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## R EPORT 2 - bromo - 3,3,3 -trifluoropropene : Dose Range Finding Study by Inhalation Administration to Rats for 2 Weeks

HLS s tudy number: WAG0024 V ersion ID: Final Issue date: 31 August 2012Huntingdon Life Sciences WAG0024

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# Details of Sponsor and Te st Facilit ies Sponsor American Paci fic Corporation 3883 Howard Hughes Parkway Suite 700 Las Vegas Nevada 89169 USA Test facility Huntingdon Life Sciences Primary location Huntingdon Research Centre Wo olley Road Alconbury Huntingdon Cambridgeshire PE28 4HS UK Test facility Huntingdon Life Sciences Histology Eye Research Centre Eye Suffolk IP23 7PX UKHuntingdon Life Sciences WAG0024

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# Ta ble of Contents Details of Sponsor and Test Facilities ................................................................................... 2 Complianc e 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 Met hod 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 “We ek” ............................................................................. 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 pro cedures .................................................. 21 2.9.1 Quality assurance ............................................................................... 21 2.9.2 Archives .............................................................................................. 21 2.10 Deviations from protocol ................................................................................. 21 3. Results ....................................................................................................................... 22Huntingdon Life Sciences WAG0024

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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 T ables Table 1 Bodyweight .................................................................................................. 29 Table 2 Organ weights .............................................................................................. 33 Table 3 Macropathology ........................................................................................... 35 Table 4 Histopathology ............................................................................................. 36 

# List of Ap pendices Appendix 1 Clinical s igns ................................................................................................ 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 ......................................................................................... 1 10 Annex 3 Certificate of analysis ................................................................................. 11 6 Annex 4 GLP compliance statement s ...................................................................... 11 8Huntingdon Life Sciences WAG0024

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# Contributing Scientist s 2-bromo- 3,3,3 -trifluoropropene: Dose Range Finding Study by Inhalation Ad ministration to Rats for 2 Weeks 

## Study management Paul J Horrell BSc (Hons) IDT Study Di rector William N Hooks MIBMS BSc (Hons) Senior Toxico lo gist 

## Formulation and inhalation analysis Caroline Finucane PhD Head of Form

ulat io n & Inhalat io n Analysis 

## Aerosol technology Simo n A Moo re BSc (Hons) PhD MRSC Head of Aerosol Techno lo gy 

## Pathology Martin Brown BSc PGCE CBio l MSB PhD Principal Patho lo gi stHuntingdon Life Sciences WAG0024

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# Summary The obj ect ive o f thi s study was to assess the lo cal

and systemic toxic potential of 2- bromo- 3,3,3 - trifluoropropene , a fire ext inguishing agent , when administered by inhalat io n to Crl:CD(SD) rats fo r 6 hours a day, 5 days a week, for 2 weeks . Three groups, each comprising three ma le and thre e female rats received 2 - bromo-3,3,3 - trifluoropropene at target exposure level s of 500, 1200 or 3 000 ppm . A similarly const itut ed Control group received clean ai r only , at the same vo lu me dose . During the study, clinical condit io n, bodyweight, food consumpt io n, organ weight, macropathology

and histopathology

invest igat io ns were undertaken. 

## R esults 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 inhalat io n of an irritant material or possible CNS effects , were ev ident during and after exposure. Im pai red body weight/ body weight gain was evident , over the 2 week treatment period , fo r treated ma les in a dosage related manner . Total food consumpt ion was slight ly reduced for treated ma les . Hi stopathological

changes related to treatment were seen mainly in the nasal turbinates and, to a m uch lesser extent, in the larynx, trachea and lungs. In the olfactory

epi thelium atrophy /disorganisat io n and inflammat io n 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 sing le fe male exposed to 2980 ppm had perivascular and peribronchio lar inflammatory infiltrate and bronchio lar epithelial hyperplasia in the lungs, epithelial hyperplasia and subepithelial inflammat io n in the larynx and epit helial hyperplasia at the tracheal b ifurcation. The relat io nship to treatment of these changes is uncertain. 

## Conclusion Hi stopathological

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 advers e 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

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# 1. Introduction 1.1 Objective The obj ect ive o f thi s study was to assess the lo cal

and systemic toxic potential of 2- bromo- 3,3,3 - tr ifluoropropene , a fire ext inguishing agent , when administered by inhalat io n to rats fo r 2 weeks , to select sui table exposure levels for a subsequent 13 week toxicit y study . 

## 1.2 Regulatory compliance Due to the preliminary nature of this study, no specific regu lat io ns 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 beca

use of the historical control data available in this laboratory . 

## 1.4 Route of administration The inhalat io n route of administration was chosen to simulate the condit io ns of potential human exposure. 

## 1.5 Treatment groups and doses In a previous 2 week dose range finding study ( Hunt ingdon 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 body weight losses at 19098 ppm, reduced bodyweight gain at 4721 or 8901 ppm, and reduced food consumpt io n and transient clinical signs at all dose levels. Hi stopathologica l 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 cavit y, wi th l ess 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 histopatho lo gical

change s at lower exposure levels.Huntingdon Life Sciences WAG0024

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# 2. Experimental Procedure 2.1 S tudy schedule and structure 2.1.1 Duration of treatment The test substance, 2- bro mo -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 com pleted in 1 day . The duration of treatment is reported as 2 weeks. 2.1.2 Time schedule Study init iat io n: (Pro tocol signed by

Study Di rector) 27 March 2012 Experimental start date: (Pre - treatment chemistry

assays) 29 March 2012 An imal arrival: 29 March 2012 Treatment commenced: 16 April 2012 Necropsy com pleted: 30 April 2012 Experimental co mp let io n date: (pathology

) 6 July 2012 Study com plet io n: 31 August 2012 2.1.3 Identity of treatment groups The study consisted of one Control and three treated groups of rats, ident ified as fo llo ws: 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 - br omo - 3,3,3 - trifluoropropene 500 3 3 4 - 6 16 - 18 2 6 3 2 - br omo - 3,3,3 - trifluoropropene 1200 3 3 7 - 9 19 - 21 3 7 4 2 - br omo - 3,3,3 - trifluoropropene 3000 3 3 10 - 12 22 - 24 4 8Huntingdon Life Sciences WAG0024

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## 2.2 Test substance and formulation 2.2.1 Test substance Informat io n supplied by the Sponsor regarding the test sub stance 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 fo llo wing informat io n is given in summary : Ident ification: 2- bromo- 3,3,3 - trifluoropropene (stabil ized) Usage : Fi

re ext inguishing agent Descript io n: Liquid below 33  C

Clear and may

b e colourless to light yellow t int Storage condit io ns: Atambient temperature or in a refrigerator. Supplier: Sponsor Batch number: Kg F080 Date of receipt: 12 Septemb er 2011 Quant it y received: 2 × 150 kg cylinders Reassay date: 1 August 2012 Puri ty : 99.9% (excluding stabilizers) Stabilisers: The Sponsor was responsible for the characterisat ion of the test substance and the documentation of

the methods of synt hesis, fabrication or derivat io n and stabilit y.Huntingdon Life Sciences WAG0024

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## 2.3 Animal management 2.3.1 Animal supply, acclimatisation and al location Crl :CD (SD) rats (a total

of 15 ma le and 15 fe male ) were received fro m Charles River (UK) Ltd . The rats were ordered at 49 to 56 days of age and wit hin a weight range of 290 to 330 g fo r ma les 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 wit h the procedure being repeated until each cage held the appropriate number of animals. Each sex was allo cated separately . Each animal was assigned a number and ident ified uniquely wit hin the study by a tail tattoo. Each cage label was colour - coded according to group and was numbered uniquely wit h cage and study n umber, as well as the identit y of the occupants. The animals were allowed to acclimat ise to the condit io ns described below for 18 days before treatment commenced. For those animals selected for this study, thei r 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 remo ved from the study room at the end of the study . 2.3.2 Animal housing, diet and water supply Animals were housed inside a r estricted access rodent facilit y (Building Y14 , Room 011) . The facilit y 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 relat ive humidit y control s were maintained wit hin the range of 19 to 23°C and 40 to 70% respectiv ely and moni tored conti

nuously. There were no de viat io ns fro m these ranges . Artificial light ing was controlled to give a cy cle of 12 hours continuous light and 12 hours continuous dark per 24 hours. Alarms were activated if there was any failure of the vent ilat io n system, or temperature limits were exc eeded. A stand - by electrici ty supply was available to be automat ically brought into operation should the public supply fail. The animals were housed th ree of one sex per cage . The cages were made of a polycarbonate body wi th a stainless steel

mesh lid. Sof

twood 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 const ituting each group were blocked tog ether by sex and the groups were di spersed in batteries so that possible environmental influences arising from their spat ia l di stribut io n were equilibrated, as far as was pract ic able. Addit io nally, batteries of cages were rotated around the room at weekly int ervals to further minimise possible spat ial variat io ns.Huntingdon Life Sciences WAG0024

