Page 1 of 84 FINAL REPORT
STUDY TITLE
ACUTE CARDIAC SENSI TI ZATION STUDY OF IN BEAGLE DOGS

STUDY NUMBER

STUDY DIRECTOR

STUDY INITIATION DATE
21 October 2009

STUDY COMPLETION DATE 3 February

2010
PERFORMING LABORATO RY

SPONSOR STUDY NUMBER

Work Request No.: Service Code No.: Study No.:
SPONSORQUALITY ASSURANCE UN IT STATEMENT

Phases Inspected

Date(s) of Inspection(s) Phase

Inspected Date(s) Findings Reported to Study Director Date(s) Findi ngs

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 Inhalatio n 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 wi th the U.S. EPA GL P Regulations (40 CFR Part 792 ), the standard operating procedures ( SOPs ), and the S ponsor'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 y earl y 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 stud y. 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 S ponsor. Acute Cardiac Sensitization Study of n Beagle Dogs

3TABLE 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

I nformation

................................ ................................ ................................ 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 Sy stem

................................ ................................ ................................ ........................ 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. Immunization s Prior to Arrival

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

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

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

................................ ................................ ................................ . 19 K. As signment 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

6Page

B. Electro cardiographic 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

7INDEX OF TABLES Page

Tabl e 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

8INDEX OF APPEND ICES 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

9STUDY INFORMATION
Substance Teste d:
Number: Purity : 99.91 413 % Known Impurities: None to be of toxicological significance at this time

Sponsor:

Study I nitiated/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

10SUMMARY

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 sensitiz ation 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 response s to the epinephrine challenge doses were collected 5 day s prior to exposure to the test substance. A predetermined challenge dose was established during this baseline prior to exposure f or 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 approximat ely

5 minutes prior to exposure to the test substance as well as approximately 5 minutes following exposure to the test substance. Two animals display ed an adv erse reaction to 70,000 ppm and were replaced for subsequent exposures . The replacemen t animals were not exposed to the first exposure level of 70,000 ppm. One animal display ed 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 selec ted 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 colon y. I ndividual body weights were recorded on exposure day s 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 display ed continuous convulsions, excessive salivation, and/or dilated pupils foll owing test substance exposure. During the next exposure of 90,000 ppm 1 animal display ed continuous convulsions, excessive salivation, and dilated pupils dur ing test substance exposure. No further animals were exposed to this concentration. Acute Cardiac Sensitization Study of in Beagle Dogs

11Duri ng the last exposure of 80,000 ppm the first animal exposed display ed

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 p pm

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 b y 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 e pinephrine. Therefore, the n o - observed -e ffect level (NOEL) for cardiac sensitization was 70,000 ppm Acute Cardiac Sensitization Study of in Beagle Dogs

12INTRODUCTION

A. General Study Information This report presents the data from the stud y entitled “Acute Car diac Sensitization Study of in Beagle Dogs”. Due to software spacing constraints, the study title appears a s “Cardiac Sensitization of by I nhalation in Dogs” on the report tables. The following computer protocols were use d 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 star ting 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 tr ansferred to cardiovascular stock colony ) Acute Cardiac Sensitization Study of in Beagle Dogs

13STUDY DESIGN

Animal receipt

Acclimation/Pre

- test ECG screening period

Animals arbitraril y assigned to study Detailed phy sical examinations conducted and body weights recorded prior to exposure; clinical observations recorded as

needed

In dividual 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 p re - 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

14EXPERIMENTAL PROCEDU RES

- MATERIALS AN D METHODS

A. Test Substance a nd 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 Phy sical

Description ( ) Lot no. Exp. date: 30 June 2011 [ ID no. ] 1 cy linder Gross weight: 15 kg Not taken

Documentation regarding the purit y and stability of the test subs tance is on file with the Sponsor and A Certificate of Anal ys is 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, an d 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 s tudy was received from Sigma

- Aldrich, In c., St. L ouis, MO on 5 October 2009 as follows:

