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FINAL REPORT

STUDY TITLE

ACUTE CARDIAC SENSITIZATION

STUDY OF IN BEAGLE DOGS

STUDY NUMBER

STUDY DIRECTOR

STUDY INITIATION DATE

21 October 2009

STUDY COMPLETION DATE

3 February 2010

PERFORMING LABORATORY

SPONSOR STUDY NUMBER

Work Request No.: 

Service Code No.: 

Study No.: 

SPONSOR
 

 

 

 
QUALITY ASSURANCE UNIT STATEMENT

Phases Inspected

Date(s) of

Inspection(s)

Phase

Inspected

Date(s) Findings

Reported

to Study Director

Date(s) Findings

Reported to 

Management Auditor(s)

18-Nov-2009 Detailed Physicals and Body 

Weights 18-Nov-2009 21-Dec-2009

16-Dec-2009 Study Records (Rx-1) 16-Dec-2009 25-Jan-2010

16-Dec-2009

17-Dec-2009

29-Dec-2009 Study Records (I-1, I-2, I-3) 29-Dec-2009 25-Jan-2010

18-Dec-2009

21-Dec-2009

29-Dec-2009 Study Records (Ex-1) 29-Dec-2009 25-Jan-2010

21-Dec-2009

29-Dec-2009 Inhalation Appendix 29-Dec-2009 25-Jan-2010

13-Jan-2010

14-Jan-2010

21-Jan-2010 Draft Report (excluding Inhalation 

Appendix) 21-Jan-2010 03-Feb-2010

This study was inspected in accordance with the U.S. EPA GLP Regulations (40 CFR Part 792), 

the standard operating procedures (SOPs), and the 

Sponsor's protocol with the following exceptions. The data concerning the test substance

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

Sponsor. Quality Assurance findings, derived from the inspections during the conduct of the 

study and from the inspections of the raw data and draft report, are documented and have been 

reported to the Study Director. Review of the amended protocol, as well as a yearly internal 

facility inspection, are conducted by the Quality Assurance Unit (QAU). 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 and SOPs.

The raw data, the retention sample, and the final report will be stored in the Archives or at 

another location specified by the Sponsor.

Acute Cardiac Sensitization 

Study of n Beagle Dogs 

3
 

 

 

 
 

 

 

 

 
TABLE OF CONTENTS

Page

GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT...................................2

QUALITY ASSURANCE UNIT STATEMENT ........................................................................3

Phases Inspected ........................................................................................................................3

Approval ....................................................................................................................................4

KEY STUDY PERSONNEL AND REPORT SUBMISSION ...................................................5

TABLE OF CONTENTS ..............................................................................................................6

INDEX OF TABLES .....................................................................................................................8

INDEX OF APPENDICES ...........................................................................................................9

STUDY INFORMATION ...........................................................................................................10

SUMMARY ..................................................................................................................................11

A. Objective ............................................................................................................................11

B. Study Design......................................................................................................................11

C. Results................................................................................................................................11

D. Conclusions........................................................................................................................12

INTRODUCTION........................................................................................................................13

A. General Study Information ................................................................................................13

B. Key Study Dates ................................................................................................................13

STUDY DESIGN..........................................................................................................................14

EXPERIMENTAL PROCEDURES - MATERIALS AND METHODS................................15

A. Test Substance and Vehicle ...............................................................................................15

1. Test Substance Identification .................................................................................................................15

2. Epinephrine Identification......................................................................................................................15

3. Epinephrine Preparation.........................................................................................................................15

B. Test System........................................................................................................................16

C. Organization of Test Groups, Dose Levels, and Treatment Regimen ...............................16

D. Exposure Methods .............................................................................................................18

E. Exposure Atmosphere Generation and Monitoring ...........................................................18

F. Animal Receipt and Acclimation/Pretest Period ...............................................................18

G. Immunizations Prior to Arrival..........................................................................................19

H. Animal Housing and Exercise ...........................................................................................19

I. Diet, Drinking Water, and Maintenance ............................................................................19

J. Environmental Conditions.................................................................................................19

K. Assignment of Animals to Treatment Groups...................................................................19

L. Animal Disposition ............................................................................................................20

PARAMETERS EVALUATED .................................................................................................21

A. Clinical Observations.........................................................................................................21

Acute Cardiac Sensitization 

Study of in Beagle Dogs 

6
Page

B. Electrocardiographic Examination.....................................................................................21

C. Statistical Methods.............................................................................................................21

Data Retention..............................................................................................................................22

RESULTS .....................................................................................................................................23

A. Clinical Observations.........................................................................................................23

B. Electrocardiographic Examinations...................................................................................23

CONCLUSIONS ..........................................................................................................................25

REFERENCES.............................................................................................................................26

DEVIATIONS FROM THE PROTOCOL................................................................................27

Acute Cardiac Sensitization 

Study of in Beagle Dogs 

7
INDEX OF TABLES

Page

Table 1 Individual Clinical Observations (Detailed Physical Examinations).......................................31

Table 2 Individual Clinical Observations (Unscheduled Observations)...............................................32

Table 3 Individual Body Weights [kg]..................................................................................................33

Table 4 Individual Cardiac Sensitization Responses ............................................................................37

Acute Cardiac Sensitization 

Study of in Beagle Dogs 

8
INDEX OF APPENDICES

Page

Appendix A Certificates of Analysis............................................................................................................38

Appendix B Exposure Atmosphere Generation Report ...............................................................................41

Appendix C Individual Electrocardiographic Data ......................................................................................57

Appendix D Example ECG Waveforms.......................................................................................................65

Appendix E Study Protocol..........................................................................................................................68

Acute Cardiac Sensitization 

Study of in Beagle Dogs 

9
STUDY INFORMATION

Substance Tested:

Number:

Purity: 99.91413%

Known Impurities: None to be of toxicological significance at this time

Sponsor:

Study Initiated/Completed: 21 October 2009 / 3 February 2010

Experimental Starting: 30 October 2009

Experimental Start/Termination 

(Completion): 13 November 2009 / 23 November 2009

Acute Cardiac Sensitization 

Study of in Beagle Dogs 

10
SUMMARY

A. 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 

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

substance to increase the sensitivity of the heart to the pharmacological effects of epinephrine.

B. Study Design

 was administered to animals via a muzzle only inhalation apparatus as acute 

exposures (gas) at concentrations of 7%, 9%, and 8% (70,000, 90,000, and 80,000 ppm [parts per 

million], respectively). One group of up to 6 male dogs per exposure level 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 responses to the epinephrine challenge doses were collected 

5 days prior to exposure to the test substance. A predetermined challenge dose was established 

during this baseline prior to exposure for each individual animal. Escalating doses of 

epinephrine (2, 3, 4, 6, or 8 μg/kg) were administered as bolus intravenous (IV) injections 

(approximately 0.1 mL/kg delivered at a rate of approximately 0.1 mL/sec) until significant or 

dangerous cardiac effects were noted or the dose of 8 μg/kg elicited no effects, whichever was 

higher. For the 70,000, 90,000, and 80,000 ppm groups, animals were administered the 

predetermined challenge dose of 8 μg/kg epinephrine via an appropriate vein beginning 

approximately 5 minutes prior to exposure to the test substance as well as approximately

5 minutes following exposure to the test substance. Two animals displayed an adverse reaction 

to 70,000 ppm and were replaced for subsequent exposures. The replacement 

animals were not exposed to the first exposure level of 70,000 ppm. One animal displayed a 

severe adverse reaction to the second exposure level of 90,000 ppm; no other animals were 

exposed to this level. The third exposure level of 80,000 ppm was selected and was discontinued 

after a similar reaction (as that observed for the 90,000 ppm exposure) was noted in the only

animal to be exposed to this level. No further exposures were conducted. Following the final 

exposure, all animals were returned to the cardiovascular stock colony. Individual body

weights were recorded on exposure days for calculation of epinephrine doses. 

Electrocardiographic (ECG) data were recorded continuously throughout the pre-exposure 

period, exposure to the test substance and throughout administration of the challenge epinephrine 

dose (up to approximately 17 minutes).

C. Results

During the first exposure of 70,000 ppm 2 of the 6 animals displayed continuous 

convulsions, excessive salivation, and/or dilated pupils following test substance exposure.