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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 . Thi s diet contained no added ant ibiot ic or other chemo therapeut ic or prophylactic agent. Potable water taken fro m the public supply was f reely available via polycarbonate bottles fi tted wi th sipper tubes, except during the dosing procedure . Each cage of animals was provided with an Aspen chew block and a plas tic shel ter fo r environmental enrichment. The soft white untreated wood blocks were provided throughout the study and were replaced when necessary; plast ic shelters were replaced at the same time as the cages. Each batch of diet was analysed routinely by the supplier for various nutri tional com ponents and chemical and microbio lo gical contaminants. Supplier’s analy ti cal certificates were scrutinised and approved before any b atch of diet was released for use. The qualit y of the water supply

i s governed by regulat io ns published by the Department f

or Envi ronment, Food and Rural Affairs. Certificates of analysis were received routinely fro m the water supplier and the sup plier s of

the bedding and A spen chew blocks. Since the results of these various analyses did not provide evidence of contaminat io n 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 int erf

ered wi th or prej udiced the outcome of

the study was known. 2.3.3 Administration Details o f 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 who le body exposure chamber. Different doses of 2- bro mo -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

f or 5 days each week (Monday to Friday ) f or 2 weeks.Huntingdon Life Sciences WAG0024

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## 2.4 Serial observations Dated and signed records of all act ivit ies relating to the day by day running and maintenance of the study wi thin the animal unit as well as to the group observat io ns and examinat io ns outlined in this experimental procedure were recorded in the Study Day Book. In addi

tion, observat io ns relat ing to individual animals made throughout the day were recorded. All observat io ns described below were performed in cage number sequence except where otherwi se indicated. 2.4.1 Clinical observations Animals were inspected visually at least twice daily for evidence of ill - healt h or reaction to treatment. Cages were inspected daily f or evidence of ill - heal

th amongst the occupant(s). Any deviat io n from normal was recorded at the time in respect of nature and severit y, date and time o f onset, duration and progress of the observed condit io n, as appropriate. Daily , on exposure days, during Week 1 of treatment, detailed observat io ns were recorded at the fo llo wing times in relation to dose administration: Pre - exposure During exposure Between one and two hours after complet io n of dosing of all groups As late as possible in the working day During Week 2 of treatment, detailed observat io ns were only recorded before exposure and between one and two hours after completion o f dosing of all groups. In addit io n, a more detailed weekl y physical examinat io n was performed on each animal to mo nit or general

heal th . During the acclimat is ation period, observat io ns 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 est imate of any spilled was recorded daily fro m Day

- 5 and th roughout the treatment period . From these records the mean dai ly

consumption per animal (g/rat/ day ) was calculated for each cage.Huntingdon Life Sciences WAG0024

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## 2.5 Necropsy and Histology 2.5.1 Method of kill Animals were killed by intra - peri toneal sodi um pentobarbi

tone, fo llo wed by

ex sanguinat io n . The sequence in which the animals were killed aft er com plet io n of treatment was selected to allow sat is factory

int er - 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 examinat io n of the tissues was per fo rmed. All

external features and orifices were examined visually . The cranial roof was removed to allow observat io n of the brain, pit ui tary gland and cranial nerves. After ventral mid - line incisio n, the neck and associated tissues and the thoracic, abdominal and pelvic cavit ies and their viscera were exposed and examined in situ. Any abnormal posit io n, mo rphology

or interaction was recorded. The requi si

te organs were weighed and external and cut surfaces of the organs and tissues were examined as appro priate. Any abnormali

ty in the appearance or size of any organ and ti ssue was recorded and the required tissue samples preserved in appropriate fixat ive. The retained tissues were checked before disposal of the carcass. 2.5.3 Organ weights The fo llo wing organs, taken from each animal killed after 2 weeks of treatment, were di ssected free o f adj acent 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 body weight, using the weight recorded before necropsy .Huntingdon Life Sciences WAG0024

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### 2.5.4 Fixation Testes were fixed in modified Davidson's fluid and eyes were fixed in Davidson's fluid. Sam ples (or the whole) of the other tissues listed below fro m all animals were preserved in 10% neutral buffered formalin: Adrenals Optic nerves Ao rta - thoracic Ovaries Brain Pancreas Caecum Pi tui tary Colon Prostate Duodenum Rectum Epididymides Salivary glands - submandibular Eyes - sublingual Femurs Sciat ic nerves Harderian glands Seminal vesicles Head# Skeletal m uscle Heart Skin wi th mammary glands (inguinal) Ileum Spinal cord Jejunum Spleen Kidneys Sternum Lachryma l glands Stomach Larynx* Testes Liver Thymus Lungs* Thy roid wi th parathy ro id s Lymph nodes - mandibular Tongue

- me senteric Trachea (including bifurcat io n)*

- tracheo - bronchial Urinary bladder Nasal turbinates* Uterus and cervix Oesophagus Vagina * Tissues examined microscopically # Including nasal cavity, paranasal sinuses and nasopharynx 

### Sam ples o f any abnormal t is sues were also retained. In those cases where a lesio n was not clearly delineated, contiguous ti ssue was fixed wit h the grossly affected region and sect io ned as appropriate. T he remaining samples were not examined histologically, but are retained against any future requi rement for microscopic examinat io n.Huntingdon Life Sciences WAG0024

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### 2.5.5 Histology For those animals specified in the Patho lo gy sect io n, the relevant tissues were subject to histological processing. Tissue samples were dehydrat ed, embedded in paraffin wax, sect io ned at approximately four to five micron thickness and stained wi th haematoxylin and eosin. Those ti ssues subject to histological processing included the fo llo wing regions: Larynx - 5 sections Lungs - sect io n from all ma jo r lobes, to include bronchi Nasal Turbinates - 3 sections Trachea - including bifurcat io n For bilateral organs, sections of both organs were prepared. A single section was prepared fro m each of the remaining tissues requ ir ed f or 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 complet io n of the scheduled treatment period. Tissues repor

ted at macroscopic examinat io n as being grossly abnormal were examined in line wit h current practice. Findings were either reported as "present" or assigned a severit y grade. In the latter case one of the fo llo wing five grades was used - minimal, slight, mo derate, marked or severe. The descript io ns listed below are an attem pt to define the grades in general t erms fo r all microscopic findings reported in a study and are not specific fo r a particular ti ssue. They are mo re strictly applicable to so me ty pe s of l esion than to others, and there may therefore be a few lesio ns fo r which these definit io ns are not completely appropriate.Huntingdon Life Sciences WAG0024

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### Thresho lds fo r the grading of l esion s fo r certain tissues may vary wi th ty pe of study

and also may vary, generally for all t is sues or the maj ori ty of ti ssues, wi

th the duration of

the study . In increasing order of degree/severit y. Minimal The lo west grade of a l esion w hich is di scernible/di st inguishable from normal and is of

ten subt le and/or invo lving a very s mall area of

the organ. Slight Clearly discernible/dist inguishable from normal wi th more extensive or mo re apparent changes than minimal. Moderate Generally invo lves a significant proportion of the affected organ or ti ssue. Marked Invo lves the majorit y of the affected organ or ti ssue. Severe Invo lves all or almo st all of an affected organ or tissue. A reviewing patho lo gi st undertook a peer review of the microscopic findings.Huntingdon Life Sciences WAG0024

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## 2.7 Computer s ystem s The computer systems wi th version numbers that were used on this study to acqu ir e and quant ify data include: Liberate Release 2 in- house system used for reporting clinical signs, dosing signs, body weights, food consumpt io n, organ weights, macropathology and histopathology . Sam ple Registry