Identification Quantity Received Phy sical

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 Anal ys is for the ep inephrine 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 glassw are 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

1580 μg/mL formulation was weighed into a tared weighing vessel. To quantitatively transfer th e 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 Corpora tion, 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 calibr ation mark. The 80 μg/mL epinephrine formulation was sterile

- filtered through a 0.22 -μm (Durapore ® ) P VDF s yringe filter (lot nos. R9KN29064 and R9MN44305, expiry dates: 1 August 2012 and 1 September 2012, respectivel y, manufactured by Millipore Corporati on, Cork, I reland) and capped with a septum. The 20, 30, 40, and 60 μg/mL epinephrine formulations were pr epared b y diluting the 80 μg/mL epinephrine formulation with the respective appropriate amounts of 0.9% saline for injection, U.S.P. The dilutions we re 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 s ys tem 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 betw een approximately

7 and 9 months of age at the initiation of exposure.
C. Organization o f Test Groups, Dose L evels , a nd 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 phy sical examinations were performed in the morning of the assessment of baseline response to the epinephrine challenges. The epi nephrine 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 epin ephrine 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 th is 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 s ubstance. Baseline responses to the epinephrine challenge doses were collected 5 day s 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 appa ratus 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 ) . I ndividual body weights were recorded and detailed ph ys ica l examinations were performed on the day s of exposure , and body weights were also recorded once after all exposures were complete. The results of the detailed phy sical Acute Cardiac Sensitization Study of in Beagle Dogs

16examinations performed on the day s 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 exp osure 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 Novem ber 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 g eneral population. The muzzle

- only method of exposure was selected to achieve precise control Acute Cardiac Sensitization Study of in Beagle Dogs

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

were e x pected.
D. Exposure Methods All animals were exposed to the test substance using a muzzle- only exposure apparatus connected to a Tedlar ® bag to suppl y the target concentration of the test substance vapor and a minimum oxy gen content of 19%. The control atmosphere (the first 7 minutes of each exposure) was filtere d room air. Each dog was comfortabl y 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 c onducted using a muzzle

- only s ystem consis ting 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 fo r each exposure. The addition of ox ygen was not required since ox ygen content of all exposure bags was above 19%. From the 200

- L Tedlar ® bag, test substance was administered thro ugh 22

- mm corrugated tubing connected to a 3

- way valve. During the pre

- expo sure 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 Far ms, Mt. Horeb, WI on 29 October 2009. Each anima l 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 considere d suitable for the stud y were housed for a 14

- day acclimation and pretest period. The animals were observed twice dail y for mortality and changes in appearance and/or behavior (moribundity ). Fecal samples were collected during the acclimation period and checked for parasites; moderate Isospora wer e observed for animal no. 7367. This was not considered to have an y impact on the study. Each dog was conditioned to the exposure/restraint apparatus for a minimum of 4 separate acclimation session s by

placing t he animal in the exposure/r estraint 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 phy sical examinations were performed during the pretest period. Observations included, but were not limited to, changes in the skin, fur, ey es and mucous membranes, respiratory , circulator y, autonomic nervous and central nervous s ys tem function, gastrointestinal changes (appetite and fecal consistency ), somatomotor activity , and behavior patterns. Acute Cardiac Sensitization Study of in Beagle Dogs

18G. Immunizations Prior to Arrival

All animals were immunized prior to arrival at The immuni zations 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 f lush 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 regu lar 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 Inte rnational (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 day s. The diet utilized at is a certified feed with appropriate anal ys es for potential contaminants performed by the manufacturer and provided to Feed lots used during the study are documented in the study records. Reverse osmosis

