## FINAL REPORT

STUDY TITLE

ACUTE CARDIAC SENSITIZATION STUDY OF HFO 1234ze AND HFO 1234yf IN DOGS

STUDY NUMBER

WIL-447008

STUDY DIRECTOR Jeffrey T. Weinberg, BS

STUDY INITIATION DATE 6 July 2004

STUDY COMPLETION DATE 23 August 2006

PERFORMING LABORATORY

WIL Research Laboratories, LLC 1407 George Road Ashland, OH 44805-9281

SPONSOR Honeywell International 101 Columbia Road Morristown, NJ 07962-1139

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## WIL-447008 Honeywell International 

## HFO 1234ze and 

## HFO 1234yf



# COMPLIANCE STATEMENT

This study, designated WIL-447008, was conducted in compliance with the United States Environmental Protection Agency (EPA) Good Laboratory Practice Standards (40 CFR Part 792), 18 September 1989; the standard operating procedures of WIL Research Laboratories, LLC, and the protocol as approved by the sponsor. Certificates 

## of Analysis were provided by the sponsor (presented in Appendix A); the characterization analyses were not conducted according to Good Laboratory Practices (GLP).

Jeffrey T. Weinberg, 

## BS Project Specialist, Inhalation Toxicology Study Director



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# T ABLE 

# O F 

# C ONTENTS



## Page
Compliance Statement .............................................................................. 2 Table Of Contents ..................................................................................... 3 Index Of Tables ......................................................................................... 5 Index Of Appendices................................................................................. 6 1. Summary.................................................................................................... 7 1.1. Objective ..................................................................................................... 7 1.2. Study Design ............................................................................................... 7 1.3. Results ......................................................................................................... 7 1.4. Conclusions ................................................................................................. 8

2. Introduction ............................................................................................... 9 2.1. General Study Information.......................................................................... 9 2.2. Key Study Dates.......................................................................................... 9

3. Study Design ............................................................................................ 10 4. Experimental Procedures - Materials And Methods ........................... 11 4.1. Test Article................................................................................................ 11 4.1.1. Test Article Identification ......................................................................... 11 4.1.2. Epinephrine Hydrochloride Identification ................................................ 11 4.1.3. Epinephrine Hydrochloride Preparation.................................................... 12

4.2. Test System ............................................................................................... 12 4.3. Exposure Methods..................................................................................... 12 4.4. Exposure Atmosphere Generation And Monitoring ................................. 13 4.5. Organization And Treatment Regimen ..................................................... 13 4.6. Animal Receipt And Acclimation/Pretest Period...................................... 16 4.7. Immunizations Prior To Arrival................................................................ 17 4.8. Animal Housing And Exercise.................................................................. 17 4.9. Diet, Drinking Water And Maintenance ................................................... 18

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Page

4.10. Environmental Conditions ........................................................................ 18 4.11. Assignment Of Animals To Treatment Groups ........................................ 18 5. Parameters Evaluated............................................................................. 20 5.1. Clinical Observations And Survival.......................................................... 20 5.2. Body Weights............................................................................................ 20 5.3. Electrocardiographic Examinations .......................................................... 20 5.4. Study Termination..................................................................................... 21 5.5. Statistical Methods .................................................................................... 21 5.6. Data Retention........................................................................................... 21

6. Results And Discussion........................................................................... 22 6.1. Clinical Observations And Survival.......................................................... 22 6.2. Body Weights............................................................................................ 22 6.3. Electrocardiographic Examinations .......................................................... 22

7. Conclusions .............................................................................................. 23 8. Key Study Personnel And Report Submission ..................................... 24 9. Quality Assurance Unit Statement ........................................................ 25 9.1. Phases Inspected........................................................................................ 25 9.2. Approval.................................................................................................... 27 10. References ................................................................................................ 28 11. Deviations From The Protocol ............................................................... 29

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# I NDEX 

# O F 

# T ABLES



## Page

1. Individual Clinical Observations (Detailed Physical Examinations/Dispositions - HFO 1234ze).................................31
2. Individual Clinical Observations (Detailed Physical Examinations/Dispositions - HFO 1234yf) .................................32 3. Individual Clinical Observations (Prior To Exposure - HFO 1234ze) ......................33 4. Individual Clinical Observations (Prior To Exposure - HFO 1234yf).......................34
5. Individual Clinical Observations (During And Post-Exposure - HFO 1234ze)..............................................................35
6. Individual Clinical Observations (During And Post-Exposure - HFO 1234yf) ..............................................................36 7. Individual Body Weights [Kg] (HFO 1234ze)...........................................................37 8. Individual Body Weights [Kg] (HFO 1234yf)...........................................................38 9. Individual Body Weight Changes [Kg] (HFO 1234ze) .............................................39 10. Individual Body Weight Changes [Kg] (HFO 1234yf)..............................................40 11. Individual Cardiac Sensitization Responses...............................................................41

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# I NDEX 

# O F 

# A PPENDICES



## Page A. Certificates Of Analysis (Sponsor-Provided Data) ....................................................42 B. Exposure Atmosphere Generation Report..................................................................45 C. Individual Evaluation Of Epinephrine Doses.............................................................63
D. Animal Room Environmental Conditions..................................................................65 E. Individual Electrocardiographic Data.........................................................................72 F. Study Protocol.............................................................................................................87

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# 1. S UMMARY 

# 1.1. O BJECTIVE 

## The objective of this study was to evaluate the cardiac sensitization potential of HFO 1234ze and HFO 1234yf during acute inhalation exposure to various concentrations of the test article. Cardiac sensitization refers to the potential for the test articles to increase sensitivity of the heart to the pharmacological effects of epinephrine (Mullin et al., 1979).



# 1.2. S TUDY 

# D ESIGN 

## HFO 1234ze and HFO 1234yf were administered via muzzle-only inhalation exposure (vapor) as acute exposures of each test article at concentrations of 2.0%, 6.0% and 12% (20,000, 60,000 and 120,000 ppm, respectively). One group of 6 male dogs was used on this study. Each dog served as its own control. Animals were administered a pre-exposure dose of epinephrine as a bolus injection via a cephalic vein approximately 5 minutes prior to exposure to the test article. Five minutes after exposure to the test article began, the animals were administered a challenge dose of epinephrine. Following the final exposure, all dogs were returned to stock.
All animals were observed twice daily for mortality and moribundity. Clinical examinations were performed prior to initiation of each exposure, continually during the exposure and immediately following each exposure period. Detailed physical examinations were performed and body weights were collected on the day prior to the first day of exposures and at the completion of all exposures. Electrocardiographic data were recorded continuously throughout the pre-exposure epinephrine dosing, exposure to the test gas and for 5 minutes following administration of the challenge epinephrine dose (total of approximately 17 minutes).



# 1.3. R ESULTS 

## All animals survived to study termination. There were no test article-related clinical observations. Body weights were unaffected by test article administration.

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## WIL-447008 HFO 1234ze and HFO 1234yf Honeywell International There was no indication of cardiac sensitization noted in any dogs at 20,000, 60,000 or 120,000 ppm HFO 1234ze or HFO 1234yf.



# 1.4. C ONCLUSIONS 

## Based on the results of this study, there was no indication of cardiac sensitization in beagle dogs exposed to muzzle-only vapor concentrations of up to 120,000 ppm (12%) of either HFO 1234ze or HFO 1234yf followed by administration of epinephrine. Therefore, the no-observed-effect level (NOEL) for both HFO 1234ze and HFO 1234yf was 120,000 ppm.

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# 2. I NTRODUCTION 

# 2.1. G ENERAL 

# S TUDY 

# I NFORMATION 

## This report presents the data from “Acute Cardiac Sensitization Study Of HFO 1234ze and HFO 1234yf In Dogs”. Due to software spacing constraints, the study title appears as “Cardiac Sensitization Study Of HFO 1234ze & HFO 1234yf In Dogs” on the report tables. On 21 April 2006, Jeffrey T. Weinberg, BS assumed the role of study director originally held by Gary R. Kiplinger, BS.
The following computer protocols were used for data collection during the study:
Computer Protocol Type of Data Collected

WIL-447008............................................Main study data (HFO 1234ze) WIL-447008A.........................................Main study data (HFO 1234yf)



# 2.2. K EY 

# S TUDY 

# D ATES 

## Date(s) Event(s)

28 June 2004 ...........................................Experimental starting date (acclimation to restraint apparatus) 20 July 2004............................................Assignment to study group 20 July 2004............................................Experimental start date (initiation of exposures) 19 August 2004.......................................Experimental termination (completion) date (animals returned to stock)

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# 3. S TUDY 

# D ESIGN

Baseline Data Collection

Randomization

6 males exposed to each test article 5 minutes after a pre-exposure dose of epinephrine

Challenge dose of epinephrine administered 5 minutes after exposure to each test article to assess cardiac sensitization

Clinical observations recorded daily; detailed

physical examinations and body weights collected on first day of exposures and at completion of all exposure

Electrocardiographic data were recorded continuously throughout the pre-exposure epinephrine dosing, exposure to the each test article and for 5 minutes following administration of the challenge epinephrine

dose (total of approximately 17 minutes)

Animals returned to stock colony following final exposure



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# 4. E XPERIMENTAL 

# P ROCEDURES 

# - M ATERIALS 

# A ND 

# M ETHODS 

# 4.1. T EST 

# A RTICLE 

# 4.1.1. T EST 

# A RTICLE 

# I DENTIFICATION 

## The test articles, HFO 1234ze and HFO 1234yf, were received from Honeywell, Buffalo, New York, on 6 July 2004, as follows:

Identification Quantity Received Physical Description Trans 1,1,1,3-Tetrafluoropropene (HFO 1234ze) Lot no. 63004, Batch 5

[WIL log no. 6214A] 1 cylinder Gross weight: 8000.8 g Clear gas

2,3,3,3,-Tetrafluoropropene (HFO 1234yf) Lot no. 62404, Batch 2

[WIL log no. 6215A] 1 cylinder Gross weight: 7796.2 g Clear gas

Data documenting the purity and stability of the test article are on file with the sponsor. Certificates of Analysis for the test articles were provided by the sponsor and are presented in Appendix A. The purity of the test articles was ≥ 99.99%. The test articles were stored at room temperature and were considered stable under this condition. Reserve samples were collected on 12 July 2004 and 14 July 2004 for HFO 1234ze and HFO 1234yf, respectively, and stored in the Archives of WIL Research Laboratories, LLC.



# 4.1.2. E PINEPHRINE 

# H YDROCHLORIDE 

# I DENTIFICATION 

## The epinephrine used on this study was received from Sigma Chemical Company, on 28 June 2004, as follows:

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Identification Quantity Received Physical Description (±)-Epinephrine Hydrochloride Lot no. 123K1187 Exp. date: 28 June 2006

[WIL log no. 6205A] 1 bottle Gross weight: 150.3 g Fine, white powder



# 4.1.3. E PINEPHRINE 

# H YDROCHLORIDE 

# P REPARATION 

## All glassware and utensils were sterilized prior to use and appropriate procedures were performed within a laminar flow hood. An approximate 80 μg/mL stock solution of epinephrine in 0.9% sodium chloride was prepared by weighing the appropriate amount of epinephrine hydrochloride into a glass vial. The appropriate amount of 0.9% sodium chloride for injection, USP was added to the vial and it was capped with a septum. The vial was then inverted/swirled several times to dissolve the epinephrine. The stock solution was then diluted to the appropriate concentrations for use on study by adding appropriate amounts of the stock solution and additional 0.9% sodium chloride for injection, USP. The formulated concentrations were prepared on the day of use and stored on ice, protected from light.



# 4.2. T EST 

# S YSTEM 

## Beagle dogs from Ridglan Farms, Mt. Horeb, Wisconsin were used as the test system on this study. This species and breed of animal is recognized as appropriate for cardiac function studies. The beagle dog was used because it is a widely used breed for which significant historical control data are available. The number of animals selected was the minimum needed to yield scientifically meaningful data.



# 4.3. E XPOSURE 

# M ETHODS 

## Each animal was exposed individually using a muzzle-only exposure apparatus operated under dynamic conditions to supply the target concentration of the test article vapor and a minimum oxygen content of 19%. The control atmosphere (the first 7 minutes of each exposure) was filtered air supplied from an in-house source. Each dog was comfortably

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## WIL-447008 HFO 1234ze and HFO 1234yf Honeywell International restrained using a canvas sling with the appropriate ECG (limb) leads attached. A detailed description of the exposure methods is presented in Appendix B. The mask used in the muzzle-only exposure system is a WIL-designed and manufactured, dual-port mask constructed of Neoprene ® 

## nylon straps and “hook-and-loop” fastening material. Based on previous experience, this combination of materials provides an air-tight seal around the muzzle to ensure proper test article exposure. There is no SOP stating the procedures for dog inhalation exposure studies. The procedure used is documented in the system description located in the study records.



# 4.4. E XPOSURE 

# A TMOSPHERE 

# G ENERATION 

# A ND 

# M ONITORING 

## Exposures were conducted using a muzzle-only system consisting of a mask connected to a 3-way valve connected to a gas bag containing the appropriate test article mixture. Prior to and following each exposure, samples were analyzed from each bag using a gas chromatographic method to assure that the atmosphere was at the target concentration. Oxygen was added to each gas bag as needed to maintain oxygen content above 19%. During the pre-treatment acclimation period, animals received room air directed through the 3-way valve. At the start of exposure, the valve was turned to the bag position for exposure to the test article. Feed and water were withheld during exposure periods. A detailed description of the generation methods is presented in Appendix B.



