REVISED FINAL REPORT

STUDY TITLE

ACUTE CARDIAC SENSITIZATION

STUDY OF IN BEAGLE DOGS

STUDY NUMBER

WIL-447023

STUDY DIRECTOR

Philip R. Atterson, MS

STUDY INITIATION DATE

20 February 2008

STUDY COMPLETION DATE

30 June 2008

REVISED FINAL REPORT DATE

21 April 2011

PERFORMING LABORATORY

WIL Research Laboratories, LLC

1407 George Road

Ashland, OH 44805-8946

SPONSOR

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TABLE OF CONTENTS

Page

Compliance Statement...............................................................................2

Revised Final Report .................................................................................3

Table Of Contents......................................................................................4

Index Of Tables..........................................................................................6

Index Of Appendices..................................................................................7

1. Summary.....................................................................................................8

1.1. Objective......................................................................................................8

1.2. Study Design................................................................................................8

1.3. Results..........................................................................................................9

1.4. Conclusions..................................................................................................9

2. Introduction..............................................................................................11

2.1. General Study Information ........................................................................11

2.2. Key Study Dates ........................................................................................11

3. Study Design.............................................................................................12

4. Experimental Procedures - Materials And Methods............................13

4.1. Test Article And Vehicle ...........................................................................13

4.1.1. Test Article Identification..........................................................................13

4.1.2. Epinephrine Identification .........................................................................13

4.1.3. Epinephrine Preparation.............................................................................14

4.2. Test System................................................................................................14

4.3. Organization Of Test Groups, Dosage Levels And Treatment

Regimen .....................................................................................................15

4.4. Exposure Methods .....................................................................................17

4.5. Exposure Atmosphere Generation and Monitoring ...................................17

4.6. Animal Receipt And Acclimation/Pretest Period ......................................17

4.7. Immunizations Prior To Arrival.................................................................18

4.8. Animal Housing And Exercise ..................................................................18

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Page

4.9. Diet, Drinking Water And Maintenance....................................................18

4.10. Environmental Conditions .........................................................................19

4.11. Assignment Of Animals To Study Groups ................................................19

5. Parameters Evaluated .............................................................................21

5.1. Clinical Observations And Survival ..........................................................21

5.2. Body Weights.............................................................................................21

5.3. Electrocardiographic Examinations...........................................................21

5.4. Study Termination .....................................................................................24

5.5. Statistical Methods.....................................................................................24

5.6. Data Retention ...........................................................................................24

6. Results And Discussion............................................................................25

6.1. Clinical Observations And Survival ..........................................................25

6.2. Body Weights.............................................................................................25

6.3. Electrocardiographic Examinations...........................................................25

7. Conclusions...............................................................................................27

8. Key Study Personnel And Report Submission......................................28

9. Quality Assurance Unit Statement.........................................................29

9.1. Phases Inspected ........................................................................................29

9.2. Approval ....................................................................................................31

10. References.................................................................................................32

11. Deviations From The Protocol................................................................33

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INDEX OF TABLES

Page

1. Summary Of Clinical Findings: Total Occurrence/No. Of Animals

(Dispositions - Main Study) .......................................................................................35

2. Summary Of Clinical Findings: Total Occurrence/No. Of Animals

(Dispositions - 50,000 PPM)......................................................................................36

3. Summary Of Clinical Findings: Total Occurrence/No. Of Animals

(Detailed Physical Examinations - Main Study)........................................................37

4. Summary Of Clinical Findings: Total Occurrence/No. Of Animals

(Detailed Physical Examinations - 50,000 PPM).......................................................38

5. Summary Of Body Weights [Kg] (Main Study)........................................................39

6. Summary Of Body Weights [Kg] (50,000 PPM) ......................................................40

7. Individual Clinical Observations (Dispositions - Main Study)..................................41

8. Individual Clinical Observations (Dispositions - 50,000 PPM) ................................42

9. Individual Clinical Observations (Detailed Physical Examinations -

Main Study) ................................................................................................................43

10. Individual Clinical Observations (Detailed Physical Examinations -

50,000 PPM)...............................................................................................................44

11. Individual Body Weights [Kg] (Main Study) ............................................................45

12. Individual Body Weights [Kg] (50,000 PPM)...........................................................48

13. Individual Cardiac Sensitization Responses (All Animals).......................................49

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INDEX OF APPENDICES

Page

A. Certificates Of Analysis (Sponsor- or Manufacturer-Provided Data) .......................50

B. Exposure Atmosphere Generation Report .................................................................53

C. Animal Room Environmental Conditions .................................................................68

D. Unscheduled Clinical Observations ...........................................................................73

E. Individual Electrocardiographic Data ........................................................................76

F. Study Protocol ............................................................................................................82

G. Signature Pages From Original Final Report.............................................................99

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1. SUMMARY

1.1. OBJECTIVE

The objective of this study was to evaluate the cardiac sensitization potential of 

during acute inhalation exposures by assessing the response to various concentrations of

the test article in Beagle dogs. Cardiac sensitization in this context refers to the potential

for the test article to increase the sensitivity of the heart to the pharmacological effects of

epinephrine.

1.2. STUDY DESIGN

 was administered via muzzle-only inhalation exposure as acute exposures (gas) at

concentrations of 2.5%, 3.5% and 5.0% (25,000, 35,000 and 50,000 ppm, respectively).

One group of 6 male dogs was used on this study with a minimum of 48 hours between

exposure concentrations for each animal. Each dog served as its own control. Baseline

response to the epinephrine challenge doses were collected 3 days prior to exposure to the

test gas. For the 25,000 and 35,000 ppm groups, animals were administered

predetermined increasing doses of epinephrine (2, 4, 6 and 8 μg/kg) as bolus injections

(approximately 0.1 ml/sec) via an appropriate vein beginning approximately 5 minutes

following exposure to the test article. Epinephrine challenge bolus injections were

administered a minimum of 3 minutes apart or until the electrocardiogram (ECG) of the

animal returned to its normal baseline rhythm. For the 50,000 ppm group, 2 animals

were exposed to the test article; however, due to behavioral responses displayed by the

animals, exposure was terminated and no other animals were exposed at 50,000 ppm.

Only 1 of the animals exposed to 50,000 ppm was administered epinephrine after

exposure, and cardiac data following epinephrine challenge were not evaluated at this

exposure level. Following the final exposure, all dogs were returned to the WIL stock

colony.

The animals were observed twice daily for mortality and moribundity. Detailed physical

examinations were performed and individual body weights were recorded approximately

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weekly during the pretest period, at randomization and on exposure days.

Electrocardiographic data were recorded continuously throughout the pre-exposure

period, exposure to the test gas and throughout administration of the challenge

epinephrine dose (total of approximately 33 minutes).

1.3. RESULTS

There were no test article-related deaths. After exposure to 50,000 ppm 

vocalization during exposure, injected sclera, excessive salivation, tremors, convulsions

and/or reddened gums were noted. After exposure to 35,000 ppm tremors,

injected sclera, reddened ears and excessive salivation and salivation-related findings

were observed.

There were no signs of cardiac sensitization in the animals challenged with epinephrine

after exposure to 25,000 and 35,000 ppm However, the presence of ECG noise

in 2 animals following administration of 35,000 ppm precluded complete

characterization of the potential cardiac sensitization response at this exposure level.

Following the initiation of exposure to 50,000 ppm 2 animals exhibited severe

clinical findings (including tremors and/or convulsions), and only 1 of these animals was

administered epinephrine after exposure; therefore, cardiac data following epinephrine

challenge were not evaluated at 50,000 ppm.

1.4. CONCLUSIONS

Based on the results of this study, inhalation exposure of to beagle dogs, clinical

findings of tremors, injected sclera and excessive salivation were observed at exposure

levels of 35,000 and 50,000 ppm. Vocalization during exposure, reddened gums and

convulsions were also noted following exposure to 50,000 ppm and evidence of

salivation and reddened ears were noted after exposure to 35,000 ppm There

was no evidence of cardiac sensitization following exposure to 25,000 ppm

(2.5%) the cardiac sensitization potential following exposure to 35,000 and

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50,000 ppm (3.5% and 5.0%, respectively) could not be definitively evaluated

due to the presence of tremors during exposure.

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2. INTRODUCTION

2.1. GENERAL STUDY INFORMATION

This report presents the data from “Acute Cardiac Sensitization Study of in

Beagle Dogs”. Due to software spacing constraints, the study title appears as “Cardiac

Sensitization of by Inhalation in Dogs” on the report tables.

The following computer protocols were used for data collection during the study:

Computer Protocol Type of Data Collected

WIL-447023 ........................................... Main study data

WIL-447023V ........................................ 50,000 ppm exposure data

WIL-447023U ........................................ Unscheduled clinical observations

2.2. KEY STUDY DATES

Date(s) Event(s)

14 February 2008.................................... Animal receipt

25 February 2008.................................... Assignment to study groups

26 February 2008.................................... Initiation of epinephrine challenge (initial

epinephrine challenge, no test-article

administration)

29 February 2008.................................... Experimental start date;

Exposure to 25,000 ppm 

(epinephrine challenge)

4 March 2008.......................................... Exposure to 50,000 ppm (limited

epinephrine challenge)

7 March 2008.......................................... Exposure to 35,000 ppm 

(epinephrine challenge)

12 March 2008........................................ Experimental termination date

(animals returned to stock colony)

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3. STUDY DESIGN

Challenge doses of epinephrine

administered beginning 5 minutes after

the start of exposure to test article

to assess cardiac sensitization

Animal receipt

Electrocardiographic data were recorded

continuously throughout the baseline

period, exposure to the test gas and for

3 minutes following administration of

each challenge epinephrine dose (up to

approximately 33 minutes)

Detailed physical examinations conducted

and body weights

recorded prior to each exposure;

clinical observations recorded when

observed

All animals returned to stock

6 males exposed to test article, once with

each applicable treatment, starting

2 minutes after initiation of ECG

Control group (Group 1)

data collection

Acclimation/Pretest ECG screening

Electrocardiographic data were recorded

continuously throughout the baseline

epinephrine response dosing

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4. EXPERIMENTAL PROCEDURES - MATERIALS AND METHODS

4.1. TEST ARTICLE AND VEHICLE

4.1.1. TEST ARTICLE IDENTIFICATION

The test article, was received from , on

21 February 2008, as follows:

Identification

Quantity

Received

Lot no. 40883-50

CAS no. 102687-65-0

[WIL log no. 7877A]

1 cylinder

Gross weight:

4273.9 g

Documentation regarding the purity of the test article is on file with the sponsor and WIL

Research Laboratories, LLC. A Certificate of Analysis for the test article was provided

by the sponsor and is presented in Appendix A. The purity of the test article was

99.978%. The test article was stored at room temperature and was considered stable

under this condition. A reserve sample of the test article (52.4 g) was collected on

26 February 2008, and stored in the Archives of WIL Research Laboratories, LLC.

4.1.2. EPINEPHRINE IDENTIFICATION

The epinephrine used on this study was received from Sigma Aldrich, Inc., St. Louis,

Missouri, on 29 May 2007, as follows:

Identification

Quantity

Received

Physical

Description

(-)-Epinephrine

Lot no. 095K1747

CAS no. 51-43-4

[WIL log no. 7488A]

1 vial

Gross weight:

19.4 g

Off-white

powder

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A Certificate of Analysis for the epinephrine was provided by Sigma Aldrich, Inc. and is

presented in Appendix A. The purity of the epinephrine was 99.6%. The epinephrine

was stored at room temperature and was considered stable under this condition.

