statistician
See attachment
for signature
Katherine A.B. Knostman, D.V.M., Ph.D., D.A.C.V.P
Pathologist
Gloria Sivko, D.V.M., Ph.D.
Technical Reviewer
Vl-v Jnn
JasMi M. Mott, D.V.M., Ph.D.
BBRC Associate Director
Stephen M.Jmfler, D.V.M..
Study Veterinarian See attachment
for signature
Sarah C. Taft, Ph.D.
Sponsor Representative
Date
Date
Date
9 /5 09
Date
Date
Date
Date
Reviewed and Registered by
Charles D. Lawrie Date
Quality Process Coordinator
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SEP-15-2009 17:31 BATTELLE 614 424 4511 P.01/01
BBRC Protocol 1020
Study Number: 1Q2Q-CG920503
Battelle Biomedical
Research Center
Date: 15 September 2009
Page 2 oflSjL
Approval Signatures
Jason E. Comer, Ph.D. 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 Sivko, D.V.M., Ph.D. Date
Technical Reviewer
Jason M. Mott, D.V.M., PhD.
BBRC Associate Director
Stephen M. Miller, D.V.M.. JDatc
Study Veterinarian
Sarah C. Taft, Ph.D. Date
Sponsor Representative
Reviewed and Registered by:
Charles D. Lawrie Date
Quality Process Coordinator
TOJPI4 P.01
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SEP-15-2009 17:02
BATTELLE
614 424 7441 P.02/02
BBRC Protocol 1020
Study Number: 1020-CG920503
Battelle Biomedical
Research Center
Date: 15 September 2009
Page 2 of 16^
Approval Signatures
Jason E. Comer, Ph.D. Date
Study Director
Gregory V. Stark, Ph.D. Date
Statistician
_ ^/i<5 13-ocrt
Katherine A.B. Knostman, D.V.M., Ph.D., D.A.C.V.P Date
Pathologist
Gloria Sivko, D.V.M., Ph.D. Date
Technical Reviewer
Jason M. Mott, D.V.M., Ph.D. Date
BBRC Associate Director
Stephen M. Miller, D.V.M.. Date
Study Veterinarian
Sarah C. Taft, Ph.D. Date
Sponsor Representative
Reviewed and Registered by:
Charles D. Lawrie Date
Quality Process Coordinator
T&Ff3_ P.02
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BBRC Protocol 1020
Study Number: 1020-CG920503
Battelle Biomedical
Research Center
Date: 15 September 2009
Page 2 of \€xl
Approval Signatures
Jason E. Comer, Ph.D. 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 Sivko, D.V.M., Ph.D. Date
Technical Reviewer
Jason M. Mott, D.V.M., Ph.D. Date
BBRC Associate Director
Stephen M. Miller, D.V.M.. Date
Study Veterinarian
i ft. _2 QCCq
Sarah C. Taft, Ph.D. t Daft
Sponsor Representative
Reviewed and Registered by:
Charles D. Lawrie Date
Quality Process Coordinator
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BBRC Protocol 1020
Study Number: 1020-CG920503
Battelle Biomedical
Research Center
Date: 15 September 2009
Page 3 of 16
1.0 INTRODUCTION
Bacillus anthracis, 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 exposure 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. anthracis spore. The
virulence of B. anthracis 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 pleural 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 1020
Study Number: 1020-CG920503
Battelle Biomedical
Research Center
Date: 15 September 2009
Page 4 of 16
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 reveal 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 a
single exposure of B. 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
surgery and histopathology and 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 a
single exposure of B. anthracis Ames strain spores.
4.0 TEST SYSTEM
Animals: Thirty-five (35) male pathogen free New Zealand White (NZW) rabbits
(Oryctolagus cuniculus) weighing at least 3.5 kg will be ordered for this study. Thirty
rabbits will be placed on study with the remaining five serving as replacements. Rabbit
age is not used as a criterion for placement on study. Rabbits will be purchased from
Covance. Prior to receipt, rabbits will be fitted with vascular access ports at Covance.
A Battelle veterinarian will implant a Data Sciences International model D70-PCTP
telemetric devices into the rabbits prior 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. Five extra animals
will be ordered and implanted with telemetric devices in case of vascular access port or
telemetric device failure.
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BBRC Protocol 1020
Study Number: 1020-CG920503
Battelle Biomedical
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Date: 15 September 2009
Page 5 of 16
5.0 STUDY DESIGN
5.1 Randomization of Animals: Prior to challenge, rabbits will be randomized by
weight into six groups of five. The rabbits within each group will also be randomized
for challenge order (based on ear tag numbers provided by supplier). The rabbits 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 6 will
be challenged last. Prior to challenge, any animals with a malfunctioning vascular
access ports or telemetric devices will be replaced with one of the five extra animals.
5.2 Aerosol Challenge Generation and Monitoring: On Study Day 0 (Study Day 0
defined as Challenge Day), rabbits will be placed into a plethysmography chamber,
passed into a Class III cabinet system, and aerosol challenged with targeted doses of
100,000, 10,000, 1,000, or 100 CFUs of B. anthracis (Ames strain) spores. A high
dose control group will be challenged with 100 LD50s and the negative control group
will exposed to an equivalent of 100LD50s of gamma irradiated spores. The Ames
LD50 value (105,000 cfu) published from USAMRIID (Zaucha, 1998) will be used.
Aqueous suspension 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.
Impinger samples that are expected to have concentrations lower than can be
enumerated by spread plating will be both plated and filtered. Briefly, 1 mL of the
sample will passed through a sterile 0.45 micron filter (Nalgene) Analytical Test Filter
Funnel, (Fisher catalog number 145-0045), the filter will placed on top of a TSA plate
and incubated for 24-72 hours at 37°C ± 2°C. The filter will then be stained with an
appropriate dye to visualize the bacteria on the white filter. 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.
To determine the affect of aerosolization on the spore coat the nebulizer and impinger
samples from the high dose group (Group 6) will be plated after the challenges with and
without heat treatment (30 minutes at 65 ± 2 °C).
Following aerosol challenge, rabbits will be held in individual stainless steel cages and
monitored for clinical signs of illness, body temperature, heart rate, respiration rate and
survivability for 21 days post-challenge.
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BBRC Protocol 1020
Study Number: 1020-CG920503
Battelle Biomedical
Research Center
Date: 15 September 2009
Page 6 of 16
Table 1. Study Design and Challenge Doses.
Group
Spore dose
(CFU)
Number of Spore
Challenges
# of Rabbits
1 (neg) control*
IOOXLD50
1
5
2
100
1
5
3
1000
1
5
4
10,000
1
5
5
100,000
1
5
6 (high dose control)
IOOXLDjo
1
5
* Negative controls will be challenge with irradiated spores.
5.3 Animal Weights: Animals will be on weighed on Study Days 0 (challenge day), 1,
2,3,7, 14 and 21.
5.4 Blood Collection Schedule: On Study Days -3, 1, 2, 3, 7 and 14 blood will be
collected into EDTA (~1.5 mL) and SST tubes (-3.5 - 4.5 mL) (Table 2). Blood taken
on Study Days 1, 2, and 3 will be drawn 24, 48, and 72 hours (± 2hours), respectively,
based on median challenge time of each group. If possible a blood sample will be taken
from animals found dead or prior to euthanasia. On Study Day 21 all surviving rabbits
with be terminally bleed 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 in EDTA tubes will be stored at room temperature if utilized
within 4 hours of collection; blood will be stored at 2-8 °C if not analyzed within 4
hours. Sera will be stored at < -70 °C until needed.
Blood samples will be collected from vascular access ports on Study Days -3,1 and 2 for
both the EDTA and SST collection tubes. After Day 2 blood collected in the EDTA
tubes will be obtained from the medial auricular artery or the marginal ear vein. Blood
collected in SST tubes will continued to be obtained from the vascular access port. If a
port fails, the medial auricular artery, the marginal ear vein, or other appropriate
vasculature may be utilized for blood collection if attainable. 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 1020
Study Number: 1020-CG920503
Battelle Biomedical
Research Center
Date: 15 September 2009
Page 7 of 16
Table 2. Blood collection schedule
Study Day
Tube Type
Day -3
Day 1
Day 2
Day 3
Day 7
Day 14
Day 21
EDTA (~ml)
1.5
1.5
1.5
1.5
1.5
1.5
1.5 *
SST (~ml)
4.5
3.5
3.5
3.5
3.5
3.5
15 *
Total per
day~(ml)
6
5
5
5
5
5
16.5
*Terminal Blood sample, minimum draw volume.
5.5 Priority usage of whole blood and sera:
Whole Blood: Bacteremia via quantitative plating »> Hematology »> Bacteremia
via Quantitative PCR
Sera: Circulating PA ELISA »> Retention sample (maximum of 750 fj.1 if possible)
»> Clinical Chemistry »> anti-PA IgG ELISA and TNA
5.6 Toxemia Assessment: A portion of all the serum collected will be analyzed for
circulating PA via the quantitative PA ELISA according to SOP BBRC. X-180.
5.7 TNA/ELISA: 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.
5.8 Bacteremia: A portion of each blood sample from the EDTA collection tubes will
be tested for bacteremia by quantitative spread plate technique (SOP BBRC. X-202),
and quantitative real-time PCR (SOP BBRC. X -146). The Day -3 samples will be
assayed for bacterema via the qualitative plating technique bacteremia (SOP BBRC. X-
096). Please note, if the blood samples cannot be plated in real-time. -400 |Ltl of blood
from the EDTA tube will be transferred to another appropriate tube and stored on ice
until plated or DNA isolation.
5.9 Hematology and Clinical Chemistry : Hematology will be performed on blood
samples collected in EDTA tubes using the Advia Hematology Analyzer according to
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
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BBRC Protocol 1020
Study Number: 1020-CG920503
Battelle Biomedical
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Date: 15 September 2009
Page 8 of 16
• Hemoglobin (HGB)
• 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)
Clinical Chemistry will be performed on all serum samples using Advia 1200
Chemistry analyzer. According to SOP BBRC. VI-077. Evaluation will include, but
not be limited to, the following parameters:
• Alanine aminotransferase (ALT)
• Asparate aminotransferase (AST)
• Total bilirubin
• Total protein
• Blood urea nitrogen (BUN)
• Creatinine
• Calcium
• Sodium
• Potassium
• Chloride
• Phosphorus
• Lactate Dehydrogenase (LDH)
• Sorbitol Dehydrogenase (SDH)
• C-Reactive Protein (CRP)
5.10 Clinical Observations: Following challenge rabbits with be observed twice daily
for clinical signs of illness and survivability due to anthrax infection (e.g., moribund,
respiratory distress, appetite, activity, and seizures).
5.11 Telemetric Monitoring: Rabbits will be surgically implanted with telemetry
units (D70-PCTP 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, ECG activity, and cardiovascular function (heart rate and respiratory
pressure) will be monitored at least 30 seconds every 15 minutes for 7 days pre-
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BBRC Protocol 1020
Study Number: 1020-CG920503
Battelle Biomedical
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Date: 15 September 2009
Page 9 of 16
challenge (baseline) and for 21 days post-challenge according to SOP MREF.VI-087.
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.
5.12 Necropsy and Histopathology: Animals that succumb to challenge, or are found
moribund and are euthanized, will undergo a gross necropsy. Surviving animals will
be euthanized and necropsied on Study Day 21. The lungs 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. 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
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BBRC Protocol 1020
Study Number: 1020-CG920503
Battelle Biomedical
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Date: 15 September 2009
Page 10 of 16
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 highly trained life sciences technician,
Battelle veterinarian, or by the Study Director will be euthanized after an individual
blind to treatment concurs with the decision to euthanize. Concurrence will not be
required in instances when the animal is observed seizing.
Rabbits that are euthanized will be sedated with Acepromazine or other approved
anesthetic and then administered an overdose of a euthanasia agent containing
pentobarbital or other American Veterinary Medical Association (AVMA) approved
method of euthanasia.
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
SOPs.
Humidity: The relative humidity of animal rooms will be maintained according to
Battelle SOPs.
Diet: PMI, 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.
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BBRC Protocol 1020
Study Number: 1020-CG920503
Battelle Biomedical
Research Center
Date: 15 September 2009
Page 11 of 16
Enrichment: To promote and enhance the psychological well being of rabbits,
enrichment will be as described in Battelle SOP No. BBRC. VII-040.
7.0 ANALYTICAL AND STATISTICAL PLAN
7.1 Sample Size:
A sample size of 5 animals per group is sufficient to detect group effects in a one-way
ANOVA model with greater than 80% power, when a two standard deviation difference
in group means is present.
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 conduced or Cox proportional hazards
model will be fitted.
For, circulating PA levels, TNA, ELISA, each hematology parameter, each clinical
chemistry parameter, quantitative bacteremia and PCR data descriptive statistics will be
produced for each animal at each sample collection time. As no negative control
animals are expected to survive, baseline values for animals 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 analysis of variance (ANOVA) model will be fitted to determine if
parameters changed significantly from baseline and whether there were significant
differences between groups. In addition, baseline data will be used to estimate a
threshold for each parameter to use in determining whether individual animals are
abnormal. Summary statistics for the proportion of animals that are abnormal in each
group will be produced for each parameter. The proportion abnormal in each group
will be compared using Fisher's exact tests for each parameter..
Telemetry endpoints may be smoothed by adjusting the post-challenge values to
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.
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BBRC Protocol 1020
Study Number: 1020-CG920503
Battelle Biomedical
Research Center
Date: 15 September 2009
Page 12 of 16
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.
8.0 RECORDS TO BE MAINTAINED
8.1 Animals
Animals surviving the challenge(s) will be euthanized on Study Day 21.
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. This model development study is intended to serve as the
model for subsequent government and non-government therapeutic efficacy studies.
9.0 BIOSAFETY CONSIDERATIONS
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Study Number: 1020-CG920503
Battelle Biomedical
Research Center
Date: 15 September 2009
Page 13 of 16
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 28 days post-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
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-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 Screening Assay using Serum."
Battelle SOP Number BBRC.VI-066, "Standard Operating Procedure (SOP) for the
Operation and Maintenance of the Siemens (formerly Bayer) Advia®120 Hematology
analyzer."
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Study Number: 1020-CG920503
Battelle Biomedical
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Date: 15 September 2009
Page 14 of 16
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 BBRC.VI-086, "Standard Operating Procedure (SOP) for Use
and Maintenance of the Data Sciences International (DSI) Telemetry System."
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-020, "Standard Operating Procedure (SOP) for the
Collection of Blood Samples from Animals."
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. VII-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."
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 Physiological Well-
Being of species other than Non-Human Primates."
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-096, "Standard Operating Procedure (SOP) for the
Qualitative Analysis of Bacteria in Blood and Tissue."
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BBRC Protocol 1020
Study Number: 1020-CG920503
Battelle Biomedical
Research Center
Date: 15 September 2009
Page 15 of 16
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 BBRC. X-146 "Standard Operating Procedure (SOP) for
Performing Quantitative Real-Time Poymerase Chain Reaction (qPCR) Using
Reference Standard Materials."
Battelle SOP Number BBRC. X-165, "Standard Operating Procedure (SOP) for
Performing Qualitative Real-Time Polymerase Chain Reaction (PCR)."
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-202, "Standard Operating Procedure (SOP) the
Enumeration of Bacteria via the Spread Plate Technique."
Battelle SOP Number BBRC. XIII-001, "Standard Operating Procedure (SOP) for the
Aerosol Exposure System to Challenge Non-Human Primates and Rabbits to
Aerosolized Agent."
Battelle SOP Number BBRC. X-146, "Standard Operating Procedure (SOP) for
Performing Quantitative Real-Time Polymerase Chain Reaction (QPCR) Using
Reference Standard Materials."
Battelle SOP Number BBRC. VI-044, "Standard Operating Procedure (SOP) for the
Operation and Maintenance of the Biomedic Data System DAS-6007 Handheld Probe
and the DAS-5002 Notebook Unit."
Battelle SOP Number BBRC. VII-040, "Standard Operating Procedure (SOP) for
Environmental Enhancement/Enrichment Plan to Promote the Psychological Well-
Being of Species Other Than Non-human Primates."
Battelle SOP Number BBRC. X-101, "Standard Operating Procedure (SOP) for
Enzyme Linked Immunosorbent Assay (ELISA) Detection of Bacillus anthracis PA-
Specific IgG in Sera."
Battelle SOP Number BBRC. X-143, "Standard Operating Procedure (SOP) for the
High Throughput Toxin Neutralization Assay (htp-TNA) Proper."
A-19
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BBRC Protocol 1020
Study Number: 1020-CG920503
Battelle Biomedical
Research Center
Date: 15 September 2009
Page 16 of 16
Inglesby, T.Y., 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.,
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-20
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BBRC Protocol 1020
Study Number: 1020-CG920503
Battelle Biomedical
Research Center
Date 21 October 2009
Page 17 of 18
Rabbit Single Dose Anthrax Telemetry Study
BBRC Protocol Amendment No. #1
Change No. # 1
Used to read:
5.5 Priority usage of whole blood and sera:
Whole Blood: Bacteremia via quantitative plating »> Hematology »> Bacteremia via
Quantitative PCR
Sera: Circulating PA ELISA »> Retention sample (maximum of 750 |_il if possible) »>
Clinical Chemistry »> anti-PA IgG ELISA and TNA.
Now reads (changes in bold):
5.5 Priority usage of whole blood and sera:
Whole Blood: Bacteremia via quantitative plating »> Hematology »> Bacteremia via
Quantitative PCR
Sera: Circulating PA ELISA >» Clinical Chemistry »> Retention sample (maximum of
750 jil if possible) »> anti-PA IgG ELISA and TNA.
Reason for Change:
The client and study director decided that clinical chemistry was more important than the
retention sample.
Impact on Study:
There is a positive impact on the study. Determining the clinical chemistry status of each animal
will help identify biomarkers of disease.
Effective Date: 10/21/09
A-21
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BBRC Protocol 1020
Study Number: 1020-CG920503
Battelle Biomedical
Research Center
Date 21 October 2009
Page 18 of 18
Approved By:
ison E. Comer, Ph.D.
Study Director
See attachment
for signature
Date
Sarah C. Taft, Ph.D.
Sponsor Representative
Date
Quality Assurance Review and Registration:
%V/C faSUJl. / rt/2 6/0^
Charles D. Lawrie Date
Quality Process Coordinator
A-22
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BBRC Protocol 1020
Study Number: 1020-CG920503
Battelle Biomedical
Research Center
Date 21 October 2009
Page lfyofl8
Approved By:
Jason E. Comer, Ph.D. Date
Study Director
jSaSsx/../J. \Q )
Sarah C.Tafi,Ph.D. {/' Dat7 1
Sponsor Representative
Quality Assurance Review and Registration:
Charles D. Lawrie Date
Quality Process Coordinator
A-23
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APPENDIX B
STUDY DEVIATIONS
B-l
-------
BATTELLE BIOMEDICAL RESEARCH CENTER
DEVIATION FORM
Deviation No. (Assigned by QAU):
CAQ No. (Assigned by QAU): //p
>rocedure Deviated:
ED Protocol (Number): 1020
~ SOP (Number):
~ Method (Number):
~ GLP (Section):
~ Other:
Type of Deviation (check one):
~ Facility
El Study (fill out study info) Study Number: 1020-CG920503
Study Title: Rabbit Single Dose Anthrax Telemetry Study
Record Affected (describe Title, Binder name, location, Form no. etc.): Microbiology Binder: Form No
Mierobio-454 and Microbio-455
Date of Deviation(s): 9/19/09
Description of Deviation:
The filtered impinger samples will not be stained with dye.
Cause of Deviation:
Colonies are visible on the filters without the use of dye.
Corrective Action:
The staff members will count the eolonies without the use of dye.
Impact of Deviation: None. The dye is not necessary to visualize the colonies.
If deviation is planned, effective date: 9/18/09
Deviation form Prepared bv/Date: TRM 9/18/09
Deviation Reviewed and Corrective Action Accepted by/Date: 7^ .
Deviation Reviewed and Registered by QAU/Date:
^ ou //
Form No. MREF Facility-035-03 (Revised 11/01/06)
Page 1 of 1
B-2
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BATTELLE BIOMEDICAL RESEARCH CE
DEVIATION FORM
Deviation No. (Assigned bv QAU): CAQ No. (Assigned by QAU): ,, J/i
~y t7j n>1 /i///f
Standard or Procedure Deviated:
~ Protocol (Number):
m SOP (Number): X-054
~ Method (Number):
~ GLP (Section):
~ Other:
Type of Deviation (check one):
~ Facility
m Study (fill out study info) Study Number: 1020-CG920503
Study Title: Rabbit Single Dose Anthrax Telemetry Study
Record Affected (describe Title, Binder name, location, Form no. etc.): Microbiology Binder; Form No
Microbio-454 and Microbio-455
Date of Deviation(s): 9/18/09
Description of Deviation:
The irradiated spores (from the challenge suspension, pre-run, nebulizer samples, and impinger samples) will
not be enumerated as per SOP X-054.
Cause of Deviation:
These spores are not viable as confirmed by testing conducted at the CDC. Enumeration of these samples
should not result in colonies.
Corrective Action:
Each sample will be directly plated onto TSA. For the challenge suspension, 0.1 niL of the neat sample will
be plated in quintuplicate. For the pre-run sample, the nebulizer sample (from each group 1 animal), and the
impinger sample (from each group 1 animal), 0.1 ml, of each neat sample will be plated onto an individual
plate.
Impact of Deviation: None. The spores are not viable and do not need to be diluted or plated for
enumeration.
If deviation is planned, effective date: 9/18/09
Deviation form Prepared by/Date: TRM 9/18/09
Deviation Reviewed and Corrective Action Accepted by/Date: ci 2^'
Deviation Reviewed and Registered by QAU/Date: ^/ / fZr 9//V /e? 9
Form No. MREF Facility-035-03 (Revised 11/01/06)
Page 1 of 1
B-3
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BATTELLE BIOMEDICAL RESEARCH CENTER
DEVIATION S ORM
CAQ No. (Assigned by QAU):
Deviation No. (Assigned by QAU): ^
St and aid or Procedure Deviated:
~ Protocol (Number):
m SOP (Number): XI-009-02
~ Method (Number):
~ GLP (Section):
~ Other:
Type of Deviation (check one):
~ Facility
13 Study (fill out study info) Study Number: 1020-CG920503
Study Title: Rabbit Single Dose Anthrax Telemetry Study
oo
Record Affected (describe Title, Binder name, location, Form no. etc.):
I. Rabbit Daily Room Activity Schedule; Form No. General-078
II. Weight and Clipping Record; Form No. Weightsht-013
III. Small Animal Daily Observation Sheet; Form No. MREF Animal Care-004
IV. Rabbit Clinical Observations (BID); Form No. obsRabbit-003
V. Collection, Transfer, and Storage of Whole Blood; Form No. Microbio-372
Vivo Binder
Date of Deviation(s):
I. 9/30/09
II. 9/18/09, 9/19/09, 9/23/09, 9/25/09, 10/9/09
III. 9/21/09
IV. 9/18/09,9/25/09, 9/27/09, 9/30/09
V. 9/19/09, 9/20/09, 9/21/09, 9/22/09, 9/23/09, 10/9/09
Description of Deviation:
SOP states, "For all records it must be absolutely clear what was done, when it was done, by whom it was
done, who entered the documentation, and when it was entered."
Cause of Deviation:
I. The technician responsible for recording PM activity failed to record time, date, initials and check the
appropriate boxes for activities performed.
II. On 9/18/09, the technician recording a correction for animal L23230 failed to use a proper footnote for a
comment. On 9/19/09, the technician responsible for study activity failed to record date and initials verifying
animals ID's, weighed by, checked sex, clipped by, and balance information on the form at the end of the
procedures. On 9/23/09, the technician recording weight identification for calibration failed to properly finish
a correction with a correction code, date, and initials. On 9/25/09 and 10/09/09, the technician responsible for
study activity failed to record date and initials for weighing and checking the sex/ID of the animals at the time
, of the activity.
TIL The technician responsible for AM observations for animals L23202 and L23209 failed to properly
record the time on the sheet but it can be verified as 0730 from the daily activity schedule.
Form No. MREF Facility-035-03 (Revised 11/01/06)
Page 1 of 2
B-4
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BATTELLE BIOMEDICAL RESEARCH. CENTER
DEVIATION FORM
Deviation No. (Assigned by QAU): g £ CAQ No. (Assigned by QAU): ^ "
IV. On 9/18/09, the technician responsible for PM observations failed to record the date at the end of study
activity for Groups 4, 5, and 6. On 9/25/09, the technician responsible for PM observations for Group 4
animals failed to record date and initials at the end of study activity. On 9/27/09, the technician responsible
for AM observations failed to record their initials at the end of study activity.
V. On 9/16/09, the technicians responsible for the blood draw on animals L23217 and L23221 failed to finish
a correction by recording date and initials for each instance. On 9/20/09, the technician responsible for the
blood draw for animal L23213 failed to record a time the blood draw actually occurred. Also on 9/20/09, the
technicians responsible for the terminal blood draw on animal L23221 failed to record date and initials for
transferring and receiving the sample, verifying the label information and a time the sample was actually
obtained. On 9/21/09, the technician responsible for the terminal blood draw on animal L23200 failed to
record date and initials for transferring the sample. On 9/22/09, the technician responsible for the terminal
blood draw for animals L23201 and L23232 failed to record the animals ID on the form. A time was recorded
for each animal, and it is being assumed based on the previous order of the animals on blood sheets that
L23201 should have been recorded first at the 0838 time and then L23232 recorded second at the 0900 time.
This is how the animals and times are being reported on the blood draw tables. On 9/23/09, the technician
responsible for the terminal blood draw on animal L23203 failed to record a time the sample was actually
obtained. On 10/9/09, the technician responsible for the blood draw on animal L23212 failed to record date
and initials for the study activity.
Corrective Action:
Technicians were asked to review SOP XI-009 for documentation and proper procedures and reminded of the
importance of recording and reviewing all study forms at the completion of study activity for accuracy and
completeness.
Impact of Deviation:
Minimal. The technicians performed the necessary assignments to carry out the study activities properly but
failed to record and/or verify all required information on the forms for accuracy and completeness at the end
of each task.
If deviation is planned, effective date:
Deviation form Prepared by/Date: i j
Deviation Reviewed and Corrective Action Accepted by/Date: i # _
3 •?" c_ i f u I (
Deviation Reviewed and Registered by QAU/Date: sf/r^/ - 7
Whrvu
Form No. MREF Facility-035-03 (Revised 11/01/06)
Page 2 of 2
B-5
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BATTELLE BIOMEDIC
DEVIATI
AL RESEARCH CENTER
ON FORM
^Deviation No. (Assigned by QAU): ^ y
CAQ No. (Assigned by QAU): fit pQ
Standard or Procedure Deviated:
SProtocol (Number): 1020
~ SOP (Number):
~ Method (Number):
~ GLP (Section):
~ Other:
Type of Deviation (check one):
~ Facility
ED Study (fill out study info) Study Number: 1020-CG920503
Study Title: Rabbit Single Dose Anthrax Telemetry Study
Record Affected (describe Title, Binder name, location, Form no. etc.):
Small Animal Daily Observation Sheet; Form No. MREF Animal Care-004,
Rabbit Clinical Observations (BID); Form No. obsRabbit-003,
Vivo Binder
Date of Deviation(s): 9/30/09, 10/9/09
Description of Deviation:
Protocol states, "Following challenge rabbits with be observed twice daily
for clinical signs of illness and survivability due to anthrax infection (e.g., moribund, respiratory distress,
appetite, activity, and seizures)."
Cause of Deviation:
On 9/30/09, the technician responsible for performing the PM observations failed to record the observations
on the proper forms for the challenged animals as well as the remaining extra animals.
On 10/9/09, the technician responsible for performing the AM observations prior to euthanasia failed to
record the observations on the proper forms.
Corrective Action:
Technicians were asked to review the protocol and documentation procedures. They were also reminded of
the importance of recording and reviewing all study forms at the completion of study activity for accuracy
and completeness.
Impact of Deviation:
Minimal. The animals that had a missed afternoon observation on 9/30/09 were all observed as normal the
morning of 9/30/09 and the following morning on 10/1/09. The animals that had a missed AM observation on
10/9/09 were scheduled for euthanasia that morning and each animal was handled individually during that
procedure so any adverse conditions would have been noted. The study was not negatively impacted in either
i instance.
If deviation is planned, effective date:
Form No. MREF Facility-035-03 (Revised 11/01/06)
Page 1 of 2
B-6
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BATTELLE BIOMEDIC
DEVIATI
AL RESEARCH CENTER
ON FORM
! Deviation No. (Assigned by QAU): -j -J
CAQ No. (Assigned by QAU): _
' ' ytoo
1 Deviation form Prepared by/Date:
/L/C £> /£>
~t3-09
Deviation Reviewed and Corrective Action Accepted by/Date; , / ,
i
Deviation Reviewed and Registered by QAU/Date:
'ff/JmruJ t/fufo
Form No, MREF Facility-035-03 (Revised 11/01/06)
Page 2 of 2
B-7
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BATTELLE BIOMEDICAL RESEARCH CENTER
DEVIATION FORM
deviation No. (Assigned by QAU): j
Standard or Procedure Dt> inled:
~ Protocol (Number and Amendment No. if applicable):
EI SOP (Number and Revision Number): X-072-09
~ Method (Number and Revision Number):
~ GLP (Section):
~ Other:
CAQ No. (Assigned by QAU):
Type of Deviation (check one):
~ Facility
t3 Study (fill out study info) Study Number: 1020-CG920503
Study Title: Rabbit Single Dose Anthrax Telemetry Study
Record Affected (describe Title, Binder name, location, Form no. etc.): Microbiology Binder; Form No.
Microbio-185, Microbio-454, and Microbio-455
Date of Deviation(s): 9/2/09, 9/3/09, and 9/4/09
Description of Deviation:
°er SOP X-072 (section V.E.), "Challenge spore suspensions may be prepared and aliquoted on the day of
challenge (for non-aerosol challenges only)" and (section Y.E.5) "The challenge suspension must be
enumerated by 2 analysis at least one day prior to the day of the challenge for all aerosol challenges." The
spray factor material with dilution IDs"1020SF-le6", "1020SF-le5" and "1020SF-le4" was not prepared and
enumerated at least 1 day prior to challenge.
Root Cause of Deviation:
It is preferred that samples at a concentration of 1 x 106 cfu/mL or less are not stored overnight. The study
director decided that it would be best to prepare these dilutions of the challenge material on the day of
challenge.
Corrective Action:
The staff members prepared the spray factor material on the day of challenge and enumerated it twice (once
by two different staff members).
Impact of Deviation: None. The challenge material described above was prepared on the day of challenge
from the "1020SF le7" material prepared on 8/27/09, which had been enumerated prior to the day of
challenge. The 3e4, 3e5 and 3e6 challenge material was then enumerated by two analysts the day of
challenge. The enumerated values were accepted by the study director and aerosol director and spray factor
calculations were determined based on these enumerated values.
If deviation is planned, effective date: n/a
Deviation Form Prepared by/Date:
~~rMi I2-2^-oi
Form No, FaciIity-035-05 (Revised 12/2/2009)
Page 1 of 2
B-8
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EARCH CENTER
deviation No. (Assigned by QAU): ^ ^
CAQ No. (Assigned by QAU):
Deviation Reviewed and Corrective Action Accepted by/Date (study Director or Responsible individual):
.'TRV.
Deviation Reviewed and Corrective Action Accepted by/Date (Supervisor, Supervisor Rep
Circle One: Vivo, Micro. Mo I Tox. Aerosol. Chemistrv. BDS. MCB. Facilitv. OA. Studv Management. Other
resentative, or Group Manager):
/
O /
Deviation Rejiejfed ai^i Registered by QAU/Date: //l 7h °Jo
IMTwrnmtwt ' " ~
/ - -
~'Category I
~ Category II
(See SOP XI-023 for details)
^ /4 " i k'/,7 ' '*• ^ZTlT, **
Form No. Facility-035-05 (Revised 12/2/2009)
Page 2 of 2
B-9
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BATTELLE BIOMEDICAL RESEARCH CENTER
DEVIATION' FORM
deviation No. (Assigned by QAl'):
CAQ No. (Assigned by QAU):
Standard or Procedure Deviated:
~ Protocol (Number and Amendment No. if applicable):
El SOP (Number and Revision Number): X-054-06
~ Method (Number and Revision Number):
~ GLP (Section):
~ Other:
Type of Deviation (check one):
~ Facility
ED Study (fill out study info) Study Number: 1020-CG920503
Study Title: Rabbit Single Dose Anthrax Telemetry Study
Record Affected (describe Title, Binder name, location, Form no. etc.): Microbiology Spray Factor
Binder; Form No. Microbio-455
Date of Deviation(s): 9/2/09. 9/3/09, and 9/4/09
Description of Deviation:
°er SOP X-054 (section V.B.19), "if no dilutions result in a majority of plates containing between 25-250
colonies or none of the dilutions yield mean counts within the 25-250 range, the sample will be repeated".
The following samples did not meet these criteria, but were not re-plated.
Spray Factor Date
Sample ID
Result (cfu/mL)
9/2/09
Pre-run
l.OOxlO1
9/2/09
Day 1 Run 4-le5
1.00x10*
9/2/09
Day 1 Run 5-le5
1.22x10*
9/2/09
Day 1 Run 6-le5
1.88x10'
9/3/09
Pre-run
1.80x10'
9/3/09
Day 2 Run 4-le5
1.80x10'
9/3/09
Day 2 Run 5-1 e5
3.80x10'
9/3/09
1020SF-le7 (starting material)
0
9/4/09
Pre-run
2.80x10'
9/4/09
Day 3 Run 4-leS
1.30x10*
9/4/09
Day 3 Run 5-le5
1.90x10*
9/4/09
Day 3 Run 6-le5
2.14x10"
Root Cause of Deviation:
Each sample except for "1020SF-le7" is of such low concentration that additional enumeration would not
yield counts within the acceptable range. The study director and aerosol director decided not to have these
samples re-enumerated or plated using a filter method. It is unknown why the enumeration of sample
I "1020SF-le7" did not yield any colonies. It appears that the sample was not appropriately diluted or plated,
Form No. Facility-035-05 (Revised 12/2/2009)
Page 1 of 2
B-10
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BATTELLE BIOMEDICAL RESEARCH CENTER
DEVIATION FORM
deviation No. (Assigned by QAU):
CAQ No. (Assigned by QAU):
Corrective Action:
A suggestion was made that samples anticipated to be very low in concentration be enumerated via X-054
and X-199 (at the study director's discretion) in attempts to obtain the best possible values which meet the
acceptance criteria.
Impact of Deviation: Minimal. The pre-run sample is enumerated as a verification of the aerosol system and
the enumeration values obtained are not utilized in any calculation. The enumeration values obtained from
runs 4, 5, and 6 will be reported and utilized in the spray factor calculations. For the starting material from
9/3/09, the challenge enumeration from the previous day (7.86x106 cfu/mL) will be utilized in the spray factor
calculations.
If deviation is planned, effective date: n/a
Deviation Form Prepared by/Date:
S#f Ml .pi
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, picrci, Mol Tox, Aerosol, Chemistry, BDS, MCB, Facility, QA, Study Management, Other , / / .
-jCfC- ij^l^
Deviation Review^ and Registered by QAU/Date:
f/jy/j 9/o
~ 'Category I
~ Category II
(See SOP XI-023 for details)
Form No. FaciIity-035-05 (Revised 12/2/2009)
Page 2 of 2
B-ll
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BATTELLE BIOMEDICAL RESEARCH CENTER
DEVIATION FORM
deviation No. (Assigned by QAU): /
CAQ No. (Assigned by QAU): ~ /
Standard or Procedure Deviated:
~ Protocol(N umber and Amendment No. if applicable):
[3 SOP (Number and Revision Number): XI-009-02
G Method (Number and Revision Number):
~ GLP (Section):
~ Other:
Type of Deviation (check one):
~ Facility
IS] Study (fill out study info) Study Number: 1020-CG920503
Study Title: Rabbit Single Dose Anthrax Telemetry Study
Record Affected (describe Title, Binder name, location, Form no. etc.): Microbiology Spray Factor
Binder;
a. Form No. Microbio-380 and Microbio-455
b. Form No. Microbio-169
c. Form No. Microbio-454
Date of Deviation(s):
a. 9/2/09, 9/3/09, and 9/4/09
b. 8/31/09
c. 9/4/09
Description of Deviation:
Per SOP XI-009 (section V.A.5), study data, including materials used, must be recorded clearly, accurately,
legibly, completely and promptly.
a. The materials used to filter the impinger samples (Runs 4, 5, and 6 (1 c5)) were not documented.
b. The date documented for the equipment/reagent verification is incorrect.
c. The dilution scheme detailed for sample "1020SF - le6 (Day 3)" is not the dilution scheme actually
created and plated. This form indicates that final dilutions plated were 103 through 105, when actually
104 through 106 were plated per Form No. Microbio-455. ^ lo ^
Root Cause of Deviation:
a. The technicians did not document this information.
b. The technician inadvertently documented 3/31/09 instead of 8/31/09.
c. The technician did not verify that the dilution scheme on Microbio-454 matched the "final dilutions
plated" column on Microbio-455.
Corrective Action:
a. The technicians have been reminded to document all relevant materials used during a procedure and
asked to review the SOP detailing documentation.
b. The technician was reminded to accurately document all dates and asked to review the SOP detailing
documentation.
———————————^—^^—-^^========^^=====^=============
Form No, Facility-035-05 (Revised 12/2/2009) Page 1 of 2 5©_
-------
BATTELLE BIOMEDICAL RESEARCH CENTER
DEVIATION FORM
Deviation No. (Assigned by QAU):
f Y CA(^ No- (Assi§ned b> QAU):
c. The technician was reminded to accurately document all relevant materials and to carefully verify all
pre-typed information. The technician was also asked to review the SOP detailing documentation.
Impact of Deviation:
a. Minimal. The filters used for the procedure cannot be verified. The impinger samples were filter and
reportable results were obtained.
b. Minimal. The equipment and reagents are accurately documented on the form. All other dates were
accurately documented on the form.
c. Minimal. The sample was plated appropriately and a reportable result was obtained. 8.62x105 cfu/mL
will be used in the spray factor calculations.
If deviation is planned, effective date: n/a
Deviation Form Prepared by/Date: |
Deviation Reviewed and Corrective Action Accepted by/Date (Study Director or Responsible Individual):
, _ 1-zC-lQ
Deviation Reviewed and Corrective Action Accepted by/Date (Supervisor, Supervisor Representative, or Group Manager):
Circle One: Vivo, .flic-ro, Mol Tox, Aerosol, Chemistry, BDS, MCB, Facility, QA, Study Management, Other
Ii o
Deviation Reviewed and Registered by QAU/Date:
ffjy/M/ p
~Category I
~ Category II
(See SOP Xl-023 for details)
Form No. Facility-035-05 (Revised 12/2/2009)
Page 2 of 2
B-13
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BATTELLE BIOMEDICAL RESEARCH CENTER
DEVIATION FORM
Deviation No. (Assigned by QAU): q, ,
CAQ No. (Assigned by QAU): //'a
//i
Standard or Procedure Deviated:
~ Protocol (Number and Amendment No. if applicable):
13 SOP (Number and Revision Number): BBRC SOP XUI-001 4C ^ 44(jL ifai/jiD/Q
~ Method (Number and Revision Number):
~ GLP (Section):
~ Other:
Type of Deviation (check one):
~ Facility
E Study (fill out study info) Study Number: 1020-CG920503
Study Title: Rabbit Single Dose Anthrax Telemetry Study
Record Affected (describe Title, Binder name, location, Form no. etc.): Form No. Aerosol-010
Date of Deviation(s): 9/18/09
Description of Deviation: SOP XlII-001 requires that, "Readjust the exhaust pump valve (if needed) so the
magnehelic gauge reads in the range of 0.0 to -0.2 inches of H20" The reading was recorded as > -.25"H20 at
various points.
Root Cause of Deviation: Magnehelic gauge actually reads from approximately 0.25 to -0.25T12O; when an
animal's muzzle is in the chamber breathing, the point in time the reading was taken, the gauge was at the -
0.25" mark. At approximately 5 minutes, this could have been due to the fact that the valve to the APS, for
taking a sample, was also open at the time. When the valve is open, the vacuum in the chamber increases,
and in turn caused the gauge to read more negative for the duration of the 10 second APS sample. Also,
rabbits can cause the readings to be in this range simply by placing their head further into the box or by
moving and creating a better seal.
Corrective Action: SOP will be r e viewed/'clarified to specify how to estimate the pressure when an animal is
breathing; when an animal breathes, the needle moves back and forth over a certain range of pressure. This
may occur at various points on the gauge itself and a median reading is taken.
Impact of Deviation: Minimal. The system is still operating at a negative pressure as is necessary for
correct operation. Due to variations in animal respiration, it is difficult to avoid some readings over -
0.20"H20.
If deviation is planned, effective date: N/A
Deviation Form Prepared by/Date: tWO)
Deviation Reviewed and Corrective Action Accepted by/Date (Study Director or Responsible Individual):
_ ate \ z~i-io
Deviation Reviewed and Corrective Action Accepted by/Date (Supervisor, Supervisor Representative, or Group Manager):
Circle One: Vivo, Micro, Mol Tox^ierosgkt:li e ini s t ry, BDS, MCB, Facility, QA, Study Management, Other
1 jlA
Deviation Reviewed and Registered by QAU/Date
1/
^//// f ^ "] /I rj 10
~ Category I
~ Category II
(See SOP Xl-023 for details)
Form No. Facility-035-05 (Revised 12/2/2009) Page lof 1
B-14
-------
BATTELLE BIOMEDICAL RESEARCH CENTER
DEVIATION FORM
Deviation No. (Assigned by QAU): CAQ No. (Assigned by QAU): y g ^
Standard or Procedure Deviated:
EUProtocol (Number and Amendment No. if applicable):
~ SOP (Number and Revision Number):
n Method (Number and Revision Number):
~ GLP (Section):
~ Other:
Type of Deviation (check one):
~ Facility
EI Study (fill out study info) Study Number: 1020-CG920503
Study Title: Rabbit Single Dose Anthrax Telemetry Study
Record Affected (describe Title, Binder name, location, Form no. etc.):
Collection, Transfer, and Storage of Whole Blood; Form No. Microbio-372
Date of Deviation(s):9/21 '09
Description of Deviation: Protocol states The critical phase auditor observed technicians taking blood
samples from the vascular access ports when they should have collected blood from the ear vein. "Blood
samples will be collected from vascular access ports on Study Days -3, 1 and 2 for both the EDTA and SST
collection tubes. After Day 2 blood collected in the EDTA tubes will be obtained from the medial auricular
artery or the marginal ear vein." During a critical phase audit Z. Willenberg noted that the Day 3 bloods were
collected from the vascular access ports. This cannot be verified in the study records as the blood collection
sites are not captured on the form.
Root Cause of Deviation: The technicians did not review the protocol prior to the start: of the Day 3 blood
draws.
Corrective Action: The technicians were reminded to review the protocol prior to study related activities.
Also the blood collection form will be reviewed to determine if the blood collection site should be captured
on the paper work. ^ j (o nK !/m/io
C3jV> tjzR ho
Impact of Deviation: None. The reason for taking blood samples from the ear after Day 2 was to avoid false
positive bacteremia results because of a colonized port. All animals with positive bacteremias on Day 3
succumbed to disease suggesting the positive results were from systemic infection and not a colonized port.
If deviation is planned, effective date: /1/" /yf
Deviation Form Prepared by/Date: ^ , 0
Deviation Reviewed and Corrective Action Accepted bv/Date (study Director or Responsible individual):
_ 31C Z-l-iO
Deviation JJeviewed and Corrective Action Accepted by/Date (Supervisor, Supervisor Representative, or Group Manager):
Circle One: VjvSTMicro, Mol "I'ox, Aerosol, Chemistry, BBS, MCB, Facility, QA, Study Management, Other
_ QaaJ 3-/l j I d
Form No. Facility-035-05 (Revised 12/2/2009)
Page 1 of 2
B-15
-------
BATTELLE BIOMEDICAL RESEARCH CENTER |
DEVIATION FORM
Deviation No. (Assigned by QAU): ^ j ^
CAQ No. (Assigned by QAU): ~J cp £
Deviation Reviewed jptd Registered by QAU/Date:
. • :4ii{iflL MfU/ £ J I fJ-0/ C?
~¦'Category I
~ Category II
(See SOP XI-023 for details)
Form No. Facility-035-05 (Revised 12/2/2009)
Page 2 of 2
B-16
-------
APPENDIX C
BORDETELLA RESULTS
-------
Printed: Friday, September 18, 2009 at 11:14
Charles River Research Animal Diagnostic Services
251 Ballardvale Street, Wilmington, MA 01887 USA
Tel: 800-338-9680 Fax: 978-658-7698
Sponsor: Battelle Memorial Institute
Accession #: 2009-040733
Diagnostic Summary Report
1425 Plain City-Georgesville Rd.
State Rt. 142
West Jefferson, OH 43162 USA
Attn: Jason Comer
Tel: 614-424-5825
Received:
Approved:
Bill Method:
Test Specimen:
15 Sep 2009
18 Sep 2009, 11:14
PO# VI14984001710
Respiratory
Sample Set Service (# Tested)
Profile Assay
Tested + +/- ?
#1 Bacteriology (34)
Bordetella Screen - B. bronchiseptica 34 9 0 0
Respiratory
Summary Remarks
+ = Positive, +/- = Equivocal, ? = Indeterminate
1
BACTERIOLOGY
Bordetella bronchiseptiea has not been demonstrated to be pathogenic in rabbits, and is considered to be of no clinical
consequence in immunocompetent animals.
Sen ice Appnnnls |
Service
Approved By*
Date
Bacteriology
Richard D. Fister
18 Sep 2009, 11:14
To assure the SPF status of your research animal colonies, it is essential that you understand the sources, pathobiology, diagnosis
and control of pathogens and other adventitious infectious agents that may cause research interference. We have summarized this
important information in infectious agent Technical Sheets, which you can view by visiting
http://www.criver.com/info/disease sheets.
*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.
CRRADSILIMSForm: FM-1741 Rev. 3
Page 1 of 2
C-2
-------
Printed: Friday, September is, 2009 at 11:14 Charles River Research Animal Diagnostic Services
251 Ballardvale Street, Wilmington, MA 01887 USA
Tel: 800-338-9680 Fax: 978-658-7698
Sponsor: Battelle Memorial Institute Accession #: 2009-040733
Product: Not Indicated Test Specimen: Respiratory Received: 15 Sep 2009
Bacteriology Results Report
Department Review: Approved by Richard D. Fister, 18 Sep 2009, 11:14*
Sample
( ode :
1
L23226
2
L23216
3
L23218
4
L23215
5
L23222
6
L23223
7
L23206
8
L23210
9
L23219
HI
L23211
IS. hrtmchiseptica
Other
2 1 1 1 - 1 - 1 - 1 1 1 - 1 - 1
Sample
( ode :
11
L23217
12
L23230
13
L23228
14
L23232
15
L23229
16
L23235
12
L23213
IS
L23225
12
L23231
20
L23207
B. bronchiseptica
-
-
2
-
-
-
-
1
1
2
Other
-
-
-
-
-
-
-
-
-
-
Sample ':
( ode :
Zi
L23201
22
L23234
23
L23212
24
L23200
25
L23214
26
L23204
27
L23203
28
L23205
29
L23221
30
L23227
B. bronchiseptica
1
-
-
-
1
-
-
-
-
-
Other i-i-i-i-i-i-i-i-i-i-
Sample ':
( ode :
31 32 33 34
L23220 L23223 L23202 L23209
B. bronchiseptica
Other
1 - 1 - 1 -
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.
Page 2 of 2
CR RADSILIMSForm: FM-1741 Rev. 3
C-3
-------
APPENDIX D
SPRAY FACTOR REPORT
1020-CG920503 Spray Factor Report
D-l
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Table of Contents
1.0 Introduction D-4
2.0 Methods D-5
3.0 Experimental Setup and Test Conditions D-7
3.1 Aerosol Generation Subsystem D-7
3.2 Delivery Subsystem D-7
3.3 Exposure Chamber D-8
3.4 Sampling/Monitoring D-8
4.0 Inhalation Results D-10
4.1 Impinger Sample Analysis D-10
4.2 Inhaled Dose Calculation D-10
5.0 Particle Size Results D-12
6.0 Conclusions D-21
7.0 References D-21
List of Tables
Table 1. Low Dose Spray Factor, 09-02-09 D-14
Table 2. Low Dose Spray Factor, 09-03-09 D-15
Table 3. Low Dose Spray Factor, 09-04-09 D-15
List of Figures
Figure 1. Exposure system diagram D-13
Figure 2. lxl04CFU/mL Spray factor log probability size distribution plot D-17
Figure 3. lxl05CFU/mL Spray factor log probability size distribution plot D-18
Figure 4. lxl06CFU/mL Spray factor log probability size distribution plot D-19
n
Figure 5. 1x10 CFU/mL Spray factor log probability size distribution plot D-20
1020-CG920503 Spray Factor Report D-2
-------
List of Acronyms
APS aerodynamic particle sizer
BBRC Battelle Biomedical Research Center
BCS III Class III biological safety cabinet
CFU colony forming unit
cm centimeter
d diameter
GSD geometric standard deviation
HEPA high efficiency particulate air
InD inhaled dose
LD50 median lethal dose
L liter
MFC mass flow controller
min minute
mL milliliter
mm millimeter
MMAD mass median aerodynamic diameter
SOP standard operating procedure
SF spray factor
TSA tryptic soy agar
|im micrometer
1020-CG920503 Spray Factor Report
D-3
-------
1. Introduction
The large animal exposure system at the Battelle Biomedical Research Center (BBRC) has been
developed by Battelle and used extensively in aerosol studies to deliver high doses of Bacillus
n
anthracis spores to animals (>1.1 xlO colony forming units [CFU] inhaled). The purpose of this
Spray Factor characterization study was to determine whether aerosol concentrations could be
2 4
achieved in the system that would allow for low doses of spores (10 -10 CFU inhaled) to be
delivered to rabbits.
1020-CG920503 Spray Factor Report D-4
-------
2. Methods
The aerosol system performance characteristics were evaluated by aerosolizing four target
nebulizer concentrations of B. anthracis spores multiple times over a three day period. The target
nebulizer concentrations tested over the three days were 1.0 xlO4, 1.0 xlO5, 1.0 xlO6, and 1.0
n
xlO CFU/milliliter (mL). Specifically, each concentration was tested a total of a total of nine
times each (three times per day over three days). Each test consisted of a 10-minute (min) period
of aerosol generation and sample collection. The overall mean SF was determined. A schematic
of the exposure system is shown in Figure 1.
The Spray Factor (SF) is a numeric correlation between nebulizer concentration and the resulting
generated aerosol concentration. The SF is calculated by dividing the aerosol concentration by
the starting nebulizer suspension concentration and is used to predict aerosol concentration for a
given starting suspension concentration. The formula for determining the Spray Factor is
presented below (Equationl):
Impinger Cone. (C'FU/mL) x Impinger Volume (mL) Aerosol Cone (CFU/L)
Spray Impinger Sample Rate (L/min) x Test Duration (mini _ ...
Factor = XT u ,• ,, , , 1AAn , „ Nebulizer Cone (CFU/L) ^ '
Nebulizer Cone (CFU/mL)_x 1000 mL/L v
Once the SF is determined, the value can be used for future studies to predict how many liters of
aerosol an animal must inhale to achieve a particular intended dose. In this case, the value was
used in study protocol 1020 which targeted inhaled doses of 1 xlO2, 1 xlO3, 1 xlO4, and 1 xlO5
CFU.
The practical application of the SF is that during an aerosol exposure when the nebulizer
concentration is known the aerosol concentration that will be produced can be calculated and the
amount an animal must inhale to reach the desired target dosage can be be determined (breathing
rate unknown). For example:
• A target total inhaled dosage of 100 CFU is chosen
• An exposure period is unknown due to individual animal breathing rate and volumes
• The starting nebulizer concentration is 1.66 x 104 CFU /mL
• Given that the aerosol concentration equation is:
ConC(aerosol) CFU/L SF X ConC(nebulizer) CFU/L
1020-CG920503 Spray Factor Report
D-5
-------
1
• Given that the SF in this study was determined experimentally to be 4.58 x 10"
Concaerosol = 1.66 x 104 CFU/L X 1000 mL/L x (4.58 x 10"7)
ConCaerosol = 7.6 CFU/L
• The target inhaled volume =100 CFU/7.6 CFU/L = 13 L to inhale.
1020-CG920503 Spray Factor Report D-6
-------
3. Experimental Setup and Test Conditions
The Battelle large animal exposure system can be divided into five subsystems. Below is a
description of each subsystem.
3.1 Aerosol Generation Subsystem
Air was supplied to the system 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 nebulizer bypass airflow of approximately 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 52% - 97%. 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 (spore lot Ames
B35) from a liquid suspension. These nebulizers are designed to generate aerosols having an
approximate mean diameter of 1-2 micrometers (|im). Each nebulizer was characterized for a
pressure that results in an approximately 7.5 L/min flow, which is approximately 25-36 pounds
per square inch, Collison nebulizer dependant.
3.2 Delivery Subsystem
After the agent aerosol was generated by the Collison nebulizer, it exited the Collison and
traveled down a 3.75 centimeter (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 -0.18 to -0.01 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.
1020-CG920503 Spray Factor Report
D-7
-------
3.3 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 material 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 aerodynamic particle sizer (APS) spectrometer and exposure of the animal all occur from the
same chamber. The aerosol system was operated within a self-contained BSC III.
3.4 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 milliliter.
Concentrations are reported in terms of 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 test using an APS spectrometer, which
draws 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). An APS was
used because of its advantages over other methods. These advantages include near real-time data
1020-CG920503 Spray Factor Report
D-8
-------
measurements, aerodynamic diameter measurements, ease of instrument operation, and the
generation of electronic data that is easy to process and export to a report.
1020-CG920503 Spray Factor Report
D-9
-------
4. Inhalation Results
The inhalation exposure system data for each exposure were 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 (CFU) was calculated from aerosol
concentration and total inhaled volume. Tables 1 through 3 show the daily aerosol testing results.
n
The mean SF value from all testing was 4.58 x 10" . The number of median lethal dose
equivalents (LD50 value) was calculated by dividing the total inhaled dose by the reported
inhalation LD50 for each particular species of animal. The reported LD50 value for rabbits is
105,000 CFU (Zaucha et al., 1998) and for Rhesus macaques is 55,000 CFU (Ivins et al., 1998).
4.1 Impinger Sample Analysis
Impinger samples were enumerated by the spread plate method 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 tryptic
soy agar (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
(C) was determined using the equation below (Equation 2):
C = (A • D) / 0.1 mL (2)
C = CFU/mL (impinger sample concentration)
A = average CFU per plate
D = dilution factor (serial dilution with countable colonies)
4.2 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. This equation assumes 100
% impinger sampling efficiency, which is based on evidence from previous studies in other
laboratories. The total number of viable spores captured during each exposure was the product of
1020-CG920503 Spray Factor Report
D-10
-------
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 was (C x V) x (S x T)"1. The inhaled dose was
the product of the aerosol concentration multiplied by the total accumulated tidal volume (see
Equation 3 below):
InD = (C x V) (S x T)-'(TATV) (3)
InD = inhaled dose (CFU)
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)
1020-CG920503 Spray Factor Report
D-ll
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5. 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
"3
diameter is the diameter of a sphere, with density = 1 g/cm , 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 <5 [j.m 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 [^m (or lower, as opposed to larger aerosols) is desired.
Figures 2 through 5 show log probability plots representing the average of all APS particle size
distributions obtained for each concentration tested over the three days of testing. The MMAD
and geometric standard deviation (GSD) are also shown.
The MMAD for the log probability plot (Figure 2) was determined from averaging the
cumulative median size (50% mass) from the aerosol size distributions obtained from the APS
for all aerosol exposures (Equation 4). 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% (4)
Where d84% is the particle size diameter in |im (d) at a cumulative % mass of 84% and d50% is
the particle size diameter (d) at a cumulative mass of 50% (Hinds, 1999).
1020-CG920503 Spray Factor Report
D-12
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Check
Valve
Valve
3-Way
Valve
Valve
Ball valve
Water
Bubbler
Exposure
Port
Ball Valve
Ball Valve
Sample
Pump
Collison
Pressure Gauge
Needle
Valve
MFM
Pressure
Regulator
~^n
MFM
Differential
I Pressure
| Gauge Q7
Pressure
Regulator
Collison
Nebulizer
Vacuum
Gauge
Transducer
APS Verification
System
Impingers
Ball Valve
Pneumotach
-7.5
MFM
Diluter
Plethysmogr iph
Exposure
Chamber
Ball valve
Temperature
And Humidity
Monitor
Valve
-in
MFM
Valve
Exhaust Pump
PC withBioSystemXA software
MaxII Preamplifier
Figure 1. Exposure system diagram. APS, aerodynamic particle sizer; MFC, mass flow controller; MFM, mass flow meter
1020-CG920503 Spray Factor Report
Ball £__l
Valve
-------
Table 1. Low Dose Spray Factor, (09-02-09)
Impinger Impinger Total Sample
Neb Impinger Volume (Total Sample Rate Sample Aerosol Volume Mortality Spray ID
(CFU/mL) (CFU/mL) (mL) CFU) (L/min) Time (min) (CFU/L) inhaled (L) Factor Run
8.55E+03
840E+00
19.00
1.60E+02
6.00
10.00
3
NA
NA
3.11E-07
1
8.55E+03
1.36E+01
19.00
2.58E+02
6.00
10.00
4
NA
NA
5.04E-07
2
8.55E+03
1.58E+01
18.50
2.92E+02
6.00
10.00
5
NA
NA
5.70E-07
3
8.17E+04
1.00E+02
19.20
1.92E+03
6.00
10.00
32
NA
NA
3.92E-07
4
8.17E+04
1.22E+02
19.20
2.34E+03
6.00
10.00
39
NA
NA
4.78E-07
5
8.17E+04
1.88E+02
19.20
3.61E+03
6.00
10.00
60
NA
NA
7.36E-07
6
7.03E+05
5.98E+02
19.00
1.14E+04
6.00
10.00
189
NA
NA
2.69E-07
7
7.03E+05
1.11E+03
18.00
2.00E+04
6.00
10.00
333
NA
NA
4.74E-07
8
7.03E+05
1.16E+03
19.00
2.20E+04
6.00
10.00
367
NA
NA
5.23E-07
9
7.86E+06
6.90E+03
19.00
1.31E+05
6.00
10.00
2185
NA
NA
2.78E-07
10
7.86E+06
7.64E+03
18.80
1.44E+05
6.00
10.00
2394
NA
NA
3.05E-07
11
7.86E+06
8.92E+03
19.00
1.69E+05
6.00
10.00
2825
NA
NA
3.59E-07
12
NA Not applicable
1020-CG920503 Spray Factor Report
D-14
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Table 2. Low Dose Spray Factor, (09-03-09)
Impinger Impinger Total Sample
Neb Impinger Volume (Total Sample Rate Sample Aerosol Volume Mortality Spray ID
(CFU/mL) (CFU/mL) (mL) CFU) (L/min) Time (min) (CFU/L) inhaled (L) Factor Run
8.55E+03
8.40E+00
19.00
1.60E+02
6.00
10.00
3
NA
NA
3.11E-07
1
8.55E+03
1.36E+01
19.00
2.58E+02
6.00
10.00
4
NA
NA
5.04E-07
2
8.55E+03
1.58E+01
18.50
2.92E+02
6.00
10.00
5
NA
NA
5.70E-07
3
8.17E+04
1.00E+02
19.20
1.92E+03
6.00
10.00
32
NA
NA
3.92E-07
4
8.17E+04
1.22E+02
19.20
2.34E+03
6.00
10.00
39
NA
NA
4.78E-07
5
8.17E+04
1.88E+02
19.20
3.61E+03
6.00
10.00
60
NA
NA
7.36E-07
6
7.03E+05
5.98E+02
19.00
1.14E+04
6.00
10.00
189
NA
NA
2.69E-07
7
7.03E+05
1.11E+03
18.00
2.00E+04
6.00
10.00
333
NA
NA
4.74E-07
8
7.03E+05
1.16E+03
19.00
2.20E+04
6.00
10.00
367
NA
NA
5.23E-07
9
7.86E+06
6.90E+03
19.00
1.31E+05
6.00
10.00
2185
NA
NA
2.78E-07
10
7.86E+06
7.64E+03
18.80
1.44E+05
6.00
10.00
2394
NA
NA
3.05E-07
11
7.86E+06
8.92E+03
19.00
1.69E+05
6.00
10.00
2825
NA
NA
3.59E-07
12
NA Not applicable
1020-CG920503 Spray Factor Report
D-15
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Table 3. Low Dose Spray Factor, (09-04-09)
Impinger Impinger Total Sample
Neb Impinger Volume (Total Sample Rate Sample Aerosol Volume Mortality Spray ID
(CFU/mL) (CFU/mL) (mL) CFU) (L/min) Time (min) (CFU/L) inhaled (L) Factor Run
8.55E+03
8.40E+00
19.00
1.60E+02
6.00
10.00
3
NA
NA
3.11E-07
1
8.55E+03
1.36E+01
19.00
2.58E+02
6.00
10.00
4
NA
NA
5.04E-07
2
8.55E+03
1.58E+01
18.50
2.92E+02
6.00
10.00
5
NA
NA
5.70E-07
3
8.17E+04
1.00E+02
19.20
1.92E+03
6.00
10.00
32
NA
NA
3.92E-07
4
8.17E+04
1.22E+02
19.20
2.34E+03
6.00
10.00
39
NA
NA
4.78E-07
5
8.17E+04
1.88E+02
19.20
3.61E+03
6.00
10.00
60
NA
NA
7.36E-07
6
7.03E+05
5.98E+02
19.00
1.14E+04
6.00
10.00
189
NA
NA
2.69E-07
7
7.03E+05
1.11E+03
18.00
2.00E+04
6.00
10.00
333
NA
NA
4.74E-07
8
7.03E+05
1.16E+03
19.00
2.20E+04
6.00
10.00
367
NA
NA
5.23E-07
9
7.86E+06
6.90E+03
19.00
1.31E+05
6.00
10.00
2185
NA
NA
2.78E-07
10
7.86E+06
7.64E+03
18.80
1.44E+05
6.00
10.00
2394
NA
NA
3.05E-07
11
7.86E+06
8.92E+03
19.00
1.69E+05
6.00
10.00
2825
NA
NA
3.59E-07
12
NA Not applicable
1020-CG920503 Spray Factor Report
D-16
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1x104cfu/mL Spray Factor log probability size distribution plot.
99.99
98 -
CO
<§ 84.14 -
50 -
-------
1x105 cfu/mL Spray Factor log probability size distribution plot.
99.99
84.14
H—'
ro
O
>-
15.87
0.1
0.5
1
2
5
10
Size [jm
• Day 1 MMAD = 0.84 p, GSD = 1.40
V Day 2 MMAD = 0.96 nm, GSD =1.46
¦ Day 3 MMAD = 0.89 nm, GSD =1.46
Figure 3. lxl05CFU/mL Spray factor log probability size distribution plot.
MMAD, mass median aerodynamic diameter; GSD, geometric standard deviation
1020-CG920503 Spray Factor Report
D-18
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1x106 cfu/mL Spray Factor log probability size distribution plot.
99.99
98 -
w
Jg 84.14 -
c
0
o
1—
50 -
0
CL
15.87 -
0.1
0.5
1
2
5
10
Size [jm
• Day 1 MMAD = 1.03 p, GSD = 1.49
V Day 2 MMAD = 0.99 p, GSD = 1.44
¦ Day 3 MMAD = 1.03 p, GSD = 1.42
Figure 4. lxl06CFU/mL Spray factor log probability size distribution plot.
MMAD, mass median aerodynamic diameter; GSD, geometric standard deviation
1020-CG920503 Spray Factor Report
D-19
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1x107 cfu/mL Spray Factor log probability size distribution plot.
99.99
98 -
84.14 -
50 -
15.87 -
0.1
0.5
1
2
5
10
Size |xm
• Day 1 MMAD = 1.04 jim, GSD = 1.34
V Day 2 MMAd = 1.05 |j,m, GSD = 1.33
¦ Day 3 MMAD = 1.06 jim, GSD = 1.39
Figure 5. lxl07CFU/mL Spray factor log probability size distribution plot.
MMAD, mass median aerodynamic diameter; GSD, geometric standard deviation
1020-CG920503 Spray Factor Report
D-20
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6. Conclusions
This testing determined that the BBRC's large animal aerosol system is capable of generating
2 4
aerosol concentrations that will allow for low doses of spores (10 -10 CFU inhaled) to be
delivered to rabbits.
7. References
1. Hinds, William C. 1999. Aerosol Technology: Properties, Behavior, and Measurement of
Airborne Particles. Second Edition. Wiley-Interscience, John Wiley & Sons Inc., New York,
NY: Pages 94-97.
2. Zaucha, G.M., Pitt, L.M., Estep, J., Ivins, B.E., and Friedlander, A.M., 1998. The pathology
of experimental rabbits exposed by inhalation and subcutaneous inoculation. Arch. Pathol.
Lab. Med. 122(11):982-992.
3. B E. Ivins, M. L. M. Pitt, P. F. Fellows, J. W. Farchaus, G. E. Benner, D. M. Waag, S. F.
Little, G. W. Anderson, Jr. , P. H. Gibbsand A. M. Friedlander (1998) Comparative efficacy
of experimental anthrax vaccine candidates against inhalation anthrax in rhesus macaques.
Vaccine 16: 1141-1148.
1020-CG920503 Spray Factor Report
D-21
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APPENDIX E
AEROSOL REPORT
1020-CG920503 Aerosol Report
E-l
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Table of Contents
Figures E-2
Tables E-2
List of Acronyms E-3
1. Methods E-4
2. Experimental Setup and Conditions E-4
2.1 Aerosol Generation Subsystem E-4
2.3 Exposure Chamber E-5
2.4 Sampling/Monitoring E-5
2.5 Plethysmography E-6
3. Inhalation Results E-7
3.1. Impinger Sample Analysis E-7
3.2 Inhaled Dose Calculation E-7
4. Particle Size Results E-9
5. References E-13
Figures
Figure 1. Exposure system diagram E-ll
Figure 2. Log probability size distribution plot E-12
Tables
Table 1. Inhalation Exposure Report for Rabbits (09-18-09) E-10
1020-CG920503 Aerosol Report E-2
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List of Acronyms
APS aerodynamic particle sizer
BBRC Battelle Biomedical Research Center
BSC III Class III biological safety cabinet
C impinger concentration
cm centimeter
CFU colony forming units
D dilution factor
d diameter
GSD geometric standard deviation
HEPA high efficiency particulate air
InD total inhaled dose
L liter
LD50 Value median lethal dose equivalent
MFC mass flow controller
MFM mass flow meter
min minute
mL milliliter
MMAD mass median aerodynamic diameter
S sampling rate
SOP standard operating procedure
T exposure time
TATV total accumulated tidal volume
TSA tryptic soy agar
|im micrometer
V impinger sampler volume
WBP whole body plethysmography
1020-CG920503 Aerosol Report
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1. Methods
Standard operating procedures (SOPs) developed by Battelle and utilized for other similar
studies were followed during animal aerosol exposure challenges.
2. Experimental Setup and Conditions
The Battelle large animal exposure system (Figure 1) can be divided into five subsystems:
Aerosol Generation Subsystem, Delivery System, Exposure Chamber, Sampling/Monitoring, and
Plethysmography. Below is a description of each subsystem.
2.1 Aerosol Generation Subsystem
Air was supplied to the system 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 liters per minute (L/min) and a nebulizer/bypass airflow of approximately 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 desired range of 74% to 83%. 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 (spore lot Ames B35) from a liquid suspension. These nebulizers are designed to generate
aerosols having an approximate mean diameter of 1 to 2 micrometer (|im). Each nebulizer was
characterized for a pressure that results in an approximately 7.5 L/min flow, which normally is
approximately 28.0 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 centimeter (cm) diameter, 40 cm long cylinder (mixing tube) that mixed 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
1020-CG920503 Aerosol Report
E-4
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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 -0.25 to -0.01
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.
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 material 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 Aerodynamic Particle Sizer (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 milliliter
(mL) of sterile water. Chamber air was drawn into and through the impinger 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 CFUs per mL
(CFU/mL) of impinger liquid. Enumeration results, along with the volume of liquid in the
1020-CG920503 Aerosol Report
E-5
-------
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
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
Whole body plethysmography (WBP) was performed real-time on each animal during agent
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 the sealed chamber (the plethysmograph). The data generated for each animal were used
to determine the TATV, which along with the aerosol concentration were used in calculating the
inhaled dose. Rabbits were physically restrained within a plethysmograph restraint device with
the head protruding out of a port that was sealed with rubber dental dam material 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 Buxco software program was calibrated to establish unit
(baseline) and air volume displacements from 5 to 40 mL to simulate animal respiration within
the plethysmograph. This calibration was performed to encompass the respiration volume range
of the animal model for accurate TATV measurements.
1020-CG920503 Aerosol Report
E-6
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3. Inhalation Results
The inhalation exposure system data for each exposure were 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 were
used to calculate the viable bioaerosol challenge concentration while plethysmography
measurements documented the total inhaled volume. Total inhaled dose (CFU) was calculated
from aerosol concentration and total inhaled volume. The number of median lethal dose
equivalents (LD50 value) was calculated by dividing the total inhaled dose by the reported
inhalation LD50 for the rabbit. The reported LD50 value for rabbits is 105,000 CFU (Zaucha et al.,
1998). Table 1 shows the inhalation results for this study.
3.1. Impinger Sample Analysis
Impinger samples were enumerated by the spread plate method 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 tryptic
soy agar (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
(C) was determined using Equation 1:
C = (A • D) / 0.1 mL (1)
C = CFU/mL
A = average CFU 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 (C) and the
impinger volume (V). The total number of viable organisms was divided by the amount that was
sampled through the impinger during the exposure time (T). The aerosol concentration was (C x
1020-CG920503 Aerosol Report
E-7
-------
V) (S x T)"1. The inhaled dose was the product of the aerosol concentration multiplied by the
total accumulated tidal volume (Equation 2).
InD = (C x V) (S x T)-'(TATV) (2)
InD = Total inhaled dose (CFU)
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)
1020-CG920503 Aerosol Report
E-8
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4. Particle Size Results
Figure 2 shows a log probability plot representing the average of all APS particle size
distributions obtained from exposure testing. The mass median aerodynamic diameter (MMAD)
and geometric standard deviation (GSD) are also shown.
The MMAD for the log probability plot (Figure 2) was determined from averaging the
cumulative median size (50% mass) from the aerosol size distributions obtained from the APS
for all aerosol exposures (Equation 3). 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).
1020-CG920503 Aerosol Report
E-9
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Table 1. Inhalation Exposure Report for Rabbits, (09-18-09)
Nebulizer Impinger Impinger Impinger Aerosol Total Accum.
Rabbit ID Cone. Cone. Volume Impinger Sample Sample Cone. Tidal Volume Inhaled Dose Ames LDS0 Spray
(CFU/mL) (CFU/mL) (mL) (CFU) Rate (L/min) Time (min) (CFU/L) Inhaled (L) (CFU/animal) Equivalents Factor
L23220
0.00E+00
0.00E+00
19.00
0.00E+00
6
7.00
0.00E+00
10.2
0
0.000
NA
L23216*
0.00E+00
3.00E+01
18.75
5.63E+01
6
9.15
1.02E+00
10.0
10
0.000
NA
L23218
0.00E+00
0.00E+00
18.75
0.00E+00
6
8.88
0.00E+00
10.0
0
0.000
NA
L23223
0.00E+00
0.00E+00
19.25
0.00E+00
6
7.77
0.00E+00
10.0
0
0.000
NA
L23222
0.00E+00
0.00E+00
19.00
0.00E+00
6
7.08
0.00E+00
10.0
0
0.000
NA
L23215
1.66E+04
5.12E+01
19.00
9.73E+02
6
7.00
2.32E+01
13.9
322
0.003
1.40E-06
L23206
1.66E+04
4.78E+01
19.40
9.27E+02
6
7.00
2.21E+01
13.5
298
0.003
1.33E-06
L23210
1.66E+04
5.34E+01
19.20
1.03E+03
6
7.85
2.18E+01
10.0
218
0.002
1.31E-06
L23219
1.66E+04
3.74E+01
19.20
7.18E+02
6
5.60
2.14E+01
15.0
321
0.003
1.29E-06
L23211
1.66E+04
3.46E+01
19.00
6.57E+02
6
6.03
1.82E+01
15.0
273
0.003
1.09E-06
L23217
1.37E+05
2.55E+02
19.40
4.95E+03
6
7.00
1.18E+02
12.6
1484
0.014
8.60E-07
L23230
1.37E+05
3.98E+02
19.40
7.72E+03
6
7.00
1.84E+02
11.0
2022
0.019
1.34E-06
L23228
1.37E+05
4.82E+02
19.40
9.35E+03
6
7.05
2.21E+02
10.1
2233
0.021
1.61E-06
L23227
1.37E+05
6.00E+02
19.20
1.15E+04
6
8.25
2.33E+02
10.0
2327
0.022
1.70E-06
L23229
1.37E+05
6.00E+02
19.00
1.14E+04
6
8.48
2.24E+02
10.0
2241
0.021
1.64E-06
L23235
1.65E+06
4.12E+03
19.20
7.91E+04
6
7.47
1.76E+03
10.0
17649
0.168
1.07E-06
L23205
1.65E+06
4.04E+03
19.20
7.76E+04
6
7.00
1.85E+03
14.8
27333
0.260
1.12E-06
L23225
1.65E+06
5.10E+03
19.60
1.00E+05
6
7.00
2.38E+03
10.9
25942
0.247
1.44E-06
L23231
1.65E+06
5.12E+03
19.00
9.73E+04
6
7.00
2.32E+03
10.4
24088
0.229
1.40E-06
L23207
1.65E+06
5.40E+03
19.40
1.05E+05
6
7.00
2.49E+03
12.8
31927
0.304
1.51E-06
L23201
1.96E+07
2.78E+04
19.20
5.34E+05
6
7.00
1.27E+04
14.0
177920
1.694
6.48E-07
L23234
1.96E+07
4.94E+04
19.20
9.48E+05
6
7.00
2.26E+04
13.1
295835
2.817
1.15E-06
L23212
1.96E+07
5.56E+04
19.40
1.08E+06
6
7.00
2.57E+04
12.8
328728
3.131
1.31E-06
L23200
1.96E+07
2.17E+04
19.60
4.25E+05
6
4.78
1.48E+04
14.8
219482
2.090
7.57E-07
L23214
1.96E+07
4.14E+04
19.40
8.03E+05
6
5.68
2.36E+04
15.0
353504
3.367
1.20E-06
L23204
1.05E+09
7.52E+05
19.40
1.46E+07
6
6.13
3.97E+05
15.0
5949755
56.664
3.78E-07
L23203
1.05E+09
1.83E+06
19.00
3.48E+07
6
7.00
8.28E+05
10.7
8858071
84.363
7.88E-07
L23213
1.05E+09
1.59E+06
19.20
3.05E+07
6
6.98
7.29E+05
10.0
7289398
69.423
6.94E-07
L23221
1.05E+09
1.85E+06
19.20
3.55E+07
6
7.00
8.46E+05
10.5
8880000
84.571
8.05E-07
L23232
1.05E+09
1.83E+06
19.20
3.51E+07
6
7.00
8.37E+05
12.4
10373486
98.795
7.97E-07
First five animal exposures were with irradiated spores, CFU/mL = 0
* Sample was likely contaminant as the aerosol system was decontaminated and showed no growth. Also all other irradiated samples were no growth
NA = not applicable. The spray factor cannot be calculated for these samples as the starting concentration enumeration is zero as there were not viable from irradiation
1020-CG920503 Aerosol Report
E-10
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Check
Valve
Valve
3-Way
Valve
Valve
Ball Valve
Water
Bubbler
Exposure
Port
Ball Valve
Ball Valve
Sample
Collison
Pressure Gauge
Needle
Valve
MFM
Pressure
Regulator
MFV
~^n
MFM
Differential
I Pressure^
| Gauge (/
, Pressure
Regulator
Collison
Nebulizer
Vacuum
Gauge
Transducer
APS Verification
System
Impingers
Ball Valve
Pneumotach
-7.5
MFM
Diluter
Plethysmogr iph
Exposure
Chamber
Ball Valve
Temperature
And Humidity
Monitor
Valve
-in
MFM
Valve
Exhaust Pump
PC with BioSystem XA Software
MaxII Preamplifier
Ball
Valve
Figure 1. Exposure system diagram.
APS = Aerodynamic Particle Sizer, MFC = mass flow controller, MFM = mass flow meter
1020-CG920503 Aerosol Report
E-ll
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Log probability size distribution plot.
99.99
98 -
w
£ 84.14 -
2
$ •
-i—•
c
CD
O
50 -
CD
CL
15.87 -
0.1
0.5
1
2
5
10
Size [im
• Group 1 MMAD = 0.96 |jm, GSD = 1.33
V Group 2 MMAD = 0.82 |jm, GSD = 1.48
¦ Group 3 MMAD = 0.92 |im, GSD = 1.57
O Group 4 MMAD = 0.87 |jm, GSD = 1.59
A Group 5 MMAD= 1.12 \im, GSD = 1.33
0 Group 6 MMAD = 1.12 |im, GSD = 1.31
Figure 2. Log probability size distribution plot.
MMAD, mass median aerodynamic diameter; GSD, geometric standard deviation
1020-CG920503 Aerosol Report
E-12
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5. References
1. Hinds, William C. Aerosol Technology: Properties, Behavior, and Measurement of Airborne
Particles. Second Edition 1999. Wiley-Interscience, John Wiley & Sons Inc., New York, NY:
Pages 94-97.
2. Zaucha, G.M., Pitt, L.M., Estep, J., Ivins, B.E., and Friedlander, A.M., 1998. The pathology
of experimental rabbits exposed by inhalation and subcutaneous inoculation. Arch. Pathol.
Lab. Med. 122(11): 982-992.
Report Prepared By
Date
David A. Fisher
Master Aerosol Technician
Report Reviewed By: {H
X! "
Roy j;. Barnewall, D.V.M, Ph. D.
Date
Manager Aerobiology
1020-CG920503 Aerosol Report
E-13
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APPENDIX F
STATISTICAL ANALYSIS OF TELEMETRY DATA
F-l
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Table of Contents
1. Introducti on F- 8
2. Statistical Methods F-9
3. Results F-ll
4. Conclusions F-16
F-2
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List of Tables
Table 1. Study Design F-8
Table 2. Summary of the ANOVA Results for the Baseline Adjusted Six-Hour Averages
for Activity (Counts/Minute) 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-25
Table 5. Summary of the ANOVA Results for the Baseline Adjusted Six-Hour Averages
forRP Inspiratory Time (Seconds) F-29
Table 6. Summary of the ANOVA Results for the Baseline Adjusted Six-Hour Averages
forRP Integral (mmHg-seconds) F-33
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
forRP respiratory rate (RCPM) F-41
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 Pairwise Log-Rank Tests Comparing the Time to Abnormality
Between Groups by Parameter F-53
Table 13. Results of Overall Log-Rank Tests Comparing the Duration of Abnormality
Between Groups by Parameter F-54
Table 14. Results of Pairwise Log-Rank Tests Comparing the Duration of Abnormality
Between Groups by Parameter F-55
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List of Figures
Figure la. Plot of baseline adjusted activity (counts/minute) for each animal in Group 1 F-49
Figure lb. Plot of baseline adjusted activity (counts/minute) for each animal in Group 2 F-49
Figure lc. Plot of baseline adjusted activity (counts/minute) for each animal in Group 3 F-50
Figure Id. Plot of baseline adjusted activity (counts/minute) for each animal in Group 4 F-50
Figure le. Plot of baseline adjusted activity (counts/minute) for each animal in Group 5 F-51
Figure If. Plot of baseline adjusted activity (counts/minute) for each animal in Group 6 F-51
Figure 2a. Plot of baseline adjusted heart rate (BPM) for each animal in Group 1 F-52
Figure 2b. Plot of baseline adjusted heart rate (BPM) for each animal in Group 2 F-52
Figure 2c. Plot of baseline adjusted heart rate (BPM) for each animal in Group 3 F-53
Figure 2d. Plot of baseline adjusted heart rate (BPM) for each animal in Group 4 F-53
Figure 2e. Plot of baseline adjusted heart rate (BPM) for each animal in Group 5 F-54
Figure 2f. Plot of baseline adjusted heart rate (BPM) for each animal in Group 6 F-54
Figure 3a. Plot of baseline adjusted RP expiratory time (seconds) for each animal in Group 1 ...F-55
Figure 3b. Plot of baseline adjusted RP expiratory time (seconds) for each animal in Group 2...F-55
Figure 3c. Plot of baseline adjusted RP expiratory time (seconds) for each animal in Group 3 ...F-56
Figure 3d. Plot of baseline adjusted RP expiratory time (seconds) for each animal in Group 4...F-56
Figure 3e. Plot of baseline adjusted RP expiratory time (seconds) for each animal in Group 5 ...F-57
Figure 3f. Plot of baseline adjusted RP expiratory time (seconds) for each animal in Group 6... F-57
Figure 4a. Plot of baseline adjusted RP inspiratory time (seconds) for each animal in Group 1 ..F-58
Figure 4b. Plot of baseline adjusted RP inspiratory time (seconds) for each animal in Group 2..F-58
Figure 4c. Plot of baseline adjusted RP inspiratory time (seconds) for each animal in Group 3 ..F-59
Figure 4d. Plot of baseline adjusted RP inspiratory time (seconds) for each animal in Group 4.. F-59
Figure 4e. Plot of baseline adjusted RP inspiratory time (seconds) for each animal in Group 5 ..F-60
Figure 4f. Plot of baseline adjusted RP inspiratory time (seconds) for each animal in Group 6.. 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 5e. Plot of baseline adjusted RP integral (mmHg-seconds) for each animal in Group 5...F-63
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Figure
5f.
Plot of baseline adjusted RP integral (mmHg-seconds) for each animal in Group 6.
.F-63
Figure
6a.
Plot of baseline adjusted RP peak amplitude (mmHg) for each animal in Group 1 ..
.F-64
Figure
6b.
Plot of baseline adjusted RP peak amplitude (mmHg) for each animal in Group 2 ..
.F-64
Figure
6c.
Plot of baseline adjusted RP peak amplitude (mmHg) for each animal in Group 3 ..
.F-65
Figure
6d.
Plot of baseline adjusted RP peak amplitude (mmHg) for each animal in Group 4 ..
.F-65
Figure
6e.
Plot of baseline adjusted RP peak amplitude (mmHg) for each animal in Group 5 ..
.F-66
Figure
6f.
Plot of baseline adjusted RP peak amplitude (mmHg) for each animal in Group 6 ..
.F-66
Figure
7a.
Plot of baseline adjusted RP respiratory rate (RCPM) for each animal in Group 1
F-67
Figure
7b.
Plot of baseline adjusted RP respiratory rate (RCPM) for each animal in Group 2
F-67
Figure
7c.
Plot of baseline adjusted RP respiratory rate (RCPM) for each animal in Group 3
F-68
Figure
7d.
Plot of baseline adjusted RP respiratory rate (RCPM) for each animal in Group 4
F-68
Figure
7e.
Plot of baseline adjusted RP respiratory rate (RCPM) for each animal in Group 5
F-69
Figure
7f.
Plot of baseline adjusted RP respiratory rate (RCPM) for each animal in Group 6
F-69
Figure
8a.
Plot of baseline adjusted temperature (Celsius) for each animal in Group 1
F-70
Figure
8b.
Plot of baseline adjusted temperature (Celsius) for each animal in Group 2
F-70
Figure
8c.
Plot of baseline adjusted temperature (Celsius) for each animal in Group 3
F-71
Figure
8d.
Plot of baseline adjusted temperature (Celsius) for each animal in Group 4
F-71
Figure
8e.
Plot of baseline adjusted temperature (Celsius) for each animal in Group 5
F-72
Figure
8f.
Plot of baseline adjusted temperature (Celsius) for each animal in Group 6
F-72
Figure
9.
Plot of mean baseline adjusted activity (counts/minute) for each group
F-73
Figure
10.
Plot of mean baseline adjusted heart rate (BPM) for each group
F-73
Figure
11.
Plot of mean baseline adjusted RP expiratory time (seconds) for each group
F-74
Figure
12.
Plot of mean baseline adjusted RP inspiratory time (seconds) for each group
F-74
Figure
13.
Plot of mean baseline adjusted RP integral (mmHg-seconds) for each group
F-75
Figure
14.
Plot of mean baseline adjusted RP peak amplitude (mmHg) for each group
F-75
Figure
15.
Plot of mean baseline adjusted RP respiratory rate (RCPM) for each group
F-76
Figure
16.