# 4.5. O RGANIZATION 

# A ND 

# T REATMENT 

# R EGIMEN 

## Prior to exposure (during the pre-study acclimation period), an individual response to intravenous epinephrine administration was established for each individual dog using epinephrine doses starting at 2 μ g/kg and increasing to 3, 4, 6 or 8 μ g/kg, if necessary. The epinephrine doses were administered as bolus injections (<5 seconds) via the cephalic vein. Time between escalating doses of epinephrine was approximately 5 minutes or until the ECG returned to baseline. During this phase of the study, the dogs were exposed to air only via the muzzle-only exposure apparatus. The information gathered was used to select dogs for use on study and to establish the specific level of

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## WIL-447008 HFO 1234ze and HFO 1234yf Honeywell International epinephrine each dog would receive during the test article exposure phase of the study. The epinephrine level selected for an individual dog was the highest dose level tested during the pre-study evaluation that did not elicit significant ECG findings (e.g., PVC). These data are presented in Appendix C.
The test group consisted of 6 dogs. Each dog was exposed to the target concentration of each test article vapor. At the end of each exposure period, the dogs were returned to their home cages. There was a minimum of 48 hours between exposure to the next exposure level. Each dog was comfortably restrained using a canvas sling or other appropriate restraint device with the appropriate ECG (limb) leads attached. The exposure duration to air was approximately 7 minutes and the exposure duration for the test gas was approximately 10 minutes. A pre-dose epinephrine exposure and a challenge dose of epinephrine were administered as noted below. In this regimen, each dog served as its own control based on pre-study and pre-exposure assessment of response to the epinephrine dose. The treatment regimen for these groups was as follows:
TIME (minutes) Event 0 Start ECG recording 2 Pre-exposure epinephrine dose 7 Test gas initiated 12 Challenge dose of epinephrine 17 Termination of exposure/ECG The epinephrine challenge doses (established for each dog as described above) were administered intravenously using an appropriately sized catheter as a bolus injection into a cephalic vein.
The exposure schedule is shown in the following tables:

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Date Test Article Animal No. Target Exposure Concentration (ppm) Epinephrine Dose(s) (μg/kg) 20-July-2004 HFO 1234yf 0136 20,000 2 20-July-2004 HFO 1234yf 0188 20,000 8 20-July-2004 HFO 1234yf 0357 20,000 4 20-July-2004 HFO 1234yf 0387 20,000 8 20-July-2004 HFO 1234yf 0409 20,000 6 20-July-2004 HFO 1234yf 0428 20,000 2 22-July-2004 HFO 1234yf 0136 60,000 2 22-July-2004 HFO 1234yf 0188 60,000 8 22-July-2004 HFO 1234yf 0357 60,000 4 22-July-2004 HFO 1234yf 0387 60,000 8 22-July-2004 HFO 1234yf 0409 60,000 6 22-July-2004 HFO 1234yf 0428 60,000 2 29-July-2004 HFO 1234yf 0136 120,000 2 29-July-2004 HFO 1234yf 0188 120,000 8 29-July-2004 HFO 1234yf 0357 120,000 4 29-July-2004 HFO 1234yf 0387 120,000 8 29-July-2004 HFO 1234yf 0409 120,000 6 29-July-2004 HFO 1234yf 0428 120,000 2

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Date Test Article Animal No. Target Exposure Concentration (ppm) Epinephrine Dose(s) (μg/kg)
10-August-2004 HFO 1234ze 0136 20,000 2 10-August-2004 HFO 1234ze 0188 20,000 8 10-August-2004 HFO 1234ze 0357 20,000 4 10-August-2004 HFO 1234ze 0387 20,000 8 10-August-2004 HFO 1234ze 0409 20,000 6 10-August-2004 HFO 1234ze 0428 20,000 2
12-August-2004 HFO 1234ze 0136 60,000 2 12-August-2004 HFO 1234ze 0188 60,000 8 12-August-2004 HFO 1234ze 0357 60,000 4 12-August-2004 HFO 1234ze 0387 60,000 8 12-August-2004 HFO 1234ze 0409 60,000 6 12-August-2004 HFO 1234ze 0428 60,000 2
16-August-2004 HFO 1234ze 0136 120,000 2 16-August-2004 HFO 1234ze 0188 120,000 8 16-August-2004 HFO 1234ze 0357 120,000 4 16-August-2004 HFO 1234ze 0387 120,000 8 16-August-2004 HFO 1234ze 0409 120,000 6 16-August-2004 HFO 1234ze 0428 120,000 2

The selected route of administration for this study was inhalation exposure because of the volatility and intended usage of the test article and because this will be the most likely route of exposure for the general population. The muzzle-only method of exposure was selected to achieve precise control of concentration levels and durations.



# 4.6. A NIMAL 

# R ECEIPT 

# A ND 

# A CCLIMATION 

# /P RETEST 

# P ERIOD 

## The male beagle dogs used on this study were obtained from the WIL Research Laboratories, LLC stock dog colony. Each animal was uniquely identified with an ear tattoo by the supplier. The animals were approximately 8 to 12 months old at the initiation of exposure. Animals considered suitable for the study were housed for an appropriate acclimation/pretest period. During this period, each animal was observed

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## WIL-447008 HFO 1234ze and HFO 1234yf Honeywell International twice daily for mortality and changes in general appearance or behavior. Fecal samples were collected prior to the initiation of dose administration and checked for parasites; none were present.
Pretest data collection began on 28 June 2004. Individual body weights were recorded and detailed physical examinations were performed periodically during the pretest period. Electrocardiographic data were also recorded for pretest animals prior to the initiation of dose administration. During the acclimation period, each dog was acclimated to exposure/restraint for several 10 to 20 minute periods. At least 10 acclimation sessions/dog were conducted. The animals used on this study were acclimated to restraint prior to the protocol being signed by the study director; this GLP deviation did not negatively impact the quality or integrity of the data nor the outcome of the study.



# 4.7. I MMUNIZATIONS 

# P RIOR 

# T O 

# A RRIVAL 

## All animals were immunized prior to arrival at WIL Research Laboratories, LLC. The immunizations included rabies, Bordetella , distemper, adenovirus type 2, Leptospira spp, parvovirus, parainfluenza and oral papilloma virus.



# 4.8. A NIMAL 

# H OUSING 

# A ND 

# E XERCISE 

## The animals were housed individually in clean, stainless steel cages elevated above stainless steel cage pans that were cleaned daily during the acclimation period and throughout the study. Animals were maintained in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council, 1996). The animals were allowed regular opportunity for exercise and social interaction in accordance with the standard operating procedures at WIL Research Laboratories, LLC, and the Animal Welfare Act (9 CFR Part 3). The animal facilities at WIL Research Laboratories, LLC, are accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC International).

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# 4.9. D IET 

# , D RINKING 

# W ATER 

# A ND 

# M AINTENANCE 

## Approximately 400 g of PMI Nutrition International, LLC, Certified Canine LabDiet  

## 5007, was offered daily. The diet utilized at WIL Research Laboratories, LLC, is a certified feed with appropriate analyses for potential contaminants performed and provided by the manufacturer. Reverse osmosis-treated (on-site) drinking water, delivered by an automatic watering system, was provided ad libitum throughout the study period. Municipal water supplying the facility was sampled for contaminants according to the standard operating procedures. The results of the diet and water analyses are maintained at WIL Research Laboratories, LLC. No contaminants were present in animal feed or water at concentrations sufficient to interfere with the objectives of this study.



# 4.10. E NVIRONMENTAL 

# C ONDITIONS 

## All animals were housed throughout the acclimation period and during the study in an environmentally controlled room. The room temperature and humidity controls were set to maintain daily averages of 68 ± 5 ° F (20 ± 3 ° C) and 50 ± 20% relative humidity. Room temperature and relative humidity were controlled and monitored using the Metasys ® 

## DDC Electronic Environmental control system. These data were recorded approximately hourly and are summarized in Appendix C. Actual mean daily temperature ranged from 67.9 ° F to 71.2 ° F (19.9 ° C to 21.8 ° C) and mean daily relative humidity ranged from 45.4% to 68.7% during the study. Light timers were set to provide 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.



# 4.11. A SSIGNMENT 

# O F 

# A NIMALS 

# T O 

# T REATMENT 

# G ROUPS 

## On 20 July 2004 (the day of initiation of dose administration), all available dogs were weighed and examined in detail for physical abnormalities. These data were collected using the WIL Toxicology Data Management System (WTDMS  ) and reviewed by the study director. The animals judged suitable for assignment to the study based on

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## WIL-447008 HFO 1234ze and HFO 1234yf Honeywell International temperament, health and acceptable baseline ECG were arbitrarily selected for use on study. The test group consisted of 6 males. The selected animals were approximately 8 to 12 months old at the initiation of dose administration; body weights ranged from 7.2 to 11.7 kg.

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# 5. P ARAMETERS 

# E VALUATED 

# 5.1. C LINICAL 

# O BSERVATIONS 

# A ND 

# S URVIVAL 

## All animals were observed twice daily, once in the morning and once in the afternoon, for mortality and moribundity.
Clinical examinations were performed on each dog prior to initiation of each exposure, continually during the exposure period and following each exposure upon return to their home cages. Detailed physical examinations were conducted on all animals on the day of the first exposure and after completion of all exposures.



# 5.2. B ODY 

# W EIGHTS 

## Individual body weights were recorded on the first day of exposure and after completion of all exposures. Body weight changes were calculated for the corresponding intervals.



# 5.3. E LECTROCARDIOGRAPHIC 

# E XAMINATIONS 

## Electrocardiograms (ECG) were recorded with a Burdick electrocardiograph. Modified leads I, II and III were used for electrocardiogram collection; ECG electrodes were placed on each limb. ECG were recorded continuously throughout the pre-exposure epinephrine dosing, exposure to the test gas and for 5 minutes following administration of the challenge epinephrine dose (total of approximately 17 minutes). The points at which epinephrine was administered and when exposure to the test article was initiated were clearly marked on each ECG recording.
ECG recordings taken during exposure to the test articles and after administration of the challenge epinephrine dose were compared to the ECG recordings made after administration of the pre-exposure epinephrine dose that was given in the absence of test article. During the epinephrine challenges (or immediately following), the following type of criteria (though not exclusively) were applied in order to determine if sensitization has occurred:

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• Eleven or more PVCs (premature ventricular contraction) in 10 seconds, with episodes of confluency

• Ventricular tachycardia

• Fibrillation


# 5.4. S TUDY 

# T ERMINATION 

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



# 5.5. S TATISTICAL 

# M ETHODS 

## Each mean was presented with the standard deviation (S.D.) and the number of animals (N) used to calculate the mean.



# 5.6. D ATA 

# R ETENTION 

## The sponsor has title to all documentation records, raw data, specimens or other work product generated during the performance of the study. All work product generated by WIL Research Laboratories, LLC, including raw paper data and specimens, are retained in the Archives at WIL Research Laboratories, LLC as specified in the study protocol.
Reserve samples of the test articles (if appropriate), pertinent electronic storage media and the original final report are retained in the Archives at WIL Research Laboratories, LLC in compliance with regulatory requirements.

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# 6. R ESULTS 

# A ND 

# D ISCUSSION 

# 6.1. C LINICAL 

# O BSERVATIONS 

# A ND 

# S URVIVAL 

## Individual Data: Tables 1, 2, 3, 4, 5, 6
All animals survived to study termination. There were no test article-related clinical observations. All clinical findings in the test article-treated groups were limited to single animals, were not observed consistently and/or were common findings for laboratory dogs of this age and breed.



# 6.2. B ODY 

# W EIGHTS 

## Individual Data: Tables 7, 8, 9, 10
Body weights were unaffected by test article administration. There were no remarkable changes over the course of the study.



# 6.3. E LECTROCARDIOGRAPHIC 

# E XAMINATIONS 

## Individual Data: Table 11, Appendix E
There was no indication of cardiac sensitization noted in any dogs at 20,000, 60,000 or 120,000 ppm HFO 1234ze or HFO 1234yf. Challenge dosing with epinephrine while the animals were under test article exposure did not result in the occurrence of arrhythmias such as ventricular fibrillation, preventricular contraction or tachycardia. Representative ECG recordings of responses to an epinephrine challenge dose in the 120,000 ppm exposure level are included in Appendix E.

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# 7. C ONCLUSIONS 

## Based on the results of this study, there was no indication of cardiac sensitization in beagle dogs exposed to muzzle-only vapor concentrations of up to 120,000 ppm (12%) of either HFO 1234ze or HFO 1234yf followed by administration of epinephrine. Therefore, the no-observed-effect level (NOEL) for both HFO 1234ze and HFO 1234yf was 120,000 ppm.

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# 8. 