4.1.3. EPINEPHRINE PREPARATION

All glassware and utensils were sterilized prior to use and appropriate procedures were

performed within a laminar flow hood. The appropriate amount of epinephrine for the

stock 100 μg/mL formulation was weighed into a tared, weigh vessel, and 0.9% saline for

injection (U.S.P.) (lot no. C727263, exp. date: 1 June 2009 and lot no. C729301, exp.

date: 1 July 2009 manufactured by Baxter Healthcare Corporation, Deerfield, Illinois)

was used to quantitatively transfer the epinephrine into a glass container. The

formulation was mixed with a magnetic stirrer until uniform. The appropriate amount of

0.9% saline for injection (U.S.P.) was then added to the container to bring the

formulation to the calibration mark. The stock epinephrine formulation was sterile

filtered through a 0.22-μm (Durapore) PVDF syringe filter and capped with a septum.

The 80 μg/mL epinephrine formulation was prepared by transferring the appropriate

amount of 0.9% saline for injection (U.S.P.) and the 100 μg/mL formulation into a sterile

vial. The vial was capped with a septum and swirled to ensure mixing. The formulated

concentration was prepared on the day of use and stored on ice.

The 20, 40 and 60 μg/mL epinephrine formulations were prepared by transferring the

appropriate amount of 0.9% saline for injection (U.S.P.) and the 80 μg/mL formulation

for each group into sterile vials. The vials were capped with septa and swirled to ensure

mixing. The formulated concentrations were prepared on the day of use and stored on

ice.

4.2. TEST SYSTEM

Beagle dogs from Ridglan Farms, Mt. Horeb, Wisconsin were used as the test system on

this study. This species and breed of animal is recognized as an appropriate model for

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assessment of potential cardiac sensitization (Mullin et al., 1979; Reinhardt et al., 1971).

The animals were approximately 7 to 9 months old at the initiation of baseline data

collection.

4.3. ORGANIZATION OF TEST GROUPS, DOSAGE LEVELS AND TREATMENT

REGIMEN

For the control exposure, the animals were exposed to room air only. The epinephrine

challenge doses were administered intravenously using an appropriately sized catheter as

a bolus injection into an appropriate vein followed by a 0.2-mL flush of 0.9% saline. The

dose volume was 0.1 mL/kg. The information gathered was used to establish the baseline

response of each dog to the epinephrine challenge doses. Baseline responses to the

epinephrine challenge doses were collected 3 days prior to exposure to the test gas.

For the 2.5%, 3.5% and 5% exposures (25,000, 35,000 and 50,000 ppm, respectively), the

exposure via the muzzle-only exposure apparatus duration to air was approximately

2 minutes, and the exposure duration for the test gas was up to approximately 33 minutes.

The treatment regimen for these groups was as follows:

Time (minutes) Event

0 Start ECG recording (baseline)

2 Test gas initiated

7 Increasing challenge doses of epinephrine

Up to approximately 33 Termination of exposure/ECG

The epinephrine challenge doses were administered intravenously using an appropriately

sized catheter as a bolus injection (approximately 0.1 ml/sec) into an appropriate vein

followed by a 0.2-mL flush of 0.9% saline. The dose volume was 0.1 mL/kg.

The exposure schedule is shown in the following table:

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Date Animal No.

Target Exposure

Concentration (ppm)

Epinephrine Dose(s)

administered (μg/kg)

26 February 2008 4935 0 2, 4, 6, 8

26 February 2008 4936 0 2, 4, 6, 8

26 February 2008 4937 0 2, 4, 6, 8

26 February 2008 4938 0 2, 4, 6, 8

26 February 2008 4941 0 2, 4, 6, 8

26 February 2008 4943 0 2, 4, 6, 8

29 February 2008 4935 25,000 2, 4, 6, 8

29 February 2008 4936 25,000 2, 4, 6, 8

29 February 2008 4937 25,000 2, 4, 6, 8

29 February 2008 4938 25,000 2, 4, 6, 8

29 February 2008 4941 25,000 2, 4, 6, 8

29 February 2008 4943 25,000 2, 4, 6, 8

4 March 2008 4935 50,000 2, 4

4 March 2008 4936 50,000 NA

4 March 2008 4937 50,000 NA

4 March 2008 4938 50,000 NA

4 March 2008 4941 50,000 NA

4 March 2008 4943 50,000 NA

7 March 2008 4935 35,000 2, 4

7 March 2008 4936 35,000 2, 4, 6, 8

7 March 2008 4937 35,000 2, 4, 6, 8

7 March 2008 4938 35,000 2

7 March 2008 4941 35,000 2, 4, 6, 8

7 March 2008 4943 35,000 2, 4, 6, 8

NA = Animal did not receive the epinephrine doses.

The selected route of administration for this study was inhalation exposure because of the

volatility and intended usage of the test article and because this was the most likely route

of exposure for the general population. The muzzle-only method of exposure was

selected to achieve precise control of concentration levels and durations.

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4.4. EXPOSURE METHODS

All animals were exposed to the test article using a muzzle-only exposure apparatus

connected to a Tedlar® bag to supply the target concentration of the test article vapor and

a minimum oxygen content of 19%. The control atmosphere (the first 2 minutes of each

exposure) was filtered air supplied from an in-house source. Each dog was comfortably

restrained using a canvas sling with the appropriate ECG (limb) leads attached. A

detailed description of the exposure methods is presented in Appendix B.

4.5. EXPOSURE ATMOSPHERE GENERATION AND MONITORING

Exposures were conducted using a muzzle only system consisting of a mask connected to

3-way valve connected to a 200-L Tedlar® gas bag of the appropriate test atmosphere.

Prior to each exposure, samples were analyzed from each bag using a gas

chromatographic method to assure that the atmosphere was at the target concentration.

The addition of oxygen was not required since oxygen content of all exposure bags was

above 19%. From the 200-L Tedlar® bag, test article was directed to a 3-way valve.

During the pre-treatment acclimation period, animals received room air directed through

a 3-way valve. At the start of exposure, the 3-way valve was turned to the 200-L bag

position for exposure to the test article. Feed and water were withheld during exposure

periods. A detailed description of the generation methods is presented in Appendix B.

4.6. ANIMAL RECEIPT AND ACCLIMATION/PRETEST PERIOD

Ten naïve male beagle dogs were received from Ridglan Farms, Mt. Horeb, Wisconsin,

on 14 February 2008. Each animal was identified uniquely with an ear tattoo by the

supplier. All dogs were approximately 7 to 9 months old upon receipt. Each animal was

examined by a qualified technician and a veterinarian upon receipt and weighed the

following day. Animals considered suitable for the study were housed for an

approximate 11-day acclimation and pretest period. The animals were observed twice

daily for mortality and changes in general appearance or behavior. Fecal samples were

collected during the acclimation period and checked for parasites; none were present.

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Each dog was conditioned to the exposure/restraint apparatus for a minimum of

4 separate acclimation sessions by placing it in the exposure/restraint apparatus for up to

a 45-minute period in order to acclimate it to test article exposure conditions.

Prior to being placed on study, individual ECG data were collected and evaluated for

abnormalities. Individual body weights were recorded and detailed physical

examinations were performed prior to randomization.

4.7. IMMUNIZATIONS PRIOR TO ARRIVAL

All animals were immunized prior to arrival at WIL Research Laboratories, LLC. The

immunizations included rabies, Bordetella, distemper, Leptospira spp, parvovirus,

parainfluenza, hepatitis and oral papilloma virus.

4.8. ANIMAL HOUSING AND EXERCISE

The animals were housed individually in clean, stainless steel cages elevated above

stainless steel cage pans that were cleaned daily during the acclimation period and

throughout the study. All animals were maintained in accordance with the Guide for the

Care and Use of Laboratory Animals (National Research Council, 1996). The animals

were allowed regular opportunity for exercise and social interaction in accordance with

the standard operating procedures at WIL Research Laboratories, LLC, and the Animal

Welfare Act (9 CFR Part 3). The animal facilities at WIL Research Laboratories, LLC,

are accredited by the Association for Assessment and Accreditation of Laboratory

Animal Care International (AAALAC International).

4.9. DIET, DRINKING WATER AND MAINTENANCE

Approximately 400 g of PMI Nutrition International, LLC, Certified Canine

LabDiet

 5007, were offered 1 to 2 hours per day, and were fed prior to baseline data

collection on exposure days. The diet utilized at WIL Research Laboratories, LLC, is a

certified feed with appropriate analyses for potential contaminants performed and

provided by the manufacturer. Drinking water, delivered by an automatic watering

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system, was provided ad libitum throughout the study period, except during acclimation

sessions and exposure periods. Municipal water supplying the facility is sampled for

contaminants according to the standard operating procedures. The results of the diet and

water analyses are maintained at WIL Research Laboratories, LLC. No contaminants

were present in animal feed or water at concentrations sufficient to interfere with the

objectives of this study.

4.10. ENVIRONMENTAL CONDITIONS

All animals were housed throughout the acclimation period and during the study in an

environmentally controlled room (study room B94). The room temperature and humidity

controls were set to maintain daily averages of 68 ± 5°F (20 ± 3°C) and 50 ± 20% relative

humidity. Room temperature and relative humidity were controlled and monitored using

the Metasys® DDC Electronic Environmental control system. These data were recorded

approximately hourly and are summarized in Appendix C. Actual mean daily

temperature ranged from 70.3°F to 70.6°F (21.3°C to 21.4°C) and mean daily relative

humidity ranged from 36.5% to 41.2% during the study. Fluorescent lighting provided

illumination for a 12-hour light (0600 hours to 1800 hours)/12-hour dark photoperiod.

