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Working for a better future

REPORT

2-bromo-3,3,3-trifluoropropene: Dose Range Finding Study by 

Inhalation Administration to Rats for 2 Weeks

HLS study number: WAG0024

Version ID: Final

Issue date: 31 August 2012
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Details of Sponsor and Test Facilities

Sponsor American Pacific Corporation

3883 Howard Hughes Parkway

Suite 700

Las Vegas

Nevada 89169

USA

Test facility Huntingdon Life Sciences

Primary location Huntingdon Research Centre

Woolley Road

Alconbury

Huntingdon

Cambridgeshire

PE28 4HS

UK

Test facility Huntingdon Life Sciences

Histology Eye Research Centre

Eye

Suffolk

IP23 7PX

UK
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Table of Contents

Details of Sponsor and Test Facilities ................................................................................... 2

Compliance with Good Laboratory Practice .......................................................................... 5

Quality Assurance Statement................................................................................................ 6

Contributing Scientists .......................................................................................................... 7

Summary .............................................................................................................................. 8

1. Introduction .................................................................................................................. 9

1.1 Objective........................................................................................................... 9

1.2 Regulatory compliance ..................................................................................... 9

1.3 Test system ...................................................................................................... 9

1.4 Route of administration..................................................................................... 9

1.5 Treatment groups and doses ............................................................................ 9

2. Experimental Procedure............................................................................................. 10

2.1 Study schedule and structure.......................................................................... 10

2.1.1 Duration of treatment........................................................................... 10

2.1.2 Time schedule..................................................................................... 10

2.1.3 Identity of treatment groups................................................................. 10

2.2 Test substance and formulation ...................................................................... 11

2.2.1 Test substance.................................................................................... 11

2.3 Animal management....................................................................................... 12

2.3.1 Animal supply, acclimatisation and allocation...................................... 12

2.3.2 Animal housing, diet and water supply ................................................ 12

2.3.3 Administration ..................................................................................... 13

2.4 Serial observations ......................................................................................... 14

2.4.1 Clinical observations ........................................................................... 14

2.4.2 Bodyweight ......................................................................................... 14

2.4.3 Food consumption............................................................................... 14

2.5 Necropsy and Histology.................................................................................. 15

2.5.1 Method of kill....................................................................................... 15

2.5.2 Macroscopic pathology........................................................................ 15

2.5.3 Organ weights..................................................................................... 15

2.5.4 Fixation ............................................................................................... 16

2.5.5 Histology ............................................................................................. 17

2.6 Pathology........................................................................................................ 17

2.6.1 Light microscopy ................................................................................. 17

2.7 Computer systems.......................................................................................... 19

2.8 Data treatment................................................................................................ 19

2.8.1 Definition of “Week”............................................................................. 19

2.8.2 Signs................................................................................................... 20

2.8.3 Bodyweight ......................................................................................... 20

2.8.4 Pathology............................................................................................ 20

2.8.5 Statistical analysis............................................................................... 20

2.9 Quality assurance and archiving procedures .................................................. 21

2.9.1 Quality assurance ............................................................................... 21

2.9.2 Archives .............................................................................................. 21

2.10 Deviations from protocol ................................................................................. 21

3. Results....................................................................................................................... 22
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3.1 Chamber atmosphere conditions .................................................................... 22

3.2 Signs associated with dosing.......................................................................... 22

3.3 Bodyweight..................................................................................................... 22

3.4 Food consumption .......................................................................................... 23

3.5 Organ weights................................................................................................. 23

3.6 Macropathology .............................................................................................. 23

3.7 Histopathology................................................................................................ 23

4. Discussion.................................................................................................................. 25

5. Conclusion ................................................................................................................. 26

6. References................................................................................................................. 27

List of Figures

Figure 1 Bodyweight ..................................................................................................28

List of Tables

Table 1 Bodyweight ..................................................................................................29

Table 2 Organ weights..............................................................................................33

Table 3 Macropathology ...........................................................................................35

Table 4 Histopathology .............................................................................................36

List of Appendices

Appendix 1 Clinical signs................................................................................................37

Appendix 2 Signs associated with dosing .......................................................................39

Appendix 3 Bodyweight ..................................................................................................58

Appendix 4 Food consumption .......................................................................................62

Appendix 5 Organ weights..............................................................................................66

Appendix 6 Macropathology and histopathology.............................................................68

List of Annexes

Annex 1 Aerosol technology and inhalation analysis report........................................93

Annex 2 Pathology report.........................................................................................110

Annex 3 Certificate of analysis.................................................................................116

Annex 4 GLP compliance statements ......................................................................118


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Contributing Scientists

2-bromo-3,3,3-trifluoropropene: Dose Range Finding Study by Inhalation 

Administration to Rats for 2 Weeks

Study management

Paul J Horrell BSc (Hons) IDT

Study Director

William N Hooks MIBMS BSc (Hons)

Senior Toxicologist

Formulation and inhalation analysis

Caroline Finucane PhD

Head of Formulation & Inhalation Analysis

Aerosol technology

Simon A Moore BSc (Hons) PhD MRSC

Head of Aerosol Technology

Pathology

Martin Brown BSc PGCE CBiol MSB PhD

Principal Pathologist
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Summary

The objective of this study was to assess the local and systemic toxic potential of

2-bromo-3,3,3-trifluoropropene, a fire extinguishing agent, when administered by inhalation

to Crl:CD(SD) rats for 6 hours a day, 5 days a week, for 2 weeks.

Three groups, each comprising three male and three female rats received 

2 -bromo-3,3,3 -trifluoropropene at target exposure levels of 500, 1200 or 3000 ppm. A 

similarly constituted Control group received clean air only, at the same volume dose. 

During the study, clinical condition, bodyweight, food consumption, organ weight, 

macropathology and histopathology investigations were undertaken. 

Results

The achieved aerosol concentrations were 533, 1171 and 2980 ppm (107, 98 and 99% of 

target) for Groups 2-4 respectively.

Treatment-related, but transient, clinical signs, related to inhalation of an irritant material or 

possible CNS effects, were evident during and after exposure. Impaired 

bodyweight/bodyweight gain was evident, over the 2 week treatment period, for treated males

in a dosage related manner. Total food consumption was slightly reduced for treated males.

Histopathological changes related to treatment were seen mainly in the nasal turbinates and, 

to a much lesser extent, in the larynx, trachea and lungs.

In the olfactory epithelium atrophy/disorganisation and inflammation were seen in males and 

females exposed to 1171 or 2980 ppm. Atrophy of the olfactory nerve fibres was seen in 

males and females exposed to 2980 ppm and in females exposed to 1171 ppm. Inflammatory

exudate in the airways was seen in males and females exposed to 2980 ppm and in a single 

male exposed to 1171 ppm.

A single female exposed to 2980 ppm had perivascular and peribronchiolar inflammatory

infiltrate and bronchiolar epithelial hyperplasia in the lungs, epithelial hyperplasia and 

subepithelial inflammation in the larynx and epithelial hyperplasia at the tracheal bifurcation. 

The relationship to treatment of these changes is uncertain.

Conclusion

Histopathological changes related to 2-bromo-3,3,3-trifluoropropene exposure were seen in 

the nasal turbinates of rats exposed to 1171 or 2980 ppm. The no observed adverse effect 

level (NOAEL) was 533 ppm, as this level was not associated with any histopathological 

changes or adverse in-life findings.
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1. Introduction

1.1 Objective

The objective of this study was to assess the local and systemic toxic potential of

2-bromo-3,3,3-trifluoropropene, a fire extinguishing agent, when administered by inhalation

to rats for 2 weeks, to select suitable exposure levels for a subsequent 13 week toxicity study.

1.2 Regulatory compliance

Due to the preliminary nature of this study, no specific regulations or guidelines are 

applicable. 

1.3 Test system

The rat was chosen as the test species because of its acceptance as a predictor of toxic change 

in man and the requirement for a rodent species by regulatory agencies. The Crl:CD (SD)

strain was used because of the historical control data available in this laboratory.

1.4 Route of administration

The inhalation route of administration was chosen to simulate the conditions of potential 

human exposure.

1.5 Treatment groups and doses

In a previous 2 week dose range finding study (Huntingdon Life Sciences Study Number 

WAG0013), 3 groups of rats were exposed to 2-bromo-3,3,3-trifluoropropene for 6 hours per 

day, 5 days per week at achieved concentrations of 4721, 8901 and 19098 ppm.

There were no unscheduled deaths at doses up to 19098 ppm. In-life findings consisted of 

bodyweight losses at 19098 ppm, reduced bodyweight gain at 4721 or 8901 ppm, and 

reduced food consumption and transient clinical signs at all dose levels.

Histopathological changes related to treatment were seen in the nasal turbinates, larynx and 

trachea and were related to exposure to an irritant material. Overall, the most marked 

changes were in the nasal cavity, with less severe changes in the larynx and trachea. Due to 

the histopathological changes evident at 4721 ppm, a no observed adverse effect level 

(NOAEL) was not established. 

The target exposure levels of 500, 1200 and 3000 ppm were selected to explore the extent of 

the previously seen histopathological changes at lower exposure levels.
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2. Experimental Procedure

2.1 Study schedule and structure

2.1.1 Duration of treatment

The test substance, 2-bromo-3,3,3-trifluoropropene, was administered over a period of 

2 consecutive weeks (6 hours daily exposure, 5 days per week). The necropsy procedures 

were completed in 1 day. The duration of treatment is reported as 2 weeks. 

2.1.2 Time schedule

Study initiation:

(Protocol signed by Study Director)

27 March 2012

Experimental start date:

(Pre-treatment chemistry assays)

29 March 2012

Animal arrival: 29 March 2012

Treatment commenced: 16 April 2012

Necropsy completed: 30 April 2012

Experimental completion date:

(pathology) 

6 July 2012

Study completion: 31 August 2012

2.1.3 Identity of treatment groups

The study consisted of one Control and three treated groups of rats, identified as follows:

Group Treatment

Target 

exposure level

(ppm)

No. of animals Animal numbers Cage numbers

Male Female Male Female Male Female

1 Control 0 3 3 1-3 13-15 1 5

2 2-bromo-3,3,3-

trifluoropropene

500 3 3 4-6 16-18 2 6

3 2-bromo-3,3,3-

trifluoropropene

1200 3 3 7-9 19-21 3 7

4 2-bromo-3,3,3-

trifluoropropene

3000 3 3 10-12 22-24 4 8
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2.2 Test substance and formulation

2.2.1 Test substance

Information supplied by the Sponsor regarding the test substance is contained in the test 

substance data sheet, which is retained in study records, and the Certificate of Analysis, 

which is presented in Annex 3.

The following information is given in summary: 

Identification: 2-bromo-3,3,3-trifluoropropene (stabilized)

Usage: Fire extinguishing agent

Description: Liquid below 33C

Clear and may be colourless to light yellow tint

Storage conditions: At ambient temperature or in a refrigerator.

Supplier: Sponsor

Batch number: KgF080

Date of receipt: 12 September 2011

Quantity received: 2 × 150 kg cylinders

Reassay date: 1 August 2012

Purity: 99.9% (excluding stabilizers)

Stabilisers:

The Sponsor was responsible for the characterisation of the test substance and the 

documentation of the methods of synthesis, fabrication or derivation and stability.
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2.3 Animal management

2.3.1 Animal supply, acclimatisation and allocation

Crl:CD (SD) rats (a total of 15 male and 15 female) were received from Charles River (UK) 

Ltd. The rats were ordered at 49 to 56 days of age and within a weight range of 290 to 330 g 

for males and 202 to 233 g for females.

On arrival, the animals were removed from the transit boxes and allocated to study cages. 

Using the sequence of cages in the battery, one animal at a time was placed in each cage with 

the procedure being repeated until each cage held the appropriate number of animals. Each 

sex was allocated separately.

Each animal was assigned a number and identified uniquely within the study by a tail tattoo. 

Each cage label was colour-coded according to group and was numbered uniquely with cage 

and study number, as well as the identity of the occupants.

The animals were allowed to acclimatise to the conditions described below for 18 days before 

treatment commenced. For those animals selected for this study, their age at the start of 

treatment was 67 to 74 days and their bodyweights were in the range of 386 to 436 g for

males and 227 to 283 g for females.

The spare animals were removed from the study room at the end of the study.

2.3.2 Animal housing, diet and water supply

Animals were housed inside a restricted access rodent facility (Building Y14, Room 011). 

The facility was designed and operated to minimise the entry of external biological and 

chemical agents and to minimise the transference of such agents between rooms. Before the 

study the room was cleaned and disinfected.

Each animal room was supplied with filtered fresh air, which was passed to atmosphere and 

not re-circulated. The temperature and relative humidity controls were maintained within the 

range of 19 to 23°C and 40 to 70% respectively and monitored continuously. There were no 

deviations from these ranges. Artificial lighting was controlled to give a cycle of 12 hours 

continuous light and 12 hours continuous dark per 24 hours.

Alarms were activated if there was any failure of the ventilation system, or temperature limits 

were exceeded. A stand-by electricity supply was available to be automatically brought into 

operation should the public supply fail.

The animals were housed three of one sex per cage. The cages were made of a polycarbonate 

body with a stainless steel mesh lid. Softwood bark-free fibre was used as bedding and was 

sterilised by autoclaving and changed at appropriate intervals each week. Cages, food 

hoppers and water bottles were changed at appropriate intervals. 

The cages constituting each group were blocked together by sex and the groups were 

dispersed in batteries so that possible environmental influences arising from their spatial 

distribution were equilibrated, as far as was practicable. Additionally, batteries of cages were 

rotated around the room at weekly intervals to further minimise possible spatial variations. 
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The animals were allowed free access to a standard rodent diet (Rat and Mouse No. 1 

Maintenance Diet), except during the dosing procedure. This diet contained no added 

antibiotic or other chemotherapeutic or prophylactic agent.

Potable water taken from the public supply was freely available via polycarbonate bottles 

fitted with sipper tubes, except during the dosing procedure.

Each cage of animals was provided with an Aspen chew block and a plastic shelter for 

environmental enrichment. The soft white untreated wood blocks were provided throughout 

the study and were replaced when necessary; plastic shelters were replaced at the same time 

as the cages.

Each batch of diet was analysed routinely by the supplier for various nutritional components 

and chemical and microbiological contaminants. Supplier’s analytical certificates were 

scrutinised and approved before any batch of diet was released for use. The quality of the 

water supply is governed by regulations published by the Department for Environment, Food 

and Rural Affairs. Certificates of analysis were received routinely from the water supplier

and the suppliers of the bedding and Aspen chew blocks. Since the results of these various 

analyses did not provide evidence of contamination that might have prejudiced the study, 

they are not presented.

