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### Working for a better future



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

HLS s tudy number: WA G0013 Version ID: Final Issue date: 21 August 2012Hun tingdon Life Sciences WA G0013

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# Details of Sponsor and Te st Facilities Sponsor American Pacific Corp oration 3883 Howard Hughes Parkway Suite 7 00 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 UKHun tingdon Life Sciences WA G0013

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# Ta ble of Contents Details of Sponsor and Test Facilities ................................................................................... 2 Compliance with Good Laboratory Practice .......................................................................... 6 Quality Assurance Statement ................................................................................................ 7 Contributing Scientists .......................................................................................................... 9 Summary ............................................................................................................................ 10 1. Introduction ................................................................................................................ 12 1.1 Obje ctive ......................................................................................................... 12 1.2 Regulatory compliance ................................................................................... 12 1.3 Test system .................................................................................................... 12 1.4 Route of administration ................................................................................... 12 1.5 Treatment groups and doses .......................................................................... 12 2. Experimental Procedure ............................................................................................. 13 2.1 Study schedule and structure .......................................................................... 13 2.1.1 Duration of treatment ........................................................................... 13 2.1.2 Time schedule ..................................................................................... 13 2 .1.3 Identity of treatment groups ................................................................ . 13 2.2 Test substance blend ...................................................................................... 14 2.2.1 Test substance .................................................................................... 14 2.3 Animal management ....................................................................................... 15 2.3.1 Animal supply, a cclimatisation and allocation ...................................... 15 2.3.2 Animal housing, diet and water supply ................................................ 15 2.3.3 Administration ..................................................................................... 16 2.4 Serial observations ......................................................................................... 16 2.4.1 Clinical observations ........................................................................... 17 2.4.2 Detailed physical examination and arena observations ....................... 17 2.4.3 Sensory reactivity and grip strength .................................................... 17 2.4.4 Motor activity ....................................................................................... 18 2.4.5 Bodyweight ......................................................................................... 19 2.4.6 Food consumption ............................................................................... 19 2.5 Necropsy and Histology .................................................................................. 19 2.5.1 Method of kill ....................................................................................... 19 2.5.2 Macrosco pic pathology ........................................................................ 19 2.5.3 Organ weights ..................................................................................... 19 2.5.4 Fixation ............................................................................................... 20 2.5.5 Histology ............................................................................................. 21 2.6 Pathology ........................................................................................................ 21 2.6.1 Light microscopy ................................................................................. 21 2.7 Computer systems .......................................................................................... 23 2.8 Data treatment ................................................................................................ 23 2.8.1 Definition of “Day” ............................................................................... 24 2.8.2 Signs ................................................................................................... 24 2. 8.3 Bodyweight ......................................................................................... 24 2.8.4 Pathology ............................................................................................ 24 2.8.5 Statistical analysis ............................................................................... 24 2.9 Quality assurance and archiving procedures .................................................. 25 2.9.1 Quality assurance ............................................................................... 25 2.9.2 Archives .............................................................................................. 25Hun tingdon Life Sciences WA G0013

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### 2.10 Deviations from protocol ................................................................................. 25 3. Results ....................................................................................................................... 26 3.1 Chamber atmosphere conditions .................................................................... 26 3.2 Signs associated with dosing .......................................................................... 26 3.3 Detailed physical examination and arena observations ................................... 26 3.4 Sensory reactivity and grip strength ................................................................ 26 3.5 Motor activity .................................................................................................. 27 3.6 Bodyweight ..................................................................................................... 27 3.7 Food consumption .......................................................................................... 27 3.8 Organ weights ................................................................................................ . 27 3.9 Macropathology .............................................................................................. 28 3.10 Histopathology ................................................................................................ 28 4. Discussion .................................................................................................................. 34 5. Conclusion ................................................................................................................. 36 6. References ................................................................................................................. 37 

# List of Figures Figure 1 Motor activity ................................................................................................ 38 Figure 2 Bodyweight .................................................................................................. 40 

# List of T ables Table 1 Sensory reactivit y and grip strength ............................................................. 41 Table 2 Motor activity ................................................................................................ 42 Table 3 Bodyweight .................................................................................................. 44 Table 4 Organ weights .............................................................................................. 48 Table 5 Macropathology ........................................................................................... 50 Table 6 Histopathology ............................................................................................. 52 

# List of Ap pendices Appendix 1 Clinical s igns ................................................................................................ 54 Appendix 2 Signs associated with dosing ....................................................................... 60 Appendix 3 Sensory reactivity and grip strength ........................................................... 1 10 Appendix 4 Motor activity .............................................................................................. 11 2 Appendix 5 Bodyweight ................................................................................................ 1 20 Appendix 6 Foo d consumption ..................................................................................... 12 4 Appendix 7 Organ weights ............................................................................................ 12 8 Appendix 8 Macropathology and histopathology ........................................................... 1 30Hun tingdon Life Sciences WA G0013

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# List of A nnexes Annex 1 Administration of 2- bromo - 3,3,3 - trifluoropropene to rats ............................ 18 8 Annex 2 Pathology report ......................................................................................... 20 6 Annex 3 Certificate of analysis ................................................................................. 21 7 Annex 4 GLP compliance statement s ...................................................................... 21 9Hun tingdon Life Sciences WA G0013

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# Quality Assurance Statement 2-bromo- 3,3,3 -trifluoropropene : Dose range finding study by inhalation administration to rats for 2 w eeks The fo llo wing inspect io ns and audits have been carried out in relation to this study : Study Phase Protocol Audit Protocol Amendment No 1 Protocol Amendment No 2 Protocol Amendment No 3 Protocol Amendment No 4 Date(s) of Inspection 25 Oct 2011 16 Nov 2011 16 Nov 2011 25 Nov 2011 10 Feb 2012 Date of Reporting to Study

Director and Management 25 Oct 2011 16 Nov 2011 16 Nov 2011 25 Nov 2011 10 Feb 2012 Report Audi

t Report Audi

t 25 Jan 2012 - 30 Jan 2012

22 Mar 2012 - 27 Mar 2012 30 Jan 2012 27 Mar 2012 Process based insp ections : Ato r about th e time this study w as in progress inspect io ns of procedures em

ploy ed on this ty

pe of study

w ere carri ed out. T hese were conducted and reported to appropriate Company Management as indicated below: Process Based Inspections Date(s) of Inspection Date of Reporting to Management Clinical signs examinat io n/palpat io ns including arena observat io ns (rodents) 17 Oct 2011 17 Oct 2011 Study management and conduct 25 Oct 2011 25 Oct 2011 Body weights and food consumption 31 Oct 2011 31 Oct 2 011 Motor activit y testing on RAMS (including ho me/ in the hand/standard arena/manipulat io ns) 01 Dec 2011 01 Dec 2011 Generat io n of Test Atmospheres and Exposure/post exposure signs, atmosphere sam

pling 13 Dec 2011 13 Dec 2011 Test Item Control and dispo si tion (including tracking and canister packing) 14 Dec 2011 14 Dec 2011 Terminal procedures 14Dec 2011 14 Dec 2011Hun tingdon Life Sciences WA G0013

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# Contributing Scientist s 2-bromo- 3,3,3 -trifluoropropene : Dose range finding study by inhalation administration to rats for 2 w eeks 

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

## Inhalation analysis Caroline Finucane PhD Head of Form

ulat io n & Inhalat io n Analysis 

## Aerosol technology Simo n A Moore BSc (Hons) PhD M RSC Head of Aerosol Techno lo gy 

## S ensory reactivity, grip strength and motor activity Martin J Co llier BSc Behavioural Scient ist 

## Pathology David Bell, BSc (Hons) BVMS MRCVS Pathologi stHun tingdon Life Sciences WA G0013

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

and systemi c 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 th ree ma le and t hree female rats received 2- bro mo - 3,3,3 - trifluoropropene at target exposure levels of 5000 , 10000 or 20000 ppm . A similarly consti tu ted Control

group received clean ai

r only, at the same vo lu me - dose. During the study, clinical condit io n, detailed physical and arena observations, sensor y reactivit y, gri

p strength, motor activit y, body weight, food consumption , organ weight, macropathology

and histopathology

invest igat io ns were undertaken . 

## Results The achieved aerosol concentrations were 4721, 8901 and 19098 ppm (94, 89 and 95% of target) fo r Groups 2 - 4 respectively. There were no unscheduled deaths at any of the dose levels. Treatment - related , but transient , clinical signs , related to inhalat io n of an irritant material or potential CNS effects , were evident during and after exposure. Body weight loses of 43g and 3g were evident for males and females exposed to 19098 ppm respectively over the treatment period. Reduced body

weight gain was evident for males exposed to 8901 or 4721 ppm (0.02X and 0.23X control respect ively). Total food consum pt io n was redu ced for all treated groups (0.66 X to 0.8 9 X Control). Atnecropsy, a reduction in the size of the thymus and depressions or roughening of the stomach were evident in animals exposed to 19098 ppm. All other organs were within the normal size a nd weight ranges. Hi stopathological

changes associ ated wi

th to treatment with 2- bro mo - 3,3,3 - trifluoropropene were seen in the nasal turbinates, larynx and trachea. Overall, the most marked changes were in the nasal cavit y, wi

th minimal to slight changes in the larynx and trachea. In th e region o f th e olfactory epi th elium of the nasal turbinates, fibrosis , wi

th or wi thout associ ated cartilage format io n, atrophy of the Bowman’s glands, atrophy of olfactory nerve fibres and epi

th elial erosion were seen in a nima ls of all test article groups. Necrosis o f th e olfactory epi th elium was seen in animals exposed to 8901 or 19098 ppm. Fusion of dorsal turbinates with associated epithelial divert icu la fo rmat io n was seen in animals o f all test article groups. In the various epithelia o f th e nasal cavit y (ol factory, respi ratory and transi tional) of animals o f all test article groups, metaplasia and combinat io ns o f di sorganisation, atrophy, degeneration and regeneration were evident. In the naso lacr imal ducts, i nflammat io n was seen in anima ls o f all test article groups. In general, the changes seen in the nasal turbinates of animals exposed to 8901 or 19098 ppm were of a similar severit y and incidence and o f a higher severit y than those seen for animals exposed to 4721 ppm. In considerat io n of the epithelial changes the olfactory epi th elium was th e most extensively

affected at all

three exposure levels.Hun tingdon Life Sciences WA G0013

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### In the larynx, epithelial metaplasia, hyperplasia and keratinisat io n, of a similar incidence and degree, were seen in anima ls of all test article groups. Inflammat io n was seen in one male and one female exposed to 19098 ppm and one male exposed to 8901 ppm. In the trachea, epithelial degenerat io n/regeneration was seen in male and female animals exposed to 19098 ppm and one female exposed to 8901 ppm. Inflammatory cell infiltrate in th e lamina propria was seen at a higher incidence and/or degree in animals exposed to 19098 ppm, compared with control animals. 

## Conclusion Hi stopathological

changes related to treatment were seen in the nasal turbinates, larynx and trachea. Although the majorit y of these changes were limited to the 8901 or 19098 ppm exposure groups, some changes in the nasal turbinates were evident at 4721 ppm. Therefore, a no observed adverse effect level (NOAEL) could not be established.Hun tingdon Life Sciences WA G0013

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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 - trifluoropropene , a fire ext inguishing agent , when administered by inhalat io n to rats for 2 weeks to s elect 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 regulat io ns or guidelines are applicable. 

## 1.3 Test system The rat was chosen as the test species because of it s acceptance as a predictor of toxic change in man and the requirement for a rodent species by regulatory agencies. The Crl:CD (SD) strain was used because of the historical control data available in this laboratory . 

## 1.4 Route of administration The 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 an acute inhalat io n toxici ty study (Laboratory

Proj ect Number 02 -5449), 0/10 rats died at 5173 ppm and 10/10 rats died at 26580 ppm. There were no intermediate groups. Clinical signs at 5173 ppm

com prising respiratory

sounds persisted for up to 8 days post - exposure in a small number of rats (maximum 4). The rats were weighed on Days 7 and 14 post exposure, th e weight gain over this pe riod was considered to be normal and unaffected by ex posure. Lung weights were normal. Based on the se findings , a target exposure le vel of 5000 ppm, f or th e low dose group , wa s considered likely

to elici t a mild to moderate clinical response wi

th no morta lit y. The starts of e xposures fo r treatment groups were staggered (i.e. the groups were exposed sequent ia lly

) by 7 days to allow assessment of the response of animals. Thus the s ubsequent target exposure levels of

10000 and 20000 ppm, selected for the int ermediate and high dose groups, were based on the response of the animals at lower dose levels .Hun tingdon Life Sciences WA G0013

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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. The necropsy procedures were com

pleted in 1 day fo r each group (staggered over 3 separate days) , during which t ime treatment continued, and serial observat io ns were recorded at appropriate intervals. The duration of treatment is repo rted as 2 weeks . 2.1.2 Time schedule Study init iat io n: (Pro tocol signed by

Study Di rector) 24 October 2011 Experimental start date: (Animal arrival) 27 October 2011 Treatment commenced: Group 2 Group 3 Groups 1 and 4 11 November 2011 18 November 2011 25 No vember 2011 Necropsy com pleted: Group 2 Group 3 Groups 1 and 4 25 November 2011 02 December 2011 09 December 2011 Experimental co mp let io n date: (pathology

) 9 March 2012 Study com plet io n: 21 August 2012 2.1.3 Identity of treatment groups The study consist ed 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 5000 3 3 4 - 6 16 - 18 2 6 3 2 - br omo - 3,3,3 - trifluoropropene 10000 3 3 7 - 9 19 - 21 3 7 4 2 - br omo - 3,3,3 - trifluoropropene 20000 3 3 10 - 12 22 - 24 4 8Hun tingdon Life Sciences WA G0013

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## 2.2 Test substance blend 2.2.1 Test substance Informat io n supplied by the Sponsor regarding t he test substance is contained in the test substance data sheet, which is retained in study records, and the Certificate of Analysis, which is presented in Annex 3. The fo llo wing informat io n is given in summary : Ident ification: 2- bromo- 3,3,3 - trifluoroprope ne (stabilized) Usage : Fi

re ext inguishing agent Appearance : Liquid at below 33  C

Clear and may

b e co lo urless to light yellow t int Storage condit io ns: Atambient temperature Supplier: Sponsor Batch number: Kg F080 Date of receipt: 12 September 2011 Qu ant 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.Hun tingdon Life Sciences WA G0013

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## 2.3 Animal management 2.3.1 Animal supply, acclimatisation and allocation Crl :C D (SD) rats (a total

of fifteen male and fift een fe male ) were received from Charles River (UK) Ltd. The rats were ordered at 49 to 56 days of age and wit hin a weight range of 290-330 g for ma les and 202 - 233 g for females . On arrival, the animals were remo ved fro m th e 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 th e procedure being repeated until each cage held the appropriate number of animals. Each sex was allo c ated separately . The cages const ituting each group were blocked together 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 equilibrat ed, as far as was pract ic able. 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 al lo wed to acclimat ise to the condit io ns described below for atleast 15 days before treatment commenced. For those animals selected for this study, thei r age at th e start of treatment was 64 to 85 days and their bodyweights were in the range of 396 to 501 g for ma les and 247 to 316 g for females. The spare animals were remo ved from the study room af ter treatment commenced. 2.3.2 Animal housing, diet and water supply Animals were housed inside a restricted access rodent facilit y (Building Y14 , Room 011 ). Sensory react ivit y, gri

p strength and motor activit y tests were performed in Room 010. 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 t he 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 4 0 to 70% respecti vely and moni tored conti

nuously. There were no deviat io ns fro m th ese 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 fa ilure of the vent ilat io n system, or temperature limits were exceeded. 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 w ere made of a polycarbonate body wi th a stainless steel

m esh lid. Wo od based material 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 int ervals.Hun tingdon Life Sciences WA G0013

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### The animals were allowed free access to a standard rodent diet (Rat and Mouse No. 1 Maintenance Diet) . This diet contained no added ant ibiotic or other chemotherapeut ic or prophylact ic agent. Potable water taken fro m th e public supply was f reely available via po lycarbonate bottles fi tted wi th sipper tubes. Each cage of animals was provided with an Aspen chew block for environmental enrichment. Chew blocks were provided throughout the study and were replaced when necessary . Each cage of animals was provided wi th a plast ic shelter for environmental enrichment, which was replaced at the same time as the cages. Each batch of diet was analysed routinely by the supplier for various nutrit io nal com ponents and chemical and microbio lo gical contaminants. Supplier’s analyt ical certificates were scrutinised and approved before any b atch of di et 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 th e water supplier and th e sup plier s of

the bedding and A spen chew blocks. Since the results of these various analyses did not provide evidence o f contaminat io n that might have prejudiced the study, th ey are not p resented. No other specific contaminants that were likely to have been present in the bedding , chew blocks, diet or water were analysed, as none that may have interfered with or prejudiced the outcome of

the study was known. 2.3.3 Administration Details o f admin istration 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 achie ved by v arying 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 t o 2- bromo-3,3,3- trifluoropropene , 6 hours /day

f or 5 days each week (Monday to Friday ) f or 2 weeks. 