System (versio n 1.0) used for Formulat io n and Inhal at io n Analysis data. Waters Empower 2, Build 2154, Feature Release 4, Service Pack D and used for Form ulat io n and Inhalat io n Analysis. Xybio n Path/Tox System (versio n 4.2.2) used for clinical signs, dosing signs, body weights, food consumpt io n, organ weight s, macropathology and histopathology . Xybio n Pri st ima (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 observat io ns relat ing to the acclimat isat io n period incl uded in this report relate to Week -1 bodyweights and fo od consumpt io n. Summary statistics (e.g. means and standard deviatio ns) presented in this report were calculated from

com puter - stored individual raw data. The summary statist ic s and the individual data were stored in the computer to a certain number of decimal places, different fo r 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 deviat io ns exactly using the presented individual data. Throughout the report the fo llo wing abbreviat io ns are used: M Male F Female N Number of animals/cages examined SD Standard deviat io n The death codes in the appendices have the fo llo wing 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 f ol lo wing. Dosing took place over the first five days of the week only . Subsequent experimental weeks of treatment were of the same duration.Huntingdon Life Sciences WAG0024

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### 2.8.2 Signs Signs are considered in two parts: observat io ns made in associat io n wi th treatment, classified as “dosing signs” and extended changes in condit ion, c la ssified as “clinical signs”. Both are presented for each animal that showed signs, providing detail of ty pe of sign, day or week of occurrence and information on the dur ation 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 abbreviat io ns used in the Appendix have the following meanings: Bifurc. Bifurcat io n Tissues which could not be examined are specified in the Appendix. The absence of a comment for a tissue scheduled for examinat io n therefore indicates that the tissue was considered to be normal. In all tabular presentations o f data the tissues specified in the protocol f or hi stopathological examinat io n pr ecede other ti ssues. 2.8.5 Statistical analysis The small sample size precludes meaningful statistical evaluat io n.Huntingdon Life Sciences WAG0024

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## 2.9 Quality assurance and archiving procedures 2.9.1 Quality assurance Details o f the Qualit y Assurance inspections and audits are presented on the Qualit y A ssurance Statement. 2.9.2 Archives Fo llo wing com

plet io n 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 o f Hunt ingdon Life Sciences. Types of sample and s pecimen which are unsuitable, by reason of instabilit y, f

or long term

retention and archiving may b e disposed of after the periods stated in Hunt ingdon Life Sciences Standard Operating Procedures. A copy of the final report and all

Quali ty A ssurance inspec tion records will be retained indefinit ely. All other appropriate specimens and records will be retained for a minimum period of one year fro m the date of i ssue of the final report. At the end of the one year retention period the Sponsor will be contacte d 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 state d that “ different concentrations of the test substance will

b e produced using different liquid feed rates controlled by the infusion

pum p and using sy

ringes of an appropriate volume” . Due to the high exposure levels required, this method of generat io n was not practical. Instead different exposure levels were achieved by varying the feed rates of the test material through metering valve s f rom pressurised reservo ir s. This deviation did not adversely affect study int egrit y.Huntingdon Life Sciences WAG0024

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# 3. Results 3.1 Chamber atmosphere conditions Annex 1 The achieved chamber concentrations are summarised as fo llo ws : 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 associ ated with dosing Appendi ces 1 and 2 There were no unscheduled deaths during the course of the study . Clinical signs seen during the fi rst 5 days of exposure included: unresponsiveness to external stimuli, underact ivit y, gasping, slow, shallow, deep and/or irregular breathing, piloerect io n, closed or partially

closed eye lid s, salivat io n, chin rubbing and lacrymat io n. These signs generally reso lved quickly on cessat io n of exposure to the test article. The number and prevalence o f signs increased wit h dose. 

## 3.3 Bodyweight Figure 1 , Table 1 and Appendix 3 Im pai red body weight/ body weight gain was evident , over the 2 week treatment period , fo r males exposed to 533, 1171 or 2980 ppm, wi th overall values of 0.54X, 0.56 X and 0.67X control , respectively . Over these firs t 5 days of exposure body weight losses of 6g, 5g and 25g were evident for males exposed to 533, 1171 or 2980 ppm, com pared wi th a body weight gain of 10g for the control males. Body

weight gains were then evident for all treated groups during the off - dose w eekend period (Days 6 and 7). During the second 5 days of exposures (Days 8 - 12) the effect of exposure on body weight was less marked, wi th body weight losses of 7g and 3g for males exposed to 533 or 1171 ppm respect ively and a slight gain of 2g evident for males exposed to 2980 ppm, com pared wi th a gain of 9g for controls. Gains were again evident for all treated groups during the second off - dose weekend period prior to necropsy .Huntingdon Life Sciences WAG0024

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### Body weight gains for females exposed to 533, 1171 or 2980 ppm, over the two w eek study period, were greater than control (2.68X, 2.91X and 2.01X control , respect ively). However, these differences were the resul t of low er overall gain for the control f emales. Females exposed to 2980 ppm lo st 4g over the first 5 days of exposure com pared wi th a gain of 3g for con

trol s (and a loss of 25g for males exposed to 2980 ppm ). The effects in the lo wer exposure groups and in the second week fo llow ed the same trends seen in males. 

## 3.4 Food consumption Appendix 4 Food consumpt io n was slightly reduc ed over the treatment period for males exposed to 2 bromo 3,3,3 trifluoropropene. Total f ood consumption (Days 1 to 14) was 0.84X, 0.87X and 0.87X con trol f or males and 0.97X, 1.00X and 0.9 1 X control for females exposed to 533, 1171 or 2980 ppm respect ivel y. 

## 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 examinat io n performed after 2 weeks of treatment revealed no intergroup differences of note. The nature and incidence of all the findings were consistent with the commo nly 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

epi thelium atrophy /disorganisat io n and inflammat io n were seen in males and females exposed to 1171 or 2980 ppm. Atrophy of the olfactory

nerve fibres was see n 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 nas al 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 Olfactor y 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 Olfactor y 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 Olfactor y ne

rve 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 relati onship to treatment A single female, Animal 22, exposed to 2980 ppm had perivascular and peribronchio lar inflammatory

infil trate and bronchiol ar epi

thelial hyperplasia in the lungs, epithelial hyperplasia and subepit helial inflammat io n in the larynx and ep ithelial hy perplasia at the tracheal bifurcat io n. 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 inhalat io n administration, using a who le - body exposure system, for 6 ho urs a day, 5 days a week, for 2 - week at achieved aerosol concentrations o f 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 irri tant effect of the test material. They are similar, but of a much reduced severit y, to the changes seen in the previous dose ranging study ( Hunt ingd on 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 relat io nship to treatment. Clinical signs of gasping, slow, shallow, deep and/or irregular breathing, piloerection, salivat io n chin rubbing and lacrymat io n are indicative of exposure to an irritant material and are consistent with the pathological findings . Whereas, the clinical signs of unresponsiveness to external st imuli, underact ivit y, and closed or partially clos ed eye lid s 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 aeroso l concentrations of 4721 to 19098 ppm. Treatment - related lo wer body weight gains were seen in males of all test article groups. These reduct io ns were exposure level related and are considered to be treatment - related and correlate to some extent wi th reduce d f

ood consumption that was also seen. Similar effects were not seen in females and this potentially represents a sex- related difference. E xposure atan aerosol concentration of 533 ppm resul ted in no histopathological changes . Sl ightly lower of body

weig htgain and food consumpt io n and transient clinical signs were seen at thi s level but were co nsidered not to be adverse.Huntingdon Life Sciences WAG0024

26 

# 5. Conclusion Hi stopathological

changes related to 2- bromo- 3,3,3 - trifluoropropene exposure were seen in the nasal turbinates of

rats expos ed to 1171 or 2980 ppm. The no observed adverse effect level (NOAEL) was 533 ppm, as this level was not associated wi

th any histopathological changes or adverse in- life findings .Huntingdon Life Sciences WAG0024

27 

# 6. References Hunt ingdon Life Sciences Study Number WAG0013: 2- bromo- 3,3,3 - trif

luoropropene: Dose Range Finding Study by Inhalat io n Administration to Rats for 2 Weeks.28Huntingdon Life SciencesWAG0024

FIGURE 1 Bodyweight - gr oup mean values (g) Group : 1 2 3 4 Compound : Control 2 - br omo - 3,3,3 - trifluoropropene 2 - br omo - 3,3,3 -

trifluoropropene 2 - br omo - 3,3,3 -

trifluoropropene Exposure (ppm) : 0 5 33 1 171 298029Huntingdon Life SciencesWAG0024