No other specific contaminants that were likely to have been present in the bedding, chew 

blocks, diet or water were analysed, as none that may have interfered with or prejudiced the 

outcome of the study was known.

2.3.3 Administration

Details of administration and analysis of the test atmospheres together with the results 

obtained are presented in Annex 1.

Each exposure system consisted of a Glass vaporiser and a whole body exposure chamber. 

Different doses of 2-bromo-3,3,3-trifluoropropene were achieved by varying the 

concentrations of the test article in the system whilst keeping the duration of exposure 

constant. Group 1 was exposed to clean air only at a target concentration the same as that 

specified for Group 4. Groups 2, 3 and 4 were exposed to 2-bromo-3,3,3-trifluoropropene, 

6 hours/day for 5 days each week (Monday to Friday) for 2 weeks.
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2.4 Serial observations

Dated and signed records of all activities relating to the day by day running and maintenance 

of the study within the animal unit as well as to the group observations and examinations 

outlined in this experimental procedure were recorded in the Study Day Book. In addition, 

observations relating to individual animals made throughout the day were recorded.

All observations described below were performed in cage number sequence except where 

otherwise indicated.

2.4.1 Clinical observations

Animals were inspected visually at least twice daily for evidence of ill-health or reaction to 

treatment. Cages were inspected daily for evidence of ill-health amongst the occupant(s). 

Any deviation from normal was recorded at the time in respect of nature and severity, date 

and time of onset, duration and progress of the observed condition, as appropriate.

Daily, on exposure days, during Week 1 of treatment, detailed observations were recorded at 

the following times in relation to dose administration:

Pre-exposure

During exposure

Between one and two hours after completion of dosing of all groups

As late as possible in the working day

During Week 2 of treatment, detailed observations were only recorded before exposure and 

between one and two hours after completion of dosing of all groups.

In addition, a more detailed weekly physical examination was performed on each animal to 

monitor general health.

During the acclimatisation period, observations of the animals and their cages were recorded

at least once per day.

2.4.2 Bodyweight

The weight of each rat was recorded daily from Day -5, throughout the treatment period and 

before necropsy. 

2.4.3 Food consumption

The weight of food supplied to each cage, that remaining and an estimate of any spilled was 

recorded daily from Day -5 and throughout the treatment period. From these records the

mean daily consumption per animal (g/rat/day) was calculated for each cage.
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2.5 Necropsy and Histology

2.5.1 Method of kill

Animals were killed by intra-peritoneal sodium pentobarbitone, followed by exsanguination. 

The sequence in which the animals were killed after completion of treatment was selected to 

allow satisfactory inter-group comparison.

2.5.2 Macroscopic pathology

All animals were subject to a detailed necropsy.

After a review of the history of each animal, a full macroscopic examination of the tissues 

was performed. All external features and orifices were examined visually. The cranial roof 

was removed to allow observation of the brain, pituitary gland and cranial nerves. After 

ventral mid-line incision, the neck and associated tissues and the thoracic, abdominal and 

pelvic cavities and their viscera were exposed and examined in situ. Any abnormal position, 

morphology or interaction was recorded.

The requisite organs were weighed and external and cut surfaces of the organs and tissues 

were examined as appropriate. Any abnormality in the appearance or size of any organ and 

tissue was recorded and the required tissue samples preserved in appropriate fixative.

The retained tissues were checked before disposal of the carcass.

2.5.3 Organ weights

The following organs, taken from each animal killed after 2 weeks of treatment, were 

dissected free of adjacent fat and other contiguous tissue and the weights recorded:

Brain Liver

Heart Lungs with mainstem bronchi 

Kidneys Spleen

Bilateral organs were weighed together. Organ weights were also adjusted for terminal 

bodyweight, using the weight recorded before necropsy.
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2.5.4 Fixation

Testes were fixed in modified Davidson's fluid and eyes were fixed in Davidson's fluid. 

Samples (or the whole) of the other tissues listed below from all animals were preserved in 

10% neutral buffered formalin:

Adrenals Optic nerves

Aorta - thoracic Ovaries

Brain Pancreas

Caecum Pituitary

Colon Prostate

Duodenum Rectum

Epididymides Salivary glands - submandibular

Eyes - sublingual

Femurs Sciatic nerves

Harderian glands Seminal vesicles

Head# Skeletal muscle 

Heart Skin with mammary glands (inguinal)

Ileum Spinal cord

Jejunum Spleen

Kidneys Sternum

Lachrymal glands Stomach

Larynx* Testes

Liver Thymus

Lungs* Thyroid with parathyroids

Lymph nodes - mandibular Tongue

- mesenteric Trachea (including bifurcation)*

- tracheo-bronchial Urinary bladder

Nasal turbinates* Uterus and cervix

Oesophagus Vagina

* Tissues examined microscopically

# Including nasal cavity, paranasal sinuses and nasopharynx

Samples of any abnormal tissues were also retained. In those cases where a lesion was not 

clearly delineated, contiguous tissue was fixed with the grossly affected region and sectioned 

as appropriate.

The remaining samples were not examined histologically, but are retained against any future 

requirement for microscopic examination.
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2.5.5 Histology

For those animals specified in the Pathology section, the relevant tissues were subject to 

histological processing.

Tissue samples were dehydrated, embedded in paraffin wax, sectioned at approximately four 

to five micron thickness and stained with haematoxylin and eosin.

Those tissues subject to histological processing included the following regions:

Larynx - 5 sections

Lungs - section from all major lobes, to include bronchi

Nasal Turbinates - 3 sections

Trachea - including bifurcation

For bilateral organs, sections of both organs were prepared. A single section was prepared 

from each of the remaining tissues required for microscopic pathology.

2.6 Pathology

2.6.1 Light microscopy

Microscopic examination was performed as follows:

Larynx, lungs, nasal turbinates, trachea and tracheo-bronchial lymph nodes were 

examined for all animals sacrificed on completion of the scheduled treatment period.

Tissues reported at macroscopic examination as being grossly abnormal were 

examined in line with current practice.

Findings were either reported as "present" or assigned a severity grade. In the latter case one 

of the following five grades was used - minimal, slight, moderate, marked or severe. 

The descriptions listed below are an attempt to define the grades in general terms for all 

microscopic findings reported in a study and are not specific for a particular tissue. They are 

more strictly applicable to some types of lesion than to others, and there may therefore be a 

few lesions for which these definitions are not completely appropriate.
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Thresholds for the grading of lesions for certain tissues may vary with type of study and also 

may vary, generally for all tissues or the majority of tissues, with the duration of the study.

In increasing order of degree/severity.

Minimal The lowest grade of a lesion which is discernible/distinguishable from 

normal and is often subtle and/or involving a very small area of the 

organ.

Slight Clearly discernible/distinguishable from normal with more extensive or 

more apparent changes than minimal.

Moderate Generally involves a significant proportion of the affected organ or 

tissue.

Marked Involves the majority of the affected organ or tissue.

Severe Involves all or almost all of an affected organ or tissue.

A reviewing pathologist undertook a peer review of the microscopic findings.
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2.7 Computer systems

The computer systems with version numbers that were used on this study to acquire and 

quantify data include: 

Liberate Release 2 in-house system used for reporting clinical signs, dosing signs, 

bodyweights, food consumption, organ weights, macropathology and 

histopathology.

Sample Registry System (version 1.0) used for Formulation and Inhalation Analysis 

data.

Waters Empower 2, Build 2154, Feature Release 4, Service Pack D and used for 

Formulation and Inhalation Analysis.

Xybion Path/Tox System (version 4.2.2) used for clinical signs, dosing signs, 

bodyweights, food consumption, organ weights, macropathology and 

histopathology.

Xybion Pristima (version 6.2) used for Pharmacy test substance management.

2.8 Data treatment

This report contains serial observations pertaining to all weeks of treatment completed, 

together with signs data collected during the necropsy period. The only serial observations 

relating to the acclimatisation period included in this report relate to Week -1 bodyweights 

and food consumption. 

Summary statistics (e.g. means and standard deviations) presented in this report were 

calculated from computer-stored individual raw data. The summary statistics and the 

individual data were stored in the computer to a certain number of decimal places, different 

for each parameter. For presentation purposes, however, they were usually rounded to fewer 

places. It is, therefore, not generally possible to reproduce the presented means and standard 

deviations exactly using the presented individual data.

Throughout the report the following abbreviations are used:

M Male

F Female

N Number of animals/cages examined

SD Standard deviation

The death codes in the appendices have the following meaning: 

T Scheduled kill

2.8.1 Definition of “Week”

The first week of the study started at midnight prior to treatment commencing and ended at 

midnight on the seventh day following. Dosing took place over the first five days of the 

week only. Subsequent experimental weeks of treatment were of the same duration.
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2.8.2 Signs

Signs are considered in two parts: observations made in association with treatment, classified

as “dosing signs” and extended changes in condition, classified as “clinical signs”. Both are 

presented for each animal that showed signs, providing detail of type of sign, day or week of

occurrence and information on the duration of the sign applicable. 

2.8.3 Bodyweight

Group mean weight changes were calculated from the weight changes of individual animals.

2.8.4 Pathology

The abbreviations used in the Appendix have the following meanings: 

Bifurc. Bifurcation

Tissues which could not be examined are specified in the Appendix. The absence of a 

comment for a tissue scheduled for examination therefore indicates that the tissue was 

considered to be normal. In all tabular presentations of data the tissues specified in the 

protocol for histopathological examination precede other tissues.

2.8.5 Statistical analysis

The small sample size precludes meaningful statistical evaluation.
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2.9 Quality assurance and archiving procedures

2.9.1 Quality assurance

Details of the Quality Assurance inspections and audits are presented on the Quality 

Assurance Statement.

2.9.2 Archives

Following completion of this study all raw data, specimens and samples, except those 

generated or used during any Sponsor’s or supplier’s analysis, were stored in the archives of 

Huntingdon Life Sciences. Types of sample and specimen which are unsuitable, by reason of 

instability, for long term retention and archiving may be disposed of after the periods stated 

in Huntingdon Life Sciences Standard Operating Procedures.

A copy of the final report and all Quality Assurance inspection records will be retained 

indefinitely. All other appropriate specimens and records will be retained for a minimum 

period of one year from the date of issue of the final report. At the end of the one year 

retention period the Sponsor will be contacted and advice sought on the above requirements. 

Under no circumstances will any item be discarded without the Sponsor’s knowledge.

2.10 Deviations from protocol

Section 5.2 of the protocol erroneously stated that “different concentrations of the test 

substance will be produced using different liquid feed rates controlled by the infusion pump 

and using syringes of an appropriate volume”. Due to the high exposure levels required, this 

method of generation was not practical. Instead different exposure levels were achieved by

varying the feed rates of the test material through metering valves from pressurised 

reservoirs.

This deviation did not adversely affect study integrity.
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3. Results

3.1 Chamber atmosphere conditions

Annex 1

The achieved chamber concentrations are summarised as follows:

Group Aerosol concentration (ppm)

Target Study mean

2 500 533

3 1200 1171

4 3000 2980

The achieved aerosol concentrations were 107, 98 and 99% of the target concentration for

Groups 2, 3 and 4, respectively.

3.2 Signs associated with dosing

Appendices 1 and 2

There were no unscheduled deaths during the course of the study.

Clinical signs seen during the first 5 days of exposure included: unresponsiveness to external 

stimuli, underactivity, gasping, slow, shallow, deep and/or irregular breathing, piloerection, 

closed or partially closed eyelids, salivation, chin rubbing and lacrymation. These signs 

generally resolved quickly on cessation of exposure to the test article. The number and 

prevalence of signs increased with dose.

3.3 Bodyweight

Figure 1, Table 1 and Appendix 3

Impaired bodyweight/bodyweight gain was evident, over the 2 week treatment period, for 

males exposed to 533, 1171 or 2980 ppm, with overall values of 0.54X, 0.56X and 0.67X 

control, respectively.

Over these first 5 days of exposure bodyweight losses of 6g, 5g and 25g were evident for

males exposed to 533, 1171 or 2980 ppm, compared with a bodyweight gain of 10g for the 

control males. Bodyweight gains were then evident for all treated groups during the off-dose 

weekend period (Days 6 and 7). During the second 5 days of exposures (Days 8-12) the 

effect of exposure on bodyweight was less marked, with bodyweight losses of 7g and 3g for 

males exposed to 533 or 1171 ppm respectively and a slight gain of 2g evident for males 

exposed to 2980 ppm, compared with a gain of 9g for controls. Gains were again evident for 

all treated groups during the second off-dose weekend period prior to necropsy.
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Bodyweight gains for females exposed to 533, 1171 or 2980 ppm, over the two week study

period, were greater than control (2.68X, 2.91X and 2.01X control, respectively). However, 

these differences were the result of lower overall gain for the control females.

Females exposed to 2980 ppm lost 4g over the first 5 days of exposure compared with a gain 

of 3g for controls (and a loss of 25g for males exposed to 2980 ppm). The effects in the 

lower exposure groups and in the second week followed the same trends seen in males.

3.4 Food consumption

Appendix 4

Food consumption was slightly reduced over the treatment period for males exposed to 

2 bromo 3,3,3 trifluoropropene.

Total food consumption (Days 1 to 14) was 0.84X, 0.87X and 0.87X control for males and 

0.97X, 1.00X and 0.91X control for females exposed to 533, 1171 or 2980 ppm respectively.

3.5 Organ weights

Table 2 and Appendix 5

There were no test article-related effects on organ weights.

3.6 Macropathology

Table 3 and Appendix 6

The macroscopic examination performed after 2 weeks of treatment revealed no intergroup 

differences of note.

The nature and incidence of all the findings were consistent with the commonly seen 

background of macroscopic changes. 

3.7 Histopathology

Table 4 and Appendix 6

Changes related to treatment with 2-bromo-3,3,3-trifluoropropene were seen mainly in the 

nasal turbinates and, to a much lesser extent, in the larynx, trachea and lungs.

Nasal turbinates

In the olfactory epithelium atrophy/disorganisation and inflammation were seen in males and 

females exposed to 1171 or 2980 ppm. Atrophy of the olfactory nerve fibres was seen in 

males and females exposed to 2980 ppm and in females exposed to 1171 ppm.