- puri fied (on

- site) municipal drinking water, delivered by an automatic watering s ys tem, was provided ad libitum, except during acclimation sessions and exposure periods. Municipal water suppl yi ng the facility is regularl y sampled for contaminants according to SOPs. The results of the diet and water anal ys es are maintained at No contaminants were present in animal feed or water at concentrations that would be expected to affect the outcome of the stud y.
J. Environmental Conditions
All animals were hou sed 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 . R oom temperature and relative humidity were controlled and monitored using the Metasy s ® DDC Electronic Environmental control sy stem. 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 stud y. 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 day s prior to the initiation of test substance exposure), all available dogs had bod y weights recorded and detailed ph ys ical examinations performed. These data were collected using and reviewed b y 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

19based upon health (body weights and detaile d physical examinations), temperament, and pre

- test ECG results. A printout containing the animal numbers and individual group assignments was generated. Bod y weights are presented in Table 3 and ranged from 6.1 kg to 8.7 kg on the day s of exposure.
L. Anim al 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

20PARAMETERS EVALUATED

A. Clinical Observations A separate co mputer protocol was used to collect any findings (unscheduled observations) outside the detailed ph ys ical examinations. Only the presence of findings was recorded and entered into . The absence of findings was not recorded.
B. Electrocardiographic Exa mination ECGs were recorded with a Burdick electrocardiograph. On ly modified lead I, II, and III ECGs were collected with alligator clips placed on each l imb. Chart speed was set at 10 mm/sec and response sensitivity was set at 10 mm/mV. ECGs were recor ded 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 an d when exposure to the test substance was initiated were clearl y 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 col lected 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 t yp es of criteria (though not exclusive) w ere applied in order to determine if sensitization ha d occurred:

 Eleven or more premature ventricular c ontractions (PVC) in 10 seconds (sec) , with episodes of confluency

 Ventricular tach yc ardia

 Fibrillation
C. Statistical Methods
For bod y weight and test atmosphere concentration da ta, 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

21DATA RETENTION

The Sponsor has title to all documentation records, raw data, specimens, or other work product generated du ring 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 regulator y requirements. Acute Cardiac Sensitization Study of in Beagle Dogs

22RESULTS

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 ey es for animal no. 7371.
A fter exposure to 90,000 ppm intermittent and then continuous convulsions, excessive salivation, and dilated pupils of both ey es were noted for animal no. 7363. Exposure was terminated due to the severit y 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. 73 64. Exposure was terminated due to the severit y 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
Individu al 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 findin gs noted during the 2 μg/kg pre -exposure epinephrine challenge for an y of the animals . During the 3 a nd 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

- exp osure epinephrine challenge, 11 episodes of escape complex (ESC) were no ted 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, a nd 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 t he other animals during the pre

- exposure epinephrine challenge.
Du ring 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
r eactions 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 a nimal 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) d id not receive the complete exposure an d also did not receive the epinephrine challenge due to the Acute Cardiac Sensitization Study of in Beagle Dogs

23adverse 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 severit y 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 shortl y after the start of test substance exposure. The epinephrine challenge dose was not administered during the exposure due to the severit y of the adverse reactions to the test substance previously discussed (see Clinical Observations )

. Due to the lack of PVCs with confluency , ventricular tachy cardia, 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 administer ed followin g 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

24CONCLUSIONS

Based on the results of this study , muzzle

- only vapor exposure to 70,000 ppm in Bea gl e dogs followed b y 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 s igns 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

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- Human Primates: W hat is Considered Normal? S afety Pharmacology

Society Meeting Poster, 2009

. Mullin, L. , Reinhardt, C., and Hemingway R. C ardiac Arrhy thmias 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, In stitute of Laboratory Animal Resources, Commission on Life Sciences; National Academy P ress: Washington, DC. 1996

. Rein hardt, C., Azar A., Marfield, M., Smith, P., and Mullin, L. C ardiac Arrhy thmias and Aerosol “Sniffing”. Arch. Envir. Health, 22:265

- 279. 1971

. Tilley , L. P. Essentials of Canine and Feline Electrocardiography: I nterpretation and Treatment , 3 rd ed., L ippincott Williams & Wilkins: Philadelphia, PA, 1992

. Acute Cardiac Sensitization Study of in Beagle Dogs

26DEVIATIONS 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 Anal ys is listed the purity as 99.91413%. The value from the Certificate of Anal ys is 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 Analy sis.

