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 



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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 
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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 status 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 exposed 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 (including 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 

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

a

The nominal concentrations in Phase 2 are the combined results of the 2 chambers for each of the 

separate male and female exposures. 
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SUMMARY 

These results indicated an acceptable agreement between the targeted and analytical and 

nominal concentrations. The particle sizing 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. 

All animals survived until their scheduled sacrifice 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 19F-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 19F-NMR spectra represented 

more than 78% of total 19F 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. 
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 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 Determinations ........................................................................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
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 TABLE OF CONTENTS 

3. RESULTS AND DISCUSSION

3.1. Chamber Monitoring Results.....................................................................34

3.2. Urine Metabolite Determinations ..............................................................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 (Phase 1, Group 1)........................................................................39

2. Chamber Generation System and 1000 Liter Whole-Body 

Exposure Chamber (Phase 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 Termination History ......................................................................84

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

E. Individual Physical Examination Findings..................................................................96

F. Individual Body Weights ...........................................................................................113
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 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 Presumed Pregnant Females .....................................................165

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

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

K. Prestudy Chamber Distribution Records ...................................................................187

L. Miran Calibration.....................................................................................................188

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

N. Protocol and Protocol Amendments ..........................................................................198

O. Scientists and Supervising Personnel ........................................................................226
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1. INTRODUCTION 

In previous studies, pregnant rabbits were found to be unexpectedly sensitive to 

an inhalation exposure of 50,000 ppm HFO-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 designed 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 determine 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 
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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) 
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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 

Levela

Test Animals 

Group (females) 

ppm mg/L 

Presumed 

Pregnant Non-Pregnant Euthanizedb

1 100,000 471 1 1 2

a

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

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-body 

inhalation exposure for 1 hour duration on GD12 with HFO1234yf at the exposure concentrations summarized below: 

Exposure 

Levela Test Animals 

Group ppm mg/L 

Presumed 

Pregnant 

Females 

NonPregnant 

Females Males Necropsyb

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. 
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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 exposure 

toxicity 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 whole-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 has 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 with this test substance. This 

data will be used as a basis for species selection in subsequent 

reproductive toxicity and/or subchronic toxicity testing. 
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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 the 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 exposure 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-pregnant females were not included in 

the Low exposure group because data are available from a 

previously conducted study (Schuster et al, TAP, 244: pp 247-253, 

2010). An n of 5 animals per sex and/or pregnancy status was 

considered by the U.S. EPA to be the minimum number of animals 

per group for acute toxicity studies. 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 
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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, fabrication 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. 
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2.5.12. DISPOSITION 

The unused portion of the test substance 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 were 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 
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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 required 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) and 

28 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 technical 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). 
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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 stabilization, fresh food was provided 

in varying amounts. After stabilization, approximately 200 grams 

of fresh food was provided to the presumed pregnant females and 

approximately 150 grams was provided 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 restriction 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 Regulations (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 
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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 within 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. 
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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 maintained, 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. 
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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 conditions 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” 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 m3 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. The oxygen was directed 

through a metering valve to the inlet 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 the inlet turret of the 1.0 m3

 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 flowmeter and then from the outlet of 

the flowmeter was directed into the turret of the 1.0 m3

 glass and 
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stainless steel exposure chamber via 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 flow 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 m3 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 flowmeter 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 m3). Each chamber was operated 

at a minimum flow rate of 200 Liters 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 T99 equilibrium time (calculated by 

multiplying the air change by the exponential factor 4.6) of at most 

23 minutes: 

Group Airflow Rate Air Change T99

(Lpm) (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 
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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 levels 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 the 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 recording 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, samples of the room air were taken 

and analyzed in the same manner for comparison to the chamber 
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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 (μg/mg) x 22.4 (μL/μmole) x 295oK

exposure duration (min) x airflow (Lpm) x MW (μg/μmole) 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 afternoon) 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. 
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2.14.3. BODY WEIGHT 

Body weights were recorded for all 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 study 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 intervals 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 
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and the container was capped. The 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 methanol 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 dry ice, to Tobias Schmidt and 

Dr. rer. nat. W. Dekant at the 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 

19F-NMR, after mixing 720 μL of urine with 80 μL D2O. 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 following the 1-hour exposure. Animals 

were not fasted prior to sacrifice. 
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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 cord; 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 
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tissues were prepared and examined microscopically for animals in 

the control and high-dose groups from 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 organs 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 methods 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 
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not monotone, Dunnett's test (Dunnett, 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 detect 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 tables 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 χ2-

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 test was still significant, then nonparametric methods were applied to mean ranks. Comparisons 

were performed using Wilcoxon rank sum tests (Wilcoxon, 1945). 
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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 Generation of report tables for the clinical 

observations and body weight data in MS 

Word formats 

Microsoft Word and/or Excel Additional 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 archive their raw data, original final 

report and any preserved urine specimens. 
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2.20. REGULATORY REFERENCES 

2.20.1. TEST GUIDELINES 

The design was not based on regulatory 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 CFR 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, Federal Register, Volume 56, No. 

32, February 15, 1991, pp. 6426-6505 effective March 18, 1991. 

2.21. PROTOCOL DEVIATIONS 

The following protocol deviations occurred 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. 
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5. Due to technician oversight, there is no documentation that the 

animals were identified by earmarking with indelible marker on the 

underside of the ear. 

6. Due to technician oversight, there 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 accordance with the Testing Facility SOP. 

On the 3rd 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. 
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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 

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

a

The nominal concentrations in Phase 2 are the 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) 

(μm) 

Geometric 

Standard 

Deviation 

(Males/Females) 

Total 

Mass 

Concentration 

(Males/Females) 

(mg/m3

) 

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 
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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 intervals for 48 hours after the end of the 

exposure and analyzed by 19F-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 19F-NMR spectra represented more than 78% of total 

19F 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. 

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. 
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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 sacrifices. 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 observed 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. 
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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 several dose levels with a zero dose control. 