The 12-hour light/12-hour dark photoperiod was interrupted as necessary to allow for the

performance of protocol-specified activities. Air handling units were set to provide a

minimum of 10 fresh air changes per hour.

4.11. ASSIGNMENT OF ANIMALS TO STUDY GROUPS

On 25 February 2008 (4 days prior to the initiation of test article exposures and 1 day

prior to the epinephrine challenge baseline), all available dogs were weighed and

examined in detail for physical abnormalities. These data were collected using the WIL

Toxicology Data Management System (WTDMSTM) and reviewed by the study director.

The animals judged most suitable based on temperament, health and acceptable pretest

ECG data were assigned by a computer program for use on study. The test group (used

for the 25,000, 35,000 and 50,000 ppm exposures) consisted of 6 males. The selected

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animals were approximately 7 to 9 months old at the initiation of exposure; body weights

ranged from 7.4 kg to 10.1 kg on the days of exposure.

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5. PARAMETERS EVALUATED

5.1. CLINICAL OBSERVATIONS AND SURVIVAL

The animals were observed twice daily, once in the morning and once in the afternoon,

for mortality and moribundity.

Detailed physical examinations were conducted on all animals on the morning of each

exposure. A separate computer protocol was used to record any observations noted

outside the above-specified interval. These unscheduled clinical observations are

presented in Appendix D.

5.2. BODY WEIGHTS

Individual body weights were recorded weekly during the pretest period, at the time of

randomization and on the day(s) of exposure.

5.3. ELECTROCARDIOGRAPHIC EXAMINATIONS

Electrocardiograms (ECG) were recorded with an Eclipse electrocardiograph. Modified

leads I, II and III were used for electrocardiogram collection; ECG electrodes were

placed on each limb. For the 25,000 ppm and 35,000 ppm test article exposures, ECGs

were recorded continuously throughout the pre-exposure epinephrine dosing, initiation of

exposure to the test gas, increasing challenge doses of epinephrine and termination of the

exposure period (total of up to approximately 33 minutes). Only 2 animals (nos. 4935

and 4936) were exposed to the 50,000 ppm due to the occurrence of tremors

and/or convulsions during exposure. Only animal no. 4935 received any challenge doses

of epinephrine (2 and 4 μg/kg), but the ECG data could not be evaluated due to artifacts

and noise from the tremors. ECG data were not recorded from animal no. 4936 after

exposure to the test article. Following exposure to 35,000 ppm animal nos. 4938

and 4935 were not challenged with all epinephrine doses due to background noise on the

ECG which precluded complete characterization of cardiac sensitization.

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The points at which epinephrine was administered and when exposure to the test article

was initiated were clearly marked on each ECG recording.

ECG recordings taken during exposure to the test article and after administration of the

challenge epinephrine doses were compared to the ECG recordings made after

administration of the pre-exposure escalating epinephrine doses (Group 1). During the

epinephrine challenges (or immediately following), the following type of criteria

(although not exclusively) were applied in order to determine if sensitization had

occurred:

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Criteria Conclusion

Normal rhythm, or evidence of a typical

pharmacological response to adrenaline, such as

bradycardia with escape beats.

No evidence of cardiac

sensitization.

A single PVC (followed by a junctional beat).

Not considered evidence of

cardiac sensitization, but

may occur with an

adrenaline challenge

without concurrent test gas

exposure.

2 to 5 PVC’s in a 10 second period that are not

confluent and multifocal. Not typically life

threatening.

Not considered evidence of

sensitization.

6 to 10 PVC’s in a 10 second period that may be

multifocal but not uniformly confluent.

Typically quickly resolve and not typically life

threatening

Typically this score is

considered equivocal, but

positive, evidence of

cardiac sensitization and

other factors, (e.g., the

dog’s baseline ECG or the

score obtained at a higher

challenge dose of

adrenaline, etc.) should be

considered in the final

interpretation.

11 or greater PVC’s that are multifocal with

periods of confluency take a relatively long time

to resolve and may occur in burst between

episodes of normal looking beats. Potentially

life threatening.

Considered unequivocal

evidence of cardiac

sensitization

Serious, life threatening events such as

ventricular tachycardia, ventricular fibrillation

or flutter

Considered unequivocal

evidence of cardiac

sensitization

Definitions

PVC = Premature ventricular contraction

Multifocal = PVC’s that originate from different parts of the ventricle and thus

have a different size and shapes in the ECG

Confluency = when a PVC occurs without any apparent baseline and/or normal

appearing beats between two consecutive PVC’s.

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Individual electrocardiographic data are presented in Appendix E.

5.4. STUDY TERMINATION

All animals were returned to the stock colony following the final exposure.

5.5. STATISTICAL METHODS

Each mean was presented with the standard deviation (S.D.) and the number of animals

(N) used to calculate the mean.

5.6. DATA RETENTION

The sponsor has title to all documentation records, raw data, specimens or other work

product generated during the performance of the study. All work product generated by

WIL Research Laboratories, LLC, including raw paper data and specimens, are retained

in the Archives at WIL Research Laboratories, LLC, as specified in the study protocol.

Reserve samples of the test article, pertinent electronic storage media and the original

final report are retained in the Archives at WIL Research Laboratories, LLC, in

compliance with regulatory requirements.

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6. RESULTS AND DISCUSSION

6.1. CLINICAL OBSERVATIONS AND SURVIVAL

Summary Data: Tables 1, 2, 3, 4, Appendix D

Individual Data: Tables 7, 8, 9 , 10, Appendix D

All animals survived to the end of the study. After exposure to 50,000 ppm 

vocalization during exposure, injected sclera and excessive salivation were noted in

animal nos. 4935 and 4936. Tremors and reddened gums were noted for animal no. 4935

after exposure to 50,000 ppm and convulsions were noted for animal no. 4936.

These were the only 2 animals exposed to 50,000 ppm After exposure to 35,000

ppm tremors, injected sclera, reddened ears, excessive salivation and salivationrelated findings (wet clear material and white frothy material around the mouth) were

observed. These findings were considered test article-related due to the number of

incidences.

There were no other test article-related clinical findings. All other observations were

limited to single animals, were not noted in a dose-related manner and/or were common

findings for laboratory dogs of this age and breed.

6.2. BODY WEIGHTS

Summary Data: Tables 5, 6

Individual Data: Table 11, 12

Body weights were collected for determination of doses only and were not analyzed for

test article effects. Body weights ranged from 7.4 to 10.1 kg on the days of dosing.

6.3. ELECTROCARDIOGRAPHIC EXAMINATIONS

Individual Data: Table 13, Appendix E

After exposure to 25,000 and 35,000 ppm there were no signs of cardiac

sensitization. Animal nos. 4938 and 4935 were not challenged with epinephrine

following exposure to 35,000 ppm background noise on the ECG precluded

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complete characterization of the potential cardiac sensitization response following

administration of 35,000 ppm 

Following the initiation of exposure to 50,000 ppm animal no. 4935 exhibited

tremors and animal no. 4936 exhibited convulsions. Only animal no. 4935 received any

challenge doses of epinephrine (2 and 4 μg/kg), but the ECG data could not be evaluated

due to artifacts and noise from the tremors. Therefore, the potential cardiac sensitization

response following administration of 50,000 ppm could not be evaluated.

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7. CONCLUSIONS

Based on the results of this study, inhalation exposure of to beagle dogs, clinical

findings of tremors, injected sclera and excessive salivation were observed at exposure

levels of 35,000 and 50,000 ppm. Vocalization during exposure, reddened gums and

convulsions were also noted following exposure to 50,000 ppm and evidence of

salivation and reddened ears were noted after exposure to 35,000 ppm There

was no evidence of cardiac sensitization following exposure to 25,000 ppm (2.5%)

 the cardiac sensitization potential following exposure to 35,000 and 50,000 ppm

(3.5% and 5.0%, respectively) could not be definitively evaluated due to the

presence of tremors during exposure.

27 of 104
 

 

 

 

 

 

 

 

 

 
WIL-447023

9. QUALITY ASSURANCE UNIT STATEMENT

9.1. PHASES INSPECTED

Date(s) of

Inspection(s) Phase Inspected

Date(s)

Findings

Reported to

Study Director

Date(s)

Findings

Reported to

Management Auditor(s)

29-Feb-2008 Test Article Exposure 29-Feb-2008 26-Mar-2008 S.Power

29-Feb-2008 Test Article Exposure

(Supplemental) 29-Feb-2008 26-Mar-2008 T.DeVan Booth

S.Power

16-May-2008 Study Records (Rx-1) 16-May-2008 23-Jun-2008 T.DeVan Booth

20-May-2008

21-May-2008 Draft Inhalation Appendix 21-May-2008 23-Jun-2008 T.DeVan Booth

20-May-2008

21-May-2008 Study Records (Ex-1) 21-May-2008 23-Jun-2008 T.DeVan Booth

22-May-2008

05-Jun-2008 Study Records (I-1, I-2, I-3) 05-Jun-2008 27-Jun-2008 T.DeVan Booth

04-Jun-2008

05-Jun-2008 Draft Report w/out Inhalation

Appendix 05-Jun-2008 27-Jun-2008 T.DeVan Booth

19-Apr-2011 Revised Final Report 19 Apr-2011 21-Apr-2011 H.Johnson

This study was inspected in accordance with the United States Environmental Protection

Agency (EPA) Good Laboratory Practice Standards (40 CFR Part 792),

18 September 1989, the standard operating procedures of WIL Research Laboratories,

LLC, and the sponsor's protocol and protocol amendments with the following exceptions.

The data located in Appendix A (Certificates of Analysis) were the responsibility of the

sponsor or manufacturer. Quality Assurance findings, derived from the inspections

during the conduct of the study and from the inspections of the raw data, draft report and

revised final report, are documented and have been reported to the study director. A

status report is submitted to management monthly.

This report accurately reflects the data generated during the study. The methods and

procedures used in the study were those specified in the protocol, its amendments and the

standard operating procedures of WIL Research Laboratories, LLC.

29 of 104
WIL-447023

The raw data, the retention sample(s), if applicable, and the final report will be stored in

the Archives at WIL Research Laboratories, LLC, or another location specified by the

sponsor.

30 of 104
 

 

 

 

 
WIL-447023

10. REFERENCES

Mullin, L., Reinhardt, C. and Hemingway, R. Cardiac Arrhythmias and Blood

Levels Associated with Inhalation of Halon 1301, Am. Ind. Hygiene Assoc. Jour.,

1979; pp.40, 653-358.

