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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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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TABLE OF CONTENTS

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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INDEX OF TABLES

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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INDEX OF APPENDICES

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. SUMMARY

1.1. OBJECTIVE

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. STUDY DESIGN

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. RESULTS

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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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. CONCLUSIONS

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. INTRODUCTION

2.1. GENERAL STUDY INFORMATION

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. KEY STUDY DATES

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. STUDY DESIGN

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. EXPERIMENTAL PROCEDURES - MATERIALS AND METHODS

4.1. TEST ARTICLE

4.1.1. TEST ARTICLE IDENTIFICATION

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. EPINEPHRINE HYDROCHLORIDE IDENTIFICATION

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. EPINEPHRINE HYDROCHLORIDE PREPARATION

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. TEST SYSTEM

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

this study. This species and breed of animal is recognized as 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. EXPOSURE METHODS

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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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. EXPOSURE ATMOSPHERE GENERATION AND MONITORING

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

a 3-way valve connected to a 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. ORGANIZATION AND TREATMENT REGIMEN

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 μg/kg and increasing to 3, 4, 6 or 8 μg/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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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. ANIMAL RECEIPT AND ACCLIMATION/PRETEST PERIOD

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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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. IMMUNIZATIONS PRIOR TO ARRIVAL

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. ANIMAL HOUSING AND EXERCISE

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

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

throughout the study. 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. DIET, DRINKING WATER AND MAINTENANCE

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. ENVIRONMENTAL CONDITIONS

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. ASSIGNMENT OF ANIMALS TO TREATMENT GROUPS

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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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. PARAMETERS EVALUATED

5.1. CLINICAL OBSERVATIONS AND SURVIVAL

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. BODY WEIGHTS

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. ELECTROCARDIOGRAPHIC EXAMINATIONS

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. STUDY TERMINATION

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

5.5. STATISTICAL METHODS

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

(N) used to calculate the mean.

5.6. DATA RETENTION

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

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

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

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

Reserve samples of the test 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. RESULTS AND DISCUSSION

6.1. CLINICAL OBSERVATIONS AND SURVIVAL

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. BODY WEIGHTS

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. ELECTROCARDIOGRAPHIC EXAMINATIONS

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. CONCLUSIONS

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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HFO 1234ze and HFO 1234yf 

8. KEY STUDY PERSONNEL AND REPORT SUBMISSION 

Report Submitted By: 

Jeffrey T. Weinberg, 

w=!, 

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. QUALITY ASSURANCE UNIT STATEMENT

9.1. PHASES INSPECTED

Date(s) of

Inspection(s) Phase Inspected

Date(s)

Findings

Reported to

Study Director

Date(s)

Findings

Reported to

Management Auditor(s)

29-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.

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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.

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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 

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10. REFERENCES

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.

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11. DEVIATIONS FROM THE PROTOCOL

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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TABLES 1 - 11

30 of 105
 TABLE 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:06/20/2006 

31 of 105
 TABLE 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:06/20/2006 

32 of 105
 TABLE 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:06/20/2006 

33 of 105
 TABLE 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:06/20/2006 

34 of 105
 TABLE 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:06/20/2006 

35 of 105
 TABLE 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:06/20/2006 

36 of 105
 TABLE 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 

37

 R:06/20/2006 

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 TABLE 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:06/20/2006 

38 of 105
 TABLE 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 

39

 R:06/20/2006 

 of 105
 TABLE 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:06/20/2006 

40 of 105
 TABLE 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:06/20/2006 

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APPENDIX A

Certificate Of Analysis (Sponsor-Provided Data)

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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) 

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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% 

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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

-1- 

46 of 105
TABLE OF CONTENTS

Page

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

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

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

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

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

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

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

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

INDEX OF FIGURES

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

INDEX OF TABLES

1. Daily Mean Of Individual Concentration [ppm]........................................................12

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1. EXPOSURE METHODS

1.1. EXPOSURE REGIMEN

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. EXPOSURE ENVIRONMENTAL CONDITIONS

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

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

temperature throughout the exposure period.