During the next exposure of 90,000 ppm 1 animal displayed continuous convulsions, 

excessive salivation, and dilated pupils during test substance exposure. No further animals were 

exposed to this concentration.

Acute Cardiac Sensitization 

Study of in Beagle Dogs 

11
During the last exposure of 80,000 ppm the first animal exposed displayed 

continuous convulsions and excessive salivation during test substance exposure similar to that 

noted in the 90,000 ppm exposure group. No further animals were exposed.

All test substance exposures were terminated as soon as convulsions were apparent.

There were no signs of cardiac sensitization after exposure to 70,000 ppm However, 

cardiac sensitization could not be evaluated following exposure to 80,000 and 90,000 ppm 

 since the adverse clinical signs observed precluded the administration of epinephrine 

challenge.

D. Conclusions

Based on the results of this study, muzzle-only vapor exposure to 70,000 ppm in 

Beagle dogs followed by intravenous administration of 8 μg/kg epinephrine did not produce 

evidence of cardiac sensitization. Evaluation for cardiac sensitization was not possible in the 

80,000 and 90,000 ppm exposure groups due to adverse clinical signs that precluded 

the administration of epinephrine. Therefore, the no-observed-effect level (NOEL) for cardiac 

sensitization was 70,000 ppm 

Acute Cardiac Sensitization 

Study of in Beagle Dogs 

12
INTRODUCTION

A. General Study Information

This report presents the data from the study entitled “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/table generation 

during the course of the study:

Computer Protocol(s) Type of Data Collected/Generated

.........................................Main study data

.........................................Unscheduled observations

B. Key Study Dates

Date(s) Event(s)

29 October 2009 .............................Experimental starting date (animal receipt)

9 November 2009 ...........................Pretest ECG screening

12 November 2009 .........................Assignment to study

13 November 2009 ......................... Experimental start date (initial epinephrine 

challenge, no test substance administration)

18 November 2009 ......................... Exposure to 70,000 ppm (epinephrine 

challenge)

20 November 2009 ......................... Exposure to 90,000 ppm (no epinephrine 

challenge)

23 November 2009 ......................... Exposure to 80,000 ppm (no epinephrine 

challenge)

23 November 2009 .........................Experimental termination (completion) date (animals 

transferred to cardiovascular stock colony)

Acute Cardiac Sensitization 

Study of in Beagle Dogs 

13
STUDY DESIGN

Animal receipt

Acclimation/Pre-test ECG screening period

Animals arbitrarily assigned to study

Detailed physical examinations conducted 

and body weights recorded prior to 

exposure; clinical observations recorded as 

needed

Individual response to IV epinephrine 

challenge evaluated

ECG recording initiated

Pre-exposure epinephrine dose administered 

2 minutes after ECG recording initiated

Test substance administered as a gas 

5 minutes after epinephrine dose

Challenge dose of epinephrine administered 

beginning 5 minutes after exposure to test 

substance to assess cardiac sensitization

ECG data recorded continuously

throughout the pre-exposure period, 

exposure to the test substance and 

throughout administration of the 

epinephrine challenge dose (up to 

approximately 17 minutes).

Animals transferred to cardiovascular 

stock colony

Acute Cardiac Sensitization 

Study of in Beagle Dogs 

14
EXPERIMENTAL PROCEDURES - MATERIALS AND METHODS

A. Test Substance and Vehicle

1. Test Substance Identification

The test substance, (also known as and 

Number was received from on 

7 October 2009, as follows:

Identification

Quantity

Received

Physical

Description

 ( )

Lot no. 

Exp. date: 30 June 2011

[ ID no. ]

1 cylinder

Gross weight: 15 kg

Not taken

Documentation regarding the purity and stability of the test substance is on file with the Sponsor 

and A Certificate of Analysis for the test substance was provided by the Sponsor and is 

presented in Appendix A. The purity of the test substance was 99.91413%. The test substance 

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

sample of the test substance was collected and stored in the Archives.

2. Epinephrine Identification

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

5 October 2009 as follows:

Identification

Quantity

Received

Physical

Description

(-)-epinephrine

Lot no. 1416374

CAS no.: 51-43-4

Exp. date: 5 October 2010

[ ID no. 

1 bottle

Net weight: 5 g

Off-white 

powder

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% and was stored refrigerated, 

protected from light, and was considered stable under these conditions.

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 

Acute Cardiac Sensitization 

Study of in Beagle Dogs 

15
80 μg/mL formulation was weighed into a tared weighing vessel. To quantitatively transfer the 

epinephrine into a glass container, 0.9% saline for injection, U.S.P. (lot nos. C782359 and 

C782748, expiry date: 1 March 2011, manufactured by Baxter Healthcare Corporation, 

Deerfield, IL; lot no. 76-432-DK, expiry date: 1 April 2011, manufactured by Hospira, Inc., 

Lake Forest, IL) was used. The formulation was mixed with a magnetic stirrer to ensure 

complete dissolution. 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 80 μg/mL

epinephrine formulation was sterile-filtered through a 0.22-μm (Durapore®) PVDF syringe filter 

(lot nos. R9KN29064 and R9MN44305, expiry dates: 1 August 2012 and 1 September 2012, 

respectively, manufactured by Millipore Corporation, Cork, Ireland) and capped with a septum.

The 20, 30, 40, and 60 μg/mL epinephrine formulations were prepared by diluting the 

80 μg/mL epinephrine formulation with the respective appropriate amounts of 0.9% saline for 

injection, U.S.P. The dilutions were prepared under sterile conditions, on the day of each use in 

sterile vials, capped with septa, swirled to ensure proper mixing, and stored refrigerated and 

maintained on ice, protected from light until administered.

B. Test System

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

This species and breed of animal is recognized as an appropriate model for assessment of 

potential cardiac sensitization (Mullin et al., 1979; Reinhardt et al., 1971). The animals were 

between approximately 7 and 9 months of age at the initiation of exposure.

C. Organization of Test Groups, Dose Levels, and Treatment Regimen

Prior to exposure (during the acclimation period), an individual response to IV epinephrine 

challenge administration was established for each individual dog. Individual body weights were 

recorded for the purpose of calculating doses and detailed physical examinations were performed 

in the morning of the assessment of baseline response to the epinephrine challenges. The 

epinephrine doses started at 2 μg/kg and increased to 3, 4, 6, and then 8 μg/kg since no 

significant or dangerous cardiac effects were noted. The dose volume for epinephrine was 

0.1 mL/kg, and doses were administered as bolus IV injections (approximately 0.1 mL/second) 

into catheters (with injection caps) placed in a cephalic vein. Each challenge dose of epinephrine 

was followed with a 0.2-mL flush of sterile 0.9% saline. The time between challenge doses of 

epinephrine was a minimum of 3 minutes. During this phase of the study, the dogs were exposed 

to air only via the nose-only exposure apparatus. The information gathered was used to establish 

that 8 μg/kg of epinephrine was appropriate for each dog to receive during the gas exposure 

phase with the test substance. Baseline responses to the epinephrine challenge doses were 

collected 5 days prior to exposure to the test gas.

For the 7%, 9%, and 8% exposures (70,000, 90,000, and 80,000 ppm, respectively), the exposure 

duration to air via the muzzle-only apparatus was scheduled to be approximately 7 minutes, and 

the exposure duration to the test substance approximately 10 minutes or less, (i.e., up to 

17 minutes from the beginning of exposure to air). Individual body weights were recorded and 

detailed physical examinations were performed on the days of exposure, and body weights were 

also recorded once after all exposures were complete. The results of the detailed physical 

Acute Cardiac Sensitization 

Study of in Beagle Dogs 

16
examinations performed on the days of exposure are presented in Table 1. The scheduled

treatment regimen was as follows:

Time (minutes) Event

0 Start ECG recording

2 Pre-exposure epinephrine dose

7 Test substance exposure initiated

12 Challenge dose of epinephrine

Up to approximately 17 Termination of exposure/ECG

The exposure schedule is shown in the following table (all epinephrine doses that were 

administered were 8 μg/kg):

Date Animal No.