Plot of mean baseline adjusted temperature (Celsius) for each group
F-76
Figure
17.
Kaplan-Meier curves for time to abnormality based on activity
F-77
Figure
18.
Kaplan-Meier curves for time to abnormality based on RP expiratory time
F-77
Figure
19.
Kaplan-Meier curves for time to abnormality based on RP inspiratory time
F-78
Figure
20.
Kaplan-Meier curves for time to abnormality based on RP integral
F-78
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Figure 21. Kaplan-Meier curves for time to abnormality based on RP peak amplitude F-79
Figure 22. Kaplan-Meier curves for time to abnormality based on RP respiratory rate F-79
Figure 23. Kaplan-Meier curves for time to abnormality based on temperature F-80
Figure 24. Kaplan-Meier curves for duration of abnormality based on heart rate F-80
Figure 25. Kaplan-Meier curves for duration of abnormality based on RP expiratory time F-81
Figure 26. Kaplan-Meier curves for duration of abnormality based on RP inspiratory time F-81
Figure 27. Kaplan-Meier curves for duration of abnormality based on RP respiratory rate F-82
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List of Acronyms
ANOVA Analysis of variance
BBRC Battelle Biomedical Research Center
BPM Beats per minute
CFU Colony forming unit
LD50 Median lethal dose
LDH Lactate Dehydrogenase
mm millimeter
N Number of animals
RCPM Respiratory cycles per minute
RP Respiratory period
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1. Introduction
This report summarizes the statistical analysis of telemetry data collected under Battelle
Biomedical Research Center (BBRC) Study No. 1020-CG920503. Thirty (30) male pathogen-
free New Zealand White rabbits (Oryctolagus ami cuius) were randomized into six Groups, with
each Group having five animals. Animals were aerosol-challenged on Study Day zero with
Bacillus cmthracis (Ames strain) spores as indicated in Table 1.
Table 1. Study Design
Group
Number of Animals
per Group
Target Spore Dose (CFU)
1a
5
100 x LD50
2
5
100
3
5
1,000
4
5
10,000
5
5
100,000
6b
5
100 x LD50
CFU Colony forming units.
a Spores are gamma-irradiated (negative control).
b High dose control.
LD50 Median lethal dose.
Telemetry data were collected for activity, heart rate, 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.
Approximately seven days of baseline data were collected prior to challenge for each animal,
while the post-challenge data were collected for surviving animals up to 21 days following
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. Furthermore, extreme observations were
identified during baseline for Animal L23234 on September 14, 2009, at 16:45; therefore, these
observations were also excluded prior to the statistical analysis.
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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. Each observation was then baseline adjusted
according to the associated clock time. 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 second 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.
In order 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:
Y dy= |i + Group, + 8(/ (1)
where Y^ is the baseline adjusted six-hour average telemetry value for the /th animal in
Group i (i= 1 to 6) at study time [i is an overall constant, and 8,, 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 t-test was performed to determine if the mean baseline adjusted telemetry
value is significantly different from zero. Additionally, Tukey's multiple comparisons procedure
F-9
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was performed to determine 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 Day combination are made at a joint
95% confidence level.
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
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.
For those parameters associated with a significant overall log-rank test, Kaplan-Meier estimates
were plotted for each Group.
All statistical analyses were conducted using SAS® (SAS Institute Inc.; Cary, NC; version 9.1)
and all results were reported at the 0.05 level of significance. Estimates and exact binomial
confidence intervals for the proportion of abnormal animals were calculated using the MEANS
procedure. All ANOVA models were fitted using the MIXED or GLM procedure. All Fisher's
exact tests were performed using the FREQ procedure, and all log-rank tests were performed
using the LIFETEST procedure. The MULTTEST procedure was used to maintain an overall
0.05 level of significance among the multiple pairwise comparisons made within each parameter.
F-10
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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 8f 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 (>l)
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
were 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 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. All animals in Group 6 died prior to the end of
Study Day 4 and all but one animal in Group 5 died prior to the end of Study Day 5. A summary
of the results listed in Tables 2 through 9 is presented below for each parameter.
• Activity (Table 2, Figures la-f): For four consecutive six-hour intervals beginning
on Study Day 3 at Noon-6PM, there were significant decreases from baseline in
Group 6. On Study Day 3 at Noon-6PM and Study Day 4 at 6AM-Noon, the mean
F-ll
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decrease from baseline in Group 6 was significantly different than the mean increases
from baseline in Groups 1 and 3. There were no significant decreases from baseline in
Group 1 following Study Day 13.
Heart Rate (Table 3, Figures 2a-f): By Study Day 1 at 6AM-Noon, all groups had
experienced significant increases from baseline. The significant increases from
baseline in Group 6 continued for most study times until Study Day 4. Groups 1 and 3
did not experience any significant increases from baseline after Study Day 4, while
Group 4 continued to experience some significant increases from baseline until Study
Day 11.
RP Expiratory Time (Table 4, Figures 3a-f): Group 1 rarely experienced
significant shifts from baseline, while Group 5 had more significant decreases from
baseline than any other Group. For eight consecutive six-hour intervals beginning on
Study Day 6 at 6AM-Noon, there were significant increases from baseline in
Group 3. For four consecutive six-hour intervals beginning on Study Day 4 at Noon-
6PM, the mean decrease from baseline in Group 5 was significantly different than the
mean change from baseline in at least one of the other lower dose groups (Groups 1
through 4).
RP Inspiratory Time (Table 5, Figures 4a-f): On Study Days 2 through 4, there
were often significant decreases from baseline in Groups 5 and 6. Groups 2 and 3
never experienced significant decreases from baseline, but did experience some
significant increases from baseline on Study Days 6 through 8. From Study Day 2
through 5, the mean decreases from baseline in Group 5 were often significantly
different than the mean changes from baseline in Group 2.
RP Integral (Table 6, Figures 5a-f): For ten consecutive six-hour intervals
beginning on Study Day 3 at 6AM-Noon, there were significant increases from
baseline in Group 5. On Study Day 4 atNoon-6PM and 6PM-Midnight, the mean
increase from baseline in Group 5 was significantly different than the mean change
F-12
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from baseline in Group 1. Groups 3 and 4 did not experience any significant shifts
from baseline during the study.
• Peak Amplitude (Table 7, Figures 6a-f): There were no significant shifts from
baseline in Groups 3 or 4, and only one significant decrease from baseline in Group 6
during the study. Groups 1, 2, and 5 experienced some significant increases from
baseline, but no significant decreases from baseline.
• RP Respiratory Rate (Table 8, Figures 7a-f): There were typically significant
increases from baseline in Group 5 from Study Day 2 at 6AM-Noon through Study
Day 5 at Noon-6PM and in Group 6 from Study Day 2 at Midnight-6AM through
Study Day 4 at Noon-6PM. The mean increase from baseline in Group 5 or 6 was
often significantly different than the mean change from baseline in at least one of the
other groups during these study times. Group 1 experienced only one significant
increase from baseline after Study Day 2.
• Temperature (Table 9, Figures 8a-f): For seven consecutive six-hour intervals
beginning on Study Day 2 at 6AM-Noon, there were significant increases from
baseline in Group 6. Excluding Study Day 3 at Noon-6PM, the mean increases from
baseline at these study times were significantly different than the mean changes from
baseline in at least three of the lower dose groups (Groups 1 through 5). On Study
Day 5 at 6AM-Noon and Noon-6PM, there were significant decreases from baseline
in Group 5 that were significantly different than the mean changes from baseline in
Groups 1 through 4.
Table 10 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 with
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. There were no significant differences between the
groups for any parameter.
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Table 11 contains the results of the overall log-rank tests for each parameter comparing the times
to abnormality within each Group. Activity, RP expiratory time, RP inspiratory time, RP
integral, RP peak amplitude, RP respiratory rate, and temperature all had significant differences
between the groups at the 0.05 level; therefore, pairwise comparisons for these parameters are
presented in Table 12. The pairwise comparisons are not dependent upon the ordering of the
groups being compared (e.g., comparing Group 1 to Group 2 is equivalent to comparing
Group 2 to Group 1); therefore, the cells in the lower left portion of the table are shaded out for
each parameter. Prior to the adjustment for multiple pairwise comparisons, the times to abnormal
activity in Groups 1, 3, and 4 were significantly greater than that in Group 5. After adjusting for
the multiple pairwise comparisons, the times to abnormal activity were not significantly different
for any pair of Groups. Prior to the adjustment for multiple pairwise comparisons, the times to
abnormal RP expiratory time in Groups 1, 2, and 4 were significantly greater than that in
Group 6. After adjusting for the multiple pairwise comparisons, the time to an abnormal RP
expiratory time in Group 4 was no longer significantly different from that in Group 6. Prior to
the adjustment for multiple pairwise comparisons, the times to abnormal RP inspiratory time in
Groups 1 through 5 were significantly greater than that in Group 6. After adjusting for the
multiple pairwise comparisons, the times to abnormal RP inspiratory time in Groups 3 and 5
were no longer significantly different from that in Group 6. Prior to the adjustment for multiple
pairwise comparisons, the times to abnormal RP integral in Groups 1 through 5 were
significantly greater than that in Group 6. After adjusting for the multiple pairwise comparisons,
the times to abnormal RP integral in Groups 2, 4, and 5 were no longer significantly different
from that in Group 6. Prior to the adjustment for multiple pairwise comparisons, the times to
abnormal RP peak amplitude in Groups 2 through 4 were significantly greater than that in
Group 6. After adjusting for the multiple pairwise comparisons, the times to abnormal RP peak
amplitude in Groups 3 and 4 were no longer significantly different from that in Group 6. Prior to
the adjustment for multiple pairwise comparisons, the times to abnormal RP respiratory rate in
Groups 2 through 5 were significantly greater than that in Group 6. After adjusting for the
multiple pairwise comparisons, the times to abnormal RP respiratory rate in Groups 3 through 5
were no longer significantly different from that in Group 6. Prior to the adjustment for multiple
pairwise comparisons, the time to abnormal temperature in Group 2 was significantly greater
than that in Group 6. After adjusting for the multiple pairwise comparisons, the times to
F-14
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abnormal temperature were not significantly different for any pair of groups. Figures 17
through 23 display the Kaplan-Meier curves associated with time to abnormality for activity, 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.
Table 13 contains the results of the overall log-rank tests for each parameter comparing the
duration of abnormality within each group. Heart rate, RP expiratory time, RP inspiratory time,
and RP respiratory rate all had significant differences between the groups at the 0.05 level;
therefore, pairwise comparisons for these parameters are presented in Table 14. Prior to the
adjustment for multiple pairwise comparisons, the duration of abnormal heart rate in Group 2
was significantly greater than those in Groups 3 and 6, and the duration of abnormal heart rate in
Group 4 was significantly greater than that in Group 3; however, these differences were no
longer significant after the adjustment for multiple comparisons. Prior to the adjustment for
multiple pairwise comparisons, the duration of abnormal RP expiratory time in Group 3 was
significantly greater than those in Groups 2, 4, and 5, and the duration of abnormal RP expiratory
time in Group 4 was significantly greater than that in Group 5; however, these differences were
no longer significant after the adjustment for multiple comparisons. No significant pairwise
differences were identified for duration of abnormal RP inspiratory time. Prior to the adjustment
for multiple pairwise comparisons, the duration of abnormal RP respiratory rates in Groups 5
and 6 were significantly greater than that in Group 1; however, these differences were no longer
significant after the adjustment for multiple comparisons. Figures 24 through 27 display the
Kaplan-Meier curves associated with duration of abnormality for heart rate, RP expiratory time,
RP inspiratory time, and RP respiratory rate, 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 its
time of death or at the end of the study, then the duration of abnormality for that animal would be
unobserved. Note the since the animals in Groups 5 and 6 died early in the study, the durations of
abnormality in the lower dose groups may appear greater than those in the higher dose Groups.
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4. Conclusions
All animals in high dose control group (Group 6) died prior to the end of Study Day 4 and all but
one animal in the 100,000 colony forming unit (CFU) dose group (Group 5) died prior to the end
of Study Day 5. Prior to the death of these animals, there were typically more significant shifts
from baseline in these two groups than there were in the lower dose groups (Groups 1 through 4).
In terms of the proportion of animals that became abnormal during the study, there were no
significant differences between the groups for any parameter. In terms of time to abnormality,
there were significant differences between the groups for RP expiratory time, RP inspiratory
time, RP integral, RP peak amplitude, RP respiratory rate, and temperature. For each of these
telemetry parameters, the time to abnormality in at least one of the lower dose groups (Groups 1
through 5) was significantly greater than that in the high dose control group (Group 6) prior to
the adjustments for multiple comparisons. The duration of abnormality was found to be
significantly different between the groups for heart rate, RP expiratory time, RP inspiratory time,
and RP respiratory rate; however, there were no significant pairwise differences between the
groups for any parameter after the adjustment for multiple comparisons.
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Table 2. Summary of the ANOVA Results for the Baseline Adjusted Six-Hour Averages for Activity (Counts/Minute)
Activity
Study
Day
Time
Mean Baseline Adjusted Value, by Group
Group Effect
P-Value
Estimated Difference (Relationship)
Tu key's P-Value#
1
2
3
4
5
6
0
6AM - Noon
1.88
1.14
2.18
20.73 t
NA
NA
0.0108*
-18.85 (1<4) 0.0218
-19.59 (2<4) 0.0169
-18.55 (3<4) 0.0242
Noon - 6PM
0.25
0.01
0.17
0.23
0.19
0.25
0.9680
6PM - Midnight
-0.13
-0.27 |
-0.39 |
-0.15
-0.34 |
0.03
0.0591
1
Midnight - 6AM
-0.10
-0.04
-0.16
-0.19
-0.18
-0.05
0.9309
6AM - Noon
1.16 t
1.01 |
0.45
0.60
1.16 t
1.00 |
0.4351
Noon - 6PM
-0.03
0.13
0.27
-0.49
-0.23
-0.56 |
0.2111
6PM - Midnight
0.06
-0.07
0.04
-0.23
-0.21
-0.43 |
0.2812
2
Midnight - 6AM
-0.08
0.07
-0.03
-0.01
-0.12
-0.26
0.7192
6AM - Noon
1.47 t
1.09 |
0.82
1.14 |
0.49
-0.31
0.3065
Noon - 6PM
-0.32
-0.46
0.05
-0.45
-0.75 |
-0.74 |
0.2913
6PM - Midnight
-0.32
-0.34
0.28
-0.09
-0.46 |
-0.24
0.1738
3
Midnight - 6AM
-0.01
-0.10
0.20
-0.12
-0.09
-0.40 |
0.3672
6AM - Noon
1.42 t
0.61
0.79
0.76
0.43
-0.62
0.0549
-2.03 (6<1) 0.0216
Noon - 6PM
0.14
-0.10
0.03
-0.22
-0.83 |
-1.14|
0.0123*
-1.27 (6<1) 0.0226
-1.17 (6<3) 0.0426
6PM - Midnight
-0.24
-0.24
-0.15
-0.28
-0.64 |
-0.78 |
0.2053
4
Midnight - 6AM
0.18
-0.16
0.24
-0.11
-0.40
-0.60 |
0.0584
6AM - Noon
0.29
-0.25
0.19
-0.30
-0.51 |
-1.25|
0.0084 *
-1.54 (6<1) 0.0086
-1.43 (6<3) 0.0159
Noon - 6PM
-0.16
0.34
-0.15
-0.31
-0.57
-0.86
0.3001
6PM - Midnight
0.07
-0.04
-0.17
-0.04
-0.55 |
NA
0.3588
5
Midnight - 6AM
0.05
-0.21
-0.14
-0.12
-0.56 |
NA
0.3593
6AM - Noon
0.10
-0.15
0.20
0.06
-0.51
NA
0.5507
Noon - 6PM
-0.11
0.12
-0.02
0.31
-0.22
NA
0.6473
6PM - Midnight
-0.17
0.45
-0.16
0.32
0.18
NA
0.2767
F-17
-------
Table 2. (Continued)
Activity
Study
Time
Mean Baseline Adjusted Value, by Group
Group Effect
Estimated Difference (Relationship)
Day
1
2
3
4
5
6
P-Value
Tu key's P-Value#
Midnight - 6AM
-0.22
0.02
-0.21
0.03
0.06
NA
0.3050
6
6AM - Noon
-0.02
-0.55 |
-0.29 |
-0.09
-0.65 |
NA
0.0546
Noon - 6PM
-0.14
-0.61 |
-0.27
-0.01
-0.44
NA
0.3610
6PM - Midnight
-0.22
0.21
0.03
0.07
-0.13
NA
0.8504
Midnight - 6AM
-0.23
-0.03
-0.45 |
-0.01
0.57
NA
0.0613
7
6AM - Noon
0.26
0.42
-0.18
0.61
-0.01
NA
0.4694
Noon - 6PM
-0.61 |
-0.90 |
-0.62 |
-0.26
-0.39
NA
0.1903
6PM - Midnight
-0.24
-0.14
-0.50 |
-0.19
0.13
NA
0.7041
Midnight - 6AM
0.17
-0.12
-0.37
0.06
0.24
NA
0.3299
8
6AM - Noon
-0.28
-0.17
-0.37
-0.30
-0.31
NA
0.9866
Noon - 6PM
-0.05
-0.86 |
-0.15
-0.17
0.26
NA
0.0738
6PM - Midnight
-0.16
-0.03
-0.35
-0.35
-0.06
NA
0.7871
Midnight - 6AM
-0.01
-0.20
-0.14
0.04
-0.15
NA
0.6375
9
6AM - Noon
-0.44
-0.40
-0.27
-0.40
-0.60
NA
0.9764
Noon - 6PM
-0.29
-0.56 |
-0.23
-0.77 |
-0.44
NA
0.6225
6PM - Midnight
-0.12
-0.25
-0.55 |
-0.29
-0.41
NA
0.5496
Midnight - 6AM
-0.18
0.16
-0.29
-0.28
-0.08
NA
0.3360
10
6AM - Noon
-0.28
-0.24
-0.11
-0.66 |
-0.49
NA
0.5618
Noon - 6PM
-0.19
-0.20
-0.24
-0.47
0.24
NA
0.8145
6PM - Midnight
-0.23
-0.25
-0.27
-0.42
-0.23
NA
0.9734
Midnight - 6AM
0.04
0.09
-0.02
-0.37
-0.19
NA
0.6544
11
6AM - Noon
-0.18
-0.41
-0.30
-0.48
-0.43
NA
0.8700
Noon - 6PM
-0.34
-0.36
-0.05
-0.41
-0.24
NA
0.8022
6PM - Midnight
-0.03
-0.23
-0.43 |
-0.11
0.11
NA
0.5817
F-18
-------
Table 2. (Continued)
Activity
Study
Day
Time
Mean Baseline Adjusted Value, by Group
Group Effect
P-Value
Estimated Difference (Relationship)
Tu key's P-Value#
1
2
3
4
5
6
12
Midnight - 6AM
0.02
-0.03
-0.26
-0.14
0.00
NA
0.7983
6AM - Noon
0.14
-0.19
-0.13
-0.17
-0.30
NA
0.9165
Noon - 6PM
-0.07
-0.26
-0.19
-0.39
-0.31
NA
0.9298
6PM - Midnight
-0.03
-0.19
-0.07
0.14
-0.45
NA
0.8764
13
Midnight - 6AM
-0.10
-0.19
-0.15
-0.12
-0.07
NA
0.9977
6AM - Noon
-0.36 |
-0.35 |
-0.43 |
-0.38
-0.66
NA
0.9379
Noon - 6PM
-0.20
-0.45 |
-0.46 |
-0.43
-0.08
NA
0.8172
6PM - Midnight
-0.10
-0.29
-0.58 |
-0.21
-0.11
NA
0.3947
14
Midnight - 6AM
-0.16
-0.29
-0.26
-0.22
0.13
NA
0.8195
6AM - Noon
0.71 t
0.42
0.22
0.73 t
0.75
NA
0.6456
Noon - 6PM
0.06
-0.47
-0.31
-0.23
-0.38
NA
0.7358
6PM - Midnight
-0.26
-0.37 |
-0.42 |
-0.39
-0.33
NA
0.9676
15
Midnight - 6AM
0.38
-0.19
-0.26
-0.18
0.20
NA
0.1260
6AM - Noon
-0.14
-0.25
-0.17
-0.45 |
-0.54
NA
0.6691
Noon - 6PM
-0.12
-0.43 |
-0.35
-0.40
-0.33
NA
0.7044
6PM - Midnight
-0.25
-0.18
-0.42 |
-0.22
-0.29
NA
0.8763
16
Midnight - 6AM
-0.09
-0.21
-0.06
-0.06
0.04
NA
0.8285
6AM - Noon
0.16
-0.57
-0.60 |
-0.39
-0.57
NA
0.3062
Noon - 6PM
-0.04
-0.42
-0.18
-0.33
-0.43
NA
0.7484
6PM - Midnight
-0.04
-0.05
-0.33
-0.27
-0.35
NA
0.6439
17
Midnight - 6AM
0.16
-0.10
0.06
-0.14
-0.10
NA
0.8302
6AM - Noon
0.03
-0.50
-0.48
-0.33
-0.74
NA
0.5444
Noon - 6PM
-0.13
-0.51 |
-0.31
0.17
-0.42
NA
0.4936
6PM - Midnight
0.27
-0.40
-0.49
-0.25
-0.40
NA
0.2189
F-19
-------
Table 2. (Continued)
Activity
Study
Day
Time
Mean Baseline Adjusted Value, by Group
Group
Effect
P-Value
Estimated Difference (Relationship)
Tukey's P-Value#
1
2
3
4
5
6
18
Midnight - 6AM
-0.18
-0.04
-0.24
0.00
0.42
NA
0.6554
6AM - Noon
-0.33
-0.41
-0.40
-0.57
-1.04
NA
0.8305
Noon - 6PM
-0.11
-0.32
-0.24
-0.11
0.08
NA
0.9095
6PM - Midnight
-0.06
-0.31
-0.32
-0.16
-0.15
NA
0.8823
19
Midnight - 6AM
-0.15
-0.19
-0.14
-0.13
-0.55
NA
0.9254
6AM - Noon
-0.12
-0.37
-0.15
-0.53
-0.56
NA
0.7316
Noon - 6PM
-0.02
-0.21
-0.23
-0.13
-0.30
NA
0.9703
6PM - Midnight
-0.20
-0.24
-0.56 |
-0.15
-0.61
NA
0.3986
20
Midnight - 6AM
-0.07
-0.11
-0.21
-0.12
0.05
NA
0.9503
6AM - Noon
0.00
-0.09
-0.38
-0.23
-0.70
NA
0.5638
Noon - 6PM
-0.13
-0.42
-0.20
0.00
-0.22
NA
0.9150
6PM - Midnight
-0.13
-0.26
-0.23
0.04
-0.23
NA
0.9008
21
Midnight - 6AM
-0.14
-0.26
-0.26
0.19
0.19
NA
0.4512
6AM - Noon
0.53
0.47
0.32
1.12
1.11
NA
0.8033
# Cells contain all pairwise comparisons that are statistically 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<6)" indicates that the mean baseline adjusted value
for Group 6 was significantly greater than that for Group 1], and (3) the Tukey-adjusted p-value.
t, | Indicate that the mean baseline adjusted value was significantly different from zero (at the 0.05 level), "t" indicates that the mean at the study time was
greater than that at baseline, while "J," indicates that the mean at the study time was less than that at baseline.
NA Data not available for this Group at this study time.
* Group effect was significant at the 0.05 level.
F-20
-------
Table 3. Summary of the ANOVA Results for the Baseline Adjusted Six-Hour Averages for Heart Rate (BPM)
Heart Rate
Study
Day
Time
Mean Baseline Adjusted Value, by Group
Group Effect
P-Value
Estimated Difference (Relationship)
Tukey's P-Value#
1
2
3
4
5
6
0
6AM - Noon
22.13
38.16 t
20.69
61.63 t
NA
NA
0.1750
Noon - 6PM
9.15
16.48
-3.19
9.69
19.16 t
10.06
0.4798
6PM - Midnight
-9.31
9.57
-2.72
1.24
10.93
11.83
0.2873
1
Midnight - 6AM
-6.05
9.65
-1.52
-6.56
0.83
15.52 t
0.1053
6AM - Noon
27.36 t
33.18 t
22.08 t
22.06 t
25.39 t
19.43 t
0.0479 *
-13.75 (6<2) 0.0377
Noon - 6PM
3.56
3.95
-6.01
1.58
-28.50 |
-4.56
0.1363
6PM - Midnight
14.82
26.04 t
9.49
36.46 t
-9.64
30.53 t
0.0369 *
-46.10 (5<4) 0.0332
2
Midnight - 6AM
3.52
17.84
-1.56
41.09 |
14.28
55.84 t
0.0269 *
-57.40 (3<6) 0.0391
6AM - Noon
28.14 t
36.44 t
27.34 t
36.90 t
24.72 t
49.64 |
0.0208 *
-22.30 (3<6) 0.0458
-24.92 (5<6) 0.0203
Noon - 6PM
11.01
4.56
4.04
7.13
11.25
19.14 |
0.6420
6PM - Midnight
-4.89
-1.46
-4.53
1.54
32.64 t
21.42 t
0.0242 *
3
Midnight - 6AM
-0.62
3.60
-4.25
6.77
36.37 t
32.30 t
0.0036 *
-36.99 (1<5) 0.0271
-40.61 (3<5) 0.0127
-36.54 (3<6) 0.0450
6AM - Noon
12.44
15.27
3.84
13.03
10.81
26.33 t
0.5870
Noon - 6PM
14.85
8.38
5.23
14.09
-15.95
5.97
0.2819
6PM - Midnight
4.37
10.68
3.13
26.41 t
18.81
42.87 t
0.1818
4
Midnight - 6AM
3.89
8.72
1.96
23.02
36.57 t
50.02 t
0.1760
6AM - Noon
-3.43
0.59
-8.08
-0.23
29.32 t
1.58
0.0063 *
-32.74 (1<5) 0.0114
-28.72 (2<5) 0.0315
-37.40 (3<5) 0.0034
-29.55 (4<5) 0.0257
Noon - 6PM
23.70 t
10.09
10.94
-1.61
24.05
-73.48 |
0.0148*
-97.18 (6<1) 0.0078
-83.57 (6<2) 0.0258
-84.42 (6<3) 0.0240
-97.53 (6<5) 0.0118
6PM - Midnight
25.82 t
23.19 t
18.23 t
30.08 t
34.23 t
NA
0.7694
F-21
-------
Table 3. (Continued)
Heart Rate
Study
Day
Time
Mean Baseline Adjusted Value, by Group
Group Effect
P-Value
Estimated Difference (Relationship)
Tukey's P-Value#
1
2
3
4
5
6
5
Midnight - 6AM
21.30
26.30 t
10.22
27.96 t
35.06 t
NA
0.6243
6AM - Noon
-6.57
-2.71
-6.06
-5.89
10.87
NA
0.2251
Noon - 6PM
1.10
-7.76
2.49
1.20
-33.34 |
NA
0.1507
6PM - Midnight
-5.01
20.59 t
5.58
20.45
17.81
NA
0.3331
6
Midnight - 6AM
-6.17
32.34 t
4.61
14.83
14.83
NA
0.0108*
-38.51 (1<2) 0.0059
6AM - Noon
-14.08|
-16.97 |
-11.64 |
-8.28
9.24
NA
0.2547
Noon - 6PM
3.29
-45.77 |
-14.09
-1.34
21.90
NA
0.0024 *
-49.06 (2<1) 0.0039
-44.42 (2<4) 0.0138
-67.67 (2<5) 0.0224
6PM - Midnight
-0.09
-11.88
-6.49
15.08
4.97
NA
0.3944
7
Midnight - 6AM
-4.65
10.34
-4.05
14.10
11.39
NA
0.2104
6AM - Noon
-6.14
-7.30 |
-12.88 |
2.48
-8.55
NA
0.0609
-15.36 (3<4) 0.0307
Noon - 6PM
6.77
-50.80 |
-21.39 |
-8.60
7.30
NA
0.0081 *
-57.57 (2<1) 0.0054
6PM - Midnight
-4.09
-26.79 |
-8.76
2.58
-1.46
NA
0.5341
8
Midnight - 6AM
-8.15
-1.49
0.05
2.38
4.92
NA
0.8162
6AM - Noon
1.99
9.44
7.29
9.28
21.83
NA
0.5319
Noon - 6PM
5.81
0.53
-1.85
-5.00
9.41
NA
0.4976
6PM - Midnight
-2.83
17.94 t
2.25
10.61
-4.43
NA
0.2638
9
Midnight - 6AM
-6.13
12.02
-2.44
26.43 t
-6.89
NA
0.1802
6AM - Noon
14.16
10.28
7.12
38.18 t
12.51
NA
0.1979
Noon - 6PM
-14.99
-26.09 |
-16.70
6.58
0.77
NA
0.4072
6PM - Midnight
-24.35
-29.06 |
-28.26 |
18.49
-18.28
NA
0.1295
10
Midnight - 6AM
-16.81
-14.29
-17.86
17.98
-7.18
NA
0.2626
6AM - Noon
-1.77
-8.47
-9.50
8.67
4.56
NA
0.0986
Noon - 6PM
-4.83
-12.67 |
-12.32 |
-9.65
14.93
NA
0.3436
6PM - Midnight
-16.81
-12.38
-10.93
23.82 t
-10.72
NA
0.0850
F-22
-------
Table 3. (Continued)
Heart Rate
Study
Day
Time
Mean Baseline Adjusted Value, by Group
Group Effect
P-Value
Estimated Difference (Relationship)
Tukey's P-Value#
1
2
3
4
5
6
11
Midnight - 6AM
-17.74
-5.32
-10.29
20.73
-6.43
NA
0.1160
6AM - Noon
-6.17
-8.31
-9.44
19.39 t
15.13
NA
0.0331 *
-28.83 (3<4) 0.0484
Noon - 6PM
4.01
-7.67 |
-12.90 |
1.38
13.35 t
NA
0.0006 *
-11.68 (2<1) 0.0317
-16.91 (3<1) 0.0019
-21.02 (2<5) 0.0248
-14.28 (3<4) 0.0219
-26.25 (3<5) 0.0049
6PM - Midnight
-7.16
-8.26
-15.36
14.77
-5.33
NA
0.2565
12
Midnight - 6AM
-14.76
-4.05
-16.15
18.67
0.06
NA
0.1212
6AM - Noon
-6.85
-5.22
-1.92
10.63
-1.49
NA
0.2468
Noon - 6PM
-13.58
-12.45
-8.20
-1.94
9.82
NA
0.5515
6PM - Midnight
-21.46 |
-15.53
-15.30
17.07
-24.99
NA
0.0791
13
Midnight - 6AM
-21.35|
-11.69
-18.55 |
13.29
5.28
NA
0.0997
6AM - Noon
-16.44|
-10.73
-16.66 |
3.90
4.48
NA
0.1082
Noon - 6PM
-16.60|
-8.58
-6.37
-1.10
7.27
NA
0.1870
6PM - Midnight
-23.27 |
-13.32
-13.81
4.51
0.71
NA
0.2204
14
Midnight - 6AM
-12.76
-8.66
-13.46
7.87
6.72
NA
0.5930
6AM - Noon
1.41
-6.29
-3.77
15.14
8.56
NA
0.2483
Noon - 6PM
8.37
-0.42
-1.26
5.20
8.90
NA
0.6183
6PM - Midnight
-8.64
-9.24
-4.24
8.87
8.51
NA
0.7039
15
Midnight - 6AM
-5.00
-8.34
-10.56
7.38
4.28
NA
0.8378
6AM - Noon
-1.98
0.19
-9.41
8.99
11.75
NA
0.2340
Noon - 6PM
-14.87|
-16.83 |
-13.12 |
-1.59
0.01
NA
0.3325
6PM - Midnight
-25.70 |
-18.09
-17.12
-0.81
-9.34
NA
0.5227
F-23
-------
Table 3. (Continued)
Heart Rate
Study
Day
Time
Mean Baseline Adjusted Value, by Group
Group Effect
P-Value
Estimated Difference (Relationship)
Tu key's P-Value#
1
2
3
4
5
6
16
Midnight - 6AM
-12.28
-12.01
-11.47
-1.12
-12.45
NA
0.9359
6AM - Noon
-2.69
2.18
-8.02
8.87
3.98
NA
0.2531
Noon - 6PM
-11.13|
-0.62
-0.96
6.92
7.58
NA
0.0915
6PM - Midnight
-24.61 |
-5.68
-0.85
7.00
-5.17
NA
0.3300
17
Midnight - 6AM
-17.43
-3.23
-4.12
4.32
6.03
NA
0.5937
6AM - Noon
-5.23
-6.63
-8.33 |
-1.39
-4.26
NA
0.7868
Noon - 6PM
-6.51
-9.24
-14.64 |
-3.37
7.35
NA
0.3718
6PM - Midnight
-5.46
-10.13
-11.76
-3.76
-1.70
NA
0.9874
18
Midnight - 6AM
-10.17
-6.82
-9.20
0.06
0.49
NA
0.9563
6AM - Noon
-12.37|
-10.43 |
-15.17 |
-5.12
-0.64
NA
0.3569
Noon - 6PM
0.05
-12.90 |
-21.89 |
-0.21
-9.31
NA
0.0744
6PM - Midnight
-20.26
-18.68
-16.27
8.53
-14.45
NA
0.4851
19
Midnight - 6AM
-18.57
-14.95
-13.30
10.71
-9.59
NA
0.3551
6AM - Noon
-5.63
-11.79
-12.21
2.38
13.55
NA
0.3871
Noon - 6PM
-11.08
-13.06
-9.37
-4.81
-2.15
NA
0.9026
6PM - Midnight
-30.22 |
-17.05
-19.29
2.58
-23.59
NA
0.4236
20
Midnight - 6AM
-28.60 |
-10.81
-17.07
23.28
-10.93
NA
0.0661
-51.88 (1<4) 0.0363
6AM - Noon
-12.21 |
-8.68 |
-15.41 |
5.73
-2.10
NA
0.0180*
-17.94 (1<4) 0.0363
-21.15 (3<4) 0.0122
Noon - 6PM
-3.55
-7.23
-18.30 |
-5.57
-7.17
NA
0.1732
6PM - Midnight
-18.76|
-9.34
-7.99
20.38
-7.68
NA
0.1357
21
Midnight - 6AM
-22.02 |
-8.19
-12.43
16.81
-6.63
NA
0.1787
6AM - Noon
-27.02 |
-28.01 |
-32.98 |
-10.38
-11.79
NA
0.1486
# Cells contain all pairwise comparisons that are statistically 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<6)" indicates that the mean baseline adjusted value
for Group 6 was significantly greater than that for Group 1], and (3) the Tukey-adjusted p-value.
t, | Indicate that the mean baseline adjusted value was significantly different from zero (at the 0.05 level), "t" indicates that the mean at the study time was
greater than that at baseline, while "J," indicates that the mean at the study time was less than that at baseline.
* Group effect was significant at the 0.05 level.
NA Data not available for this Group at this study time.
F-24
-------
Table 4. Summary of the ANOVA Results for the Baseline Adjusted Six-Hour Averages for RP Expiratory Time
(Seconds)
RP Expiratory Time
Study
Day
Time
Mean Baseline Adjusted Value, by Group
Group Effect
P-Value
Estimated Difference (Relationship)
Tu key's P-Value#
1
2
3
4
5
6
0
6AM - Noon
-0.12 |
-0.17 |
-0.11 |
-0.26 |
NA
NA
0.2401
Noon - 6PM
-0.05
-0.06
0.03
-0.03
-0.04
-0.02
0.4223
6PM - Midnight
0.01
0.02
0.07
0.06
-0.01
0.03
0.7305
1
Midnight - 6AM
-0.01
-0.04
0.07
0.01
-0.02
0.02
0.4677
6AM - Noon
-0.06
-0.12 |
-0.04
-0.07 |
-0.10|
-0.03
0.2536
Noon - 6PM
0.02
-0.03
-0.02
0.03
0.08
0.02
0.5652
6PM - Midnight
0.01
-0.04
-0.03
-0.07
0.01
-0.08
0.6755
2
Midnight - 6AM
-0.01
-0.08
0.00
-0.13|
-0.01
-0.22 |
0.0754
6AM - Noon
-0.08
-0.14 |
-0.11 |
-0.17|
-0.10
-0.17|
0.7225
Noon - 6PM
0.03
0.02
-0.02
0.00
-0.05
-0.12
0.4864
6PM - Midnight
0.01
0.00
-0.03
-0.01
-0.15|
-0.17|
0.1569
3
Midnight - 6AM
0.00
-0.09
-0.04
-0.08
-0.20 |
-0.21 |
0.1083
6AM - Noon
-0.07
-0.15 |
-0.08
-0.12|
-0.16|
-0.17|
0.5627
Noon - 6PM
-0.01
-0.03
0.00
-0.04
-0.03
-0.07
0.9725
6PM - Midnight
-0.01
-0.01
0.01
-0.08
-0.19|
-0.12
0.4552
4
Midnight - 6AM
-0.06
-0.11
-0.02
-0.11
-0.23 |
-0.18
0.4925
6AM - Noon
-0.07
-0.09
0.00
-0.10
-0.05
-0.14
0.8742
Noon - 6PM
-0.03
-0.05
-0.01
-0.01
-0.22 |
-0.12
0.0291 *
-0.19 (5<1) 0.0496
-0.21 (5<3) 0.0294
-0.21 (5<4) 0.0230
6PM - Midnight
-0.08 |
-0.07
-0.08 |
-0.07
-0.27 |
NA
0.0215*
-0.19 (5<1) 0.0312
-0.20 (5<2) 0.0233
-0.19 (5<3) 0.0355
-0.20 (5<4) 0.0354
F-25
-------
Table 4. (Continued)
RP Expiratory Time
Study
Day
Time
Mean Baseline Adjusted Value, by Group
Group Effect
P-Value
Estimated Difference (Relationship)
Tukey's P-Value#
1
2
3
4
5
6
5
Midnight - 6AM
-0.04
-0.13 |
-0.07
0.00
-0.29 |
NA
0.0235 *
-0.25 (5<1) 0.0442
-0.30 (5<4) 0.0185
6AM - Noon
-0.02
-0.11 |
-0.05
0.01
-0.18|
NA
0.0375*
-0.19 (5<4) 0.0422
Noon - 6PM
-0.01
0.03
0.01
0.00
-0.10|
NA
0.2616
6PM - Midnight
0.01
-0.04
0.05
-0.03
0.00
NA
0.7478
6
Midnight - 6AM
-0.03
0.05
0.10
0.00
-0.03
NA
0.8364
6AM - Noon
0.05
0.26 t
0.22 t
0.07
0.09
NA
0.3494
Noon - 6PM
0.02
0.32 t
0.25 t
0.05
-0.05
NA
0.1491
6PM - Midnight
-0.02
0.18
0.27 t
-0.01
-0.04
NA
0.2888
7
Midnight - 6AM
0.00
0.13
0.26 t
-0.03
-0.08
NA
0.3826
6AM - Noon
0.01
0.19
0.30 t
0.02
-0.07
NA
0.2783
Noon - 6PM
0.01
0.43 t
0.32 t
0.04
-0.02
NA
0.1932
6PM - Midnight
-0.06
0.35 t
0.35 t
-0.05
-0.06
NA
0.2118
8
Midnight - 6AM
-0.08
0.22
0.36 t
-0.07
-0.09
NA
0.2848
6AM - Noon
-0.01
0.06
0.09
-0.02
-0.03
NA
0.5805
Noon - 6PM
-0.06
0.01
0.00
0.01
-0.02
NA
0.8485
6PM - Midnight
-0.09 |
-0.06
-0.01
-0.09 |
-0.08
NA
0.6223
9
Midnight - 6AM
-0.09 |
-0.13 |
-0.05
-0.12|
-0.04
NA
0.6489
6AM - Noon
-0.03
-0.05
-0.03
-0.06
-0.06
NA
0.9538
Noon - 6PM
-0.02
0.02
0.00
0.01
0.02
NA
0.9842
6PM - Midnight
-0.05
-0.05
0.08
-0.08
0.04
NA
0.1717
10
Midnight - 6AM
-0.04
-0.08
-0.05
-0.10
-0.08
NA
0.9062
6AM - Noon
-0.01
-0.05
0.00
0.00
0.11
NA
0.2995
Noon - 6PM
-0.04
-0.06
0.03
0.02
-0.04
NA
0.5821
6PM - Midnight
-0.01
-0.01
0.02
0.15
0.00
NA
0.4683
F-26
-------
Table 4. (Continued)
RP Expiratory Time
Study
Day
Time
Mean Baseline Adjusted Value, by Group
Group Effect
P-Value
Estimated Difference (Relationship)
Tu key's P-Value#
1
2
3
4
5
6
11
Midnight - 6AM
-0.04
-0.10
0.02
0.09
0.05
NA
0.2024
6AM - Noon
-0.01
-0.04
-0.03
0.15 t
0.04
NA
0.0499 *
-0.19 (2<4) 0.0494
Noon - 6PM
-0.04
-0.03
-0.02
0.06
0.00
NA
0.7158
6PM - Midnight
-0.02
-0.04
0.02
-0.04
-0.02
NA
0.8422
12
Midnight - 6AM
-0.05
-0.21 |
-0.01
-0.04
-0.06
NA
0.0151 *
-0.16 (2<1) 0.0470
-0.19 (2<3) 0.0128
6AM - Noon
0.06
-0.06
0.01
0.04
0.08
NA
0.2259
Noon - 6PM
0.03
-0.03
-0.01
0.07
0.00
NA
0.7445
6PM - Midnight
0.00
-0.02
0.01
-0.08
0.12
NA
0.6903
13
Midnight - 6AM
0.04
-0.10
-0.05
-0.09
-0.01
NA
0.6185
6AM - Noon
0.04
-0.06
-0.02
0.05
0.05
NA
0.5210
Noon - 6PM
0.03
-0.03
-0.02
0.01
0.03
NA
0.8851
6PM - Midnight
-0.02
-0.01
-0.02
-0.04
0.05
NA
0.9753
14
Midnight - 6AM
0.02
-0.08
-0.03
-0.08
-0.06
NA
0.6841
6AM - Noon
-0.04
-0.06
-0.04
-0.06
-0.05
NA
0.9880
Noon - 6PM
-0.04
-0.02
-0.01
0.00
0.07
NA
0.8709
6PM - Midnight
-0.02
0.05
-0.02
-0.01
-0.03
NA
0.8871
15
Midnight - 6AM
-0.04
-0.09
-0.08
-0.03
-0.07
NA
0.9737
6AM - Noon
-0.02
-0.06
-0.03
0.00
0.08
NA
0.6182
Noon - 6PM
0.00
0.02
-0.02
0.01
0.01
NA
0.9869
6PM - Midnight
-0.03
-0.05
-0.04
-0.02
-0.01
NA
0.9935
16
Midnight - 6AM
-0.05
-0.08
-0.06
-0.09
0.07
NA
0.8983
6AM - Noon
0.03
-0.08
-0.01
0.04
0.11
NA
0.3066
Noon - 6PM
0.01
-0.01
-0.02
0.00
-0.01
NA
0.9973
6PM - Midnight
-0.02
-0.04
-0.04
-0.06
0.09
NA
0.8114
F-27
-------
Table 4. (Continued)
RP Expiratory Time
Study
Day
Time
Mean Baseline Adjusted Value, by Group
Group Effect
P-Value
Estimated Difference (Relationship)
Tu key's P-Value#
1
2
3
4
5
6
17
Midnight - 6AM
0.00
-0.12 |
-0.05
-0.02
0.08
NA
0.4033
6AM - Noon
-0.04
0.00
0.02
0.04
0.07
NA
0.4028
Noon - 6PM
-0.04
-0.01
0.01
0.01
0.04
NA
0.9455
6PM - Midnight
-0.05
0.02
0.01
-0.01
0.00
NA
0.8740
18
Midnight - 6AM
-0.04
-0.09
-0.01
-0.03
-0.01
NA
0.8591
6AM - Noon
-0.01
-0.05
0.06
0.05
0.06
NA
0.4214
Noon - 6PM
-0.05
0.01
-0.02
0.00
0.00
NA
0.8972
6PM - Midnight
-0.05
0.02
-0.02
-0.05
-0.01
NA
0.9006
19
Midnight - 6AM
-0.06
-0.11
-0.01
-0.08
-0.09
NA
0.8350
6AM - Noon
-0.03
-0.02
0.01
0.03
-0.02
NA
0.8810
Noon - 6PM
0.00
-0.02
-0.02
-0.02
0.00
NA
0.9941
6PM - Midnight
0.03
-0.02
0.05
-0.05
0.01
NA
0.4663
20
Midnight - 6AM
0.08
-0.11
0.04
-0.06
0.06
NA
0.1805
6AM - Noon
0.00
-0.06 |
0.02
0.01
0.07
NA
0.2019
Noon - 6PM
-0.03
-0.06
-0.04
-0.02
0.00
NA
0.9120
6PM - Midnight
-0.03
-0.05
-0.01
-0.06
0.04
NA
0.8750
21
Midnight - 6AM
-0.04
-0.11
-0.03
-0.04
-0.02
NA
0.8630
6AM - Noon
-0.02
-0.09
0.00
-0.04
0.04
NA
0.7086
# Cells contain all pairwise comparisons that are statistically 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<6)" indicates that the mean baseline adjusted value
for Group 6 was significantly greater than that for Group 1], and (3) the Tukey-adjusted p-value.
t, | Indicate that the mean baseline adjusted value was significantly different from zero (at the 0.05 level), "t" indicates that the mean at the study time was
greater than that at baseline, while "J," indicates that the mean at the study time was less than that at baseline.
* Group effect was significant at the 0.05 level.
NA Data not available for this Group at this study time.
F-28
-------
Table 5. Summary of the ANOVA Results for the Baseline Adjusted Six-Hour Averages for RP Inspiratory Time (Seconds)
RP Inspiratory Time
Study
Day
Time
Mean Baseline Adjusted Value, by Group
Group Effect
P-Value
Estimated Difference (Relationship)
Tukey's P-Value *
1
2
3
4
5
6
0
6AM - Noon
-0.12|
-0.05
-0.05
-0.13
NA
NA
0.5769
Noon - 6PM
-0.08 |
0.02
0.01
-0.01
-0.04
-0.02
0.3054
6PM - Midnight
-0.01
0.01
0.02
0.00
-0.04
-0.01
0.8324
1
Midnight - 6AM
-0.02
0.04
-0.03
-0.02
0.02
0.10
0.5210
6AM - Noon
-0.10|
-0.03
-0.02
-0.07
-0.05
0.03
0.5017
Noon - 6PM
-0.03
0.02
0.00
0.00
0.06
0.08
0.5192
6PM - Midnight
-0.07
0.01
0.00
-0.02
-0.02
-0.03
0.9754
2
Midnight - 6AM
-0.03
-0.02
-0.02
-0.03
-0.10
-0.15|
0.6075
6AM - Noon
-0.09
-0.06
-0.03
-0.07
-0.14 i
-0.15|
0.3835
Noon - 6PM
-0.04
0.00
-0.01
-0.02
-0.10 i
-0.13|
0.2892
6PM - Midnight
-0.03
0.00
0.01
-0.07
-0.19 i
-0.14|
0.0163*
-0.19 (5<2) 0.0450
-0.20 (5<3) 0.0264
3
Midnight - 6AM
-0.02
-0.02
-0.03
-0.08
-0.21 i
-0.21 |
0.0471 *
6AM - Noon
-0.07 |
-0.04
-0.01
-0.09 |
-0.20 i
-0.14|
0.0030 *
-0.16 (5<2) 0.0153
-0.19 (5<3) 0.0027
Noon - 6PM
-0.05
0.02
0.02
-0.02
-0.01
-0.08
0.9027
6PM - Midnight
-0.03
0.02
-0.01
0.03
-0.17 i
-0.14|
0.0238 *
-0.19 (5<2) 0.0417
-0.19 (5<4) 0.0403
4
Midnight - 6AM
-0.03
0.05
-0.04
-0.04
-0.21 i
-0.11
0.0158*
-0.26 (5<2) 0.0059
6AM - Noon
-0.05
0.03
-0.04
-0.04
0.42
-0.14
0.6463
Noon - 6PM
-0.06 |
0.03
0.01
-0.02
-0.11 i
-0.15|
0.0083 *
-0.14 (5<2) 0.0222
6PM - Midnight
-0.05
-0.02
-0.03
-0.02
-0.15 i
NA
0.2084
5
Midnight - 6AM
-0.02
0.00
-0.07
-0.03
-0.15 i
NA
0.1974
6AM - Noon
-0.07
0.03
-0.01
-0.04
-0.20 i
NA
0.0661
-0.23 (5<2) 0.0400
Noon - 6PM
0.01
0.05
0.06
0.01
-0.12 i
NA
0.0875
6PM - Midnight
-0.02
0.01
0.09
-0.04
0.00
NA
0.5178
F-29
-------
Table 5. (Continued)
RP Inspiratory Time
Study
Day
Time
Mean Baseline Adjusted Value, by Group
Group Effect
P-Value
Estimated Difference (Relationship)
Tukey's P-Value *
1
2
3
4
5
6
6
Midnight - 6AM
0.04
0.14 |
0.10
-0.09
-0.02
NA
0.1870
6AM - Noon
0.01
0.20 t
0.13
0.00
-0.03
NA
0.1424
Noon - 6PM
-0.01
0.24 t
0.13
0.00
0.05
NA
0.1743
6PM - Midnight
0.01
0.15
0.18 |
-0.04
0.00
NA
0.3579
7
Midnight - 6AM
0.08
0.25 t
0.18
-0.04
-0.02
NA
0.4037
6AM - Noon
0.02
0.16
0.15
-0.06
-0.05
NA
0.3044
Noon - 6PM
0.02
0.35 t
0.23
0.07
0.04
NA
0.3027
6PM - Midnight
0.02
0.25
0.31 t
0.00
0.02
NA
0.3127
8
Midnight - 6AM
0.01
0.25 t
0.21
0.04
0.01
NA
0.4975
6AM - Noon
0.01
0.12 t
0.12 t
-0.07
-0.01
NA
0.0755
Noon - 6PM
-0.01
0.08 |
0.01
0.03
0.00
NA
0.3558
6PM - Midnight
-0.03
0.04
-0.02
-0.02
-0.01
NA
0.9100
9
Midnight - 6AM
-0.04
-0.04
-0.03
-0.01
0.02
NA
0.9855
6AM - Noon
0.00
0.04
0.00
-0.05
-0.02
NA
0.6900
Noon - 6PM
0.00
0.06
0.00
0.14 |
-0.01
NA
0.1542
6PM - Midnight
-0.02
0.07
0.06
0.07
0.00
NA
0.7119
10
Midnight - 6AM
0.05
-0.02
-0.03
-0.02
0.04
NA
0.7881
6AM - Noon
-0.01
0.03
0.00
-0.02
0.06
NA
0.9130
Noon - 6PM
0.03
0.02
-0.02
0.06
0.01
NA
0.5486
6PM - Midnight
0.01
0.01
0.01
0.40 |
0.04
NA
0.3972
11
Midnight - 6AM
0.08
-0.02
0.02
0.33 t
-0.05
NA
0.3772
6AM - Noon
0.00
0.00
-0.02
0.48 |
-0.04
NA
0.3051
Noon - 6PM
0.01
0.03
0.02
0.06
0.00
NA
0.9059
6PM - Midnight
0.03
-0.01
0.02
0.04
-0.03
NA
0.8926
12
Midnight - 6AM
0.04
-0.01
0.00
0.03
-0.05
NA
0.9776
6AM - Noon
0.01
0.01
0.03
0.05
-0.02
NA
0.9734
Noon - 6PM
0.00
0.04
-0.01
0.05
0.00
NA
0.7948
6PM - Midnight
-0.01
-0.01
-0.01
0.05
-0.02
NA
0.8308
F-30
-------
Table 5. (Continued)
RP Inspiratory Time
Study
Day
Time
Mean Baseline Adjusted Value, by Group
Group Effect
P-Value
Estimated Difference (Relationship)
Tukey's P-Value *
1
2
3
4
5
6
13
Midnight - 6AM
0.01
-0.04
0.02
0.05
-0.01
NA
0.9004
6AM - Noon
-0.03
0.01
0.07
0.01
-0.02
NA
0.6330
Noon - 6PM
0.03
0.02
0.02
0.08
-0.03
NA
0.7101
6PM - Midnight
0.04
0.02
0.04
0.11
-0.03
NA
0.8018
14
Midnight - 6AM
0.02
0.05
0.00
0.07
-0.03
NA
0.9191
6AM - Noon
-0.04
0.00
0.01
-0.03
-0.04
NA
0.8979
Noon - 6PM
0.04
0.04
0.05
0.10 |
-0.01
NA
0.7026
6PM - Midnight
0.02
0.06
0.02
0.11
-0.02
NA
0.6394
15
Midnight - 6AM
0.01
-0.01
0.02
0.05
-0.02
NA
0.9720
6AM - Noon
0.02
0.04
0.06
0.07
-0.03
NA
0.9512
Noon - 6PM
0.00
0.04
0.04
0.09
-0.04
NA
0.6844
6PM - Midnight
0.01
0.05
0.03
0.11
-0.05
NA
0.6423
16
Midnight - 6AM
0.01
0.01
0.01
0.08
-0.01
NA
0.9477
6AM - Noon
-0.01
0.05
0.07
0.06
-0.03
NA
0.6528
Noon - 6PM
-0.01
0.05
0.04
0.07
-0.02
NA
0.5922
6PM - Midnight
0.02
0.00
0.00
0.04
-0.03
NA
0.9458
17
Midnight - 6AM
0.02
0.02
-0.02
0.05
-0.02
NA
0.9622
6AM - Noon
-0.02
0.03
0.04
0.06
-0.04
NA
0.6357
Noon - 6PM
-0.01
0.02
0.00
0.07
-0.04
NA
0.5859
6PM - Midnight
-0.05
0.04
0.01
0.13
-0.03
NA
0.3056
18
Midnight - 6AM
-0.01
0.00
-0.01
0.03
0.00
NA
0.9878
6AM - Noon
0.02
0.01
0.01
0.06
-0.03
NA
0.8818
Noon - 6PM
-0.01
0.03
0.02
0.09
-0.04
NA
0.4446
6PM - Midnight
0.02
0.01
0.02
0.11
-0.02
NA
0.8310
F-31
-------
Table 5. (Continued)
RP Inspiratory Time
Study
Day
Time
Mean Baseline Adjusted Value, by Group
Group Effect
P-Value
Estimated Difference (Relationship)
Tukey's P-Value *
1
2
3
4
5
6
19
Midnight - 6AM
0.03
-0.04
0.05
0.05
0.04
NA
0.8401
6AM - Noon
-0.01
0.03
0.05
0.04
-0.02
NA
0.8834
Noon - 6PM
-0.01
0.05
-0.01
0.06
0.01
NA
0.4585
6PM - Midnight
0.02
0.01
0.03
0.11
0.03
NA
0.8963
20
Midnight - 6AM
0.05
0.01
0.05
0.04
-0.02
NA
0.9567
6AM - Noon
0.05
0.06
0.05
0.05
-0.02
NA
0.9282
Noon - 6PM
-0.04
0.02
0.01
0.00
-0.02
NA
0.7146
6PM - Midnight
0.02
0.02
0.02
0.05
-0.02
NA
0.9783
21
Midnight - 6AM
0.09
0.00
0.01
0.07
-0.02
NA
0.7896
6AM - Noon
0.05
0.05
0.04
0.03
-0.05
NA
0.9663
# Cells contain all pairwise comparisons that are statistically 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<6)" indicates that the mean baseline adjusted value
for Group 6 was significantly greater than that for Group 1], and (3) the Tukey-adjusted p-value.
t, | Indicate that the mean baseline adjusted value was significantly different from zero (at the 0.05 level), "t" indicates that the mean at the study time was
greater than that at baseline, while "J," indicates that the mean at the study time was less than that at baseline.
* Group effect was significant at the 0.05 level.
NA Data not available for this Group at this study time.
F-32
-------
Table 6. Summary of the ANOVA Results for the Baseline Adjusted Six-Hour Averages for RP Integral (mmHg-seconds)
RP Integral
Study
Day
Time
Mean Baseline Adjusted Value, by Group
Group Effect
P-Value
Estimated Difference (Relationship)
Tukey's P-Value *
1
2
3
4
5
6
0
6AM - Noon
2.24
3.49 t
1.28
1.66
NA
NA
0.7033
Noon - 6PM
0.51
3.29 t
-0.37
0.00
0.44
-0.74
0.2196
6PM - Midnight
-1.54
1.37
0.01
-1.20
-0.74
-1.80
0.6757
1
Midnight - 6AM
-0.44
1.99
0.50
0.11
0.18
-2.39 |
0.1565
6AM - Noon
1.76
2.94 t
-0.09
1.10
0.92
-0.73
0.2933
Noon - 6PM
-0.66
2.59
0.10
-0.96
0.17
-1.03
0.4688
6PM - Midnight
-0.94
1.58
-0.12
0.41
0.89
0.68
0.8248
2
Midnight - 6AM
0.44
2.13
0.75
-0.15
0.00
2.70 t
0.4145
6AM - Noon
1.58
2.56 t
0.81
1.52
1.17
2.64 t
0.7702
Noon - 6PM
0.67
2.02
0.60
-0.25
1.17
2.55
0.6164
6PM - Midnight
-0.66
1.88
0.50
-0.93
1.63
2.98
0.5503
3
Midnight - 6AM
-0.44
3.21
0.52
-0.63
2.11
3.64
0.5136
6AM - Noon
0.36
3.11
1.66
0.95
4.74 t
2.95
0.3794
Noon - 6PM
-1.59
2.89
-0.14
0.13
4.44 |
1.63
0.2926
6PM - Midnight
-1.66
1.49
-0.04
-0.99
8.74 t
1.57
0.0843
4
Midnight - 6AM
1.86
2.97
0.64
-1.20
7.42 t
1.40
0.2447
6AM - Noon
2.38
3.52
0.84
1.46
5.41 t
1.34
0.6689
Noon - 6PM
-1.02
3.71
1.19
1.82
8.54 t
2.59
0.0905
-9.56 (1 <5) 0.0468
6PM - Midnight
0.22
2.52
0.98
1.13
9.26 t
NA
0.0443 *
-9.04 (1 <5) 0.0347
5
Midnight - 6AM
1.42
3.09
1.13
-0.24
8.81 |
NA
0.0814
6AM - Noon
-0.02
3.27
1.10
-0.80
7.90 t
NA
0.0762
Noon - 6PM
0.97
2.36
0.87
-0.31
6.00 |
NA
0.1822
6PM - Midnight
-2.20
1.49
0.53
0.16
0.09
NA
0.8268
F-33
-------
Table 6. (Continued)
RP Integral
Study
Time
Mean Baseline Adjusted Value, by Group
Group Effect
Estimated Difference (Relationship)
Day
1
2
3
4
5
6
P-Value
Tukey's P-Value *
Midnight - 6AM
1.00
1.07
0.14
-0.99
0.67
NA
0.9048
6
6AM - Noon
-0.34
-0.30
-1.07
-1.45
-1.13
NA
0.9871
Noon - 6PM
-0.87
0.19
-2.65
0.04
0.53
NA
0.7906
6PM - Midnight
-1.52
-0.86
-1.99
0.21
0.98
NA
0.9254
Midnight - 6AM
-0.93
-0.17
-1.17
-0.33
2.09
NA
0.9647
7
6AM - Noon
0.37
-0.24
-1.54
-0.49
0.31
NA
0.9631
Noon - 6PM
-0.49
-0.71
-2.10
-0.39
0.57
NA
0.9477
6PM - Midnight
-0.85
-1.90
-2.46
0.74
0.73
NA
0.8496
Midnight - 6AM
3.59
-0.25
-1.93
1.64
2.07
NA
0.3266
8
6AM - Noon
2.72
0.01
0.25
1.00
0.71
NA
0.7167
Noon - 6PM
1.36
1.87
0.78
0.90
0.29
NA
0.9726
6PM - Midnight
1.53
1.66
0.17
1.26
1.67
NA
0.9604
Midnight - 6AM
3.93 t
2.31
1.74
1.65
1.08
NA
0.8573
9
6AM - Noon
-0.66
2.40
0.88
1.18
1.20
NA
0.8766
Noon - 6PM
-0.16
1.57
0.62
0.47
-0.29
NA
0.9570
6PM - Midnight
2.10
1.44
1.01
1.22
0.24
NA
0.9660
Midnight - 6AM
4.31 t
2.86
1.61
1.76
1.82
NA
0.8578
10
6AM - Noon
2.61
1.86
1.54
0.79
0.66
NA
0.9014
Noon - 6PM
1.50
2.84 t
1.12
0.99
0.94
NA
0.8673
6PM - Midnight
0.73
0.93
0.66
0.68
0.59
NA
0.9999
Midnight - 6AM
3.25
2.64
0.85
1.11
0.13
NA
0.7956
11
6AM - Noon
0.76
1.82
0.96
0.53
-0.75
NA
0.9554
Noon - 6PM
0.40
2.77
1.41
0.49
-0.11
NA
0.8094
6PM - Midnight
0.23
1.90
0.78
1.00
-0.23
NA
0.9543
F-34
-------
Table 6. (Continued)
RP Integral
Study
Day
Time
Mean Baseline Adjusted Value, by Group
Group Effect
P-Value
Estimated Difference (Relationship)
Tukey's P-Value *
1
2
3
4
5
6
12
Midnight - 6AM
2.89
3.72 t
0.99
1.86
0.36
NA
0.7254
6AM - Noon
-0.84
1.95
1.19
0.27
-2.10
NA
0.8306
Noon - 6PM
-0.87
1.65
1.15
0.33
-0.18
NA
0.8695
6PM - Midnight
2.93 t
1.28
1.27
1.04
-1.59
NA
0.6757
13
Midnight - 6AM
3.35
2.80
0.81
1.56
-0.49
NA
0.7574
6AM - Noon
1.17
1.94
0.83
0.37
-2.10
NA
0.7661
Noon - 6PM
0.49
1.46
1.05
0.70
-0.85
NA
0.9543
6PM - Midnight
1.40
1.38
0.69
0.50
-0.99
NA
0.9485
14
Midnight - 6AM
0.62
2.08
0.28
1.45
0.60
NA
0.9190
6AM - Noon
1.18
2.27
0.94
1.55
-0.18
NA
0.9459
Noon - 6PM
0.92
1.45
0.16
0.53
-0.09
NA
0.9362
6PM - Midnight
2.14
0.18
0.24
0.31
-0.36
NA
0.8027
15
Midnight - 6AM
4.25
1.82
0.21
1.57
0.45
NA
0.6991
6AM - Noon
3.38 t
1.09
-0.04
0.80
-1.05
NA
0.5057
Noon - 6PM
1.82
1.89
0.56
0.56
-1.05
NA
0.8633
6PM - Midnight
1.71
0.90
0.50
0.61
-0.61
NA
0.9418
16
Midnight - 6AM
3.02
2.16
0.93
1.41
-0.39
NA
0.8231
6AM - Noon
1.32
2.18
0.57
0.15
-1.24
NA
0.8094
Noon - 6PM
-3.94
1.37
0.78
0.32
-0.23
NA
0.6457
6PM - Midnight
-2.14
0.39
0.61
0.36
-0.15
NA
0.8928
17
Midnight - 6AM
2.14
2.00
0.60
0.82
0.19
NA
0.9301
6AM - Noon
2.20
0.76
0.55
0.23
-1.11
NA
0.7516
Noon - 6PM
1.20
1.79
0.80
0.30
-0.11
NA
0.9522
6PM - Midnight
2.28
0.10
0.60
0.52
-0.08
NA
0.8214
F-35
-------
Table 6. (Continued)
RP Integral
Study
Day
Time
Mean Baseline Adjusted Value, by Group
Group Effect
P-Value
Estimated Difference (Relationship)
Tukey's P-Value *
1
2
3
4
5
6
18
Midnight - 6AM
3.23
1.50
0.60
0.69
1.24
NA
0.8738
6AM - Noon
3.09
1.65
0.43
0.00
-1.09
NA
0.6480
Noon - 6PM
1.82
0.92
1.41
0.57
-0.24
NA
0.9351
6PM - Midnight
2.75
-1.56
0.64
1.09
0.02
NA
0.6083
19
Midnight - 6AM
4.88
-1.16
0.76
1.41
0.90
NA
0.6568
6AM - Noon
1.79
1.16
0.88
0.74
0.73
NA
0.9853
Noon - 6PM
-2.78
2.25
0.94
0.69
0.76
NA
0.7263
6PM - Midnight
-1.11
1.82
0.31
1.50
0.86
NA
0.8321
20
Midnight - 6AM
2.65
2.74
0.81
2.97
0.50
NA
0.8471
6AM - Noon
3.26
2.77
0.72
0.88
-0.24
NA
0.7397
Noon - 6PM
1.91
2.75 t
1.10
1.36
1.11
NA
0.8947
6PM - Midnight
2.55
1.68
-0.01
1.54
0.11
NA
0.7967
21
Midnight - 6AM
4.23
2.54
0.44
3.16
0.19
NA
0.8189
6AM - Noon
2.95
2.93
0.34
1.46
-0.24
NA
0.7819
# Cells contain all pairwise comparisons that are statistically 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<6)" indicates that the mean baseline adjusted value
for Group 6 was significantly greater than that for Group 1], and (3) the Tukey-adjusted p-value.
t, | Indicate that the mean baseline adjusted value was significantly different from zero (at the 0.05 level), "t" indicates that the mean at the study time was
greater than that at baseline, while "J," indicates that the mean at the study time was less than that at baseline.
* Group effect was significant at the 0.05 level.
NA Data not available for this Group at this study time.
F-36
-------
Table 7. Summary of the ANOVA Results for the Baseline Adjusted Six-Hour Averages for RP Peak Amplitude (mmHg)
RP Integral
Study
Day
Time
Mean Baseline Adjusted Value, by Group
Group Effect
P-Value
Estimated Difference (Relationship)
Tukey's P-Value *
1
2
3
4
5
6
0
6AM - Noon
1.67
3.74
1.04
2.10
NA
NA
0.8270
Noon - 6PM
-0.24
5.33 t
-0.80
-0.07
0.87
-2.05
0.1343
6PM - Midnight
-1.67
2.57
0.38
-1.40
-0.35
-2.90
0.5993
1
Midnight - 6AM
-0.97
2.83
0.94
0.42
1.30
-4.24 |
0.1878
6AM - Noon
1.45
4.09 |
-0.17
1.19
2.01
-1.55
0.2983
Noon - 6PM
-2.12
5.11 t
0.44
-0.78
0.58
-1.59
0.3334
6PM - Midnight
-2.01
2.95
-0.57
0.56
1.17
0.22
0.7236
2
Midnight - 6AM
-2.04
3.22
1.31
-0.45
0.85
2.69
0.4724
6AM - Noon
0.67
3.00
0.71
2.11
1.48
3.27
0.8323
Noon - 6PM
0.57
4.15 t
1.04
0.28
0.00
3.80
0.5169
6PM - Midnight
-3.48
3.62
0.74
-1.51
-1.65
4.53
0.5000
3
Midnight - 6AM
-2.23
4.51
0.64
-1.26
-0.22
4.82
0.4630
6AM - Noon
-2.84
4.40
2.63
0.99
4.30
4.27
0.3589
Noon - 6PM
-6.29
5.23
-0.31
0.91
5.04
2.65
0.3355
6PM - Midnight
-5.12
3.06
-0.49
-1.34
10.09
1.82
0.3083
4
Midnight - 6AM
-0.23
4.48
0.65
-2.82
8.29
1.71
0.4069
6AM - Noon
2.65
5.65
1.54
2.40
5.90
1.88
0.8614
Noon - 6PM
-6.63
6.52
2.04
3.37
10.48
5.22
0.2266
6PM - Midnight
-2.71
3.88
0.85
1.87
11.46 t
NA
0.1547
5
Midnight - 6AM
-1.89
4.22
1.50
0.28
10.93 t
NA
0.1543
6AM - Noon
-4.31
5.35
1.90
-0.36
8.59
NA
0.2867
Noon - 6PM
0.21
5.01 t
1.93
0.26
8.09 |
NA
0.2393
6PM - Midnight
-6.51
3.12
1.37
0.54
0.09
NA
0.6691
6
Midnight - 6AM
-0.43
3.56
1.46
-1.06
1.31
NA
0.6959
6AM - Noon
-2.56
4.12
-0.08
-1.39
-1.46
NA
0.6643
Noon - 6PM
-4.30
5.15
-2.84
0.83
0.93
NA
0.4539
6PM - Midnight
-7.48
1.89
-2.16
0.16
1.83
NA
0.7760
F-37
-------
Table 7. (Continued)
RP Peak Amplitude
Study
Day
Time
Mean Baseline Adjusted Value, by Group
Group Effect
P-Value
Estimated Difference (Relationship)
Tukey's P-Value *
1
2
3
4
5
6
7
Midnight - 6AM
-7.03
3.11
-0.27
-1.17
3.07
NA
0.7142
6AM - Noon
-0.98
3.50
-0.15
-0.81
-0.39
NA
0.8695
Noon - 6PM
-2.60
5.39
-1.25
0.50
1.38
NA
0.5821
6PM - Midnight
-3.33
0.88
-2.45
1.06
1.51
NA
0.9415
8
Midnight - 6AM
5.06
2.86
-1.88
2.59
3.02
NA
0.5989
6AM - Noon
4.68
1.40
0.79
1.76
0.80
NA
0.8114
Noon - 6PM
2.26
4.44
1.16
2.34
0.77
NA
0.8554
6PM - Midnight
2.47
2.65
-0.82
1.63
2.50
NA
0.9027
9
Midnight - 6AM
4.79
2.63
2.42
2.18
1.53
NA
0.9250
6AM - Noon
-0.13
4.08
0.84
1.48
1.26
NA
0.8598
Noon - 6PM
0.46
3.45
0.85
2.04
-0.86
NA
0.8841
6PM - Midnight
3.20
2.51
1.93
2.16
0.31
NA
0.9819
10
Midnight - 6AM
5.94 |
3.53
2.24
2.48
2.64
NA
0.8881
6AM - Noon
4.23
3.17
2.69
1.77
1.30
NA
0.9395
Noon - 6PM
2.86
5.19 t
1.87
2.71
1.51
NA
0.8556
6PM - Midnight
1.97
1.77
1.15
2.20
0.86
NA
0.9978
11
Midnight - 6AM
4.91
3.39
1.46
2.72
-0.07
NA
0.8516
6AM - Noon
2.15
3.20
1.70
2.13
-1.99
NA
0.9077
Noon - 6PM
0.99
5.35 t
2.52
1.75
-0.44
NA
0.7146
6PM - Midnight
1.01
3.46
1.22
1.33
-0.63
NA
0.9427
12
Midnight - 6AM
4.07
5.18 t
1.36
2.28
0.15
NA
0.7681
6AM - Noon
0.01
3.49
2.14
0.92
-3.18
NA
0.8399
Noon - 6PM
-0.12
3.89
1.83
1.64
-0.56
NA
0.8342
6PM - Midnight
5.41 t
2.61
1.79
1.31
-2.45
NA
0.6653
F-38
-------
Table 7. (Continued)
RP Peak Amplitude
Study
Day
Time
Mean Baseline Adjusted Value, by Group
Group Effect
P-Value
Estimated Difference (Relationship)
Tukey's P-Value *
1
2
3
4
5
6
13
Midnight - 6AM
5.49
3.33
1.14
1.91
-1.22
NA
0.7669
6AM - Noon
3.62
3.49
1.48
0.75
-3.71
NA
0.6213
Noon - 6PM
2.37
3.27
1.57
1.90
-1.47
NA
0.9361
6PM - Midnight
2.82
2.80
0.57
0.60
-1.33
NA
0.8746
14
Midnight - 6AM
1.03
3.04
-0.23
1.83
0.48
NA
0.8635
6AM - Noon
1.40
3.87
1.35
2.20
-1.02
NA
0.8799
Noon - 6PM
2.14
3.62
0.12
1.91
-0.19
NA
0.8278
6PM - Midnight
3.61
1.42
-0.21
0.90
-0.97
NA
0.7984
15
Midnight - 6AM
5.79
2.54
0.16
2.50
-0.03
NA
0.7381
6AM - Noon
5.17
2.19
-0.46
1.71
-1.94
NA
0.5767
Noon - 6PM
3.48
4.46
0.22
1.67
-2.01
NA
0.7417
6PM-Midnight
3.40
1.87
0.37
1.41
-1.47
NA
0.8627
16
Midnight - 6AM
4.47
2.86
1.26
1.83
-0.55
NA
0.8353
6AM - Noon
3.21
3.73
1.18
0.45
-2.16
NA
0.7434
Noon - 6PM
-6.28
3.53
0.78
1.11
-0.74
NA
0.5414
6PM - Midnight
-3.14
1.04
0.34
0.27
-0.35
NA
0.9131
17
Midnight - 6AM
4.33
2.80
0.34
0.85
0.48
NA
0.8213
6AM - Noon
3.82
0.90
0.79
0.76
-2.15
NA
0.7241
Noon - 6PM
2.00
3.38
0.80
0.98
-0.50
NA
0.9208
6PM - Midnight
2.99
0.51
0.63
1.12
-0.20
NA
0.9219
18
Midnight - 6AM
3.84
1.70
0.69
0.73
2.38
NA
0.9228
6AM - Noon
4.71
2.10
0.87
0.45
-2.28
NA
0.7303
Noon - 6PM
3.16
1.56
2.08
1.68
-1.00
NA
0.9537
6PM - Midnight
4.61
-4.09
0.82
1.93
0.13
NA
0.5928
F-39
-------
Table 7. (Continued)
RP Peak Amplitude
Study
Day
Time
Mean Baseline Adjusted Value, by Group
Group Effect
P-Value
Estimated Difference (Relationship)
Tukey's P-Value *
1
2
3
4
5
6
19
Midnight - 6AM
6.87
-4.85
1.03
1.83
0.94
NA
0.6274
6AM - Noon
2.45
1.96
1.55
1.36
1.07
NA
0.9974
Noon - 6PM
-5.26
5.13
1.09
1.85
1.35
NA
0.5935
6PM - Midnight
-2.05
3.15
0.82
2.61
1.51
NA
0.8228
20
Midnight - 6AM
3.42
3.78
1.87
4.36
0.74
NA
0.9380
6AM - Noon
5.36
4.80
2.04
1.63
-0.50
NA
0.8250
Noon - 6PM
3.15
5.16 t
1.82
2.91
2.06
NA
0.8839
6PM - Midnight
4.42
3.13
0.18
2.58
0.34
NA
0.8237
21
Midnight - 6AM
6.14
3.52
0.71
4.76
0.52
NA
0.8258
6AM - Noon
4.30
5.13
0.85
2.40
0.41
NA
0.8081
# Cells contain all pairwise comparisons that are statistically 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<6)" indicates that the mean baseline adjusted value
for Group 6 was significantly greater than that for Group 1], and (3) the Tukey-adjusted p-value.
t, | Indicate that the mean baseline adjusted value was significantly different from zero (at the 0.05 level), "t" indicates that the mean at the study time was
greater than that at baseline, while "J," indicates that the mean at the study time was less than that at baseline.
* Group effect was significant at the 0.05 level.
NA Data not available for this Group at this study time.
F-40
-------
Table 8. Summary of the ANOVA Results for the Baseline Adjusted Six-Hour Averages for RP Respiratory Rate (RCPM)
RP Respiratory Rate
Study
Day
Time
Mean Baseline Adjusted Value, by Group
Group Effect
P-Value
Estimated Difference (Relationship)
Tukey's P-Value *
1
2
3
4
5
6
0
6AM - Noon
14.94 |
14.20 t
13.27 t
23.99 t
NA
NA
0.3889
Noon - 6PM
7.35 t
1.52
-0.74
1.71
5.19
3.14
0.3478
6PM - Midnight
-0.28
-1.08
-2.89
-2.03
1.11
-1.39
0.7682
1
Midnight - 6AM
-0.08
-1.66
-0.67
-0.07
-0.23
-5.58 |
0.5608
6AM - Noon
7.97 t
7.74 t
4.89
6.77 t
8.47 t
2.23
0.6242
Noon - 6PM
0.50
-0.08
1.01
0.22
-5.01
-4.24
0.6622
6PM - Midnight
3.00
-0.33
2.43
8.52
1.98
12.70
0.6897
2
Midnight - 6AM
1.64
2.59
1.18
8.74
6.16
29.74 t
0.0098 *
-28.10 (1<6) 0.0156
-27.16 (2<6) 0.0207
-28.56 (3<6) 0.0136
6AM - Noon
8.22 t
9.99 |
8.51 t
15.64 t
15.51 t
24.62 t
0.0082 *
-16.40 (1<6) 0.0112
-14.64 (2<6) 0.0283
-16.12 (3<6) 0.0131
Noon - 6PM
-0.03
-1.77
2.68
2.83
10.96 t
22.17 t
0.0188*
-22.20 (1<6) 0.0353
-23.94 (2<6) 0.0199
6PM - Midnight
0.67
-0.39
-0.17
4.21
19.32 t
26.12 t
0.0047 *
-25.45 (1<6) 0.0304
-26.51 (2<6) 0.0223
-26.29 (3<6) 0.0237
3
Midnight - 6AM
0.71
2.81
3.57
9.15
22.00 t
35.03 t
0.0056 *
-34.32 (1<6) 0.0109
-32.23 (2<6) 0.0186
-31.46 (3<6) 0.0225
6AM - Noon
6.78
8.79 t
5.52
13.83 t
23.19 t
25.19 t
0.0024 *
-16.41 (1<5) 0.0366
-18.41 (1<6) 0.0242
-17.67 (3<5) 0.0211
-19.67 (3<6) 0.0142
Noon - 6PM
2.85
-0.31
-1.09
5.15
14.63 t
21.16 t
0.0116*
-21.46 (2<6) 0.0302
-22.25 (3<6) 0.0232
6PM - Midnight
1.43
-2.13
2.36
4.88
20.13 t
24.15 t
0.0096 *
-22.26 (2<5) 0.0353
-26.28 (2<6) 0.0286
F-41
-------
Table 8. (Continued)
RP Respiratory Rate
Study
Day
Time
Mean Baseline Adjusted Value, by Group
Group Effect
P-Value
Estimated Difference (Relationship)
Tukey's P-Value *
1
2
3
4
5
6
4
Midnight - 6AM
4.86
1.13
3.26
8.46
24.46 t
24.11 t
0.0379 *
6AM - Noon
5.77
2.72
3.44
8.78
9.05
23.74 t
0.5764
Noon - 6PM
4.34
1.34
0.38
4.48
25.55 t
23.32 t
<0.0001 *
-21.21 (1<5) 0.0008
-24.22 (2<5) 0.0002
-21.98 (2<6) 0.0236
-25.17 (3<5) 0.0001
-22.94 (3<6) 0.0172
-21.08 (4<5) 0.0009
6PM - Midnight
5.80
3.06
7.54 t
5.19
26.45 t
NA
0.0094 *
-20.65 (1<5) 0.0179
-23.39 (2<5) 0.0068
-18.92 (3<5) 0.0327
-21.26 (4<5) 0.0200
5
Midnight - 6AM
2.39
3.83
7.53 t
1.29
22.15 t
NA
0.0079 *
-19.76 (1<5) 0.0094
-18.32 (2<5) 0.0168
-20.86 (4<5) 0.0087
6AM - Noon
4.41
3.39
3.91
3.32
24.47 t
NA
0.0181 *
-20.06 (1<5) 0.0302
-21.08 (2<5) 0.0215
-20.56 (3<5) 0.0256
-21.14 (4<5) 0.0286
Noon - 6PM
-1.30
-5.17
-4.19
-0.97
15.71 t
NA
0.0350 *
-20.87 (2<5) 0.0274
-19.90 (3<5) 0.0375
6PM - Midnight
0.53
-1.02
-3.85
4.72
-0.17
NA
0.7259
6
Midnight - 6AM
-1.56
-3.88
-5.14
3.48
1.60
NA
0.4260
6AM - Noon
-2.73
-15.63 |
-9.40 |
-3.44
-4.56
NA
0.1580
Noon - 6PM
-2.05
-17.84 |
-13.37 |
-3.05
0.00
NA
0.2067
6PM - Midnight
-0.13
-9.67
-10.92
3.85
0.80
NA
0.3063
F-42
-------
Table 8. (Continued)
RP Respiratory Rate
Study
Day
Time
Mean Baseline Adjusted Value, by Group
Group Effect
P-Value
Estimated Difference (Relationship)
Tukey's P-Value *
1
2
3
4
5
6
7
Midnight - 6AM
-3.26
-7.53
-9.24 |
3.15
4.46
NA
0.2431
6AM - Noon
-1.60
-12.62 |
-10.26|
3.98
7.21
NA
0.0952
Noon - 6PM
-2.09
-21.23 |
-13.81 |
-5.28
-0.36
NA
0.2698
6PM - Midnight
0.60
-13.98 |
-13.45|
2.99
3.84
NA
0.1553
8
Midnight - 6AM
2.19
-9.46 |
-9.21
1.88
3.86
NA
0.1832
6AM - Noon
-0.65
-5.76 |
-4.29
5.41
1.66
NA
0.0812
Noon - 6PM
3.88
-2.90
-0.31
-2.25
1.67
NA
0.4326
6PM - Midnight
6.07
0.80
2.57
8.59 t
3.46
NA
0.5806
9
Midnight - 6AM
5.53
7.64
4.17
8.09
1.57
NA
0.9467
6AM - Noon
0.83
-0.52
2.20
9.31 t
3.66
NA
0.4782
Noon - 6PM
1.05
-3.21
0.31
-5.30
-0.55
NA
0.7769
6PM - Midnight
3.51
-1.04
-6.44 |
2.34
-1.07
NA
0.0557
-9.96 (3<1) 0.0432
10
Midnight - 6AM
-0.19
4.51
3.53
8.76
1.82
NA
0.6950
6AM - Noon
-0.03
-0.17
1.02
3.51
-8.48
NA
0.6532
Noon - 6PM
0.32
2.87
1.11
0.18
2.85
NA
0.9668
6PM - Midnight
0.19
0.30
-1.61
-11.95 |
-1.13
NA
0.3108
11
Midnight - 6AM
-1.33
4.97
-0.71
-9.62 |
0.24
NA
0.2549
6AM - Noon
0.06
1.10
2.93
-13.21 |
-1.08
NA
0.1188
Noon - 6PM
1.71
-0.73
0.41
-5.67
0.43
NA
0.6131
6PM - Midnight
-0.52
2.35
-1.66
0.62
1.40
NA
0.8203
12
Midnight - 6AM
-0.01
9.82 t
0.07
1.16
3.14
NA
0.2730
6AM - Noon
-3.26
0.43
-2.02
-2.26
-3.78
NA
0.5419
Noon - 6PM
-1.31
-0.97
1.02
-5.20
0.57
NA
0.7670
6PM - Midnight
1.90
1.57
0.39
2.15
-4.47
NA
0.7925
F-43
-------
Table 8. (Continued)
RP Respiratory Rate
Study
Day
Time
Mean Baseline Adjusted Value, by Group
Group Effect
P-Value
Estimated Difference (Relationship)
Tukey's P-Value *
1
2
3
4
5
6
13
Midnight - 6AM
-1.70
7.36
2.05
1.23
0.93
NA
0.5224
6AM - Noon
-0.42
2.82
-0.09
-1.08
-1.52
NA
0.9446
Noon - 6PM
-2.99
0.56
0.36
-2.95
-0.63
NA
0.8384
6PM - Midnight
-0.90
-0.31
-0.16
-2.36
-0.28
NA
0.9885
14
Midnight - 6AM
-1.54
2.00
1.79
1.21
3.94
NA
0.6784
6AM - Noon
4.06
2.59
2.88
7.51 t
5.76
NA
0.7714
Noon - 6PM
0.19
-0.86
-2.27
-3.67
-2.15
NA
0.8935
6PM - Midnight
-0.11
-3.93
0.55
-3.81
2.70
NA
0.5351
15
Midnight - 6AM
0.75
4.84
4.15
-0.94
4.40
NA
0.8602
6AM - Noon
-0.80
-0.29
-0.79
-1.67
-3.25
NA
0.9898
Noon - 6PM
0.13
-2.60
-0.12
-4.33
1.96
NA
0.8740
6PM - Midnight
2.02
0.05
0.78
-3.90
1.95
NA
0.6715
16
Midnight - 6AM
1.14
2.71
4.02
0.63
-1.41
NA
0.8788
6AM - Noon
-1.28
-1.24
-2.52
-3.24
-5.39
NA
0.9069
Noon - 6PM
1.46
-2.29
-0.10
-3.72
2.20
NA
0.9234
6PM - Midnight
0.53
0.52
2.98
1.39
-2.93
NA
0.6619
17
Midnight - 6AM
-1.94
3.17
3.24
-1.51
-2.65
NA
0.1515
6AM - Noon
2.68
-2.44
-1.72
-3.66
-1.99
NA
0.2958
Noon - 6PM
3.21
-1.20
-0.17
-3.43
0.35
NA
0.6888
6PM - Midnight
5.54 |
-3.51
-1.20
-4.22
2.52
NA
0.0584
18
Midnight - 6AM
3.92
2.68
0.80
0.04
-0.76
NA
0.8647
6AM - Noon
-0.08
0.16
-5.15
-4.22
-3.02
NA
0.5430
Noon - 6PM
3.01
-2.80
0.24
-4.22
2.27
NA
0.3684
6PM - Midnight
1.49
-2.18
-0.31
-1.31
1.01
NA
0.6707
F-44
-------
Table 8. (Continued)
RP Respiratory Rate
Study
Day
Time
Mean Baseline Adjusted Value, by Group
Group Effect
P-Value
Estimated Difference (Relationship)
Tukey's P-Value *
1
2
3
4
5
6
19
Midnight - 6AM
1.97
5.36 t
-0.94
2.32
2.68
NA
0.3552
6AM - Noon
1.17
-2.01
-2.11
-2.45
1.76
NA
0.1728
Noon - 6PM
1.00
-1.77
1.55
-1.56
-0.07
NA
0.8515
6PM - Midnight
-1.87
-0.58
-4.31 |
-1.02
-2.06
NA
0.7114
20
Midnight - 6AM
-4.99 |
3.55
-3.26
0.09
-1.75
NA
0.0913
6AM - Noon
-3.01 |
-1.43
-3.82 |
-2.57
-4.66
NA
0.6915
Noon - 6PM
3.37
1.90
3.84
2.49
1.72
NA
0.9639
6PM - Midnight
0.00
0.33
0.48
0.32
-1.78
NA
0.9815
21
Midnight - 6AM
-1.47
3.42 t
1.41
-0.97
1.32
NA
0.2172
6AM - Noon
-0.72
2.79
-0.67
3.81
3.61
NA
0.2952
# Cells contain all pairwise comparisons that are statistically 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<6)" indicates that the mean baseline adjusted value
for Group 6 was significantly greater than that for Group 1], and (3) the Tukey-adjusted p-value.
t, | Indicate that the mean baseline adjusted value was significantly different from zero (at the 0.05 level), "t" indicates that the mean at the study time was
greater than that at baseline, while "J," indicates that the mean at the study time was less than that at baseline.
