### HLS STUDY NO. 10-2226
SPONSOR STUDY NO. MA- 252E-93-23

HFO-1234yf:

A 2-PHASE INHALATION SCREENING STUDY AND SINGLE EXPOSURE

STUDY IN RABBITS VIA WHOLE-BODY INHALATION EXPOSURE (GLP)

Final Report

Author: Gary M. Hoffman, BA, DABT

Performed by: Huntingdon Life Sciences
100 Mettlers Road East Millstone, New Jersey 08875-2360

Submitted to: Honeywell
101 Columbia Road

PO Box 2245R Morristown, New Jersey 07962-1057

Attn: George M. Rusch, PhD, DABT

Date: 28 February 2011

Page 1 of 226

### Huntingdon Life Sciences 10-2226 Page 5
MA-252E-93-23 Final Report

LIST OF ABBREVIATIONS

Abbreviation Term hr(s) hour(s) min(s) minute(s)
M Male
F Female GD Gestation Day Lpm Liters per minute mg/L milligrams of test substance per Liter of air
np non-pregnant ppm parts per million
n number

### Huntingdon Life Sciences 10-2226 Page 6

MA-252E-93-23 Final Report

HFO-1234yf: A 2-Phase Inhalation Screening Study and Single Exposure Study in Rabbits via Whole-Body Inhalation Exposure (GLP)

SUMMARY

New Zealand White rabbits received a single whole-body inhalation exposure of HFO-1234yf for 1 hour in this 2-phase study.

For Phase 1, one non-pregnant and one presumed pregnant female rabbit were exposed at a target exposure level of 100,000 ppm on the presumed Gestation Day 12 (for the presumed pregnant female rabbit) followed by a 2-day post-exposure period. At the end of the 2-day post-exposure period, animals were euthanized and discarded without examination except evaluation of pregnancy st atus for the presumed pregnant female. Parameters evaluated during Phase 1 were : viability, clinical observations and body weights.

For Phase 2, 15 males (5/group) and 28 females (6/group presumed pregnant and 5/Groups 1 and 3 non-pregnant) rabbits were expos ed at target exposure levels of 0 (Air control), 50,000 or 100,000 ppm on the presumed Gestation Day 12 (for the presumed pregnant female rabbit) followed by a 14-day post-exposure period. Urine samples were

collected for the determination of metabolite concentrations within approximately 12 hours before and for approximately 48 hours after the exposure at approximately 12 hour intervals. At the end of the 14-day post-exposure period, all animals were euthanized and necropsied. Parameters evaluated during the study were: viability, clinical observations, body weights, macroscopic observations (inc luding evaluation of pregnancy status for presumed pregnant females) and microscopic pathology.

The target and mean analytical (IR) and nominal concentrations are summarized as

follows:

Group Target Concentration (Males/Females) (ppm) Analytical Concentration (Males/Females) (ppm) Nominal Concentration (Males/Females) (ppm)

Phase 1 100,000 110,000 100,000

Phase 2

1 0 0.0/0.0 NA

2 50,000 45,000/47,000

3 100,000 100,000/102,000 150,000/140,000 a

a The nominal concentrations in Ph ase 2 are the combined results of the 2 chambers for each of the separate male and female exposures.

### Huntingdon Life Sciences 10-2226 Page 7

MA-252E-93-23 Final Report

SUMMARY

These results indicated an acceptable agreemen t between the targeted and analytical and nominal concentrations. The particle sizing results indicated that the atmospheres were essentially gas only, as expected, since th ere was no substantial difference between the

test substance chambers and the air control chamber.

All animals survived until their scheduled s acrifice and there were no test substancerelated clinical signs or differences in body weights or macroscopic/microscopic examinations (heart, kidneys, liver and lungs) in any of the test animals.

Urine was collected in 12 hour intervals for 48 hours after the end of the exposure and analyzed by 19 

### F-NMR and LC/MS-MS. The predominant metabolites were S -(3,3,3- trifluoro-2-hydroxypropanyl)-mercaptolactic acid and N -acetyl- S -(3,3,3-trifluoro-2- hydroxypropanyl)- L -cysteine whose signal intensities in 19 

### F-NMR spectra represented

more than 78% of total 19 

### F related signals in all urine analyzed samples. Quantification of the HFO-1234yf-mercapturic acid isomers by LC /MS-MS showed no difference in the mean recovery of this metabolite between female (43.11 ± 22.35 μmol) and pregnant female rabbits (50.47 ± 19.72 μmol) excreted in urine within 48 hours. With the exception of one yet unidentified metabolite (12-17% female rabbits and < 7% male rabbits of the six main metabolites), no differences in urinary metabolite pattern or quantity of metabolites excreted were observed between the different groups.

In conclusion, no lethality and no clinical signs were observed in the exposed animals. No substantial differences in urinary metabolite pattern or quantity of metabolites excreted were observed between the different groups. The results suggest that the previously seen lethality of HFO-1234yf in pregnant rabbits after inhalation exposures is unlikely due to changes in biotransformation patterns or capacity of pregnant rabbits.

### Huntingdon Life Sciences 10-2226 Page 8

MA-252E-93-23 Final Report

TABLE OF CONTENTS

COVER PAGE..................................................................................................................... 1

STATEMENT OF COMPLIANCE..................................................................................... 2

SIGNATURE PAGE ........................................................................................................... 3 QUALITY ASSURANCE STATEMENT .......................................................................... 4

LIST OF ABBREVIATIONS.............................................................................................. 5

SUMMARY........................................................................................................................

. 6

TABLE OF CONTENTS..................................................................................................... 8

1. 

### INTRODUCTION

................................................................................................ 11

2. 

### MATERIALS AND METHODS

2.1. Study Management .................................................................................... 11

2.2. Study Dates ................................................................................................ 12

2.3. Experimental Outline................................................................................. 13

2.4. Justifications .............................................................................................. 14

2.5. Test Substance ........................................................................................... 15

2.6. Test Animals .............................................................................................. 17

2.7. Animal Assignment ................................................................................... 18

2.8. Animal Identification................................................................................. 18

2.9. Veterinary Care.......................................................................................... 18

2.10. Husbandry During the Non-Exposure Periods .......................................... 18

2.11. Husbandry During the Exposure Periods................................................... 21

2.12. Test Substance Administration and Chamber Operation........................... 21

2.13. Exposure Chamber Sampling .................................................................... 24

2.14. Experimental Evaluations .......................................................................... 25

2.15. Metabolite Dete rminations ........................................................................ 26

2.16. Postmortem ................................................................................................ 27

2.17. Statistical Analysis..................................................................................... 29

2.18. Major Computer Systems .......................................................................... 31

2.19. Data Storage............................................................................................... 31

2.20. Regulatory References............................................................................... 32

2.21. Protocol Deviations.................................................................................... 32

### Huntingdon Life Sciences 10-2226 Page 9

MA-252E-93-23 Final Report

TABLE OF CONTENTS

3. 

### RESULTS AND DISCUSSION

3.1. Chamber Monito ring Results..................................................................... 34

3.2. Urine Metabolite De terminations .............................................................. 35

3.3. Mortality .................................................................................................... 35

3.4. Clinical Observations................................................................................. 35

3.5. Body Weights............................................................................................. 35

3.6. Anatomic Pathology................................................................................... 36

4. 

### CONCLUSION

.................................................................................................... 36

REFERENCES .................................................................................................................. 37

GENERAL PREFACE ...................................................................................................... 38

FIGURES

1. Chamber Generation System and 1000 Liter Whole-Body Exposure Chamber (Pha se 1, Group 1)........................................................................ 39

2. Chamber Generation System and 1000 Liter Whole-Body Exposure Chamber (Pha se 2, Group 1)........................................................................ 40

3. Chamber Generation System and 1000 Liter Whole-Body Exposure Chamber (Phase 2, Groups 2 and 3) ............................................................ 41

TABLES

1. Summary of In-Chamber Observations ....................................................................... 42

2. Summary of Daily Observations.................................................................................. 43

3. Summary of Physical Examination Findings............................................................... 49

4. Mean Body Weights .................................................................................................... 58

5. Mean Body Weight Change from Baseline ................................................................. 61

6. Incidence Summary Report for Gross Necropsy Observations ................................... 64

7. Incidence Summary of Microscopic Findings with Severity Levels........................... 67

APPENDICES

A. Phase 1 Screening Study ........................................................................................................

. 71

B. Chamber Monitoring Results (Phase 2) .................................................................................. 78



### C. Individual Animal Term ination History ...................................................................... 84

D. Individual Daily Observations..................................................................................... 86

E. Individual Physical Ex amination Findings.................................................................. 96

F. Individual Body Weights ........................................................................................... 113

### Huntingdon Life Sciences 10-2226 Page 10

MA-252E-93-23 Final Report

TABLE OF CONTENTS

G. Individual Body Weight Change from Baseline........................................................ 116

H. Anatomic Pathology................................................................................................... 119 Anatomic Pathology Report....................................................................................... 120

Individual Animal Gross and Microscopic Observations.......................................... 122 Pregnancy Status of Presum ed Pregnant Females ..................................................... 165

I. Urine Metabolite Report ............................................................................................ 166

J. Equipment List........................................................................................................... 185

K. Prestudy Chamber Dist ribution Records ................................................................... 187

L. Miran  

### Calibration..................................................................................................... 188

M. Certificate of Analysis ............................................................................................... 197

N. Protocol and Protoc ol Amendments .......................................................................... 198

O. Scientists and Supe rvising Personnel ........................................................................ 226

### Huntingdon Life Sciences 10-2226 Page 11

MA-252E-93-23 Final Report

1. INTRODUCTION
In previous studies, pregnant rabbits we re found to be unexpectedly sensitive to an inhalation exposure of 50,000 ppm HF O-1234yf (an industrial chemical for possible use as a refrigerant) when compared with pregnant and non-pregnant rats and with male rats. This study was de signed to assess the acute toxicity and biotransformative effects of a single 1 hour whole-body inhalation exposure of HFO-1234yf in pregnant and non-pregnant female rabbits and in male rabbits. Also, the purpose of this study was to de termine whether there is a pregnancybased or gender-based difference in sensitivity in rabbits with this test substance.

This data will be used as a basis for risk assessment.

A screening study (Phase 1) evaluated the highest intended exposure level in a

minimal number of test animals in order to confirm the safety of that same exposure level for the main study (Phase 2) using a full complement of test

animals.

2. MATERIALS AND METHODS

2.1. STUDY MANAGEMENT

2.1.1. SPONSOR

Honeywell

101 Columbia Road

PO Box 2245R Morristown, New Jersey 07962-1057

2.1.2. SPONSOR REPRESENTATIVE

George M. Rusch, PhD, DABT

2.1.3. TESTING FACILITY

Huntingdon Life Sciences

100 Mettlers Road East Millstone, New Jersey 08875-2360

2.1.4. STUDY DIRECTOR

Gary M. Hoffman, BA, DABT

### Huntingdon Life Sciences 10-2226 Page 12

MA-252E-93-23 Final Report

2.2. STUDY DATES

2.2.1. STUDY INITIATION

26 October 2010 (Date Study Director signed the Protocol)

2.2.2. DATE OF ANIMAL RECEIPT

29 October 2010

2.2.3. DATES OF EXPOSURE

Phase 1: 3 November 2010 (Experimental Start Date)

Phase 2 Females: 8 November 2010

Phase 2 Males: 10 November 2010

2.2.4. TERMINAL SACRIFICES

Phase 1: 5 November 2010

Phase 2 Females: 22 November 2010

Phase 2 Males: 24 November 2010

2.2.5. EXPERIMENTAL TERMINATION DATE

24 February 2011 (Last date on which data was collected directly from the study)

2.2.6. STUDY COMPLETION

28 February 2011 (Date Final Report is signed by the Study Director)

### Huntingdon Life Sciences 10-2226 Page 13

MA-252E-93-23 Final Report

2.3. EXPERIMENTAL OUTLINE

2.3.1. SCREENING STUDY (PHASE 1)

One experimental group (one non-pregnant and one presumed pregnant female on Gestation Day (GD) 12 received whole-body inhalation exposure once for 1 hour with HFO-1234yf:

Exposure

Level a 

### Test Animals (females) Group

ppm mg/L Presumed
Pregnant Non-Pregnant Euthanized b



### 1 100,000 471 

### 1 1 2

a Exposure was 1 hour for 1 day via whole-body e xposure.

b Animals were euthanized 2 days following the exposure and discarded without
examination. Only the pregnancy status was determined for the presumed pregnant

animal.



### 2.3.2. MAIN STUDY (PHASE 2)

Three experimental groups received a single whole-bodyinhalation exposure for 1 hour duration on GD12 with HFO1234yf at the exposure concentrations summarized below:

Exposure

Level a 

### Test Animals

Group ppm mg/L Presumed
Pregnant

Females NonPregnant

Females Males Necropsy b



### 1 – Air control 0 0 

### 6 5 5 16

2 - Low 50,000 236 

### 6 0 5 11
3 - High 100,000 471 

### 6 5 5 16

a Exposure was 1 hour for 1 day via whole-body exposure.

b Complete postmortem evaluations was performed 14 days following the exposure and
the pregnancy status was determined for the presumed pregnant animals.

### Huntingdon Life Sciences 10-2226 Page 14

MA-252E-93-23 Final Report

2.4. JUSTIFICATIONS

2.4.1. ROUTE, DURATION AND FREQUENCY OF

ADMINISTRATION

The inhalation route is a potential route of human exposure to this test substance. The duration and frequency of the exposure was requested by the U.S. EPA to insure adequate acute exposuretoxicity characterization for the test substance in pregnant and nonpregnant female rabbits and in male rabbits.

2.4.2. EXPOSURE LEVEL SELECTION

Mortality was seen in previous rabbit developmental toxicity studies with multiple 6-hour w hole-body inhalation exposure from 5500 ppm up to 50,000 ppm HFO-1234yf. This study was designed to evaluate the acute toxicity effects from a single 1-hour exposure to a similar 50,000 ppm level and a 100,000 ppm level that might be briefly experienced during an accidental release of the test substance. Since this was a single exposure at a reduced duration (1 hour), severe toxicity such as lethality was not anticipated.

2.4.3. TEST ANIMAL SELECTION

The rabbit is accepted by Regulatory Authorities (including the U.S. EPA) as a non-rodent surrogate for humans in the detection of toxic effects. In prior testing via inhalation exposure with this test substance, pregnant rabbits were found to be more sensitive to the test substance when compared with pregnant and non-pregnant rats. Limited or no testing ha s been conducted in non-pregnant female or male rabbits, respectively. This study was designed to determine whether there is a pregnancy-based or gender-based difference in sensitivity in rabbits w ith this test substance. This data will be used as a basis for species selection in subsequent reproductive toxicity and/or subchronic toxicity testing.

### Huntingdon Life Sciences 10-2226 Page 15

MA-252E-93-23 Final Report

2.4.4. NUMBER OF ANIMALS

The number of animals in this study was in general accordance with U.S EPA acute inhalation test guideline (OPPTS 870.1300) and was considered the minimum necessary to allow for meaningful interpretation of th e study. For the Screening Study(Phase 1), a single exposure with 1 presumed pregnant and 1 nonpregnant rabbit was considered adequate to provide an initial assessment of the test substance general toxicity. For the Main Study (Phase 2), a single exposur e with two exposure groups and an air control group were considered sufficient to allow assessment of any toxicity that may be present. The Control and High exposure groups are each comprised of 6 presumed pregnant rabbits, 5 non-pregnant female animals, and 5 males. The Low exposure group was comprised of 6 presumed pregnant animals and 5 male animals. Non-pregna nt females were not included in the Low exposure group because data are available from a previously conducted study (Schus ter et al, TAP, 244: pp 247-253, 2010). An n of 5 animals per se x and/or pregnancy status was considered by the U.S. EPA to be the minimum number of animals per group for acute toxicity studi es. A group size of 6 presumed pregnant rabbits per group in the Main Study was based on the Testing Facility’s historical control data showing that the expected pregnancy rate for this strain of rabbit is approximately 85-95%. The additional presumed pregnant rabbit was intended to ensure the availability of at least 5 pregnant rabbits per group for

evaluation.

2.5. TEST SUBSTANCE

HFO-1234yf (2,3,3,3 – Tetrafluoropropene)

MW = 114 grams/mole

2.5.1. CAS NUMBER

754-12-1

2.5.2. CATEGORY

Industrial chemical used as a possible refrigerant

### Huntingdon Life Sciences 10-2226 Page 16

MA-252E-93-23 Final Report

2.5.3. MANUFACTURER/SUPPLIER

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

2.5.4. LOT NUMBER

BZ129B

2.5.5. PURITY

99.99%

2.5.6. DESCRIPTION

Clear gas

2.5.7. DATE RECEIVED

24 June 2010

2.5.8. EXPIRATION DATE

3 June 2011

2.5.9. ANALYSIS

Documentation of the identity, strength, composition, stability and method of synthesis, fabricati on and/or derivation of the test

substance and the maintenance of these records were the responsibility of the Sponsor. A Certificate of Analysis is presented in Appendix M

.

2.5.10. STORAGE

Room temperature

2.5.11. ARCHIVAL SAMPLE

An archival sample of the test substance was not taken for this study.

### Huntingdon Life Sciences 10-2226 Page 17

MA-252E-93-23 Final Report

2.5.12. DISPOSITION

The unused portion of the test s ubstance was returned to the Sponsor on 9 December 2010.

2.6. TEST ANIMALS

2.6.1. SPECIES

Albino Rabbits

New Zealand White

2.6.2. SUPPLIER

Robinson Services, Inc. Mocksville, North Carolina 27028

2.6.3. NUMBER OF ANIMALS

Received/Placed on test: 45 total (15 males, 30 females [19 presumed pregnant, 11 non-pregnant])

2.6.4. AGE AT RECEIPT AND INITIATION OF DOSING

Approximately 5 to 6 months

Presumed pregnant females we re received on GD 7 for Phase 1 and GD 2 for Phase 2, where GD 0 was the day of mating.

2.6.5. WEIGHT AT INITIATION OF DOSING (GRAMS)

Phase 1 Individual Weight Female (non-pregnant): 3178.1 Female (presumed pregnant): 3153.7

Phase 2 Mean Range

Male: 2994.4 2621.6 – 3676.6 Female (non-pregnant): 3258.2 3023.8 – 3547.5 Female (presumed pregnant): 3444.5 3118.5 – 3699.3

### Huntingdon Life Sciences 10-2226 Page 18

MA-252E-93-23 Final Report

2.6.6. STABILIZATION PERIOD

Animals were stabilized for at least 5 days. All animals were examined during the stabilization period to confirm suitability for study.

2.7. ANIMAL ASSIGNMENT

The exact number of animals require d for the study were purchased and stabilized. Animals were arbitrarily assigned to study phases and groups upon arrival. Two females (1 presumed pregnant, 1 non-pregnant) were assigned to Screening Study (Phase 1); while 15 males (5/group) and28 females (6/group presumed pregnant and 5/Groups 1 and 3 nonpregnant) were assigned to the Main Study (Phase 2).

2.8. ANIMAL IDENTIFICATION

Each animal bore an ear tattoo with a USDA number. Upon allocation to study, each animal was assigned a new identification number. This number comprised the unique identification for each animal and served as the animal’s permanent identification for the duration of the study. Each study animal’s cage was ascribed a cage card, color-coded for exposure level identification and bearing the study number and animal number.

2.9. VETERINARY CARE

Animals were monitored by the t echnical staff for any conditions requiring possible veterinary care and treated as necessary. Miscellaneous, non-test substance-related veterinarian evaluations and treatments (as necessary) for individual animals were reviewed by the Study Director and are documented in the study file.

2.10. HUSBANDRY DURING NON-EXPOSURE PERIODS

2.10.1. FACILITIES MANAGEMENT/ANIMAL HUSBANDRY

Currently acceptable practices of good animal husbandry were followed e.g., Guide for the Care and Use of Laboratory Animals ; National Academy Press, 1996. Huntingdon Life Sciences, East Millstone, New Jersey is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC).

### Huntingdon Life Sciences 10-2226 Page 19

MA-252E-93-23 Final Report

2.10.2. HOUSING

All animals were housed individually in suspended cages with grid bottoms. A stainless steel food hopper was attached to each cage.