## 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 we re performed in cage number sequence except where otherwi se indicated.Hun tingdon Life Sciences WA G0013

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### 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 th roughout the treatment period, detailed observat io ns were performed immediately

b ef ore dosing and between one and two hours after complet io n of dosing of all groups. F urther observat io n was performed during exposure and as late as possible in the working day during the first week of tr eatment. During the acclimat is ation period, observat io ns of the animals and their cages were recorded once per day . 2.4.2 Detailed physical examination and arena observations Before treatment commenced and on Day 10 of treatment, detailed physical examinat io n and arena observat io ns were performed on each animal. On each occasion, the examinat io ns were performed at approximately the same t ime of day

(before dosing during the treatment period) . After removal

f rom the home cage, animals were assessed for physical c ondi tion and behaviour during handling and after being placed in a standard arena. Any deviation fro m normal was recorded wi

th respect to the nature and, where appropriate, degree of severit y. Particular attention was paid to possible signs of neurotoxic it y, such as convulsio ns, tremor and abnormalit ies of gait or behaviour. 2.4.3 Senso ry reactivity and grip strength Sensory react ivit y and gri

p strength assessments were performed (before dosing ) on all animals on Day

10 of treatment. The fo llo wing measuremen ts, reflexes and responses were recorded: Approach response A blunt probe was brought towards the animal’s head unt il itwas close to the animal’s nose (but not touching the vibrissae). The animal’s reaction was recorded as:

1 No reaction or ignores probe 2 Normal

awareness and react io n e.g. approaches and/or sniffs probe 3 Abnormally

f earful or aggressive reactionHun tingdon Life Sciences WA G0013

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### Touch response The animal was stroked gently on the nape of the neck with a blunt probe and the reaction recorded as:

1 No reaction or igno res probe 2 Normal

awareness and react io n e.g. turns towards or moves away 3 Abnormally

f earful or aggressive reaction Auditory startle reflex The animal’s response to a sudden loud no ise was assessed and scored as:

1 No response 2 Weak response e.g. ear twitch only 3 Normal

response e.g. obvious flinch or startle 4 Exaggerated response e.g. all feet off floor Tail pinch response The animal’s tail was pinched sharply wit h fo rceps approximately one third fro m th e ti p and th e response graded as:

1 No response 2 Weak response e.g. turns around slowly or weak vocalisat io n wi thout mo ving away 3 Normal

response e.g. j umps f

orward or turns around sharply, usually wi th vocalisat io n 4 Exaggerated response e.g. excessive vocalisat io n, body movement or aggr essio n Grip strength Forelimb and hindlimb grip strength was measured using Mecmesin Force Indicators. Three tri als were performed. Atany point during the observat io ns, addi tional comments were made as free text where considered appropriate. 2.4.4 M otor activ ity On Day 10 of

treatment (before dosing), the motor activit y of each animal was measured using a Rodent Activit y Monitoring System (Version 2.0.3), wit h hardware supplied by Pearson Technical Services and so ft ware developed and maintained by Hunt ingdon L ife Sci ences. Animals were tested individually in clear polycarbonate cages and motor activit y was measured by counting infra - red beam breaks over ten 6 -minute intervals (one hour total). Ten beams were set at two height levels (five low and five high) to detect cage floor and rearing activit y respect ively .Hun tingdon Life Sciences WA G0013

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### 2.4.5 Bodyweight The weight of each rat was recorded daily from Day -5 , daily th

roughout the treatment, and before necropsy . 2.4.6 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 each day th

roughout the treatment period. 

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

tone, fo llo wed by

ex sanguinat io n. 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 performed. All external features and orifices were examined visua lly . The cranial roof was removed to allow obse rv at 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, abdo minal 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 appropriate. Any abnormalit y 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 ti ssue and the weights recorded: Brain Liver Heart Lungs with mainstem bronchi Kidneys Spleen Bilater al organs were weighed together . Organ weights were also adjusted for terminal body weight, using the weight recorded before necropsy .Hun tingdon Life Sciences WA G0013

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### 2.5.4 Fixation T estes 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 Ovari es Brain Pancreas Caecum Pi tui tary Colon Prostate Duodenum Rectum Epididymides Salivary glands - submandibular Eyes - sublingual Femurs Sci at ic nerves Harderi an glands Seminal vesicles Head Skeletal m uscle Heart Skin wi th m ammary glands Ileum Spinal cord Jejunum Spleen Kidneys Sternum Lachryma l glands Stomach Larynx* Testes Liver Thymus Lungs including bronchi* Thy roid wi th parathy roids Lymph nodes - ma ndibular Tongue

- me senteric Trachea *

- trache o- bronchial Urinary bladder Nasal turbinates * Uterus and cervix Oesophagus Vagina * Tissues examined microscopically 

### 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 appropri ate. Sam ples o f th e head (including nasal cavit y, paranasal sinuses and nasopharynx) and the remaining femur, salivary gland, sciat ic nerve and skeletal muscle were not examined histologically, but are retained against any f uture requi

rement for microscopic examinat io n.Hun tingdon Life Sciences WA G0013

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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 dehydrated, embedded in paraffin wax, sect io ned at approximately fo ur to five micron thickness and stained with 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 major lobes, to include bronchi Nasal Turbinates - 3 sec tions Trachea - including bifurcat io n 

## 2.6 Pathology 2.6.1 Light microscopy Microscopic examination was performed as follows: Larynx, lungs, nasal turbinates and trachea were examined for all animals o f Groups 1 (Control) and 4 ( 20000 ppm ) sacrificed on com

pletion o f th e scheduled treatment period. Larynx, nasal turbinates and trachea were examined for all animals of Groups 2 (5000 ppm) and 3 (10000 ppm) sacrificed on complet io n of the scheduled treatment period. Tissues reported 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, moderate, marked or severe. The desc ript io ns listed below are an attem pt to define th e 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 pes of l esion t han to oth ers, and th ere may th erefore be a few lesio ns fo r which these definit io ns are not completely appropriate.Hun tingdon Life Sciences WA G0013

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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 th e 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 a nd is of

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

t he organ. Slight Clearly discernible/dist ingui shable from normal wi th more extensive or mo re apparent changes than minimal. Moderate Generally invo lves a significant proportion of t he affected organ or ti ssue. Marked Invo lves the majorit y of the affected organ or tissue. 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.Hun tingdon Life Sciences WA G0013

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## 2.7 Computer s ystem s The computer systems wi th v ersion 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, mac ropathology and histopathology . RAMS Rodent Activit y Moni toring System

(version 2. 0) used for activit y mo nit oring. Responder - X (version 5. 11) used for startle response. Sam ple Registry

System (versio n 1.0) used for Formulat io n and Inhalat 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 S ystem (v ersion 4.2.2) used for clinical signs, dosing signs, body weights, food consumpt io n, organ weights, 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 o bservations 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 the detailed physical examinat io n and arena observat io ns, 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 appendi ces have the fo llo wing mea ning: T Scheduled killHun tingdon Life Sciences WA G0013

24 

### 2.8.1 Definition of “ Day ” The first day of

treatment started at midnight prior to treatment commencing and ended at midnight on the day

f ol lo wing. Subsequent experimental days of treatment were of the same duration. 2.8.2 Signs Signs are consi dered 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, da y of occurrence and information on the duration of the sign applicable . 2.8.3 Bodyweight Group mean weight changes were calculated from the weight changes of individual animals. 2.8.4 Pathology The abbreviat io ns used in the Appendix have the following meanings: Ln/LN Lymph nodes 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 precede other tissues. 2.8.5 Statistical analysis The small sample size precludes meaningful statistical evaluat io n.Hun tingdon Life Sciences WA G0013

25 

## 2.9 Quality assurance and archiving procedures 2.9.1 Quality assurance Details o f th e Qualit y Assurance inspections and audits (undertaken at Huntingdon Life Sci ences) are presented on the Qualit y Assurance 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 us ed during any Sponsor’s or supplier’s analysis, were stored in the archives o f Hunt ingdon Life Sciences. Types of sample and specimen which are unsuitable, by reason o f instabilit y, f

or long term

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

Quali ty A ssurance inspect io n records will be retained indefinit ely. All other appropriate specimens and records will be retained for a minimum period of one year fro m th e date of i ssue of the final report. At the end of the one year retention period the Sponsor will be contacted and advice sought on the above requirements. Under no circumstances will any item be discarded without the Sponsor’s knowledge . 

## 2.10 Deviations from protocol There were no deviat io ns from protocol .Hun tingdon Life Sciences WA G0013

26 

# 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 5000 4721 3 10000 8901 4 20000 19098 

### The achieved aerosol concentrations were 94 , 89 and 9 5 % of the target concentration for Groups 2, 3 and 4, respectively. 

## 3.2 Signs associated with dosing Appendix 2 There were no unscheduled deaths during the course of the study . Clinical signs seen during exposure included: underactivit y, shallow breathing, pilo erection, unsteady gai t, parti ally

clos ed eye lid s, salivat io n and chin rubbing. These signs generally resolved quickly

on cessat io n of exposure to the test article. The num ber and prevalence of signs increased with dose. 

## 3.3 Detailed physical examination and arena observations Appendix 1 Detailed physical examinat io ns during Week 2 of exposures showed underact ivit y fo r 3 out 6 rats and piloerect io n fo r 2 out of 6 rats exposed t o 19098 ppm . Abnormal gait and reduced body tem perature were also evident for a single female exposed to 19098 ppm . 

## 3.4 Sensory reactivity and grip strength Table 1 and Appendix 3 Atleast one of the males or females exposed to 8901 or 19098 ppm fa iled to res pond during approach response, touch response and auditory startle reflex tests, although there was no consistent dose - response. However, all animals showed a tail pinch response and sensory reactivit y responses for males and females exposed to 4721 ppm w ere unaffected. There were no clear treatment - related changes in grip strength values.Hun tingdon Life Sciences WA G0013

27 

## 3.5 Motor activity Figure 1 , Table 2 and Appendix 4 High beam

and low beam

motor activi

ty scores (rearing and cage floor activit y respect ively) fo r males and females expose d to 8901 or 19098 ppm were markedly reduced throughout the 1-hour recording period. The effect was more marked in females, although a more consistent dose - relat io nship was evident in males. Motor activit y scores for females exposed to 4721 ppm were also reduced but activit y fo r males at thi s level was considered to be unaffected. 

## 3.6 Bodyweight Figure 2 , Table 3 and Appendix 5 Body weight loses of 43g and 3g were evident for males and females exposed to 19098 ppm respectively

over the treatment period. Reduc ed body weight gain was evident for males exposed to 8901 or 4721 ppm (0.02X and 0.23X co nt rol respectively). Body

weight gains for females exposed to 8901 or 4721 ppm greater than control ( 1.68X and 1.35X control respectively

). 

## 3.7 Food consumption Appendix 6 Food consumpt io n was reduced over the treatment period fo r rats exposed to 2- bromo- 3,3,3 - trifluoropropene . Total f ood consumption (Days 1 to 14) was 0.87X , 0.89X and 0.67X con trol f or males and 0.73X , 0. 85X and 0.66X of control f or f emales exposed to 47 21, 8901 or 19098 ppm respectively . 

## 3.8 Organ weights Table 4 and Appendix 7 There were no test article - related effects on o rgan weights.Hun tingdon Life Sciences WA G0013

28 

## 3.9 Macropathology Table 5 and Appendix 8 The macroscopic examinat io n performed after 2 weeks of treatment revealed the fo llo w ing int ergroup differences: Thymus A reduction in the size of the thymus was noted in animals treated with 19098 ppm. Summary of findings in the thymus for animals killed after 2 weeks of treatment Group/sex 1M 2M 3M 4M 1F 2F 3F 4F Achieved Exposure Level (ppm) 0 4721 8901 19098 0 4721 8901 19098 Small 0 0 0 3 0 0 0 3 Number of animals examined 3 3 3 3 3 3 3 3 

### Stomach Depressio ns or roughening of the stomach was observed in animals treated with 19098 ppm. Summary of findings in the stomach for animals killed after 2 weeks of treatment Group/sex 1M 2M 3M 4M 1F 2F 3F 4F Achieved Exposure Level (ppm) 0 4721 8901 19098 0 4721 8901 19098 Forestomach Depression(s) 0 0 0 2 0 0 0 0 Forestomach Roughened 0 0 0 1 0 0 0 2 Number of animals examined 3 3 3 3 3 3 3 3 

### The nature and incidence of all oth er findings were consistent with the commo nly seen background of macroscopic changes in rats at these laboratories . 