* Group effect was significant at the 0.05 level.
NA Data not available for this Group at this study time.
F-45
-------
Table 9. Summary of the ANOVA Results for the Baseline Adjusted Six-Hour Averages for Temperature (Celsius)
Temperature
Study
Day
Time
Mean Baseline Adjusted Value, by Group
Group Effect
P-Value
Estimated Difference (Relationship)
Tukey's P-Value *
1
2
3
4
5
6
0
6AM - Noon
0.00
0.25 t
0.19 |
0.66 |
NA
NA
0.0007*
-0.66 (1<4) 0.0004
-0.41 (2<4) 0.0181
-0.47 (3<4) 0.0068
Noon - 6PM
-0.01
0.01
0.02
0.02
-0.01
-0.54 |
0.5165
6PM - Midnight
-0.10
-0.05
-0.09
-0.01
0.02
-0.43
0.6981
1
Midnight - 6AM
-0.06
0.00
0.01
-0.02
0.07
-0.48 |
0.3151
6AM - Noon
0.09
0.12
0.10
0.10
0.12
-0.34
0.5419
Noon - 6PM
0.12
0.00
0.05
0.35
-0.12
-0.05
0.9181
6PM - Midnight
0.03
0.00
0.03
0.50
0.18
0.63
0.8355
2
Midnight - 6AM
-0.02
0.04
0.00
0.54
0.44
0.74
0.7995
6AM - Noon
0.13
0.16
0.15
0.51 t
0.77 t
1.80 |
0.0001 *
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Noon - 6PM
0.00
-0.03
0.02
0.14
0.75 t
1.61 t
<0.0001 *
-1.61 (1<6) 0.0001
-1.64 (2<6) 0.0001
-1.59 (3<6) 0.0002
-1.47 (4<6) 0.0004
6PM - Midnight
-0.11
-0.07
-0.02
0.14
1.05 t
1.50 t
<0.0001 *
-1.16 (1<5) 0.0036
-1.61 (1<6) 0.0001
-1.12 (2<5) 0.0051
-1.57 (2<6) 0.0002
-1.07 (3<5) 0.0077
-1.52 (3<6) 0.0003
-0.91 (4<5) 0.0292
-1.37 (4<6) 0.0011
F-46
-------
Table 9. (Continued)
Temperature
Study
Day
Time
Mean Baseline Adjusted Value, by Group
Group Effect
P-Value
Estimated Difference (Relationship)
Tukey's P-Value *
1
2
3
4
5
6
3
Midnight - 6AM
-0.06
0.01
0.02
0.33
0.62 t
1.60 t
0.0021 *
-1.66 (1 <6) 0.0028
-1.60 (2<6) 0.0041
-1.58 (3<6) 0.0045
-1.27 (4<6) 0.0302
6AM - Noon
0.07
0.09
0.16
0.45
0.31
1.95 t
0.0017*
-1.88 (1 <6) 0.0021
-1.86 (2<6) 0.0023
-1.79 (3<6) 0.0035
-1.51 (4<6) 0.0171
-1.64 (5<6) 0.0081
Noon - 6PM
0.00
-0.11
-0.02
0.39
-0.03
1.54 t
0.3398
6PM - Midnight
-0.07
-0.09
-0.05
0.47
1.15 t
1.98 |
0.0042 *
-2.05 (1 <6) 0.0107
-2.07 (2<6) 0.0097
-2.02 (3<6) 0.0119
4
Midnight - 6AM
-0.04
-0.01
0.00
0.30
1.17 t
0.58
0.0260 *
-1.21 (1 <5) 0.0286
-1.19 (2<5) 0.0336
-1.18 (3<5) 0.0354
6AM - Noon
0.00
0.05
0.10
-0.22
-3.63
-1.45
0.6945
Noon - 6PM
0.05
-0.07
0.02
-0.57
1.25 t
-3.55 |
0.0033 *
-3.60 (6<1) 0.0101
-3.48 (6<2) 0.0134
-3.57 (6<3) 0.0107
-2.98 (6<4) 0.0412
-4.80 (6<5) 0.0011
6PM - Midnight
0.02
0.04
0.05
0.12
0.54 |
NA
0.0084 *
-0.52 (1 <5) 0.0079
-0.50 (2<5) 0.0114
-0.49 (3<5) 0.0128
F-47
-------
Table 9. (Continued)
Temperature
Study
Day
Time
Mean Baseline Adjusted Value, by Group
Group Effect
P-Value
Estimated Difference (Relationship)
Tukey's P-Value *
1
2
3
4
5
6
5
Midnight - 6AM
0.01
0.01
0.03
0.03
-0.08
NA
0.9962
6AM - Noon
-0.01
0.01
0.03
0.04
-0.80 |
NA
0.0108*
-0.79 (5<1) 0.0194
-0.82 (5<2) 0.0154
-0.83 (5<3) 0.0138
-0.84 (5<4) 0.0173
Noon - 6PM
-0.07
-0.12
0.03
0.04
-2.26 |
NA
0.0027 *
-2.19 (5<1) 0.0050
-2.14 (5<2) 0.0061
-2.29 (5<3) 0.0034
-2.30 (5<4) 0.0048
6PM - Midnight
-0.02
0.03
0.04
0.05
0.08
NA
0.9382
6
Midnight - 6AM
-0.06
-0.04
-0.03
0.01
-0.08
NA
0.7980
6AM - Noon
-0.12|
-0.16|
-0.09 |
-0.08
-0.09
NA
0.5666
Noon - 6PM
-0.09
-0.23
-0.12
0.39
-0.06
NA
0.3240
6PM - Midnight
-0.05
-0.05
-0.04
0.53
0.04
NA
0.4099
7
Midnight - 6AM
-0.07
-0.14
-0.03
0.37
0.00
NA
0.3235
6AM - Noon
-0.04
-0.18|
-0.07
0.21 t
-0.07
NA
0.0858
-0.38 (2<4) 0.0481
Noon - 6PM
-0.04
-0.29 |
-0.16
-0.07
-0.07
NA
0.1967
6PM - Midnight
-0.11
-0.13
-0.09
0.00
0.05
NA
0.8877
8
Midnight - 6AM
-0.12
-0.09
-0.13
0.16
-0.02
NA
0.4590
6AM - Noon
-0.03
-0.12
-0.09
0.54 |
-0.02
NA
0.3058
Noon - 6PM
-0.05
-0.07
-0.03
0.43
-0.05
NA
0.4042
6PM - Midnight
-0.08
0.08
0.07
0.70 t
-0.06
NA
0.4330
9
Midnight - 6AM
-0.07
0.07
0.01
0.58
-0.03
NA
0.4842
6AM - Noon
0.01
0.08
0.02
0.64 |
0.06
NA
0.4569
Noon - 6PM
-0.13
-0.10
-0.07
0.34
-0.04
NA
0.5217
6PM - Midnight
-0.15
-0.05
-0.07
0.30
-0.05
NA
0.3076
F-48
-------
Table 9. (Continued)
Temperature
Study
Day
Time
Mean Baseline Adjusted Value, by Group
Group Effect
P-Value
Estimated Difference (Relationship)
Tukey's P-Value *
1
2
3
4
5
6
10
Midnight - 6AM
-0.15|
0.04
-0.07
0.00
-0.06
NA
0.1684
6AM - Noon
-0.08
0.07
-0.01
-0.42 |
-0.05
NA
0.4244
Noon - 6PM
-0.10
-0.12
0.00
-0.93 |
-0.01
NA
0.4623
6PM - Midnight
-0.15
-0.08
-0.03
-2.47 |
-0.07
NA
0.4942
11
Midnight - 6AM
-0.10
-0.03
-0.07
-3.85 |
-0.02
NA
0.4563
6AM - Noon
-0.05
0.01
-0.02
-4.09 |
-0.01
NA
0.4337
Noon - 6PM
-0.08
-0.19|
-0.09
-0.08
-0.02
NA
0.7444
6PM - Midnight
-0.12
-0.02
-0.01
0.06
-0.07
NA
0.4544
12
Midnight - 6AM
-0.14|
-0.01
-0.07
0.06
-0.12
NA
0.3074
6AM - Noon
0.02
0.06
0.00
0.06
-0.06
NA
0.6222
Noon - 6PM
-0.02
-0.15|
-0.04
-0.03
-0.03
NA
0.4517
6PM - Midnight
-0.14 |
-0.06
-0.01
0.13
-0.16
NA
0.1897
13
Midnight - 6AM
-0.18|
-0.05
-0.02
0.05
-0.07
NA
0.3537
6AM - Noon
-0.04
0.03
0.02
0.03
-0.06
NA
0.6696
Noon - 6PM
-0.08
-0.14
0.03
-0.03
0.03
NA
0.5769
6PM - Midnight
-0.19|
-0.11
-0.06
0.01
0.02
NA
0.3118
14
Midnight - 6AM
-0.20 |
-0.08
-0.07
-0.01
0.03
NA
0.2490
6AM - Noon
0.03
0.07
0.03
0.14 |
0.07
NA
0.6757
Noon - 6PM
-0.07
-0.10
-0.01
0.00
-0.04
NA
0.9268
6PM - Midnight
-0.11
-0.08
-0.02
-0.03
0.03
NA
0.8349
15
Midnight - 6AM
-0.13
-0.04
0.00
0.02
-0.02
NA
0.5560
6AM - Noon
0.00
0.03
0.02
-0.06
-0.03
NA
0.9247
Noon - 6PM
-0.11
-0.12
0.07
-0.02
-0.03
NA
0.3484
6PM - Midnight
-0.17
-0.11
0.04
0.05
0.03
NA
0.4481
F-49
-------
Table 9. (Continued)
Temperature
Study
Day
Time
Mean Baseline Adjusted Value, by Group
Group Effect
P-Value
Estimated Difference (Relationship)
Tukey's P-Value *
1
2
3
4
5
6
16
Midnight - 6AM
-0.13
-0.03
0.02
0.00
-0.05
NA
0.6024
6AM - Noon
-0.06
0.02
0.07
-0.01
-0.10
NA
0.6122
Noon - 6PM
-0.12
-0.09
0.02
0.01
-0.12
NA
0.7182
6PM - Midnight
-0.19 |
-0.07
0.00
0.04
-0.03
NA
0.4142
17
Midnight - 6AM
-0.18|
0.00
-0.03
0.05
-0.04
NA
0.1124
6AM - Noon
-0.01
0.10
-0.04
-0.02
-0.02
NA
0.4603
Noon - 6PM
-0.05
-0.07
-0.04
0.02
-0.04
NA
0.9732
6PM - Midnight
-0.08
-0.08
0.00
-0.01
0.04
NA
0.9265
18
Midnight - 6AM
-0.12
-0.04
-0.09
0.02
-0.04
NA
0.7082
6AM - Noon
0.01
0.04
-0.02
-0.02
-0.05
NA
0.8193
Noon - 6PM
-0.01
-0.06
0.04
-0.10
0.07
NA
0.8422
6PM - Midnight
-0.11
-0.07
0.03
-0.01
0.00
NA
0.8641
19
Midnight - 6AM
-0.16
-0.05
-0.05
0.05
0.01
NA
0.5508
6AM - Noon
0.01
-0.01
0.05
0.00
0.05
NA
0.9691
Noon - 6PM
-0.07
-0.08
0.00
-0.01
0.07
NA
0.8634
6PM - Midnight
-0.17 |
-0.09
-0.04
0.04
-0.03
NA
0.4749
20
Midnight - 6AM
-0.18|
-0.07
-0.13
0.12
-0.04
NA
0.1707
6AM - Noon
-0.04
0.07
-0.01
-0.02
0.01
NA
0.5116
Noon - 6PM
-0.04
-0.05
0.00
0.02
0.10
NA
0.6889
6PM - Midnight
-0.13
-0.07
-0.04
0.09
0.07
NA
0.4001
21
Midnight - 6AM
-0.17 |
-0.06
-0.13
0.08
-0.07
NA
0.2912
6AM - Noon
-0.13|
0.10 |
-0.02
-0.04
0.04
NA
0.0275 *
-0.22 (1 <2) 0.0136
# Cells contain all pairwise comparisons that are statistically 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<6)" indicates that the mean baseline adjusted value
for Group 6 was significantly greater than that for Group 1], and (3) the Tukey-adjusted p-value.
t, | Indicate that the mean baseline adjusted value was significantly different from zero (at the 0.05 level), "t" indicates that the mean at the study time was
greater than that at baseline, while "J," indicates that the mean at the study time was less than that at baseline.
* Group effect was significant at the 0.05 level.
NA Data not available for this Group at this study time.
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
Group
Number
Abnormal/ N
Proportion Abnormal
(95% Confidence Interval)
Mean Duration of
Abnormal (Days)#
Fisher's Group
Effect P-Value
1
0/5
0.00 (0.00, 0.52)
NA
2
1/5
0.20 (0.01, 0.72)
0.00
Activity
3
0/5
0.00 (0.00, 0.52)
NA
0.0558
4
0/5
0.00 (0.00, 0.52)
NA
5
3/5
0.60 (0.15, 0.95)
0.00
6
0/5
0.00 (0.00, 0.52)
NA
1
4/5
0.80 (0.28, 0.99)
8.69
2
4/5
0.80 (0.28, 0.99)
11.63
Heart Rate
3
3/5
0.60 (0.15, 0.95)
0.42
1.0000
4
4/5
0.80 (0.28, 0.99)
8.81
5
4/5
0.80 (0.28, 0.99)
1.31
6
4/5
0.80 (0.28, 0.99)
1.31
1
2/5
0.40 (0.05, 0.85)
8.38
2
4/5
0.80 (0.28, 0.99)
2.75
RP Expiratory
3
4/5
0.80 (0.28, 0.99)
11.19
0.8253
Time
4
4/5
0.80 (0.28, 0.99)
3.75
5
3/5
0.60 (0.15, 0.95)
0.75
6
4/5
0.80 (0.28, 0.99)
1.25
1
2/5
0.40 (0.05, 0.85)
10.63
2
4/5
0.80 (0.28, 0.99)
5.38
RP Inspiratory
3
3/5
0.60 (0.15, 0.95)
6.00
0.4096
Time
4
2/5
0.40 (0.05, 0.85)
7.75
5
3/5
0.60 (0.15, 0.95)
2.08
6
5/5
1.00 (0.48, 1.00)
1.05
1
4/5
0.80 (0.28, 0.99)
14.94
2
3/5
0.60 (0.15, 0.95)
7.42
RP Integral
3
5/5
1.00 (0.48, 1.00)
4.20
0.0586
4
3/5
0.60 (0.15, 0.95)
2.75
5
1/5
0.20 (0.01, 0.72)
1.00
6
5/5
1.00 (0.48, 1.00)
1.10
1
4/5
0.80 (0.28, 0.99)
16.00
2
4/5
0.80 (0.28, 0.99)
7.44
RP Peak
3
4/5
0.80 (0.28, 0.99)
8.81
0.9079
Amplitude
4
3/5
0.60 (0.15, 0.95)
3.33
5
3/5
0.60 (0.15, 0.95)
0.58
6
5/5
1.00 (0.48, 1.00)
0.70
F-51
-------
TablelO. (Continued)
Parameter
Group
Number
Abnormal/ N
Proportion Abnormal
(95% Confidence Interval)
Mean Duration of
Abnormal (Days)#
Fisher's Group
Effect P-Value
1
2/5
0.40 (0.05, 0.85)
0.00
RP
Respiratory
Rate
2
4/5
0.80 (0.28, 0.99)
1.06
3
2/5
0.40 (0.05, 0.85)
5.38
0.2832
4
2/5
0.40 (0.05, 0.85)
2.25
5
3/5
0.60 (0.15, 0.95)
2.08
6
5/5
1.00 (0.48, 1.00)
1.50
1
4/5
0.80 (0.28, 0.99)
6.19
2
3/5
0.60 (0.15, 0.95)
4.17
Temperature
3
3/5
0.60 (0.15, 0.95)
1.67
0.8752
4
2/5
0.40 (0.05, 0.85)
3.63
5
4/5
0.80, (0.28, 0.99)
2.00
6
4/5
0.80 (0.28, 0.99)
2.06
# Means exclude those animals that were never abnormal.
NA There were no abnormal animlas for this group.
Table 11. Results of Overall Log-Rank Tests Comparing the Time to Abnormality Between
Groups by Parameter
Parameter
Group Effect
P-Value
Activity
0.0307 *
Heart Rate
0.2572
RP Expiratory Time
0.0083 *
RP Inspiratory Time
<0.0001 *
RP Integral
0.0002 *
RP Peak Amplitude
0.0388 *
RP Respiratory Rate
0.0252 *
Temperature
0.0259 *
* Group effect was significant at the 0.05 level.
F-52
-------
Table 12. Results of Pairwise Log-Rank Tests Comparing the Time to Abnormality Between Groups by Parameter
Parameter
Group
Pairwise Log-Rank Test P-Values
Unadjusted P-Value
Bonferroni-Holm Adjusted P-Value
Activity
0.3173
1.0000
1.0000
0.0486 *
1.0000
1.0000
1.0000
1.0000
0.7286
1.0000
0.3173
0.3173
0.3186
0.3173
1.0000
0.0486
1.0000
0.0486
1.0000
0.0584
1.0000
1.0000
1.0000
1.0000
1.0000
0.7286
1.0000
0.7286
1.0000
0.7286
RP
Expiratory
Time
0.0720
0.1599
0.1740
0.1662
0.0033
0.7917
1.0000
1.0000
1.0000
0.0495
0.6724
0.5276
0.4664
0.0033*
0.8701
0.8226
0.0615
~~~
~~
0.8226
0.0483
0.1086
1.0000
1.0000
1.0000
0.0495 *
7,
1.0000
1.0000
0.7379
1.0000
0.6278
1.0000
RP
Inspiratory
Time
0.1991
0.4713
0.7649
0.1955
0.0021
1.0000
1.0000
1.0000
1.0000
0.0309
0.6201
0.1435
0.4719
0.0021 *
0.6714
0.6249
0.0374
ttttt
m
0.2361
0.0021 *
T777777:.
1.0000
1.0000
1.0000
0.0309 *
0.0072
1.0000
1.0000
0.4110
1.0000
0.0309 *
0.0869
RP Integral
0.5784
0.9629
0.8285
0.2851
0.0021 *
1.0000
1.0000
1.0000
1.0000
0.0309 *
~
0.3589
0.9143
0.5822
0.0112
y
0.4661
0.2846
0.0021 *
2
~
1.0000
1.0000
1.0000
0.1345
0.4046
0.0391
~~
0.0072 *
1.0000
1.0000
0.0309 *
1.0000
0.4299
0.0941
RP Peak
Amplitude
0.5447
0.6820
0.5336
0.9896
0.1689
1.0000
1.0000
1.0000
1.0000
1.0000
~~~
0.8190
0.9885
0.2348
0.0018*
0.7704
0.5178
0.0377
0.6889
W///A
0.0471 *
0.0578
1.0000
1.0000
1.0000
0.0276 *
1.0000
1.0000
0.5280
1.0000
777m
0.6122
0.6935
F-53
-------
Table 12. (Continued)
TTTTT
0.1809
rTTTTT—
F77771
[TTTTT1
Pairwise Log-Rank Test P-Values
Parameter Group
Unadjusted P-Value
Bonferroni-Holm Adjusted P-Value
1.0000
0.0020 *
0.0303 *
Respiratory
0.0399*
0.4783
0.0128*
0.1668
0.0052 *
0.0727
0.8012
0.0070*
0.1056
Temperature
0.0907
0.9972
0.0572
0.8012
0.0572
0.8012
0.2257
0.0572
* Significant at the 0.05 level.
Table 13. Results of Overall Log-Rank Tests Comparing the Duration of Abnormality Between Groups by Parameter
Parameter
Group Effect P-Value
Activity
NA
Heart Rate
0.0278 *
RP Expiratory Time
0.0032 *
RP Inspiratory Time
0.0213*
RP Integral
0.1414
RP Peak Amplitude
0.4127
RP Respiratory Rate
0.0074 *
Temperature
0.2958
NA No animals had a duration of abnormality greater than zero days.
* Group effect was significant at the 0.05 level.
F-54
-------
Table 14. Results of Pairwise Log-Rank Tests Comparing the Duration of Abnormality Between Groups by Parameter
Parameter
Group
Pairwise Log-Rank Test P-Values
Unadjusted P-Value
Bonferroni-Holm Adjusted P-Value
Heart Rate
0.6787
0.0515
0.7419
0.6490
0.3393
1.0000
0.6178
1.0000
1.0000
0.0100
0.6361
0.2971
0.0423
0.0100*
0.2590
0.0515
0.2971
0.1161
0.8606
0.1493
1.0000
1.0000
0.1493
1.0000
1.0000
RP
Expiratory
Time
0.0772
0.5151
0.1036
0.0634
0.4795
0.7722
1.0000
0.9327
0.6974
0.0266 *
0.1585
0.4000
0.5904
0.0476
0.0101
0.6394
~~~
0.0101
0.9183
~~
~~
0.1897
0.3456
1.0000
1.0000
0.5709
0.1509
0.1509
RP
Inspiratory
Time
0.1051
0.2807
0.3173
0.4142
0.1095
1.0000
1.0000
1.0000
1.0000
0.5269
0.1051
0.5936
0.1435
0.2807
0.7055
0.1325
ttttt
m
0.4142
0.1095
T777777:.
1.0000
1.0000
1.0000
T,
0.1325
1.0000
1.0000
1.0000
RP
Respiratory
Rate
0.1138
0.0833
0.0833
0.0455 *
0.0143*
1.0000
0.9159
0.9159
0.6370
0.0520
0.0520
0.1986
0.2314
y
0.3173
0.4142
0.3431
0.6757
0.6757
1.0000
0.8864
0.8849
rTTTTT
0.8702
1.0000
1.0000
iiiM
1.0000
TTTTT
Significant at the 0.05 level.
F-55
-------
L23216
L23218
L23220
L23222
L23223
Days from Midnight of Challenge Day
Figure la. Plot of baseline adjusted activity (counts/minute) for each animal in Group 1.
OJ
13
(=
IZ
D
O
O
o
<
-O
CO
ij
<
CD
c
0
(/>
ro
m
¦L23206
¦ L23210
-L23211
-L23215
¦L23219
7 14 21
Days from Midnight of Challenge Day
28
Figure lb. Plot of baseline adjusted activity (counts/minute) for each animal in Group 2.
F-56
-------
7
CD
U
6
C
s
5
c
o
4
O
>
3
o
<
2
73
O
CO
1
3
"a
<
0
0
C
Q)
to
-1
ro
DO
-2
¦L23217
¦L23227
L23228
¦L23229
¦L23230
14
21
28
Figure lc.
Days from Midnight of Challenge Day
Plot of baseline adjusted activity (counts/minute) for each animal in Group 3.
50
CD
|,o
CO
c
ZJ
O
10
t) 20
<
"O
B
00
<
Q)
1 »
in
CO
00
-10
¦L23205
¦L23207
L23225
¦L23231
•L23235
14
21
28
Days from Midnight of Challenge Day
Figure Id. Plot of baseline adjusted activity (counts/minute) for each animal in Group 4.
F-57
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¦L23200
-L23201
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L23214
-L23234
14
21
28
Figure le.
Days from Midnight of Challenge Day
Plot of baseline adjusted activity (counts/minute) for each animal in Group 5.
.22
=3
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(1)
-40
to
CO -50
¦L23216
•L23218
¦L23220
L23222
•L23223
7 14 21
Days from Midnight of Challenge Day
28
Figure 2a. Plot of baseline adjusted heart rate (BPM) for each animal in Group 1.
Q)
03
Q1
n
co
CD
I
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3
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c
100
90
80
70
60
50
40
30
20
10
0
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-20
cd -30
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m
-50
-60
-70
-80
-L23206
L23210
L23211
-L23215
¦L23219
7 14 21
Days from Midnight of Challenge Day
28
Figure 2b. Plot of baseline adjusted heart rate (BPM) for each animal in Group 2.
F-59
-------
7j -20
(D -30
S -50
•L23217
¦L23227
¦L23228
L23229
•L23230
7 14 21
Days from Midnight of Challenge Day
28
Figure 2c. Plot of baseline adjusted heart rate (BPM) for each animal in Group 3.
L23205
L23207
L23225
L23231
L23235
d) -30
CC -40
7 14 21
Days from Midnight of Challenge Day
Figure 2d. Plot of baseline adjusted heart rate (BPM) for each animal in Group 4.
F-60
-------
L23200
L23201
L23212
L23214
L23234
Days from Midnight of Challenge Day
Figure 2e. Plot of baseline adjusted heart rate (BPM) for each animal in Group 5.
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110
100
90
80
70
60
50
40
30
20
10
0
-10
-20
-30
-40
-50
-60
-70
-80
-L23203
-L23204
L23213
¦L23221
-L23232
14
21
28
Days from Midnight of Challenge Day
Figure 2f. Plot of baseline adjusted heart rate (BPM) for each animal in Group 6.
F-61
-------
123216
L23218
L23220
L23222
L23223
1 ' ' ' ' ' ' 1 ' ' ' ' ' ' 1 r_
7 14 21
Days from Midnight of Challenge Day
Plot of baseline adjusted RP expiratory time (seconds) for each animal in
Group 1.
-L23206
-L23210
L23211
-L23215
¦L23219
Figure 3b. Plot of baseline adjusted RP expiratory time (seconds) for each animal in
Group 2.
Days from Midnight of Challenge Day
Figure 3a.
F-62
-------
L23217
L23227
L23228
L23229
L23230
-> 1 i ' . . . ' 1 1 ' i > T , , ¦—
7 14 21
Days from Midnight of Challenge Day
Plot of baseline adjusted RP expiratory time (seconds) for each animal in
Group 3.
-L23205
¦L23207
L23225
¦L23231
¦L23235
7 14 21 28
Days from Midnight of Challenge Day
Figure 3c.
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Figure 3d.
Plot of baseline adjusted RP expiratory time (seconds) for each animal in
Group 4.
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7 14 21
Days from Midnight of Challenge Day
¦L23200
-L23201
L23212
•L23214
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28
Figure 3e. Plot of baseline adjusted RP expiratory time (seconds) for each animal in
Group 5.
0 2
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L23213
¦L23221
¦L23232
fi
7 14 21
Days from Midnight of Challenge Day
28
Figure 3f. Plot of baseline adjusted RP expiratory time (seconds) for each animal in
Group 6.
F-64
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L23216
L23218
L23220
L23222
L23223
— 1 i ' i ' i ' 1 1 ' i 1 • ' 1
7 14 21
Days from Midnight of Challenge Day
Figure 4a. Plot of baseline adjusted RP inspiratory time (seconds) for each animal in
Group 1.
L23206
L23210
L23211
L23215
L23219
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L23217
L23227
L23228
L23229
L23230
a: 0 4
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8> 00
7 14 21
Days from Midnight of Challenge Day
Figure 4c. Plot of baseline adjusted RP inspiratory time (seconds) for each animal in
Group 3.
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cc
T3
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tn
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03
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-1
¦L23205
¦L23207
L23225
¦L23231
¦L23235
7 14 21
Days from Midnight of Challenge Day
28
Figure 4d. Plot of baseline adjusted RP inspiratory time (seconds) for each animal in
Group 4.
F-66
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¦L23200
-L23201
L23212
•L23214
¦L23234
7 14 21
Days from Midnight of Challenge Day
28
Figure 4e. Plot of baseline adjusted RP inspiratory time (seconds) for each animal in
Group 5.
0 6
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ra
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-10
-20
-30
¦L23216
L23218
L23220
¦L23222
¦L23223
7 14 21
Days from Midnight of Challenge Day
28
Figure 5a. Plot of baseline adjusted RP integral (niniHg-seconds) for each animal in
Group 1.
20
03
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tr
to 0
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-------
L23217
L23227
L23228
L23229
L23230
Days from Midnight of Challenge Day
Figure 5c. Plot of baseline adjusted RP integral (mniHg-seconds) for each animal in
Group 3.
L23205
L23207
L23225
L23231
L23235
Figure 5d. Plot of baseline adjusted RP integral (inmHg-seconds) for each animal in
Group 4.
Days from Midnight of Challenge Day
F-69
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-10
¦L23200
L23201
L23212
¦L23214
¦L23234
7 14 21
Days from Midnight of Challenge Day
28
Figure 5e. Plot of baseline adjusted RP integral (nnnHg-seconds) for each animal in
Group 5.
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¦L23203
¦L23204
L23213
•L23221
¦L23232
7 14 21
Days from Midnight of Challenge Day
28
Figure 5f. Plot of baseline adjusted RP integral (mmHg-seconds) for each animal in
Group 6.
F-70
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30
3 20
n
m
10
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-20
-30
-40
-L23206
¦ L23210
L23211
-L23215
¦ L23219
7 14 21
Days from Midnight of Challenge Day
28
Figure 6b. Plot of baseline adjusted RP peak amplitude (mmHg) for each animal in
Group 2.
F-71
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20
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¦L23205
•L23207
L23225
L23231
¦L23235
7 14 21
Days from Midnight of Challenge Day
28
Figure 6d. Plot of baseline adjusted RP peak amplitude (mmHg) for each animal in
Group 4.
F-72
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30
20
10
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e?
o
-M
03
I—
Q_
CO
00
-20
¦L23216
L23218
L23220
¦L23222
¦L23223
7 14 21
Days from Midnight of Challenge Day
28
Figure 7 a. Plot of baseline adjusted RP respiratory rate (RCPiM) for each animal in Group 1.
L23206
L23210
L23211
L23215
L23219
ro ?0
CD
f> -30
7 14 21
Days from Midnight of Challenge Day
Figure 7b. Plot of baseline adjusted RP respiratory rate (RCPM) for each animal in
Group 2.
F-74
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0
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0Q
-50
¦L23217
-L23227
-L23228
•L23229
¦L23230
7 14 21
Days from Midnight of Challenge Day
28
Figure 7c. Plot of baseline adjusted RP respiratory rate (RCPM) for each animal in
Group 3.
50
S 40
30
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10
-10
-40
¦L23205
-L23207
-L23225
¦L23231
-L23235
14
21
28
Days from Midnight of Challenge Day
Figure 7d. Plot of baseline adjusted RP respiratory rate (RCPM) for each animal in
Group 4.
F-75
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50
£ 40
30
10
a>
ro
CC
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co
a. 20
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8 -30
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¦L23200
L23201
L23212
¦L23214
¦L23234
V
14
21
28
Days from Midnight of Challenge Day
Figure 7e. Plot of baseline adjusted RP respiratory rate (RCPM) for each animal in
Group 5.
50
40
30
Q
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cc
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ct:
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L23216
L23218
L23220
L23222
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0.6
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0.2
0.1
0.0
-0.1
-0.2
-0.3
-0.4
-0.5
-0 6
7 14 21
Days from Midnight of Challenge Day
Figure 8a. Plot of baseline adjusted temperature (Celsius) for each animal in Group 1.
L23206
L23210
L23211
L23215
L23219
"O
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-o
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C
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m
0.5
0.4
0.3
0.2
0.1
0 0
-0.1
-0.2
-0.3
-0.4
-0.5
-0 6
7 14 21
Days from Midnight of Challenge Day
Figure 8b. Plot of baseline adjusted temperature (Celsius) for each animal in Group 2.
F-77
-------
L23217
L23227
L23228
L23229
L23230
(/>
2
CD
O
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0.3
0.2
0.1
0 0
-0.1
-0.2
-0.3
-0 4
-0.5
-0 6
7 14 21
Days from Midnight of Challenge Day
Figure 8c. Plot of baseline adjusted temperature (Celsius) for each animal in Group 3.
L23205
L23207
L23225
L23231
L23235
Days from Midnight of Challenge Day
Figure 8d. Plot of baseline adjusted temperature (Celsius) for each animal in Group 4.
co
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o
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3
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-8
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-11
-12
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F-78
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V)
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0 7 14 21 28
Days from Midnight of Challenge Day
Figure 8f. Plot of baseline adjusted temperature (Celsius) for each animal in Group 6.
F-79
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0)
3
C
c
3
o
O
30
20
Group 1
Group 2
Group 3
Group 4
Group 5
Group 6
o
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d)
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CD
C
a>
CO
CO
m
10
-101
14
21
28
Days from Midnight of Challenge Day
Figure 9. Plot of mean baseline adjusted activity (counts/minute) for each group.
70
60
50
0) 40
™ 30
20
10
0
-10
-20
-30
¦c
CO
(1)
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C
« -40
m -50
-60
-70
-80
Group 1
Group 2
Group 3
Group 4
Group 5
Group 6
Days from Midnight of Challenge Day
Figure 10. Plot of mean baseline adjusted heart rate (BPM) for each group.
F-80
-------
Group 1
Group 2
Group 3
Group 4
Group 5
Group 6
£•
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LU
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01
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0 5
0.4
0.3
0 2
0 1
0 0
-0.1
-0.2
"0'3|
— 1 . . 1 1 1 1 . . . . 1 1 r-
7 14 21
Days from Midnight of Challenge Day
Figure 11. Plot of mean baseline adjusted RP expiratory time (seconds) for each group.
0)
w
ro -0.2
DQ
Group 1
Group 2
Group 3
Group 4
Group 5
Group 6
Days from Midnight of Challenge Day
Figure 12. Plot of mean baseline adjusted RP inspiratory time (seconds) for each group.
F-81
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10
n
y
8
CD
7
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-1
CD
-2
-3
-4
Group 1
Group 2
Group 3
Group 4
Group 5
Group 6
7 14 21
Days from Midnight of Challenge Day
28
Figure 13. Plot of mean baseline adjusted RP integral (minHg-seconds) for each group.
G>
T5
3
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o
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CL
££
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Group 1
Group 2
Group 3
Group 4
Group 5
Group 6
Group 1
Group 2
Group 3
Group 4
Group 5
Group 6
Figure 15. Plot of mean baseline adjusted RP respiratory rate (RCPM) for each group.
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L_
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a:
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T3
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7 14 21
Days from Midnight of Challenge Day
40
30
20
10
0
-10
-20
-30^
7 14 21
Days from Midnight of Challenge Day
Figure 16. Plot of mean baseline adjusted temperature (Celsius) for each group.
F-83
-------
1.0
0.9
CO
0.8
o
0.7
c
.Q
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0.6
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0.5
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c
0.4
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Q.
0.3
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0.2
0.1
0.0
— Group 1
— Group 2
¦ - Group 3
• ¦ Group 4
— Group 5
- Group 6
7 14 21
Days From Midnight of Challenge Day
28
Figure 17. Kaplan-Meier curves for time to abnormality based on activity.
1.0
0.9
CO
0 8
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0.7
c
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<
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0.5
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c
0.4
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0.3
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0.2
0.1
0.0
1
4
L.
— Group 1
— - Group 2
¦ — Group 3
• • Group 4
— Group 5
- Group 6
7 14 21
Days From Midnight of Challenge Day
28
Figure 18. Kaplan-Meier curves for time to abnormality based on RP expiratory time.
F-84
-------
1.0
0.9
0 8
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o
0 7
c
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0 6
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>
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o
c
04
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0.3
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0.2
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00
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I1,
— Group 1
- - Group 2
- — Group 3
¦ ¦ Group 4
— Group 5
- Group 6
7 14 21
Days From Midnight of Challenge Day
28
Figure 19. Kaplan-Meier curves for time to abnormality based on RP inspiratory time.
1.0
0.9
15 0.8
O 0.7
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0.6
0.5
i
<= 0.4
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fc
o 0.3
o
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0.1
0.0 .
— Group 1
_ - Group 2
¦ - Group 3
- - Group 4
— Group 5
- Group 6
7 14 21
Days From Midnight of Challenge Day
28
Figure 20. Kaplan-Meier curves for time to abnormality based on RP integral.
F-85
-------
1.0
0.9
15 08
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c
n
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>-
z 05
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ITT
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1
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1
i.,
— Group 1
- - Group 2
- — Group 3
¦ ¦ Group 4
— Group 5
- Group 6
7 14 21
Days From Midnight of Challenge Day
28
Figure 21. Kaplan-Meier curves for time to abnormality based on RP peak amplitude.
1.0
0.9
CO
0 8
E
o
07
c
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<
06
d)
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Oh
o
c
04
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0.3
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0.2
0.1
00
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i
I
i
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Group 1
Group 2
Group 3
Group 4
Group 5
Group 6
7 14 21
Days From Midnight of Challenge Day
28
Figure 22. Kaplan-Meier curves for time to abnormality based on RP respiratory rate.
F-86
-------
1.0
0.9
0 8
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0 /
c
o
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0 6
0)
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0.6
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c
04
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0.3
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0.2
0.1
00
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— Group 1
— - Group 2
* — Group 3
¦ ¦ Group 4
— Group 5
- Group 6
7 14 21
Days From Midnight of Challenge Day
28
Figure 23. Kaplan-Meier curves for time to abnormality based on temperature.
CO
£
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c
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tr
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Cl
0
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0.5
Group 1
Group 2
Group 3
— • ¦ Group 4
Group 5
Group 6
0.9
0 8
02
0.0
7 14
Duration of Abnormality (Days)
21
Figure 24. Kaplan-Meier curves for duration of abnormality based on heart rate.
F-87
-------
Group 1
Group 2
Group 3
Group 4
Group o
Group 6
7 14
Duration of Abnormality (Days)
Figure 25. Kaplan-Meier Curves for duration of abnormality based on RP expiratory time.
tr
-Q 0 6
O 0.3
Group 1
- Group 2
Group 3
• ¦ — ¦ • — ¦ • Group 4
Group 5
Group 6
7 14
Duration of Abnormality (Days)
21
Figure 26. Kaplan-Meier curves for duration of abnormality based on RP inspiratory time.
F-88
-------
1.0
0.9
0 8
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<
105
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0.1
0.0.
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I i i.
7 14
Duration of Abnormality (Days)
— Group 1
— Group 2
¦ — Group 3
¦ • Group 4
— Group 5
- Group 6
21
Figure 27. Kaplan-Meier curves for duration of abnormality based on RP respiratory rate.
F-89
-------
APPENDIX G
BLOOD DRAW TIMES
G-l
-------
9
to
Prepared By:.
1020-CG920503
Blood Draw Times
A = See DR# tfilG m'/< <->
a - Unable to obtain EDTA sample
P = Unable to obtain EDTA and SST samples
kih-
1
h)i
C"*\
QC/Tech Reviewed By: f I - 7" IQ
' f
QA Audit Comotop
8v/Date:
Animal ID
Group#
Day -3 Date
Day -3 Time
Day 1 Date
Day 1 Time
Day 2 Date
Day 2 Time
L23220
1
9/15/2009
1006
9/19/2009
0822
9/20/2009
0842
L23216
1
9/15/2009
0930
9/19/2009
0816
9/20/2009
0823
L23218
1
9/15/2009
0940
9/19/2009
0818
9/20/2009
0828
L23223
1
9/15/2009
1031
9/19/2009
0830
9/20/2009
0846
L23222
1
9/15/2009
1017
9/19/2009
0845
9/20/2009
0902
L23215
2
9/15/2009
0948
9/19/2009
0834
9/20/2009
0835
L23206
2
9/15/2009
1028
9/19/2009
0840
9/20/2009
0855
L23210
2
9/15/2009
1040
9/19/2009
0900
9/20/2009
0910
L23219
2
9/15/2009
1051
9/19/2009
0849
9/20/2009
0835
L23211
2
9/15/2009
1047
9/19/2009
0855
9/20/2009
0840
L23217
3
9/15/2009
1056
9/19/2009
0857
9/20/2009
0914
L23230
3
9/15/2009
1057
9/19/2009
0902
9/20/2009
0915
L23228
3
9/15/2009
1100
9/19/2009
0901
9/20/2009
0917
L23227
3
9/15/2009
1230
9/19/2009
0910
9/20/2009
0920
L23229
3
9/15/2009
1107
9/19/2009
0908
9/20/2009
0920
L23235
4
9/15/2009
1112
9/19/2009
1040
9/20/2009
1019
L2.3205
4
9/15/2009
1217
9/19/2009
1045
9/20/2009
1020
L23225
4
9/15/2009
1130
9/19/2009
1042
9/20/2009
1024
L23231
4
9/15/2009
1145
9/19/2009
1047
9/20/2009
1024
L23207
4
9/15/2009
1149
9/19/2009
1050
9/20/2009
1027
L23201
5
9/15/2009
1148
9/19/2009
1051
9/20/2009
1042
L23234
5
9/15/2009
1154
9/19/2009
1054
9/20/2009
1042
L23212
5
9/15/2009
1156
9/19/2009
1056
9/20/2009
1047
L23200
5
9/15/2009
1206
9/19/2009
1100
9/20/2009
1056
L23214
5
9/15/2009
1207
9/19/2009
1104
9/20/2009
1050
L23204
6
9/15/2009
1208
9/19/2009
1132
9/20/2009
1128
L23203
6
9/15/2009
1211
9/19/2009
1133
9/20/2009
1128
L23213
6
9/15/2009
P
9/19/2009
1138
9/20/2009
****a
L23221
6
9/15/2009
1224
9/19/2009
1209
J
L23232
6
9/15/2009
P
9/19/2009
1200
0/2009
1137 j
1/7/2010
'/c;
1 of 3
-------
1020-CG920503
Blood Draw Times
1/7/2010
Animal ID
Group#
Day 3 Date
Day 3 Time
Day 7 Date
Day 7 Time
Day 14 Date
Day 14 Time
L23220
1
9/21/2009
0912
9/25/2009
0903
10/2/2009
0945
L23216
1
9/21/2009
0855
9/25/2009
0908
10/2/2009
0934
L23218
1
9/21/2009
0905
9/25/2009
0912
10/2/2009
0951
L23223
1
9/21/2009
0928
9/25/2009
0917
10/2/2009
1000
L2322.2
1
9/21/2009
0936
9/25/2009
0940
10/2/2009
0955
L23215
2
9/21/2009
0913
9/25/2009
0926
10/2/2009
1005
L23206
2
9/21/2009
1011
9/25/2009
0934
10/2/2009
1004
L23210
2
9/21/2009
0953
9/25/2009
1005
10/2/2009
1010
L23219
2
9/21/2009
0922
9/25/2009
0948
10/2/2009
1019
L23211
2
9/21/2009
0927
9/25/2009
1000
10/2/2009
1017
L23217
3
9/21/2009
0934
9/25/2009
1019
10/2/2009
1019
L23230
3
9/21/2009
0940
9/25/2009
1023
10/2/2009
1032
L23228
3
9/21/2009
0945
9/25/2009
1020
10/2/2009
1031
L23227
3
9/21/2009
0949
9/25/2009
1024
10/2/2009
1026
L23229
3
9/21/2009
0955
9/25/2009
1024
10/2/2009
1039
L23235
4
9/21/2009
1000
9/25/2009
1036
t—
L23205
4
9/21/2009
1143
9/25/2009
1033
I 10/2/2009
1105
L23225
4
9/21/2009
1007
:-S'
L23231
4
9/21/2009
1018
9/25/2009
1040
10/2/2009
1041
L23207
4
9/21/2009
1019
9/25/2009
1041
10/2/2009
1043
L23201
5
9/21/2009
1047
- -
L23234
5
9/21/2009
1048
L23212
5
9/21/2009
1128
9/25/2009
105
10/2/2009
105-- " 1
L23200
5
9/21/2009
L23214
5
9/21/2009
: ] ici
12.3204
6
9/21/2009
1132
L23203
6
9/21/2009
1137
L23213
6
9/21/2009
P
L23221
6 !
6
9/21/2009
1154 |
Not Applicable
Prepared By: >hJ/P
QC/Tech Reviewed By.
'Mi
No time recorded for sample, see DR # 8V?Co !M>e-
A = See DR# ^ ^ L' I n t>u
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p = Unable to obtain EDTA and SST samples
QA Audit Ccipptetsc
Bv/Date: //
2 of 3
-------
9
Prepared Bv: .-n*- tHh*
V
QC/Tech Reviewed By:
1020-CG920503
Blood Draw Times
1/7/2010
Animal ID
Group#
Day 21 Date
Day 21 Time
Terminal Date
Terminal Time
L23220
1
10/9/2009
0920
NA
NA
L23216
1
10/9/2009
0918
NA
NA
L23218
1
10/9/2009
0926
NA
NA
L23223
1
10/9/2009
0937
NA
NA
L23222
1
10/9/2009
0945
NA
NA
L23215
2
10/9/2009
0952
NA
NA
L23206
2
10/9/2009
1002
NA
NA
L23210
2
10/9/2009
1003
NA
NA
L23219
2
10/9/2009
1011
NA
NA
L23211
2
10/9/2009
1016
NA
NA
L23217
3
10/9/2009
1022
NA
NA
L23230
3
10/9/2009
1042
NA
NA
L23228
3
10/9/2009
1040
NA
NA
123227
3
10/9/2009
1058
NA
NA
L23229
3
10/9/2009
1112
NA
NA
L23235
4
9/29/2009
0822
L23205
4
: ''79/2009
1132
NA
NA
L23225
4
9/22/2009
1443
L23231
4
10/9/2009
112!)
NA
NA
L23207
4
10/9/2009
1119
NA
NA
L23201
5
9/22/2009
Q838A
L23234
5
1 9/24/2009
0901
L23212
5
: 0/9/2009
1136m |
NA
NA
L23200
5
1 9/21/2009
1116
L23214
5
I 9/24/2009
0907
L23204
6
9/22/2009
1506
L23203
6
9/23/2009
* * *
L23213
6
9/21/2009
1252
L23221
6
9/20/2009
* * # *
L23232
6
1 9/22/2009
0900A
Not Applicable
A = See DR# f:H1 (* -M <- *&
a = Unable to obtain EDTA sample
P = Unable to obtain EDTA and SST samples
, hi-1ik
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8v/Date:
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3 of 3
-------
APPENDIX H
FINAL REPORT ON STATISTICAL ANALYSIS OF
PA-ELISA, QPCR, AND QUANTITATIVE
BACTEREMIA DATA
H-l
-------
Table of Contents
1. Introduction H-4
2. Statistical Methods H-5
3. Results H-6
4. Conclusions H-8
List of Tables
Table 1. Study Design H-4
Table 2. Descriptive Statistics and Test Results for PA-ELISA (ng/mL) by Group and Study
Day 11-9
Table 3. Descriptive Statistics and Test Results for Quantitative Bacteremia (CFU/mL) by
Group and Study Day H-13
H-2
-------
List of Acronyms
BBRC Battelle Biomedical Research Center
BD Below detection limit
CFU Colony forming unit
DNA Deoxyribonucleic acid
LD50 Median lethal dose
LOD limit of detection
LOQ limit of quantification
N Number of animals
PA-ELISA Protective antigen enzyme-linked immunosorbent assay
qPCR Quantitative real-time polymerase chain reaction
H-3
-------
1. Introduction
This report summarizes the statistical analysis of survival data collected under Battelle
Biomedical Research Center (BBRC) Study No. 1020-CG920503. Thirty (30) male pathogen-
free New Zealand White rabbits (Oryctolagus cuniculus) were randomized into six Groups, with
each Group having five animals. Animals were aerosol challenged on Study Day zero with
Bacillus anthracis (Ames strain) spores as indicated in Table 1. Blood samples were analyzed on
the Study Days listed in Table 1 for circulating protective antigen via the quantitative protective
antigen enzyme-linked immunosorbent assay (PA-ELISA), quantitative real-time polymerase
chain reaction (qPCR), and bacteremia via the quantitative spread plate techniques.
Table 1. Study Design
Group
Number of Animals
per Group
Spore Dose (CFU)
Blood Collection Study Days
1a
5
100 x LD50
2
5
100
3
5
1,000
-3, 1, 2, 3, 7, 14, 21, and
4
5
10,000
Terminal
5
5
100,000
6b
5
100 x LD50
CFU Colony forming units.
a Spores are gamma-irradiated (negative control).
LD50 Median lethal dose,
b High dose control.
H-4
-------
2. Statistical Methods
All three parameters (PA-ELIS A, qPCR, and quantitative bacteremia) were log-transformed for
the statistical analyses. The limit of detection (LOD) for the PA-ELISA assay was 2 ng/mL. All
PA-ELISA measurements less than the LOD or recorded as "BD" (below detection limit) were
replaced with one half of the LOD. The LOD for the qPCR assay was two copies/(j,L of
deoxyribonucleic acid (DNA). All qPCR measurements equal to zero copies/|iL were replaced
with 0.5 copies/(j,L and all measurements recorded as "<2" were replaced with one half of the
LOD. The LOD and limit of quantification (LOQ) for the quantitative bacteremia assay were
100 CFU/mL and 2,500 CFU/mL, respectively. All quantitative bacteremia measurements equal
to zero CFU/mL were replaced with one half of the LOD. Furthermore, if an observation was
positive for Bacillus anthracis but was less than the LOQ, then it was replaced with one half of
the LOQ.
For each parameter (PA-ELISA, qPCR, and quantitative bacteremia), summary statistics
including geometric means with 95% confidence intervals were computed for each Group and
Study Day. Additionally, the proportion of PA-ELISA observations greater than the LOD, the
proportion of qPCR observations greater than zero, and the proportion of quantitative bacteremia
observations that were positive for Bacillus anthracis were computed for each Group and Study
Day. Furthermore, a t-test was performed to determine if the geometric mean was significantly
greater than the LOD for each parameter, Group, and Study Day. All analyses were performed in
SAS® (SAS Institute Inc.; Cary, NC; version 9.1) or Stata (StataCorp LP; College Station, TX;
version 11.1).
H-5
-------
3. Results
Tables 2, 3, and 4 contain summary statistics and test results for PA-ELISA, qPCR,
and quantitative bacteremia, respectively. For each parameter, Group, and Study Day, the
summary statistics include the sample size, the geometric mean with corresponding 95%
confidence interval, and the p-value associated with a t-test used to determine if the geometric
mean was significantly greater than the LOD. 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.
Additionally for each Group and Study Day, Table 2 contains the proportion of PA-ELISA
observations greater than the LOD, Table 3 contains the proportion of qPCR observations greater
than zero, and Table 4 contains the proportion of observations that were positive for quantitative
bacteremia. All animals in the high dose control Group (Group 6) died prior to Study Day 7.
For PA-ELISA, all animals in Groups 1 through 3 had observations less than the LOD on all
Study Days. One animal in Group 4 had an observation above the LOD on Study Day 3 and on
the terminal day. Some animals in Group 5 had observations greater than the LOD on Study
Days 2 and 3, while some animals in Group 6 had observations greater than the LOD on Study
Days 2 and 3 and on the terminal day. The geometric mean PA-ELISA measurements were not
significantly greater than the LOD for any Group on any Study Day.
For qPCR, one animal in each of Groups 1 and 2 had an observation greater than zero on Study
Days 1 and 3, respectively. All animals in Group 3 had observations equal to zero on all Study
Days. One animal in Group 4 had observations greater than zero on Study Days -3 through 3.
Some animals in Groups 5 and 6 had observations greater than zero on Study Days 1, 2, and 3
and on the terminal day. The geometric mean qPCR measurements in Groups 5 and 6 were
significantly greater than the LOD at the terminal sample and Study Day 3, respectively.
For quantitative bacteremia, all animals in Groups 1 through 3 had observations negative for
Bacillus anthracis on all Study Days. At least one animal in Group 4 had observations that were
positive for Bacillus anthracis on Study Days 2 and 3 and on the terminal day. Some animals in
Groups 5 and 6 had observations that were positive for Bacillus anthracis on Study Days 1, 2,
and 3 and on the terminal day. The geometric mean quantitative bacteremia measurement in
Group 4 was significantly greater than the LOD at the terminal sample, and the geometric mean
H-6
-------
quantitative bacteremia measurement in Group 6 was significantly greater than the LOD on
Study Day 3 and at the terminal sample.
H-7
-------
4. Conclusions
All animals in the high dose control Group (Group 6) died prior to Study Day 7. All animals in
the three lowest dose Groups (Groups 1 through 3) had PA-ELISA observations less than the
LOD, and quantitative bacteremia observations negative for Bacillus anthracis on all Study
Days. The geometric mean PA-ELISA measurements were not significantly greater than the
LOD for any Group on any Study Day. The geometric mean qPCR measurements were
significantly greater than the LOD in the 100,000 colony forming unit (CFU) dose Group
(Group 5) at the terminal sample and in the high dose control Group (Group 6) on Study Day 3.
The geometric mean quantitative bacteremia measurement in the 10,000 CFU dose Group
(Group 4) was significantly greater than the LOD at the terminal sample, and the geometric mean
quantitative bacteremia measurement in the high dose control Group (Group 6) was significantly
greater than the LOD on Study Day 3 and at the terminal sample.
H-8
-------
Table 2. Descriptive Statistics and Test Results for PA-ELISA (ng/mL) by Group and
Study Day
Group
Study Day
N
Proportion Greater
Than LOD
Geometric Mean
(95% Confidence Interval)
P-Valuea
-3
5
0.00
-------
Table 2. (Continued)
Group
Study Day
N
Proportion
Greater
Than LOD
Geometric Mean
(95% Confidence Interval)
P-Valuea
-3
5
0.00
-------
Table 3. Descriptive Statistics and Test Results for Quantitative PCR (qPCR, copies/jiL of
DNA) by Group and Study Day
Group
Study Day
N
Proportion Greater
Than Zero
Geometric Mean
(95% Confidence Interval)
P-Valuea
-3
5
0.00
-------
Table 3. (Continued)
Group
Study Day
N
Proportion
Greater
Than Zero
Geometric Mean
(95% Confidence Interval)
P-Valuea
-3
5
0.00
-------
Table 4. Descriptive Statistics and Test Results for Quantitative Bacteremia (CFU/mL) by
Group and Study Day
Group
Study
Day
N
Proportion Positive
for Bacillus anthracis
Geometric Mean
(95% Confidence Interval)
P-Valuea
-3
5
0.00
-------
Table 4. (Continued)
Group
Study Day
N
Proportion Positive
for Bacillus anthracis
Geometric Mean
(95% Confidence Interval)
P-Valuea
-3
5
0.00
-------
APPENDIX I
STATISTICAL ANALYSIS OF HEMATOLOGY DATA
1-1
-------
Table of Contents
1. Introduction 1-7
2. Statistical Methods 1-9
3. Results 1-12
2.1. Red blood cell parameters 1-13
2.2. Platelet counts and volume 1-15
2.3. Total and differential white blood cell parameters 1-15
4. Conclusions 1-19
5. Hematology Parameter 1-20
6. Group Effect P-Value 1-20
Attachment 1 1-1
1-2
-------
List of Tables
Table 1. Study Design 1-7
Table 2. List of Potential Hematology Outliers 1-20
Table 3. Summary of ANOVA Results for Baseline Data (Study Day -3) 1-20
Table 4a. Descriptive Statistics for Red Blood Cell Count (RBC, 106 cells/|iL), by
Group and Study Day 1-21
Table 4b. Test Results for Red Blood Cell Count (RBC, 106 cells/|iL) 1-22
Table 5a. Descriptive Statistics for Hemoglobin (HGB, g/dL) by Group and Study Day. ...1-23
Table 5b. Test Results for Hemoglobin (HGB, g/dL) 1-24
Table 6a. Descriptive Statistics for Hematocrit (HCT, %) by Group and Study Day 1-25
Table 6b. Test Results for Hematocrit (HCT, %) 1-26
Table 7a. Descriptive Statistics for Mean Corpuscular Volume (MCV, fL) by Group
and Study Day 1-27
Table 7b. Test Results for Mean Corpuscular Volume (MCV, fL) 1-28
Table 8a. Descriptive Statistics for Mean Corpuscular Hemoglobin (MCH, pg) by
Group and Study Day 1-29
Table 8b. Test Results for Mean Corpuscular Hemoglobin (MCH, pg) 1-30
Table 9a. Descriptive Statistics for Mean Corpuscular Hemoglobin
Concentration (MCHC, g/dL) by Group and Study Day 1-31
Table 9b. Test Results for Mean Corpuscular Hemoglobin Concentration
(MCHC, g/dL) 1-32
Table 10a. Descriptive Statistics for Red Cell Distribution Width (RDW, %) by Group
and Study Day 1-33
Table 10b. Test Results for Red Cell Distribution Width (RDW, %) 1-34
Table 11a. Descriptive Statistics for Platelet Count (PLT, 103 cells/|iL) by Group
and Study Day 1-35
Table 1 lb. Test Results for Platelet Count (PLT, 103 cells/|iL) 1-36
Table 12a. Descriptive Statistics for Mean Platelet Volume (MPV, fL) by Group
and Study Day 1-37
Table 12b. Test Results for Mean Platelet Volume (MPV, fL) 1-38
Table 13a. Descriptive Statistics for White Blood Cell Count (WBC, 103 cells/|iL) by
Group and Study Day 1-39
Table 13b. Test Results for White Blood Cell Count (WBC, 103 cells/|iL) 1-40
"3
Table 14a. Descriptive Statistics for Neutrophils (10 cells/|iL) by Group and Study
I- 3
-------
Day 1-41
Table 14b. Test Results for Neutrophils (103 cells/|iL) 1-42
Table 15a. Descriptive Statistics for Lymphocytes (10 cells/|iL) by Group and
Study Day 1-43
Table 15b. Test Results for Lymphocytes (103 cells/|iL) 1-44
Table 16a. Descriptive Statistics for Neutrophils/Lymphocytes Ratio by Group and
Study Day 1-45
Table 16b. Test Results for Neutrophils/Lymphocytes Ratio 1-46
"3
Table 17a. Descriptive Statistics for Monocytes (10 cells/|iL) by Group and Study Day 1-47
Table 17b. Test Results for Monocytes (103 cells/|iL) 1-48
"3
Table 18a. Descriptive Statistics for Eosinophils (10 cells/|iL) by Group and Study
Day 1-49
Table 18b. Test Results for Eosinophils (103 cells/|iL) 1-50
"3
Table 19a. Descriptive Statistics for Basophils (10 cells/|iL) by Group and Study Day 1-51
Table 19b. Test Results for Basophils (103 cells/|iL) 1-52
Table 20. Proportion of Animals that were Abnormal and with Exact 95% Confidence
Interval by Parameter and Group 1-53
Table 21. Results of Overall Two-sided Fisher's Exact Tests for the Proportion of
Animals that were Abnormal by Parameter 1-55
Table 22. Results of One-sided Pairwise Fisher's Exact Tests for the Proportion of
Animals with Abnormal Platelet Counts 1-55
Table 23. Results of Overall Log-rank Tests for Time to Abnormality by Parameter 1-56
Table 24. Results of Pairwise Log-rank Tests for Time to Abnormal Platelet Count 1-56
Table 1-1. Test Results for Red Blood Cell Count (RBC, 106 cells/|iL) with Potential
Outliers Excluded 1-4
Table 1-2. Test Results for Hematocrit (HCT, %) with Potential Outliers Excluded 1-4
Table 1-3. Test Results for Mean Platelet Volume (MPV, fL) with Potential Outliers
Excluded 1-5
Table 1-4. Test Results for Neutrophils (103 cells/|iL) with Potential Outliers
Excluded 1-6
Table 1-5. Test Results for Neutrophils/Lymphocytes Ratio with Potential Outliers
Excluded 1-6
1-4
-------
List of Figures
Figure 1. Plot of Red Blood Cell Count over time 1-50
Figure 2. Plot of Hemoglobin over time 1-50
Figure 3. Plot of Hematocrit over time 1-51
Figure 4. Plot of Mean Corpuscular Volume (MCV) over time 1-51
Figure 5. Plot of Mean Corpuscular Hemoglobin (MCH) over time 1-52
Figure 6. Plot of Mean Corpuscular Hemoglobin Concentration (MCHC)
overtime 1-52
Figure 7. Plot of Red Cell Distribution Width (RDW) over time 1-53
Figure 8. Plot of Platelet Count over time 1-53
Figure 9. Plot of Mean Platelet Volume (MPV) over time 1-54
Figure 10. Plot of White Blood Cell Count over time 1-54
Figure 11. Plot of Neutrophils over time 1-55
Figure 12. Plot of Lymphocytes over time 1-55
Figure 13. Plot of Neutrophils/Lymphocytes (N/L) Ratio over time 1-56
Figure 14. Plot of Monocytes over time 1-56
Figure 15. Plot of Eosinophils over time 1-57
Figure 16. Plot of Basophils over time 1-57
Figure 17. Kaplan-Meier curves representing time to abnormal Platelet
Count for each Group 1-58
I- 5
-------
List of Acronyms
ANOVA Analysis of Variance
BBRC Battelle Biomedical Research Center
CFU Colony forming unit
dL deciliter
fL femotoliter
gram
HCT Hematocrit
HGB Hemoglobin
LD50 Median lethal dose
MCH Mean corpuscular hemoglobin
MCHC Mean corpuscular hemoglobin concentration
MCV Mean corpuscular volume
MPV Mean platelet volume
Number of animals
ratio Neutrophils/lymphocytes ratio
Platelet count
picogram
" red blood cell count
V red cell distribution width
C white blood cell count
microliter
micrometer
I- 6
-------
1 Introduction
This report summarizes the statistical analysis of hematology data collected under Battelle
Biomedical Research Center (BBRC) Study No. 1020-CG920503. Thirty (30) male
pathogen-free New Zealand White rabbits (Oryctolagus cunicuius) were randomized into six
Groups, with each Group having five animals. Animals were aerosol challenged on Study
Day zero with Bacillus cmthracis (Ames strain) spores as indicated in Table 1.
Table 1. Study Design
Group
Number of Animals
per Group
Spore Dose (CFU)
Hematology Blood
Collection Study Days
1a
5
100 x LD50
2
5
100
3
5
1,000
-3, 1,2, 3, 7, 14, 21, and
4
5
10,000
Terminal
5
5
100,000
6b
5
100 x LD50
CFU Colony forming units.
a Spores are gamma-irradiated (negative control),
b High-dose control.
LD50 Median lethal dose.
Blood samples were collected for hematology analysis as indicated in Table 1. Blood collection
on Study Day -3 served as a pre-challenge baseline for each animal. There were only
two animals (from different Groups) with terminal samples; therefore, all terminal measurements
were excluded from the statistical analysis.
The hematology parameters that were included in this analysis are:
• Red blood cell count (RBC, 106 cells/[xL)
• Hemoglobin (HGB, 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 (RDW, %)
1-7
-------
• Platelet count (PLT, 103 cells/[xL)
• Mean platelet volume (MPV, £L)
• White blood cell count (WBC, 103 cells/[xL)
• Neutrophils (103 cells/[j,L)
-3
• Lymphocytes (10 cells/[j,L)
• Monocytes (103 cells/[j,L)
• Eosinophils (10 cells/[xL)
• Basophils (103 cells/[j,L)
• Neutrophils/lymphocytes ratio (N/L ratio)
Hematology analysis was performed at six levels:
1. Descriptive statistics (arithmetic or geometric means with 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 in order 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.
5. For each parameter, the proportion abnormal in each Group was obtained and
evaluated to identify significant differences between the Groups.
6. For each parameter, the time to an abnormal measurement for each animal was
obtained and evaluated to identify significant differences between the Groups.
1-8
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2 Statistical Methods
Analysis of variance (ANOVA) models fitted separately to each hematology parameter 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 in order 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
by 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 in order 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, in order to evaluate their effect on the results.
For each hematology parameter, the following ANOVA model was fitted to the data at Study
Day -3 in order to determine if there were significant differences between the Groups at baseline:
Yi, = |i + Group, + By (1)
where Y(/ is the observed hematology result for the /th animal in Group i (/= 1 to 6) at the
baseline, |i is an overall constant, and zL, is the random error left unexplained by the model.
Tukey's multiple comparisons procedure was also performed for each parameter in order to
determine which pairs of Groups had baseline means that were significantly different from each
other; however, the results will only be presented if significant differences are identified. If the
1-9
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parameter was log-transformed for analysis, then the same model was used with Yy replaced by
Log(Yy), the base-10 log-transformed parameter value for the /th animal in Group i (/'= 1 to 6).
In order 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 on
each post-challenge Study Day:
Y6/y - Y/,y = (i + Group, + 8y (2)
where Yaj is the observed hematology result for the /th animal in Group i (i= 1 to 6) on Study
Day d (d= 1, 2, 3, 7, 14, and 21), Yuj is the observed hematology result for the /th animal in
Group i (/'= 1 to 6) at baseline, |i is an overall constant, and z,, 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 Ydy and Yuj replaced by their base-10 log-transformed counterparts Log(Y^) and Log(Yby),
respectively.
Least square mean estimates from the above shift from baseline ANOVA models were calculated
and approximate t-tests were performed to determine if, for each Group, there was a significant
shift in hematology results between baseline and each post-challenge Study Day. For
untransformed data, this tests whether the difference in 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.
For each parameter, the threshold for an abnormal parameter value was defined as each
individual animal's baseline (Study Day -3) parameter value plus or minus two standard
deviations. Since each animal had only one baseline value for each parameter, the standard
deviation associated with each parameter was calculated using the baseline values of all animals.
Animals were determined to have an abnormal parameter value when their observed value was
above the upper threshold or below the lower threshold for that respective parameter.
1-10
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For each parameter, an overall two-sided Fisher's exact test was performed to determine if there
was a significant difference between the abnormal rates in each Group. If the overall Fisher's
exact test was significant, then one-sided pairwise Fisher's exact tests were performed to
evaluate all pairwise Group comparisons. The Bonferroni-Holm adjustment was used to maintain
an overall 0.05 significance level among the multiple pairwise comparisons made within each
parameter.
For each parameter, an overall log-rank test was performed to determine if there was a
significant difference between the times to abnormality in each Group. If the overall log-rank test
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 significance
level among the multiple pairwise comparisons made within each parameter. For those
parameters that had significant overall log-rank test, Kaplan-Meier curves were plotted by
Group.
All statistical analyses were conducted using SAS® (SAS Institute Inc., Cary, NC, version 9.1)
and all results are reported at the 0.05 level of significance. All ANOVA models were fitted
using the MIXED or GLM procedure. All Fisher's exact tests were performed using the FREQ
procedure, and all log-rank tests were performed using the LIFETEST procedure. The
MULTTEST procedure was used to maintain an overall 0.05 significance level among the
multiple pairwise comparisons made within each parameter.
1-11
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3 Results
The model assumption of normality was more reasonable for 6 of the 16 parameters when
models were fitted to the base-10 log-transformed data. These parameters were: RDW, MPV,
N/L ratio, monocytes, eosinophils, and basophils. Therefore, models were fitted to base-10
log-transformed values in the final analysis for these parameters. Table 2 contains a list of nine
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 in order to evaluate their effect on the results. Attachment A contains 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 not significant at the baseline for any of the hematology parameters. This is important
to the interpretation of subsequent analysis results of differing shifts from baseline in the Groups.
The lack of differences between Groups at baseline implies that differing shifts from baseline for
different Groups are entirely associated with the effects of treatment after challenge and not with
inherent differences between the Groups prior to challenge.
Descriptive statistics, Group comparisons, and abnormal counts for each parameter are presented
in pairs of tables, where Tables 4 through 19 are associated with the parameter of interest. For
each parameter, Table "a" contains the descriptive statistics, Table "b" contains the test results
for comparing the mean shifts from baseline within each Group at each Study Day.
Tables 4a through 19a contain descriptive statistics (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 at each Study Day.
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 the means and
confidence intervals over the course of the study for each hematology parameter.
Tables 4b through 19b contain test results for the mean shift from baseline within each Group at
each Study Day. These tables contain test results that indicate if the mean difference between the
Study Day and baseline was significantly different from zero for untransformed parameters, or if
1-12
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the ratio of geometric means was significantly different from one for base-10 log-transformed
parameters (at the 0.05 level). In each cell, the estimate of the shift from baseline (difference or
ratio) is shown for that parameter, Group, and 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 Group effect p-values for each
Study Day, as well as test results from the Tukey's pairwise comparisons procedure that was
used to identify pairs of Groups with significantly different shifts from baseline. Under the
Tukey procedure, the set of comparisons within each parameter and Study Day is made at a joint
95% confidence level. Each significant difference is shown as the estimated comparison between
the shifts from baseline for the pair of Groups under consideration, the direction of the
comparison (i.e., which Group experienced a larger shift from baseline), and the corresponding
Tukey-adjusted p-value. P-values less than 0.05 provide evidence of a significant difference.
The results from Tables 4b through 19b are discussed below in Groups of related parameters.
3.1 Red blood cell parameters
• RBC (Tables 4a-b, Figure 1): RBC levels were significantly decreased from
baseline for Group 1 on Study Day 2. Group 2 had significant decreases from
baseline on Study Days 2, 3, and 21. Group 4 had significant decreases from baseline
on Study Days 2 and 3. Group 5 had significant decreases from baseline on Study
Days 1 through 3. Group 6 had significant decreases from baseline on Study Days 2
and 3. There were no significant differences in the shifts from baseline for any pair of
Groups on any Study Day.
• HGB (Tables 5a-b, Figure 2): HGB levels were significantly decreased from
baseline for Group 1 on Study Days 2, 3, and 21. Group 2 had significant decreases
from baseline on Study Days 1 through 3 and 21. Group 3 had significant decreases
from baseline on Study Days 2, 3, and 21. Group 4 had a significant decrease from
baseline on Study Days 1 through 3 and on Study Day 21. Groups 5 and 6 had
significant decreases from baseline on Study Days 1 through 3. There were no
significant differences in the shifts from baseline for any pair of Groups on any Study
Day.
1-13
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HCT (Tables 6a-b, Figure 3): HCT levels were significantly decreased from
baseline for Groups 1 and 4 on Study Day 2. Group 2 had significant decreases from
baseline on Study Days 2 and 21. Group 3 had a significant decrease from baseline on
Study Day 21. Group 5 had significant decreases from baseline on Study Days 1
through 3. Group 6 had significant decreases from baseline on Study Days 2 and 3.
There were no significant differences in the shifts from baseline for any pair of
Groups on any Study Day.
MCV (Tables 7a-b, Figure 4): There were significant decreases from baseline for
Group 1 on Study Day 21. Groups 2 and 3 had a significant increase from baseline on
Study Day 7. Group 4 had significant decreases from baseline on Study Days 1, 2,
and 21. Group 5 had significant decreases from baseline on Study Days 1 through 3.
Group 6 had significant decreases from baseline on Study Days 2 and 3. There was a
significant Group effect on Study Day 2; however, Tukey's multiple pairwise
comparisons procedure did not identify significant differences between any pair of
Groups.
MCH (Tables 8a-b, Figure 5): There were significant decreases from baseline for
Groups 1, 3, and 4 on all Study Days. Group 2 had significant decreases from
baseline on Study Days 1 through 3 and Study Day 14. Group 5 had significant
decreases from baseline on Study Days 1 through 3 and on Study Day 14. Group 6
had significant decreases from baseline on Study Days 1 through 3. There were no
significant differences in the shifts from baseline for any pair of Groups on any Study
Day.
MCHC (Tables 9a-b, Figure 6): MCHC levels were significantly decreased from
baseline for Groups 1 and 2 on Study Days 1 through 7. Groups 3 and 4 had
significant decreases from baseline on Study Days 1 through 14. Group 5 and 6 had
significant decreases from baseline on Study Day 1. There were no significant
differences in the shifts from baseline for any pair of Groups on any Study Day.
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• RDW (Tables lOa-b, Figure 7): RDW levels were significantly decreased as a
proportion of baseline for Group 1 on Study Day 21. Group 2 had significant
increases as a proportion of baseline on Study Days 2 through 7 and a significant
decrease as a proportion of baseline on Study Day 21. Group 3 had significant
increases as a proportion of baseline on Study Days 3 and 7 and a significant decrease
as a proportion of baseline on Study Day 21. Group 4 had significant increases as a
proportion of baseline on Study Days 3 and 7. On Study Day 3, the mean increase as
a proportion of baseline for Group 2 was significantly different than the mean
decrease as a proportion of baseline for Group 6.
Platelet counts and volume
• PLT (Tables lla-b, Figure 8): PLT levels were significantly decreased for Group 1
on Study Days 3 and 14. Group 2 had a significant decrease from baseline on Study
Day 14. Group 3 had a significant decrease from baseline on Study Day 21. Group 5
had significant decreases from baseline on Study Days 2, 3, and 21. Group 6 had a
significant decrease from baseline on Study Day 2. There were no significant
differences in the shifts from baseline for any pair of Groups on any Study Day.
• MPV (Tables 12a-b, Figure 9): There were significant decreases as a proportion of
baseline for Group 1 on Study Days 1, 2, 7, and 21. Groups 2, 3, and 4 had significant
decreases as a proportion of baseline on Study Days 1, 2, and 21. Group 5 had
significant decreases as a proportion of baseline on Study Days 1 and 2. Group 6 had
significant decreases as a proportion of baseline on Study Days 1 and 2 and a
significant increase as a proportion of baseline on Study Day 3. On Study Day 3, the
mean increase as a proportion of baseline for Group 6 was significantly different than
the mean changes as a proportion of baseline for Groups 1, 2, 3 and 4. On Study
Day 7, the mean decrease as a proportion of baseline for Group 1 was significantly
different than the mean increase as a proportion of baseline for Group 4.
Total and differential white blood cell parameters
• WBC (Tables 13a-b, Figure 10): WBC counts were significantly decreased for
Group 1 on all Study Days. Groups 2, 3, 4, and 5 had a significant decrease from
1-15
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baseline on Study Day 21. Group 6 had a significant decrease from baseline on Study
Day 3. There was a significant Group effect on Study Day 1; however, Tukey's
multiple pairwise comparisons procedure did not identify significant differences
between any pair of Groups. On Study Day 2, the mean decrease from baseline for
Group 1 was significantly different from the mean increase for Group 4. On Study
Day 3, the mean decrease from baseline for Group 6 was significantly different from
the mean increase for Group 5.
• Neutrophils (Tables 14a-b, Figure 11): Neutrophil counts were significantly
decreased for Group 1 on Study Days 1 through 3 and Study Days 14 and 21.
Groups 2 and 3 had a significant decrease from baseline on Study Day 21. Group 4
had a significant decrease from baseline on Study Day 21. Group 5 had a significant
decrease from baseline on Study Day 1. Group 6 had a significant increase from
baseline on Study Day 2. On Study Day 1 the mean decrease from baseline for
Group 1 was significantly different from the mean increase for Groups 4 and 6. On
Study Day 2,the mean increase from baseline for Group 6 was significantly different
from the mean change from baseline for Groups 1, 2, 3, 4, and 5. On Study Day 3 the
mean decrease from baseline for Group 1 was significantly different from the mean
increase for Group 5.
• Lymphocytes (Tables 15a-b, Figure 12): Lymphocyte counts were significantly
decreased for Group 1 on Study Days 14 and 21. Group 2 had significant decreases
from baseline on Study Days 7 and 21. Groups 3, 4, and 5 had a significant decrease
from baseline on Study Day 21. Group 6 had significant decreases from baseline on
Study Days 2 and 3. There was a significant Group effect on Study Day 2; however,
Tukey's multiple pairwise comparisons procedure did not identify significant
differences between any pair of Groups. On Study Day 3, the mean decrease from
baseline for Group 6 was significantly different from the mean increase for Groups 2,
3, 4, and 5.
• N/L Ratio (Tables 16a-b, Figure 13): There were significant decreases as a
proportion of baseline for Group 1 on Study Days 1, 3, 14, and 21. Group 2 had
1-16
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significant decreases as a proportion of baseline on Study Days 3 and 21. Group 4 had
a significant decrease as a proportion of baseline on Study Days 3 and 21. Group 5
had a significant decrease as a proportion of baseline on Study Day 1. Group 6 had a
significant increase as a proportion of baseline on Study Day 2. On Study Day 2, the
mean increase as a proportion of baseline for Group 6 was significantly different than
the mean decrease as a proportion of baseline for Groups 1, 2, 3, 4, and 5. On Study
Day 3, the mean decrease as a proportion of baseline for Group 1 was significantly
different than the mean increase as a proportion of baseline for Group 6.
• Monocytes (Tables 17a-b, Figure 14): There were significant decreases as a
proportion of baseline for Group 1 on Study Days 1, 2, and 21. Groups 2, 3, and 4 had
a significant decrease as a proportion of baseline on Study Day 21. Group 5 had a
significant decrease as a proportion of baseline on Study Day 3. Groups 6 had
significant decreases as a proportion of baseline on Study Days 2 and 3. On Study
Day 2, the mean decrease as a proportion of baseline for Group 6 was significantly
different than the mean increases as a proportion of baseline for Groups 2 and 3.
• Eosinophils (Tables 18a-b, Figure 15): There was a significant decrease as a
proportion of baseline for Group 1 on Study Day 21. Group 3 had significant
increases as a proportion of baseline on Study Days 1 and 7. Group 4 had a
significant increase as a proportion of baseline on Study Day 7. Group 5 had
significant increases as a proportion of baseline on Study Days 2 and 3. Group 6 had
a significant decrease as a proportion of baseline on Study Day 2. On Study Day 2,
the mean decrease as a proportion of baseline for Group 5 was significantly different
than the mean increase as a proportion of baseline for Group 3. On Study Day 7, the
mean increase as a proportion of baseline for Group 4 was significantly different than
the mean decrease as a proportion of baseline for Groupl.
• Basophils (Tables 19a-b, Figure 16): There were significant decreases as a
proportion of baseline for Group 1 on Study Days 7 through 21. Groups 2 and 3 had a
significant decrease as a proportion of baseline on Study Day 21. Group 4 had a
significant increase as a proportion of baseline on Study Day 7. Group 5 had a
1-17
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significant decrease as a proportion of baseline on Study Day 2 and a significant
increase as a proportion of baseline on Study Day 7. Group 6 had significant
increases as a proportion of baseline on Study Days 2 and 3. On Study Days 2 and 3,
the mean decrease as a proportion of baseline for Group 6 was significantly different
than the mean increase as a proportion of baseline for Group 3. On Study Day 7, the
mean decrease as a proportion of baseline for Group 1 was significantly different than
the mean increase as a proportion of baseline for Group 4.
Table 20 contains descriptive statistics associated with the proportion of animals that were
abnormal at any point during the study by Group. Table 21 contains the results of the overall
two-sided Fisher's exact tests for each parameter and Table 22 contains the pairwise Fisher's
exact tests comparing the abnormal rates for each pair of Groups with respect to those
parameters that had significant overall tests. The pairwise comparisons are not dependent upon
the ordering of the Groups being compared (e.g., comparing Group 1 to Group 2 is equivalent to
comparing Group 2 to Group 1); therefore, the cells in the lower left portion of Table 22 are
shaded out. A significant Group effect was identified for PLT, with the proportion of abnormal
animals in Group 5 being significantly greater than that in Group 4; however, the comparison
between Groups 4 and 5 was no longer significant after the Bonferroni-Holm adjustment for
multiple comparisons.
Table 23 contains the results of the overall log-rank tests for each parameter and Table 24
contains the results of the pairwise log-rank tests comparing the time to abnormality for each pair
of Groups with respect to those parameters that had significant overall tests. Again, the pairwise
comparisons are not dependent upon the ordering of the Groups being compared (e.g., comparing
Group 1 to Group 2 is equivalent to comparing Group 2 to Group 1); therefore, the cells in the
lower left portion of Table 24 are shaded out. A significant Group effect was identified for PLT.
For PLT, the times to abnormality in Groups 3, 4, and 6 were significantly greater than that in
Group 5 and the time to abnormality in Group 4 was also significantly greater than those in
Groups 1 and 2. After the Bonferroni-Holm adjustment for multiple comparisons, only the time
to abnormality in Group 4 was significantly greater than that in Group 5. Figure 17 displays the
Kaplan-Meier curves associated with time to abnormality for PLT in each Group.
1-18
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4 Conclusions
All animals in the high dose control Group (Group 6), and all but one animal in the 100,000
colony forming unit (CFU) Group (Group 5), died prior to Study Day 7. Among the red blood
cell parameters, MCH and MCHC had similar results in terms of significant shifts from baseline
with the lowest dose Groups (Groups 1 through 4) all experiencing significant decreases from
baseline on Study Days 1 through 3. Only RDW had a significant result in terms of the pairwise
Group comparisons, with the mean increase from baseline in the 100 CFU dose Group (Group 2)
being significantly different than mean decrease from baseline in the high dose control Group
(Group 6) on Study Day 3.
Among the platelet counts and volume, the mean shifts as a proportion of baseline for MPV were
significantly different between the Groups on Study Days 3 and 7. On Study Day 3, the mean
increase as a proportion of baseline in the high dose control Group (Group 6) was significantly
different than the mean changes as a proportion of baseline in the lowest dose Groups (Groups 1
through 4). On Study Day 7, the mean decrease as a proportion of baseline in the negative
control Group (Group 1) was significantly different than the mean change as a proportion of
baseline in the 10,000 CFU dose Group (Group 4). For PLT, the proportion of abnormal animals
in the 100,000 CFU dose Group (Group 5) was significantly greater than that in the 10,000 CFU
dose Group (Group 4); however, this comparison was no longer significant after the
Bonferroni-Holm adjustment for multiple comparisons. Also for PLT, the times to abnormality
in the 1,000 CFU and 10,000 CFU dose Groups (Groups 3 and 4) and in the high dose control
Group (Group 6) were significantly greater than that in the 100,000 CFU dose Group (Group 5),
and the time to abnormality in the 10,000 CFU dose Group (Group 4) was significantly greater
than those in the two lowest dose Groups (Groups 1 and 2). However, after the Bonferroni-Holm
adjustment for multiple comparisons, only the time to abnormality in the 10,000 CFU dose
Group (Group 4) was significantly greater than that in the 100,000 CFU dose Group (Group 5).
Among the white blood cell parameters, the mean increases from baseline in the high dose
control Group (Group 6) were significantly different than the mean changes from baseline in all
other Groups (Groups 1 through 5) on Study Day 2 for neutrophils and N/L ratio. For
lymphocytes, the mean decrease from baseline in the high dose control Group (Group 6) was
1-19
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significantly different than the mean changes from baseline in most of the lower dose Groups
(Groups 2 through 5) on Study Day 3.
Table 2. List of Potential Hematology Outliers
Parameter
Animal
Group
Study
Day
Parameter
Value
Deleted Studentized
Residual
Red Blood Cell Count
L23218
1
2
3.67
-4.979
Hemoglobin
L23218
1
2
7.60
-5.289
Hematocrit
L23218
1
2
23.80
-4.787
L23223
1
3
13.10
4.375
Mean Platelet Volume T
L23230
3
14
13.10
4.398
L23225
4
3
15.20
4.437
Neutrophils
L23212
5
3
1.06
-4.177
Neutrophils/Lymphocytes
L23200
5
1
0.03
-4.503
Ratio1
L23214
5
2
3.00
4.363
¦f 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 Data (Study Day -3)
Hematology Parameter
Group
Effect
P-Value
Red Blood Cell Count
0.4155
Hemoglobin
0.2456
Hematocrit
0.1475
Mean Corpuscular Volume
0.4034
Mean Corpuscular Hemoglobin
0.6842
Mean Corpuscular Hemoglobin
Concentration
0.5803
Red Cell Distribution Width T
0.3691
Platelet Count
0.3199
Mean Platelet Volume T
0.4595
White Blood Cell Count
0.5139
Neutrophils
0.2069
Lymphocytes
0.9845
Neutrophils/Lymphocytes Ratio T
0.7187
Monocytes T
0.2357
Eosinophils T
0.1076
Basophils T
0.8744
¦f Indicates that values for this parameter were log-transformed for the analysis.