Target Exposure 

Concentration (ppm)

13 November 2009 7363 0

13 November 2009 7366 0

13 November 2009 7368 0

13 November 2009 7370 0

13 November 2009 7371 0

13 November 2009 7372 0

18 November 2009 7364 0

18 November 2009 7367 0

18 November 2009 7363 70,000

18 November 2009 7366 70,000

18 November 2009 7368 70,000

18 November 2009 7370 70,000

18 November 2009 7371 70,000

18 November 2009 7372 70,000

20 November 2009 7363 90,000

23 November 2009 7364 80,000

The 90,000 and 80,000 ppm exposures were stopped prior to the challenge dose of epinephrine 

due to adverse clinical signs associated with the test substance exposure.

Because of the volatility and intended usage of the test substance, the route of administration for 

this study was via inhalation exposure, as this was the most likely route of exposure for the 

general population. The muzzle-only method of exposure was selected to achieve precise control 

Acute Cardiac Sensitization 

Study of in Beagle Dogs 

17
of concentration levels and durations. Only males were used as no significant gender differences 

were expected.

D. Exposure Methods

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

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

minimum oxygen content of 19%. The control atmosphere (the first 7 minutes of each exposure) 

was filtered room air. 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.

E. Exposure Atmosphere Generation and Monitoring

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

3-way valve connected to a 200-L Tedlar®

gas bag. A known volume of the test substance was 

diluted with sufficient filtered room air to achieve the appropriate target concentration for each 

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

was above 19%. From the 200-L Tedlar®bag, test substance was administered through 

22-mm corrugated tubing connected to a 3-way valve. During the pre-exposure 

acclimation/baseline period, animals received filtered room air directed through the 3-way valve. 

At the start of exposure, the 3-way valve was turned to the 200-L Tedlar®bag position for 

exposure to the test substance. Feed and water were withheld during exposure periods. A 

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

F. Animal Receipt and Acclimation/Pretest Period

Ten naïve male Beagle dogs were received from Ridglan Farms, Mt. Horeb, WI on 

29 October 2009. Each animal was uniquely identified with an ear tattoo by the supplier. All 

dogs were approximately 6 to 8 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 a 14-day acclimation and pretest period. The 

animals were observed twice daily for mortality and changes in appearance and/or behavior 

(moribundity). Fecal samples were collected during the acclimation period and checked for 

parasites; moderate Isospora were observed for animal no. 7367. This was not considered to 

have any impact on the study.

Each dog was conditioned to the exposure/restraint apparatus for a minimum of 4 separate 

acclimation sessions by placing the animal in the exposure/restraint apparatus for up to 

45 minutes and the muzzle-only mask for 30 minutes in order to acclimate it to test substance 

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 during the pretest period. Observations included, but were not limited to, changes in 

the skin, fur, eyes and mucous membranes, respiratory, circulatory, autonomic nervous and 

central nervous system function, gastrointestinal changes (appetite and fecal consistency), 

somatomotor activity, and behavior patterns.

Acute Cardiac Sensitization 

Study of in Beagle Dogs 

18
G. Immunizations Prior to Arrival

All animals were immunized prior to arrival at The immunizations included rabies, 

Bordetella, oral papilloma virus, distemper, hepatitis, Leptospira spp, Parvo, and parainfluenza.

H. Animal Housing and Exercise

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

cage flush 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 SOPs and the Animal Welfare Act 

(9 CFR Part 3). The animal facilities at are fully accredited by the Association for 

Assessment and Accreditation of Laboratory Animal Care International (AAALAC 

International).

I. 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 the animals were fed prior to baseline data collection on 

exposure days. The diet utilized at is a certified feed with appropriate analyses for potential 

contaminants performed by the manufacturer and provided to Feed lots used during the 

study are documented in the study records. Reverse osmosis-purified (on-site) municipal 

drinking water, delivered by an automatic watering system, was provided ad libitum, except 

during acclimation sessions and exposure periods. Municipal water supplying the facility is 

regularly sampled for contaminants according to SOPs. The results of the diet and water 

analyses are maintained at No contaminants were present in animal feed or water at 

concentrations that would be expected to affect the outcome of the study. 

J. Environmental Conditions

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

environmentally controlled room (B19). The room temperature and humidity controls were set 

to maintain environmental conditions of 68°F ± 5°F (20°C ± 3°C) and 50% ± 20% relative 

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

Metasys

® DDC Electronic Environmental control system. Actual mean temperature ranged from 

67.8°F to 68.2°F (19.9°C to 20.1°C) and mean relative humidity ranged from 39.3% to 48.8% 

during the study. Fluorescent lighting provided illumination for a 12-hour light (0600 hours to 

1800 hours)/12-hour dark photoperiod. Air handling units were set to provide a minimum of 

10 fresh air changes per hour.

K. Assignment of Animals to Treatment Groups

On 12 November 2009 (6 days prior to the initiation of test substance exposure), all available 

dogs had body weights recorded and detailed physical examinations performed. These data were 

collected using and reviewed by the Study Director. Following acclimation, animals 

that were judged suitable for testing were assigned to treatment groups using a computer 

randomization program. The animals were arbitrarily assigned to study. The assignment was 

Acute Cardiac Sensitization 

Study of in Beagle Dogs 

19
based upon health (body weights and detailed physical examinations), temperament, and pre-test 

ECG results. A printout containing the animal numbers and individual group assignments was 

generated. Body weights are presented in Table 3 and ranged from 6.1 kg to 8.7 kg on the days 

of exposure.

L. Animal Disposition

This study was non-terminal. Upon completion of the study, all animals were transferred to and 

maintained in the cardiovascular stock colony for possible use on future studies.

Acute Cardiac Sensitization 

Study of in Beagle Dogs 

20
PARAMETERS EVALUATED

A. Clinical Observations

A separate computer protocol was used to collect any findings (unscheduled observations) 

outside the detailed physical examinations. Only the presence of findings was recorded and 

entered into . The absence of findings was not recorded.

B. Electrocardiographic Examination

ECGs were recorded with a Burdick electrocardiograph. Only modified lead I, II, and III ECGs 

were collected with alligator clips placed on each limb. Chart speed was set at 10 mm/sec and 

response sensitivity was set at 10 mm/mV. ECGs were recorded continuously throughout the 

pre-exposure epinephrine dosing, exposure to the test substance, and for 5 minutes following 

administration of the challenge epinephrine dose (total of approximately 17 minutes). The points 

at which epinephrine was given and when exposure to the test substance was initiated were 

clearly marked on each ECG recording.

ECG recordings collected during exposure to the test substance and after administration of the 

challenge epinephrine dose were compared to the ECG recordings collected after administration 

of the pre-exposure epinephrine dose that was given in the absence of test substance. During the 

epinephrine challenges (or immediately following), the following types of criteria (though not 

exclusive) were applied in order to determine if sensitization had occurred:

 Eleven or more premature ventricular contractions (PVC) in 10 seconds (sec), with 

episodes of confluency

 Ventricular tachycardia

 Fibrillation

C. Statistical Methods

For body weight and test atmosphere concentration data, each mean was presented with the 

standard deviation (S.D.) and the number of animals or samples (N) used to calculate the mean.

Acute Cardiac Sensitization 

Study of in Beagle Dogs 

21
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 for data collected at 

including raw paper data, pertinent electronic storage media, and specimens, are retained in the 

Archives as specified in the study protocol and in compliance with regulatory requirements.

Acute Cardiac Sensitization 

Study of in Beagle Dogs 

22
RESULTS

A. Clinical Observations

Individual Data: Table 2

After exposure to 70,000 ppm continuous convulsions and excessive salivation were 

noted for animal nos. 7371 and 7372 as well as dilated pupils in both eyes for animal no. 7371.

After exposure to 90,000 ppm intermittent and then continuous convulsions, 

excessive salivation, and dilated pupils of both eyes were noted for animal no. 7363. Exposure 

was terminated due to the severity of the observed clinical signs. No other animal was exposed 

to the test substance at 90,000 ppm due to the adverse reaction shown by animal no. 7363.

After exposure to 80,000 ppm intermittent and then continuous convulsions and 

excessive salivation were noted for animal no. 7364. Exposure was terminated due to the 

severity of the observed clinical signs. No other animal was exposed to the test substance at 

80,000 ppm due to the adverse clinical observations noted for animal no. 7364.

B. Electrocardiographic Examinations

Individual Data: Table 4; Appendix C

During the pretest screening, no abnormal ECG findings were noted for any of the animals. 