2.10.3. FEED

Global High Fiber Diet # 2031C (Harlan Teklad, Madison, Wisconsin) was provided in measured amounts, up to 200 grams, to the rabbits daily. During stab ilization, fresh food was provided in varying amounts. After stab ilization, approximately 200 grams of fresh food was provided to the presumed pregnant females and approximately 150 grams was provi ded to males and non-pregnant females. Food was presented at approximately the same time each day.

2.10.4. FEED ANALYSIS

Analysis of each feed lot used during this study was performed by the manufacturer. Results were provided to the Testing Facility and are maintained on file at the Testing Facility. There were no known contaminants in the feed that were expected to interfere with the results of this study.

2.10.5. WATER

Water (New Jersey-American Water Company, Cherry Hill, New Jersey) was available without rest riction via an automated watering
system.

2.10.6. WATER ANALYSIS

Water analyses are conducted by New Jersey-American Water Company, Cherry Hill, New Jersey (Raritan-Millstone Plant) to ensure that water meets standards specified under the EPA Federal Safe Drinking Water Act Regula tions (40 CFR Part 141). In addition, water samples are collected biannually from representative rooms in the Testing Facility; chemical and microbiological water analyses are conducted on these samples by a subcontract laboratory. Results of all water analyses are maintained on file at the Testing Facility. There were no known

### Huntingdon Life Sciences 10-2226 Page 20

MA-252E-93-23 Final Report

contaminants in the water which were expected to interfere with the results of this study.

2.10.7. ENVIRONMENTAL CONDITIONS

Light/Dark Cycle

A twelve hour light/dark cycle controlled via an automatic timer was provided.

Temperature

Temperature was monitored in accordance with Testing Facility

SOPs and maintained within the specified range to the maximum extent possible.

Desired Range: 16 to 22 ° C Daily Average Range: 17 to 21 °C

Relative Humidity

Relative humidity was monitored in accordance with Testing Facility SOPs and maintained with in the specified range to the maximum extent possible.

Desired Range: 30 to 70% Daily Average Range: 32 to 64%

Air Changes

Animal rooms had 10 to 15 air changes per hour. The actual number of air changes per hour in each animal room is recorded at least twice each year, and the Testing Facility retains these records.

2.10.8. ENVIRONMENTAL ENRICHMENT

The animals were provided with environmental enrichment following the Testing Facility’s Standard Operating Procedures including hay which was given once.

### Huntingdon Life Sciences 10-2226 Page 21

MA-252E-93-23 Final Report

2.11. HUSBANDRY DURING EXPOSURE PERIODS

Animals were individually housed in suspended, stainless steel cages.

2.11.1. FEED

None was provided during exposure.

2.11.2. WATER

None was provided during exposure.

2.11.3. ENVIRONMENTAL CONDITIONS

Chamber temperature and relative humidity were recorded every half-hour during exposure and main tained, to the maximum extent possible, within the ranges presented below. Excursions outside the specified range did not affect the integrity of the study.

Temperature

Desired: 16 to 24 ° C

Actual: 19 to 22 ° C

Relative Humidity

Desired: 40 to 60%

Actual: 49 to 66%

2.12. TEST SUBSTANCE ADMINISTRATION AND

CHAMBER OPERATION

2.12.1. ROUTE OF ADMINISTRATION

Inhalation as a gas by whole-body exposure.

2.12.2. FREQUENCY AND DURATION OF ADMINISTRATION

Animals were exposed for 1 hour once. The presumed pregnant animals in Phase 1 and Phase 2 were exposed on GD 12, where GD 0 is the day of mating.

### Huntingdon Life Sciences 10-2226 Page 22

MA-252E-93-23 Final Report

2.12.3. PRE-STUDY TRIALS

The test atmosphere was generated by an appropriate procedure determined during pre-study trials, which were performed to evaluate the optimal set of conditi ons and equipment to generate a stable atmosphere at the target exposure levels and maintain uniform conditions throughout the exposure chambers.

2.12.4. EXPOSURE PROCEDURE

Group 1 (Phase 1)

The outlet of the stainless steel “T” was connected to a second “T”

that divided the flow via 1⁄4” tubi ng to 2 metering valves connected

to 2 mass flowmeters. The test substance was directed from the

outlet of each flowmeter into the turret of the 1.0 m 3 

### glass and stainless steel exposure chamber via additional 1⁄4” tubing. As the test substance was drawn into the chamber, it was mixed with room air to create the exposure atmosphere.

Supplementary oxygen was delivered from an oxygen cylinder through a toggle valve to 1⁄4” t ubing. The oxygen was directed through a metering valve to the in let of the flowmeter and then from the outlet of the flowmeter, was directed via 1⁄4” tubing to the

turret of the chamber.

Group 1 (Phase 2)

Room air was drawn into th e inlet turret of the 1.0 m 3 

### glass and stainless steel exposure chamber, which was operated using clean air only at approximately the same total flow rate as the test substance-exposed groups.

Groups 2 and 3 (Phase 2)

The test substance was delivered directly from the Sponsor cylinder via a backpressure gauge through a toggle valve to 1⁄4” tubing to a stainless steel “T”. From the “T”, the test substance wad directed via 1⁄4” tubing to the Groups 2 and 3 exposure
systems.

For Group 2, the test substance was directed through a metering

valve connected to a mass flowmete r and then from the outlet of

the flowmeter was directed into the turret of the 1.0 m 3 

### glass and

### Huntingdon Life Sciences 10-2226 Page 23

MA-252E-93-23 Final Report

stainless steel exposure chamber vi a additional 1⁄4” tubing. As the test substance was drawn into the chamber, it mixed with room air to create the exposure atmosphere.

For Group 3, the outlet of the stainless steel “T” was connected to

a second “T” that divided the fl ow via 1⁄4” tubing to 2 metering

valves connected to 2 mass flowmeters. The test substance was

directed from the outlet of each flowmeter into the turret of the

1.0 m 3 

### glass and stainless steel exposure chamber via additional 1⁄4” tubing. As the test substance was drawn into the chamber, it was mixed with room air to create the exposure atmosphere.

Supplementary oxygen was delivered from an oxygen cylinder through a toggle valve to 1⁄4” tubing where a stainless steel “T” was used to split the delivery line to 2 flowmeters (one each from Groups 2 and 3). The oxygen was directed through a metering

valve to the inlet of each flowme ter and then from the outlet of each flowmeter, was directed via 1⁄4” tubing to the turret of the Group 2 and 3 chambers.

2.12.5. CHAMBER OPERATION

The whole-body exposure chambers each had a volume of approximately 1000 Liters (1.0 m 3 

### ). Each chamber was operated

at a minimum flow rate of 200 Lite rs per minute. The final airflow was set to provide at least one air change (calculated by dividing the chamber volume by the airflow rate) in 5.0 minutes (12 air changes/hour) and a T 99 

### equilibrium time (calculated by multiplying the air change by the expone ntial factor 4.6) of at most

23 minutes:

Grou p Airflow Rate Air Chan g e T 99 

### ( L p m ) ( min ) ( min )

Phase 1 224 4.5 23

Phase 2

1 210 4.8 23

2 211 4.7 23

3 224 4.5 23

The chamber size and airflow rates (and supplementary oxygen as prior descibed) were considered adequate to maintain the oxygen level at least 19% and the animal loading factor below 5%. At the

### Huntingdon Life Sciences 10-2226 Page 24

MA-252E-93-23 Final Report

end of each exposure, all animals generally remained in the chamber for a minimum of 23 minutes. During this time, each chamber was operated at approximately the same flow rate using clean air only. The chambers were exhausted through the in-house filtering system, which consisted of a coarse filter, a HEPA filter

and activated charcoal.

See Figures 1

, 2 and 3 and Appendix J for equipment details.

2.13. EXPOSURE CHAMBER SAMPLING

2.13.1. CHAMBER SAMPLING

Determination of the exposure le vels were made using a MIRAN 


### Ambient Air analyzer equipped with a strip chart recorder. The test atmosphere was monitored continuously by the MIRAN ® 

### and measurements, from the normal sampling portal, were recorded 3 times during each exposure. The exposure levels were determined by comparison of th e measured absorbance to a calibrated response curve constructed using the same instrument settings.

See Appendix J for equipment details and Appendix L for

MIRAN  

### calibration.

2.13.2. OXYGEN MONITORING

The chamber oxygen was monitored every 30 minutes using an oxygen analyzer. Prior to recordi ng the reading, the instrument was allowed to equilibrate for a few seconds. The chamber atmosphere was drawn from a separate sampling port.

2.13.3. PARTICLE SIZE DISTRIBUTION ANALYSIS

During each exposure, particle size determinations were performed once using a TSI Aerodynamic Particle Sizer to characterize the aerodynamic particle size distribution of any aerosol present. The samples were drawn for 20 seconds at a flow rate of 5.0 Lpm. The mass median aerodynamic diameter, geometric standard deviation

and total mass concentration were calculated based on the amount of particles collected. Also, samp les of the room air were taken and analyzed in the same manne r for comparison to the chamber

### Huntingdon Life Sciences 10-2226 Page 25

MA-252E-93-23 Final Report

samples. Results of the particle size distribution analysis of the room air samples were not reported.

See Appendix J for equipment details.

2.13.4. NOMINAL CONCENTRATION

The nominal concentration was determined by weighing the cylinder containing the test substance before and after the exposure and calculating as follows:

Conc (ppm) = amount consumed (g) x 1000 (mg/g) x 1000 ( μ g/mg) x 22.4 ( μ L/ μ mole) x 295oK exposure duration (min) x airflow (Lpm) x MW ( μ g/ μ mole) x 273oK

MW = 114 grams/mole



### The nominal concentration for Phase 2 was calculated for both test substance-treated groups combined using the average airflow of both chambers. For Phase 1, the Group 1 airflow only was used.

2.14. EXPERIMENTAL EVALUATIONS

2.14.1. VIABILITY CHECKS (CAGE-SIDE)

Generally, animals were observed in their cages twice daily (once in the morning and once in the af ternoon) for mortality, morbidity and signs of severe toxicity.

2.14.2. PHYSICAL EXAMINATIONS

Exposure Observations

All animals were observed as a group once during the exposure.

Detailed Physical Examinations

Animals were examined once daily during the stabilization period and once daily (post-exposure on the day of exposure) during the study. Examinations included observations of general condition, skin and fur, eyes, nose, oral cavity, abdomen, as well as evaluations of respiration.

### Huntingdon Life Sciences 10-2226 Page 26

MA-252E-93-23 Final Report

2.14.3. BODY WEIGHT

Body weights were recorded for a ll animals on the day of arrival for health monitoring purposes. This information is maintained in the raw data but is not reported.

Phase 1

Body weights were recorded on day of exposure (prior to exposure), and daily for 2 days.

Phase 2

Body weights were recorded on day of exposure (prior to exposure), and on Study Days 3, 5, 8 and 15 (GDs 14, 16, 19 and 26 for the presumed pregnant females).

2.14.4. FOOD CONSUMPTION

Food was available without restriction 7 days/week. A visual check was made each day of the st udy for all animals. This was performed as part of the daily viability checks and any decreased food consumption recorded.

2.15. METABOLITE DETERMINATIONS (PHASE 2 ONLY)

Urine samples were obtained for the determination of metabolites of HFO-1234yf.

2.15.1. COLLECTION TIMES AND NUMBER OF ANIMALS

Urine was collected at approximately 12 hours before the inhalation exposure and for approximately 48 hours after exposure, at approximately 12 hour interv als from 5 males/group, up to 6 presumed pregnant females/group and 5 non-pregnant females/Groups 1 and 3.

2.15.2. COLLECTION PROCEDURES

Urine was collected, via urine pans beneath each animal’s cage, into ice-chilled containers, and the volume was measured for each

interval. Animals had access to food and water during the collection periods. At the time of sample collection, the collection system was checked to ensure no urine was retained in the system

### Huntingdon Life Sciences 10-2226 Page 27

MA-252E-93-23 Final Report

and the container was capped. Th e urine pans were cleaned during the exposure and at the 24 hour collection interval. At time points when one sample was collected, the next sampling period began immediately after the collection system was checked.

2.15.3. STORAGE AND DISPOSITION OF SAMPLES

Samples were processed by removing samples from wet ice, measuring the volume collected and when possible based on volume collected, split into 2, up to 10 mL/sample aliquots; however, the 24-36 hour sample volume for Animal No. 3501 was not split although the total sample volume was 9 mL. When the total sample volume was 5 mL or less, the volume was not split. Urine samples were frozen in meth anol and dry ice and stored at approximately -20 °C±10 °C until shipped. Each sample was labeled with Testing Facility labels. One sample, in a Sponsor supplied tube, was shipped, on dr y ice, to Tobias Schmidt and

Dr. rer. nat. W. Dekant at th e address below. The remaining sample, in a Testing Facility supplied tube, was stored frozen at approximately -20 °C±10 °C as a backup sample and discarded following issuance of the final report.

University of Würzburg
Versbacher Straße 9 97078 Würzburg, Germany

2.15.4. SAMPLE ANALYSIS AND REPORTING

For metabolite identification, urine samples were analyzed by

19 

### F-NMR, after mixing 720 μL of urine with 80 μL D 2 

### O. Known metabolites were identified and quantified by GC or LC/MS ( Schuster et al., 2010 ). Results of the analysis were provided in a report presented in Appendix I

.

2.16. POSTMORTEM

2.16.1. NECROPSY INFORMATION

Phase 1

Necropsy was performed on 1 presumed pregnant and 1 nonpregnant female, 2 days followi ng the 1-hour exposure. Animals were not fasted prior to sacrifice.

### Huntingdon Life Sciences 10-2226 Page 28

MA-252E-93-23 Final Report

Phase 2

Necropsy was performed on 5 males/group and 6 presumed pregnant females/group and 5 non- pregnant females/Groups 1 and 3, 14 days following the 1-hour exposure (GD 26 for the presumed pregnant females). Animals were not fasted overnight prior to

necropsy.

Method of Euthanasia

All adult females and males were euthanized by intravenous injection of sodium pentobarbital. Live fetuses were euthanized by intraperitoneal injection of sodium pentobarbital on GD 26.

2.16.2. MACROSCOPIC EXAMINATIONS

Phase 1

Animals were euthanized and discarded without examination, except the pregnancy status was determined for the presumed pregnant animal.

Phase 2

Complete macroscopic examinations were performed on all animals and the pregnancy status was determined for the presumed pregnant animals. The macroscopic examination included examination of the external surface and all orifices; the external surfaces of the brain and spinal co rd; the organs and tissues of the cranial, thoracic, abdominal and pelvic cavities and neck; and the remainder of the carcass for the presence of macroscopic morphologic abnormalities. Minor sporadic lesions that are common to this strain of animal, such as localized patchy hair loss, were not sampled or recorded.

No evaluation of the fetuses was performed.

2.16.3. TISSUES PRESERVED AND EXAMINED HISTOPATHOLOGICALLY

The heart, kidneys, liver, lungs (with mainstem bronchi) and gross

lesions were obtained at the scheduled sacrifice interval and preserved for all animals from Phase 2. In addition, slides of these

### Huntingdon Life Sciences 10-2226 Page 29

MA-252E-93-23 Final Report

tissues were prepared and examined microscopically for animals in the control and high-dose groups fr om Phase 2. Any abnormalities not noted during macroscopic examinations which were seen during histology processing were recorded.

Preservatives

All tissues - 10% neutral buffered formalin.

Processing

After fixation, the tissues and orga ns from animals in the control and high-dose groups from Phase 2 were routinely processed, embedded in paraffin, cut at a microtome setting of 4-7 microns, mounted on glass slides, stained with hematoxylin and eosin and examined by light microscopy.

2.17. STATISTICAL ANALYSIS

The following parameters were analyzed statistically from data collected during Phase 2:

body weight cumulative body weight change from baseline

2.17.1. METHOD OF ANALYSIS

The parameters to analyze were identified as continuous. Testsubstance treated groups were then compared to the control using the following procedures.

For all parameters, significant differences between control and test substance-treated groups were expressed at the 5% (p<0.05), 1% (p<0.01) or the 0.1% (p<0.001) level.

For comparisons involving more than two groups, if Bartlett's test for variance homogeneity ( Bartlett, 1937 ) was not significant at the 1% level, then parametric met hods were applied. If the F1 approximate test for monotonicity ( described below ) of exposureresponse was not significant at the 1% level, Williams' test (Williams’, 1971

, 1972 ) for a monotonic trend was applied. If the F1 test was significant, suggesting that the exposure-response was

### Huntingdon Life Sciences 10-2226 Page 30

MA-252E-93-23 Final Report

not monotone, Dunnett's test ( Dunn ett, 1955

, 1964 ) was performed instead. If Bartlett's test was significant at the 1% level, then logarithmic and square-root transformations were tried. If Bartlett's test was still significant, then non-parametric methods were applied to mean ranks. If the H1 approximate test for monotonicity ( described below ) was not significant at the 1% level, Shirley's test for a monotonic trend ( Shirley, 1977 ) was applied. If the H1 test was significant, then Steel's test ( Steel, 1959 ) was performed instead.

The F1 test is designed to de tect significant departure from monotonicity of an ordered set of means. The test statistic compares the mean square, NMS, for the deviations of the absolute or adjusted means from the maximum likelihood means, calculated under a constraint of monotonicity, with the usual error mean square, EMS. The null hypothesis is that the true means are monotonically ordered. The test statistic is F1 = NMS/EMS which can be compared with standard ta bles of the F-distribution with 1 and error degrees of freedom.

The H1 test is the non-parametric equivalent of the F1 test. The test statistic compares the non-monotonicity sums of squares, NRSS, for the deviations of the mean ranks from the maximum likelihood mean ranks, with the non-parametric equivalent of the error sums of squares, ERSS=N(N+1)/ 12. The test statistic is H1 = NRSS/ERSS which can be compared to standard tables of the χ 2- distribution with 1 degree of freedom.

For comparisons involving only two groups, if Bartlett's test for variance homogeneity ( Bartlett, 1937 ) was not significant at the 1% level, then parametric methods were applied. Comparisons were performed using t -tests. If Bartlett's test was significant at the 1% level, then logarithmic and square-root transformations

were tried. If Bartlett's te st was still significant, then nonparametric methods were applied to mean ranks. Comparisons were performed using Wilcoxon rank sum tests ( Wilcoxon, 1945 ).

### Huntingdon Life Sciences 10-2226 Page 31

MA-252E-93-23 Final Report

2.18. MAJOR COMPUTER SYSTEMS

The following major computer/software systems were utilized at Huntingdon Life Sciences, East Millstone, New Jersey.

System Version Use

Liberate Reporting System 2.2 Generati on of report tables for the clinical observations and body weight data in MS

Word formats

Microsoft Word and/or Excel Add itional formatting of report tables.

Quasar 1.1 Statistical analysis of data

REES Scientific Environmental Monitoring
System WIN 1.5 Monitor and record temperature, humidity and/or light intensity readings of applicable rooms as well as the temperature readings of applicable refrigerators and/freezers

TSI Aerodynamic Particle Sizer Spectrometer 2.9 Acquisition of particle size data

Xybion Path/Tox System 4.2.2 Direct online capture of clinical observations, body weights and anatomic pathology data; and generation of report tables for the anatomic pathology data



### 2.19. DATA STORAGE

All raw data, preserved specimens, and retained samples, as well as the original study protocol and the original final report are to be maintained in the Archives of the Testing Facility upon completion of the study. The Sponsor will determine the final disposition of these materials.

The Subcontractor laboratory will arch ive their raw data, original final report and any preserved urine specimens.

### Huntingdon Life Sciences 10-2226 Page 32

MA-252E-93-23 Final Report

2.20. REGULATORY REFERENCES

2.20.1. TEST GUIDELINES

The design was not based on regul atory guidelines for toxicity studies but followed the Sponsor requirements for this evaluation as per guidance of the U.S. EPA.

2.20.2. GOOD LABORATORY PRACTICES

This study was conducted in compliance with the United States EPA Good Laboratory Practices as set forth in 40 CFR Part 792 (TSCA) with the exception that the urine metabolite determinations were performed as a separate non-GLP investigation.