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Table 4a. Descriptive Statistics for Red Blood Cell Count (RBC, 106 cells/jiL), by Group and Study Day
Group
Study
Day
N
Mean
(95% Confidence
Interval)
Group
Study
Day
N
Mean
(95% Confidence
Interval)
Group
Study
Day
N
Mean
(95% Confidence
Interval)
1
-3
5
5.80 (5.18, 6.42)
2
-3
5
6.27 (5.76, 6.78)
3
-3
5
6.02 (5.71, 6.32)
1
5
5.87 (5.66, 6.08)
1
5
6.06 (5.64, 6.48)
1
5
5.93 (5.54, 6.32)
2
5
5.24 (4.10, 6.38)
2
5
5.85 (5.59, 6.12)
2
5
5.68 (5.43, 5.93)
3
5
5.45 (4.93, 5.98)
3
4
5.85 (5.29, 6.42)
3
5
5.75 (5.55, 5.94)
7
5
5.83 (5.52, 6.13)
7
4
6.23 (5.33, 7.12)
7
5
6.22 (5.80, 6.64)
14
5
5.94 (5.38, 6.51)
14
5
6.29 (5.84, 6.74)
14
5
6.15 (5.69, 6.60)
21
5
5.79 (5.51, 6.08)
21
4
5.77 (5.18, 6.36)
21
5
5.72 (5.15, 6.28)
Group
Study
Day
N
Mean
(95% Confidence
Interval)
Group
Study
Day
N
Mean
(95% Confidence
Interval)
Group
Study
Day
N
Mean
(95% Confidence
Interval)
4
-3
5
5.87 (5.10, 6.64)
5
-3
5
6.05 (5.86, 6.24)
6
-3
3
5.66 (4.71, 6.61)
1
5
5.69 (5.20, 6.18)
1
5
5.77 (5.55, 5.99)
1
4
5.82 (5.41, 6.24)
2
5
5.46 (4.89, 6.04)
2
5
5.49 (5.22, 5.77)
2
3
5.22 (5.06, 5.38)
3
5
5.38 (4.74, 6.02)
3
3
5.34 (4.72, 5.96)
3
3
5.06 (4.38, 5.75)
7
4
5.69 (5.40, 5.98)
7
1
5.86 (--)
7
NA
NA
14
3
5.98 (4.90, 7.07)
14
1
6.07 (-)
14
NA
NA
21
3
5.46 (4.92, 6.01)
21
1
5.90 (-)
21
NA
NA
N Number of animals.
Confidence interval could not be calculated since only one observation was available for this Group on this Study Day.
NA Data not available for this Group at this Study Day.
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Table 4b. Test Results for Red Blood Cell Count (RBC, 106 cells/jiL)
Red Blood Cell Count
Study
Day
Mean Shift from Baseline, by Group
Group Effect
P-Value
Estimated Difference
(Relationship) Tukey's P-Value #
1
2
3
4
5
6
1
0.07
-0.20
-0.09
-0.18
-0.28 |
-0.09
0.5495
2
-0.56 |
-0.41 |
-0.33
-0.41 |
-0.56 |
-0.68 |
0.7742
3
-0.34
-0.52 |
-0.27
-0.49 |
-0.72 |
-0.84 |
0.5576
7
0.03
-0.14
0.20
-0.03
-0.10
NA
0.7840
14
0.14
0.02
0.13
0.41
0.11
NA
0.7823
21
0.00
-0.60 |
-0.30
-0.11
-0.06
NA
0.1734
# Cells contain all significant pairwise 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.
1.1 "T" 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).
NA Data not available for this Group at this Study Day.
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Table 5a. Descriptive Statistics for Hemoglobin (HGB, g/dL) by Group and Study Day
Group
Study
Day
N
Mean
(95% Confidence
Interval)
Group
Study
Day
N
Mean
(95% Confidence
Interval)
Group
Study
Day
N
Mean
(95% Confidence
Interval)
1
-3
5
12.2 (11.3, 13.1)
2
-3
5
13.0 (12.5, 13.5)
3
-3
5
12.5 (12.0, 13.1)
1
5
11.8 (11.3, 12.3)
1
5
12.2 (11.6, 12.7)
1
5
12.0 (11.3, 12.7)
2
5
10.5 (8.4, 12.6)
2
5
11.8 (11.3, 12.3)
2
5
11.5 (11.0, 12.0)
3
5
11.0 (10.0, 11.9)
3
4
11.7 (10.9, 12.6)
3
5
11.6 (11.0, 12.2)
7
5
11.8 (11.3, 12.3)
7
4
12.7 (10.6, 14.7)
7
5
12.7 (12.1, 13.3)
14
5
12.0 (11.0, 13.1)
14
5
12.8 (12.1, 13.5)
14
5
12.5 (11.5, 13.5)
21
5
11.6 (11.1, 12.1)
21
4
11.6 (10.6, 12.5)
21
5
11.5 (10.6, 12.4)
Group
Study
Day
N
Mean
(95% Confidence
Interval)
Group
Study
Day
N
Mean
(95% Confidence
Interval)
Group
Study
Day
N
Mean
(95% Confidence
Interval)
4
-3
5
12.6 (11.4, 13.9)
5
-3
5
12.9 (12.3, 13.4)
6
-3
3
12.0 (10.6, 13.3)
1
5
11.7 (11.3, 12.2)
1
5
11.8 (11.4, 12.2)
1
4
11.7 (10.3, 13.1)
2
5
11.3 (10.4, 12.1)
2
5
11.2 (10.6, 11.8)
2
3
10.5 (9.1, 11.8)
3
5
11.2 (10.0, 12.4)
3
3
11.1 (9.7, 12.5)
3
3
10.3 (8.0, 12.5)
7
4
12.0 (11.1, 12.9)
7
1
12.4 (-)
7
NA
NA
14
3
12.5 (12.2, 12.9)
14
1
12.7 (-)
14
NA
NA
21
3
11.1 (10.3, 11.8)
21
1
12.3 (-)
21
NA
NA
N Number of animals.
Confidence interval could not be calculated since only one observation was available for this Group on this Study Day.
NA Data not available for this Group at this Study Day.
1-23
-------
Table 5b. Test Results for Hemoglobin (HGB, g/dL)
Hemoglobin
Study
Day
Mean Shift from Baseline, by Group
Group Effect
P-Value
Estimated Difference
(Relationship) Tukey's P-Value #
1
2
3
4
5
6
1
-0.4
-0.8 |
-0.6
-0.9 1
-1.1 1
-0.6 1
0.5533
2
-1-7 4
-1.2 |
-1.1 1
-1.4 1
-1.7 1
-1.8 1
0.6658
3
-1.2 4
-1.4 |
-0.9 1
-1.4 1
-2.0 1
-2.0 1
0.5408
7
-0.4
-0.5
0.1
-0.5
-0.5
NA
0.8298
14
-0.2
-0.2
0.0
0.5
-0.2
NA
0.7165
21
-0.6 |
-1.6 1
-1.0 i
-0.9 1
-0.6
NA
0.1997
# Cells contain all significant pairwise 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.
1.1 "T" 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).
NA Data not available for this Group at this Study Day.
1-24
-------
Table 6a. Descriptive Statistics for Hematocrit (HCT, %) by Group and Study Day
Group
Study
Day
N
Mean
(95% Confidence
Interval)
Group
Study
Day
N
Mean
(95% Confidence
Interval)
Group
Study
Day
N
Mean
(95% Confidence
Interval)
1
-3
5
36.8 (34.5, 39.1)
2
-3
5
39.4 (37.2, 41.7)
3
-3
5
37.7 (36.5, 38.9)
1
5
37.2 (35.0, 39.4)
1
5
38.2 (36.6, 39.7)
1
5
37.5 (34.5, 40.5)
2
5
33.2 (26.6, 39.7)
2
5
36.5 (34.2, 38.8)
2
5
35.6 (34.0, 37.2)
3
5
34.4 (30.8, 38.1)
3
4
36.7 (33.0, 40.3)
3
5
36.0 (33.8, 38.3)
7
5
37.3 (34.8, 39.8)
7
4
40.3 (33.1, 47.4)
7
5
40.5 (38.9, 42.1)
14
5
37.3 (33.4, 41.1)
14
5
39.6 (37.8, 41.4)
14
5
38.5 (34.9, 42.0)
21
5
35.7 (33.8, 37.6)
21
4
35.5 (31.8, 39.1)
21
5
35.2 (32.0, 38.4)
Group
Study
Day
N
Mean
(95% Confidence
Interval)
Group
Study
Day
N
Mean
(95% Confidence
Interval)
Group
Study
Day
N
Mean
(95% Confidence
Interval)
4
-3
5
38.1 (34.0, 42.1)
5
-3
5
39.4 (37.9, 41.0)
6
-3
3
36.1 (32.0, 40.2)
1
5
36.2 (34.9, 37.5)
1
5
36.9 (34.8, 39.1)
1
4
36.5 (32.2, 40.7)
2
5
34.8 (31.4, 38.2)
2
5
34.4 (32.2, 36.7)
2
3
32.0 (27.4, 36.6)
3
5
35.2 (30.3, 40.0)
3
3
33.6 (29.4, 37.7)
3
3
30.9 (25.3, 36.5)
7
4
38.2 (36.0, 40.4)
7
1
37.9 (-)
7
NA
NA
14
3
38.9 (37.3, 40.5)
14
1
38.5 (-)
14
NA
NA
21
3
34.1 (31.8, 36.3)
21
1
37.3 (-)
21
NA
NA
N Number of animals.
Confidence interval could not be calculated since only one observation was available for this Group on this Study Day.
NA Data not available for this Group at this Study Day.
1-25
-------
Table 6b. Test Results for Hematocrit (HCT, %)
Hematocrit
Study
Day
Mean Shift from Baseline, by Group
Group Effect
P-Value
Estimated Difference
(Relationship) Tukey's P-Value #
1
2
3
4
5
6
1
0.4
-1.3
-0.2
-1.9
-2.5 |
-1.0
0.3718
2
-3.6 |
-2.9 |
-2.1
-3.3 |
-5.0 |
-5.2 |
0.5290
3
-2.4
-3.0
-1.7
-2.9
-6.2 |
-6.3 |
0.3582
7
0.5
0.6
2.8
0.7
-1.0
NA
0.6659
14
0.5
0.2
0.8
2.8
-0.4
NA
0.7256
21
-1.1
-4.2 |
-2.5 |
-2.0
-1.6
NA
0.4060
# Cells contain all significant pairwise 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.
1.1 "T" 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).
NA Data not available for this Group at this Study Day.
1-26
-------
Table 7 a. Descriptive Statistics for Mean Corpuscular Volume (MCV, fL) by Group and Study Day
Group
Study
Day
N
Mean
(95% Confidence
Interval)
Group
Study
Day
N
Mean
(95% Confidence
Interval)
Group
Study
Day
N
Mean
(95% Confidence
Interval)
1
-3
5
63.7 (60.6, 66.8)
2
-3
5
63.0 (60.2, 65.7)
3
-3
5
62.8 (59.8, 65.8)
1
5
63.3 (60.0, 66.5)
1
5
63.1 (59.9, 66.4)
1
5
63.3 (60.0, 66.6)
2
5
63.4 (61.4, 65.5)
2
5
62.3 (59.3, 65.4)
2
5
62.7 (60.0, 65.3)
3
5
63.1 (59.6, 66.5)
3
4
62.6 (58.0, 67.2)
3
5
62.7 (60.2, 65.2)
7
5
64.1 (60.2, 68.0)
7
4
64.6 (61.2, 68.1)
7
5
65.2 (62.6, 67.9)
14
5
62.7 (60.3, 65.1)
14
5
63.0 (60.6, 65.4)
14
5
62.6 (60.1, 65.0)
21
5
61.6 (59.7, 63.6)
21
4
61.5 (58.4, 64.7)
21
5
61.7 (59.7, 63.7)
Group
Study
Day
N
Mean
(95% Confidence
Interval)
Group
Study
Day
N
Mean
(95% Confidence
Interval)
Group
Study
Day
N
Mean
(95% Confidence
Interval)
4
-3
5
65.0 (61.4, 68.5)
5
-3
5
65.2 (64.4, 66.0)
6
-3
3
63.9 (58.4, 69.4)
1
5
63.9 (60.0, 67.8)
1
5
64.0 (62.4, 65.6)
1
4
62.6 (59.5, 65.6)
2
5
63.7 (59.9, 67.5)
2
5
62.7 (61.3, 64.1)
2
3
61.4 (54.3, 68.5)
3
5
65.4 (60.7, 70.0)
3
3
62.9 (59.8, 65.9)
3
3
61.1 (56.3, 65.8)
7
4
67.1 (62.5, 71.8)
7
1
64.7 (-)
7
NA
NA
14
3
65.2 (56.2, 74.2)
14
1
63.5 (-)
14
NA
NA
21
3
62.4 (52.0, 72.9)
21
1
63.2 (--)
21
NA
NA
N Number of animals.
Confidence interval could not be calculated since only one observation was available for this Group on this Study Day.
NA Data not available for this Group at this Study Day.
1-27
-------
Table 7b. Test Results for Mean Corpuscular Volume (MCV, fL)
Mean Corpuscular Volume
Study
Day
Mean Shift from Baseline, by Group
Group Effect
P-Value
Estimated Difference
(Relationship) Tukey's P-Value #
1
2
3
4
5
6
1
-0.4
0.2
0.5
-1.1 |
-1.2 |
-0.8
0.0650
2
-0.2
-0.6
-0.1
-1.3 |
-2.5 |
-1-7 4
0.0485 *
3
-0.6
0.3
-0.1
0.4
-2.8 |
-2.0 |
0.0572
7
0.4
2.3 t
2.4 t
1.5
-0.7
NA
0.1821
14
-1.0
0.0
-0.2
0.3
-1.9
NA
0.4295
21
-2.1 |
-0.8
-1.1
-2.5 |
-2.2
NA
0.6018
# Cells contain all significant pairwise 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.
1.1 "T" 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.
NA Data not available for this Group at this Study Day.
1-28
-------
Table 8a. Descriptive Statistics for Mean Corpuscular Hemoglobin (MCH, pg) by Group and Study Day
Group
Study
Day
N
Mean
(95% Confidence
Interval)
Group
Study
Day
N
Mean
(95% Confidence
Interval)
Group
Study
Day
N
Mean
(95% Confidence
Interval)
1
-3
5
21.1 (20.2, 21.9)
2
-3
5
20.8 (19.9, 21.7)
3
-3
5
20.9 (19.7, 22.1)
1
5
20.1 (19.4, 20.7)
1
5
20.1 (19.4, 20.8)
1
5
20.2 (19.1, 21.3)
2
5
20.1 (19.4, 20.9)
2
5
20.1 (19.5, 20.8)
2
5
20.2 (19.4, 21.1)
3
5
20.2 (19.3, 21.0)
3
4
20.1 (19.5, 20.6)
3
5
20.3 (19.5, 21.1)
7
5
20.3 (19.4, 21.1)
7
4
20.4 (19.5, 21.3)
7
5
20.4 (19.5, 21.2)
14
5
20.3 (19.6, 20.9)
14
5
20.3 (19.8, 20.9)
14
5
20.3 (19.4, 21.2)
21
5
20.1 (19.5, 20.6)
21
4
20.1 (19.4, 20.9)
21
5
20.2 (19.3, 21.0)
Group
Study
Day
N
Mean
(95% Confidence
Interval)
Group
Study
Day
N
Mean
(95% Confidence
Interval)
Group
Study
Day
N
Mean
(95% Confidence
Interval)
4
-3
5
21.6 (20.3, 22.9)
5
-3
5
21.3 (20.6, 21.9)
6
-3
3
21.2 (18.7, 23.7)
1
5
20.7 (19.2, 22.2)
1
5
20.4 (19.9, 21.0)
1
4
20.1 (19.0, 21.2)
2
5
20.7 (19.3, 22.0)
2
5
20.3 (19.8, 20.8)
2
3
20.1 (18.0, 22.2)
3
5
20.9 (19.5, 22.3)
3
3
20.8 (20.5, 21.1)
3
3
20.3 (17.6, 22.9)
7
4
21.1 (19.1, 23.2)
7
1
21.2 (--)
7
NA
NA
14
3
21.0 (17.4, 24.5)
14
1
20.9 (--)
14
NA
NA
21
3
20.3 (16.9, 23.7)
21
1
20.9 (--)
21
NA
NA
N Number of animals.
Confidence interval could not be calculated since only one observation was available for this Group on this Study Day.
NA Data not available for this Group at this Study Day.
1-29
-------
Table 8b. Test Results for Mean Corpuscular Hemoglobin (MCH, pg)
Mean Corpuscular Hemoglobin
Study
Day
Mean Shift from Baseline, by Group
Group Effect
P-Value
Estimated Difference
(Relationship) Tukey's P-Value #
1
2
3
4
5
6
1
-1.0 4
-0.7 |
-0.8 |
-0.9 |
-0.8 |
-0.8 |
0.6054
2
-0.9 |
-0.7 |
-0.7 |
-0.9 |
-1.0 |
-0.7 |
0.5213
3
-0.9 |
-0.6 |
-0.6 |
-0.7 |
-0.9 |
-0.6 |
0.6367
7
-0.8 |
-0.3
-0.6 |
-0.7 |
-0.5
NA
0.4259
14
-0.8 |
-0.4 |
-0.6 |
-0.6 |
-0.8 |
NA
0.4576
21
-1.0 |
-0.5
-0.8 |
-1.3 |
-0.8
NA
0.5371
# Cells contain all significant pairwise 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.
1.1 "T" 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).
NA Data not available for this Group at this Study Day.
1-30
-------
Table 9a. Descriptive Statistics for Mean Corpuscular Hemoglobin Concentration (MCHC, g/dL) by Group and Study Day
Group
Study
Day
N
Mean
(95% Confidence
Interval)
Group
Study
Day
N
Mean
(95% Confidence
Interval)
Group
Study
Day
N
Mean
(95% Confidence
Interval)
1
-3
5
33.1 (32.5, 33.8)
2
-3
5
33.0 (31.8, 34.2)
3
-3
5
33.3 (32.5, 34.1)
1
5
31.8 (31.0, 32.6)
1
5
31.9 (31.2, 32.7)
1
5
32.0 (30.6, 33.3)
2
5
31.7 (31.1, 32.4)
2
5
32.3 (31.6, 33.0)
2
5
32.3 (31.7, 32.9)
3
5
32.0 (31.0, 33.0)
3
4
32.1 (30.6, 33.6)
3
5
32.4 (31.6, 33.2)
7
5
31.7 (30.8, 32.5)
7
4
31.6 (31.0, 32.1)
7
5
31.2 (30.1, 32.4)
14
5
32.3 (31.7, 33.0)
14
5
32.3 (31.7, 32.9)
14
5
32.5 (31.3, 33.6)
21
5
32.6 (32.1, 33.0)
21
4
32.7 (31.2, 34.2)
21
5
32.7 (31.9, 33.6)
Group
Study
Day
N
Mean
(95% Confidence
Interval)
Group
Study
Day
N
Mean
(95% Confidence
Interval)
Group
Study
Day
N
Mean
(95% Confidence
Interval)
4
-3
5
33.2 (32.8, 33.7)
5
-3
5
32.6 (31.9, 33.3)
6
-3
3
33.2 (32.1, 34.2)
1
5
32.4 (31.8, 33.0)
1
5
31.9 (30.8, 33.0)
1
4
32.2 (31.6, 32.7)
2
5
32.5 (31.6, 33.3)
2
5
32.4 (31.7, 33.1)
2
3
32.7 (32.4, 33.1)
3
5
32.0 (30.5, 33.5)
3
3
33.0 (31.4, 34.6)
3
3
33.2 (31.5, 34.9)
7
4
31.5 (29.8, 33.1)
7
1
32.7 (-)
7
NA
NA
14
3
32.1 (30.7, 33.5)
14
1
32.9 (-)
14
NA
NA
21
3
32.5 (32.5, 32.5)
21
1
33.0 (--)
21
NA
NA
N Number of animals.
Confidence interval could not be calculated since only one observation was available for this Group on this Study Day.
NA Data not available for this Group at this Study Day.
1-31
-------
Table 9b. Test Results for Mean Corpuscular Hemoglobin Concentration (MCHC, g/dL)
Mean Corpuscular Hemoglobin Concentration
Study
Day
Mean Shift from Baseline, by Group
Group Effect
P-Value
Estimated Difference
(Relationship) Tukey's P-Value #
1
2
3
4
5
6
1
-1.4 4
-1.1 4
-1.3 1
-0.8 1
-0.7 1
-0.9 1
0.4264
2
-1.4 4
-0.7 |
-1.0 1
-0.8 1
-0.3
-0.3
0.0904
3
-1.1 |
-1.1 |
-0.9 1
-1.3 1
0.0
0.2
0.1866
7
-1.5 4
-1.6 1
-2.1 1
-1.8 1
-0.5
NA
0.5186
14
-0.8
-0.7
-0.8 1
-1.1 1
-0.3
NA
0.9328
21
-0.6
-0.5
-0.6
-0.8
-0.2
NA
0.9873
# Cells contain all significant pairwise 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.
1.1 "T" 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).
NA Data not available for this Group at this Study Day.
1-32
-------
Table 10a. Descriptive Statistics for Red Cell Distribution Width (RDW, %) by Group and Study Day
Group
Study
Day
N
Geometric Mean
(95% Confidence
Interval)
Group
Study
Day
N
Geometric Mean
(95% Confidence
Interval)
Group
Study
Day
N
Geometric Mean
(95% Confidence
Interval)
1
-3
5
12.2 (11.3, 13.1)
2
-3
5
12.7 (12.2, 13.2)
3
-3
5
12.8 (12.3, 13.4)
1
5
12.2 (11.4, 13.1)
1
5
12.9 (12.0, 14.0)
1
5
12.9 (12.4, 13.4)
2
5
12.1 (11.4, 12.9)
2
5
13.1 (12.2, 14.0)
2
5
13.1 (12.5, 13.7)
3
5
12.3 (11.4, 13.3)
3
4
13.6 (12.5, 14.7)
3
5
13.5 (12.9, 14.1)
7
5
12.7 (12.4, 13.0)
7
4
13.7 (12.5, 15.0)
7
5
14.0 (13.4, 14.6)
14
5
11.8 (11.4, 12.2)
14
5
12.4 (11.9, 13.0)
14
5
12.6 (12.1, 13.2)
21
5
11.4 (11.0, 11.9)
21
4
11.6 (10.9, 12.3)
21
5
12.1 (11.7, 12.4)
Group
Study
Day
N
Geometric Mean
(95% Confidence
Interval)
Group
Study
Day
N
Geometric Mean
(95% Confidence
Interval)
Group
Study
Day
N
Geometric Mean
(95% Confidence
Interval)
4
-3
5
12.5 (11.9, 13.1)
5
-3
5
12.8 (12.2, 13.5)
6
-3
3
12.8 (11.5, 14.3)
1
5
12.6 (12.2, 12.9)
1
5
12.9 (12.1, 13.6)
1
4
12.9 (12.1, 13.8)
2
5
12.6 (12.1, 13.1)
2
5
13.0 (12.1, 13.8)
2
3
12.9 (11.7, 14.2)
3
5
12.9 (12.5, 13.3)
3
3
13.3 (11.6, 15.2)
3
3
12.7 (12.1, 13.4)
7
4
13.5 (12.5, 14.5)
7
1
13.8 (--)
7
NA
NA
14
3
12.4 (11.8, 13.1)
14
1
12.3 (--)
14
NA
NA
21
3
12.1 (11.6, 12.7)
21
1
12.0 (--)
21
NA
NA
N Number of animals.
Confidence interval could not be calculated since only one observation was available for this Group on this Study Day.
NA Data not available for this Group at this Study Day.
1-33
-------
Table 10b. Test Results for Red Cell Distribution Width (RDW, %)
t
#
t.4
NA
Red Cell Distribution Width +
Study
Day
Mean Shift as a Proportion of Baseline, by Group
Group Effect
P-Value
Estimated Ratio (Relationship)
Tukey's P-Value *
1
2
3
4
5
6
1
1.00
1.02
1.00
1.01
1.00
1.01
0.9304
2
0.99
1.03 t
1.02
1.01
1.01
1.00
0.2352
3
1.01
1.07 t
1.05 t
1.03 t
1.02
0.98
0.0227 *
0.92 (6<2) 0.0261
7
1.04
1.08 |
1.09 |
1.06 t
1.10
NA
0.6415
14
0.97
0.98
0.98
0.98
0.98
NA
0.9513
21
0.94 |
0.92 |
0.94 |
0.96
0.95
NA
0.4860
Indicates that values for this parameter were log-transformed for the analysis.
Cells contain all significant pairwise 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).
The overall Group effect was significant at the 0.05 level.
Data not available for this Group at this Study Day.
1-34
-------
Table 11a. Descriptive Statistics for Platelet Count (PLT, 103 cells/jiL) by Group and Study Day
Group
Study
Day
N
Mean
(95% Confidence
Interval)
Group
Study
Day
N
Mean
(95% Confidence
Interval)
Group
Study
Day
N
Mean
(95% Confidence
Interval)
1
-3
5
545 (331, 759)
2
-3
5
473 (312, 635)
3
-3
5
437 (341, 534)
1
5
628 (175, 1082)
1
5
472 (280, 664)
1
5
396 (348, 445)
2
5
540 (53, 1027)
2
5
397 (232,561)
2
5
355 (325, 384)
3
5
372 (15, 729)
3
4
422 (205, 638)
3
5
415 (373, 457)
7
5
484 (187, 782)
7
4
265 (5, 525)
7
5
353 (265, 441)
14
5
191 (0*, 445)
14
5
263 (68, 459)
14
5
284 (81,488)
21
5
473 (245, 702)
21
4
401 (49, 753)
21
5
284 (154, 413)
Group
Study
Day
N
Mean
(95% Confidence
Interval)
Group
Study
Day
N
Mean
(95% Confidence
Interval)
Group
Study
Day
N
Mean
(95% Confidence
Interval)
4
-3
5
457 (337, 577)
5
-3
5
580 (468, 692)
6
-3
3
493 (336, 649)
1
5
451 (261, 641)
1
5
504 (373, 634)
1
4
509 (404, 614)
2
5
403 (262, 544)
2
5
360 (206, 514)
2
3
249 (124, 373)
3
5
473 (342, 605)
3
3
307 (13, 601)
3
3
290 (0*, 749)
7
4
384 (144, 624)
7
1
251 (-)
7
NA
NA
14
3
524 (439, 610)
14
1
325 (-)
14
NA
NA
21
3
453 (442, 463)
21
1
170(-)
21
NA
NA
N Number of animals.
Confidence interval could not be calculated since only one observation was available for this Group on this Study Day.
* Lower bound of confidence interval set to zero since a negative parameter value is not possible.
NA Data not available for this Group at this Study Day.
1-35
-------
Table lib. Test Results for Platelet Count (PLT, 103 cells/jiL)
Platelet Count
Study
Day
Mean Shift from Baseline, by Group
Group Effect
P-Value
Estimated Difference
(Relationship)
Tukey's P-Value *
1
2
3
4
5
6
1
83
-1
-41
-6
-77
20
0.3600
2
-5
-77
-83
-54
-220 |
-237 |
0.1052
3
-173 |
-17
-23
16
-242 |
-196
0.1998
7
-61
-174
-85
-93
-175
NA
0.9493
14
-354 |
-210 |
-153
4
-101
NA
0.2296
21
-72
-38
-154 |
-67
-256 |
NA
0.3360
# Cells contain all significant pairwise 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.
1.1 "T" 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).
NA Data not available for this Group at this Study Day.
1-36
-------
Table 12a.Descriptive Statistics for Mean Platelet Volume (MPV, fL) by Group and Study Day
Group
Study
Day
N
Geometric Mean
(95% Confidence
Interval)
Group
Study
Day
N
Geometric Mean
(95% Confidence
Interval)
Group
Study
Day
N
Geometric Mean
(95% Confidence
Interval)
1
-3
5
8.4 (7.3, 9.7)
2
-3
5
8.1 (7.4, 8.9)
3
-3
5
8.2 (7.3, 9.3)
1
5
7.2 (6.4, 8.2)
1
5
7.1 (6.6, 7.7)
1
5
7.0 (6.5, 7.5)
2
5
6.8 (6.5, 7.0)
2
5
6.8 (5.9, 7.7)
2
5
6.3 (6.1, 6.4)
3
5
7.8 (5.3, 11.3)
3
4
6.9 (5.6, 8.4)
3
5
7.2 (6.4, 8.0)
7
5
6.8 (5.5, 8.4)
7
4
7.8 (6.7, 9.1)
7
5
8.2 (7.7, 8.7)
14
5
8.5 (6.2, 11.7)
14
5
7.2 (6.7, 7.8)
14
5
7.7 (5.3, 11.2)
21
5
6.5 (5.9, 7.2)
21
4
6.6 (5.8, 7.4)
21
5
6.6 (5.9, 7.4)
Group
Study
Day
N
Geometric Mean
(95% Confidence
Interval)
Group
Study
Day
N
Geometric Mean
(95% Confidence
Interval)
Group
Study
Day
N
Geometric Mean
(95% Confidence
Interval)
4
-3
5
8.0 (7.1, 8.9)
5
-3
5
7.5 (6.8, 8.3)
6
-3
3
8.2 (7.1, 9.5)
1
5
6.2 (5.8, 6.7)
1
5
6.4 (5.7, 7.1)
1
4
6.1 (5.5, 6.9)
2
5
6.6 (5.5, 8.0)
2
5
6.6 (6.0, 7.4)
2
3
6.9 (5.8, 8.1)
3
5
8.9 (6.1, 13.2)
3
3
9.2 (5.2, 16.3)
3
3
16.0 (8.2, 31.2)
7
4
8.2 (5.9, 11.5)
7
1
8.4 (-)
7
NA
NA
14
3
6.3 (5.1, 7.8)
14
1
7.6 (-)
14
NA
NA
21
3
5.7 (5.2, 6.2)
21
1
7.9 (-)
21
NA
NA
N Number of animals.
Confidence interval could not be calculated since only one observation was available for this Group on this Study Day.
NA Data not available for this Group at this Study Day.
1-37
-------
Table 12b. Test Results for Mean Platelet Volume (MPV, fL)
Mean Platelet Volume +
Study
Day
Mean Shift as a Proportion of Baseline, by Group
Group Effect
P-Value
Estimated Ratio
(Relationship)
Tukey's P-Value *
1
2
3
4
5
6
1
0.86 |
0.88 |
0.85|
0.78|
0.85 |
0.77 |
0.0642
2
0.81 |
0.83 |
0.76 |
0.83 |
0.88 |
0.86 |
0.3934
3
0.92
0.86
0.87
1.12
1.23
2.00 t
0.0002 *
0.46 (1 <6) 0.0007
0.43 (2<6) 0.0004
0.43 (3<6) 0.0003
0.56 (4<6) 0.0114
7
0.81 |
0.98
0.99
1.06
1.11
NA
0.0431 *
0.76 (1 <4) 0.0462
14
1.02
0.89
0.94
0.80
1.00
NA
0.5882
21
0.78 |
0.82 |
0.80 |
0.72 |
1.04
NA
0.2611
f Indicates that values for this parameter were log-transformed for the analysis.
# Cells contain all significant pairwise 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.
1.1 "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.
NA Data not available for this Group at this Study Day.
1-38
-------
Table 13a. Descriptive Statistics for White Blood Cell Count (WBC, 103 cells/jiL) by Group and Study Day
Group
Study
Day
N
Mean
(95% Confidence
Interval)
Group
Study
Day
N
Mean
(95% Confidence
Interval)
Group
Study
Day
N
Mean
(95% Confidence
Interval)
-3
5
8.05 (4.84, 11.26)
-3
5
6.77 (5.37, 8.16)
-3
5
7.20 (5.34, 9.07)
1
5
6.38 (4.00, 8.76)
1
5
6.45 (5.06, 7.83)
1
5
7.81 (5.95, 9.66)
2
5
5.55 (3.48, 7.62)
2
5
6.67 (5.31, 8.02)
2
5
7.73 (5.41, 10.05)
1
3
5
6.46 (3.39, 9.54)
2
3
4
6.57 (4.96, 8.17)
3
3
5
7.66 (5.08, 10.25)
7
5
5.94 (3.24, 8.65)
7
4
4.97 (3.56, 6.38)
7
5
6.48 (4.82, 8.15)
14
5
5.03 (2.13, 7.92)
14
5
5.70 (4.27, 7.13)
14
5
6.59 (3.97, 9.21)
21
5
4.14 (3.23, 5.05)
21
4
3.90 (1.98, 5.82)
21
5
4.40 (2.77, 6.04)
Group
Study
Day
N
Mean
(95% Confidence
Interval)
Group
Study
Day
N
Mean
(95% Confidence
Interval)
Group
Study
Day
N
Mean
(95% Confidence
Interval)
-3
5
6.78 (5.67, 7.89)
-3
5
8.31 (6.58, 10.03)
-3
3
6.84 (1.85, 11.83)
1
5
7.34 (6.58, 8.11)
1
5
7.69 (4.58, 10.79)
1
4
7.87 (4.56, 11.19)
2
5
7.59 (6.18, 9.00)
2
5
7.60 (2.94, 12.26)
2
3
7.51 (4.76, 10.27)
4
3
5
6.56 (4.36, 8.76)
5
3
3
10.10 (2.86, 17.35)
6
3
3
3.89 (0.00 *, 9.00)
7
4
7.99 (3.48, 12.51)
7
1
6.88 (--)
7
NA
NA
14
3
6.70 (2.60, 10.80)
14
1
5.25 (-)
14
NA
NA
21
3
3.61 (0.41, 6.80)
21
1
4.25 (-)
21
NA
NA
N Number of animals.
Confidence interval could not be calculated since only one observation was available for this Group on this Study Day.
* Lower bound of confidence interval set to zero since a negative parameter value is not possible.
NA Data not available for this Group at this Study Day.
1-39
-------
Table 13b. Test Results for White Blood Cell Count (WBC, 103 cells/jiL)
White Blood Cell Count
Study
Day
Mean Shift from Baseline, by Group
Group Effect
P-Value
Estimated Difference
(Relationship)
Tukey's P-Value *
1
2
3
4
5
6
1
-1.67|
-0.32
0.61
0.56
-0.62
0.57
0.0444 *
2
-2.50 |
-0.10
0.53
0.81
-0.71
0.23
0.0530
-3.31 (1<4) 0.0447
3
-1.59|
-0.13
0.46
-0.22
1.20
-3.40 |
0.0292 *
-4.60 (6<5) 0.0342
7
-2.11 |
-1.73
-0.72
0.97
-0.87
NA
0.2541
14
-3.03 |
-1.07
-0.61
-0.37
-2.50
NA
0.1939
21
-3.91 |
-2.79 |
-2.80 |
-3.47 |
-3.50 |
NA
0.5997
# Cells contain all significant pairwise 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.
1.1 "T" 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.
NA Data not available for this Group at this Study Day.
1-40
-------
Table 14a. Descriptive Statistics for Neutrophils (103 cells/jiL) by Group and Study Day
Group
Study
Day
N
Mean
(95% Confidence
Interval)
Group
Study
Day
N
Mean
(95% Confidence
Interval)
Group
Study
Day
N
Mean
(95% Confidence
Interval)
1
-3
5
2.50 (0.81, 4.20)
2
-3
5
1.73 (1.31, 2.15)
3
-3
5
1.64 (1.19, 2.09)
1
5
1.34 (0.38, 2.30)
1
5
1.48 (1.22, 1.73)
1
5
1.52 (1.07, 1.98)
2
5
1.30 (0.66, 1.93)
2
5
1.40 (0.99, 1.81)
2
5
1.46 (1.00, 1.92)
3
5
1.30 (0.48, 2.13)
3
4
1.08 (0.77, 1.39)
3
5
1.40 (0.93, 1.87)
7
5
1.50 (0.62, 2.39)
7
4
1.09 (0.80, 1.37)
7
5
1.44 (1.00, 1.89)
14
5
1.17 (0.22, 2.11)
14
5
1.34 (0.75, 1.94)
14
5
1.45 (0.69, 2.21)
21
5
0.91 (0.68, 1.14)
21
4
0.60 (0.38, 0.83)
21
5
0.75 (0.54, 0.96)
Group
Study
Day
N
Mean
(95% Confidence
Interval)
Group
Study
Day
N
Mean
(95% Confidence
Interval)
Group
Study
Day
N
Mean
(95% Confidence
Interval)
4
-3
5
1.80 (1.36, 2.23)
5
-3
5
2.59 (1.30, 3.89)
6
-3
3
1.68 (1.01, 2.35)
1
5
1.87 (1.04, 2.69)
1
5
1.94 (0.41, 3.47)
1
4
2.07 (1.67, 2.46)
2
5
1.99 (0.66, 3.32)
2
5
2.17 (0.23, 4.11)
2
3
4.37 (0.56, 8.18)
3
5
1.22 (0.33, 2.11)
3
3
3.48 (0.00*, 8.71)
3
3
1.28 (0.00*, 3.00)
7
4
2.32 (0.34, 4.30)
7
1
1.32 (--)
7
NA
NA
14
3
1.40 (1.12, 1.69)
14
1
0.82 (--)
14
NA
NA
21
3
0.57 (0.11, 1.03)
21
1
0.56 (--)
21
NA
NA
N Number of animals.
Confidence interval could not be calculated since only one observation was available for this Group on this Study Day.
* Lower bound of confidence interval set to zero since a negative parameter value is not possible.
NA Data not available for this Group at this Study Day.
1-41
-------
Table 14b. Test Results for Neutrophils (103 cells/jiL)
Neutrophils
Study
Day
Mean Shift from Baseline, by Group
Group Effect
P-Value
Estimated Difference
(Relationship)
Tukey's P-Value *
1
2
3
4
5
6
1
-1.16|
-0.25
-0.12
0.07
-0.66 |
0.29
0.0061 *
-1.23 (1<4) 0.0185
-1.45 (1<6) 0.0075
2
-1.21 |
-0.33
-0.18
0.19
-0.42
2.68 t
0.0002 *
-3.88 (1<6) <0.0001
-3.01 (2<6) 0.0011
-2.86 (3<6) 0.0019
-2.48 (4<6) 0.0077
-3.10 (5<6) 0.0008
3
-1.20|
-0.53
-0.24
-0.58
0.91
-0.41
0.0459 *
-2.12 (1<5) 0.0171
7
-1.00
-0.52
-0.20
0.61
0.06
NA
0.4099
14
-1.34|
-0.39
-0.19
-0.40
-0.44
NA
0.4795
21
-1.59|
-1.00 |
-0.89 |
-1.24 |
-0.70
NA
0.5883
# Cells contain all significant pairwise 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.
t, | "T" 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.
NA Data not available for this Group at this Study Day.
1-42
-------
Table 15a. Descriptive Statistics for Lymphocytes (103 cells/jiL) by Group and Study Day
Group
Study
Day
N
Mean
(95% Confidence
Interval)
Group
Study
Day
N
Mean
(95% Confidence
Interval)
Group
Study
Day
N
Mean
(95% Confidence
Interval)
-3
5
4.82 (1.93, 7.72)
-3
5
4.48 (3.37, 5.59)
-3
5
4.91 (3.40, 6.43)
1
5
4.40 (2.77, 6.03)
1
5
4.49 (3.29, 5.69)
1
5
5.57 (3.89, 7.25)
2
5
3.77 (2.00, 5.54)
2
5
4.75 (3.61, 5.89)
2
5
5.51 (3.61, 7.42)
1
3
5
4.63 (2.43, 6.83)
2
3
4
5.07 (3.32, 6.81)
3
3
5
5.54 (3.19, 7.90)
7
5
3.90 (2.05, 5.75)
7
4
3.42 (2.52, 4.32)
7
5
4.27 (3.08, 5.46)
14
5
3.42 (1.66, 5.18)
14
5
3.82 (3.08, 4.56)
14
5
4.50 (2.61, 6.39)
21
5
2.82 (2.11, 3.54)
21
4
2.95 (1.35, 4.55)
21
5
3.20 (2.06, 4.35)
Group
Study
Day
N
Mean
(95% Confidence
Interval)
Group
Study
Day
N
Mean
(95% Confidence
Interval)
Group
Study
Day
N
Mean
(95% Confidence
Interval)
-3
5
4.39 (3.20, 5.59)
-3
5
4.94 (3.54, 6.35)
-3
3
4.61 (0.00 *, 10.20)
1
5
4.93 (4.02, 5.85)
1
5
5.13 (3.31, 6.96)
1
4
5.13 (2.39, 7.87)
2
5
5.04 (3.14, 6.93)
2
5
4.98 (1.54, 8.42)
2
3
2.89 (1.86, 3.92)
4
3
5
4.75 (3.07, 6.43)
5
3
3
6.21 (0.64, 11.79)
6
3
3
2.33 (0.00 *, 5.75)
7
4
4.93 (2.48, 7.37)
7
1
4.67 (-)
7
NA
NA
14
3
4.70 (1.21, 8.19)
14
1
3.93 (-)
14
NA
NA
21
3
2.69 (0.17, 5.21)
21
1
3.23 (--)
21
NA
NA
N Number of animals.
Confidence interval could not be calculated since only one observation was available for this Group on this Study Day.
* Lower bound of confidence interval set to zero since a negative parameter value is not possible.
NA Data not available for this Group at this Study Day.
1-43
-------
Table 15b. Test Results for Lymphocytes (103 cells/jiL)
Lymphocytes
Study
Day
Mean Shift from Baseline, by Group
Group Effect
P-Value
Estimated Difference
(Relationship)
Tukey's P-Value *
1
2
3
4
5
6
1
-0.42
0.01
0.65
0.54
0.19
0.22
0.6180
2
-1.06
0.27
0.60
0.64
0.04
-2.09 |
0.0396 *
3
-0.20
0.51
0.63
0.36
0.56
-2.65 |
0.0206 *
-3.15 (6<2) 0.0304
-3.27 (6<3) 0.0164
-3.00 (6<4) 0.0314
-3.21 (6<5) 0.0425
7
-0.92
-1.14 |
-0.64
0.18
-1.3
NA
0.3713
14
-1.40 |
-0.66
-0.42
-0.01
-2.04
NA
0.2958
21
-2.00 |
-1.61 |
-1.71 |
-2.03 |
-2.74 |
NA
0.8896
# Cells contain all significant pairwise 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.
1.1 "T" 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.
NA Data not available for this Group at this Study Day.
1-44
-------
Table 16a. Descriptive Statistics for Neutrophils/Lymphocytes Ratio by Group and Study Day
Group
Study
Day
N
Geometric Mean
(95% Confidence
Interval)
Group
Study
Day
N
Geometric Mean
(95% Confidence
Interval)
Group
Study
Day
N
Geometric Mean
(95% Confidence
Interval)
1
-3
5
0.511 (0.198, 1.319)
2
-3
5
0.387 (0.299, 0.500)
3
-3
5
0.334 (0.230, 0.486)
1
5
0.260 (0.111, 0.606)
1
5
0.332 (0.244, 0.453)
1
5
0.273 (0.166, 0.448)
2
5
0.342 (0.195, 0.602)
2
5
0.293 (0.202, 0.424)
2
5
0.266 (0.164, 0.430)
3
5
0.231 (0.092, 0.580)
3
4
0.214 (0.124, 0.367)
3
5
0.257 (0.137, 0.480)
7
5
0.378 (0.239, 0.598)
7
4
0.317 (0.266, 0.378)
7
5
0.336 (0.231, 0.488)
14
5
0.280 (0.139, 0.563)
14
5
0.338 (0.245, 0.464)
14
5
0.310 (0.188, 0.513)
21
5
0.321 (0.232, 0.443)
21
4
0.211 (0.153, 0.291)
21
5
0.236 (0.172, 0.325)
Group
Study
Day
N
Geometric Mean
(95% Confidence
Interval)
Group
Study
Day
N
Geometric Mean
(95% Confidence
Interval)
Group
Study
Day
N
Geometric Mean
(95% Confidence
Interval)
4
-3
5
0.412 (0.264, 0.642)
5
-3
5
0.497 (0.247, 1.001)
6
-3
3
0.388 (0.090, 1.674)
1
5
0.363 (0.203, 0.651)
1
5
0.241 (0.051, 1.132)
1
4
0.418 (0.278, 0.628)
2
5
0.370 (0.142, 0.967)
2
5
0.394 (0.070, 2.228)
2
3
1.468 (0.447, 4.823)
3
5
0.226 (0.116, 0.441)
3
3
0.484 (0.037, 6.349)
3
3
0.596 (0.201, 1.772)
7
4
0.438 (0.267, 0.720)
7
1
0.283 (--)
7
NA
NA
14
3
0.307 (0.178, 0.530)
14
1
0.209 (--)
14
NA
NA
21
3
0.214 (0.183, 0.251)
21
1
0.173 (--)
21
NA
NA
N Number of animals.
Confidence interval could not be calculated since only one observation was available for this Group on this Study Day.
NA Data not available for this Group at this Study Day.
1-45
-------
Table 16b. Test Results for Neutrophils/Lymphocytes Ratio
Neutrophils/Lymphocytes Ratio T
Study
Day
Mean Shift as a Proportion of Baseline, by Group
Group Effect
P-Value
Estimated Ratio
(Relationship)
Tukey's P-Value *
1
2
3
4
5
6
1
0.51 |
0.86
0.82
0.88
0.49 |
1.06
0.2663
2
0.67
0.76
0.79
0.90
0.79
3.95 t
0.0076 *
0.17 (1 <6) 0.0050
0.19 (2<6) 0.0097
0.20 (3<6) 0.0126
0.23 (4<6) 0.0241
0.20 (5<6) 0.0125
3
0.45 |
0.60 |
0.77
0.55 |
1.16
1.60
0.0200 *
0.28 (1<6) 0.0224
7
0.74
0.89
1.01
1.23
1.34
NA
0.5146
14
0.55 |
0.87
0.93
0.80
0.99
NA
0.6374
21
0.63 |
0.59 |
0.71
0.56 |
0.82
NA
0.8576
f Indicates that values for this parameter were log-transformed for the analysis.
# Cells contain all significant pairwise 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.
1.1 "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.
NA Data not available for this Group at this Study Day.
1-46
-------
Table 17a. Descriptive Statistics for Monocytes (103 cells/jiL) by Group and Study Day
Group
Study
Day
N
Geometric Mean
(95% Confidence
Interval)
Group
Study
Day
N
Geometric Mean
(95% Confidence
Interval)
Group
Study
Day
N
Geometric Mean
(95% Confidence
Interval)
1
-3
5
0.15 (0.08, 0.29)
2
-3
5
0.09 (0.08, 0.11)
3
-3
5
0.11 (0.06, 0.22)
1
5
0.07 (0.02, 0.25)
1
5
0.08 (0.06, 0.10)
1
5
0.11 (0.06, 0.22)
2
5
0.06 (0.02, 0.19)
2
5
0.12 (0.06, 0.22)
2
5
0.13 (0.07, 0.24)
3
5
0.08 (0.02, 0.38)
3
4
0.09 (0.06, 0.15)
3
5
0.11 (0.06, 0.20)
7
5
0.08 (0.02, 0.25)
7
4
0.08 (0.03, 0.21)
7
5
0.13 (0.06, 0.25)
14
5
0.06 (0.02, 0.20)
14
5
0.06 (0.03, 0.16)
14
5
0.09 (0.03, 0.26)
21
5
0.04 (0.03, 0.07)
21
4
0.03 (0.02, 0.05)
21
5
0.05 (0.02, 0.10)
Group
Study
Day
N
Geometric Mean
(95% Confidence
Interval)
Group
Study
Day
N
Geometric Mean
(95% Confidence
Interval)
Group
Study
Day
N
Geometric Mean
(95% Confidence
Interval)
4
-3
5
0.18 (0.12, 0.27)
5
-3
5
0.18 (0.07, 0.43)
6
-3
3
0.14 (0.04, 0.46)
1
5
0.13 (0.08, 0.20)
1
5
0.09 (0.03, 0.30)
1
4
0.14 (0.10, 0.18)
2
5
0.11 (0.04, 0.31)
2
5
0.11 (0.04, 0.30)
2
3
0.03 (0.01, 0.09)
3
5
0.20 (0.11, 0.37)
3
3
0.05 (0.00, 1.53)
3
3
0.04 (0.01, 0.21)
7
4
0.16 (0.09, 0.29)
7
1
0.08 (--)
7
NA
NA
14
3
0.14 (0.01, 1.36)
14
1
0.07 (-)
14
NA
NA
21
3
0.05 (0.01, 0.17)
21
1
0.03 (-)
21
NA
NA
N Number of animals.
Confidence interval could not be calculated since only one observation was available for this Group on this Study Day.
NA Data not available for this Group at this Study Day.
1-47
-------
Table 17b. Test Results for Monocytes (103 cells/jiL)
Monocytes T
Study
Day
Mean Shift as a Proportion of Baseline, by Group
Group Effect
P-Value
Estimated Ratio
(Relationship)
Tukey's P-Value *
1
2
3
4
5
6
1
0.48 |
0.85
0.98
0.73
0.54
0.87
0.5115
2
0.38 |
1.27
1.17
0.63
0.61
0.20 |
0.0211 *
0.16 (6<2) 0.0291
0.17 (6<3) 0.0408
3
0.53
1.08
0.99
1.14
0.24 |
0.23 |
0.1599
7
0.51
0.90
1.13
1.02
1.14
NA
0.5768
14
0.40
0.69
0.78
0.82
1.00
NA
0.7679
21
0.29 |
0.39 |
0.44|
0.29 |
0.43
NA
0.7359
f Indicates that values for this parameter were log-transformed for the analysis.
# Cells contain all significant pairwise 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, | "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.
NA Data not available for this Group at this Study Day.
1-48
-------
Table 18a. Descriptive Statistics for Eosinophils (103 cells/jiL) by Group and Study Day
Group
Study
Day
N
Geometric Mean
(95% Confidence
Interval)
Group
Study
Day
N
Geometric Mean
(95% Confidence
Interval)
Group
Study
Day
N
Geometric Mean
(95% Confidence
Interval)
1
-3
5
0.16 (0.14, 0.18)
2
-3
5
0.13 (0.10, 0.16)
3
-3
5
0.11 (0.06, 0.19)
1
5
0.18 (0.15, 0.21)
1
5
0.14 (0.10, 0.20)
1
5
0.15 (0.09, 0.24)
2
5
0.13 (0.10, 0.17)
2
5
0.11 (0.09, 0.14)
2
5
0.14 (0.07, 0.25)
3
5
0.12 (0.06, 0.25)
3
4
0.10 (0.08, 0.14)
3
5
0.14 (0.08, 0.22)
7
5
0.15 (0.10, 0.24)
7
4
0.13 (0.10, 0.17)
7
5
0.16 (0.09, 0.28)
14
5
0.12 (0.07, 0.20)
14
5
0.15 (0.12, 0.20)
14
5
0.13 (0.08, 0.23)
21
5
0.12 (0.10, 0.15)
21
4
0.11 (0.07, 0.17)
21
5
0.09 (0.05, 0.17)
Group
Study
Day
N
Geometric Mean
(95% Confidence
Interval)
Group
Study
Day
N
Geometric Mean
(95% Confidence
Interval)
Group
Study
Day
N
Geometric Mean
(95% Confidence
Interval)
4
-3
5
0.10 (0.08, 0.13)
5
-3
5
0.15 (0.10, 0.23)
6
-3
3
0.10 (0.03, 0.28)
1
5
0.11 (0.09, 0.14)
1
5
0.16 (0.11, 0.25)
1
4
0.13 (0.08, 0.21)
2
5
0.13 (0.09, 0.17)
2
5
0.09 (0.05, 0.16)
2
3
0.06 (0.03, 0.11)
3
5
0.10 (0.07, 0.14)
3
3
0.09 (0.03, 0.29)
3
3
0.07 (0.02, 0.26)
7
4
0.17 (0.13, 0.22)
7
1
0.27 (-)
7
NA
NA
14
3
0.10 (0.06, 0.19)
14
1
0.14 (-)
14
NA
NA
21
3
0.10 (0.06, 0.19)
21
1
0.15 (-)
21
NA
NA
N Number of animals.
Confidence interval could not be calculated since only one observation was available for this Group on this Study Day.
NA Data not available for this Group at this Study Day.
1-49
-------
Table 18b. Test Results for Eosinophils (103 cells/jiL)
Eosinophils T
Study
Day
Mean Shift as a Proportion of Baseline, by Group
Group Effect
P-Value
Estimated Ratio
(Relationship)
Tukey's P-Value *
1
2
3
4
5
6
1
1.12
1.1
1.41 |
1.04
1.05
1.24
0.3456
2
0.83
0.88
1.30
1.22
0.57 |
0.58 |
0.0270 *
0.44 (5<3) 0.0495
3
0.77
0.76
1.27
0.97
0.58 |
0.73
0.2324
7
0.96
0.94
1.54 t
1.75 t
1.80
NA
0.0153*
0.55 (1 <4) 0.0470
14
0.76
1.20
1.27
0.99
0.93
NA
0.1025
21
0.79 |
0.82
0.84
0.75
1.00
NA
0.8583
f Indicates that values for this parameter were log-transformed for the analysis.
# Cells contain all significant pairwise 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, | "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.
NA Data not available for this Group at this Study Day.
1-50
-------
Table 19a. Descriptive Statistics for Basophils (103 cells/jiL) by Group and Study Day
Group
Study
Day
N
Geometric Mean
(95% Confidence
Interval)
Group
Study
Day
N
Geometric Mean
(95% Confidence
Interval)
Group
Study
Day
N
Geometric Mean
(95% Confidence
Interval)
1
-3
5
0.30 (0.17, 0.55)
2
-3
5
0.27 (0.17, 0.41)
3
-3
5
0.33 (0.16, 0.65)
1
5
0.33 (0.19, 0.56)
1
5
0.24 (0.19, 0.30)
1
5
0.37 (0.17, 0.78)
2
5
0.25 (0.18, 0.36)
2
5
0.26 (0.19, 0.34)
2
5
0.37 (0.15, 0.91)
3
5
0.21 (0.07, 0.60)
3
4
0.21 (0.15, 0.29)
3
5
0.36 (0.15, 0.89)
7
5
0.23 (0.12, 0.44)
7
4
0.21 (0.12, 0.38)
7
5
0.39 (0.19, 0.78)
14
5
0.17 (0.07, 0.46)
14
5
0.28 (0.18, 0.42)
14
5
0.29 (0.11, 0.80)
21
5
0.21 (0.13, 0.37)
21
4
0.18 (0.10, 0.34)
21
5
0.22 (0.08, 0.62)
Group
Study
Day
N
Geometric Mean
(95% Confidence
Interval)
Group
Study
Day
N
Geometric Mean
(95% Confidence
Interval)
Group
Study
Day
N
Geometric Mean
(95% Confidence
Interval)
4
-3
5
0.24 (0.18, 0.33)
5
-3
5
0.29 (0.19, 0.44)
6
-3
3
0.27 (0.18, 0.41)
1
5
0.27 (0.18, 0.41)
1
5
0.29 (0.18, 0.45)
1
4
0.37 (0.20, 0.68)
2
5
0.25 (0.14, 0.45)
2
5
0.20 (0.14, 0.28)
2
3
0.14 (0.07, 0.29)
3
5
0.24 (0.16, 0.38)
3
3
0.18 (0.07, 0.50)
3
3
0.11 (0.03, 0.42)
7
4
0.33 (0.20, 0.55)
7
1
0.52 (-)
7
NA
NA
14
3
0.30 (0.19, 0.47)
14
1
0.28 (-)
14
NA
NA
21
3
0.19 (0.11, 0.34)
21
1
0.28 (--)
21
NA
NA
N Number of animals.
Confidence interval could not be calculated since only one observation was available for this Group on this Study Day.
NA Data not available for this Group at this Study Day.
1-51
-------
Table 19b. Test Results for Basophils (103 cells/jiL)
Basophils T
Study
Day
Mean Shift as a Proportion of Baseline, by Group
Group Effect
P-Value
Estimated Ratio
(Relationship)
Tukey's P-Value *
1
2
3
4
5
6
1
1.09
0.89
1.12
1.10
1.00
1.23
0.4322
2
0.84
0.96
1.12
1.02
0.68 |
0.47 |
0.0488 *
0.42 (6<3) 0.0476
3
0.70
0.80
1.10
1.00
0.72
0.37 |
0.0611
0.34 (6<3) 0.0385
7
0.76 |
0.80
1.19
1.38 t
1.86 t
NA
0.0091 *
0.55 (1 <4) 0.0382
14
0.58 |
1.04
0.89
1.31
1.00
NA
0.1333
21
0.71 |
0.70 |
0.68|
0.84
1.00
NA
0.6564
f Indicates that values for this parameter were log-transformed for the analysis.
# Cells contain all significant pairwise 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, | "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.
NA Data not available for this Group at this Study Day.
1-52
-------
Table 20. Proportion of Animals that were Abnormal with Exact 95 % Confidence Interval
by Parameter and Group
Parameter
Group
Number
Abnormal / N
Proportion Abnormal
(95% Confidence Interval)
Red Blood Cell Count
1
2/5
0.40 (0.05, 0.85)
2
0/5
0.00 (0.00, 0.52)
3
0/5
0.00 (0.00, 0.52)
4
1/5
0.20 (0.01, 0.72)
5
1/5
0.20 (0.01, 0.72)
6
1/4
0.25 (0.01, 0.81)
Hemoglobin
1
5/5
1.00 (0.48, 1.00)
2
4/5
0.80 (0.28, 0.99)
3
2/5
0.40 (0.05, 0.85)
4
3/5
0.60 (0.15, 0.95)
5
4/5
0.80 (0.28, 0.99)
6
2/4
0.50 (0.07, 0.93)
Hematocrit
1
3/5
0.60 (0.15, 0.95)
2
2/5
0.40 (0.05, 0.85)
3
3/5
0.60 (0.15, 0.95)
4
4/5
0.80 (0.28, 0.99)
5
3/5
0.60 (0.15, 0.95)
6
2/4
0.50 (0.07, 0.93)
Mean Corpuscular Volume
1
0/5
0.00 (0.00, 0.52)
2
0/5
0.00 (0.00, 0.52)
3
0/5
0.00 (0.00, 0.52)
4
1/5
0.20 (0.01, 0.72)
5
1/5
0.20 (0.01, 0.72)
6
0/4
0.00 (0.00, 0.60)
Mean Corpuscular
Hemoglobin
1
0/5
0.00 (0.00, 0.52)
2
0/5
0.00 (0.00, 0.52)
3
0/5
0.00 (0.00, 0.52)
4
1/5
0.20 (0.01, 0.72)
5
0/5
0.00 (0.00, 0.52)
6
0/4
0.00 (0.00, 0.60)
Mean Corpuscular
Hemoglobin Concentration
1
4/5
0.80 (0.28, 0.99)
2
3/5
0.60 (0.15, 0.95)
3
5/5
1.00 (0.48, 1.00)
4
3/5
0.60 (0.15, 0.95)
5
2/5
0.40 (0.05, 0.85)
6
0/4
0.00 (0.00, 0.60)
Red Cell Distribution Width
1
4/5
0.80 (0.28, 0.99)
2
4/5
0.80 (0.28, 0.99)
3
3/5
0.60 (0.15, 0.95)
4
2/5
0.40 (0.05, 0.85)
5
1/5
0.20 (0.01, 0.72)
6
0/4
0.00 (0.00, 0.60)
Platelet Count
1
4/5
0.80 (0.28, 0.99)
2
4/5
0.80 (0.28, 0.99)
3
2/5
0.40 (0.05, 0.85)
4
0/5
0.00 (0.00, 0.52)
5
5/5
1.00 (0.48, 1.00)
6
1/4
0.25 (0.01, 0.81)
1-53
-------
Table 20. (Continued)
Parameter
Group
Number
Abnormal / N
Proportion Abnormal
(95% Confidence Interval)
Mean Platelet Volume
1
5/5
1.00 CO.48. 1.00)
2
5/5
1.00 CO.48. 1.00)
3
4/5
0.80 CO.28. 0.99)
4
5/5
1.00 CO.48. 1.00)
5
3/5
0.60 CO.15. 0.95)
6
2/4
0.50 CO.07. 0.93)
White Blood Cell Count
1
4/5
0.80 CO.28. 0.99)
2
0/5
0.00 fO.OO. 0.52)
3
1/5
0.20 fO.01. 0.72)
4
2/5
0.40 CO.05. 0.85)
5
3/5
0.60 CO.15. 0.95)
6
1/4
0.25 fO.01. 0.81)
Neutrophils
1
2/5
0.40 CO.05. 0.85)
2
0/5
0.00 fO.OO. 0.52)
3
0/5
0.00 fO.OO. 0.52)
4
3/5
0.60 CO.15. 0.95)
5
2/5
0.40 CO.05. 0.85)
6
2/4
0.50 CO.07. 0.93)
Lymphocytes
1
1/5
0.20 fO.01. 0.72)
2
0/5
0.00 fO.OO. 0.52)
3
0/5
0.00 fO.OO. 0.52)
4
1/5
0.20 fO.01. 0.72)
5
2/5
0.40 CO.05. 0.85)
6
1/4
0.25 fO.01. 0.81)
Neutrophils/Lymphocytes
Ratio
1
3/5
0.60 CO.15. 0.95)
2
0/5
0.00 fO.OO. 0.52)
3
0/5
0.00 fO.OO. 0.52)
4
1/5
0.20 fO.01. 0.72)
5
2/5
0.40 CO.05. 0.85)
6
1/4
0.25 fO.01. 0.81)
Monocytes
1
5/5
1.00 CO.48. 1.00)
2
2/5
0.40 CO.05. 0.85)
3
2/5
0.40 CO.05. 0.85)
4
3/5
0.60 CO.15. 0.95)
5
3/5
0.60 CO.15. 0.95)
6
2/4
0.50 CO.07. 0.93)
Eosinophils
1
3/5
0.60 CO.15. 0.95)
2
0/5
0.00 fO.OO. 0.52)
3
2/5
0.40 CO.05. 0.85)
4
1/5
0.20 fO.01. 0.72)
5
3/5
0.60 CO.15. 0.95)
6
0/4
0.00 fO.OO. 0.60)
Basophils
1
2/5
0.40 CO.05. 0.85)
2
0/5
0.00 fO.OO. 0.52)
3
1/5
0.20 fO.01. 0.72)
4
0/5
0.00 fO.OO. 0.52)
5
1/5
0.20 fO.01. 0.72)
6
1/4
0.25 fO.01. 0.81)
N = number of animals
1-54
-------
Table 21. Results of Overall Two-sided Fisher's Exact Tests for the Proportion of Animals
that were Abnormal by Parameter
Parameter
Group Effect
P-Value
Red Blood Cell Count
0.6579
Hemoglobin
0.3947
Hematocrit
0.9499
Mean Corpuscular Volume
1.0000
Mean Corpuscular Hemoglobin
1.0000
Mean Corpuscular Hemoglobin
Concentration
0.0638
Red Cell Distribution Width
0.1029
Platelet Count
0.0081 *
Mean Platelet Volume
0.1821
White Blood Cell Count
0.1361
Neutrophils
0.1937
Lymphocytes
0.6579
Neutrophils/Lymphocytes Ratio
0.2039
Monocytes
0.4344
Eosinophils
0.1757
Basophils
0.6579
* Comparison significant at the 0.05 level.
Table 22. Results of One-sided Pairwise Fisher's Exact Tests for the Proportion of
Animals with Abnormal Platelet Counts
One-Sided Pairwise Fisher's Exact Test P-Values
Parameter
Group
Unadjusted P-Values
Bonferroni-Holm Adjusted P-Values
1.0000
Platelet
Count
0.9921
1.0000
0.8810
0.4444
1.0000
1.0000
1.0000
1.0000
1.0000
0.9921
a A p-value of 1.0000 was substituted when all animals in both Groups experienced the same abnormality
result.
* Comparison significant at the 0.05 level.
1-55
-------
Table 23. Results of Overall Log-rank Tests for Time to Abnormality by Parameter
Parameter
Group Effect
P-value
Red Blood Cell Count
0.5648
Hemoglobin
0.2279
Hematocrit
0.8633
Mean Corpuscular Volume
0.5460
Mean Corpuscular Hemoglobin
0.4408
Mean Corpuscular Hemoglobin
Concentration
0.0830
Red Cell Distribution Width
0.1375
Platelet Count
0.0027 *
Mean Platelet Volume
0.4142
White Blood Cell Count
0.0685
Neutrophils
0.2612
Lymphocytes
0.5447
Neutrophils/Lymphocytes Ratio
0.1539
Monocytes
0.0921
Eosinophils
0.0817
Basophils
0.5524
Comparison significant at the 0.05 level.
Table 24. Results of Pairwise Log-rank Tests for Time to Abnormal Platelet Count
Pairwise Log-rank Test P-values
Parameter
Group
Unadjusted P-Values
Bonferroni-Holm Adjusted P-Values
0.4305
0.0854
0.0133
0.4221
0.1844
1.0000
1.0000
0.3851
1.0000
1.0000
0.1639
0.0135
0.0927
0.2297
1.0000
0.3851
1.0000
1.0000
Platelet
Count
0.1343
0.0142
0.7710
1.0000
0.3851
1.0000
0.0015
0.2636
0.0463
1.0000
0.0374
0.9343
* Comparison significant at the 0.05 level.
1-56
-------
Group 1 (Negative Control)
Group 3 (1.000 CFU)
Group 5 (100,000 CFU)
Group 2 (100 CFU)
Group 4 {10,000 CFU)
Group 6 (High Dose Control)
3
°o
c
=3
O
O
5
"D
O
o
CQ
~D
o
a:
i—
-3
2 3
Study Day
Figure 1. Plot of Red Blood Cell Count over time.
"O
cz
-Q
o
CD
O
16
15
14
-3
Group 1 (Negative Control)
Group 3(1.000 CFU)
Group 5 (100,000 CFU)
Group 2 (100 CFU)
Group 4 (10,000 CFU)
Group 6 (High Dose Control)
2 3
Study Day
14
21
Figure 2. Plot of Hemoglobin over time.
1-57
-------
45
Group 1 (Negative Control) * * * Group 2 (100 CFU)
* Group 4 (10,000 CFU)
Group 6 (High Dose Control)
Group 3 (1.000 CFU)
Group 5 (100,000 CFU)
42
¦3
1
2
3
7
14
21
Study Day
Figure 3. Plot of Hematocrit over time.
74
Group 2 (100 CFU)
Group 4 (10,000 CFU)
Group 6 (High Dose Control)
Group 1 (Negative Control)
Group 3 (1,000 CFU)
Group 5 (100,000 CFU)
68
58
¦3
1
2
3
7
14
21
Study Day
Figure 4. Plot of Mean Corpuscular Volume (MCV) over time.
1-58
-------
Group 1 (Negative Control) * * * Group 2 (100 CFU)
* Group 4 (10,000 CFU)
Group 6 (High Dose Control)
Group 3 (1.000 CFU)
Group 5 (100,000 CFU)
¦3
1
2
3
7
14
21
Study Day
Figure 5. Plot of Mean Corpuscular Hemoglobin (MCH) over time.
~ Group 1 (Negative Control) * * * Group 2 (100 CFU)
> Group 3 (1,000 CFU) A-A-* Group 4 (10,000 CFU)
I Group 5 (100,000 CFU) ~ 'l* *• Group 6 (High Dose Control)
-3 1 2 3 7 14 21
Study Day
Figure 6. Plot of Mean Corpuscular Hemoglobin Concentration (MCHC) over time.
1-59
-------
100
Group 1 (Negative Control)
Group 3 (1,000 CPU)
Group 5 (100,000 CFU)
r Group 2 (100 CFU)
t Group 4 (10,000 CFU)
• Group 6 (High Dose Control)
Q
cr
iiL
10
-3
3 7
Study Day
14
21
1-60
-------
Figure 7. Plot of Red Cell Distribution Width (RDW) over time.
1100
1000
900
800
5
o
700
-—"
c
600
Z3
o
o
500
CD
400
CL
300
200
100
0
-3
Group 1 (Negative Control)
Group 3 (1,000 CFU)
Group 5 (100,000 CFU)
Group 2 (100 CFU)
Group 4 (10,000 CFU)
Group 6 (High Dose Control)
2 3 7
Study Day
Figure 8.
Plot of Platelet Count over time.
100
Group 1 (Negative Control)
Group 3 (1,000 CFU)
Group 5 (100,000 CFU)
Group 2 (100 CFU)
Group 4 (10,000 CFU)
Group 6 (High Dose Control)
—i—
14
3 7
Study Day
21
1-61
-------
Figure 9.
Plot of Mean Platelet Volume (MPV) over time.
I Group 1 (Negative Control) * * * Group 2 (100 CFU)
~ Group 3 (1,000 CFU) AAA Group 4 (10.000 CFU)
I Group 5 (100,000 CFU) * * "» Group 6 (High Dose Control)
0tr r r t r
-31237
Study Day
Figure 10. Plot of White Blood Cell Count over time.
Group 2 (100 CFU)
Group 4 (10,000 CFU)
Group 6 (High Dose Control)
Group 1 (Negative Control)
Group 3 (1,000 CFU)
Group 5 (100,000 CFU)
¦3
2
3
7
14
21
Study Day
1-62
-------
Figure 11. Plot of Neutrophils over time.
Group 1 (Negative Control)
Group 3 (1,000 CFU)
Group 5 (100,000 CFU)
Group 2 (100 CFU)
Group 4 (10,000 CFU)
Group 6 (High Dose Control)
3
1
2
3
7
14
21
Study Day
Figure 12. Plot of Lymphocytes over time.
1-63
-------
10
0.001
Group 1 (Negative Control)
Group 3 (1,000 CFU)
Group 5 (100,000 CFU)
Group 2 (100 CFU)
Group 4 (10,000 CFU)
Group 6 (High Dose Control)
Study Day
Figure 14. Plot of Monocytes over time.
Figure 13. Plot of Neutrophils/Lympliocytes (N/L) Ratio over time.
2 3
Study Day
Group 1 (Negative Control) * Group 2 (100 CFU)
Group 3 (1,000 CFU) AAA Group 4 (10,000 CFU)
Group 5 (100,000 CFU) 'I' » *' Group 6 (High Dose Control)
1-64
-------
Group 1 (Negative Control)
Group 3(1,000 CFU)
Group 5 (100.000 CFU)
r Group 2 (100 CFU)
t Group 4 (10,000 CFU)
* Group 6 (High Dose Control)
p
Q.
O
c
CO
o
LU
0.01
IF"
14
~T
21
Study Day
Figure 15. Plot of Eosinophils over time.
no
a.
(2
0.01
Group 1 (Negative Control)
Group 3 (1,000 CFU)
Group 5 (100,000 CFU)
-3
Group 2 (100 CFU)
Group 4 (10,000 CFU)
Group 6 (High Dose Control)
14
21
Study Day
Figure 16. Plot of Basophils over time.
1-65
-------
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0 1
0.0
¦Group 1 (Negative Control)
-Group 3 (1,000 CFU)
¦Group 5 (100,000 CFU)
Group 2 (100 CFU)
Group 4 (10,000 CFU)
Group 6 (High Dose Control)
14
21
Time to Abnormal (Days Post-Challenge)
Figure 17. Kaplan-Meier curves representing time to abnormal Platelet Count
for each Group.
1-66
-------
ATTACHMENT 1:
RESULTS OF ANALYSIS REPEATED
WITH POTENTIAL OUTLIERS EXCLUDED
-------
ATTACHMENT 1: RESULTS OF ANALYSIS REPEATED WITH POTENTIAL
OUTLIERS EXCLUDED
Nine 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 1-1 through 1-5 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:
• RBC (Table 1-1): On Study Day 3 there was a significant decrease from baseline in
Group 3.
• HCT (Table 1-2): On Study Day 2 there was a significant decrease from baseline in
Group 3 and there was no longer a significant decrease from baseline in Group 1.
• MPV (Table 1-3): On Study Day 3 there was a significant decrease as a proportion
of baseline in Group 1 and a significant increase as a proportion in Group 5.
Additionally, the mean increase as a proportion of baseline for Group 5 was
significantly different from the mean decrease as a proportion of baseline for
Groups 1 and 3, and the mean increase as a proportion of baseline for Group 6 was
significantly greater than that for Group 5. On Study Day 14 there was a significant
decrease as a proportion of baseline in Groups 2, 3, and 4.
• Neutrophils (Table 1-4): On Study Day 3 there was a significant increase from
baseline in Group 5, and the mean increase from baseline in Group 5 was
significantly different from the mean decrease from baseline in Group 4.
1-2
-------
• N/L Ratio (Table 1-5): On Study Day 1 there was no longer a significant decrease as
a proportion of baseline in Groups. Additionally, the mean decrease as a proportion of
baseline for Group 1 was significantly different from the mean increase as a
proportion of baseline for Group 6. On Study Day 2, Group 5 had a significant
decrease as a proportion of baseline.
1-3
-------
Table 1-1. Test Results for Red Blood Cell Count (RBC, 106 cells/jiL) with Potential Outliers Excluded
Red Blood Cell Count
Study
Day
Mean Shift from Baseline, by Group
Group Effect
P-Value
Estimated Difference
(Relationship)
Tukey's P-Value *
1
2
3
4
5
6
2
-0.33 |
-0.41 |
-0.33 |
-0.41 |
-0.56 |
-0.68 |
0.5729
# Cells contain all significant pairwise 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.
1.1 "T" 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).
Table 1-2. Test Results for Hematocrit (HCT, %) with Potential Outliers Excluded
Hematocrit
Study
Day
Mean Shift from Baseline, by Group
Group Effect
P-Value
Estimated Difference
(Relationship)
Tu key's P-Value *
1
2
3
4
5
6
2
-2.0
-2.9 |
-2.1 |
-3.3 |
-5.0 |
-5.2 |
0.2033
# Cells contain all significant pairwise 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.
1.1 "T" 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).
1-4
-------
Table 1-3. Test Results for Mean Platelet Volume (MPV, fL) with Potential Outliers Excluded
Mean Platelet Volume +
Study
Day
Mean Shift as a Proportion of Baseline, by Group
Group Effect
P-Value
Estimated Ratio
(Relationship)
Tukey's P-Value *
1
2
3
4
5
6
3
0.84|
0.86
0.87
1.01
1.23 t
2.00 t
<0.0001*
0.68 (1<5) 0.0376
0.42 (1 <6) <0.0001
0.43 (2<6) <0.0001
0.70 (3<5) 0.0491
0.43 (3<6) <0.0001
0.50 (4<6) 0.0002
0.62 (5<6) 0.0107
14
1.02
0.89 |
0.82 |
0.80 |
1.00
NA
0.0605
f Indicates that values for this parameter were log-transformed for the analysis.
# Cells contain all significant pairwise 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.
1.1 "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.
NA Data not available for this Group at this Study Day.
1-5
-------
Table 1-4. Test Results for Neutrophils (103 cells/jiL) with Potential Outliers Excluded
Neutrophils
Study
Day
Mean Shift from Baseline, by Group
Group Effect
P-Value
Estimated Difference
(Relationship)
Tukey's P-Value *
1
2
3
4
5
6
3
-1.20|
-0.53
-0.24
-0.58
1.47 t
-0.41
0.0156*
-2.67 (1 <5) 0.0049
-2.05 (4<5) 0.0398
# Cells contain all significant pairwise 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.
1.1 "T" 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.
Table 1-5. Test Results for Neutrophils/Lymphocytes Ratio with Potential Outliers Excluded
Neutrophils/Lymphocytes Ratio T
Study
Day
Mean Shift as a Proportion of Baseline, by Group
Group Effect
P-Value
Estimated Ratio
(Relationship)
Tukey's P-Value *
1
2
3
4
5
6
1
0.51 |
0.86
0.82
0.88
0.82
1.06
0.0684
0.48 (1<6) 0.0429
2
0.67
0.76
0.79
0.90
0.55|
3.95 t
0.0005*
0.17 (1<6) 0.0006
0.19 (2<6) 0.0014
0.20 (3<6) 0.0019
0.23 (4<6) 0.0043
0.14 (5<6) 0.0003
f Indicates that values for this parameter were log-transformed for the analysis.
# Cells contain all significant pairwise 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, | "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.
1-6
-------
APPENDIX J
CLINICAL CHEMISTRY STATISTICAL REPORT
j-i
-------
Table of Contents
List of Acronyms J-6
1. Introduction J-7
2. Statistical Methods J-9
3. Results J-12
4. Conclusions J-20
Attachment 1: Results of Analysis Repeated with Potential Outliers Excluded 1-1
J-2
-------
List of Tables
Table 1. Study Design J-7
Table 2. List of Potential Clinical Chemistry Outliers J-22
Table 3. Summary of ANOVA Results for Baseline Data (Study Day -3) J-23
Table 4a. Descriptive Statistics for C-Reactive Protein (mg/dL) by Group and
Study Day J-24
Table 4b. Summary of C-Reactive Protein Test Results for the Shifts between
Study Days and Baseline (Study Day -3) J-25
Table 5a. Descriptive Statistics for Aspartate Aminotransferase (AST, U/L) by Group
and Study Day J-26
Table 5b. Summary of AST Test Results for the Shifts between Study Days and
Baseline (Study Day -3) J-27
Table 6a. Descriptive Statistics for Alanine Aminotransferase (ALT, U/L) by Group
And Study Day J-28
Table 6b. Summary of ALT Test Results for the Shifts between Study Days and
Baseline (Study Day -3) J-29
Table 7a. Descriptive Statistics for Lactate Dehydrogenase (LDH, U/L) by Group
and Study Day J-30
Table 7b. Summary of LDH Test Results for the Shifts between Study Days and
Baseline (Study Day -3) J-31
Table 8a. Descriptive Statistics for Sorbitol Dehydrogenase (SDH, U/L) by Group
and Study Day J-32
Table 8b. Summary of SDH Test Results for the Shifts between Study Days and
Baseline (Study Day -3) J-33
Table 9a. Descriptive Statistics for Total Protein (g/dL) by Group and Study Day J-34
Table 9b. Summary of Total Protein Test Results for the Shifts between Study Days
And Baseline (Study Day -3) J-35
Table 10a. Descriptive Statistics for Blood Urea Nitrogen (BUN, mg/dL) by Group
and Study Day J-36
Table 10b. Summary of BUN Test Results for the Shifts between Study Days and
Baseline (Study Day -3) J-37
Table 11a. Descriptive Statistics for Creatinine (mg/dL) by Group and Study Day J-38
Table 1 lb. Summary of Creatinine Test Results for the Shifts between Study Days and
Baseline (Study Day -3) J-39
Table 12a. Descriptive Statistics for BUN/Creatinine Ratio by Group and Study Day J-40
Table 12b. Summary of BUN/Creatinine Ratio Test Results for the Shifts between
Study Days and Baseline (Study Day -3) J-41
Table 13a. Descriptive Statistics for Sodium (mEq/L) by Group and Study Day J-42
Table 13b. Summary of Sodium Test Results for the Shifts between Study Days
and Baseline (Study Day -3) J-43
Table 14a. Descriptive Statistics for Potassium (mEq/L) by Group and Study Day J-44
Table 14b. Summary of Potassium Test Results for the Shifts between Study Days
J-3
-------
and Baseline (Study Day -3) J-45
Table 15a. Descriptive Statistics for Chloride (mEq/L) by Group and Study Day J-46
Table 15b. Summary of Chloride Test Results for the Shifts between Study Days
and Baseline (Study Day -3) J-47
Table 16a. Descriptive Statistics for Calcium (mg/dL) by Group and Study Day J-48
Table 16b. Summary of Calcium Test Results for the Shifts between Study Days
and Baseline (Study Day -3) J-49
Table 17a. Descriptive Statistics for Phosphorus (mg/dL) by Group and Study Day J-50
Table 17b. Summary of Phosphorus Test Results for the Shifts between Study Days
and Baseline (Study Day -3) J-51
Table 18. Proportion of Animals that were Abnormal with Exact 95% Confidence
Interval by Parameter and Group J-52
Table 19. Results of Overall Two-sided Fisher's Exact Test for the Proportion of
Animals that were Abnormal by Parameter J-54
Table 20. Results of One-sided Pairwise Fisher's Exact Tests for the Proportion of
Animals with Abnormal Lactate Dehydrogenase and Phosphorus J-54
Table 21. Results of Overall Log-rank Tests for Time to Abnormality by Parameter J-55
Table 22. Results of the Pairwise Log-rank Tests for Time to Abnormal
Lactate Dehydrogenase and Phosphorus J-55
Table 1-1. Summary of Test Results for Aspartate Aminotransferase (AST, U/L) with
Potential Outliers Excluded 1-4
Table 1-2. Summary of Test Results for Alanine Aminotransferase (ALT, U/L) with
Potential Outliers Excluded 1-4
Table 1-3. Summary of Test Results for Sorbitol Dehydrogenase (SDH, U/L) with
Potential Outliers Excluded 1-5
Table 1-4. Summary of Test Results for Blood Urea Nitrogen (mg/dL) with
Potential Outliers Excluded 1-5
Table 1-5. Summary of Test Results for Creatinine (mg/dL) with Potential Outliers
Excluded 1-6
Table 1-6. Summary of Test Results for BUN/Creatinine Ratio with Potential Outliers
Excluded 1-6
Table 1-7. Summary of Test Results for Sodium (mEq/L) with Potential
Outliers Excluded 1-6
Table 1-8. Summary of Test Results for Chloride (mEq/L) with Potential Outliers
Excluded 1-7
Table 1-9. Summary of Test Results for Calcium (mg/dL) with Potential Outliers
Excluded 1-7
J-4
-------
List of Figures
Figure 1. Plot of C-Reactive Protein over time J-56
Figure 2. Plot of Aspartate Aminotransferase over time J-57
Figure 3. Plot of Alanine Aminotransferase over time J-58
Figure 4. Plot of Lactate Dehydrogenase over time J-59
Figure 5. Plot of Sorbitol Dehydrogenase over time J-60
Figure 6. Plot of Total Protein over time J-61
Figure 7. Plot of Blood Urea Nitrogen over time J-62
Figure 8. Plot of Creatinine over time J-63
Figure 9. Plot of BUN/Creatinine ratio over time J-64
Figure 10. Plot of Sodium over time J-65
Figure 11. Plot of Potassium over time J-66
Figure 12. Plot of Chloride over time J-67
Figure 13. Plot of Calcium over time J-68
Figure 14. Plot of Phosphorus over time J-69
Figure 15. Kaplan-Meier curves representing time to abnormal Lactate Dehydrogenase
data for each group J-70
Figure 16. Kaplan-Meier curves representing time to abnormal Phosphorus data for
each group J-71
J-5
-------
List of Acronyms
ALT Alanine Aminotransferase
ANOVA Analysis of variance
AST Aspartate Aminotransferase
BBRC Battelle Biomedical Research Center
BUN Blood Urea Nitrogen
CFU Colony forming unit
CRP C-Reactive Protein
dL deciliter
g gram
LD50 Median lethal dose
LDH Lactate Dehydrogenase
L liter
LOD limit of detection
mEq milliequivalents
mg milligram
N Number of animals
SDH Sorbitol Dehydrogenase
U/L units/liter
J-6
-------
1. Introduction
This report summarizes the statistical analysis of clinical chemistry data collected under Battelle
Biomedical Research Center (BBRC) Study No. 1020-CG920503. Thirty (30) male
pathogen-free New Zealand White rabbits (Oryctolagus cuniculus) were randomized into
six Groups, with each Group having five animals. Animals were aerosol challenged on Study
Day 0 with Bacillus anthracis (Ames strain) spores as indicated in Table 1.
Table 1. Study Design
Group
Number of Animals
per Group
Spore Dose Colony
Forming Units
(CFU)
Clinical Chemistry Blood
Collection Study Days
1a
5
100 x LD50
2
5
100
3
5
1,000
-3, 1,2, 3, 7, 14, 21, and
4
5
10,000
Terminal
5
5
100,000
6b
5
100 x LD50
a Spores are gamma-irradiated (negative control)
b High dose control
LD50 Median lethal dose
Blood samples were collected for clinical chemistry analysis as indicated in Table 1. Blood
collection on study Day -3 served as a pre-challenge baseline for each animal.