## 3.10 Histopathology Table 6 and Appendix 8 Changes related to treatment with 2 - bromo- 3,3,3 - trifluoropropene were seen in the nasal turbinates, larynx and trachea . Nasal turbinates In the olfactory

epi th elium of male and female animals exposed to concentrations of 4721, 8901 or 19098 ppm, the test article - relat ed findings seen included the fo llo wing. Minimal to marked fibrosis wit h or without associated cartilage formation in the dorsal lamina propria. Minimal to marked atrophy of the Bowman’s glands in the dorsal lamina propria.Hun tingdon Life Sciences WA G0013

29 

### Minimal to moderate atrophy of o lfactory nerve fibres. Minimal epit helial necrosis was seen in two male animals exposed to 19098 ppm and one female exposed to 8901 ppm. Minimal erosion of the epithelium. Minimal to moderate degeneration/atrophy /disorganisation of

the epi thelium. Minimal squamous metaplasia of the epi th elium . Minimal or slight respiratory m etaplasia of the epithelium wit h or wi thout associ ated inflammatory

cell infil trate. In the turbinates, minimal to moderate fusio n of dorsal turbinates with associated epithelial diverti cula formation was seen in male and female animals exposed to 4721, 8901 or 19098 ppm. In the respiratory

epi th elium of male and female animals exposed to 4721, 8901 or 19098 ppm, the test article - related findings seen included the fo llo wing. Minimal or sl ight disorganisat io n/degenerat io n/regenerat io n of the epithelium. Minimal squamous metaplasia of the epithelium was only seen in animals exposed to 19098 ppm. In the transit io nal epit helium, minimal or slight disorganisat io n/regenerat io n of the epi th elium was seen in male and female animals exposed to 4721, 8901 or 19098 ppm. In the naso la crimal ducts, minimal epit helial degeneration was seen in one female exposed to 19098 ppm and minimal or slight inflammat io n was seen in animals exposed to 4721, 8901 or 1 9098 ppm. In the airway, minimal or slight debris and inflammatory cells were also seen in animals exposed to 4721, 8901 or 19098 ppm.Hun tingdon Life Sciences WA G0013

30 Summary of treatment related findings in the nasal turbinates for animals killed after 2 weeks of treatment Group/sex 1M 2M 3M 4M 1F 2F 3F 4F Achieved Exposure Level (ppm) 0 4721 8901 19098 0 4721 8901 19098 Olfactory epithelium Fibrosis with or without associated cartilage formation in dorsal lamina propria Minimal Slight Moderate Marked 0

0 0 0 2 0 0 0 0 1 2 0 0 1 0 2 0 0 0 0 3 0 0 0 0 0 3 0 0 0 3 0 Total 0 2 3 3 0 3 3 3 Bowman’s gland atrophy in dorsal lamina propria Minimal Slight Moderate Marked 0

0 0 0 2 1 0 0 0 3 0 0 0 0 1 2 0 0 0 0 0 0 3 0 0 0 3 0 0 0 3 0 Total 0 3 3 3 0 3 3 3 Olfactor y ne

rve fibres - atrophy

Minimal Slight Moderate 0

0 0 1 2 0 0 3 0 0 3 0 0 0 0 2 1 0 0 2 1 0 2 1 Total 0 3 3 3 0 3 3 3 Necrosis Minimal 0 0 0 2 0 0 1 0 Total 0 0 0 2 0 0 1 0 Erosi on Minimal 0 1 1 2 0 1 1 1 Total 0 1 1 2 0 1 1 1 Degeneration/atrophy/disorganisation Minimal Slight Moderate 0

0 0 3 0 0 1 1 1 0 2 1 0 0 0 1 1 0 0 3 0 0 1 2 Total 0 3 3 3 0 2 3 3 Squamous metaplasia Minim al 0 3 2 1 0 3 3 2 Total 0 3 2 1 0 3 3 2 Respiratory

metaplasia with or without associated inflammatory cell infiltrate Minimal Slight 0

0 2 0 1 2 1 2 0 0 3 0 3 0 2 0 Total 0 2 3 3 0 3 3 2Hun tingdon Life Sciences WA G0013

31 Summary of treatment related findings in the nasal turbinates for animals killed after 2 weeks of treatment Group/sex 1M 2M 3M 4M 1F 2F 3F 4F Achieved Exposure Level (ppm) 0 4721 8901 19098 0 4721 8901 19098 Turbinates Fusion of dors al turbinate with associated epithelial diverticula formatio n Minimal

Slight Moderate 0

0 0 0 0 0 0 0 3 0 1 2 0 0 0 1 0 0 0 0 3 0 0 3 Total 0 0 3 3 0 1 3 3 Respiratory epithelium Disorganisation/Degeneration/ Regeneration Minimal Slight 0

0 1 0 3 0 3 0 0 0 1 0 1 1 2 1 Total 0 1 3 3 0 1 2 3 Squamous metaplasia Minimal 0 0 0 3 0 0 0 1 Total 0 0 0 3 0 0 0 1 Transitional epithelium Disorganisation/Regeneration Minimal Slight 0

0 2 0 2 0 2 1 0 0 2 0 2 0 2 1 Total 0 2 2 3 0 2 2 3 Nasolacrimal ducts Epithelial degeneration Minimal 0 0 0 0 0 0 0 1 Total 0 0 0 0 0 0 0 1 Inflammation Minimal Slight 0

0 1 0 1 0 0 2 0 0 1 0 0 0 2 0 Total 0 1 1 2 0 1 0 2 Airway Debris and inflammatory cells Minimal Slight 0

0 2 0 1 0 0 1 0 0 0. 0 2 0 1 0 Total 0 2 1 1 0 0 2 1 Number of tissues examined 3 3 3 3 3 3 3 3Hun tingdon Life Sciences WA G0013

32 

### Larynx In the ventral larynx, minimal squamous metaplasia was seen in male and female animals exposed to 8901 or 19098 ppm and minimal epit helial hyperplasia was seen in one male exposed to 19098 ppm. In the ventrolateral larynx, minimal epit helial keratinisat io n was seen in male and female animals o f all groups exposed to 2- bromo - 3,3,3 - trifluoropropene . In the ary teno ids, minimal or slight squamous metaplasia and minimal epit helial hyperplasia were seen in animals o f all test article groups. Minimal or slight inflammat io n was seen in one male and one female exposed to 19098 ppm and one male exposed to 8901 ppm. Summary of treatment related findings in the larynx for animals killed after 2 weeks of treatment Group/sex 1M 2M 3M 4M 1F 2F 3F 4F Achieved Exposure Level (ppm) 0 4721 8901 19098 0 4721 8901 19098 Squamous metaplasia - ventral Minimal 0 0 1 2 0 0 3 3 Total 0 0 1 2 0 0 3 3 Epithelial hyperplasia - ventral Minimal 0 0 0 1 0 0 0 0 Total 0 0 0 1 0 0 0 0 Epithelial keratinisation - ventrolateral Minimal 0 2 2 3 0 2 2 3 Total 0 2 2 3 0 2 2 3 Squamous metaplasia - ary tenoid Minimal Slight 0

0 1 1 3 0 1 1 0 0 2 0 3 0 3 0 Total 0 2 3 2 0 2 3 3 Epithelial hyperplasia - ary tenoid Minim al 0 1 1 0 0 0 0 2 Total 0 1 1 0 0 0 0 2 Inflammation Minimal Slight 0

0 0 0 1 0 1 0 0 0 0 0 0 0 0 1 Total 0 0 1 1 0 0 0 1 Number of tissues examined 3 3 3 3 3 3 3 3Hun tingdon Life Sciences WA G0013

33 

### Trachea Minimal or slight epithelial degeneration/regeneratio n was seen in male and female animals exposed to 19098 ppm and one female exposed to 8901 ppm. Minimal or slight inflammatory

cell infil trate in the lamina propri a was seen at a higher incidence and/or degree in animals exposed to 19098 pp m, than in control animals. Summary of treatment related findings in the trachea for animals killed after 2 weeks of treatment Group/sex 1M 2M 3M 4M 1F 2F 3F 4F Achieved Exposure Level (ppm) 0 4721 8901 19098 0 4721 8901 19098 Epithelial degeneration/regeneration Minimal Slight 0

0 0 0 0 0 1 2 0 0 0 0 1 0 2 0 Total 0 0 0 3 0 0 1 2 Inflammato

ry cell infiltrate

– lamina propria Minimal Slight 1

0 1 0 0 0 1 1 0 0 0 0 1 0 0 1 Total 1 1 0 2 0 0 1 1 Number o f tissues examined 3 3 3 3 3 3 3 3Hun tingdon Life Sciences WA G0013

34 

# 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, fo r 6 hours a day, 5 days a week, for 2 we ek at achieved aerosol concentrations o f 4721 , 8901 and 19098 ppm . A control group of rats received air only at the same ai rflow as the high dose group. Hi stopathology

f indings related to treatment in the nasal turbinates, were generally seen in th e tissu es of

the dorsal

half of the nasal cavi ty , with the severit y of

f indings increasing in a rostral to caudal di rect io n and the olfactory

epi th elium b eing the most extensively affected. In the region o f th e olfactory epi th elium, fibrosi s wi

th or wi thout assoc iat ed cartilage fo rmat io n, atrophy

of the Bowman’s glands and atrophy of olfactory nerve fibres were seen in the dorsal lamina propria and erosion of the epithelium were seen in animals of all groups exposed to 2 - bromo-3,3,3- trifluoropropene. Necrosis of th e olfactory epi th elium was seen in animals exposed to 8901 or 19098 ppm. The fibrosis with or without associated cartilage fo rmat io n is considered a protective response to irritat io n by the test substance, which would have caused the necrosis and erosio n, wi th the atrophy

of the Bowman’s glands and olfactory nerves being either a direct effect of the irritation or a consequence of the epithelial degeneration. The fusio n of dorsal turbinates with associated epithelial diverticula format io n seen in animals of

all test article groups, was considered a result of damage to the epithelium overlying the turbinates and dorsal aspect of the nasal cavit y wi th subsequent fusio n of the turbinates and formation o f epi th elial diverticula. In the various epithelia o f th e nasal cavit y (olfactory, respiratory and transit io nal) of animals of all test article groups similar findings were seen. These included metaplasia and combinat io ns o f di sorganisation, atrophy, degeneration and regenerat io n. The metaplasia was an adapt ive change to produce a ty pe of epi th elium which was more resistant to the irri tation. The combinat io ns of disorganisat io n, atrophy and degeneration were due to the irri tation of the epi th elium and the regenerat io n was part of the tissue repair process. In th e naso la crimal ducts, inflammation was seen in animals of all test article groups. Alt hough epithelial degenerat io n was seen in only one female exposed to 19098 ppm, both th e inflammat io n and epithelial degenerat io n are considered due to the irritancy of the test substance. In the ai rway, debris and inflammatory cells were seen in of all test article groups and are considered a consequence of the other changes in the nasal cavit y. In general, the changes seen in the nasal turbinates of animals exposed t o 8901 or 19098 ppm were of a similar severit y and incidence and o f a higher severit y than those seen for animals exposed to 4721 ppm. In considerat io n of the epithelial changes the olfactory epi th elium was th e most extensively

affected at all

three expos ure levels.Hun tingdon Life Sciences WA G0013

35 

### In the larynx, epithelial metaplasia, hyperplasia and keratinisat io n were seen in animals o f all test article groups. As with the nasal epit helia, these changes were an adapt ive response to produce an epithelium more resistant to irritation. Inflammat io n was seen in one male and one female exposed to 19098 ppm and one male exposed to 8901 ppm and was considered a consequence o f irri

tancy. In general, the findings in the larynx were of a similar incidence and degree in all groups exposed to 2 - bromo-3,3,3- trifluoropropene. In the trachea, epithelial degenerat io n/regeneration was seen in male and female animals exposed to 19098 ppm and one female exposed to 8901 ppm. As in the nasal cavit y, the degeneration was due to the irritation o f th e epi th elium and the regeneration was part of the ti ssue repai

r process. Inflammatory

cell infiltrate in the lamina propria was seen at a higher incidence and/or degree in animals exposed to 19098 ppm, compared with control animals, which again was considered a consequence of irritancy. Overall, the most marked changes were in the nasal cavit y, wi th minima l to slight changes in th e larynx and trachea. For the nasal cavit y, in general the changes seen in animals exposed to 8901 or 19098 ppm were of a similar seve ri ty and incidence. As rodents are obligate nose breathers, this distribution o f findings is considered accounted for by the nasal cavit y being th e first part of the respiratory tract to have contact with the test article. In addit io n, the nasal epi th elium, and especially the olfactory epi thelium which is anatomically confined to the dorsal half o f th e nasal cavit y, i s a si te of m etabo lic enzymes and so metabo lism o f th e test substance may have contributed to the severit y of changes seen. Atnecropsy, a r eduction in the size of the thymus and depressions or roughening of the stomach were noted in animals exposed to 19098 ppm. These tissues were not required to be examined microscopically. Treatment - related body

weight losses or reductions in bodyweight gai n were seen at all test article groups. These reduct io ns wer e exposure level related and are considered to be treatment - related and correlate to some extent with reduced food consumpt io n that was al so seen. Clinical signs of shallow breathing, piloerect io n, salivat io n and chin rubbing evident during and after exposure are indicat ive of exposure to an irritant material. Whereas, the clinical signs of underactivit y, unsteady

gai t and partially closed eye lid s, together with the reduced sensory and motor activit y responses seen at 8901 or 19098 ppm represent potential CNS effects. Ho wever , all o f th ese signs were transient in nature.Hun tingdon Life Sciences WA G0013

36 

# 5. Conclusion Hi stopathological

changes related to treatment were seen in the nasal turbinates, larynx and trachea. Alt hough the majo ri ty of these changes were limited to the 8901 or 19098 ppm exposure groups, some changes in the nasal turbinates were evident at 4721 ppm. Therefore, a no observed adverse effect level (NOAEL) could not be established.Hun tingdon Life Sciences WA G0013

37 

# 6. References 2- Bromo-3,3,3- triflu oropropene, E -1- Bromo - 3,3,3 - trifluoropropene and 1 - Bromo-2- trifluoromethyl - 3,3,3 - trifluoropropene: an acute (4 - hour) inhalation toxicit y study in the rat via nose - only

exposure. (Laboratory

Proj ect Number 02 -5449)38Huntingdon Life SciencesWAG0013

FIGURE 1 Motor activity - gr oup mean scores (beam breaks) for males on Day 10 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 4721 8901 1 9098 1M 2M 3M 4M

0 20 40 60 80 100 120 140 160

6 12 18 24 30 36 42 48 54 60Number of beam breaks

Ti me (m in utes) High b ea ms

0 50 100 150 200 250 300 350

6 12 18 24 30 36 42 48 54 60Number of beam breaks

Tim e (m in utes)

Low b ea ms

0 250 500 750

1 2 3 4Beam breaks

Group

Total scores

0 500 1000 1500

1 2 3 4Beam breaks

Group

Total sc ores39Huntingdon Life SciencesWAG0013

FIGURE 1 - continued Motor activity - gr oup mean scores (beam breaks) for females on Day 10 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 Expo sure (ppm) : 0 4721 8901 19098 1F 2F 3F 4F