There were no abnormal ECG findings noted during the 2 μg/kg pre-exposure epinephrine 

challenge for any of the animals. During the 3 and 4 μg/kg pre-exposure epinephrine challenge, 

1 episode of second degree atrioventricular block (AVB2) during each challenge was noted for 

animal no. 7368. During the 6 μg/kg pre-exposure epinephrine challenge, 11 episodes of escape 

complex (ESC) were noted for animal no. 7370 and 2 episodes of ESC were noted for animal 

no. 7372. During the 8μg/kg pre-exposure epinephrine challenge, multiple episodes of ESC 

were noted for animal nos. 7363, 7370, and 7372 (10, 19, and 3 episodes, respectively) along 

with 2 episodes of PVC noted for animal no. 7372. These findings can be common in dogs of 

this breed (Tilley, 1992; Atterson et al., 2009). There were no other ECG findings present for 

any of the other animals during the pre-exposure epinephrine challenge.

During the 70,000 ppm exposure, a single episode of premature atrial 

contraction (PAC) was noted for animal no. 7363 after the epinephrine challenge. All other 

animals exposed to at this level presented normal ECGs. Based on the adverse 

reactions noted for both animal nos. 7371 and 7372 previously discussed, exposure to the test 

substance was prematurely halted for both animals. The epinephrine challenge dose was not 

administered to animal no. 7372 due to the above-listed findings. Both of these animals were 

replaced; however, the replacement animals, nos. 7364 and 7367, did not receive the 

70,000 ppm exposure.

The only animal that was exposed to the test substance at 90,000 ppm (no. 7363) did not receive 

the complete exposure and also did not receive the epinephrine challenge due to the 

Acute Cardiac Sensitization 

Study of in Beagle Dogs 

23
adverse reaction to the test substance as previously discussed (see Clinical Observations). 

Exposure was suspended for all other animals at this exposure level due to the severity of the 

adverse reactions for animal no. 7363.

At the 80,000 ppm exposure level, animal no. 7364 presented 4 episodes of AVB2 

after the pre-exposure epinephrine dose was administered and 1 episode of AVB2 shortly after 

the start of test substance exposure. The epinephrine challenge dose was not administered during 

the exposure due to the severity of the adverse reactions to the test substance previously

discussed (see Clinical Observations). 

Due to the lack of PVCs with confluency, ventricular tachycardia, and fibrillation following the 

exposure of 70,000 ppm where animals were challenged with epinephrine, it was 

determined that exposure at this level did not result in cardiac sensitization. Because the 

epinephrine challenge was unable to be administered following exposure of 80,000 and 

90,000 ppm the potential for cardiac sensitization at these exposure levels was 

unable to be assessed.

Acute Cardiac Sensitization 

Study of in Beagle Dogs 

24
CONCLUSIONS

Based on the results of this study, muzzle-only vapor exposure to 70,000 ppm in 

Beagle dogs followed by intravenous administration of 8 μg/kg epinephrine did not produce 

evidence of cardiac sensitization. Evaluation for cardiac sensitization was not possible in the 

80,000 and 90,000 ppm exposure groups due to adverse clinical signs that precluded 

the administration of epinephrine challenge. Therefore, the no-observed-effect level (NOEL) for 

cardiac sensitization was 70,000 ppm 

Acute Cardiac Sensitization 

Study of in Beagle Dogs 

25
REFERENCES

Atterson, P., Voss, K., and Kopp, G. Occurrence and Variance of ECG Waveform 

Abnormalities in Canines and Non-Human Primates: What is Considered Normal? Safety

Pharmacology Society Meeting Poster, 2009.

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

Associated with Inhalation of Halon 1301. Am. Ind. Hygiene Assoc. Jour. 40:353-358. 

1979.

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 A., Marfield, M., Smith, P., and Mullin, L. Cardiac Arrhythmias and 

Aerosol “Sniffing”. Arch. Envir. Health, 22:265-279. 1971.

Tilley, L.P. Essentials of Canine and Feline Electrocardiography: Interpretation and 

Treatment, 3rd ed., Lippincott Williams & Wilkins: Philadelphia, PA, 1992.

Acute Cardiac Sensitization 

Study of in Beagle Dogs 

26
DEVIATIONS FROM THE PROTOCOL

This study was conducted in accordance with the protocol except for the following:

 Protocol Section 5.3 states that the purity of the test substance was 99.98%. 

The Certificate of Analysis listed the purity as 99.91413%. The value from the 

Certificate of Analysis was reported. This deviation had no impact on the study because 

the exposure concentrations were not corrected for purity and was within 0.1% of the

value in the protocol.

Reason for the deviation: The protocol did not match the Certificate of Analysis.

 Protocol Section 5.5 states that the physical description of the test substance 

was to be documented by Due to the volatility of the test substance and the fact 

that it was protected from light, a physical description was not documented. The 

Certificate of Analysis did not list the physical description; therefore, the lack of a 

physical description had no impact on the study since it could not be compared to the 

Certificate of Analysis.

Reason for the deviation: Physical description was not documented due to the volatility

of the test substance.

 Protocol Section 5.7 states that approximately 3 g of the test substance was to be 

withdrawn from the liquid line of the supplied cylinder into an appropriately sealed 

container. A Study Director Notification (SDN) dated 28 October 2009 directed that the 

test substance retention sample was to be collected by expelling a volume of gas into a 

1-L Tedlar®

bag. The sample was collected as directed by the original protocol which 

was not in adherence to the SDN. This deviation had no impact on the study since a 

retention sample was collected.

Reason for the deviation: Retention sample collection did not adhere to the SDN due to 

technician error.

 Protocol Section 8.6 states that controls were to be set to maintain test atmosphere 

exposure temperature at approximately 22°C (± 2°C) and humidity between 

approximately 40% and 60%. Exposure atmospheres in the test substance gas bags were 

prepared under ambient conditions. Temperature and humidity were not monitored. This 

deviation had no impact on the study since the protocol-prescribed temperature and 

humidity were similar to the ambient conditions in the area where the test substance gas 

bags were prepared.

Reason for the deviation: Temperature and humidity were not monitored for test 

substance exposure atmosphere due to technician error.

Acute Cardiac Sensitization 

Study of in Beagle Dogs 

27
 Protocol Section 8.6 states that all (6) animals were to be exposed to the test substance 

using muzzle-only exposure apparatus. For the 90,000 and 80,000 ppm 

exposures, only 1 dog was exposed to the test substance due to the severity of the adverse 

clinical signs observed in that animal. No other animals were exposed to those 

concentrations. This deviation had no impact on the study since appropriate study data 

were collected following the 70,000 ppm exposure.

Reason for the deviation: Not all animals were exposed due to the severity of adverse 

clinical signs.

 Protocol Section 8.9 states that the exposure duration to air was to be approximately

7 minutes and the exposure duration for the test substance was to be approximately

10 minutes. Animal nos. 7371 and 7372 were exposed to the 70,000 ppm 

concentration for approximately 8 and 2 minutes, respectively. Animal no. 7363 was 

exposed to the 90,000 ppm concentration for approximately 1 minute. 

Animal no. 7364 was exposed to the 80,000 ppm concentration for 

approximately 1 minute. The exposures for these animals were terminated prematurely

due to the severity of the adverse clinical signs observed. These deviations had no impact 

on the study since appropriate study data were collected following the 70,000 ppm 

 exposure.

Reason for the deviation: Exposure duration did not match the protocol due to the 

severity of the adverse clinical signs observed; exposure was halted.

 Protocol Section 8.9 states that the epinephrine challenge dose (established for each dog 

as described in section 8.8) was to be administered intravenously, into an appropriate 

vein (e.g., cephalic, saphenous, etc.) via a catheter with injection cap, using a dose 

volume of 0.1 mL/kg. The previously established epinephrine challenge dose for each 

animal was determined to be 8 μg/kg. Animal nos. 7372, 7363, and 7364 did not receive 

the epinephrine challenge during exposure of 70,000, 90,000, and 80,000 ppm

 respectively due to the severity of the adverse clinical observations noted for 

each animal. Animal no. 7364 was administered the incorrect dose volume of 0.87 mL

epinephrine. The animal was to receive 0.69 mL, resulting in an overdose of 0.18 mL. 