Biometrics 28: 519-531. 
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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 collecting 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 ppm 
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Phase 1 

Group 1 

Chamber Generation System 

and 1000 Liter Whole-Body Exposure Chambera 

aSampling port H3 is on the front of the chamber; while, sampling port H4 is on the back of the chamber. Oxyge

the chamber using a similar set-up as used to deliver the test substance. 
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Phase 2 

Group 1 

Chamber Generation System 

and 1000 Liter Whole-Body Exposure Chambera 

aSampling port H3 is on the front of the chamber; while, sampling port H4 is on the back of the chamber. 
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Phase 2 

Groups 2 and 3 

Chamber Generation System 

and 1000 Liter Whole-Body Exposure Chambera 

aSampling port H3 is on the front of the chambers; while, sampling port H4 is on the back of the chambers. Oxy

the chambers using a similar set-up as used to deliver the test substance. 
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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 
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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) Observation 
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Presumed Pregnant 

Females Summary of Daily Observations - Phase 2 

 Phase PRE PH2 

Day 2 3 4 5 6 7 8 9 11 3 4 7 

Session AM AM AM AM AM AM AM AM AM AM AM AM A

Observations Group Gastrointestinal, Decreased Fecal Volume 1 2 1 1 

2 1 2 1 1 1 1 1 3 3 1 1 1 

Gastrointestinal, No Stool 2 1 Miscellaneous, Decreased Food Consumption 1 1 1 1 1 

2 2 1 1 1 1 3 2 2 1 2 
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Non-Pregnant 

Females Summary of Daily Observations - Phase 2 

 Phase PRE 

Day 3 4 5 6 7 7 8 8 9 9 10 10 

Session AM AM AM AM AM PM AM PM AM PM AM PM A

Observations Group Gastrointestinal, Decreased Fecal Volume 1 2 

3 2 1 

Gastrointestinal, No Stool 1 1 1 1 

3 

Miscellaneous, Decreased Food Consumption 1 1 1 1 

3 
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Non-Pregnant 

Females Summary of Daily Observations - Phase 2 

 Phase PRE PH2 

Day 12 12 13 1 2 4 5 6 7 8 10 

Session AM PM AM PM AM AM AM AM AM AM AM 

Observations Group Gastrointestinal, Decreased Fecal Volume 1 1 

3 1 
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Males Summary of Daily Observations - Phase 2 

 Phase PRE 

Day 3 4 5 6 7 7 8 8 9 9 10 10 

Session AM AM AM AM AM PM AM PM AM PM AM PM A

Observations Group Dermal General, Ano-Genital Staining 2 1 2 1 2 2 1 1 1 1 Dermal General, Stains on Fur (Head/Neck) 1 1 Gastrointestinal, Decreased Fecal Volume 1 1 1 

2 1 2 1 1 1 2 1 3 1 1 

Gastrointestinal, No Stool 2 1 1 1 1 Gastrointestinal, Unformed Stool 2 1 General Appearance, Thin 2 1 1 1 1 1 1 Miscellaneous, Decreased Food Consumption 1 1 1 

2 1 2 2 3 2 2 3 1 
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Males Summary of Daily Observations - Phase 2 

 Phase PRE PH2 

Day 12 12 13 1 2 4 5 6 7 8 10 

Session AM PM AM PM AM AM AM AM AM AM AM 

Observations Group Dermal General, Ano-Genital Staining 2 1 1 1 1 1 

3 2 

Dermal General, Stains on Fur (Head/Neck) 3 1 Dermal General, Stains on Fur (Torso) 3 1 Gastrointestinal, Decreased Fecal Volume 1 2 

3 1 1 1 

Gastrointestinal, No Stool 2 1 

3 1 

Gastrointestinal, Unformed Stool 3 1 1 1 General Appearance, Thin 2 1 1 1 1 Miscellaneous, Decreased Food Consumption 1 1 

2 1 3 1 1 2 
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Summary of Physical Examination Findings 

Preface Table 3 

Detailed physical examinations were performed for all animals on study once daily 

during the pretest period and 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 ppm 
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Presumed Pregnant 

Females Summary of Physical Examination Findings - Phase 2 

 Phase PRE PH2 

Day 1 2 3 4 5 6 7 8 9 10 1 2 

Observations Group Within Normal Limits 1 6 4 6 5 5 5 5 5 5 5 5 5 

2 5 4 5 6 5 5 5 5 6 6 6 6 3 6 3 5 6 6 6 6 4 6 6 6 6 

Dermal General, Ano-Genital Staining 2 1 1 1 Dermal General, Scab(s) 1 1 1 1 1 1 1 1 1 1 

3 

Gastrointestinal, Decreased Fecal Volume 1 2 1 

2 1 1 1 1 1 3 3 1 1 

Miscellaneous, Decreased Food Consumption 1 1 

2 1 1 1 1 3 2 
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Presumed Pregnant 

Females Summary of Physical Examination Findings - Phase 2 

 Phase PH2 

Day 6 7 8 9 10 11 12 13 14 15 

Observations Group Within Normal Limits 1 5 4 6 6 6 6 6 5 5 5 

2 6 5 6 6 6 6 6 5 5 5 3 5 4 5 5 5 5 5 4 4 4 

Dermal General, Ano-Genital Staining 1 1 1 1 

2 1 1 1 3 2 2 2 

Dermal General, Scab(s) 1 1 

3 1 1 1 1 1 1 1 1 1 1 

Gastrointestinal, Decreased Fecal Volume 1 1 

2 1 3 1 

Miscellaneous, Decreased Food Consumption 1 1 

3 1 
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Non-Pregnant 

Females Summary of Physical Examination Findings - Phase 2 

 Phase PRE PH2 

Day 1 2 3 4 5 6 7 8 9 10 1 2 

Observations Group Within Normal Limits 1 5 5 3 5 4 4 2 3 3 3 4 4 

3 5 5 5 5 4 5 5 5 3 3 5 5 

Dermal General, Alopecia (Head/Neck) 3 1 1 Dermal General, Scab(s) 1 2 2 2 2 1 1 

3 1 1 

Gastrointestinal, Decreased Fecal Volume 1 2 

3 1 

Gastrointestinal, No Stool 1 1 1 1 Miscellaneous, Decreased Food Consumption 1 1 1 1 
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 MA-252E-93-23 