TABLE 1 Request ID: 269565 Bodyweight - gr oup mean values (g) Group : 1 2 3 4 Compound : Control 2 - br omo - 3,3,3 - trifluoropropene 2 - br omo - 3,3,3 -

trifluoropropene 2 - br omo - 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 1 M 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 2 6.2 29.3 25.9 N 3 3 3 3 3 3 3 3 3 3 3 3 2 M 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 3 M 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 4 M 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 330Huntingdon Life SciencesWAG0024

TABLE 1 - continued Reque st ID: 269565 Bodyweight - gr oup mean values (g) Group : 1 2 3 4 Compound : Control 2 - br omo - 3,3,3 - trifluoropropene 2 - br omo - 3,3,3 -

trifluoropropene 2 - br omo - 3,3,3 -

trifluoropropene Exposure (ppm) : 0 533 1171 2980 Group Day Day Day Day Day D ay Day Day Change As % of /Sex 8 9 10 11 12 13 14 15 1 - 15 Control 1 M 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 2 M 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 3 M Mean 428 428 423 42 5 425 428 436 439 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 4 M 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 331Huntingdon Life SciencesWAG0024

TABLE 1 - continued Request ID: 269565 Bodyweight - gr oup mean values (g) Group : 1 2 3 4 Compound : Control 2 - br omo - 3,3,3 - trifluoropropene 2 - br omo - 3,3,3 -

trifluoropropene 2 - br omo - 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 1 F Mean 243 242 246 2 48 250 251 250 253 255 254 254 258

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 2 F 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 3 F 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 4 F 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 332Huntingdon Life SciencesWAG0024

TABLE 1 - continued Request ID: 269565 Bodyweight - gr oup mean values (g) Group : 1 2 3 4 Compound : Control 2 - br omo - 3,3,3 - trifluoropropene 2 - br omo - 3,3,3 -

trifluoropropene 2 - br omo - 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 1 F 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 2 F 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 3 F 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 4 F 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 333Huntingdon Life SciencesWAG0024

TABLE 2 Request ID: 269563 Organ weights - gr oup mean unadjusted and adjusted value s (g) for animals killed after 2 weeks of treatment Group : 1 2 3 4 Compound : Control 2 - br omo - 3,3,3 - trifluoropropene 2 - br omo - 3,3,3 -

trifluoropropene 2 - br omo - 3,3,3 -

trifluoropropene Exposure (ppm) : 0 533 1171 2980 Group /Sex Terminal

Bodyweight B rain Heart Kidneys Liver Lungs + Br onchi Spleen Un adjusted 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 0.06 4 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 0.82634Huntingdon Life SciencesWAG0024

TABLE 2 - continued Request ID: 269563 Organ weights - gr oup mean unadjusted and adjusted values (g) for animals killed after 2 weeks of treatment Group : 1 2 3 4 Compound : Control 2 - br omo - 3,3,3 - trifluoropropene 2 - br omo - 3,3,3 -

trifluoropropene 2 - br omo - 3,3,3 -

trifluoropropene Exposure (ppm) : 0 533 1171 2980 Group /Sex Terminal

Bodyweight Br ain Heart Kidneys Liver Lungs + Br onchi Spleen Un adjusted 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 0.73635Huntingdon Life SciencesWAG0024

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 - br omo - 3,3,3 - trifluoropropene 2 - br omo - 3,3,3 -

trifluoropropene 2 - br omo - 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 Thy mus Firm 0 0 0 0 0 0 0 136Huntingdon Life SciencesWAG0024

TABLE 4 Request ID: 274747 Histopathology - gr oup distribution of findings for animals killed after 2 weeks of treatment Group : 1 2 3 4 Compound : Control 2 - br omo - 3,3,3 - trifluoropropene 2 - br omo - 3,3,3 -

trifluoropropene 2 - br omo - 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 Br onchiolar Epithelial Hyperplasia 0 0 0 0 0 0 0 1 Perivascular/Peribronchiolar Inflammatory Infiltrate 0 0 0 0 0 0 0 1 Lary nx Number Examined: 3 3 3 3 3 3 3 3 Epithelial Hyperplasia, Ventral 0 0 0 0 0 0 0 1 Squamous Metaplasia - Aryt enoid 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 337Huntingdon Life SciencesWAG0024

APPENDIX 1 Request ID: 269558 Clinical signs - individual observations Group : 1 2 3 4 Compound : Control 2 - br omo - 3,3,3 - trifluoropropene 2 - br omo - 3,3,3 -

trifluoropropene 2 - br omo - 3,3,3 -

trifluoropropene Exposure (ppm) : 0 533 1171 2980 Group Anima l 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 presented38Huntingdon Life SciencesWAG0024

APPENDIX 1 - continued Request ID: 269558 Clinical sign s - individual observations Group : 1 2 3 4 Compound : Control 2 - br omo - 3,3,3 - trifluoropropene 2 - br omo - 3,3,3 -

trifluoropropene 2 - br omo - 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 presented39Huntingdon Life SciencesWAG0024

APPENDIX 2 Request ID: 269838 Signs associated with dosing - individual observations Group : 1 2 3 4 Compound : Control 2 - br omo - 3,3,3 - trifluoropropene 2 - br omo - 3,3,3 -

trifluoropropene 2 - br omo - 3,3,3 -

trifluoropropene Exposure (ppm) : 0 533 1171 2980Days 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 Br eathing Gasping 1 Slow 1, 5 1 - 2, 5 Coat Piloerection 3 4 Eyelids (Left) Clos ed 2, 5 Partially Closed 1 - 5 1 - 5 1 - 5 Eyelids (Right) Closed 2, 5 Partially Closed 1 - 5 1 - 5 1 - 5 Posture Hun ched 1, 3 - 5 2 - 540Huntingdon Life SciencesWAG0024

APPENDIX 2 - continued Request ID: 269838 Signs associated wi th dosing - individual observations Group : 1 2 3 4 Compound : Control 2 - br omo - 3,3,3 - trifluoropropene 2 - br omo - 3,3,3 -

trifluoropropene 2 - br omo - 3,3,3 -

trifluoropropene Exposure (ppm) : 0 533 1171 2980Days Group /Sex Animal Number Sign 1 - 2 hour s 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 Br eathing 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 Hun ched 3 3 341Huntingdon Life SciencesWAG0024

APPENDIX 2 - continued Request ID: 269838 Signs associated with dosing - individual observations Group : 1 2 3 4 Compound : Control 2 - br omo - 3,3,3 - trifluoropropene 2 - br omo - 3,3,3 -

trifluoropropene 2 - br omo - 3,3,3 -

trifluoropropene Exposure (ppm) : 0 533 1171 2980Days 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 Br eathing 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 Hun ched 3 242Huntingdon Life SciencesWAG0024

APPENDIX 2 - continued Request ID: 269838 Signs associated with dosing - individual observations Group : 1 2 3 4 Compound : Cont ro l 2 - br omo - 3,3,3 -

trifluoropropene 2 - br omo - 3,3,3 -

trifluoropropene 2 - br omo - 3,3,3 -

trifluoropropene Exposure (ppm) : 0 533 1171 2980Days 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 Br eathing 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 Hun ched 2 - 4 2 - 443Huntingdon Life SciencesWAG0024

APPENDIX 2 - continued Request ID: 269838 Signs associated with dosing - individual observations Group : 1 2 3 4 Compound : Control 2 - br omo - 3,3,3 - trifluoropropene 2 - br omo - 3,3,3 -

trifluorop ropene 2 - br omo - 3,3,3 -

trifluoropropene Exposure (ppm) : 0 533 1171 2980Days 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 Br eathing 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 Hun ched 144Huntingdon Life SciencesWAG0024

APPENDIX 2 - continued Request ID: 269838 Signs associated with dosing - individual observations Group : 1 2 3 4 Compound : Control 2 - br omo - 3,3,3 - trifluoropropene 2 - br omo - 3,3,3 -

trifluoropropene 2 - br omo - 3,3,3 -

trifluoropropene Exposure (ppm) : 0 533 1171 2980Days Group /Sex Animal Number Sign 1 - 2 hours during exposure 3 - 4 hours during

exposure 5 - 6 hours during

exposure 3M 9 Behaviour

Ch in Rubbing 1 Underactive 1 - 5 1 - 5 1 - 5 Salivation 3 Br eathing 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 Hun ched 2, 4 1 - 2, 4 - 545Huntingdon Life SciencesWAG0024