The actual epinephrine dose for this animal (based on a concentration of 80 μg/mL and a 

dose volume of 0.1 mL/kg) was approximately 10 μg/kg. This deviation did not have an 

impact on this study because inspection of the ECGs following the approximate 

10 μg/kg dose did not indicate any excessive response.

Reason for the deviation: The dose volume of epinephrine administered to animal 

no. 7364 did not match the protocol (technician did not check the animal identification 

prior to dosing).

 Protocol Section 9.1 states that all animals were to have a detailed physical examination 

weekly during the pretest period, at the time of animal selection, on the morning of 

baseline response to the epinephrine challenge, and on the morning of exposure and the 

data were to be entered into A detailed physical examination was not 

performed during study week -1. Additionally, animal nos. 7364 and 7367 were selected 

Acute Cardiac Sensitization 

Study of in Beagle Dogs 

28
to act as replacement animals on 18 November 2009 following the 70,000 ppm 

 exposure where 2 animals (nos. 7371 and 7372) had exposure terminated 

prematurely due to adverse clinical signs. The decision to use these animals as 

replacements occurred in the afternoon. The detailed physical examinations were not 

performed on the morning of baseline response to the epinephrine challenge and could 

not be performed thoroughly since the cage pans had already been flushed therefore 

precluding any cage-side observations. These deviations did not have any impact on the 

study since no adverse observations were noted during pretest or during the study.

Reasons for the deviation: Detailed physical examinations were not collected according 

to protocol specifications due to technician error and study schedule constraints.

 Protocol Section 9.2 states that individual body weights were to be recorded weekly 

during the pretest period, at the time of animal selection, on the morning of baseline 

response to the epinephrine challenge, and on the day(s) of exposure and after the 

completion of all exposures. Body weights were not recorded during study week -1. 

Additionally, animal nos. 7364 and 7367 were selected to act as replacement animals on 

18 November 2009 following the 70,000 ppm exposure where 2 animals 

(nos. 7371 and 7372) had exposure terminated prematurely due to adverse clinical signs. 

The decision to use these animals as replacements occurred in the afternoon. The body

weights were not recorded on the morning of baseline response to the epinephrine 

challenge. These deviations did not have any impact on the study since body weights 

were used mostly for the calculation of doses (which was done in the afternoon of 

baseline response to the epinephrine challenge) and were not specifically evaluated for 

health assessment.

Reasons for the deviation: Body weights were not collected according to protocol 

specifications due to technician error and study schedule constraints.

These deviations did not negatively impact the quality or integrity of the data or the outcome of 

the study.

Acute Cardiac Sensitization 

Study of in Beagle Dogs 

29
TABLES 1 - 4

Acute Cardiac Sensitization 

Study of in Beagle Dogs 

30
 TABLE 1 (DETAILED PHYSICAL EXAMINATIONS)

PROJECT NO. CARDIAC SENSITIZATION OF BY INHALATION IN DOGS PAGE 1

SPONSOR: INDIVIDUAL CLINICAL OBSERVATIONS

SPONSOR NO.

 TABLE RANGE: 11-13-09 TO 11-23-09

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

ANIMAL SEX GROUP CATEGORY DATE TIME OBSERVATIONS

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

7363M1 M 0 PPM EXCRETA 11-13-09 6:19 SOFT FECES

7364M1 M 0 PPM NORMAL 11-18-09 15:01 NO SIGNIFICANT CLINICAL OBSERVATIONS

7366M1 M 0 PPM NORMAL 11-13-09 6:20 NO SIGNIFICANT CLINICAL OBSERVATIONS

7367M1 M 0 PPM NORMAL 11-18-09 15:03 NO SIGNIFICANT CLINICAL OBSERVATIONS

7368M1 M 0 PPM NORMAL 11-13-09 6:21 NO SIGNIFICANT CLINICAL OBSERVATIONS

7370M1 M 0 PPM NORMAL 11-13-09 6:22 NO SIGNIFICANT CLINICAL OBSERVATIONS

7371M1 M 0 PPM EYES/EARS/NOSE 11-13-09 6:22 CLEAR DISCHARGE RIGHT EYE

7372M1 M 0 PPM NORMAL 11-13-09 6:23 NO SIGNIFICANT CLINICAL OBSERVATIONS

7363M2 M 70,000 PPM NORMAL 11-18-09 6:21 NO SIGNIFICANT CLINICAL OBSERVATIONS

7366M2 M 70,000 PPM EXCRETA 11-18-09 6:22 SOFT FECES

7368M2 M 70,000 PPM NORMAL 11-18-09 6:23 NO SIGNIFICANT CLINICAL OBSERVATIONS

7370M2 M 70,000 PPM NORMAL 11-18-09 6:24 NO SIGNIFICANT CLINICAL OBSERVATIONS

7371M2 M 70,000 PPM NORMAL 11-18-09 6:25 NO SIGNIFICANT CLINICAL OBSERVATIONS

7372M2 M 70,000 PPM NORMAL 11-18-09 6:26 NO SIGNIFICANT CLINICAL OBSERVATIONS

7363M3 M 90,000 PPM NORMAL 11-20-09 7:44 NO SIGNIFICANT CLINICAL OBSERVATIONS

7364M3 M 90,000 PPM NORMAL 11-20-09 7:46 NO SIGNIFICANT CLINICAL OBSERVATIONS

7366M3 M 90,000 PPM NORMAL 11-20-09 7:47 NO SIGNIFICANT CLINICAL OBSERVATIONS

7367M3 M 90,000 PPM NORMAL 11-20-09 7:45 NO SIGNIFICANT CLINICAL OBSERVATIONS

7368M3 M 90,000 PPM NORMAL 11-20-09 7:48 NO SIGNIFICANT CLINICAL OBSERVATIONS

7370M3 M 90,000 PPM NORMAL 11-20-09 7:46 NO SIGNIFICANT CLINICAL OBSERVATIONS

7364M4 M 80,000 PPM NORMAL 11-23-09 7:38 NO SIGNIFICANT CLINICAL OBSERVATIONS

7366M4 M 80,000 PPM NORMAL 11-23-09 7:39 NO SIGNIFICANT CLINICAL OBSERVATIONS

7367M4 M 80,000 PPM NORMAL 11-23-09 7:41 NO SIGNIFICANT CLINICAL OBSERVATIONS

7368M4 M 80,000 PPM NORMAL 11-23-09 7:40 NO SIGNIFICANT CLINICAL OBSERVATIONS

7370M4 M 80,000 PPM NORMAL 11-23-09 7:42 NO SIGNIFICANT CLINICAL OBSERVATIONS

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

 

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Study of in Beagle Dogs Acute Cardiac Sensitization 

31
 TABLE 2 (UNSCHEDULED OBSERVATIONS)

PROJECT NO. CARDIAC SENSITIZATION OF BY INHALATION IN DOGS PAGE 1

SPONSOR: INDIVIDUAL CLINICAL OBSERVATIONS

SPONSOR NO.

 TABLE RANGE: 11-13-09 TO 11-23-09

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

ANIMAL SEX GROUP CATEGORY DATE TIME OBSERVATIONS

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

7371M2 M 70,000 PPM BEHAVIOR/CNS 11-18-09 11:47 CONVULSIONS, CONTINUOUS

7371M2 M 70,000 PPM EYES/EARS/NOSE 11-18-09 11:47 PUPIL DILATED LEFT EYE

 11-18-09 11:47 PUPIL DILATED RIGHT EYE

7371M2 M 70,000 PPM ORAL/DENTAL 11-18-09 11:50 EXCESSIVE SALIVATION

7372M2 M 70,000 PPM BEHAVIOR/CNS 11-18-09 12:40 CONVULSIONS, CONTINUOUS

7372M2 M 70,000 PPM ORAL/DENTAL 11-18-09 12:42 EXCESSIVE SALIVATION

7363M3 M 90,000 PPM BEHAVIOR/CNS 11-20-09 9:56 CONVULSIONS, INTERMITTENT

 11-20-09 9:57 CONVULSIONS, CONTINUOUS

7363M3 M 90,000 PPM EYES/EARS/NOSE 11-20-09 10:02 PUPIL DILATED LEFT EYE

 11-20-09 10:02 PUPIL DILATED RIGHT EYE

7363M3 M 90,000 PPM ORAL/DENTAL 11-20-09 10:00 EXCESSIVE SALIVATION

7364M4 M 80,000 PPM BEHAVIOR/CNS 11-23-09 9:32 CONVULSIONS, INTERMITTENT

 11-23-09 9:32 CONVULSIONS, CONTINUOUS

7364M4 M 80,000 PPM ORAL/DENTAL 11-23-09 9:33 EXCESSIVE SALIVATION

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

 

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Study of in Beagle Dogs Acute Cardiac Sensitization 

32
 TABLE 3

PROJECT NO. CARDIAC SENSITIZATION OF BY INHALATION IN DOGS PAGE 1

SPONSOR: INDIVIDUAL BODY WEIGHTS [KG]

SPONSOR NO.