Non-Pregnant 

Females Summary of Physical Examination Findings - Phase 2 

 Phase PH2 

Day 4 5 6 7 8 9 10 11 12 13 14 15 

Observations Group Within Normal Limits 1 4 4 4 4 4 4 5 5 5 5 5 5 

3 5 5 5 5 5 5 5 5 5 5 5 5 

Dermal General, Scab(s) 1 1 1 1 1 1 1 
Huntingdon Life Sciences 10-2226 

 MA-252E-93-23 

Males Summary of Physical Examination Findings - Phase 2 

 Phase PRE P

Day 1 2 3 4 5 6 7 8 9 10 11 12 

Observations Group Within Normal Limits 1 1 1 1 1 1 1 1 1 3 3 3 4 

2 1 1 1 1 1 1 1 3 4 4 3 4 4 4 4 4 3 3 4 4 

Dermal General, Alopecia (Head/Neck) 2 1 1 1 1 1 1 1 1 1 1 Dermal General, Alopecia (Torso) 1 Dermal General, Ano-Genital Staining 1 1 1 1 1 1 1 1 1 1 1 1 1 

2 2 2 2 3 3 3 3 3 2 2 2 2 3 1 1 1 1 1 1 1 1 1 1 1 1 

Dermal General, Scab(s) 1 1 1 1 1 

2 1 1 1 1 1 1 1 1 1 1 1 1 3 1 1 

Dermal General, Stains on Fur (Head/Neck) 1 1 1 1 1 1 1 1 1 

2 2 2 2 2 2 2 2 2 2 2 2 2 3 1 1 1 1 1 1 1 1 1 1 1 1 

Dermal General, Stains on Fur (Torso) 1 2 2 2 2 2 2 2 2 

2 1 1 1 1 1 1 1 1 1 1 1 1 3 1 1 1 1 1 1 1 1 1 1 1 1 
Huntingdon Life Sciences 10-2226 

 MA-252E-93-23 

Males Summary of Physical Examination Findings - Phase 2 

 Phase PRE P

Day 1 2 3 4 5 6 7 8 9 10 11 12 

Observations Group Gastrointestinal, Decreased Fecal Volume 2 1 2 2 2 2 

3 1 

Gastrointestinal, No Stool 3 1 Gastrointestinal, Unformed Stool 2 1 General Appearance, Thin 2 1 1 1 1 1 Miscellaneous, Decreased Food Consumption 2 1 1 2 3 

3 1 

Oral/Buccal, Incisor(s) Broken/Missing 2 1 1 
Huntingdon Life Sciences 10-2226 

 MA-252E-93-23 

Males Summary of Physical Examination Findings - Phase 2 

 Phase PH2 

Day 4 5 6 7 8 9 10 11 12 13 14 15 

Observations Group Within Normal Limits 1 2 3 3 3 4 4 4 4 3 3 3 3 

2 1 1 1 1 1 2 2 2 2 2 2 2 3 4 3 3 3 3 3 3 3 3 3 3 3 

Dermal General, Alopecia (Head/Neck) 1 1 1 1 1 Dermal General, Alopecia (Torso) 1 1 Dermal General, Ano-Genital Staining 1 1 1 1 1 1 1 1 1 2 2 2 2 

2 2 2 2 2 2 2 2 2 2 2 2 2 3 1 1 2 2 2 2 2 2 2 2 2 2 

Dermal General, Scab(s) 2 1 1 1 1 1 1 1 1 1 1 1 1 Dermal General, Stains on Fur (Head/Neck) 2 2 2 2 2 2 1 1 1 1 1 

3 1 1 1 1 1 

Dermal General, Stains on Fur (Torso) 2 1 1 1 1 1 1 1 1 1 1 1 1 

3 1 1 1 1 1 1 1 1 1 1 1 1 

Gastrointestinal, No Stool 3 1 Gastrointestinal, Unformed Stool 3 1 
Huntingdon Life Sciences 10-2226 

 MA-252E-93-23 

Males Summary of Physical Examination Findings - Phase 2 

 Phase PH2 

Day 4 5 6 7 8 9 10 11 12 13 14 15 

Observations Group Miscellaneous, Decreased Food Consumption 3 1 
Huntingdon 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 mean 
Huntingdon 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 mean 
Huntingdon 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 mean 
Huntingdon 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 mean 
Huntingdon 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 mean 
Huntingdon 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.............................................................................................................65

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 ppm 


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.............................................................................................................68 

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 ppm 



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 Termination History ......................................................................73

3. Individual Physical Examination 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/m3) 

1001 3.648 1.662 9.41E-03 

 

Chamber Environment 

Exposure Total 

Hour Temperature Relative Humidity Chamber Airflow 

 (°C) (%) (Lpm) 

T99 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 Daya 

 

Group 1 - 100,000 ppm 

4501b 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 included observations of general condition, skin 

and fur, eyes, nose, oral cavity, abdomen, as well as evaluations of respiration. 

In-chamber observations were performed for all animals as a group once during the 

exposure period. All animals were within normal limits. 

In addition, 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. 

On the day of exposure, animals were observed before and after exposure. All animals 

were considered to be within normal limits at all time points. 
Huntingdon Life Sciences 10-2226 

 MA-252E-93-23 

Phase 1 

Females Individual Physical Examination Findings 

 Phase PRE PH1 Animal Day 1 2 3 4 5 1 2 3 Number Observations Group 1 – 100,000 ppm 4501 Within normal limits P  Dermal general, scab(s), mouth - on P P P P P P P 4502 Within normal limits P P P P P P P P P = present 
Huntingdon 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 2 

 
Huntingdon 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/m3) 

1001 4.398 1.967 8.80E-3 

 

Chamber Environment 

Exposure Total 

Hour Temperature Relative Humidity Chamber Airflow 

 (°C) (%) (Lpm) 

T99 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/m3) 

1002 3.883 1.452 7.35E-3 

 

Chamber Environment 

Exposure Total 

Hour Temperature Relative Humidity Chamber Airflow 

 (°C) (%) (Lpm) 

T99 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/m3

) 

2001 3.764 1.437 3.59E-2 

 

Chamber Environment 

Exposure Total 

Hour Temperature Relative Humidity Chamber Airflow 

 (°C) (%) (Lpm) 

T99 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/m3

) 

2002 18.490 2.139 3.78E-2 

 

Chamber Environment 

Exposure Total 

Hour Temperature Relative Humidity Chamber Airflow 

 (°C) (%) (Lpm) 

T99 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/m3

) 

3001 6.079 2.030 0.152 

 

Chamber Environment 

Exposure Total 

Hour Temperature Relative Humidity Chamber Airflow 

 (°C) (%) (Lpm) 

T99 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/m3

) 

3002 4.988 1.681 1.51E-2 

 

Chamber Environment 

Exposure Total 

Hour Temperature Relative Humidity Chamber Airflow 

 (°C) (%) (Lpm) 

T99 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 0.0 
Huntingdon 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 Daya