# KEY 

# STUDY PERSONNEL AND 

# REPORT SUBMISSION Report Submitted 

## By: 

# w=!, Jeffrey T. Weinberg, 

## BS Project Specialist, Inhalation Toxicology Study Director
Report Prepared By: I

.I



## Group Supervkor,%dy Analysis And Reports Report Reviewed 

## By:

Philip R. Atterson, 

## MS Head, Cardiovascular Pharmacology Study Personnel: Sally A. Keets, AS Teresa 

## D. Morris, 

## BS Theresa M. Rafeld Robert A. Wally, 

## BS, RAC 

# 2%%+%?ZaoG Date

DateJ

Date

Senior Operations Manager, Vivarium Operations Manager, Toxicology Group Manager, Formulations Laboratory Manager, Reporting and Regulatory Technical

Services



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# 9. Q UALITY 

# A SSURANCE 

# U NIT 

# S TATEMENT 

# 9.1. P HASES 

# I NSPECTED

Date(s) of Inspection(s) Phase Inspected Date(s) Findings Reported to Study Director Date(s) Findings Reported to Management Auditor(s) 29-Jul-2004 ECG’S 29-Jul-2004 31-Aug-2004 K.Dobbs 02-Aug-2004 Animal Care and Equipment 02-Aug-2004 29-Sep-2004 K.Dobbs 07-Oct-2004, 08-Oct-2004 Study Records (Ex-1) 08-Oct-2004 30-Nov-2004 E.Crawford
07-Oct-2004, 08-Oct-2004 Study Records (I-1) 11-Oct-2004 30-Nov-2004 T.DeVan Booth 08-Oct-2004 Study Records (I-2) 11-Oct-2004 30-Nov-2004 T.DeVan Booth 08-Oct-2004 Study Records (I-3) 11-Oct-2004 30-Nov-2004 T.DeVan Booth 11-Oct-2004 Study Records (I-5) 11-Oct-2004 30-Nov-2004 T.DeVan Booth 11-Oct-2004 Study Records (I-8) 11-Oct-2004 30-Nov-2004 T.DeVan Booth 11-Oct-2004 Study Records (I-9) 11-Oct-2004 30-Nov-2004 T.DeVan Booth 11-Oct-2004 Study Records (I-4) 11-Oct-2004 30-Nov-2004 T.DeVan Booth 11-Oct-2004 Study Records (I-6) 11-Oct-2004 30-Nov-2004 T.DeVan Booth 11-Oct-2004 Study Records (I-7) 11-Oct-2004 30-Nov-2004 T.DeVan Booth 19-Oct-2004 Study Records (Rx-1) 19-Oct-2004 30-Nov-2004 E.Crawford 16-Jan-2005 Draft Inhalation Appendix 17-Jan-2005 16-Feb-2005 E.Crawford 14-Jun-2006 Draft Inhalation Appendix 14-Jun-2006 25-Jul-2006 T.DeVan Booth 15-Jun-2006 Draft Report without Inhalation Appendix 15-Jun-2006 25-July-2006 T.DeVan Booth



## This study was inspected in accordance with the U.S. EPA Good Laboratory Practice Standards (40 CFR Part 792), the standard operating procedures of WIL Research Laboratories, LLC and the sponsor's protocol and protocol amendments, with the following exception. The data 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. A status report is submitted to management monthly.
This report accurately reflects the data generated during the study. The methods and procedures used in the study were those specified in the protocol, its amendments and the standard operating procedures of WIL Research Laboratories, LLC.

25 of 105

## WIL-447008 HFO 1234ze and HFO 1234yf Honeywell International The raw data, the retention sample(s), if applicable, and the final report will be stored in the Archives at WIL Research Laboratories, LLC or another location specified by the
sponsor.

26 of 105

## WIL-447008 Honeywell International 

## HFO 1234ze and 

## HFO 1234yf



# 9.2. APPROVAL This study was inspected according to the criteria discussed in Section 

## 9.1,

Q' 

## Compliance Specialist Report Released By:

Heather L. 

## Osborn, 

## BS, RQAP-GLP Manager, Quality Assurance



## 27 of 105

## WIL-447008 HFO 1234ze and HFO 1234yf Honeywell International


# 10. R EFERENCES 

## Mullin, L., Reinhardt, C. and Hemingway R. Cardiac arrhythmias and blood levels associated with inhalation of halon 1301. American Industrial Hygiene Association Journal , 1979 ; pp 40, 653-358.
National Research Council. Guide for the Care and Use of Laboratory Animals, Institute of Laboratory Animal Resources, Commission on Life Sciences; National Academy Press: Washington, DC, 1996

.

28 of 105

## WIL-447008 HFO 1234ze and HFO 1234yf Honeywell International


# 11. D EVIATIONS 

# F ROM 

# T HE 

# P ROTOCOL 

## This study was conducted in accordance with the protocol and protocol amendments, except for the following.

• Protocol Section 7.4. states that the animals will be offered food daily for approximately 1-2 hours. Although the animals were fed daily, the duration of the daily feeding period was not recorded.

• Protocol Section 9.2. states that detailed physical examinations will be performed on all animals on the day prior to the first day of exposures (study day -1). Detailed physicals were actually conducted on the first day of exposure (study day 0).

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

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## WIL-447008 HFO 1234ze and HFO 1234yf Honeywell International



# TABLES 1 - 11

30 of 105TABLE 1 (DETAILED PHYSICAL EXAMINATIONS/DISPOSITIONS - HFO 1234ze) PROJECT NO.:WIL-447008A CARDIAC SENSITIZATION STUDY OF HFO 1234ze & HFO 1234yf IN DOGS PAGE 1 SPONSOR:HONEYWELL INDIVIDUAL CLINICAL OBSERVATIONS
TABLE RANGE: 08-10-04 TO 08-19-04

----------------------------------------------------------------------------------------------------------------------------------- ANIMAL SEX GROUP CATEGORY DATE TIME GRADE OBSERVATIONS

----------------------------------------------------------------------------------------------------------------------------------- 0136 M HFO 1234ze NORMAL 08-15-04 8:56 P NO SIGNIFICANT CLINICAL OBSERVATIONS 08-19-04 13:38 P NO SIGNIFICANT CLINICAL OBSERVATIONS 0136 M HFO 1234ze DISPOSITION 08-19-04 13:51 P SENT TO STOCK COLONY 0188 M HFO 1234ze NORMAL 08-19-04 13:38 P NO SIGNIFICANT CLINICAL OBSERVATIONS 0188 M HFO 1234ze DISPOSITION 08-19-04 13:51 P SENT TO STOCK COLONY 0188 M HFO 1234ze EYES/EARS/NOSE 08-15-04 8:58 P CLEAR DISCHARGE LEFT EYE 08-15-04 8:58 P CLEAR DISCHARGE RIGHT EYE 0357 M HFO 1234ze DISPOSITION 08-19-04 13:51 P SENT TO STOCK COLONY 0357 M HFO 1234ze EYES/EARS/NOSE 08-15-04 8:59 P CLEAR DISCHARGE LEFT EYE 08-15-04 8:59 P CLEAR DISCHARGE RIGHT EYE 08-15-04 8:59 P INJECTED SCLERA LEFT EYE 08-15-04 8:59 P INJECTED SCLERA RIGHT EYE 0357 M HFO 1234ze ORAL/DENTAL 08-19-04 13:39 P REDDENED GUMS 0387 M HFO 1234ze NORMAL 08-19-04 13:40 P NO SIGNIFICANT CLINICAL OBSERVATIONS 0387 M HFO 1234ze DISPOSITION 08-19-04 13:51 P SENT TO STOCK COLONY 0387 M HFO 1234ze EYES/EARS/NOSE 08-15-04 9:01 P CLEAR DISCHARGE LEFT EYE 0409 M HFO 1234ze NORMAL 08-15-04 9:02 P NO SIGNIFICANT CLINICAL OBSERVATIONS 0409 M HFO 1234ze DISPOSITION 08-19-04 13:51 P SENT TO STOCK COLONY 0409 M HFO 1234ze ORAL/DENTAL 08-19-04 13:40 P WET CLEAR MATERIAL AROUND MOUTH 0428 M HFO 1234ze NORMAL 08-15-04 9:04 P NO SIGNIFICANT CLINICAL OBSERVATIONS 0428 M HFO 1234ze DISPOSITION 08-19-04 13:51 P SENT TO STOCK COLONY 0428 M HFO 1234ze ORAL/DENTAL 08-19-04 13:40 P WET CLEAR MATERIAL AROUND MOUTH 08-19-04 13:41 P WET CLEAR MATERIAL VENTRAL NECK

----------------------------------------------------------------------------------------------------------------------------------- GRADE CODE: 1 - SLIGHT 2 - MODERATE 3 - SEVERE P - PRESENT PCRDv4.1 05/11/2006 R:06/20/200631of105TABLE 2 (DETAILED PHYSICAL EXAMINATIONS/DISPOSITIONS - HFO 1234yf) PROJECT NO.:WIL-447008A CARDIAC SENSITIZATION STUDY OF HFO 1234ze & HFO 1234yf IN DOGS PAGE 1 SPONSOR:HONEYWELL INDIVIDUAL CLINICAL OBSERVATIONS
TABLE RANGE: 07-20-04 TO 08-09-04

----------------------------------------------------------------------------------------------------------------------------------- ANIMAL SEX GROUP CATEGORY DATE TIME GRADE OBSERVATIONS

----------------------------------------------------------------------------------------------------------------------------------- 0136 M HFO 1234yf NORMAL 07-20-04 7:43 P NO SIGNIFICANT CLINICAL OBSERVATIONS 0188 M HFO 1234yf NORMAL 07-20-04 7:45 P NO SIGNIFICANT CLINICAL OBSERVATIONS 0357 M HFO 1234yf NORMAL 07-20-04 7:46 P NO SIGNIFICANT CLINICAL OBSERVATIONS 0377 M HFO 1234yf NORMAL 07-20-04 7:48 P NO SIGNIFICANT CLINICAL OBSERVATIONS 0387 M HFO 1234yf EYES/EARS/NOSE 07-20-04 7:52 P INJECTED SCLERA RIGHT EYE 0387 M HFO 1234yf EYES/EARS/NOSE 07-20-04 7:52 P INJECTED SCLERA LEFT EYE 0387 M HFO 1234yf EYES/EARS/NOSE 07-20-04 7:52 P REDDENED EAR(S) 0409 M HFO 1234yf EYES/EARS/NOSE 07-20-04 7:54 P INJECTED SCLERA RIGHT EYE 0409 M HFO 1234yf EYES/EARS/NOSE 07-20-04 7:54 P INJECTED SCLERA LEFT EYE 0428 M HFO 1234yf EYES/EARS/NOSE 07-20-04 7:57 P REDDENED EAR(S) 0428 M HFO 1234yf ORAL/DENTAL 07-20-04 7:58 P WET CLEAR MATERIAL AROUND MOUTH 0459 M HFO 1234yf NORMAL 07-20-04 7:59 P NO SIGNIFICANT CLINICAL OBSERVATIONS

----------------------------------------------------------------------------------------------------------------------------------- GRADE CODE: 1 - SLIGHT 2 - MODERATE 3 - SEVERE P - PRESENT MANUALv1.0 05/11/2006 R:06/20/200632of105TABLE 3 (PRIOR TO EXPOSURE - HFO 1234ze) PROJECT NO.:WIL-447008A CARDIAC SENSITIZATION STUDY OF HFO 1234ze & HFO 1234yf IN DOGS PAGE 1 SPONSOR:HONEYWELL INDIVIDUAL CLINICAL OBSERVATIONS
TABLE RANGE: 08-10-04 TO 08-19-04

----------------------------------------------------------------------------------------------------------------------------------- ANIMAL SEX GROUP CATEGORY DATE TIME GRADE OBSERVATIONS

----------------------------------------------------------------------------------------------------------------------------------- 0188 M HFO 1234ze EXCRETA 08-10-04 9:04 P SOFT FECES 0387 M HFO 1234ze EYES/EARS/NOSE 08-10-04 10:08 P CLEAR DISCHARGE LEFT EYE 0387 M HFO 1234ze EXCRETA 08-10-04 10:08 P DIARRHEA 0409 M HFO 1234ze EYES/EARS/NOSE 08-12-04 11:56 P INJECTED SCLERA RIGHT EYE 08-16-04 12:06 P INJECTED SCLERA RIGHT EYE 0409 M HFO 1234ze EXCRETA 08-10-04 11:59 P SOFT FECES

----------------------------------------------------------------------------------------------------------------------------------- GRADE CODE: 1 - SLIGHT 2 - MODERATE 3 - SEVERE P - PRESENT PCRDv4.1 05/11/2006 R:06/20/200633of105TABLE 4 (PRIOR TO EXPOSURE - HFO 1234yf) PROJECT NO.:WIL-447008 CARDIAC SENSITIZATION STUDY OF HFO 1234ze & HFO 1234yf IN DOGS PAGE 1 SPONSOR:HONEYWELL INDIVIDUAL CLINICAL OBSERVATIONS
TABLE RANGE: 07-20-04 TO 07-29-04

----------------------------------------------------------------------------------------------------------------------------------- ANIMAL SEX GROUP CATEGORY DATE TIME GRADE OBSERVATIONS

----------------------------------------------------------------------------------------------------------------------------------- 0387 M HFO 1234yf EYES/EARS/NOSE 07-20-04 12:01 P CLEAR DISCHARGE LEFT EYE 07-22-04 13:14 P CLEAR DISCHARGE LEFT EYE 07-29-04 12:24 P CLEAR DISCHARGE LEFT EYE 0409 M HFO 1234yf EYES/EARS/NOSE 07-20-04 12:23 P INJECTED SCLERA LEFT EYE 07-20-04 12:23 P INJECTED SCLERA RIGHT EYE 07-22-04 13:40 P INJECTED SCLERA LEFT EYE 07-22-04 13:40 P INJECTED SCLERA RIGHT EYE 07-29-04 12:48 P INJECTED SCLERA RIGHT EYE