National Research Council. Guide for the Care and Use of Laboratory Animals,

Institute of Laboratory Animal Resources, Commission on Life Sciences; National

Academy Press: Washington, DC, 1996.

Reinhardt, C., Azar, Alk Marfield, M., Smith, P. and Mullin, L. Cardiac

Arrhythmias and Aerosol “Sniffing,” Arch. Envir. Health, 1971, pp. 22, 265-279.

32 of 104
WIL-447023

11. DEVIATIONS FROM THE PROTOCOL

This study was conducted in accordance with the protocol and protocol amendments,

except for the following.

• Protocol Section 5.5 states that a physical description of the test article will be

documented by WIL Research Laboratories, LLC. Due to the test article being a

liquefied gas, a physical description could not be determined.

• Protocol Section 5.7 states that approximately 3.0 grams of test article will be

collected as a retention sample. The retention sample collected was 52.4 grams.

• Protocol Section 7.4 states that food will be offered for approximately 1-2 hours

daily. There was no documentation of the animals being fed for 1-2 hours on

non-exposure days.

• Protocol Section 8.2 states that animals will be arbitrarily assigned to the study.

However, the animals were assigned to study via an unstratified straight

randomization.

• Protocol Section 8.5 states that the exposure atmosphere temperature was to be

approximately 22°C. The exposure atmosphere was prepared at room temperature

and exposed at room temperature.

• Protocol Section 9.4 states that body weights will be collected following the

completion of all exposures. However, the body weights were not collected

following the completion of all exposures.

These deviations did not negatively impact the quality or integrity of the data nor the

outcome of the study.

33 of 104
WIL-447023

TABLES 1 - 13

34 of 104
 TABLE 1 (DISPOSITIONS - MAIN STUDY) 

 PROJECT NO.:WIL-447023 CARDIAC SENSITIZATION OF BY INHALATION IN DOGS PAGE 1 

 SPONSOR: SUMMARY OF CLINICAL FINDINGS: TOTAL OCCURRENCE/NO. OF ANIMALS 

 

 ----- M A L E ----- 

 ----------------------------------------------------------------------------------------------------------------------------------- 

 TABLE RANGE: 02-26-08 TO 03-12-08 

 GROUP: 1 2 3 

 ----------------------------------------------------------------------------------------------------------------------------------- 

 

 DISPOSITION 

 -TRANSFERRED TO 25,000 PPM GROUP 6/ 6 0/ 0 0/ 0 

 -TRANSFERRED TO 50,000 PPM GROUP 0/ 0 6/ 6 0/ 0 

 -ANIMAL TRANSFERRED TO WIL STOCK COLONY 0/ 0 0/ 0 6/ 6 

 (WIL-SPDOG08) 

 ----------------------------------------------------------------------------------------------------------------------------------- 

 1- EPINEPHRINE 2- 25,000 PPM 3- 35,000 PPM 

 PCSUv4.07 

 05/22/2008 

 R:04/13/2011 

35 of 104
 TABLE 2 (DISPOSITIONS - 50,000 PPM) 

 PROJECT NO.:WIL-447023V CARDIAC SENSITIZATION OF BY INHALATION IN DOGS PAGE 1 

 SPONSOR: SUMMARY OF CLINICAL FINDINGS: TOTAL OCCURRENCE/NO. OF ANIMALS 

 

 ----- M A L E ----- 

 ----------------------------------------------------------------------------------------------------------------------------------- 

 TABLE RANGE: 03-07-08 

 GROUP: 1 

 ----------------------------------------------------------------------------------------------------------------------------------- 

 

 DISPOSITION 

 -TRANSFERRED TO 35,000 PPM GROUP 6/ 6 

 ----------------------------------------------------------------------------------------------------------------------------------- 

 1- 50,000 PPM 

 PCSUv4.07 

 05/22/2008 

 R:04/13/2011 

36 of 104
 TABLE 3 (DETAILED PHYSICAL EXAMINATIONS - MAIN STUDY) 

 PROJECT NO.:WIL-447023 CARDIAC SENSITIZATION OF BY INHALATION IN DOGS PAGE 1 

 SPONSOR: SUMMARY OF CLINICAL FINDINGS: TOTAL OCCURRENCE/NO. OF ANIMALS 

 

 ----- M A L E ----- 

 ----------------------------------------------------------------------------------------------------------------------------------- 

 TABLE RANGE: 02-26-08 TO 03-12-08 

 GROUP: 1 2 3 

 ----------------------------------------------------------------------------------------------------------------------------------- 

 

 NORMAL 

 -NO SIGNIFICANT CLINICAL OBSERVATIONS 6/ 6 6/ 6 4/ 4 

 

 EYES/EARS/NOSE 

 -REDDENED EAR(S) 0/ 0 0/ 0 1/ 1 

 

 ORAL/DENTAL 

 -EMESIS CONTAINING FOOD 0/ 0 0/ 0 1/ 1 

 

 SPECIAL II 

 -TOENAILS LONG, TRIMMED 0/ 0 0/ 0 1/ 1 

 ----------------------------------------------------------------------------------------------------------------------------------- 

 1- EPINEPHRINE 2- 25,000 PPM 3- 35,000 PPM 

 PCSUv4.07 

 05/22/2008 

 R:04/13/2011 

37 of 104
 TABLE 4 (DETAILED PHYSICAL EXAMINATIONS - 50,000 PPM) 

 PROJECT NO.:WIL-447023V CARDIAC SENSITIZATION OF BY INHALATION IN DOGS PAGE 1 

 SPONSOR: SUMMARY OF CLINICAL FINDINGS: TOTAL OCCURRENCE/NO. OF ANIMALS 

 

 ----- M A L E ----- 

 ----------------------------------------------------------------------------------------------------------------------------------- 

 TABLE RANGE: 03-04-08 

 GROUP: 1 

 ----------------------------------------------------------------------------------------------------------------------------------- 

 

 NORMAL 

 -NO SIGNIFICANT CLINICAL OBSERVATIONS 6/ 6 

 

 BEHAVIOR/CNS 

 -TREMORS, INTERMITTENT 1/ 1 

 -CONVULSIONS, INTERMITTENT 1/ 1 

 -VOCALIZATION DURING DOSING 2/ 2 

 

 EYES/EARS/NOSE 

 -INJECTED SCLERA LEFT EYE 2/ 2 

 -INJECTED SCLERA RIGHT EYE 2/ 2 

 

 ORAL/DENTAL 

 -EXCESSIVE SALIVATION 2/ 2 

 -REDDENED GUMS 1/ 1 

 ----------------------------------------------------------------------------------------------------------------------------------- 

 1- 50,000 PPM 

 PCSUv4.07 

 05/05/2008 

 R:04/13/2011 

38 of 104
 TABLE 5 (MAIN STUDY) 

 PROJECT NO.:WIL-447023 CARDIAC SENSITIZATION OF BY INHALATION IN DOGS PAGE 1 

 SPONSOR: SUMMARY OF BODY WEIGHTS [KG] 

 

 ----------------------------------------------------------------------------------------------------------------------------------- 

 MALES 

 GROUP: EPINEPHRINE 25,000 PPM 35,000 PPM 

 ----------------------------------------------------------------------------------------------------------------------------------- 

 MEAN 8.6 8.6 8.8 

 S.D. 0.99 1.03 1.00 

 N 6 6 6 

 

 ----------------------------------------------------------------------------------------------------------------------------------- 

 PJTBWSUv5.14 

 05/05/2008 

 R:04/13/2011 

39 of 104
 TABLE 6 (50,000 PPM) 

 PROJECT NO.:WIL-447023V CARDIAC SENSITIZATION OF BY INHALATION IN DOGS PAGE 1 

 SPONSOR: SUMMARY OF BODY WEIGHTS [KG] 

 

 ----------------------------------------------------------------------------------------------------------------------------------- 

 MALES 

 GROUP: 50,000 PPM 

 ----------------------------------------------------------------------------------------------------------------------------------- 

 MEAN 8.9 

 S.D. 0.99 

 N 6 

 

 ----------------------------------------------------------------------------------------------------------------------------------- 

 PBFSTv5.28 

 05/05/2008 

 R:04/13/2011 

40 of 104
 TABLE 7 (DISPOSITIONS - MAIN STUDY) 

 PROJECT NO.:WIL-447023 CARDIAC SENSITIZATION OF BY INHALATION IN DOGS PAGE 1 

 SPONSOR: INDIVIDUAL CLINICAL OBSERVATIONS 

 

 TABLE RANGE: 02-26-08 TO 03-12-08 

 ----------------------------------------------------------------------------------------------------------------------------------- 

 ANIMAL SEX GROUP CATEGORY DATE TIME GRADE OBSERVATIONS 

 ----------------------------------------------------------------------------------------------------------------------------------- 

 4935M1 M EPINEPHRINE DISPOSITION 02-29-08 0:00 P TRANSFERRED TO 25,000 PPM GROUP 

 4936M1 M EPINEPHRINE DISPOSITION 02-29-08 0:00 P TRANSFERRED TO 25,000 PPM GROUP 

 4937M1 M EPINEPHRINE DISPOSITION 02-29-08 0:00 P TRANSFERRED TO 25,000 PPM GROUP 

 4938M1 M EPINEPHRINE DISPOSITION 02-29-08 0:00 P TRANSFERRED TO 25,000 PPM GROUP 

 4941M1 M EPINEPHRINE DISPOSITION 02-29-08 0:00 P TRANSFERRED TO 25,000 PPM GROUP 

 4943M1 M EPINEPHRINE DISPOSITION 02-29-08 0:00 P TRANSFERRED TO 25,000 PPM GROUP 

 4935M2 M 25,000 PPM DISPOSITION 03-04-08 0:00 P TRANSFERRED TO 50,000 PPM GROUP 

 4936M2 M 25,000 PPM DISPOSITION 03-04-08 0:00 P TRANSFERRED TO 50,000 PPM GROUP 

 4937M2 M 25,000 PPM DISPOSITION 03-04-08 0:00 P TRANSFERRED TO 50,000 PPM GROUP 

 4938M2 M 25,000 PPM DISPOSITION 03-04-08 0:00 P TRANSFERRED TO 50,000 PPM GROUP 

 4941M2 M 25,000 PPM DISPOSITION 03-04-08 0:00 P TRANSFERRED TO 50,000 PPM GROUP 

 4943M2 M 25,000 PPM DISPOSITION 03-04-08 0:00 P TRANSFERRED TO 50,000 PPM GROUP 

 4935M3 M 35,000 PPM DISPOSITION 03-12-08 12:40 P ANIMAL TRANSFERRED TO WIL STOCK COLONY (WIL-SPDOG08) 