2.20.3. ANIMAL WELFARE ACT COMPLIANCE

This study complied with all appropriate parts of the Animal Welfare Act Regulations: 9 CF R Parts 1 and 2 Final Rules, Federal Register, Volume 54, No. 168, August 31, 1989, pp. 36112-36163 effective October 30, 1989 and 9 CFR Part 3 Animal Welfare Standards; Final Rule, Fe deral Register, Volume 56, No. 32, February 15, 1991, pp. 6426-6505 effective March 18, 1991.

2.21. PROTOCOL DEVIATIONS

The following protocol deviations o ccurred during the study but were not considered to have compromised the validity or integrity of the study:

1. Urine samples were not collected at the 24 to 36 hour and 36 to 48 hour post-exposure intervals from presumed pregnant Animal Nos. 1506, 2506 and 3506 because of a shortage of pans.

2. Due to technician oversight, documentation that hay was presented to the animals was recorded as performed only on Phase 2, Pretest Day 8 (Phase 1 animals were euthanized on this day).

3. Due to technician oversight, a daily viability check was performed only once on Phase 2, Day 10 (males) and Day 12 (females).

4. The urine sample collected during the 24 to 36 hours post dose interval for Animal No. 3501 was 9 mL; however, due to technician oversight, this sample was not split into 2 aliquots.

### Huntingdon Life Sciences 10-2226 Page 33

MA-252E-93-23 Final Report

5. Due to technician oversight, th ere is no documentation that the animals were identified by earmarking with indelible marker on the

underside of the ear.

6. Due to technician oversight, th ere is no documentation that the animals were fed at the same time on each day.

7. The protocol required the amount of feed presented to presumed pregnant females be done in accordan ce with the Testing Facility SOP.

On the 3 rd 

### day following receipt, the amount presented to the presumed pregnant females was not according to the Testing Facility

SOP.

8. The protocol required the urine assays be performed as described by the protocol; however, several of the details were conducted differently in order to improve the analysis.

### Huntingdon Life Sciences 10-2226 Page 34

MA-252E-93-23 Final Report

3. RESULTS AND DISCUSSION

3.1. CHAMBER MONITORING RESULTS

( Appendices A
, B
, K and L )

Pre-study chamber distribution analyses showed the test substance was evenly distributed within each chamber. The target and mean analytical (IR) and nominal concentrations are summarized as follows:

Group Target Concentration (Males/Females) (ppm) Analytical Concentration (Males/Females) (ppm) Nominal Concentration (Males/Females) (ppm)

Phase 1 100,000 110,000 100,000

Phase 2

1 0 0.0/0.0 NA

2 50,000 45,000/47,000

3 100,000 100,000/102,000 150,000/140,000 a

a The nominal concentrations in Phase 2 are th e combined results of the 2 chambers for each of the separate male and female exposures.



### These results indicated an acceptable agreement between the targeted and analytical and nominal concentrations.

Mean particle size distribution measurements for the exposures are

summarized as follows:

Group Test
Substance Mass Median Aerodynamic

Diameter (Males/Females) ( μ

m) Geometric
Standard
Deviation (Males/Females) Total

Mass
Concentration (Males/Females) (mg/m 3 

### )

Phase 1 HFO-1234yf 3.648 1.662 (females only) 9.41 x 10 -3



### Phase 2

1 Air Control 3.883/4.398 1.452/1.967 7.35 x 10 -3 

### /8.80 x 10 -3



### 2 HFO-1234yf 18.490/3.764 2.139/1.437 3.78 x 10 -2 

### /3.59 x 10 -2



### 3 HFO-1234yf 4.988/6.079 1.681/2.030 1.51 x 10 -2 

### /0.152

### Huntingdon Life Sciences 10-2226 Page 35

MA-252E-93-23 Final Report

These results indicated that the atmospheres were essentially gas only, as expected, since there was no substantial difference between the test

substance chambers and the air control chamber.

3.2. URINE METABOLITE DETERMINATIONS

( Appendix I )

Urine was collected in 12 hour interv als for 48 hours after the end of the exposure and analyzed by 19 

### F-NMR and LC/MS-MS. The predominant

metabolites were S -(3,3,3-trifluoro-2-hydroxypropanyl)-mercaptolactic

acid and N -acetyl- S -(3,3,3-trifluoro-2-hydroxypropanyl)- L -cysteine whose signal intensities in 19 

### F-NMR spectra represented more than 78% of total

19 

### F related signals in all urine analyzed samples. Quantification of the HFO-1234yf-mercapturic acid isomers by LC/MS-MS showed no difference in the mean recovery of this metabolite between female (43.11 ± 22.35 μmol) and pregnant fe male rabbits (50.47 ± 19.72 μmol)

excreted in urine within 48 hours. With the exception of one yet unidentified metabolite (12-17% female rabbits and < 7% male rabbits of the six main metabolites), no differences in urinary metabolite pattern or quantity of metabolites excreted were observed between the different

groups.

3.3. MORTALITY

( Appendices A and C )

All animals survived until their scheduled sacrifice.

3.4. CLINICAL OBSERVATIONS

( Tables 1
, 2 and 3
, Appendices A
, D and E )

There were no test substance-related clinical signs in any of the test

animals

3.5. BODY WEIGHTS

( Tables 4 and 5
, Appendices A
, F and G )

There were no test substance-related effects on body weights in any of the test groups of animals.

### Huntingdon Life Sciences 10-2226 Page 36

MA-252E-93-23 Final Report

3.6. ANATOMIC PATHOLOGY

3.6.1. MACROSCOPIC

( Table 6
, Appendix H )

In Phase 1, the presumed pregnant female was confirmed pregnant. In Phase 2, the presumed pregnant females were confirmed pregnant except 1 female in Group 1 (Animal No. 1502) and 1 female in Group 3 (Animal No. 3506).

In Phase 2, there were no test substance-related macroscopic findings at the terminal sacrifi ces. Macroscopic findings were of low incidence and considered common incidental findings in the test species. This includes the finding of discolored lungs in a small number of females given control or test substance.

3.6.2. MICROSCOPIC

( Table 7
, Appendix H )

In Phase 2, there were no test substance-related microscopic findings (heart, kidneys, liver and lungs) at the terminal sacrifices. Microscopic findings were sporadic or present in control and test substance exposed animals, and considered unrelated to the test substance. This includes the findings of interstitial inflammatory cell infiltrates in the lungs of some animals given the control or

test substance and minimal focal alveolar ossification in two females given the test substance.

4. CONCLUSION
No lethality and no clinical signs were obs erved in the exposed animals. With the exception of one yet unidentified metabolite (12-17% female rabbits and < 7% male rabbits of the six main metabolites), no differences in urinary metabolite pattern or quantity of metabolites excreted were observed between the different groups. The results suggest that the previously seen lethality of HFO-1234yf in pregnant rabbits after inhalation exposures is unlikely due to changes in biotransformation patterns or capacity of pregnant rabbits.

### Huntingdon Life Sciences 10-2226 Page 37
MA-252E-93-23 Final Report

REFERENCES

Bartlett, M.S. 1937. Properties of sufficiency and statistical tests. Proceedings of the Royal Society, Series A , 160: 268-282.

Dunnett, C.W. 1955. A multiple comparison procedure for comparing several treatments
with a control. Journal of the American Statistical Association 50: 1096-1121.

Dunnett, C.W. 1964. New tables for multiple comparisons with a control. Biometrics 20-
3: 482-491.

Shirley, E.A.C. 1977. A non-parametric equivalent of Williams’ test for contrasting increasing dose levels of a treatment. Biometrics 33: 386-389.

Schuster et al. 2010. Biotransformation of 2,3,3,3-tetrafluoropropene (HFO-1234yf) in

rats. Original Research Article 1: 247-253.

Steel, R.G.D. 1959. A multiple comparison rank sum test: treatments versus control.

Biometrics 15: 560-572.

Wilcoxon, F. 1945. Individual comparisons by ranking methods. Biometrics Bulletin 1:
80-83.

Williams, D.A. 1971. A test for differences between treatment means when several dose levels are compared with a zero dose control. Biometrics 27: 103-117.

Williams, D.A. 1972. The comparison of severa l dose levels with a zero dose control.

Biometrics 28: 519-531.

### Huntingdon Life Sciences 10-2226 Page 38
MA-252E-93-23 Final Report

General Preface

General Notes Individual animal data presented in this report may be rounded. Unrounded individual animal data were used to calculate the reported mean and standard deviation values. Therefore, use of the reported individual values to reproduce means, standard deviations and/or to perform any subsequent calculations may produce minor discrepancies between the calculated values and those presented in this report.

The data collection system used for collecti ng the in-life and post-life data divides the study into phases. The start of each phase begins with Day 1/Week1. The phases utilized on this study were:

Pretest Phase (PRE): Begins the day the animals arrive. During this phase, animals are not being dosed. Animals are sorted into groups and assigned their permanent identification numbers on the day of arrival.

Dosing Phase (PH1): Phase 1 begins for each animal on the day it was exposed to the test substance as specified in the protocol. The target exposure level for the Phase 1 (Group 1) animals was 100,000 ppm.

Dosing Phase (PH2): Phase 2 begins for each animal on the day it was exposed to the test substance as specified in the protocol.

Corresponding target exposure levels for each group in Phase 2 were as follows:

Group 1 - 0 ppm Group 2 - 50,000 ppm Group 3 - 100,000 ppmHuntingdon Life Sciences10-2226Page 39MA-252E-93-23FinalReportPhase 1Group 1Chamber Generation Systemand 1000 Liter Whole-Body Exposure ChamberaFigure 1aSampling port H3 is on the front of the chamber; while, sampling port H4 is on the back of the chamber. Oxygen was delivered tothe chamber using a similar set-up as used to deliver the test substance.Huntingdon Life Sciences10-2226Page 40MA-252E-93-23FinalReportPhase 2Group 1Chamber Generation Systemand 1000 Liter Whole-Body Exposure ChamberaFigure 2aSampling port H3 is on the front of the chamber; while, sampling port H4 is on the back of the chamber.Huntingdon Life Sciences10-2226Page 41MA-252E-93-23FinalReportPhase 2Groups 2 and 3Chamber Generation Systemand 1000 Liter Whole-Body Exposure ChamberaFigure 3aSampling port H3 is on the front of the chambers; while, sampling port H4 is on the back of the chambers. Oxygen was deliveredtothe chambers using a similar set-up as used to deliver the test substance.

### Huntingdon Life Sciences 10-2226 Page 42
MA-252E-93-23 Final Report

Summary of In-Chamber Observations – Phase 2 Table 1

All = 100% of the animals exhibiting a given observation. Exposure Day 1 1
Males Females Group 1 – 0 ppm

Within Normal Limits All All

Group 2 – 50,000 ppm

Within Normal Limits All All

Group 3 – 100,000 ppm

Within Normal Limits All All

### Huntingdon Life Sciences 10-2226 Page 43
MA-252E-93-23 Final Report

Summary of Daily Observations
Preface Table 2

Cage-side observations were performed twice daily (once in the morning and once in the afternoon) for mortality, morbidity and signs of severe toxicity. Food consumption was also visually checked each day as part of the cage-side observations, and any decrease in food consumption was recorded. On the day of exposure, animals were observed before and after exposure. For summarization purposes, only time-points with animals exhibiting abnormal findings are presented.

Corresponding exposure levels for each group in Phase 2 were as follows:

Group 1 - 0 ppm Group 2 - 50,000 ppm Group 3 - 100,000 ppm

Key to Abbreviations:

AM = Morning (Pre-exposure on the day of exposure) Observation PM = Afternoon (Post-exposure on the day of exposure) ObservationHuntingdon Life Sciences10-2226Page 44MA-252E-93-23FinalReportPresumed PregnantFemalesSummary of Daily Observations - Phase 2Table 2Phase PRE PH2Day 2 3 4 5 6 7 8 9 11 3 4 7 10Session AM AM AM AM AM AM AM AM AM AM AM AM AMObservationsGroupGastrointestinal, Decreased Fecal Volume 1 2 1 1 12 1 2 1 1 1 1 13 3 1 1 1Gastrointestinal, No Stool2 1Miscellaneous, Decreased Food Consumption 1 1 1 1 12 2 1 1 1 13 2 2 1 2Huntingdon Life Sciences10-2226Page 45MA-252E-93-23FinalReportNon-PregnantFemalesSummary of Daily Observations - Phase 2Table 2Phase PREDay 3 4 5 6 7 7 8 8 9 9 10 10 11 11Session AM AM AM AM AM PM AM PM AM PM AM PM AM PMObservationsGroupGastrointestinal, Decreased Fecal Volume 1 2 23 2 1 3Gastrointestinal, No Stool1 1 1 1 13 1Miscellaneous, Decreased Food Consumption 1 1 1 1 13 2Huntingdon Life Sciences10-2226Page 46MA-252E-93-23FinalReportNon-PregnantFemalesSummary of Daily Observations - Phase 2Table 2Phase PRE PH2Day 12 12 13 1 2 4 5 6 7 8 10Session AM PM AM PM AM AM AM AM AM AM AMObservationsGroupGastrointestinal, Decreased Fecal Volume 1 13 1Huntingdon Life Sciences10-2226Page 47MA-252E-93-23FinalReportMalesSummary of Daily Observations - Phase 2Table 2Phase PREDay 3 4 5 6 7 7 8 8 9 9 10 10 11 11Session AM AM AM AM AM PM AM PM AM PM AM PM AM PMObservationsGroupDermal General, Ano-Genital Staining2 1 2 1 2 2 1 1 1 1 1 1Dermal General, Stains on Fur (Head/Neck) 1 1Gastrointestinal, Decreased Fecal Volume 1 1 12 1 2 1 1 1 2 1 13 1 1Gastrointestinal, No Stool2 1 1 1 1 1Gastrointestinal, Unformed Stool2 1General Appearance, Thin2 1 1 1 1 1 1 1 1Miscellaneous, Decreased Food Consumption 1 1 12 1 2 2 3 2 2 13 1Huntingdon Life Sciences10-2226Page 48MA-252E-93-23FinalReportMalesSummary of Daily Observations - Phase 2Table 2Phase PRE PH2Day 12 12 13 1 2 4 5 6 7 8 10Session AM PM AM PM AM AM AM AM AM AM AMObservationsGroupDermal General, Ano-Genital Staining2 1 1 1 1 13 2Dermal General, Stains on Fur (Head/Neck) 3 1Dermal General, Stains on Fur (Torso)3 1Gastrointestinal, Decreased Fecal Volume 1 23 1 1 1Gastrointestinal, No Stool2 13 1Gastrointestinal, Unformed Stool3 1 1 1General Appearance, Thin2 1 1 1 1Miscellaneous, Decreased Food Consumption 1 12 13 1 1 2

### Huntingdon Life Sciences 10-2226 Page 49
MA-252E-93-23 Final Report

Summary of Physical Examination Findings
Preface Table 3

Detailed physical examinations were pe rformed for all animals on study once daily during the pretest period a nd once daily (post exposure on the day of exposure) during the dosing phase. For summarization purposes, descriptive comments [i.e., location of scab(s) and/or sore(s), etc.] are not presented in this table. These data are contained in the study raw data if needed.

Corresponding exposure levels for each group in Phase 2 were as follows:

Group 1 - 0 ppm Group 2 - 50,000 ppm Group 3 - 100,000 ppmHuntingdon Life Sciences10-2226Page 50MA-252E-93-23FinalReportPresumed PregnantFemalesSummary of Physical Examination Findings - Phase 2Table 3Phase PRE PH2Day 1 2 3 4 5 6 7 8 9 10 1 2 3 4 5ObservationsGroupWithin Normal Limits1 6 4 6 5 5 5 5 5 5 5 5 5 5 5 52 5 4 5 6 5 5 5 5 6 6 6 6 6 6 63 6 3 5 6 6 6 6 4 6 6 6 6 5 5 5Dermal General, Ano-Genital Staining2 1 1 1Dermal General, Scab(s)1 1 1 1 1 1 1 1 1 1 1 1 13 1 1 1Gastrointestinal, Decreased Fecal Volume 1 2 12 1 1 1 1 13 3 1 1Miscellaneous, Decreased Food Consumption 1 12 1 1 1 13 2Huntingdon Life Sciences10-2226Page 51MA-252E-93-23FinalReportPresumed PregnantFemalesSummary of Physical Examination Findings - Phase 2Table 3Phase PH2Day 6 7 8 9 10 11 12 13 14 15ObservationsGroupWithin Normal Limits1 5 4 6 6 6 6 6 5 5 52 6 5 6 6 6 6 6 5 5 53 5 4 5 5 5 5 5 4 4 4Dermal General, Ano-Genital Staining1 1 1 12 1 1 13 2 2 2Dermal General, Scab(s)1 13 1 1 1 1 1 1 1 1 1 1Gastrointestinal, Decreased Fecal Volume 1 12 13 1Miscellaneous, Decreased Food Consumption 1 13 1Huntingdon Life Sciences10-2226Page 52MA-252E-93-23FinalReportNon-PregnantFemalesSummary of Physical Examination Findings - Phase 2Table 3Phase PRE PH2Day 1 2 3 4 5 6 7 8 9 10 1 2 3ObservationsGroupWithin Normal Limits1 5 5 3 5 4 4 2 3 3 3 4 4 43 5 5 5 5 4 5 5 5 3 3 5 5 5Dermal General, Alopecia (Head/Neck)3 1 1Dermal General, Scab(s)1 2 2 2 2 1 1 13 1 1Gastrointestinal, Decreased Fecal Volume 1 23 1Gastrointestinal, No Stool1 1 1 1Miscellaneous, Decreased Food Consumption 1 1 1 1Huntingdon Life Sciences10-2226Page 53MA-252E-93-23FinalReportNon-PregnantFemalesSummary of Physical Examination Findings - Phase 2Table 3Phase PH2Day 4 5 6 7 8 9 10 11 12 13 14 15ObservationsGroupWithin Normal Limits1 4 4 4 4 4 4 5 5 5 5 5 53 5 5 5 5 5 5 5 5 5 5 5 5Dermal General, Scab(s)1 1 1 1 1 1 1Huntingdon Life Sciences10-2226Page 54MA-252E-93-23FinalReportMalesSummary of Physical Examination Findings - Phase 2Table 3Phase PRE PH2Day 1 2 3 4 5 6 7 8 9 10 11 12 1 2 3ObservationsGroupWithin Normal Limits1 1 1 1 1 1 1 1 1 3 3 3 4 3 3 32 1 1 1 1 1 1 1 1 1 13 4 4 3 4 4 4 4 4 3 3 4 4 4 4 4Dermal General, Alopecia (Head/Neck)2 1 1 1 1 1 1 1 1 1 1Dermal General, Alopecia (Torso)1 1 1 1Dermal General, Ano-Genital Staining1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 12 2 2 2 3 3 3 3 3 2 2 2 2 2 2 23 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1Dermal General, Scab(s)1 1 1 1 12 1 1 1 1 1 1 1 1 1 1 1 1 1 1 13 1 1Dermal General, Stains on Fur (Head/Neck) 1 1 1 1 1 1 1 1 12 2 2 2 2 2 2 2 2 2 2 2 2 2 2 23 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1Dermal General, Stains on Fur (Torso)1 2 2 2 2 2 2 2 22 1 1 1 1 1 1 1 1 1 1 1 1 1 1 13 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1Huntingdon Life Sciences10-2226Page 55MA-252E-93-23FinalReportMalesSummary of Physical Examination Findings - Phase 2Table 3Phase PRE PH2Day 1 2 3 4 5 6 7 8 9 10 11 12 1 2 3ObservationsGroupGastrointestinal, Decreased Fecal Volume2 1 2 2 2 23 1Gastrointestinal, No Stool3 1Gastrointestinal, Unformed Stool2 1General Appearance, Thin2 1 1 1 1 1 1Miscellaneous, Decreased Food Consumption 2 1 1 2 33 1Oral/Buccal, Incisor(s) Broken/Missing2 1 1Huntingdon Life Sciences10-2226Page 56MA-252E-93-23FinalReportMalesSummary of Physical Examination Findings - Phase 2Table 3Phase PH2Day 4 5 6 7 8 9 10 11 12 13 14 15ObservationsGroupWithin Normal Limits1 2 3 3 3 4 4 4 4 3 3 3 32 1 1 1 1 1 2 2 2 2 2 2 23 4 3 3 3 3 3 3 3 3 3 3 3Dermal General, Alopecia (Head/Neck)1 1 1 1 1Dermal General, Alopecia (Torso)1 1Dermal General, Ano-Genital Staining1 1 1 1 1 1 1 1 1 2 2 2 22 2 2 2 2 2 2 2 2 2 2 2 23 1 1 2 2 2 2 2 2 2 2 2 2Dermal General, Scab(s)2 1 1 1 1 1 1 1 1 1 1 1 1Dermal General, Stains on Fur (Head/Neck) 2 2 2 2 2 2 1 1 1 1 13 1 1 1 1 1Dermal General, Stains on Fur (Torso)2 1 1 1 1 1 1 1 1 1 1 1 13 1 1 1 1 1 1 1 1 1 1 1 1Gastrointestinal, No Stool3 1Gastrointestinal, Unformed Stool3 1Huntingdon Life Sciences10-2226Page 57MA-252E-93-23FinalReportMalesSummary of Physical Examination Findings - Phase 2Table 3Phase PH2Day 4 5 6 7 8 9 10 11 12 13 14 15ObservationsGroupMiscellaneous, Decreased Food Consumption 3 1Huntingdon Life Sciences 10-2226 Page 58
MA-252E-93-23 Final Report