----------------------------------------------------------------------------------------------------------------------------------- GRADE CODE: 1 - SLIGHT 2 - MODERATE 3 - SEVERE P - PRESENT PCRDv4.1 05/11/2006 R:06/20/200634of105TABLE 5 (DURING AND POST-EXPOSURE - HFO 1234ze) PROJECT NO.:WIL-447008A CARDIAC SENSITIZATION STUDY OF HFO 1234ze & HFO 1234yf IN DOGS PAGE 1 SPONSOR:HONEYWELL INDIVIDUAL CLINICAL OBSERVATIONS
TABLE RANGE: 08-10-04 TO 08-19-04

----------------------------------------------------------------------------------------------------------------------------------- ANIMAL SEX GROUP CATEGORY DATE TIME GRADE OBSERVATIONS

----------------------------------------------------------------------------------------------------------------------------------- 0136 M HFO 1234ze BEHAVIOR/CNS 08-16-04 8:53 P VOCALIZATION DURING EXPOSURE 0188 M HFO 1234ze BEHAVIOR/CNS 08-16-04 9:14 P VOCALIZATION DURING EXPOSURE 0188 M HFO 1234ze EYES/EARS/NOSE 08-10-04 9:23 P INJECTED SCLERA LEFT EYE 08-10-04 9:23 P INJECTED SCLERA RIGHT EYE 08-16-04 9:17 P INJECTED SCLERA RIGHT EYE 0409 M HFO 1234ze BEHAVIOR/CNS 08-16-04 12:19 P VOCALIZATION DURING EXPOSURE

----------------------------------------------------------------------------------------------------------------------------------- GRADE CODE: 1 - SLIGHT 2 - MODERATE 3 - SEVERE P - PRESENT PCRDv4.1 05/11/2006 R:06/20/200635of105TABLE 6 (DURING AND POST-EXPOSURE - HFO 1234yf) PROJECT NO.:WIL-447008 CARDIAC SENSITIZATION STUDY OF HFO 1234ze & HFO 1234yf IN DOGS PAGE 1 SPONSOR:HONEYWELL INDIVIDUAL CLINICAL OBSERVATIONS
TABLE RANGE: 07-20-04 TO 07-29-04

----------------------------------------------------------------------------------------------------------------------------------- ANIMAL SEX GROUP CATEGORY DATE TIME GRADE OBSERVATIONS

----------------------------------------------------------------------------------------------------------------------------------- 0188 M HFO 1234yf EYES/EARS/NOSE 07-20-04 10:31 P CLEAR DISCHARGE LEFT EYE 07-20-04 10:31 P INJECTED SCLERA LEFT EYE 07-20-04 10:31 P INJECTED SCLERA RIGHT EYE 07-29-04 9:23 P INJECTED SCLERA RIGHT EYE 0357 M HFO 1234yf BEHAVIOR/CNS 07-22-04 11:45 P VOCALIZATION DURING DOSING 0357 M HFO 1234yf EYES/EARS/NOSE 07-20-04 10:54 P CLEAR DISCHARGE LEFT EYE 07-20-04 10:54 P CLEAR DISCHARGE RIGHT EYE 07-29-04 10:19 P INJECTED SCLERA LEFT EYE 07-29-04 10:19 P INJECTED SCLERA RIGHT EYE 0387 M HFO 1234yf BEHAVIOR/CNS 07-22-04 13:30 P STRUGGLING DURING DOSING 0387 M HFO 1234yf EYES/EARS/NOSE 07-20-04 12:18 P CLEAR DISCHARGE LEFT EYE 07-22-04 13:30 P CLEAR DISCHARGE LEFT EYE

----------------------------------------------------------------------------------------------------------------------------------- GRADE CODE: 1 - SLIGHT 2 - MODERATE 3 - SEVERE P - PRESENT PCRDv4.1 05/11/2006 R:06/20/200636of105TABLE 7 (HFO 1234ze) PROJECT NO.:WIL-447008A CARDIAC SENSITIZATION STUDY OF HFO 1234ze & HFO 1234yf IN DOGS PAGE 1 SPONSOR:HONEYWELL INDIVIDUAL BODY WEIGHTS [KG]

MALE GROUP: HFO 1234ze WEEK 0 1 1’

----------------------------------------------------------------------------------------------------------------------------------- ANIMAL 0136 7.5 7.6 7.7 0188 9.6 9.8 9.9 0357 9.3 9.7 9.8 0387 10.0 10.1 10.3 0409 11.9 12.3 12.4 0428 9.7 10.3 10.4

MEAN 9.7 10.0 10.1 S.D. 1.41 1.48 1.51 N 6 6 6

’ = SECOND WEEKLY BODY WEIGHT PBFTSv4.44 05/11/2006 R:06/20/200637of105TABLE 8 (HFO 1234yf) PROJECT NO.:WIL-447008 CARDIAC SENSITIZATION STUDY OF HFO 1234ze & HFO 1234yf IN DOGS PAGE 1 SPONSOR:HONEYWELL INDIVIDUAL BODY WEIGHTS [KG]
MALE GROUP: HFO 1234yf WEEK 0 1 2 3

----------------------------------------------------------------------------------------------------------------------------------- ANIMAL 0136 7.2 7.3 7.3 7.5 0188 9.5 9.5 9.5 9.6 0357 9.2 9.3 9.3 9.3 0387 9.7 9.9 9.7 10.0 0409 11.7 11.7 11.8 11.9 0428 9.8 10.1 9.8 9.7

MEAN 9.5 9.6 9.6 9.7 S.D. 1.46 1.43 1.42 1.41 N 6 6 6 6 PBFTSv4.44 05/11/2006 R:06/20/200638of105TABLE 9 (HFO 1234ze) PROJECT NO.:WIL-447008A CARDIAC SENSITIZATION STUDY OF HFO 1234ze & HFO 1234yf IN DOGS PAGE 1 SPONSOR:HONEYWELL INDIVIDUAL BODY WEIGHT CHANGES [KG]

MALE GROUP: HFO 1234ze WEEK 0 TO 1 1 TO 1’

----------------------------------------------------------------------------------------------------------------------------------- ANIMAL 0136 0.2 0.0 0188 0.2 0.0 0357 0.4 0.1 0387 0.1 0.2 0409 0.4 0.1 0428 0.6 0.1

MEAN 0.3 0.1 S.D. 0.18 0.08 N 6 6

’ = SECOND WEEKLY BODY WEIGHT PBFTSv4.44 05/11/2006 R:06/20/200639of105TABLE 10 (HFO 1234yf) PROJECT NO.:WIL-447008 CARDIAC SENSITIZATION STUDY OF HFO 1234ze & HFO 1234yf IN DOGS PAGE 1 SPONSOR:HONEYWELL INDIVIDUAL BODY WEIGHT CHANGES [KG]
MALE GROUP: HFO 1234yf WEEK 0 TO 1 1 TO 2 2 TO 3

----------------------------------------------------------------------------------------------------------------------------------- ANIMAL 0136 0.2 0.0 0.2 0188 -0.1 0.0 0.1 0357 0.1 0.0 0.0 0387 0.2 -0.2 0.4 0409 0.0 0.0 0.1 0428 0.3 -0.3 0.0

MEAN 0.1 -0.1 0.1 S.D. 0.15 0.13 0.15 N 6 6 6 PBFTSv4.44 05/11/2006 R:06/20/200640of105TABLE 11 PROJECT NO.:WIL-447008 CARDIAC SENSITIZATION STUDY OF HFO 1234ze & HFO 1234yf IN DOGS PAGE 1 SPONSOR:HONEYWELL INDIVIDUAL CARDIAC SENSITIZATION RESPONSES

----------------------------------------------------------------------------------------------------------------------------------- EXPOSURE LEVEL (PPM) ANIMAL NO. SEX RESPONSE-A NO. POSITIVE/NO. EXPOSED

----------------------------------------------------------------------------------------------------------------------------------- 20,000 0136 M - 0188 M - 0357 M - 0387 M - 0409 M - 0428 M - 0/6
60,000 0136 M - 0188 M - 0357 M - 0387 M - 0409 M - 0428 M - 0/6
120,000 0136 M - 0188 M - 0357 M - 0387 M - 0409 M - 0428 M - 0/6

----------------------------------------------------------------------------------------------------------------------------------- A = FOR EITHER TEST ARTICLE M = MALE + = RESPONSE INDICATIVE OF CARDIAC SENSITIZATION - = NO RESPONSE INDICATIVE OF CARDIAC SENSITIZATION MANUALv1.0 05/12/2006 R:06/20/200641of105

## WIL-447008 HFO 1234ze and HFO 1234yf Honeywell International



# APPENDIX A

Certificate Of Analysis (Sponsor-Provided Data)

42 of 105

## Honeywell International Buffalo Research Laboratory 20 Peabody Street Buffalo, New York 14210 USA

Sample ID # Material Puritv-Area%

AR #34105 Lot #62404 Batch #2 2,3,3,3-tetrafluoropropene (HFO-1234yf) CAS# 754-12-1 99.996%

Reference: Notebook #40183 Pg. 43

Certified by:

S

.

A. Ferguson Research Chemist 6/30/04 Unknown-0.004% (Not detected by GCMS)



## 43 of 105

## Honeywell International Buffalo Research Laboratory 20 Peabody Street Buffalo, New York 14210 USA

Sample ID # Material
AR#33749 tram 1 ,1 ,1,3-tetrafluoropropene LAM63004 (tram-HFO-1234ze) Batch#5 CAM 1645-83-6

Reference: Notebook #40 183 Pg. 30

Certified by:

S.A. Ferguson Research Chemist 5/20/04 

### Purity-Area%
>99.99%



## 44 of 105

## WIL-447008 HFO 1234ze and HFO 1234yf Honeywell International



# APPENDIX B

Exposure Atmosphere Generation Report

45 of 105

## ACUTE CARDIAC SENSITIZATION STUDY OF HFO 1234ze AND HFO 1234yf IN DOGS

Exposure Atmosphere Generation Report

Inhalation Department
WIL Research Laboratories, LLC WIL-447008 Honeywell International HFO 1234ze and HFO 1234yf

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# T ABLE 

# O F 

# C ONTENTS 

## Page

Table Of Contents ..................................................................................... 2 Index Of Figures........................................................................................ 2 Index Of Tables ......................................................................................... 2 1. Exposure Methods..................................................................................... 3 1.1. Exposure Regimen ...................................................................................... 3 1.2. Exposure Environmental Conditions .......................................................... 3

2. Test Atmosphere Generation Methods ................................................... 3 3. Methods Of Characterization Of Exposure Atmospheres .................... 4 3.1. Analytical Methods ..................................................................................... 4 3.2. Gas Chromatograph Calibration.................................................................. 5

4. Results Of Characterization Of Exposure Atmosphere ........................ 7 4.1. Analyzed Exposure Concentrations ............................................................ 7

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



# I NDEX 

# O F 

# F IGURES


## 1. Exposure Diagram ......................................................................................................10



# I NDEX 

# O F 

# T ABLES


## 1. Daily Mean Of Individual Concentration [ppm]........................................................12 WIL-447008 Honeywell International HFO 1234ze and HFO 1234yf

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# 1. E XPOSURE 

# M ETHODS 

# 1.1. E XPOSURE 

# R EGIMEN 

## The test articles, HFO 1234ze and HFO 1234yf, were administered as muzzle-only inhalation exposures at various concentrations of each test article gas in air. Animals were exposed individually to each test article concentration for a duration of 10 minutes for each exposure. Each animal received target concentrations of 2% (20,000 ppm), 6% (60,000 ppm) and 12% (120,000 ppm) with each test article. The following table presents the overall schedule of the general inhalation exposures:

Test Article Identification Exposure Number Date HFO 1234yf 1 20 July 2004 HFO 1234yf 2 22 July 2004 HFO 1234yf 3 29 July 2004 HFO 1234ze 1 10 August 2004 HFO 1234ze 2 12 August 2004 HFO 1234ze 3 16 August 2004



# 1.2. E XPOSURE 

# E NVIRONMENTAL 

# C ONDITIONS 

## The test atmospheres were not monitored for temperature and humidity. The test atmospheres were prepared using dry compressed air and were maintained at room temperature throughout the exposure period.



# 2. T EST 

# A TMOSPHERE 

# G ENERATION 

# M ETHODS 

## Exposures were conducted using a muzzle-only system consisting of a mask connected to 2-way valve connected to a 200-L Tedlar gas bag containing the appropriate test article mixture. The test atmosphere and exposure system is shown schematically in Figure 1 of this appendix. Mixtures of test article in compressed air/oxygen were prepared in the 200-L Tedlar gas bag by metering a pre-determined amount of air through a calibrated WIL-447008 Honeywell International HFO 1234ze and HFO 1234yf

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## Gilmont Flowmeter (Barnet/Gilmont Instruments, Barrington, IL) into a known volume of compressed air. Samples were analyzed from each bag using a gas chromatograph (GC) equipped with flame ionization detection (FID), and if necessary, additional compressed air or test article was added to the bag to achieve the appropriate target concentration. In addition to compressed air, oxygen was added as needed to maintain oxygen content above 19%. Oxygen was metered from an oxygen cylinder through a calibrated Cole Parmer Flowmeter (Cole Parmer, Vernon Hills, IL). If required, additional test article was added to the 200-L Tedlar bag through a calibrated Omega Flowmeter (Omega Engineering, Inc., Stamford, CT). From the 200-L Tedlar bag, test article was administered through corrugated tubing connected to a 3-way valve. During the pre-treatment acclimation period, animals received room air directed through a 3-way valve. At the start of exposure the 3-way valve was turned to the 200-L bag position for exposure to the test article. Food and water were withheld during exposure periods.