 Protocol Section 5.5 states that the phy sical description of the test substan ce was to be documented b y Due to the volatility of the t est substance and the fact that it was protected from light, a phy sical description was not documented. T he Certificate of Anal ys is did no t list the phy sical description; therefore , the lack of a phy sical description had no impact on the study since it co uld not be compared to the Certificate of Anal ys is. Reason for the deviation: Phy sical 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 cy linder into an appropriately sealed container. A Stud y Director Notification (SDN) dated 28 October 2009 directed that the test substance retention sample was to be collected by exp elling 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 t he area where the test sub stance 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 expo sed 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 severit y of the adverse clinical signs observed in that animal. No other animals were exposed to those concentrations. Th i s 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 t o 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, respectivel y. 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 severit y 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 expo sure. Reason for the deviation: Exposure duration did not match the protocol due to the severit y of the adverse clinical signs observed; exposure was halted.

 Protocol Section 8.9 states that the epinephrine challenge dose (established for each dog as desc ribed 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 previousl y established epinephrine challenge dose for each anim al 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

respectivel y due to the severit y of the adverse clinical observations noted for each an imal. Animal no. 736 4 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 t he approximate 10 μg/kg dose did not indicate any excessive response.
Reason for the deviation: The dose volum e 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 ph ys ical examination weekl y during the pr etest period, at the time of animal selection, on the morning of baseline response to the epinephrine chall enge, and on the morning of exposure and the data were to be entered into A detailed phy sical examination was not performed during study week

- 1. Additionally , animal nos. 7364 and 7367 were selected Acute Cardiac Sensitization Study of in Beagle Dogs

28to act as replacement animals on 18 November 2 009 fo llowing the 70,000 ppm exposure where 2 animals (nos. 7371 and 7372) had exposure terminated prematurel y due to adverse clinical signs. The decision to use these animals as replacements occurred in the afternoon. The detailed phy sical exam inations were not performed on the morning of baseline response to the epinephrine challenge and could not be performed thoroughl y since the cage pans had alread y been flushed therefore precluding an y 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 ph ys ical examinations were not collected according to protocol specifications due to technician error and study schedu le constraints.

 Protocol Section 9.2 states that individual body weights were to be recorded weekl y 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. Bod y 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 ani mals (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 epinep hrine challenge. These deviations did not have an y 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: Bod y weights were not collected according to protocol specifications due to technician error and study schedule constraints. These deviations did not negativel y impact the quality or integrit y of the data or the outcome of the study

. Acute Cardiac Sensitization Study of in Beagle Dogs

29TABLES 1 - 4 Acute Cardiac Sensitization Study of in Beagle Dogs

30TABLE 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

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

01/05/2010 R:01/05/2010Acute Cardiac SensitizationStudy of in Beagle Dogs31TABLE 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

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

01/05/2010 R:01/05/2010Acute Cardiac SensitizationStudy of in Beagle Dogs32TABLE 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 EXPOSURESAcute Cardiac SensitizationStudy of in Beagle Dogs33TABLE 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 EXPOSURESAcute Cardiac SensitizationStudy of in Beagle Dogs34TABLE 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 EXPOSURESAcute Cardiac SensitizationStudy of in Beagle Dogs35TABLE 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 R:01/06/2010Acute Cardiac SensitizationStudy of in Beagle Dogs36TABLE 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

01/05/2010 R:01/26/2010Acute Cardiac SensitizationStudy of in Beagle Dogs37

### APPENDIX A
Certificates of Analy sis Acute Cardiac Sensitization Study of in Beagle Dogs

38Acute Cardiac Sensitization Study of in Beagle Dogs

39Acute Cardiac Sensitization Study of in Beagle Dogs

40

### APPENDIX B
Exposure Atmosphere Generation Report Acute Cardiac Sensitization Study of in Beagle Dogs

41ACUTE CARDIAC SENSI TI ZATION STUDY OF IN BEAGLE DOGS Exposure Atmosphere Generation Report