Presumed Pregnant Mean Body Weights Females (grams) Table 4

Phase PH2 Day 1 3 5 8 15

Group 1 - 0 ppm

Mean 3439.2 3475.6 3465.6 3583.4 3648.4
SD 113.28 109.10 83.46 102.03 161.72
N 6 6 6 6 6

Group 2 – 50,000 ppm

Mean 3520.3 3584.8 3574.4 3664.1 3744.3
SD 123.49 140.83 107.67 123.09 163.64
N 6 6 6 6 6

Group 3 – 100,000 ppm

Mean 3373.9 3452.8 3450.7 3567.7 3651.2
SD 239.63 254.33 252.50 290.32 303.93
N 6 6 6 6 6

No statistically significant differences from control meanHuntingdon Life Sciences 10-2226 Page 59
MA-252E-93-23 Final Report

Non-Pregnant Mean Body Weights Females (grams) Table 4

Phase PH2 Day 1 3 5 8 15

Group 1 - 0 ppm

Mean 3285.9 3239.5 3285.4 3378.0 3453.4
SD 211.55 201.25 199.15 206.70 218.06

N 5 5 5 5 5

Group 3 – 100,000 ppm

Mean 3230.5 3208.1 3179.6 3274.6 3368.6
SD 167.13 164.92 167.15 215.11 172.52

N 5 5 5 5 5

No statistically significant differences from control meanHuntingdon Life Sciences 10-2226 Page 60
MA-252E-93-23 Final Report

Mean Body Weights Males (grams) Table 4

Phase PH2 Day 1 3 5 8 15

Group 1 - 0 ppm

Mean 3071.4 3040.6 3092.5 3088.0 3184.8
SD 257.16 225.30 184.76 183.13 164.59

N 5 5 5 5 5

Group 2 – 50,000 ppm

Mean 2834.6 2905.7 2991.8 2992.1 3112.1
SD 161.70 135.58 167.21 157.17 159.39

N 5 5 5 5 5

Group 3 – 100,000 ppm

Mean 3077.3 3089.2 3082.8 3116.5 3244.1
SD 354.67 337.97 266.70 224.25 283.27

N 5 5 5 5 5

No statistically significant differences from control meanHuntingdon Life Sciences 10-2226 Page 61
MA-252E-93-23 Final Report

Presumed Pregnant Mean Body Weight Change from Baseline Females (grams) Table 5

Phase PH2 Day 3 5 8 15

Group 1 - 0 ppm

Mean 36.3 26.4 144.2 209.2
SD 45.15 67.59 69.34 133.56
N 6 6 6 6

Group 2 – 50,000 ppm

Mean 64.4 54.1 143.7 224.0
SD 51.38 68.06 58.33 110.22
N 6 6 6 6

Group 3 – 100,000 ppm

Mean 79.0 76.8 193.8 277.4
SD 28.64 42.07 71.37 79.56
N 6 6 6 6

No statistically significant differences from control meanHuntingdon Life Sciences 10-2226 Page 62
MA-252E-93-23 Final Report

Non-Pregnant Mean Body Weight Change from Baseline Females (grams) Table 5

Phase PH2 Day 3 5 8 15

Group 1 - 0 ppm

Mean -46.5 -0.5 92.1 167.4
SD 63.71 41.56 70.18 67.59

N 5 5 5 5

Group 3 – 100,000 ppm

Mean -22.4 -50.9 44.1 138.1
SD 24.24 28.45 50.97 15.51

N 5 5 5 5

No statistically significant differences from control meanHuntingdon Life Sciences 10-2226 Page 63
MA-252E-93-23 Final Report

Mean Body Weight Change from Baseline Males (grams) Table 5

Phase PH2 Day 3 5 8 15

Group 1 - 0 ppm

Mean -30.8 21.1 16.6 113.4
SD 46.84 78.38 75.97 98.24

N 5 5 5 5

Group 2 – 50,000 ppm

Mean 71.1 157.2 157.5 277.5
SD 97.78 112.58 114.00 94.31

N 5 5 5 5

Group 3 – 100,000 ppm

Mean 11.9 5.5 39.2 166.7
SD 30.86 110.04 158.39 79.79

N 5 5 5 5

No statistically significant differences from control mean

### Huntingdon Life Sciences 10-2226 Page 64
MA-252E-93-23 Final Report

Incidence Summary Report for Gross Necropsy Observations
Preface Table 6

Table of Contents:

Presumed Pregnant............................................................................................................ .6 5 Males and Non-Pregnant Females ..................................................................................... 66

Corresponding exposure levels for each group in Phase 2 were as follows:

Group 1 - 0 ppm Group 2 - 50,000 ppm Group 3 - 100,000 ppmPage 65Page 66

### Huntingdon Life Sciences 10-2226 Page 67
MA-252E-93-23 Final Report

Incidence Summary of Microscopic Findings with Severity Levels
Preface Table 7

Table of Contents:

Presumed Pregnant............................................................................................................ .6 8 Males and Non-Pregnant Females ..................................................................................... 69

Key to Abbreviations:

Ctls = Controls - = Finding not present or observed

Histopathology grading:

Grade 1: MINIMAL = the change is barely discernible and/or very few/very
small foci or areas are affected

Grade 2: SLIGHT = the change is more noticeable but only evident as
few/small foci or areas affected

Grade 3: MODERATE = the change is obviously present, and of appreciable
size and/or number

Grade 4: MARKED = the change is abundant in many areas of the section and/or is of prominent size

Grade 5: SEVERE = the change affects a large proportion of the tissue and/or is of a large size

Corresponding exposure levels for each group were as follows:

Group 1 - 0 ppm Group 2 - 50,000 ppm Group 3 - 100,000 ppmPage 68Page 69Page 70

### Huntingdon Life Sciences 10-2226 Page 71
MA-252E-93-23 Final Report

Phase 1 Screening Study
Preface Appendix A

Exposure Level:

Group 1 – 100,000 ppm

TABLES:

1. Chamber Monitoring Records ..................................................................................... 72

2. Individual Animal Term ination History ...................................................................... 73

3. Individual Physical Ex amination Findings.................................................................. 74

4. Individual Body Weights ............................................................................................ .76

5. Individual Body Weight Change from Baseline......................................................... .77

### Huntingdon Life Sciences 10-2226 Page 72
MA-252E-93-23 Final Report

Group 1
Females Chamber Monitoring Records
Phase 1 Appendix A
Table 1

Chamber Concentration Sample Analytical Nominal Number (ppm) (ppm)

1001 120,000 100,000 1002 110,000 1003 100,000

Mean 110,000 S.D. 10,000

Particle Size Determinations Sample
Number MMAD GSD TMC (μm) (mg/m 3 

### )

1001 3.648 1.662 9.41E-03

Chamber Environment Exposure Total Hour Temperature Relative Humidity Chamber Airflow
(°C) (%) (Lpm)

T 99 

### 19 56 224
0.0 19 51 224
0.5 19 51 224
1.0 19 49 224

Mean 19 52 224
S.D. 0.0 3.0 0.0

### Huntingdon Life Sciences 10-2226 Page 73
MA-252E-93-23 Final Report

Phase 1
Females Individual Animal Termination History Appendix A
Table 2

a 

### First day of each phase defined as Day 1. Day of exposure defined as Dosing Day 1.

b 

### Confirmed pregnant at necropsy. Animal Date of Phase
Number Death Type of Death Phase Day a



### Group 1 - 100,000 ppm

4501 b 

### 5 Nov 2010 Terminal sacrifice DOSING PHASE 3
4502 5 Nov 2010 Terminal sacrifice DOSING PHASE 3

### Huntingdon Life Sciences 10-2226 Page 74
MA-252E-93-23 Final Report

Phase 1 Individual Physical Examination Findings
Preface Appendix A
Table 3

Detailed physical examinations were performed for all animals once daily during the stabilization (pretest) period and once daily (post-exposure on the day of exposure) during the dosing phase. Examinations include d observations of gene ral condition, skin and fur, eyes, nose, oral cavity, abdomen, as well as evaluations of respiration.

In-chamber observations were performed fo r all animals as a group once during the exposure period. All animals were within normal limits.

In addition, cage-side observations were pe rformed twice daily (once in the morning and once in the afternoon) for mortality, morbid ity and signs of seve re toxicity. Food consumption was also visually checked each da y as part of the cage-side observations. On the day of exposure, animals were observe d before and after exposure. All animals were considered to be within normal limits at all time points.Huntingdon Life Sciences10-2226Page 75MA-252E-93-23FinalReportPhase 1Appendix AFemales IndividualPhysicalExamination FindingsTable 3Phase PRE PH1AnimalDay 1 2 3 4 5 1 2 3Number ObservationsGroup 1 – 100,000 ppm4501 Within normal limitsPDermal general, scab(s), mouth - onP P P P P P P4502 Within normal limitsP P P P P P P PP = presentHuntingdon Life Sciences 10-2226 Page 76
MA-252E-93-23 Final Report

Phase 1 Individual Body Weights Appendix A Females (grams) Table 4

Animal Phase PH1 Number Day 1 2 3

Group 1 – 100,000 ppm

4501 3153.7 3260.9 3354.6
4502 3178.1 3238.4 3232.5

Mean 3165.9 3249.7 3293.6
SD 17.25 15.91 86.34
N 2 2 2Huntingdon Life Sciences 10-2226 Page 77
MA-252E-93-23 Final Report

Phase 1 Individual Body Weight Change from Baseline Appendix A Females (grams) Table 5

Animal Phase PH1 Number Day 2 3

Group 1 – 100,000 ppm

4501 107.2 200.9
4502 60.3 54.4

Mean 83.8 127.6
SD 33.16 103.59
N 2 2

### Huntingdon Life Sciences 10-2226 Page 78
MA-252E-93-23 Final Report

Group 1
Females Chamber Monitoring Records
Phase 2 Appendix B

Chamber Concentration Sample Analytical Number (ppm)

1001 0

1002 0

1003 0

Mean 0
S.D. 0.0

Particle Size Determinations Sample
Number MMAD GSD TMC (μm) (mg/m 3 

### )

1001 4.398 1.967 8.80E-3

Chamber Environment Exposure Total Hour Temperature Relative Humidity Chamber Airflow
(°C) (%) (Lpm)

T 99 

### 21 64 210
0.0 21 66 210
0.5 22 63 210
1.0 22 63 210

Mean 22 64 210
S.D. 0.6 1.4 0.0

### Huntingdon Life Sciences 10-2226 Page 79
MA-252E-93-23 Final Report

Group 1

Males Chamber Monitoring Records
Phase 2 Appendix B

Chamber Concentration Sample Analytical Number (ppm)

1004 0

1005 0

1006 0

Mean 0
S.D. 0.0

Particle Size Determinations Sample
Number MMAD GSD TMC (μm) (mg/m 3 

### )

1002 3.883 1.452 7.35E-3

Chamber Environment Exposure Total Hour Temperature Relative Humidity Chamber Airflow
(°C) (%) (Lpm)

T 99 

### 20 59 210
0.0 20 58 210
0.5 20 58 210
1.0 20 56 210

Mean 20 58 210
S.D. 0.0 1.3 0.0

### Huntingdon Life Sciences 10-2226 Page 80
MA-252E-93-23 Final Report

Group 2
Females Chamber Monitoring Records
Phase 2 Appendix B

a 

### Combined nominal for Groups 2 and 3 Chamber Concentration Sample Analytical Nominal Number (ppm) (ppm)

2001 45,000 140,000 a 

### 2002 48,000 2003 47,000

Mean 47,000
S.D. 1530

Particle Size Determinations Sample
Number MMAD GSD TMC (μm) (mg/m 3 

### )
2001 3.764 1.437 3.59E-2

Chamber Environment Exposure Total Hour Temperature Relative Humidity Chamber Airflow (°C) (%) (Lpm)

T 99 

### 20 57 211
0.0 21 58 211
0.5 21 55 211
1.0 20 55 211

Mean 21 56 211
S.D. 0.6 1.5 0.0

### Huntingdon Life Sciences 10-2226 Page 81
MA-252E-93-23 Final Report

Group 2

Males Chamber Monitoring Records
Phase 2 Appendix B

a 

### Combined nominal for Groups 2 and 3 Chamber Concentration Sample Analytical Nominal Number (ppm) (ppm)

2004 41,000 150,000 a 

### 2005 45,000 2006 49,000

Mean 45,000
S.D. 4000

Particle Size Determinations Sample
Number MMAD GSD TMC (μm) (mg/m 3 

### )
2002 18.490 2.139 3.78E-2

Chamber Environment Exposure Total Hour Temperature Relative Humidity Chamber Airflow (°C) (%) (Lpm)

T 99 

### 20 54 211
0.0 19 53 211
0.5 19 52 211
1.0 19 52 211

Mean 19 53 211
S.D. 0.5 1.0 0.0

### Huntingdon Life Sciences 10-2226 Page 82
MA-252E-93-23 Final Report

Group 3
Females Chamber Monitoring Records
Phase 2 Appendix B

a 

### Combined nominal for Groups 2 and 3 Chamber Concentration Sample Analytical Nominal Number (ppm) (ppm)

3001 110,000 140,000 a 

### 3002 100,000 3003 95,000

Mean 102,000
S.D. 7640

Particle Size Determinations Sample
Number MMAD GSD TMC (μm) (mg/m 3 

### )
3001 6.079 2.030 0.152

Chamber Environment Exposure Total Hour Temperature Relative Humidity Chamber Airflow (°C) (%) (Lpm)

T 99 

### 20 58 224
0.0 20 58 224
0.5 21 57 224
1.0 21 56 224

Mean 21 57 224
S.D. 0.6 1.0 0.0

### Huntingdon Life Sciences 10-2226 Page 83
MA-252E-93-23 Final Report

Group 3

Males Chamber Monitoring Records
Phase 2 Appendix B

a 

### Combined nominal for Groups 2 and 3 Chamber Concentration Sample Analytical Nominal Number (ppm) (ppm)

3004 100,000 150,000 a 

### 3005 100,000 3006 100,000

Mean 100,000
S.D. 0.0

Particle Size Determinations Sample
Number MMAD GSD TMC (μm) (mg/m 3 

### )
3002 4.988 1.681 1.51E-2

Chamber Environment Exposure Total Hour Temperature Relative Humidity Chamber Airflow (°C) (%) (Lpm)

T 99 

### 19 57 224
0.0 19 54 224
0.5 20 51 224
1.0 20 51 224

Mean 20 53 224
S.D. 0.6 2.9 0.0Huntingdon Life Sciences 10-2226 Page 84
MA-252E-93-23 Final Report

Males Individual Animal Termination History – Phase 2 Appendix C

Animal Date of Phase Number Death Type of Death Phase Day a

Group 1 - 0 ppm

1001 24 Nov 10 Terminal sacrifice DOSING PHASE 15
1002 24 Nov 10 Terminal sacrifice DOSING PHASE 15
1003 24 Nov 10 Terminal sacrifice DOSING PHASE 15
1004 24 Nov 10 Terminal sacrifice DOSING PHASE 15
1005 24 Nov 10 Terminal sacrifice DOSING PHASE 15

Group 2 – 50,000 ppm

2001 24 Nov 10 Terminal sacrifice DOSING PHASE 15
2002 24 Nov 10 Terminal sacrifice DOSING PHASE 15
2003 24 Nov 10 Terminal sacrifice DOSING PHASE 15
2004 24 Nov 10 Terminal sacrifice DOSING PHASE 15
2005 24 Nov 10 Terminal sacrifice DOSING PHASE 15

Group 3 – 100,000 ppm

3001 24 Nov 10 Terminal sacrifice DOSING PHASE 15
3002 24 Nov 10 Terminal sacrifice DOSING PHASE 15
3003 24 Nov 10 Terminal sacrifice DOSING PHASE 15
3004 24 Nov 10 Terminal sacrifice DOSING PHASE 15
3005 24 Nov 10 Terminal sacrifice DOSING PHASE 15

a First day of each phase defined as Day 1. Day of exposure defined as Dosing Day 1Huntingdon Life Sciences 10-2226 Page 85
MA-252E-93-23 Final Report

Females Individual Animal Termination History – Phase 2 Appendix C

Animal Date of Phase Number Death Type of Death Phase Day a

Group 1 - 0 ppm

1501 22 Nov 10 Terminal sacrifice DOSING PHASE 15
1502 22 Nov 10 Terminal sacrifice DOSING PHASE 15
1503 22 Nov 10 Terminal sacrifice DOSING PHASE 15
1504 22 Nov 10 Terminal sacrifice DOSING PHASE 15
1505 22 Nov 10 Terminal sacrifice DOSING PHASE 15
1506 22 Nov 10 Terminal sacrifice DOSING PHASE 15
1507 22 Nov 10 Terminal sacrifice DOSING PHASE 15
1508 22 Nov 10 Terminal sacrifice DOSING PHASE 15
1509 22 Nov 10 Terminal sacrifice DOSING PHASE 15
1510 22 Nov 10 Terminal sacrifice DOSING PHASE 15
1511 22 Nov 10 Terminal sacrifice DOSING PHASE 15

Group 2 – 50,000 ppm

2501 22 Nov 10 Terminal sacrifice DOSING PHASE 15
2502 22 Nov 10 Terminal sacrifice DOSING PHASE 15
2503 22 Nov 10 Terminal sacrifice DOSING PHASE 15
2504 22 Nov 10 Terminal sacrifice DOSING PHASE 15
2505 22 Nov 10 Terminal sacrifice DOSING PHASE 15
2506 22 Nov 10 Terminal sacrifice DOSING PHASE 15

Group 3 – 100,000 ppm

3501 22 Nov 10 Terminal sacrifice DOSING PHASE 15
3502 22 Nov 10 Terminal sacrifice DOSING PHASE 15
3503 22 Nov 10 Terminal sacrifice DOSING PHASE 15
3504 22 Nov 10 Terminal sacrifice DOSING PHASE 15
3505 22 Nov 10 Terminal sacrifice DOSING PHASE 15
3506 22 Nov 10 Terminal sacrifice DOSING PHASE 15
3507 22 Nov 10 Terminal sacrifice DOSING PHASE 15
3508 22 Nov 10 Terminal sacrifice DOSING PHASE 15
3509 22 Nov 10 Terminal sacrifice DOSING PHASE 15
3510 22 Nov 10 Terminal sacrifice DOSING PHASE 15
3511 22 Nov 10 Terminal sacrifice DOSING PHASE 15

a First day of each phase defined as Day 1. Day of exposure defined as Dosing Day 1

### Huntingdon Life Sciences 10-2226 Page 86
MA-252E-93-23 Final Report

Individual Daily Observations
Preface Appendix D

Cage-side observations were performed twice daily (once in the morning and once in the afternoon) for mortality, morbidity and signs of severe toxicity. Food consumption was also visually checked each day as part of the cage-side observations, and any decrease in food consumption was recorded. On the day of exposure, animals were observed before and after exposure. For summarization purposes, only time-points with animals exhibiting abnormal findings are presented.