# 3. M ETHODS 

# O F 

# C HARACTERIZATION 

# O F 

# E XPOSURE 

# A TMOSPHERES 

# 3.1. A NALYTICAL 

# M ETHODS 

## Analyzed exposure concentrations were obtained using a GC. Samples of the exposure atmospheres were collected from each Tedlar bag containing test article prior and following each animal exposure using a sample loop and a multi-position valve (Valco Instruments Co., Inc., Houston, TX). The following table summarizes the GC conditions:

GC Manufacturer: Hewlett-Packard Model No. 5890 Series II Detector Type: FID Detector Temperature: 200°C Column Diameter × Length: 30 m × 0.53 mm ID Column Type: J & W DB-5 Megabore Fuel Gas: Hydrogen
Fuel Flowrate: 35 mL/minute
Air Flowrate: 334 mL/minute WIL-447008 Honeywell International HFO 1234ze and HFO 1234yf

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## Carrier Gas: Helium Carrier Gas Flowrate: 22 mL/minute Injector Temperature: 250°C Injection Volume: 0.25 mL Retention Time: Approximately 0.665 minute Integrator Manufacturer: HP Model 3396A Series II
Chart Zero Offset: 0 Chart Attenuation: 5 Chart Speed: 2.0 cm/minute Peak Area Rejection Value: 0
Peak Threshold: 5
Peak Width: 0.04
For the 20,000 and 60,000 ppm HFO 1234yf target exposures, the integrator chart speed was set to 1.0 cm/min.



# 3.2. G AS 

# C HROMATOGRAPH 

# C ALIBRATION 

## The GC was calibrated using gas-phase standards prepared to contain known gas concentrations of test article in 10-L Tedlar gas bags. A separate GC calibration was prepared for each test article. Standards were prepared by injecting a known volume of gas into a 10-L Tedlar gas bag. Compressed air was delivered to the 10-L Tedlar bag through a dry test meter (Model DTM-200A, American Metering Co., Horshham, PA). The following concentrations of the gas-phase standards were prepared for GC calibration: WIL-447008 Honeywell International HFO 1234ze and HFO 1234yf

- 5 - 50 of 105

## Standard Concentration (ppm) Volume of Test Article Used (mL) Volume of Air Used (L)
7937 20 2.5 19,608 50 2.5 38,462 100 2.5 56,604 150 2.5 84,249 230 2.5 107,143 300 2.5 122,807 350 2.5
Formula for calculation of standards prepared:
mL Test Article × 10 6 

## Concentration (ppm) = mL Air + mL Test Article Prior to initiation of animal exposures, the GC was calibrated with standards prepared and analyzed in triplicate. The calibration curve was defined using standard bags prepared at 7 gas concentrations spanning an appropriate range relative to the target exposure concentrations for the study. The prime calibration curve was prepared using the quadratic formula and was used to calculate exposure concentrations for all chambers. The peak area (y) and the theoretical concentrations of the calibration standards (x) were fit with least-squares regression analysis to the ln-quadratic function:
ln (y) = a × [ln (x)]2 + b × ln (x) + c
Each day, prior to the initiation of exposures, 1 of the 7 standards was prepared and analyzed to perform a calibration check of the GC. The GC was considered to be within normal calibration limits if the analyzed concentration of the prepared standard was within ± 10% of the known concentration. WIL-447008 Honeywell International HFO 1234ze and HFO 1234yf

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# 4. R ESULTS 

# O F 

# C HARACTERIZATION 

# O F 

# E XPOSURE 

# A TMOSPHERE 

# 4.1. A NALYZED 

# E XPOSURE 

# C ONCENTRATIONS 

## The overall mean analyzed concentrations for test article HFO 1234yf were 20,159 (Exposure 1), 60,522 (Exposure 2) and 120,189 ppm (Exposure 3) for the 2%, 6% and 12% target levels, respectively. The overall mean analyzed concentration for test article HFO 1234ze were 20,037 (Exposure 1), 60,271 (Exposure 2) and 119,826 ppm (Exposure 3) for the 2%, 6% and 12% target levels, respectively. Individual analyzed concentrations for each test article are presented in Table 1 of this appendix. WIL-447008 Honeywell International HFO 1234ze and HFO 1234yf

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## WIL-447008 Honey we1 1 

## International HFO 1234ze and HFO 1234yf



# 5. KEY 

# STUDY PERSONNEL AND 

# REPORT SUBMISSION

Report Submitted By: 

# &&-SLd+ / 

## Jeffkdy T. Weinberg, BS Project Specialist, Inhalation Toxicology Study Director
Report Prepared By: 

# do lL Michael S. 

## Cockbum Project Leader, Inhalation Toxicology

Study Personnel: Randy M. Barrett, BS Jay G. Henson, 

## BS Robert 

## A. Wally, BS, RAC Publishing Specialist, Regulatory Affairs and Services Group Supervisor, Study Analysis and Reports Manager, Reporting and Regulatory Technical Services

-8-



## 53 of 105

# FIGURE 1 WIL-447008 Honeywell International HFO 1234ze and HFO 1234yf

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# F IGURE 

# 1. E XPOSURE 

# D IAGRAM

200 L TEDLAR BAG 3- WAY VALVE TO MASK

TO EXHAUST DOG MASK One Way

Valve

One Way

Valve 

## WIL-447008 Honeywell International HFO 1234ze and HFO 1234yf

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# TABLE 1 WIL-447008 Honeywell International HFO 1234ze and HFO 1234yf

- 11 - 56 of 105INHALATION TABLE 1 PROJECT NO.:WIL-447008 CARDIAC SENSITIZATION STUDY OF HFO 1234ze & HFO 1234yf IN DOGS PAGE 1 SPONSOR:HONEYWELL DAILY MEAN OF INDIVIDUAL CONCENTRATION [PPM]

--------------------------------------------------------------------------------------------------------------------------------- TEST ARTICLE: HFO 1234ze EXPOSURE DATE: 10-AUG-2004 TARGET CONCENTRATION 20000 PPM

--------------------------------------------------------------------------------------------------------------------------------- ANIMAL NO. CONCENTRATION

--------------------------------------------------------------------------------------------------------------------------------- 0136 19860 20633

0188 19999 20214

0357 20449 20405

0387 19809 19537

0409 19843 20027

0428 19870 19801

-------------------------------------------------------------------------------------------------------------------------------- GRAND MEAN 20037 S.D. 323.0 C.V.(%) 1.61 N 12WIL-447008Honeywell InternationalHFO 1234ze and HFO 1234yf-12-57of105INHALATION TABLE 1 PROJECT NO.:WIL-447008 CARDIAC SENSITIZATION STUDY OF HFO 1234ze & HFO 1234yf IN DOGS PAGE 2 SPONSOR:HONEYWELL DAILY MEAN OF INDIVIDUAL CONCENTRATION [PPM]

--------------------------------------------------------------------------------------------------------------------------------- TEST ARTICLE: HFO 1234ze EXPOSURE DATE: 12-AUG-2004 TARGET CONCENTRATION 60000 PPM

--------------------------------------------------------------------------------------------------------------------------------- ANIMAL NO. CONCENTRATION

--------------------------------------------------------------------------------------------------------------------------------- 0136 60230 60237

0188 59639 60906

0357 59520 59831

0387 60642 60540

0409 60512 60466

0428 59328 61404

-------------------------------------------------------------------------------------------------------------------------------- GRAND MEAN 60271 S.D. 606.2 C.V.(%) 1.01 N 12WIL-447008Honeywell InternationalHFO 1234ze and HFO 1234yf-13-58of105INHALATION TABLE 1 PROJECT NO.:WIL-447008 CARDIAC SENSITIZATION STUDY OF HFO 1234ze & HFO 1234yf IN DOGS PAGE 3 SPONSOR:HONEYWELL DAILY MEAN OF INDIVIDUAL CONCENTRATION [PPM]

--------------------------------------------------------------------------------------------------------------------------------- TEST ARTICLE: HFO 1234ze EXPOSURE DATE: 16-AUG-2004 TARGET CONCENTRATION 120000 PPM

--------------------------------------------------------------------------------------------------------------------------------- ANIMAL NO. CONCENTRATION

--------------------------------------------------------------------------------------------------------------------------------- 0136 120305 120822

0188 120939 110253

0357 120509 120667

0387 121333 121070

0409 120538 119865

0428 121382 120226

-------------------------------------------------------------------------------------------------------------------------------- GRAND MEAN 119826 S.D. 3047.9 C.V.(%) 2.54 N 12WIL-447008Honeywell InternationalHFO 1234ze and HFO 1234yf-14-59of105INHALATION TABLE 1 PROJECT NO.:WIL-447008 CARDIAC SENSITIZATION STUDY OF HFO 1234ze & HFO 1234yf IN DOGS PAGE 4 SPONSOR:HONEYWELL DAILY MEAN OF INDIVIDUAL CONCENTRATION [PPM]

--------------------------------------------------------------------------------------------------------------------------------- TEST ARTICLE: HFO 1234yf EXPOSURE DATE: 20-JULY-2004 TARGET CONCENTRATION 20000 PPM

--------------------------------------------------------------------------------------------------------------------------------- ANIMAL NO. CONCENTRATION

--------------------------------------------------------------------------------------------------------------------------------- 0136 20125 20200

0188 20201 20201

0357 19434 18929

0387 20609 20841

0409 20879 20826

0428 19634 20033

-------------------------------------------------------------------------------------------------------------------------------- GRAND MEAN 20159 S.D. 600.6 C.V.(%) 2.98 N 12WIL-447008Honeywell InternationalHFO 1234ze and HFO 1234yf-15-60of105INHALATION TABLE 1 PROJECT NO.:WIL-447008 CARDIAC SENSITIZATION STUDY OF HFO 1234ze & HFO 1234yf IN DOGS PAGE 5 SPONSOR:HONEYWELL DAILY MEAN OF INDIVIDUAL CONCENTRATION [PPM]

--------------------------------------------------------------------------------------------------------------------------------- TEST ARTICLE: HFO 1234yf EXPOSURE DATE: 22-JULY-2004 TARGET CONCENTRATION 60000 PPM

--------------------------------------------------------------------------------------------------------------------------------- ANIMAL NO. CONCENTRATION

--------------------------------------------------------------------------------------------------------------------------------- 0136 61937 61389

0188 61351 60603

0357 59174 58088

0387 60016 64263

0409 59560 59695

0428 60192 60000

-------------------------------------------------------------------------------------------------------------------------------- GRAND MEAN 60522 S.D. 1579.2 C.V.(%) 2.61 N 12WIL-447008Honeywell InternationalHFO 1234ze and HFO 1234yf-16-61of105INHALATION TABLE 1 PROJECT NO.:WIL-447008 CARDIAC SENSITIZATION STUDY OF HFO 1234ze & HFO 1234yf IN DOGS PAGE 6 SPONSOR:HONEYWELL DAILY MEAN OF INDIVIDUAL CONCENTRATION [PPM]

--------------------------------------------------------------------------------------------------------------------------------- TEST ARTICLE: HFO 1234yf EXPOSURE DATE: 29-JULY-2004 TARGET CONCENTRATION 120000 PPM

--------------------------------------------------------------------------------------------------------------------------------- ANIMAL NO. CONCENTRATION

--------------------------------------------------------------------------------------------------------------------------------- 0136 119479 119918

0188 121091 120768

0357 120837 121042

0387 120215 119845

0409 119172 119147

0428 119703 121050

-------------------------------------------------------------------------------------------------------------------------------- GRAND MEAN 120189 S.D. 744.1 C.V.(%) 0.62 N 12 MANUALv1.0 11/11/2004 R:06/20/2006WIL-447008Honeywell InternationalHFO 1234ze and HFO 1234yf-17-62of105

## WIL-447008 HFO 1234ze and HFO 1234yf Honeywell International



# APPENDIX C

Individual Evaluation Of Epinephrine Doses

63 of 105

### WIL-447008 The following are the results observed from the baseline ECG and epinephrine doses conducted on 7/14/04 (** = conducted on 7/16/04):

Animal # Observations noted: 134 No effects were observed through the 6 ug/kg epinephrine dose level. At the 8 ug/kg epinephrine dose level starting approximately 1.5 minutes after injection, 2 AV blocks were observed (AV blocks generally considered common in young beagle dogs). 188 No effects observed through the 8 ug/kg epinephrine dose level. 387 No effects observed through the 8 ug/kg epinephrine dose level. 409 No effects observed through the 6 ug/kg epinephrine dose level. At the 8 ug/kg epinephrine dose level, approximately 30 seconds after the epinephrine injection there were a few instances where it appeared that the p wave occurred prior to the t wave (significance not understood). 459 No effects observed through the 6 ug/kg epinephrine dose level. At the 8 ug/kg epinephrine dose level starting at approximately 12 seconds after the injection, 2 PVCs were observed within a 5-second time frame. 136 No effects observed at the 2 ug/kg epinephrine dose level. At the 3 ug/kg epinephrine dose level starting at approximately 40 seconds after injection, 4 PVCs were observed within a 10-second time frame. At the 4 ug/kg epinephrine dose level starting at approximately 20 seconds after injection, 12 PVCs were observed within a 15-second time frame. No effects observed at the 6 ug/kg epinephrine dose level . ** At the 8 ug/kg epinephrine dose level starting at approximately 15 seconds after injection, 13 PVCs were observed within a 30-second time frame. ** 357 No effects observed through the 4 ug/kg epinephrine dose level. At the 6 ug/kg epinephrine dose level starting at approximately 15 seconds after injection, 29 PVCs were observed within a 40-second time frame. At the 8 ug/kg epinephrine dose level starting at approximately 15 seconds after injection, 21 PVCs were observed within a 45-second time frame. ** 377 At the 2 ug/kg epinephrine dose level starting at approximately 10 seconds after injection, 5 PVCs were observed within a 35-second time frame. At the 3 ug/kg epinephrine dose level starting at approximately 12 seconds after injection, 18 PVCs were observed within a 35-second time frame. No effects observed at the 4 ug/kg epinephrine dose level. ** At the 6 ug/kg epinephrine dose level starting at approximately 5 seconds after injection, 21 PVCs were observed within a 40-second time frame. ** At the 8 ug/kg-epinephrine dose level starting at approximately 25 seconds after injection, there were 11 PVCs in <10 seconds. ** 428 No effects observed at the 2 ug/kg epinephrine dose level. At the 3 ug/kg epinephrine dose level starting at approximately 20 seconds after injection, 13 PVCs were observed within a 25-second time frame. At the 4 ug/kg epinephrine dose level starting at approximately 30 seconds after injection, 11 PVCs were observed within a 25-second time frame. ** At the 6 ug/kg epinephrine dose level starting at approximately 20 seconds after injection, 22 PVCs were observed within a 35-second time frame. ** At the 8 ug/kg epinephrine dose level starting at approximately 20 seconds after injection, 35 PVCs were observed within a 50-second time frame. **