Inhalation Department

1 Acute Cardiac Sensitization Study of in Beagle Dogs

42TABLE 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 Sy stem Description

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

................................ ................................ ................... 4 TEST ATMOSPHERE PREPARATION METHO DS
................................ .............................. 5 METHODS OF CHARACTERIZATION OF EXPOSURE ATMOSPHERES ..................... 6 A. Analy zed Exposure Concentrations

................................ ................................ ..................... 6 1. Gas Chromatograph Calibration

................................ ................................ ............................................... 7 RESU LTS OF CHARACTERIZATION OF ACTUAL EXPOSURE CONCENTRATIONS
................................ ................................ ................................ ................... 9 A. Analy zed Exposure Concentrations

................................ ................................ ..................... 9


### INDEX OF FIGURES
Figure 1 Atmosphere Generation a nd Exposure Sy stem

................................ ........................ . 1 1



### INDEX OF TABLES

Tabl e 1 Individual Test Atmosphere Concentrations

............................................................ 13

3 Acute Cardiac Sensitization Study of in Beagle Dogs

44EXPOSURE 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 sc hedule 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, I nc., Eight y- Four, P ennsy lvania, U.S.A. ) attached to a specialized muzzle placed on the animal. The muzzle allowed the animal to inhal e directly from the gas bag and exhale directl y to exhaust through a one

- way breathing valve as shown schematicall y 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 Safet y 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. Ox ygen content of the test substance atmosphere was measured prior to each exposure using a Drager Pac III equipped with a calibrated oxygen sensor (Draeger Safet y Inc., Pittsburgh, Penns yl vania, U.S.A. ) . Ox ygen content was 20.1% to 20.9% for all exposures.

4 Acute Cardiac Sensitization Study of in Beagle Dogs

45TEST 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 ( m odel DTM

- 200A, Elster American Meter Co., Nebraska Cit y, N ebraska, 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 cy linder 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 , C alifornia, U.S.A. ). During the test substance delivery , the Top Trak was appro ximately 8 to 12 indicated for approxim ately 7, 12, and 10 minutes for Groups 2, 3, and 4, respectively . The gas cy linder was heated using an Omega heat tape (model no. SRT -101

- 100) and controlled using a thermocouple and CN370 temperature controller (Ome ga Engine ering, Inc., Stamford, C onnecticut, U.S.A. ) set to 50°C. I f necessary , additional test substance or air was added to the gas bag to provide the desired anal yz ed concentration.

5 Acute Cardiac Sensitization Study of in Beagle Dogs

46METHODS OF CHARACTERIZATION OF EXPOSURE ATMOSPHERES

A. Analyzed Exposur e Concentrations Analy zed 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 chromatograph y column occurred via an internal gas

- sampling valve with a sample loop. The chromatograph was display ed 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 chromatograph y methods:

Instrument: Hewlett Packard 5890 Series II Detector: Flame Ionization Detector (FID) Column: J&W Scient ific, DB

- 5, 30 m × 0.530 mm I D, 1 .5

- micron film thickness
Gases (mL/minute) Carrier - Helium: 30 Fuel

- Hy drogen: 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

47Run Para meters

Chart Zero Offset: 0 Chart Attenuation: 0 Chart Speed: 1.5 cm/min ute 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 b y metering the test substance gas from the bulk test substance cy linder to a 1

- L Tedlar ® bag. Standard bags were prepared b y m etering a known volume of compressed air into the bag using a 1000

- mL gas s yringe (model S

- 1000, Hamilton Co., Reno, N evada, 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) = 10 6 × V (A + V)
Where: V = Volume of test substance injected into bag, mL 10 6 = Basis bag concentration, ppm A = Volume of air used to prepare bag, mL
Prior to initiation of anim al exposures, the GC was calibrated with standards prepared and anal yz ed 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 calib ration 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 regress ion anal ys is to the ln -quadratic function: ln (y ) = a × [ln (x)]2 + b × ln (x) + c