 4936M3 M 35,000 PPM DISPOSITION 03-12-08 12:40 P ANIMAL TRANSFERRED TO WIL STOCK COLONY (WIL-SPDOG08) 

 4937M3 M 35,000 PPM DISPOSITION 03-12-08 12:40 P ANIMAL TRANSFERRED TO WIL STOCK COLONY (WIL-SPDOG08) 

 4938M3 M 35,000 PPM DISPOSITION 03-12-08 12:40 P ANIMAL TRANSFERRED TO WIL STOCK COLONY (WIL-SPDOG08) 

 4941M3 M 35,000 PPM DISPOSITION 03-12-08 12:40 P ANIMAL TRANSFERRED TO WIL STOCK COLONY (WIL-SPDOG08) 

 4943M3 M 35,000 PPM DISPOSITION 03-12-08 12:40 P ANIMAL TRANSFERRED TO WIL STOCK COLONY (WIL-SPDOG08) 

 ----------------------------------------------------------------------------------------------------------------------------------- 

 GRADE CODE: 1 - SLIGHT 2 - MODERATE 3 - SEVERE P - PRESENT 

 PCRDv4.11 

 05/22/2008 

 R:04/13/2011 

41 of 104
 TABLE 8 (DISPOSITIONS - 50,000 PPM) 

 PROJECT NO.:WIL-447023V CARDIAC SENSITIZATION OF BY INHALATION IN DOGS PAGE 1 

 SPONSOR: INDIVIDUAL CLINICAL OBSERVATIONS 

 

 TABLE RANGE: 03-07-08 TO 03-07-08 

 ----------------------------------------------------------------------------------------------------------------------------------- 

 ANIMAL SEX GROUP CATEGORY DATE TIME GRADE OBSERVATIONS 

 ----------------------------------------------------------------------------------------------------------------------------------- 

 4935M3 M 50,000 PPM DISPOSITION 03-07-08 0:00 P TRANSFERRED TO 35,000 PPM GROUP 

 4936M3 M 50,000 PPM DISPOSITION 03-07-08 0:00 P TRANSFERRED TO 35,000 PPM GROUP 

 4937M3 M 50,000 PPM DISPOSITION 03-07-08 0:00 P TRANSFERRED TO 35,000 PPM GROUP 

 4938M3 M 50,000 PPM DISPOSITION 03-07-08 0:00 P TRANSFERRED TO 35,000 PPM GROUP 

 4941M3 M 50,000 PPM DISPOSITION 03-07-08 0:00 P TRANSFERRED TO 35,000 PPM GROUP 

 4943M3 M 50,000 PPM DISPOSITION 03-07-08 0:00 P TRANSFERRED TO 35,000 PPM GROUP 

 ----------------------------------------------------------------------------------------------------------------------------------- 

 GRADE CODE: 1 - SLIGHT 2 - MODERATE 3 - SEVERE P - PRESENT 

 PCRDv4.11 

 05/05/2008 

 R:04/13/2011 

42 of 104
 TABLE 9 (DETAILED PHYSICAL EXAMINATIONS - MAIN STUDY) 

 PROJECT NO.:WIL-447023 CARDIAC SENSITIZATION OF BY INHALATION IN DOGS PAGE 1 

 SPONSOR: INDIVIDUAL CLINICAL OBSERVATIONS 

 

 TABLE RANGE: 02-26-08 TO 03-12-08 

 ----------------------------------------------------------------------------------------------------------------------------------- 

 ANIMAL SEX GROUP CATEGORY DATE TIME GRADE OBSERVATIONS 

 ----------------------------------------------------------------------------------------------------------------------------------- 

 4935M1 M EPINEPHRINE NORMAL 02-26-08 8:00 P NO SIGNIFICANT CLINICAL OBSERVATIONS 

 4936M1 M EPINEPHRINE NORMAL 02-26-08 8:01 P NO SIGNIFICANT CLINICAL OBSERVATIONS 

 4937M1 M EPINEPHRINE NORMAL 02-26-08 8:02 P NO SIGNIFICANT CLINICAL OBSERVATIONS 

 4938M1 M EPINEPHRINE NORMAL 02-26-08 8:04 P NO SIGNIFICANT CLINICAL OBSERVATIONS 

 4941M1 M EPINEPHRINE NORMAL 02-26-08 8:05 P NO SIGNIFICANT CLINICAL OBSERVATIONS 

 4943M1 M EPINEPHRINE NORMAL 02-26-08 8:06 P NO SIGNIFICANT CLINICAL OBSERVATIONS 

 4935M2 M 25,000 PPM NORMAL 02-29-08 6:39 P NO SIGNIFICANT CLINICAL OBSERVATIONS 

 4936M2 M 25,000 PPM NORMAL 02-29-08 6:40 P NO SIGNIFICANT CLINICAL OBSERVATIONS 

 4937M2 M 25,000 PPM NORMAL 02-29-08 6:41 P NO SIGNIFICANT CLINICAL OBSERVATIONS 

 4938M2 M 25,000 PPM NORMAL 02-29-08 6:43 P NO SIGNIFICANT CLINICAL OBSERVATIONS 

 4941M2 M 25,000 PPM NORMAL 02-29-08 6:44 P NO SIGNIFICANT CLINICAL OBSERVATIONS 

 4943M2 M 25,000 PPM NORMAL 02-29-08 6:45 P NO SIGNIFICANT CLINICAL OBSERVATIONS 

 4935M3 M 35,000 PPM NORMAL 03-07-08 6:38 P NO SIGNIFICANT CLINICAL OBSERVATIONS 

 4936M3 M 35,000 PPM NORMAL 03-07-08 6:40 P NO SIGNIFICANT CLINICAL OBSERVATIONS 

 4937M3 M 35,000 PPM NORMAL 03-07-08 6:42 P NO SIGNIFICANT CLINICAL OBSERVATIONS 

 4938M3 M 35,000 PPM ORAL/DENTAL 03-07-08 6:44 P EMESIS CONTAINING FOOD 

 4941M3 M 35,000 PPM EYES/EARS/NOSE 03-07-08 6:46 P REDDENED EAR(S) 

 4941M3 M 35,000 PPM SPECIAL II 03-07-08 6:46 P TOENAILS LONG, TRIMMED 

 4943M3 M 35,000 PPM NORMAL 03-07-08 6:48 P NO SIGNIFICANT CLINICAL OBSERVATIONS 

 ----------------------------------------------------------------------------------------------------------------------------------- 

 GRADE CODE: 1 - SLIGHT 2 - MODERATE 3 - SEVERE P - PRESENT 

 PCRDv4.11 

 05/22/2008 

 R:04/13/2011 

43 of 104
 TABLE 10 (DETAILED PHYSICAL EXAMINATIONS - 50,000 PPM) 

 PROJECT NO.:WIL-447023V CARDIAC SENSITIZATION OF BY INHALATION IN DOGS PAGE 1 

 SPONSOR: INDIVIDUAL CLINICAL OBSERVATIONS 

 

 TABLE RANGE: 03-04-08 TO 03-04-08 

 ----------------------------------------------------------------------------------------------------------------------------------- 

 ANIMAL SEX GROUP CATEGORY DATE TIME GRADE OBSERVATIONS 

 ----------------------------------------------------------------------------------------------------------------------------------- 

 4935M3 M 50,000 PPM NORMAL 03-04-08 8:32 P NO SIGNIFICANT CLINICAL OBSERVATIONS 

 4935M3 M 50,000 PPM BEHAVIOR/CNS 03-04-08 0:00 P VOCALIZATION DURING DOSING 

 03-04-08 0:00 P TREMORS, INTERMITTENT 

 4935M3 M 50,000 PPM EYES/EARS/NOSE 03-04-08 0:00 P INJECTED SCLERA LEFT EYE 

 03-04-08 0:00 P INJECTED SCLERA RIGHT EYE 

 4935M3 M 50,000 PPM ORAL/DENTAL 03-04-08 0:00 P EXCESSIVE SALIVATION 

 03-04-08 0:00 P REDDENED GUMS 

 4936M3 M 50,000 PPM NORMAL 03-04-08 8:34 P NO SIGNIFICANT CLINICAL OBSERVATIONS 

 4936M3 M 50,000 PPM BEHAVIOR/CNS 03-04-08 11:43 P VOCALIZATION DURING DOSING 

 03-04-08 11:43 P CONVULSIONS, INTERMITTENT 

 4936M3 M 50,000 PPM EYES/EARS/NOSE 03-04-08 11:45 P INJECTED SCLERA LEFT EYE 

 03-04-08 11:45 P INJECTED SCLERA RIGHT EYE 

 4936M3 M 50,000 PPM ORAL/DENTAL 03-04-08 11:43 P EXCESSIVE SALIVATION 

 4937M3 M 50,000 PPM NORMAL 03-04-08 8:36 P NO SIGNIFICANT CLINICAL OBSERVATIONS 

 4938M3 M 50,000 PPM NORMAL 03-04-08 8:38 P NO SIGNIFICANT CLINICAL OBSERVATIONS 

 4941M3 M 50,000 PPM NORMAL 03-04-08 8:40 P NO SIGNIFICANT CLINICAL OBSERVATIONS 

 4943M3 M 50,000 PPM NORMAL 03-04-08 8:42 P NO SIGNIFICANT CLINICAL OBSERVATIONS 

 ----------------------------------------------------------------------------------------------------------------------------------- 

 GRADE CODE: 1 - SLIGHT 2 - MODERATE 3 - SEVERE P - PRESENT 

 PCRDv4.11 

 05/05/2008 

 R:04/13/2011 

44 of 104
 TABLE 11 (MAIN STUDY) 

 PROJECT NO.:WIL-447023 CARDIAC SENSITIZATION OF BY INHALATION IN DOGS PAGE 1 

 SPONSOR: INDIVIDUAL BODY WEIGHTS [KG] 

 

 ----------------------------------------------------------------------------------------------------------------------------------- 

 MALES 

 ----------------------------------------------------------------------------------------------------------------------------------- 

 ANIMALS FROM GROUP 1: EPINEPHRINE 

 ------------------------------------------- 

 4935M1 8.1 

 4936M1 9.9 

 4937M1 9.7 

 4938M1 7.5 

 4941M1 7.9 

 4943M1 8.7 

 

 MEAN 8.6 

 S.D. 0.99 

 N 6 

 

45 of 104
 TABLE 11 (MAIN STUDY) 