Inflammatory exudate in the airways was seen in males and females exposed to 2980 ppm 

and in a single male exposed to 1171 ppm.
Huntingdon Life Sciences

WAG0024

24

Summary of treatment related findings in the nasal turbinates for animals killed after 2 weeks of 

treatment

Group/sex 1M 2M 3M 4M 1F 2F 3F 4F

Achieved Exposure Level. (ppm) 0 533 1171 2980 0 533 1171 2980

Olfactory epithelium -

atrophy/disorganisation 

Minimal 0 0 3 0 0 0 0 0

Slight 0 0 0 3 0 0 3 3

Total 0 0 3 3 0 0 3 3

Olfactory epithelium -

inflammation 

Minimal 0 0 0 3 0 0 3 2

Slight 0 0 1 0 0 0 0 1

Total 0 0 1 3 0 0 3 3

Olfactory nerve fibres - atrophy 

Minimal 0 0 0 1 0 0 1 1

Slight 0 0 0 2 0 0 1 2

Total 0 0 0 3 0 0 2 3

Airways - inflammatory exudate 

Minimal 0 0 1 2 0 0 0 1

Slight 0 0 0 1 0 0 0 0

Total 0 0 1 3 0 0 0 1

Number of tissues examined 3 3 3 3 3 3 3 3

Findings of uncertain relationship to treatment

A single female, Animal 22, exposed to 2980 ppm had perivascular and peribronchiolar 

inflammatory infiltrate and bronchiolar epithelial hyperplasia in the lungs, epithelial 

hyperplasia and subepithelial inflammation in the larynx and epithelial hyperplasia at the 

tracheal bifurcation. The relationship to treatment of these changes is uncertain.

All other changes were considered to be incidental.
Huntingdon Life Sciences

WAG0024

25

4. Discussion

The test article, 2-bromo-3,3,3-trifluoropropene, was administered to 3 groups of 6 rats by

inhalation administration, using a whole-body exposure system, for 6 hours a day, 5 days a 

week, for 2-week at achieved aerosol concentrations of 533, 1171 and 2980 ppm. A control 

group of rats received air only at the same airflow as the high dose group.

The histopathological changes seen in the nasal turbinates are consistent with a localised 

irritant effect of the test material. They are similar, but of a much reduced severity, to the 

changes seen in the previous dose ranging study (Huntingdon Life Sciences Study Number: 

WAG0013).

The changes seen in the respiratory tract of Animal 22, exposed to 2980 ppm, were not seen 

in any other animals and were of uncertain relationship to treatment.

Clinical signs of gasping, slow, shallow, deep and/or irregular breathing, piloerection, 

salivation chin rubbing and lacrymation are indicative of exposure to an irritant material and 

are consistent with the pathological findings. Whereas, the clinical signs of unresponsiveness 

to external stimuli, underactivity, and closed or partially closed eyelids represent possible

CNS effects. Clinical signs were evident for all treated animals over the first 5 days of 

treatment, with a dose-related increase in number and prevalence, however all signs were 

transient in nature. Signs during exposure were not recorded during the second week of 

exposure. However, in the previous dose ranging study (Huntingdon Life Sciences Study

Number: WAG0013), clinical signs did not worsen during the second week of exposures at 

aerosol concentrations of 4721 to 19098 ppm.

Treatment-related lower bodyweight gains were seen in males of all test article groups. 

These reductions were exposure level related and are considered to be treatment-related and 

correlate to some extent with reduced food consumption that was also seen. Similar effects 

were not seen in females and this potentially represents a sex-related difference.

Exposure at an aerosol concentration of 533 ppm resulted in no histopathological changes. 

Slightly lower of bodyweight gain and food consumption and transient clinical signs were 

seen at this level but were considered not to be adverse.
Huntingdon Life Sciences

WAG0024

26

5. Conclusion

Histopathological changes related to 2-bromo-3,3,3-trifluoropropene exposure were seen in 

the nasal turbinates of rats exposed to 1171 or 2980 ppm. The no observed adverse effect 

level (NOAEL) was 533 ppm, as this level was not associated with any histopathological

changes or adverse in-life findings.
Huntingdon Life Sciences

WAG0024

27

6. References

Huntingdon Life Sciences Study Number WAG0013:

2-bromo-3,3,3-trifluoropropene: Dose Range Finding Study by Inhalation Administration to 

Rats for 2 Weeks. 
28

Huntingdon Life Sciences

WAG0024

FIGURE 1

Bodyweight - group mean values (g)

Group : 1 2 3 4

Compound : Control 2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

Exposure (ppm) : 0 533 1171 2980
29

Huntingdon Life Sciences

WAG0024

TABLE 1 Request ID: 269565

Bodyweight - group mean values (g)

Group : 1 2 3 4

Compound : Control 2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

Exposure (ppm) : 0 533 1171 2980

Group Day Day Day Day Day Day Day Day Day Day Day Day

/Sex -5 -4 -3 -2 -1 1 2 3 4 5 6 7

1M Mean 384 392 395 401 406 408 415 414 417 418 421 420

SD 14.7 17.2 15.4 18.8 19.4 18.9 23.7 19.8 24.7 26.2 29.3 25.9

N 3 3 3 3 3 3 3 3 3 3 3 3

2M Mean 376 382 386 392 397 406 398 398 400 400 401 411

SD 10.0 15.6 13.5 19.2 19.7 21.4 22.9 22.8 24.7 22.3 24.7 27.4

N 3 3 3 3 3 3 3 3 3 3 3 3

3M Mean 390 394 398 405 408 418 408 402 405 413 418 423

SD 16.6 14.0 13.5 11.7 11.8 10.6 14.7 20.0 17.3 15.9 13.4 13.0

N 3 3 3 3 3 3 3 3 3 3 3 3

4M Mean 395 397 401 406 411 418 404 400 393 393 396 409

SD 11.4 8.2 10.5 17.2 16.6 19.5 20.1 16.3 20.0 20.4 21.3 21.5

N 3 3 3 3 3 3 3 3 3 3 3 3
30

Huntingdon Life Sciences

WAG0024

TABLE 1 - continued Request ID: 269565

Bodyweight - group mean values (g)

Group : 1 2 3 4

Compound : Control 2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

Exposure (ppm) : 0 533 1171 2980

Group Day Day Day Day Day Day Day Day Change As % of

/Sex 8 9 10 11 12 13 14 15 1-15 Control

1M Mean 427 430 433 433 436 437 443 446 38 -

SD 28.5 31.7 34.3 31.5 34.5 33.7 36.6 38.3 19.7

N 3 3 3 3 3 3 3 3 3

2M Mean 419 413 413 411 412 414 420 427 21 54

SD 25.2 29.4 27.0 28.8 31.2 30.2 34.4 35.9 14.7

N 3 3 3 3 3 3 3 3 3

3M Mean 428 428 423 425 425 428 436 439 21 56

SD 13.7 14.6 14.3 15.6 14.2 14.9 16.0 15.2 7.1

N 3 3 3 3 3 3 3 3 3

4M Mean 419 421 418 419 421 421 437 443 26 67

SD 23.4 28.6 29.1 29.9 30.6 32.2 30.8 32.8 13.4

N 3 3 3 3 3 3 3 3 3
31

Huntingdon Life Sciences

WAG0024

TABLE 1 - continued Request ID: 269565

Bodyweight - group mean values (g)

Group : 1 2 3 4

Compound : Control 2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

Exposure (ppm) : 0 533 1171 2980

Group Day Day Day Day Day Day Day Day Day Day Day Day

/Sex -5 -4 -3 -2 -1 1 2 3 4 5 6 7

1F Mean 243 242 246 248 250 251 250 253 255 254 254 258

SD 15.3 17.5 13.7 14.1 14.5 20.5 12.8 15.4 14.8 15.9 13.6 10.0

N 3 3 3 3 3 3 3 3 3 3 3 3

2F Mean 252 249 247 258 260 260 254 260 266 260 258 262

SD 21.4 22.5 24.4 25.5 26.1 28.7 28.2 30.5 28.8 29.5 31.0 32.5

N 3 3 3 3 3 3 3 3 3 3 3 3

3F Mean 235 238 234 240 243 244 239 242 246 247 246 249

SD 12.2 13.6 13.2 11.5 12.6 8.8 13.8 6.2 4.7 4.1 6.1 5.4

N 3 3 3 3 3 3 3 3 3 3 3 3

4F Mean 253 253 255 258 260 258 250 252 253 254 252 259

SD 3.6 6.2 4.3 7.8 6.2 10.2 5.8 9.6 6.4 3.8 5.7 6.8

N 3 3 3 3 3 3 3 3 3 3 3 3
32

Huntingdon Life Sciences

WAG0024

TABLE 1 - continued Request ID: 269565

Bodyweight - group mean values (g)

Group : 1 2 3 4

Compound : Control 2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

Exposure (ppm) : 0 533 1171 2980

Group Day Day Day Day Day Day Day Day Change As % of

/Sex 8 9 10 11 12 13 14 15 1-15 Control

1F Mean 257 258 260 259 263 264 265 259 8 -

SD 13.5 16.9 11.3 9.6 13.2 14.5 15.5 10.6 12.5

N 3 3 3 3 3 3 3 3 3

2F Mean 272 265 271 265 270 273 280 283 23 268

SD 37.5 33.9 32.9 35.8 35.9 35.4 36.2 37.9 10.9

N 3 3 3 3 3 3 3 3 3

3F Mean 255 257 253 251 253 260 266 268 25 291

SD 2.6 1.9 5.4 6.0 4.1 2.3 2.3 5.1 4.3

N 3 3 3 3 3 3 3 3 3

4F Mean 265 267 264 264 264 268 273 275 17 201

SD 12.5 9.1 5.1 11.0 12.5 9.2 8.5 4.6 8.8

N 3 3 3 3 3 3 3 3 3
33

Huntingdon Life Sciences

WAG0024

TABLE 2 Request ID: 269563

Organ weights - group mean unadjusted and adjusted values (g) for animals killed after 2 weeks of treatment

Group : 1 2 3 4

Compound : Control 2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

Exposure (ppm) : 0 533 1171 2980

Group

/Sex

Terminal 

Bodyweight

Brain Heart Kidneys Liver Lungs + 

Bronchi

Spleen

Unadjusted Means

1M Mean 446 2.08 1.838 3.16 18.53 1.434 0.806

SD 40 0.05 0.227 0.12 3.18 0.150 0.090

N 3 3 3 3 3 3 3

2M Mean 426 1.98 1.349 2.41 15.23 1.448 0.680

SD 37 0.04 0.189 0.32 2.29 0.039 0.103

N 3 3 3 3 3 3 3

3M Mean 439 1.95 1.472 2.85 17.29 1.465 0.739

SD 15 0.07 0.205 0.04 1.97 0.066 0.081

N 3 3 3 3 3 3 3

4M Mean 442 1.96 1.735 2.77 17.15 1.566 0.834

SD 33 0.02 0.113 0.13 1.18 0.064 0.103

N 3 3 3 3 3 3 3

Adjusted Means

1M Mean 2.08 1.828 3.13 18.06 1.420 0.789

2M Mean 1.97 1.364 2.46 15.99 1.470 0.707

3M Mean 1.95 1.471 2.85 17.24 1.463 0.737

4M Mean 1.96 1.730 2.76 16.92 1.559 0.826
34

Huntingdon Life Sciences

WAG0024

TABLE 2 - continued Request ID: 269563

Organ weights - group mean unadjusted and adjusted values (g) for animals killed after 2 weeks of treatment

Group : 1 2 3 4

Compound : Control 2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

Exposure (ppm) : 0 533 1171 2980

Group

/Sex

Terminal 

Bodyweight

Brain Heart Kidneys Liver Lungs + 

Bronchi

Spleen

Unadjusted Means

1F Mean 259 1.94 1.039 1.67 9.45 1.115 0.482

SD 10 0.06 0.119 0.09 0.84 0.023 0.030

N 3 3 3 3 3 2 3

2F Mean 282 1.91 1.108 1.78 11.64 1.245 0.570

SD 38 0.07 0.125 0.26 2.98 0.113 0.173

N 3 3 3 3 3 3 3

3F Mean 268 1.83 1.090 1.87 10.65 1.217 0.548

SD 6 0.02 0.264 0.08 0.26 0.093 0.064

N 3 3 3 3 3 3 3

4F Mean 274 1.84 1.176 1.82 13.79 1.467 0.753

SD 6 0.03 0.113 0.10 4.53 0.336 0.348

N 3 3 3 3 3 3 3

Adjusted Means

1F Mean 1.96 1.045 1.75 10.44 1.172 0.538

2F Mean 1.89 1.102 1.72 10.73 1.209 0.518

3F Mean 1.83 1.092 1.89 10.87 1.227 0.561

4F Mean 1.83 1.174 1.79 13.49 1.456 0.736
35

Huntingdon Life Sciences

WAG0024

TABLE 3 Request ID: 274746

Macropathology - group distribution of findings for animals killed after 2 weeks of treatment

Group : 1 2 3 4

Compound : Control 2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

Exposure (ppm) : 0 533 1171 2980

Group/Sex: 1M 2M 3M 4M 1F 2F 3F 4F

Tissue and Finding Number Examined: 3 3 3 3 3 3 3 3

Liver

Enlarged 0 0 0 0 0 0 0 1

Lungs + bronchi

Dark area(s) 0 0 0 0 0 0 0 1

Pale area(s) 0 0 0 0 1 0 0 1

Spleen

Capsule thickened 0 0 0 1 0 0 0 0

Enlarged 0 0 0 0 0 0 0 1

Thymus

Firm 0 0 0 0 0 0 0 1
36

Huntingdon Life Sciences

WAG0024

TABLE 4 Request ID: 274747

Histopathology - group distribution of findings for animals killed after 2 weeks of treatment