 

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 1 
Huntingdon 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 Daya

 

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) Observation 
Huntingdon Life Sciences 10-2226 

 MA-252E-93-23 

Presumed Pregnant 

Females Individual Daily Observations - Phase 2 

 Phase PRE PH2 Animal Day 2 3 4 5 6 7 8 9 11 3 4 Number Observations Session AM AM AM AM AM AM AM AM AM AM AGroup 1 - 0 ppm 1501 Gastrointestinal, decreased fecal volume P  Miscellaneous, decreased food consumption P 1502 Gastrointestinal, decreased fecal volume P P  Miscellaneous, decreased food consumption P P 1503 Gastrointestinal, decreased fecal volume P 1505 Gastrointestinal, decreased fecal volume Group 2 - 50,000 ppm 2503 Gastrointestinal, no stool P 2504 Gastrointestinal, decreased fecal volume P  Miscellaneous, decreased food consumption P 2506 Gastrointestinal, decreased fecal volume P P P P P P P  Miscellaneous, decreased food consumption P P P P P P = present 
Huntingdon Life Sciences 10-2226 

 MA-252E-93-23 

Presumed Pregnant 

Females Individual Daily Observations - Phase 2 

 Phase PRE PH2 Animal Day 2 3 4 5 6 7 8 9 11 3 4 Number Observations Session AM AM AM AM AM AM AM AM AM AM AGroup 3 - 100,000 ppm 3501 Miscellaneous, decreased food consumption P P 3502 Gastrointestinal, decreased fecal volume P P  Miscellaneous, decreased food consumption P 3503 Miscellaneous, decreased food consumption 3504 Miscellaneous, decreased food consumption P 3505 Gastrointestinal, decreased fecal volume P 3506 Gastrointestinal, decreased fecal volume P P P  Miscellaneous, decreased food consumption P P = present 
Huntingdon Life Sciences 10-2226 

 MA-252E-93-23 

Non-Pregnant 

Females Individual Daily Observations - Phase 2 

 Phase PRE Animal Day 3 4 5 6 7 7 8 8 9 9 1Number Observations Session AM AM AM AM AM PM AM PM AM PM AGroup 1 - 0 ppm 1507 Gastrointestinal, decreased fecal volume P  Gastrointestinal, no stool 1509 Gastrointestinal, decreased fecal volume  Miscellaneous, decreased food consumption 1510 Gastrointestinal, no stool P P P  Miscellaneous, decreased food consumption P P P 1511 Gastrointestinal, decreased fecal volume P Group 3 - 100,000 ppm 3508 Gastrointestinal, decreased fecal volume P P  Miscellaneous, decreased food consumption 3509 Gastrointestinal, decreased fecal volume P  Gastrointestinal, no stool  Miscellaneous, decreased food consumption 3510 Gastrointestinal, decreased fecal volume P = present 
Huntingdon Life Sciences 10-2226 

 MA-252E-93-23 

Non-Pregnant 

Females Individual Daily Observations - Phase 2 

 Phase PRE Animal Day 3 4 5 6 7 7 8 8 9 9 1Number Observations Session AM AM AM AM AM PM AM PM AM PM AGroup 3 - 100,000 ppm 3511 Gastrointestinal, decreased fecal volume P = present 
Huntingdon Life Sciences 10-2226 

 MA-252E-93-23 

Non-Pregnant 

Females Individual Daily Observations - Phase 2 

 Phase PRE PH2 Animal Day 12 12 13 1 2 4 5 6 7 8 1Number Observations Session AM PM AM PM AM AM AM AM AM AM AGroup 1 - 0 ppm 1507 Gastrointestinal, decreased fecal volume P Group 3 - 100,000 ppm 3508 Gastrointestinal, decreased fecal volume P P = present 
Huntingdon Life Sciences 10-2226 

 MA-252E-93-23 

Males Individual Daily Observations - Phase 2 

 Phase PRE Animal Day 3 4 5 6 7 7 8 8 9 9 1Number Observations Session AM AM AM AM AM PM AM PM AM PM AGroup 1 - 0 ppm 1002 Dermal general, stains on fur (head/neck), snout 1 

1005 Gastrointestinal, decreased fecal volume P P  Miscellaneous, decreased food consumption P P Group 2 - 50,000 ppm 

2003 Gastrointestinal, decreased fecal volume P  Miscellaneous, decreased food consumption P 2004 General appearance, thin P P P P P  Dermal general, ano-genital staining 1 1 1 1 1 1 1  Gastrointestinal, decreased fecal volume P P P P  Gastrointestinal, no stool P P P  Miscellaneous, decreased food consumption P P P P P P 2005 Dermal general, ano-genital staining 1 1 1  Gastrointestinal, unformed stool P  Gastrointestinal, decreased fecal volume P P P P  Gastrointestinal, no stool P  Miscellaneous, decreased food consumption P P P P P P = present, 1 = slight 
Huntingdon Life Sciences 10-2226 

 MA-252E-93-23 

Males Individual Daily Observations - Phase 2 

 Phase PRE Animal Day 3 4 5 6 7 7 8 8 9 9 1Number Observations Session AM AM AM AM AM PM AM PM AM PM AGroup 3 - 100,000 ppm 3002 Gastrointestinal, decreased fecal volume P  Miscellaneous, decreased food consumption P 3005 Gastrointestinal, decreased fecal volume P P = present 
Huntingdon Life Sciences 10-2226 

 MA-252E-93-23 

Males Individual Daily Observations - Phase 2 

 Phase PRE PH2 Animal Day 12 12 13 1 2 4 5 6 7 8 1Number Observations Session AM PM AM PM AM AM AM AM AM AM AGroup 1 - 0 ppm 1004 Gastrointestinal, decreased fecal volume P 1005 Gastrointestinal, decreased fecal volume P  Miscellaneous, decreased food consumption P Group 2 - 50,000 ppm 2004 General appearance, thin P P P P  Dermal general, ano-genital staining 1 1 1 1 1 2005 Gastrointestinal, no stool P  Miscellaneous, decreased food consumption P Group 3 - 100,000 ppm 3001 Dermal general, ano-genital staining 2  Gastrointestinal, unformed stool P P P 

 Gastrointestinal, decreased fecal volume P P  Gastrointestinal, no stool P  Miscellaneous, decreased food consumption P P P P = present, 1 = slight, 2 = moderate 
Huntingdon Life Sciences 10-2226 