 PROJECT NO.:WIL-447023 CARDIAC SENSITIZATION OF BY INHALATION IN DOGS PAGE 2 

 SPONSOR: INDIVIDUAL BODY WEIGHTS [KG] 

 

 ----------------------------------------------------------------------------------------------------------------------------------- 

 MALES 

 ----------------------------------------------------------------------------------------------------------------------------------- 

 ANIMALS FROM GROUP 2: 25,000 PPM 

 ------------------------------------------- 

 4935M2 8.1 

 4936M2 10.0 

 4937M2 9.7 

 4938M2 7.4 

 4941M2 7.8 

 4943M2 8.6 

 

 MEAN 8.6 

 S.D. 1.03 

 N 6 

 

46 of 104
 TABLE 11 (MAIN STUDY) 

 PROJECT NO.:WIL-447023 CARDIAC SENSITIZATION OF BY INHALATION IN DOGS PAGE 3 

 SPONSOR: INDIVIDUAL BODY WEIGHTS [KG] 

 

 ----------------------------------------------------------------------------------------------------------------------------------- 

 MALES 

 ----------------------------------------------------------------------------------------------------------------------------------- 

 ANIMALS FROM GROUP 3: 35,000 PPM 

 ------------------------------------------- 

 4935M3 8.3 

 4936M3 9.9 

 4937M3 10.0 

 4938M3 7.7 

 4941M3 7.9 

 4943M3 8.8 

 

 MEAN 8.8 

 S.D. 1.00 

 N 6 

 PJTBWv4.22 

 05/05/2008 

 R:04/13/2011 

47 of 104
 TABLE 12 (50,000 PPM) 

 PROJECT NO.:WIL-447023V CARDIAC SENSITIZATION OF BY INHALATION IN DOGS PAGE 1 

 SPONSOR: INDIVIDUAL BODY WEIGHTS [KG] 

 

 MALE GROUP: 50,000 PPM 

 ----------------------------------------------------------------------------------------------------------------------------------- 

 ANIMAL 

 4935M3 8.4 

 4936M3 10.1 

 4937M3 10.0 

 4938M3 7.8 

 4941M3 8.1 

 4943M3 9.0 

 

 

 MEAN 8.9 

 S.D. 0.99 

 N 6 

 PBFTSv4.44 

 05/05/2008 

 R:04/13/2011 

48 of 104
 TABLE 13 (ALL ANIMALS) 

 PROJECT NO.:WIL-447023 CARDIAC SENSITIZATION OF BY INHALATION IN DOGS PAGE 1 

 SPONSOR: INDIVIDUAL CARDIAC SENSITIZATION RESPONSES 

 

 ----------------------------------------------------------------------------------------------------------------------------------- 

 EXPOSURE LEVEL (PPM) ANIMAL NO. RESPONSE: 2.0 UG/KG 4.0 UG/KG 6.0 UG/KG 8.0 UG/KG 

 ----------------------------------------------------------------------------------------------------------------------------------- 

 0 4935 - - - - 

 4936 - - - - 

 4937 - - - - 

 4938 - - - - 

 4941 - - - - 

 4943 - - - - 

 

 25,000 4935 - - - - 

 4936 - - - - 

 4937 - - - - 

 4938 - - - - 

 4941 - - - - 

 4943 - - - - 

 

 50,000 4935 - - NA NA 

 4936-A NA NA NA NA 

 4937-B NA NA NA NA 

 4938-B NA NA NA NA 

 4941-B NA NA NA NA 

 4943-B NA NA NA NA 

 

 35,000 4935 - - NA NA 

 4936 - - - - 

 4937 - - - - 

 4938 - NA NA NA 

 4941 - - - - 

 4943 - - - - 

 

 ----------------------------------------------------------------------------------------------------------------------------------- 

 - = NO RESPONSE INDICATIVE OF CARDIAC SENSITIZATION 

 NA = NOT APPLICABLE 

 A = ANIMAL NO. 4936 EXPOSED TO TEST ARTICLE, BUT NOT EPINEPHRINE 

 B = ANIMAL NOT EXPOSED TO TEST ARTICLE 

 MANUALv1.00 

 04/30/2008 

 R:04/13/2011 

49 of 104
WIL-447023

APPENDIX A

Certificates Of Analysis (Sponsor- or Manufacturer-Provided Data)

50 of 104
 

 

Material: 

 

Sample ID # : Lot # 40883-50 

AR#43699 

Assay: 99.978 area% 

 Organic Impurities in area % 

 Trifluoropropyne 0.0008 

 HFC-245fa 0.0085 

 HFO-1234ze 0.0090 

 HCFC-244bb 0.0011 

 HCFO-1233xf 0.0023 

 HCFO-1233 isomer 0.0008 

 

Certified by: 

Sr. Technical Manager 

Analytical Services 

2/20/08 

 

51 of 104
 

 

52 of 104
WIL-447023

APPENDIX B

Exposure Atmosphere Generation Report

53 of 104
ACUTE CARDIAC SENSITIZATION

STUDY OF IN BEAGLE DOGS

Exposure Atmosphere Generation Report

Inhalation Department

WIL Research Laboratories, LLC

WIL-447023

 -1- 

54 of 104
TABLE OF CONTENTS

Page

Table Of Contents......................................................................................2

Index Of Figures ........................................................................................2

Index Of Tables..........................................................................................2

1. Exposure Methods .....................................................................................3

1.1. Exposure Regimen.......................................................................................3

1.2. Exposure Environmental Conditions ...........................................................3

2. Test Atmosphere Generation Methods ....................................................3

3. Methods Of Characterization Of Exposure Atmospheres.....................5

3.1. Analytical Methods......................................................................................5

3.2. Gas Chromatograph Calibration ..................................................................6

4. Results Of Characterization Of Exposure Atmosphere.........................7

4.1. Analyzed Exposure Concentrations.............................................................7

5. Key Study Personnel And Report Submission........................................8

INDEX OF FIGURES

1. Atmosphere Generation And Exposure System ........................................................10

INDEX OF TABLES

1. Individual Test Atmosphere Concentrations..............................................................12

WIL-447023

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55 of 104
1. EXPOSURE METHODS

1.1. EXPOSURE REGIMEN

The test article, was administered as muzzle-only inhalation exposures at

multiple concentrations. Animals were exposed individually to the test article

concentration for durations of 2 to 21 minutes for each exposure. Due to unexpected

behavioral responses, only 2 of the 6 animals on study received all 3 target concentrations

of 2.5% (25000 ppm), 5% (50000 ppm) and 3.5% (35000 ppm) of test article. The

following table presents the overall schedule of the general inhalation exposures:

Test Article

Concentration (PPM)

Exposure

Number Date

25,000 1 29 February 2008

50,000 2 4 March 2008

35,000 3 7 March 2008

1.2. EXPOSURE ENVIRONMENTAL CONDITIONS

The test atmospheres were not monitored for temperature and humidity. The test

atmospheres were prepared using dry compressed air and were maintained at room

temperature throughout the exposure period. Oxygen content was performed on each

generation bag prior to each animal’s exposure using either a AMI Oxygen Analyzer

Model 201RSP (Advanced Micro Instruments, Inc., Huntington Beach, CA) or a Drager

Pac III (Drager Safety Inc., Pittsburgh, PA). The oxygen content was between 20.0% and

20.6%.

2. TEST ATMOSPHERE GENERATION METHODS

Exposures were conducted using a muzzle-only system consisting of a mask connected to

a 3-way valve which was connected to a 200-L Tedlar gas bag of the test atmosphere.

The test atmosphere and exposure system is shown schematically in Figure 1. Mixtures

of test article and compressed air were prepared in a 200-L Tedlar gas bag by metering a

WIL-447023

 -3- 

56 of 104
pre-determined amount of test article through a calibrated rotameter-type flowmeter

(Cole-Parmer, Vernon Hills, IL) into a known volume of compressed air. The bag was

sampled using a gas chromatograph (GC), and, if necessary, additional compressed air or

test article was added to the bag to achieve the appropriate target concentration. The

addition of oxygen was not required since the oxygen content of all exposure bags was

above 19%. Test article was administered from the 200-L Tedlar bag connected to the

3-way valve. During the pre-treatment acclimation period, animals received room air

directed through a 3-way valve. At the start of exposure, the 3-way valve was turned to

the 200-L bag position for exposure to the test article. Feed and water were withheld

during exposure periods.

WIL-447023

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57 of 104
3. METHODS OF CHARACTERIZATION OF EXPOSURE ATMOSPHERES

3.1. ANALYTICAL METHODS

Analyzed exposure concentrations were obtained using a gas chromatograph. Samples of

the exposure atmospheres were collected from each Tedlar bag containing test article

prior to and following each animal exposure using a sample loop. Test article exposure

concentration averages were within ± 5% of each target concentration. The following

table summarizes the GC conditions:

GC Manufacturer: Hewlett-Packard

Model Number: 5890 Series II

Detector Type: Flame ionization detection

Detector Temperature: 200°C

Column Diameter × Length: 30 m × 0.53 mm ID

Column Type: J & W DB-5 Megabore

Fuel Gas: Hydrogen

Fuel Flowrate: 27 mL/minute

Air Flowrate: 262 mL/minute

Carrier Gas: Helium

Carrier Gas Flowrate: 10 mL/minute

Injector Temperature: 150°C

Injection Volume: 0.25 mL

Retention Time: Approximately 0.770 minutes

Integrator Manufacturer: HP Model 3396A Series II

Chart Zero Offset: 0

Chart Attenuation: 10

Chart Speed: 1.0 cm/minute

Peak Area Rejection Value: 0

Peak Threshold: 2

Peak Width: 0.04

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3.2. GAS CHROMATOGRAPH CALIBRATION

The GC was calibrated using gas-phase standards prepared to contain known vapor

concentrations of the test article. A neat sample of test article was drawn from the test

article cylinder into a 1-L Tedlar® bag (SKC, Inc., Eighty Four, PA). Each standard was

prepared by removing a known volume of test article from the neat sample bag using a

gas tight syringe (SGE International Analytical Science PTY LTD, Ringwood, Australia)

and injecting it into a 1-L Tedlar bag filled with a known volume of air. A dry test meter

(Model DTM-200A, Elster American Meter Co., Nebraska City, NE) was used to

measure the total volume of air in the standard bag. Concentrations of the gas-phase

standards were calculated as follows:

TA × 106

Concentrations (ppm) = Air + TA

TA = Volume of test article, mL

Air = Volume of air, mL

Prior to initiation of animal exposures, the GC was calibrated with standards prepared

and analyzed in triplicate. The calibration curve constructed was defined using standards

at 3 vapor concentrations for each target exposure concentration. The prime calibration

curve was using a quadratic formula and used to calculate exposure concentrations for all

animal exposure bags. The peak area (y) and the theoretical concentrations of the

calibration standards (x) were fit with least squares-regression analysis to the ln-quadratic

function:

ln (y) = a × [ln (x)]2 + b × ln (x) + c

Each day, prior to the initiation of exposures, 1 of the 3 standards was prepared and

analyzed, and the prime calibration curve was used to perform a calibration check of the

GC. The GC was considered to be within normal calibration limits if the analyzed

concentration of the prepared standard was within ± 10% of the known concentration.