Key to Abbreviations:

AM = Morning (Pre-exposure on the day of exposure) Observation PM = Afternoon (Post-exposure on the day of exposure) ObservationHuntingdon Life Sciences10-2226Page 87MA-252E-93-23FinalReportPresumed PregnantFemalesIndividual Daily Observations - Phase 2Appendix DPhase PRE PH2AnimalDay 2 3 4 5 6 7 8 9 11 3 4 7 10Number ObservationsSession AM AM AM AM AM AM AM AM AM AM AM AM AMGroup 1 - 0 ppm1501 Gastrointestinal, decreased fecal volumePMiscellaneous, decreased food consumptionP P1502 Gastrointestinal, decreased fecal volumeP PMiscellaneous, decreased food consumptionP P1503 Gastrointestinal, decreased fecal volumeP1505 Gastrointestinal, decreased fecal volumePGroup 2 - 50,000 ppm2503 Gastrointestinal, no stoolP2504 Gastrointestinal, decreased fecal volumePMiscellaneous, decreased food consumptionP2506 Gastrointestinal, decreased fecal volumeP P P P P P PMiscellaneous, decreased food consumptionP P P P PP = presentHuntingdon Life Sciences10-2226Page 88MA-252E-93-23FinalReportPresumed PregnantFemalesIndividual Daily Observations - Phase 2Appendix DPhase PRE PH2AnimalDay 2 3 4 5 6 7 8 9 11 3 4 7 10Number ObservationsSession AM AM AM AM AM AM AM AM AM AM AM AM AMGroup 3 - 100,000 ppm3501 Miscellaneous, decreased food consumptionP P P3502 Gastrointestinal, decreased fecal volumeP PMiscellaneous, decreased food consumptionP3503 Miscellaneous, decreased food consumptionP3504 Miscellaneous, decreased food consumptionP3505 Gastrointestinal, decreased fecal volumeP3506 Gastrointestinal, decreased fecal volumeP P PMiscellaneous, decreased food consumptionPP = presentHuntingdon Life Sciences10-2226Page 89MA-252E-93-23FinalReportNon-PregnantFemalesIndividual Daily Observations - Phase 2Appendix DPhase PREAnimalDay 3 4 5 6 7 7 8 8 9 9 10 10 11 11Number ObservationsSession AM AM AM AM AM PM AM PM AM PM AM PM AM PMGroup 1 - 0 ppm1507 Gastrointestinal, decreased fecal volumePGastrointestinal, no stoolP1509 Gastrointestinal, decreased fecal volumePMiscellaneous, decreased food consumptionP1510 Gastrointestinal, no stoolP P PMiscellaneous, decreased food consumptionP P P1511 Gastrointestinal, decreased fecal volumeP PGroup 3 - 100,000 ppm3508 Gastrointestinal, decreased fecal volumeP P PMiscellaneous, decreased food consumptionP3509 Gastrointestinal, decreased fecal volumePGastrointestinal, no stoolPMiscellaneous, decreased food consumptionP3510 Gastrointestinal, decreased fecal volumePP = presentHuntingdon Life Sciences10-2226Page 90MA-252E-93-23FinalReportNon-PregnantFemalesIndividual Daily Observations - Phase 2Appendix DPhase PREAnimalDay 3 4 5 6 7 7 8 8 9 9 10 10 11 11Number ObservationsSession AM AM AM AM AM PM AM PM AM PM AM PM AM PMGroup 3 - 100,000 ppm3511 Gastrointestinal, decreased fecal volumePP = presentHuntingdon Life Sciences10-2226Page 91MA-252E-93-23FinalReportNon-PregnantFemalesIndividual Daily Observations - Phase 2Appendix DPhase PRE PH2AnimalDay 12 12 13 1 2 4 5 6 7 8 10Number ObservationsSession AM PM AM PM AM AM AM AM AM AM AMGroup 1 - 0 ppm1507 Gastrointestinal, decreased fecal volumePGroup 3 - 100,000 ppm3508 Gastrointestinal, decreased fecal volumePP = presentHuntingdon Life Sciences10-2226Page 92MA-252E-93-23FinalReportMalesIndividual Daily Observations - Phase 2Appendix DPhase PREAnimalDay 3 4 5 6 7 7 8 8 9 9 10 10 11 11Number ObservationsSession AM AM AM AM AM PM AM PM AM PM AM PM AM PMGroup 1 - 0 ppm1002 Dermal general, stains on fur (head/neck), snout11005 Gastrointestinal, decreased fecal volumeP PMiscellaneous, decreased food consumptionP PGroup 2 - 50,000 ppm2003 Gastrointestinal, decreased fecal volumePMiscellaneous, decreased food consumptionP2004 General appearance, thinP P P P P P P PDermal general, ano-genital staining1 1 1 1 1 1 1 1 1 1 1Gastrointestinal, decreased fecal volumeP P P P PGastrointestinal, no stoolP P PMiscellaneous, decreased food consumptionP P P P P P2005 Dermal general, ano-genital staining1 1 1Gastrointestinal, unformed stoolPGastrointestinal, decreased fecal volumeP P P PGastrointestinal, no stoolP PMiscellaneous, decreased food consumptionP P P P P PP = present, 1 = slightHuntingdon Life Sciences10-2226Page 93MA-252E-93-23FinalReportMalesIndividual Daily Observations - Phase 2Appendix DPhase PREAnimalDay 3 4 5 6 7 7 8 8 9 9 10 10 11 11Number ObservationsSession AM AM AM AM AM PM AM PM AM PM AM PM AM PMGroup 3 - 100,000 ppm3002 Gastrointestinal, decreased fecal volumePMiscellaneous, decreased food consumptionP3005 Gastrointestinal, decreased fecal volumePP = presentHuntingdon Life Sciences10-2226Page 94MA-252E-93-23FinalReportMalesIndividual Daily Observations - Phase 2Appendix DPhase PRE PH2AnimalDay 12 12 13 1 2 4 5 6 7 8 10Number ObservationsSession AM PM AM PM AM AM AM AM AM AM AMGroup 1 - 0 ppm1004 Gastrointestinal, decreased fecal volumeP1005 Gastrointestinal, decreased fecal volumePMiscellaneous, decreased food consumptionPGroup 2 - 50,000 ppm2004 General appearance, thinP P P PDermal general, ano-genital staining1 1 1 1 12005 Gastrointestinal, no stoolPMiscellaneous, decreased food consumptionPGroup 3 - 100,000 ppm3001 Dermal general, ano-genital staining2Gastrointestinal, unformed stoolP P PGastrointestinal, decreased fecal volumeP PGastrointestinal, no stoolPMiscellaneous, decreased food consumptionP P PP = present, 1 = slight, 2 = moderateHuntingdon Life Sciences10-2226Page 95MA-252E-93-23FinalReportMalesIndividual Daily Observations - Phase 2Appendix DPhase PRE PH2AnimalDay 12 12 13 1 2 4 5 6 7 8 10Number ObservationsSession AM PM AM PM AM AM AM AM AM AM AMGroup 3 - 100,000 ppm3002 Dermal general, ano-genital staining1Dermal general, stains on fur (head/neck), head1Dermal general, stains on fur (torso), dorsal1Miscellaneous, decreased food consumptionP3003 Gastrointestinal, decreased fecal volumePP = present, 1 = slight

### Huntingdon Life Sciences 10-2226 Page 96
MA-252E-93-23 Final Report

Individual Physical Examination Findings
Preface Appendix E

Detailed physical examinations were pe rformed for all animals on study once daily during the stabilization (pretest) period a nd once daily (post exposure on the day of exposure) during the dosing phase. For summ arization purposes, descriptive comments [i.e., color of stains on fur and/or ano-genital area] are not presented in this table. These
data are contained in the study raw data if needed.Huntingdon Life Sciences10-2226Page 97MA-252E-93-23FinalReportPresumed PregnantFemales IndividualPhysicalExamination Findings - Phase 2Appendix EAnimal Phase PRE PH2Number ObservationsDay 1 2 3 4 5 6 7 8 9 10 1 2 3 4 5Group 1 - 0 ppm1501 Within normal limitsP P P P PDermal general, scab(s), ear(s)P P P P P P P P PGastrointestinal, decreased fecal volumeP1502 Within normal limitsP P P P P P P P P P P PDermal general, scab(s), ear(s)P P PGastrointestinal, decreased fecal volumePMiscellaneous, decreased food consumptionP1503 Within normal limitsP P P P P P P P P P P P P PGastrointestinal, decreased fecal volumeP1504 Within normal limitsP P P P P P P P P P P P P P P1505 Within normal limitsP P P P P P P P P P P P P P P1506 Within normal limitsP P P P P P P P P P P P P P PGroup 2 – 50,000 ppm2501 Within normal limitsP P P P P P P P P P P P P P PP = presentHuntingdon Life Sciences10-2226Page 98MA-252E-93-23FinalReportPresumed PregnantFemales IndividualPhysicalExamination Findings - Phase 2Appendix EAnimal Phase PRE PH2Number ObservationsDay 1 2 3 4 5 6 7 8 9 10 1 2 3 4 5Group 2 – 50,000 ppm2502 Within normal limitsP P P P P P P P P P P P P P P2503 Within normal limitsP P P P P P P P P P P P P P P2504 Within normal limitsP P P P P P P P P P P P P P P2505 Within normal limitsP P P P P P P P P P P PDermal general, ano-genital staining1 1 12506 Within normal limitsP P P P P P P P P PGastrointestinal, decreased fecal volumeP P P P PMiscellaneous, decreased food consumptionP P P PGroup 3 – 100,000 ppm3501 Within normal limitsP P P P P P P P P P P P P PMiscellaneous, decreased food consumptionPP = present, 1 = slightHuntingdon Life Sciences10-2226Page 99MA-252E-93-23FinalReportPresumed PregnantFemales IndividualPhysicalExamination Findings - Phase 2Appendix EAnimal Phase PRE PH2Number ObservationsDay 1 2 3 4 5 6 7 8 9 10 1 2 3 4 5Group 3 – 100,000 ppm3502 Within normal limitsP P P P P P P P P PDermal general, scab(s), ear(s)P P PGastrointestinal, decreased fecal volumeP PMiscellaneous, decreased food consumptionP3503 Within normal limitsP P P P P P P P P P P P P P P3504 Within normal limitsP P P P P P P P P P P P P P P3505 Within normal limitsP P P P P P P P P P P P P PGastrointestinal, decreased fecal volumeP3506 Within normal limitsP P P P P P P P P P P P PGastrointestinal, decreased fecal volumeP PP = presentHuntingdon Life Sciences10-2226Page 100MA-252E-93-23FinalReportPresumed PregnantFemales IndividualPhysicalExamination Findings - Phase 2Appendix EAnimal Phase PH2Number ObservationsDay 6 7 8 9 10 11 12 13 14 15Group 1 - 0 ppm1501 Within normal limitsP P P P P P P PDermal general, scab(s), ear(s)PMiscellaneous, decreased food consumptionP1502 Within normal limitsP P P P P P P P P P1503 Within normal limitsP P P P P P P P P P1504 Within normal limitsP P P P P P P P PGastrointestinal, decreased fecal volumeP1505 Within normal limitsP P P P P P P P P P1506 Within normal limitsP P P P P P PDermal general, ano-genital staining1 1 1Group 2 - 50,000 ppm2501 Within normal limitsP P P P P PDermal general, ano-genital staining1 1 1Gastrointestinal, decreased fecal volumePP = present, 1 = slightHuntingdon Life Sciences10-2226Page 101MA-252E-93-23FinalReportPresumed PregnantFemales IndividualPhysicalExamination Findings - Phase 2Appendix EAnimal Phase PH2Number ObservationsDay 6 7 8 9 10 11 12 13 14 15Group 2 - 50,000 ppm2502 Within normal limitsP P P P P P P P P P2503 Within normal limitsP P P P P P P P P P2504 Within normal limitsP P P P P P P P P P2505 Within normal limitsP P P P P P P P P P2506 Within normal limitsP P P P P P P P P PGroup 3 - 100,000 ppm3501 Within normal limitsP P P P P P P P PGastrointestinal, decreased fecal volumePMiscellaneous, decreased food consumptionP3502 Dermal general, ano-genital staining2 2 2Dermal general, scab(s), ear(s)P P P P P P P P P P3503 Within normal limitsP P P P P P P P P PP = present, 2 = moderateHuntingdon Life Sciences10-2226Page 102MA-252E-93-23FinalReportPresumed PregnantFemales IndividualPhysicalExamination Findings - Phase 2Appendix EAnimal Phase PH2Number ObservationsDay 6 7 8 9 10 11 12 13 14 15Group 3 - 100,000 ppm3504 Within normal limitsP P P P P P P P P P3505 Within normal limitsP P P P P P PDermal general, ano-genital staining1 1 13506 Within normal limitsP P P P P P P P P PP = present, 1 = slightHuntingdon Life Sciences10-2226Page 103MA-252E-93-23FinalReportNon-PregnantFemales IndividualPhysicalExamination Findings - Phase 2Appendix EAnimal Phase PRE PH2Number ObservationsDay 1 2 3 4 5 6 7 8 9 10 1 2 3Group 1 - 0 ppm1507 Within normal limitsP P P P P P P PDermal general, scab(s), ear(s)P P P PGastrointestinal, decreased fecal volumeP1508 Within normal limitsP P P P P P P P P P P P P1509 Within normal limitsP P P P P PDermal general, scab(s), ear(s)P P P P P P P1510 Within normal limitsP P P P P P P P P PGastrointestinal, no stoolP P PMiscellaneous, decreased food consumptionP P P1511 Within normal limitsP P P P P P P P P P P PGastrointestinal, decreased fecal volumePGroup 3 - 100,000 ppm3507 Within normal limitsP P P P P P P P P P P P PP = presentHuntingdon Life Sciences10-2226Page 104MA-252E-93-23FinalReportNon-PregnantFemales IndividualPhysicalExamination Findings - Phase 2Appendix EAnimal Phase PRE PH2Number ObservationsDay 1 2 3 4 5 6 7 8 9 10 1 2 3Group 3 - 100,000 ppm3508 Within normal limitsP P P P P P P P P PDermal general, scab(s), ear(s)P PGastrointestinal, decreased fecal volumeP3509 Within normal limitsP P P P P P P P P P P P P3510 Within normal limitsP P P P P P P P P P P P P3511 Within normal limitsP P P P P P P P P P PDermal general, alopecia (head/neck), head3 3P = present, 3 = extremeHuntingdon Life Sciences10-2226Page 105MA-252E-93-23FinalReportNon-PregnantFemales IndividualPhysicalExamination Findings - Phase 2Appendix EAnimal Phase PH2Number ObservationsDay 4 5 6 7 8 9 10 11 12 13 14 15Group 1 - 0 ppm1507 Within normal limitsP P P P P P P P P P P P1508 Within normal limitsP P P P P P P P P P P P1509 Within normal limitsP P P P P PDermal general, scab(s), ear(s)P P P P P P1510 Within normal limitsP P P P P P P P P P P P1511 Within normal limitsP P P P P P P P P P P PGroup 3 - 100,000 ppm3507 Within normal limitsP P P P P P P P P P P P3508 Within normal limitsP P P P P P P P P P P P3509 Within normal limitsP P P P P P P P P P P P3510 Within normal limitsP P P P P P P P P P P PP = presentHuntingdon Life Sciences10-2226Page 106MA-252E-93-23FinalReportNon-PregnantFemales IndividualPhysicalExamination Findings - Phase 2Appendix EAnimal Phase PH2Number ObservationsDay 4 5 6 7 8 9 10 11 12 13 14 15Group 3 - 100,000 ppm3511 Within normal limitsP P P P P P P P P P P PP = presentHuntingdon Life Sciences10-2226Page 107MA-252E-93-23FinalReportMalesIndividual Physical Examination Findings - Phase 2Appendix EAnimal Phase PRE PH2Number ObservationsDay 1 2 3 4 5 6 7 8 9 10 11 12 1 2 3Group 1 - 0 ppm1001 Dermal general, ano-genital staining1 1 1 1 1 1 1 1 1 1 1 1 1 1 11002 Within normal limitsP P P PDermal general, stains on fur (head/neck), snout1 1 1 1 1 1 1 1Dermal general, scab(s), ear(s)P P P P1003 Within normal limitsP P P P P P P P P P P P P P P1004 Within normal limitsP P P P P P PDermal general, stains on fur (torso), ventral1 1 1 1 1 1 1 11005 Within normal limitsP P P PDermal general, alopecia (torso)2 2 2Dermal general, stains on fur (torso), ventral1 1 1 1 1 1 1 1Group 2 - 50,000 ppm2001 Dermal general, scab(s), ear(s)P P P P P P P P P P P P P P POral/buccal, incisor(s) broken/missingP P2002 Dermal general, stains on fur (head/neck), ear(s)1 1 1 1 1 1 1 1 1 1 1 1 1 1 1P = present, 1 = slight, 2 = moderateHuntingdon Life Sciences10-2226Page 108MA-252E-93-23FinalReportMalesIndividual Physical Examination Findings - Phase 2Appendix EAnimal Phase PRE PH2Number ObservationsDay 1 2 3 4 5 6 7 8 9 10 11 12 1 2 3Group 2 - 50,000 ppm2003 Dermal general, alopecia (head/neck)2 2 2 2 2 2 2 2 2 2Dermal general, ano-genital staining1 1 1 1 1 1 1 1 1 1 1 1 1 1 1Dermal general, stains on fur (head/neck), head1 1 1 1 1 1 1 1 1 1 1 1 1 1 1Dermal general, stains on fur (torso), dorsal1 1 1 1 1 1 1 1 1 1 1 1 1 1 1Gastrointestinal, decreased fecal volumePMiscellaneous, decreased food consumptionP2004 General appearance, thinP P P P P PDermal general, ano-genital staining1 1 1 1 1 1 1 1 1 1 1 1 1 1 1Gastrointestinal, decreased fecal volumeP P P P PMiscellaneous, decreased food consumptionP P P P2005 Within normal limitsP P P P P P P P P PDermal general, ano-genital staining1 1 1 1 1Gastrointestinal, unformed stoolPGastrointestinal, decreased fecal volumeP P PMiscellaneous, decreased food consumptionP PGroup 3 - 100,000 ppm3001 Within normal limitsP P P P P P P P P P P P P P PP = present, 1 = slight, 2 = moderateHuntingdon Life Sciences10-2226Page 109MA-252E-93-23FinalReportMalesIndividual Physical Examination Findings - Phase 2Appendix EAnimal Phase PRE PH2Number ObservationsDay 1 2 3 4 5 6 7 8 9 10 11 12 1 2 3Group 3 - 100,000 ppm3002 Dermal general, ano-genital staining1 1 1 1 1 1 1 1 1 1 1 1 1 1 1Dermal general, stains on fur (head/neck), head1 1 1 1 1 1 1 1 1 1 1 1 1 1 1Dermal general, stains on fur (torso), dorsal1 1 1 1 1 1 2 2 2 2 2 1 1 1 1Gastrointestinal, no stoolPMiscellaneous, decreased food consumptionP3003 Within normal limitsP P P P P P P P P P P P P P P3004 Within normal limitsP P P P P P P P P P P P PDermal general, scab(s), ear(s)P P3005 Within normal limitsP P P P P P P P P P P P P PGastrointestinal, decreased fecal volumePP = present, 1 = slight, 2 = moderateHuntingdon Life Sciences10-2226Page 110MA-252E-93-23FinalReportMalesIndividual Physical Examination Findings - Phase 2Appendix EAnimal Phase PH2Number ObservationsDay 4 5 6 7 8 9 10 11 12 13 14 15Group 1 - 0 ppm1001 Dermal general, ano-genital staining2 2 2 2 2 1 1 1 2 2 1 11002 Within normal limitsP P P P P P P P P P P P1003 Within normal limitsP P P P P P P PDermal general, alopecia (head/neck)2 2 2 21004 Within normal limitsP P P P P P P PDermal general, ano-genital staining1 1 1 11005 Within normal limitsP P P P P P P P P P PDermal general, alopecia (torso)2Group 2 - 50,000 ppm2001 Dermal general, scab(s), ear(s)P P P P P P P P P P P P2002 Within normal limitsP P P P P P PDermal general, stains on fur (head/neck), ear(s) 1 1 1 1 1P = present, 1 = slight, 2 = moderateHuntingdon Life Sciences10-2226Page 111MA-252E-93-23FinalReportMalesIndividual Physical Examination Findings - Phase 2Appendix EAnimal Phase PH2Number ObservationsDay 4 5 6 7 8 9 10 11 12 13 14 15Group 2 - 50,000 ppm2003 Dermal general, ano-genital staining1 1 1 1 1 1 1 1 1 1 1 1Dermal general, stains on fur (head/neck), head1 1 1 1 1 1 1 1 1 1Dermal general, stains on fur (torso), dorsal1 1 1 1 1 1 1 1 1 1 1 12004 Dermal general, ano-genital staining1 1 1 1 1 1 1 1 1 1 1 12005 Within normal limitsP P P P P P P P P P P PGroup 3 - 100,000 ppm3001 Within normal limitsPDermal general, ano-genital staining2 2 2 2 2 2 2 2 1 1Gastrointestinal, unformed stoolPGastrointestinal, no stoolPMiscellaneous, decreased food consumptionP3002 Dermal general, ano-genital staining1 1 1 1 1 1 1 1 1 1 1 1Dermal general, stains on fur (head/neck), head1 1 1 1 1Dermal general, stains on fur (torso), dorsal1 1 1 1 1 1 1 1 1 1 1 13003 Within normal limitsP P P P P P P P P P P PP = present, 1 = slight, 2 = moderateHuntingdon Life Sciences10-2226Page 112MA-252E-93-23FinalReportMalesIndividual Physical Examination Findings - Phase 2Appendix EAnimal Phase PH2Number ObservationsDay 4 5 6 7 8 9 10 11 12 13 14 15Group 3 - 100,000 ppm3004 Within normal limitsP P P P P P P P P P P P3005 Within normal limitsP P P P P P P P P P P PP = presentHuntingdon Life Sciences 10-2226 Page 113
MA-252E-93-23 Final Report