AV = Atrioventricular block PVC = Preventricular contraction



## 64 of 105

## WIL-447008 HFO 1234ze and HFO 1234yf Honeywell International



# APPENDIX D

Animal Room Environmental Conditions

65 of 105TEMPERATURE/HUMIDITY - DAILY SUMMARY REPORT BY STUDY447008HONEYWELLCARDIAC SENSITIZATION STUDY OF HFO 1234ZE & HFO 1234YF IN DOGSSPONSOR:PROJECT NO.:WILPAGE 144700806/20/047:0016:0008/19/04STUDY SPECIFICATIONS:DATE IN:TIME IN:DATE OUT:TIME OUT:B ROOM 17DOGLOW TEMPERATURE oF:63.0 HIGH TEMPERATURE oF:73.0LOW HUMIDITY:30.0HIGH HUMIDITY:70.0ROOM SPECIFICATIONS:SPECIES:TEMPERATURE HUMIDITYMEAN (oF)DATEMEAN (%RH)LOW TEMPERATURE oC:17.2 HIGH TEMPERATURE oC:22.8MEAN (oC)67.9 45.420-Jun-04 19.968.0 50.021-Jun-04 20.068.0 52.622-Jun-04 20.068.0 48.923-Jun-04 20.068.2 52.524-Jun-04 20.168.0 50.525-Jun-04 20.068.0 46.626-Jun-04 20.067.9 48.227-Jun-04 20.068.0 50.728-Jun-04 20.067.9 50.729-Jun-04 20.068.1 50.830-Jun-04 20.169.2 56.001-Jul-04 20.769.2 53.802-Jul-04 20.768.4 51.803-Jul-04 20.268.6 56.504-Jul-04 20.468.8 56.205-Jul-04 20.571.2 61.606-Jul-04 21.868.5 56.307-Jul-04 20.368.8 53.608-Jul-04 20.569.4 52.309-Jul-04 20.868.9 52.710-Jul-04 20.570.3 57.211-Jul-04 21.369.3 58.912-Jul-04 20.76/20/2006 11:12REPORT 4VERSION 1.09NOTE: + = VALUE WAS GREATER THAN HIGH RANGE- = VALUE WAS LESS THAN LOW RANGENOTE: MEANS REPRESENT THE MEAN OF THE DAILY VALUES



## 66 of 105TEMPERATURE/HUMIDITY - DAILY SUMMARY REPORT BY STUDY447008HONEYWELLCARDIAC SENSITIZATION STUDY OF HFO 1234ZE & HFO 1234YF IN DOGSSPONSOR:PROJECT NO.:WILPAGE 244700806/20/047:0016:0008/19/04STUDY SPECIFICATIONS:DATE IN:TIME IN:DATE OUT:TIME OUT:B ROOM 17DOGLOW TEMPERATURE oF:63.0 HIGH TEMPERATURE oF:73.0LOW HUMIDITY:30.0HIGH HUMIDITY:70.0ROOM SPECIFICATIONS:SPECIES:TEMPERATURE HUMIDITYMEAN (oF)DATEMEAN (%RH)LOW TEMPERATURE oC:17.2 HIGH TEMPERATURE oC:22.8MEAN (oC)69.2 57.313-Jul-04 20.768.3 55.014-Jul-04 20.268.1 53.115-Jul-04 20.168.5 51.116-Jul-04 20.367.9 51.817-Jul-04 20.068.1 51.518-Jul-04 20.068.4 52.919-Jul-04 20.268.6 54.320-Jul-04 20.369.0 55.421-Jul-04 20.669.7 58.322-Jul-04 21.068.1 54.623-Jul-04 20.168.5 47.424-Jul-04 20.368.2 49.025-Jul-04 20.169.3 56.326-Jul-04 20.768.0 53.427-Jul-04 20.068.0 51.828-Jul-04 20.067.9 51.529-Jul-04 20.068.8 58.230-Jul-04 20.468.4 58.131-Jul-04 20.269.0 55.301-Aug-04 20.668.8 55.502-Aug-04 20.469.8 61.003-Aug-04 21.068.2 56.204-Aug-04 20.16/20/2006 11:12REPORT 4VERSION 1.09NOTE: + = VALUE WAS GREATER THAN HIGH RANGE- = VALUE WAS LESS THAN LOW RANGENOTE: MEANS REPRESENT THE MEAN OF THE DAILY VALUES



## 67 of 105TEMPERATURE/HUMIDITY - DAILY SUMMARY REPORT BY STUDY447008HONEYWELLCARDIAC SENSITIZATION STUDY OF HFO 1234ZE & HFO 1234YF IN DOGSSPONSOR:PROJECT NO.:WILPAGE 344700806/20/047:0016:0008/19/04STUDY SPECIFICATIONS:DATE IN:TIME IN:DATE OUT:TIME OUT:B ROOM 17DOGLOW TEMPERATURE oF:63.0 HIGH TEMPERATURE oF:73.0LOW HUMIDITY:30.0HIGH HUMIDITY:70.0ROOM SPECIFICATIONS:SPECIES:TEMPERATURE HUMIDITYMEAN (oF)DATEMEAN (%RH)LOW TEMPERATURE oC:17.2 HIGH TEMPERATURE oC:22.8MEAN (oC)67.9 50.905-Aug-04 20.068.0 48.606-Aug-04 20.067.9 46.107-Aug-04 19.968.0 46.608-Aug-04 20.068.0 50.709-Aug-04 20.068.0 54.410-Aug-04 20.068.2 54.011-Aug-04 20.168.1 49.412-Aug-04 20.169.8 55.213-Aug-04 21.068.3 49.314-Aug-04 20.269.1 49.615-Aug-04 20.669.1 54.916-Aug-04 20.669.2 53.117-Aug-04 20.769.5 56.618-Aug-04 20.870.2 68.719-Aug-04 21.26/20/2006 11:12REPORT 4VERSION 1.09NOTE: + = VALUE WAS GREATER THAN HIGH RANGE- = VALUE WAS LESS THAN LOW RANGENOTE: MEANS REPRESENT THE MEAN OF THE DAILY VALUES



## 68 of 105TEMPERATURE/HUMIDITY - DAILY SUMMARY REPORT BY STUDY447008HONEYWELLCARDIAC SENSITIZATION STUDY OF HFO 1234ZE & HFO 1234YF IN DOGSSPONSOR:PROJECT NO.:WILPAGE 444700806/20/047:0016:0008/19/04STUDY SPECIFICATIONS:DATE IN:TIME IN:DATE OUT:TIME OUT:B ROOM 17DOGLOW TEMPERATURE oF:63.0 HIGH TEMPERATURE oF:73.0LOW HUMIDITY:30.0HIGH HUMIDITY:70.0ROOM SPECIFICATIONS:SPECIES:TEMPERATURE HUMIDITYMEAN (oF)DATEMEAN (%RH)LOW TEMPERATURE oC:17.2 HIGH TEMPERATURE oC:22.8MEAN (oC)68.6TEMPERATURE oF 67.9 71.268.745.453.361MEAN MINMAX20.3TEMPERATURE oC 19.9 21.8HUMIDITY (%RH)**GRAND STATS**N DAYS6/20/2006 11:12REPORT 4VERSION 1.09NOTE: + = VALUE WAS GREATER THAN HIGH RANGE- = VALUE WAS LESS THAN LOW RANGENOTE: MEANS REPRESENT THE MEAN OF THE DAILY VALUES



## 69 of 105TEMPERATURE/HUMIDITY - END OF STUDY SUMMARY REPORT447008HONEYWELLCARDIAC SENSITIZATION STUDY OF HFO 1234ZE & HFO 1234YF IN DOGSSPONSOR:PROJECT NO.:WIL-11:1120-Jun-06PAGE 1B ROOM 17DOGLOW TEMPERATURE: 63.0HIGH TEMPERATURE: 73.0LOW HUMIDITY: 30.0HIGH HUMIDITY: 70.0ROOM SPECIFICATIONS:SPECIES:TEMPERATURE HUMIDITY06/20/047:0016:0008/19/04DATE IN:TIME IN:DATE OUT:TIME OUT:**ROOM B ROOM 17 SUMMARY**MEANMINMAX68.666.175.753.243.484.41.246.37SDNSAMPLES14401440NDAYSFIRST DAYLAST DAY06/20/0408/19/04616/20/2006 11:11REPORT 5VERSION 1.10NOTE: TEMPERATURE UNITS = DEGREES FAHRENHEITHUMIDITY UNITS = % RELATIVE HUMIDITYNOTE: MEANS REPRESENT THE MEAN OF ALL VALUES



## 70 of 105TEMPERATURE/HUMIDITY - END OF STUDY SUMMARY REPORT447008HONEYWELLCARDIAC SENSITIZATION STUDY OF HFO 1234ZE & HFO 1234YF IN DOGSSPONSOR:PROJECT NO.:WIL-11:1120-Jun-06PAGE 2**STUDY 447008 SUMMARY**MEANMINMAX68.666.175.753.243.484.41.246.37SDNSAMPLES14401440FIRST DAYLAST DAY06/20/0408/19/04NDAYS616/20/2006 11:11REPORT 5VERSION 1.10NOTE: TEMPERATURE UNITS = DEGREES FAHRENHEITHUMIDITY UNITS = % RELATIVE HUMIDITYNOTE: MEANS REPRESENT THE MEAN OF ALL VALUES



## 71 of 105

## WIL-447008 HFO 1234ze and HFO 1234yf Honeywell International



# APPENDIX E

Individual Electrocardiographic Data

72 of 105

## WIL –447008

ECG results from the 2% (20,000 ppm) exposure to HFO 1234ze conducted on August 10, 2004 are as follows. The mean analyzed exposure concentration for all six dogs was 20,037 ppm. The o xygen content of all exposure atmospheres was 19.6%.

Animal # EPI dose ECG Responses

136 2 ug/kg Pre-exposure: no effect 2 ug/kg Exposure: no effect

188 8 ug/kg Pre-exposure: no effect 8 ug/kg Exposure: no effect 357 4 ug/kg Pre-exposure: no effect

4 ug/kg Exposure: no effect

387 8 ug/kg Pre-exposure: 32 seconds after injection 5 PVC’s in 22 seconds 8 ug/kg Exposure: 27 seconds after injection 8 PVC’s in 33 seconds

409 6 ug/kg Pre-exposure: no effect 6 ug/kg Exposure: 24 seconds after injection 28 PVC’s in 49 seconds

428 2 ug/kg Pre-exposure: no effect 2 ug/kg Exposure: no effect

PVC = Preventricular contraction



## 73 of 105

## WIL –447008

ECG results from the 2% (20,000-ppm) exposure to HFO 1234yf conducted on July 20, 2004 are as follows. The mean analyzed exposure concentration for all six dogs was 20,159 ppm. The o xygen content of all exposure atmospheres was 19.7-19.8%.

Animal # EPI dose ECG Responses

136 2 ug/kg Pre-exposure: no effect 2 ug/kg Exposure: no effect

188 8 ug/kg Pre-exposure: no effect 8 ug/kg Exposure: no effect 357 4 ug/kg Pre-exposure: 22 seconds after injection 3 PVCs in 25 seconds

4 ug/kg Exposure: 40 seconds after injection 5 PVCs in 14 seconds

387 8 ug/kg Pre-exposure: 48 seconds after injection 4 PVCs in 23 seconds 8 ug/kg Exposure: 24 seconds after injection 7 PVCs in 30 seconds

409 6 ug/kg Pre-exposure: 29 seconds after injection 35 PVCs in 48 seconds 6 ug/kg Exposure: no effects

428 2 ug/kg Pre-exposure: no effect 2 ug/kg Exposure: no effect

PVC = Preventricular contraction



## 74 of 105

## WIL –447008

ECG results from the 6% (60,000-ppm) exposure to HFO 1234ze conducted on August 12, 2004 are as follows. The mean analyzed exposure concentration for all six dogs was 60,271 ppm. The o xygen content of all exposure atmospheres was 19.5-19.7%.