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The following table summarizes the standard parameters:

Standard

Concentration (ppm)

Volume of

Test Article (mL)

Volume of

Air (mL)

12,346 10 800

24,390 20 800

36,145 30 800

53,254 45 800

80,460 70 800

4. RESULTS OF CHARACTERIZATION OF EXPOSURE ATMOSPHERE

4.1. ANALYZED EXPOSURE CONCENTRATIONS

The overall mean analyzed concentrations for test article were 25,610, 50,836

and 34,930 ppm. Individual test atmosphere concentrations for each level of test article

are presented in Table 1 of this appendix.

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FIGURE 1

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FIGURE 1: ATMOSPHERE GENERATION AND EXPOSURE SYSTEM

200 L TEDLAR

BAG

3- WAY VALVE

TO MASK

TO

EXHAUST

One Way DOG MASK Valve

One Way

Valve

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 -10- 

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TABLE 1

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64 of 104
 INHALATION TABLE 1 

 PROJECT NO.:WIL-447023 CARDIAC SENSITIZATION OF BY INHALATION IN DOGS PAGE 1 

 SPONSOR: INDIVIDUAL TEST ATMOSPHERE CONCENTRATIONS --------------------------------------------------------------------------------------------------------------------------------- 

 TARGET CONCENTRATION: 25,000 PPM EXPOSURE DATE: 29-FEB-2008 

 --------------------------------------------------------------------------------------------------------------------------------- 

 ANIMAL NO. CONCENTRATION (PPM) 

 --------------------------------------------------------------------------------------------------------------------------------- 

 4935M2 26204 

 26245 

 

 4936M2 24666 

 26131 

 

 4937M2 25362 

 25391 

 

 4938M2 25917 

 25874 

 

 4941M2 25548 

 25518 

 

 4943M2 25176 

 25293 

 -------------------------------------------------------------------------------------------------------------------------------- 

 GRAND MEAN 25610 

 S.D. 476.1 

 C.V. (%) 1.86 

 N 12 

 ---------------------------------------------------------------------------------------------------------------------------------- 

 PPM = PARTS PER MILLION 

 

 

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 INHALATION TABLE 1 

 PROJECT NO.:WIL-447023 CARDIAC SENSITIZATION OF BY INHALATION IN DOGS PAGE 2 

 SPONSOR: INDIVIDUAL TEST ATMOSPHERE CONCENTRATIONS --------------------------------------------------------------------------------------------------------------------------------- 

 TARGET CONCENTRATION: 35,000 PPM EXPOSURE DATE: 07-MAR-2008 

 --------------------------------------------------------------------------------------------------------------------------------- 

 ANIMAL NO. CONCENTRATION (PPM) 

 --------------------------------------------------------------------------------------------------------------------------------- 

 4935M3 35049 

 35363 

 

 4936M3 34998 

 35175 

 

 4937M3 35400 

 35445 

 

 4938M3 35242 

 34223 

 

 4941M3 35125 

 35241 

 

 4943M3 35871 

 32029 

 --------------------------------------------------------------------------------------------------------------------------------- 

 GRAND MEAN 34930 

 S.D. 990.2 

 C.V. (%) 2.83 

 N 12 

 ---------------------------------------------------------------------------------------------------------------------------------- 

 PPM = PARTS PER MILLION 

 

 

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 INHALATION TABLE 1 

 PROJECT NO.:WIL-447023 CARDIAC SENSITIZATION OF BY INHALATION IN DOGS PAGE 3 

 SPONSOR: INDIVIDUAL TEST ATMOSPHERE CONCENTRATIONS --------------------------------------------------------------------------------------------------------------------------------- 

 TARGET CONCENTRATION: 50,000 PPM EXPOSURE DATE: 04-MAR-2008 

 --------------------------------------------------------------------------------------------------------------------------------- 

 ANIMAL NO. CONCENTRATION (PPM) 

 --------------------------------------------------------------------------------------------------------------------------------- 

 4935M3 51496 

 49702 

 

 4936M3 51255 

 50891 

 

 --------------------------------------------------------------------------------------------------------------------------------- 

 GRAND MEAN 50836 

 S.D. 795.9 

 C.V. (%) 1.57 

 N 4 

 ---------------------------------------------------------------------------------------------------------------------------------- 

 PPM = PARTS PER MILLION 

 

 MANUALv1.0 

 04/15/2008 

 R:04/13/2011 

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WIL-447023

APPENDIX C

Animal Room Environmental Conditions

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447023 TEMPERATURE/HUMIDITY - DAILY SUMMARY REPORT BY STUDY

CARDIAC SENSITIZATION OF BY INHALATION IN DOGS

SPONSOR:PROJECT NO.:WIL

447023 02/14/08 

03/12/08 

STUDY SPECIFICATIONS: DATE IN: TIME IN:

DATE OUT: TIME OUT:

B ROOM 94DOG

LOW TEMPERATURE oF: 63.0 HIGH TEMPERATURE oF: 73.0 

H

ROOM SPECIFICATIONS:SPECIES:

TEMPERATURE HUMIDITY

MEAN (oF)DATE MEAN (%RH)

LOW TEMPERATURE oC: 17.2 HIGH TEMPERATURE oC: 22.8

MEAN (oC)

14-Feb-08 21.4 70.6 36.5

15-Feb-08 21.4 70.4 38.7

16-Feb-08 21.3 70.3 38.1

17-Feb-08 21.4 70.6 40.8

18-Feb-08 21.4 70.5 38.5

19-Feb-08 21.4 70.5 38.0

20-Feb-08 21.4 70.5 38.1

21-Feb-08 21.4 70.5 37.5

22-Feb-08 21.4 70.5 38.3

23-Feb-08 21.4 70.5 38.4

24-Feb-08 21.4 70.5 38.2

25-Feb-08 21.4 70.5 38.7

26-Feb-08 21.4 70.4 39.1

27-Feb-08 21.4 70.5 38.8

28-Feb-08 21.4 70.5 38.7

29-Feb-08 21.4 70.5 38.7

01-Mar-08 21.4 70.5 38.7

02-Mar-08 21.4 70.5 38.7

03-Mar-08 21.4 70.6 41.2

04-Mar-08 21.4 70.6 39.1

05-Mar-08 21.4 70.5 39.3

06-Mar-08 21.4 70.5 39.2

07-Mar-08 21.4 70.5 39.2

4

NOTE: + = VALUE WAS GREATER THAN HIGH RANGE - = VALUE WAS LESS THAN LOW RANGENOTE: MEANS REPRESENT THE MEAN OF THE DAILY VALUES

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447023 TEMPERATURE/HUMIDITY - DAILY SUMMARY REPORT BY STUDY

CARDIAC SENSITIZATION OF BY INHALATION IN DOGS

SPONSOR:PROJECT NO.:WIL

447023 02/14/08 

03/12/08 

STUDY SPECIFICATIONS: DATE IN: TIME IN:

DATE OUT: TIME OUT:

B ROOM 94DOG

LOW TEMPERATURE oF: 63.0 HIGH TEMPERATURE oF: 73.0 

H

ROOM SPECIFICATIONS:SPECIES:

TEMPERATURE HUMIDITY

MEAN (oF)DATE MEAN (%RH)

LOW TEMPERATURE oC: 17.2 HIGH TEMPERATURE oC: 22.8

MEAN (oC)

08-Mar-08 21.4 70.5 38.4

09-Mar-08 21.4 70.5 38.5

10-Mar-08 21.4 70.5 39.1

11-Mar-08 21.4 70.6 38.5

12-Mar-08 21.4 70.6 38.6

70.5TEMPERATURE oF 70.3 70.6

41.236.538.7

28MEAN MIN MAX

TEMPERATURE oC 21.4 21.3 21.4

HUMIDITY (%RH)GRAND STATSN DAYS

4

NOTE: + = VALUE WAS GREATER THAN HIGH RANGE - = VALUE WAS LESS THAN LOW RANGENOTE: MEANS REPRESENT THE MEAN OF THE DAILY VALUES

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447023 TEMPERATURE/HUMIDITY - END OF STUDY SUMMARY REPORT

CARDIAC SENSITIZATION OF BY INHALATION IN DOGS

SPONSOR:PROJECT NO.:WIL- 9:43 14-Apr-

B ROOM 94DOG

LOW TEMPERATURE: 63.0HIGH TEMPERATURE: 73.0LOW HUMIDITY: 30.0HIGH HUMIDITY: 70.0ROOM SPECIFICATIONS:SPECIES:

TEMPERATURE HUMIDITY

02/14/086:0016:0003/12/08

DATE IN:TIME IN:DATE OUT:TIME OUT:

ROOM B ROOM 94 SUMMARY

MEANMINMAX

70.568.872.4

38.729.559.0

SD 1.03 2.59N SAMPLES 656 656N DAYSFIRST DAYLAST DAY

02/14/0803/12/08

28

4

NOTE: TEMPERATURE UNITS = DEGREES FAHRENHEIT HUMIDITY UNITS = % RELATIVE HUMIDITYNOTE: MEANS REPRESENT THE MEAN OF ALL VALUES

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447023 TEMPERATURE/HUMIDITY - END OF STUDY SUMMARY REPORT

CARDIAC SENSITIZATION OF BY INHALATION IN DOGS

SPONSOR:PROJECT NO.:WIL- 9:43 14-Apr-

STUDY 447023 SUMMARY

MEANMINMAX

70.568.872.4

38.729.559.0

SD 1.03 2.59N SAMPLES 656 656FIRST DAYLAST DAY

02/14/0803/12/08

N DAYS 28

4

NOTE: TEMPERATURE UNITS = DEGREES FAHRENHEIT HUMIDITY UNITS = % RELATIVE HUMIDITYNOTE: MEANS REPRESENT THE MEAN OF ALL VALUES