Presumed Pregnant Individual Body Weights Females (grams) Appendix F

Animal Phase PH2 Number Day 1 3 5 8 15

Group 1 - 0 ppm

1501 3435.3 3452.7 3492.8 3559.1 3647.9
1502 NP 3323.9 3331.6 3333.4 3521.1 3521.7
1503 3446.7 3549.0 3502.4 3644.2 3664.2
1504 3338.1 3423.2 3464.5 3540.2 3732.9
1505 3450.9 3449.6 3420.0 3477.0 3430.7
1506 3640.3 3647.2 3580.4 3758.6 3893.2

Mean 3439.2 3475.6 3465.6 3583.4 3648.4
SD 113.28 109.10 83.46 102.03 161.72
N 6 6 6 6 6

Group 2 – 50,000 ppm

2501 3521.3 3555.2 3535.5 3594.5 3755.7
2502 3656.8 3692.4 3627.6 3806.3 3883.2
2503 3342.8 3337.8 3387.6 3475.3 3449.6
2504 3427.7 3555.0 3601.9 3618.8 3717.6
2505 3652.7 3734.6 3708.0 3742.9 3751.2
2506 3520.5 3633.5 3585.6 3746.5 3908.2

Mean 3520.3 3584.8 3574.4 3664.1 3744.3
SD 123.49 140.83 107.67 123.09 163.64
N 6 6 6 6 6

Group 3 – 100,000 ppm

3501 3135.0 3209.1 3201.2 3267.3 3432.1
3502 3699.3 3770.8 3754.4 3891.9 4037.6
3503 3303.1 3406.1 3446.0 3522.7 3570.8
3504 3381.2 3442.7 3452.1 3653.1 3636.7
3505 3606.0 3727.4 3710.6 3863.2 3972.3
3506 NP 3118.5 3160.9 3139.7 3207.9 3257.8

Mean 3373.9 3452.8 3450.7 3567.7 3651.2
SD 239.63 254.33 252.50 290.32 303.93
N 6 6 6 6 6

NP - Non-PregnantHuntingdon Life Sciences 10-2226 Page 114
MA-252E-93-23 Final Report

Non-Pregnant Individual Body Weights Females (grams) Appendix F

Animal Phase PH2 Number Day 1 3 5 8 15

Group 1 - 0 ppm

1507 3482.9 3333.4 3423.8 3470.2 3549.6
1508 3101.3 3081.6 3083.8 3257.8 3273.8
1509 3547.5 3547.1 3561.0 3699.1 3792.6
1510 3159.2 3161.9 3166.0 3262.2 3304.7
1511 3138.8 3073.3 3192.5 3200.7 3346.1

Mean 3285.9 3239.5 3285.4 3378.0 3453.4
SD 211.55 201.25 199.15 206.70 218.06

N 5 5 5 5 5

Group 3 – 100,000 ppm

3507 3088.9 3039.8 3028.9 3064.5 3220.1
3508 3341.8 3337.2 3283.4 3429.5 3506.5
3509 3283.6 3243.9 3193.5 3338.6 3421.9
3510 3414.2 3386.5 3392.9 3507.7 3539.9
3511 3023.8 3032.9 2999.1 3032.7 3154.6

Mean 3230.5 3208.1 3179.6 3274.6 3368.6
SD 167.13 164.92 167.15 215.11 172.52

N 5 5 5 5 5Huntingdon Life Sciences 10-2226 Page 115
MA-252E-93-23 Final Report

Individual Body Weights Males (grams) Appendix F

Animal Phase PH2 Number Day 1 3 5 8 15

Group 1 - 0 ppm

1001 3016.0 2949.5 3031.3 3074.2 3116.0
1002 2830.6 2815.7 2930.3 2908.4 3028.1
1003 3326.6 3269.6 3310.3 3263.4 3373.8
1004 2829.7 2874.3 2923.6 2909.0 3056.9
1005 3354.1 3293.7 3267.2 3285.2 3349.1

Mean 3071.4 3040.6 3092.5 3088.0 3184.8
SD 257.16 225.30 184.76 183.13 164.59

N 5 5 5 5 5

Group 2 – 50,000 ppm

2001 2951.4 2952.7 3034.7 3054.8 3168.2
2002 2849.3 2871.0 2972.4 2897.8 3070.9
2003 3020.3 3044.2 3149.6 3160.2 3286.7
2004 2621.6 2689.6 2716.4 2768.3 2861.9
2005 2730.3 2970.8 3086.0 3079.5 3172.9

Mean 2834.6 2905.7 2991.8 2992.1 3112.1
SD 161.70 135.58 167.21 157.17 159.39

N 5 5 5 5 5

Group 3 – 100,000 ppm

3001 3676.6 3646.7 3490.6 3435.5 3708.6
3002 2749.8 2739.4 2772.0 2837.8 2942.2
3003 3052.7 3075.7 3115.1 3178.2 3214.1
3004 2900.8 2943.7 2940.0 2985.8 3131.1
3005 3006.7 3040.5 3096.5 3145.2 3224.3

Mean 3077.3 3089.2 3082.8 3116.5 3244.1
SD 354.67 337.97 266.70 224.25 283.27

N 5 5 5 5 5Huntingdon Life Sciences 10-2226 Page 116
MA-252E-93-23 Final Report

Presumed Pregnant Individual Body Weight Change from Baseline Females (grams) Appendix G

Animal Phase PH2 Number Day 3 5 8 15

Group 1 - 0 ppm

1501 17.4 57.5 123.8 212.6
1502 NP 7.7 9.5 197.2 197.8
1503 102.3 55.7 197.5 217.5
1504 85.1 126.4 202.1 394.8
1505 -1.3 -30.9 26.1 -20.2
1506 6.9 -59.9 118.3 252.9

Mean 36.3 26.4 144.2 209.2
SD 45.15 67.59 69.34 133.56
N 6 6 6 6

Group 2 – 50,000 ppm

2501 33.9 14.2 73.2 234.4
2502 35.6 -29.2 149.5 226.4
2503 -5.0 44.8 132.5 106.8
2504 127.3 174.2 191.1 289.9
2505 81.9 55.3 90.2 98.5
2506 113.0 65.1 226.0 387.7

Mean 64.4 54.1 143.7 224.0
SD 51.38 68.06 58.33 110.22
N 6 6 6 6

Group 3 – 100,000 ppm

3501 74.1 66.2 132.3 297.1
3502 71.5 55.1 192.6 338.3
3503 103.0 142.9 219.6 267.7
3504 61.5 70.9 271.9 255.5
3505 121.4 104.6 257.2 366.3
3506 NP 42.4 21.2 89.4 139.3

Mean 79.0 76.8 193.8 277.4
SD 28.64 42.07 71.37 79.56
N 6 6 6 6

NP - Non-PregnantHuntingdon Life Sciences 10-2226 Page 117
MA-252E-93-23 Final Report

Non-Pregnant Individual Body Weight Change from Baseline Females (grams) Appendix G

Animal Phase PH2 Number Day 3 5 8 15

Group 1 - 0 ppm

1507 -149.5 -59.1 -12.7 66.7
1508 -19.7 -17.5 156.5 172.5
1509 -0.4 13.5 151.6 245.1
1510 2.7 6.8 103.0 145.5
1511 -65.5 53.7 61.9 207.3

Mean -46.5 -0.5 92.1 167.4
SD 63.71 41.56 70.18 67.59

N 5 5 5 5

Group 3 – 100,000 ppm

3507 -49.1 -60.0 -24.4 131.2
3508 -4.6 -58.4 87.7 164.7
3509 -39.7 -90.1 55.0 138.3
3510 -27.7 -21.3 93.5 125.7
3511 9.1 -24.7 8.9 130.8

Mean -22.4 -50.9 44.1 138.1
SD 24.24 28.45 50.97 15.51

N 5 5 5 5Huntingdon Life Sciences 10-2226 Page 118
MA-252E-93-23 Final Report

Individual Body Weight Change from Baseline Males (grams) Appendix G

Animal Phase PH2 Number Day 3 5 8 15

Group 1 - 0 ppm

1001 -66.5 15.3 58.2 100.0
1002 -14.9 99.7 77.8 197.5
1003 -57.0 -16.3 -63.2 47.2
1004 44.6 93.9 79.3 227.2
1005 -60.4 -86.9 -68.9 -5.0

Mean -30.8 21.1 16.6 113.4
SD 46.84 78.38 75.97 98.24

N 5 5 5 5

Group 2 – 50,000 ppm

2001 1.3 83.3 103.4 216.8
2002 21.7 123.1 48.5 221.6
2003 23.9 129.3 139.9 266.4
2004 68.0 94.8 146.7 240.3
2005 240.5 355.7 349.2 442.6

Mean 71.1 157.2 157.5 277.5
SD 97.78 112.58 114.00 94.31

N 5 5 5 5

Group 3 – 100,000 ppm

3001 -29.9 -186.0 -241.1 32.0
3002 -10.4 22.2 88.0 192.4
3003 23.0 62.4 125.5 161.4
3004 42.9 39.2 85.0 230.3
3005 33.8 89.8 138.5 217.6

Mean 11.9 5.5 39.2 166.7
SD 30.86 110.04 158.39 79.79

N 5 5 5 5

### Huntingdon Life Sciences 10-2226 Page 119
MA-252E-93-23 Final Report

Individual Animal Gross and Microscopic Observations
Preface Appendix H

Table of Contents:

Anatomic Pathology Report............................................................................................. 120
Individual Animal Gross and Microscopic Observations................................................ 122 Pregnancy Status.............................................................................................................. 1 65

Corresponding exposure levels for each group were as follows:

Group 1 - 0 ppm Group 2 - 50,000 ppm Group 3 - 100,000 ppm

Note: Unless otherwise specified in a histopathology note, the organ/tissue examined was the required (routine) section.

### Huntingdon Life Sciences 10-2226 Page 120

MA-252E-93-23 Final Report

Anatomic Pathology Report Appendix H

1. PATHOLOGY
This study was designed to assess the acute toxicity and biotransformative effects of single 1 hour whole-body inhalation exposure of HFO-1234yf in pregnant and non-pregnant female rabb its and in male rabbits. A purpose of this study was to determine whether there is pregnancy-based or gender-based difference in sensitivity in rabbits with this test substance.

Unscheduled Decedents

There were no unscheduled deaths during any study phase. All animals survived to their respective scheduled sacrifices.

Terminal Sacrifice

1.1. MACROSCOPIC

In Phase 1, the presumed pregnant female was confirmed pregnant. In Phase 2, the presumed pregnant females were confirmed pregnant except 1 female in Group 1 (Animal No . 1502) and 1 female in Group 3 (Animal No. 3506).



### In Phase 2, there were no test substance-related macroscopic findings at the terminal sacrifi ces. Macroscopic findings were of low incidence and considered common incidental findings in the test species. This includes the finding of discolored lungs in a small number of females given control or test substance.

### Page 121Page 122Page 123Page 124Page 125Page 126Page 127Page 128Page 129Page 130Page 131Page 132Page 133Page 134Page 135Page 136Page 137Page 138Page 139Page 140Page 141Page 142Page 143Page 144Page 145Page 146Page 147Page 148Page 149Page 150Page 151Page 152Page 153Page 154Page 155Page 156Page 157Page 158Page 159Page 160Page 161Page 162Page 163Page 164

### Huntingdon Life Sciences 10-2226 Page 165
MA-252E-93-23 Final Report

Females Pregnancy Status of Presumed Pregnant Females
Phase 2 Appendix H

Animal
Number Pregnancy Status

Group 1 - 0 ppm

1501 Pregnant 1502 Non-Pregnant 1503 Pregnant 1504 Pregnant 1505 Pregnant 1506 Pregnant

Group 2 – 50,000 ppm

2501 Pregnant 2502 Pregnant 2503 Pregnant 2504 Pregnant 2505 Pregnant 2506 Pregnant

Group 3 – 100,000 ppm

3501 Pregnant 3502 Pregnant 3503 Pregnant 3504 Pregnant 3505 Pregnant 3506 Non-PregnantProf. W.Dekant; T.Schmidt: Biotransformation of 2,3,3,3

- tetrafluoropropene (1234yf) page 1 of 19



# Study Report

– Rabbit urine analysis –

Biotransformation and toxicokinetics of 2,3,3,3

- tetrafluoropropene

( HFO

- 1234yf) in male, pregnant and non

- pregnant female rabbits

Authors: Prof. Dr. Wolfgang Dekant,

Tobias Schmidt

Department of Toxicology

Univer sity of Würzburg

Sponsor: Honeywell International Inc.

P.O. Box 1057

Morristown, NJ 07962-1057, USA

Test facility: Institute of Toxicology

University of Würzburg

Versbacher Str. 9

97078 Würzburg, Germany Appendix I Page 166Prof. W.Dekant; T.Schmidt: Biotransformation of 2,3,3,3

- tetrafluoropropene (1234yf) page 2 of 19



# Signature page

I hereby declare that the work described in this report was performed under my

supervision, as Principal Investigator, and that the report provides a true and

accurate record of the results obtained. No unforeseen circumstances were observed

which might have affected the validity of the data and the quality and integrity of the

study.

Principal Investigator .............................. February 22, 2010

Prof. Dr. W. Dekant Date Page 167Prof. W.Dekant; T.Schmidt: Biotransformation of 2,3,3,3

- tetrafluoropropene (1234yf) page 3 of 19



# Responsible personnel

Principal Investigator Prof. Dr. W. Dekant

Tel. +49 (0) 931 201 48449

Email: dekant@toxi.uni

- wuerzburg.de

Technical coordination Tobias Schmidt Page 168Prof. W.Dekant; T.Schmidt: Biotransformation of 2,3,3,3

- tetrafluoropropene (1234yf) page 4 of 19



# Table of Content s

1 Summary

...................................................................................................... 5

2 Introduction

................................................................................................... 6

3 Material and Methods

................................................................................... 6

3 .1 Synthesis of N -acetyl- S -(3,3,3-trifluoro-2-hydroxypropanyl)- L -cys teine ....... 6

3 .2 19 

### F- NMR method

.......................................................................................... 7

3 .3 LC/MS -MS method

....................................................................................... 7

4 Results

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

4 .1 Inhalation exposure and urine sampling

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

4 .2 19 

### F- NMR spectra.

........................................................................................ 10

4 .3 Urine levels of N -acetyl- S -(3,3,3-trifluoro-2-hydroxypropanyl)- L -cysteine

.. 13

5 Discussion

.................................................................................................. 17

References

................................................................................................ . 19 Page 169Prof. W.Dekant; T.Schmidt: Biotransformation of 2,3,3,3

- tetrafluoropropene (1234yf) page 5 of 19



# 1. Summ a ry

In order to examine a possible gender or pregnancy-based difference in HFO- 1234yf

metabolism in rabbits, male (n=5/group), non-pregnant female (n=5/group) and

pregnant female (n=6/group) New Zealand White rabbits were exposed to 50,000

ppm or 100,000 p pm of HFO

- 1234yf for one hour in an open dynamic chamber. Non

pregnant rabbits were not exposed to 50,000 ppm as this data had previously been

determined [1

- 2]

.

Urine analysis by 19 

### F-NMR indicate N -acetyl- S -(3,3,3-trifluoro-2-hydroxypropanyl)- L

cysteine and S -(3,3,3-trifluoro-2-hydroxypropanyl)-mercaptolactic acid as major

metabolites, representing more than 78 % of total 19 

### F related signals in all analyzed

urine samples. In contrast to earlier biotransformation analysis [1]

, S -(3,3,3-trifluoro2-hydroxypropanyl)-mercaptolactic acid was identified as predominant

biotransformation product represen ting more than 46 % of total 19 

### F related signals

(100,00 ppm, 0-48 hours).

Quan tification of the HFO- 1234yf -mercapturic acid isomers by LC/MS -MS s howed no

significant difference (P = 0.58, S tudent ’s t -test) in the mean recovery of this

metabolite between female (43.11 ± 22.35 μ mol) and pregnant female rabbits (50.47

± 19.72 μ mol) excreted in urine within 48 hours. More than 83% of recovered N

acetyl- S -(3,3,3-trifluoro-2-hydroxypropanyl)- L -cysteine in male and more than 93% in

non -pregnant female/pregnant female animals was excreted within 24 hours after

inhalation exposure (50,000 ppm and 100,000 ppm exposures), suggesting an

elimination half life of approximately 6 hours for HFO

- 1234yf -mercapturic acid.

Furthermore, th e t otal amount of metabolized HFO-1234yf was calculated to be less

than 0.1 % of received dose in all groups.

Lethality and clinical signs were not observed in the exposed animals. There is one

yet unidentified metabolite, 11.9 -17% in female rabbits and < 7.2% in male rabbits,

accordi ng to 19F -NMR signal intensity (signal intensity of the six main metabolites

was set to 100%). No further differences in urinary metabolite patterns were

obs erved between the different groups. Page 170Prof. W.Dekant; T.Schmidt: Biotransformation of 2,3,3,3

- tetrafluoropropene (1234yf) page 6 of 19



### The results suggest that lethality of HFO-1234yf in pregnant rabbits after inhalation

exposure is unlikely due to changes in biotransformation patterns or capacity of

pregnant rabbits.



# 2. Introduction

Despite repeated dose toxicity studies of HFO 1234yf in rodents with inhalation

NOAELS > 10,000 ppm and a rat developmental toxicity study with a NOAEL of

50,000 ppm, a developmental toxicity study in rabbits showed a mortality and

morbundity at exposure levels of 5,500 ppm and above. In contrast, n o lethality was

observed at exposure levels of 2,500 and 4,000 ppm of HFO

- 1234yf [3]

.
The higher sensitivity of rabbits, in comparison to rats, may be explained due to

biotransformation differences between the species

. However

, s tudies e xamining this

hypothesis did not find major variations. In r abbit s, rats and mice the predominant

biotransformation product of HFO

- 1234yf was determined as N -acetyl- S -(3,3,3-

trifluoro-2-hydroxypropanyl)- L -cysteine (approx. 44, 90 and 32% of total 19 

### F- NMR

signals) . W hereas S -(3,3,3-trifluoro-2-hydroxypropanyl)-mercaptolactic acid, 3,3,3

-

trifluoro- 1,2 -dihydroxypropane, the sulfoxides of the mercapturic and mercaptolactic

acid S -conjugates represented minor metabolites of HFO- 1234yf. The only species

differences observed, were the absence of 3,3,3-trifluorolactic acid, 3,3,3-

trifluoroacetic acid and 3,3,3-trifluoro-1-hydroxyacetone in rabbit urine [2]

.