 MALE GROUP: 0 PPM

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

DAY 0 5 7 10 10*

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

ANIMAL

7363M1 6.1 NA NA NA NA

7364M1 7.1 NA NA NA NA

7366M1 8.2 NA NA NA NA

7367M1 8.4 NA NA NA NA

7368M1 8.2 NA NA NA NA

7370M1 7.3 NA NA NA NA

7371M1 7.3 NA NA NA NA

7372M1 8.6 NA NA NA NA

MEAN 7.7 NA NA NA NA

S.D. 0.84 NA NA NA NA

 N 8 NA NA NA NA

NA = NOT APPLICABLE

 * = BODY WEIGHTS RECORDED ON THE LAST DAY OF THE STUDY AFTER THE COMPLETION OF ALL TEST SUBSTANCE EXPOSURES

Study of in Beagle Dogs Acute Cardiac Sensitization 

33
 TABLE 3

PROJECT NO. CARDIAC SENSITIZATION OF BY INHALATION IN DOGS PAGE 2

SPONSOR: INDIVIDUAL BODY WEIGHTS [KG]

SPONSOR NO.

 MALE GROUP: 70,000 PPM

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

DAY 0 5 7 10 10*

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

ANIMAL

7363M2 NA 7.2 NA NA NA

7366M2 NA 8.3 NA NA NA

7368M2 NA 8.2 NA NA NA

7370M2 NA 7.3 NA NA NA

7371M2 NA 7.2 NA NA 7.7

7372M2 NA 8.7 NA NA 9.5

MEAN NA 7.8 NA NA 8.6

S.D. NA 0.67 NA NA 1.29

 N NA 6 NA NA 2

NA = NOT APPLICABLE

 * = BODY WEIGHTS RECORDED ON THE LAST DAY OF THE STUDY AFTER THE COMPLETION OF ALL TEST SUBSTANCE EXPOSURES

Study of in Beagle Dogs Acute Cardiac Sensitization 

34
 TABLE 3

PROJECT NO. CARDIAC SENSITIZATION OF BY INHALATION IN DOGS PAGE 3

SPONSOR: INDIVIDUAL BODY WEIGHTS [KG]

SPONSOR NO.

 MALE GROUP: 90,000 PPM

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

DAY 0 5 7 10 10*

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

ANIMAL

7363M3 NA NA 7.4 NA 7.8

7364M3 NA NA 6.7 NA NA

7366M3 NA NA 8.4 NA NA

7367M3 NA NA 8.1 NA NA

7368M3 NA NA 8.4 NA NA

7370M3 NA NA 7.6 NA NA

MEAN NA NA 7.8 NA 7.8

S.D. NA NA 0.67 NA 0.00

 N NA NA 6 NA 1

NA = NOT APPLICABLE

 * = BODY WEIGHTS RECORDED ON THE LAST DAY OF THE STUDY AFTER THE COMPLETION OF ALL TEST SUBSTANCE EXPOSURES

Study of in Beagle Dogs Acute Cardiac Sensitization 

35
 TABLE 3

PROJECT NO. CARDIAC SENSITIZATION OF BY INHALATION IN DOGS PAGE 4

SPONSOR: INDIVIDUAL BODY WEIGHTS [KG]

SPONSOR NO.

 MALE GROUP: 80,000 PPM

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

DAY 0 5 7 10 10*

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

ANIMAL

7364M4 NA NA NA 6.9 7.0

7366M4 NA NA NA 8.7 9.2

7367M4 NA NA NA 8.0 8.3

7368M4 NA NA NA 8.4 8.8

7370M4 NA NA NA 7.7 8.0

MEAN NA NA NA 7.9 8.3

S.D. NA NA NA 0.70 0.83

 N NA NA NA 5 5

NA = NOT APPLICABLE

 * = BODY WEIGHTS RECORDED ON THE LAST DAY OF THE STUDY AFTER THE COMPLETION OF ALL TEST SUBSTANCE EXPOSURES

 

 01/05/2010

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Study of in Beagle Dogs Acute Cardiac Sensitization 

36
 TABLE 4 (ALL ANIMALS)

PROJECT NO. CARDIAC SENSITIZATION OF BY INHALATION IN DOGS PAGE 1

SPONSOR: INDIVIDUAL CARDIAC SENSITIZATION RESPONSES

SPONSOR 

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

EXPOSURE LEVEL (PPM) ANIMAL NO. RESPONSE: 2 UG/KG 3 UG/KG 4 UG/KG 6 UG/KG 8 UG/KG

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

0 7363M1 - - - - -

 7364M1 - - - - -

 7366M1 - - - - -

 7367M1 - - - - -

 7368M1 - - - - -

 7370M1 - - - - -

 7371M1 - - - - -

 7372M1 - - - - -

70,000 7363M2 NA NA NA NA -

 7366M2 NA NA NA NA -

 7368M2 NA NA NA NA -

 7370M2 NA NA NA NA -

 7371M2-A NA NA NA NA -

 7372M2-B NA NA NA NA NA

90,000 7363M3-B NA NA NA NA NA

 7364M3 NA NA NA NA NA

 7366M3 NA NA NA NA NA

 7367M3 NA NA NA NA NA

 7368M3 NA NA NA NA NA

 7370M3 NA NA NA NA NA

80,000 7364M4-B NA NA NA NA NA

 7366M4 NA NA NA NA NA

 7367M4 NA NA NA NA NA

 7368M4 NA NA NA NA NA

 7370M4 NA NA NA NA NA

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

NA = NOT APPLICABLE

 - = NO RESPONSE INDICATIVE OF CARDIAC SENSITIZATION

 A = EXPOSURE TO TEST SUBSTANCE HALTED DUE TO ADVERSE CLINICAL OBSERVATIONS; EPINEPHRINE CHALLENGE WAS ADMINISTERED

 B = EXPOSURE TO TEST SUBSTANCE HALTED DUE TO ADVERSE CLINICAL OBSERVATIONS; EPINEPHRINE CHALLENGE WAS NOT ADMINISTERED

 

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Study of in Beagle Dogs Acute Cardiac Sensitization 

37
APPENDIX A

Certificates of Analysis

Acute Cardiac Sensitization 

Study of in Beagle Dogs 

38
Acute Cardiac Sensitization 

Study of in Beagle Dogs 

39
Acute Cardiac Sensitization 

Study of in Beagle Dogs 

40
APPENDIX B 

Exposure Atmosphere Generation Report

Acute Cardiac Sensitization 

Study of in Beagle Dogs 

41
ACUTE CARDIAC SENSITIZATION

STUDY OF IN BEAGLE DOGS

Exposure Atmosphere Generation Report

Inhalation Department

1

Acute Cardiac Sensitization 

Study of in Beagle Dogs 

42
 

 

 
TABLE OF CONTENTS

Page

KEY STUDY PERSONNEL AND REPORT SUBMISSION ...................................................2

TABLE OF CONTENTS ..............................................................................................................3

INDEX OF FIGURES ...................................................................................................................3

INDEX OF TABLES .....................................................................................................................3

EXPOSURE METHODS ..............................................................................................................4

A. Exposure Regimen...............................................................................................................4

B. Inhalation Exposure System Description.............................................................................4

C. Exposure Environmental Conditions...................................................................................4

TEST ATMOSPHERE PREPARATION METHODS..............................................................5

METHODS OF CHARACTERIZATION OF EXPOSURE ATMOSPHERES .....................6

A. Analyzed Exposure Concentrations.....................................................................................6

1. Gas Chromatograph Calibration...............................................................................................................7

RESULTS OF CHARACTERIZATION OF ACTUAL EXPOSURE 

CONCENTRATIONS ...................................................................................................................9