The clinical chemistry parameters that were included in this analysis are:
• C-reactive protein (CRP, mg/dL)
• Total bilirubin (mg/dL)
• Aspartate aminotransferase (AST, U/L)
• Alanine aminotransferase (ALT, U/L)
• Lactate dehydrogenase (LDH, U/L)
• Sorbitol dehydrogenase (SDH, U/L)
• Total protein (g/dL)
• Blood urea nitrogen (BUN, mg/dL)
• Creatinine (mg/dL)
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• BUN/creatinine ratio
• Sodium (mEq/L)
• Potassium (mEq/L)
• Chloride (mEq/L)
• Calcium (mg/dL)
• Phosphorus (mg/dL)
Clinical chemistry analysis was performed at six levels:
1. Descriptive statistics (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 in order 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.
5. For each parameter, the proportion abnormal in each Group was obtained and
evaluated to identify significant differences between the Groups.
6. For each parameter, the time to an abnormal measurement for each animal was
obtained and evaluated to identify significant differences between the Groups.
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2. Statistical Methods
Analysis of variance (ANOVA) models fitted separately to each clinical chemistry parameter
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 in order 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
by 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 in order 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, in order to evaluate their effect on the results.
For each clinical chemistry parameter, the following ANOVA model was fitted to the data at
Study Day -3 in order to determine if there were significant differences between the Groups at
baseline:
Yi, = (i + Group, + ztj (1)
where Yy is the observed clinical chemistry result for the /th animal in Group i (/= 1 to 6) at the
baseline, [i is an overall constant, and 8(/ is the random error left unexplained by the model.
Tukey's multiple comparisons procedure was also performed for each parameter in order to
determine which pairs of Groups had baseline means that were significantly different from each
other; however, the results will only be presented if significant differences are identified. If the
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parameter was log-transformed for analysis, then the same model was used with Yy replaced by
Log(Yy), the base-10 log-transformed parameter value for the /th animal in Group i (/'= 1 to 6).
In order to determine if the mean shifts from baseline were significantly different between the
Groups, the following ANOVA model was fitted separately for each clinical chemistry
parameter on each Study Day:
Ydij-Ybi, = + Group, + 8(/ (2)
where Yaj is the observed clinical chemistry result for the /th animal in Group i (i= 1 to 6) on
Study Day d (d= 1, 2, 3, 7, and 14), Yuj is the observed clinical chemistry result for the /th animal
in Group i (/'= 1 to 6) at the baseline, [i is an overall constant, and 8(/ 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 Ydy and Yuj replaced by their base-10 log-transformed counterparts
Log(Y^) and Log(Ybij), respectively.
Least square mean estimates from the above shift from baseline ANOVA models were calculated
and approximate t-tests were performed to determine if, for each Group, there was a significant
shift in clinical chemistry results between baseline and each Study Day. For untransformed data,
this tests whether the difference in 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.
For each parameter, excluding CRP, the threshold for an abnormal parameter value was defined
as each individual animal's baseline (Study Day -3) parameter value plus or minus two standard
deviations. Since each animal had only one baseline value for each parameter, the standard
deviation associated with each parameter was calculated using the baseline values of all animals.
Animals were determined to have an abnormal parameter value when their observed value was
above the upper threshold or below the lower threshold for that respective parameter. For CRP,
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an animal was considered abnormal if it had a value above the limit of detection (LOD), which
was 0.5.
For each parameter, an overall two-sided Fisher's exact test was performed to determine if there
was a significant difference between the abnormal rates in each Group. If the overall Fisher's
exact test was significant, then one-sided pairwise Fisher's exact tests were performed to
evaluate all pairwise Group comparisons. The Bonferroni-Holm adjustment was used to maintain
an overall 0.05 significance level among the multiple pairwise comparisons made within each
parameter.
For each parameter, an overall log-rank test was performed to determine if there was a
significant difference between the times to abnormality in each Group. If the overall log-rank test
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 significance
level among the multiple pairwise comparisons made within each parameter. For those
parameters that had significant overall log-rank test, Kaplan-Meier curves were plotted by
Group.
All statistical analyses were conducted using SAS® (SAS Institute, Inc.; Cary, NC; version 9.1)
and all results are reported at the 0.05 level of significance. All ANOVA models were fitted
using the MIXED or GLM procedure. All Fisher's exact tests were performed using the FREQ
procedure, and all log-rank tests were performed using the LIFETEST procedure. The
MULTTEST procedure was used to maintain an overall 0.05 significance level among the
multiple pairwise comparisons made within each parameter.
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3. Results
The model assumption of normality was more reasonable for 10 of the 15 parameters when
models were fitted to the base-10 log-transformed data. These parameters were: CRP, AST,
ALT, LDH, SDH, total protein, BUN, creatinine, potassium, and phosphorus. Therefore, models
were fitted to base-10 log-transformed values in the final analysis for these parameters. Since the
total bilirubin measurements for each animal were equal to zero at all Study Days, total bilirubin
was removed from the analysis. Table 2 contains a list of 18 measurements that were identified
as potential outliers using the procedure described above. The statistical analyses of the clinical
chemistry data were performed both with and without the potential outliers in order to evaluate
their effect on the results. Attachment 1 contains 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). There was a
significant Group effect for AST (p-value=0.0038), with the negative control Group (Group 1)
having significantly higher levels than Groups 2, 3, 4, and 6. There was a significant group effect
for ALT (p-value=0.0057), with Group 5 having significantly higher levels than Group 4. There
was a significant group effect for LDH (p-value=0.0074), with the negative control group
(Group 1) having significantly higher levels than Groups 3, 4, 5, and 6. There was a significant
group effect for total protein (p-value=0.0096), with Group 3 having significantly lower levels
than Groups 1 and 5. There was a significant group effect for calcium (p-value=0.0268), with
Group 5 having significantly higher levels than Group 2. There was a significant group effect for
phosphorus (p-value=0.0011), with the negative control group (Group 1) having significantly
higher levels than all other groups (Groups 2 through 6). 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.
Descriptive statistics and group comparisons for each parameter are presented in pairs of tables,
where Tables 4 through 17 are associated with the parameter of interest. For each parameter,
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Table "a" contains the descriptive statistics, while Table "b" contains the test results for
comparing the mean shifts from baseline within each Group at each Study Day.
Tables 4a through 17a contain descriptive statistics (means with 95% confidence intervals for
untransformed data, or geometric means with 95% confidence intervals for base-10
log-transformed data) for the clinical chemistry parameter results within each group at each
Study Day. 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 14 display the means
and confidence intervals over the course of the study for each clinical chemistry parameter.
Tables 4b through 17b contain test results for the mean shift from baseline within each group at
each Study Day. These tables contain test results that indicate if the mean difference between the
Study Day and baseline was significantly different from zero for untransformed parameters, or if
the ratio of geometric means was significantly different from one for base-10 log-transformed
parameters (at the 0.05 level). In each cell, the estimate of the shift from baseline (difference or
ratio) is shown for that parameter, Group, and 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 group effect p-values for each Study
Day, as well as test results from the Tukey's pairwise comparisons procedure that was used to
identify pairs of groups with significantly different shifts from baseline. Under the Tukey
procedure, the set of comparisons within each parameter and Study Day is made at a joint 95%
confidence level. Each significant difference is shown as the estimated comparison between the
shifts from baseline for the pair of groups under consideration, the direction of the comparison
(i.e., which group experienced a larger shift from baseline), and the corresponding
Tukey-adjusted p-value. P-values less than 0.05 provide evidence of a significant difference. The
results from Tables 4b through 17b are discussed below in groups of related parameters.
3.1 Liver Function Parameters
• CRP (Tables 4a-b, Figure 1): There were significant increases as a proportion of
baseline in Groups 2, 3, 5, and 6 at Study Day 1, and the increases in Groups 2 and 5
were both significantly different than the decrease in Group 1. As a proportion of
baseline, Groups 5 and 6 had significant increases at Study Day 2, and the increase in
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Group 6 was significantly greater than those in Groups 1, 2, 3, and 4. There were
significant increases as a proportion of baseline in Groups 5 and 6 at Study Day 3.
The increase in Group 6 was significantly different than the changes in Groups 1, 2,
3, and 4, and the increase in Group 5 was significantly different than the decrease in
Group 1. There was a significant decrease as a proportion of baseline in Group 1 at
Study Day 21.
• AST (Tables 5a-b, Figure 2): There was a significant increase as a proportion of
baseline in Groups 5 and 6 at Study Day 2, and in Group 5 at Study Day 3. The
increase in Group 5 at Study Day 3 was significantly different than the changes in
Groups 1, 2, 3, 4, and 6. There were significant increases as a proportion of baseline
in Groups 2, 3, 4, and 5 at Study Day 3, and the increase in Group 4 was significantly
different than the decrease in Group 1. As a proportion of baseline, there was a
significant increase in Group 3 at Study Day 14, and in Groups 2 and 3 at Study
Day 21.
• ALT (Tables 6a-b, Figure 3): There were significant increases as a proportion of
baseline in Groups 2, 3, 4, and 5 at Study Day 1, in Groups 5 and 6 at Study Day 2,
and in Groups 4 and 5 at Study Day 3. The increase in Group 5 at Study Day 3 was
significantly greater than those in Groups 1, 2, 3, 4, and 6. As a proportion of
baseline, Groups 2, 3, and 4 had significant increases at Study Day 7, as did Groups 2
and 3 at Study Day 14.
• LDH (Tables 7a-b, Figure 4): As a proportion of baseline, there was a significant
increase in Group 5 at Study Day 1. There was a significant decrease as a proportion
of baseline in Group 1, and significant increases as a proportion of baseline in
Groups 5 and 6 at Study Day 2. The increase in Group 5 at Study Day 2 was
significantly different than the decrease in Group 1. There was a significant decrease
as a proportion of baseline in Group 1 and significant increases as a proportion of
baseline in Groups 5 and 6 at Study Day 3. The increase in Groups 5 was
significantly different than the decreases in Groups 1, 2, 3, and 4, and the increase in
Group 6 was significantly different than the decreases in Groups 1 and 3. There was a
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significant decrease as a proportion of baseline in Group 1 and significant increases
as a proportion of baseline in Groups 4 and 5 at Study Day 7. The decrease in
Group 1 was significantly different than the increases in Groups 2, 3, 4, and 5. As a
proportion of baseline, there was a significant increase in Group 4 at Study Day 21.
• SDH (Tables 8a-b, Figure 5): There was a significant increase as a proportion of
baseline in Group 6 at Study Day 1. There were significant increases as a proportion
of baseline in Groups 5 and 6 at Study Day 2, and the increase in Group 6 was
significantly different than the decrease in Group 1. At Study Day 3, there was a
significant increase as a proportion of baseline in Group 5 that was significantly
different than the changes in all other groups. There were significant increases as a
proportion of baseline in Groups 2, 3, 4, and 5 at Study Day 7. The increases in
Groups 2, 3, and 4 were all significantly greater than that in Group 1. There were
significant increases as a proportion of baseline in Group 3 at Study Day 14.
3.2 Serum Protein Parameters
• Total Protein (Tables 9a-b, Figure 6): There were significant increases as a
proportion of baseline in Groups 2, 3, and 4 at Study Day 1, and the increase in
Group 2 was significantly greater than that in Group 5. There were significant
increases as a proportion of baseline in Groups 2 and 3 at Study Days 2 and 3, and the
increase in Group 2 at Study Day 3 was significantly different than the decrease in
Group 5. As a proportion of baseline, there were significant increases in Groups 1, 2,
3, and 4 at Study Days 7 and 14. There were significant decreases as a proportion of
baseline in Groups 1 and 4 at Study Day 21, and the decrease in Group 1 was
significantly different than the increases in Groups 2 and 3.
3.3 Kidney Function Parameters
• BUN (Tables lOa-b, Figure 7): There were significant increases as a proportion of
baseline in Groups 1, 2, and 3 at Study Day 1, and the increases in Groups 1 and 2
were both significantly different than the decrease in Group 6. There were significant
decreases as a proportion of baseline in Groups 4 and 6 at Study Day 2, both of which
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were significantly different than the increases in Groups 1 and 2. Group 4 had a
significant decrease as a proportion of baseline again at Study Day 3. There was a
significant increase as a proportion of baseline in Group 2 at Study Day 7, and in
Groups 1 and 2 at Study Day 14.
• Creatinine (Tables lla-b, Figure 8): There was a significant decrease as a
proportion of baseline in Group 4 at Study Day 1, and in Group 3 at Study Day 2.
There was a significant increase as a proportion of baseline in Group 5 at Study
Day 3, and in Group 2 at Study Days 7 and 21.
• BUN/Creatinine Ratio (Tables 12a-b, Figure 9): There were significant increases
from baseline in Groups 3, 4, and 5 at Study Day 1. There was a significant increase
from baseline in Group 1 and a significant decrease from baseline in Group 6 at Study
Day 2, and the decrease from baseline in Group 6 was significantly different than the
increases in Groups 1, 2, and 3. There was a significant decrease from baseline in
Group 6 at Study Day 3, and in Group 4 at Study Day 21.
Electrolytes, Ions
• Sodium (Tables 13a-b, Figure 10): There were significant increases from baseline
in Groups 3 and 4 at Study Days 1, 2, and 3, and the increases in both groups at Study
Day 3 were significantly different than the decreases in Groups 5 and 6. There were
significant increases from baseline in Groups 2 and 3 at Study Day 7, and in Group 3
at Study Day 14.
• Potassium (Tables 14a-b, Figure 11): There were significant increases as a
proportion of baseline in Group 1 at Study Days 7 and 14, in Group 2 at Study
Days 1,3, and 14, in Group 3 at Study Days 7 and 14, and in Group 4 at Study
Days 7 and 14. There were significant decreases as a proportion of baseline in
Groups 1, 2, and 4 at Study Day 21, in Group 5 at Study Day 3, and in Group 6 at
Study Day 2. There was a significant group effect at Study Day 2; however, there
were no significant differences in shifts as a proportion of baseline between any two
groups.
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• Chloride (Tables 15a-b, Figure 12): There were significant increases from baseline
in Groups 1 and 3 at Study Day 2, and while the group effect was significant, there
were no significant differences between any pair of groups. There were significant
decreases from baseline in Groups 5 and 6 at Study Day 3, and the decrease in
Group 6 was significantly different than the increases in Group 3 and 4. There was a
significant increase from baseline in Group 3 at Study Day 14.
• Calcium (Tables 16a-b, Figure 13): There were significant increases from baseline
in Groups 2 and 3 at Study Day 1, and the increase in Group 2 was significantly
greater than that in Group 5. There was a significant increase from baseline in
Group 2 and a significant decrease from baseline in Group 6 at Study Day 2. The
decrease in Group 6 was significantly different than the increase in Group 2. There
was a significant increase from baseline in Group 2 and a significant decrease from
baseline in Group 5 at Study Day 3. The decrease in Group 5 was significantly
different than the increases in Groups 2 and 3. There were significant increases from
baseline in Groups 1, 2, and 3 at Study Day 7, and in Groups 1, 2, 3, and 4 at Study
Day 14. There was a significant decrease from baseline in Group 1 at Study Day 21.
• Phosphorus (Tables 17a-b, Figure 14): There was a significant decrease as a
proportion of baseline in Group 1 and a significant increase as a proportion of
baseline in Group 3 at Study Day 1. Group 1 had a significant decrease as a
proportion of baseline at Study Day 2, while Groups 2, 5, and 6 had significant
increases as a proportion of baseline. The increases in Groups 2, 5, and 6 were all
significantly different than the decrease in Group 1. There were significant increases
as a proportion of baseline in Groups 2, 3, 5, and 6 at Study Day 3, and in Groups 1
through 5 at Study Day 7. The increases in Groups 2 and 3 were both significantly
greater than those in Groups 1 and 4 at Study Day 7. There were significant increases
as a proportion of baseline in Groups 2, 3, and 4 at Study Day 14, and the increases in
Groups 2 and 3 were both significantly greater than that in Group 1. There were
significant increases as a proportion of baseline in Groups 1 through 5 at Study
Day 21, and the increases in Group 2 and 3 were both significantly greater than that
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in Group 1. There were significant increases as a proportion of baseline in Groups 4,
5, and 6 at the terminal blood draw.
For each parameter, Table 18 contains the proportion of animals that were abnormal at any point
during the study by Group. Table 19 presents the results of the overall Fisher's exact tests for
each parameter. The overall tests identified significant differences between the Groups for LDH
(p-value=0.0046) and phosphorus (p-value=0.0020). Table 20 contains the results of pairwise
Fisher's exact tests comparing the proportion of abnormal animals associated with each pair of
Groups for those parameters that had significant overall tests (LDH and phosphorus). The
pairwise comparisons are not dependent upon the ordering of the Groups being compared
(e.g. comparing Group 1 to Group 2 is equivalent to comparing Group 2 to Group 1); therefore,
the cells in the lower left portion of the table are shaded out for each parameter. For LDH,
Group 5 had a significantly greater proportion abnormal than Groups 2 and 3. However, after the
Bonferroni-Holm adjustment for multiple comparisons, the proportion abnormal in Group 5 was
no longer significantly greater than that in any other Group. For phosphorus, the proportion
abnormal in Groups 2, 3, 4, and 5 were significantly greater than that in Group 1. However, these
comparisons were no longer significant after the Bonferroni-Holm adjustment for multiple
comparisons.
Table 21 contains the results of the overall log-rank tests for each parameter. There were
significant differences between the Groups for LDH (p-value=0.0046) and phosphorus
(p-value=0.0028); therefore, the results of their pairwise comparisons are displayed in Table 22.
Again, the pairwise comparisons are not dependent upon the ordering of the Groups being
compared (e.g., comparing Group 1 to Group 2 is equivalent to comparing Group 2 to Group 1);
therefore, the cells in the lower left portion of the table are shaded out for each parameter. Prior
to adjusting for the multiple pairwise comparisons within each parameter, the time to an
abnormal LDH level in Groups 2 and 3 was significantly greater than that in Groups 4 and 5, and
the time to an abnormal LDH level in Group 4 was significantly greater than that in Group 5.
Prior to adjusting for the multiple pairwise comparisons within each parameter, the time to an
abnormal phosphorus level in Group 1 was significantly greater than that in Groups 2, 3, 4 and 5,
and the time to an abnormal phosphorus level in Group 4 was significantly greater than that in
Groups 2 and 3. After adjusting for the multiple pairwise comparisons made within each
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parameter, the times to an abnormal LDH level in Groups 2 and 3 remained significantly greater
than that in Group 5. The time to an abnormal phosphorus level in Group 1 remained
significantly greater than those in Groups 2 and 3, and the time to an abnormal phosphorus level
in Group 4 remained significantly greater than that in Group 3. Figures 15 and 16 display the
Kaplan-Meier curves associated with time to abnormality for LDH and phosphorus, respectively.
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4. Conclusions
All animals in the high dose control group (Group 6), and all but one animal in the 100,000 CFU
group (Group 5), died prior to Study Day 7. Among the liver function parameters, there were
similar results in terms of significance at Study Days 2, 3, and 7. At Study Day 2, the increase as
a proportion of baseline in the high dose control group (Group 6) was significantly different than
the change as a proportion of baseline in the negative control group (Group 1) for CRP, AST,
and SDH. At Study Day 3, the increase as a proportion of baseline in the 100,000 CFU group
(Group 5) was significantly different than the changes as a proportion of baseline in the lower
dose groups (Groups 1 through 4) for AST, ALT, LDH, and SDH. At Study Day 7, the increases
as a proportion of baseline in the higher dose groups with surviving animals (Groups 2
through 5) were often significantly different than the change as a proportion of baseline in the
negative control group (Group 1) for LDH and SDH.
For total protein, all but two of the significant shifts as a proportion of baseline were increases.
In the 100 CFU and 1,000 CFU groups (Groups 2 and 3), there were significant increases as a
proportion of baseline on Study Days 1 through 21, while the highest dose groups (Groups 5
and 6) experienced no significant shifts as a proportion of baseline at any Study Day. At Study
Days 1 and 3, the increase as a proportion of baseline in the 100 CFU dose group (Group 2) was
significantly different than the changes in the 100,000 CFU dose group (Group 5). At Study
Day 21, the decrease as a proportion of baseline in the negative control group (Group 1) was
significantly different than the increases in the 100 CFU and 1,000 CFU groups (Groups 2
and 3).
Among the kidney function parameters, the decrease as a proportion of baseline in the high dose
control group (Group 6) was significantly different than the increases as a proportion of baseline
in the negative control group (Group 1) and in the 100 CFU dose group (Group 2) at Study
Days 1 and 2 for BUN. The decrease as a proportion of baseline in the 10,000 CFU group
(Group 4) was also significantly different than the increases as a proportion of baseline in the
negative control group (Group 1) and in the 100 CFU dose group (Group 2) at Study Day 2 for
BUN. Furthermore, for BUN/creatinine ratio, the decrease from baseline in the high dose control
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group (Group 6) was significantly different than the increases in the lowest dose groups
(Groups 1 through 3) at Study Day 2.
Among the electrolytes and ions, sodium, chloride, and calcium exhibited some similarities in
terms of significant differences. At Study Day 3, the increases from baseline in the 1,000 CFU
and 10,000 CFU groups (Groups 3 and 4) were significantly different than the decrease from
baseline in the high dose control group (Group 6) for sodium and chloride. Also at Study Day 3,
the increase from baseline in the 1,000 CFU group (Group 3) was significantly different than the
decrease from baseline in the 100,000 CFU group (Group 5) for sodium and calcium. For
phosphorus, the change as a proportion of baseline in the negative control group (Group 1) was
significantly different than the changes in at least two of the higher dose groups (Groups 2
through 6) at Study Days 2 and 7 through 21.
LDH and phosphorus were the only parameters that had significant differences between the
groups in terms of the proportion that were ever abnormal during the study. According to the
unadjusted pairwise comparisons between groups within LDH, the proportion of abnormal
animals in the 100,000 CFU group (Group 5) was significantly greater than that in the 100 CFU
and 1,000 CFU groups (Groups 2 and 3). For phosphorus, the proportions of abnormal animals in
most of the higher dose groups (Groups 2 through 5) were significantly greater than those in the
negative control group (Group 1). After adjusting for the multiple pairwise comparisons made
within each parameter, there were no significant differences between any two groups for either
parameter.
LDH and phosphorus were also the only two parameters that had significant differences between
groups in terms of time to abnormality. For LDH, the times to abnormality in the 100 CFU and
1,000 CFU groups (Groups 2 and 3) were significantly greater than those in the 10,000 CFU and
100,000 CFU groups (Groups 4 and 5), and the 10,000 CFU group (Group 4) had a significantly
greater time to abnormality than the 100,000 CFU group (Group 5). For phosphorus, the time to
abnormality in the negative control group (Group 1) was significantly greater than that in most of
the higher dose groups (Groups 2 through 5), and the 10,000 CFU group (Group 4) had a
significantly greater time to abnormality than the 100 CFU and 1,000 CFU groups (Groups 2
and 3). After adjusting for the multiple pairwise comparisons made within each parameter, the
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time to an abnormal LDH level in the 100 CFU and 1,000 CFU groups (Groups 2 and 3,
respectively) remained significantly greater than that in the 100,000 CFU group (Group 5), the
time to an abnormal phosphorous level in negative control group (Group 1) remained
significantly greater than those in the 100 CFU and 1,000 CFU groups (Groups 2 and 3,
respectively), and the time to an abnormal phosphorous level in the 10,000 CFU group (Group 4)
remained significantly greater than that in the 1,000 CFU group (Group 3).
Table 2. List of Potential Clinical Chemistry Outliers
Parameter
Animal
Group
Study Day
Result
Deleted Studentized
Residual
C-Reactive ProteinT
L23225
4
3
9.59
4.283
Aspartate
L23201
5
2
701.80
7.353
Aminotransferase1
L23212
5
3
21.50
-6.241
Alanine
Aminotransferase1
L23201
5
2
290.60
5.749
L23201
5
3
588.10
4.486
L23212
5
3
37.00
-5.482
Sorbitol Dehydrogenase1
L23201
5
2
331.10
5.655
L23212
5
3
24.50
-5.000
Blood Urea Nitrogen1
L23201
5
3
60.30
6.644
Creatinine1
L23204
6
Terminal
5.01
5.232
L23213
6
Terminal
1.65
-5.232
BUN/Creatinine Ratio
L23201
5
3
31.40
4.206
Sodium
L23204
6
Terminal
121.00
-10.511
L23213
6
Terminal
150.00
10.511
Chloride
L23204
6
Terminal
77.00
-6.258
L23213
6
Terminal
94.00
6.258
Calcium
L23201
5
3
6.45
-5.810
L23212
5
3
13.99
4.030
f Distribution was log-normal for these parameters. Parameter values are reported on the original scale, while
the residuals are reported on the log-transformed scale.
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Table 3. Summary of ANOVA Results for Baseline Data (Study Day -3)
Parameter
Group
Effect
P-value
Estimated Difference or Ratio
(Relationship)
Tukey's P-value#
C-Reactive ProteinT
0.3631
Aspartate Aminotransferase1
0.0038 *
0.55 (2<1) 0.0124
0.51 (3<1) 0.0051
0.58 (4<1) 0.0286
0.53 (6<1) 0.0091
Alanine Aminotransferase1
0.0057 *
0.57 (4<5) 0.0380
Lactate Dehydrogenase1
0.0074 *
0.45 (3d) 0.0351
0.42 (4<1) 0.0202
0.39 (5<1) 0.0110
0.40 (6<1) 0.0137
Sorbitol Dehydrogenase1
0.1322
Total Protein1
0.0096 *
0.90 (3d) 0.0261
0.89 (3<5) 0.0119
Blood Urea Nitrogen1
0.1073
Creatinine1
0.4196
BUN/Creatinine Ratio
0.1397
Sodium
0.1059
Potassium1
0.3524
Chloride
0.3057
Calcium
0.0268 *
-1.41 (2<5) 0.0435
Phosphorus1
0.0011 *
0.71 (2d) 0.0035
0.68 (3d) 0.0011
0.73 (4<1) 0.0082
0.72 (5<1) 0.0049
0.76 (6<1) 0.0279
# Cells contain all pairwise comparisons that are significant at the 0.05 level. The format within each cell is:
(1) the difference of means (or ratio of geometric means if parameter is log-transformed), (2) the relationship
between the corresponding pair of group means shown in parentheses, and (3) the Tukey-adjusted p-value.
f Indicates that values for this parameter were log-transformed for the analysis.
* Group effect was significant at the 0.05 level.
J-23
-------
Table 4a. Descriptive Statistics for C-Reactive Protein (mg/dL) by Group and Study Day
Group
Study Day
N
Geometric Mean
(95% Confidence Interval)
-3
5
0.75 (0.11, 5.02)
1
5
0.60 (0.18, 2.01)
2
5
0.89 (0.23, 3.40)
1
3
5
0.46 (0.15, 1.38)
7
5
0.41 (0.10, 1.61)
14
5
0.40 (0.11, 1.47)
21
5
0.25 (-)
Terminal
NA
NA
-3
5
0.25 (-)
1
5
1.29 (0.79, 2.11)
2
5
0.45 (0.22, 0.95)
2
3
5
0.33 (0.15, 0.73)
7
4
0.32 (0.14, 0.71)
14
3
0.25 (-)
21
5
0.25 (-)
Terminal
NA
NA
-3
5
0.32 (0.16, 0.61)
1
5
0.73 (0.27, 1.96)
2
5
0.33 (0.15, 0.72)
3
3
5
0.29 (0.19, 0.44)
7
5
0.25 (-)
14
5
0.32 (0.16, 0.64)
21
5
0.29 (0.19, 0.45)
Terminal
NA
NA
-3
5
0.36 (0.13, 0.95)
1
5
0.76 (0.24, 2.35)
2
5
0.68 (0.12, 4.01)
4
3
5
0.52 (0.07, 3.93)
7
4
0.58 (0.04, 8.73)
14
3
0.25 (-)
21
3
0.25 (-)
Terminal
1
6.28 (-)
-3
5
0.33 (0.15, 0.71)
1
5
1.65 (0.92, 2.97)
2
5
1.15 (0.48, 2.78)
5
3
4
1.92 (0.22, 16.66)
7
1
0.25 (-)
14
1
0.25 (-)
21
1
0.25 (-)
Terminal
1
3.27 (-)
J-24
-------
Table 4a. (Continued)
Group
Study Day
N
Geometric Mean
(95% Confidence Interval)
-3
5
0.33 (0.23, 0.48)
1
4
1.62 (0.64, 4.11)
2
3
6.22 (2.68, 14.45)
6
3
3
11.14 (7.76, 16.01)
7
NA
NA
14
NA
NA
21
NA
NA
Terminal
2
7.86 (3.08, 20.06)
N Number of animals.
Confidence intervals could not be calculated since only one observation was available.
NA Data not available for this Group at this Study Day.
Table 4b. Summary of C-Reactive Protein Test Results for the Shifts between Study Days
and Baseline (Study Day -3)
C-Reactive Protein1
Study
Day
Me«
1
in Shift
2
as a Pr<
by (
3
)portior
3 roup
4
of Base
5
line,
6
Group
Effect
P-value
Estimated Ratio
(Relationship)
Tukey's P-value#
1
0.80
5.15 t
2.29 t
2.13
5.01 t
4.09 |
0.0255 *
0.15 (1<2) 0.0316
0.16 (1<5) 0.0351
2
1.18
1.81
1.04
1.91
3.49 t
18.29 t
0.0056 *
0.06 (1<6) 0.0064
0.10 (2<6) 0.0268
0.06 (3<6) 0.0043
0.10 (4<6) 0.0318
3
0.61
1.33
0.92
1.46
5.43 t
32.76 t
0.0002 *
0.11 (1<5) 0.0288
0.02 (1<6) 0.0002
0.04 (2<6) 0.0021
0.03 (3<6) 0.0006
0.04 (4<6) 0.0028
7
0.54
1.28
0.79
2.34
1.00
NA
0.6596
14
0.53
1.00
1.01
1.00
1.00
NA
0.9172
21
0.33 i
1.00
0.92
1.00
1.00
NA
0.2318
Terminal
NA
NA
NA
4.27
13.08
19.80
0.4009
f Indicates that values for this parameter were log-transformed for the analysis.
# Cells contain all pairwise comparisons that are significant at the 0.05 level. The format within each cell is:
(1) the ratio of shifts, (2) the relationship between the corresponding pair of group mean shifts shown in
parentheses [For example, "(1<2)" indicates that the mean shift as a proportion of baseline for Group 1 was
less than that for Group 2], and (3) the Tukey-adjusted p-value.
t, | Indicate that the ratio of the geometric mean at baseline and the geometric mean at the Study Day was
significantly different from one (at the 0.05 level), "t" indicates that the geometric mean at the Study Day was
greater than that at baseline, while "J," indicates that the geometric mean at the Study Day was less than that at
baseline.
* Group effect was significant at the 0.05 level.
NA Data not available for this Group at this Study Day.
J-25
-------
Table 5a. Descriptive Statistics for Aspartate Aminotransferase (AST, U/L) by Group and
Study Day
Group
Study Day
N
Geometric Mean
(95% Confidence Interval)
-3
5
24.1 (15.0, 38.5)
1
5
19.1 (15.2, 24.0)
2
5
19.9 (15.2, 26.0)
1
3
5
20.0 (16.3, 24.6)
7
5
21.7 (16.3, 28.9)
14
5
28.5 (17.7, 46.1)
21
5
24.3 (17.2, 34.2)
Terminal
NA
NA
-3
5
13.1 (10.4, 16.6)
1
5
15.7 (12.8, 19.1)
2
5
14.8 (12.7, 17.2)
2
3
5
17.5 (14.6, 20.9)
7
4
18.4 (12.3, 27.4)
14
3
18.1 (14.3, 22.9)
21
5
17.7 (14.6, 21.4)
Terminal
NA
NA
-3
5
12.4 (10.2, 15.0)
1
5
14.3 (10.9, 18.8)
2
5
15.6 (12.1, 20.1)
3
3
5
13.1 (8.9, 19.3)
7
5
16.2 (12.4, 21.3)
14
5
30.8 (8.7, 109.5)
21
5
16.6 (11.8, 23.3)
Terminal
NA
NA
-3
5
13.9 (10.9, 17.8)
1
5
15.7 (11.1, 22.2)
2
5
19.0 (7.0, 51.4)
4
3
5
20.2 (16.2, 25.3)
7
4
22.9 (16.3, 32.2)
14
3
19.0 (11.5, 31.2)
21
3
19.6 (9.6, 40.3)
Terminal
1
944.0 (-)
-3
5
16.3 (10.6, 25.3)
1
5
19.1 (12.8, 28.6)
2
5
36.1 (4.6, 286.1)
5
3
4
278.8 (16.4, 4735.3)
7
1
23.7 (-)
14
1
20.5 (-)
21
1
21.8 (-)
Terminal
1
121.2 (-)
J-26
-------
Table 5a. (Continued)
Group
Study Day
N
Geometric Mean
(95% Confidence Interval)
-3
5
12.9 (10.3, 16.1)
1
4
15.1 (12.2, 18.6)
2
3
66.8 (27.2, 164.1)
6
3
3
22.0 (13.1, 37.2)
7
NA
NA
14
NA
NA
21
NA
NA
Terminal
2
301.4 (4.9, 18510.0)
N Number of animals.
Confidence intervals could not be calculated since only one observation was available.
NA Data not available for this Group at this Study Day.
Table 5b. Summary of AST Test Results for the Shifts between Study Days and Baseline
(Study Day -3)
Aspartate AminotransferaseT
Study
Day
Me.
1
in Shift
2
as a Pr<
by <
3
sportior
Broup
4
of Base
5
line,
6
Group
Effect
P-value
Estimated Ratio
(Relationship)
Tu key's P-value#
1
0.79
1.19
1.16
1.13
1.17
1.10
0.2086
2
0.83
1.13
1.26
1.36
2.21 t
5.13 t
0.0233 *
0.16 (1 <6) 0.0155
3
0.83
1.33
1.06
1.45
17.20 t
1.69
<0.0001*
0.05 (1<5) <0.0001*
0.08 (2<5) <0.0001*
0.06 (3<5) <0.0001*
0.08 (4<5) 0.0001
0.10 (6<5) 0.0014
7
0.90
1.37 t
1.31 t
1.69 t
1.71 t
NA
0.0133*
0.53 (1 <4) 0.0097
14
1.19
1.24
2.49 t
1.32
1.47
NA
0.4649
21
1.01
1.34 t
1.35 t
1.36
1.57
NA
0.3843
Terminal
NA
NA
NA
60.13
7.21
23.83
0.465
f Indicates that values for this parameter were log-transformed for the analysis.
# Cells contain all pairwise comparisons that are significant at the 0.05 level. The format within each cell is:
(1) the ratio of shifts, (2) the relationship between the corresponding pair of Group mean shifts shown in
parentheses [For example, "(1<5)" indicates that the mean shift as a proportion of baseline for Group 1 was
less than that for Group 5], and (3) the Tukey-adjusted p-value.
t, | Indicate that the ratio of the geometric mean at baseline and the geometric mean at the Study Day was
significantly different from one (at the 0.05 level), "t" indicates that the geometric mean at the Study Day was
greater than that at baseline, while "J," indicates that the geometric mean at the Study Day was less than that at
baseline.
* Group effect was significant at the 0.05 level.
NA Data not available for this Group at this Study Day.
J-27
-------
Table 6a. Descriptive Statistics for Alanine Aminotransferase (ALT, U/L) by Group and
Study Day
Group
Study Day
N
Geometric Mean
(95% Confidence Interval)
-3
5
35.1 (21.3, 57.7)
1
5
35.4 (23.7, 52.7)
2
5
37.3 (26.1, 53.4)
1
3
5
38.0 (28.3, 51.0)
7
5
37.2 (27.4, 50.4)
14
5
40.9 (25.3, 66.3)
21
5
37.6 (28.7, 49.2)
Terminal
NA
NA
-3
5
22.1 (15.9, 30.6)
1
5
28.3 (21.3, 37.6)
2
5
27.5 (21.7, 34.7)
2
3
5
29.9 (22.3, 40.0)
7
4
34.5 (26.1, 45.4)
14
3
28.8 (21.6, 38.3)
21
5
26.2 (19.9, 34.3)
Terminal
NA
NA
-3
5
22.4 (18.1, 27.6)
1
5
29.2 (23.0, 36.9)
2
5
28.7 (22.3, 36.9)
3
3
5
28.0 (23.0, 34.0)
7
5
30.4 (25.4, 36.4)
14
5
39.2 (24.3, 63.3)
21
5
26.3 (20.8, 33.3)
Terminal
NA
NA
-3
5
21.5 (16.1, 28.8)
1
5
27.1 (19.6, 37.5)
2
5
28.8 (16.8, 49.2)
4
3
5
30.3 (22.3, 41.1)
7
4
31.4 (21.1, 46.8)
14
3
26.5 (13.5, 52.1)
21
3
22.3 (15.6, 32.0)
Terminal
1
143.8 (-)
-3
5
38.0 (24.2, 59.7)
1
5
45.2 (30.7, 66.6)
2
5
58.6 (18.9, 181.8)
5
3
4
165.0 (27.0, 1009.4)
7
1
45.7 (-)
14
1
39.1 (-)
21
1
34.7 (-)
Terminal
1
56.4 (-)
J-28
-------
Table 6a. (Continued)
Group
Study Day
N
Geometric Mean
(95% Confidence Interval)
-3
5
22.5 (18.6, 27.1)
1
4
25.3 (19.7, 32.5)
2
3
43.6 (37.1, 51.3)
6
3
3
29.5 (20.7, 42.2)
7
NA
NA
14
NA
NA
21
NA
NA
Terminal
2
133.0 (56.3, 314.0)
N Number of animals.
Confidence intervals could not be calculated since only one observation was available.
NA Data not available for this Group at this Study Day.
Table 6b. Summary of ALT Test Results for the Shifts between Study Days and Baseline
(Study Day -3)
Alanine Aminotransferase1
Study
Day
Mean S
1
hift as a
2
Proport
3
ion of Be
4
iseline, b
5
y Group
6
Group
Effect
P-value
Estimated Ratio
(Relationship)
Tukey's P-value#
1
1.01
1.28 t
1.30 t
1.26 t
1.19 t
1.05
0.1921
2
1.06
1.24
1.28
1.34
1.54 t
1.91 t
0.2148
3
1.08
1.35
1.25
1.41 |
4.02 t
1.29
0.0005 *
0.27 (1 <5) 0.0003
0.34 (2<5) 0.0026
0.31 (3<5) 0.0012
0.35 (4<5) 0.0037
0.32 (6<5) 0.0061
7
1.06
1.44 t
1.36 t
1.45 t
1.38
NA
0.0861
14
1.17
1.41 t
1.76 t
1.22
1.18
NA
0.1715
21
1.07
1.19
1.18
1.03
1.05
NA
0.7789
Terminal
NA
NA
NA
6.82
2.01
5.76
0.3590
¦f Indicates that values for this parameter were log-transformed for the analysis.
# Cells contain all pairwise comparisons that are significant at the 0.05 level. Hie format within each cell is: (1) the ratio of
shifts, (2) the relationship between the corresponding pair of Group mean shifts shown in parentheses [For
example, "(1<5)" indicates that the mean shift as a proportion of baseline for Group 1 was less than that for Group 5], and
(3) the Tukey-adjusted p-value.
t, | Indicate that the ratio of the geometric mean at baseline and the geometric mean at the Study Day was significantly
different from one (at the 0.05 level), "f" indicates that the geometric mean at the Study Day was greater than that at
baseline, while "J," indicates that the geometric mean at the Study Day was less than that at baseline.
* Group effect was significant at the 0.05 level.
NA Data not available for this Group at this Study Day.
J-29
-------
Table 7 a. Descriptive Statistics for Lactate Dehydrogenase (LDH, U/L) by Group and
Study Day
Group
Study Day
N
Geometric Mean
(95% Confidence Interval)
-3
5
134 (78, 230)
1
5
108 (52, 223)
2
5
78 (50, 122)
1
3
5
51 (33, 77)
7
5
70 (40, 123)
14
5
105 (47, 237)
21
5
118 (79, 178)
Terminal
NA
NA
-3
5
74 (62, 89)
1
5
76 (63, 92)
2
5
71 (43, 117)
2
3
5
63 (37, 108)
7
4
76 (56, 103)
14
3
55 (22, 134)
21
5
87 (64, 118)
Terminal
NA
NA
-3
5
60 (37, 96)
1
5
59 (47, 75)
2
5
57 (50, 66)
3
3
5
40 (31, 53)
7
5
73 (53, 101)
14
5
74 (37, 146)
21
5
90 (61, 134)
Terminal
NA
NA
-3
5
56 (26, 120)
1
5
67 (34, 135)
2
5
52 (26, 106)
4
3
5
53 (31, 91)
7
4
67 (62, 73)
14
3
62 (34, 115)
21
3
112 (41, 306)
Terminal
1
5189 (-)
-3
5
53 (36, 77)
1
5
93 (66, 132)
2
5
118 (35, 401)
5
3
4
341 (82, 1415)
7
1
64 (-)
14
1
113(-)
21
1
70 (-)
Terminal
1
447 (-)
J-30
-------
Table 7 a. (Continued)
Group
Study Day
N
Geometric Mean
(95% Confidence Interval)
-3
5
54 (36, 80)
1
4
62 (33, 118)
2
3
90 (53, 151)
6
3
3
113 (5, 2507)
7
NA
NA
14
NA
NA
21
NA
NA
Terminal
2
2011 (0, 120932398)
N Observation was available.
NA Data not available for this Group at this Number of animals.
Confidence intervals could not be calculated since only one Study Day.
Table 7b. Summary of LDH Test Results for the Shifts between Study Days and Baseline
(Study Day -3)
Lactate Dehydrogenase1
Study
Day
Mean S
1
hift as a
2
Proport
3
ion of Ba
4
iseline, b
5
y Group
6
Group
Effect
P-Value
Estimated Ratio
(Relationship)
Tu key's P-Value#
1
0.81
1.02
0.99
1.20
1.77 t
1.14
0.2734
2
0.58 |
0.95
0.96
0.93
2.24 t
1.85 t
0.0068 *
0.26 (1<5) 0.0049
3
0.38 |
0.85
0.67
0.94
6.58 t
2.33 t
<0.0001*
0.06 (1<5) <0.0001*
0.16 (1<6) 0.0017
0.13 (2<5) 0.0002
0.10 (3<5) <0.0001*
0.29 (3<6) 0.0463
0.14 (4<5) 0.0003
7
0.52 |
1.01
1.22
1.50 t
2.00 t
NA
0.0007*
0.52 (1<2) 0.0416
0.43 (1<3) 0.0043
0.35 (1<4) 0.0011
0.26 (1<5) 0.0091
14
0.78
0.68
1.23
1.37
3.53
NA
0.267
21
0.88
1.18
1.51
2.45 t
2.19
NA
0.1799
Terminal
NA
NA
NA
37.60
7.98
35.37
0.7843
¦f Indicates that values for this parameter were log-transformed for the analysis.
# Cells contain all pairwise comparisons that are significant at the 0.05 level. Hie format within each cell is: (1) the ratio of
shifts, (2) the relationship between the corresponding pair of Group mean shifts shown in parentheses [For
example, "(1<5)" indicates that the mean shift as a proportion of baseline for Group 1 was less than that for Group 5], and
(3) the Tukey-adjusted p-value.
t, | Indicate that the ratio of the geometric mean at baseline and the geometric mean at the Study Day was significantly
different from one (at the 0.05 level), "f" indicates that the geometric mean at the Study Day was greater than that at
baseline, while "J," indicates that the geometric mean at the Study Day was less than that at baseline.
* Group effect was significant at the 0.05 level.
NA Data not available for this Group at this Study Day.
J-31
-------
Table 8a. Descriptive Statistics for Sorbitol Dehydrogenase (SDH, U/L) by Group and
Study Day
Group
Study Day
N
Geometric Mean
(95% Confidence Interval)
-3
5
28.6 (22.0, 37.1)
1
5
23.3 (15.4, 35.2)
2
5
21.0 (12.8, 34.3)
1
3
5
24.8 (18.1, 33.9)
7
5
28.6 (21.9, 37.2)
14
5
30.0 (18.0, 50.0)
21
5
23.1 (15.8, 33.9)
Terminal
NA
NA
-3
5
17.7 (14.1, 22.3)
1
5
20.8 (15.7, 27.6)
2
5
20.7 (17.0, 25.2)
2
3
5
23.5 (17.8, 31.0)
7
4
28.7 (19.4, 42.4)
14
3
22.2 (20.1, 24.4)
21
5
20.4 (17.6, 23.6)
Terminal
NA
NA
-3
5
21.5 (17.3, 26.7)
1
5
26.7 (18.3, 39.0)
2
5
24.9 (14.6, 42.7)
3
3
5
21.9 (14.5, 33.0)
7
5
31.4 (24.1, 40.8)
14
5
41.0 (18.4, 91.5)
21
5
20.8 (15.6, 27.7)
Terminal
NA
NA
-3
5
22.7 (14.6, 35.4)
1
5
25.1 (21.3, 29.6)
2
5
30.2 (14.2, 64.3)
4
3
5
34.1 (21.7, 53.5)
7
4
35.9 (18.8, 68.7)
14
3
24.6 (10.0, 60.6)
21
3
19.4 (6.1, 61.4)
Terminal
1
210.5 (-)
-3
5
24.9 (14.3, 43.2)
1
5
31.5 (21.9, 45.2)
2
5
42.6 (10.1, 179.6)
5
3
4
148.5 (19.4, 1136.5)
7
1
26.7 (-)
14
1
22.9 (-)
21
1
19.0 (-)
Terminal
1
135.9 (-)
J-32
-------
Table 8a. (Continued)
Group
Study Day
N
Geometric Mean
(95% Confidence Interval)
-3
5
18.2 (12.4, 26.7)
1
4
28.5 (20.0, 40.5)
2
3
51.8 (25.0, 107.3)
6
3
2
23.9 (1.1, 517.9)
7
NA
NA
14
NA
NA
21
NA
NA
Terminal
2
120.8 (0.0, 2780073.5)
N Number of animals.
Confidence intervals could not be calculated since only one observation was available.
NA Data not available for this Group at this Study Day.
Table 8b. Summary of SDH Test Results for the Shifts between Study Days and Baseline
(Study Day -3)
Sorbitol Dehydrogenase1
Study
Day
Mean S
1
hift as a
2
Proport
3
ion of Be
4
iseline, b
5
y Group
6
Group
Effect
P-Value
Estimated Ratio
(Relationship)
Tukey's P-Value#
1
0.82
1.17
1.24
1.10
1.27
1.45 t
0.0969
2
0.73
1.16
1.16
1.33
1.71 t
2.78 t
0.0161 *
0.26 (1 <6) 0.0084
3
0.87
1.33
1.02
1.5
6.50 |
1.02
<0.0001*
0.13 (1<5) <0.0001*
0.20 (2<5) 0.0009
0.16 (3<5) 0.0001
0.23 (4<5) 0.0022
0.16 (6<5) 0.0028
7
1.00
1.50 t
1.46 t
1.77 t
1.63 t
NA
0.0031 *
0.67 (1 <2) 0.0268
0.69 (1 <3) 0.0299
0.57 (1 <4) 0.0021
14
1.05
1.24
1.91 t
1.37
1.4
NA
0.3296
21
0.81
1.15
0.97
1.08
1.16
NA
0.2881
Terminal
NA
NA
NA
5.93
3.88
7.33
0.7737
f Indicates that values for this parameter were log-transformed for the analysis.
# Cells contain all pairwise comparisons that are significant at the 0.05 level. The format within each cell is: (1) the
ratio of shifts, (2) the relationship between the corresponding pair of Group mean shifts shown in parentheses [For
example, "(1<5)" indicates that the mean shift as a proportion of baseline for Group 1 was less than that for
Group 5], and (3) the Tukey-adjusted p-value.
|, I Indicate that the ratio of the geometric mean at baseline and the geometric mean at the Study Day was significantly
different from one (at the 0.05 level), "f" indicates that the geometric mean at the Study Day was greater than that at
baseline, while "J," indicates that the geometric mean at the Study Day was less than that at baseline.
* Group effect was significant at the 0.05 level.
NA Data not available for this Group at this Study Day.
J-33
-------
Table 9a. Descriptive Statistics for Total Protein (g/dL) by Group and Study Day
Group
Study Day
N
Geometric Mean
(95% Confidence Interval)
-3
5
5.83 (5.71,
5.95)
1
5
6.00 (5.61,
6.42)
2
5
5.86 (5.55,
6.18)
1
3
5
6.10 (5.71,
6.53)
7
5
6.25 (5.84,
6.68)
14
5
6.19 (5.81,
6.59)
21
5
5.39 (5.16,
5.63)
Terminal
NA
NA
-3
5
5.46 (5.29,
5.64)
1
5
6.18 (6.00,
6.36)
2
5
6.03 (5.80,
6.27)
2
3
5
6.40 (6.05,
6.77)
7
4
6.67 (6.22,
7.15)
14
3
6.25 (5.59,
6.98)
21
5
5.53 (5.33,
5.74)
Terminal
NA
NA
-3
5
5.24 (4.97,
5.52)
1
5
5.72 (5.43,
6.03)
2
5
5.65 (5.34,
5.97)
3
3
5
5.67 (5.27,
6.09)
7
5
6.05 (5.59,
6.55)
14
5
6.06 (5.72,
6.41)
21
5
5.32 (5.18,
5.47)
Terminal
NA
NA
-3
5
5.69 (5.27,
6.16)
1
5
6.02 (5.80,
6.25)
2
5
5.92 (5.38,
6.51)
4
3
5
5.92 (5.14,
6.83)
7
4
6.27 (5.91,
6.65)
14
3
6.32 (5.60,
7.15)
21
3
5.30 (4.99,
5.63)
Terminal
1
4.30 (-
-)
-3
5
5.89 (5.36,
6.48)
1
5
5.99 (5.60,
6.39)
2
5
5.90 (5.55,
6.27)
5
3
4
5.43 (4.67, 6.32)
7
1
6.08 (-)
14
1
6.21 (-)
21
1
5.52 (-)
Terminal
1
5.96 (-)
J-34
-------
Table 9a. (Continued)
Group
Study Day
N
Geometric Mean
(95% Confidence Interval)
-3
5
5.51 (5.17, 5.87)
1
4
5.65 (5.24, 6.10)
2
3
5.74 (4.88, 6.76)
6
3
3
5.36 (4.10, 7.00)
7
NA
NA
14
NA
NA
21
NA
NA
Terminal
2
5.06 (0.91, 28.12)
N Number of animals.
Confidence intervals could not be calculated since only one observation was available.
NA Data not available for this Group at this Study Day.
Table 9b. Summary of Total Protein Test Results for the Shifts between Study Days and
Baseline (Study Day -3)
Total Protein+
Study
Day
Mean S
1
hift as a
2
Proport
3
ion of Ba
4
iseline, b
5
y Group
6
Group
Effect
P-Value
Estimated Ratio
(Relationship)
Tu key's P-Value#
1
1.03
1.13 t
1.09 t
1.06 t
1.02
1.04
0.0216*
0.90 (5<2) 0.0246
2
1.00
1.10 t
1.08 t
1.04
1.00
1.07
0.0579
3
1.05
1.17 t
1.08 t
1.04
0.92
0.99
0.0096 *
0.78 (5<2) 0.0038
7
1.07 t
1.21 t
1.16 t
1.10 t
1.09
NA
0.1346
14
1.06 t
1.15 t
1.16 t
1.12 t
1.11
NA
0.218
21
0.92 |
1.01
1.02
0.94 |
0.99
NA
0.0036 *
0.91 (1<2) 0.0110
0.91 (1<3) 0.0086
Terminal
NA
NA
NA
0.76
1.02
0.91
0.6399
¦f Indicates that values for this parameter were log-transformed for the analysis.
# Cells contain all pairwise comparisons that are significant at the 0.05 level. Hie format within each cell is: (1) the ratio of
shifts, (2) the relationship between the corresponding pair of Group mean shifts shown in parentheses [For
example, "(1<2)" indicates that the mean shift as a proportion of baseline for Group 1 was less than that for Group 2], and
(3) the Tukey-adjusted p-value,
t, | Indicate that the ratio of the geometric mean at baseline and the geometric mean at the Study Day was significantly
different from one (at the 0.05 level), "f" indicates that the geometric mean at the Study Day was greater than that at
baseline, while "J," indicates that the geometric mean at the Study Day was less than that at baseline.
* Group effect was significant at the 0.05 level.
NA Data not available for this Group at this Study Day.
J-35
-------
Table 10a. Descriptive Statistics for Blood Urea Nitrogen (BUN, mg/dL) by Group and
Study Day
Group
Study Day
N
Geometric Mean
(95% Confidence Interval)
-3
5
17.4 (13.5, 22.6)
1
5
20.0 (16.4, 24.4)
2
5
19.2 (16.4, 22.6)
1
3
5
15.8 (12.8, 19.4)
7
5
18.7 (14.7, 23.7)
14
5
21.3 (16.1, 28.1)
21
5
18.6 (15.6, 22.0)
Terminal
NA
NA
-3
5
15.1 (12.6, 18.0)
1
5
17.7 (16.1, 19.4)
2
5
16.2 (12.7, 20.8)
2
3
5
15.1 (11.4, 19.9)
7
4
18.9 (16.4, 21.8)
14
3
20.3 (12.6, 32.7)
21
5
16.6 (13.3, 20.9)
Terminal
NA
NA
-3
5
18.6 (15.7, 22.0)
1
5
20.6 (17.9, 23.6)
2
5
17.6 (15.2, 20.4)
3
3
5
15.7 (13.7, 18.0)
7
5
19.1 (16.8, 21.7)
14
5
21.4 (17.9, 25.5)
21
5
17.2 (15.5, 19.1)
Terminal
NA
NA
-3
5
20.6 (15.9, 26.5)
1
5
20.4 (16.6, 25.1)
2
5
17.8 (14.4, 22.0)
4
3
5
16.2 (12.0, 21.9)
7
4
20.9 (14.4, 30.4)
14
3
24.1 (12.6, 45.9)
21
3
19.4 (13.8, 27.1)
Terminal
1
66.9 (-)
-3
5
20.0 (14.9, 26.8)
1
5
19.7 (15.9, 24.4)
2
5
18.8 (14.5, 24.4)
5
3
4
21.8 (7.3, 65.1)
7
1
18.0 (-)
14
1
20.8 (-)
21
1
17.0 (-)
Terminal
1
20.5 (-)
J-36
-------
Table 10a. (Continued)
Group
Study Day
N
Geometric Mean
(95% Confidence Interval)
-3
5
19.5 (17.0, 22.4)
1
4
18.1 (14.1, 23.1)
2
3
14.5 (10.9, 19.4)
6
3
3
15.1 (8.2, 28.0)
7
NA
NA
14
NA
NA
21
NA
NA
Terminal
2
43.6 (0.2, 8376.0)
N Number of animals.
Confidence intervals could not be calculated since only one observation was available.
NA Data not available for this Group at this Study Day.
Table 10b. Summary of BUN Test Results for the Shifts between Study Days and Baseline
(Study Day -3)
Blood Urea Nitrogen1
Study
Day
Mean S
1
hift as a
2
Proport
3
ion of Ba
4
iseline, b
5
y Group
6
Group
Effect
P-Value
Estimated Ratio
(Relationship)
Tu key's P-Value#
1
1.15 t
1.17 t
1.11 |
0.99
0.98
0.93
0.0058*
0.81 (6<1) 0.0403
0.79 (6<2) 0.0176
2
1.10
1.08
0.95
0.86|
0.94
0.74|
0.0005 *
0.78 (4<1) 0.0233
0.67 (6<1) 0.0008
0.80 (4<2) 0.0470
0.69 (6<2) 0.0015
3
0.91
1.00
0.84
0.79 i
1.12
0.77
0.2116
7
1.07
1.23 t
1.02
0.98
1.03
NA
0.1982
14
1.22 t
1.30 t
1.15
1.11
1.19
NA
0.8626
21
1.06
1.11
0.93
0.89
0.97
NA
0.3365
Terminal
NA
NA
NA
3.72
0.91
2.10
0.4028
t Indicates that values for this parameter were log-transformed for the analysis.
# Cells contain all pairwise comparisons that are significant at the 0.05 level. The format within each cell is:
(1) the ratio of shifts, (2) the relationship between the corresponding pair of Group mean shifts shown in
parentheses [For example, "(6<1)" indicates that the mean shift as a proportion of baseline for Group 6 was
less than that for Group 1], and (3) the Tukey-adjusted p-value.
t, | Indicate that the ratio of the geometric mean at baseline and the geometric mean at the Study Day was
significantly different from one (at the 0.05 level), "t" indicates that the geometric mean at the Study Day was
greater than that at baseline, while "J," indicates that the geometric mean at the Study Day was less than that at
baseline.
* Group effect was significant at the 0.05 level.
NA Data not available for this Group at this Study Day.
J-37
-------
Table 11a. Descriptive Statistics for Creatinine (mg/dL) by Group and Study Day
Group
Study Day
N
Geometric Mean
(95% Confidence Interval)
-3
5
0.96 (0.81, 1.13)
1
5
0.90 (0.72, 1.11)
2
5
0.82 (0.75, 0.91)
1
3
5
0.98 (0.93, 1.04)
7
5
1.06 (0.94, 1.19)
14
5
1.05 (0.90, 1.23)
21
5
1.11 (0.96, 1.27)
Terminal
NA
NA
-3
5
0.78 (0.65, 0.94)
1
5
0.81 (0.64, 1.04)
2
5
0.74 (0.64, 0.86)
2
3
5
0.90 (0.72, 1.12)
7
4
1.15 (0.86, 1.55)
14
3
0.90 (0.64, 1.27)
21
5
1.06 (0.92, 1.23)
Terminal
NA
NA
-3
5
1.03 (0.64, 1.68)
1
5
0.82 (0.73, 0.92)
2
5
0.79 (0.65, 0.94)
3
3
5
0.88 (0.75, 1.04)
7
5
1.06 (0.79, 1.42)
14
5
1.00 (0.88, 1.14)
21
5
1.16 (1.02, 1.32)
Terminal
NA
NA
-3
5
0.98 (0.53, 1.81)
1
5
0.72 (0.63, 0.83)
2
5
0.80 (0.64, 0.99)
4
3
5
0.87 (0.66, 1.16)
7
4
0.93 (0.80, 1.07)
14
3
1.08 (0.98, 1.19)
21
3
1.14 (0.86, 1.51)
Terminal
1
4.92 (-)
-3
5
0.86 (0.77, 0.95)
1
5
0.71 (0.69, 0.73)
2
5
0.88 (0.70, 1.11)
5
3
4
1.29 (0.82, 2.03)
7
1
0.87 (-)
14
1
0.97 (-)
21
1
1.14 (-)
Terminal
1
1.04 (-)
J-38
-------
Table 11a. (Continued)
Group
Study Day
N
Geometric Mean
(95% Confidence Interval)
-3
5
0.75 (0.61, 0.94)
1
4
0.73 (0.60, 0.89)
2
3
0.92 (0.64, 1.33)
6
3
3
0.96 (0.50, 1.85)
7
NA
NA
14
NA
NA
21
NA
NA
Terminal
2
2.88 (0.00, 3335.06)
N Number of animals.
Confidence intervals could not be calculated since only one observation was available.
NA Data not available for this Group at this Study Day.
Table lib. Summary of Creatinine Test Results for the Shifts between Study Days and
Baseline (Study Day -3)
Creatinine+
Study
Day
Mean S
1
hift as a
2
Proport
3
ion of Be
4
iseline, b
5
y Group
6
Group
Effect
P-Value
Estimated Ratio
(Relationship)
Tukey's P-Value#
1
0.94
1.04
0.79
0.74 i
0.83
0.89
0.4206
2
0.86
0.94
0.76 i
0.81
1.03
1.17
0.3001
3
1.03
1.15
0.85
0.89
1.50 t
1.22
0.0777
7
1.11
1.53 t
1.03
1.15
1.10
NA
0.316
14
1.10
1.17
0.97
1.32
1.23
NA
0.6993
21
1.16
1.36 t
1.13
1.39
1.44
NA
0.6842
Terminal
NA
NA
NA
2.31
1.22
3.40
0.7401
f Indicates that values for this parameter were log-transformed for the analysis.
# There were no significant Tukey's pairwise comparisons.
t, | Indicate that the ratio of the geometric mean at baseline and the geometric mean at the Study Day was
significantly different from one (at the 0.05 level), "t" indicates that the geometric mean at the Study Day was
greater than that at baseline, while "J," indicates that the geometric mean at the Study Day was less than that at
baseline.
NA Data not available for this Group at this Study Day.
J-39
-------
Table 12a. Descriptive Statistics for BUN/Creatinine Ratio by Group and Study Day
Group
Study Day
N
Mean
(95% Confidence Interval)
-3
5
184 (15.5, 21.3)
1
5
22.4 (20.1, 24.6)
2
5
23.4 (21.3, 25.5)
1
3
5
16.2 (13.5, 18.9)
7
5
17.8 (14.6, 21.0)
14
5
20.7 (14.8, 26.6)
21
5
16.9 (14.0, 19.8)
Terminal
NA
NA
-3
5
19.5 (14.7, 24.4)
1
5
21.9 (18.3, 25.6)
2
5
22.0 (19.4, 24.7)
2
3
5
16.8 (14.7, 19.0)
7
4
16.5 (12.5, 20.5)
14
3
22.6 (18.5, 26.7)
21
5
15.7 (13.8, 17.6)
Terminal
NA
NA
-3
5
18.8 (11.5, 26.0)
1
5
25.3 (19.9, 30.8)
2
5
22.9 (17.0, 28.8)
3
3
5
18.0 (14.5, 21.4)
7
5
18.4 (12.6, 24.2)
14
5
21.7 (16.0, 27.4)
21
5
14.9 (13.2, 16.6)
Terminal
NA
NA
-3
5
22.9 (12.1, 33.8)
1
5
28.5 (22.2, 34.8)
2
5
22.5 (17.9, 27.2)
4
3
5
19.0 (13.8, 24.2)
7
4
22.8 (16.4, 29.1)
14
3
22.8 (8.0, 37.6)
21
3
17.0 (15.3, 18.7)
Terminal
1
13.6 (-)
-3
5
23.6 (18.7, 28.4)
1
5
28.2 (21.6, 34.8)
2
5
21.8 (15.8, 27.8)
5
3
4
18.3 (3.9, 32.6)
7
1
20.7 (-)
14
1
214 (--)
21
1
14.9 (-)
Terminal
1
19.7 (-)
J-40
-------
Table 12a. (Continued)
Group
Study Day
N
Mean
(95% Confidence Interval)
-3
5
26.9 (19.6, 34.1)
1
4
25.2 (14.6, 35.8)
2
3
15.8 (14.7, 16.9)
6
3
3
15.9 (10.3, 21.4)
7
NA
NA
14
NA
NA
21
NA
NA
Terminal
2
15.4 (0.0a, 42.7)
N Number of animals.
NA Data not available for this Group at this Study Day.
Confidence intervals could not be calculated since only one observation was available,
a Lower bound of confidence interval set to zero as a negative parameter value is not possible.
Table 12b. Summary of BUN/Creatinine Ratio Test Results for the Shifts between Study
Days and Baseline (Study Day -3)
BUN/Creatinine Ratio
Study
Day
Mean S
1
hift as a
2
Proport
3
ion of Ba
4
iseline, b
5
y Group
6
Group
Effect
P-Value
Estimated Difference
(Relationship)
Tukey's P-Value#
1
4.0
2.4
6.5 t
5.6 t
4.6 t
0.5
0.5081
2
5.0 |
2.5
4.1
-0.4
-1.8
-10.0 |
0.0119*
-15.0 (6<1) 0.0098
-12.4 (6<2) 0.0419
-14.1 (6<3) 0.0168
3
-2.2
-2.7
-0.8
-3.9
-4.6
-9.9 i
0.2346
7
-0.6
-4.2
-0.4
-3.8
-1.5
NA
0.6311
14
2.3
1.8
2.9
-4.1
-0.8
NA
0.6404
21
-1.5
-3.8
-3.9
-9.9 i
-7.3
NA
0.2624
Terminal
NA
NA
NA
5.1
-6.9
-10.5
0.5690
# Cells contain all pairwise comparisons that are statistically significant at the 0.05 level. The format within each
cell is: (1) the difference of shifts, (2) the relationship between the corresponding pair of Group mean shifts
shown in parentheses [For example, "(6<1)" indicates that the mean shift from baseline for Group 6 was less
than that for Group 1], and (3) the Tukey-adjusted p-value.
t, | Indicate that the difference between the mean at baseline and the mean at the Study Day was significantly
different from zero (at the 0.05 level), "t" indicates that the mean at the Study Day was greater than that at
baseline, while "J," indicates that the mean at the Study Day was less than that at baseline.
* Group effect was significant at the 0.05 level.
NA Data not available for this Group at this Study Day.
J-41
-------
13a. Descriptive Statistics for Sodium (mEq/L) by Group and Study
Group
Study Day
N
Mean
(95% Confidence Interval)
-3
5
144 (142, 146)
1
5
145 (144, 147)
2
5
145 (144, 146)
1
3
5
144 (141, 147)
7
5
145 (142, 148)
14
5
145 (143, 146)
21
5
141 (140, 142)
Terminal
NA
NA
-3
5
144 (142, 146)
1
5
146 (145, 148)
2
5
145 (144, 147)
2
3
5
146 (144, 147)
7
4
151 (144, 158)
14
4
145 (141, 148)
21
5
142 (140, 143)
Terminal
NA
NA
-3
5
138 (128, 148)
1
5
145 (143, 147)
2
5
146 (144, 147)
3
3
5
146 (144, 147)
7
5
148 (144, 151)
14
5
145 (143, 146)
21
5
142 (141, 142)
Terminal
NA
NA
-3
5
140 (131, 150)
1
5
146 (145, 147)
2
5
146 (144, 149)
4
3
5
148 (142, 155)
7
4
146 (145, 147)
14
3
146 (-)
21
3
141 (134, 148)
Terminal
1
139 (-)
-3
5
146 (143, 149)
1
5
146 (144, 147)
2
5
145 (143, 147)
5
3
4
145 (144, 146)
7
1
143 (-)
14
1
145 (-)
21
1
141 (-)
Terminal
1
143 (-)
-------
Table 13a. (Continued)
Group
Study Day
N
Mean
(95% Confidence Interval)
-3
5
144 (142, 145)
1
4
146 (145, 147)
2
3
143 (136, 151)
6
3
3
141 (138, 144)
7
NA
NA
14
NA
NA
21
NA
NA
Terminal
2
136 (0a, 320)
N Number of animals.
NA Data not available for this Group at this Study Day.
Confidence intervals could not be calculated since only one observation was available,
a Lower bound of confidence interval set to zero as a negative parameter value is not possible.
Table 13b. Summary of Sodium Test Results for the Shifts between Study Days and
Baseline (Study Day -3)
Sodium
Study
Day
Mean S
1
hift as a
2
Proport
3
ion of Be
4
iseline, b
5
y Group
6
Group
Effect
P-Value
Estimated Difference
(Relationship)
Tu key's P-Value#
1
1
2
7t
6 t
-1
2
0.145
2
1
2
8 t
6 t
-1
-1
0.0504
3
0
2
8 |
8 |
-2
-3
0.0017*
-9 (5<3) 0.0256
-10 (6<3) 0.0240
-10 (5<4) 0.0158
-11 (6<4) 0.0154
7
1
7 t
10 t
3
-1
NA
0.1681
14
1
1
7t
3
1
NA
0.2442
21
-3
-2
4
-2
-3
NA
0.1981
Terminal
NA
NA
NA
12
-2
-9
0.7947
# Cells contain all pairwise comparisons that are statistically significant at the 0.05 level. The format within each
cell is: (1) the difference of shifts, (2) the relationship between the corresponding pair of Group mean shifts
shown in parentheses [For example, "(5<3)" indicates that the mean shift from baseline for Group 5 was less
than that for Group 3], and (3) the Tukey-adjusted p-value.
t, | Indicate that the difference between the mean at baseline and the mean at the Study Day was significantly
different from zero (at the 0.05 level), "t" indicates that the mean at the Study Day was greater than that at
baseline, while "J," indicates that the mean at the Study Day was less than that at baseline.
* Group effect was significant at the 0.05 level.
NA Data not available for this Group at this Study Day.
J-43
-------
Table 14a. Descriptive Statistics for Potassium (mEq/L) by Group and Study Day
Group
Study Day
N
Geometric Mean
(95% Confidence Interval)
-3
5
4.3 (3.5, 5.4)
1
5
4.9 (4.2, 5.7)
2
5
4.7 (4.3, 5.2)
1
3
5
4.8 (4.3, 5.5)
7
5
5.3 (4.8, 5.9)
14
5
5.5 (5.4, 5.6)
21
5
3.7 (3.4, 3.9)
Terminal
NA
NA
-3
5
4.5 (4.3, 4.8)
1
5
5.2 (4.4, 6.1)
2
5
5.1 (4.4, 5.9)
2
3
5
5.3 (4.6, 6.0)
7
4
5.2 (4.6, 5.9)
14
4
6.0 (4.8, 7.5)
21
5
3.8 (3.5, 4.1)
Terminal
NA
NA
-3
5
4.2 (3.9, 4.4)
1
5
4.5 (4.3, 4.8)
2
5
4.6 (4.1, 5.1)
3
3
5
4.4 (4.2, 4.7)
7
5
5.1 (3.9, 6.9)
14
5
5.3 (4.9, 5.8)
21
5
3.8 (3.6, 4.0)
Terminal
NA
NA
-3
5
4.7 (4.1, 5.5)
1
5
4.7 (4.2, 5.1)
2
5
4.7 (4.2, 5.3)
4
3
5
5.0 (4.0, 6.4)
7
4
5.8 (4.8, 7.0)
14
3
6.0 (5.1, 6.9)
21
3
3.9 (3.5, 4.3)
Terminal
1
21.2 (-)
-3
5
4.8 (3.7, 6.2)
1
5
4.6 (4.1, 5.3)
2
5
4.4 (3.6, 5.2)
5
3
4
4.0 (3.3, 4.8)
7
1
4.8 (-)
14
1
4.9 (-)
21
1
3.9 (-)
Terminal
1
11.4 (-)
J-44
-------
Table 14a. (Continued)
Group
Study Day
N
Geometric Mean
(95% Confidence Interval)
-3
5
4.9 (4.5, 5.3)
1
4
4.8 (4.3, 5.4)
2
3
3.7 (3.2, 4.3)
6
3
3
4.5 (3.0, 6.6)
7
NA
NA
14
NA
NA
21
NA
NA
Terminal
2
22.5 (0.8, 640.0)
N Number of animals.
NA Data not available for this Group at this Study Day.
Confidence intervals could not be calculated since only one observation was available.
Table 14b. Summary of Potassium Test Results for the Shifts between Study Days and
Baseline (Study Day -3)
Potassium+
Study
Day
Mean S
1
hift as a
2
Proport
3
ion of Ba
4
iseline, b
5
y Group
6
Group
Effect
P-Value
Estimated Ratio
(Relationship)
Tukey's P-Value#
1
1.13
1.14 t
1.09
0.99
0.97
1.01
0.2939
2
1.09
1.12
1.10
0.99
0.91
0.77 i
0.0392 *
3
1.12
1.16 t
1.06
1.06
0.85 i
0.92
0.0543
7
1.22 t
1.16
1.24 t
1.24 t
1.14
NA
0.9793
14
1.27 t
1.30 t
1.29 t
1.28 t
1.17
NA
0.9584
21
0.85 i
0.83 i
0.91
0.83 i
0.93
NA
0.6789
Terminal
NA
NA
NA
4.24
2.24
4.74
0.4641
f Indicates that values for this parameter were log-transformed for the analysis.
# There were no significant Tukey's pairwise comparisons.
1.1 Indicate that the ratio of the geometric mean at baseline and the geometric mean at the Study Day was
significantly different from one (at the 0.05 level), "t" indicates that the geometric mean at the Study Day was
greater than that at baseline, while "J," indicates that the geometric mean at the Study Day was less than that at
baseline.
* Group effect was significant at the 0.05 level.
NA Data not available for this Group at this Study Day.
J-45
-------
Table 15a. Descriptive Statistics for Chloride (mEq/L) by Group and Study Day
Mean
Group
Study Day
N
(95%
Confidence
Interval)
-3
5
103 (101, 105)
1
5
107 (105, 108)
2
5
108 (106, 110)
1
3
5
102 (98, 105)
7
5
104 (102, 107)
14
5
106 (104, 108)
21
5
103 (101, 104)
Terminal
NA
NA
-3
5
107 (105, 109)
1
5
106 (105, 108)
2
5
107 (106, 108)
2
3
5
105 (103, 106)
7
4
110 (103, 117)
14
4
107 (105, 108)
21
5
105 (101, 108)
Terminal
NA
NA
-3
5
103 (95, 111)
1
5
107 (105, 109)
2
5
107 (106, 109)
3
3
5
106 (104, 108)
7
5
108 (104, 113)
14
5
108 (107, 109)
21
5
105 (103, 107)
Terminal
NA
NA
-3
5
103 (95, 111)
1
5
106 (105, 107)
2
5
105 (103, 108)
4
3
5
106 (103, 110)
7
4
107 (104, 110)
14
3
109 (105, 113)
21
3
104 (102, 106)
Terminal
1
86 (-)
-3
5
106 (103, 110)
1
5
104 (101, 107)
2
5
104 (103, 106)
5
3
4
102 (99, 104)
7
1
106 (-)
14
1
108 (-)
21
1
103 (-)
Terminal
1
95 (-)
J-46
-------
Table 15a. (Continued)
Group
Study Day
N
Mean
(95% Confidence Interval)
-3
5
107 (103, 112)
1
4
106 (104, 108)
2
3
105 (101, 108)
6
3
3
102 (99, 105)
7
NA
NA
14
NA
NA
21
NA
NA
Terminal
2
86 (0a, 194)
N Number of animals.
NA Data not available for this Group at this Study Day.
Confidence intervals could not be calculated since only one observation was available,
a Lower bound of confidence interval set to zero as a negative parameter value is not possible.
Table 15b. Summary of Chloride Test Results for the Shifts between Study Days and
Baseline (Study Day -3)
Chloride
Study
Day
Mean S
1
hift as a
2
Proport
3
ion of Be
4
iseline, b
5
y Group
6
Group
Effect
P-Value
Estimated Difference
(Relationship)
Tu key's P-Value#
1
4
-1
4
3
-2
-2
0.1233
2
5 t
0
4 t
2
-2
-4
0.0393*
3
-1
-2
3
3
-5|
"7 I
0.0102*
-10 (6<3) 0.0451
-10 (6<4) 0.0393
7
1
3
5
2
2
NA
0.8904
14
3
-1
5 t
4
4
NA
0.4181
21
0
-2
2
-1
-1
NA
0.6619
Terminal
NA
NA
NA
-6
-10
-23
0.7402
# Cells contain all pairwise comparisons that are statistically significant at the 0.05 level. The format within each
cell is: (1) the difference of shifts, (2) the relationship between the corresponding pair of Group mean shifts
shown in parentheses [For example, "(6<3)" indicates that the mean shift from baseline for Group 6 was less
than that for Group 3], and (3) the Tukey-adjusted p-value.
t, | Indicate that the difference between the mean at baseline and the mean at the Study Day was significantly
different from zero (at the 0.05 level), "t" indicates that the mean at the Study Day was greater than that at
baseline, while "J," indicates that the mean at the Study Day was less than that at baseline.
* Group effect was significant at the 0.05 level.
NA Data not available for this Group at this Study Day.
J-47
-------
Table 16a. Descriptive Statistics for Calcium (mg/dL) by Group and Study Day
Group
Study Day
N
Mean
(95% Confidence Interval)
1
-3
5
13.65 (12.42, 14.88)
1
5
14.20 (13.33, 15.08)
2
5
14.07 (13.24, 14.91)
3
5
14.01 (13.15, 14.86)
7
5
14.72 (14.12, 15.32)
14
5
15.11 (14.47, 15.75)
21
5
12.48 (12.08, 12.89)
Terminal
NA
NA
2
-3
5
12.56 (11.87, 13.26)
1
5
14.54 (13.60, 15.48)
2
5
14.11 (13.39, 14.82)
3
5
14.40 (13.58, 15.22)
7
4
14.33 (13.29, 15.38)
14
4
15.02 (13.70, 16.34)
21
5
12.63 (12.24, 13.02)
Terminal
NA
NA
3
-3
5
12.72 (11.88, 13.56)
1
5
13.84 (13.02, 14.66)
2
5
13.86 (12.74, 14.98)
3
5
13.55 (12.56, 14.54)
7
5
14.47 (13.13, 15.80)
14
5
14.91 (13.80, 16.03)
21
5
12.45 (12.24, 12.67)
Terminal
NA
NA
4
-3
5
13.59 (12.76, 14.43)
1
5
14.35 (13.99, 14.71)
2
5
14.04 (12.58, 15.49)
3
5
14.14 (11.95, 16.33)
7
4
14.86 (13.94, 15.78)
14
3
15.51 (13.36, 17.66)
21
3
12.58 (11.19, 13.97)
Terminal
1
16.09 (-)
5
-3
5
13.97 (13.00, 14.95)
1
5
14.22 (13.78, 14.66)
2
5
13.03 (10.52, 15.54)
3
4
10.78 (5.72, 15.84)
7
1
13.72 (-)
14
1
14.48 (-)
21
1
12.59 (-)
Terminal
1
15.59 (-)
J-48
-------
Table 16a. (Continued)
Group
Study Day
N
Mean
(95% Confidence Interval)
-3
5
13.41 (12.89, 13.93)
1
4
13.90 (12.95, 14.84)
2
3
11.48 (9.50, 13.46)
6
3
3
11.36 (10.35, 12.37)
7
NA
NA
14
NA
NA
21
NA
NA
Terminal
2
12.71 (7.81, 17.60)
N Number of animals.
NA Data not available for this Group at this Study Day.
Confidence intervals could not be calculated since only one observation was available.
Table 16b. Summary of Calcium Test Results for the Shifts between Study Days and
Baseline (Study Day -3)
Calcium
Study
Day
Mean S
1
hift as a
2
Proport
3
ion of Be
4
iseline, b
5
y Group
6
Group
Effect
P-Value
Estimated Difference
(Relationship)
Tu key's P-Value#
1
0.56
1.97 t
1.12 t
0.76
0.25
0.64
0.0522
-1.73 (5<2) 0.0351
2
0.42
1.54 t
1.14
0.44
-0.95
-1.76 i
0.0186*
-3.30 (6<2) 0.0316
3
0.36
1.84 |
0.83
0.55
-3.11 |
-1.88
0.0060 *
-4.95 (5<2) 0.0054
-3.94 (5<3) 0.0354
7
1.07 t
1.62 t
1.74 t
1.13
0.6
NA
0.7459
14
1.46 t
2.60 t
2.19 t
1.88 t
1.36
NA
0.5546
21
-1.16 X
0.07
-0.27
-1.05
-0.53
NA
0.2304
Terminal
NA
NA
NA
3.04
1.29
-0.73
0.1228
# Cells contain all pairwise comparisons that are statistically significant at the 0.05 level. The format within each
cell is: (1) the difference of shifts, (2) the relationship between the corresponding pair of Group mean shifts
shown in parentheses [For example, "(5<2)" indicates that the mean shift from baseline for Group 5 was less
than that for Group 2], and (3) the Tukey-adjusted p-value.
t, | Indicate that the difference between the mean at baseline and the mean at the Study Day was significantly
different from zero (at the 0.05 level), "t" indicates that the mean at the Study Day was greater than that at
baseline, while "J," indicates that the mean at the Study Day was less than that at baseline.
* Group effect was significant at the 0.05 level.
NA Data not available for this Group at this Study Day.
J-49
-------
Table 17a. Descriptive Statistics for Phosphorus (mg/dL) by Group and Study Day
Group
Study Day
N
Geometric Mean
(95% Confidence Interval)
-3
5
4.38 (4.19, 4.58)
1
5
3.81 (2.97, 4.87)
2
5
3.71 (3.00, 4.60)
1
3
5
4.90 (4.31, 5.58)
7
5
5.26 (4.37, 6.34)
14
5
4.60 (3.71, 5.69)
21
5
5.59 (5.00, 6.25)
Terminal
NA
NA
-3
5
3.10 (2.35, 4.10)
1
5
3.53 (3.09, 4.02)
2
5
3.55 (2.96, 4.27)
2
3
5
4.30 (3.59, 5.15)
7
4
5.57 (4.72, 6.58)
14
3
4.66 (3.75, 5.80)
21
5
5.76 (5.03, 6.60)
Terminal
NA
NA
-3
5
2.98 (2.64, 3.36)
1
5
3.42 (3.07, 3.80)
2
5
3.24 (2.98, 3.52)
3
3
5
3.85 (3.59, 4.12)
7
5
4.83 (3.95, 5.92)
14
5
4.33 (3.85, 4.86)
21
5
5.83 (5.49, 6.19)
Terminal
NA
NA
-3
5
3.19 (2.85, 3.57)
1
5
3.21 (2.61, 3.95)
2
5
3.26 (2.45, 4.34)
4
3
5
3.58 (2.62, 4.91)
7
4
3.82 (2.78, 5.25)
14
3
4.47 (3.52, 5.66)
21
3
5.57 (5.14, 6.04)
Terminal
1
16.39 (-)
-3
5
3.13 (2.54, 3.86)
1
5
3.18 (2.68, 3.77)
2
5
3.68 (2.90, 4.67)
5
3
4
4.82 (2.98, 7.81)
7
1
4.13 (-)
14
1
4.26 (-)
21
1
5.36 (-)
Terminal
1
6.38 (-)
J-50
-------
Table 17a. (Continued)
Group
Study Day
N
Geometric Mean
(95% Confidence Interval)
-3
5
3.33 (3.13, 3.54)
1
4
3.68 (3.02, 4.49)
2
3
4.57 (2.83, 7.36)
6
3
3
4.44 (3.07, 6.42)
7
NA
NA
14
NA
NA
21
NA
NA
Terminal
2
18.59 (8.95, 38.61)
N Number of animals.
NA Data not available for this Group at this Study Day.
Confidence intervals could not be calculated since only one observation was available.
Table 17b. Summary of Phosphorus Test Results for the Shifts between Study Days and
Baseline (Study Day -3)
Phosphorus+
Study
Day
Mean S
1
hift as a
2
Proport
3
ion of Be
4
iseline, b
5
y Group
6
Group
Effect
P-Value
Estimated Ratio
(Relationship)
Tukey's P-Value#
1
0.87 i
1.14
1.15 t
1.01
1.02
1.09
0.0578
2
0.85|
1.15 t
1.09
1.02
1.17 t
1.35 t
0.0038*
0.74 (1<2) 0.0341
0.72 (1 <5) 0.0193
0.63 (1 <6) 0.0028
3
1.12
1.39 t
1.29 t
1.12
1.56 t
1.31 t
0.0935
7
1.20 t
1.64 t
1.62 t
1.22 t
1.30 t
NA
0.0021 *
0.73 (1<2) 0.0105
0.74 (1 <3) 0.0090
0.74 (4<2) 0.0216
0.75 (4<3) 0.0200
14
1.05
1.53 t
1.45 t
1.36 t
1.34
NA
0.0350*
0.69 (1<2) 0.0473
0.72 (1 <3) 0.0471
21
1.28 t
1.86 t
1.96 t
1.70 t
1.69 t
NA
0.0011 *
0.69 (1<2) 0.0027
0.65 (1 <3) 0.0008
Terminal
NA
NA
NA
4.76 t
1.91 |
5.68 t
0.0398*
0.34 (5<6) 0.0386
f Indicates that values for this parameter were log-transformed for the analysis.
# Cells contain all pairwise comparisons that are significant at the 0.05 level. The format within each cell is:
(1) the ratio of shifts, (2) the relationship between the corresponding pair of Group mean shifts shown in
parentheses [For example, "(1<2)" indicates that the mean shift as a proportion of baseline for Group 1 was
less than that for Group 2], and (3) the Tukey-adjusted p-value.
t, | Indicate that the ratio of the geometric mean at baseline and the geometric mean at the Study Day was
significantly different from one (at the 0.05 level), "t" indicates that the geometric mean at the Study Day was
greater than that at baseline, while "J," indicates that the geometric mean at the Study Day was less than that at
baseline.
* Group effect was significant at the 0.05 level.
NA Data not available for this Group at this Study Day.