 MA-252E-93-23 

Males Individual Daily Observations - Phase 2 

 Phase PRE PH2 Animal Day 12 12 13 1 2 4 5 6 7 8 1Number Observations Session AM PM AM PM AM AM AM AM AM AM AGroup 3 - 100,000 ppm 3002 Dermal general, ano-genital staining 1  Dermal general, stains on fur (head/neck), head 1  Dermal general, stains on fur (torso), dorsal 1 

 Miscellaneous, decreased food consumption P 3003 Gastrointestinal, decreased fecal volume P P = 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 performed for all animals on study once daily 

during the stabilization (pretest) period and once daily (post exposure on the day of 

exposure) during the dosing phase. For summarization 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 Sciences 10-2226 

 MA-252E-93-23 

Presumed Pregnant 

Females Individual Physical Examination Findings - Phase 2 

Animal Phase PRE PHNumber Observations Day 1 2 3 4 5 6 7 8 9 10 Group 1 - 0 ppm 1501 Within normal limits P P P P P  Dermal general, scab(s), ear(s) P P P P P  Gastrointestinal, decreased fecal volume P 1502 Within normal limits P P P P P P P P  Dermal general, scab(s), ear(s) P P P  Gastrointestinal, decreased fecal volume P  Miscellaneous, decreased food consumption P 1503 Within normal limits P P P P P P P P P P  Gastrointestinal, decreased fecal volume P 1504 Within normal limits P P P P P P P P P P P 1505 Within normal limits P P P P P P P P P P P 1506 Within normal limits P P P P P P P P P P P Group 2 – 50,000 ppm 2501 Within normal limits P P P P P P P P P P P P = present 
Huntingdon Life Sciences 10-2226 

 MA-252E-93-23 

Presumed Pregnant 

Females Individual Physical Examination Findings - Phase 2 

Animal Phase PRE PHNumber Observations Day 1 2 3 4 5 6 7 8 9 10 Group 2 – 50,000 ppm 2502 Within normal limits P P P P P P P P P P P 2503 Within normal limits P P P P P P P P P P P 2504 Within normal limits P P P P P P P P P P P 2505 Within normal limits P P P P P P P P  Dermal general, ano-genital staining 1 1 1 2506 Within normal limits P P P P P P  Gastrointestinal, decreased fecal volume P P P P P  Miscellaneous, decreased food consumption P P P P Group 3 – 100,000 ppm 3501 Within normal limits P P P P P P P P P P  Miscellaneous, decreased food consumption P P = present, 1 = slight 
Huntingdon Life Sciences 10-2226 

 MA-252E-93-23 

Presumed Pregnant 

Females Individual Physical Examination Findings - Phase 2 

Animal Phase PRE PHNumber Observations Day 1 2 3 4 5 6 7 8 9 10 Group 3 – 100,000 ppm 3502 Within normal limits P P P P P P P P P  Dermal general, scab(s), ear(s)  Gastrointestinal, decreased fecal volume P P  Miscellaneous, decreased food consumption P 3503 Within normal limits P P P P P P P P P P P 3504 Within normal limits P P P P P P P P P P P 3505 Within normal limits P P P P P P P P P P  Gastrointestinal, decreased fecal volume P 3506 Within normal limits P P P P P P P P P  Gastrointestinal, decreased fecal volume P P P = present 
Huntingdon Life Sciences 10-2226 

 MA-252E-93-23 

Presumed Pregnant 

Females Individual Physical Examination Findings - Phase 2 

Animal Phase PH2 Number Observations Day 6 7 8 9 10 11 12 13 14 15 Group 1 - 0 ppm 1501 Within normal limits P P P P P P P P  Dermal general, scab(s), ear(s) P  Miscellaneous, decreased food consumption P 1502 Within normal limits P P P P P P P P P P 1503 Within normal limits P P P P P P P P P P 1504 Within normal limits P P P P P P P P P  Gastrointestinal, decreased fecal volume P 1505 Within normal limits P P P P P P P P P P 1506 Within normal limits P P P P P P P  Dermal general, ano-genital staining 1 1 1 Group 2 - 50,000 ppm 2501 Within normal limits P P P P P P  Dermal general, ano-genital staining 1 1 1  Gastrointestinal, decreased fecal volume P P = present, 1 = slight 
Huntingdon Life Sciences 10-2226 

 MA-252E-93-23 

Presumed Pregnant 

Females Individual Physical Examination Findings - Phase 2 

Animal Phase PH2 Number Observations Day 6 7 8 9 10 11 12 13 14 15 Group 2 - 50,000 ppm 2502 Within normal limits P P P P P P P P P P 2503 Within normal limits P P P P P P P P P P 2504 Within normal limits P P P P P P P P P P 2505 Within normal limits P P P P P P P P P P 2506 Within normal limits P P P P P P P P P P Group 3 - 100,000 ppm 3501 Within normal limits P P P P P P P P P  Gastrointestinal, decreased fecal volume P  Miscellaneous, decreased food consumption P 3502 Dermal general, ano-genital staining 2 2 2  Dermal general, scab(s), ear(s) P P P P P P P P P P 3503 Within normal limits P P P P P P P P P P P = present, 2 = moderate 
Huntingdon Life Sciences 10-2226 

 MA-252E-93-23 

Presumed Pregnant 

Females Individual Physical Examination Findings - Phase 2 

Animal Phase PH2 Number Observations Day 6 7 8 9 10 11 12 13 14 15 Group 3 - 100,000 ppm 3504 Within normal limits P P P P P P P P P P 3505 Within normal limits P P P P P P P  Dermal general, ano-genital staining 1 1 1 3506 Within normal limits P P P P P P P P P P P = present, 1 = slight 
Huntingdon Life Sciences 10-2226 

 MA-252E-93-23 

Non-Pregnant 

Females Individual Physical Examination Findings - Phase 2 

Animal Phase PRE PHNumber Observations Day 1 2 3 4 5 6 7 8 9 10 Group 1 - 0 ppm 1507 Within normal limits P P P P P P  Dermal general, scab(s), ear(s) P P P P  Gastrointestinal, decreased fecal volume P 1508 Within normal limits P P P P P P P P P P P 1509 Within normal limits P P P P P P  Dermal general, scab(s), ear(s) P P P P P 1510 Within normal limits P P P P P P P P  Gastrointestinal, no stool P P P  Miscellaneous, decreased food consumption P P P 1511 Within normal limits P P P P P P P P P P  Gastrointestinal, decreased fecal volume P Group 3 - 100,000 ppm 3507 Within normal limits P P P P P P P P P P P P = present 
Huntingdon Life Sciences 10-2226 