2. TEST ATMOSPHERE GENERATION METHODS

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

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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. METHODS OF CHARACTERIZATION OF EXPOSURE ATMOSPHERES

3.1. ANALYTICAL METHODS

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

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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. GAS CHROMATOGRAPH CALIBRATION

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:

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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 × 106

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.

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4. RESULTS OF CHARACTERIZATION OF EXPOSURE ATMOSPHERE

4.1. ANALYZED EXPOSURE CONCENTRATIONS

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

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HFO 1234ze and HFO 1234yf

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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

-9- 

54 of 105
FIGURE 1. EXPOSURE DIAGRAM

200 L TEDLAR

BAG

3- WAY VALVE

TO MASK

TO

EXHAUST

One Way DOG MASK Valve

One Way

Valve

WIL-447008

Honeywell International

HFO 1234ze and HFO 1234yf

-10- 

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TABLE 1

WIL-447008

Honeywell International

HFO 1234ze and HFO 1234yf

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56 of 105
 INHALATION 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 12 

 

 

Honeywell InternationalWIL-447008 HFO 1234ze and HFO 1234yf

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 INHALATION 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 12 

 

 

Honeywell InternationalWIL-447008 HFO 1234ze and HFO 1234yf

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58 of 105
 INHALATION 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 12 

 

 

Honeywell InternationalWIL-447008 HFO 1234ze and HFO 1234yf

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 INHALATION 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 12 

 

 

Honeywell InternationalWIL-447008 HFO 1234ze and HFO 1234yf

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60 of 105
 INHALATION 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 12 

 

 

Honeywell InternationalWIL-447008 HFO 1234ze and HFO 1234yf

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 INHALATION 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:06/20/2006 

 

 

Honeywell InternationalWIL-447008 HFO 1234ze and HFO 1234yf

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WIL-447008 HFO 1234ze and HFO 1234yf

Honeywell International

APPENDIX C

Individual Evaluation Of Epinephrine Doses

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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

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WIL-447008 HFO 1234ze and HFO 1234yf

Honeywell International

APPENDIX D

Animal Room Environmental Conditions

65 of 105
447008 TEMPERATURE/HUMIDITY - DAILY SUMMARY REPORT BY STUDY

HONEYWELL

CARDIAC SENSITIZATION STUDY OF HFO 1234ZE & HFO 1234YF IN DOGS

SPONSOR:PROJECT NO.:WIL

447008 06/20/04 

08/19/04 

STUDY SPECIFICATIONS: DATE IN: TIME IN:

DATE OUT: TIME OUT:

B ROOM 17DOG

LOW TEMPERATURE oF: 63.0 HIGH TEMPERATURE oF: 73.0 

H

ROOM SPECIFICATIONS:SPECIES:

TEMPERATURE HUMIDITY

MEAN (oF)DATE MEAN (%RH)

LOW TEMPERATURE oC: 17.2 HIGH TEMPERATURE oC: 22.8

MEAN (oC)