Group : 1 2 3 4

Compound : Control 2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

Exposure (ppm) : 0 533 1171 2980

Group/Sex: 1M 2M 3M 4M 1F 2F 3F 4F

Tissue and Finding Number: 3 3 3 3 3 3 3 3

Trachea Number Examined: 3 3 3 3 3 3 3 3

Lungs + Bronchi Number Examined: 3 3 3 3 3 3 3 3

Aggregations of Alveolar Macrophages 0 1 0 0 2 0 0 1

Alveolar Haemorrhage 0 1 0 0 0 0 0 0

Alveolitis 1 0 0 0 1 0 0 0

Bronchiolar Epithelial Hyperplasia 0 0 0 0 0 0 0 1

Perivascular/Peribronchiolar Inflammatory Infiltrate 0 0 0 0 0 0 0 1

Larynx Number Examined: 3 3 3 3 3 3 3 3

Epithelial Hyperplasia, Ventral 0 0 0 0 0 0 0 1

Squamous Metaplasia - Arytenoid 0 0 0 0 0 0 1 0

Subepithelial Inflammation 0 0 0 0 0 0 0 1

Tracheal Bifurc. Number Examined: 3 3 3 3 3 3 3 3

Epithelial Hyperplasia 0 0 0 0 0 0 0 1

Nasal turbinates Number Examined: 3 3 3 3 3 3 3 3

Inflammatory Exudate in Airways 0 0 1 3 0 0 0 1

Olfactory Epithelium - Atrophy/Disorganisation 0 0 3 3 0 0 3 3

Olfactory Epithelium - Inflammation 0 0 1 3 0 0 3 3

Olfactory Nerve Fibres - Atrophy 0 0 0 3 0 0 2 3
37

Huntingdon Life Sciences

WAG0024

APPENDIX 1 Request ID: 269558

Clinical signs - individual observations

Group : 1 2 3 4

Compound : Control 2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

Exposure (ppm) : 0 533 1171 2980

Group Animal Death Week of

/Sex Number Code Death Category Observation Week(s)

1F 15 T 3 Behaviour Irritable 2

Vocalisation 2

Only animals with observations are presented
38

Huntingdon Life Sciences

WAG0024

APPENDIX 1 - continued Request ID: 269558

Clinical signs - individual observations

Group : 1 2 3 4

Compound : Control 2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

Exposure (ppm) : 0 533 1171 2980

Group Animal Death Week of

/Sex Number Code Death Category Observation Week(s)

2F 17 T 3 Behaviour Irritable 2-3

Vocalisation 2-3

Only animals with observations are presented
39

Huntingdon Life Sciences

WAG0024

APPENDIX 2 Request ID: 269838

Signs associated with dosing - individual observations

Group : 1 2 3 4

Compound : Control 2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

Exposure (ppm) : 0 533 1171 2980

Days

Group 

/Sex

Animal 

Number Sign

1-2 hours during

exposure

3-4 hours during

exposure

5-6 hours during

exposure

2M 4 Behaviour

 Underactive 1, 4-5 1-5 1-5

 Salivation 4

Breathing

 Gasping 1

 Slow 1, 5 1-2, 5

Coat

 Piloerection 3 4

Eyelids (Left)

 Closed 2, 5

 Partially Closed 1-5 1-5 1-5

Eyelids (Right)

 Closed 2, 5

 Partially Closed 1-5 1-5 1-5

Posture

 Hunched 1, 3-5 2-5
40

Huntingdon Life Sciences

WAG0024

APPENDIX 2 - continued Request ID: 269838

Signs associated with dosing - individual observations

Group : 1 2 3 4

Compound : Control 2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

Exposure (ppm) : 0 533 1171 2980

Days

Group 

/Sex

Animal 

Number Sign

1-2 hours during

exposure

3-4 hours during

exposure

5-6 hours during

exposure

2M 5 Behaviour

 Underactive 1, 3-4 1-2, 4-5 1-2, 4-5

 Unresponsive 2

Breathing

 Deep 4

 Shallow 1 1

 Slow 5 5

Coat

 Piloerection 1

Eyelids (Left)

 Closed 2

 Partially Closed 1-4 1-5 1-5

Eyelids (Right)

 Closed 2

 Partially Closed 1-4 1-5 1-5

Eye (Left)

 Lacrimation 1

Eye (Right)

 Lacrimation 1

Posture

 Hunched 3 3 3
41

Huntingdon Life Sciences

WAG0024

APPENDIX 2 - continued Request ID: 269838

Signs associated with dosing - individual observations

Group : 1 2 3 4

Compound : Control 2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

Exposure (ppm) : 0 533 1171 2980

Days

Group 

/Sex

Animal 

Number Sign

1-2 hours during

exposure

3-4 hours during

exposure

5-6 hours during

exposure

2M 6 Behaviour

 Underactive 1, 3-4 1-5 1-5

Breathing

 Slow 5 5

Coat

 Piloerection 3 3

Eyelids (Left)

 Partially Closed 1, 3-4 1-5 1-5

Eyelids (Right)

 Partially Closed 1, 3-4 1-5 1-5

Eye (Left)

 Lacrimation 1

Eye (Right)

 Lacrimation 1

Posture

 Hunched 3 2
42

Huntingdon Life Sciences

WAG0024

APPENDIX 2 - continued Request ID: 269838

Signs associated with dosing - individual observations

Group : 1 2 3 4

Compound : Control 2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

Exposure (ppm) : 0 533 1171 2980

Days

Group 

/Sex

Animal 

Number Sign

1-2 hours during

exposure

3-4 hours during

exposure

5-6 hours during

exposure

3M 7 Behaviour

 Underactive 1, 4-5 1-5 1-5

Breathing

 Gasping 1 1

 Slow 5 2, 4-5 4-5

Eyelids (Left)

 Partially Closed 1, 4-5 1-5 1-5

Eyelids (Right)

 Partially Closed 1, 4-5 1-5 1-5

Posture

 Hunched 2-4 2-4
43

Huntingdon Life Sciences

WAG0024

APPENDIX 2 - continued Request ID: 269838

Signs associated with dosing - individual observations

Group : 1 2 3 4

Compound : Control 2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

Exposure (ppm) : 0 533 1171 2980

Days

Group 

/Sex

Animal 

Number Sign

1-2 hours during

exposure

3-4 hours during

exposure

5-6 hours during

exposure

3M 8 Behaviour

 Underactive 1-5 1-2, 4-5 1-5

 Unresponsive 3 3 3

Breathing

 Gasping 1

 Irregular 2

 Shallow 1

 Slow 1 4-5 1, 4-5

Coat

 Piloerection 4 4

Eyelids (Left)

 Closed 1

 Partially Closed 1-5 1-5 1-5

Eyelids (Right)

 Closed 1

 Partially Closed 1-5 1-5 1-5

Posture

 Hunched 1
44

Huntingdon Life Sciences

WAG0024

APPENDIX 2 - continued Request ID: 269838

Signs associated with dosing - individual observations

Group : 1 2 3 4

Compound : Control 2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

Exposure (ppm) : 0 533 1171 2980

Days

Group 

/Sex

Animal 

Number Sign

1-2 hours during

exposure

3-4 hours during

exposure

5-6 hours during

exposure

3M 9 Behaviour

 Chin Rubbing 1

 Underactive 1-5 1-5 1-5

 Salivation 3

Breathing

 Gasping 1

 Slow 1, 5 4-5 2, 4-5

Coat

 Piloerection 1 1-2, 4

Eyelids (Left)

 Closed 1 5

 Partially Closed 1-5 1-5 1-5

Eyelids (Right)

 Closed 1 5

 Partially Closed 1-5 1-5 1-5

Posture

 Hunched 2, 4 1-2, 4-5
45

Huntingdon Life Sciences

WAG0024

APPENDIX 2 - continued Request ID: 269838

Signs associated with dosing - individual observations

Group : 1 2 3 4

Compound : Control 2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

Exposure (ppm) : 0 533 1171 2980

Days

Group 

/Sex

Animal 

Number Sign

1-2 hours during

exposure

3-4 hours during

exposure

5-6 hours during

exposure

4M 10 Behaviour

 Underactive 2-5 1-5 1-5

 Unresponsive 1 1

Breathing

 Gasping 1 1

 Slow 1-2, 5 1-2, 4-5 2, 4-5

Eyelids (Left)

 Closed 1 1

 Partially Closed 1-5 1-5 1-5

Eyelids (Right)

 Closed 1 1

 Partially Closed 1-5 1-5 1-5

Eye (Left)

 Lacrimation 1 1

Eye (Right)

 Lacrimation 1 1

Posture

 Flat 1 1 1

 Hunched 2, 4-5 2, 4 4-5
46

Huntingdon Life Sciences

WAG0024

APPENDIX 2 - continued Request ID: 269838

Signs associated with dosing - individual observations

Group : 1 2 3 4

Compound : Control 2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

Exposure (ppm) : 0 533 1171 2980

Days

Group 

/Sex

Animal 

Number Sign

1-2 hours during

exposure

3-4 hours during

exposure

5-6 hours during

exposure

4M 11 Behaviour

 Underactive 1-5 1-5 1-5

 Unresponsive 1 1 1

 Salivation 1

Breathing

 Gasping 1

 Shallow 1

 Slow 1, 5 1-2, 4-5 2, 4-5

Coat

 Piloerection 1, 3

Eyelids (Left)

 Closed 1 1 1

 Partially Closed 1-5 1-5 1-5

Eyelids (Right)

 Closed 1 1 1

 Partially Closed 1-5 1-5 1-5

Eye (Left)

 Lacrimation 1 1

Eye (Right)

 Lacrimation 1 1

Posture

 Flat 1

 Hunched 1 4 1-2
47

Huntingdon Life Sciences

WAG0024

APPENDIX 2 - continued Request ID: 269838

Signs associated with dosing - individual observations

Group : 1 2 3 4

Compound : Control 2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

Exposure (ppm) : 0 533 1171 2980

Days

Group 

/Sex

Animal 

Number Sign

1-2 hours during

exposure

3-4 hours during

exposure

5-6 hours during

exposure

4M 12 Behaviour

 Underactive 1-5 1-5 1-5

 Salivation 1

Breathing

 Gasping 1

 Slow 1, 5 1-2, 4-5 1, 4-5

Coat

 Piloerection 5 5

Eyelids (Left)

 Closed 1

 Partially Closed 1-5 1-5 1-5

Eyelids (Right)

 Closed 1

 Partially Closed 1-5 1-5 1-5

Eye (Left)

 Lacrimation 1

Eye (Right)

 Lacrimation 1

Posture

 Flat 4

 Hunched 3 2-4 1-4
48

Huntingdon Life Sciences

WAG0024

APPENDIX 2 - continued Request ID: 269838

Signs associated with dosing - individual observations

Group : 1 2 3 4

Compound : Control 2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

Exposure (ppm) : 0 533 1171 2980

Days

Group 

/Sex

Animal 

Number Sign

1-2 hours during

exposure

3-4 hours during

exposure

5-6 hours during

exposure

1F 14 Coat

 Piloerection 2
49

Huntingdon Life Sciences

WAG0024

APPENDIX 2 - continued Request ID: 269838

Signs associated with dosing - individual observations

Group : 1 2 3 4

Compound : Control 2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

Exposure (ppm) : 0 533 1171 2980

Days

Group 

/Sex

Animal 

Number Sign

1-2 hours during

exposure

3-4 hours during

exposure

5-6 hours during

exposure

2F 16 Behaviour

 Underactive 1, 4-5 1, 4-5 1-2, 4-5

Breathing

 Deep 4

 Slow 5 5 2, 5

Coat

 Piloerection 1 4 2, 4

Eyelids (Left)

 Closed 1 1 1

 Partially Closed 1-5 1-5 2-5

Eyelids (Right)

 Closed 1 1 1

 Partially Closed 1-5 1-5 2-5

Posture

 Hunched 1-2 1-4 1-5
50

Huntingdon Life Sciences

WAG0024

APPENDIX 2 - continued Request ID: 269838

Signs associated with dosing - individual observations

Group : 1 2 3 4

Compound : Control 2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

Exposure (ppm) : 0 533 1171 2980

Days

Group 

/Sex

Animal 

Number Sign

1-2 hours during

exposure

3-4 hours during

exposure

5-6 hours during

exposure

2F 17 Behaviour

 Underactive 1, 3-5 1-5 2-5

Breathing

 Slow 5 1-2, 5 1-2

Coat

 Piloerection 1, 4 1-2, 4

Eyelids (Left)

 Closed 1 1, 5 1, 5

 Partially Closed 1-5 1-5 2-4

Eyelids (Right)

 Closed 1 1, 5 1, 5

 Partially Closed 1-5 1-5 2-4

Eye (Left)

 Lacrimation 1

Eye (Right)

 Lacrimation 1

Posture

 Hunched 1-5 1-2, 4
51

Huntingdon Life Sciences

WAG0024

APPENDIX 2 - continued Request ID: 269838

Signs associated with dosing - individual observations

Group : 1 2 3 4

Compound : Control 2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

Exposure (ppm) : 0 533 1171 2980

Days

Group 

/Sex

Animal 

Number Sign

1-2 hours during

exposure

3-4 hours during

exposure

5-6 hours during

exposure

2F 18 Behaviour

 Chin Rubbing 3

 Underactive 4-5 1-5 1-5

 Unresponsive 1 1

Breathing

 Shallow 1 1

 Slow 5 5 5

Coat

 Piloerection 1, 5 1, 4-5 1-2, 5

Eyelids (Left)

 Closed 1 1

 Partially Closed 1, 4-5 1-5 2-5

Eyelids (Right)

 Closed 1 1

 Partially Closed 1, 4-5 1-5 2-5

Posture

 Hunched 5 1, 3-5 1-2, 4-5
52

Huntingdon Life Sciences

WAG0024

APPENDIX 2 - continued Request ID: 269838

Signs associated with dosing - individual observations

Group : 1 2 3 4

Compound : Control 2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

Exposure (ppm) : 0 533 1171 2980

Days

Group 

/Sex

Animal 

Number Sign

1-2 hours during

exposure

3-4 hours during

exposure

5-6 hours during

exposure

3F 19 Behaviour

 Underactive 1-2, 4-5 1-2, 4-5 1-5

 Unresponsive 3 1, 3 1, 3

Breathing

 Gasping 1

 Slow 1 2, 4-5 2, 4-5

Coat

 Piloerection 1, 4 1, 4 1-2, 4-5

Eyelids (Left)

 Closed 2, 5 2

 Partially Closed 1-2, 4 1-5 1-5

Eyelids (Right)

 Closed 2, 5 2

 Partially Closed 1-2, 4 1-5 1-5

Posture

 Hunched 1-2, 4 1-2, 4 1-2, 4-5
53

Huntingdon Life Sciences

WAG0024

APPENDIX 2 - continued Request ID: 269838

Signs associated with dosing - individual observations

Group : 1 2 3 4

Compound : Control 2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

Exposure (ppm) : 0 533 1171 2980

Days

Group 

/Sex

Animal 

Number Sign

1-2 hours during

exposure

3-4 hours during

exposure

5-6 hours during

exposure

3F 20 Behaviour

 Underactive 1-2, 4-5 1-5 1-5

 Unresponsive 3 1

 Salivation 1 1, 3

Breathing

 Gasping 1 1

 Slow 1 1-2, 4-5 4-5

Coat

 Piloerection 3, 5 1

Eyelids (Left)