0 20 40 60 80 100 120 140 160 180 200

6 12 18 24 30 36 42 48 54 60Number of beam breaks

Tim e (m in utes)

Hi gh b ea ms

0 50 100 150 200 250 300 350 400

6 12 18 24 30 36 42 48 54 60Number of beam breaks

Ti me (m inutes) Low b ea ms

0 250 500 750 1000

1 2 3 4Beam breaks

Group

Total scores

0 500 1000 1500 2000 2500

1 2 3 4Beam breaks

Group

Total scores40Huntingdon Life SciencesWAG0013

FIGURE 2 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 4721 8901 1909841Huntingdon Life SciencesWAG0013

TABLE 1 Sensory reactivity observations and grip strength - summary of findings on Day 10 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 4721 8901 19098 Group/sex: 1M 2M 3M 4M 1F 2F 3F 4F Number of animals: 3 3 3 3 3 3 3 3 Parameter Grade Approach response (1 - 3) 1 0 0 1 3 0 0 1 2 2 3 3 2 0 3 3 2 1 Touch response (1 - 3) 1 0 0 2 1 0 0 2 2 2 3 3 1 2 3 3 1 1 Auditory startle reflex (1 - 4) 1 0 0 0 2 0 0 0 0 2 1 0 1 0 0 0 0 1 3 2 3 2 1 3 3 3 2 Tail pinch response (1 - 4) 2 0 0 0 0 0 0 0 1 3 3 3 3 3 2 3 3 2 4 0 0 0 0 1 0 0 0 Forelimb grip strength (kg) Mean 0.94 1.14 1.06 0.95 0.92 0.94 0.81 1.04 SD 0.09 0.10 0.25 0.22 0.04 0.10 0.08 0.19 Hindlimb grip strength (kg) Mean 0.58 0.52 0.53 0.49 0.49 0.39 0.47 0.45 SD 0.01 0.05 0.03 0.06 0.11 0.07 0.03 0.1142Huntingdon Life SciencesWAG0013

TABLE 2 Motor activity - gr oup mean scores (b eam breaks) on Day 10 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 4721 8901 19098 Group /Sex Number of animals Beam

level Time (minutes)

6 12 18 24 30 36 42 48 54 60 Total 1M 3 High 138.7 97.7 81.7 48.3 50.7 41.3 10.3 34.3 38.3 2.7 544.0

SD 18.3 12.7 60.9 47.9 38.0 43.7 13.8 32.0 65.5 2.5 297.8 2M 3 High 119.3 76.0 49.0 43.3 22.0 21.7 22.3 1 1.7 23.3 20.7 409.3

SD 8.4 19.1 13.1 39.2 7.0 14.8 10.1 3.2 2.1 11.0 92.4 3M 3 High 83.0 47.3 33.3 20.0 13.7 2.0 11.0 10.0 7.0 10.3 237.7

SD 11.3 28.9 30.5 19.5 19.5 2.0 9.6 11.8 6.2 17.9 108.9 4M 3 High 70.0 11.3 6.0 0.3 0.0 0.0 0.3 0.0 0.0 2.7 90.7

SD 20.7 8.1 8.7 0.6 0.0 0.0 0.6 0.0 0.0 4.6 28.7 1M 3 Low 262.3 184.3 147.7 112.3 145.0 54.3 21.0 36.3 46.0 14.7 1024.0

SD 51.0 72.6 73.5 9.1 54.1 40.4 20.5 11.7 57.7 13.3 105.3 2M 3 Lo w 274.7 140.3 124.3 83.3 95.0 50.3 57.0 37.7 84.3 36.7 983.7

SD 96.7 49.3 25.7 24.1 11.4 15.3 27.9 12.7 18.2 12.1 197.8 3M 3 Low 197.7 97.7 80.3 42.7 21.0 9.0 32.3 15.0 22.0 16.0 533.7

SD 52.6 50.9 62.2 25.6 19.9 4.4 31.5 13.7 26.2 27 .7 95.1 4M 3 Low 179.3 63.3 20.0 1.3 0.0 0.0 1.7 4.3 2.0 23.0 295.0

SD 19.3 66.5 26.2 2.3 0.0 0.0 1.5 7.5 3.5 39.8 154.243Huntingdon Life SciencesWAG0013

TABLE 2 - continued Motor activity - gr oup mean scores (beam breaks) on Day 10 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 4721 8901 19098 Group /Sex Number of animals Beam

level Time (minutes)

6 12 18 24 30 36 42 48 54 60 Total 1F 3 High 174.7 110.7 108.0 90.3 52.3 73.0 27.7 41.0 22.0 36.3 736.0

SD 29.0 35.7 56.9 23.5 7.8 18.3 23.7 35.7 22.5 24.8 137.4 2F 3 High 101.3 73.7 44.3 22.3 24.3 16.0 6.0 2.7 24.7 27.7 343.0

SD 12.4 39.1 2 .5 16.8 15.9 14.7 9.5 4.6 21.6 27.5 91.0 3F 3 High 138.3 53.3 15.7 0.0 0.0 0.0 0.0 0.0 8.7 4.3 220.3

SD 41.2 47.4 4.0 0.0 0.0 0.0 0.0 0.0 7.6 7.5 92.8 4F 3 High 121.7 26.3 13.7 0.0 3.7 9.7 0.0 0.0 0.0 0.0 175.0

SD 61.9 39.7 11.1 0.0 6.4 16.7 0.0 0.0 0.0 0.0 131.2 1F 3 Low 309.7 254.3 203.0 166.7 155.0 179.7 108.0 100.3 103.3 90.7 1670.7

SD 93.3 101.1 103.9 16.7 31.5 19.9 31.2 95.5 101.5 30.7 506.6 2F 3 Low 239.0 177.3 113.0 91.3 107.3 69.0 38.3 12.7 65.7 50.7 964.3

SD 41.1 34.7 12.5 32.7 43.3 79.6 42.1 11.7 59.2 44.0 56.6 3F 3 Low 231.3 132.0 50.0 0.7 0.0 0.3 0.0 3.0 9.0 25.3 451.7

SD 54.4 36.9 50.7 1.2 0.0 0.6 0.0 5.2 11.5 43.0 94.4 4F 3 Low 219.3 76.3 59.7 4.0 19.7 53.7 3.3 0.0 0.0 0.0 436.0

SD 88.2 114.1 60.0 6.9 34.1 93.0 5.8 0.0 0.0 0.0 374.544Huntingdon Life SciencesWAG0013

TABLE 3 Request ID: 257064 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 4721 8901 19098 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 44 3 444 448 450 451 454 455 457 460 461 466 466

SD 20.2 15.7 16.2 13.6 15.9 17.3 20.0 17.0 19.4 15.4 20.7 18.9 N 3 3 3 3 3 3 3 3 3 3 3 3 2 M Mean 391 398 404 408 418 424 406 412 4 20 414 414 415

SD 20.8 21.9 24.9 24.8 25.8 27.5 20.9 26.0 29.3 30.9 29.2 28.1 N 3 3 3 3 3 3 3 3 3 3 3 3 3 M Mean 444 448 454 456 461 469 452 455 466 457 448 448

SD 27.3 27.2 26 .8 27.2 29.6 31.3 30.9 28.7 31.7 28.7 29.1 29.6 N 3 3 3 3 3 3 3 3 3 3 3 3 4 M Mean 450 450 454 453 456 453 438 436 452 438 426 417

SD 18.9 17.2 16.3 18.6 18.0 22.9 13.7 19.6 18.9 18.5 28.2 34.9 N 3 3 3 3 3 3 3 3 3 3 3 345Huntingdon Life SciencesWAG0013

TABLE 3 - continued Request ID: 257064 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 -

tri fluoropropene 2 - br omo - 3,3,3 -

trifluoropropene Exposure (ppm) : 0 4721 8901 19098 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 M Mean 467 467 472 476 475 477 477 480 27 -

SD 21.1 24.8 20.8 22.5 23.1 22.6 24.7 23.1 5.8 N 3 3 3 3 3 3 3 3 3 2 M Mean 425 422 438 443 439 437 429 430 6 23

SD 27.4 28.6 27.1 30.6 28.9 29 .1 26.8 26.4 6.2 N 3 3 3 3 3 3 3 3 3 3 M Mean 448 449 471 483 476 473 463 470 0 2

SD 27.7 23.6 30.9 29.5 33.9 26.7 24.7 29.3 5.2 N 3 3 3 3 3 3 3 3 3 4 M Mean 406 400 417 434 427 419 410 410 - 43 -

SD 38.4 44.9 53.6 41.6 38.3 34.3 35.2 36.3 13.5 N 3 3 3 3 3 3 3 3 346Huntingdon Life SciencesWAG0013

TABLE 3 - continued Request ID: 257064 Bodyweigh t - 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 4721 8901 19098 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 303 298 300 292 302 304 309 298 312 315 319 304

SD 10.1 10.0 12.1 11.5 12.6 10.4 6.7 6.4 8.1 8.0 6.2 4.4 N 3 3 3 3 3 3 3 3 3 3 3 3 2 F Mean 240 243 243 247 246 250 242 245 249 244 250 247

SD 9.5 6.0 3.0 7.8 9.9 3.3 7.7 10.5 12.8 13.3 10.2 11.5 N 3 3 3 3 3 3 3 3 3 3 3 3 3 F Me an 258 259 257 264 265 267 261 267 271 269 267 271

SD 7.3 11.2 12.1 10.2 6.3 15.8 9.3 11.5 9.4 11.4 7.7 10.2 N 3 3 3 3 3 3 3 3 3 3 3 3 4 F Mean 263 265 271 269 266 269 260 263 2 76 265 261 266

SD 10.2 14.0 12.4 9.2 9.4 10.0 2.4 6.6 10.3 7.3 1.9 4.4 N 3 3 3 3 3 3 3 3 3 3 3 347Huntingdon Life SciencesWAG0013

TABLE 3 - continued Request ID: 257064 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 4721 8901 19098 Group Day Day Day Day Day Day Day Day Change As % of /Sex 8 9 10 11 12 13 14 1 5 1 - 15 Control 1 F Mean 320 318 317 306 315 323 315 314 10 -

SD 8.3 7.9 9.9 5.0 12.4 7.2 9.9 12.2 13.1 N 3 3 3 3 3 3 3 3 3 2 F Mean 251 253 265 269 2 63 265 261 263 13 135

SD 12.0 10.7 16.3 12.3 6.3 10.9 13.1 11.6 8.7 N 3 3 3 3 3 3 3 3 3 3 F Mean 272 274 288 292 281 287 280 284 17 168

SD 14.8 14.4 15.6 14.0 10.9 12.2 16.2 14.9 6.9 N 3 3 3 3 3 3 3 3 3 4 F Mean 255 258 276 282 277 261 260 265 - 3 -

SD 12.2 12.5 5.3 5.1 5.5 6.1 15.5 6.5 16.5 N 3 3 3 3 3 3 3 3 348Huntingdon Life SciencesWAG0013

TABLE 4 Request ID: 257053 Organ weights - gr oup mean unadjusted and adjusted 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 4 721 8901 19098 Group /Sex Terminal

Bodyweight Br ain Heart Kidneys Liver Lungs + Br onchi Spleen Un adjusted Means 1M Mean 483 2.04 1.690 3.15 17.30 1.640 0.790 SD 24 0.05 0.155 0.53 1.54 0.166 0.017 N 3 3 3 3 3 3 3 2M Me an 432 2.01 1.456 3.44 15.72 1.634 0.663 SD 25 0.02 0.264 0.25 2.20 0.099 0.090 N 3 3 3 3 3 3 3 3M Mean 469 2.07 1.635 3.21 19.75 1.658 0.891 SD 28 0.10 0.262 0.35 2.20 0.282 0.164 N 3 3 3 3 3 3 3 4M Mean 412 2.09 1.569 2.97 1 6.15 1.730 0.636 SD 36 0.11 0.199 0.54 1.30 0.258 0.055 N 3 3 3 3 3 3 3 Adjusted Means 1M Mean 1.96 1.714 2.74 15.93 1.517 0.728 2M Mean 2.05 1.444 3.65 16.41 1.696 0.694 3M Mean 2.02 1.650 2.96 18.92 1.584 0.854 4M Mean 2.18 1.542 3.42 17.65 1.864 0.70449Huntingdon Life SciencesWAG0013

TABLE 4 - continued Request ID: 257053 Organ weights - gr oup mean unadjusted and adjusted values (g) 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 4721 8901 19098 Group /Sex Terminal

Bodyweight Br ain Heart Kidneys Liver Lungs + Br onchi Spleen Un adjusted Means 1F Mean 314 1.97 1.325 1.91 10.12 1.368 0.563 SD 12 0.02 0.100 0.07 0.83 0.097 0.046 N 3 3 3 3 3 3 3 2F Mean 264 1.84 0.927 1.89 9.30 1.190 0.510 SD 11 0.14 0.111 0.18 1.23 0.100 0.084 N 3 3 3 3 3 3 3 3F Mean 283 1.88 1.049 1.91 11.51 1.298 0.502 SD 15 0.13 0.112 0.13 0.88 0.202 0.105 N 3 3 3 3 3 3 3 4F Mean 265 1.87 1.029 1.72 10.08 1.328 0.484 SD 7 0.03 0.036 0.05 0.25 0.117 0.031 N 3 3 3 3 3 3 3 Adjusted Means 1F Mean 1.79 1.250 1.91 8.67 1.349 0.712 2F Mean 1.93 0.967 1.90 10.07 1.200 0.430 3F Mean 1.87 1.046 1.91 11.44 1.297 0.510 4F Mean 1.96 1.068 1.72 10.82 1.337 0.40850Huntingdon Life SciencesWAG0013

TABLE 5 Request ID: 264436 Macropathology - group distribution of findings Group : 1 2 3 4 Compound : Control 2 - br omo - 3,3,3 - trifluoropropene 2 - bromo - 3,3,3 -

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

trifluoropropene Exposure (ppm) : 0 4721 8901 19098 Group/Sex: 1M 2M 3M 4M 1F 2F 3F 4F Tissue and Finding Number Examined: 3 3 3 3 3 3 3 3 Kidneys Pale area(s) 0 0 0 1 0 0 0 0 Pelvic dil atation 0 0 0 1 0 0 0 0 Ln mandibular Enlarged 0 0 0 0 0 1 0 0 Lungs + bronchi Dark area(s) 0 0 1 0 0 0 0 0 Pale area(s) 0 1 0 1 0 1 0 0 Skin Hair loss 2 0 2 0 0 0 0 0 Scab(s) 0 0 1 0 0 0 0 0 Spleen Capsule thickened 0 2 0 0 0 0 0 0 Stomach Forestomach depression(s) 0 0 0 2 0 0 0 0 Forestomach roughened 0 0 0 1 0 0 0 251Huntingdon Life SciencesWAG0013