APPENDIX 2 - continued Request ID: 2 69838 Signs associated with dosing - individual observations Group : 1 2 3 4 Compound : Control 2 - br omo - 3,3,3 - trifluoropropene 2 - br omo - 3,3,3 -

trifluoropropene 2 - br omo - 3,3,3 -

trifluoropropene Exposure (ppm) : 0 533 1171 2980Days 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 Br eathing 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) Lacrimatio

n 1 1 Posture Flat 1 1 1 Hun ched 2, 4 - 5 2, 4 4 - 546Huntingdon Life SciencesWAG0024

APPENDIX 2 - continued Request ID: 269838 Signs associated with dosing - individual observations Group : 1 2 3 4 Compound : Control 2 - br omo - 3,3,3 - trifluoropropene 2 - br omo - 3,3,3 -

trifluoropropene 2 - br omo - 3,3,3 -

trifluoropropene Exposure (ppm) : 0 533 1171 2980Days 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 Br eathing 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 Fla t 1 Hun ched 1 4 1 - 247Huntingdon Life SciencesWAG0024

APPENDIX 2 - continued Request ID: 269838 Signs associated with dosing - individual observations Group : 1 2 3 4 Compound : Control 2 - br omo - 3,3,3 - trifluoropropene 2 - br omo - 3,3,3 -

trifluoropropene 2 - br omo - 3,3,3 -

trifluoropropene Exposure (ppm) : 0 533 1171 2980Days 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 Sal ivation 1 Br eathing 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 Hun ched 3 2 - 4 1 - 448Huntingdon Life SciencesWAG0024

APPENDIX 2 - continued Request ID: 269838 Signs associate d with dosing - individual observations Group : 1 2 3 4 Compound : Control 2 - br omo - 3,3,3 - trifluoropropene 2 - br omo - 3,3,3 -

trifluoropropene 2 - br omo - 3,3,3 -

trifluoropropene Exposure (ppm) : 0 533 1171 2980Days Group /Sex Animal Number Sign 1 - 2 hours during exposure 3 - 4 hours during

exposure 5 - 6 hours during

exposure 1F 14 Coat

Piloerection 249Huntingdon Life SciencesWAG0024

APPENDIX 2 - continued Request ID: 269838 Signs associated with dosing - individual observations Group : 1 2 3 4 Compound : Control 2 - br omo - 3,3,3 - trifluoropropene 2 - br omo - 3,3,3 -

trifluoropropene 2 - br omo - 3,3,3 -

trifluoropropene Exposure (ppm) : 0 533 1171 2980Days Group /Sex Animal Number Sign 1 - 2 hours during exposure 3 - 4 hours during

exposure 5 - 6 h our s during

exposure 2F 16 Behaviour

Underactive 1, 4 - 5 1, 4 - 5 1 - 2, 4 - 5 Br eathing 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 Hun ched 1 - 2 1 - 4 1 - 550Huntingdon Life SciencesWAG0024

APPENDIX 2 - continued Request ID: 269838 Signs associated with dosing - individual observations Group : 1 2 3 4 Compound : Control 2 - br omo - 3,3,3 - trifluoropropene 2 - br omo - 3,3,3 -

trifluoropropene 2 - br omo - 3,3,3 -

trifluoropropene Exposure (ppm) : 0 533 1171 2980Days 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 Br eathing 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 Hun ched 1 - 5 1 - 2, 451Huntingdon Life SciencesWAG0024

APPENDIX 2 - continued Request ID: 269838 Signs associated with dosing - individual observations Group : 1 2 3 4 Compound : Control 2 - br omo - 3,3,3 - trifluoropropene 2 - br omo - 3,3,3 -

trifluoropropene 2 - br omo - 3,3,3 -

trifluoropropene Exposure (ppm) : 0 533 1171 2980Days Group /Sex Animal Number Sign 1 - 2 hours during exposure 3 - 4 hours during

exposure 5 - 6 hours during

exposure 2F 18 Behaviour

Ch in Rubbing 3 Underactive 4 - 5 1 - 5 1 - 5 Unresponsive 1 1 Br eathing 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) Close d 1 1 Partially Closed 1, 4 - 5 1 - 5 2 - 5 Posture Hun ched 5 1, 3 - 5 1 - 2, 4 - 552Huntingdon Life SciencesWAG0024

APPENDIX 2 - continued Request ID: 269838 Signs associated with dosing - individual observations Group : 1 2 3 4 Compound : Control 2 - br omo - 3,3,3 - trifluoropropene 2 - br omo - 3,3,3 -

trifluoropropene 2 - br omo - 3,3,3 -

trifluoropropene Exposure (ppm) : 0 533 1171 2980Days 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 Br eathing Gasping 1 Slow 1 2, 4 - 5 2, 4 - 5 Coat Piloerection 1, 4 1, 4 1 - 2, 4 - 5 Eyelids (Left) C losed 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 Hun ched 1 - 2, 4 1 - 2, 4 1 - 2, 4 - 553Huntingdon Life SciencesWAG0024

APPENDIX 2 - continued Request ID: 269838 Signs associated with dosing - individual observations Group : 1 2 3 4 Compound : Control 2 - br omo - 3,3,3 - trifluoropropene 2 - br omo - 3,3,3 -

trifluoropropene 2 - br omo - 3,3,3 -

trifluoropropene Exposure (ppm) : 0 533 1171 2980Days 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 Br eathing 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 Hun ched 4 - 5 1 - 2, 4 - 5 1 - 2, 4 - 554Huntingdon Life SciencesWAG0024

APPENDIX 2 - continued Request ID: 269838 Signs associated with dosing - individual observations Group : 1 2 3 4 Compound : Control 2 - br omo - 3,3,3 - trifluoropropene 2 - br omo - 3,3,3 -

trifluoropropene 2 - br omo - 3,3,3 -

trifluoropropene Exposure (ppm) : 0 533 1171 2980Days 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 Br eathing 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 Hun ched 5 1 1, 455Huntingdon Life SciencesWAG0024

APPENDIX 2 - continued Request ID: 269838 Signs associated with dosing - individual observations Group : 1 2 3 4 Compound : Control 2 - br omo - 3,3,3 - trifluoropropene 2 - br omo - 3,3,3 -

trifluoroprop ene 2 - br omo - 3,3,3 -

trifluoropropene Exposure (ppm) : 0 533 1171 2980Days 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 Br eathing 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 Hun ched 1 - 5 1 - 5 1 - 2, 456Huntingdon Life SciencesWAG0024

APPENDIX 2 - continued Request ID: 269838 Signs associated with dosing - individual observations Group : 1 2 3 4 Compound : Control 2 - br omo - 3,3,3 - trifluoropropene 2 - br omo - 3,3,3 -

trifluoropropene 2 - br omo - 3,3,3 -

trifluoropropene Exposure (ppm) : 0 533 1171 2980Days 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 Br eathing Gasping 1 Slow 1, 5 2, 4 - 5 4 - 5 Coat Piloerection 1, 4 1 Eyeli ds (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 Hun ched 1 - 4 1 - 2, 4 - 5 1 - 557Huntingdon Life SciencesWAG0024

APPENDIX 2 - continued Request ID: 269838 Signs associated with dosing - individual observations Group : 1 2 3 4 Compound : Control 2 - br omo - 3,3,3 - trifluoropropene 2 - br omo - 3,3,3 -

trifluoropropene 2 - br omo - 3,3,3 -

trifluoropropene Exposure (ppm) : 0 533 1171 2980Days 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 Br eathing 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 Hun ched 1, 3 - 4 1 - 2, 4 1 - 3, 558Huntingdon Life SciencesWAG0024

APPENDIX 3 Request ID: 269560 Bodyweight - individual values (g) Group : 1 2 3 4 Compound : Control 2 - br omo - 3,3,3 - trifluoropropene 2 - br omo - 3,3,3 -

trifluoropropene 2 - br omo - 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 1 M 1 392 394 399 404 408 411 417 415 416 419 418 418

2 367 374 378 381 385 388 391 393 392 391 39 3 395 3 393 409 407 418 424 426 438 433 442 443 451 447 2 M 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 3 M 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 4 M 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 43159Huntingdon Life SciencesWAG0024