7 Acute Cardiac Sensitization Study of in Beagle Dogs

48The 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

49RESULTS OF CHARACTERIZATION OF ACTUAL EXPOSURE CONCENTRATIONS

A. Analyzed Exposure Concentrations
Individual anal yz ed concentrations for each group are presented in Table 1 of this appendix. The following tab le summarizes the mean anal yz ed concentrations for each exposure:

Group: 2 3 4 Mean Concentration (ppm): 71,477 90,416 82,286 Standard Deviation: 3 238 1 179 571 N: 12 2 2

9 Acute Cardiac Sensitization Study of in Beagle Dogs

50

### F IGURE 

### 1

10 Acute Cardiac Sensitization Study of in Beagle Dogs

51

### F IGURE 

### 1: A TMOSPHERE 

### G ENERA TION 

### A ND 

### E XPOSURE 

### S YSTEM



### 200 -L Tedlar Bag 3- Way

Valve 1- Wa y

Valve

1- Wa y 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

53INHALAT 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 1213Acute Cardiac SensitizationStudy of in Beagle Dogs54INHALAT 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 214Acute Cardiac SensitizationStudy of in Beagle Dogs55INHALAT 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:12/10/200915Acute Cardiac SensitizationStudy of in Beagle Dogs56

### 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) betw een approximately 15 and 34 seconds postepinephrine challenge 7368M1 2 μg/kg Epinephrine: NSCO 3 μg/kg Epinephrine: AVB2 (1) approxi mately 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) be tween approximately 17 and 45 seconds post-epinephrine challenge 8 μg/kg Epinephrine: ESC (19) be tween 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: NSCO6 μg/kg Epinephrine: ESC (2) between approximately 34 and 35 seconds postepinephrine challenge8 μg/kg Epinephrine: PVC (2) between approximately 11 and 15 seconds postepinephrine challengeESC (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

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## 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

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## 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

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## Acute Cardiac Sensitization Study –

90,000 ppm Collection Date: 20 November 2009

Animal No. FINDINGS

7363M3 Animal did not receive complete test substan ce exposure or the epinephrine challenge during exposure Acute Cardiac Sensitization Study of in Beagle Dogs

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## 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

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### APPENDIX D
Example ECG Waveforms Acute Cardiac Sensitization Study of in Beagle Dogs

65N ORMAL ECG T RACINGAcute Cardiac SensitizationStudy of in Beagle Dogs66M ULTIPLE P REMATURE V ENTRICULAR C ONTRACTIONS (PVC)Acute Cardiac SensitizationStudy of in Beagle Dogs67

### APPENDIX E
Study Protocol Acute Cardiac Sensitization Study of in Beagle Dogs

68Acute Cardiac Sensitization Study of in Beagle Dogs

69Acute Cardiac Sensitization Study of in Beagle Dogs

70Acute Cardiac Sensitization Study of in Beagle Dogs

71Acute Cardiac Sensitization Study of in Beagle Dogs

72Acute Cardiac Sensitization Study of in Beagle Dogs

73Acute Cardiac Sensitization Study of in Beagle Dogs

74Acute Cardiac Sensitization Study of in Beagle Dogs

75Acute Cardiac Sensitization Study of in Beagle Dogs

76Acute Cardiac Sensitization Study of in Beagle Dogs

77Acute Cardiac Sensitization Study of in Beagle Dogs

78Acute Cardiac Sensitization Study of in Beagle Dogs

79Acute Cardiac Sensitization Study of in Beagle Dogs

80Acute Cardiac Sensitization Study of in Beagle Dogs

81Acute Cardiac Sensitization Study of in Beagle Dogs

82Acute Cardiac Sensitization Study of in Beagle Dogs

83Acute Cardiac Sensitization Study of in Beagle Dogs

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