Animal # EPI dose ECG Responses

136 2 ug/kg Pre-exposure: no effect 2 ug/kg Exposure: no effect

188 8 ug/kg Pre-exposure: no effect 8 ug/kg Exposure: no effect 357 4 ug/kg Pre-exposure: no effect

4 ug/kg Exposure: no effect

387 8 ug/kg Pre-exposure: 23 seconds after injection 10 PVC’s in 37 seconds 8 ug/kg Exposure: 25 seconds after injection 17 PVC’s in 45 seconds

409 6 ug/kg Pre-exposure: no effect 6 ug/kg Exposure: no effect

428 2 ug/kg Pre-exposure: no effect 2 ug/kg Exposure: no effect

PVC = Preventricular contraction



## 75 of 105

## WIL –447008

ECG results from the 6% (60,000-ppm) exposure to HFO 1234yf conducted on July 22, 2004 are as follows. The mean analyzed exposure concentration for all six dogs was 60,522 ppm. The o xygen content of all exposure atmospheres was 19.5-19.6%.

Animal # EPI dose ECG Responses

136 2 ug/kg Pre-exposure: no effect 2 ug/kg Exposure: no effect

188 8 ug/kg Pre-exposure: no effect 8 ug/kg Exposure: no effect 357 4 ug/kg Pre-exposure: 42 seconds after injection 6 PVCs in 21 seconds

4 ug/kg Exposure: no effects

387 8 ug/kg Pre-exposure: 63 seconds after injection 6 PVCs in 14 seconds 8 ug/kg Exposure: no effect

409 6 ug/kg Pre-exposure: 23 seconds after injection 44 PVCs in 56 seconds 6 ug/kg Exposure: 23 seconds after injection 39 PVCs in 63 seconds

428 2 ug/kg Pre-exposure: no effect 2 ug/kg Exposure: no effect

PVC = Preventricular contraction



## 76 of 105

## WIL –447008

ECG results from the 12% (120,000-ppm) exposure to HFO 1234ze conducted on August 16, 2004 are as follows. The mean analyzed exposure concentration for all six dogs was 119,826 ppm. The oxygen content of all exposure atmospheres was 19.3-19.5%.

Animal # EPI dose ECG Responses

136 2 ug/kg Pre-exposure: no effect 2 ug/kg Exposure: no effect

188 8 ug/kg Pre-exposure: no effect 8 ug/kg Exposure: 24 seconds after injection 1 PVC 357 4 ug/kg Pre-exposure: 33 seconds after injection 4 PVC’s in 24 seconds

4 ug/kg Exposure: 29 seconds after injection 3 PVC’s in 6 seconds

387 8 ug/kg Pre-exposure: 24 seconds after injection 14 PVC’s in 37 seconds 8 ug/kg Exposure: 22 seconds after injection 16 PVC’s in 57 seconds

409 6 ug/kg Pre-exposure: no effect 6 ug/kg Exposure: no effect

428 2 ug/kg Pre-exposure: no effect 2 ug/kg Exposure: no effect

PVC = Preventricular contraction



## 77 of 105

## WIL –447008

ECG results from the 12% (120,000-ppm) exposure to HFO 1234yf conducted on July 29, 2004 are as follows. The mean analyzed exposure concentration for all six dogs was 120,189 ppm. The oxygen content of all exposure atmospheres was 19.4-19.8%.

Animal # EPI dose ECG Responses

136 2 ug/kg Pre-exposure: no effect 2 ug/kg Exposure: no effect

188 8 ug/kg Pre-exposure: no effect 8 ug/kg Exposure: no effect 357 4 ug/kg Pre-exposure: no effect

4 ug/kg Exposure: 21 seconds after injection 1 PVC

387 8 ug/kg Pre-exposure: 32 seconds after injection 2 PVC’s in 11 seconds 8 ug/kg Exposure: 38 seconds after injection 1 PVC

409 6 ug/kg Pre-exposure: no effect 6 ug/kg Exposure: no effect

428 2 ug/kg Pre-exposure: no effect 2 ug/kg Exposure: no effect

PVC = Preventricular contraction



## 78 of 105

## Representative Electrocardiogram

No Effect Animal No. 409

HFO 1234ze 6.0 μg/kg Challenge Dose 120,000 ppm Test Article

79 of 1056.0μg/kgchallenge doseof epinephrine



## 80 of 105

## 81 of 1050
c

d0 

## * c



## 82 of 105

## Representative Electrocardiogram

No Effect Animal No. 409

HFO 1234yf 6.0 μ g/kg Challenge Dose 120,000 ppm Test Article

83 of 1056.0μg/kgchallenge doseof epinephrine.



## 84 of 105

## 85 of 1050

Iz **Lu** 0 nz w **a m**  cy0 --d

-0

**a 3**  0 

### -- IYO

**1 a** 



## 86 of 105

## WIL-447008 HFO 1234ze and HFO 1234yf Honeywell International



# APPENDIX F

Study Protocol

87 of 105

### Study Number: WIL-447008



### PROTOCOL AMENDMENT I1
Sponsor: Honeywell International

A. Title of Study:

Acute Cardiac Sensitization Study of 

### HFO 1234ze and 

### HFO 1234yf in Dogs

B



### . Protocol Modifications:
1) 

### 2.2 WIL Study Director:
Change this section to: Jeffrey T. Weinberg, BS Project Specialist, Inhalation Tel: 419-289-8700 Fax: 419-289-3650 E-mail: j 

### weinberg @ 

### wilresearch.com

C. 

### Reasons for Protocol Modifications: 

### 1) The original study director is no longer employed by WE Research Laboratories, LLC. Approved By: 

### HoneywellJJ?fternational 

## ,/

- 

# --

**a** 

### Gehe M. RuscKPhD, DABT, FATS 

# ' Sponsor Representative Prepared By: 

### WIL Research Laboratories, LLC

45kLaLA-y 

### y T. Weinberg, 

### BS Study Director

WIL 

### RESEARCH LABORATORIES, LI,C 1407 GEORGE ROAD ASHLAND, OM 44805-9281 (419) 289 8700 FAX (419) 289-3650

I -..*nv Improving human health and protecting the environment through scientific research services.@



## 88 of 105Study Number: WL-447008

PROTOCOL AMENDMENT I Sponsor: Honeywell International

A. Title of Studv:

Acute Cardiac Sensitization Study of HFO 1234ze and HFO 1234yfin Dogs

B. Protocol Modifications:
1) 3 STUDY SCHEDULE:
Proposed Experimental Start Date: July 20,2004
Proposed Experimental Termination Date: Week of August 16,2004 Proposed Audited Draft Report Date: Week of November 8,2004



### 2) 8 EXPERIMENTAL DESIGN:

8.2 Assicwment to Study:

The following paragraph is added:

Seven of nine animals were considered suitable based on health, temperament, pretest ECG results and individual sensitivity to epinephrine. A straight randomization using the WIL Toxicology Data Management System (WTDMSM) will be conducted to assign six animals to the study.
3) 8.5 Evaluation of EuineDhrine Dose:

The units of epinephrine doses are changed from “mg/kg” to “@kg.”
4) 8.7 Exposure Methods:

The test article atmosphere will not be monitored for temperature and humidity. The atmosphere will be prepared using dry compressed air, thereby, resulting in the exposure atmosphere containing <lo% relative humidity. The test atmosphere will be maintained at room temperature throughout the exposure period.

WIL RESEARCH LABORATORIESINC 1407GEORGEROAD ASHLAND,OH 448059281 (419)2898700 FAXt419)2893630 Improving human health and protecting the environment through scientific research services.@



## 89 of 105WIL-447008 Protocol Amendment I Page Two 

### 5) 9 PARAMETERS TO BE EVALUATED

9.3 Individual Bodv Weights:

This section has been changed to the following:

Individual body weights will be recorded just prior to the first exposure, approximately once weekly thereafter and after completion of all exposures. 

### 6) 9.4 Electrocardiograahic Examinations:
During the pretreatment period it was determined that a chart speed of 25 mm/set will be employed throughout the study.

C. Reasons for Protocol Modifications: 1) Study schedule dates are added. 2) A randomization was considered appropriate since more than six dogs were considered suitable for study assignment. 3) Correction of a typographical error. 4) Based on the method of test atmosphere generation and exposure using Tedlar bags, temperature and relative humidity will not be monitored during the exposures. 5) Body weights will be collected more frequently to allow for weekly adjustments to the epinephrine volumes.



## 90 of 105WL-447008 Protocol Amendment I Page Three 6) During the pretreatment period it was determined that a chart speed of 25 mrnkec. allowed for better assessment of cardiovascular condition and responses to epinephrine doses.
Approved By:

I Sponsor Representative
Prepared By:

WIL Research Laboratories, Inc.

U &ary R. Kip%ger, B.S. Study Director



## 91 of 105Page I of 14

PROTOCOL

ACUTE CARDIAC SENSITIZATION STUDY OF HFO 1234ze AND HFO 1234yf IN DOGS

Submitted To:

Honeywell International 101 Columbia Rd. Morristown, NJ 07962-l 139 WIL-447008 June 22,2004

WIL Research Laboratories, Inc. 1407 George Road Ashland. OH 448059281



## 92 of 105Pagelof14 WIL-447008 June 22.2004

1 OBJECTIVE:
The objective of this study is to evaluate the cardiac sensitization potential of HFO 1234ze and HFO 1234yf during acute inhalation exposures to various concentrations of each test article. Cardiac sensitization in this context refers to the potential for the test articles to increase the sensitivity of the heart to the pharmacological effects of epinephrine.
The study will be conducted in compliance with the EPA (Environmental Protection Agency) Good Laboratory Practice Standards, 40 CFR Part 792.

2 PERSONNEL INVOLVED IN THE STUDY:

2.1 Sponsor Rkwesentative: George M. Rusch, Ph.D., D.A.B.T., F.A.T.S. Director of Toxicology and Risk Assessment Tel: 973-455-3672 Fax: 973-455-4857 E-mail: george.rusch@honeywell.com

2.2 WIL Studv Director: Gary R. Kiplinger, B.S. Manager II, Inhalation Toxicology Tel: 419-289-8700 Fax: 419-289-3650 E-mail: gkiplinger@wilresearch.com

2.3 WIL Deputy Director:
Daniel T. Kirkpatrick, Ph.D., D.A.B.T. Director, Inhalation Tel: 419-289-8700 Fax: 419-289-3650 E-mail: dkirkpatrick@wilresearch.com

2.4 WIL Departmental Responsibilities: Christopher P. Chengelis, Ph.D., D.A.B.T. Director, Toxicology

Jonathan M. Hurley, B.S. Project Specialist, General Toxicology



## 93 of 105Page3 of 14 WIL-t47038 June 22.2004

Maxim V. Soloviev, M.D., Ph.D. Staff Toxicologist

Lewis E. Kaufman, M.S., RAC RAQP-GLP Director, Regulatory Affairs and Services

Lisa T. Snyder, D.V.M. Clinical Veterinarian

Teresa D. Morris, B.S. Operations Manager, Toxicology

Carol A. Kopp, B.S., L.A.T. Manager, Gross Pathology and

Developmental Toxicology Laboratory

Susan C. Haley, B.S. Manager, Clinical Pathology

Ronald E. Wilson, B.S. Director, Informational Systems Sally A. Keets, A..% Senior Operations Manager, Vivarium

Heather L. Osbom, B.S., RQAP-GLP Manager, Quality Assurance

3 STUDY SCHEDULE: Proposed Experimental Start Date: To be added by protocol amendment Proposed Experimental Termination Date: To be added by protocol amendment Proposed Audited Draft Report Date: To be added by protocol amendment

4 TEST ARTICLE INFORMATION:

4.1 Identification:
HFO 1234ze



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4.1.1 Lot Number:

4.1.2 To be provided by the Sponsor and documented in the study records. Purity:
Test article purity data are the responsibility of the Sponsor. ’ 4.1-j Stability:
Test article stability data are the responsibility of the Sponsor.

4.1.4 Physical Description:
To be documented by WIL Research Laboratories, Inc.

4.1.5

4J.6 Storage Conditions:
To be provided by the Sponsor. Reserqe Samples:
A reserve sample will be collected and stored in the Archives at WIL Research Laboratories, Inc.

4.1.7 Personnel Safety Data: Long sleeves (lab coat), eye protection, latex or nitrile gloves and organic respirator required when handling material or when potential high exposure may occur. Refer to Material Safety Data Sheet for complete available precautions.

5 TEST ARTICLE INFORMATION:

5.1 Identification: \

HFO 1234yf
51.1 Lot Number:
To be provided by the Sponsor and documented in the study records.

51.2 Purity:
Test article purity data are the responsibility of the Sponsor.



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5.1.3 Stability:

Test article stability data are the responsibility of the Sponsor.

5.1.4 Physical Description:
To be documented by WIL Research Laboratories, Inc.

5.1.5 Storage Conditions:
To be provided by the Sponsor.

5.1.6 Reserve Samples:
A reserve sample will be collected and stored in the Archives at WIL Research Laboratories, Inc.

5.1.7 Personnel Safety Data: Long sleeves (lab coat), eye protection, latex or nitrile gloves and organic respirator required when handling material or when potential high exposure may occur. Refer to Material Safety Data Sheet for complete available precautions.

6 TEST SYSTEM:
6.1 Species:
Dog
6.2 Strain: Beagle

6.3 Source: Ridglan Farms, Inc. Mt. Horeb, WI

6.4 Number of Animals: Up to ten males will be assigned to the acclimation phase of this study. Six males will be placed on study.



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6.5 Approximate Weight:

6.6 Animals will weigh approximately 7-12 kg at initiation of exposure. Approximate Age:

6.7 Animals will be approximately 8 to 12 months of age when placed on study.
Ider&ication System:
Animals will be uniquely identified by anear tattoo. Individual cage cards will be affixed to each cage and will display the animal number and study number.