TABLE 5 - continued Request ID: 264436 Macropathology - group di stribution of 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 4721 8901 19098 Group/Sex: 1M 2M 3M 4M 1F 2F 3F 4F Tissue and Finding Number E xamined: 3 3 3 3 3 3 3 3 Thy mus Small 0 0 0 3 0 0 0 3 Ln b ronc hial Enlarged 0 0 0 0 0 1 0 052Huntingdon Life SciencesWAG0013

TABLE 6 Request ID: 264437 Histopathology - gr oup distribution of 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 4721 8901 19098 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 Epithelial Degeneration/Regeneration 0 0 0 3 0 0 1 2 Inflammatory Cell Infiltrate - Lamina Propria 1 1 0 2 0 0 1 1 Tracheal Bifurc. Number Examined: 3 3 3 3 3 3 3 3 Inflammatory Cell Infiltrate in Lamina Propria 2 1 0 2 0 0 1 1 Lungs + Bronchi Number Examined: 3 0 0 3 3 0 0 3 Alveolar Eosinophilic Crystals with Associated Inflammatory Cell Infiltrate 0 0 0 0 2 0 0 0 Alveolar Macrophages 1 0 0 1 1 0 0 3 Alveolitis 1 0 0 1 0 0 0 0 Ar terial Mural Min er alisation 2 0 0 2 2 0 0 1 Perivascular Inflammatory Cell Infiltrate 2 0 0 0 0 0 0 0 Lary nx Number Examined: 3 3 3 3 3 3 3 3 Epithelial Hyperplasia - Ar yt enoid 0 1 1 0 0 0 0 2 Epithelial Hyperplasia - Ventral 0 0 0 1 0 0 0 0 Epithelial Keratinisation - Ventrolateral 0 2 2 3 0 2 2 3 Inflammation 0 0 1 1 0 0 0 1 Squamous Metaplasia - Ar yt enoid 0 2 3 2 0 2 3 3 Squamous Metaplasia - Ventral 0 0 1 2 0 0 3 3 Submucosal Foreign Body Granulomata - Ventral 0 1 1 0 0 0 0 053Huntingdon Life SciencesWAG0013

TABLE 6 - continued Request ID: 264437 Histopathology - gr oup distribution of 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 4721 8901 19098 Group/Sex: 1M 2M 3M 4M 1F 2F 3F 4F Tissue and Finding Number: 3 3 3 3 3 3 3 3 Nasal turbinates Number Examined: 3 3 3 3 3 3 3 3 Debris and Inflammatory Cells in Airway 0 2 1 1 0 0 2 1 Fusion of Dorsal Turbinate Wi th Associated Epithelial Diverticula Formation 0 0 3 3 0 1 3 3 Nasolacrimal Ducts - Epithelial Degeneration 0 0 0 0 0 0 0 1 Nasolacrimal Ducts - Inflammation 0 1 1 2 0 1 0 2 Olfactory Epithelium - Bowman's Gland Atrophy in Dorsal Lamina Propria 0 3 3 3 0 3 3 3 Olfactory Epithelium - Degeneration/Atrophy/Disorganisation 0 3 3 3 0 2 3 3 Olfactory Epithelium - Erosion 0 1 1 2 0 1 1 1 Olfactory Epithelium - Fibrosis With or Without Associated Cartilage Formation in Dorsal Lamina Propria 0 2 3 3 0 3 3 3 Olfactory Epithelium - Necrosis 0 0 0 2 0 0 1 0 Olfactory Epithelium - Respiratory Metaplasia With or Without Associated Inflammatory Cell Infiltrate 0 2 3 3 0 3 3 2 Olfactory Epithelium - Squamous Metaplasia 0 3 2 1 0 3 3 2 Olfactory Nerve Fibres - Atrophy 0 3 3 3 0 3 3 3 Respiratory Epithelium - Disorganisation/Degeneration/ Regeneration 0 1 3 3 0 1 2 3 Respiratory Epithelium - Goblet Cell Hyperplasia 0 1 1 0 0 1 1 0 Respiratory Epithelium - Squamous Metaplasia 0 0 0 3 0 0 0 1 Submucosal Foreign Body Granulomata - Dorsal 0 0 1 1 0 0 0 0 Transitional Epithelium - Disorganisation/Regeneration 0 2 2 3 0 2 2 354Huntingdon Life SciencesWAG0013

APPENDIX 1 Request ID: 258669 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 4721 8901 19098 Group Animal Death Day of /Sex Number Code Death Category Observation Day (s) 1M 1 T 15 Coat Hair loss, Forelimbs - 4, 3, 10, 14 - 15 3 T 15 Coat Hair loss, Forelimbs 14 - 15 Only animals with observations are presented55Huntingdon Life SciencesWAG0013

APPENDIX 1 - continued Request ID: 258669 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 4721 8901 19098 Group Animal Death Day of /Sex Number Code Death Category Observation Day (s) 2M 5 T 15 Coat Hair loss, Forepaws - 4, 3, 10 Only animals with observations are presented56Huntingdon Life SciencesWAG0013

APPENDIX 1 - continued Request ID: 258669 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 4721 8901 19098 Group Animal Death Day of /Sex Number Code Death Category Observation Day (s) 3M 7 T 15 Coat Hair loss, Head - 4, 3, 10, 14 - 15 9 T 15 Coat Hair loss, Head 3, 10, 14 - 15 Only animals with observations are presented57Huntingdon Life SciencesWAG0013

APPENDIX 1 - continued Request ID: 258669 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 4721 8901 19098 Group Animal Death Day of /Sex Number Code Death Category Observation Day (s) 4M 10 T 15 Behaviour Un deractive 15 11 T 15 Coat Piloerection 10 Only animals with observations are presented58Huntingdon Life SciencesWAG0013

APPENDIX 1 - continued Request ID: 258669 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 4721 8901 19098 Group Animal Death Day of /Sex Number Code Death Category Observation Day (s) 1F 14 T 15 B ehaviour Irritable 10, 14 Only animals with observations are presented59Huntingdon Life SciencesWAG0013

APPENDIX 1 - continued Request ID: 258669 Clinical signs - individual observations 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 4721 8901 19098 Group Animal Death Day of /Sex Number Code Death Category Observation Day (s) 4F 22 T 15 Behaviour Un deractive 14 - 15 Coat Piloerection 14 - 15 Staining Abnormal Colour, Red, Head 14 - 15 23 T 15 Body temperature Reduced 14 Coat Hair loss, Forelimbs - 4, 3, 10, 14 - 15 Muscle Reaction Abnormal Gait, Elevated 14 - 15 Staining Abnormal Colour, Brown, Anus 14 24 T 15 Behaviour Un deractive 14 - 15 Only animals with observations are presented60Huntingdon Life SciencesWAG0013

APPENDIX 2 Request ID: 259116 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 -

t rifluoropropene 2 - br omo - 3,3,3 -

trifluoropropene Exposure (ppm) : 0 4721 8901 19098 Days Group /Sex Animal Number Sign Pre - dose 1 to 2 hours during exposure 3 to 4 hours

during exposure 5 to 6 hours

during exposure 1 to 2 hours after

completion of dosing 1M 1 Coat

Piloerection 5 1, 4 - 5 1, 4 - 5 2 Coat Piloerection 1 161Huntingdon Life SciencesWAG0013

APPENDIX 2 - continued Request ID: 259116 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 4721 8901 19098 Days Group /Sex Animal Number Sign Additional observation As late as

possible in the working day 1M 1 Coat

Piloerection 2 Coat Piloerection62Huntingdon Life SciencesWAG0013

APPENDIX 2 - continued Request ID: 259116 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 4721 8901 19098 Days Group /Sex Animal Number Sign Pre - dose 1 to 2 hours during exposure 3 to 4 hours

during exposure 5 to 6 hours

during exposure 1 to 2 hours after

complet ion of dosing 2M 4 Behaviour

Ch in Rubbing 13 - 14 12 12 11 Salivation 1, 11, 13 13 Br eathing Gasping 1 Shallow 1 1 1, 12 Slow 1 Coat Piloerection 1, 4 1, 12 1, 12 14 E yelids (Left) Closed 1 Par tially Closed 11, 14 11 - 12, 14 12, 14 14 Eyelids (Right) Closed 1 Par tially Closed 11, 14 11 - 12, 14 12, 14 14 Eye (Left) Lacrimation 1 Eye (Right) Lacrimation 1 Posture Hun ched 1 1 163Huntingdon Life SciencesWAG0013

APPENDIX 2 - continued Request ID: 259116 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 -

tri fluoropropene 2 - br omo - 3,3,3 -

trifluoropropene Exposure (ppm) : 0 4721 8901 19098 Days Group /Sex Animal Number Sign Additional observation As late as

possible in the working day 2M 4 Behaviour

Ch in Rubbing Salivation Br e athing Gasping Shallow Slow Coat Piloerection Eyelids (Left) Closed Par tially Closed Eyelids (Right) Closed Par tially Closed Eye (Left) Lacrimation Eye (Right) Lacrimation Posture Hun ched64Huntingdon Life SciencesWAG0013

APPENDIX 2 - continued Request ID: 259116 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 -

tr ifluoropropene 2 - br omo - 3,3,3 -

trifluoropropene Exposure (ppm) : 0 4721 8901 19098 Days Group /Sex Animal Number Sign Pre - dose 1 to 2 hours during exposure 3 to 4 hours

during exposure 5 to 6 hours

during exposure 1 to 2 hours after

completion of dosing 2M 5 Behaviour

Ch in Rubbing 8, 12 - 14 12 7 - 8, 11 - 14 Irritable 13 Salivation 1, 8, 11 - 14 8, 11 - 14 7, 12 - 14 7, 12 - 14 Br eathing Gasping 1 Shallow 1, 12 1, 12 1, 12 Slow 1 Coat Piloerection 1 1 1 14 Eyelids (Left) Closed 1 Par tially Closed 14 12 - 14 12 - 14 14 Eyelids (Right) Closed 1 Par tially Closed 14 12 - 14 12 - 14 14 Eye (Left) Lacrimation 1 Eye (Right) Lacrimation 1 Posture Hun ched 1 1 1 Staining Post Salivation Staining 8 8, 11, 1465Huntingdon Life SciencesWAG0013

APPENDIX 2 - continued Request ID: 259116 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 4721 8901 19098 Days Group /Sex Animal Number Sign Additional observation As late as

possible in the working day 2M 5 Behaviour

Ch in Rubbing Irritable Salivation Br eathing Gasping Shallow Slow Coat Piloerection Eyelids (Left) Closed Par tially Clo sed Eyelids (Right) Closed Par tially Closed Eye (Left) Lacrimation Eye (Right) Lacrimation Posture Hun ched Staining Post Salivation Staining 7, 11 7, 1166Huntingdon Life SciencesWAG0013

APPENDIX 2 - continued Request ID: 259116 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 4721 8901 19098 Days Group /Sex Animal Number Sign Pre - dose 1 to 2 hours during exposure 3 to 4 hours

during exposure 5 to 6 hours

during exposure 1 to 2 hours after

completion of dosing 2M 6 Behaviour

Ch in Rubbing 8, 11, 13 - 14 Salivation 1, 11, 13 Br eathing Gasping 1 Shallow 1 1, 12 1, 12 Slow 1 Coat Piloerection 1, 14 1, 12, 14 1, 12, 14 Eyelids (Left) Closed 1 Par tially Closed 14 11 - 14 12, 14 Eyelids (Right) Closed 1 Par tially Closed 14 11 - 14 12, 14 Eye (Left) Lacrimation 1 Eye (Right) Lacrimation 1 Posture Hun ched 1 1 1 Staining Post Salivation Stain ing 1167Huntingdon Life SciencesWAG0013

APPENDIX 2 - continued Request ID: 259116 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 4721 8901 19098 Days Group /Sex Animal Number Sign Additional observation As late as

possible in the working day 2M 6 Behaviour

Ch in Rubbing Salivation Br eathing Gasping Shallow Slow Coat Piloerection Eyelids (Left) Closed Par tially Closed Eyelids (Right) Closed Par tially Closed Eye (Left) Lacrimation Eye (Right) Lacrimation Posture Hun ched Staining Post Salivation Staining68Huntingdon Life SciencesWAG0013

APPENDIX 2 - continued Request ID: 259116 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 -

tr ifluoropropene 2 - br omo - 3,3,3 -

trifluoropropene Exposure (ppm) : 0 4721 8901 19098 Days Group /Sex Animal Number Sign Pre - dose 1 to 2 hours during exposure 3 to 4 hours

during exposure 5 to 6 hours

during exposure 1 to 2 hours after

completion of dosing 3M 7 Behaviour

Ch in Rubbing 5 - 8, 11 5 8 Irritable 6 Underactive 8, 12, 14 8, 12 - 14 12 - 14 11 Unresponsive 6 - 7 6 Salivation 1, 5 - 6, 11 - 14 5, 13 - 14 5, 13 - 14 13 Br eathing Gaspin g 1 Irregular 1 Shallow 1, 4 - 5, 7, 11 1, 4 - 7, 11 1, 5 - 7, 11 Slow 1 1 Coat Piloerection 8, 11 8, 11 - 12 8, 11 - 12 8 Eyelids (Left) Par tially Closed 1, 6 - 8, 11 - 14 6 - 8, 11 - 14 6 - 8, 11 - 14 Eyeli ds (Right) Par tially Closed 1, 6 - 8, 11 - 14 6 - 8, 11 - 14 6 - 8, 11 - 14 Eye (Left) Lacrimation 1 Eye (Right) Lacrimation 1 Muscle Reaction Un steady 1, 8 8 469Huntingdon Life SciencesWAG0013

APPENDIX 2 - continued Request ID: 259116 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 4721 8901 19098 Days Group /Sex Animal Number Sign Additional observation As late as

possible in the working day 3M 7 Behaviour

Ch in Rubbing Irritable Underactive Unresponsive Salivation Br eathing Gasping Irregular Shallow Slow Coat Piloerection Eyelids (Left) Par tially Closed Eyelids (Right) Par tially Closed Eye (Left) Lacrimation Eye (Right) Lacrimation Muscle React ion Un steady70Huntingdon Life SciencesWAG0013

APPENDIX 2 - continued Request ID: 259116 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 -

trifluorop ropene Exposure (ppm) : 0 4721 8901 19098 Days Group /Sex Animal Number Sign Pre - dose 1 to 2 hours during exposure 3 to 4 hours

during exposure 5 to 6 hours

during exposure 1 to 2 hours after

completion of dosing 3M 7 Staining

Post Salivation Staining 5 - 8, 1171Huntingdon Life SciencesWAG0013

APPENDIX 2 - continued Request ID: 259116 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 4721 8901 19098 Days Group /Sex Animal Number Sign Additional observation As late as

possible in the working day 3M 7 Staining

Post Salivation Staining72Huntingdon Life SciencesWAG0013

APPENDIX 2 - continued Request ID: 259116 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 4721 8901 19098 Days Group /Sex Animal Number Sign Pre - dose 1 to 2 hours during exposure 3 to 4 hours

during exposure 5 to 6 hours

during exposure 1 to 2 hours after

completion of dosing 3M 8 Behaviour

Ch in Rubbing 5 - 7, 11 5 - 6 6 6 Underactive 8, 12 - 14 8, 12 - 14 12 - 14 Unresponsive 6 - 7 6 Salivation 1 5 5 Br eathing Gasping 1 Irregular 1 Shallow 1, 4 - 5, 7, 11 1, 4 - 7, 11 1, 5 - 7, 11 7 Slow 1 Coat Piloerection 8, 11 8, 1 1 - 12 7 - 8, 11 - 12 7 Eyelids (Left) Par tially Closed 1, 7 - 8, 11 - 14 1, 6 - 8, 11, 1, 6 - 8, 11, 7 13 - 14 13 - 14 Eyelids (Right) Par tially Closed 1, 7 - 8, 11 - 14 1, 6 - 8, 11, 1, 6 - 8, 11, 7 13 - 14 13 - 14 Eye (Left) Lacrimation 1 Eye (Right) Lacrimation 1 Muscle Reaction Un steady 873Huntingdon Life SciencesWAG0013