 MA-252E-93-23 

Non-Pregnant 

Females Individual Physical Examination Findings - Phase 2 

Animal Phase PRE PHNumber Observations Day 1 2 3 4 5 6 7 8 9 10 Group 3 - 100,000 ppm 3508 Within normal limits P P P P P P P P  Dermal general, scab(s), ear(s) P P  Gastrointestinal, decreased fecal volume P 3509 Within normal limits P P P P P P P P P P P 3510 Within normal limits P P P P P P P P P P P 3511 Within normal limits P P P P P P P P P  Dermal general, alopecia (head/neck), head 3 3 P = present, 3 = extreme 
Huntingdon Life Sciences 10-2226 

 MA-252E-93-23 

Non-Pregnant 

Females Individual Physical Examination Findings - Phase 2 

Animal Phase PH2 Number Observations Day 4 5 6 7 8 9 10 11 12 13 1Group 1 - 0 ppm 1507 Within normal limits P P P P P P P P P P P 1508 Within normal limits P P P P P P P P P P P 1509 Within normal limits P P P P P  Dermal general, scab(s), ear(s) P P P P P P 1510 Within normal limits P P P P P P P P P P P 1511 Within normal limits P P P P P P P P P P P Group 3 - 100,000 ppm 3507 Within normal limits P P P P P P P P P P P 3508 Within normal limits P P P P P P P P P P P 3509 Within normal limits P P P P P P P P P P P 3510 Within normal limits P P P P P P P P P P P P = present 
Huntingdon Life Sciences 10-2226 

 MA-252E-93-23 

Non-Pregnant 

Females Individual Physical Examination Findings - Phase 2 

Animal Phase PH2 Number Observations Day 4 5 6 7 8 9 10 11 12 13 1Group 3 - 100,000 ppm 3511 Within normal limits P P P P P P P P P P P P = present 
Huntingdon Life Sciences 10-2226 

 MA-252E-93-23 

Males Individual Physical Examination Findings - Phase 2 

Animal Phase PRE Number Observations Day 1 2 3 4 5 6 7 8 9 10 1Group 1 - 0 ppm 1001 Dermal general, ano-genital staining 1 1 1 1 1 1 1 1 1 1 1002 Within normal limits  Dermal general, stains on fur (head/neck), snout 1 1 1 1 1 1 1 1 

 Dermal general, scab(s), ear(s) P P P P 1003 Within normal limits P P P P P P P P P P P 1004 Within normal limits P P P  Dermal general, stains on fur (torso), ventral 1 1 1 1 1 1 1 1 

1005 Within normal limits P P P  Dermal general, alopecia (torso)  Dermal general, stains on fur (torso), ventral 1 1 1 1 1 1 1 1 Group 2 - 50,000 ppm 

2001 Dermal general, scab(s), ear(s) P P P P P P P P P P P  Oral/buccal, incisor(s) broken/missing P P 2002 Dermal general, stains on fur (head/neck), ear(s) 1 1 1 1 1 1 1 1 1 1 

P = present, 1 = slight, 2 = moderate 
Huntingdon Life Sciences 10-2226 

 MA-252E-93-23 

Males Individual Physical Examination Findings - Phase 2 

Animal Phase PRE Number Observations Day 1 2 3 4 5 6 7 8 9 10 1Group 2 - 50,000 ppm 2003 Dermal general, alopecia (head/neck) 2 2 2 2 2 2 2 2 2 2  Dermal general, ano-genital staining 1 1 1 1 1 1 1 1 1 1 

 Dermal general, stains on fur (head/neck), head 1 1 1 1 1 1 1 1 1 1 

 Dermal general, stains on fur (torso), dorsal 1 1 1 1 1 1 1 1 1 1 

 Gastrointestinal, decreased fecal volume P  Miscellaneous, decreased food consumption P 2004 General appearance, thin P P P P  Dermal general, ano-genital staining 1 1 1 1 1 1 1 1 1 1 

 Gastrointestinal, decreased fecal volume P P P P P  Miscellaneous, decreased food consumption P P P P 2005 Within normal limits P P P P P P  Dermal general, ano-genital staining 1 1 1 1 1  Gastrointestinal, unformed stool P  Gastrointestinal, decreased fecal volume P P P  Miscellaneous, decreased food consumption P P Group 3 - 100,000 ppm 3001 Within normal limits P P P P P P P P P P P P = present, 1 = slight, 2 = moderate 
Huntingdon Life Sciences 10-2226 

 MA-252E-93-23 

Males Individual Physical Examination Findings - Phase 2 

Animal Phase PRE Number Observations Day 1 2 3 4 5 6 7 8 9 10 1Group 3 - 100,000 ppm 3002 Dermal general, ano-genital staining 1 1 1 1 1 1 1 1 1 1  Dermal general, stains on fur (head/neck), head 1 1 1 1 1 1 1 1 1 1 

 Dermal general, stains on fur (torso), dorsal 1 1 1 1 1 1 2 2 2 2 2 

 Gastrointestinal, no stool P  Miscellaneous, decreased food consumption P 3003 Within normal limits P P P P P P P P P P P 3004 Within normal limits P P P P P P P P P  Dermal general, scab(s), ear(s) P P 3005 Within normal limits P P P P P P P P P P  Gastrointestinal, decreased fecal volume P P = present, 1 = slight, 2 = moderate 
Huntingdon Life Sciences 10-2226 

 MA-252E-93-23 

Males Individual Physical Examination Findings - Phase 2 

Animal Phase PH2 Number Observations Day 4 5 6 7 8 9 10 11 12 13 1Group 1 - 0 ppm 1001 Dermal general, ano-genital staining 2 2 2 2 2 1 1 1 2 2 1002 Within normal limits P P P P P P P P P P P 1003 Within normal limits P P P P P P P  Dermal general, alopecia (head/neck) 2 2 2 2 1004 Within normal limits P P P P P P P P  Dermal general, ano-genital staining 1 1 1005 Within normal limits P P P P P P P P P P  Dermal general, alopecia (torso) 2 Group 2 - 50,000 ppm 2001 Dermal general, scab(s), ear(s) P P P P P P P P P P P 2002 Within normal limits P P P P P P  Dermal general, stains on fur (head/neck), ear(s) 1 1 1 1 1 P = present, 1 = slight, 2 = moderate 
Huntingdon Life Sciences 10-2226 