APPENDIX 3 - continued Request ID: 269560 Bodyweight - individual values (g) Group : 1 2 3 4 Compound : Control 2 - br omo - 3,3,3 - trifluoropropene 2 - br omo - 3,3,3 -

trifluoropropene 2 - br omo - 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 1 M 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 2 M 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 3 M 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 4 M 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 47560Huntingdon Life SciencesWAG0024

APPENDIX 3 - continued Request ID: 269560 Bodyweight - individual values (g) Group : 1 2 3 4 Compound : Control 2 - br omo - 3,3,3 - trifluoropropene 2 - br omo - 3,3,3 -

trifluoropropene 2 - br omo - 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 1 F 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 2 F 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 3 F 19 238 242 241 238 242 245 244 243 248 252 251 253

20 222 223 218 230 231 234 223 235 24 0 245 239 243 21 246 249 242 252 256 251 249 247 249 245 248 250 4 F 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 25261Huntingdon Life SciencesWAG0024

APPENDIX 3 - continued Request ID: 269560 Bodyweight - individual values (g) Group : 1 2 3 4 Compound : Control 2 - br omo - 3,3,3 - trifluoropropene 2 - br omo - 3,3,3 -

trifluoropropene 2 - br omo - 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 1 F 13 243 239 247 249 250 250 250 2 50

14 257 263 264 258 264 264 265 258 15 270 271 269 269 276 279 281 271 2 F 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 3 F 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 4 F 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 27062Huntingdon Life SciencesWAG0024

APPENDIX 4 Request ID: 269561 Food consumption - individual values (g/animal/day) Group : 1 2 3 4 Compound : Control 2 - br omo - 3,3,3 - trifluorop ropene 2 - br omo - 3,3,3 -

trifluoropropene 2 - br omo - 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 3563Huntingdon Life SciencesWAG0024

APPENDIX 4 - continued Request ID: 269561 Food consumptio

n - individual values (g/animal/day) Group : 1 2 3 4 Compound : Control 2 - br omo - 3,3,3 - trifluoropropene 2 - br omo - 3,3,3 -

trifluoropropene 2 - br omo - 3,3,3 -

trifluoropropene Exposure (ppm) : 0 533 1171 2980 Group Cage Day Day Day Day Day Day Day /S ex 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 3464Huntingdon Life SciencesWAG0024

APPENDIX 4 - continued Request ID: 269561 Food consumptio

n - individual values (g/animal/day) Group : 1 2 3 4 Compound : Control 2 - br omo - 3,3,3 - trifluoropropene 2 - br omo - 3,3,3 -

trifluoropropene 2 - br omo - 3,3,3 -

trifluoropropene Exposure (ppm) : 0 533 1171 2980 Group Cage Day Day Day Day Day Day Day Day Day D ay 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 2565Huntingdon Life SciencesWAG0024

APPENDIX 4 - continued Request ID: 269561 Food consumption - individual values (g/animal/day) Group : 1 2 3 4 Compound : Control 2 - br omo - 3,3,3 - trifluoropropene 2 - br omo - 3,3,3 -

trifluoropropene 2 - brom o - 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 2466Huntingdon Life SciencesWAG0024

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 - br omo - 3,3,3 - trifluoropropene 2 - br omo - 3,3,3 -

trifluoropropene 2 - br omo - 3,3,3 -

trifluoropropene Exposure (ppm) : 0 533 1171 2980 Group /Sex Animal Number Terminal Bodyweight Br ain Heart Kidneys Liver Lungs + Br onchi 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 0.93167Huntingdon Life SciencesWAG0024

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 - br omo - 3,3,3 - trifluoropropene 2 - br omo - 3,3,3 -

trifluoropropene 2 - br omo - 3,3,3 -

trifluoropropene Exposure (ppm) : 0 533 1171 2980 Group /Sex Animal Number Terminal Bodyweight Br ain Heart Kidneys Liver Lungs + Br onchi 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 Un suitable

– lungs and bronchi inflated with fixative prior to weighing in error.68Huntingdon Life SciencesWAG0024

APPENDIX 6 Request ID: 274748 Macropathology and histopathology - individual findings Group : 1 2 3 4 Compound : Control 2 - br omo - 3,3,3 - trifluoropropene 2 - br omo - 3,3,3 -

trifluoropropene 2 - br omo - 3,3,3 -

trifluoropropene Exposure (pp m) : 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 MACROPA THOLOGY 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.69Huntingdon Life SciencesWAG0024

APPENDIX 6 - continued Request ID: 274748 Macropathology and histopathology - individual findings Group : 1 2 3 4 Compound : Control 2 - br omo - 3,3,3 - trifluoropropene 2 - br omo - 3,3,3 -

trifluoropropene 2 - br omo - 3,3,3 -

trifluoropropene Exposure (ppm) : 0 533 1171 2980 Animal Number: 2 Sex: Male Dose Group: 1 Phase: Treatment Sacrifice Status: Fi nal 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 + Bron chi, Nasal turbinates, Trachea, Tracheal Bifurc.70Huntingdon Life SciencesWAG0024

APPENDIX 6 - continued Request ID: 274748 Macropathology and histopathology - individual findings Group : 1 2 3 4 Compound : Control 2 - br omo - 3,3,3 - trifluoropropene 2 - br omo - 3,3,3 -

trifluoropropene 2 - br omo - 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 Body weight: 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.71Huntingdon Life SciencesWAG0024

APPENDIX 6 - continued Request ID: 274748 Macropathology and histopathology - individual findings Group : 1 2 3 4 Compound : Control 2 - br omo - 3,3,3 - trifluoropropene 2 - br omo - 3,3,3 -

trifluoropropene 2 - br omo - 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 microsc opically: Larynx, Lungs + Bronchi, Nasal turbinates, Trachea, Tracheal Bifurc.72Huntingdon Life SciencesWAG0024

APPENDIX 6 - continued Request ID: 274748 Macropathology and histopathology - individual findings Group : 1 2 3 4 Compound : Control 2 - br omo - 3,3,3 - triflu or opropene 2 - br omo - 3,3,3 -

trifluoropropene 2 - br omo - 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 FIND INGS RECORDED*** Lungs + Bronchi Alveolar Haemorrhage, Minimal The following examined tissues had no gross observations and no findings microscopically: Larynx, Nasal turbinates, Trachea, Tracheal Bifurc.73Huntingdon Life SciencesWAG0024

APPENDIX 6 - continued Request ID: 274748 Macropathology and histopathology - individual findings Group : 1 2 3 4 Compound : Control 2 - br omo - 3,3,3 - trifluoropropene 2 - br omo - 3,3,3 -

trifluoropropene 2 - br omo - 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 follo wing examined tissues had no gross observations and no findings microscopically: Larynx, Nasal turbinates, Trachea, Tracheal Bifurc.74Huntingdon Life SciencesWAG0024

APPENDIX 6 - continued Request ID: 274748 Macropathology and histopathology - individual findings Group : 1 2 3 4 Compound : Control 2 - br omo - 3,3,3 - trifluoropropene 2 - br omo - 3,3,3 -

trifluoropropene 2 - br omo - 3,3,3 -

trifluoropropene Exposure (ppm) : 0 533 1171 2980 Animal Number: 7 Sex: Male Dose Group: 3 Phase: Treatment Sacrifice Status: Final phase sa cr ifice 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 - Atr ophy/Disorganisation, Minimal The following examined tissues had no gross observations and no findings microscopically: Larynx, Lungs + Bronchi, Trachea, Tracheal Bifurc.75Huntingdon Life SciencesWAG0024

APPENDIX 6 - continued Request ID: 274748 Macropathology and histopathology - individual findings Group : 1 2 3 4 Compound : Control 2 - br omo - 3,3,3 - trifluoropropene 2 - br omo - 3,3,3 -

trif luoropropene 2 - br omo - 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 - Atr ophy/Disorganisation, Minimal The following examined tissues had no gross observations and no findings microscopically: Larynx, Lungs + Bronchi, Trachea, Tracheal Bifurc.76Huntingdon Life SciencesWAG0024

APPENDIX 6 - continued Request ID: 274748 Macropathology and histopathology - individual findings Group : 1 2 3 4 Compound : Control 2 - br omo - 3,3,3 - trifluoropropene 2 - br omo - 3,3,3 -

trifluoropropene 2 - br omo - 3,3,3 -

trifluoropropene Exposure (ppm) : 0 533 1171 2980 Ani mal 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 - Atr ophy/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.77Huntingdon Life SciencesWAG0024