APPENDIX 2 - continued Request ID: 259116 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 4721 8901 19098 Days Group /Sex Animal Number Sign Additional observation As late as

possible in the working day 3M 8 Behaviour

Ch in Rubbing Underactive Unresponsive Salivation Br eathing Gasping Irregular Shallow Slow Coat Piloerection Eyelids (Left) Par tially Closed Eyelids (Right) Par tially Closed Eye (Left) Lacrimation Eye (Right) Lacrimation Muscle Reaction Un steady74Huntingdon Life SciencesWAG0013

APPENDIX 2 - continued Request ID: 259116 Signs associated with dosing - indivi dual 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 4721 8901 19098 Days Group /Sex Animal Number Sign Pre - dose 1 to 2 hours during exposure 3 to 4 hours

during exposure 5 to 6 hours

during exposure 1 to 2 hours after

completion of dosing 3M 8 Staining

Post Salivation Staining 5 - 775Huntingdon Life SciencesWAG0013

APPENDIX 2 - continued Request ID: 259116 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 4721 8901 19098 Days Group /Sex Animal Number Sign Additional obs ervation As late as

possible in the working day 3M 8 Staining

Post Salivation Staining76Huntingdon Life SciencesWAG0013

APPENDIX 2 - continued Request ID: 259116 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 4721 8901 19098 Days Group /Sex Animal Number Sign Pre - dose 1 to 2 hours during exposure 3 to 4 hours

during exposure 5 to 6 hours

during exposure 1 to 2 hours after

completion of dosing 3M 9 Behaviour

Ch in Rubbing 5 - 7 5 4 Underactive 8, 12, 14 8, 12 - 14 12 - 14 11 Unresponsive 6 - 7 6 Salivation 1 Br eathing Gasping 1 Irregular 1 Shallow 1, 4, 7, 11 1, 4 - 7, 11 1, 5 - 6, 11 Slow 1 1 Coat Piloerection 8, 12 5, 8, 12 5, 8, 12 7 Eyelids (Left) Closed 1 Par tially Closed 1, 5 - 8, 11 - 14 5 - 8, 11 - 14 5 - 6, 8, 11 - 14 Eyelids (Right) Closed 1 Par tially Closed 1, 5 - 8, 11 - 14 5 - 8, 11 - 14 5 - 6, 8, 11 - 14 Eye (Left) Lacrimation 1 Eye (Right) Lacrimation 1 Muscle Reaction Un steady 1 8 477Huntingdon Life SciencesWAG0013

APPENDIX 2 - continued Request ID: 259116 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 4721 89 01 19098 Days Group /Sex Animal Number Sign Additional observation As late as

possible in the working day 3M 9 Behaviour

Ch in Rubbing Underactive Unresponsive Salivation Br eathing Gasping Irregular Shallow Slow Coat Piloerection Eyelids (Left) Closed Par tially Closed Eyelids (Right) Closed Par tially Closed Eye (Left) Lacrimation Eye (Right) Lacrimation Muscle Reaction Un steady78Huntingdon Life SciencesWAG0013

APPENDIX 2 - continued Request ID: 259116 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 -

tri fluoropropene 2 - br omo - 3,3,3 -

trifluoropropene Exposure (ppm) : 0 4721 8901 19098 Days Group /Sex Animal Number Sign Pre - dose 1 to 2 hours during exposure 3 to 4 hours

during exposure 5 to 6 hours

during exposure 1 to 2 hours after

completion of dosing 4M 10 Behaviour

Irritable 4 Underactive 1, 5 - 7 5 - 7 4 - 7 Unresponsive 1, 4 4, 7 7 Salivation 1 Br eathing Gasping 1 Shallow 1, 4 - 5 1, 4 - 5, 7 1, 4 - 5, 7 Slow 1 7 7 Coat Piloerection 1, 6 - 7 1, 5 - 7 1, 4 - 7 1, 4 Eyelids (Left) Par tially Closed 1, 4 - 7 1, 4 - 7 1, 4 - 7 1 Eyelids (Right) Par tially Closed 1, 4 - 7 1, 4 - 7 1, 4 - 7 1 Eye (Left) Lacrimation 1 Eye ( Right) Lacrimation 1 Muscle Reaction Un steady 1, 6 1, 5 - 6 1, 6 679Huntingdon Life SciencesWAG0013

APPENDIX 2 - continued Request ID: 259116 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 4721 8901 19098 Days Group /Sex Animal Number Sign Additional observation As late as

possible in the working day 4M 10 Behaviour

Irritable Underactive Unresponsive Salivation Br eathing Gasping Shallow Slow Coat Piloerection Eyelids (Left) Par tially Closed Eyelids (Right) Par tially Clos ed Eye (Left) Lacrimation Eye (Right) Lacrimation Muscle Reaction Un steady80Huntingdon Life SciencesWAG0013

APPENDIX 2 - continued Request ID: 259116 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 4721 8901 19098 Days Group /Sex Animal Number Sign Pre - dose 1 to 2 hours during exposure 3 to 4 hours

during exposure 5 to 6 hours

during exposure 1 to 2 hours after

completion of dosing 4M 11 Behaviour

Underactive 1, 5 - 7 5 - 7 4 - 7 5 Unresponsive 1, 7 7 7 Salivation 1 Body Temperature Reduced Br eathing Gasping 1 Shallow 1, 4 - 5, 7 1, 4 - 5, 7 1, 4 - 5, 7 Slow 1 Coat Piloerection 1, 6 1, 5 - 6 1, 4 - 6 4, 6 - 7 Eyelids (Left) Par tially Closed 1, 4 - 7 1, 4 - 7 1, 4 - 7 4 - 5 Eyelids (Right) Par tially Clo sed 1, 4 - 7 1, 4 - 7 1, 4 - 7 4 - 5 Eye (Left) Lacrimation 1 Eye (Right) Lacrimation 1 Muscle Reaction Bod y Tremo

rs 7 Un steady 1, 6 1, 6 1, 6 6 - 781Huntingdon Life SciencesWAG0013

APPENDIX 2 - continued Request ID: 2591 16 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 4721 8901 19098 Days Group /Sex Animal Number Sign Additional observation As late as

possible in the working day 4M 11 Behaviour

Underactive Unresponsive Salivation Body Temperature Reduced 8 8 Br eathing Gasping Shallow Slow Coat Piloerection Eyelids (Left) Par tially Closed Eyelids (Right) Par tially Closed Eye (Left) Lacrimation Eye (Right) Lacrimation Muscle Reaction Bod y Tremo

rs Un steady82Huntingdon Life SciencesWAG0013

APPENDIX 2 - continued Request ID: 259116 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 4721 8901 19098 Days Group /Sex Animal Number Sign Pre - dose 1 to 2 hours during exposure 3 to 4 hours

during exposure 5 to 6 hours

during exposure 1 to 2 hours after

completion of dosing 4M 11 Posture

Hun che d 7 12 Behaviour Irritable 4 Underactive 1, 5 - 7 5 - 7 4 - 7 5 Unresponsive 1 Salivation 1 Br eathing Gasping 1 Shallow 1, 4 - 5 1, 4 - 5 1, 4 - 5 Slow 1 Coat Piloerection 1, 5 - 6 1, 5 - 6 1, 4 - 6 4, 6 Eyelids (Left) Par tially Closed 1, 4 - 7 1, 4 - 7 1, 4 - 7 4 - 5 Eyelids (Right) Par tially Closed 1, 4 - 7 1, 4 - 7 1, 4 - 7 4 - 5 Eye (Left) Lacrimation 1 Eye (Right) Lacrimation 1 Muscle Reaction Un steady 1, 6 1, 5 - 6 1, 6 683Huntingdon Life SciencesWAG0013

APPENDIX 2 - continued Request ID: 259116 Signs associated with dosing - individual observations 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 4721 8901 19098 Days Group /Sex Animal Number Sign Additional observation As late as

possible in the working day 4M 11 Posture

Hun ched 12 Behaviour Irritable Underactive Unresponsive Salivation Br eathing Gasping Shallow Slow Coat Piloerection Eyelids (Left) Par tially Closed Eyelids (Righ t) Par tially Closed Eye (Left) Lacrimation Eye (Right) Lacrimation Muscle Reaction Un steady84Huntingdon Life SciencesWAG0013

APPENDIX 2 - continued Request ID: 259116 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 4721 8901 19098 Days Group /Sex Animal Number Sign Pre - dose 1 to 2 hours during exposure 3 to 4 hours

during exposure 5 to 6 hours

during exposure 1 to 2 hours after

completion of dosing 1F 13 Coat

Piloerection 7 1, 4 - 5, 7 1, 4 - 7 Eyelids (Left) Par tially Closed 7 7 Eyelids (Right) Par tially Close d 7 7 14 Coat Piloerection 1, 6 15 Coat Piloerection 4 4, 685Huntingdon Life SciencesWAG0013

APPENDIX 2 - continued Request ID: 259116 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 4721 8901 19098 Days Group /Sex Animal Number Sign Additional observation As late as

possible in the working day 1F 13 Co at

Piloerection Eyelids (Left) Par tially Closed Eyelids (Right) Par tially Closed 14 Coat Piloerection 15 Coat Piloerection86Huntingdon Life SciencesWAG0013

APPENDIX 2 - continued Request ID: 259116 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 4721 8901 19098 Days Group /Sex Animal Numb er Sign Pre - dose 1 to 2 hours during exposure 3 to 4 hours

during exposure 5 to 6 hours

during exposure 1 to 2 hours after

completion of dosing 2F 16 Behaviour

Ch in Rubbing 8, 11 - 14 11 - 12 12 8, 13 - 14 Irritable 12 12 Underactive 7 Unresponsive 13 13 Salivation 1, 11 - 13 12 12 Body Temperature Reduced 7 7 Br eathing Gasping 1 Irregular 7 Shallow 1, 11 - 12, 14 1, 7, 11 - 14 1, 7, 12 - 14 7 Coat Piloerection 1, 4, 7, 14 1, 7, 12 - 14 1, 7, 12 - 14 7, 14 Eyelids (Left) Closed 1 Par tially Closed 7, 11, 13 - 14 7, 11, 13 - 14 7, 12 - 14 7 Eyelids (Right) Closed 1 Par tially Closed 7, 11, 13 - 14 7, 11, 13 - 14 7, 12 - 14 7 Eye (Left) Dull 7 Lacrimation 187Huntingdon Life SciencesWAG0013

APPENDIX 2 - continued Request ID: 259116 Signs associated with dosing - individual observations 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 4721 8901 19098 Days Group /Sex Animal Number Sign Additional observation As late as

possible in the working day 2F 16 Behaviour

Ch in Rubbing Irritable Underactive 7 7 Unresponsive Salivation Body Temperature Reduced Br eathing Gasping Irregular Shallow Coat Piloerection Eyelids (Left) Closed Par tially Closed Eyelids (Right) Closed Par tially Closed Eye (Left) Dull 7 7 Lacrimation88Huntingdon Life SciencesWAG0013

APPENDIX 2 - continued Request ID: 259116 Signs associated with dosing - individual observations Gr oup : 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 4721 8901 19098 Days Group /Sex Animal Number Sign Pre - dose 1 to 2 hours during exposure 3 to 4 hours

during exposure 5 to 6 hours

during exposure 1 to 2 hours after

completion of dosing 2F 16 Eye (Right)

Dull 7 Lacrimation 1 Muscle Reaction Abn or mal Gait Un steady 7 Posture Hun ched 1 1 1 7 Skin Colour Pallor 7 7 7 Staining Post Salivation Staining 8, 12 Comments: Elevated89Huntingdon Life SciencesWAG0013

APPENDIX 2 - continued Request ID: 259116 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 4721 8901 19098 Days Group /Sex Animal Number Sign Additional observation A s late as

possible in the working day 2F 16 Eye (Right)

Dull 7 7 Lacrimation Muscle Reaction Abn or mal Gait 7 7 Un steady 7 7 Posture Hun ched 7 7 Skin Colour Pallor 7 7 Staining Post Salivation Staining Comments: Elevated 7 790Huntingdon Life SciencesWAG0013

APPENDIX 2 - continued Request ID: 259116 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 4721 8901 19098 Days Group /Sex Animal Number Sign Pre - dose 1 to 2 hours during exposure 3 to 4 hours

during exposure 5 to 6 hours

during exposure 1 to 2 hours after

completion of dosing 2F 17 Behaviour

Ch in Rubbing 8, 11 - 14 11 11, 14 Unresponsive 13 13 Salivation 1, 11, 13 Br eathing Gasping 1 Shallow 1, 11 1, 11 - 13 1, 12 - 13 Coat Piloerection 1 1, 12 1, 12, 14 14 Eyelids (Left) Closed 1 Par tially Closed 11, 13 - 14 11, 13 12 - 13 14 Eyelids (Right) Closed 1 Par tially Closed 11, 13 - 14 11, 13 12 - 13 14 Eye (Left) Lacrimation 1 Eye (Right) Lacrimation 1 Posture Hun ched 1 1 1 Staining Post Salivation Staining 1491Huntingdon Life SciencesWAG0013

APPENDIX 2 - continued Request ID: 259116 Signs associated with dosing - individual observations Gr oup : 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 4721 8901 19098 Days Group /Sex Animal Number Sign Additional observation As late as

possible in the working day 2F 17 Behaviour

Ch in Rubbing Unresponsive Salivation Br eathing Gasping Shallow Coat Piloerection Eyelids (Left) Closed Par tially Closed Eyelids (Ri ght) Closed Par tially Closed Eye (Left) Lacrimation Eye (Right) Lacrimation Posture Hun ched Staining Post Salivation Staining92Huntingdon Life SciencesWAG0013