 MA-252E-93-23 

Males Individual Physical Examination Findings - Phase 2 

Animal Phase PH2 Number Observations Day 4 5 6 7 8 9 10 11 12 13 1Group 2 - 50,000 ppm 2003 Dermal general, ano-genital staining 1 1 1 1 1 1 1 1 1 1  Dermal general, stains on fur (head/neck), head 1 1 1 1 1 1 1 1 1 1 

 Dermal general, stains on fur (torso), dorsal 1 1 1 1 1 1 1 1 1 1 

2004 Dermal general, ano-genital staining 1 1 1 1 1 1 1 1 1 1 2005 Within normal limits P P P P P P P P P P P Group 3 - 100,000 ppm 3001 Within normal limits P  Dermal general, ano-genital staining 2 2 2 2 2 2 2 2  Gastrointestinal, unformed stool P  Gastrointestinal, no stool P  Miscellaneous, decreased food consumption P 3002 Dermal general, ano-genital staining 1 1 1 1 1 1 1 1 1 1  Dermal general, stains on fur (head/neck), head 1 1 1 1 1 

 Dermal general, stains on fur (torso), dorsal 1 1 1 1 1 1 1 1 1 1 

3003 Within normal limits P P P P P P P P P P P P = present, 1 = slight, 2 = moderate 
Huntingdon Life Sciences 10-2226 

 MA-252E-93-23 

Males Individual Physical Examination Findings - Phase 2 

Animal Phase PH2 Number Observations Day 4 5 6 7 8 9 10 11 12 13 1Group 3 - 100,000 ppm 3004 Within normal limits P P P P P P P P P P P 3005 Within normal limits P P P P P P P P P P P P = present 
Huntingdon 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-Pregnant 
Huntingdon 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 5 

 
Huntingdon 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 5 

 
Huntingdon 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-Pregnant 
Huntingdon 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 5 

 
Huntingdon 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..............................................................................................................165 

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 rabbits 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 sacrifices. 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 121











































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-Pregnant 
Prof. 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 

University 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 166
Prof. 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 167
Prof. 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 168
Prof. W.Dekant; T.Schmidt: Biotransformation of 2,3,3,3-tetrafluoropropene (1234yf) page 4 of 19

Table of Contents 

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-cysteine ....... 6 

3.2 19F-NMR method .......................................................................................... 7 

3.3 LC/MS-MS method ....................................................................................... 7 

4 Results.......................................................................................................... 9 

4.1 Inhalation exposure and urine sampling....................................................... 9 

4.2 19F-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 169
Prof. W.Dekant; T.Schmidt: Biotransformation of 2,3,3,3-tetrafluoropropene (1234yf) page 5 of 19 

1. Summary

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 ppm of HFO-1234yf for one hour in an open dynamic chamber. Nonpregnant rabbits were not exposed to 50,000 ppm as this data had previously been 

determined [1-2]. 

Urine analysis by 19F-NMR indicate N-acetyl-S-(3,3,3-trifluoro-2-hydroxypropanyl)-Lcysteine and S-(3,3,3-trifluoro-2-hydroxypropanyl)-mercaptolactic acid as major 

metabolites, representing more than 78% of total 19F 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 representing more than 46% of total 19F related signals 

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

Quantification of the HFO-1234yf-mercapturic acid isomers by LC/MS-MS showed no 

significant difference (P = 0.58, Student’s t-test) 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. More than 83% of recovered Nacetyl-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, the total 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, 

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

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

observed between the different groups. 

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Prof. 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, no 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, studies examining this 

hypothesis did not find major variations. In rabbits, 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 19F-NMR 

signals). Whereas 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. Urine was collected for 48 

hours in 12 hours intervals for biotransformation analysis. 

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Prof. 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)-Lcysteine

3,3,3-Trifluoro-1,2-epoxypropane was slowly added to a well stirred solution of Nacetyl-L-cysteine in ethanol/water (1/2 v/v) adjusted to pH > 9 with triethylamine. The 

reaction mixture was maintained to 4 0C 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, [D6]-DMSO): δ = 1.02 

– 1.99 (m, 20 H, cyclohexane -CH2-), 1.82 (s, 3 H, -COCH3), 2.60 (dd, JAB = 13.6 Hz, 

JAX = 10.0 Hz, 1H, HA), 2.73 (dd, JBA = 13.6 Hz, JBX = 3.3 Hz, 1H, HB), 2.82 (dd, JAB = 

13.5 Hz, JAX1 = 6.1 Hz, 1H, HA), 2.97 (dd, JBA = 13.5 Hz, JBX1 = 5.1 Hz, 1H, HB), 3.92- 

3.00 (m, 2 H, cyclohexane -NCH-), 4.01 (m, 1 H,HX), 4.1 (m, 1 H, HX1), 7.58 (d, J = 

7.25 Hz, 1H, NHCOCH3). 

3.2 19F-NMR method 

19F-NMR spectra were recorded with a Bruker DRX 300 NMR spectrometer with a 5 

mm fluoride probe operating at 282 MHz. 19F chemical shifts were referenced to 

external CFCl3. Spectra were recorded with a 30 0 pulse, a pulse length of 11 μsec 

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

recorded to obtain a good signal to noise ratio (S/N). The 19F 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 μL) of the obtained supernatant was added to 80 μL of deuterium oxide. No 

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Prof. W.Dekant; T.Schmidt: Biotransformation of 2,3,3,3-tetrafluoropropene (1234yf) page 8 of 19

further sample workup was performed before 19F-NMR analysis. NMR spectra were 

analyzed with TopSpin 3.0 (Bruker). 

3.3 LC-MS/MS method – quantification 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 μm; Maisch, 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 μL/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-

hydroxypropanyl)-L-cysteine. 

Substance Transition

[m/z]

Declustering 

potential [V]

Collision 

energy [V]

Metabolite 274-145

N-Acetyl-S-(3,3,3-trifluoro-2-

hydroxypropanyl)-L-cysteine 274-75 -26 -30

Internal standard 278-149

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

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Prof. 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 μL of the dilutions were added to 

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

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

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

additionally 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 R2

-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 0.45μg/L (1.63 nmol/L) and limit of 

detection (LOD) was 0.14 μg/L (0.51 nmol/L) in urine. Standard deviations (mean ± 

SD) were calculated using MicrosoftExcel® spreadsheets and default setting. 