 Closed 1 1

 Partially Closed 1-2, 4-5 2-5 1-5

Eyelids (Right)

 Closed 1 1

 Partially Closed 1-2, 4-5 2-5 1-5

Eye (Left)

 Lacrimation 1

Eye (Right)

 Lacrimation 1

Posture

 Hunched 4-5 1-2, 4-5 1-2, 4-5
54

Huntingdon Life Sciences

WAG0024

APPENDIX 2 - continued Request ID: 269838

Signs associated with dosing - individual observations

Group : 1 2 3 4

Compound : Control 2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

Exposure (ppm) : 0 533 1171 2980

Days

Group 

/Sex

Animal 

Number Sign

1-2 hours during

exposure

3-4 hours during

exposure

5-6 hours during

exposure

3F 21 Behaviour

 Underactive 1-2, 4-5 1-5 1-5

 Unresponsive 1

 Salivation 3

Breathing

 Gasping 1

 Slow 1 1, 4-5 4

Coat

 Piloerection 5 1, 5 1, 4

Eyelids (Left)

 Closed 1 1

 Partially Closed 1-5 1-5 1-5

Eyelids (Right)

 Closed 1 1

 Partially Closed 1-5 1-5 1-5

Eye (Left)

 Lacrimation 1

Eye (Right)

 Lacrimation 1

Posture

 Hunched 5 1 1, 4
55

Huntingdon Life Sciences

WAG0024

APPENDIX 2 - continued Request ID: 269838

Signs associated with dosing - individual observations

Group : 1 2 3 4

Compound : Control 2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

Exposure (ppm) : 0 533 1171 2980

Days

Group 

/Sex

Animal 

Number Sign

1-2 hours during

exposure

3-4 hours during

exposure

5-6 hours during

exposure

4F 22 Behaviour

 Underactive 1, 3-5 1, 3-5 2-5

 Unresponsive 1 1-3, 5 1-2, 5

Breathing

 Deep 5

 Gasping 1

 Slow 5 4-5 4

Coat

 Piloerection 1 3 2, 5

Eyelids (Left)

 Closed 1 4-5 1, 4-5

 Partially Closed 1, 3-5 1-5 1-3

Eyelids (Right)

 Closed 1 4-5 1, 4-5

 Partially Closed 1, 3-5 1-5 1-3

Posture

 Hunched 1-5 1-5 1-2, 4
56

Huntingdon Life Sciences

WAG0024

APPENDIX 2 - continued Request ID: 269838

Signs associated with dosing - individual observations

Group : 1 2 3 4

Compound : Control 2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

Exposure (ppm) : 0 533 1171 2980

Days

Group 

/Sex

Animal 

Number Sign

1-2 hours during

exposure

3-4 hours during

exposure

5-6 hours during

exposure

4F 23 Behaviour

 Underactive 1-5 1-5 1-5

 Unresponsive 1 1 1, 3

Breathing

 Gasping 1

 Slow 1, 5 2, 4-5 4-5

Coat

 Piloerection 1, 4 1

Eyelids (Left)

 Closed 1 1

 Partially Closed 1-5 1-5 1-5

Eyelids (Right)

 Closed 1 1

 Partially Closed 1-5 1-5 1-5

Eye (Left)

 Lacrimation 1

Eye (Right)

 Lacrimation 1

Posture

 Hunched 1-4 1-2, 4-5 1-5
57

Huntingdon Life Sciences

WAG0024

APPENDIX 2 - continued Request ID: 269838

Signs associated with dosing - individual observations

Group : 1 2 3 4

Compound : Control 2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

Exposure (ppm) : 0 533 1171 2980

Days

Group 

/Sex

Animal 

Number Sign

1-2 hours during

exposure

3-4 hours during

exposure

5-6 hours during

exposure

4F 24 Behaviour

 Underactive 1-5 1-5 1-3, 5

 Unresponsive 1, 3 1-2 1-2

 Salivation 1, 3

Breathing

 Gasping 1

 Slow 1 2, 4 1-2, 5

Coat

 Piloerection 1, 5 1

Eyelids (Left)

 Closed 1 4 1

 Partially Closed 1-5 1-5 1-3, 5

Eyelids (Right)

 Closed 1 4 1

 Partially Closed 1-5 1-5 1-3, 5

Eye (Left)

 Lacrimation 4

Eye (Right)

 Lacrimation 4

Posture

 Hunched 1, 3-4 1-2, 4 1-3, 5
58

Huntingdon Life Sciences

WAG0024

APPENDIX 3 Request ID: 269560

Bodyweight - individual values (g)

Group : 1 2 3 4

Compound : Control 2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

Exposure (ppm) : 0 533 1171 2980

Group Animal Day Day Day Day Day Day Day Day Day Day Day Day

/Sex Number -5 -4 -3 -2 -1 1 2 3 4 5 6 7

1M 1 392 394 399 404 408 411 417 415 416 419 418 418

2 367 374 378 381 385 388 391 393 392 391 393 395

3 393 409 407 418 424 426 438 433 442 443 451 447

2M 4 366 366 373 373 380 386 373 375 376 378 375 383

5 386 397 400 411 418 429 418 420 425 423 425 437

6 377 382 386 391 392 403 402 399 398 399 402 412

3M 7 388 392 396 398 401 412 396 380 387 395 403 409

8 408 409 412 419 421 430 424 419 422 425 428 434

9 375 382 385 399 400 411 403 407 407 419 423 427

4M 10 386 389 391 391 395 398 384 384 375 373 375 389

11 392 397 400 403 409 420 405 398 389 392 394 408

12 408 406 412 425 428 436 424 417 414 414 418 431
59

Huntingdon Life Sciences

WAG0024

APPENDIX 3 - continued Request ID: 269560

Bodyweight - individual values (g)

Group : 1 2 3 4

Compound : Control 2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

Exposure (ppm) : 0 533 1171 2980

Group Animal Day Day Day Day Day Day Day Day

/Sex Number 8 9 10 11 12 13 14 15

1M 1 428 428 433 435 439 438 446 445

2 398 400 399 400 400 403 405 409

3 455 463 468 463 469 470 478 485

2M 4 395 384 385 383 382 383 385 392

5 446 443 439 441 444 444 453 463

6 418 411 415 409 411 415 423 425

3M 7 416 415 408 410 411 414 419 426

8 443 444 436 441 439 443 451 456

9 427 426 427 425 424 427 438 436

4M 10 397 392 389 390 391 390 407 410

11 417 422 419 416 421 419 437 445

12 443 449 447 450 452 454 468 475
60

Huntingdon Life Sciences

WAG0024

APPENDIX 3 - continued Request ID: 269560

Bodyweight - individual values (g)

Group : 1 2 3 4

Compound : Control 2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

Exposure (ppm) : 0 533 1171 2980

Group Animal Day Day Day Day Day Day Day Day Day Day Day Day

/Sex Number -5 -4 -3 -2 -1 1 2 3 4 5 6 7

1F 13 225 222 230 232 234 227 235 237 239 236 239 248

14 251 253 253 254 255 261 256 255 258 260 257 257

15 252 251 254 258 262 265 259 267 269 267 265 268

2F 16 228 223 219 229 231 228 222 226 234 226 224 226

17 268 263 266 276 280 283 274 283 289 280 283 288

18 261 261 256 269 271 270 267 273 275 273 268 273

3F 19 238 242 241 238 242 245 244 243 248 252 251 253

20 222 223 218 230 231 234 223 235 240 245 239 243

21 246 249 242 252 256 251 249 247 249 245 248 250

4F 22 256 260 255 265 266 270 254 258 256 259 259 261

23 254 251 259 260 259 252 253 257 258 254 250 265

24 249 248 250 250 254 252 243 241 246 251 248 252
61

Huntingdon Life Sciences

WAG0024

APPENDIX 3 - continued Request ID: 269560

Bodyweight - individual values (g)

Group : 1 2 3 4

Compound : Control 2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

Exposure (ppm) : 0 533 1171 2980

Group Animal Day Day Day Day Day Day Day Day

/Sex Number 8 9 10 11 12 13 14 15

1F 13 243 239 247 249 250 250 250 250

14 257 263 264 258 264 264 265 258

15 270 271 269 269 276 279 281 271

2F 16 229 226 233 225 231 233 238 239

17 297 290 294 293 302 300 304 307

18 289 279 286 278 279 287 297 302

3F 19 257 259 256 258 257 263 267 271

20 252 255 247 246 249 259 264 262

21 256 258 257 250 252 258 268 271

4F 22 267 265 261 260 261 267 275 278

23 275 276 269 277 278 277 280 277

24 251 258 261 256 254 259 263 270
62

Huntingdon Life Sciences

WAG0024

APPENDIX 4 Request ID: 269561

Food consumption - individual values (g/animal/day)

Group : 1 2 3 4

Compound : Control 2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

Exposure (ppm) : 0 533 1171 2980

Group Cage Day Day Day Day Day Day Day Day Day Day Day Day

/Sex Number -5 -4 -3 -2 -1 1 2 3 4 5 6 7

1M 1 34 33 29 32 31 32 30 29 30 31 31 31

2M 2 31 33 33 32 33 20 20 23 22 28 30 34

3M 3 33 33 35 32 33 19 21 22 26 32 32 32

4M 4 33 34 33 33 34 12 20 24 18 26 33 35
63

Huntingdon Life Sciences

WAG0024

APPENDIX 4 - continued Request ID: 269561

Food consumption - individual values (g/animal/day)

Group : 1 2 3 4

Compound : Control 2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

Exposure (ppm) : 0 533 1171 2980

Group Cage Day Day Day Day Day Day Day

/Sex Number 8 9 10 11 12 13 14

1M 1 28 31 27 30 27 33 31

2M 2 19 27 22 23 24 31 30

3M 3 24 26 24 23 26 31 30

4M 4 27 26 25 27 26 35 34
64

Huntingdon Life Sciences

WAG0024

APPENDIX 4 - continued Request ID: 269561

Food consumption - individual values (g/animal/day)

Group : 1 2 3 4

Compound : Control 2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

Exposure (ppm) : 0 533 1171 2980

Group Cage Day Day Day Day Day Day Day Day Day Day Day Day

/Sex Number -5 -4 -3 -2 -1 1 2 3 4 5 6 7

1F 5 18 22 22 22 19 19 22 20 20 23 23 21

2F 6 20 20 24 21 22 12 19 21 15 21 21 27

3F 7 23 20 22 21 23 14 18 19 18 23 22 26

4F 8 20 21 22 21 20 10 14 14 17 17 24 25
65

Huntingdon Life Sciences

WAG0024

APPENDIX 4 - continued Request ID: 269561

Food consumption - individual values (g/animal/day)

Group : 1 2 3 4

Compound : Control 2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

Exposure (ppm) : 0 533 1171 2980

Group Cage Day Day Day Day Day Day Day

/Sex Number 8 9 10 11 12 13 14

1F 5 18 22 20 22 18 21 19

2F 6 14 21 17 19 19 26 28

3F 7 19 20 19 16 24 25 25

4F 8 19 19 19 18 20 23 24
66

Huntingdon Life Sciences

WAG0024

APPENDIX 5 Request ID: 269562

Organ weights - individual absolute values (g) for animals killed after 2 weeks of treatment

Group : 1 2 3 4

Compound : Control 2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

Exposure (ppm) : 0 533 1171 2980

Group

/Sex

Animal

Number

Terminal 

Bodyweight

Brain Heart Kidneys Liver Lungs + 

Bronchi

Spleen

1M 1 444 2.05 1.587 3.22 19.29 1.436 0.824

2 407 2.05 2.031 3.03 15.05 1.283 0.709

3 487 2.14 1.895 3.25 21.27 1.584 0.886

2M 4 389 1.98 1.142 2.24 12.76 1.425 0.564

5 463 1.93 1.513 2.78 17.30 1.493 0.758

6 426 2.01 1.393 2.21 15.64 1.426 0.717

3M 7 425 2.02 1.334 2.81 16.90 1.539 0.767

8 456 1.89 1.707 2.88 19.43 1.415 0.647

9 437 1.94 1.375 2.87 15.55 1.440 0.801

4M 10 409 1.98 1.857 2.67 16.41 1.497 0.726

11 443 1.95 1.636 2.73 16.53 1.623 0.847

12 474 1.94 1.711 2.92 18.51 1.578 0.931
67

Huntingdon Life Sciences

WAG0024

APPENDIX 5 - continued Request ID: 269562

Organ weights - individual absolute values (g) for animals killed after 2 weeks of treatment

Group : 1 2 3 4

Compound : Control 2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

Exposure (ppm) : 0 533 1171 2980

Group

/Sex

Animal

Number

Terminal 

Bodyweight

Brain Heart Kidneys Liver Lungs + 

Bronchi

Spleen

1F 13 248 1.91 0.943 1.61 8.48 1.099 0.450

14 260 1.90 1.002 1.63 9.89 1.131 0.509

15 269 2.02 1.172 1.77 9.98 U 0.485

2F 16 238 1.84 1.084 1.49 8.21 1.119 0.370

17 305 1.92 0.996 1.91 13.06 1.339 0.668

18 301 1.98 1.243 1.96 13.64 1.277 0.672

3F 19 272 1.81 0.948 1.79 10.49 1.110 0.571

20 262 1.84 1.395 1.96 10.51 1.265 0.598

21 270 1.84 0.928 1.86 10.95 1.277 0.476

4F 22 279 1.84 1.304 1.74 18.97 1.854 1.150

23 277 1.81 1.134 1.93 10.56 1.254 0.504

24 267 1.86 1.089 1.78 11.85 1.293 0.605

U Unsuitable – lungs and bronchi inflated with fixative prior to weighing in error.
68

Huntingdon Life Sciences

WAG0024

APPENDIX 6 Request ID: 274748

Macropathology and histopathology - individual findings

Group : 1 2 3 4

Compound : Control 2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

Exposure (ppm) : 0 533 1171 2980

Animal Number: 1 Sex: Male Dose Group: 1 Phase: Treatment Sacrifice Status: Final phase sacrifice

Date of Death: 30-Apr-12 Day of Death: 15 Week of Death: 3 Subgroup: 1 Terminal Bodyweight: 444.3 g