J-51
-------
Table 18. Proportion of Animals that were Abnormal with Exact 95% Confidence Interval
by Parameter and Group
Parameter
Group
Number
Abnormal / N
Proportion Abnormal
(95% Confidence Interval)
C-Reactive Protein
1
5/5
1.00 (0.48, 1.00)
2
5/5
1.00 (0.48, 1.00)
3
4/5
0.80 (0.28, 0.99)
4
4/5
0.80 (0.28, 0.99)
5
5/5
1.00 (0.48, 1.00)
6
2/4
0.50 (0.07, 0.93)
Aspartate
Aminotransferase
1
2/5
0.40 (0.05, 0.85)
2
0/5
0.00 (0.00, 0.52)
3
2/5
0.40 (0.05, 0.85)
4
1/5
0.20 (0.01, 0.72)
5
4/5
0.80 (0.28, 0.99)
6
2/4
0.50 (0.07, 0.93)
Alanine Aminotransferase
1
0/5
0.00 (0.00, 0.52)
2
0/5
0.00 (0.00, 0.52)
3
2/5
0.40 (0.05, 0.85)
4
1/5
0.20 (0.01, 0.72)
5
3/5
0.60 (0.15, 0.95)
6
1/4
0.25 (0.01, 0.81)
Lactate Dehydrogenase
1
2/5
0.40 (0.05, 0.85)
2
0/5
0.00 (0.00, 0.52)
3
0/5
0.00 (0.00, 0.52)
4
3/5
0.60 (0.15, 0.95)
5
5/5
1.00 (0.48, 1.00)
6
2/4
0.50 (0.07, 0.93)
Sorbitol Dehydrogenase
1
2/5
0.40 (0.05, 0.85)
2
0/5
0.00 (0.00, 0.52)
3
2/5
0.40 (0.05, 0.85)
4
3/5
0.60 (0.15, 0.95)
5
4/5
0.80 (0.28, 0.99)
6
2/4
0.50 (0.07, 0.93)
Total Protein
1
2/5
0.40 (0.05, 0.85)
2
4/5
0.80 (0.28, 0.99)
3
4/5
0.80 (0.28, 0.99)
4
3/5
0.60 (0.15, 0.95)
5
1/5
0.20 (0.01, 0.72)
6
1/4
0.25 (0.01, 0.81)
Blood Urea Nitrogen
1
1/5
0.20 (0.01, 0.72)
2
1/5
0.20 (0.01, 0.72)
3
1/5
0.20 (0.01, 0.72)
4
2/5
0.40 (0.05, 0.85)
5
1/5
0.20 (0.01, 0.72)
6
1/4
0.25 (0.01, 0.81)
J-52
-------
Table 18. (Continued)
Parameter
Group
Number
Abnormal / N
Proportion Abnormal
(95% Confidence Interval)
Creatinine
1
0/5
0.00 (0.00, 0.52)
2
1/5
0.20 (0.01, 0.72)
3
1/5
0.20 (0.01, 0.72)
4
2/5
0.40 (0.05, 0.85)
5
1/5
0.20 (0.01, 0.72)
6
1/4
0.25 (0.01, 0.81)
BUN/Creatinine Ratio
1
0/5
0.00 (0.00, 0.52)
2
0/5
0.00 (0.00, 0.52)
3
2/5
0.40 (0.05, 0.85)
4
2/5
0.40 (0.05, 0.85)
5
0/5
0.00 (0.00, 0.52)
6
1/4
0.25 (0.01, 0.81)
Sodium
1
0/5
0.00 (0.00, 0.52)
2
0/5
0.00 (0.00, 0.52)
3
2/5
0.40 (0.05, 0.85)
4
1/5
0.20 (0.01, 0.72)
5
0/5
0.00 (0.00, 0.52)
6
1/4
0.25 (0.01, 0.81)
Potassium
1
5/5
1.00 (0.48, 1.00)
2
4/5
0.80 (0.28, 0.99)
3
3/5
0.60 (0.15, 0.95)
4
3/5
0.60 (0.15, 0.95)
5
2/5
0.40 (0.05, 0.85)
6
2/4
0.50 (0.07, 0.93)
Chloride
1
0/5
0.00 (0.00, 0.52)
2
0/5
0.00 (0.00, 0.52)
3
1/5
0.20 (0.01, 0.72)
4
1/5
0.20 (0.01, 0.72)
5
1/5
0.20 (0.01, 0.72)
6
1/4
0.25 (0.01, 0.81)
Calcium
1
3/5
0.60 (0.15, 0.95)
2
5/5
1.00 (0.48, 1.00)
3
4/5
0.80 (0.28, 0.99)
4
3/5
0.60 (0.15, 0.95)
5
2/5
0.40 (0.05, 0.85)
6
2/4
0.50 (0.07, 0.93)
Phosphorus
1
0/5
0.00 (0.00, 0.52)
2
5/5
1.00 (0.48, 1.00)
3
5/5
1.00 (0.48, 1.00)
4
4/5
0.80 (0.28, 0.99)
5
4/5
0.80 (0.28, 0.99)
6
2/4
0.50 (0.07, 0.93)
N Number of animals.
J-53
-------
Table 19. Results of Overall Two-sided Fisher's Exact Test for the Proportion of Animals
that were Abnormal by Parameter
Parameter
Group Effect P-Value
C-Reactive Protein
0.1737
Aspartate Aminotransferase
0.1892
Alanine Aminotransferase
0.2039
Lactate Dehydrogenase
0.0046 *
Sorbitol Dehydrogenase
0.2411
Total Protein
0.3341
Blood Urea Nitrogen
1.0000
Creatinine
0.9079
BUN/Creatinine Ratio
0.2158
Sodium
0.3421
Potassium
0.4908
Chloride
0.8684
Calcium
0.4908
Phosphorus
0.0020 *
*Group effect was significant at the 0.05 level.
Table 20. Results of One-sided Pairwise Fisher's Exact Tests for the Proportion of Animals
with Abnormal Lactate Dehydrogenase and Phosphorus
One-Sided Pairwise Fisher's Exact Test P-Values
Parameter
Group
Unadjusted P-Values
Bonferroni-Holm Adjusted P-Values
1.0000
1.0000
0.5000
0.0833
0.6429
1.0000
1.0000
1.0000
1.0000
1.0000
Lactate
Dehydrogenase
Phosphorus
0.1667
,0000a
1.0000
0.0556
1.0000
1 0.0040*
0.0040*
0.0238*
0.0238*
0.1667
0.0556
1.0000
0.0556
1.0000
•m.
0.0556
0.2857
0.2857
1.0000
1.0000
1.0000
0.9524
0.9524
0.1667
0.8333
1.0000
,0000a
1.0000
1.0000
1.0000
)
1.0000
1.0000
1.0000
1.0000
1.0000
1.0000
1.0000
1.0000
a A p-value of 1.0000 was substituted when all animals in both Groups experienced the same abnormality result.
* Comparison significant at the 0.05 level.
J-54
-------
Table 21. Results of Overall Log-rank Tests for Time to Abnormality by Parameter
Parameter
Group Effect P-Value
C-Reactive Protein
0.2267
Aspartate Aminotransferase
0.1726
Alanine Aminotransferase
0.1810
Lactate Dehydrogenase
0.0046 *
Sorbitol Dehydrogenase
0.2478
Total Protein
0.0532
Blood Urea Nitrogen
0.9773
Creatinine
0.7911
BUN/Creatinine Ratio
0.2258
Sodium
0.3132
Potassium
0.2245
Chloride
0.7951
Calcium
0.0764
Phosphorus
0.0028 *
* Group effect was significant at the 0.05 level.
Table 22. Results of the Pairwise Log-rank Tests for Time to Abnormal Lactate
Dehydrogenase and Phosphorus
* Comparison significant at the 0.05 level.
Pairwise Log-Rank Test P-Values
0.3253
0.1198
1.0000
0.8478
Phosphorus
0.1558
0.1060
0.9346
0.8478
0.6438
0.5881
1.0000
1.0000
0.5076
1.0000
Parameter Group
Unadjusted P-Values
Bonferroni-Holm Adjusted P-Values
0.8957
1.0000
Lactate
Dehydrogenase
0.1120
0.0019*
0.8348
0.0888
1.0000
0.0019*
0.0888
0.0284*
0.8881
0.0284*
0.8881
0.1618
1.0000
0.1734
0.9400
0.7993
0.0124*
0.0144*
0.7317
0.1758
0.0888
0.0303*
J-55
-------
### Group 1:
Negative Control
+*~ Group 2: 100 CFU
~+~ Group 3:
1,000 CFU
Group 4: 10,000 CFU
¦¦¦ Group 5:
100,000 CFU
Group 6: High-Dose Control
Study Day
Figure 1. Plot of C-Reactive Protein over time.
J-56
-------
10000
1000
100
10
Group 1: Negative Control
Group 3: 1,000 CFU
Group 5: 100,000 CFU
Group 2: 100 CFU
AAA Group 4: 10,000 CFU
Group 6: High-Dose Control
-3
1 2 3
14
21
Study Day
Figure 2. Plot of Aspartate Aminotransferase over time.
J-57
-------
1000CH
Group 1: Negative Control
Group 3: 1,000 CFU
Group 5: 100,000 CFU
>!< v X*
Group 2: 100 CFU
Group 4: 10,000 CFU
Group 6: High-Dose Control
1000:
100
10i
1 2 3
14
21
Study Day
Figure 3. Plot of Alanine Aminotransferase over time.
J-58
-------
10000
Group 1: Negative Control
Group 3: 1,000 CFU
Group 5: 100,000 CFU
v X* V
Group 2: 100 CFU
Group 4: 10,000 CFU
Group 6: High-Dose Control
1000:
100
10J
1 2 3
14
21
Study Day
Figure 4. Plot of Lactate Dehydrogenase over time.
J-59
-------
10000J
Group 1: Negative Control
Group 3: 1,000 CFU
Group 5: 100,000 CFU
X'
Group 2: 100 CFU
Group 4: 10,000 CFU
Group 6: High-Dose Control
1000-
100
10:
1 2 3
14
21
Study Day
Figure 5. Plot of Sorbitol Dehydrogenase over time.
J-60
-------
10
••• Group 1: Negative Control
~~~ Group 3: 1,000 CFU
¦¦¦ Group 5: 100,000 CFU
Group 2: 100 CFU
Group 4: 10,000 CFU
'i' * * Group 6: High-Dose Control
-it
-3
1 2 3
14
21
Study Day
Figure 6. Plot of Total Protein over time.
J-61
-------
100
Group 1: Negative Control
Group 3: 1,000 CFU
Group 5: 100,000 CFU
Group 2: 100 CFU
Group 4: 10,000 CFU
*-*-* Group 6: High-Dose Control
10
-3
1 2 3
14
21
Study Day
Figure 7. Plot of Blood Urea Nitrogen over time.
J-62
-------
10
•++ Group 1:
Negative Control
Group 2:
100 CFU
~+~ Group 3:
1,000 CFU
Group 4:
10,000 CFU
¦M Group 5:
100,000 CFU
W Group 6:
High-Dose Control
0.1
II
-3
1 2 3
14
21
Study Day
Figure 8. Plot of Creatinine over time.
J-63
-------
55
50:
45
40
35
30
25
20
15;
10
5
0
Group 1: Negative Control
Group 3: 1,000 CFU
Group 5: 100,000 CFU
AAA
Group 2: 100 CFU
Group 4: 10,000 CFU
Group 6: High-Dose Control
-3
1 2 3
14
21
Study Day
Figure 9.
Plot of BUN/Creatinine Ratio over time.
J-64
-------
160
Group 1: Negative Control
Group 3: 1,000 CFU
Group 5: 100,000 CFU
Group 2: 100 CFU
Group 4: 10,000 CFU
Group 6: High-Dose Control
Study Day
Figure 10. Plot of Sodium over time.
J-65
-------
++# Group 1: Negative Control
~+~ Group 3: 1,000 CFU
umm Group 5: 100,000 CFU
Group 2: 100 CFU
Group 4: 10,000 CFU
'I' * * Group 6: High-Dose Control
-3
1 2 3
14
21
Study Day
Plot of Potassium over time.
J-66
-------
120
Group 1: Negative Control
Group 3: 1,000 CFU
Group 5: 100,000 CFU
Group 2: 100 CFU
Group 4: 10,000 CFU
Group 6: High-Dose Control
Study Day
Figure 12. Plot of Chloride over time.
J-67
-------
18;
16;
14
12
10-
8:
6;
4
2
0
Group 1: Negative Control
Group 3: 1,000 CFU
Group 5: 100,000 CFU
Group 2: 100 CFU
Group 4: 10,000 CFU
*> Group 6: High-Dose Control
Study Day
Plot of Calcium over time.
J-68
-------
10
••• Group 1: Negative Control
Group 2: 100 CFU
~~~ Group 3: 1,000 CFU 1
Group 4: 10,000 CFU
1
—I // 1—
¦Wl Group 5: 100,000 CFU *
I I =1=
Group 6: High-Dose Control
I I
Study Day
Figure 14. Plot of Phosphorus over time.
J-69
-------
Group 1: Negative Control
Group 3: 1,000 CFU
Group 5: 100,000 CFU
Group 2: 100 CFU
Group 4: 10,000 CFU
Group 6: High-Dose Control
~~r~
0
~T~
7
14
—r
21
Figure 15.
Days to Abnormal (Post Challenge)
Kaplan-Meier curves representing time to abnormal Lactate Dehydrogenase data
for each group.
J-70
-------
"I
¦Group 1: Negative Control
¦Group 2: 100 CFU
Group 3: 1,000 CFU
Group 4: 10,000 CFU
Group 5: 100,000 CFU
Group 6: High-Dose Control
-1
0
I
T
7
14
—r
21
Days to Abnormal (Post Challenge)
Figure 16. Kaplan-Meier curves representing time to abnormal Phosphorus data for each
group.
1-71
-------
Attachment 1:
Results of Analysis Repeated with
Potential Outliers Excluded
-------
Attachment 1: Results of Analysis Repeated with Potential Outliers Excluded
The clinical chemistry parameters contained a total of 18 potential outliers that are displayed 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 1-1 through 1-9 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 17b 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 17b. With the potential outliers excluded, the following changes in
significance were noted:
• AST (Table 1-1): There was no longer a significant increase as a proportion of
baseline in Group 5 at Study Day 2. The increase in Group 6 at Study Day 2 was
significantly different than the changes in Groups 2, 3, 4, and 5. There were
significant increases as a proportion of baseline in Groups 2, 4, and 6 at Study Day 3.
The increases in Groups 2, 4, and 6 were significantly different than the decrease in
Group 1.
• ALT (Table 1-2): The increase as a proportion of baseline in Group 5 at Study
Day 2 was no longer significant. There were significant increases as a proportion of
baseline in Groups 2, 3, and 4 at Study Day 2. The increase in Group 6 was
significantly greater than that in Group 1. There were significant increases as a
proportion of baseline in Groups 2, 3, and 6 at Study Day 3.
• SDH (Table 1-3): There was no longer a significant increase as a proportion of
baseline in Group 5 at Study Day 2. There was a significant decrease as a proportion
of baseline in Group 1 at Study Day 2. The increase in Group 6 at Study Day 2 was
significantly greater than those in Groups 2, 3, 4, and 5. There were significant
increases as a proportion of baseline in Groups 2 and 4 at Study Day 3.
1-2
-------
BUN (Table 1-4): At Study Day 3, there was a significant decrease as a proportion
of baseline in Groups 3 and 6.
Creatinine (Table 1-5): There was a significant increase as a proportion of baseline
in Groups 4 and 5 at the terminal blood draw.
BUN/Creatinine Ratio (Table 1-6): There was a significant decrease from baseline
in Group 5 at Study Day 3.
Sodium (Table 1-7): There was a significant increase from baseline in Group 4, and
a significant decrease from baseline in Group 5 at the terminal blood draw.
Chloride (Table 1-8): There were significant decreases from baseline in Groups 4
and 5 at the terminal blood draw.
Calcium (Table 1-9): There was a significant decrease from baseline in Group 6 at
Study Day 3 that was significantly different than the changes in Groups 2, 3, and 4.
The decrease from baseline in Group 5 was significantly different than the increases
in Groups 1 and 4 at Study Day 3.
1-3
-------
Table 1-1. Summary of Test Results for Aspartate Aminotransferase (AST, U/L) with
Potential Outliers Excluded
Aspartate Aminotransferase1
Study
Day
Mean S
1
hift as a
2
Proport
3
ion of Be
4
iseline, b
5
y Group
6
Group
Effect
P-Value
Estimated Ratio
(Relationship)
Tukey's P-Value#
2
0.83
1.13
1.26
1.36
1.21
5.13 t
<0.0001*
0.16 (1 <6) <0.0001*
0.22 (2<6) 0.0003
0.25 (3<6) 0.0008
0.27 (4<6) 0.0014
0.24 (5<6) 0.0009
3
0.83
1.33 t
1.06
1.45 t
38.39 t
1.69 t
<0.0001*
0.62 (1<2) 0.0225
0.57 (1<4) 0.0054
0.02 (1 <5) <0.0001*
0.49 (1<6) 0.0020
0.03 (2<5) <0.0001*
0.03 (3<5) <0.0001*
0.04 (4<5) <0.0001*
0.04 (6<5) <0.0001*
¦f Indicates that values for this parameter were log-transformed for the analysis.
# Cells contain all pairwise comparisons that are significant at the 0.05 level. Hie format within each cell is: (1) the ratio of
shifts, (2) the relationship between the corresponding pair of Group mean shifts shown in parentheses [For
example, "(1<6)" indicates that the mean shift as a proportion of baseline for Group 1 was less than that for Group 6], and
(3) the Tukey-adjusted p-value,
t, | Indicate that the ratio of the geometric mean at baseline and the geometric mean at the Study Day was significantly
different from one (at the 0.05 level), "f" indicates that the geometric mean at the Study Day was greater than that at
baseline, while "J," indicates that the geometric mean at the Study Day was less than that at baseline.
* Group effect was significant at the 0.05 level.
Table 1-2. Summary of Test Results for Alanine Aminotransferase (ALT, U/L) with
Potential Outliers Excluded
Aspartate Aminotransferase1
Study
Day
Mean S
1
hift as a
2
Proport
3
ion of Be
4
iseline, b
5
y Group
6
Group
Effect
P-Value
Estimated Ratio
(Relationship)
Tukey's P-Value#
2
1.06
1.24 t
1.28 t
1.34|
1.21
1.91 |
0.0443 *
0.56 (1<6) 0.0170
3
1.08
1.35 t
1.25 t
1.41 |
5.33 t
1.29 t
<0.0001*
0.20 (1 <5) <0.0001*
0.25 (2<5) <0.0001*
0.23 (3<5) <0.0001*
0.26 (4<5) <0.0001*
0.24 (6<5) <0.0001*
¦f Indicates that values for this parameter were log-transformed for the analysis.
# Cells contain all pairwise comparisons that are significant at the 0.05 level. The format within each cell is: (1) the ratio of
shifts, (2) the relationship between the corresponding pair of Group mean shifts shown in parentheses [For
example, "(1<6)" indicates that the mean shift as a proportion of baseline for Group 1 was less than that for Group 6], and
(3) the Tukey-adjusted p-value,
t, | Indicate that the ratio of the geometric mean at baseline and the geometric mean at the Study Day was significantly
different from one (at the 0.05 level), "f" indicates that the geometric mean at the Study Day was greater than that at
baseline, while "J," indicates that the geometric mean at the Study Day was less than that at baseline.
* Group effect was significant at the 0.05 level.
1-4
-------
Table 1-3. Summary of Test Results for Sorbitol Dehydrogenase (SDH, U/L) with Potential
Outliers Excluded
Sorbitol Dehydrogenase1
Study
Day
Mean S
1
hift as a
2
Proport
3
ion of Be
4
iseline, b
5
y Group
6
Group
Effect
P-Value
Estimated Ratio
(Relationship)
Tukey's P-Value#
2
0.73|
1.16
1.16
1.33
1.19
2.78 t
0.0006 *
0.26 (1 <6) 0.0001
0.42 (2<6) 0.0123
0.42 (3<6) 0.0117
0.48 (4<6) 0.0428
0.43 (5<6) 0.0214
3
0.87
1.33 t
1.02
1.50 t
10.61 t
1.02
<0.0001*
0.08 (1 <5) <0.0001*
0.12 (2<5) <0.0001*
0.10 (3<5) <0.0001*
0.14 (4<5) <0.0001*
0.10 (6<5) <0.0001*
¦f Indicates that values for this parameter were log-transformed for the analysis.
# Cells contain all pairwise comparisons that are significant at the 0.05 level. Hie format within each cell is: (1) the ratio of
shifts, (2) the relationship between the corresponding pair of Group mean shifts shown in parentheses [For
example, "(1<6)" indicates that the mean shift as a proportion of baseline for Group 1 was less than that for Group 6], and
(3) the Tukey-adjusted p-value,
t, | Indicate that the ratio of the geometric mean at baseline and the geometric mean at the Study Day was significantly
different from one (at the 0.05 level), "f" indicates that the geometric mean at the Study Day was greater than that at
baseline, while "J," indicates that the geometric mean at the Study Day was less than that at baseline.
* Group effect was significant at the 0.05 level.
Table 1-4. Summary of Test Results for Blood Urea Nitrogen (mg/dL) with Potential
Outliers Excluded
Blood Urea Nitrogen1
Study
Day
Mean S
1
hift as a
2
Proport
3
ion of Ba
4
iseline, b
5
y Group
6
Group
Effect
P-Value
Estimated Ratio
(Relationship)
Tukey's P-Value#
3
0.91
1.00
0.84|
0.79 |
0.90
0.77|
0.2364
¦f Indicates that values for this parameter were log-transformed for the analysis.
# Cells contain all pairwise comparisons that are significant at the 0.05 level. The format within each cell is: (1) the ratio of
shifts, (2) the relationship between the corresponding pair of Group mean shifts shown in parentheses [For
example, "(1<6)" indicates that the mean shift as a proportion of baseline for Group 1 was less than that for Group 6], and
(3) the Tukey-adjusted p-value.
t, | Indicate that the ratio of the geometric mean at baseline and the geometric mean at the Study Day was significantly
different from one (at the 0.05 level), "f" indicates that the geometric mean at the Study Day was greater than that at
baseline, while "J," indicates that the geometric mean at the Study Day was less than that at baseline.
1-5
-------
Table 1-5. Summary of Test Results for Creatinine (mg/dL) with Potential Outliers Excluded
Creatinine+
Study
Day
Mean S
1
hift as a
2
Proport
3
ion of Be
4
iseline, b
5
y Group
6
Group
Effect
P-Value
Estimated Ratio
(Relationship)
Tukey's P-Value#
Terminal
NA
NA
NA
2.31 t
1.22 t
NA
NA
¦f Indicates that values for this parameter were log-transformed for the analysis.
# Cells contain all pairwise comparisons that are significant at the 0.05 level. Hie format within each cell is: (1) the ratio of
shifts, (2) the relationship between the corresponding pair of Group mean shifts shown in parentheses [For
example, "(3<5)" indicates that the mean shift as a proportion of baseline for Group 3 was less than that for Group 5], and
(3) the Tukey-adjusted p-value.
NA Data not available for this Group at this Study Day, or not enough animals to perform analysis,
t, | Indicate that the ratio of the geometric mean at baseline and the geometric mean at the Study Day was significantly
different from one (at the 0.05 level), "f" indicates that the geometric mean at the Study Day was greater than that at
baseline, while "J," indicates that the geometric mean at the Study Day was less than that at baseline.
Table 1-6. Summary of Test Results for BUN/Creatinine Ratio with Potential Outliers
Excluded
BUN/Creatinine Ratio
Study
Day
Mean S
1
hift as a
2
Proport
3
ion of Be
4
iseline, b
5
y Group
6
Group
Effect
P-Value
Estimated Difference
(Relationship)
Tukey's P-Value#
3
-2.2
-2.7
-0.8
-3.9
-7.1 |
-9.9 |
0.1235
# There were no significant Tukey's pairwise comparisons.
t, | Indicate that the difference between the mean at baseline and the mean at the Study Day was significantly different from
zero (at the 0.05 level), "f" indicates that the mean at the Study Day was greater than that at baseline, while "J," indicates
that the mean at the Study Day was less than that at baseline.
Table 1-7. Summary of Test Results for Sodium (mEq/L) with Potential Outliers Excluded
Sodium
Study
Day
Mean S
1
hift as a
2
Proport
3
ion of Be
4
iseline, b
5
y Group
6
Group
Effect
P-Value
Estimated Difference
(Relationship)
Tukey's P-Value#
Terminal
NA
NA
NA
12 t
-2|
NA
NA
# Cells contain all pairwise comparisons that are statistically significant at the 0.05 level. The format within each cell is:
(1) the difference of shifts, (2) the relationship between the corresponding pair of Group mean shifts shown in parentheses
[For example, "(1<4)" indicates that the mean shift from baseline for Group 1 was less than that for Group 4], and (3) the
Tukey-adjusted p-value.
t, | Indicate that the difference between the mean at baseline and the mean at the Study Day was significantly different from
zero (at the 0.05 level), "f" indicates that the mean at the Study Day was greater than that at baseline, while "J," indicates
that the mean at the Study Day was less than that at baseline.
NA Data not available for this Group at this Study Day, or not enough animals to perform analysis.
1-6
-------
Table 1-8. Summary of Test Results for Chloride (mEq/L) with Potential Outliers Excluded
Chloride
Study
Day
Mean S
1
hift as a
2
Proport
3
ion of Be
4
iseline, b
5
y Group
6
Group
Effect
P-Value
Estimated Difference
(Relationship)
Tu key's P-Value#
Terminal
NA
NA
NA
"6 I
-10 |
NA
NA
# Cells contain all pairwise comparisons that are statistically significant at the 0.05 level. Hie format within each cell is:
(1) the difference of shifts, (2) the relationship between the corresponding pair of Group mean shifts shown in parentheses
[For example, "(5<1)" indicates that the mean shift from baseline for Group 5 was less than that for Group 1], and (3) the
Tukey-adjusted p-value.
t, | Indicate that the difference between the mean at baseline and the mean at the Study Day was significantly different from
zero (at the 0.05 level), "f" indicates that the mean at the Study Day was greater than that at baseline, while "J," indicates
that the mean at the Study Day was less than that at baseline.
NA Data not available for this Group at this Study Day, or not enough animals to perform analysis.
Table 1-9. Summary of Test Results for Calcium (mg/dL) with Potential Outliers Excluded
Calcium
Study
Day
Mean S
1
hift as a
2
Proport
3
ion of Ba
4
iseline, b
5
y Group
6
Group
Effect
P-Value
Estimated Difference
(Relationship)
Tukey's P-Value#
3
0.36
1.84 |
0.83
0.55
-2.36 |
-1.88|
0.0003 *
-2.72 (5<1) 0.0467
-4.19 (5<2) 0.0011
-3.72 (6<2) 0.0009
-3.18 (5<3) 0.0148
-2.71 (6<3) 0.0180
-2.90 (5<4) 0.0298
-2.43 (6<4) 0.0397
# Cells contain all pairwise comparisons that are statistically significant at the 0.05 level. The format within each cell is:
(1) the difference of shifts, (2) the relationship between the corresponding pair of Group mean shifts shown in parentheses
[For example, "(5<2)" indicates that the mean shift from baseline for Group 5 was less than that for Group 2], and (3) the
Tukey-adjusted p-value.
t, | Indicate that the difference between the mean at baseline and the mean at the Study Day was significantly different from
zero (at the 0.05 level), "f" indicates that the mean at the Study Day was greater than that at baseline, while "J," indicates
that the mean at the Study Day was less than that at baseline.
* Group effect was significant at the 0.05 level.
1-7
-------
APPENDIX K
HEAT SHOCK RESULTS
K-l
-------
1020-CG920503 Heat Shock Comparison (cfu/mL)
Sample Type
Sample ID
Date of First
Heat Shock
Enumeration
Prior to First
Heat Shock
Enumeration
Post First
Heat Shock
Date of Second
Heat Shock
Enumeration
Prior to Second
Heat Shock
Enumeration
Post Second Heat
Shock
Nebulizer
L23204
9/30/2009
9.30E+08
8.80E+08
N/A
N/A
N/A
Nebulizer
1.23203
9/30/2009
1.48E+09
1.26E+09
N/A
N/A
N/A
Nebulizer
L23213
9/30/2009
1.22E+09
1.18E+09
N/A
N/A
N/A
Nebulizer
L23221
9/30/2009
1.33E+09
1.27E+09
N/A
N/A
N/A
Nebulizer
L23232
9/30/2009
1.27E+09
1.44E+09
N/A
N/A
N/A
Impinger
L23204
9/18/2009
7.52E+05
7.80E+05
9/30/2009
7.28E+05
6.16E+05
Impinger
L23203
9/18/2009
1.83E+06
1.88E+06
9/30/2009
1.72E+06
1.I6E+06
Impinger
L23213
9/18/2009
1.59E+06
1.83E+06
9/30/2009
1.68 Ht 06
1.34E+06
Impinger
L23221
9/18/2009
1.85E+06
1.73E+06
9/30/2009
1.60E+06
1.43E+06
Impinger
L23232
9/18/2009
1.83E+06
2.73E+06
9/30/2009
1.73E+06
1.35E+06
Printed By/Date: J £)*?
QC/Technical Review By/Date: I I I U> 0 °l
QA Auefl1
Bv/Date:^_
Page I of 1
-------
APPENDIX L
CLINICAL OBSERVATIONS
L-l
-------
1020-CG920503
Pre Challenge Observations
11/8/2009
Animal ID
123220"
Sex
M
Group
1
1216
M
1
123218
M
i 7
L23223
M
1
123222
M
1
L23215
M
-z
123206
M
r
123210
M
/
L23219
1 M
2
L23211 ~1
M
«
L2321?
M
3
123230
M
3
228
M
3
¦227
M
3
.'1229
M
3
L23235
M
' 4
1-23 C">
M
4
M |
1
L23231
M
4
123207
M
4
1.23201
M 1
5
L23:
M
5
L23- V
M
5
L23200
M
5
123214
M
5
L232Q4 1
M
6
1.23203
__
6
123213 t
M
6
. -221 I
M
t
232 J
M |
6
9/8/2009
9/9/2009
9/10/2009
9/11/2009
9112/2009
9/13/2009
AH
PM
N
AM
N
PM
N
AM
PM
AM
PM
AM
PM
AM
AM
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
__
N
N
N
N
N
N
N
N
I Animals arrived in AM COMMENTS:
| Animals challenged 'Animal chewed on right rear foot, sloughing skin off
KEY:
N=Normal
NE=Not Eating
A=Abnorma! w/comment
r
to
1 of 2
QC/Tech Reviewed By:
Prepared
-------
1020-CG920503
Pre Challenge Observations
11/6/2009
9/15/2009
9/16/2009
9/17/2009
9/18/2009
Animal ID
Sex
Group
AM
PM
AM
PM
AM
PM
AM
PM
1.23220 M
1
N
N
N
N
N
N
N
123216
I M
1
N
N
N
N
N
N
N
L23218
1 M
1
N
N
N
N
N
N
N
L23223
I M
1
N
N
N
N
N
N
N
L23222
M
__
1
N
N
N
N
N
N
N
1
L23215
2
N
N
N
N
N
N
N
I
123206
M
2
N
N
N
N
N
N
N
J
123210
M
_ _
2
N
N
N
N
N
N
N
—I
L23219
2
N
N
N
N
N
N
N
...J
L23211
M
2
N
N
N
N
N
N
N
¦ -j
L23217
M
3
N
N
N
N
N
N
N
j
123230
M
3
N
N
N
N
N
N
N
j
123228
_ _j
3
N
N
N
N
N
N
N
1
L23227
M
3
N
N
N
N
N
N
N
3BHH
123229
M
3
N
N
N
N
N
N
N
I
L23235
4
N
N
N
N
N
N
N
j
123205
M
4
N
N
N
N
N
N
N
j
225
M
4
N
N
N
N
N
N
N
—1
L23231 M
4
N
N
N
N
N
N
N
flHVB
L23207
M
4
N
N
N
N
N
N
N
-j
L23201
S~' M
r _ .
5
N
N
N
N
N
N
N
¦ -J
L23234
5
N
N
N
N
N
N
N
—1
1 12 ~!
M
5
N
N
N
N
N
N
N
j
L23200
M
5
N
N
N
N
N
N
N
i
1.321^ i
M
5
N
N
N
N
N
N
N
j
123, u4
M
6
N
N
N
N
N
N
N
j
123203
M
—
6
N
N
N
N
N
N
N
j
L2321.. 1
6
N
N
N
N
N
N
N
—I
123221
M |
6
N
N
N
N
N
N
N
¦ ¦ J
1.23232
M |
8
N
N
N
N
N
N
N
I
wals arrived in AM COMMENTS:
MiilHwi Animals challenged 1 Animal chewed on right rear foot, sloughing stein off
KEY:
N=Normaf
NE=Not Eating
A-'Afanormai w/com merit
r
2 of 2
Prepared By:_ag
QC/Tsch Reviewed By 0
-------
1020-CG920503
Post-Challenge Clinical Observations
1/6/2010
8
mm
9/19/2009
9/20/2009
9/21/2009
9/2212009
9/23/2009
8S4I2009
812512009
PM
AM
PM
AM
PM
AM
PM
AM
PM
AM
PM
AM
PM
AM
PM
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
H
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
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
N
N
N
N
H
N
N
N
N
N
N
N
N
N
N
N
N
N
N
M
N
N
N
N
N
N
N
N
N
N
j™N)
N
N
N
H
N
N
N
N
N
N
N
N
N
N
N
N
N
N
H
N
N
N
N
N
N
N
N
N
N
N
N
NE
N
N
N
N
N
N
N
N
N
N
N
I N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
r n
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
SS
N
N
N
l*_ N
N
N
N
N
N
N
N
N
N
N
N
N
. —
W
N
N
N
N
N
N
N
N
N
N
N
N
N I
N
N ~'
N
\
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
NE
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
NE
L
L,RA
FD
N
N
N
N
N
N
N
N
"JNINIniN
N
N
N
N
N
N
N
N
N
N
; J \ 1 ; I
N
N
N
N
N
N
N
NE,L
I
FD
N
N
N
N
N
N
L
N
N
N r —
FO
..
N
\
N
N
N
N
N
N
L
N
M
K» I ti
f,*
N
N
N
N
N
N
FD
|
j,-jt
N
N
N
N
N
NE
L.O1
L
\
t!) hWKSWI
N
N
N
N
N
NE
N
L
N
N
N
N
N
NE
L
\ I)
N
N N
N
L
L NE
FD I
N
N
NE I L
N I N
FD pMMNWU
N | N|
neTi
_ | —|
•
¦ "
Animal 10
123220"
Sex
Group
AM
L23216
L23218
L23223
1,23222
L23215
123206
L23210
L23219
L23211
123217
L23235
L23205
123225
123231
123207
L23201
L23234
L23212
123200
1.23214
L23204
123203
L23213
123221
L23232
Precbailersge obs
Animal deceased
N = Normal
MS = No Stool
NE = Not Ealing
I = Lethargic
RA = Respiratory Abnormalities
FD = Found Dead
O = Other w/ comment
COMMENTS: ' ht,h o
' Little stool present
'Large scab on top of right htrtd foot- appears tender
3E-CQllar placed on animal due to chewing
"Scab between shoulder blades
""Water line discovered disconnected
*Ear tag fell out- multiple scabs and swollen areas on left ear- very tender
'left ear tip bruised, sore and notched
a u Animals were euthanize! before am observations, please reference OR «
QA Audit Comprise, Prepares! By:
i-y. K;
/ '
-------
1G20-CG92G503
Post-Challenge Clinical Observations
1/8/2010
I 912612009
i 9/27/2009
I 9/28/2009
[ 9/29/2009
| 9130/2009
1 1011/2009
1 10/2/2009 I
j Animal ID
Sex
| Group
AM
PM
AM
PM
AM
PM
AM
PM
AM
PM
AM
m
AM
PM
izdzm i m 1
N
N
N
N
N
N
N
N
N
****
N
H
N
I N
123216
M
1
N
N
N
N
N
N
N
N
N
*«~*
N
N
N
t >18
r m
1
O2
N
N
N
N
N
N
N
G3
~***
N
N
223
¥_—L_J—
N
N
N
N
N
N
N
N
N
*«*
N
N
N
•,
122
M
1
N
N
N
N
N
N
N
N
N
****
N
;
N !
N
¦, . "
[ M
I 2
O"
N
N
N
N
N
N
N
n ;
***»
N
N ™1
™ N "
N
I. .*>08
M
t———
NE.O5
N
H
N
N
N
N
N
N
****
N
N
N
_—2
N
L23210
Lm
_
NE.O5
N
N
N
N
N
N
N
N
##•**
N
H
N
L23219
1 M
2
NE.G5'5
1 N
N
N
N
N
N
N
N
\
N
h*
•
123211
_ M
NE.O5
N
N
N
N
N
N
N
N
\
N
H
N
L23217
M
3
NE.O5,7
: |sj
N
N
N
N
N
N
N
N
n
N
N
123230
*'
3
NE.O5
-
N
N
N
N
N
N
N
N
r~ N
N
N
L23228
3
N
N
N
N
N
N
N
N
.
H
N
N
123227
"
3
N
_
N
N
N
N
N
N
N
N
....
V
¦.
N
N
L23229
m"
3
N
N
N
N
N
N
N
N
4 *¦*,*
w
n
N
L23235
"
; 4
N
N
NE,L
N
NE,l
L.O'
NE.NS.FD
.
L23205
v 1
4
N
N
N
sums
N
N
•33
3
CKgacaii
*«** 1
iaiigiiiasiiM
M ""1
lf ¦ "1
_ jTj
L23225
* 1
4
.
f 1.23231
1 M
4
1 1.23207 1 M
4
M
N
123201
M
123234
L23212
~¥
5^j
N
N
123200
5
123214
5
123204
"
1
oTj
M
f.
'13
f,"
6
123221
"
6
... 132
M
6
I Prechaltenge tsbs
(Animal deceased
N = Normal
NS = No Stool
ME = Not Eating
t = Lethargic
RA = Respiratory Abnormalities
FD -¦ Found Dead
O = Other w/ comment
¦ Observation missing please reference DR # mil
COMMENTS:
-------
1Q20-CG92Q503
Post-Challenge Clinical Observations
1/8/20ti
10/3/2009
10/4/2009
10/5/2009
10/6/2OO9
10/7/2008
10/8/2009
Animal ID
Group
123216
L23218
123222
123215
L2320S
L23210
L23213
123211
123217
123225
123231
123207
1.23201
L.23200
123214
L23204
L23203
L23213
123221
Prechaltersge obs
Animal deceased
N = Normal
NS = No Stool
NE = Not Eating
L = Lethargic
RA = Respiratory Abnormalities
FD = Found Dead
O = Other w/ comment
* Observation missing p.'ease reference DR #
COMMENTS:
' little stool present
\arge scab on top of right hind foot- appears tender
3E-eoilar placed on animal due to chewing
4Scab between shoulder blades
sWater line discovered disconnected
"Ear tag fell out- multiple scabs and swollen areas on left ear- very tender
'Left ear tip bruised, sore and notched
Animals were euthanized before am observations, please reference DR # f _ ,
r
On
3 of 3
Prepat. J By: a-a-, JJ,
OC/Tech Reviewed f., ,
-------
APPENDIX M
BODY WEIGHTS
-------
1020-CG920503 Body Weight Summary Table
1 of 1
Body Weight (kg)
Animal
ID
Group
Day 0
Day 1
Day 2
Day 3
Day 7
Day 14
Day 21
Moribund/
Terminal
L23216
1
3.3
3,4
3.4
3,0
3.3
3,39
3.42
NA
L2321S
1
3.3
3.3
3,4
3,3
3.3
3.29
3.29
NA
L23220
1
NA
3,0
3,0
3.0
3.0
3.13
3.10
NA
L23222
1
3.5
3,5
3.6
3.6
3.5
3.67
3.54
NA
123223
1
3.0
3.0
3.0
3,0
3.0
3.10
3.08
NA
L23206
2
3.2
3,3
3.2
3,2
3.0
3.30
3.42
NA
L23210
2
3.0
3,5
3.1
3,1
2,9
3.24
3.29
NA
L2321I
2
3.3
3.2
3.3
3.3
3.1
3.43
3.40
NA
1 23215
2
3.1
3.1
3.1
3.0
3.1
3.24
3.15
NA
I 23210
2
3.1
3.1
3.2
3.1
2.9
3.29
3.29
NA
L23217
3
3.2
3.2
3.2
3.3
3.1
3.30
3.29
NA
1.2322"
3
3.3
3.3
3.3
3.3
3.3
3.37
3,31
NA
L23228
3
3.2
3.1
3.1
3.2
3.2
3.33
3.29
NA
L23229
3
3.4
3.4
3.4
3.5
3.4
3,48
3,55
NA
L23230
3
3.0
3.0
3.1
3.1
2.8
3.11
3.06
NA
1 23205
4
3.3
3.3
3.3
3,4
3.4
3.37
3.44
NA
L23207
4
3.5
3.5
3.4
3.5
3.4
3.58
3.50
NA
1 23225
4
3.3
3.1
3.1
3.1
2.7
L23231
4
3.2
3.1
3.2
3.3
3.2
3.30
3.22
NA
L23235
4
3.3
3.2
3.3
3.3
3.2
3.13
L23200
5
3.4
3.2
3.2
3.3
3.3
1 2320!
5
3.2
3.2
3.0
3.0
2.8
1 23212
5
3.2
3.2
3.2
3.3
3.3
3.33
3.31
NA
1 23214
5
3.2
3.2
3.0
3.0
2.9
L23234
5
3.2
3.3
3.2
3.2
3
L23203
6
3.0
2.9
3.0
3.0
2.7
1.23204
6
3.1
3.1
3.0
3.0
2,7
L23213
6
3.3
2.9
2.8
3.0
L23221
6
3.1
3.2
3.2
i 23232
6
3.1
3.1
3,0
2.9
2.9
* = A moribund/terminal weight was not recorded for rabbit L23213
OA AU! )MPLETED
BY/DATE:"Wvufe atoj
Prepared
QC reviewed by/dat
-------
APPENDIX N
INDIVIDUAL MORTALITY RESULTS
N-l
-------
1020-CG920503
Mortality Table
1/6/2010
Animal ID
Dose Group
Challenge Date &
Time
Date & Time of Death
Time to Death
(days)
Found Dead or
Moribund/Euthanized/
Survived
123220
1
9/18/09 8
19
NA
NA
Survived
L23216
1
9/18/09 8
32
NA
NA
Survived
L23218
1
9/18/09 8
44
NA
NA
Survived
L23223
1
9/18/09 8
55
NA
NA
Survived
L23222
1
9/18/09 9
05
NA
NA
Survived
L23215
2
9/18/09 9
37
NA
NA
Survived
123206
2
9/18/09 9
47
NA
NA
Survived
L23210
2
9/18/09 9
59
NA
NA
Survived
123219
2
9/18/09 10
07
NA
NA
Survived
L23211
2
9/18/09 10
17
NA
NA
Survived
L23217
3
9/18/09 10
27
NA
NA
Survived
L23230
3
9/18/09 10
37
NA
NA
Survived
L23228
3
9/18/09 10
48
NA
NA
Survived
L23227
3
9/18/09 10
59
NA
NA
Survived
L23229
3
9/18/09 11
11
NA
NA
Survived
L23235
4
9/18/09 11
27
9/29/2009 7:56
11
Found Dead
L23205
4
9/18/09 11
37
NA
NA
Survived
L23225
4
9/18/09 11
48
9/22/2009 15:14
4
Found Dead
L23231
4
9/18/09 11
58
NA
NA
Survived
L23207
4
9/18/09 12
08
NA
NA
Survived
L23201
5
9/18/09 12
18
9/22/2009 8:34
4
Found Dead
L23234
5
9/18/09 12
29
9/24/2009 8:32
6
Found Dead
L23212
5
9/18/09 12
39
NA
NA
Survived
L23200
5
9/18/09 12
47
9/21/2009 11:16
3
Found Dead
L23214
5
9/18/09 12
56
9/24/2009 8:32
6
Found Dead
L23204
6
9/18/09 13
07
9/22/2009 15:14
4
Found Dead
L23203
6
9/18/09 13
17
9/23/2009 8:25
5
Found Dead
L23213
6
9/18/09 13
27
9/21/2009 13:05
3
Found Dead
1.23221
6
9/18/09 13
37
9/20/2009 6:33
2
Found Dead
L23232
6
9/18/09 13
48
9/22/2009 8:34
4
Found Dead
NA = Not Applicable
Prepared By: srr- ilbL.
QC/Tech Reviewed
i-
N-2
bvC42iofi
if
-------
APPENDIX O
FINAL SURVIVAL STATISTICAL REPORT
o-i
-------
Table of Contents
1. Introduction 0-4
2. Statistical Methods 0-5
3. Results 0-6
4. Conclusions 0-8
List of Tables
Table 1. Study Design 0-4
Table 2. Proportion of Surviving Animals with Exact 95% Confidence Interval by Group 0-9
Table 3. Results of Two-Sided Pairwise Fisher's Exact Tests 0-9
Table 4. Results of Pairwise Log-Rank Tests 0-9
List of Figures
Figure 1. Estimated logistic regression curve and observed survival or mortality O-IO
Figure 2. Kaplan-Meier curves representing time to death and survival data for each group. .0-10
0-2
-------
List of Acronyms
BBRC Battelle Biomedical Research Center
CFU Colony forming unit
LD50 Median lethal dose
N Number of animals
0-3
-------
1. Introduction
This report summarizes the statistical analysis of survival data collected under Battelle
Biomedical Research Center (BBRC) Study No. 1020-CG920503. Thirty (30) male
pathogen-free New Zealand White rabbits (Oryctolagus cuniculus) were randomized into
six groups, with each group having five animals. Animals were aerosol challenged on study
day zero with Bacillus anthracis (Ames strain) spores as indicated in Table 1. Animals were
observed twice daily post-challenge and survival data was recorded.
Table 1. Study Design
Group
Number of Animals
per Group
Target
Spore Dose (CFU)
1a
5
100 x LD50
2
5
100
3
5
1,000
4
5
10,000
5
5
100,000
6b
5
100 x LD50
CFU Colony forming units
a Spores are gamma-irradiated (negative control)
b High-dose control
LD50 Median lethal dose
0-4
-------
2. Statistical Methods
Estimates with exact 95% binomial confidence intervals for the proportion of surviving animals
within each group were calculated using the FREQ procedure in SAS® (SAS Institute, Cary, NC,
version 9.1). 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. The Fisher's
exact tests were performed using the SAS® FREQ procedure. The SAS® MULTTEST procedure
was used to perform the Bonferroni-Holm adjustment to maintain an overall 0.05 level of
significance for the multiple pairwise comparisons.
Excluding the negative control group (Group 1), a logistic regression model was fitted to the
survival data as a function of the base-10 logarithm of the estimated 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 logistic
regression models were fitted using the SAS® LOGISTIC procedure, while the Fieller's 95%
confidence intervals were obtained by refitting the models using the SAS® PROBIT procedure as
a matter of convenience.
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. Using the
SAS® LIFETEST procedure, Kaplan-Meier curves were plotted 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.
0-5
-------
3. Results
Table 2 contains the estimated proportion of surviving animals within each group, along with
exact binomial 95% confidence intervals. All animals in the negative control group (Group 1)
and the two lowest spore dose groups (Groups 2 and 3) survived. Three of the five animals in the
targeted 10,000 colony forming unit (CFU) dose group (Group 4) survived, and only one of the
five animals in the targeted 100,000 CFU dose group (Group 5) survived. No animals in the high
dose control group (Group 6) survived.
The overall Fisher's exact test was significant (p-value<0.0001), indicating that the proportion of
surviving animals in at least one of the groups was significantly different from those in the other
groups. 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. Based on the unadjusted Fisher's exact
tests, the proportions of surviving animals in the negative control group (Group 1) and in the two
lowest dose groups (Groups 2 and 3) were significantly greater than those in the targeted
100,000 CFU dose group (Group 5) and in the high dose control group (Group 6). However,
after adjusting for the 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 -2.21 (p-value=0.0147). The estimated LD50 was 51,789 CFU with a 95% Fieller confidence
interval ranging from 6,142 CFU to 726,639 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<0.0001), 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
0-6
-------
significantly different from each other. Based on the unadjusted log-rank tests, the times to death
in the negative control group (Group 1) and in the two lowest target dose groups (Groups 2
and 3) were significantly greater than those in the targeted 100,000 CFU dose group (Group 5)
and in the high dose control group (Group 6). Furthermore, the targeted 10,000 CFU dose group
(Group 4) also experienced significantly greater times to death than the high dose control group
(Group 6). However, after adjusting for the multiple pairwise comparisons, only the times to
death in the negative control group (Group 1) and in the two lowest dose groups (Groups 2
and 3) were significantly greater than that in the high dose control group (Group 6). Figure 2
displays the Kaplan-Meier curves for each of the six dose groups. Since all animals in Groups 1
through 3 survived the length of the study, Groups 2 and 3 were plotted with a slight offset so
that their 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.
0-7
-------
4. Conclusions
The proportion of surviving animals decreased for groups that received higher spore doses. All
animals in the negative control and the lower dose groups (Groups 1 through 3) survived, while
none of the animals in the high dose control group (Group 6) survived. Prior to adjusting for
multiple pairwise comparisons, the proportions of surviving animals in the negative control and
the two lower dose groups (Groups 1 through 3) were significantly greater than those in the
targeted 100,000 CFU dose and high dose control groups (Groups 5 and 6). However, these
differences were no longer significant after adjusting for the multiple pairwise comparisons.
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 LD50 was 51,789 CFU with a 95% Fieller confidence
interval ranging from 6,142 CFU to 726,639 CFU.
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 pairwise
comparisons, the times to death in the negative control and the two lowest dose groups (Groups 1
through 3) were significantly greater than those in the targeted 100,000 CFU dose and the high
dose control groups (Groups 5 and 6). Furthermore, the time to death in the targeted 10,000 CFU
dose group (Group 4) was significantly greater than that in the high dose control group
(Group 6). After adjusting for the multiple pairwise comparisons, the times to death in the
negative control and the two lowest dose groups (Groups 1 through 3) were still significantly
greater than that in the high dose control group (Group 6). 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.
0-8
-------
Table 2. Proportion of Surviving Animals with Exact 95% Confidence Interval by Group
Group
Number of Surviving
Animals / N
Proportion Survived
(95% Confidence Interval)
1
5/5
1.00 (0.48, 1.00)
2
5/5
1.00 (0.48, 1.00)
3
5/5
1.00 (0.48, 1.00)
4
3/5
0.60 (0.15, 0.95)
5
1/5
0.20 (0.01, 0.72)
6
0/5
0.00 (0.00, 0.52)
N Number of animals.
Table 3. Results of Two-Sided Pairwise Fisher's Exact Tests
Two-Sided Pairwise Fisher's Exact Test P-Values
Group
Unadjusted P-Values
Bonferroni-Holm Adjusted P-Values
0.4444
0.0476*
0.0079*
1.00003
1.00003
1.0000
0.5714
0.1190
1.00003
0.0079*
0.0079*
0.1667
1.0000
1.00003
0.1190
0.1190
1.0000
1.0000
1.00003
0.4444
0.0476*
1.0000
0.5714
1.0000
0.5714
0.4444
0.0476*
1.0000
a A p-value of 1.0000 was substituted since all animals in both groups survived.
* Comparison significant at the 0.05 level.
Table 4. Results of Pairwise Log-Rank Tests
a A p-value of 1.0000 was substituted since all animals in both groups survived.
* Comparison significant at the 0.05 level
Pairwise Log-rank Test P-Values
Group
Unadjusted P-Values
Bonferroni-Holm Adjusted P-Values
0.0133*
0.0018*
1.00003
1.00003
0.9400
0.1461
0.0276*
0.1343
0.0018*
0.0018*
0.0064*
0.0943
0.0276*
0.0276*
0.0770
0.7544
0.1343
0.0133*
0.1343
0.0133*
0.1403
0-9
-------
TO
>
>
3
CO
T3
CD
U 0.2
IS 0.1
0.8
0.7
0.6
0.5
0.4
0.3
103 104 105 106
Inhaled Dose (CFU)
Figure 1. Estimated logistic regression curve and observed survival or mortality.
o
o
£=
o
CO
Q
"ro
>
>
1
=>
CO
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0 .
™T
1
— Negative Control (Group 1)
— 100 CFU (Group 2)
1,000 CFU (Group 3)
— 10,000 CFU (Group 4)
— 100,000 CFU (Group 5)
- High Dose Control (Group 6)
7 14
Time to Death (Days)
21
Figure 2. Kaplan-Meier curves representing time to death and survival data for each
group.
O
-------
APPENDIX P
INDIVIDUAL CIRCULATING PA ELISA RESULTS
P-l
-------
Animal ID| (irniiji
1.23220 7
~ 1.23216 1
1.23218
L23223
L23222
1.23215
1
Dciy -3
BD
_lZ_Dayl
1020-CG920503 PA ELISA Results
BD
BD
BD
BD
BD
BD
BD
BD
BD
Day 2
BD
BD
Day 3
BD
BD
BD
BD
BD
BD
BD
BD
Day 7
BD
BD
BD
BD
BD
Day 14_
BD
BD
BD
BD
BD
Day 21^
BD
BD
BD
BD
BD
Terminal
*
BD
BD
BD
BD
BD
BD
BD
BD
BD
BD
BD
BD
BD
BD
1.23219
L23211
L23217
1.23230
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
BD
BD
BD
BD
L23227
L23229
BD
BD
BD
BD
BD
BD
BD
BD
BD
BD
BD
BD
BD
L23235
1.23205
L23225
JL
4
BD
BD
BD
BD
BD
BD
BD
BD
BD
BD
BD
BD
BD
BD
BD
51.199
BD
BD
BD
32964.272
*
1.23231
BD
BD
BD
BD
BD
L23207
L23201
BD
BD
BD
64.857
BD
BD
BD
471.046
L23212
L23200
L23214
L23204
L23203
BD
BD
BD
BD
20.082
BD
BD
BD
BD
28.579
BD
BD
BD
2.833
9.637
BD
BD
BD
25344.927
*
1.23213
1.23221
L23232
L23202
1.23209
6
Extra
BD
*
BD
125.095
1.631
*
BD
BD
BD
2.107
383.515
BD
BD
Extra
to
[PA] is reported in ng/rnL
* indicates that no sample was received,
BD indicates that the result was below the detection limit of the assay
Optical LOD is set at 0.139 per QD-186 results
LOD, LLGQ and ULOQ are set at 2.0ng/mL, 4.9ng/mL and 10000ng/mL respectively per QD-186 results
QCTech Review by:.
-------
APPENDIX Q
INDIVIDUAL BACTEREMIA CULTURE RESULTS
Q-i
-------
1020-CG920503 Bacteremia (efu/mL)1
Animal ID
Group
Targeted
Challenge
Dose
(cfu)
Day -3 2
Day 1
(24h PC)
Day 2
(48h PC)
Day 3
(72h PC)
Day 7
Day 14
Day 21
Terminal
L23220
1
lOOXLD,,3
0
0
0
0
0
0
0
1.23216
1
ioox l.iv'
0
0
0
0
0
0
0
1.23218
1
looxijv'
0
0
0
0
0
0
0
I 23223
1
100XLD5)13
0
0
0
0
0
0
0
1.23222
1
100X I.D„,J
0
0
0
0
0
0
0
"
1.23215
2
100
0
0
0
0
0
0
0
1.23206
2
100
0
0
0
0
0
0
0
1.23210
2
100
0
0
0
0
0
0
0
1.23219
2
100
0
0
0
0
0
0
0
1.23211
2
100
0
0
0
0
0
0
0
1.23217
3
1000
0
0
0
0
0
0
0
1.23230
3
1000
0
0
0
0
0
0
0
1 23228
3
1000
0
0
0
0
0
0
0
1.23227
3
1000
0
0
0
0
0
0
0
1.23229
3
1000
0
0
0
0
0
0
0
1.23235
4
10,000
0
0
0
0
0
—
I.70E+06
1.23205
4
10,000
0
0
0
0
0
0
0
^ - ~
1.23225
4
10,000
0
0
4.53E+03
+
8.03E+05
1.23231
4
10,000
0
0
0
0
0
0
0
1.23207
4
10,000
0
0
0
0
0
0
0
L23201
5
100,000
0
0
+
9.73E+05
9.80E+05
I 23234
5
100,000
0
0
0
+
5.43E+06
1.23212
5
100,000
0
0
0
0
0
0
0
L23200
5
100,000
0
+
+
+
1 23214
5
100,000
0
0
8.23E+03
o4
0
I 23204
6
100X l.l>„
0
+
¦f
2.80E+03
1.15E+05
1.23203
6
lOOXLDs,
0
0
+
+
3.03E+07
1.23213
6
100X LDjq
NS
0
+
1.31E+05
1.23221
6
I00XLD„
0
NS
2.58E+06
1.23232
6
100XLD»
NS
+
0
2.77E+03
05
|0 * Negative; + = Positive for B. anthracis but less than quantitative level of detection (2500 cfu/mL); NS = No sample received; Refer to DR-8178.
" Qualitative bacteremia performed.
,0 ' Negative control (Group 1) will be challenged with irradiated spores.
i'j Qualitative bacteremia performed; Sample was directly plated onto solid media from the cell pellet of the serum separation tube; Refer to DR-8170.
Qualitative bacteremia performed; Due to nature of the sample, the sample could not be diluted for quantitative bacteremia; Refer to DR-8170.
Printed By/Date yfa
QC'Technical Review By/Date: ftfil fi lt#o1 n , fl
rage 1 ot I
-------
APPENDIX R
INDIVIDUAL BACTEREMIA QPCR RESULTS
R-l
-------
I
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c c; c o c w c c; c C- o tz o o o o c: $ c c c c.
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Illilllllslllslslllissi
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lllllslllllllililllllll
II-
' V , «• V , V , ' ^ - ' p'' ^ ^ ^ <*"¦ ^ <""¦ J""' <"*¦ _
;; .' - ^*tifiiitiffillIiiitills
sa sllllllliyi
IIIIISIIIIIIIIIIIIIIIII2IIJ
NA
iou
HX?
i "4
0
« ! \ \ ' 3
NA
F
NA
NA
KM)
im
, < /«
570756
" t
NA
i 2^1^
Day 3
Blood
100
100
1020-005
0
i » N\ ^
NA
I.23223
Day 3
Biood
I
100
100
1020-1X15
0
i-\ \ >,
NA
L23222
Day 3
Biood
1
100
HK!
1020-005
0
1. O 1 l\ * 1,J !
NA
Day 3
Bbod
100
100
1020-005
0
NA
i .i..'1 'n
Day 3
Blood
2
100
\m
1020-005
0
i » « \ \ 102
NA
Day 3
Blood
100
100
I020-0ii5
< 2
.u' - ,\ \ im
NA
1,23219
Day 3
Blood
2
100
100
I020-(Ki5
0
t •. ¦-1JN A-104
NA
1,23 2 i J
Day 3
Blood
2
100
KX)
1020-005
0
1020-DNA-105
NA
1.23217
Day 3
Blood
3
KX)
HX)
1020-005
0
1020-DNA-106
NA
1,23230
Day 3
Blood
3
100
100
1020-005
0
U»20-I)NA-107
NA
1,23228
Day 3
Blood
3
100
KM)
1020-005
0
1020-DNA-108
NA
L23227
Day 3
Blood
3
100
HK)
1020-005
0
1020-DNA-109
NA
1.23229
Day 3
Blood
3
100
HK)
1020-005
0
1020-DNA-110
NA
1.23235
Day 3
Blood
4
HX)
I CKi
1020-005
0
1020-DNA-1! 1
NA
1.23205
Day 3
Blood
4
100
100
1020-tK)5
0
it'20-UNA-! 12.
NA
1.23225
Day 3
Blood
4
100
HK)
1020-!K)5
109
1020-DNA-l13
NA
1.2323 i
Day 3
Blood
4
100
100
1020-005
t)
1020-DNA-l14
NA
1,23207
Day 3
Blood
4
100
KX)
1020-(K)5
0
1020-DNA-115
NA
Negiilivc Control
NA
Water
NA
100
HK)
1020-005
0
1020-DNA-I 16
NA
Positive Control
NA
Culture
NA
100
100
1020-006
548818
1020-DNA-l17
NA
1.23201
Day 3
Blood
5
100
lOt)
1020-N \ 32
NA
1,23211
Day 7
Blood
2
100
100
1020-006
0
1 !N\ 33
NA
1,23217
Day 7
Biood
3
100
100
1070 f»)f^
0
i 1N \ 134
NA
1.23230
Day 7
Blood
3
100
HK)
1020-tX)6
0
1 )N\i35
NA
1.23228
Day 7
Blood
3
100
100
10 IMI )f,
0
DN A 136
NA
1.23227
Day 7
Biood
3
100
100
lO.'O ('lift
0
tO i) D\ \ 37
NA
1,23229
Day 7
Biood
3
10(1
100
1020-006
0
* - p\ « _1H
NA
Se
NA
Water
too
HK)
I020-«X)6
0
39
NA
P.
NA
Culture
NA
100
HX)
H)2f!-tX)7
387793
< 0 A\ 40
NA
1.23235
Day 7
Blood
4
100
100
It 0/
0
i *. v 41
NA
1,23 205
Day 7
Blood
4
100
100
1020-007
0
* ' -v t42
NA
1.23231
Day 7
Blood
4
HK)
100
1020-007
0
! » * * j43
NA
1,23207
Day 7
Blood
4
UK)
KX)
10204)07
0
! 1 s \ 144
NA
1,23212
Day 7
Biood
5
HX)
100
1020-007
0
I *• 1 45
NA
1.23220
Day 14
Blood
i
too
100
H)20-(K)7
0
i N,» 4ft
NA
I 2321ft
Day 14
Blood
i
too
100
1020-tX)7
0
1 W <47
NA
1.23218
Day 14
Blood
1
100
100
1020-007
f)
A * 48
NA
Day 14
1
HK!
100
102t)-(X)7
0
) » » 4y
NA
Day 14
1
100
KX)
I020-(X)7
0
f ' \ v 50
NA
2
100
10t>
K- 'O ,;.f
0
x 51
NA
HX)
100
I020-iMs7
0
if * 4 52
NA
Day i 4
2
HK!
100
1020-00?
0
' * 53
NA
2
100
100
0
- .54
NA
Day 14
2
HK)
HK!
0
/ " ' 55
NA
3
100
HX)
'O .
0
* 1 56
NA
HK)
100
102I!-(X)7
0
P N 57
NA
Day 14
3
100
100
: ¦¦¦ : , 1. . ''
0
^ \ 5H
NA
! ,23227
Blood
3
HX)
/
0
I * * 59
NA
Blood
3
100
t
0
!/ v 60
NA
I
Day 14
... <
4
100
Hi20-i)i)7
0
, v V 6!
NA
* «
" x
NA
100
!02t)-iX)7
u
'»2
NA
•
NA
HX)
,
< .1
si
NA
Day 14
Blood
4
HX)
H)20-tKW
tl
> Vt
NA
1,23207
Day 14
Biood
4
im
B>2
-------
I "O
I
SS8S2S8S3R
Hi
H ~ —; ri H ~~ .
1IIIIIH
??
a-, c? c> \s> ^ ¦-
»!H§H
/ 7 s / 7 s s ' ' / ' / j ~ ' , ~
' > ' ^ .
;8§S15SSS1SI
|l|l|!|||||lf,rlllll
ff »-¦
illslsslil! i
lisslliiis! - - "I - i >" - - " . . i
= f; = B c c ^ § !d !
;5S?S5SJ5;*:
x 5 x k 3. x 5r 3? x x 3a .
if*
1
If
III
ff:
>i=
-------
APPENDIX S
INDIVIDUAL TNA RESULTS
S-l
-------
1020-CG920503 TNA PRESCREEN RESULTS
Animal ID Information
Plate ID Information
Prescreen Test
Results
Animal ID
Animal Group#
Bleed Day
TNA Sample ID#
Plate ID
Positive or Negative
123223
1
Day -3
L23223 Day-3
111309-376
Negative
123222
Day -3
L23222 Day-3
111809-376
Negative
123230
Day -3
123230 Day-3
111909-376
Negative
L23227
Day -3
L23227 Day -3
111809-376
Neqaltve
L23205
Day -3
L23205 Day-3
111809-376
Negative
L23225 n
4
Day -3
L23225 Day -3
111809-376
Negative
L23234
Day -3
L23234 Day-3
111809-378
Negative
123212
Day -3
L23212 Day-3
111809-376
Negative
123220
Day 1
L23220 Day 1
111809-376
Negative
123216
Day 1
L23216 Day 1
111809-376
Negative
123218
Day 1
123218 Day 1
111809-376
Negative
L23223
Day 1
L23223 Day 1
111809-376
Negative
L23222
Day 1
123222 Day 1
111809-376
Negative
L23215
Day 1
L23215 Day 1
111809-376
Negative
123206
Day 1
L232G6 Day 1
111809-376
Negative
123210
Day 1
L23210 Day 1
111809-376
Negative
L23211
2
Day 1
L2321 Day 1
1118Q9-37S
Negative
1.2321?
Day 1
L23217 Day 1
111809-378
Negative
L23230
Day 1
L2323Q Day 1
111809-376
Negative
123228
Day 1
L.23228 Day 1
111809-376
Negative
L23227
Day 1
1.23227 Day 1
111809-376
Negative
1.23229 ~~1
Day 1
L23229 Day 1
111809-376
Negative
L23235
4
Day 1
L23235 Day 1
111809-376
rvwaai've
Animal ID Information
Plate ID Information
Prescreen Test
Results
Animal ID
Animal Group#
Bleed Day
TNA Sample ID#
Plate ID
Positive or Negative
123205
4
Day 1
L23205Day 1
111809-377
Negative
123225 H
4
Day 1
123225 Day 1
111809-377
Negative
123231
4
Day 1
L23231 Day 1
111809-377
Negative
L23207
4
Day 1
L23207 Day 1
111809-377
Negative
L23201
5
Day 1
L23201 Day 1
111809-377
Negative
123234
Day 1
L23234 Day 1
111809-377
Negative
L23212
Day 1
L23212 Day 1
111809-377
Neqative
L2320G
5
Day 1
L23200 Day 1
111809-377
Negative
L23214
Day 1
L23214 Day 1
111809-377
Negative
1.23204
6
Day 1
123204 Day 1
111809-377
Negative
L232.03
6
Day 1
L232G3 Day 1
111809-377
Negative
L23213
6
Day 1
L23213 Day 1
111809-377
Negative
L23232
6
Day 1
L23232 Day 1
111809-377
Negative
123220
Day 2
L23220 Day2
111809-377
Negative
L23218
Day 2
L23216 Day2
111809-377
N'v., -fiv"
123223
Day 2
L23223 Day2
111809-377
Neqative
1.23210
Day 2
L232I0 Day2
111809-377
Neqative
L23211
2
Day 7.
L23211 Day2
111809-377
Negative
L2323Q
Day 2
L2323Q Day2
111809-377
Negative
L23235
4
Day 2
L23235 Day2
111809-377
Negative
L23225
Day 2
L23225 Day2
111809-377
Negative
L23201
Day 2
1.23201 Day2
111809-377
Neqative
1.23214
5
Day 2
L23214 Day2
111809-377
Negative
, .--3^ IDiT COMPLETED
i By/Date:^-su^. ^ ,,
-------
1020-CG920503 TNA PRESCREEN RESULTS
Animal ID Information
Plate ID Information
Prescreen Test
Results
Animal ID
Animal Group#
Bleed Day
TNA Sample ID#
Plate ID
Positive or Negative
123203
8
Day 2
123203 Day2
1118G9-378
Negative
123232
6
Day 2
L23232 Day2
111309-378
Negative
L23220
1
Day 3
123220 Day 3
111809-378
Negative
L23218
1
Day 3
L23218 Day 3
111809-378
Neqj
L23223
Day 3
L23223 Day 3
111809-378
Negative
123222
Day 3
L23222 Day 3
111809-378
*•
123215
2
Day 3
L23215 Day 3
111809-378
Negative
123206
2
Day 3
L23206 Day 3
111809-378
Negative
L.23210
2
Day 3
L23210 Day 3
111809-378
Negative
L23217
3
Day 3
L23217 Day 3
111809-378
Negative
L23230
3
Day 3
L23230 Day 3
111809-378
Negative
L23228
Day 3
L23228 Day 3
111809-378
Negative
1.23227
Day 3
L23227 Day 3
111809-378
Negative
123235
4
Day 3
L23235Day 3
111809-378
Negative
L232Q5
4
Day 3
L23205 Day 3
111809-378
Negative
123225
Day 3
L23225 Day 3
111809-378
Negative
L23231
Day 3
L23231 Day 3
111809-378
Negative
L23201
Day 3
L23201 Day 3
111809-378
Negative
L23234
Day 3
L23234 Day 3
111809-378
Negative
L2320G
Terminal
123200 Terminal
111809-378
Negative
123214
5
Day 3
L23214 Day 3
111809-378
Negative
L232G3
6
Day 3
L23203 Day 3
111809-378
Negative
L23213
6
Terminal
L23213 Terminal
111809-378
Negative
Animal ID Information
Plate ID Information
Prescreen Test
Results
Animal ID
Animal Group#
Bleed Day
TNA Sample iD#
Plate ID
Positive or Negative
L23220
1
Day 7
L23220 Day 7
111809-379
Negative
L23216
1
Day 7
123216 Day 7
111809-379
Negative
1.23218
1
Day 7
L23218 Day 7
111809-379
Negative
123223
1
Day 7
L23223 Day 7
111809-379
Negative
123222
1
Day 7
L23222 Day 7
111809-379
Negative
123206
2
Day 7
1,23208 Day 7
111809-379
Negative
123211
2
Day 7
L23211 Day 7
111809-379
Negative
123217
Day 7
L23217 Day 7
111809-379
Negative
L.23228
Day 7
L23228Day 7
111809-379
Negative
L23227
Day 7
L23227 Day 7
111809-379
Negative
L23229
Day 7
L23229 Day 7
111809-379
Negative
123235
4
Day 7
L23235 Day 7
111809-379
Negative
L.23205
4
Day 7
L232Q5 Day 7
111809-379
Negative
L23231
4
Day 7
L23231 Day 7
111809-379
Negative
L23207
Day 7
L23207 Day 7
111809-379
'
L23212
5
Day 7
L23212 Day 7
111809-379
Neqatsve
1.23220
Day 14
L23220 Day 14
111809-379
Nega*
123205
2
Day 14
L23206 Day 14
111809-379
Negative
L 23219
2
Day 14
L23219 Day 14
111809-379
Negative
L23230
3
Day 14
L23230 Day 14
111809-379
Negative
L.23228
Day 14
L23228 Day 14
111809-379
Negative
L23229
3
Day 14
123229 Day 14
111809-379
Negative
L.23231
Day 14
L23231 Day 14
111809-379
Negative
, hpev.By/Oat OA AUDIT COMP
1 ^ BY/D
-ETED r~7^d/to
alio
-------
1020-CG920503 TNA PRESCREEN RESULTS
Animal ID Information
Plate ID Information
Prescreen Test
Results
Animal ID
Animal Group#
TNA Sample ID#
Plate ID
Positive or Negative
L232G7
4
L232D7 Day 14
111809-380
Negat
ve
L23212
L23212 Day 14
111809-380
Negat
ve
L23220
123220 Day 21
111809-380
Negal
ve
L23216
L23216 Day 21
111809-380
Neqative
L23218
123218 Day 21
111809-380
Neqalive
L23223
L.23223 Day 21
111809-380
Negative
L23222
L23222 Day 21
111809-380
Negat
ve
L23215
2
L23215 Day 21
111809-360
Negat
ve
1.23206
L232GS Day 21
111809-380
Neqat
ve
L23210
L23210 Day 21
111809-380
Negat
ve
L23219
L23219 Day 21
111809-380
Negative
L23211
L23211 Day 21
111809-380
Neqat
ve
L2321?
L23217 Day 21
111809-380
Neqat
ve
L23230
L23230 Day 21
111809-380
Neqat
ve
L23228
L23228 Day 21
111809-380
Negat
ve
L23227
L23227 Day 21
111809-380
Neqat
ve
L23229
L23229 Day 21
111809-380
Neqat
ve
L23205
4
123205 Day 21
1118Q9-38Q
Neqat
ve
L23231
L23231 Day 21
111809-380
Neqat
ve
L232Q7
4
L232Q7 Day 21
111809-380
Negative
L23212
L23212 Day 21
111809-380
Negative
L232Q4
6
L23204 Terminal
111809-380
Negative
-to
m mv. ' m\ . .v
?-v i""Ww (ffalio
fll
-------
Animal ID Information
Plate ID Information
ED50 I
} 4aSjHis| | 1
AntaiitiBj (;rmFf Time Point | TNA Simple IB#
Test!
Test 2 '
Test 1
Test 2
Mean ED50
Median j
ED5Q
, Pint ID
AnalysTf Plate ID
ED50
ED50
123212
123214
L23215
123222
123225
123230
Day 2
Day 2
Day -3
Day 7
Day 2
T emiinal
Day 7
L232Q4 Day 2
123212 Day 2
123214 Day -3
123215 Day 7
123222 Day 2
2123225 Termini
L23230 Day 7
PKItl
PKflE
PKill
PKill
PKH!
PKill
PKIU
111809-382
1118014-382
111809-382
111809-382
111809-382
111809-382
111809-382
KN
KN
KN
KN
KN
KN
KN
111809-381
111809-381
111803-381
111809-381
111809-381
111809-381
111809-381
QAAUL-, , JIF .
BY/P^-1 r (Z/zlto
-------
APPENDIX T
INDIVIDUAL ANTI-PA IGG ELISA RESULTS
T-l
-------
pRge 1 of 5
1020-CG920503 ELISA Results
Group
(Uay)
mBHam
Oitt- Tike n
lest Opi ftitur 1
Plate Numbe*
'est Gpcratoi ?
Plate Number
TfStOpCMtOf 3
Plato Number
1 est Operator 4
Plate Number
Results
of test
operator
1
?-159 KAS
J-163 KAS
-102109-176 LAN
-102109-177 LAN
-102109-177 LAN
1881
¦WBBI
ml I
Results
oftest :
operator
1—
Results
of test
operator
—j—
Results
of test
opetator
4
Mean fcLISA
anti PA IgG
(MC/mL)
Std Olv of
the Test
Operators
KtV
between
Test
tare'v
Median
FUSA anf
PA IfiCi
iue/mL\
ELfSA
Reportable
Values
3.000
02109-176 LAN
02109-177 LAN
r C';C
0.000
" GTJ
:oco
f coc
,or,
i,.i
2n
000 0,000
*
-102109-177 LAN
:: « n
i :r-
¦h
02109-176 LAN
02109-177 LAN
02109-177 LAN
33"
iji1
0.000 0.000
rrrr
J-176 LAN
Pre Screen -102109-177 LAN
>-177 LAN
) I rcoc |
Pi Sv t c» ' 1/i „A\ j ' q'jc" | C
I ";-1c i
as
0 003 [
QC/TR by
QAAUi:'-:''."»MF:.
,v •'.!* tffaho
-------
Page 2 of 5
1020-CG920503 ELISA Results
Group
• USA
('Kir
TcM Operi9-1
u.uau
0 000
0.000
109-167 0?
0.000
0.000
0,000
0.000
Terminal
0.000
Pre-Screen -102109-176 LAN
0,000
O.OOO
0,000
101205-106 Pre-Screen GEM
101209-109 AMB | 102109-160 KAS
102109-lb' fvT"1-12109-171JNG
0.000
ctQQ
clOQ
0.000
0.000
Q.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
Termma
Pre-Screen -102109-176 LAN
Pm-Woon - imino_i
0.000
0.000 |
0.000
0.000
0.000
u.QQO
0.000
-------
Page 3 of 5
102G-CG92Q503 ELISA Results
ELBA
< odf
(»ay)
Results
of test
operatoi
Medtan
EliSAdnti
PAlgG
Mcdf) niSA
ant>-PA IgC
fog/ml)
Std Dev of
the Ie«:t
Oner i
Test Operator ?
Plate Number
Test Opcrjtor4
Number
fcl'SA
Reportable
Animal ID
yperato
Screen -102109-176 LAN
0.000
0.000
0.000
0-000
.23228
0-000 0.000
0,000 0.000
0.000
0.000
0.000
t" L 2109^T72jNG
0.000
0.000
0.000
0.000
0,000
0.000 | 0.000
0.000 I 0,000
0.000 1
-------
1020-CG920503 ELISA Results
GG/TR by
Results I Results
uftest I of lest
apcrdtoi! oprr.it o<
Results
of tPSt
i
Result*;
of test
jper.itor
'
Median
ELISA
PA IpCj
Mean El ISA
iinti PA l|:G
(Wi/ml)
std [)rv of
the Test
0»<*rritors
¦ pst Oprrntor i
Number
T est Opcr jtor 4
Number
EUSA
Animal 10
wj"ort,in'c
0.000
0.000
0.000
-------
Paye 5 of 5
1020-CG920503 EUSA Results
QG/TR by:
Results
Results
EUSA
anli-PA l(;G
Iw/mU
lest OptTiUof 1
F'ljtr N»m hi»i
Stcl Ut>v of
theT
Uptir
• Ml Operator ?
PUte Number
f I'M Operator J
Pl.itc Number
TcstOpcrjtorft
Plate Number
EUSA
iMhie
Animat >0
of tost
of test
Ddte Ijkcn
operator
111909-283 LAN
111909-285 ALS
0.000
0,000
0,000
101209-105 Pre~Scre Pre-Screen GtM
n -102109-177 LAN j q.qqq | q.qqq j
0,000
0,000
0.000
..23232
J
j%CV >30% but sample is
-------
APPENDIX U
INDIVIDUAL HEMATOLOGY RESULTS
U-l
-------
Study 1020-CG920503 Hematology
c
to
Bold > Normal Range
Underscore « Normal Range
* Clot Removed
- No Value
Parameter
Group ID
Animal ID
Sex
Day-3
Day 1
Day 2
Day 3
Day 7
Day 14
Day 21
4.36
Terminal
White Blood Cell Count (2.90-8.10 1 CP/uL)
1
L23216
M
7.61
4.61
4.56
7.40
8.16
6.51
1
L23218
M
5.86
5.51
3.49
6.35
6.03
5.44 •
3.71 1 --
1
I" L23220
M
7.74
7.83
5.81
6.23
2.76*
I 2.47*
4.05" j -
1
L23222
M
12.47
8.98
7.94*
9.59
7.71
7.87
5.25
1
I L23223
M
6.58
4.99
5.95
2.75
5.05
2.84*
3 32
Average
8.05
6.38
5.55
6.46
5 94
5.03
4.14" 1 --
Std Dev
2.59
1.92
1.67
2.48
2.18
2.33
0.73
2
L23206
M
i 7.12
7.94
7.16*
5.99
4.12 *
7.39
4S6 1 --
2
L23210
M
7.06
6.73
7.34
-
..
5.02 *
|— g
L23211
M
4.97
5.61
5.09
5.83
4.62
6.40 '
2.21 j ' -
2
I L23215
M
6.66
5.11
6.01
6.41
4.93
4.86*
3.89 j
2
L23219
M
h g o2
6.84
7.74
8.03*
6.20
4.82 *
4.94 1
Average
6.77
6.45
6.67
6.57
4.97
5.70
3.90 1
Std Dev
1.12
1.11
1.09
1.01
0.89
1.15
~Tz\ t -
3
L23217
M
9.44
10.18
10.87
11.21
8.28
8.49
6.64 ' j" " --
3
L23227
M
8.27
I 7.67
7.31
7 09
8.19
4.39 i
l_ _
L23228
M
6.72
7.38
7.49
6.80
6.36
6.58
3.58 1
r 3
L23229
M
5.90
6.53
6.15
7.27
6.03
6.52
4.09
~ "3I2
...
3
L23230
M
5.98
6.68
6.49
5.73
4.65
3.18*
-
Average
7.20
7.81
7.73
7.66
6.48
6.59
4.40
-
Std Dev
1.50
1.49
1.87
2.08
1.34
2.11
1.32
-
4
L23205
M
7.59
7.63
8.14
7.53
6.83
8.47
5.09
4
L23207
M
5.93
6.93
5.80
6.05
5.39
5.20
2.79
4
L23225
M
5.80
6.96
7.20
3.67
-
--
-
4
L23231
M
7.70
8.30
8.09
7.74
7.76
6.44
2.94
4
L23235
M
6.89
6.90
8.72
7.82
11.99
-
Average
6.78
7.34
r 7.59
6.56
7.99
6.70
3 61
Std Dev
0.89
0.61
1.14
1.77
2.84
1.65
1.29
5
L23200
M
6.20
3.43
4.33
-
-
-
8.91
5
h L23201
M
9.73
10.02
12.64
13.47
-
-
5
L23212
M
7.75
8.03
8.33
8.33
6.88*
5.25
4.25
5
L23214
M
8.64 1
8.23
3.47
-
--
--
-
5
L23234
M
9.22
8.72
9.23
8.51
-
-
..
Average
8.31
7.69
7.60
10.10
6.88
5.25
4.25
8 91
Std Dev
1.39
2.50
3.75
2.92
-
-
6
L23203
M
9.13
8.80
6.71
4.68
-
-
6
L23204
M
5.37
6.68
7.05
5.43
--
-
...
6
L23213
M
-
10.32
-
-
-
--
12.85
6
L23221
M
6.02
..
-
-
_
..
..
6
L23232
M
-
5.69
8.78
1.55
..
-
--
-
Average
6.84
7.87
7.51
3.89
..
--
..
12.85
Std Dev
2.01
2.08
1.11
2.06
-
[
f
Page 1
QCfftch!«», By/Date: 2 >'oCt
-------
Study 1020-CG920503 Hematology
c
oo
Parameter
Group ID
Animal ID
Sex
Day-3
Day 1
Day 2
Day 3
Day 7 | Day 14
Day 21 I Terminal
6.10 j
Red Blood Cell Count (4.20-6.70 10*6i/uL)
1
L23216
M
6.01
6.03
5.52
5.08
5.80 6.45
1
L23218
M
5.17
5.62
3.67
5.89
r 6.00
6.05*
5.46*
6.27
5.66 |
5.60 I
1
L23220
M
5.64
5.79
[ 5.28
5.38
] 5.53 *
r 1
L23222
M
6.51
| 5.92
6.04*
5.90
6.14
5.98
5.63
..
t
L23223
M
5.66
[ 5.99
5.67
5.02
5.66 t 5.48*
Average
5.80
5.87
5.24
5.45
0.43
5.83 1 5.94
5.79
Std Dev
0.50
0.17
0.92
0.25
0.45 ' 0.23 '
2
L23206
M
5.83
5.56
5.76*
5.53
6.02*
6.04
5.29
2
L23210
M
5.85
5.97
5.67
-
-
5.85*
-
-
2
L23211
M
6.47
'6.45
5.75
5.84
5.65
6.79*
5.66
-
2
L23215
M
6.42
6.29
5.87
5.69
6.26
6.42*
65TT --
6i08 »
2
L23219 j M
6.76
6.04
6.22
6.35*
6.98
6.35*
Average
) 6.27
6.06
5.85
5.85
6.23
6.29
5.77
Std Dev
0.41
0.34
0.22
0.36
0.56
0.36
0.37
3
L23217
M
6.14
5.76
5.56
5.70
6.63 1 6.41
5.83
3
L23227
M
6.29
6.42
J- 6 Q2
5.88
6.24 6.20
6.11
—
3
L23228
M
5.70
5.72
5.56
5.67
5.71 6.01
4.94
-
3
L23229
M
5.82
5.68
5.55
5.93
6.17 [ 6.52
5.97
—
3
L23230
M
6.13
6.06
5.72
5.55
6.35
5.59*
5.73
—
Average
6.02
5.93
5.68
5.75
6.22
6.15
5.72
--
Std Dev
0.25
0.31
0.20
0.16
0.33
0.37
0.46
-
4
L23205
M
5.00
5.10
4.90
4.98
5.42
5.57
5 ?1
-
4
L23207
M
6.28
5.95
6.13
6.19
5.81
6.44
5.60
-
4
L23225
M
6.47
6.14
5.26
4.93
-
-
-
-
4
L23231
M
5.45
5,66
5.34
5.27 5 77
5.94
5.58
—
4
L23235
M
6.15
5.59
5.69
5.53 5 76
--
Average
5.87
5.69
5.46
5.38 ! 5.69
5.98
5 46
-
Std Dev
0.62
0.40
0.47
0.51
0.18
0.44
0.22
~
5
L23200
M
6.06
5.70
5.66 '
-
_
-
-
5.60
5
L23201
M
6.31
5.63
5.21
5.29
--
-
-
5
L23212
M
5.96
5.80
5.76
5.61 • 5.86
6.07
5.90
-
5
L23214
M
5.99
6.07
5.38
-
-
-
-
5
L23234
M
5.92
5.65
5.45
5.12
_
_
...
Average
6.05
5.77
5.49
5.34
5.86
6.07
5.90
5.60
Std Dev
0.16
0.18
0.22
0.25
I
..
6
L23203
M
5.97
6.06
5.19
4.78
!
-
6 I
L23204
M
5.77
5.55
5.17
5.08
-
-
-
--
6
L23213
M
-
5.65
-
-
--
..
_
6.07
6
L23221 1
M
5.23
-
-
-
_
-
..
..
6
L23232
M
-
6.03
5.29
5.33
-
-
-
-
Average
5.66
5.82
5.22
5.06
-
-
..