To investigate whether there is a gender -based or pregnancy-based difference in

toxic response and biotransformation of HFO- 1234yf in rabbits, an acute inhalation

toxicity study was designed. Male (5 per group), non-pregnant female (5 per group)

and pregnant female (GD12 – 6 per group) rabbits were exposed to 50,000 ppm or

100,000 ppm of HFO -1234yf for one hour

. Non -pregnant rabbits were not exposed to

50,000 ppm as this data had previously been determined. U rine was collected for 48

hours in 12 hours intervals for biotransformation analysis. Page 171Prof. W.Dekant; T.Schmidt: Biotransformation of 2,3,3,3

- tetrafluoropropene (1234yf) page 7 of 19



# 3. Material and Methods

3 .1 Synthesis of N

- acetyl

- S

- (3,3,3

- trifluoro -2- hydroxypropanyl)

- L

cystei ne

3,3,3 -Trifluoro- 1,2 -epoxypropane was slowly added to a well stirred solution of N

acetyl- L -cysteine in ethanol/water (1/2 v/v) adjusted to pH > 9 with triethylamine . The

reaction mixture was maintained to 4 0 C during the reaction. After neutralizing by

addition of 2 N HCL and evaporation of the ethanol under reduced pressure, the

water phase was extracted with ethyl acetate [4] . For further purification, the reaction

products were precipitated as dicyclohexylammonium salts and recrystallized from

ethyl acetate.

Two diastereomers of N -acetyl- S -(3,3,3-trifluoro-2-hydroxypropanyl)-L-cysteine were

obtained in a ratio of 5:1 according to HPLC-separation with UV-detection at 225 nm.

Purity was determined > 96% based on on 1 

### H- NMR ( 500 MHz, [D 6 

### ]-DMSO): δ = 1.02

– 1.99 (m, 20 H, cyclohexane - CH 2 

### -), 1.82 (s, 3 H, - COC H 3 

### ), 2.60 (dd, J AB 

### = 13.6 Hz,

J AX 

### = 10.0 Hz, 1H, H A 

### ), 2.73 (dd, J BA 

### = 13.6 Hz, J BX 

### = 3.3 Hz, 1H, H B 

### ), 2.82 (dd, J AB 

### =

13.5 Hz, J AX 1 

### = 6.1 Hz, 1H, H A 

### ), 2.97 (dd, J BA 

### = 13.5 Hz, J BX1 

### = 5.1 Hz, 1 H, H B 

### ), 3.92-

3.00 (m, 2 H, cyclohexane - NC H-), 4.01 (m, 1 H,H X 

### ), 4.1 (m, 1 H, H X1 

### ), 7.58 (d, J =

7.25 Hz, 1H, N H COCH 3 

### ).



## 3 .2 19 

## F- NMR me thod

19 

### F-NMR spectra were recorded with a Bruker DRX 300 NMR spectrometer with a 5

mm fluoride probe operating at 282 MHz. 19 

### F chemical shifts were referenced to

external CFCl 3 

### . Spectra were recorded with a 30 0 pulse, a pulse length of 11 μ sec

and a cycle delay of 6 μ sec. The acquisition time was 2.9 sec and 10240 scans were

recorded to obtain a good signal to noise ratio (S/N). The 19 

### F spectra were acquired

with proton coupling and a spectral with of 100 ppm

. Before integration of peak

areas, an accurate baseline correction and spectra phasing was performed between -

70 and -85 ppm chemical shift.

Sample preparation

. After thawing at 4°C, 1 mL of a rabbit urine sample was

vortexed and then centrifuged for 10 minutes at 14,000 rpm and 4°C. An aliquot

( 720 μ L ) of the obtained supernatant was added to 80 μ L of deuterium oxide. No Page 172Prof. W.Dekant; T.Schmidt: Biotransformation of 2,3,3,3

- tetrafluoropropene (1234yf) page 8 of 19



### further sample workup was performed before 19 

### F-NMR analysis. NMR spectra were

analyzed with TopSpin 3.0 (Bruker).



## 3 .3 LC

- MS/MS me thod

– q uantification of N

- acetyl

- S

- (3,3,3

- trifluoro -2-

hydroxypropanyl)

- L

- cysteine

Rabbit urine was analyzed by LC -MS/MS using electrospray ionization. Processed

urine samples (see below) were separated on a Reprosil Pur C18 AQ HPLC column

(2 mm x 150 mm; 5 μ m ; M aisch, Ammerbuch, Germany) using an Agilent 1100

autosampler and an Agilent 1100 HPLC-pump (Agilent, Waldbronn, Germany).

Separation was performed by gradient elution with water containing 0.1% formic acid

(solvent A) and acetonitrile (solvent B) using the following conditions: 98% A, 2% B,

isocratic for 1 min, linear increase to 90% B within 5 min, followed by 90% B for 5

min, at a flow -rate of 300 μ L/min. The HPLC-system was directly coupled to a triple

stage quadrupole mass spectrometer (API 3000, Applied Biosystems, Darmstadt,

Germany). Analytes were detected in the negative-ion mode at a vaporizer

temperature of 400°C and a TurboIon ® 

### Spray voltage of -4.0 kV. Spectral data were

recorded with nitrogen as collision gas (CAD = 4) in the multiple reaction monitoring

(MRM) mode. Mass transitions monitored during the separation are listed in Table 1.

Table 1 : Mass spectrometric parameters (MS- MS -transitions, declustering potential and collision energy) used to quantify N -acetyl- S -(3,3,3-trifluoro-2- hydroxypro panyl)

- L -cysteine.

Substance Transition [m/z] Declustering potential [V] Collision energy [V]

274

- 145 Metabolite

N

- Acetyl

- S

- (3,3,3

- trifluoro

- 2

- hydroxypropanyl)

- L

- cysteine 274

- 75 - 26

- 30

278

- 149 Internal standard

N

- Acetyl

- S

- (2

- chloro

- 1,1,2

- trifluoroethyl)

- L

- cysteine 278

- 129 - 26

- 30

The following mass transitions were used for quantification: m/z 278-149 for the

internal standard N -acetyl- S -(2-chloro- 1,1,2 -trifluoroethyl)- L -cysteine, m/z 274-145 for

the analyt e N -acetyl- S -(3,3,3-trifluoro-2-hydroxypropanyl)- L -cysteine. Page 173Prof. W.Dekant; T.Schmidt: Biotransformation of 2,3,3,3

- tetrafluoropropene (1234yf) page 9 of 19



### Sample preparation

. After thawing at 4°C, 1 mL of a rabbit urine sample was

vortexed and then centrifuged for 10 minutes at 14,000 rpm and 4°C. The obtained

supernatants were diluted with water (1:10) and 10 μ L of the dilutions were added to

90 μ L of a 500pg/ μ L internal standard solution, vortexed, transferred into glass

autosampler vials, sealed and aliquots ( 10 μ L ) were analyzed by LC-MS/MS. If

sample values were outside the linear range of the calibration curve, they wer e

additiona lly diluted 100 or 1, 000 fold.

Quantification

. Quantification was based on the peak area of N -acetyl- S -(3,3,3-

trifluoro-2-hydroxypropanyl)- L -cysteine relative to the peak area of the internal

standard N -acetyl- S -(2-chloro- 1,1,2 -trifluoroethyl)- L -cysteine. Quantification was

based on calibration curves obtained after fortification of urine samples from

unexposed rabbits with the internal standard. Calibration curves were calculated from

eight to ten data points using Analyst 1.4.1 (Applied Biosystems). The R 2 

### -values of

the calibration curves were > 0.99. Limit of quantification (LOQ) for N -acetyl- S -(3,3,3-

trifluoro-2-hydroxypropanyl)- L -cysteine was 0.45 μ g/L (1.63 nmol/L) and l imit of

detection (LOD) was 0.14 μ g/ L (0.51 n mol/L) in urine. Standard deviations (m ean ±
SD) were calculated using MicrosoftExcel® spreadsheets and default setting.

Statistical analysis was performed by GraphPad Prism 5.



# 4 Results

4 .1 Inhalation ex posure and urine sampl ing

Single exposure study in rabbits (whole-body inhalation) and urine sampling was

conducted by Huntingdon Life Sciences (100 Mettlers Road, East Millstone, New

Jersey 08875 -2360).

Non -pregnant female, presumed pregnant female (GD 12) and male New Zealand

White Rabbits received a single whole-body inhalation exposure for 1 hour duration

with 2,3,3,3 -tetrafluoropropene (HFO-1234yf) at two different exposure

concentrations: Page 174Prof. W.Dekant; T.Schmidt: Biotransformation of 2,3,3,3

- tetrafluoropropene (1234yf) page 10 of 19



### Table 2 : Study design: exposure levels and number of animals.

Test Animals

Group Exposure

Level [ppm] Presumed

Pregnant

Females Non

- Pregnant

Females Males

Air control 0 6 5 5

Low 50, 000 6 0 5

High 100, 000 6 5 5

Urine was collected at approximately 12 hours before initiation of the inhalation

exposure and for approximately 48 hours after exposure, at approximately 12 hour

intervals from 5 males/gro up, up to 6 pregnant females/group and 5 non-pregnant

females /group. Urine was collected in ice chilled containers and volume was

measured for each interval. Obtained samples were then split into aliquots

(approximately 10 mL), frozen in methanol and dry ice and shipped to Germany on

dry ice.

Samples were received frozen and without damage at the Institute of Toxicology,

Versbacher Str. 9, 97078 Würzburg and stored at - 20 0 C until analysis.



## 4 .2 19 

## F- NMR – m etabolites identification

Biotransformation products of HFO-1234yf were identified by 19F -NMR spectrometry,

comparing the chemical shifts and F-H coupling constants with spectra of standard

substances already available in the I nstitute for a previous study [1]

.

Fig. 1 illustrates that there are no significant differences between the urine 19 

### Fspectra of female, pregnant female and male rabbits . A ll spectra show the same

major peaks with very similar relative intensities. Page 175Prof. W.Dekant; T.Schmidt: Biotransformation of 2,3,3,3

- tetrafluoropropene (1234yf) page 11 of 19



### Fig. 1 : 19 

### F-NMR spectra of (A) pregnant female, (B) female, male (C) rabbit urine (100,000pmm, 0-12h).

Signals found in spectra of different urine fractions (Fig. 2 and 3) were assigned to

3,3,3 -trifluoropropionic acid ( δ = -75.4 ppm, s) , 3,3,3 -trifluoro- 1,2 -dihydroxypropane ( δ

= - 77.2 ppm; d, J HF 

### = 7.4Hz)

, N -acetyl- S -(3,3,3-trifluoro-2-hydroxypropanyl)- L

cysteine ( δ = - 78.7 ppm; d, J HF 

### = 6.6Hz) and S -(3,3,3-trifluoro-2-hydroxypropanyl)-

mercaptolactic acid ( δ = - 78.9 ppm; d, J HF 

### = 6.9Hz). Beside these known metabolites

,
ther e were two unidentified metabolites present.

Fig. 2 : 19 

### F-NMR spectrum of 0- 12 h post inhalation exposure urine; resonances were assigned to (A) unidentified metabolite ( δ = - 77.1 ppm; d, J HF 

### = 7.2 Hz)

, (B) 3,3,3 -trifluoro- 1,2 -dihydroxyprop ane ( δ = - 77.2 pp m; d, J HF 

### = 7.4Hz) , ( C)
unidentified metabolite ( δ = - 78.4 ppm; d, J HF 

### = 6.8Hz) , ( D) N -acetyl- S -(3,3,3- trifluoro-2-hydroxypropanyl)- L -cysteine ( δ = - 78.7 ppm; d, J HF 

### = 6.6Hz), (E) S

- (3,3,3-trifluoro-2-hydroxypropanyl)-mercaptolactic acid ( δ = - 78.9 ppm; d, J HF 

### = 6.9Hz). Page 176Prof. W.Dekant; T.Schmidt: Biotransformation of 2,3,3,3

- tetrafluoropropene (1234yf) page 12 of 19



### Fig. 3 : 19 

### F-NMR spectrum of 36-48h post inhalation exposure urine; resonances were assigned to (A) 3,3,3 -trifluoropropionic acid ( δ = -75.4 ppm , s ), (B)
unidentified metabolite ( δ = - 78. 4 ppm; d, J HF 

### = 6. 8Hz), (C) N -acetyl- S -(3,3,3- trifluoro-2-hydroxypropanyl)- L -cysteine ( δ = - 78.7 ppm; d, J HF 

### = 6.6Hz)

, (D) S

- (3,3,3-trifluoro-2-hydroxypropanyl)-mercaptolactic acid ( δ = - 78.9 ppm; d, J HF 

### = 6.9Hz).

For further characterization of female, pregnant female and male rabbits metabolism

, relative peak areas (related to area of the signal of S -(3,3,3-trifluoro-2-

hydroxypropanyl)-mercaptolactic acid) of the six major metabolic products were

determined and compared between the different study groups. As shown in Fig. 4

, there were only minor differ ences in metabolic pattern between male and female

animals as well as between dose groups 50,000 ppm and 100,000 ppm but no

differences between non-pregnant female and pregnant female animals in all major

metabolites at all time intervals collected. Page 177Prof. W.Dekant; T.Schmidt: Biotransformation of 2,3,3,3

- tetrafluoropropene (1234yf) page 13 of 19



### Fig. 4 : Relative amount of metabolite excretion in 12 hour intervals for (one animal per group): (A) S -(3,3,3-trifluoro-2-hydroxypropanyl)-mercaptolactic acid, (B)
N

-acetyl- S -(3,3,3-trifluoro-2-hydroxypropanyl)- L -cysteine, (C) unidentified metabolite ( δ = -78.4 ppm), (D) 3,3,3 -trifluoro- 1,2

- dihy droxypropane, (E) unidentified metabolite ( δ = -77.1 ppm), (F) 3,3,3 -trifluoropropionic acid.



## 4 .3 LC

- MS/MS – u rine levels of N

- acetyl

- S

- (3,3,3

- trifluoro -2-

hydroxypropanyl)

- L

- cysteine

Urine concentrations of N -acetyl- S -(3,3,3-trifluoro-2-hydroxypropanyl)- L -cysteine

were determined in all samples received. Concentrations in all samples except one

(presumed pregnant female, 50.000 ppm group, 36-48 h collection interval) were well

above LOQ (0.45 μ g/L ). A typical chromatogram of a urine sample is shown in Fig. 5.

A single peak was obtained showing the typical mass transitions (qualifier 274- 75;

quantifier 274-145) at the expected retention time (8.1 min). Page 178Prof. W.Dekant; T.Schmidt: Biotransformation of 2,3,3,3

- tetrafluoropropene (1234yf) page 14 of 19



### Fig.5 : Quantifier: mass transition - 274

- 145 (A) ; qua lifier: mass transition 274- 75 (B). B A Page 179Prof. W.Dekant; T.Schmidt: Biotransformation of 2,3,3,3

- tetrafluoropropene (1234yf) page 15 of 19



### Table 3 : Excretion of N -acetyl- S -(3,3,3-trifluoro-2-hydroxypropanyl)- L -cysteine by time intervals ( μ mol); male (M), pregnant female (P), non-pregnant female (F).

Time
interval Group Animal 1 [ μ mol] Animal 2 [ μ mol] Animal 3 [ μ mol] Animal 4 [ μ mol] Animal 5 [ μ mol] Animal 6 [ μ mol] Sample
number Average
amount
excreted

[ μ mol] SDV

0

- 12 h M 50,000 ppm 70.93 57.56 46.23 64.58 29.49

- 5 53.76 16.37

0

- 12 h P 50,000 ppm 52.87 62.30 44.34 50.31 34.58 25.99 6 45.07 13.11

0

- 12 h M 1 00,000 ppm 96.80 34.10 32.07 74.63 80.17

- 5 63.55 29.00

0

- 12 h P 100,000 ppm

- * 31.89 1.49 22.88 34.13 9.18 5 19.91 14.89

0

- 12 h F 100,000 ppm 51.09 47.89 4.60 41.69 17.29

- 5 32.51 20.47

12

- 24 h M 50,000 ppm 5.07 12.45 11.35 9.82 5.37

- 5 8.81 3.41

12

- 24 h P 50,000 ppm 22.95 3.38 0.52 8.80 21.24 22.14 6 13.17 10.16

12

- 24 h M 100,000 ppm 25.33 17.56 6.52 8.67 13.19

- 5 14.25 7.51

12

- 24 h P 100,000 ppm 18.75 11.14 39.90 39.38 32.05 21.77 6 27.17 11.76

12

- 24 h F 100,000 ppm 11.40 2.62 5.12 9.70 11.61

- 5 8.09 4.02

24

- 36 h M 50,000 ppm 7.12 11.81 9.44 4.26 13.14

- 5 9.15 3.57

24

- 36 h P 50,000 ppm 3.00 0.76 0.02 1.45 6.42

- * 5 2.33 2.54

24

- 36 h M 100,000 ppm 10.01 2.04 3.79 2.03 12.02

- 5 5.98 4.71

24

- 36 h P 100,000 ppm 9.57 1.30 4.81 7.09 3.49

- * 5 5.25 3.20

24

- 36 h F 100,000 ppm 3.71 1.05 0.45

- * 2.83

- 4 2.01 1.52

36

- 48 h M 50,000 ppm 5.52 4.75 2.97 1.95 2.38

- 5 3.51 1.55

36

- 48 h P 50,000 ppm 1.92 0.46 < LOQ 0.53 1.83

- * 5 1.19 0.80

36

- 48 h M 100,000 ppm 5.88 0.03 2.13 1.72 3.31

- 5 2.61 2.17

36

- 48 h P 100,000 ppm 0.93 1.53 2.91 5.00 3.60

- * 5 2.79 1.63

36

- 48 h F 100,000 ppm 2.53 0.22 0.14 0.68 0.87

- 5 0.89 0.97

* no urine could be collected



### The time courses of urinary excretion of N -acetyl- S -(3,3,3-trifluoro-2-

hydroxyprop anyl)

- L -cysteine in rabbits are shown in Table 3 and Figure 6. Peak urine

concentrations were reached 12 h after termination of the exposures (mean values:

male 100,000 ppm

– 63.55 μmol, male 50,000 ppm

– 53.76 μmol, non -pregnant

female 100,000 ppm

– 32.51 μmol, pregnant female 50,000 ppm – 45.07 μmol)

except for the study group pregnant females 100,000 ppm. For this group, the peak

level was reached 24 h after end of exposure (mean value: 27.17 μmol).The Page 180Prof. W.Dekant; T.Schmidt: Biotransformation of 2,3,3,3

- tetrafluoropropene (1234yf) page 16 of 19



### met abolite elimination followed 1 st 

### order kinetics w ith a half

- life of app rox. 6 hours in

all five groups. Hence, N -acetyl- S -(3,3,3-trifluoro-2-hydroxypropanyl)- L -cysteine is

rapidly excreted with >93% recovered within 24 hours in female and pregnant female

rabbits and >83% within 24 hours in male rabbi ts f ollowing the inhalation exposure.

E limination was slower in the 100,000 ppm pregnant female group (39,5% of total

metabolite excretion) within the first 12 hours compared to all other groups (mean

value: 73,3% of total metabolite excretion).

Fig. 6 : Mea n concentrations of N -acetyl- S -(3,3,3-trifluoro-2-hydroxypropanyl)- L

cysteine (mean ± SD) in urine samples from male (100.000 ppm and 50.000 ppm), pregnant female (100.000 ppm and 50.000 ppm ) and non-pregnant female (100.000 ppm) after whole- body inhalat ion of 100.000 or 50.000 pm 1234yf for one hour.