Statistical analysis was performed by GraphPad Prism 5. 

4 Results

4.1 Inhalation exposure and urine sampling

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: 

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Prof. 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/group, 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 0C until analysis. 

4.2 19F-NMR – metabolites 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 Institute for a previous study [1]. 

Fig. 1 illustrates that there are no significant differences between the urine 19Fspectra of female, pregnant female and male rabbits. All spectra show the same 

major peaks with very similar relative intensities. 

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Prof. W.Dekant; T.Schmidt: Biotransformation of 2,3,3,3-tetrafluoropropene (1234yf) page 11 of 19

Fig. 1: 19F-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, JHF = 7.4Hz), N-acetyl-S-(3,3,3-trifluoro-2-hydroxypropanyl)-Lcysteine (δ = -78.7 ppm; d, JHF = 6.6Hz) and S-(3,3,3-trifluoro-2-hydroxypropanyl)-

mercaptolactic acid (δ = -78.9 ppm; d, JHF = 6.9Hz). Beside these known metabolites, 

there were two unidentified metabolites present. 

Fig. 2: 19F-NMR spectrum of 0-12h post inhalation exposure urine; resonances were 

assigned to (A) unidentified metabolite (δ = -77.1 ppm; d, JHF = 7.2Hz), (B) 

3,3,3-trifluoro-1,2-dihydroxypropane (δ = -77.2 ppm; d, JHF = 7.4Hz), (C) 

unidentified metabolite (δ = -78.4 ppm; d, JHF = 6.8Hz), (D) N-acetyl-S-(3,3,3-

trifluoro-2-hydroxypropanyl)-L-cysteine (δ = -78.7 ppm; d, JHF = 6.6Hz), (E) S-

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

= 6.9Hz). 

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Prof. W.Dekant; T.Schmidt: Biotransformation of 2,3,3,3-tetrafluoropropene (1234yf) page 12 of 19

Fig. 3: 19F-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, JHF = 6.8Hz), (C) N-acetyl-S-(3,3,3-

trifluoro-2-hydroxypropanyl)-L-cysteine (δ = -78.7 ppm; d, JHF = 6.6Hz), (D) S-

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

= 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 differences 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. 

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Prof. 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-dihydroxypropane, (E) 

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

4.3 LC-MS/MS – urine 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 μg/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). 

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Prof. W.Dekant; T.Schmidt: Biotransformation of 2,3,3,3-tetrafluoropropene (1234yf) page 14 of 19

Fig.5: Quantifier: mass transition - 274-145 (A); qualifier: mass transition 274-75

(B). 

B

A

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Prof. 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 (μmol); male (M), pregnant female (P), non-pregnant female 

(F). 

Time 

interval Group Animal 1 [μmol]

Animal 2 

[μmol]

Animal 3 

[μmol]

Animal 4

[μmol]

Animal 5 

[μmol]

Animal 6 

[μmol]

Sample 

number

Average 

amount 

excreted 

[μmol]

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 100,000 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-

hydroxypropanyl)-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 

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Prof. W.Dekant; T.Schmidt: Biotransformation of 2,3,3,3-tetrafluoropropene (1234yf) page 16 of 19

metabolite elimination followed 1st order kinetics with a half-life of approx. 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 rabbits following the inhalation exposure. 

Elimination 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: Mean concentrations of N-acetyl-S-(3,3,3-trifluoro-2-hydroxypropanyl)-Lcysteine (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 inhalation 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 μmol; male 50,000 ppm – 86.40 

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

50.47 μmol; pregnant 50,000 ppm – 60.97 μmol; non-pregnant 100,000 ppm – 43,10 

Page 181
Prof. W.Dekant; T.Schmidt: Biotransformation of 2,3,3,3-tetrafluoropropene (1234yf) page 17 of 19

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

non-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)-Lcysteine (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 (pregnant female 100,000 

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

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

and pregnant female rabbits (Tabl. 4). 

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Prof. 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 the 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 in this study, in general support the 

results of earlier metabolic studies of this compound, showing a biotransformation of 

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

with glutathione. Metabolites identified by 19F-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-dihydroxypropane and 3,3,3-trifluoropropionic acid. In contrast 

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

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

trifluoro-2-hydroxypropanyl)-L-cysteine. Comparison of 19F 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 excreted 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)-Lcysteine mean 

excretion within 48 

hours [μmol]

N-acetyl-S-(3,3,3-

trifluoro-2-

hydroxypropanyl)-Lcysteine mean fraction 

of the five main 

metabolites [%] *

Total fluoro 

metabolites 

excretion 

[μmol]

Inhaled

gas dose 

[μmol]**

Metabolised

amount [%]

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 0,04

F 100,000 ppm 43,10 29,10 148,11 414,755 0,04

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Prof. 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. 233(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 TrakTM mass flowmeter, size 0-10 Lpm, Model 822-13-OVI-PVI-VI (Sierra 

Instruments) 

Top TrakTM 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® MicrofibreTM Disposable Filter Units, Grade DQ 
Huntingdon Life Sciences 10-2226 Page 186 

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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 O2 (Oxigraf) 

Miscellaneous 

Stop watch, Fisher Scientific, Model 51439788 

Balance 

Pelouze 4040 
Huntingdon Life Sciences 10-2226 Page 187 

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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 

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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 calibration 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 Concentration1 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 

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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 injection. 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% range, the original was accepted. If the value 

of the average was outside the 15% range, the Study Director decided if a new graph was 

to be prepared. 
Huntingdon Life Sciences 10-2226 Page 190 

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Miran® Calibration Appendix L 

Calibration Curve for HFO-1234yf 

y = 110.72x

3

 + 43.952x2 + 463.97x

R2

 = 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 

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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 calibration 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 Concentration1 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 

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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 injection. 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% range, the original was accepted. If the value 

of the average was outside the 15% range, the 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 = 94388x3 + 6018.7x2 + 125250x

R2

 = 0.9986

0

20000

40000

60000

80000

100000

120000

140000

0.000 0.200 0.400 0.600 0.800 1.000

Absorbance (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 calibration 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 Concentration1 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 injection. 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% range, the original was accepted. If the value 

of the average was outside the 15% range, the 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

 + 

R2

 = 

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
Appendix N Page 198
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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