APPENDIX 2 - continued Request ID: 259116 S igns 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 4721 8901 19098 Days Group /Sex Animal Number Sign Pre - dose 1 to 2 hours during exposure 3 to 4 hours

during exposure 5 to 6 hours

during exposure 1 to 2 hours after

completion of dosing 2F 18 Behaviour

Ch in Rubbing 8, 11 - 14 8, 11 12 14 Irritable 13 Unresp onsive 13 Salivation 1, 11 - 13 12 - 13 12 Br eathing Gasping 1 Shallow 1, 11 1, 11 - 12 1, 12 Coat Piloerection 1, 4, 14 1, 7, 12 - 14 1, 4, 7, 11 - 14 11, 14 Eyelids (Left) Closed 1 Par tially Closed 11, 13 - 14 11, 13 - 14 13 - 14 Eyelids (Right) Closed 1 Par tially Closed 11, 13 - 14 11, 13 - 14 13 - 14 Eye (Left) Lacrimation 1 Eye (Right) Lacrimation 1 Posture Hun ched 1 1 193Huntingdon Life SciencesWAG0013

APPENDIX 2 - continued Request ID: 259116 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 4721 8901 19098 Days Group /Sex Animal Number Sign Additional observation As late as

possible in the working day 2F 18 Behaviour

Ch in Rubbing Irritable Unresponsive Salivation Br eathing Gasping Shallow Coat Piloerection Eyelids (Left) Closed Par tially Closed Eyelids (Right) Closed Par tially Closed Eye (Left) Lacrimation Eye (Right) Lacrimation Posture Hun ched94Huntingdon Life SciencesWAG0013

APPENDIX 2 - continued Request ID: 259116 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 - bromo - 3,3,3 -

trifluoropropene Exposure (ppm) : 0 4721 8901 19098 Days Group /Sex Animal Number Sign Pre - dose 1 to 2 hours during exposure 3 to 4 hours

during exposure 5 to 6 hours

during exposure 1 to 2 hours after

completion of dosing 3 F 19 Behaviour

Ch in Rubbing 4 - 8, 11 - 13 5 6, 8 Irritable 4 Underactive 8, 12 - 14 8, 12 - 14 12 - 14 7, 11 Unresponsive 6 - 7 6 - 7 Salivation 1, 6 Br eathing Gasping 1 Irregular 1 Shallow 1, 4 - 5, 7, 11 1, 4 - 7, 11 1, 5 - 7, 11 Slow 1 Coat Piloerection 7 - 8, 11 - 14 1, 5, 7 - 8, 1, 5 - 8, 11 - 14 4, 7 - 8, 11 11 - 14 Eyelids (Left) Par tially Closed 1, 4 - 8, 12 - 14 1, 5 - 8, 12 - 14 1, 5 - 8, 12 - 14 7 Eyelids (Right) Par tially Closed 1, 4 - 8, 12 - 14 1, 5 - 8, 12 - 14 1, 5 - 8, 12 - 14 7 Eye (Left) Lacrimation 1 Eye (Right) Lacrimation 1 Muscle Reaction Un steady 195Huntingdon Life SciencesWAG0013

APPENDIX 2 - contin ued Request ID: 259116 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 4721 8901 19098 Days Group /Sex Animal Number Sign Additional observation As late as

possible in the working day 3F 19 Behaviour

Ch in Rubbing Irritable 4 4 Underactive Unresponsive Salivation Br eathing Gasping Irregular Shallow Slow Coat Piloerection Eyelids (Left) Par tially Closed Eyelids (Right) Par tially Closed Eye (Left) Lacrimation Eye (Right) Lacrimation Muscle Reaction Un steady96Huntingdon Life SciencesWAG0013

APPENDIX 2 - continued Request ID: 259116 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 4721 8901 19098 Days Group /Sex Animal Number Sign Pre - dose 1 to 2 hours during exposure 3 to 4 hours

during exposure 5 to 6 hours

during exposure 1 to 2 hours after

completion of dosing 3F 20 Behaviour

Ch in Rubbing 6 - 8, 11 6 6, 13 Irritable 4 Underactive 8, 12 - 14 8, 12 - 14 12 - 14 7, 11 - 12 Unresponsive 6 6 - 7 6 - 7 Salivation 1 Br eathing Gasping 1 Irregular 1 Sh allow 1, 4 - 5, 7, 11 1, 4 - 7, 11 - 12 1, 5 - 7, 11 - 12 Slow 1 Coat Piloerection 8 5, 8, 12 - 13 5, 8, 11 - 13 Eyelids (Left) Par tially Closed 1, 7 - 8, 12 - 14 1, 7 - 8, 12 - 14 1, 5, 7 - 8, 7 12 - 14 Eyelids (Right) Par tially Closed 1, 7 - 8, 12 - 14 1, 7 - 8, 12 - 14 1, 5, 7 - 8, 7 12 - 14 Eye (Left) Lacrimation 1 13 Eye (Right) Lacrimation 1 1397Huntingdon Life SciencesWAG0013

APPENDIX 2 - continued Request ID: 259116 Signs associated with dosin g - 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 4721 8901 19098 Days Group /Sex Animal Number Sign Additional observati on As late as

possible in the working day 3F 20 Behaviour

Ch in Rubbing Irritable Underactive Unresponsive Salivation Br eathing Gasping Irregular Shallow Slow Coat Piloerection Eyelids (Left) Par tially Closed Eyelids (Right) Par tially Closed Eye (Left) Lacrimation Eye (Right) Lacrimation98Huntingdon Life SciencesWAG0013

APPENDIX 2 - continued Request ID: 259 116 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 4721 8901 19098 Days Group /Sex An imal Number Sign Pre - dose 1 to 2 hours during exposure 3 to 4 hours

during exposure 5 to 6 hours

during exposure 1 to 2 hours after

completion of dosing 3F 20 Muscle Reaction

Un steady 1 Staining Post Salivation Staining 199Huntingdon Life SciencesWAG0013

APPENDIX 2 - continued Request ID: 259116 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 4721 8901 19098 Days Group /Sex Animal Number Sign Additional observation As late as

possible in the working day 3F 20 Muscle Reaction

Un steady Staining Post Salivation Staining100Huntingdon Life SciencesWAG0013

APPENDIX 2 - c ontinued Request ID: 259116 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 4721 8901 19098 Days Group /Sex Animal Number Sign Pre - dose 1 to 2 hours during exposure 3 to 4 hours

during exposure 5 to 6 hours

during exposure 1 to 2 hours after

completion of dosing 3F 21 Behaviour

Ch in Rubbing 6 - 8, 11 - 12 6, 13 Irritable 4 Underactive 8, 12, 14 8, 12 - 14 12 - 14 11 Unresponsive 6 - 7 6 - 7 Salivation 1 Br eathing Gasping 1 Irregular 1 Shallow 1, 4 - 5, 7, 11 1, 4 - 7, 11 - 12 1, 5 - 7, 11 - 12 Slow 1 Coat Piloerection 7 - 8, 11 - 14 1, 5, 7 - 8, 1, 5 - 8, 11 - 14 7 - 8 11 - 14 Eyelids (Left) Closed 1 Par tially Closed 1, 5 - 8, 11 - 14 1, 5 - 8, 11 - 14 1, 5 - 8, 12 - 14 8 Eyelids (Right) Closed 1 Par tially Closed 1, 5 - 8, 11 - 14 1, 5 - 8, 11 - 14 1, 5 - 8, 12 - 14 8 Eye (Left) Lacrimation 1 Eye (Right) Lacrimation 1101Huntingdon Life SciencesWAG0013

APPENDIX 2 - continued Request ID: 259116 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 4721 8901 19098 Days Group /Sex Animal Number Sign Additional observation As late as

possible in t

he working day 3F 21 Behaviour

Ch in Rubbing Irritable Underactive Unresponsive Salivation Br eathing Gasping Irregular Shallow Slow Coat Piloerection Eyelids (Left) Closed Par tially Closed Eyelids (Right) Closed Par tially Closed Eye (Left) Lacrimation Eye (Right) Lacrimation102Huntingdon Life SciencesWAG0013

APPENDIX 2 - continued Request ID: 2591 16 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 4721 8901 19098 Days Group /Sex Ani mal Number Sign Pre - dose 1 to 2 hours during exposure 3 to 4 hours

during exposure 5 to 6 hours

during exposure 1 to 2 hours after

completion of dosing 3F 21 Muscle Reaction

Un steady 1103Huntingdon Life SciencesWAG0013

APPENDIX 2 - continued Request ID : 259116 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 4721 8901 19098 Days Group /Sex Animal Number Sign Additional observation As late as

possible in the working day 3F 21 Muscle Reaction

Un steady104Huntingdon Life SciencesWAG0013

APPENDIX 2 - continued Request ID: 259116 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 4721 8901 19098 Days Group /Sex Animal Number Sign Pre - dose 1 to 2 hours during exposure 3 to 4 hours

during expos ure 5 to 6 hours

during exposure 1 to 2 hours after

completion of dosing 4F 22 Behaviour

Irritable 4 Underactive 1, 5, 7 1, 5, 7 4 - 5 Unresponsive 1, 4 1, 4 Salivation 1 1 Br eathing Gasping 1 Shallow 1, 4 - 7 1, 4 - 7 1, 4 - 7 4 Slow 1 1 Coat Piloerection 1, 4 - 7 1, 4 - 7 1, 4 - 7 1, 4, 6 Eyelids (Left) Par tially Closed 1, 4 - 7 1, 4 - 7 1, 4 - 7 Eyelids (Right) Par tially Closed 1, 4 - 7 1, 4 - 7 1, 4 - 7 Eye (Left) Lacrimation 1 1 Eye (Right) Lacrimation 1 1 Muscle Reaction Un steady 1, 4 - 7 1, 5 - 7 1, 6 6105Huntingdon Life SciencesWAG0013

APPENDIX 2 - continued Request ID: 259116 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 4721 8901 19098 Days Group /Sex Animal Number Sign Additional observation As late as

possible in the working day 4F 22 Behaviour

Irritable Underactive Unresponsive Salivation Br eathing Gasping Shallow Slow Coat Piloerection Eyelids (Left) Par tially Closed Eyelids (Right) Par tially Closed Eye (Left) Lacrimation Eye (Right) Lacrimation Muscle Reaction Un steady106Huntingdon Life SciencesWAG0013

APPENDIX 2 - continued Request ID: 259116 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 4721 8901 19098 Days Group /Sex Animal Number Sign Pr e- dose 1 to 2 hours during exposure 3 to 4 hours

during exposure 5 to 6 hours

during exposure 1 to 2 hours after

completion of dosing 4F 23 Behaviour

Irritable 4 Underactive 1, 5 - 7 1, 5 - 7 5 - 7 Unresponsive 1, 4 1, 4 Salivation 1 1 Br eathing Shallow 1, 4 - 7 1, 4 - 7 1, 4 - 7 4 Slow 1 1 Coat Piloerection 1, 4 - 7 1, 4 - 7 1, 4 - 7 1, 4, 6 Eyelids (Left) Par tially Closed 1, 4 - 7 1, 4 - 7 1, 4 - 7 5 Eyelids (Right ) Par tially Closed 1, 4 - 7 1, 4 - 7 1, 4 - 7 5 Eye (Left) Lacrimation 1 1 Eye (Right) Lacrimation 1 1 Muscle Reaction Un steady 1, 4, 6 1, 6 1, 4, 6 6107Huntingdon Life SciencesWAG0013

APPENDIX 2 - continued Request ID: 259116 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 4721 8901 19098 Days Group /Sex An imal Number Sign Additional observation As late as

possible in the working day 4F 23 Behaviour

Irritable Underactive Unresponsive Salivation Br eathing Shallow Slow Coat Piloere ction Eyelids (Left) Par tially Closed Eyelids (Right) Par tially Closed Eye (Left) Lacrimation Eye (Right) Lacrimation Muscle Reaction Un steady108Huntingdon Life SciencesWAG0013

APPENDIX 2 - continued Request I D: 259116 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 4721 8901 19098 Days Group / Sex Animal Number Sign Pre - dose 1 to 2 hours during exposure 3 to 4 hours

during exposure 5 to 6 hours

during exposure 1 to 2 hours after

completion of dosing 4F 24 Behaviour

Irritable 4 Underactive 1, 5 - 7 1, 5 - 7 5 - 7 5 - 6 Unresponsive 4 4 Salivation 1 1 Br eathing Shallow 1, 4, 6 1, 4, 6 1, 4, 6 4 Slow 1 1 Coat Piloerection 1, 5 - 7 1, 5 - 7 1, 4 - 7 1, 4, 6 Eyelids (Left) Par tially Closed 1, 4 - 7 1, 4 - 7 1 , 4 - 7 4 - 5 Eyelids (Right) Par tially Closed 1, 4 - 7 1, 4 - 7 1, 4 - 7 4 - 5 Eye (Left) Lacrimation 1 1 Eye (Right) Lacrimation 1 1 Muscle Reaction Un steady 1, 4, 6 1, 6 1, 6 6109Huntingdon Life SciencesWAG0013

APPENDIX 2 - continued Request ID: 259116 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 4721 8901 1909 8 Days Group /Sex Animal Number Sign Additional observation As late as

possible in the working day 4F 24 Behaviour

Irritable Underactive Unresponsive Salivation Br eathing Shallow Slow Coat Piloerection Eyelids (Left) Par tially Closed Eyelids (Right) Par tially Closed Eye (Left) Lacrimation Eye (Right) Lacrimation Muscle Reaction Un steady110Huntingdon Life SciencesWAG0013

APPEND IX 3 Sensory reactivity observations and grip strength - individual findings on Day 10 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) : 0 472 1 8901 19098 Group/sex: 1M 2M 3M 4M Animal number: 1 2 3 4 5 6 7 8 9 10 11 12 Parameter (Grade range) Approach response (1 - 3) 2 2 2 2 2 2 1 2 2 1 1 1 Touch response (1 - 3) 2 2 2 2 2 2 1 2 1 1 2 2 Auditory startle reflex (1 - 4) 2 3 3 3 3 3 2 3 3 3 1 1 Tail pinch response (1 - 4) 3 3 3 3 3 3 3 3 3 3 3 3 Forelimb grip strength (kg) Trial 1 1.01 1.14 0.88 1.36 1.22 1.07 1.52 0.90 0.98 0.65 0.95 1.18 Trial 2 0.85 0.98 0.71 1.21 1.15 0.98 1.18 0 .86 0.93 0.68 0.85 1.07 Trial 3 1.04 0.89 0.94 1.18 0.91 1.17 1.34 0.91 0.90 0.82 1.12 1.21 Mean 0.97 1.00 0.84 1.25 1.09 1.07 1.35 0.89 0.94 0.72 0.97 1.15 Hindlimb grip strength (kg) Trial 1 0.51 0.61 0.62 0.51 0.58 0.50 0.56 0.49 0.60 0.39 0.41 0.58 Trial 2 0.58 0.59 0.58 0.44 0.55 0.60 0.57 0.50 0.51 0.57 0.45 0.62 Trial 3 0.65 0.53 0.56 0.48 0.59 0.42 0.56 0.50 0.52 0.51 0.46 0.45 Mean 0.58 0.58 0.59 0.48 0.57 0.51 0.56 0.50 0.54 0.49 0.44 0.55 Additional observations - - - - - - - - - - - -111Huntingdon Life SciencesWAG0013