PATHOLOGY OBSERVATIONS

MACROPATHOLOGY HISTOPATHOLOGY

***ANIMAL HAS NO MACROSCOPIC FINDINGS RECORDED*** ***ANIMAL HAS NO MICROSCOPIC FINDINGS RECORDED***

The following examined tissues had no gross observations and no findings microscopically:

 Larynx, Lungs + Bronchi, Nasal turbinates, Trachea, Tracheal Bifurc.
69

Huntingdon Life Sciences

WAG0024

APPENDIX 6 - continued Request ID: 274748

Macropathology and histopathology - individual findings

Group : 1 2 3 4

Compound : Control 2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

Exposure (ppm) : 0 533 1171 2980

Animal Number: 2 Sex: Male Dose Group: 1 Phase: Treatment Sacrifice Status: Final phase sacrifice

Date of Death: 30-Apr-12 Day of Death: 15 Week of Death: 3 Subgroup: 1 Terminal Bodyweight: 406.9 g

PATHOLOGY OBSERVATIONS

MACROPATHOLOGY HISTOPATHOLOGY

***ANIMAL HAS NO MACROSCOPIC FINDINGS RECORDED*** ***ANIMAL HAS NO MICROSCOPIC FINDINGS RECORDED***

The following examined tissues had no gross observations and no findings microscopically:

 Larynx, Lungs + Bronchi, Nasal turbinates, Trachea, Tracheal Bifurc.
70

Huntingdon Life Sciences

WAG0024

APPENDIX 6 - continued Request ID: 274748

Macropathology and histopathology - individual findings

Group : 1 2 3 4

Compound : Control 2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

Exposure (ppm) : 0 533 1171 2980

Animal Number: 3 Sex: Male Dose Group: 1 Phase: Treatment Sacrifice Status: Final phase sacrifice

Date of Death: 30-Apr-12 Day of Death: 15 Week of Death: 3 Subgroup: 1 Terminal Bodyweight: 486.5 g

PATHOLOGY OBSERVATIONS

MACROPATHOLOGY HISTOPATHOLOGY

***ANIMAL HAS NO MACROSCOPIC FINDINGS RECORDED***

Lungs + Bronchi

 Alveolitis, Focal, Minimal

The following examined tissues had no gross observations and no findings microscopically:

 Larynx, Nasal turbinates, Trachea, Tracheal Bifurc.
71

Huntingdon Life Sciences

WAG0024

APPENDIX 6 - continued Request ID: 274748

Macropathology and histopathology - individual findings

Group : 1 2 3 4

Compound : Control 2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

Exposure (ppm) : 0 533 1171 2980

Animal Number: 4 Sex: Male Dose Group: 2 Phase: Treatment Sacrifice Status: Final phase sacrifice

Date of Death: 30-Apr-12 Day of Death: 15 Week of Death: 3 Subgroup: 1 Terminal Bodyweight: 388.6 g

PATHOLOGY OBSERVATIONS

MACROPATHOLOGY HISTOPATHOLOGY

***ANIMAL HAS NO MACROSCOPIC FINDINGS RECORDED*** ***ANIMAL HAS NO MICROSCOPIC FINDINGS RECORDED***

The following examined tissues had no gross observations and no findings microscopically:

 Larynx, Lungs + Bronchi, Nasal turbinates, Trachea, Tracheal Bifurc.
72

Huntingdon Life Sciences

WAG0024

APPENDIX 6 - continued Request ID: 274748

Macropathology and histopathology - individual findings

Group : 1 2 3 4

Compound : Control 2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

Exposure (ppm) : 0 533 1171 2980

Animal Number: 5 Sex: Male Dose Group: 2 Phase: Treatment Sacrifice Status: Final phase sacrifice

Date of Death: 30-Apr-12 Day of Death: 15 Week of Death: 3 Subgroup: 1 Terminal Bodyweight: 463.3 g

PATHOLOGY OBSERVATIONS

MACROPATHOLOGY HISTOPATHOLOGY

***ANIMAL HAS NO MACROSCOPIC FINDINGS RECORDED***

Lungs + Bronchi

 Alveolar Haemorrhage, Minimal

The following examined tissues had no gross observations and no findings microscopically:

 Larynx, Nasal turbinates, Trachea, Tracheal Bifurc.
73

Huntingdon Life Sciences

WAG0024

APPENDIX 6 - continued Request ID: 274748

Macropathology and histopathology - individual findings

Group : 1 2 3 4

Compound : Control 2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

Exposure (ppm) : 0 533 1171 2980

Animal Number: 6 Sex: Male Dose Group: 2 Phase: Treatment Sacrifice Status: Final phase sacrifice

Date of Death: 30-Apr-12 Day of Death: 15 Week of Death: 3 Subgroup: 1 Terminal Bodyweight: 425.8 g

PATHOLOGY OBSERVATIONS

MACROPATHOLOGY HISTOPATHOLOGY

***ANIMAL HAS NO MACROSCOPIC FINDINGS RECORDED***

Lungs + Bronchi

 Aggregations of Alveolar Macrophages, Minimal

The following examined tissues had no gross observations and no findings microscopically:

 Larynx, Nasal turbinates, Trachea, Tracheal Bifurc.
74

Huntingdon Life Sciences

WAG0024

APPENDIX 6 - continued Request ID: 274748

Macropathology and histopathology - individual findings

Group : 1 2 3 4

Compound : Control 2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

Exposure (ppm) : 0 533 1171 2980

Animal Number: 7 Sex: Male Dose Group: 3 Phase: Treatment Sacrifice Status: Final phase sacrifice

Date of Death: 30-Apr-12 Day of Death: 15 Week of Death: 3 Subgroup: 1 Terminal Bodyweight: 424.9 g

PATHOLOGY OBSERVATIONS

MACROPATHOLOGY HISTOPATHOLOGY

***ANIMAL HAS NO MACROSCOPIC FINDINGS RECORDED***

Nasal turbinates

 Olfactory Epithelium - Atrophy/Disorganisation, Minimal

The following examined tissues had no gross observations and no findings microscopically:

 Larynx, Lungs + Bronchi, Trachea, Tracheal Bifurc.
75

Huntingdon Life Sciences

WAG0024

APPENDIX 6 - continued Request ID: 274748

Macropathology and histopathology - individual findings

Group : 1 2 3 4

Compound : Control 2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

Exposure (ppm) : 0 533 1171 2980

Animal Number: 8 Sex: Male Dose Group: 3 Phase: Treatment Sacrifice Status: Final phase sacrifice

Date of Death: 30-Apr-12 Day of Death: 15 Week of Death: 3 Subgroup: 1 Terminal Bodyweight: 455.6 g

PATHOLOGY OBSERVATIONS

MACROPATHOLOGY HISTOPATHOLOGY

***ANIMAL HAS NO MACROSCOPIC FINDINGS RECORDED***

Nasal turbinates

 Olfactory Epithelium - Atrophy/Disorganisation, Minimal

The following examined tissues had no gross observations and no findings microscopically:

 Larynx, Lungs + Bronchi, Trachea, Tracheal Bifurc.
76

Huntingdon Life Sciences

WAG0024

APPENDIX 6 - continued Request ID: 274748

Macropathology and histopathology - individual findings

Group : 1 2 3 4

Compound : Control 2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

Exposure (ppm) : 0 533 1171 2980

Animal Number: 9 Sex: Male Dose Group: 3 Phase: Treatment Sacrifice Status: Final phase sacrifice

Date of Death: 30-Apr-12 Day of Death: 15 Week of Death: 3 Subgroup: 1 Terminal Bodyweight: 436.7 g

PATHOLOGY OBSERVATIONS

MACROPATHOLOGY HISTOPATHOLOGY

***ANIMAL HAS NO MACROSCOPIC FINDINGS RECORDED***

Nasal turbinates

 Olfactory Epithelium - Atrophy/Disorganisation, Minimal

 Inflammatory Exudate in Airways, Minimal

 Olfactory Epithelium - Inflammation, Diffuse, Slight

The following examined tissues had no gross observations and no findings microscopically:

 Larynx, Lungs + Bronchi, Trachea, Tracheal Bifurc.
77

Huntingdon Life Sciences

WAG0024

APPENDIX 6 - continued Request ID: 274748

Macropathology and histopathology - individual findings

Group : 1 2 3 4

Compound : Control 2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

Exposure (ppm) : 0 533 1171 2980

Animal Number: 10 Sex: Male Dose Group: 4 Phase: Treatment Sacrifice Status: Final phase sacrifice

Date of Death: 30-Apr-12 Day of Death: 15 Week of Death: 3 Subgroup: 1 Terminal Bodyweight: 408.9 g

PATHOLOGY OBSERVATIONS

MACROPATHOLOGY HISTOPATHOLOGY

***ANIMAL HAS NO MACROSCOPIC FINDINGS RECORDED***

Nasal turbinates

 Olfactory Epithelium - Atrophy/Disorganisation, Slight

 Olfactory Nerve Fibres - Atrophy, Slight

 Inflammatory Exudate in Airways, Minimal

 Olfactory Epithelium - Inflammation, Diffuse, Minimal

The following examined tissues had no gross observations and no findings microscopically:

 Larynx, Lungs + Bronchi, Trachea, Tracheal Bifurc.
78

Huntingdon Life Sciences

WAG0024

APPENDIX 6 - continued Request ID: 274748

Macropathology and histopathology - individual findings

Group : 1 2 3 4

Compound : Control 2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

Exposure (ppm) : 0 533 1171 2980

Animal Number: 11 Sex: Male Dose Group: 4 Phase: Treatment Sacrifice Status: Final phase sacrifice

Date of Death: 30-Apr-12 Day of Death: 15 Week of Death: 3 Subgroup: 1 Terminal Bodyweight: 442.6 g

PATHOLOGY OBSERVATIONS

MACROPATHOLOGY HISTOPATHOLOGY

Nasal turbinates

 Olfactory Epithelium - Atrophy/Disorganisation, Slight

 Olfactory Nerve Fibres - Atrophy, Slight

 Inflammatory Exudate in Airways, Slight

 Olfactory Epithelium - Inflammation, Diffuse, Minimal

Spleen

 Capsule thickened, Anterior pole, A few, areas, up to 1mm.

The following examined tissues had no gross observations and no findings microscopically:

 Larynx, Lungs + Bronchi, Trachea, Tracheal Bifurc.
79

Huntingdon Life Sciences

WAG0024

APPENDIX 6 - continued Request ID: 274748

Macropathology and histopathology - individual findings

Group : 1 2 3 4

Compound : Control 2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

Exposure (ppm) : 0 533 1171 2980

Animal Number: 12 Sex: Male Dose Group: 4 Phase: Treatment Sacrifice Status: Final phase sacrifice

Date of Death: 30-Apr-12 Day of Death: 15 Week of Death: 3 Subgroup: 1 Terminal Bodyweight: 474.4 g

PATHOLOGY OBSERVATIONS

MACROPATHOLOGY HISTOPATHOLOGY

***ANIMAL HAS NO MACROSCOPIC FINDINGS RECORDED***

Nasal turbinates

 Olfactory Epithelium - Atrophy/Disorganisation, Slight

 Olfactory Nerve Fibres - Atrophy, Minimal

 Inflammatory Exudate in Airways, Minimal

 Olfactory Epithelium - Inflammation, Diffuse, Minimal

The following examined tissues had no gross observations and no findings microscopically:

 Larynx, Lungs + Bronchi, Trachea, Tracheal Bifurc.
80

Huntingdon Life Sciences

WAG0024

APPENDIX 6 - continued Request ID: 274748

Macropathology and histopathology - individual findings

Group : 1 2 3 4

Compound : Control 2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

Exposure (ppm) : 0 533 1171 2980

Animal Number: 13 Sex: Female Dose Group: 1 Phase: Treatment Sacrifice Status: Final phase sacrifice

Date of Death: 30-Apr-12 Day of Death: 15 Week of Death: 3 Subgroup: 1 Terminal Bodyweight: 248.2 g

PATHOLOGY OBSERVATIONS

MACROPATHOLOGY HISTOPATHOLOGY

Lungs + Bronchi Lungs + Bronchi

 Pale area(s), Punctate, Multiple, subpleural. Alveolitis, Minimal

 Aggregations of Alveolar Macrophages, Slight

The following examined tissues had no gross observations and no findings microscopically:

 Larynx, Nasal turbinates, Trachea, Tracheal Bifurc.
81

Huntingdon Life Sciences

WAG0024

APPENDIX 6 - continued Request ID: 274748

Macropathology and histopathology - individual findings

Group : 1 2 3 4

Compound : Control 2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

Exposure (ppm) : 0 533 1171 2980

Animal Number: 14 Sex: Female Dose Group: 1 Phase: Treatment Sacrifice Status: Final phase sacrifice

Date of Death: 30-Apr-12 Day of Death: 15 Week of Death: 3 Subgroup: 1 Terminal Bodyweight: 260.4 g

PATHOLOGY OBSERVATIONS

MACROPATHOLOGY HISTOPATHOLOGY

***ANIMAL HAS NO MACROSCOPIC FINDINGS RECORDED***

Lungs + Bronchi

 Aggregations of Alveolar Macrophages, Minimal

The following examined tissues had no gross observations and no findings microscopically:

 Larynx, Nasal turbinates, Trachea, Tracheal Bifurc.
82

Huntingdon Life Sciences

WAG0024

APPENDIX 6 - continued Request ID: 274748

Macropathology and histopathology - individual findings

Group : 1 2 3 4

Compound : Control 2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

Exposure (ppm) : 0 533 1171 2980

Animal Number: 15 Sex: Female Dose Group: 1 Phase: Treatment Sacrifice Status: Final phase sacrifice

Date of Death: 30-Apr-12 Day of Death: 15 Week of Death: 3 Subgroup: 1 Terminal Bodyweight: 268.8 g

PATHOLOGY OBSERVATIONS

MACROPATHOLOGY HISTOPATHOLOGY

***ANIMAL HAS NO MACROSCOPIC FINDINGS RECORDED*** ***ANIMAL HAS NO MICROSCOPIC FINDINGS RECORDED***

The following examined tissues had no gross observations and no findings microscopically:

 Larynx, Lungs + Bronchi, Nasal turbinates, Trachea, Tracheal Bifurc.
83

Huntingdon Life Sciences

WAG0024

APPENDIX 6 - continued Request ID: 274748

Macropathology and histopathology - individual findings

Group : 1 2 3 4

Compound : Control 2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

Exposure (ppm) : 0 533 1171 2980

Animal Number: 16 Sex: Female Dose Group: 2 Phase: Treatment Sacrifice Status: Final phase sacrifice