6.8 Justification for Selection and Number of Animals:
This species and breed of animal is recognized as an appropriate model for cardiac function studies. The number of animals selected is the minimum needed to yield statistically and scientifically meaningful data.

7 SPECIFIC (NONEXPOSURE PERIOD) MAINTENANCE SCHEDULE:

7.1

7.2

7.3 Animal Housitw:
All animals will be housed individually in clean suspended stainless steel cages in an environmentally controlled room. The cages will be elevated above stainless steel flush pans, which will be cleaned daily. Dogs will be given frequent and regular opportunities for exercise and socialization in accordance with standard operating procedures.
Environmental Conditions:
Controls will be set to maintain an average daily temperature of 68 + 5°F (22 +- 3°C) and an average daily relative humidity of 50 2 20%. Temperature and relative humidity will be monitored continuously. Data for these two parameters will be scheduled for automatic collection on an hourly basis. Fluorescent lighting controlled by light timers will provide illumination for a 12-hour light/dark photoperiod. Temporary adjustments to the light/dark cycles may be made to accommodate protocol-specified activities. The ventilation rate will be set at a minimum of 10 room air changes per hour, 100% fresh air. Drinking Water:
Reverse osmosis-purified water will be available ad Iibitum. Filters servicing the automatic watering system are changed regularly. The municipal water supplying the laboratory is analyzed on a routine basis to assure that contaminants are not



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

7.4 Basal Diet: Approximately 400 g of PM1 Nutrition International, LLC Certified Canine Diet 5007 will be offered daily for approximately l-2 hours. Analyses of the certified feed for the presence of contaminants will be provided by the manufacturer to ensure that none are present at concentrations that would be expected to affect the outcome of the study.

8 EXPERIMENTAL DESIGN:

8.1

8.2

8.3 Animal Receipt and Acclimation:
The dogs used on this study will be selected from available stock based upon age, health and acceptable pretest ECG.
Each animal will be inspected by qualified personnel upon receipt. Dogs judged to be in good health and suitable as test animals (see section 7.2) will be acclimated for at least 14 days. Dogs will be weighed periodically during acclimation until study start, including approximately one week prior to initiation of the exposures and on the day prior to the first day of exposures. During the acclimation period, each dog will be observed twice daily for changes in general appearance and behavior. Immunizations prior to arrival of the dogs will include: rabies, Bordetella, oral papilloma, distemper, hepatitis, leptospirosis (DHL), Parvo and parainfluenza. Stool samples will be collected during the acclimation period and checked for parasites. Each dog will be given a thorough pretest ECG evaluation.
Prior to the start of the study, each dog will be placed in the exposure/restraint apparatus for several 10 to 20 minute periods in order to acclimate it to test article exposure conditions. At least ten acclimation sessions will be conducted. Assignment to Study:
Animals judged to be suitable for testing will be assigned to the study from available stock based on health, temperament, pretest ECG results and individual sensitivity to epinephrine.

Route and Rationale of Test Article Administration:
Because of the volatility and intended usage of the test article, the route of administration will be by inhalation exposure as this is the most likely route of



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8.4 Exposure Levels: Initially, one test article will be tested at a target exposure concentration of 2% 

### (20,000 ppm). Based on these results, at least two additional exposure concentrations with the same test article will be selected and tested. Additional test article exposure concentrations with this test article may be evaluated as necessary to define a dose-response and/or clarify previous responses (by amendment at additional cost). Following completion of all exposure levels using the initial test article, the animals will be provided at least a 7-day” washout period prior to proceeding with exposures using the second test article. Exposure concentrations with the second test article will be conducted similarly as indicated above.

8.5 Evaluation of Epinephrine Dose:
Prior to exposure (during the acclimation period) an individual response to intravenous epinephrine alone will be established for each individual dog using epinephrine doses starting at 2 mgkg and increasing to 3, 4, 6 or 8 mgkg, if necessary. The epinephrine doses will be administered as a bolus injection (~5 seconds) via the cephalic vein. During this phase of the study, the dogs will be exposed to air only via the nose-only exposure apparatus. The information gathered will be used to establish the specific level of epinephrine each dog will receive during the gas exposure phase with both test articles (see Section 10, Evaluation of Results).

8.6 Organization and Treatment Rekmen: exposure for the general population. The nose-only method of exposure was selected to achieve precise control of concentration levels and durations.

There will be one group of six dogs. Each dog will be exposed to the same concentration of test materials. At the end of each exposure period the dogs will be returned to their home cages. If additional exposure concentrations to the same test article are concluded to be necessary, a minimum of 48 hours between exposure periods will be made available for each animal.
Each dog will be comfortably restrained using a canvas sling with the appropriate ECG (limb) leads attached.
The exposure duration to air will be approximately 7 minutes and the exposure duration for the test gas will be approximately 10 minutes. In this regimen, a dog will serve as its own control based on pre-study and pre-exposure assessment of response to the epinephrine dose. The treatment regimen will be:



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8.7 (minutes) TIME

0 2 7 12 17 Exposure Methods:
All animals will be exposed to the test articles using a nose-only exposure apparatus. Test article exposure atmospheres will be prepared in 200 liter Tedlar bags. A known volume of the test article will be diluted with sufficient air to achieve the appropriate target concentration for each expos,ure. Oxygen will be supplemented as necessary to maintain a level of 19% or above. The method of exposure will be documented in the study records and described in detail in the final report. Feed and water will be withheld during the exposure period. Controls will be set to maintain test atmosphere exposure temperature at approximately 22°C (*2”) and humidity between approximately 40 to 60 percent.
8.8 Test Atmosphere Monitoring: Exposure concentrations in the Tedlar@ bags will be measured by gas chromatography prior to initiation and after completion of each exposure. Oxygen content of each exposure atmosphere will be recorded prior to initiation of exposure.
8.9 Epinephrine Challenge: &g.g

Start ECG recording Pre-exposure epinephrine dose Test gas initiated Challenge dose of epinephrine Termination of exposure/EGG

The epinephrine challenge doses (established for each dog as described in Section 7.5) will be given intravenously as a bolus injection into a cephalic vein. The dose volume will be 0.1 rnIJkg. Epinephrine solutions will be prepared fresh daily, as needed, and will be kept on ice. The vehicle will be sterile 0.9% saline (U.S.P.) for injection. Catheters may be used, if necessary, to accommodate the epinephrine dosings.
The epinephrine used will be the epinephrine hydrochloride salt (supplier to be documented in the study records). The epinephrine hydrochloride salt will be prepared as a solution in 0.9% sterile saline at a concentration of 80 yg/mL and diluted to yield epinephrine concentrations of 20, 30, 40 or 60 pg/mL, as appropriate. All glassware and utensils used for preparation of the epinephrine will be autoclaved prior to use.



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9 PARAMETERS TO BE EVALUATED:

9.1 Viability Checks:

All animals will be checked for mortality and moribundity each morning and afternoon not less than four hours apart.
9.2 Clinical Observations:
The dogs will be observed for clinical signs prior to initiation of each exposure, continually during the exposure period, and following each exposure when they are returned to their cages.
Detailed pliysical examinations will be conducted on all animals on the day prior to the,first day of exposures and after completion of all exposures.
9.3

9.4 Individual Bodv Weights:
Individual body weights will be recorded on the day prior to the first day of exposures and, after completion of all exposures. Electrocardiographic Examinations:
ECGs will be recorded with a Burdick electrocardiograph or equivalent. Only modified lead 1, 2, 3 electrocardiograms will be collected. Alligator clip electrodes will be placed on each limb. Chart speed will be 10 mm/set and response sensitivity will be set at 10 mm/mV. ECGs will be recorded continuously throughout the pre-exposure epinephrine dosing, exposure to the test gas and for five 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 article was initiated will be clearly marked on each ECG recording. During the pretreatment periods different chart speeds may be used to more fully evaluate the cardiovascular condition of the animals.

9.5 Anatomic Patholoav:
Animals in exrremis will be anesthetized by intravenous injection of sodium pentobarbital and exsanguinated. Gross necropsies will be conducted on animals that die or are euthanized in extremis. Tissues will be collected at the discretion of the pathologist and/or study director. Microscopic examination may be performed at the discretion of the pathologist and study director. Animals that survive the treatment regimen will not be subjected to necropsy



## 101 of 105and will be returned to stock. Animals not selected for use on study’will be returned to stock.

9.6 Blood/Test Article Concentration:

Will not be performed.

10 EVALUATION OF RESULTS:
ECG recordings taken during exposure to the test article and after administration of the challenge epinephrine dose will be compared to the ECG recordings made after administration of the pre-exposure epinephrine dose that was given in the absence of test article. During the epinephrine challenges (or immediately following), the following type of criteria (though not exclusively) will be applied in order to determine if sensitization has occurred:

l Eleven or more PVCs (premature ventricular contraction) in 10 seconds, with episodes of confluency.

l Ventricular tachycardia

l Fibrillation

11 QUALITY ASSURANCE:
The study will be audited by the WIL Quality Assurance Unit while in progress to assure compliance with EPA Good Laboratory Practice regulations, adherence to the protocol and to WIL standard operating procedures. The final report will be audited by the WIL Quality Assurance Unit prior to submission to the sponsor to assure that the final report accurately describes the conduct and the findings of the study.
This study is an EPA regulated study and shall be included on the master schedule.

12 RECORDS TO BE MAINTAINED:

All original raw data records, as defined by GLPs and WIL SOPS, will be stored as described in Section 13 in the Archives at WIL Research Laboratories, Inc. for a period of 10 years. At that time, in accordance with the Sponsor’s instructions, the data will be destroyed or sent to the Sponsor.

13 WORK PRODUCT: Sponsor will have title to all documentation records, raw data, slides, specimens, or other work product generated during the performance of the study. All work product



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including raw. paper data, pertinent electronic storage media and specimens will be retained at no charge for a period of 10 years following issuance of the final report in the Archives at WIL Research Laboratories, Inc. Thereafter, in accordance with the Sponsor’s instructions, the data will be destroyed or sent to the Sponsor. All work product will be stored in compliance with regulatory requirements. Any work product, including documents, specimens, and samples, that are required by this protocol, its amendments, or other written instructions of the Sponsor, to be shipped by WIL Research Laboratories, Inc. to another location will be appropriately packaged and labeled as defined by WIL’s SOPS and delivered to a common carrier for shipment. WIL. Research Laboratories, Inc. will not be responsible for shipment following delivery to the common carrier.

14 REPORTS:
The final report will contain a summary, test material data, methods and procedures, and an interpretation and discussion of the study results. The report will contain all information necessary to conform with current EPA TSCA specifications. Three copies (one unbound, two bound) of the final report will be supplied.

WIL Research Laboratories will submit one copy of an audited draft report in a timely manner upon completion of data collection prior to issuance of the final report. One revision will be permitted as part of the cost of the study, from which the Sponsor’s reasonable revisions and suggestions will be incorporated into the final report, as appropriate. Additional changes or revisions may be made, at extra cost. It is expected that the Sponsor will review the draft report and provide comments to WIL within a two-month time frame following submission. WIL will submit the final report within one month following receipt of comments. If the Sponsor’s comments and/or authorization to finalize the report have not been received at WIL within one year following submission of the draft report, WIL may elect to finalize the report following appropriate written notification to the Sponsor. Two electronic copies (PDF’) of the final report on CD-R will be provided. Requests for additional paper copies of the final report may result in additional charges.

15 PROTOCOL MODIFICATION:
Modification of the protocol may be accomplished during the course of this investigation. However, no changes will be made in the study design without the verbal or written permission of the Sponsor. In the event that the Sponsor verbally requests or approves changes in the protocol, such changes will be made by appropriate documentation in the form of protocol amendments. All alterations of the protocol and reasons for the modification(s) will be signed by the Study Director.



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16 ANIMAL WELFARE ACT COMPLIANCE:
This study will comply with all applicable sections of the Final Rules of the Animal Welfare Act regulations (9 CFR). The Sponsor should make particular note of the following:

l The Sponsor signature on this protocol documents for the Study Director the Sponsor’s assurance that the study described in this protocol does not unnecessarily duplicate previous experiments.

l Whenever possible, procedures used in this study have been designed to avoid or minimize discomfort, distress or pain to animals. All methods are described in this study protocol or in written laboratory standard operating procedures.

l Animals that experience severe or chronic pain or distress that cannot be relieved will be painlessly euthanized as deemed appropriate by the veterinary staff and Study Director. The Sponsor will be advised by the Study Director of all circumstances which could lead to this action in as timely a manner as possible.

. Methods of euthanasia used during this study are in conformance with the above referenced regulation.

. The Sponsor/Study Director has considered alternatives to procedures that may cause more than momentary or slight pain or distress to the animals and has provided a written narrative description (AWA covered species only) of the methods and sources used to determine that alternatives are not available.

17 REFERENCES: Mullin, L., Reinhardt, C. and Hemingway R. Cardiac Arrhythmias and Blood Levels Associated with Jnhalation of Halon 1301, Am. Jnd. Hygiene Assoc. Jour., 1979;~~. 40, 653-358.



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18 PROTOCOL APPROVAL: Reinhardt, C.,.Azar A., Marfield, M., Smith, P., and Mullin, L., Cardiac Arrhythmias and Aerosol “Sniffing,” Arch. Envir. Health, 1971, pp. 22,265-279.

Sponsor approval received via /vi# on N4. ,-

WIL Research Laboratories, Inc.

Date Study Director



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