A. Analyzed Exposure Concentrations.....................................................................................9

INDEX OF FIGURES

Figure 1 Atmosphere Generation and Exposure System.........................................................11

INDEX OF TABLES

Table 1 Individual Test Atmosphere Concentrations ............................................................13

3

Acute Cardiac Sensitization 

Study of in Beagle Dogs 

44
EXPOSURE METHODS

A. Exposure Regimen

The test substance, was administered as muzzle-only inhalation exposures at 3 target 

concentrations. A detailed description of the study design and exposure schedule is presented in 

the main report. The following table presents the overall schedule for inhalation exposures:

Group

Number

Target Exposure

Concentration (ppm)

Exposure

Date

2 70,000 18 November 2009

3 90,000 20 November 2009

4 80,000 23 November 2009

B. Inhalation Exposure System Description

Each inhalation exposure was conducted using a 200-L Tedlar®gas bag

(SKC, Inc., Eighty-Four, Pennsylvania, U.S.A.) attached to a specialized muzzle placed on the 

animal. The muzzle allowed the animal to inhale directly from the gas bag and exhale directly to 

exhaust through a one-way breathing valve as shown schematically in Figure 1. The animal 

received room air during non-exposure periods via a 3-way valve which was in-line between the 

gas bag and muzzle. Animal exposures were performed by the Safety Pharmacology 

Department.

C. Exposure Environmental Conditions

The atmosphere was not monitored for temperature and humidity. The test substance

atmosphere was prepared using dry compressed air and was maintained at room temperature 

throughout the exposure period. Oxygen content of the test substance atmosphere was measured 

prior to each exposure using a Drager Pac III equipped with a calibrated oxygen sensor 

(Draeger Safety Inc., Pittsburgh, Pennsylvania, U.S.A.). Oxygen content was 20.1% to 20.9% 

for all exposures.

4

Acute Cardiac Sensitization 

Study of in Beagle Dogs 

45
TEST ATMOSPHERE PREPARATION METHODS

The test substance atmosphere was prepared in 200-L Tedlar®gas bags as follows: A mixture of 

test substance and air was prepared by metering test substance in a gas bag containing a known 

volume of compressed air. Using a dry test meter (model DTM-200A, Elster American Meter 

Co., Nebraska City, Nebraska, U.S.A.), approximately 150 to 166 L of air was added to each gas 

bag. The test substance was delivered as a gas from the bulk test substance cylinder to the gas 

bag using a needle valve and Top Trak mass flowmeter (model no. 822S-L-2-OK1-PV1-V1, 

Sierra Instruments, Inc., Monterey, California, U.S.A.). During the test substance delivery, the 

Top Trak was approximately 8 to 12 indicated for approximately 7, 12, and 10 minutes for

Groups 2, 3, and 4, respectively. The gas cylinder was heated using an Omega heat tape

(model no. SRT-101-100) and controlled using a thermocouple and CN370 temperature 

controller (Omega Engineering, Inc., Stamford, Connecticut, U.S.A.) set to 50°C. If necessary, 

additional test substance or air was added to the gas bag to provide the desired analyzed 

concentration.

5

Acute Cardiac Sensitization 

Study of in Beagle Dogs 

46
METHODS OF CHARACTERIZATION OF EXPOSURE ATMOSPHERES

A. Analyzed Exposure Concentrations

Analyzed exposure concentrations were determined prior to and following each animal exposure 

using a gas chromatograph (GC). Samples were manually collected from each gas bag. Gas 

sample injection onto the chromatography column occurred via an internal gas-sampling valve 

with a sample loop. The chromatograph was displayed and the area under the sample peak was 

calculated and stored. The concentration, in parts per million (ppm), was calculated using an 

ln-quadratic equation based on the GC calibration curve. The following table summarizes the 

operational parameters for the gas chromatography methods:

Instrument: Hewlett Packard 5890 Series II

Detector: Flame Ionization Detector (FID)

Column: J&W Scientific, DB-5, 30 m × 0.530 mm ID,

1.5-micron film thickness

Gases (mL/minute)

Carrier - Helium: 30

Fuel - Hydrogen: 23

Air: 301

Temperatures (oC)

Column: 120

Detector: 250

Auxiliary 120

Integrator: Hewlett Packard Model 3396 Series II

6

Acute Cardiac Sensitization 

Study of in Beagle Dogs 

47
Run Parameters 

Chart Zero Offset: 0

Chart Attenuation: 0

Chart Speed: 1.5 cm/minute

Peak Area Rejection Value: 0

Peak Threshold: 0

Peak Width: 0.04

Retention Time (minutes): Approximately 0.4

1. Gas Chromatograph Calibration

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

concentrations of the test substance in 1-L Tedlar®gas bags. A 1,000,000 ppm basis bag was 

prepared by metering the test substance gas from the bulk test substance cylinder to a 1-L 

Tedlar®bag. Standard bags were prepared by metering a known volume of compressed air into 

the bag using a 1000-mL gas syringe (model S-1000, Hamilton Co., Reno, Nevada, U.S.A.), and 

then injecting a known volume of test substance from the basis bag. Concentrations of the 

gas-phase standards are calculated as follows:

Concentration (ppm) = 106

× V

(A + V)

Where: V = Volume of test substance injected into bag, mL

106

= Basis bag concentration, ppm

A = Volume of air used to prepare bag, mL

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

analyzed in triplicate. The curve was defined using standards prepared at 3 vapor concentrations 

spanning an appropriate range relative to the target exposure concentrations. The prime 

calibration curve was prepared using the quadratic formula and was used to calculate exposure 

concentrations for all exposure atmospheres. 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

7

Acute Cardiac Sensitization 

Study of in Beagle Dogs 

48
The following table summarizes the standard parameters used in the exposures:

Standard

(ppm)

Volume of Test 

Substance (mL)

Volume of

Air (mL)

58,824 50 800

90,909 80 800

130,435 120 800

8

Acute Cardiac Sensitization 

Study of in Beagle Dogs 

49
RESULTS OF CHARACTERIZATION OF ACTUAL EXPOSURE CONCENTRATIONS

A. Analyzed Exposure Concentrations

Individual analyzed concentrations for each group are presented in Table 1 of this appendix. The 

following table summarizes the mean analyzed concentrations for each exposure:

Group: 2 3 4

Mean Concentration (ppm): 71,477 90,416 82,286

Standard Deviation: 3238 1179 571

N: 12 2 2

9

Acute Cardiac Sensitization 

Study of in Beagle Dogs 

50
FIGURE 1

10

Acute Cardiac Sensitization 

Study of in Beagle Dogs 

51
FIGURE 1: ATMOSPHERE GENERATION AND EXPOSURE SYSTEM

200-L

Tedlar Bag

3- Way

Valve 1-Way

Valve

1-Way

Valve

Dog Mask

To Exhaust

Room Air

 11

Acute Cardiac Sensitization 

Study of in Beagle Dogs 

52
TABLE 1

12

Acute Cardiac Sensitization 

Study of in Beagle Dogs 

53
 INHALAT E 1

PROJECT CARDIAC SENSITIZATION OF BY INHALATION IN DOGS PAGE 1

SPONSOR INDIVIDUAL TEST AT CONCENTRATIONS

SPONSOR 

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

 TARGET CONCENTRATION: 70,000 PPM GROUP 2

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

 ANIMAL NO. CONCENTRATION (PPM)

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

 7363M2 71597

 75418

 7366M2 69922

 68884

 7368M2 75418

 74644

 7370M2 68884

 68057

 7371M2 74644

 74609

 7372M2 68057

 67587

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

 MEAN 71477

 SD 3238

 CV (%) 4.5

 N 12

13

Study of in Beagle Dogs Acute Cardiac Sensitization 

54
 INHALAT E 1

PROJECT CARDIAC SENSITIZATION OF BY INHALATION IN DOGS PAGE 2

SPONSOR INDIVIDUAL TEST AT CONCENTRATIONS

SPONSOR 

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

 TARGET CONCENTRATION: 90,000 PPM GROUP 3

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

 ANIMAL NO. CONCENTRATION (PPM)