Date of Death: 30-Apr-12 Day of Death: 15 Week of Death: 3 Subgroup: 1 Terminal Bodyweight: 238.3 g

PATHOLOGY OBSERVATIONS

MACROPATHOLOGY HISTOPATHOLOGY

***ANIMAL HAS NO MACROSCOPIC FINDINGS RECORDED*** ***ANIMAL HAS NO MICROSCOPIC FINDINGS RECORDED***

The following examined tissues had no gross observations and no findings microscopically:

 Larynx, Lungs + Bronchi, Nasal turbinates, Trachea, Tracheal Bifurc.
84

Huntingdon Life Sciences

WAG0024

APPENDIX 6 - continued Request ID: 274748

Macropathology and histopathology - individual findings

Group : 1 2 3 4

Compound : Control 2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

Exposure (ppm) : 0 533 1171 2980

Animal Number: 17 Sex: Female Dose Group: 2 Phase: Treatment Sacrifice Status: Final phase sacrifice

Date of Death: 30-Apr-12 Day of Death: 15 Week of Death: 3 Subgroup: 1 Terminal Bodyweight: 305.3 g

PATHOLOGY OBSERVATIONS

MACROPATHOLOGY HISTOPATHOLOGY

***ANIMAL HAS NO MACROSCOPIC FINDINGS RECORDED*** ***ANIMAL HAS NO MICROSCOPIC FINDINGS RECORDED***

The following examined tissues had no gross observations and no findings microscopically:

 Larynx, Lungs + Bronchi, Nasal turbinates, Trachea, Tracheal Bifurc.
85

Huntingdon Life Sciences

WAG0024

APPENDIX 6 - continued Request ID: 274748

Macropathology and histopathology - individual findings

Group : 1 2 3 4

Compound : Control 2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

Exposure (ppm) : 0 533 1171 2980

Animal Number: 18 Sex: Female Dose Group: 2 Phase: Treatment Sacrifice Status: Final phase sacrifice

Date of Death: 30-Apr-12 Day of Death: 15 Week of Death: 3 Subgroup: 1 Terminal Bodyweight: 301.5 g

PATHOLOGY OBSERVATIONS

MACROPATHOLOGY HISTOPATHOLOGY

***ANIMAL HAS NO MACROSCOPIC FINDINGS RECORDED*** ***ANIMAL HAS NO MICROSCOPIC FINDINGS RECORDED***

The following examined tissues had no gross observations and no findings microscopically:

 Larynx, Lungs + Bronchi, Nasal turbinates, Trachea, Tracheal Bifurc.
86

Huntingdon Life Sciences

WAG0024

APPENDIX 6 - continued Request ID: 274748

Macropathology and histopathology - individual findings

Group : 1 2 3 4

Compound : Control 2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

Exposure (ppm) : 0 533 1171 2980

Animal Number: 19 Sex: Female Dose Group: 3 Phase: Treatment Sacrifice Status: Final phase sacrifice

Date of Death: 30-Apr-12 Day of Death: 15 Week of Death: 3 Subgroup: 1 Terminal Bodyweight: 272.5 g

PATHOLOGY OBSERVATIONS

MACROPATHOLOGY HISTOPATHOLOGY

***ANIMAL HAS NO MACROSCOPIC FINDINGS RECORDED***

Larynx

 Squamous Metaplasia - Arytenoid, Slight

Nasal turbinates

 Olfactory Epithelium - Atrophy/Disorganisation, Slight

 Olfactory Nerve Fibres - Atrophy, Minimal

 Olfactory Epithelium - Inflammation, Diffuse, Minimal

The following examined tissues had no gross observations and no findings microscopically:

 Lungs + Bronchi, Trachea, Tracheal Bifurc.
87

Huntingdon Life Sciences

WAG0024

APPENDIX 6 - continued Request ID: 274748

Macropathology and histopathology - individual findings

Group : 1 2 3 4

Compound : Control 2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

Exposure (ppm) : 0 533 1171 2980

Animal Number: 20 Sex: Female Dose Group: 3 Phase: Treatment Sacrifice Status: Final phase sacrifice

Date of Death: 30-Apr-12 Day of Death: 15 Week of Death: 3 Subgroup: 1 Terminal Bodyweight: 262.1 g

PATHOLOGY OBSERVATIONS

MACROPATHOLOGY HISTOPATHOLOGY

***ANIMAL HAS NO MACROSCOPIC FINDINGS RECORDED***

Nasal turbinates

 Olfactory Epithelium - Atrophy/Disorganisation, Slight

 Olfactory Epithelium - Inflammation, Diffuse, Minimal

The following examined tissues had no gross observations and no findings microscopically:

 Larynx, Lungs + Bronchi, Trachea, Tracheal Bifurc.
88

Huntingdon Life Sciences

WAG0024

APPENDIX 6 - continued Request ID: 274748

Macropathology and histopathology - individual findings

Group : 1 2 3 4

Compound : Control 2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

Exposure (ppm) : 0 533 1171 2980

Animal Number: 21 Sex: Female Dose Group: 3 Phase: Treatment Sacrifice Status: Final phase sacrifice

Date of Death: 30-Apr-12 Day of Death: 15 Week of Death: 3 Subgroup: 1 Terminal Bodyweight: 270.4 g

PATHOLOGY OBSERVATIONS

MACROPATHOLOGY HISTOPATHOLOGY

***ANIMAL HAS NO MACROSCOPIC FINDINGS RECORDED***

Nasal turbinates

 Olfactory Epithelium - Atrophy/Disorganisation, Slight

 Olfactory Nerve Fibres - Atrophy, Slight

 Olfactory Epithelium - Inflammation, Diffuse, Minimal

The following examined tissues had no gross observations and no findings microscopically:

 Larynx, Lungs + Bronchi, Trachea, Tracheal Bifurc.
89

Huntingdon Life Sciences

WAG0024

APPENDIX 6 - continued Request ID: 274748

Macropathology and histopathology - individual findings

Group : 1 2 3 4

Compound : Control 2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

Exposure (ppm) : 0 533 1171 2980

Animal Number: 22 Sex: Female Dose Group: 4 Phase: Treatment Sacrifice Status: Final phase sacrifice

Date of Death: 30-Apr-12 Day of Death: 15 Week of Death: 3 Subgroup: 1 Terminal Bodyweight: 279.0 g

PATHOLOGY OBSERVATIONS

MACROPATHOLOGY HISTOPATHOLOGY

Liver

 Enlarged

Lungs + Bronchi Lungs + Bronchi

 Dark area(s), Left, Two, subpleural, up to 5mm. Bronchiolar Epithelial Hyperplasia, Moderate

 Perivascular/Peribronchiolar Inflammatory Infiltrate, Marked

Larynx

 Epithelial Hyperplasia, Ventral, Minimal

 Subepithelial Inflammation, Diffuse, Minimal

Nasal turbinates

 Olfactory Epithelium - Atrophy/Disorganisation, Slight

 Olfactory Nerve Fibres - Atrophy, Minimal

 Olfactory Epithelium - Inflammation, Diffuse, Minimal

Spleen

 Enlarged
90

Huntingdon Life Sciences

WAG0024

APPENDIX 6 - continued Request ID: 274748

Macropathology and histopathology - individual findings

Group : 1 2 3 4

Compound : Control 2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

Exposure (ppm) : 0 533 1171 2980

Animal Number: 22 Sex: Female Dose Group: 4 Phase: Treatment Sacrifice Status: Final phase sacrifice

Date of Death: 30-Apr-12 Day of Death: 15 Week of Death: 3 Subgroup: 1 Terminal Bodyweight: 279.0 g

PATHOLOGY OBSERVATIONS

MACROPATHOLOGY HISTOPATHOLOGY

continued from previous page...

Tracheal Bifurc.

 Epithelial Hyperplasia, Minimal

Thymus

 Firm, Left lobe

The following examined tissues had no gross observations and no findings microscopically:

 Trachea
91

Huntingdon Life Sciences

WAG0024

APPENDIX 6 - continued Request ID: 274748

Macropathology and histopathology - individual findings

Group : 1 2 3 4

Compound : Control 2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

Exposure (ppm) : 0 533 1171 2980

Animal Number: 23 Sex: Female Dose Group: 4 Phase: Treatment Sacrifice Status: Final phase sacrifice

Date of Death: 30-Apr-12 Day of Death: 15 Week of Death: 3 Subgroup: 1 Terminal Bodyweight: 277.0 g

PATHOLOGY OBSERVATIONS

MACROPATHOLOGY HISTOPATHOLOGY

***ANIMAL HAS NO MACROSCOPIC FINDINGS RECORDED***

Nasal turbinates

 Olfactory Epithelium - Atrophy/Disorganisation, Slight

 Olfactory Nerve Fibres - Atrophy, Slight

 Inflammatory Exudate in Airways, Minimal

 Olfactory Epithelium - Inflammation, Diffuse, Minimal

The following examined tissues had no gross observations and no findings microscopically:

 Larynx, Lungs + Bronchi, Trachea, Tracheal Bifurc.
92

Huntingdon Life Sciences

WAG0024

APPENDIX 6 - continued Request ID: 274748

Macropathology and histopathology - individual findings

Group : 1 2 3 4

Compound : Control 2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

2-bromo-3,3,3-

trifluoropropene

Exposure (ppm) : 0 533 1171 2980

Animal Number: 24 Sex: Female Dose Group: 4 Phase: Treatment Sacrifice Status: Final phase sacrifice

Date of Death: 30-Apr-12 Day of Death: 15 Week of Death: 3 Subgroup: 1 Terminal Bodyweight: 267.3 g

PATHOLOGY OBSERVATIONS

MACROPATHOLOGY HISTOPATHOLOGY

Lungs + Bronchi Lungs + Bronchi

 Pale area(s), Left, Punctate, One, subpleural. Aggregations of Alveolar Macrophages, Minimal

Nasal turbinates

 Olfactory Epithelium - Atrophy/Disorganisation, Slight

 Olfactory Nerve Fibres - Atrophy, Slight

 Olfactory Epithelium - Inflammation, Diffuse, Slight

The following examined tissues had no gross observations and no findings microscopically:

 Larynx, Trachea, Tracheal Bifurc.
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Annex 1 Aerosol technology and inhalation analysis report
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Working for a better future

Aerosol Technology Report

HLS study number: WAG0024

Authors A Allen

Simon A Moore

Version ID: Final

Issue date: 31 August 2012
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Table of Contents

1. Pre-study Atmosphere Characterization ...................................................................... 97

2. Test Article Handling and Processing for Inhalation Generation .................................. 97

3. Exposure System ........................................................................................................ 97

4. Administration ............................................................................................................. 97

5. Atmosphere Analysis................................................................................................... 98

5.1 Aerosol Concentration................................................................................. 98

5.2 Flow diagram of the analytical method for the analysis of 2-bromo-3,3,3-

trifluoropropene in test atmosphere samples................................................. 98

5.3 Nominal Concentration................................................................................ 98

5.4 Chamber Temperature................................................................................ 98

5.5 Chamber Humidity ...................................................................................... 98

6. Data and Calculations ................................................................................................. 99

7. Results and Discussion ............................................................................................... 99

7.1 Aerosol Concentrations............................................................................... 99

List of Tables

Table 1 Chamber atmosphere concentration of 2-bromo-3,3,3-trifluoropropene -

individual exposure values for Group 1 ......................................................100

Table 2 Chamber atmosphere concentration of 2-bromo-3,3,3-trifluoropropene – daily 

means and individual exposure values for Group 2....................................101

Table 3 Chamber atmosphere concentration of 2-bromo-3,3,3-trifluoropropene - daily 

means and individual exposure values for Group 3....................................102

Table 4 Chamber atmosphere concentration of 2-bromo-3,3,3-trifluoropropene - daily 

means and individual exposure values for Group 4....................................103

Table 5 Chamber Temperatures - daily mean.........................................................104

Table 6 Chamber Humidity - daily mean.................................................................105

Table 7 Nominal Chamber atmosphere concentration of 

2-bromo-3,3,3-trifluoropropene - individual exposure values for Group 2 ...106

Table 8 Nominal Chamber atmosphere concentration of 

2-bromo-3,3,3-trifluoropropene - individual exposure values for Group 3 ...107

Table 9 Nominal Chamber atmosphere concentration of 

2-bromo-3,3,3-trifluoropropene - individual exposure values for Group 4 ...108

List of Figures

Figure 1 Schematic of Inhalation Exposure System .................................................109
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Signature Page

Aerosol Technology Report
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97

1. Pre-study Atmosphere Characterization

Inhalation exposure system characterized including determinations of:

Achieved vapour concentration;

Spatial distribution around the chamber. 

2. Test Article Handling and Processing for Inhalation 

Generation

As supplied, 2-Bromo-3,3,3-trifluoro-1-propene was easily vapourised using warmed air 

through a sintered glass vapouriser.

3. Exposure System

A schematic of the exposure system is presented in Figure 1. Components of the exposure 

system:

Directed flow Whole body exposure chamber:

0.75 m

3 chamber of stainless–steel and glass construction

Animal Restraint:

Stainless mesh compartments

Aerosol Generator:

Sintered glass vaporiser for Groups 2 to 4

Liquid reservoir and feed rate used to alter concentration

Test groups 30 L/min

Temperature 40 C

Diluent Airflow:

From in-house compressed air system – breathing quality

Group 1 – 149 L/min

Group 2 to 4 119 – L/min

Extract Airflow:

Drawn by a fan extract system

Drawn from base of exposure system

Controlled by a gate clamp situated at the rear of the chamber

Airflow Monitoring:

In-line flowmeters monitored continuously for generation and diluent 

airflows

Chamber pressure differential monitored by Magnahelic set at -1 to -5

mm/H2O to ensure a slight negative pressure within the chamber ~

150 L/min.

4. Administration

Animals exposed for 6 hours per day, 5 days a week for 2 weeks.

Exposure system operational parameters monitored continuously.