6.07
Std Dev
0.38
0.26
0.06
0.28
-
—
Bold > Normal Range
Underscore < Normal Range
* Clot Removed
- No Value
Page 2
fHe- l"
QCrtech Iw, iiPiti; 1 - ° ^
-------
Study 1020-CG920503 Hematology
Bold > Normal Range
Underscore < Normal Range
* Clot Removed
- No Value
Parameter
Group ID
Animal ID
Sex
Day-3
Day 1
Day 2
Day 3
| Day 7
I 11.9
I Day 14
| 13.2
1 TZA~
| Day 21
l 12.3
1 Terminal
Hemoglobin (9.5-14.5 g/dL)
1
L23216
M
12.8
12.3
11.4
10.4
1
L23218
M
11.3
11.5
7.6
12.2
| i2A
r tt.5
1
L23220
M
12.0
11.6
10.6
10.8
HT.4*'*
11.2 *
[ iT.4
.
L23222
M
13.0
11.4
11.6 '
11.2
pT.8
i 12.1
f Tils
...
1
L23223
M
11.9
12.2
I 11.4
10.3
] 115
f 1T2 *
1 11-4
1
Average
12.2
11.8
10.5
11.0
TT.8
j "12.0
11.6
[
Std Dev
0.7
0.4
0.8
! o.4
08
t 6.4
i
j ~
2
r L23206
M
12.7
11.5
12.0*
11.4
'lie*'"
1 12.6
1CL9
!
2
L23210
M
12.5
12.4
11.6
-
-
1' 1Z2"*
..
i
2
L23211
M
13.2
12.7
11.3
11.6
11.3
137*
j lT3
»
2
123215
M
13.2
12.3
11.6
11.4
12.4
12.8*
--
2
L23219
M
13.5
I 12.0
12.4
12.5 •
f 14.4
12.7*'"
F 12J-
Average
13.0
12.2
11.8
11.7
12.7
12.8
1 11.6
Std Dev
0.4
0.5
0.4
0.5
1.3
0.6 "
0.'6
f
3
L23217
M
12.0
11.1
10.8
11.2
13.0
12.4
11.3
3
L23227
M
12.7
12.5
11.8
11.5
12.3
12.3
11.9
....
3
L23228
M
12.2
11.9
11.6
11.8
12.0
12.6
10.4
—
3
I L23229
M
12.8
12.1
11.6
12.4
12.9
13.7
L 122
—
3
i L23230
M
13.0
12.3
11.6
11.3
13.1
11.5*
"11". 7
Average
I 12.5
12.0
11.5
11.6
12.7
12.5
11.5
--
Std Dev
0.4
0.5
0.4
0.5
0.5
6.8
QJ
—
4
L23205
M
11.4
11.3
10.7
11.0
12.0
12.4
11.4
4
L23207
M
12.7
11.4
11.8
12.1
11.2 "
12.5
109
—
4
L23225
M
13.4
12.1
10.4
9.8
-
-
4
L23231
M
11.9
11.9
11.3
11.1
12.1
12,7
10.9
—
4
123235
M
' 13.8
12.0
12.1
12.0
12.6
...
—
Average
11.7
11.3
11.2
12.0
12.5
' "IT"1
—
Std Dev
1.0
0.4
0.7
0.9
0.6
0.2
03
—
5 ~1
L23200
M
12.4
11.4
11.1
--
-
-
11.0
5 ~i
L23201
M
13.6
11.6
10.6
11.0
-
-
..
—
5
L23212
M
12.9
12.1
11.9
11.7
12.4*
12.7
12.3
—
5
L23214 1
M
12.5
12.1
10.9
-
..
-
-
5
L23234
M
12.9
11.7
11.3
10.6
-
-
-
Average
12.9
11.8
11.2
11.1
12.4
12.7
12.3
11.0
Std Dev
0.5
0.3
0.5
0.6
--
-
6
L23203
M
12.6
12.3
10.5
9.8
-
-
-
6
L23204
M
11.7
10.6
9.9
9.7
-
-
6
L23213
M
-
11.5
-
_
-
12.3
6
L23221
M
11.6
--
-
-
-
6
L23232
M
--
12.5 I
11.0
11.3
--
--
Average
12.0
11.7
10.5
10.3
--
-
12.3
Std Dev
0.6
0.9
0.6
0.9
_
--
—
..
Page 3
pH-c,. KZ.-ZX-O'X
QCITtcli R«v, By/Pate; |z- *24 ¦ oCt
-------
Study 1020-CG920503
Hematology
Bold > Normal Range
Underscore < Normal Range
* Clot Removed
-- No Value
Parameter
Group ID
Animal ID
Sex
Day-3
Day 1
Day 2
Day 3
I Day 7
! Day 14
1 41.3
i Day 21
t 38.4™
i Terminal
Hematocrit (27.2-45.9 %)
1
L23216
M
38.4
38.7
35.1
32.2
P3&3
1
L23218
M
34.1
36.2
23.8
38.9
40.3
] 39.7*
1 3&3
;
1
L23220
M
36.0
37.0
34.3
I 35.0
i 35.6 '
33^8*""
1 34^6
;¦
1
L23222
M
38.7
34.8
36.6 "
34 6
! 36.3
I 37.5
j 35.2
—
1
L23223
M
36.8
39.1
I 36.0
31 4
36.0
i ' 34.6 *
34.9
1
Average
f 36.8
37.2
33.2
34.4
37.3
1 37.3
F 35.7~~
i
Std Dev
r 1.9
1.8
5.3
2.9
t 2.0
3.1
j 1.5
!
i
2
F L23206
M
37.9
36.2
37.8 *
36.9
39.9 *
39.4
! ' * 33.7
1
2
L23210
M
' 38.3
39.4
36.1
-
t
] 37.9
i
2
L23211
M
h 39.9
38.9
34.1
r 35.5
35.8
| 41.6*
! 33.3
—
2
L23215
M
38.6
38.0
35.6
34.5
38.9
| 40.3 '
37 5
—
2
L23219
M
r 42.4
38.3
38.9
39.8*
46.5
38.7 *
37.4
1
Average
39,4
I 38.2
36.5
36.7
40.3
1 39.6
~35.5
--
Std Dev
1.8
1.2
1.9
2.3
4.5
1 1.4
2.3
—
r
3
L23217
M
36.5
34.4
33.7
35.0
41.0
{ 38-9
1 34^9
3
' L23227
M
39.0
41.2
37.1
r- 3g j
41.3
' 38.7
' 37\2
—
3
I L23228
M
37.1
f- 37 6
36.3
r 36.6
38.5
39.1
31,3
—
3
L23229
M
37.9
37.1
35.8
38.7
40.0
41.8
37.8
...
1" 3
L23230
M
r 38.1
37.1
I" 35.0
33.8
41.8
33.9 *
34.8
Average
r 37.7
37.5
35.6
36.0
40 5
38.5
35.2
—
Std Dev
1.0
h 2.4
1.3
1.8
1.3
2.9
2.6
—
4
L23205
M
33.9
34.5
32.8
^ 34.7
37.6
38.3
35.1
..
4
L23207
M
38.8
36 2
37.6
39.9
I 36.6
39.6
33.6
4
L23225
M
40.3
37.2
31.5
29.4
-
-
--
4
L23231
M
35.6
36.3
34 5
34.6
39.8
J 38.9
33,5
4
L23235
M
h _
36.9
37.5
37.2
38.7
-
-
—
Average
38.1
36.2
34.8
35.2
38.2
38.9
34.1
—
Std Dev
3.3
1.0
2.7
3.9
1.4
0.7
0.9
--
5
L23200
M
38.9
35.4
35.2
--
--
-
36.9
5
L23201
M
41.7
35.7
32.0
32.5
-
-
~
5
L23212
M
38.9
37.3
36.8
35.5
37.9 *
38.5
37,3
-
5
L23214
M
38.9
39.7
33.5
-
-
..
-
-
5
L23234
M
38.8
36.6
34.7
32.7
-
-
-
Average
39.4
36.9
34.4
33.6
37.9
38.5
37.3
36.9
Std Dev
1.3
1.7
1.8
1.7
--
-
-
6 i
L23203
M
37.9 1
38.3
32.0
29,3
--
..
--
6
L23204
M
35.7
33.3
30.2
30.0
-
-
-
6 ~1
L23213
M
-
35.2
-
-
-
-
44.7
6
L23221
M
34.7
--
-
-
-
__
--
6
123232
M
--
39.0
33.9
33.5
--
..
--
-
Average
36.1
36.5 1
32.0
30.9 T -
-
-
44.7
Std Dev
1.6
2.7
1.9
2.3
-
-
-
—
Page 4
-------
Study 1020-CG920503
Hematology
c
On
Bold > Normal Range
Underscore < Normal Range
* Clot Removed
- No Value
Parameter
Group ID
Animal ID
Sex
Day-3
Day 1
Day 2
Day 3 I Day 7
I Day 14
1 64.0
I Day 21
| 62.9
i Terminal
MCV (59.1 -75.4 fL)
1
L23216
M
64.0
64.1
63.5
63.3
Peai
1
L23218
M
65.9
64.4
l 64.7
65.9
67.2
t 64.7 ^
i 62.5
!
t- i
L23220
M
63.9
64.0
64.9
65.0
I 64.4*
1 62.0*""
1 61.8
..
1
L23222
M
59.5
58.7
60.7 *
58.7
! 59.0
! 591'"
! 58.9
i
1
L23223
M
65.1
65.2
63.4
62.5
63.7
[ 63.1 *
62 0
;
Average
f 63.7
63.3
63.4
63.1
64.1
] ' 6Z7
61 6
;
Std Dev
2.5
2.6
1.7
2.8
I 3.2
I 1.9
| 1.6
!
1
!
2
L23206
M
64.9
65.1
65.6*
66.7
66.3 *
1 65 ri
! 63.7
^ 2
L23210
M
65.4
66.1
63.6
-
-
r-648"*""
i
—
2
L23211
M
61.6
60.4
59.3
60.7
6374 '
61.2 *
58.9
—
2
L23215
M
60.1
60.5
60.7
60.5
62.2
I 62.8*
62.0
!
2
L23219
M
62.8
63.5
62.5
62 6 *
66.6
61.0*
t 61.5
I
Average
63.0
63.1
62.3
62.6
64.6
| 63.0
r 61. 5'
!
Std Dev
2.2
2.6
2.5
2.9
22
I 1.9
2.0
I
3
L23217
M
59.5
59.8
60.6
61.4
61.8
'60.7 "
59~8
!
3
^ L23227
M
62.0
64.3
61.6
61.4 J 66.2
62.4
60.9
—
3
L23228
M
65.2
65.8
65.4
t 64.4
67.5
65.0 "
63.4
—
3
L23229
M
65.2
65.4
64.5
65.3
64.9
64.1
63.3
3
L23230
M
62.1
61.2
61.2
60.9
65.7
60.6*
60.9
Average
62.8
63.3
62.7
62.7
65.2
62i
61.7
—
Std Dev
2.4
2.7
2.1
2.0
2.1
2.0
16
—
4
L23205
M
67.6
ri7.7
67.0
69.7
69.3
68.7
67'5
—
4
L23207
M
61.8
60.9
61.3
64.5
I 63.0
61.5
59"9~"
—
4
L23225
M
62.3
60.5 "
59.8
59.6
-
-
..
4
L-23231
M
65.3
64.2
64.6
65.7
69.1
65.4
60 1
—
4
L23235
M
67.9
66.0
65.9
67 3
67,1
-
—
Average
65.0
63.9
63.7
65.4
67.1
t 65.2
""62.4
--
Std Dev
2.9
3.1
3.1
3.8
2.9
3.6
4.2
5
L23200
M
64.2
62.1 ^
62,1
-
-
-
-
65.9
5 I
L23201
M
66.0
63.5
61.4
61.5
-
-
-
-
5
L23212
M
65.4
64.2
64.0
63.2
64.7*
63.5
63.2
-
5
L23214\
M
64.9
65.4
62.3
-
-
-
5
L23234
M
65.5
64.8
63.7
63.9
-
-
Average
65.2
64.0
62.7
62.9
64.7
63.5
63.2
65.9
Std Dev
0.7
1.3
1.1
1.2
--
--
-
-
6 ^
L23203
M
63.5
63.2
61.7
61.2
-
-
-
-
6
L23204
M
61.9
60.1
58.4
59.1
-
-
6
L23213
M
-
62.3
-
..
-
..
--
73.7
6
L23221
M
66.3
-
-
_
-
-
-
6
L23232
M
-
64.6
64.1
62.9
-
-
-
-
Average
63.9
62.6
61.4
61.1
--
-
-
73.7
Std Dev
2.2
1.9
2.9
1.9
..
-
-
-
Page 5
fltc. \z-Z\-oct
QCfTech Rev. ifftiti:
-------
Study 1020-CG920503 Hematology
c
Parameter
Group ID
Animal ID
Sex
Day-3
Day 1
Day 2
Day 3
Day 7
Day 14
Day 21
20T2
Terminal
MCH (19.7-24.6 pg)
1
L23216
M
I 21.2
20.4
20.6
20.5
f "20.5
20.5
..
r i
L23218
M
I 21.9
20.5
20.7
20.7
20.8
r 20.5*
| 203
—
1
L23220
M
21.3
20.0
' 20.1
20.1
20.7 *
20.5 •
h 20.4
r 1
L23222
M
20.0
19.2
19.2*
19.0
T ig^
19.3
19.3
—
1
L23223
M
21.0
20.3
20.1
20.5
20.3
20.5'
20.2
Average
I 21.1
20.1
20.1
20.2
20.3
20.3
20.1
—
Std Dev
0.7
0.5
0.6
id
0.7
0.5
0.4
2
F L23206
M
21.7
20.6
20.9*
20.6
21.0 '
20.8
20.7
2
L23210
M
21.3
20.8
20.4
-
20 8 '
-
~
2
L23211
M
2Q4
19.7
19.7
19.9
20.0
20.2 *
19.9
-
2
I L23215
M
20.6
19.6
19.7
20.0
19.9
19.9*
19.6
—
2
L23219
M
19.9
19.9
19.9
19.8*
20V7
20.0 '
20.3
Average
20.8
20.1
I 20.1
20.1
20.4
20 3
20,1
Std Dev
r 0.7
0.5
0.5
0.4
0.5
0.4
0.5
-
3
L23217
M
19.6
19.2
19.5
19.7
19.6
19.3
19.5
—
3
L23227
M
20.3
f 19.4
19.6
h 19.6
19.7
19.8
19.4
-
3
r L23228
M
21.5
20.8
20.9
20.8
21.0
20.9
—
3
L23229
M
22.0
21.3
20.9
21.0
20.9
210
20.5
-
3
L23230
M
21.3
20.2
20.2
20.4
20.6
20.6 *
20.5
-
Average
20.9
20.2
20.2
20.3
20.4
20.3
20.2
..
Std Dev
1.0
0.9
0.7
L °-6
0.7
0.7
0.7
4
L23205
M
22.7
22.1
21.9
22 2
22 2
22.2
21.9
-
4
L23207
M
20.2
19.2
19.3
19.5
19.3
19.4
19.5
-
4
123225
M
20.8
19.6
19.8
20.0
-
--
-
4
L23231
M
21.9
21.1
21.1
21.2
21.1
21 3
19.5
..
4
L23235
M
22.4
21.5
21.3
21.6
21.9
--
Average
21.6
20.7
20.7
20.9
21.1
21 0
20.3
-
Std Dev
1.1
1.2
1.1
1.1
1.3
1.4
-
5
L23200
M
20.5
20.1
19.6
-
--
19,6
5
L23201
M
21.5
20.6
20.4
20.7
--
-
-
5
L23212
M
21.7
20.8
20.6
20.9
21.2 *
20.9
20.9
5 1
L23214
M
20.9
19.9
20.2
--
--
...
..
5
L23234 ~1
M
21.7
20.8
20.7
20.7
-
--
-
-
Average
21.3 '
20.4
20.3
20.8
21.?
20.9
20.9
19.6
Std Dev
0.5
0.4
0.4 ,
0.1
-
--
6
L23203 ^
M
21.2
20.3 H
20.2
20.5
--
_
-
-
6
L23204
M
20.2
19.1
19.2
19.1
¦¦
-
--
6
L23213
M
--
20.3
-
-
--
--
20.3
6
1.23221
M
22.2
-
-
-
--
..
-
6
L23232
M
-
20.7
20.9 1
21.2
--
..
--
Average
21.2
20.1
20.1
20.3
-
--
20.3
Std Dev
1.0
0.7
0.9
1.1
-
..
-
Bold > Normal Range
Underscore < Normal Range
* Clot Removed
- No Value
Page 6
QCITech Rev. u • z v • ^
-------
Study 102Q-CG920503 Hematology
Parameter
Group ID
Animal ID
Sex
Day -3
Day 1
Day 2
Day 3
Day 7
! Day 14
! Day 21
32'. 1
I Terminal
MCHC (31.0-34.9 g/dL)
t- j
L23216
M
33.2
t 31.9
32.4
32.4
31.0
r 32.i
1
f L23218
M
33.2
31.9
32.0
31.3
30.9 '
t" 31J *
3274
1
1
L23220
M
33.4
31.2
31.0
31.0
32.1 '
j 33.1 *
33.0
1
L23222
M
33.6
32.7
31.6*
I 32.5
32.4
32.2
32 8
;
1
i L23223
M
32.3
31.1
31.7
32.8
31.9 '
" 32.5 *
32.6
Average
33.1
31.8
31.7
32.0
31.7
32.3
32.6
—
Std Dev
0.5
0.6
0.5
0.8
0.7
0.5
073
2
L23206
M
33.5
31.7
31.9 *
31.0
31.6*
31.9
32.4
2
L23210
M
t- 32 e
31.5
32.1
-
-
32.1 *
-
~
2
L23211
M
^ 33.1
32.7
33.2
32.8
31.5
32.9 *
33.9~~
-
2
r ^21-
M
34.3
32.4
32.5
33.0
32.0
31.8*
.___
2
f L23219
M
31.7
31.3
31.8
31.6*
31.1
32.8*
32.9
—
Average
33.0
31.9
32.3
32.1
31.6
32.3
32.7
Std Dev
1.0
0.6
0.6
1.0
0.4
0.5
1.0
—
—
3
L23217
M
r 33.0
32.1
32.1
32.0
31.7
31.8
3275
r
3
r L23227
M
32.7
30.3
31.9
31.9
29.8
31.7
31.9
r ¦ ¦
3
L23228
M
r 32.9
31.7
31.9
32.4
31 1
32.2
33.1
3
L23229
M
33.7
32.6
32.5
32.1
32.2
32.7
32.4
3
L23230
M
34.2
33.1
33.1
[ 33.5
31.4
33.9*
33.7
Average
h 33.3
32.0
32.3
32.4
31.2
'
-
Std Dev
0.6
1.1
0.5
0.7
0.9
0.9
0.7
4
L23205
M
r 33.6
32.6
32.7
31.8
32.0
32.3
32.5
-
4
L23207
M
r 32.7
31.6
31.4
30.2
30.7
31.5
32.5
-
4
L23225
M
33.4
32.5
33.2
33.5
-
-
«
¦
4
L23231
M
33.5
32.8
32.8
32.2
30.5
32.6
32.5
4
L23235
M
33.0
32.6
32.3
32.2
32.6
-
-
-
Average
33.2
32.4
32.5
32.0
31.5
32.1
32.5
Std Dev
0.4
0.5
0.7
1.2
1.0
0.6
0.0
-
5
L23200
M
31.9
32.3
31.6
--
-
-
-
29.7
5
L23201 1
M
32.6
32.4
33.2
33.7
-
-
-
„
5
L23212
M
33.2
32.4
32.2
33.0
32.7*
32.9
33.0
-
5
L23214
M
32.3
30.4
32.4
-
-
-
-
5
L23234
M
33.2
32.0
32.5
32.4
--
-
_
Average
32.6
31.9
32.4
33.0
32.7
32.9
33.0
29.7
Std Dev
0.6
0.9
0.6
0.7
-
-
-
..
6
L23203
M
33.3
32.1'
32.7
33.5 1
--
-
6
L23204
M
32.7
31.8
32.9
32.4
--
-
-
--
6 1
L23213
M
-
32.6
-
--
-
-
-
27.5
6
L23221
M
33.5
-
-
i
--
6 1
L23232
M
-
32.1
32.6
33.7
-
-
-
Average
33.2
32.2
32.7
33.2
..
-
--
27.5
Std Dev
0.4
0.3
0.2
0.7
-
-
--
-
I
Bold > Normal Range
Underscore < Normal Range
* Clot Removed
-- No Value
fWd \z-Z\-cFt
Page 7
-------
Study 1020-CG92O5O3 Hematology
c
Parameter
Group ID
Animal ID
Sex
Day-3
Day 1
Day 2
Day 3
I Day 7
I Day 14
Day 21
! Terminal
RDW (10.3-14.4%)
r i
L23216
M
11.7
11.7
11.4
11.4 I 13.0
' 11.5 "
1 11.3
i
1
L23218
M
12.2
12.4
12.4
12.4
1£L4
' 11.5*
HI
—
1
L23220
M
13.4
13.4
13.1
13.5
I 12.9 *
11.7*
1 i iTa
!
1
L23222
M
r 11.5
11.7
11.8*
I 12.1
12.6
12.0
11.2
__
1
L23223
M
12.3
12.0
12.0
12.2
12.8
12.2 *
12.0
Average
12.2
12.2
12.1
12.3
12.7
11.8
1 11.4
—
Std Dev
0.7
0.6
0.8
0.2
0.3
f 0.4
2
L23206
M
13.2
14.2
14.1 *
14.2
13.4 •
12.6
11.8
—
2
L23210
M
12.7
12.3
12.7
-
12.7*
-
-
2
(_ L2321!
M
12.8
13.1
13.1
^ o
CO < Normal Range
Underscore < Normal Range
* Clot Removed
- No Value
Page 8
pHc. tz-Zl-e*!
-------
Study 102Q-CG920503 Hematology
c
o
Bold > Normal Range
Underscore < Normal Range
* Ciot Removed
- No Value
Parameter
Group ID
Animal ID
Sex
Day-3
Day 1
Day 2
Day 3
Day 7
Day 14
I Day 21
1 Terminal
Platelet Count (137-558 W/uL)
f 1
L23216
M
426
339
257
270
733
451
f 248 1 --
1
L23218
M
j_ 35Q
355
224
364
408
~"73 *
t 451
1
L23220
M
741
1137
1087
806
120'
r 26 *
685
1
L23222
M
' 501
r '415
306 '
410
509
| 373
f 352 1 -
1
L23223
M
707
896
826
10
652
31 *
631
Average
545
628
540
372
484
191
473
...
Std Dev
365
F 392
288
239
205
' 184
..
I
^ 2
L23206
M
616
693
435 "
623
62*
520
591
-
2
L23210
M
611
569
580
-
-
258 *
-
-
r g
L23211
M
381
328
266
325
211
240 *
114
..
|- g
L23215
M
413
355
267
362
208*
558
-
2
L23219
M
r~346
417
r 435
377*
358
91 *
340 I
Average
473
472
r 397
422
265
263
401 ""| ~ -
Std Dev
130
155
133
136
163
157
221
I
L23217
M
440
363
326
370
233
296 I --
3
L23227
M
474
416
342
416
390
324
353
3
L23228
M
307
352
351
399
257
113
3
L23229
M
510
r 446
366
461
f 393
470
275 r --
3
l_ L23230
M
456
405
388
428
404
26*
381 !
Average
437
396
355
r 4^5
353
284
284' "T --
Std Dev
77
39
24
34
71
164
104
4
L23205
M
486
476
454
513
414
506
454
..
4
L23207
M
486
425
479
549
512
503
456
-
4
L23225
M
380
563
299
408
-
-
-
4
L23231
M
588
588
517
575
443
564
448
„
4
L23235 1
M
345
203
264
322
166
-
-
Average
457
451
403 n
473
384
524
453
--
Std Dev
97
153
113 1
106
151
34
4
5
L23200
M
653
457
-
--
„
408
5
L23201
M
634
555
220
173
--
--
-
5
L23212
M
426
349
359
396
251 *
325
170
--
5
123214
M
600
626
270
-
..
-
-
5
L23234
M
588
531
534
353
-
..
..
Average
580
504
360
307
251
325
170
408
Std Dev
gQ -i
105
124
118
--
-
6
L23203
M
421 i
426
198
288
--
..
-
-
6
L23204
M
539
486
250
476
-
-
6 ~1
L23213
M
-
568
..
-
-
-
193
6
L23221
M
518
-
-
-
_
-
-
6
L23232
M
-
555
298
107
-
-
Average
493
509
249
290
«
-
193
Std Dev
63
66
50
185
-
-
-
-
inUuiii CowMQfc/
tfv/Datf: "//> ///
Page 9
|Nc_
(ICiTeARev.Iffiitt; v*
-------
Study 1020-CG920503
Hematology
Parameter
Group ID
Animal ID
Sex
Day-3
Day 1
Day 2
Day 3
Day 7
Day 14
Day 21
Terminal
MPV (5.2-7.7 fL)
1
L23216
M
f 8.2
7.1
7.0
I 7.0
6.1
6.7
7.5
1
h L232ig
M
8,4
6.6
r 6.7
7.5
7.0
8.5 *
6.1
—
1
L23220
M
8.7
7.7
6.9
6.7
9.0*
10.6*
6.6
...
r i
L23222
M
7.1
6.6
6.5 •
6.1
57
6.6
6.3
—
1
' L23223
M
r 9.8
8.4
6.8
13.1
6.6
11.4 ¦
6.2
Average
8.4
7.3
6.8
8.1
6,9
8.8
"63
Std Dev
1.0
0,8
0,2
2.9
1.3
2.2
0.6
2
h L23206
M
8.6
I 7.5
7.5*
8.1
9.0*
6.7
6.6
-
2
L23210
M
8.7
7.6
6.0
--
-
7.3 *
-
2
L23211
M
7.3
r 6.5
6.1
' 6.1
7.6
7.4 *
7.3
--
[- g
L23215
M
7.7
' 7.0
7.1
6.4
73
7.9 ¦
63
2
L23219
M
8.3
7.1
7.2
7.1 *
6.9"
6.1
Average
8.1
I 7.1
6.8
6.9
7.8
7.2
6.6
Std Dev
0.6
0,4
0.7
0.9
08
0.5
0.5
—
3
L23217
M
9.0
6.9
6.4
6.3
8.6
6.9
62
-
3
L23227
M
8.3
7.5
6.3
7.7
8.5
6.7
6.5
-
3
L23228
M
9.0
7.2
6.2
7.8
8.3
7.4
7.5
-
3
L23229
M
r 7.1
6.4
6.1
6.7
7.7
6.2
7.0
3
L23230
M
7.9
7.0
6.4
7.4
78
13.1 *
6.0
-
Average
^ 8.3
7.0
6.3
7.2
8.2
8.1
66
Std Dev
0.8
0.4
0.1
0.7
0.4
2.9
0.6
--
4
L23205
M
8.4
6.0
r 6.0
8.1
8.1
6.5
5.9
-
4
L23207^
M
7.0
6.0
5.7
7.0
6.2
5.7
5.6
..
4
L23225
M
8.7
6.1
8.3
15.2
--
_
-
..
r 4
L23231
M
8.2
6.3
6.3
9.1
9.2
6.7
5.5
4
L23235
M
7.6
6.8
7.2
7.3
10.0
..
...
Average
8.0
6.2
6.7
9.3
8.4
6.3
5.7
-
Std Dev
0.7
0.3
1.1
3.4
1.6
0.5
0.2
5
L23200
M
8.5
6.5
6.6
-
-
-
...
9.2
5
L23201
M
7.1
5.8
6.7
11.8
-
-
-
-
5
L23212
M
7.6
7.3
7.6
8.7
8.4 *
7.6
7.9
..
5
L23214
M
6.9
6.0
6.3
--
--
-
...
-
5 1
L23234
M
7.6
6.4
6 1
7.5
-
-
-
Average
7.5 I
6.4
6 7
9.3 ^
8.4
7.6
7.9
9.2
Std Dev
0.6
0.6 "
0.6
2.2
-
„
-
6
L23203
M
7.9
6.8
6.7
14.4
--
..
6
L23204
M
8.0
6.1
¦
13.1
-
-
..
6
L23213
M
-
5.9
-
..
-
-
-
14.4
6
L23221
M
8.8
-
-
..
--
„
...
6
L23232
M
-
5.8
6.5
21.7
--
-
-
Average
8.2
6.2
6.9
16.4
-
--
-
14.4
Std Dev
0.5
0.5
0.5
4.6
-
-
--
Bold > Normal Range
Underscore < Normal Range
* Clot Removed
- No Value
12. - ^ I -
Page 10
-------
Study 1020-CG920503
Hematology
Parameter
Group ID
Animal ID
Sex
Day-3
Day 1
Day 2
Day 3
Day 7
Day 14
Day 21
Terminal
%NEUT (17.2-40.0 %)
1
L23216
M
23.2
7.2
17.1
17.7
31.6
24.5
20.3
-
,
L23218
M
27.4
27.5
26.0
23.5
29.9
30.3*
2ai
..
1
L23220
M
60.9
30.4
36.0
29.9
26.4 *
15.7*
24.6
—
1
L23222
M
23.6
|_ i7g
17.2*
17.6
16.6
24.1
19.5
..
1
f L23223
M
22.6
17.7
22.6
6.3
22.4
10.3*
17.9
..
Average
31.5
20.1
23.8
19.0
25.4
21.0
22.1
—
Std Dev
16.5
I 9.2
7 8
8.7
6.0
7.9
4.2
..
2
L23206
M
24.0
20.2
16.4*
14.9
20.5*
26.0
15.5
—
2
r- L23210
M
31.7
25.3
25.9
-
-
23.2 *
..
2
r 123211
M
' 26.3
27.6
26.4
20.8
24.7
27.9 •
18.1
-
2
L23215
M
24.8
25.6
17.4
19.9
22.2
19.9*
17.5
-
2
L23219
M
21.9
r 17.7
19.5
11.5 *
20.3
18.3 * i 12.6
-
Average
25.7
23.3
21.1
16.8
21.9
23.1
15.9
-
Std Dev
3.7
4.1
4.7
44
2.0
4.0
2.5
-
^ 3
L23217
M
18.6
14.0
12.0
11.4
20.1
18.6
14.6
-
3
' L23227
M
23.1
n i5 g
16.9
15.9
18.4
19.4
17.8
-
3
L23228
M
30.8
27.4
28.2
30.1
30.2
34,2
23.5
3
L23229
M
20.2
16.8
19.6
15.2
21.8
19.4
15.1
-
3
L23230
M
22.5
26.3
21.3
24.6
21.5
18.0 *
16.3
Average
23.0
20.1
19.6
19.4
22,4
21.9
17.5
Std Dev
4.7
6.3
6.0
7.7
4.6
6.9
3.6
4
L23205
M
21.8
16.9
14.4
14.0
20.9
24.0
17.8
15.4
4
L23207
M
26.5
24.0
r 26 7 -j
21.6
24.6
15.9
4
L23225
M
36.9
39.9
51.1
9.5
..
4
L23231
M
28.6
28.0
29.6 -1
30.2
33.6
22.0
18.8
4
L23235
M
20.6
18.6
13.2
136
33.1
Average
26.9
25.5
27.0
17.8
27.9
21.5
r 16.0
Std Dev
6.5
9.2
15.3
8.2
6.4
3.4
0.7
5
L23200
M
30.0 '
2.7
6.5
-
--
--
--
14.2
5
L23201
M
27.5
26.5
34.3
36 1
--
-
-
-
^ 5
L23212 ^
M
16.2
15.6
13.4
12.7
19.2*
15.6
13.2
..
5
L23214
M
39.4
33.2
69.3
-
-
--
5
L23234
M
40.9
34.0 29.6
53.3
-
-
...
Average
30.8
22.4
30.6
34.0
19.2
15.6
13.2
14.2
Std Dev
10.0
13.3
24.4
20.4
-
--
j
6
L23203 "1
M
15.6
23.0
49.1
25.1
--
-
__
6
L23204
M
30.9
31.3
52 3
37.2
-
--
-
6
123213
M
--
23.0
--
--
--
29.3
6
L23221
M
32.5
-
-
--
--
--
--
-
g ^
L23232
M
-
31.0
69.8
42.3
--
-
-
--
Average
26.3
27.1
57,1
34.9
-
-
-
29.3
Std Dev
9.3
4.7
11.1
8.8
-
-
—
Bold > Normal Range
Underscore < Normal Range
* Clot Removed
- No Value
Page 11
{*CI. 12'Z
¦ \ (\te \
¦ ¦ ¦¦¦¦¦ ."I'. ¦' ¦¦¦
-------
Study 1020-CG920503
Hematology
c
Bold > Normal Range
Underscore < Normal Range
* Clot Removed
- No Value
Parameter
Group ID
Animal ID
Sex
Day-3
Day 1
Day 2
Day 3
Day 7
Day 14
Day 21
eas
Terminal
"oLYMPH (57.5-81.1 %)
1
L23216
M
h 65 5
78.7
72.7
72.9
56.8
64,2
—
1
L23218
M
65.2
63.2
65.3
69.1
61.2
* 60.1
62.3
1
t- L23220
M
30.6
58.5
60.1
64.6*
76.7*
64.2
—
1
L23222
M
69.1
73.7
75.6*
' 74.5
75.5
68.8
71.5
—
1
L23223
M
65.9
74.2
70.8
89.1
70.6
82.5*
74.1
Average
59.3
I 69.7
67.3
73.1
65.7
70.5
68,1
—
Std Dev
16.1
8.4
9.3
10.5
7.4
9.1
419
—
2
L23206
M
66.9
71.9
76.0*
78.6
71.0*
65.5
"76.3
-
2
L23210
M
59.1
67.1
66.7
-
__
67.5*
..
-
2
L23211
M
64.3
64.6 T 63.9
70.7
66.4
' 59.7 •
69.7
2
L23215
M
69.8
67.6
77 1
74.6
71.1
73.4*
mi
2
L23219
M
"" 70.2
H75.3
71.5
82.8*
67.3
72.1 "
77.2
...
Average
66.1
69.3
71.0
76.7
67.6
74.8
Std Dev
4.6
4.3
5.7
5.2
5.5
-
3
L23217
M
71.7
75.3
74.7
76.8
63.4
67.9
70.9
3
L23227
M
69.1
74.6
74.0
r __ _
73.5
73.1
72.8
3
L23228
M
55.3
60.7
60.4
57.7
f 53.9
53.6
64.6
-
3
L23229
M
73.3
76.6
72.9
77.6
70.4
73.8
77.6
-
3
123230
M
71.4
67.8
73.4
I 70.1
69.1
75.2 *
79.1
~
Average
h 6g 2
71.0
71.1
71.5
66.1
68.7
73.0
-
Std Dev
7.3
6.7
6.0
CO
00
7.7
8.9
5.8 ~"
~
4
L23205
M
70.0
73.9
75.6
79.5
67.9
72.9
75.8
-
4
L23207
M
64.9
68.7
65.5
68.8
64.7
64.9
74.1
—
4
L23225
M
51.1
54.0
45.1
75.8
-
-
-
4
L23231
M
64.9
65.3
63.3
62.3
57.7
71.0
72.8
-
4
L23235
M
70.2
73.7
78.7
76.6
59.2
...
Average
64.2
67.1
65.6
72.6
62.4
69.6
74.2
...
Std Dev
7.8
8.2
13.2
7.0
r 4.8
4.2
1 5
-
5
L23200
M
60.6
87.5
85.6
-
-
__
73.6
5
L23201
M
64.3
67.3
61.3
60.5
--
-
-
5
L23212
M
77.1
76.9
80.7
81.1
\ 67g *
74.8
76.0
5
L23214
M
46.4
54.7
r 23.0
-
--
5
L23234
M
51.2
60.2
64.2
44.1
..
Average
59.9
69.3
63.0
61.9
67.9
74.8
76.0
73.6
Std Dev
12.0
13.1
24.6
18.5
-
-
6
L23203
M
78.7
68.8
46.3
68.5
-
..
6
L23204
M
56.8
58.7
44.7
56.0
__
--
6 1
L23213
M
-
68.6
-
-
--
-
__
59.6
6
L23221
M
59.5
--
-
-
..
„
..
6 1
L23232
M
-
60.9
27.5
48.4
-
Average
65.0
64.3
39.5
57.6
...
..
59.6
Std Dev
11.9
5.2
10.4
10.1
-
...
..
Page 12
t-o^
-------
Study 1Q20-CG920503 Hematology
Parameter
Group ID
Animal ID
Sex
Day-3
Day 1
Day 2
Day 3
Day 7 ! Day 14
Day 21
Terminal
%MONO (0.0-6.1 °o)
r f
L23216
M
1.3
0.4
0.5
3.2
4.3
1"1
0.9
—
1
L23218
M
1.9
2.3
0.8
1.3
1.8
4.1 *
1.8
—
1
L23220
M
2.2
3.2
3.8
2.1
1.4 •
0.9*
1.1
—
1
L23222
M
r i.o
0.9
1.1 *
r 1.6
0.7
1.2
0.8
—
1
1 L23223
M
5.4
0.8
1.0
0.5
0.9
1.1 *
0.8
--
Average
2.4
1.5
1.4
1.7
1.8
1.7
1,1 "
--
Std Dev
1.8
1.2
^ 1.3
1.0
1.5
1.4
0,4
...
2
L23206
M
1.3
1.3
2.4*
2.2
1,1 *
0.8
0.6
—
2
L23210
M
1.8
1.3
1.3
-
-
1.0 *
-
__
2
L23211
M
1.8
1.5
2.3
1.8
1,8
3.6 *
1.2
-
2
L23215
M
1.2
1.3
r i.o
1.1
1.0
0.8 *
0.9
-
2
L23219
M
1.2
0.8
2.7
1.0*
3.0
0.8*
1.0
-
Average
1.5
1.2
1.9
1.5
1.7
1.4
0.9
-
Std Dev
0.3
0.3
0.7
0.6
0.9
1.2
0.3
-
3
L23217
M
1.4
1.0
1.9
1.7
2 2
3.1
1.5
-
3
L23227
M
1.3
r~ 2.8
2.4
1.0
1.5
2.0
1.0
-
3
L23228
M
3.8
1.3
I 1.6
1.7
3.8
1.2
1.6
-
3
L23229
M
1.4
2.0
r 2.3
2.3
0.9
0.8
1.3
-
3
L23230
M
1.1
0.7
1.0
L 1-1
2.3
1.1 *
0.7
.
Average
1.8
1.6
1.8
1.6
2.1
1.6
1.2
--
Std Dev
1.1
0.8
0,6
0.5
1.1
0.9
0.4
„
4
L23205
M
1.8
2.3
r 3.1
1.2
1.5
3.3
1,7
_
4
L23207
M
3.5
2.6
1.4
3.6
3.9
3.8
1.2
4
L23225
M
4.8
1.5
0.4
8.3
--
--
--
4
L23231
M
2.2
1.5
2.3
2.5
2.8
0.8
--
4
L23235
M
1.7
1.2
1.7
3.7
1.2
--
--
Average
2.8
1.8
1.8
3.9
2.4
2.6
1.5
Std Dev
1.3
0.6
1.0
2.7
1.2
1.6
0.3
5
L23200
M
1.6
0.5
0.6
-
--
--
--
6.0
5
L23201
M
4.0
1.6
1.5
1.0
--
--
--
5 H
L23212
M
0.9
1.2
1.5
0.9
1.1 *
1.4
0.6
5
L23214
M
2.5
2.7
2.5
--
--
--
--
5
L23234
M
3.0
1.4
2.6
0.1
--
-
--
Average
2.4
1.5
1.7
0.7
1.1
1.4
0.6
6.0
Std Dev
1.2
0.8
0,8
0.5
-
-
-
6
L23203
M
1.3
2.0
0.4
0.6
-
--
-
6
L23204
M
4.5
2.0
0.6
1.6
--
-
--
6
L23213 H
M
-
1.2
-
-
--
-
--
7 4
6
L23221
M
1.7
-
-
-
--
--
-
6
L23232
M
-
2.3
0.4
1.4
--
--
-
Average
2.5
1.9
0.5
1.2
-
-
-
7.4
Std Dev
1.7
0.5
0.1
0.5
-
--
-
Bold > Normal Range
Underscore < Normal Range
* Clot Removed
— No Value Page 13 ^ ^ ^
-------
Study 1020-CG920503 Hematology
'Jl
Parameter
Group ID
Animal ID
Sex
Day-3
Day 1
Day 2
Day 3
! Day 7
i Day 14
I Day 21
| ~2.7
! Terminal
%EOS (0.1-7.
1
L23216
M
2.4
' 3.9
2.7
1.2
2?
r 3.0
i
1
_
L23218
M
^ 2.3
3.2
3.4
2.7
3.7
! 2J5"*
t 4.3
t
123220
M
2.1
2.1
2.8
3.0
f 3.1 *
3.1 *
3.2
!
1
L23222
M
1.3
2.4
r 2.2 *
1.9
2.3
t 1.8
233
!
1
I L23223
M
2.2
3.1
1.6
2.0
I 2.4
j 2.8 *
3.0
!
Average
2.1
2.9
2.5
2.2
I 2.7
I 2.6
3.1
Std Dev
0.4
0.7
0.7
0.7
0.6
0.5
0.8
¦
2
L23206
M
2.3
2.7
2,0*
1.5
2.6*
2.6
3.1
I
2
L23210
M
r~ 1.4
2.6
1.6
-
-
3.0 *
..
2
L23211
M
2.8
2.2
2.0
2.1
2.9
2.4*
3.9
2
L23215
M
1.6
1.9
1.4
1.4
2.4
2.3 •
2.2
2
r L23219
M
1.8
2.0
1.5
1.5*
2.5
3.6*
2.8
-
Average
2.0
2.3
1.7
1.6
2.6
2.8
3.0
-
Std Dev
0.6
0.4
0.3
0.3
0.2
0.5
0.7'
3
L23217
M
l" 1.9
2.4
2.9
1.8
3.7
2.3
2.8
..
3
L23227
M
0.9
1.4
1.2
1.7
1.5
1.5
T.4
—
3
I L23228
M
2.3
2.4
2.1
r 2.9
3.5
'3.3
...
3
h L2322g
M
1.1
1.3
' 1.6
1.3
2.2
1.4
T "1
--
r 3
L23230
M
2.1
2.4
1.9
1.6
2.6
2.9 -
1.7
—
Average
r 1.7
2.0
1.9
1.9
2.7
2.3
2.2
Std Dev
0.6
0.6
0.6
0.6
0.9
0.9
0.8
r 4
L23205
M
1.6
1.2
1.9
1.2
2.9
1.5
1.7
4
L23207
M
1.7
1.9
2.2
2.5
1.6
2.8
__
4
L23225
M
23
1.7
1.3
1.9
-
-
-
—
4
L23231 "
M
1.3
1.6
1.9
1.4
2.4
1.9
..
4
L23235
M
1.2
1.3
1.7
1.5
1.4
-
-
-
Average
1.6
1.5
1.7
1.6
2.3
1.7
2.3
..
Std Dev
0.4
0.2 ^
0.3
0.4
0.6
0.2
0.6 ""
...
5
L23200
M
1.8
3.6
3.3
-
-
-
1.5
5
L23201
M
1.3
1.6
0.9
0.4
--
-
...
-
5
L23212
M
2.0 1
1.8
1.1
1.7
4.0 *
2.7
3.5
-
5
L23214
M
2.8
3.6
1.3 1
-
-
-¦
-
5
L23234 ~t
M
1.9
1.7
io
0.8
..
-
-
-
Average
2.0
2.5
1.5
1.0
4.0
2.7
3.5
1.5
Std Dev
0.5
1.0
1.0
0.7
-
-
6
L23203
M
1.4
2.1
1.3
2.8
-
-
g —1
L23204
M
1.0
1.6
0.7
1.2
-
-
-
-
6
L23213
M
--
1.6
-
-
-
-
-
6.9
6
L23221 1
M
2.0
--
-
--
-
-
..
6 "1
L23232
M
-
1.8
0.5
3.2
-
-
Average
1.5
1.8
0.8
2.4
--
--
-
0.9
Std Dev
0.5
0.2
04
1.1
-
-
~
..
Bold > Normal Range . 2. i - e.^
Underscore < Normal Range
* Clot Removed . * //¦'? ' , A "1 ///
-------
Study 1020-CG92Q503 Hematology
c
On
Parameter
Group ID
Animal ID
Sex
Day-3
Day1
Day 2
Day 3
Day?
Day 14
Day 21
Terminal
r oBASO (2.2-9.7 %)
1
L23216
M
7.2
9.6
6.9
4.9
5.0
7.0
7.4
-
1
L23218
M
3.0
3.8
4.5
3.2
3.2
2.8*
3.4
..
1
L23220
M
3.6
5.7
5.1
4.5
4.5*
•i.: ¦
> ¦
-
^ 1
L23222
M
I 3.5
5.1
3.8*
4.3
4.8
4.2
5.6
-
1
L23223
M I 3.3
4.1
r 3.9
2.0
3.7
3.1 *
4,2
-
Average
r i 4.1
5.7
4.8
3.8
4.2
4.1
5.5
-
Std Dev
1.7
i 2.3
1.3
1.2
0.8
1.7
1.7
-
2
L23206
M
4.8
3.2*
2.8
45 *
5.0
4.4
-
2 i L23210
M
• 3.9"
3.6
3.4
-
-
5.2*
-
-
2
L23211
M
4.6
4.1
5.5
4.4
4.0
6.2*
7.2
-
2
L23215
M
2.4
3.5
3.1
3.0
3.2
3.2
-
2
L23219
M
4.9
4.0
4.7
3.0*
5.8
52*
6.0
--
Average
r 4.1
3.8
4.0
3.3
4.4
r 5.0
5.2
-
Std Dev
1.0
0.3
1.1
0.7
r 1.1
1.0
1.8
-
3
L23217
M
5.7
r 7.o
8.1
7.8
10.3
7.9
10.1
-
3
L23227
M
5.1
5.5
59 49
3.4
6,5
-
3
L23228
M
7.5
8.0
7.6
7 4 8 3
7.4
6.7
_
3
L23229
M
3.3
3.2
3.5
[_ 3.5 4.6
4.5
4.1
_
3
123230
M
2.8
2.7
2.2
2.4 i 4.3
2.6 *
2X1
-
Average
4.9
5.2
5.4
5.4
6.5
5.2
-
Std Dev
1.9
2.3
2.5
2.4
2.7
2.4
3.0
4
L23205
M
4.3
5.2
4.9
3.9
6.5
4.4
4.9
-
4
L23207
M
> 3.1
3.1
4.4
3.7
4.8
5.1
5.7
-
4
L23225
M
4.3 "I
2.5
1.8
3.7
-
-
-
..
4
L23231
M
2.7
3.1
2.5
3.4
3.3
6.1
-
4
L23235
M
4.2
5.2
4.3
4.5
3.6
--
--
Average
3.7
3.8
3.6
3.8
46
4.6
5.6
-
Std Dev
0.8
1.3
1.3
0.4
1.5
0.5
0.6
-
5
L23200
M
3.8
5.5
3.8
-
-
--
..
4.1
5
L23201
M
2.7
2.9
1.9
1.1
76*
-
-
...
5
L23212
3.7
4.3
3.1
3.4
5.4
6.7
...
5 1
L23214
M
6.0
5.7
3.8
--
-
-
¦ ¦
5
L23234
M
2.4
2.5
2.4 1
1.7
-
-
-
...
Average
3.7
4.2
3.0
2.1
7.6
5.4
6.7
4.1
Std Dev
1.4
1.5
0.8
1.2
-
-
-
6
L23203
M
2.9
4.0
2.7
2.8
-
-
-
6
L23204
M
6.1
6.3
1.4
3.2
-
-
-
-
6
L23213
M
-
5.5
-
-
-
-
1 8
6
L23221
M
4.0
-
-
-
--
-
--
6
L23232
M
_
3.8
1.7
3.9
_
-
-
Average
4.3
4.9
1.9
3.3
--
..
-
1.8
Std Dev
1.6
1.2
0.7
0.6
-
-
-
Bold > Normal Range
Underscore < Normal Range
* Clot Removed
- No Value
Page 15
2.-2\-o
-------
¦"mfr
Study 1020-CG920503 Hematology
c
Parameter
Group ID
Animal ID
Sex
Day-3
Day 1
Day 2
Day 3
Day 7
Day 14
Day 21
Terminal
#NEUT (0.80-2.90 W/uL)
1
L23216
M
1.77
0.33
0.78
1.31
2.58
1.60
0.89
—
r 1
L23218
M
1.61
h 1.52
0.91
h 1.49
1.80
1.65 *
1.05
...
1
L23220
M
4.71
2.38
2.09
1.86
0.73*
0.39 *
1.00
....
1
123222
M
2.94
1.59
1.36*
1.68
1.28
1.90
1.02
-
1
1 L23223
M
1.49
0.88
1.34
0.17
1.13
0.59
Average
2.50
1.34
1.30
1.30
1.50
1 17
0.91
-
Std Dev
1.36
0.78
0.51
0.67
0.71
0.76
0.19
-
2
L23206
M
1.71
1.60
1.18*
0.89
0.84*
1.92
0,71
-
2
L23210
M
2.24
1.71
1.90
-
-
1.16 •
-
2
L23211
M
1.30
ri.55
1.34
1.21
1.14
1.79 *
0.40
h 2
L23215
M
1.65
1^31
1.05
1.28
1.10
0 68
..
2
L23219
M
1.75
1.21
1.51
X 0 93 ¦
1.26
0.88*
-
Average
1.73
1.48
1.40
1.08
1.09
1.34
0.60
Std Dev
0.34
0.21
0.33
0.20
0.18
0.48
0 14
3
L23217
M
1.76
1.42
1.30
1.28
1.67
1.58
r OiT-
r 3
L23227
M
1.84
1.31
1.29
1.16
1.31
1.59
0.78
3
L23228
M
2.07
2.02
2.11
2.05
1.92
2.25
0.84
3
L23229
M
1.19
1.10
1.20
1.10
1.31
1.26
0.62
3
L23230
M
1 34
1.76
1.38
1.41
1.00
0.57*
0.54
Average
1.64
1.52
1.46
1.40
1.44
1.45
0.75
Std Dev
0.36
0.37
0.37
0.38
0.36
0.61
0.17
-
4
L23205
M
1.65
1.29
1.17
1.05
1.43
1.51
0.78
4
123207
M
1.57
h 1.66
1 55
1.30
1.29
" r
0.44
-
4
L23225
M
2.14
r 2.78
3.68
035.
--
--
4
L23231
M
2.20
2.32
2.40
2.34
1.06
2.60
1.42
0.49
r 4
L23235
M
1.42
1.28
1.15
3.97
-
Average
1.80
1.87
1.99
1.22
2.32
1.40
f 0.57
-
Std Dev
0.35
0.66
1.07
0.72
1.25
0.12
0.18
..
5
L23200
M
1.86
0.09
0.28
-
-
-
__
1.27
5
L23201 ^
M
2.68
2.66
4.33
4.86
-
5
123212
M
1.26
r 1.25
1.11
1 06
1.32*
* 0.82
0.56
5
L23214
M
3.40
2.73
2.40
-
--
5
L23234
M
3.77
2.96
2 73
4.53
--
..
Average
2.59
1.94
2 17
3.48
1.32
0.82
1.27
Std Dev
1.04
1.23
1.56
2.11
-
..
-
6
L23203
M
1.42
2.02
3.30
1.18
-
..
6
L23204
M
1.66
2.09
3.69
2.02
-
6
L23213
M
-
2.38
¦-
-
--
--
3.77
6
L23221
M
1.96
-
-
-
-
..
..
6
L23232
M
-
1.77
[_ 6.13
0.65
-
__
..
Average
1.68
2.07
4.37
1.28
--
-
3.77
Std Dev
0.27 1
0.25
h 1 ^
0.69
--
-
Bold > Normal Range
Underscore < Normal Range
* Clot Removed
-- No Value
(He. iz-2 I • oCf
'a
Page 16
z. -c\ • o
-------
Study 1020-CG920503
Hematology
Parameter
Group ID
Animal ID
Sex
Day-3
Day 1
Day 2
Day 3
Day 7 | Day 14 Day 21 ! Terminal
4j64 1 4.18 ! 2.99 t -
#LYMPH (2.20-5.30 W/uL)
1
L23216
M
4.98
3.63
3.31
5.40
1
L23218
M
3.82
3.48
2.28
4.39 3.69 I 3.27* | 2,32
1
L23220
M
2.37
4 58 3.03
3.75
7.15
1.79*1 1.89 '
2.60 I
3.75 !
1
L23222
M
8.62
6.62 6.00*
5.83 5.41
1
L23223
M
l 4.33
r 3.70
4.22 j 2.45
3.57 1 2.35* I 2,46 |
3.90 f 3.42 I 2.82 I -
1.49 1.42 1 0T57 |
Average
4.82
r~4.40
3.77 | 4.63
Std Dev
2.33
1.31
1.43
1.77
] ) i !
2
L23206
M
r 4 7g
5.71
5.44*
4.71 t 2,92 * t 4.84 1 3.48 1
-1-1 3.39* |-1
2
L23210
M
4.17
4.51
4.90
2
L23211
M
3.19
3.62
3.25
4.12
3.07 J 3.82* J 1.54 j
3.51 | 3.57-* I 2.96 ::
2
L23215
M
4.65
3.46
4.63
4.78
2
123219
M
5.63
5.15
5.53
6.65 * J 4.18 3.48 * ! 3.82 I
5.07 ! 3.42 3.82 | 2.95 1
Average
4.48
4.49
4.75
Std Dev
^ 0.89
0.97
0.92
1.10
0.57 0.59 1.00 j
5 25 5.76
3
L23217
M
6,76
7.66
8.12
8.61
4.71 1
3
L23227
M
5.50
6.17
5.67
5.52 5 21 5.99 3 19
3.93 j 3.43 I 3.53 j 2.31 !
5.64 !' 4.25 1 4.81 3.18 j
3
L23228
M
3.72
4.48
4.52
3
L23229
M
4.32
5.00
4.48
3
L23230
M
4.27
4.53
f 4.77
4.01
5.54
3.21 2.40* ! 2.62 ]
i27 4.50 1 3^20 t -
Average
4.91
5.57
5.51
Std Dev
1.22
1.35
1.54
1.90
0.96
1.52 j 0.92 j -
4
L23205
M
5.31
5.64
6.16
5.99
4.64
6.17
3.86
-
4
L23207
M
3.85 H
4.76
3.80
4.16
3.49
3.37
2.07
-
4
L23225
M
2.97 1
3.76
3.24
2.78
-
--
..
4
L23231
M
4.99
5.42
5.12
4.82
4.48
4.57
2.14
4
L23235
M
4.84
5.09
6.86
f 5.99
7.10
Average
4.39
4.93
h 5.04
4.75
4.93
4.70
2.69
Std Dev
0.96 1
0.74
1.53
1.35
1 54
1.40
1 01
-
5
L23200
M
3.76
3.00
3.71
-
-
-
--
6.56
5
L23201
M
6.26
6.74
7.75
8.14
-
5
L23212
M
5.97
6.18
6,72
6.75
3 93
3.23
..
5
L23214
M
4.00
4.50
0.80
-
-
-
5
L23234
M
4.72
5.25
5.93
3.75
-
..
..
Average
4.94
5.13
4.98
6.21
4.67
3.23
6.56
Std Dev
1.13
1.47
2.77
2.24
-
6
L23203
M
7.19
6.05
3.11
h 321
-
6
L23204
M
3.05
3.92
3.15
3.04
-
__
6
L23213 n
M
-
7.08
-
-
-
-
7.65
6
L23221
M
3.58
-
-
-
--
-
-
6
L23232
M
-
3.47
2.41
^ 0.75
--
-
..
Average
4.61
5.13
2.89
2.33
--
„
-
7.65
Std Dev
2.25
1.72
0.42
1.37
-
-
Bold > Normal Range
Underscore < Normal Range
* Clot Removed
- No Value
|>tC_ l2-Z|-oe
," , I
-------
Study 1020-CG920503 Hematology
c
vo
Parameter
Group ID
Animal ID
Sex
Day-3
Day 1
Day 2
Day 3
Day 7
Day 14
Day 21
Terminal
#MONO (0.00-0.40 103/uL)
1
L23216
M
0.10
0.02
0.02
0.23
0.35
0.07
0.04
-
1
L23218
M
0.11
0.13
0.03
0.08
0.11
0,22*
0.07
-
1
L23220
M
0.17
0.25
0.22
0.13
0.04*
0.02 *
0.05
-
1
L23222
M
0.13
r 6.08
0.09*
0.15
0.05
0.09
0 04
-
1
L23223
M
0.35
0.04
0.06
0.01
0.04
0.03*
0.03
...
Average
0.17
0.10
L0.08
0.12
0.12
0.09
0.05
...
Std Dev
0.10
r 0.09
0.08
0.08
0.13
0.08
0.02
2
L23206
M
0.09
0.10
0.17*
0.13
0.05 *
0.06
0.03
2
L23210
M
0.12
0.09
0.09
-
0.05 *
2
L232"! 1
M
0.09
i 0.08
0.12
0.11
0 08
0.23 *
0.03
...
2
r L23215
M
0.08
0X)7
0.06
0.07
0.05
0.04 *
0.03
...
2
f L23219
M
0.09
0.06
0.21
0.08*
0.19
0.04 *
0.05
Average
0.09
0.08
0.13
0.10
0.09
0.08
0.04
-
Std Dev
0.02
h 0.02
l~0.06
0.03
0.07
0.08
0.01
3
L23217
M
0.13
0.11
0.20
I 0.19
ro.19
0.26
0.10
3
L23227
M
0.11
0.23
0.19
0.08
I 0.11
0.17
0.05
-
3
L23228
M
0.26
0.10
0.12
' 0.11
r 0.24
0.08
0.06
3
L23229
M
0.08
0.13
I 0.14
0.17
0.06
0.05
0.05
3
L23230
M
0.06
0.05
0.06
0.06
0.11
0.03*
0.02
Average
0.13
0.12
0.14
h 0.12
0.14
V
0.06
Std Dev
0.08
0.07
0.06
0.06
0.07
0.10
0.03
4
L23205
M
0.14
0.18
0.25
0.09
0.10
0.28
0.08
4
L23207
M
0.21
0.18
0.08
0.22
0.21
0.20
0.03
4
L23225
M
0.28
0.10
0.03
0.30
-
-
"
4
L23231
M
0.17
0.13
0.18
0.19
0.22
0.05
4
L23235
M
0.12
0.08
0.15
0.29
0.14
--
Average
0.18
I 0.13
0.14
0.22
0.17
0.18
0 05
Std Dev
0.06
0.05
0.09
0.09
0.06
0.12
0.03
5
L23200
M
r 0.10
0.02
0.03
-
-
„
0.54
5
L23201
M
0.39
0.16
0.19
r 0.14
-
..
-
5
L23212
M
0.07
0.09
0.12
0.08
0.08*
0.07
0.03
5
L23214
M
0.22
0.22
0.09
--
-
-
--
5
L23234
M
0.28
0.12
0.24
0.01
--
--
-
Average
0.21
0.12
[_ 0.13
0.08
0.08
0.07
0.03
0.54
Std Dev
0.13
0.07
0.08
0.07
--
..
-
6
L23203
M
0.11
0.18
0.02
0.03
--
--
-
6
L23204
M
0.24
0.13
0.04
0.08
--
--
6
L23213
M
-
0.12
-
-
--
--
-
0 95
6
L23221
M
0.10
-
-
-
--
-
6
L23232
M
--
0.13
0.04
0.02
--
-
--
Average
0.15
0.14
0.03
0.04
-
--
--
0.95
Std Dev
0.08
0.03
0.01
0.03
--
..
Bold > Normal Range
Underscore < Normal Range
* Clot Removed
- No Value
Page 18
OflPjith Rw fWRsfe:
(>tc \Z-Z\-oq
-------
Study 1020-CG920503 Hematology
c
to
o
Parameter
Group ID
Animal ID
Sex
Day-3
Day 1
Day 2
Day 3
Day 7
Day 14
Day 21
I Terminal
#EOS (0.00-0.40 W/i
1
L23216
M
0.18
r o.i8
0.12
0.09
0.18
0.20
0.12
-
l~_ 1
L23218
M
0.14
0.17
r o.i2
0.17
0.22
0.14*
0.16
-
1
' L23220
M
0.16
0.16
0.19
0.09 *
0.08 *
0.13
...
1
L23222
M
0.16
0.22
0.17*
0.18
0.18
0.14
0.12
i
r 1
L23223
M
0.15
0.15
0.10
0.05
0.12
0.08 1
0.10
f
Average
0.16
0.18
0.13
0.14
0.16
0.13
0.13
.....
Std Dev
h_0.01
0.03
'0.03
0.06
0.05
0.05
0.02
i
t 2
L23206
M
0.16
0.21
0.14*
0.09
0.11 *
0.20
0.14
L23210
M
0.10
0.17
0.12
-
0.12
0.15*
-
-
r L23211
M
0.14
0.12
0.10
0.13
0.15 *
0.09
«
1 2
L23215
M
0.11
0.10
0.09
0.09
0.12
0.11 *
0,09
...
2
L23219
M
0.14
0.13
0.12
0.12 *
0.16
0 1 / ¦
6.14
Average
0.13
0.15
0.11
0.11
0.13
0.16
0.12
_
Std Dev
0.02
0.04
0.02
0.02
0.02
0.03
0.03
3
L23217
M
0.18
0.24
r 0.31
0.21
0.30
0.20
6.19
3
L23227
M
r o.o7
0.12
0.09
0.12
0.11
0.12
0.06
3
L23228
M
0.15
0.18
0.15
0.20
0.23
0.12
3
L23229
M
0.06
0.09
r 0.10
0.10 ! 0.13
0 09
0.07
3
L23230
M
r o.i2
0.16
0.12
0.09
0.12
0.09 *
0.06
Average
0.12
0.16
0.15
0.14
0.18
0.15
0.10
..
Std Dev
0.05
0.06
0.09
0.06
0.08
0.07
0.06
..
4
L23205
M
0.12
0.09
0.15
0.09
0.20
0.12
0.09
_
4
L23207
M
0.10
0.12
0.11
0.13
0.14
0.08
0.08
«
4
L23225
M
r 0.13
0.12
0.09
0.07
-
-
-
I 4
L23231
M
0.10
0.13
0.15
0.11
0.19
0.12
0.07
4
L23235
M
0.08
" 0.09
0.15
0.12
-
-
Average
0.11
0.11
0.13
0.10
0.18
0.11
0.08
..
Std Dev
r o.o2
0.02
0.03
0.02
0.03
0.02
0.01
5
L23200
M
0.11
0.12
0.14
-
--
-
..
0.14
5
L23201
M
0.12
0.16
0.11
0.06
..
--
..
_
L23212
M
0.15
0.14
0.09
0.15
0.27 *
0.14
0.15
5
L23214
M
^ 0.24
0.29
0.04
--
--
..
..
5
L23234
M
0.18
0.14
0.09
0.07
--
Average
0.16
0.17
0.09
0.09
0.27
0.14
0.15
Std Dev
0.05
0.07
0.04
0.05
6
L23203
M
0.13
0.18
0.08
0.13
--
...
6
L23204
M
0.06
0.11
0.05
0.06
--
--
-
6
L23213
M
-
0.16
-
-
..
6.11
6
L23221
M
--
-
-
-
--
--
r~ 6
L23232
M
-
0.10
0.05
0.05
--
-
-
Average
0.10
0.14
0.06
0.08
-
0.11
Std Dev
0.04
0.04
0.02
0.04
--
Bold > Normal Range
Underscore < Normal Range
* Clot Removed
- No Value
Page 19
(Vfc. la-zfoq
-------
Study 1020-CG920503
Hematology
c
to
Parameter
Group ID
Animal ID
Sex
Day-3
Day 1
Day 2
Day 3
Day?
Day 14
Day 21
Terminal
#BASO (0.10-0,60 W/uL)
1
h L23216
M
r 0.55
0.44
0.32
h 0.36
0.41
6.46
0.32
-
1
L23218
M
0.17
0.21
0.16
0.20
0.19
0.15*
0.13
-
1
r~ L23220
M
0.28
r 0.45
0.30
0.28
0.12 *
0.08*
0.27
1
L23222
M
0.44
0.46
0.30 *
0.41
0.37
0.33
0.29
-
1
r 123223
M
0.22
0.20
0.23
0.05
0.19
0.09 '
0.14
Average
0.33
0.35
0.26
0.26
0.26
0.22
0.23
Std Dev
0.16
0.13
0.07
0.14
0.13
0.17
0.09
-
2
L23206
M
0.34
0.29
0.23 *
0.17
0.19*
0.37
0.20
-
2
f L23210
M
0.28
0.24
0.25
-
-
0.26 *
-
2
L23211
M
0.23
0.23
0.28
0.26
0.18
0 40 1
0.16
2
L23215
M
0.16
0.18
0.19
0.19
0.16
0 17 *
0.12
2
L23219
M
0.39
0.27
0.36
0.24 '
0.36
0.25 *
0.30
..
Average
0.28
0.24
0.26
0.22
0.22
0.29
0.20
Std Dev
0.09
P 0.04
0.06
0.04
0.09
0.09
0.08
3
L23217
M
0.54
0.71
0,88
0.88
0 85
0.67
0.67
..
3
L23227
M
0.43
i 0.42
0.42
0.43
0.35
0.27
0.29
-
3
L23228
M
0.50
0.59
0.57
0.50
0.53
0.49
0.24
3
L23229
M
0.19
0.21
0.22
0.25
0.28
0.29
0.17
-
3
L23230
M
0.17
0.18
I 0.14
0.13
0.20
0.08 -
0.07
--
Average
0.37
0.42
r 0.45
0.44
0.44
0.36
0.29
--
Std Dev
0.17
0.23
0.30
0.29
0.26
0.23
0.23
..
4
L23205
M
0.32
0.40
h 0 4Q
0.30
0.45
0.37
0.25
4
L23207
M
0.18
0.21
0.25
r 0.23
0.26
0.27
0.16
4
L23225
M
r 0.25
0.18
0.13
0.14
4
L23231
M
0.21
0.26
I 0.20
0.26
0.25
0.27
r o.i8
4
L23235
M
0.29
0.36
r 0.38
0.35
0.43
Average
0.25
0.28
' 0.27
0.26
0.35
0.30
6.20
n Std Dev
0.06
0.09
0.12
0.08
0.11
0.06
0.05
5
L23200
M
0.23
0.19
0.16
--
-
0.37
5
L23201
M
0.26
0.29
0.24
0.15
--
-
5
L23212
M
0.28
0.34
0.26
0.29
0.28
6.28
5
L23214
M
0.52
0.47
0.13
i
-
5
L23234
M
^ 0.22
0.22
0.22
r o.i41 -
-
Average
0.30
0.30
0.20
0.19
0.52
0.28
6.28
0.37
Std Dev
l~~ 0.12
0.11
0.05
0.08
..
6
L23203
M
0.26
0.35
0.18
0.13
6
L23204
M
0.33
0.42
0.10
0.17
--
6
L23213
M
--
0.56
-
--
--
-
-
0.24
6
L23221
M
0.24
-
--
--
-
-
6
L23232
M
..
0.22
0.15
0 06
..
--
-
Average
0.28
0.39
0.14
0.12
--
--
0.24
Std Dev
0.05
0.14
0.04
0.06
Bold > Normal Range
Underscore < Normal Range
* Clot Removed
-------
Study 102Q-CG920503 Hematology
c
to
to
Parameter
Group ID
Animal ID
Sex
Day-3
Day 1
Day 2
Day 3
Day 7
0.556
Day 14
Day 21
Terminal
Neut#/Lymph« Ratio
r 1
L23216
M
0.355
0.091
0.236
0.243
0.383
0.298
—
1
L23218
M
0.421
0.437
0.399
0.339
0.488
0 505 *
0.453
-
i
L23220
M
1.987
0.520
0.690
0.496
0.408*
0 206 •
0.385
1
L23222
M
0.341
0.240
0.227 *
0.235
0.220
0.351
0.272
-
1
F 123223
M
0.344
0.238
0.318
0.069
0.317
0 123 '
0.240
-
Average
0.690
0.305
0.374
0.276
0.398
0.314
0.330
Std Dev
0.726
0.172
0.190
0.157
0.134
0.151
0.088
2
1 L23206
M
0.359
0.280
0.217*
0.189
0.288 *
0.397
0.204
2
i L23210
M
0.537
0.379
0.388
-
--
0.342 *
2
L23211
M
0.408
0.428
i 0.412
0.294
0.371
0.469 *
0.260
2
L23215
M
0.355
0.379
0.227
0.268
0.313
0.272 *
0.230
-
2
L23219
M
0.311
0.235
0.273
0.140 *
0.301
0.253 *
0.162
Average
0.394
0.340
10.303
0.223
0.318
0.347
0.214
Std Dev
0.087
0.080
0.091
0.071
0.037
0.089
0.042
3
L23217
M
h 0 26Q
0.185
0.160
0.149
0.318
0.274
0 206
3
L23227
M
0.335^
0.212
0.228
0.210
0.251
0.265
0.245
3
L23228
M
0.556
0,451
0.467
0.522
0.560
0.637
0.364
3
L23229
M
0.275
0.220
0.268
0.195
0.308
0.262
0.195
3
L23230
M
0.314
0.389
0.289
0.352
0.312
0.238 *
0.206
Average
0.348
0.291
0.282
0.286
0.350
0.335
0.243
-
Std Dev
0.120
0.120
0.114
0.152
0.121
0.169
0.070
4
' L23205
M
0.311
0.229
0.190
0.175
0.308
0.245
0.202
4
L23207
M
0.408
0.349
0.408
0.313
0.370
0.380
0.213
..
4
L23225
M
0.721
0.739
1.136
0.126
--
" "
- -
-
4
L23231
M
0.441
0.428
0.469
0.485
0.580
0.311
0.229
-
4
L23235
M
0.293
0.251
0.168
0.177
0.559
-
-
Average
0.435
0.399
0.474
0.255
0.454
0.312
0.215
-
Std Dev
0.172
0.206
0.393
0.146
0.136
0.068
0.014
5
L23200
M
0.495 1
0.030
0.075
-
--
::
-
0.194
5
L23201
M
0.428
0.395
0.559
^0.597
--
-
5
L23212
M
oifP
0.202
0.165
0.157
0.283 *
0.209
0.173
r
5
L23214
M
0.850
0.607
3.000
-
-
..
5
L23234
M
0.799
0.564
0.460
1.208
_
Average
0.557
0.360
0.852
0.654
0.283
0.209
0,173
0.194
Std Dev
0.267
0.244
1.217
0.528
-
-
-
6
L23203
M
0.197
0.334
1.061
0.368
--
6
I L23204
M
0.544
0.533
1.171
0.664
-
-
6
L23213
M
-
0.336
-
_
--
0.493
6
L23221
M
0.547
-
-
-
-
-
6
L23232
M
-
0.510
2.544
0.867
--
-
Average
0.429
0.428
1.592
0.633
-
0.493
Std Dev
0.201
0.108
0.826
0.251
--
-
Bold > Normal Range 0*C_ I s- ¦ Z-l • °C1
Underscore < Normal Range
Page21 ¦ U
-------
Study 1020-CG920503 Hematology
c
to
oo
Parameter
Group ID
Animal ID
Sex
Day-3
Day 1
Day 2
Day 3
Day 7
Day 14
Day 21
Terminal
NeufVLymph°b Ratio
r- |
L23216
M
0.354
0.091
0.235
0.243
0.556
0.382
0.296
-
1
L23218
M
0.420~"
0.435
0.398
0.340
0.489
0.504 *
0.451
-
1
L23220
M
1.990
0,520
0.691
0.498
0.409 *
0.205 '
0.383 F
1
L23222
M
0.342
0.242
0.228 *
0.236
0.220
0.350
0.273
1
L23223
M
r 0.343
0.239
0.319
0.071
0.317
0.125*
0.242
-
Average
0.690
0.305
0.374
0.278
0.398
0.313
0.329
Std Dev
0.728
0.171
O.igo
0.157
0.134
0.150
0.086
..
2
L23206
M
0.359
0.281
0.216*
0.190
0.289 *
0.397
0.203
2
L23210
M
0.536
0.377
0 388
--
__
0,344*
__
--
2
L23211
M
0.409
0.427
0.413
0.294
0.372
0.467*
0.260
2
L23215
M
0.355
0.379
0.226
0.267
0.312
0,271 *
0.230
..
2
L23219
M
0.312
0.235
0.273
0.139 *
0.302
0.254 '
0.163
-
Average
0.394 "I
0.340
0.303
0.223
0.319
0.347
0.214
Std Dev
0.086
0.079
0.092
0.071
0.037
0.089
0.041
3
L23217
M
0.259
0.186
0.161
0.148
0.317
0.274
0.206
-
3 ~1
L23227
M
0.334
0.212
0.228
0.211
0.250
0.265
0.245
3
L23228
M
0.557
0.451
0.467
0.522
0.560
0.638
0.364
3
L23229
M
0.276
0.219
0.269
0.196
0.310
0.263
0.195
„
3
L23230
M
0.315
0.388
0.290
0.351
0.311
_____
0.239 *
0.206
Average
0.348
0.291
0.283
0.286
0.336
0.243
Std Dev
0.120
0.120
0,114
0.152
r 0i2i
0.169
0.070
4
L23205
M
0.311
0.229
0.190
0.176
0.308
0 244
0.203
4
L23207
M
0.408
0.349
0.408
0.314
0.371
0.379
0.215
.
4
L23225
M
0.722
0.739
1.133
0.125
--
4
L23231
M
0.441
0.429
0.468
0.485
0.582
0.310
0.231
4
L23235
M
0.293
0.252
0.168
0.178
0.559
--
"
Average
0.435
0.400
0.4731
0.256
0.455
0.311
0.216
-
Std Dev
0.172
0.206
0.391
0.146
0 136
0.068
0.014
..
l_ 5
L23200
M
0.495
0.031
0.076
-
-
. . . .
0 193
5
L23201
M
0.428
0.394
0.560
0.597
-
5
L23212
M
0.210
^3.203
0.166
0.157
0.283 *
0.209
0.174
..
5
L23214
M
0.849
^0.607
3.013
..
--
--
„
5
L23234
M
0.799
0.565
0.461
1.209
--
-
Average
0.556
0.360
0.855
0.654
0.283
0.209
0.174
0.193
Std Dev
0.267
F 0.243
1.223
0.528
--
..
6
L23203
M
0.198
0.334
1.060
0.366
-
-
-
6
L23204
M
0.544
0.533
1.170
0.664
--
-
6
L23213
M
-
0.335
..
-
--
--
0.492
6
L23221
M
0.546
-
..
-
-
--
6
L23232
M
-
0.509
2.538
0.874
-
Average
0.429
0.4128
1.589
0.635
-
„
-
0.492
Std Dev
0.200
0.108
0.823
0.255
--
-
-
-
Bold > Normal Range
Underscore < Normal Range
* Clot Removed
- No Value
CIA Audit Corni^letac
17*»©•»!//'J7//C
Page 22
-------
APPENDIX V
INDIVIDUAL CLINICAL CHEMISTRY RESULTS
V-l
-------
Study 102Q-GG92O5O3
Clinical Chemistry
to
' Severe Hemolysis
Bold » Nor
* Slight Hemolysis; *'
** Moderate Lipemia
Instrument unable to calculate result
+ Sample quantity insufficient
- No Value
Parameter
I Group ID
Animal ID
Sex
Day-3
Day 1
Day 2
Day 3
Day 7
Day 14
Day 21
Terminal
Blood Urea Nitrogen (5.0-30.0 mg/dL)
| 1
L23216
y
15.3
T 20.3
17.1
15.2
20.1
23.0
16 7
I 1
r- L2321g
M
15.6
16.8
17.2
14.7
14.0
f 14.3
\ 15.5
i
I 1
L23220
M
15.6
17.9
19.0
128
17.4
l 22.6
| 19.7
„
1 1
f L23222
M
25.1
25.4
23.4*
¦
23.4
24,1
] 21.6
__
1 1
L23223
M
17.3
20.6
' 20.1
17.4
| 19.9
I 24.4
f 20.0
)
Average
17.8
20.2
19.4
16.0
j TsuT
[ 21J
187
-
- J
Std Dev
4.2
3.3
r 2.6
2.7
~"3l5
4.2
J 25
!
_.L.
1
i 2
L23206
M
18.0
f ,8 ,
19.4
19.8
| 211
I 205™"
t 175
!
i 2
L23210
M
13.8
19.1
19.8 *
17.5
...
1 24.3
i 20 8
:
2
L23211
M
^ 16.2
18.7
16.7
14.9
i 19.1
] +
| 17.9'
!
123215
M
I 12.5
16,0
f 12.7
11.1
1 17j0
1 +
Tie
-
! 2
L23219
M
15.4
16.7
\>fc
13.5
| 18.'6~
1 165
J Ts',4
—
Average
15.2
17.7
16.5
15,4
I 19.0
1 20.6
| 16.9
—
Std Dev
2.1
1.3
3.2
3.4
1.7
i 3.9
( 3.0
....
3
L23217
M
16.5
19.1
15.4
14.0
19,3
J 18.4
I 19'.4
i
r 3
L23227
M
22.1
22.9
17,9
13.9
22Xf
f~22J'
f 17.1"
3
" L23228
M
19.5
19.9
21.0
t- t7,2
ll9
25.9
I Wa
(
r g
L23229
M
15.9
18.1
16.4
16.3
17,2
r Ti-'s
15.3
3
L23230
M
19.7 •
23.3
' 17.9
17.4
I 17^3
"22.5"
17 1
Average
18.7
I 20.7
17.7
15.8
Tal
21.5
r 17.3
'
Std Dev
2.5
2.3
2.1
1.7
2.6
[ TTsT
" _
.
4
L23205
M
18.5
20.5
h ___
18.4
20.8
18.5
16.6
..
4
L23207
M
29.5
26,4
24.0
22.8
28.2
31.1
21.4
..
4
I" L23225
M
18.0
18.9
16.9
12.8
...
-
...
66.9
4
L23231
M
18.8
' 20.5
17.1
15.9
20.5
24.3
20,4
..
4
L23235
M
19.9
16.8
16.4
13,6
15.9
..
__
..
!
Average
20.9
20.6
18.0
16.6
21.4
24.6
19.5
66.9
L J
Std Dev
4.8
3.6
3.4
4.2
5.1
6.3
2.5
..
5
L23200
M
22.6
21.0
22.7
--
..
...
..
20.5
5
L23201
M
27.2
25.0
21.9*, a*
60.3 *,
-
—
„
I 5
L23212
M
17.5
18.4
17.6
16.6
18.0
20.8
r 17.0
..
5
L23214
M
20.1
19.5
20.0 *
17.0 «
-
—
..
5 1
153234
M
1477 1
15.6
13.5
13.2
..
..
...
Average i
20.4
19.9
19.1
26.8
18.0
20 8
17rQ
20,5
Std Dev
4.8
3.5
3.7
22.4
..
~
..
|
I 6
L23203 f
M
17.5
17.1 |
12.9
12.3
-
-
—
..
! 6
L23204
M
23.0
22.7
16.3*
14.1 ****
..
65.9
6
L23213
M
-
16.2
--
--
..
..
28.8 '
i 6
L23221
M
20.8
--
-
-
-
-
..
..
[ 6
123232 ~t
M
--
16.9
14.6
19.9
--
..
;
Average
20.4
18.2
14.6
15.4
-
--
47.4
L
Std Dev
2.8
3.0
1.7
4.0
¦
-
..
26.2
_j
Wirt M
-------
Study 1Q20-CG920503
Clinical Chemistry
oo
Bold
* Slight Hemolysis; *"
AA Moderate Lipemia
Parameter
! Group ID
Animal ID
Sex
Day-3
Day 1
I Day 2
Day 3
Day 7
Day 14
Day 21
Terminal
Creatinine (0.5-3.0 mg/dL)
r 1
L23216
M
0.92
f 0.89
I 0.82
: 0.93
1.07
1 13
1 21
..
I 1
L23218
M
0.93
0.76
0.75
I 0.97
1 03
1.07
0.99
..
I 1
L23220
M
0.94
0.75
I 0.82
0...".
1.00
0.85
0.98
-
! 1
L23222
M
' 1.19
r 1.07
0.93 *
1.04
1.24
1.10
1.25
—
! 1
L23223
M
r 0.83
1.06
! 0.81
1.01
i 0.98
1 1.15
[ 'Tu
!
t
Average
h 0.96
0.91
| 0.83
0.98
j 1.06
1.06
1.11
:
Std Dev
r o.i3
f ^.jg
I 0.07
J o.04
! olo"
I QJ2
! 0 12
i
I 2
L23206
M
0.75
0.74
| 0.78
I 1.00
I lis
096
1.13
i
I 2
L23210
M
0.92
1.11
[ 0.88 *
1.09
0.99
1J3
I 2
L23211
M
0.91
0.86
0.74
0.90
T.27
+
1 1.17
...
2
L23215
M
0.71
0.66
0,66
0.68
" 0.88
+
| 0J7
I 2
L23219
M
0.66
" 0.76
0.66
0.88
f 1"34
0.77
1.05
...
]
Average
0.79
0.83
0.74
0.91
1.17
0.91
1.07
—
Std Dev
0.12
0.17
0.09
0.15
0.20
0.T2
0.12
3
L23217
M
0.92
0.94
0.94
1.03
1S7'"
m
1.37
3
L23227
M
1.24
0.73
0.72
0.73
0.89
0.89
1.05
_
3
L23228
M
0.73
0.80
0.82
0.88
0*99"
0.96
1 14
! 3
r 123229
M
0.76
0.81
0.84
0.96
0.89
1.01
OsT"
...
I 3
L23230
M
"1.86*
0.84
0.64
0.84
iTio"
TToo
f 1".09
Average
1.10
0.82
0.79
0,' •
1.09
TToi
f 1717
Std Dev
0.47
0.08
0,12
0.11
0.28
ail
0J2
__
j
I 4
L23205
M
0.58
0.65
0.70
0.78
0.86
1.05
1.00
..
I 4
L23207
M
1.13
0.74
r o.9o
1.'
1.02
i.06~
1.20
...
I 4
L23225
M
2.13
0.83
1.01
1.05
..
..
4 92 "**
4
L23231
M
0.83
0.78
0.77
0,77
0.99
1.13 _
T.zi
L23235
M
0.78
0.64
0.66
0768
"aae
.
Average
1.09
0.73
0.81
0.89
0.93
Ids
4.92::;
Std Dev
0.61
0.08
0.15
0.21
0.08
0.04
QJ3
r 5
L23200 '
M
0.85
0.69
0.77
-
::
1.04
5
L23201
M
0.96
0.70
1.18*,™
1.92 *, ^
-
_
..
5
L23212
M
0.79
0.71
0.78
0.99
0.87
0.97
1 14
...
5
L23214
M
0.90 1
0.71
0.95 *
1.29 «
-
—
5
L23234
M
0.80
0.73
0.79
1.14
..
..
..
j
Average
0.86
0.71
0.89
1.34
0.87
0.97
1.14
1.04
Std Dev
0.07
0.01
0.18
41
-
-
..
[
6
L23203
M
0.68
0.76
0.79
0.71
-
..
..
! 6
L23204
M
0.77
0.65
1.06 *
1.06
-
-
5.01 **"
6
L23213
M
-
0.68
--
..
-
..
—
1.65 *
I 6
L23221
M
0.59
--
-
-
-
--
—
i 6
L23232
M
--
0.86
0.93
1.17
..
..
...
...
Average
0.68
0.74
0.93
0.98
-
-
..
3.33
Std Dev
0.09
0.09
0.14
0.24
-
-
2 38
Severe Hemolysis
Instrument unable to calculate result
+ Sample quantity insufficient
-- No Value
(WC- tO-SA.-©^
Q Ac.
-------
Study 1020-CG920503
Clinical Chemistry
Parameter
Group ID
Animal ID
Sex
Day-3
Day 1
Day 2
Day 3
' Day 7
Day 14
' Day 21
Terminal
BUN/Creatinme Ratio
!
L23216
M
16.6
22.8
20.9
I 1f
[ 18.8 '
20.4
13.8
i
1
L23218
M
16.8
22.1
22.9
15.2
13.6
Tai
15.7
1
1
L23220
M
16.6
23.9
23.2
13.3
{ --j-
~26.'6 1 20.1
i
1
L23222
M
21.1
23.7
25 2*
19.0
{ 18.9
21.9
17J3
1
1
L23223
M
20.8
r 19.4
24.8
17.2
I '203
' 212
17.5
i
Average
18.4
22.4
23.4
16.2
17.8 '
20.7
16.9
Std Dev
r 1.8
1.7
2.1
2.6
4.7
2.3
-
2
L23206
M
24.0
24.5
24.9
19.8
17.9
21.7
m4
..