APPENDIX 6 - continued Request ID: 274748 Macropathology and histopathology - individual findings Group : 1 2 3 4 Compound : Control 2 - br omo - 3,3,3 - trifluoropropene 2 - br omo - 3,3,3 -

trifluoropropene 2 - br omo - 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 - Atr ophy /Disorganisation, Slight Olfactory Nerve Fibres - Atr ophy, Slight Inflammatory Exudate in Airways, Minimal Olfactory Epithelium - Inflammation, Diffuse, Minimal The following examined tissues had no gross observation s and no findings microscopically: Larynx, Lungs + Bronchi, Trachea, Tracheal Bifurc.78Huntingdon Life SciencesWAG0024

APPENDIX 6 - continued Request ID: 274748 Macropathology and histopathology - individual findings Group : 1 2 3 4 Compound : Control 2 - br omo - 3,3,3 - trifluoropropene 2 - br omo - 3,3,3 -

trifluoropropene 2 - br omo - 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 - Atr ophy/Disorganisation, Slight Olfactory Nerve Fibres - Atr ophy, 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.79Huntingdon Life SciencesWAG0024

APPENDIX 6 - continued Request ID: 274748 Macropathology and histopathology - individual findings Group : 1 2 3 4 Compound : Control 2 - br omo - 3,3,3 - trifluoropropene 2 - br omo - 3,3,3 -

trifluoropropene 2 - br omo - 3,3,3 -

trifluoropropene Exposure (ppm) : 0 533 1171 2980 Animal Number: 12 Sex: M ale 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 - Atr ophy/Disorganisation, Slight Olfactory Nerve Fibr es - Atr ophy, Minimal Inflammatory Exudate in Airways, Minimal Olfactory Epithelium - Inflammation, Diffuse, Minimal The following examined tissues had no gross observations and no findings microscopically: Larynx, Lun gs + Bronchi, Trachea, Tracheal Bifurc.80Huntingdon Life SciencesWAG0024

APPENDIX 6 - continued Request ID: 274748 Macropathology and histopathology - individual findings Group : 1 2 3 4 Compound : Control 2 - br omo - 3,3,3 - trifluoropropene 2 - br omo - 3,3,3 -

trifluoropropene 2 - brom o - 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.81Huntingdon Life SciencesWAG0024

APPENDIX 6 - continued Request ID: 274748 Macropathology and histopathology - individual findings Group : 1 2 3 4 Compound : Control 2 - br omo - 3,3,3 - trifluoropropene 2 - br omo - 3,3,3 -

trifluoropropene 2 - br omo - 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 MACROPATHO LOGY HISTOPATHOLOGY ***ANIMAL HAS NO MACROSCOPIC FINDINGS RECORDED*** Lungs + Bronchi Aggregations of Alveolar Macrophages, Mini mal The following examined tissues had no gross observations and no findings microscopically: Larynx, Nasal turbinates, Trachea, Tracheal Bifurc.82Huntingdon Life SciencesWAG0024

APPENDIX 6 - continued Request ID: 274748 Macropathology and histopathology - individu al findings Group : 1 2 3 4 Compound : Control 2 - br omo - 3,3,3 - trifluoropropene 2 - br omo - 3,3,3 -

trifluoropropene 2 - br omo - 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.83Huntingdon Life SciencesWAG0024

APPENDIX 6 - continued Request ID: 274748 Macropathology and histopathology - individual findings Group : 1 2 3 4 Compound : Control 2 - br omo - 3,3,3 - trifluoropropene 2 - br omo - 3,3,3 -

tri fluoropropene 2 - br omo - 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.84Huntingdon Life SciencesWAG0024

APPENDIX 6 - continued Request ID: 2 74748 Macropathology and histopathology - individual findings Group : 1 2 3 4 Compound : Control 2 - br omo - 3,3,3 - trifluoropropene 2 - br omo - 3,3,3 -

trifluoropropene 2 - br omo - 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.85Huntingdon Life SciencesWAG0024

APPENDIX 6 - continued Request ID: 274748 Macropathology and histopathology - individual findings Group : 1 2 3 4 Compound : Cont ro l 2 - br omo - 3,3,3 -

trifluoropropene 2 - br omo - 3,3,3 -

trifluoropropene 2 - br omo - 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 ***ANI MAL 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.86Huntingdon Life SciencesWAG0024

APPENDIX 6 - continued Request ID: 274748 Macropathology and histopathology - individual findings Group : 1 2 3 4 Compound : Control 2 - br omo - 3,3,3 - trifluoropropene 2 - br omo - 3,3,3 -

trifluoropropene 2 - br omo - 3,3,3 -

trifluoropropene Expo sure (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 OBSERVATI ONS MACROPATHOLOGY HISTOPATHOLOGY ***ANIMAL HAS NO MACROSCOPIC FINDINGS RECORDED*** Lary nx Squamous Metaplasia - Aryt enoid, Slig ht Nasal turbinates Olfactory Epithelium - Atr ophy/Disorganisation, Slight Olfactory Nerve Fibres - Atr ophy, Minimal Olfactory Epithelium - Inflammation, Diffuse, Minimal The following examined tissues had no gr oss observations and no findings microscopically: Lungs + Bronchi, Trachea, Tracheal Bifurc.87Huntingdon Life SciencesWAG0024

APPENDIX 6 - continued Request ID: 274748 Macropathology and histopathology - individual findings Group : 1 2 3 4 Compound : Control 2 - brom o - 3,3,3 - trifluoropropene 2 - br omo - 3,3,3 -

trifluoropropene 2 - br omo - 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 - Atr ophy/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.88Huntingdon Life SciencesWAG0024

APPENDIX 6 - continued Request ID: 274748 Macropathology and histopathology - individual findings Group : 1 2 3 4 Compound : Control 2 - br omo - 3,3,3 - trifluor opropene 2 - br omo - 3,3,3 -

trifluoropropene 2 - br omo - 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 We ek of Death: 3 Subgroup: 1 Terminal Bodyweight: 270.4 g

PATHOLOGY OBSERVATIONS MACROPATHOLOGY HISTOPATHOLOGY ***ANIMAL HAS NO MACROSCOPIC FINDINGS RECORDED*** Nasal turbinates Olfactory Epithelium - Atr ophy/Disorganisation, Slight Olfactory Nerve Fibres - Atr ophy, Slight Olfactory Epithelium - Inflammation, Diffuse, Minimal The following examined tissu es had no gross observations and no findings microscopically: Larynx, Lungs + Bronchi, Trachea, Tracheal Bifurc.89Huntingdon Life SciencesWAG0024

APPENDIX 6 - continued Request ID: 274748 Macropathology and histopathology - individual findings Group : 1 2 3 4 Compound : Control 2 - br omo - 3,3,3 - trifluoropropene 2 - br omo - 3,3,3 -

trifluoropropene 2 - br omo - 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. Br onchiolar Epithelial Hyperplasia, Moderate Per ivascular/Peribronchiolar Inflammatory Infiltrate, Marked Lary nx Epithelial Hyperplasia, Ventral, Minimal Subepithelial Inflammation, Diffuse, Minimal Nasal turbinates Olfactory Epithelium - Atr ophy/Disorganisation, Slight Olfactory Nerve Fibres - Atr ophy, Minimal Olfactory Epithelium - Inflammation, Diffuse, Minimal Spleen Enlarged90Huntingdon Life SciencesWAG0024

APPENDIX 6 - continued Request ID: 274748 Macropathology and histopathology - individual findings Group : 1 2 3 4 Compound : Control 2 - br omo - 3,3,3 - trifluoropropene 2 - br omo - 3,3,3 -

trifluoropropene 2 - br omo - 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 Deat h: 15 Week of Death: 3 Subgroup: 1 Terminal Bodyweight: 279.0 g

PATHOLOGY OBSERVATIONS MACROPATHOLOGY HISTOPATHOLOGY continued from previous pag e... Tracheal Bifurc. Epithelial Hyperplasia, Minimal Thy mus Firm, Left lobe The following examined tissues had no gross observations and no findings microscopically: Trachea91Huntingdon Life SciencesWAG0024

APPENDIX 6 - continued Request ID: 274748 Macropathology and histopathology - individual findings Group : 1 2 3 4 Compound : Control 2 - br omo - 3,3,3 - trifluoropropene 2 - br omo - 3,3,3 -

trifluoropropene 2 - br omo - 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 - Atr ophy/Disorganisation, Slight Olfactory Nerve Fibres - Atr ophy, 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.92Huntingdon Life SciencesWAG0024