The total urinary recovery of N -acetyl- S -(3,3,3-trifluoro-2-hydroxypropanyl)- L -cysteine

within 48 hours after exposure is illustrated in Figure 7

. Recovery in males within 48

hours (mean values: male 100,000 ppm

– 75.24 μ mol; male 50,000 ppm

– 86.40

μ mol) was higher as compared to females (mean values: pregnant 100,000 ppm –

50. 47 μ mol; pregnant 50,000 ppm – 60.97 μ mol; non -pregnant 100,000 ppm – 43,10 Page 181Prof. W.Dekant; T.Schmidt: Biotransformation of 2,3,3,3

- tetrafluoropropene (1234yf) page 17 of 19



### μ mol). No significant difference (P = 0.58, Student’s t-test) was observed between

n on -pregnant females and pregnant females in the 100, 000 ppm group. Given that

the quantity of metabolite recovered at 100,000 ppm is lower or at least equal to the

quantity recovered at 50,000 ppm, it is supposed that the gas uptake and metabolism

is already saturated at an exposure level of 100,000 ppm of HFO- 1234yf , like it was

observed for similar substances [5]

.

Fig. 7 : Total urine excretion of N -acetyl- S -(3,3,3-trifluoro-2-hydroxypropanyl)- L

cysteine (mean ± SD) within 48 hours after whole body inhalation exposure

Based on mean body weights of 2, 994 g in male, 3

, 258 g in non -pregnant female and

3, 444 g in pregnant female rabbits, respiratory minute volume is determined as 1.548

L min -1 

### , 1.663 L min -1 

### and 1,744 L min -1 

### respectively [6] . Therefore the total dose of

HFO -1234yf received within one hour of whole -body inhalation exposure is

calculated as 386 mmol (male 100,000 ppm), 193 mmol (male 50,000 ppm), 415

mmol (non-pregnant female 100,000 ppm), 435 mmol (preg nant female 100,000

ppm) and 217 mmol (pregnant female 50,000 ppm). The calculated extents of

biotransformation of HFO- 1234yf do not indicate di fference between non -pregnant

and pregnant female rabbits (Tabl. 4). Page 182Prof. W.Dekant; T.Schmidt: Biotransformation of 2,3,3,3

- tetrafluoropropene (1234yf) page 18 of 19



### Tabl. 4 : Biotransformation extent of HFO- 1234yf in male (M) , female (F) and pregnant female (P) rabbits after one hour exposure.

* based on th e metabolic pattern of one rabbit per group
** mole volume of HFO

- 1234yf was calculated to be 24.06 L/ mol under standard conditions (273.15 K, 1013.25hPa)



# 5 Discussion

Biotransformation products of HFO- 1234yf, found i n this study, in general support the

results of earlier metabolic studies of this compound, showing a biotransfor mation of

the fluoro- olefine by CYP 450 2E1 oxidation and further conjugation of the epoxide

with glutathione. Metabolites identified by 19 

### F- NMR were N -acetyl- S -(3,3,3-trifluoro-2-

hydroxypropanyl)- L -cysteine, S -(3,3 ,3 -trifluoro-2-hydroxypropanyl)-mercaptolactic

acid, 3,3,3

- trifluoro

- 1,2

- dihy droxyprop ane and 3,3,3

- trifluoropropionic acid. In c ontrast

to earlier rabbit urine analysis [1] , the predominant metabolite was determined as S

- (3,3,3-trifluoro-2- hyd roxypropanyl)-mercaptolactic acid instead of N -acetyl- S -(3,3,3-

trifluoro-2-hydroxypropanyl)- L -cysteine. Comparison of 19 

### F NMR spectra showed no

major differences in metabolite pattern in any urine fraction between non-pregnant

and pregnant female animals.

Based on urine recovery of N -acetyl- S -(3,3,3-trifluoro-2-hydroxypropanyl)- L -cysteine

, HFO -1234yf is rapidly metabolized in all study groups . More than 83% of the

metabolite in male and more than 93% in non -pregnant female/pregnant female

animals was excr eted within 24 hours after inhalation exposure (50,000 ppm and

100,000 ppm exposures)

. However, pregnant females in the 100,000 ppm group

eliminated approximately only half of the metabolite amount within the first 12 hours

after exposure compared to non -pregnant females exposed to the same

concentration of HFO

- 1234yf . On the other hand total excretion of N -acetyl- S -(3,3,3- Group N

- acetyl

- S

- (3,3,3

- trifluoro

- 2

- hydroxypropanyl)

- L

cysteine mean
excretion within 48 hours [ μ mol] N

- acetyl

- S

- (3,3,3

- trifluoro

- 2

- hydroxypropanyl)

- L

cysteine mean fraction
of

the five main metabolites [%] * Total fluoro
metabolites

excretion

[ μ mol] Inhale d gas dose [ μ mol] ** Metabolised

am ount [%]

M 50,000 ppm 75,24 35,83 210,00 192,976 0, 11

P 50,000 ppm 60,97 37,27 188,94 217,415 0, 09

M 100,000 ppm 86,40 32,70 264,22 385,952 0, 07

P 100,000 ppm 50,47 31,33 161,10 434,830 0,0 4

F 100,000 ppm 43,10 29,10 148,11 414,755 0,0 4 

### Page 183Prof. W.Dekant; T.Schmidt: Biotransformation of 2,3,3,3

- tetrafluoropropene (1234yf) page 19 of 19



### trifluoro-2-hydroxypropanyl)- L -cysteine within 48 hours was not significantly (P = 0.58,

Student’s t-test) different between non -pregnant female and pregnant female animals.

Beside the slightly slower metabolite elimination within the first 12 hours after

inhalation exposure to 100,000 ppm of HFO

- 1234yf, no differences in urinary

metabolite pattern or quantity of metabolites excreted were observed between the

different groups.



# References

1. Schuster, P., et al., Biotransformation of 2,3,3,3-tetrafluoropropene (HFO1234yf) in rabbits. Toxicol Appl Pharmacol, 2010. 244 (3): p. 247- 53.

2. Schuster, P., Biotransformation of trans- 1,1,1,3 -tetrafluoropropene, 2,3,3,3- tetrafluoropropene and 1,2,3,3,3-pentafluoropropene . 2009, University of Würzburg: Wuerzburg. p. 107.

3. Honeywell, An inhalation range-finding prenatal developmental toxicity study
ofHFO -1234yf (2,3,3,3-tetrafluoropropene) in rabbits, Report no. WIL- 447021

. 2008, WIL Research Laboratories, LLC, 1407 George Road, Ashland, OH
44805

- 8946.

4. Schuster, P., et al., Biotransformation of 2,3,3,3-tetrafluoropropene (HFO1234yf). Toxicol Appl Pharmacol, 2008. 23 3 (2): p. 323- 32.

5. Gargas, M.L., M.E. Andersen, and H.J. Clewell, 3rd, A physiologically based simulation approach for determining metabolic constants from gas uptake
data. Toxicol Appl Pharmacol, 1986. 86 (3): p. 341- 52.

6. Alexander, D.J., et al., Association of Inhalation Toxicologists (AIT) Working Party Recommendation for Standard Delivered Dose Calculation and Expression in Non-Clinical Aerosol Inhalation Toxicology Studies with
Pharmaceuticals. Inhalation Toxicology, 2008. 20 (13): p. 1179- 1189. Page 184

### Huntingdon Life Sciences 10-2226 Page 185

MA-252E-93-23 Final Report

Equipment List Appendix J

Exposure Chamber 1000 Liter glass and stainless steel chamber (Wahmann)

Compound Reservoir Sponsor Cylinder with backpressure guage and toggle valve

Flowmeters Top Trak TM mass flowmeter, size 0-10 Lpm, Model 822-13-OVI-PVI-VI (Sierra

Instruments) Top Trak TM mass flowmeter, size 0-50 Lpm, Model 826 (Sierra Instruments) Flowmeter, size 0-20 Lpm (Dwyer ® 

### Instruments Inc.), calibrated prestudy with the

DryCal, Model ML-350-44, SN 106387 (Bios International Corporation)

Pressure/Vacuum Gauges

GAST ® 

### vacuum gauge, 0-30 in Hg

Valves Metering Valve, Model SS-4L Series, (Nupro ® 

### Co.)

Tubing

Teflon ® 

### , size 1⁄4” Size 1⁄4” PVC clear plastic (MasterKleer)

Stainless steel “T” Stainless steel, 1⁄2”

Vacuum Pump Thomas Industries Inc., Model 707CM 50

Air Analyzer

MIRAN ® 

### 1A-CVF Ambient Air Analyzer (Wilks) and recorded by a Cole Parmer strip

chart recorder, Model 201 and a Micronta ® 

### LCD Benchtop Digital Multimeter, Model

22-195

Filters

Balston ® 

### Microfibre TM Disposable Filter Units, Grade DQ

### Huntingdon Life Sciences 10-2226 Page 186

MA-252E-93-23 Final Report

Equipment List Appendix J

Particle Sizer/Analyzer TSI Aerodynamic Particle Sizer, Model 331001 and a DELL computer, Model 486P/25,

equipped with an Epson LQ-570+ Dot matrix printer, Model P630B

Chamber Air Flow Dwyer ® 

### Magnehelic ® 

### gauge, (Dwyer ® 

### Instruments Inc.); calibrated prestudy with a Dry

Gas Meter , Model 2M (Singer)

Chamber Static Pressure Dwyer ® 

### Magnehelic ® 

### gauge, calibrated prestudy with Dwyer  

### Mark II Manometer,

Model 25 (Dwyer  

### Instruments Inc.)

Environmental Monitoring Temperature and Humidity Gauge (VWR) calibrated prestudy with a Digital

Hygrometer-Thermometer, Model 11-661-7b (Fisher Scientific) Oxygen Analyzer, Model O 2 

### (Oxigraf)

Miscellaneous Stop watch, Fisher Scientific, Model 51439788

Balance

Pelouze 4040

### Huntingdon Life Sciences 10-2226 Page 187
MA-252E-93-23 Final Report

Prestudy Chamber Distribution Records Appendix K

Group (target ppm) Date Port Concentration (ppm) Ratio to H-1

2 (50,000) 26 Oct 10 H-1 55,000 - H-2 55,000 1.00 H-7 55,000 1.00 H-8 54,000 0.98 H-4 54,000 0.98

3 (100,000) 27 Oct 10 H-1 100,000 - H-2 100,000 1.00 H-7 100,000 1.00 H-8 99,000 0.99 H-4 98,000 0.98

### Huntingdon Life Sciences 10-2226 Page 188
MA-252E-93-23 Final Report

Miran ® 

### Calibration Appendix L

Methodology for HFO-1234yf (Group 1, Phase 2):

Settings: The instrument settings for the unit (Miran ® 

### 06) are summarized below:

wavelength, microns 8.2 pathlength, dial setting 1.00 slit width, mm 1 range, absorbance 1 response, seconds 40 gain High chart speed, cm/min 1
chart volts 1

Calibrations: The Miran ® 

### was turned on and allowed to warm up for approximately
10 minutes. The cell was flushed with room air for approximately one minute. The loop was closed, the unit was zeroed and the calibra tion series was performed as shown below. The resultant data were plotted to obtain a calibration curve. Each observer used a separate syringe for calibration.

Injection Calculated Absorbance
Volume Concentration 1 

### Operator 1 Operator 2 Average

(μL) (ppm) (volts) (volts) (volts)

2,250 399 0.713 0.727 0.720 2,850 505 0.861 0.872 0.867 3,400 603 0.972 0.989 0.981

56 9.93 0.0161 0.0241 0.0201
560 99.3 0.2057 0.2110 0.2084

1 

### Calculated Conc. (ppm) = Injection volume (μL)

5.64 L

where: 5.64 L = volume of Miran cell

Syringe: 0-5000 μL Hamilton gas-tight syringe, Model No. 1005 used for injections.

### Huntingdon Life Sciences 10-2226 Page 189
MA-252E-93-23 Final Report

Miran ® 

### Calibration Appendix L

Calibration Checks: A one point calibration check of the Miran ® 

### was performed for each exposure prior to sampling the chambers. The parameters are shown below:

Expected Acceptable Injection Calculated Absorbance Absorbance Volume Concentration Reading Range

(μL) (ppm) (volts) (volts)

560 99.3 0.2084 0.1771 - 0.2397

The absorbance was recorded after each inj ection. The absorbance was considered satisfactory if it was within 15% of the original calibration series. If any of the absorbance values fell outside the 15% range, the injection was rechecked as follows. The volume for the value that was out of range was re-injected twice. The closer pair of the three injections were averaged and the results were compared to the original curve. If the average of the pair was within the 15% ra nge, the original was accepted. If the value of the average was outside the 15% range, th e Study Director decided if a new graph was to be prepared.

### Huntingdon Life Sciences 10-2226 Page 190
MA-252E-93-23 Final Report

Miran ® 

### Calibration Appendix L

Calibration Curve for HFO-1234yf

y = 110.72 x 3 + 43.952x 2 + 463.97x

R 2 = 0.9996

0 100 200 300 400 500 600 700 800

0.000 0.100 0.200 0.300 0.400 0.500 0.600 0.700 0.800 0.900 1.000

Absorbance (Volts)

### Huntingdon Life Sciences 10-2226 Page 191
MA-252E-93-23 Final Report

Miran ® 

### Calibration Appendix L

Methodology for HFO-1234yf (Group 2, Phase 2):

Settings: The instrument settings for the unit (Miran ® 

### 04) are summarized below:

wavelength, microns 3.24 pathlength, dial setting 3.18 slit width, mm 1 range, absorbance 1 response, seconds 1 gain High chart speed, cm/min 1
chart volts 1

Calibrations: The Miran ® 

### was turned on and allowed to warm up for approximately
10 minutes. The cell was flushed with room air for approximately one minute. The loop was closed, the unit was zeroed and the calibra tion series was performed as shown below. The resultant data were plotted to obtain a calibration curve. Each observer used a separate syringe for calibration.

Injection Calculated Absorbance
Volume Concentration 1 

### Operator 1 Operator 2 Average

(μL) (ppm) (volts) (volts) (volts)

220,000 39,007 0.301 0.2770 0.289 280,000 49,645 0.366 0.348 0.357 560,000 99,291 0.602 0.609 0.606 680,000 120,567 0.690 0.693 0.692

1 

### Calculated Conc. (ppm) = Injection volume (μL)

5.64 L

where: 5.64 L = volume of Miran cell

Syringe: 0-1500 μL Hamilton gas-tight syringe, Model No. S-1500 used for injections.

### Huntingdon Life Sciences 10-2226 Page 192
MA-252E-93-23 Final Report

Miran ® 

### Calibration Appendix L

Calibration Checks: A one-point calibration check of the Miran ® 

### was performed for each exposure prior to sampling the chambers. The parameters are shown below:

Expected Acceptable Injection Calculated Absorbance Absorbance Volume Concentration Reading Range

(μL) (ppm) (volts) (volts)

280,000 49,645 0.357 0.303 - 0.411

The absorbance was recorded after each inj ection. The absorbance was considered satisfactory if it was within 15% of the original calibration series. If any of the absorbance values fell outside the 15% range, the injection was rechecked as follows. The volume for the value that was out of range was reinjected twice. The closer pair of the three injections were averaged and the results were compared to the original curve. If the average of the pair was within the 15% ra nge, the original was accepted. If the value of the average was outside the 15% range, th e Study Director decided if a new graph was to be prepared.

### Huntingdon Life Sciences 10-2226 Page 193
MA-252E-93-23 Final Report

Miran ® 

### Calibration Appendix L

Calibration Curve for HFO-1234yf

y = 94388x 3 + 6018.7x 2 + 125250x

R 2 = 0.9986

0 20000 40000 60000 80000 100000 120000 140000

0.000 0.200 0.400 0.600 0.800 1.000

A bsorbance (Volts)

### Huntingdon Life Sciences 10-2226 Page 194
MA-252E-93-23 Final Report

Miran ® 

### Calibration Appendix L

Methodology for HFO-1234yf (Group 1, Phase 1 and Group 3, Phase 2):

Settings: The instrument settings for the unit (Miran ® 

### 1A-2315) are summarized below:

wavelength, microns 3.24 pathlength, dial setting 3.44 slit width, mm 1 range, absorbance 1 response, seconds 1 gain High chart speed, cm/min 1
chart volts 1

Calibrations: The Miran ® 

### was turned on and allowed to warm up for approximately
10 minutes. The cell was flushed with room air for approximately one minute. The loop was closed, the unit was zeroed and the calibra tion series was performed as shown below. The resultant data were plotted to obtain a calibration curve. Each observer used a separate syringe for calibration.

Injection Calculated Absorbance
Volume Concentration 1 

### Operator 1 Operator 2 Average

(μL) (ppm) (volts) (volts) (volts)

220,000 39,007 0.341 0.358 0.350 280,000 49,645 0.420 0.442 0.431 560,000 99,291 0.720 0.744 0.732 680,000 120,567 0.832 0.866 0.849

1 

### Calculated Conc. (ppm) = Injection volume (μL)

5.64 L

where: 5.64 L = volume of Miran cell

Syringe: 0-1500 μL Hamilton gas-tight syringe, Model No. S-1500 used for injections.

### Huntingdon Life Sciences 10-2226 Page 195
MA-252E-93-23 Final Report

Miran ® 

### Calibration Appendix L

Calibration Checks: A one-point calibration check of the Miran ® 

### was performed for each exposure prior to sampling the chambers. The parameters are shown below:

Expected Acceptable Injection Calculated Absorbance Absorbance Volume Concentration Reading Range

(μL) (ppm) (volts) (volts)

560 99.3 0.2084 0.1771 - 0.2397

The absorbance was recorded after each inj ection. The absorbance was considered satisfactory if it was within 15% of the original calibration series. If any of the absorbance values fell outside the 15% range, the injection was rechecked as follows. The volume for the value that was out of range was reinjected twice. The closer pair of the three injections were averaged and the results were compared to the original curve. If the average of the pair was within the 15% ra nge, the original was accepted. If the value of the average was outside the 15% range, th e Study Director decided if a new graph was to be prepared.

### Huntingdon Life Sciences 10-2226 Page 196
MA-252E-93-23 Final Report

Miran ® 

### Calibration Appendix L

Calibration Curve for HFO-1234yf

y = - 3 + 2 +

R 2

=

0 2000 4000 6000 8000 100000 120000 140000

0.00 0.20 0.40 0.60 0.80 1.00

Absorbance (Volts)

### Appendix M Page 197

### Page 198 Appendix N

### Page 199

### Page 200

### Page 201

### Page 202

### Page 203

### Page 204

### Page 205

### Page 206

### Page 207

### Page 208

### Page 209

### Page 210

### Page 211

### Page 212

### Page 213

### Page 214

### Page 215

### Page 216

### Page 217

### Page 218

### Page 219

### Page 220

### Page 221

### Page 222

### Page 223

### Page 224

### Page 225

### Huntingdon Life Sciences 10-2226 Page 226
MA-252E-93-23 Final Report

Scientists and Supervising Personnel Appendix O

TITLE/DEPARTMENT NAME/DEGREE

SENIOR VICE PRESIDENT,
SAFETY ASSESSMENT Sylvie J. Gosselin, D.V.M., PhD, Diplomate A.C.V.P.

DIRECTOR, DEVELOPMENTAL AND

REPRODUCTIVE TOXICOLOGY Robert M. Parker, PhD, DABT

DIRECTOR, ANALYTICAL SERVICES Barbara A. Litzenberger, B.S., M.T. (ASCP)

DIRECTOR, TOXICOLOGY
OPERATIONS Ian Vanterpool, F.I.A.T.

DIRECTOR, QUALITY ASSURANCE Melissa Elliott, B.S., RQAP-GLP

STUDY DIRECTOR Gary M. Hoffman, B.A., D.A.B.T.

PATHOLOGIST Alric Lopez-Martinez PhD, D.V.M., Diplomate A.C.V.P.

REPORT WRITERS Sonya Gray Kathleen P. Smith, A.A.S.

VETERINARIAN Teresa S. Kusznir, V.M.D.

MANAGER/SUPERVISOR

Rodent Toxicology Inhalation Toxicology Pharmacy Necropsy and Fetal Pathology

Histology Thomas Jones, BS, AALAS LATG Stuart Cracknell, CBiol, MIBiol. Michael S. McCarthy Donna G. Chuddley, B.S., CLA (ASCP) HT Sally Wilcox, B.Sc.

PRINCIPAL INVESTIGATOR

URINE METABOLITE ANALYSIS Tobias Schmidt & Dr. rer. Nt. W. Dekant University of Würzburg
Versbacher Straße 9 97078 Würzburg Germany