Airflows documented initially and monitored during exposure.
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5. Atmosphere Analysis

5.1 Aerosol Concentration

Aerosol samples collected as follows:

Sample frequency – minimum of 3 samples/group/exposure 

Sample location – representative animal level

Sample position remained constant

Sample analysis

Infra-red spectrophotometer

5.2 Flow diagram of the analytical method for the analysis of 

2-bromo-3,3,3-trifluoropropene in test atmosphere samples

Sample Assay

Test atmosphere sample (Gas bag or chamber)



Sample directly using the IR spectrophotometer



Perform a single measurement on the IR 

spectrophotometer

Calibration

Prepare a series of calibration standards by direct 

addition of a suitable volume of test item directly into 

gas bags containing a suitable volume of air



Prepare a set of calibration standards at nominal 

concentrations of 388, 779, 1459, 1946, 2918 and 

3880 ppm



Measure the calibration range standards using the IR 

spectrophotometer for calibration

IR conditions

Instrument: MIRAN SapphIRe model 205B

Path length: Short (0.5 m)

Wavelength: 10.699 μm

The IR spectrophotometer was calibrated using external standards. Absorbance 

measurements acquired by the IR spectrophotometer were used to calculate analysed 

concentration (ppm) using a 2nd order fit subjected to least squares regression analysis.

5.3 Nominal Concentration

Monitored and summarised in Table 7 to Table 9.

5.4 Chamber Temperature 

Monitored using a liquid in glass thermometer, daily means presented in Table 5.

5.5 Chamber Humidity

Monitored using wet and dry liquid in glass thermometers, with humidity calculated using 

proprietary tables, daily means presented in Table 6.
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6. Data and Calculations

To minimize the cumulative errors from repeated rounding of numbers, data in this report has 

been calculated continuously using unrounded numbers and only rounded for printing. 

Consequently, presented data may include rounding errors in the last significant figure.

All data not presented in this report is retained in the study data files and archives. 

7. Results and Discussion

7.1 Aerosol Concentrations

Individual exposure concentrations and daily means are presented in Table 1 to Table 4 and 

summarized below:

Group Concentration (ppm)

Target Mean

1 0 0

2 500 533

3 1200 1171

4 3000 2980

Additional samples were taken for confirmation of atypical results and after minor generation

rate adjustments.

All groups were close to target.
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Table 1 Chamber atmosphere concentration of 

2-bromo-3,3,3-trifluoropropene - individual exposure values for 

Group 1

Exposure

No.

Chamber concentration (ppm)

Sample 1 Sample 2 Sample 3

1 ND ND ND

2 ND ND ND

3 ND ND ND

4 ND ND ND

5 ND ND ND

6 ND ND ND

7 ND ND ND

8 ND ND ND

9 ND ND ND

10 ND ND ND

ND No test item detected
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Table 2 Chamber atmosphere concentration of 

2-bromo-3,3,3-trifluoropropene – daily means and individual 

exposure values for Group 2

Exposure Chamber concentration (ppm)

No. Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Daily mean

1 549 530 446 418 486

2 426 664 543 337 493

3 538 677 594 603

4 640 679 543 508 451 564

5 465 608 461 511

6 459 535 792 595

7 624 562 451 425 516

8 558 513 548 540

9 455 575 550 527

10 490 505 484 493

Mean 533

SD 42.3

CV (%) 7.9

sd Standard deviation

CV (%) Coefficient of variation (sd × 100/mean)
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Table 3 Chamber atmosphere concentration of 

2-bromo-3,3,3-trifluoropropene - daily means and individual 

exposure values for Group 3

Exposure Chamber concentration (ppm)

No. Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Daily mean

1 1020 1381 1154 1204 1190

2 860 1339 1005 1288 1182 1135

3 898 1250 1160 1103

4 1126 1220 1290 1212

5 1297 1301 1080 1226

6 1317 1240 1262 1273

7 706 1095 1147 1119 1017

8 1134 1084 1166 1128

9 1192 1227 1205 1208

10 1276 1160 1227 1221

Mean 1171

SD 75.2

CV (%) 6.4

sd Standard deviation

CV (%) Coefficient of variation (sd × 100/mean)
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Table 4 Chamber atmosphere concentration of 

2-bromo-3,3,3-trifluoropropene - daily means and individual 

exposure values for Group 4

Exposure Chamber concentration (ppm)

No. Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Sample 6 Daily 

mean

1 1970 3129 3086 3145 2833

2 2411 2162 3110 1992 4631 2577 2814

3 3977 3652 2920 3516

4 2757 1627 3414 3173 2743

5 3110 3048 2992 3050

6 2835 2818 3344 2999

7 2243 3059 2856 2925 2771

8 2956 3195 2717 2956

9 3051 3171 2909 3044

10 3028 3296 2914 3079

Mean 2980

SD 225

CV (%) 7.6

sd Standard deviation

CV (%) Coefficient of variation (sd × 100/mean)
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Table 5 Chamber Temperatures - daily mean

Exposure

No.

Chamber Temperature (°C)

Group 1 Group 2 Group 3 Group 4

1 20.9 20.9 20.7 20.3

2 20.2 20.0 19.9 19.8

3 20.0 20.0 20.0 19.8

4 20.0 20.0 19.9 19.0

5 20.1 19.3 19.6 19.6

6 20.8 20.0 20.0 20.0

7 20.2 20.0 20.0 19.9

8 20.3 20.0 20.0 20.0

9 20.2 19.6 19.8 19.7

10 20.0 20.0 19.7 19.7
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Table 6 Chamber Humidity - daily mean

Exposure

No.

Chamber Temperature (%RH)

Group 1 Group 2 Group 3 Group 4

1 28.5 24.2 28.4 37.0

2 31.7 26.8 27.8 36.2

3 32.4 29.5 31.4 37.5

4 31.6 27.9 30.2 44.0

5 33.5 35.6 39.4 38.0

6 23.2 27.2 32.6 32.6

7 31.3 29.8 29.2 35.5

8 31.2 30.3 29.6 34.5

9 32.5 35.1 34.2 33.9

10 32.0 29.5 32.1 37.8
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Table 7 Nominal Chamber atmosphere concentration of 

2-bromo-3,3,3-trifluoropropene - individual exposure values for 

Group 2

Exposure 

No

Barometric 

Pressure 

P(mmHg)

Test 

Compound

usage TCU

(g)

Volume of 

Test

Compound 

V (L)

Analysed 

Conc.(ppm)

Nominal 

Conc.(ppm)

A/N1

ratio%

1 768 251 34.2 486 634 76.7

2 747 236 33.0 493 611 80.7

3 736 314 44.6 603 825 73.1

4 738 266 37.6 564 697 81.0

5 743 230 32.3 511 597 85.6

6 741 271 38.2 595 707 84.2

7 749 259 36.1 516 668 77.2

8 735 275 39.1 540 723 74.7

9 744 243 34.1 527 631 83.6

10 768 274 37.7 493 697 70.7

1 Analysed/Nominal (A/N) ratio (%) = Analysed concentration (ppm) / Nominal Concentration 

(ppm)x100

 

P(mmHg)

760

MW (g/mol)

TCU (g) R T( C) 273 )( 

LV 

(F D) V(L)

6

)( 10

NominalConc (ppm)







LV

V =Volume of test article 

R =Gas constant (0.08206 Latm/mol°K)

MW =Molecular weight (174.95 g/mol)

F =Chamber airflow (150 L/min)

D =Duration 6 Hours (360 mins)
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Table 8 Nominal Chamber atmosphere concentration of 

2-bromo-3,3,3-trifluoropropene - individual exposure values for 

Group 3

Exposure 

No.

Barometric 

Pressure 

P(mmHg)

Test 

Compound

usage TCU

(g)

Volume of 

Test

Compound 

V (L)

Analysed 

Conc.(ppm)

Nominal 

Conc.(ppm)

A/N1

ratio%

1 768 480 65.4 1190 1210 98.3

2 747 480 67.1 1135 1241 91.5

3 736 480 68.1 1103 1260 87.6

4 738 450 63.7 1212 1178 102.9

5 743 520 73.0 1226 1350 90.8

6 741 490 69.1 1273 1277 99.7

7 749 430 60.0 1017 1109 91.7

8 735 430 61.1 1128 1130 99.8

9 744 430 60.3 1208 1116 108.3

10 760 350 48.1 1221 889 137.3

1 Analysed/Nominal (A/N) ratio (%) = Analysed concentration (ppm) / Nominal Concentration 

(ppm)x100

 

P(mmHg)

760

MW (g/mol)

TCU(g) R T( C) 273 )( 



LV 

(F D) V(L)

6

)( 10 NominalConc.(ppm)







LV

V =Volume of test article 

R =Gas constant (0.08206 Latm/mol°K)

MW =Molecular weight (174.95 g/mol)

F =Chamber airflow (150 L/min)

D =Duration 6 Hours (360 mins)
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Table 9 Nominal Chamber atmosphere concentration of 

2-bromo-3,3,3-trifluoropropene - individual exposure values for 

Group 4

Exposure 

No 

Barometric 

Pressure 

P(mmHg)

Test 

Compound

usage TCU

(g)

Volume of 

Test

Compound 

V (L)

Analysed 

Conc.(ppm)

Nominal 

Conc.(ppm)

A/N1

ratio%

1 768 1160 157.9 2833 2916 97.1

2 747 1390 194.2 2814 3583 78.5

3 736 1330 188.6 3516 3481 101

4 738 1310 184.8 2743 3410 80.4

5 743 1270 178.3 3050 3291 92.7

6 741 1260 177.6 2999 3278 91.5

7 749 1300 181.2 2771 3345 82.8

8 735 1320 187.6 2956 3462 85.4

9 744 1320 185.1 3044 3417 89.1

10 760 1240 170.2 3079 3142 98.0

1 Analysed/Nominal (A/N) ratio (%) = Analysed concentration (ppm) / Nominal Concentration 

(ppm)x100

 

P(mmHg)

760

MW (g/mol)

TCU(g) R T( C) 273

)( 



LV 

(F D) V(L)

6

)( 10 NominalConc.(ppm)







LV

V =Volume of test article 

R =Gas constant (0.08206 Latm/mol°K)

MW =Molecular weight (174.95 g/mol)

F =Chamber airflow (150 L/min)

D =Duration 6 Hours (360 mins)
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Figure 1 Schematic of Inhalation Exposure System

D

A

B

F

C

E

F

H

I

J

K

Front view Side view

L

Generation Trolley

G

M

Key

A Whole body chamber H Air-line coil

B Test material delivery tubing I Sintered glass vaporiser

C Elutriator J Water bath

D Secondary generation trolley K Generation flowmeters

E Magnahelics L Control valves

F Pressure Vessels M Balance for more accurate weighing

G Load cells
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Annex 2 Pathology report
111
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1. Introduction 

1.1 Objective 

Assessment of local and systemic toxic potential in a 14 day inhalation study in rats to select 

suitable exposure levels for a subsequent 13 week inhalation toxicity study. 

Group Treatment Achieved Exposure Level 

(ppm) 

Number of animals 

Main study 

Male Female 

 

1 Control 0 3 3 

2 2-bromo-3,3,3-trifluoropropene 533 3 3 

3 2-bromo-3,3,3-trifluoropropene 1171 3 3 

4 2-bromo-3,3,3-trifluoropropene 2980 3 3 

 

2. Results 

2.1 Decedents 

There were no premature deaths in this study. 

2.2 Macroscopic Pathology 

2.2.1 Animals killed after 2 weeks of treatment 

The macroscopic examination performed after 2 weeks of treatment revealed no intergroup 

differences of note. 

The nature and incidence of all the findings were consistent with the commonly seen 

background of macroscopic changes. 

2.3 Microscopic Pathology 

2.3.1 Animals killed after 2 weeks of treatment 

Treatment related findings 

Changes related to treatment with 2-bromo-3,3,3-trifluoropropene were seen mainly in the 

nasal turbinates and, to a much lesser extent, in the larynx, trachea and lungs. 

Nasal turbinates 

In the olfactory epithelium atrophy/disorganisation and inflammation were seen in males and 

females at 1171 and 2980 ppm. Atrophy of the olfactory nerve fibres was seen in males and 

females at 2980 ppm and in females only at 1171 ppm. 

Inflammatory exudate in the airways was seen in males and females at 2980 ppm and in a 

single male at 1171 ppm. 

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Summary of treatment related findings in the nasal turbinates for animals killed after 2 weeks of 

treatment 

Group/sex 1M 2M 3M 4M 1F 2F 3F 4F 

Achieved Exposure Level. (ppm) 0 533 1171 2980 0 533 1171 2980 

 

Olfactory epithelium - 

atrophy/disorganisation 

Minimal 0 0 3 0 0 0 0 0 

Slight 0 0 0 3 0 0 3 3 

Total 0 0 3 3 0 0 3 3 

 

Olfactory epithelium - 

inflammation 

 

Minimal 0 0 0 3 0 0 3 2 

Slight 0 0 1 0 0 0 0 1 

Total 0 0 1 3 0 0 3 3 

 

Olfactory nerve fibres - atrophy 

Minimal 0 0 0 1 0 0 1 1 

Slight 0 0 0 2 0 0 1 2 

Total 0 0 0 3 0 0 2 3 

 

Airways - inflammatory exudate 

Minimal 0 0 1 2 0 0 0 1 

Slight 0 0 0 1 0 0 0 0 

Total 0 0 1 3 0 0 0 1 

 

Number of tissues examined 3 3 3 3 3 3 3 3 

 

2.3.2 Findings of uncertain relationship to treatment 

A single female, Animal 22, exposed to 2980 ppm had perivascular and peribronchiolar 

inflammatory infiltrate and bronchiolar epithelial hyperplasia in the lungs, epithelial 

hyperplasia and subepithelial inflammation in the larynx and epithelial hyperplasia at the 

tracheal bifurcation. 

The relationship to treatment of these changes is uncertain. 

Incidental findings 

All other changes were considered to be incidental. 

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3. Discussion 

The changes seen in the nasal turbinates are consistent with a localised toxic effect of the test 

material. They are similar, but of a much reduced severity, to the changes seen in the 

previous dose ranging study (Huntingdon Life Sciences Study Number: WAG0013). 

The changes seen in the respiratory tract of Animal 22, exposed to 2980 ppm, were not seen 

in any other animals and were of uncertain relationship to treatment. 

4. Conclusion 

Treatment with 2-bromo-3,3,3-trifluoropropene for 2 weeks by the inhalation route produced 

changes, predominantly, in the nasal turbinates. 

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5. References 

Huntingdon Life Sciences Study Number: WAG0013 

2-bromo-3,3,3-trifluoropropene: Dose Range Finding Study by Inhalation Administration to 

Rats for 2 Weeks. 

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Annex 3 Certificate of analysis
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Annex 4 GLP compliance statements 
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HUNTINGDON RESEARCH CENTRE GLP COMPLIANCE STATEMENT 2011 
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EYE RESEARCH CENTRE GLP COMPLIANCE STATEMENT 2011