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WIL-447023

APPENDIX D

Unscheduled Clinical Observations

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 UNSCHEDULED CLINICAL OBSERVATIONS (MAIN STUDY) 

 PROJECT NO.:WIL-447023U CARDIAC SENSITIZATION OF BY INHALATION IN DOGS PAGE 1 

 SPONSOR: SUMMARY OF CLINICAL FINDINGS: TOTAL OCCURRENCE/NO. OF ANIMALS 

 

 ----- M A L E ----- 

 ----------------------------------------------------------------------------------------------------------------------------------- 

 TABLE RANGE: 02-26-08 TO 03-12-08 

 GROUP: 1 2 3 

 ----------------------------------------------------------------------------------------------------------------------------------- 

 

 BEHAVIOR/CNS 

 -TREMORS, INTERMITTENT 0/ 0 0/ 0 5/ 5 

 

 EYES/EARS/NOSE 

 -INJECTED SCLERA LEFT EYE 0/ 0 0/ 0 2/ 2 

 -INJECTED SCLERA RIGHT EYE 0/ 0 0/ 0 2/ 2 

 -REDDENED EAR(S) 0/ 0 0/ 0 2/ 2 

 

 EXCRETA 

 -SOFT FECES 0/ 0 0/ 0 1/ 1 

 

 ORAL/DENTAL 

 -WET CLEAR MATERIAL AROUND MOUTH 0/ 0 0/ 0 4/ 4 

 -EXCESSIVE SALIVATION 0/ 0 0/ 0 2/ 2 

 -WHITE FROTHY MATERIAL AROUND MOUTH 0/ 0 0/ 0 2/ 2 

 ----------------------------------------------------------------------------------------------------------------------------------- 

 1- EPINEPHRINE 2- 25,000 PPM 3- 35,000 PPM 

 PCSUv4.07 

 05/05/2008 

 R:04/13/2011 

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 UNSCHEDULED CLINICAL OBSERVATIONS (MAIN STUDY) 

 PROJECT NO.:WIL-447023U CARDIAC SENSITIZATION OF BY INHALATION IN DOGS PAGE 1 

 SPONSOR: INDIVIDUAL CLINICAL OBSERVATIONS 

 

 TABLE RANGE: 02-26-08 TO 03-12-08 

 ----------------------------------------------------------------------------------------------------------------------------------- 

 ANIMAL SEX GROUP CATEGORY DATE TIME GRADE OBSERVATIONS 

 ----------------------------------------------------------------------------------------------------------------------------------- 

 4936M3 M 35,000 PPM BEHAVIOR/CNS 03-07-08 13:53 P TREMORS, INTERMITTENT 

 4936M3 M 35,000 PPM EYES/EARS/NOSE 03-07-08 13:59 P REDDENED EAR(S) 

 03-07-08 14:00 P INJECTED SCLERA LEFT EYE 

 03-07-08 14:00 P INJECTED SCLERA RIGHT EYE 

 4936M3 M 35,000 PPM ORAL/DENTAL 03-07-08 14:02 P EXCESSIVE SALIVATION 

 4937M3 M 35,000 PPM BEHAVIOR/CNS 03-07-08 12:19 P TREMORS, INTERMITTENT 

 4937M3 M 35,000 PPM EXCRETA 03-07-08 12:25 P SOFT FECES 

 4937M3 M 35,000 PPM ORAL/DENTAL 03-07-08 12:30 P WHITE FROTHY MATERIAL AROUND MOUTH 

 03-07-08 12:30 P WET CLEAR MATERIAL AROUND MOUTH 

 4938M3 M 35,000 PPM BEHAVIOR/CNS 03-07-08 11:11 P TREMORS, INTERMITTENT 

 4938M3 M 35,000 PPM EYES/EARS/NOSE 03-07-08 11:13 P REDDENED EAR(S) 

 03-07-08 11:14 P INJECTED SCLERA LEFT EYE 

 03-07-08 11:14 P INJECTED SCLERA RIGHT EYE 

 4938M3 M 35,000 PPM ORAL/DENTAL 03-07-08 11:13 P EXCESSIVE SALIVATION 

 03-07-08 11:13 P WET CLEAR MATERIAL AROUND MOUTH 

 03-07-08 11:13 P WHITE FROTHY MATERIAL AROUND MOUTH 

 4941M3 M 35,000 PPM BEHAVIOR/CNS 03-07-08 10:04 P TREMORS, INTERMITTENT 

 4941M3 M 35,000 PPM ORAL/DENTAL 03-07-08 10:20 P WET CLEAR MATERIAL AROUND MOUTH 

 4943M3 M 35,000 PPM BEHAVIOR/CNS 03-07-08 9:39 P TREMORS, INTERMITTENT 

 4943M3 M 35,000 PPM ORAL/DENTAL 03-07-08 9:43 P WET CLEAR MATERIAL AROUND MOUTH 

 ----------------------------------------------------------------------------------------------------------------------------------- 

 GRADE CODE: 1 - SLIGHT 2 - MODERATE 3 - SEVERE P - PRESENT 

 PCRDv4.11 

 05/05/2008 

 R:04/13/2011 

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APPENDIX E

Individual Electrocardiographic Data

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Acute Cardiac Sensitization Study – WIL-447023 

Pretest Screening 22 February 2008 

 DOG # FINDINGS

4941 NSCO 

4943 NSCO 

4938 NSCO 

4936 NSCO 

4935 NSCO 

4937 NSCO 

NSCO = No Significant Clinical Observations 

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Acute Cardiac Sensitization Study – WIL-447023 

0 PPM (0%) Exposure Findings 26 February 2008 

 DOG # FINDINGS

4941M1 NSCO 

4943M1 NSCO 

4938M1 NSCO 

4936M1 

6 μg/kg Epinephrine

ESC(2) approximately 15 seconds to 20 seconds post-epinephrine. 

ESC(6) from 20 seconds post-dose to 47 second post-epinephrine. 

ESC rhythm of 18 beats from 25 seconds to 40 seconds post-epinephrine. 

8 μg/kg Epinephrine

AVB2(4) from 30 seconds to 40 seconds post-epinephrine. 

ESC(2) from 50 seconds to 55 seconds post-epinephrine.

4935M1 NSCO 

4937M1 NSCO 

NSCO = No Significant Clinical Observations 

ESC = Escape Complex 

AVB2 = Atrioventricular Block 2nd Degree 

( ) = Number in parentheses indicates the total number of incidences 

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25,000 PPM (2.5%) Exposure Findings 29 February 2008 

 DOG # FINDINGS

4941M2 NSCO 

4943M2 8 μg/kg Epinephrine

ESC(2) 11 and 12 seconds post-epinephrine. 

4938M2 

6 μg/kg Epinephrine

AVB2 from 98 seconds post-epinephrine. 

8 μg/kg Epinephrine

AVB2 72 seconds post-epinephrine. 

ESC(8) from 28 seconds to 57 second post-epinephrine.

4936M2 

6 μg/kg Epinephrine

ESC(4) 29 seconds to 39 seconds post-epinephrine. 

ESC(2) from 40 seconds to 50 second post-epinephrine. 

ESC(2) from 50 seconds to 60 second post-epinephrine. 

8 μg/kg Epinephrine

Escape rhythm of 3 beats from 18 seconds to 20 seconds post-epinephrine. 

ESC(3) from 21 seconds to 26 seconds post-epinephrine. 

Escape rhythm of 4 beats from 28 seconds to 32 seconds post-epinephrine. 

Escape rhythm of 3 beats from 35 seconds to 38 seconds post-epinephrine. 

ESC(4) from 40 seconds to 50 seconds post-epinephrine. 

PAC(2) from 40 seconds to 50 seconds post-epinephrine. 

ESC from 50 seconds to 60 seconds post-epinephrine. 

4935M2 8 μg/kg Epinephrine

AVB2(2) from 48 seconds to 54 seconds post-epinephrine. 

4937M2 

6 μg/kg Epinephrine

ESC 22 seconds post-epinephrine. 

Escape rhythm of 5 beats from 25 seconds to 29 seconds post-epinephrine. 

ESC(5) from 30 seconds to 40 second post-epinephrine. 

ESC(3) from 40 seconds to 50 second post-epinephrine. 

AVB2 (2) from 40 seconds to 50 seconds post-epinephrine. 

ESC(2) from 50 seconds to 60 second post-epinephrine. 

ESC(3) from 60 seconds to 70 second post-epinephrine. 

AVB2(2) from 60 seconds to 70 seconds post- epinephrine. 

8 μg/kg Epinephrine

ESC 20 seconds post-epinephrine. 

Escape rhythm of 10 beats from 23 seconds to 32 seconds post-epinephrine. 

AVB2(2) from 23 seconds to 32 seconds post-epinephrine. 

ESC(4) from 34 seconds to 40 seconds post-epinephrine. 

AVB2 from 34 seconds to 40 seconds post-epinephrine. 

ESC(4) from 41 seconds to 51 seconds post-epinephrine. 

AVB2(2) from 41 seconds to 51 seconds post-epinephrine. 

NSCO = No significant clinical observations 

ESC = Escape complex 

AVB2 = Atrioventricular block 2nd degree 

PAC = Premature atrial contraction 

( ) = Number in parentheses indicates the total number of incidences 

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35,000 PPM (3.5%) Exposure Findings 7 March 2008 

 DOG # FINDINGS

4941M3 NSCO 

4943M3 NSCO 

4938M3 

Prior to exposure

AVB2 

Epinephrine challenges

Epinephrine challenge not completed and was not evaluated. 

4936M3 6 μg/kg Epinephrine

PVC approximately 39 seconds post-epinephrine. 

4935M3 Epinephrine challenges

Epinephrine challenge not completed and was not evaluated. 

4937M3 NSCO 

NSCO = No significant clinical observations 

AVB2 = Atrioventricular block 2nd degree 

PVC = Premature ventricular contraction 

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50,000 PPM (5.0%) Exposure Findings 4 March 2008 

 DOG # FINDINGS

4941M3 Epinephrine challenges

No epinephrine challenge performed. 

4943M3 Epinephrine challenges

No epinephrine challenge performed. 

4938M3 Epinephrine challenges

No epinephrine challenge performed. 

4936M3 Epinephrine challenges

No epinephrine challenge performed. 

4935M3 After administration of 0.2 and 0.4 μg/kg epinephrine doses the epinephrine challenge was 

discontinued and the ECG waveform was unreadable 

4937M3 Epinephrine challenges

No epinephrine challenge performed. 

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APPENDIX F

Study Protocol

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APPENDIX G

Signature Pages From Original Final Report

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