__
L23210
M
•15.0
17.2
22 5 *
16.1
—
24i>
"isTT
2
L23211
M
17.8
21.7
16.6
+
15.3
2
123215
M
17.6
24.2
19.2
16.3
19.3
+
14.7
2
' L23219
M
23.3
22.0
20.9
15.3
T3L9
2l6~
| 14 I
Average
19.5
21.9
22.0
16.8
16.5
22.6
1577
..
Std Dev
3.9
2.9
2.1
1.7
2.5
1.6
T7s
i
3
L23217
M
17.9
20.3
16.4
13.6
12.3 "
15^~
14 2
—
3
L23227
M
17.8
31.4
24.9
19.0
24.7"
. ; ¦!
16 3
...
3
M
26.7
24.9
25.6
19.5
20?i
27.0'
15 3
3
L23229
M
20.9
22.3
19.5
17.0
19.3
Tae
..
3
L23230
M
10.6*
27.7
28 0
20.7
|"T5?7
~~2j£5
15.7
..
Average
18.8
25.3
22 9
18.0
18.4
2?7
14.9
__
_
Std Dev
5.8
n4;4
CO
2.8
" 477
1.3
..
4
L23205
M
31,9
31.5
22.3
23 6
24.2
17.6
16.6"'
~
4
L23207
M
26.1
35.7
26.7
19.5
27.6"
29.3
1/8
4
L23225
M
8.5
22.8
16.7
12.2
--
--
-
13.6 ""
4
L23231
M
22.7
26.3
22.2
20.6
20 j
21.5
16.6
...
4
L23235
M
25.5
26.3
24.8
19.1
18.5
--
..
...
Average
22.9
28.5
22.5
19.0
22.8
22.8
17.0
13,6
Std Dev
8.7
5.1
3.8
4.2
4.0
6.0
0.7
—
5 H
L23200
M
26.6 1
30.4
29.5
-
..
19.7
5
L23201
M
28.3
35.7 ^
18.6 *,w
31.4*,**
--
--
-
5
L23212
M
22.2
25.9
22.6
16.8
20.7
21.4
14.9
—
5
L23214
M
22.3
27.5
21.1 *
13.2 «
-
..
..
5
L23234
M
18.4
21.4
17.1
11.6
..
-
..
Average
23.6 n
28.2
21.8
18.3
20.7
14.9
19.7
Std Dev
3.9
5.3
4.8
9.0
-
-
--
..
6
L23203 1
M
25.7
22.5
16.3
17.3
..
-
..
...
6
L23204
M
29.9
34.9
15.4*
13.3 ****
--
..
-
! 6
L23213
M
--
23.8
--
--
--
-
17.5 *
.. ,
6
L23221
M
3573
--
--
--
--
-
-
6 H
L23232
M
--
19.7
15.7
17.0
„
—
-I
Average
30.3
25.2 1
15.8
15.9
15.4
Std Dev
4.8
6.7
0.5
.
..
3.0
— —
t
* Slight Hemolysis; **** Severe Hemolysis
M Moderate Upemia
Instrument unable to calculate result
+ Sample quantity insufficient
-- No Value
mm Ik BWSate; fa- >° ^
-------
Study 1Q20-GG92O5O3
Clinical Chemistry
Parameter
Group ID
Animal ID
Sex
Day-3
Day 1
Day 2
Day 3
Day 7
Day 14
Day 21
Terminal
Sodium (125-160 mEq/L)
1
L23216
M
143
145
146
141
147
145
141
-
1
L23218
M
143
145
146
148
148'
146
142
--
1
L23220
M
146
145
145
145
144
143
140
...
1
L23222
M
143
145
144*
143
142
143
140
1
L23223
M
145
147
145
144
144
146
141
—
Average
144
145
145
144
145
145
141
Std Dev
1
l_ ..
3
2
2
1
...
i 2
L232G6
M
143
145
145
147
153
146
143
I 2
123210
M
142
147
144*
145
-
143
141
..
2
L23211
M
146
148
L 147
146
151
r""147
142
I 2
L23215
M
143
145
144
| 144
145
+
142
I 2
L23219
M
' 145
146
r 147
147
155
143
140
Average
144
146
145
146
151
145
142
Std Dev
2
1
2
1
4
2
1
..
! 3
L23217
M
U1
144
145
L 145
148
" 143
142
..
! 3
L23227
M
135
148
146
146
149"
j 145
141
..
3
L23228
M
r 146
145
146
146
148
146
142
I 3
L23229
M
142
144
145
147
143
146
"Til"
...
. ! - 3
L23230
M
126 *
144
147
144
151
144-"
141
..
I
Average
r 145
146
146
148
145
142
..
Std Dev
8
2
1
1
3" "
1
1
I 4
L23205
M
143
147
146
150
146
146
143
—
4
L23207
y
146
147
150
156
' 147
146
138
..
4
L23225
M
127
146
144
142
—
..
139^
4
L23231
M
141
145
146
147
146
146
142 "
4
L23235
M
144
146
146
147
145
...
..
!
Average
140
146
146
148
146
146
141
139
Std Dev
8
1
2
5
'i
0
~ 3"
..
I 5
[23200 H
M
145
144
146
-
...
143
I 5
L23201
M
150
146
146 *, ^
145*.«
5
L23212
M
144
145
144
145
143
145
141
...
5
L23214
M
146
147
143*
145 aa
-
-
..
I 5
L23234
M
146 I
146 1
146
144
--
...
..
—
Average
146
146
145
145
143
145
141
143
:
Std Dev
2
1
1
-
..
„
..
i
! 6
^23203
M
144'
147
145
140
-
..
-
..
I 6
L23204
M
145 1
146
140*
142 *'**
--
-
121 ¦**'**
I 6
L23213
M
145
--
-
-
-
-
150*
I 6
L23221
M
143
--
-
-
-
--
-
6
L23232
M
--
146 1
145
142
-
-
-
..
!
Average
144
146
143
141
-
...
136
i
Std Dev
1
3
1
-
—
21
* Slight Hemolysis; **** Severe Hemolysis
Aft Moderate Lipemia
~~ instrument unable to calculate result
+ Sample quantity insufficient
-- No Value
fttc_
CICllSft 111 !©/«* <*i
-------
Study 1020-CG920503
Clinical Chemistry
On
Bold > Normal Range; Underscore < Normal Range
* Slight Hemolysis; **** Severe Hemolysis
M Moderate Lipemia
— Instrument unable to calculate result
+ Sample quantity insufficient
-- No Value
Parameter Group ID
Animal ID
Sex
Day-3
Day 1
Day 2
Day 3
Day 7
Day 14
Day 21
Terminal
Potassium (3,0-7.0 rnEq/L)
- 1-
L23216
M
3.7
4.8
I 4.3
4.3
5.6
b 5
3.7
-
i 1
L23218
M
4.0
4.2
4.4
4.8
5.9
5.4
3.5
--
! 1
L23220
M
r 3.9
4.7
4_7
4.6
5,2
5.5
3.5
--
I 1
L23222
M
5.8
4.9
5.0 *
5.7
4.8
5.4
3.6
--
I 1
L23223
,M
4.5
5.9
5.2
4.9
5.0
5.6
4.0
-
Average
4.4
4.9
4.,'
4.9
5.3
5.5
3.7
-
Std Dev
0.8
0.6
0.4
0
6.4
0.1
0.2
2
L23206
M
4.7
5.2
5.7
6.0
4.8
6.3
3.8
2
L23210
M
4.7
6.3
~ 5.2 *
5,3
-
6.0
3 9
..
2
L23211
M
4.7
5.4
4.2
5
5.4
7.0
3^9
..
2
L23215
M
4.2
4.7
4.9
4.6
5.7
+
3,4
2
L23219
M
44
4.5
5.6
5.5
5j0
5.0
3.9
Average
4.5
5.2
5J
b 3
5.2
6l
378
...
Std Dev
0.2
0.7
0.6
0.5
o74
as
0.2
__
i
3
L23217
M
4.2
4.6
4.5
4.3
4.1
5.0
37s
...
3
L23227
M
4.4
4,5
" 474
4.2
6.6 "
5.6 "
376
3
123228
M
4.3
I 4,8
5.2
4.3
6.6
5 y
3.9
..
3
L23229
M
4.0
4.5
I-
4.6
' 4T
4.9
3.8
..
3
L23230
M
3.9*
4.2
4.2
4.7
4~3
56
3.9
••
Average
I 4.2
4.5
r 46
4.4
5.3
5.4
! -
..
Std Dev
0.2
0.2
0.4
' 0.2
{.2
0.4
0.1
4
L23205
M
4.3
4.8
^ 4.6
4.9
5.2
576
3.7
4
L23207
M
5.6
5.0
5.5
6.9
6.3
673
3.9
...
4
L23225
M
5.0
4,8
¦I 3
5.0
-
-
-
21.2 **"
4
L23231
M
4.7
7
r 4.4
(i
6.0
4.0
..
4
L23235
M
4.7
¦!
45
4.3
5.2
--
-
-
Average
r 4.8
4.7
4.7
5.1
5.8
6.0
3.9
21.2
Std Dev
0.6
0.3
0.5
1.1
0 7
:
0.2
-
! 5
L23200
M
5.1
4.8
h.t
--
--
--
..
11.4
L _
L23201
M
6.5
4.5
3.7 *.
3.7 *,«
..
..
-
..
5
L232-j2
M
4.2
4.3
5.0
4.7
4.8
4.9
3,9
..
5
L23214
M
4.7
5.5
3.8 *
3.7 v-
-
-
..
! 5
L23234
M
3.8
4.2
4.4
3.9
-
-
...
Average
4.9
4.7
4.4
4.0
4.8
4.9
3.9
11.4
Std Dev
1.0
0.5
0.7
0.5
--
--
-
-
I
I 6
[23203
M
5.0
5.2
3.5
3.8
-
..
..
; - -6 - _
L23204
M
49
4.5
3.8*
5.2 ****
--
--
-
29 3 "**"
! 6
L23213
M
--
4.6
--
..
-
--
17.3*
6
L23221
M
5.3
--
--
-
--
..
-
-
; e _
•£23232
M
--
5.1
3.9
4.5
-
..
-
Average
5.1
4.9
3,7
4.5
--
--
..
23.3
!
Std Dev
0.2
0.4
0.2
0.7
-
-
-
8.5
fHc_ to-zz-o^
QOTechRev.ByH' ;
-------
Study 1020-CG920503
Clinical Chemistry
Parameter
Group ID
I Animal ID
Sex
Day -3
Day 1
Day 2
Day 3
Day 7
I 107 "
Day 14
I "106'
Day 21
| 103
Terminal
Chloride (92-120 mEq/l)
1
P 123216
M
104
106
108
98
1
L23218
M
101
108
109
I 101
! 105
I 106
t 102
1
L2322G
M
102
107
105
[ 102 ~
t 102
I 105
1 105
..
1
L23222
M
105
107
109*
1 105
! 103
I 108
] 102
__
•
r
M
102
105
107
1 102
j 104
I 105
f 102
i
Average
103
107
108
| 102
[ 104
1 106
t 103
!
Std Dev
2
1
" 2
3
2
1
1 1
t~~
2
L23206
M
106
108
108
I 106
! 114
| 108
[ 105
I
I 2
L23210
M
105
105
106 * 1 103
i
! 106
! 102
;
I 2
L23211
Ml 109
107
107
104
I 111
! 106
1 105
i
I 2
L23215
M T 107
107
106
r 106
1 104
! +
! 109
i
2
L23219
M
108
105
108
104 "
112
j 106
1 103
| -
Average
107
106
107
f 105
| 110
1 107
1 105
!
Std Dev
2
^ 1
1 "
1
' 4 ¦
! i
f 3
I
;
! 3
L23217
M
*"" 108
107
108
107
! 104
! 109
106
;
! 3
L23227
M
101
109
106
108
t 109
I 109
104
1
3
L23228
M
t- 1Q7
j- 10/
107
104
107
I 108
i 103
1
I 3
L23229
M
107
104
107
106
107
I 107
106
]
! 3
L23230
M
92*
108
109
106
114
! 107
107
:
Average
[_ 1Q3
107
107
106
108
f 108
105
)
I
Std Dev
7
2
1
1
4
1
2
..
! 4
L23205
M
r 109
107
108
108
107
108
105
...
I 4
L23207
M
104
107
105
106
110
Ill
104
..
! 4
L23225
M
92
105
103
102
__ "
...
86""***'*'
! 4
L23231 ~1
M
102
105
106
~ 104
106
106
108
103
4
L23235
M
107
106
109
r 106
..
i
Average
103
106
105
" T06
107
109
104
86
Std Dev
7
1
2
~ 3
2
2
1
!
| 5
L23200
M
105
101
102
-
95
i 5
L23201
M
111
107
105 *,«
102 *,"«
—
j 5 ~l
L23212
M
104
103
105
102
106
108
103
__
5
L23214
M
105
106
104*
99 w
—
...
I 5
L23234
M
107
105
106
103
-
...
..
Average
106
104
104
102
" 106
108
103
95
Std Dev
3
2
2
2
...
6
L23203
M
111
107
106
103
-
__
I 6
[23204
M
111
106
103*
101 ****
-
..
77 '**'**
i 6
L23213
M
--
107
--
-
-
..
..
94*"
. . 6 _
L23221
M
^-f
-
-
--
-
-
-
..
I 6
L23232
M
-
104
105
103
--
-
-
..
Average
109
106
105
102
-
..
-
86
Std Dev
4 1
1
2
1
-
—
„
12
- — - "i
i
* Slight Hemolysis; **** Severe Hemolysis
w Moderate Lipemia
~~ Instrument unable to calculate result
+ Sample quantity insufficient
- No Value
HUiM Coil
ftte... io-tr-oq
QC/Ttcli In IfPite fit- >o -zz \
-------
Study 102O-CG92Q5O3
Clinical Chemistry
Parameter
Group ID
Animal ID
Sex
Day-3
Day 1
Day 2
Day 3
Day 7
Day 14
Day 21
Terminat
Calcium (5.50-16,50 mg/dL)
1
L23216
M
t 12.44
14.43
14.25
{ ' ""13733
j mil"
| 15.78
1 12.71
:
1
L23218
M
f 13.26
13.26
13.08
r Tier
! 15.21
r iW'
p__
!
1
L23220
M
13.44
13.68
13 73
['" 13.20
14 23
(—14.67
j 11 95
;
1
123222
M
1 1512
14.74
14.64*
| "" "" USES
! 14J20
1 15.T9
1 12.69
1
1
L23223
M
13.98
14.91 | "14jB6
! 14T32
14784"
( 15.37
| 12.68
¦i
Average
13.65
14.20
14.07
r 14.01
\~VW2
1 15Ti
t 12748""
1
StdDev
0.99
0.71
f 0.67
t ¦ 0769
1 6748"
! 6752
0.33
1
*
2
L23206
M
t_
14.03
14.48
15.20
nw
15.38'"
1 12.67
! 2
L23210
M
11.97
15.84
14.93 *
14.65
..
15:37
13765
L23211
M
12.62
14.39
r
13.98
13.84
!' 15.54
! 12.78
1
2
L23215
M
13.14
14.46
r 1364
13.51
! 15.30
+
12726"
|
2
L23219
M
12.01
13.97
13.81
14.67
! 14.13"
13.78
' 12.39
i
j
Average
12.56
14.54
Till
14.40
14.33
15762
•¦¦¦
:
I
r StdDev
0.56
0.76
0.57
0.66
0.66
0.83
0.31
'
!
3
L23217
M
12.02
13.37
13.19
12.59
13.06
13799"''
12.39
I 3
L23227
M
13.61
13.46
13.92
12.82
14.98"
14724
12,65'
—
3
L23228
M
12.30
14.34
14.41
14.14
"15.95"
16.27
12.47
...
3
L23229
M
I" 13.25
14.75
15.01 T 14.35
14.27"
14.88
12.56
..
3
L23230
M
12.43 *
h 13.29
12777
13.85
14.07
15719"'"
12.20
1
Average
r 1272
13.84
13.86
13.55
14.47"
14.91
12.45
..
I
^ Std Dev
0.68
0.66
0.90
0.80
1.08
"6790
0.17
...
i
4
L23205
M
13.06
14.19
14.08
14.44
14.63
14.51
12.60
4
L23207
M
14.57
14.31
15.57
16.93
15^22"
15797
12.01
..
' 4
L23225
M
h 13Q5
14.23
12.27
12 !
-
..
16.09""
4
L23231
M
13.27
I" 14.86
i" 14.23
13.68
j. -
16.05
13.13
—
- ! .4
L23235
M
14.01
r 14.16
14.03
13.47
14.16
—
--
..
Average
13.59
14.35
14.04
t"l4.86"
15751""'
r" 12758
f'1'8-09
Std Dev
0.67
0.29
1.17
r 1,76
0.58
6787
036
..
i _ .
'
I 5
L232CX)
M
14.30
13.98
14.70
-
.»
15.59
| 5
L23201
M
15.06
14.39
9.90 *,«
6.45 *,«
..
5
L23212
M
13.12
14.01
14.19
13.99
13.72
~14.48~
—
I 5
L23214
M
14.07
14.77
12.09*
10.79 «
..
-
..
i 5
L23234
M
13,32
13.95
14.26
11.89
..
-
...
..
Average
13.97
14.22
13.03
10.78
13.72
14.48
12.59
15.59
Std Dev
0.78
0.36
2.02
3.18
--
-
..
....
6
L23203
M
12.86
13 09
10.56
11.00
-
-
..
6
L23204
M
13.54
14.22 1
11.97*
11.27 ""
-
-
-
13.09 ****
I 6
L23213 1
M
--
13.82
-
...
--
--
-
12.32 *
I 6
L23221
M
14.01
--
--
--
-
-
..
..
¦-
L23232
M
14.45
11.91
11.80
-
...
...
!
Average
13.47
13.90
11.48
11.36
-
-
-
12.71
Std Dev
0.58
0.60
0.80
0 41
--
-
-
0.54
!
Bold > Normal Range; Underscore < Normal Range
* Slight Hemolysis; **** Severe Hemolysis
M Moderate Lipemia
— Instrument unable to calculate result
+ Sample quantity insufficient
-- No Value
ptc_
TO# In SfiitL
•cf'\
-------
Study 1020-CG920503
Clinical Chemistry
Parameter
Group ID
Animal ID
Sex
Day-3
Day 1
Day 2
Day 3
Day 7
Day 14
Day 21
Terminal
Phosphorus (2.0-9.0 mg/dL)
1
L23216
M
4.34
' 3.73
3.09
4.75
5.90
¦1 ).!
¦1
-
1
L23218
M
4.41
3.38
3.45
4.93
5.12
3.91
4.94
..
1
L23220
M
4.59
3.20
4.34
5.46
6.11
4.94
5.31
1
[.23222
M
4.16
3.71
3.34*
4.19
4.17
3.79
5.79
-
1
L23223
M
4.41
5.33
4.57
5.!''9
5.24
5.68
6.26
..
Average •
4.38
3.87
3.76
4.92
5.31
4.65
5.61
..
r StdDev
0.15
0.85
0.65
0 50
0.76
0 79
0.50
..
2
L23206
M
4.01
4.12
4.45
5. ¦;
5.57
5.14
f 6.83
...
i 2
L23210
M
2.16
3.65
3.43*
4. '
-
4.56
5.43
-
! 2
L23211
M
3.16
3.14
2.98
3.67
5.91
+
5.76
...
i 2
L23215
M
f 3.18
3.27
3.69
3 89
4.81
+
5.09
( 2. _
L23219
M
3.29
3.53
3.38
4.26
6.08
4.33
5.84
-
Average
3.16
3.54
3.59
4.33
5.59
4,68
5./9
-
Std Dev
0.66
0.38
0.55
0.65
0.56
0.42
ois
3
L23217
M
3.13
13.43
3.60
3.71
5.38
_____
5.43
__
3
L23227
M
h 3.05
3.76
3.33
3.58
~484
4.64
5.88
..
3
L23228
M
2.73
3.14
3.09
4.11
4.62
4.26
i 8.12
„
3
L23229
M
2.67
3.13
3.15
3.88
3.78 '
3.72
5.70
..
3
[^230
M
3.37 *
3.68
r 3.0/
3.97
5.79
4 38
6.05
..
Average
2.99
3.43
3.25
3.85
4.88
4.34
5.84
..
StdDev
0.29
0.29
0.22
0.21
0.77
0,39
"6728
..
4
L23205
M
3.17
3.21
296
3.38
4.01
5/74
..
1 4
L23207
M
3.37
3.33
3 95
4.54
4.10
4 39
5.59
..
L23225
M
3.44
3.86
4.32
4.77
-
..
—
16.39
L23231
M
h 3 2g ^
3.37
2.95
3.03
4.52
4.10
5.38
_
{ 4
L23235
M
2.74
2.45
2.47
2,i
2,86
..
..
!
Average
3.20
3.24
3.33
3.68
3v87
4 48
5 57
16.39
i
Std Dev
0.28
0.51 ^
0.77
0.93
0.71
0 43
0,18
....
1
5
L23200
M
3.34
3.63
3.46
-
__
-
..
6.38
! 5
L23201 '
M
3.48
2.97
4.40 *, ™
7.35 *,'
-
-
..
. - 1-5
L23212
M
3.17
3.29
3.51
3.69
4.13
4"26
5.36
...
! 5
L23214
M
3.51
3.54
4.48 *
4.87 «
--
...
..
5
L23234
M
2.34
2.60
2.82
4.10
-
-
-
~
Average
3.17
3.21 "I
3.73
5.00
4.13
4.26
5.36
6,38
StdDev
0.48
0.42
0.70
1.64
..
-
...
1
! 6
L23203
M
3.61
3.95
5.28
3.99
-
-
-
...
; - e._
L23204
M
3.17
3.32
3.67 *
4.17 ««
--
-
17.55
1 6
L23213
M
-
3.31
--
--
--
-
19.69 *
1 6
L23221
M
3.24
--
-
-
--
-
-
..
! 6
M
-
4.23
4.91
5.26
--
-
Average
3.34
3.70
4.62
4.47'
--
1
--
18.62
Std Dev
0.24
0.46
0.84
0.69
-
--
-
1.51
Bold > Normal Range; Underscore < Normal Range
* Slight Hemolysis; **** Severe Hemolysis
AA Moderate Lipemia
~~ Instrument unable to calculate result
+ Sample quantity insufficient
- No Value
!€>.z.Z'CJ*=\
~A Humi Qomf:
' S"'
QCJTecbRev,!
\o -
•c^
-------
Study 1020-CG920503
Clinical Chemistry
Parameter
Group ID
Animal ID
Sex
Day-3
Day 1
Day 2
Day 3
Day 7
Day 14
Day 21
Terminal
Total Protein (3.5-8.5 g/dL)
1
L23216
M
I 5.90
6.29
6.09
6.20
6.77
6.62
5.68
-
1
L23218
M
5.84
I 5.96
5.75
6.58
6,44
6.21
b.4b
..
1
£23220
M
5.69
5.54
5.52
5.76
5.97
5.82
5.23
...
1
L23222
M
5.94
5.90
5.83 *
5.82
5.99
5.98
5.21
..
1
L23223
M
5.79
6.34
6.12
6.20
6.10
6.34
5.40
..
Average
5.83
6.01
r 5.86
6.11
6,25
6.19
5.39
..
Std Dev
0.10
0.33
r 0.25
0.33
0,34
031
0.19
..
7 " -
2
L23206
M
5.59
6.14
6.19
6.75
6.75
6.57
5.72
2
L23210
M
5.31
6.14
6.10*
6.27
-
6,03
5J50
—
2
r L23211
M
5.47
6.42
5.89
6.23
6.68
+
_____
-
2
L23215
M
5.61
' 6.02
5.78
6.09
6.28
+
5.70
..
2
L23219
M
I 5.34
6.18
6.20
6 67
6.98
6.15
5^37
-
Average
5.46
6 ig
6.03
6.40
6.67
6.25
~5£4
..
Std Dev
0.14
I 0.15
r Q )g
0.29
0.29
0.28
0J7
-
3
L23217
M
5.00
5.68
5.59
5.63
5.89
5.94
5.24
...
3
L23227
M
5.39
5.58
5.51
5.13
5.91
5765
5722
...
3
L23228
M
I 5.00
5.83
5,89
5.84
6.37
6.29
5.26
...
3
L23229
M
r 5.40
6.08
5.93
5.91
5.59
6.30
5^40
..
3
L23230
M
5.42*
5.45
5.33
5,86
6 55
6.13
5.50
...
Average
5,24
5^72
5 65
5.67
6.06
6.06 *
5.32
Std Dev
0.22
0.24
0.26
0.32
0 39
0.27
o7i2
....
I A
L23205
M
5.35
5.73
5.48
5.91
5.98
5.98
5,25
4
L23207
M
6.26
6.22
6.71
7.14
6.23
"~&45
5.45
—
L23225
M
5.66
6.04
5.90
5.23
-
4.30
4
L23231
M
5,42
6.10
5.66
5 80
6.53
6.56
5^21
...
4
L23235
M
5.82
6.03
5.92
5,69
6.35
-
-
—
Average
5.70
6.02
5.93
5.95
6.27
6 33
5"30
4.30
Std Dev
0.36
0.18
0.47
0.71
0.23
0.31
67i3
i 5
L23200
M
5.83 H
5.98
6.20
-
-
~
—
5.96
i
5
L23201
M
6.69
6,18
5.97 *,«
5.09 *,«
-
-
.
5
L23212
M
5.57
5.90
6.14
5.87
6.08
6.21
5.52
—
5
L23214
M
5.90
6.37
') 15C. '
5.91 "
-
„
-
..
5
L23234
M
5.55
5.53
5 55
4.92
~
--
Average
5.91
5.99
5.90
5.45
6.08
6.21
5 5?
5.96
Std Dev
0.46
0.32 1
0.29
0.52
-
-
-
6
L23203
M
5.07
5.28
5.34
4.84
-
..
..
-
6
L23204
M
5.50
5.87
5.84*
5.29"**
--
--
..
4.42 ****
i 6
L23213
M
-
5.66
--
-
--
--
..
5.79 *
6
L23221
M
5.82
--
--
--
--
--
-
6
L23232
M
--
5.82
6.07
6.00
--
--
..
„
Average
5.46
5.66
5.75
5.38
--
..
--
5.11
Std Dev
0.38
0.27
0.37
0.58
--
--
-
0.97
(Hc_ i o • 2.2 • oq
QCITalilifJfl:1 ? i -.
Bold > Normal Range; Underscore < Normal Range
* Slight Hemolysis; **** Severe Hemolysis
AA Moderate lipemia
- Instrument unable to calculate result
+ Sample quantity Insufficient
-- No Value
-------
Study 1020-CG920503 Clinical Chemistry
<
Parameter
Group ID
Animal ID
Sex
Day-3
Day 1
Day 2
Day 3
Day 7
Day 14
Day 21
Terminal
Total Bilirubin (0.0-1.0 mg/dL)
1
L23216
M
r 0.0
0.0
0.0
00
0.0
0.0
o.o
__
1
L23218
M
0.0
0.0
0.0
0.0
0.0
o.o
0.0
1
L23220
M
0.0
0.0
0.0
r 0.0
0.0
o.o
0.0
..
1
L23222
M
0.0
0.0
0.0 *
0.0
0.0
0.0
0.0
1
L23223
M
0.0
0.0
r o.o
0.0
0.0
0.0
0.0
-
Average
0.0
0,0
0.0
0.0
o.o
6.6
6.6
Std Dev
0.0
I 0.0
0.0
0.0
0.0
0.0
0.0
-
r—
2
L23206
M
0.0
0.0
0.0
0.0
00
0.0
0,0
-
L23210
M
0.0
0.0
0.0*
0.0
0.0
0,0
«
2
L23211
M
0.0
0.0
0.0
0.0
o.o
+
0.0
.
2
L23215
M
0.0
0.0
0.0
0.0
0.0
+
0.0
2
L23219
M
0.0
0,0
0.0
0.0
0.0
0.0
0.0
Average
0.0
0.0
0.0
0.0
0.0
oJT
0.0
Std Dev
0.0
0.0
0.0
0.0
6.6
0.0
0.0
-
3
I L23217
M
0.0
0.0
0.0
0.0
o.o
0,0
-
3
L23227
M
0.0
0.0
0.0
0
0.0
0.0
..
3
L23228
M
0.0
0.0
0.0
0.0
0.0
0.0
6.6
-
3
L23229
M
h
0.0
0.0
0
00
0.0
6.6
-
3
L23230
M
0.0*
0.0
0.0
0
6.6
0.0
0.0
-
Average
0.0
0.0
0.0
0.0
0.0
0.0
6.6
Std Dev
0.0
0.0
0.0
0,0
o.o
0.0
0.0
-
4
L23205
M
0.0
0.0
0.0
0.0
6.6
0.0
0.0
4
L23207
M
0.0
0.0
0.0
0.0
0,0
0.0
0.0
4
L23225
M
0.0
h 0.0
0.0
0.0
..
-
....
4
L23231
M
0.0
0.0
0.0
0.0
0.0
6.6
0.0
4
L23235
M
0.0
0.0
0.0
0.0
0.0
..
..
Average
0.0
0.0
0.0
0'
6,6
6.6
0.0
Std Dev
0.0
0.0
0.0
o.o
o.o
0.0
0.0
—
5
L23200
M
0.0
0.0
0.0
-
-
—
0.0
5
L23201
M
0.0
0.0
0.0
0.0 ¦ ¦
--
..
..
5
L23212
M
0.0
0.0
0.0
0,0
0.0
0.0
0.0
..
5
L23214
M
0.0
0.0
0.0*
*
o
o
--
-
5
!34
M
0.0
0.0
0.0
0.0
-
--
-
Average
0.0
0.0
0.0
0.0
6.6
0.0
0.0
0.6
Std Dev
0.0
0.0
0.0
0.0
..
--
i
. I 6 ,
L23203
M
0,0
0.0
0.0
0.0
..
..
--
..
6
L23204
M
0.0
0.0
0.0*
0.0 ****
-
--
6
L23213
M
--
0.0
-
--
--
-
0.0 *
6
L23221
M
0.0
--
--
-
..
_
-
-
6
L23232
M
--
0.0
0.0
0.0
-
-
..
-
Average
0.0
0.0
0.0
0.0
--
--
0.0
Std Dev
0.0
0.0
0.0
0.0
..
..
Bold > Normal Range; Underscore < Normal Range
* Slight Hemolysis; **** Severe Hemolysis
M Moderate Lipemia
— Instrument unable to calculate result
+ Sample quantity insufficient
- No Valun
~A Auoit CoNmiirtSf
-UilS
QCfFechit*,Bfl'Dati i-
-------
Study 1020-CG920503
Clinical Chemistry
Parameter ! Group ID
Animal ID
Sex
Day-3
Day 1
Day 2
Day 3
Day 7
Day 14
Day 21
Terminal
Aspartate Aminotransferase (0.0-120,0 U/L)
1
L23216
M
18.5
r 2o,o
20.4
18 V
15.2
53.2
f 27,2
! 1
L23218
M
18.1
16.7
15.0
1«
24.0
21.1
t ____
L23220
M
42.5
15.6
27.0
25.1
25.6
23.2
28.9
I 1
L23222
M
19.0
L 194
17.8 *
19.2
26.4
22.4
15.3
L23223
M
29.8
25.1
21.1
21.9
194
32.5
f 23.3
..
Average
25.6
19.4
20.3
20.2
22.1
30.5
25.0
-
Std Dev
r 10.6
3.7
4.5
3.4
4.7
13.5
6.0
...
"
! 2
L23206
M
15.8
17.2
16.8
18.9
18.3
16.4
19 7
...
I 2
L23210
M
12.0
15,9
14.1 *
17.9
-
19.8
13J9
—
! 2
L23211
M
10.8
13,7
"• '
15.3
15.5
*+
18.8
- 2 _
L23215
M
^ 11.7
13,0
I" 13.0
J 14.9
1 21~i
15,3
+
16.5
..
i 2
L23219
M
16.3
I" 19.3
16.9
26.2
18.2
r
...
Average
13 3
15.8
14.9
11 '¦
18.8
1.7
175
—
Std Dev
2.5
2.6
1.8
2.li
5.1
2.6
...
f 3
L23217
M
11.1
16.2
r 142
13.2
17.3
16.6
14.0
-
I 3
L23227
M
13.1
16.7
r 13.8
18,1
18.0
50.1
r 21.9
—
I 3
L23228
M
12.7
13.2
22.1
12.2
13.2
146.8
13.7"'"
—
I 3
h L23229
M
15,3
167
15.8
16,3
21.4
18,8
22.9
..
3
L23230
M
10.2 *
10.t
13.4
8.1
12.8
. ...
13.2
...
Average
12.5
14.6
15.9
13.6
16.5
48,9
17.1 ""
..
I
r Std Dev
2.0
2.9
3.6
3.9
3.6
56.8
4.8
—
! ' '
: 4
L23205
M
12.1
13.7
r 10.7
f 21.6
19.5
16.7
23.9 "
-
I 4
h L23207
M
13.5
19.2
22.3
20 6
25.1
17.1
14.1
~
r
M
15.7
23.1
r 72.4
20.7
--
...
-
944.0 "•*
L23231
M
18.3
13.0
12.2
24,4
29.7
23.9
22.5
--
I 4
L23235
M
11.2
12.0
11.6
15
19.0
..
..
--
t"
Average
14.2
16.2
25.8
20.5
23.3
19.2
20.2
944.0
Std Dev
2.9
4.8
26.5
3.4
5.1
4,6
5.3
-
5
L23200
M
16.8
15.8
15.0
--
--
..
..
121.2
5 ~l
L23201
M
28.8
25.4
701 8 ¦.
1292.8 *, AA
--
-
-
5
L23212
M
13.9
15.9
15.1
r 21.5
23.7
20.5
21.8
--
! 5
L23214
M
11.2
14.0
22.6*
394.6 **
-
-
-
I 5
L23234
r M
15.4
28.8
17.1
551
-
-
-
-
Average
17.2 '
20.0
154.3
565.0
23.7
20.5
21.8
r 121.2
Std Dev
6.8
6.7
306 1
533.6
-
..
i
6
L23203 I M
13.8
12.6
83.9
18.3
-
..
...
6
L23204
M
10.0
14.8
44.0 *
27.7 *"*
-
-
-
416.8
! 6
123213
M
--
17.1
--
--
--
..
-
218.0 *
! 6
L23221
M
11.3
-
--
--
-
..
-
..
6
L23232
M
--
16.1
80.7
21.1
-
--
..
—
Average
11.7
15.2
69.5
22.4
...
-
-
317.4
Std Dev
1,9
1.9
22.2
4.8
-
-
140.0
Bold > Normal Range; Underscore < Normal Range
* Slight Hemolysis; "" Severe Hemolysis
AA Moderate Lipemia
-- Instrument unable to calculate result
+ Sample quantity insufficient
- No Value
fH-c. io-zz-o^
QS/TisI RiiJviifc: foe.
11
-------
Study 1020-CG920503
Clinical Chemistry
Parameter
Group ID
Animal ID
Sex
Day -3
Day 1
Day 2
Day 3
Day 7
Day 14
Day 21
Terminal
Alanine Aminotransferase (7.5-85.0 U/L)
1
L23216
M
36.2
44.9
44.7
44.1
I 42,6
64.3
49.2
...
1
L23218
M
19.4
20.3
22.5
25.2
! 25.5
24.1
: 30.7
-
1
L23220
M
37.8
37.8
41.2
40.6
I 33.1
33.8
33.9
-
1
L23222
M
33.6
36.9
38.8 *
39.0
I 43.3
40.1
32.1
...
1
L23223
M
59.6
43.5
44 9
45.1
t 45.6
54.5
45.8
-
Average
37.3
36.7
38.4
38.8
i 38.6
43.4
' 38.3
~
Std Dev
14.4
9.8
9.3
8.0
I 8.5
16.1
! 8.5
...
2 ~
L23306~~
~ M
" 24.5
28?4
28.8
30,8
! 35.5
32.6
26.9"
2
L23210
M
16.1
19.8
20.6*
20.8
!
26.0
t g-y
-
2
L23211
M
19.2
28.5
25.6
27.8
I 26.8
+
22.3
..
2
L23215
M
32.3
37.5
33.6
37.8
I 39.8
+
36.9
..
2
L23219
M
21.4
30.0
30.6
35.4
I 37.2
28.1
26.2
Average
22.7
28.8
' 27.8
30.5
34.8
28.9
26.7
-
Std Dev
6.2
r 6.3
5.0
6.7
! 5.6
3.4
6.2
3
L2321?
M
I" 26.9
34.1
30.5
32.0
1 34.8
43.1
33.8
...
3
1.23227
M
26.9
29.0
30.3
31.1
' 31.2
58.3
' 30.6
-
3
L23228
M
19.6
36.4
38.0
30.7
i 35.6
53.8
25.7
...
3
L23229
M
I 19.2
23.7
23.0
22.7
1 26.0
25.1
21.9
-
3
L23230
M
p__ „ .
r 24.7
h 24 i
24.6
| 26.3
27.4
22.2
...
Average
22.6
29.6
29.2
28.2
41,5
26.7
-
Std Dev
3.9
5.6
6.0
4.3
! 4.4
15.0
5.1
-
4
L23205
M
15.5
19.0
18.3
24.4
I 22.1
19.4
20 6
-
4
L23207
M
29.9
34.0
40.2
41.1
] 38.8
32.0
26.4
-
4
L23225
M
21.1
36.2
51.0
37.3
i
-
-
143.8
4
L23231
M
22.0
25.0
23.1
28.5
' 36 1
30.0
20,5
...
4
L23235
M
21.6
r 24.9
22.8
24.6
31.6
..
..
Average
22.0
27.8
31.1
' 311
32 2
27.1
2275
14378
Std Dev
5.1
7.1
13.9
' .'5
1 773 1
6.8
3.4
I
5
L23200
M T 28.0
30.9
31.6
..
..
56.4
5
L23201
M
71.5
67.2
290.6
588.1 *, AA
—
..
..
5
L23212
M
33.1
36.5
35.8
37.0
! 4577 !
39.1
34,7
..
5
L23214
M
34.6
44.8
41.4*
171.8 **
..
--
..
...
5
L23234
M
34.7
55.5
50.6
198.4
..
--
..
..
Average
40.4
47.0
90.0
248.8
1 45.7 " I
39.1
34.7
56.4
Std Dev
17.6
14.6
112.4
237.0
..
1
I 6
[23203
M
22.4
21.0
46.3
3l'.2
1
-
..
..
! 6
L23204
M
19.0
26.3
40.7*
25.1 **"
-
..
142.3 ****
T 6
L23213
M
-
30.6
-
-
--
..
124.3*
! 6
123221
M
19.7
-
-
--
...
6
L23232
M
-
24.3
44.1
32.9
—
...
..
Average
20.4
25.6
43.7
29.7
-
-
133.3
Std Dev
1.8
4.0
2.8
4.1 I - I
-
12.7
I
Bold > Normal Range; Underscore < Normal Range
* Slight Hemolysis; **** Severe Hemolysis
w Moderate Lipemia
~~ Instrument unable to calculate result
+ Sample quantity insufficient
- No Value
12
(CNr<_
CJCfTech in ByfMg. (He. tc- -ix- c.q
-------
Study 1020-CG920503
Clinical Chemistry
Parameter
Group ID
Animal ID
Sex
Day-3
Day 1
Day 2
Day 3
Day 7 Day 14
Day 21
Terminal
Lactate Dehydrogenase (0.0-260 0 U/L)
1
L23216
y
93
138
60
39
38 1 58
86
1
L23218
M
121
97
59
59
76 I 123
156
1
L23220
M
285
50
61
86
128
76
86
..
1
L23222
M
120
89
118 *
41
54
77
115
..
1
L23223
M
112
245
114
I 41
83
305
175
Average
146
124
82
f 53
76
128
124
Std Dev
78
75
31
20
34
102
41
2
123206
M
89
95
111
99
63
39
64
2
L23210
M
69
74
88*
91
_
52
70
-
2
L23211
M
64
71
39
35
99
+
106
—
2
L23215
M
68
63
80
65
71
+
104
-
2
L.23219
M
84
80
58
48
75
80
103
..
Avera9?
75
77
75
68
77
57
89
Std Dev
11
12
| 28
27
15
21
21
-
I 3
L23217
M
59
54
52
36
83
35
53
-
3
L23227
M
67
65
58
51
68
116
113
...
3
L23228
M
55
77
70
40
64
136
iTT"
-
3
L23229
M
35
46
54
30 ! 54
58
90
—
3
L23230
M
100*
59
g5
48 ; 107
68
96
—
Average
63
60
58
41 75
83
94
Std Dev
24
12
7
9
21
42
25
4
L23205
M
32
48
31
41
67
58
127
4
h 123207
M
80 1
90
67
46
63
51
71
4
L23225
M
138
158
121
114
_
..
5189
4
L23231
M
37
45
50
47
69 n
82
158 1
4
L23235 ^
M
43
45
31
41
71 1
..
..
Average
66
77
60
58
68
64
118
6189
Std Dev
44
49
37
32
3
16
43
..
5
L23200
M
56
116
85
--
..
447
5
L23201
M
75
128
574 *,
602 \
-
..
..
5
L23212
M
32
65
49
90
64
113
70
—
5
L23214
M
53
78
155*
493 AA
--
-
—
5
L23234
M
57
93
62
509
..
_
Average
55
96
185
424
64
113
70
447
Std Dev
15
26
221
227
_
..
—
6
L23203
M
35
55
106
43
..
-
—
4782
6
. L23204
M
42
105
71 *
462 ""
..
-
6
L23213
M
-
40
-
-
..
..
—
S4@*
6
L23221
M
68
--
--
--
..
..
6
L23232
M
--
64
96
72
_
-
-
—
Average
48
66
91
192
-
..
..
2814
Std Dev
17
28
18
234
-
_
..
2783
Bold > Normal Range; Underscore < Normal Range , zZ-'C^
* Slight Hemolysis; **** Severe Hemolysis 1
** Moderate Lipefma
-- Instrument unable to calculate result
+ Sample quantity insufficient <••• >' C|CrfTf\, t'W •»
- No Value 13 ~
-------
Study 1020-CG920503
Clinical Chemistry
<
Parameter
Group ID
Animal ID
Sex
Day-3
Day 1
Day 2
Day 3
Day 7
Day 14
Day 21
Terminal
Sorbitol Dehydrogenase (U/L)
1 I L23216
M
. 24.4
27.7
29.0
27.2
28 4
40.6
23.2
--
L23218
M
, 25.3
17.7
12.2
20.5
21.8
18.3
28.9
...
1
L23220
M
36.3
16.5
17.8
22.4
26.2
24.6
17.4
1
L23222
M
23.9
22.8
19.7 *
20.3
! 2979 "
2670
t 16.8
..
1
j~ 1.23223
M
35.5
37.2
32.8
36.9
! 39.2
! 51.1
I 33.8
Average
29.1
24.4
22.3
25.5
| "29.1
i 32.1
24.0
..
Std Dev
I 6.3
r 8.4
8.4
7.0
6.4
1 1374""'
I 7.3
__
"
i 2
L23206
M
20.0
20.0
20.4
27.1
27.0
22.8
22.9
..
I 2
L23210
M
13.3
14.9
18.2*
23.7
! 22.5
j 19.2
..
I 2
L23211
M
18.0
23.2
' 20.6
20.4
24.5
1 +""
1 18.0
I 2 " ~
L23215
M
17.0
20.3
18.3
17 f
1 24.8
+
1971'
i 2
L23219
M
21.6
I 27.7
26.9
31.1
f 4172
21.2
! 23.4
- { - - -
Average
18.0
21.2
20.9
24.0
29.4
22.2
'"'"""2075
..
Std Dev
3.2
4.7
3.5
5.3
876
0.9
r 2.5
—
I
I 3
L23217
M
21.7
28.0
29.1
25.9
34.4
33.9
i 3
L23227
M
23.3
28.3
24.7
22.:'i
3ll
877!
f '227
..
I 3
L23228
M
24.5
r 42.1
47.1
* "3377
41.8
75.4
t"""""277l
3 _
L23229
M
15.9
h 20.9
18.5
18.1
2379""
2475"
15.9
i 3
[23230
M
23.4 "
1 19.5
15.4
U
2779
2l'.3
16J
Average
21.8
27.8
27.0
22.8
32.0
4874
2172
..
Std Dev
r~ 3.4
r 9.0
12.5
7.5
as
30.6
478
f
I 4
L23205
M
17.4
22.3
20.0
49.7
30.2
25.2
26.8
—
4
L232Q7
M
15.0
21.6
23.2
19.7
22.0""
16.9
1"T."4
....
i 4
L23225
M
35.5
26.2
87.2
40.7
--
2 1 075""*'*'**'"
L23231
M
22.3
26.7
21.5
40.0
4678
34.9
23-9l„
L23235
M
29.4
29.7
29.0
29.0
53.5
'
Average
23.9
25.3
36.2
35.8
387T
2577
20-7
210.5
Std Dev <
8.5
3.3
28.7
11.6
14.5
9.0
8.2
_
"
5
L23200
M
35.0
36.7
33.2
-
—
..
135.9
! 5
L23201
M
44.9
42.7
331.1 *, **
497.2 *,«
—
...
I 5
L23212
M
16.4
21.9
20.6
24.5
26.7
2279
19,0
—
- | - 5
L23214
M
21.0
24.3
24.5 *
178.9 «
-
-
-
..
I 5
L23234
M
17.6
37.0
25.3
223.1
--
-
-
..
i _
Average
27.0
32.5
86 9
230.9
26.7
22.9
19.0
135.9
Std Dev
12.5
9.0
136.6
196.9
¦
¦ ¦
..
i
I 6
L23203
M
23.8
24.2
69.9
3C
-
..
-
—
! 6 _
L23204
M
11.7
25.7
38.9*
—
-
...
...
54.8 ****
6
L23213
M
-
39.5
--
-
-
-
-
268.3*
I 6
L23221
M
14.9
-
-
--
-
--
-
6
L23232
M
-
26.8
51.1
18.8
--
-
-
...
( -
Average
16.8
29.1
53.3
24.7
-
-
..
160.8
l
Std Dev
6.3
7.0
15.6
8.3
--
..
-
149.6
i
* Slight Hemolysis; **** Severe Hemolysis
^ Moderate Lipemia
-- Instrument unable to calculate result
+ Sample quantity insufficient
-• No Value
Audit CatiWfeisc
! ru-ojioj,,:'
14
fer£.Z.-Oc?
QCITech Rftv, BvfDate: Ph i
-------
Study 1020-CG92O5O3 Clinical Chemistry
Parameter
Group ID
Animal ID
Sex
Day -3
Day 1
Day 2
Day 3
Day 7
Day 14
Day 21
Terminal
C-Reactive Protein (mg/dL)
1 I L23216
M
<0.5
<0.5
1.89
0.73
j <0.5
-.0 5
<0.5
I
1 ! L23218
M
<0.5
0.56
<0.5
<0.5
'2.94 ~
1 2.62
<0.5
i -
1
L23220
M
2.98
2.57
3.75
1.77
<65"
!o
¦¦ V
f <0.5
,
1
L23222
M
<0.5
0.87
0.56 *
<0.5
i <0.5
! <6^5
r <65
1
L23223
M
5.26
<0.5
0.56
<0.5
: Normal Range; Underscore < Normal Range
* Slight Hemolysis;""" Severe Hemolysis
** Moderate Lipemia
Instrument unable to calculate result ».• »
+ Sample quantity insufficient
- No Value
-------
APPENDIX W
ANATOMIC PATHOLOGY
-------
Table of Contents
1.0 Introduction W-5
2.0 Pathology W-6
2.1 Necropsy W-6
2.2 Histopathology W-7
4.0 Conclusions W-27
5.0 References W-28
List of Tables
Table 1. Study Design and Challenge Doses W-5
Table 2. Mortality (Found Dead) Due to B. anthracis Infection (Males only) W-6
Table 3. Incidence Summary of Microscopic Nonneoplastic Graded Observations with
Average Severity - Males, Day 21 W-9
Table 4. Incidence Summary of Microscopic Nonneoplastic Graded Observations with
Average Severity - Unscheduled Death Males W-12
Table 5. Summary of Individual Gross and Microscopic Observations, Males W-15
Table 6. Individual Gross and Microscopic Observations, Males W-19
List of Figures
Figure 1 Animal 103 (L23218): Lung, Normal. HE. 4X 1-2
Figure 2 Animal 402 (L23225): Lung, alveoli. Interstitial inflammation and
intravascular and interstitial anthrax bacilli (arrow). HE. 20X 1-3
Figure 3 Animal 601(L23204) -Lung, alveolus. Multinucleated Giant Cells (arrow).
HE. 10X 1-4
Figure 4 Animal 601 (L23204): Lung, alveolus: Multinucleated Giant Cell Foreign
Body (arrow). HE. 20X 1-5
Figure 5. Animal 605 (L23213): Lymph node, sinuses: Anthrax bacilli (arrow head).
HE. 4X 1-6
Figure 6. Animal 605 (L23213): Lymph node, sinuses: Hemorrhage and Lymphocyte
Depletion HE. 4X 1-7
Figure 7. Animal 605 (L23213): Lymph node, sinuses: Anthrax bacilli (arrow head).
HE. 40X 1-8
W-2
-------
Figure 8.. Animal 502 (L23234): Thymus: Lymphocyte depletion (arrow heads). HE.
4X 1-9
List of Appendices
Attachment 1 1-1
W-3
-------
List of Acronyms
CFU colony forming units
LD50 median lethal dose
min minute
mL milliliter
mm millimeter
NZW New Zealand White
W-4
-------
1.0 Introduction
The objective of this study was to determine physiological markers of disease following a
single exposure to Bacillus cmthracis Ames strain spores. Thirty (30) male pathogen-free
New Zealand White (NZW) rabbits were aerosol challenged with targeted doses of 100,000,
10,000, 1,000, or 100 colony forming units (CFUs) of B. cmthracis (Ames strain).
Additionally, a high dose control group was challenged with 100 LD50 and a negative control
group was exposed to an equivalent of 100 LD50 of gamma irradiated spores as outlined in
Table 1.
Table 1. Study Design and Challenge Doses
Group
Spore Dose
(CFU)
Number of Spore
Challenges
# of Rabbits
1 (negative) control*
100 x LD50
1
5
2
100
1
5
3
1000
1
5
4
10,000
1
5
5
100,000
1
5
6 (high dose control)
100 x LD50
1
5
*Negative controls were challenged with irradiated spores
Complete necropsies were performed on all rabbits following spontaneous death or
euthanasia, including rabbits surviving to study termination on Day 21. The lungs and gross
lesions from each animal were collected. The meninges, ependyma, ventricles, frontal cortex,
hippocampus, thalamus, cerebellum, and brain stem were included when gross lesions were
present in the brain. The brain and standard sections of all other collected tissues were placed
in 10% neutral buffered formalin, processed to approximately 5 micron 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) 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. Gross and microscopic diagnoses were entered into the PATH/TOX SYSTEM
W-5
-------
(Xybion Medical Systems Corporation, Cedar Knolls New Jersey) for data tabulation and
analysis.
2.0 Pathology
2.1 Necropsy
Two (40%) rabbits in Group 4, four (80%) rabbits in Group 5, and five (100%) rabbits in
Group 6 died following challenge with B. cmthrcicis spores. Mortality rates are summarized
in Table 2.
Table 2. Mortality (Found Dead) Due to B. anthracis Infection
Group
Mortality Rate
per Group (Males)
Mortality Rate
per Group (Total)
(%)
r (ioo x ld50)
0/5
0
2 (100 CFU)
0/5
0
3 (1,000 CFU)
0/5
0
4 (10,000 CFU)
2/5
40
5 (100,000 CFU)
4/5
80
6 (IOOxLDjo)
5/5
100
aGroup 1 animals were challenged with irradiated spores
Gross lesions in unscheduled death rabbits (rabbits that succumbed to challenge with
B. anthracis) included: discoloration of the brain (meninges), crusting of the skin, abdominal
and thoracic cavity fluid, bronchial and mediastinal lymph node enlargement, small intestinal
fluid, and thymic fluid. These gross lesions were typical of anthrax in rabbits (Zaucha et al.,
1998) and correlated histologically with hemorrhage, necrosis, edema, and/or suppurative
inflammation. A gross lesion was present in only one scheduled death (Day 21) rabbit
(L23231) consisting of a skin crust of the left hind limb correlating microscopically to
dermal and epidermal necrosis. No intralesional anthrax bacilli were found; hence this lesion
was not attributed to anthrax. Gross lesions are detailed in the Individual Gross and
Microscopic Observation tables (Attachment 1 to Appendix W).
W-6
-------
2.2 Histopathology
The lungs and all gross lesions were examined microscopically for evidence of anthrax.
Microscopic lesions typical of anthrax (Zaucha, et al., 1998) were present in all unscheduled
death rabbits, and included minimal to moderate suppurative inflammation (predominately
degenerate and viable heterophils [polymorphonuclear cells]), necrosis, hemorrhage, fibrin
and/or large rod-shaped bacteria in the lungs, bronchial and mediastinal lymph nodes, skin,
small intestine, brain (meninges), and thymus. Lung lesions consisted of suppurative
inflammation and bacteria generally found in the pulmonary interstitium and associated with
alveolar capillaries or larger pulmonary blood vessels. Scheduled death rabbits (Day 21) had
little or no inflammation in the lungs (inflammation when present was generally
suppurative); additional anthrax-related lesions were found in lymph nodes, meninges, skin,
thymus, and small intestine. Lymph node findings included hemorrhage, lymphoid necrosis,
fibrin, and bacteria. There was minimal to mild intravascular bacteria, suppurative
inflammation, and hemorrhage (with vascular necrosis) primarily in the meninges of the
brain of one rabbit (L23232). The skin of rabbit L23234 had areas of suppurative
inflammation/edema, hemorrhage, necrosis, and intra-/extravascular bacteria. The thymus
had mild to moderate atrophy of lymphoid tissue, intravascular bacteria, and evidence of
edema. Additionally, there was suppurative inflammation and intra- and extravascular
bacteria in the small intestine (jejunum) of one rabbit (L23221). Where suppurative
inflammation occurred in tissues (other than the lung), it was generally associated with
bacteria (bacilli).
Multinucleated giant cells (occasionally organizing towards granuloma formation) were
present in the lungs of some rabbits in all groups, including control rabbits. These
multinucleated giant cells were randomly distributed throughout the lung in ruptured or intact
airways or less frequently in or near pulmonary blood vessels or lymphatics. The
multinucleated giant cells often surrounded or contained birefringent foreign debris. These
cells and debris were also seen in the lungs of two control animals; but occurred with greater
frequency and severity in anthrax challenged rabbits. Cell aggregates of this type were not
described in a previous acute anthrax rabbit study (Zaucha et al., 1998); hence, it is uncertain
W-7
-------
if anthrax is contributory to the development of this lesion. While many pulmonary
macrophages in challenged rabbits contained foamy or granular cytoplasm and cellular
debris, bacilli were not identifiable in most cases. However, it is possible that these lesions
were caused by an indirect effect of prolonged anthrax septicemia on macrophage function.
Macrophage dysfunction has been described as occurring late in sepsis (Pahuja et al., 2008).
Inhaled debris or emboli from indwelling vascular access ports may have also contributed to
the development of multinucleated giant cell infiltrate/granuloma formation. Pulmonary
granulomas/multinucleated giant cells, thromboemboli, and perivascular eosinophils have
been described in animals fitted with indwelling vascular access ports (Taketoh et al., 2009).
Microscopically the skin of rabbit (L23231) had superficial and dermal necrosis with
underlying granulation tissue. Coccoid bacteria were present but there were no bacteria
characteristic of anthrax in this lesion, so it was not attributed to anthrax. The incidence
summary of microscopic observations is presented in Table 3 for survivors humanely
terminated on Day 21 and in Table 4 for unscheduled-death rabbits. In both 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).
Photomicrographs representative of anthrax are included in Attachment 1.
W-8
-------
Table 3. Incidence Summary of Microscopic Nonneoplastic Graded Observations with Average Severity - Day 21
Number Observed Per Group
1 issue/Observation
Group:
1*
2
3
4
5
6
Brain
Number Examined:
0
0
0
0
0
0
Bacteria
-
-
-
-
-
-
Average Severity:
-
-
-
-
-
-
Hemorrhage
-
-
-
-
-
-
Average Severity:
-
-
-
-
-
-
Inflammation, Suppurative
-
-
-
-
-
-
Average Severity:
-
-
-
-
-
-
Intestine, Small
Number Examined:
0
0
0
0
0
0
Bacteria (bacilli)
-
-
-
-
-
-
Average Severity:
-
-
-
-
-
-
Inflammation, Suppurative
-
-
-
-
-
-
Average Severity:
-
-
-
-
-
-
Lung
Number Examined:
5
5
5
3
1
0
Bacteria (bacilli)
0
0
1
1
0
-
Average Severity:
0.0
0.0
0.2
0.3
0.0
-
Inflammation, Nonsuppurative
0
0
0
3
0
-
Average Severity:
0.0
0.0
0.0
1.0
0.0
-
Inflammation, Suppurative
0
3
4
0
1
-
Average Severity:
0.0
0.6
1.2
0.0
1.0
-
Multi-Nucleated Giant Cells
2
2
2
1
0
-
Average Severity:
0.4
0.4
0.8
0.7
0.0
-
Perivascular Eosinophils
0
0
1
1
0
-
Average Severity:
0.0
0.0
0.6
0.3
0.0
-
* Irradiated spores
Group Legend: 1=100 X LDS0; 2=100 CFU; 3=1,000 CFU; 4=10,000 CFU; 5=100,000 CFU; 6=100 x LDS0
W-9
-------
Table 3. Incidence Summary of Microscopic Nonneoplastic Graded Observations with Average Severity - Day 21 (Continued)
Tissue/Observation
Group:
Number Observed Per Group
Lymph Node, Bronchial
Number Examined:
0
0
0
0
0
0
Bacteria (bacilli)
Average Severity:
-
-
-
-
-
-
Fibrin
Average Severity:
~
~
~
~
~
~
Hemorrhage
Average Severity:
~
~
~
~
~
~
Necrosis, Lymphoid
-
-
-
-
-
-
Average Severity:
-
-
-
-
-
-
Lymph Node, Mediastinal
Number Examined:
0
0
0
0
0
0
Bacteria (bacilli)
Average Severity:
-
-
-
-
-
-
Fibrin
Average Severity:
~
~
~
~
~
~
Hemorrhage
Average Severity:
~
~
~
~
~
~
Histiocytosis
Average Severity:
~
~
~
~
~
~
Necrosis, Lymphoid
Average Severity:
~
~
~
~
~
~
Necrosis, Vascular
Average Severity:
~
~
~
~
~
~
* Irradiated spores
Group Legend: 1=100 X LDS0; 2=100 CFU; 3=1,000 CFU; 4=10,000 CFU; 5=100,000 CFU; 6=100 x LDS0
W-10
-------
Table 3. Incidence Summary of Microscopic Nonneoplastic Graded Observations with Average Severity Day 21 (Continued)
Tissue/Observation
Group:
Number Observed Per Group
Skin
Number Examined:
0
0
0
1
0
0
Bacteria (bacilli)
Average Severity:
-
-
-
0
0.0
-
-
Hemorrhage
Average Severity:
~
~
~
0
0.0
~
~
Inflammation, Suppurative
Average Severity:
~
~
~
0
0.0
~
~
Necrosis
-
-
-
1
-
-
Average Severity:
-
-
-
3.0
-
-
Thymus
Number Examined:
0
0
0
0
0
0
Atrophy, Lymphoid
Average Severity:
-
-
-
-
-
-
Bacteria
Average Severity:
~
~
~
~
~
~
Edema
-
-
-
-
-
-
Average Severity:
* Irradiated spores
Group Legend: 1=100 X LD50; 2=100 CFU; 3=1,000 CFU; 4=10,000 CFU; 5=100,000 CFU; 6=100 x LD50
W-ll
-------
Table 4. Incidence Summary of Microscopic Nonneoplastic Graded Observations with Average Severity - Unscheduled Death
Tissue/Observation
Group:
Number Observed Per Group
Brain
Number Examined:
0 0 0
0
0
1
Bacteria
.
-
-
1
Average Severity:
-
-
-
1.0
Hemorrhage
-
-
-
1
Average Severity:
-
-
-
2.0
Inflammation, Suppurative
-
-
-
1
Average Severity:
-
-
-
3.0
Intestine, Small
Number Examined:
0 0 0
0
0
1
Bacteria (bacilli)
-
-
-
1
Average Severity:
-
-
-
2.0
Inflammation, Suppurative
-
-
-
1
Average Severity:
-
-
-
2.0
Lung
Number Examined:
0 0 0
2
4
5
Bacteria (bacilli)
-
1
3
5
Average Severity:
-
1.5
1.8
1.8
Inflammation, Nonsuppurative
-
0
0
0
Average Severity:
-
0.0
0.0
0.0
Inflammation, Suppurative
-
2
4
5
Average Severity:
-
1.5
2.0
2.4
Multi-Nucleated Giant Cells
.
0
4
2
Average Severity:
-
0.0
1.8
0.6
Perivascular Eosinophils
-
0
0
0
Average Severity:
.
0.0
0.0
0.0
* Irradiated spores
Group Legend: 1=100 X LD50; 2=100 CFU; 3=1,000 CFU; 4=
=10,000 CFU; 5=100,000 CFU; 6=100 xLD50
W-12
-------
Table 4. Incidence Summary of Microscopic Nonneoplastic Graded Observations with Average Severity - Unscheduled Death
(Continued)
Tissue/Observation
Group:
Number Observed Per Group
Lymph Node, Bronchial
Number Examined: 0 0 0
0
0
1
Bacteria (bacilli)
-
-
-
1
Average Severity: ...
-
-
1.0
Fibrin
.
-
-
1
Average Severity: ...
-
-
1.0
Hemorrhage
-
-
-
1
Average Severity: ...
-
-
2.0
Necrosis, Lymphoid
-
-
-
1
Average Severity: ...
-
-
2.0
Lymph Node, Mediastinal
Number Examined: 0 0 0
0
0
2
Bacteria (bacilli)
-
-
-
2
Average Severity: ...
-
-
2.5
Fibrin
.
-
-
2
Average Severity: ...
-
-
2.0
Hemorrhage
-
-
-
1
Average Severity: ...
-
-
1.5
Histiocytosis
-
-
-
1
Average Severity: ...
-
-
1.0
Necrosis, Lymphoid
-
-
-
2
Average Severity: ...
-
-
3.0
Necrosis, Vascular
.
-
-
1
Average Severity: ...
-
-
1.0
*Irradiated spores
Group Legend: 1=100 X LD50; 2=
=100 CFU; 3=1,000 CFU; 4=10,000 CFU; 5=100,000 CFU; 6=100 xLD50
W-13
-------
Table 4. Incidence Summary of Microscopic Nonneoplastic Graded Observations with Average Severity
- Unscheduled Death
(Continued)
Number Observed Per Group
Tissue/Observation
Group:
1*
2
3
4
5
6
Skin
Number Examined:
0
0
0
0
1
0
Bacteria (bacilli)
-
-
-
-
1
-
Average Severity:
-
-
-
-
3.0
-
Hemorrhage
-
-
-
-
1
-
Average Severity:
-
-
-
-
2.0
-
Inflammation, Suppurative
-
-
-
-
1
-
Average Severity:
-
-
-
-
2.0
-
Necrosis
-
-
-
-
1
-
Average Severity:
-
-
-
-
2.0
-
Thymus
Number Examined:
0
0
0
0
1
1
Atrophy, Lymphoid
-
-
-
-
1
1
Average Severity:
-
-
-
-
3.0
2.0
Bacteria
-
-
-
-
1
0
Average Severity:
-
-
-
-
2.0
0.0
Edema
-
-
-
-
1
1
Average Severity:
-
-
-
-
2.0
2.0
*Irradiated spores
Group Legend: 1=100 x LD50; 2=100 CFU; 3=1,000 CFU; 4=10,000 CFU; 5=100,000 CFU; 6=100 xLD50
W-14
-------
Table 5. Summary of Individual Gross and Microscopic Observations
Group
Number
Animal Number/
Death Status
Gross Findings
Microscopic Findings
1
100 LD50
(Irradiated)
101 (L23220)/FS
Lung: Unremarkable
102 (L23216)/FS
Lung: Unremarkable
103 (L23218)/FS
Lung: Multi-nucleated giant cells, minimal
104 (L23223)/FS
Lung: Unremarkable
105 (L23222)/FS
Lung: Multi-nucleated giant cells, minimal
2
100 CFU
201 (L23215)/FS
Lung: Inflammation, suppurative, minimal
Lung: Multi-nucleated giant cells, minimal
202 (L23206)/FS
Lung: Inflammation, suppurative, minimal
203 (L23210)/FS
Lung: Unremarkable
204 (L23219)/FS
Lung: Inflammation, suppurative, minimal
Lung: Multi-nucleated giant cells, minimal
205 (L23211)/FS
Lung: Unremarkable
3
1000 CFU
301 (L23217)/FS
Lung: Inflammation, suppurative, moderate
Lung: Multi-nucleated giant cells, moderate
Lung: Perivascular eosinophils, moderate
302 (L23230)/FS
Lung: Inflammation, suppurative, minimal
303 (L23228)/FS
Lung: Inflammation, suppurative, minimal
Lung: Multi-nucleated giant cells, minimal
304 (L23227)/FS
Lung: Bacteria (bacilli), minimal
305 (L23229)/FS
Lung: Inflammation, suppurative, minimal
FD = Found Dead, FS = Final Phase Sacrifice
W-15
-------
Table 5. Summary of Individual Gross and Microscopic Observations (Continued)
Group
Number
Animal Number/
Death Status
Gross Findings
Microscopic Findings
401 L23235)/FD
Lung: Inflammation, suppurative, minimal
402 (L23205)/FS
Lung: Inflammation, nonsuppurative, minimal
Lung: Perivascular eosinophils, minimal
4
10,000
CFU
403 (L23225)/FD
Cavity, Abdominal: Fluid, red, -30 mL.
Cavity, Thoracic: Fluid, red, -15 mL.
Lung: Inflammation, suppurative, mild
Lung: Bacteria (bacilli), moderate
404 (L23231)/FS
Skin: Crust(s), hindlimb, red, left hind limb,
20 mm x 20 mm.
Lung: Inflammation, nonsuppurative, minimal
Skin: Necrosis, moderate
405 (L23207)/FS
Lung: Multi-nucleated giant cells, mild
Lung: Bacteria (bacilli), minimal
Lung: Inflammation, nonsuppurative, minimal
501 (L23201)/FD
Cavity, Thoracic: Fluid, red, -15 mL.
Lung: Inflammation, suppurative, mild
Lung: Multi-nucleated giant cells, mild
Lung: Bacteria (bacilli), mild
5
100,000
CFU
502 (L23234)/FD
Cavity, Thoracic: Fluid, red, -10 mL.
Skin: Crust(s), hindlimb, dark, left hindlimb,
30 mm 5 mm.
Skin: Crust(s), dark, back, 5 mm 5 mm.
Skin: Fluid, abdominal, clear, ventral abdomen,
-8 mL.
Thymus: Fluid, clear, -8 mL.
Lung: Inflammation, suppurative, moderate
Lung: Multi-nucleated giant cells, mild.
Lung: Bacteria (bacilli), moderate
Skin: Necrosis, mild
Skin: Bacteria (bacilli), moderate
Skin: Inflammation, suppurative, mild
Skin: Hemorrhage, mild
Thymus: Atrophy, lymphoid, moderate
Thymus: Edema, mild
Thymus: Bacteria, mild
503 (L23212)/FS
Lung: Inflammation, suppurative, minimal
FD = Found Dead, FS = Final Phase Sacrifice
W-16
-------
Table 5. Summary of Individual Gross and Microscopic Observations (Continued)
Group
Number
Animal Number/
Death Status
Gross Findings
Microscopic Findings
504 (L23200)/FD
Cavity, Thoracic: Fluid, red, -20 mL.
Lung: Inflammation, suppurative, minimal
Lung: Multi-nucleated giant cells, minimal
505 (L23214)/FD
Lung: Inflammation, suppurative, mild
Lung: Multi-nucleated giant cells, mild
Lung: Bacteria (bacilli), mild
601 (L23204)/FD
Cavity, Thoracic: Fluid, red, -15 mL.
Lung: Inflammation, suppurative, mild
Lung: Multi-nucleated giant cells, mild
Lung: Bacteria (bacilli), mild
602 (L23203)/FD
Lung: Inflammation, suppurative, mild
Lung: Multi-nucleated giant cells, mild
Lung: Bacteria (bacilli), mild
6
100 LDjo
603 (L23232)/FD
Brain: Discoloration(s), meninges, diffuse, red,
affects all lobes.
Lymph Node, Bronchial: Enlarged, dark, 3x.
Lymph Node, Mediastinal: Enlarged, dark, 3x.
Brain: Inflammation, suppurative, moderate
Brain: Hemorrhage, mild
Brain: Bacteria, minimal
Lung: Inflammation, suppurative, moderate
Lung: Bacteria (bacilli), minimal
Lymph Node, Bronchial:
Fibrin, minimal
Hemorrhage, mild
Necrosis, lymphoid, mild
Bacteria (bacilli), minimal
Lymph Node, Mediastinal:
Fibrin, minimal
Histiocytosis, mild
Necrosis, lymphoid, mild
Necrosis, vascular, mild
Bacteria (bacilli), minimal
Thymus: Atrophy, lymphoid, mild
Thymus: Edema, mild
FD = Found Dead, FS = Final Phase Sacrifice
W-17
-------
Table 5. Summary of Individual Gross and Microscopic Observations (Continued)
Group
Number
Animal Number/
Death Status
Gross Findings
Microscopic Findings
604 (L23221)/FD
Cavity, Abdominal: Fluid, red, -60 mL.
Intestine, Small: Fluid, jejunum, green, -50
mL. Abdomen was distended.
Intestine, Small:
Inflammation, suppurative, mild
Bacteria (bacilli), mild
Lung: Inflammation, suppurative, mild
Lung: Bacteria (bacilli), moderate
605 (L23213)/FD
Lymph Node, Mediastinal: Enlarged, dark, 3x.
Lung: Inflammation, suppurative, mild
Lung: Bacteria (bacilli), minimal
Lymph Node Mediastinal:
Fibrin, moderate
Hemorrhage, moderate
Necrosis, lymphoid, marked
Bacteria (bacilli), marked
FD = Found Dead, FS = Final Phase Sacrifice
Blank Space = No gross lesions observed on tissue
W-18
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Table 6. Individual Gross and Microscopic Observations
Animal ID: 101 (L23220)
Group: 100xLD50*
Day of Death: 21 (Final Phase Sacrifice)
Tissue Gross Observation(s)
Microscopic Observation(s)
Lung No gross lesions observed on tissue.
Unremarkable.
Animal ID: 102 (L23216)
Group: lOOxLDso*
Day of Death: 21 (Final Phase Sacrifice)
Tissue Gross Observation(s)
Microscopic Observation(s)
Lung No gross lesions observed on tissue.
Unremarkable.
Animal ID: 103 (L23218)
Group: lOOxLDso*
Day of Death: 21 (Final Phase Sacrifice)
Tissue Gross Observation(s)
Microscopic Observation(s)
Lung No gross lesions observed on tissue.
Multi-nucleated giant cells, minimal.
Animal ID: 104 (L23223)
Group: lOOxLDso*
Day of Death: 21 (Final Phase Sacrifice)
Tissue Gross Observation(s)
Microscopic Observation(s)
Lung No gross lesions observed on tissue.
Unremarkable.
Animal ID: 105 (L23222) Group: 100 x LD50*
Day of Death: 21 (Final Phase Sacrifice)
Tissue Gross Observation(s)
Microscopic Observation(s)
Lung No gross lesions observed on tissue.
Multi-nucleated giant cells, minimal.
Animal ID: 201 (L23215)
Group: 100 CFU
Day of Death: 21 (Final Phase Sacrifice)
Tissue Gross Observation(s)
Microscopic Observation(s)
Lung No gross lesions observed on tissue.
Inflammation, suppurative, minimal.
Multi-nucleated giant cells, minimal.
* Irradiated spores.
W-19
-------
Table 6. Individual Gross and Microscopic Observations (Continued)
Animal ID: 202 (L23206)
Day of Death: 21 (Final Phase Sacrifice)
Group: 100 CFU
Tissue Gross Observation(s)
Microscopic Observation(s)
Lung No gross lesions observed on tissue.
Inflammation, suppurative, minimal.
Animal ID: 203 (L23210)
Day of Death: 21 (Final Phase Sacrifice)
Group: 100 CFU
Tissue Gross Observation(s)
Microscopic Observation(s)
Lung No gross lesions observed on tissue.
Unremarkable.
Animal ID: 205 (L23211)
Day of Death: 21 (Final Phase Sacrifice)
Group: 100 CFU
Tissue Gross Observation(s)
Microscopic Observation(s)
Lung No gross lesions observed on tissue.
Inflammation, suppurative, minimal.
Multi-nucleated giant cells, minimal.
Animal ID: 301 (L23217)
Day of Death: 21 (Final Phase Sacrifice)
Group: 1,000 CFU
Tissue Gross Observation(s)
Microscopic Observation(s)
Lung No gross lesions observed on tissue.
Inflammation, suppurative, moderate.
Multi-nucleated giant cells, moderate.
Perivascular eosinophils, moderate.
Animal ID: 302 (L23230)
Day of Death: 21 (Final Phase Sacrifice)
Group: 1,000 CFU
Tissue Gross Observation(s)
Microscopic Observation(s)
Lung No gross lesions observed on tissue. Inflammation, suppurative, minimal.
W-20
-------
Table 6. Individual Gross and Microscopic Observations (Continued)
Animal ID: 303 (L23228)
Day of Death: 21 (Final Phase Sacrifice)
Group: 1,000 CFU
Tissue Gross Observation(s)
Microscopic Observation(s)
Lung No gross lesions observed on tissue.
Inflammation, suppurative, minimal.
Multi-nucleated giant cells, minimal.
Animal ID: 304 (L23227)
Day of Death: 21 (Final Phase Sacrifice)
Group: 1,000 CFU
Tissue Gross Observation(s)
Microscopic Observation(s)
Lung No gross lesions observed on tissue.
Bacteria (bacilli), minimal.
Animal ID: 305 (L23229)
Day of Death: 21 (Final Phase Sacrifice)
Group: 1,000 CFU
Tissue Gross Observation(s)
Microscopic Observation(s)
Lung No gross lesions observed on tissue.
Inflammation, suppurative, minimal.
Animal ID: 402 (L23205) Group: 10,000 CFU
Day of Death: 21 (Final Phase Sacrifice)
Tissue
Gross Observation(s)
Microscopic Observation(s)
Lung
No gross lesions observed on tissue.
Inflammation, nonsuppurative, minimal.
Perivascular eosinophils, minimal.
Animal ID: 403 (L23225)
Day of Death: 4 (Found Dead)
Group: 10,000 CFU
Tissue
Gross Observation(s)
Microscopic Observation(s)
Cavity, Abdominal
Fluid, red, Gl/ Gl= -30 mL; no section
taken.
Cavity, Thoracic
Fluid, red, G2/ G2= -15 mL; no section
taken.
Lung
No gross lesions observed on tissue.
Inflammation, suppurative, mild.
Bacteria (bacilli), moderate.
W-21
-------
Table 6. Individual Gross and Microscopic Observations (Continued)
Animal ID: 404(L23231)
Day of Death: 21 (Final Phase Sacrifice)
Group: 10,000 CFU
Tissue Gross Observation(s)
Microscopic Observation(s)
Lung No gross lesions observed on tissue.
Inflammation, nonsuppurative, minimal.
Skin Crust(s), hindlimb, red, Gl/ Gl=left hind Necrosis, moderate.
limb; 20mm x 20mm.
Note: Gl = necrosis.
Animal ID: 405 (L23207)
Day of Death: 21 (Final Phase Sacrifice)
Group: 10,000 CFU
Tissue Gross Observation(s)
Microscopic Observation(s)
Lung No gross lesions observed on tissue.
Multi-nucleated giant cells, mild.
Bacteria (bacilli), minimal.
Inflammation, nonsuppurative, minimal.
Animal ID: 501 (L23201)
Day of Death: 4 (Found Dead)
Group: 100,000 CFU
Tissue Gross Observation(s)
Microscopic Observation(s)
Cavity, Thoracic Fluid, red, Gl/ Gl= ~15mL; no sample
taken.
Lung No gross lesions observed on tissue.
Inflammation, suppurative, mild.
Multi-nucleated giant cells, mild.
Bacteria (bacilli), mild.
"G" numbers are internal tracking numbers used to correlate gross and microscopic lesions (e.g., Gl, G2, etc.).
W-22
-------
Table 6. Individual Gross and Microscopic Observations (Continued)
Animal ID: 502 (L23234)
Day of Death: 6 (Found Dead)
Group: 100,000 CFU
Tissue Gross Observation(s)
Microscopic Observation(s)
Cavity, Thoracic Fluid, red, G5/ G5= -10 mL; no sample
taken.
Lung No gross lesions observed on tissue.
Inflammation, suppurative, moderate.
Multi-nucleated giant cells, mild.
Bacteria (bacilli), moderate.
Skin Crust(s), hindlimb, dark, Gl/ Gl=left
Necrosis, mild.
hindlimb; 30mmx5mm.
Bacteria (bacilli), moderate.
Crust(s), dark, G2/ G2=back; 5mmx5mm. Inflammation, suppurative, mild.
Fluid, abdominal, clear, G3/ G3=ventral Hemorrhage, mild.
abdomen; ~8 mL.
Note: G1-G3 = necrosis (escar formation).
Thymus Fluid, clear, G4/ G4= ~8 mL.
Atrophy, lymphoid, moderate.
Edema, mild.
Bacteria, mild.
Note: G4 = edema.
Animal ID: 503 (L23212)
Day of Death: 21 (Final Phase Sacrifice)
Group: 100,000 CFU
Tissue Gross Observation(s)
Microscopic Observation(s)
Lung No gross lesions observed on tissue.
Inflammation, suppurative, minimal.
Animal ID: 504 (L23200)
Day of Death: 3 (Found Dead)
Group: 100,000 CFU
Tissue Gross Observation(s)
Microscopic Observation(s)
Cavity, Thoracic Fluid, red, Gl/ Gl= -20 mL; no section
taken.
Lung No gross lesions observed on tissue. Inflammation, suppurative, minimal.
Multi-nucleated giant cells, minimal.
W-23
-------
Table 6. Individual Gross and Microscopic Observations (Continued)
Animal ID: 505 (L23214)
Day of Death: 6 (Found Dead)
Group: 100,000 CFU
Tissue
Gross Observation(s)
Microscopic Observation(s)
Lung
No gross lesions observed on tissue.
Inflammation, suppurative, mild.
Multi-nucleated giant cells, mild.
Bacteria (bacilli), mild.
Animal ID: 601 (L23204)
Day of Death: 4 (Found Dead)
Group: 100 x LD50
Tissue
Gross Observation(s)
Microscopic Observation(s)
Cavity, Thoracic
Fluid, red, Gl/ Gl= ~15mL; no sample
taken.
Lung
No gross lesions observed on tissue.
Inflammation, suppurative, mild.
Multi-nucleated giant cells, mild.
Bacteria (bacilli), mild.
Animal ID: 602 (L23203)
Day of Death: 5 (Found Dead)
Group: 100 x LD50
Tissue
Gross Observation(s)
Microscopic Observation(s)
Lung
No gross lesions observed on tissue.
Inflammation, suppurative, moderate.
Multi-nucleated giant cells, minimal.
Bacteria (bacilli), mild.
"G" numbers are internal tracking numbers used to correlate gross and microscopic lesions (e.g., Gl, G2, etc.).
W-24
-------
Table 6. Individual Gross and Microscopic Observations (Continued)
Animal ID: 603 (L23232)
Day of Death: 4 (Found Dead)
Group: 100 x LD50
Tissue
Gross Observation(s)
Microscopic Observation(s)
Brain
Discoloration(s), meninges, diffuse, red,
G3/ G3=affects all lobes.
Inflammation, suppurative, moderate.
Hemorrhage, mild.
Bacteria, minimal.
Note: G3 = hemorrhage.
Lung
No gross lesions observed on tissue.
Inflammation, suppurative, moderate.
Bacteria (bacilli), minimal.
Lymph Node, Bronchial
Enlarged, dark, Gl/ Gl=3x.
Fibrin, minimal.
Hemorrhage, mild.
Necrosis, lymphoid, mild.
Bacteria (bacilli), minimal.
Note: Gl = necrosis and hemorrhage.
Lymph Node, Mediastinal
Enlarged, dark, G2/ G2=3x.
Fibrin, minimal.
Histiocytosis, mild.
Necrosis, lymphoid, mild.
Necrosis, vascular, mild.
Bacteria (bacilli), minimal.
Note: G2 = necrosis and hemorrhage.
Thymus
No gross lesions observed on tissue.
Atrophy, lymphoid, mild.
Edema, mild.
W-25
-------
Table 6. Individual Gross and Microscopic Observations (Continued)
Animal ID: 604 (L23221)
Day of Death: 2 (Found Dead)
Group: 100 x LD50
Tissue
Gross Observation(s)
Microscopic Observation(s)
Cavity, Abdominal
Fluid, red, Gl/ Gl= -60 mL; no section
taken.
Intestine, Small
Fluid, jejunum, green, G2/ G2= -50 mL;
abdomen was distended due to G2.
Inflammation, suppurative, mild.
Bacteria (bacilli), mild.
Note: G2 = inflammation, suppurative.
Lung
No gross lesions observed on tissue.
Inflammation, suppurative, mild.
Bacteria (bacilli), moderate.
Animal ID: 605 (L23213)
Day of Death: 3 (Found Dead)
Group: 100 x LD50
Tissue
Gross Observation(s)
Microscopic Observation(s)
Lung
No gross lesions observed on tissue.
Inflammation, suppurative, mild.
Bacteria (bacilli), minimal.
Lymph Node, Mediastinal
Enlarged, dark, Gl/ Gl=3x.
Fibrin, moderate.
Hemorrhage, moderate.
Necrosis, lymphoid, marked.
Bacteria (bacilli), marked.
Note: Gl = necrosis and hemorrhage.
"G" numbers are internal tracking numbers used to correlate gross and microscopic lesions (e.g., Gl, G2, etc.).
W-26
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4.0 CONCLUSIONS
Exposure to a single dose of B. anthracis resulted in 0, 0, 0, 40, 80, and 100 % mortality in the
negative control, 100 CFU, 1,000 CFU, 10,000 CFU, 100,000 CFU, and 100 x LD50 treatment groups,
respectively. Death in these rabbits was attributed to B. anthracis. Gross and microscopic lesions were
consistent with inhalation anthrax and generally increased in severity (see Individual Gross and
Microscopic Observations in Table 5 and Table 6) as the dose increased. The most severe microscopic
lesions occurred in the 100 x LD50 treatment group. Inflammation was generally more suppurative
(versus nonsuppurative) in tissues where bacteria were present. Multinucleated giant cells were present
in all groups, but occurred with greater frequency and severity in treated rabbits (when compared to
controls). The relationship of these cellular aggregates to anthrax is unclear.
W-27
-------
5.0 REFERENCES
Pahuja, M., Tran, C., Haichoa, W., and Yin, K. 2008. Alveolar macrophage suppression in sepsis is
associated with high mobility group Box 1 Transmigration. Shock 29(6)754-760.
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. J of
Toxicol Sci 34(1)39-52.
Zaucha, G.M., Pitt. L.M., Estep, J., Ivins, B.E., and Friedlander, A.M. 1998. The pathology of
experimental anthrax in rabbits exposed by inhalation and subcutaneous inoculation. Arch Pathol Lab
Med 122(11)982-992.
W-28
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Attachment 1
1-1
-------
Figure 1. Animal 103 (L23218): Lung, Normal. HE. 4X
1-2
-------
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Figure 2. Animal 402 (L23225): Lung, alveoli. Interstitial inflammation and intravascular and
interstitial anthrax bacilli (arrow). HE. 20X
1-3
-------
Figure 3. Animal 601(L23204) -Lung, alveolus. Multinucleated Giant Cells (arrow). HE. 10X
1-4
-------
-------
~
4
4
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Figure 5. Animal 605 (L23213): Lymph node, sinuses: Anthrax bacilli (arrow head). HE. 4X
1-6
-------
Figure 6. Animal 605 (L23213): Lymph node, sinuses: Hemorrhage and Lymphocyte Depletion
HE. 4X
1-7
-------
®53Bv,
Figure 7. Animal 605 (L23213): Lymph node, sinuses: Anthrax bacilli (arrow head). HE. 40X
1-8
-------
4
I
Figure 8. Animal 502 (L23234): Thymus: Lymphocyte depletion (arrow heads). HE. 4X
1-9
-------
&EPA
United States
Environmental Protection
Agency
PRESORTED STANDARD
POSTAGE & FEES PAID
EPA
PERMIT NO. G-35
Office of Research and Development (8101R)
Washington, DC 20460
Official Business
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$300
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