APPENDIX 6 - continued Request ID: 274748 Macropathology and histopathology - individual findings Group : 1 2 3 4 Compound : Control 2 - br omo - 3,3,3 - trifluoropropene 2 - br omo - 3,3,3 -

trifluoropr opene 2 - br omo - 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 Termina l 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 - Atr ophy/Disorganisation, Slight Olfactory Nerve Fibres - Atr ophy, Slight Olfactory Epithelium - Inflammation, Diffuse, Slight The following examined tissues had no gross observations and no findings microscopically: Larynx, Trachea, Tracheal Bifurc.Huntingdon Life Sciences WAG0024

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### Annex 1 Aerosol technology and inhalation analysis reportHuntingdon Life Sciences WAG0024

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### Working for a better future



## Aerosol Technology R eport

HLS s tudy number: WAG00 24 Authors A Allen 

### Simo n A Moore Version ID: Final Issue date: 31 August 2012Huntingdon Life Sciences WAG0024

95 

# Ta ble 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 Nom inal 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 T ables Table 1 Chamber atmosphere concentration of 2 - bromo - 3,3,3 - trifluoropropene -

individual exposure values for Group 1 ...................................................... 100 Table 2 Chamber atm osphere 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 exp osure values for Group 4 ... 108 

# List of Figures Figure 1 Schematic of Inhalation Exposure System ................................................. 109Huntingdon Life Sciences WAG0024

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# Signature Page Aerosol Technology R eportHuntingdon Life Sciences WAG0024

97 

# 1. Pre -study

A tmosphere Characterization Inhalat io n exposure system characterized including determinat io ns of: Achieved vapour concentration; Spatial distribut io n around the chamber. 

# 2. Te st Article Handling and Processing for Inhalation Generation As supplied, 2 - Bromo- 3,3,3 - trifluoro -1- propene was easily vapo uri sed using warmed air th rough a sintered glass vapo uri ser. 

# 3. Exposure Sy

stem A schemat ic o f the exposure system is presented in Figure 1 . Components of the exposure system : Di

rected flow Whole body exposure chamber: 0.75 m

3 

### chamber of s tainless

– steel and glass construction Animal Restraint: Stainless mesh compartments Aerosol Generator: Sint ered glass vaporiser for Groups 2 to 4 Liquid reservoi

r and feed rate used to alter concentration Test groups 30 L/min Tem perature 40  C Diluent Airflow: From in- house co mpressed air system

– breathing qualit y Group 1 – 149 L/min Group 2 to 4 119 – L/min Extract Ai

rflow: Drawn by a fan extract system Drawn fro m base of exposure system Controlled by a gate clamp s ituated at the rear of the chamber Airflow Monitoring: In- line flowmeters monitored continuously for generation and diluent ai rflows Chamber pressure different ial mo nit ored by Magnahelic set at - 1 to -5 mm/H2O to ensure a slight negative pressure within the chamber ~ 150 L/min. 

# 4. Ad ministration Animals exposed for 6 hours per day, 5 days a week for 2 weeks . Exposure system

operational

parameters moni

tore d conti

nuously . Airflows documented init ially and moni

tored during exposure .Huntingdon Life Sciences WAG0024

98 

# 5. At mosphere Analysis 5.1 Aerosol C oncentration Aerosol samples collected as fo llo ws: Sam ple f requency – minimum o f 3 sam ples/group/exposure Sam ple location – representative animal level Sam ple posi tion remained constant Sam ple analysis Infra - red spectrophotometer 

## 5.2 Flow diagram of the anal ytical 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, 19 46, 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 c alibrated using external standards. Absorbance measurements acquired by the IR spectrophotometer were used to calculate analysed concentration (ppm) using a 2 nd 

### order fi t subjected to least squares regression analysis. 

## 5.3 Nominal Concentration Moni tored and summarised in Table 7 to Table 9. 

## 5.4 Chamber Temperature Moni tored using a liquid in glass thermometer, daily means presented in Table 5. 

## 5.5 Chamber Humidity Moni tored using wet and dry

liquid in glass thermometers, wi th h umidit y calculated using proprietary

tables, daily means presented in Table 6.Huntingdon Life Sciences WAG0024

99 

# 6. Data and Calculations To minimize the cumulat ive errors from repeated rounding of numbers, data in this report has been calculated continuously using unrounded numbers and only rounded for print ing. Consequent ly, presented data may include rounding errors in the last significant figure. All data not prese nt ed 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 C oncentration ( ppm )

Target Mean 1 0 0 2 500 533 3 1200 1171 4 3000 2980 

### Addit io nal samples were taken for confirmat io n of atypical resul ts and after minor generat io n rate adjust me nt s. All groups were close to target.Huntingdon Life Sciences WAG0024

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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 detectedHuntingdon Life Sciences WAG0024

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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)Huntingdon Life Sciences WAG0024

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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)Huntingdon Life Sciences WAG0024

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### Table 4 Cham ber 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)Huntingdon Life Sciences WAG0024

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### Table 5 Chamber Temperatures - daily mean Exposure No. Cha mber 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 1 0 20.0 20.0 19.7 19.7Huntingdon Life Sciences WAG0024

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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 1 0 32.0 29.5 32.1 37.8Huntingdon Life Sciences WAG0024

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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/N 1

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 (g/mol) MW 273

C) T( R (g) TCU )( 

  LV V(L) D) (F 6 10 )( (ppm) Conc Nominal    LV

V =Volume of test article R =Gas constant (0.08206 Latm/mol°K) MW =Molecular weight (174.95 g/mol) F = Ch amber airflow (150 L/min) D =Duration 6 Hours (360 mins)Huntingdon Life Sciences WAG0024

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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/N 1

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 1 350 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 Concent ration (ppm) x100 

#   P(mmHg) 760 (g/mol) MW 273 C) T( R (g) TCU )( 

  LV 

## V(L) D) (F 6 10 )( Conc.(ppm) Nominal    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)Huntingdon Life Sciences WAG0024

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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/N 1

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 (g/mol) MW 273

C) T( R (g) TCU )( 

  LV 

## V(L) D) (F 6 10 )( c.(ppm ) Nom inalCon    LV

V =V olume 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)Huntingdon Life Sciences WAG0024

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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 Who le body

chamber H Air - line co il B T est material delivery tubing I Sint ered glass vaporiser C El utriator J Water bath D Secondary

generation trolley K Generat io n flo wmeters E Magnaheli cs L Control valves F Pressure Vessels M Balance for mo re accurate weighing G Load cellsHuntingdon Life Sciences WAG0024

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### Annex 2 Pathology reportHuntingdon Life Sciences WAG0024

Issue No: 2 Page 2 of 5 

# 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 subseque nt 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 afte r 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 afte r 2 weeks of treatment Treatment related findings Changes related to treatment w ith 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 ol factory 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.Huntingdon Life Sciences WAG0024

Issue No: 2 Page 3 of 5 Summary of treatment related findings in the nasa l 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 pp m had perivascular and peribronchiolar inflammatory infiltrate and bronchiolar ep ithelial hyperplasia inthe 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 cons idered to be incidental.Huntingdon Life Sciences WAG0024

Issue No: 2 Page 4 of 5 

# 3. Discussion The changes seen in the nasal turbinates are consis tent 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 un certain relationship to treatment. 

# 4. Conclusion Treatment with 2-bromo-3,3,3-trifluoropropene fo r 2 weeks by the inhalation route produced changes, predominantly, in the nasal turbinates.Huntingdon Life Sciences WAG0024

Issue No: 2 Page 5 of 5 

# 5. References Huntingdon Life Sciences Study Number: WAG0013 2-bromo-3,3,3-trifluoropropene: Dose Range Fi nding Study by Inhalati on Administration to Rats for 2 Weeks.Huntingdon Life Sciences WAG0024

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### Annex 3 Certificate of analysisHuntingdon Life Sciences WAG0024

117Huntingdon Life Sciences WAG0024

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### Annex 4 GLP compliance statementsHuntingdon Life Sciences WAG0024

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### HUNTINGDON RESEARCH CENTRE GLP COMPLIANCE STATEMENT 2011Huntingdon Life Sciences WAG0024

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### EYE RESEARCH CENTRE GLP COMPLIANCE STATEMENT 2011