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

 7363M3 91249

 89582

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

 MEAN 90416

 SD 1179

 CV (%) 1.3

 N 2

14

Study of in Beagle Dogs Acute Cardiac Sensitization 

55
 INHALAT E 1

PROJECT CARDIAC SENSITIZATION OF BY INHALATION IN DOGS PAGE 3

SPONSOR INDIVIDUAL TEST AT CONCENTRATIONS

SPONSOR 

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

 TARGET CONCENTRATION: 80,000 PPM GROUP 4

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

 ANIMAL NO. CONCENTRATION (PPM)

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

 7364M4 82689

 81882

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

 MEAN 82286

 SD 571

 CV (%) 0.7

 N 2

 

 R:12/10/2009

15

Study of in Beagle Dogs Acute Cardiac Sensitization 

56
APPENDIX C

Individual Electrocardiographic Data

Acute Cardiac Sensitization 

Study of in Beagle Dogs 

57
Acute Cardiac Sensitization Study – 

Pretest Screening 

Collection Date: 9 November 2009 

Animal 

No. FINDINGS

7363 NSCO 

7364 NSCO 

7365 NSCO 

7366 NSCO 

7367 NSCO 

7368 NSCO 

7369 NSCO 

7370 NSCO 

7371 NSCO 

7372 NSCO 

NSCO: No Significant Clinical Observations 

Acute Cardiac Sensitization 

Study of in Beagle Dogs 

58
Acute Cardiac Sensitization Study – 

Epinephrine Challenge 

Collection Date: 13 November 2009 

Animal 

No. FINDINGS

7366M1 2 μg/kg Epinephrine: NSCO 

3 μg/kg Epinephrine: NSCO 

4 μg/kg Epinephrine: NSCO 

6 μg/kg Epinephrine: NSCO 

8 μg/kg Epinephrine: NSCO 

7363M1 2 μg/kg Epinephrine: NSCO 

3 μg/kg Epinephrine: NSCO 

4 μg/kg Epinephrine: NSCO 

6 μg/kg Epinephrine: NSCO 

8 μg/kg Epinephrine: ESC (10) between approximately 15 and 34 seconds postepinephrine challenge 

7368M1 2 μg/kg Epinephrine: NSCO 

3 μg/kg Epinephrine: AVB2 (1) approximately 30 seconds post-epinephrine 

challenge 

4 μg/kg Epinephrine: AVB2 (1) between approximately 15 and 16 seconds 

post-epinephrine challenge 

6 μg/kg Epinephrine: NSCO 

8 μg/kg Epinephrine: NSCO 

AVB2: 2nd Degree Atrioventricular Block 

ESC: Escape Complex 

NSCO: No Significant Clinical Observations 

( ): Denotes the Total Number of Incidences 

Acute Cardiac Sensitization 

Study of in Beagle Dogs 

59
Acute Cardiac Sensitization Study – 

Epinephrine Challenge 

Collection Date: 13 November 2009 

Animal 

No. FINDINGS

7370M1 2 μg/kg Epinephrine: NSCO 

3 μg/kg Epinephrine: NSCO 

4 μg/kg Epinephrine: NSCO 

6 μg/kg Epinephrine: ESC (11) between approximately 17 and 45 seconds 

post-epinephrine challenge 

8 μg/kg Epinephrine: ESC (19) between approximately 17 and 52 seconds 

post-epinephrine challenge 

7371M1 2 μg/kg Epinephrine: NSCO 

3 μg/kg Epinephrine: NSCO 

4 μg/kg Epinephrine: NSCO 

6 μg/kg Epinephrine: NSCO 

8 μg/kg Epinephrine: NSCO 

7372M1 2 μg/kg Epinephrine: NSCO 

3 μg/kg Epinephrine: NSCO 

4 μg/kg Epinephrine: NSCO 

6 μg/kg Epinephrine: ESC (2) between approximately 34 and 35 seconds postepinephrine challenge 

8 μg/kg Epinephrine: PVC (2) between approximately 11 and 15 seconds postepinephrine challenge 

ESC (3) between approximately 24 and 41 seconds postepinephrine challenge 

ESC: Escape Complex 

NSCO: No Significant Clinical Observations 

PVC: Premature Ventricular Contraction 

( ): Denotes the Total Number of Incidences 

Acute Cardiac Sensitization 

Study of in Beagle Dogs 

60
Acute Cardiac Sensitization Study – 

Epinephrine Challenge 

Collection Date: 18 November 2009 

Animal 

No. FINDINGS

7364M1 2 μg/kg Epinephrine: NSCO 

3 μg/kg Epinephrine: NSCO 

4 μg/kg Epinephrine: NSCO 

6 μg/kg Epinephrine: NSCO 

8 μg/kg Epinephrine: NSCO 

7367M1 2 μg/kg Epinephrine: NSCO 

3 μg/kg Epinephrine: NSCO 

4 μg/kg Epinephrine: NSCO 

6 μg/kg Epinephrine: NSCO 

8 μg/kg Epinephrine: NSCO 

NSCO: No Significant Clinical Observations 

Acute Cardiac Sensitization 

Study of in Beagle Dogs 

61
Acute Cardiac Sensitization Study – 

70,000 ppm 

Collection Date: 18 November 2009 

Animal 

No. FINDINGS

7371M2 Animal did not receive complete exposure to test substance 

7372M2 Animal did not receive complete exposure to test substance or epinephrine challenge 

7363M2 PAC (1) between approximately 5 and 6 seconds post-epinephrine challenge during exposure 

to test substance 

7366M2 NSCO 

7368M2 NSCO 

7370M2 NSCO 

NSCO: No Significant Clinical Observations 

PAC: Premature Atrial Contraction 

( ): Denotes the Total Number of Incidences 

Acute Cardiac Sensitization 

Study of in Beagle Dogs 

62
Acute Cardiac Sensitization Study – 

90,000 ppm 

Collection Date: 20 November 2009

Animal 

No. FINDINGS

7363M3 Animal did not receive complete test substance exposure or the epinephrine challenge during 

exposure 

Acute Cardiac Sensitization 

Study of in Beagle Dogs 

63
Acute Cardiac Sensitization Study – 

80,000 ppm 

Collection Date: 23 November 2009 

Animal 

No. FINDINGS

7364M4 

AVB2 (1) approximately 55 seconds following the pre-exposure epinephrine challenge 

AVB2 (3) between approximately 2 to 5 minutes following the pre-exposure epinephrine 

challenge 

AVB2 (1) approximately 8 seconds after the start of the test substance exposure 

During test substance exposure the epinephrine challenge was not given due to an adverse 

reaction 

AVB2: 2nd Degree Atrioventricular Block 

( ): Denotes the Total Number of Incidences 

Acute Cardiac Sensitization 

Study of in Beagle Dogs 

64
APPENDIX D

Example ECG Waveforms

Acute Cardiac Sensitization 

Study of in Beagle Dogs 

65
NORMAL ECG

TRACING

Study of in Beagle Dogs 

Acute Cardiac Sensitization 

66
MULTIPLE 

PREMATURE VENTRICULAR CONTRACTIONS (PVC) 

Study of in Beagle Dogs 

Acute Cardiac Sensitization 

67
APPENDIX E

Study Protocol

Acute Cardiac Sensitization 

Study of in Beagle Dogs 

68
Acute Cardiac Sensitization 

Study of in Beagle Dogs 

69
Acute Cardiac Sensitization 

Study of in Beagle Dogs 

70
Acute Cardiac Sensitization 

Study of in Beagle Dogs 

71
Acute Cardiac Sensitization 

Study of in Beagle Dogs 

72
Acute Cardiac Sensitization 

Study of in Beagle Dogs 

73
Acute Cardiac Sensitization 

Study of in Beagle Dogs 

74
Acute Cardiac Sensitization 

Study of in Beagle Dogs 

75
Acute Cardiac Sensitization 

Study of in Beagle Dogs 

76
Acute Cardiac Sensitization 

Study of in Beagle Dogs 

77
Acute Cardiac Sensitization 

Study of in Beagle Dogs 

78
Acute Cardiac Sensitization 

Study of in Beagle Dogs 

79
Acute Cardiac Sensitization 

Study of in Beagle Dogs 

80
Acute Cardiac Sensitization 

Study of in Beagle Dogs 

81
Acute Cardiac Sensitization 

Study of in Beagle Dogs 

82
Acute Cardiac Sensitization 

Study of in Beagle Dogs 

83
Acute Cardiac Sensitization 

Study of in Beagle Dogs 

84