20-Jun-04 19.9 67.9 45.4

21-Jun-04 20.0 68.0 50.0

22-Jun-04 20.0 68.0 52.6

23-Jun-04 20.0 68.0 48.9

24-Jun-04 20.1 68.2 52.5

25-Jun-04 20.0 68.0 50.5

26-Jun-04 20.0 68.0 46.6

27-Jun-04 20.0 67.9 48.2

28-Jun-04 20.0 68.0 50.7

29-Jun-04 20.0 67.9 50.7

30-Jun-04 20.1 68.1 50.8

01-Jul-04 20.7 69.2 56.0

02-Jul-04 20.7 69.2 53.8

03-Jul-04 20.2 68.4 51.8

04-Jul-04 20.4 68.6 56.5

05-Jul-04 20.5 68.8 56.2

06-Jul-04 21.8 71.2 61.6

07-Jul-04 20.3 68.5 56.3

08-Jul-04 20.5 68.8 53.6

09-Jul-04 20.8 69.4 52.3

10-Jul-04 20.5 68.9 52.7

11-Jul-04 21.3 70.3 57.2

12-Jul-04 20.7 69.3 58.9

6

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

66 of 105
447008 TEMPERATURE/HUMIDITY - DAILY SUMMARY REPORT BY STUDY

HONEYWELL

CARDIAC SENSITIZATION STUDY OF HFO 1234ZE & HFO 1234YF IN DOGS

SPONSOR:PROJECT NO.:WIL

447008 06/20/04 

08/19/04 

STUDY SPECIFICATIONS: DATE IN: TIME IN:

DATE OUT: TIME OUT:

B ROOM 17DOG

LOW TEMPERATURE oF: 63.0 HIGH TEMPERATURE oF: 73.0 

H

ROOM SPECIFICATIONS:SPECIES:

TEMPERATURE HUMIDITY

MEAN (oF)DATE MEAN (%RH)

LOW TEMPERATURE oC: 17.2 HIGH TEMPERATURE oC: 22.8

MEAN (oC)

13-Jul-04 20.7 69.2 57.3

14-Jul-04 20.2 68.3 55.0

15-Jul-04 20.1 68.1 53.1

16-Jul-04 20.3 68.5 51.1

17-Jul-04 20.0 67.9 51.8

18-Jul-04 20.0 68.1 51.5

19-Jul-04 20.2 68.4 52.9

20-Jul-04 20.3 68.6 54.3

21-Jul-04 20.6 69.0 55.4

22-Jul-04 21.0 69.7 58.3

23-Jul-04 20.1 68.1 54.6

24-Jul-04 20.3 68.5 47.4

25-Jul-04 20.1 68.2 49.0

26-Jul-04 20.7 69.3 56.3

27-Jul-04 20.0 68.0 53.4

28-Jul-04 20.0 68.0 51.8

29-Jul-04 20.0 67.9 51.5

30-Jul-04 20.4 68.8 58.2

31-Jul-04 20.2 68.4 58.1

01-Aug-04 20.6 69.0 55.3

02-Aug-04 20.4 68.8 55.5

03-Aug-04 21.0 69.8 61.0

04-Aug-04 20.1 68.2 56.2

6

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

67 of 105
447008 TEMPERATURE/HUMIDITY - DAILY SUMMARY REPORT BY STUDY

HONEYWELL

CARDIAC SENSITIZATION STUDY OF HFO 1234ZE & HFO 1234YF IN DOGS

SPONSOR:PROJECT NO.:WIL

447008 06/20/04 

08/19/04 

STUDY SPECIFICATIONS: DATE IN: TIME IN:

DATE OUT: TIME OUT:

B ROOM 17DOG

LOW TEMPERATURE oF: 63.0 HIGH TEMPERATURE oF: 73.0 

H

ROOM SPECIFICATIONS:SPECIES:

TEMPERATURE HUMIDITY

MEAN (oF)DATE MEAN (%RH)

LOW TEMPERATURE oC: 17.2 HIGH TEMPERATURE oC: 22.8

MEAN (oC)

05-Aug-04 20.0 67.9 50.9

06-Aug-04 20.0 68.0 48.6

07-Aug-04 19.9 67.9 46.1

08-Aug-04 20.0 68.0 46.6

09-Aug-04 20.0 68.0 50.7

10-Aug-04 20.0 68.0 54.4

11-Aug-04 20.1 68.2 54.0

12-Aug-04 20.1 68.1 49.4

13-Aug-04 21.0 69.8 55.2

14-Aug-04 20.2 68.3 49.3

15-Aug-04 20.6 69.1 49.6

16-Aug-04 20.6 69.1 54.9

17-Aug-04 20.7 69.2 53.1

18-Aug-04 20.8 69.5 56.6

19-Aug-04 21.2 70.2 68.7

6

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

68 of 105
447008 TEMPERATURE/HUMIDITY - DAILY SUMMARY REPORT BY STUDY

HONEYWELL

CARDIAC SENSITIZATION STUDY OF HFO 1234ZE & HFO 1234YF IN DOGS

SPONSOR:PROJECT NO.:WIL

447008 06/20/04 

08/19/04 

STUDY SPECIFICATIONS: DATE IN: TIME IN:

DATE OUT: TIME OUT:

B ROOM 17DOG

LOW TEMPERATURE oF: 63.0 HIGH TEMPERATURE oF: 73.0 

H

ROOM SPECIFICATIONS:SPECIES:

TEMPERATURE HUMIDITY

MEAN (oF)DATE MEAN (%RH)

LOW TEMPERATURE oC: 17.2 HIGH TEMPERATURE oC: 22.8

MEAN (oC)

68.6TEMPERATURE oF 67.9 71.2

68.745.453.3

61MEAN MIN MAX

20.3TEMPERATURE oC 19.9 21.8

HUMIDITY (%RH)GRAND STATSN DAYS

6

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

69 of 105
447008 TEMPERATURE/HUMIDITY - END OF STUDY SUMMARY REPORT

HONEYWELL

CARDIAC SENSITIZATION STUDY OF HFO 1234ZE & HFO 1234YF IN DOGS

SPONSOR:PROJECT NO.:WIL- 11:11 20-Jun-

B ROOM 17DOG

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

TEMPERATURE HUMIDITY

06/20/047:0016:0008/19/04

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

ROOM B ROOM 17 SUMMARY

MEANMINMAX

68.666.175.7

53.243.484.4

SD 1.24 6.37N SAMPLES 1440 1440N DAYSFIRST DAYLAST DAY

06/20/0408/19/04

61

6

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

70 of 105
447008 TEMPERATURE/HUMIDITY - END OF STUDY SUMMARY REPORT

HONEYWELL

CARDIAC SENSITIZATION STUDY OF HFO 1234ZE & HFO 1234YF IN DOGS

SPONSOR:PROJECT NO.:WIL- 11:11 20-Jun-

STUDY 447008 SUMMARY

MEANMINMAX

68.666.175.7

53.243.484.4

SD 1.24 6.37N SAMPLES 1440 1440FIRST DAYLAST DAY

06/20/0408/19/04

N DAYS 61

6

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

71 of 105
WIL-447008 HFO 1234ze and HFO 1234yf

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APPENDIX E

Individual Electrocardiographic Data

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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 oxygen

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 oxygen

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 oxygen

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 oxygen

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 105
6.0 μg/kg

challenge dose of epinephrine 

80 of 105
81 of 105
c 

0 

* 

c 

0 

d82 of 105
Representative Electrocardiogram 

No Effect 

Animal No. 409 

HFO 1234yf 

6.0 μg/kg Challenge Dose 

120,000 ppm Test Article 

83 of 105
6.0 μg/kg

challenge dose of epinephrine. 

84 of 105
85 of 105
0 

Lu 

z 

I0

a 

w 

nz 

m 

--d 

cy0 

-0 

0 

3 

a 

-- IYO 

1 a86 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 105
Study 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.@ 

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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. 

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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 

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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 

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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 

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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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WIL447008 

June 22.2034 

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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WIL-447008 

June 22.2’3X 

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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June 22,2004 

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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WIL-447008 

Page 7 of 14 June 22.2004 

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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WIL-447008 

June 22. 2004 

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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WIL-447008 

June 22.2001 

8.7 

TIME (minutes) 

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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June 22.2004 

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 

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and 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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WIL-447008 

June 22,2M)4 

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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WIL-447008 

June 22.2co4 

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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WIL-447cal 

June 22‘2oM 

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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