APPENDIX 3 - continued Sensory reactivity observations and grip strength - individual findings on Day 10 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 4721 8901 19098 Group/sex: 1F 2F 3F 4F Animal number: 13 14 15 16 17 18 19 20 21 22 23 24 Parameter (Grade range) Approach response (1 - 3) 2 2 2 2 2 2 2 2 1 1 1 2 Touch response (1 - 3) 2 2 2 2 2 2 2 1 1 1 1 2 Auditory startle reflex (1 - 4) 3 3 3 3 3 3 3 3 3 3 3 2 Tail pinch response (1 - 4) 4 3 3 3 3 3 3 3 3 2 3 3 Forelimb grip strength (kg) Trial 1 0.94 0.88 0.89 1.08 0.80 1. 04 0.97 0.82 0.72 0.94 1.10 1.21 Trial 2 0.99 0.96 0.96 1.03 0.76 0.95 0.76 0.72 0.78 0.86 1.12 1.04 Trial 3 0.94 0.84 0.85 0.84 0.89 1.03 0.94 0.89 0.73 0.70 1.07 1.34 Mean 0.96 0.89 0.90 0.98 0.82 1.01 0.89 0.81 0.74 0.83 1.10 1.20 Hindlimb grip strength (kg) Trial 1 0.62 0.45 0.37 0.38 0.31 0.47 0.44 0.48 0.44 0.47 0.39 0.50 Trial 2 0.58 0.47 0.44 0.40 0.32 0.45 0.43 0.42 0.62 0.42 0.32 0.54 Trial 3 0.62 0.48 0.36 0.40 0.32 0.42 0.46 0.50 0.42 0.44 0.34 0.66 Mean 0 .61 0.47 0.39 0.39 0.32 0.45 0.44 0.47 0.49 0.44 0.35 0.57 Additional observations - - - - - - - - - - - -112Huntingdon Life SciencesWAG0013

APPENDIX 4 Motor activity - individual scores (beam breaks) on Day 10 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 4721 8901 19098 Group /Sex Animal Beam

level Time (minutes) number 6 12 18 24 30 36 42 48 54 60 Total 1M 1 High 145 83 37 28 23 37 26 20 0 5 404

2 118 105 57 14 35 0 0 12 1 0 342 3 153 105 151 103 94 87 5 71 114 3 886 1 Low 261 160 97 102 197 89 41 23 21 18 1009 2 314 266 232 119 149 10 0 41 5 0 1136 3 212 127 114 116 89 64 22 45 11 2 26 927113Huntingdon Life SciencesWAG0013

APPENDIX 4 - continued Motor activity - individual scores (beam breaks) on Day 10 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 -

trifluoropropen e Exposure (ppm) : 0 4721 8901 19098 Group /Sex Animal Beam

level Time (minutes) number 6 12 18 24 30 36 42 48 54 60 Total 2M 4 High 129 88 64 87 15 38 34 8 21 32 516

5 115 54 43 32 22 18 16 13 25 20 358 6 114 86 40 11 29 9 17 14 24 10 354 4 Low 192 97 111 106 82 68 25 23 64 24 792 5 251 130 108 86 103 42 70 44 90 48 972 6 381 194 154 58 100 41 76 46 99 38 1187114Huntingdon Life SciencesWAG0013

APPENDIX 4 - continued Motor activity - individual scores (beam breaks) o n D ay 10 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 4721 8901 19098 Group /Sex Animal Beam

level Time (minutes) number 6 12 18 24 30 36 42 48 54 60 Total 3M 7 High 89 62 33 39 36 4 15 23 9 31 341

8 90 66 64 21 5 2 0 0 0 0 248 9 70 14 3 0 0 0 18 7 12 0 124 7 Low 230 62 49 31 44 6 34 27 15 48 546 8 226 156 152 72 9 7 0 0 0 0 622 9 137 75 40 25 10 14 63 18 51 0 433115Huntingdon Life SciencesWAG0013

APPENDIX 4 - continued Motor activity - individual scores (beam breaks) on Day 10 of treatment Group : 1 2 3 4 Compound : Control 2 - br omo - 3,3,3 - trifluoropropene 2 - br omo - 3,3,3 -

trifluoropropene 2 - bromo - 3,3,3 -

trifluoropropene Exposure (ppm) : 0 4721 8901 19098 Group /Sex Animal Beam

level Time (minutes) number 6 12 18 24 30 36 42 48 54 60 Total 4M 10 High 48 10 1 0 0 0 1 0 0 0 60

11 89 4 1 1 0 0 0 0 0 0 95 12 73 2 0 16 0 0 0 0 0 0 8 117 10 Low 157 29 2 0 0 0 2 0 0 0 190 11 190 21 8 4 0 0 0 0 0 0 223 12 191 140 50 0 0 0 3 13 6 69 472116Huntingdon Life SciencesWAG0013

APPENDIX 4 - continued Motor activity - individual scores (beam breaks) on Day 10 of treatme nt 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 4721 8901 19098 Group /Sex Animal Beam

level Time (minutes) number 6 12 18 24 30 36 42 4 8 54 60 Total 1F 13 High 145 98 67 67 50 77 55 0 0 23 582

14 203 151 84 90 61 53 14 58 45 21 780 15 176 83 173 114 46 89 14 65 21 65 846 13 Low 224 160 92 148 124 173 141 5 0 70 1137 14 409 361 298 180 154 164 104 196 203 76 2145 15 296 242 219 172 187 202 79 100 107 126 1730117Huntingdon Life SciencesWAG0013

APPENDIX 4 - continued Motor activity - individual scores (beam breaks) on Day 10 of treatment Group : 1 2 3 4 Compound : Control 2 - br omo - 3,3,3 - trifluoropropene 2 - brom o - 3,3,3 -

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

trifluoropropene Exposure (ppm) : 0 4721 8901 19098 Group /Sex Animal Beam

level Time (minutes) number 6 12 18 24 30 36 42 48 54 60 Total 2F 16 High 108 116 47 26 6 0 0 0 40 55 398

17 109 66 42 37 33 19 17 8 34 28 393 18 87 39 44 4 34 29 1 0 0 0 238 16 Low 286 173 127 61 73 2 14 15 82 66 899 17 210 145 103 87 93 48 87 23 115 85 996 18 221 214 109 126 156 157 14 0 0 1 998118Huntingdon Life SciencesWAG0013

APPENDIX 4 - continue d Motor activity - individual scores (beam breaks) on Day 10 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 4721 8901 19098 Group /Sex Animal Beam

level Time (minutes) number 6 12 18 24 30 36 42 48 54 60 Total 3F 19 High 184 108 18 0 0 0 0 0 14 0 324

20 127 29 11 0 0 0 0 0 12 13 192 21 104 23 18 0 0 0 0 0 0 0 145 19 Low 211 147 33 2 0 1 0 9 5 0 408 20 190 90 10 0 0 0 0 0 22 75 387 21 293 159 107 0 0 0 0 0 0 1 560119Huntingdon Life SciencesWAG0013

APPENDIX 4 - continued Motor activity - individual scores (beam breaks) on Day 10 of treatment Group : 1 2 3 4 Compound : Control 2 - br omo - 3,3,3 - trifl uoropropene 2 - br omo - 3,3,3 -

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

trifluoropropene Exposure (ppm) : 0 4721 8901 19098 Group /Sex Animal Beam

level Time (minutes) number 6 12 18 24 30 36 42 48 54 60 Total 4F 22 High 187 72 24 0 11 29 0 0 0 0 323

23 64 7 2 0 0 0 0 0 0 0 73 24 114 0 15 0 0 0 0 0 0 0 129 22 Low 296 208 120 0 59 161 10 0 0 0 854 23 123 8 0 0 0 0 0 0 0 0 131 24 239 13 59 12 0 0 0 0 0 0 323120Huntingdon Life SciencesWAG0013

APPENDIX 5 Request ID: 257057 Bodywei ght - 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 4721 8901 19098 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 1 419 426 429 434 432 434 432 437 437 444 442 445

2 452 453 455 457 458 460 463 465 468 469 472 473 3 457 454 459 459 461 466 469 468 473 471 482 482 2 M 4 369 375 380 384 393 396 384 385 393 385 387 391

5 395 400 403 407 417 424 409 413 416 411 410 408 6 410 419 430 434 445 451 425 437 45 1 446 445 446 3 M 7 474 477 483 486 492 501 485 485 501 487 477 479

8 420 423 429 433 434 438 424 428 439 430 419 420 9 438 445 451 450 458 468 445 451 458 452 446 447 4 M 10 467 465 468 468 473 471 442 449 461 449 449 441

11 429 431 436 432 437 427 423 413 430 417 394 377 12 452 454 458 459 459 461 449 444 465 448 434 432121Huntingdon Life SciencesWAG0013

APPENDIX 5 - continued Request ID: 257057 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 4721 8901 19098 Group Animal Day Day Day Day Day Day Day Day /Sex Number 8 9 10 11 12 13 14 15 1 M 1 443 439 448 450 448 451 448 454

2 477 479 483 488 487 489 490 490 3 481 484 485 489 489 491 492 497 2 M 4 401 395 415 420 416 410 404 407

5 419 420 431 433 431 433 425 424 6 455 452 468 478 472 468 457 459 3 M 7 477 473 502 513 513 502 491 502

8 422 426 440 454 4 46 450 444 444 9 444 448 469 482 469 467 454 463 4 M 10 433 431 450 458 449 439 431 436

11 362 349 355 386 383 379 369 369 12 424 420 445 458 449 438 429 426122Huntingdon Life SciencesWAG0013

APPENDIX 5 - continued Request ID: 257057 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 4721 8901 19098 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 13 314 309 313 305 316 316 316 305 320 323 325 309

14 302 297 293 284 298 299 305 297 312 314 318 304 15 294 289 292 286 292 297 304 292 304 308 313 300 2 F 16 242 244 246 250 247 249 246 252 255 252 258 243

17 230 237 240 239 236 247 233 233 234 229 23 8 238 18 248 249 243 253 256 254 247 250 257 252 253 260 3 F 19 255 255 248 260 263 267 254 271 271 273 265 268

20 266 272 271 276 272 283 272 276 280 277 276 283 21 253 251 253 257 259 252 257 254 262 256 261 263 4 F 22 252 260 264 260 256 266 259 261 272 262 259 271

23 272 281 285 278 275 280 263 270 288 273 263 265 24 263 255 264 267 267 260 259 258 269 259 261 262123Huntingdon Life SciencesWAG0013

APPENDIX 5 - continued Request ID: 257057 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 -

t rifluoropropene Exposure (ppm) : 0 4721 8901 19098 Group Animal Day Day Day Day Day Day Day Day /Sex Number 8 9 10 11 12 13 14 15 1 F 13 324 325 321 309 320 326 323 318

14 327 320 325 309 324 329 319 324 15 311 309 306 300 301 315 304 300 2 F 16 252 256 279 281 264 267 263 265

17 238 241 247 256 256 253 247 251 18 262 262 269 270 269 275 273 274 3 F 19 268 267 285 288 274 280 271 276

20 288 290 305 307 294 301 299 301 21 260 263 274 280 276 279 271 275 4 F 22 265 270 281 277 272 269 267 267

23 241 245 271 285 277 258 242 258 24 258 260 276 285 283 258 270 271124Huntingdon Life SciencesWAG0013

APPENDIX 6 Request ID: 257049 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 - bromo - 3,3,3 -

trifluoropropene Exposure (ppm) : 0 4721 8901 19098 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 31 30 31 31 28 28 29 30 31 30 29 30 2M 2 36 35 33 35 35 17 31 28 20 24 24 29 3M 3 34 33 35 35 35 17 27 29 22 21 22 21 4M 4 32 32 30 33 26 15 21 27 20 18 13 12125Huntingdon Life SciencesWAG0013

APPENDIX 6 - continued Request ID: 257049 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 - br omo - 3,3,3 -

trifluoropropene Exposure (ppm) : 0 4721 8901 19098 Group Cage Day Day Day Day Day Day Day /Sex Number 8 9 10 11 12 13 14 1M 1 30 31 32 28 31 30 30 2M 2 26 36 33 25 27 23 23 3M 3 24 37 36 31 30 30 27 4M 4 14 21 31 24 24 23 19126Huntingdon Life SciencesWAG0013

APPENDIX 6 - continued Request ID: 257049 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 - br omo - 3,3,3 -

trifluoropropene Exposure (ppm) : 0 4721 8901 19098 Group Cage Day Day Day Day Day Day Day Day Day Day Day Day /Sex Number - 5 - 4 - 3 - 2 - 1 1 2 3 4 5 6 7 1F 5 25 26 20 29 21 25 20 29 27 27 19 30 2F 6 24 20 22 21 21 13 18 19 14 18 16 16 3F 7 30 19 26 26 27 12 21 24 18 20 19 17 4F 8 24 26 24 21 20 8 19 26 14 13 17 11127Huntingdon Life SciencesWAG0013

APPENDIX 6 - continued Request ID: 257049 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 - br omo - 3,3,3 -

trifluoropropene Exposure (ppm) : 0 4721 8901 19098 Group Cage Day Day Day Day Day Day Day /Sex Number 8 9 10 11 12 13 14 1F 5 27 26 20 24 28 26 21 2F 6 19 25 25 18 22 15 16 3F 7 22 28 32 22 23 20 20 4F 8 13 24 27 17 17 11 14128Huntingdon Life SciencesWAG0013

APPEN DIX 7 Request ID: 257052 Organ weights - individual absolute 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 4721 8901 19098 Group /Sex An imal Number Terminal Bodyweight Br ain Heart Kidneys Liver Lungs + Br onchi Spleen 1M 1 455 1.98 1.697 2.82 17.47 1.588 0.794

2 493 2.07 1.531 3.77 18.75 1.506 0.804 3 500 2.07 1.841 2.87 15.68 1.825 0.770 2M 4 411 1.98 1.758 3.20 1 4.48 1.667 0.738

5 424 2.03 1.269 3.43 14.41 1.522 0.563 6 460 2.00 1.342 3.71 18.26 1.713 0.687 3M 7 501 2.15 1.489 3.54 21.68 1.976 1.079

8 446 1.96 1.937 2.84 17.35 1.435 0.817 9 462 2.10 1.478 3.24 20.21 1.563 0.779 4M 10 437 2.19 1.770 3.25 17.65 1.987 0.669

11 370 1.97 1.373 2.34 15.29 1.731 0.573 12 427 2.11 1.563 3.32 15.51 1.472 0.667129Huntingdon Life SciencesWAG0013

APPENDIX 7 - continued Request ID: 257052 Organ weights - individual absolute 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 4721 8901 19098 Group /Sex Animal Number Terminal Bodyweight Br ain Heart Kidneys Liver Lungs + Br onchi Spleen 1F 1 3 319 1.95 1.297 1.85 10.00 1.480 0.587 14 323 1.99 1.437 1.98 11.00 1.308 0.510 15 301 1.96 1.243 1.92 9.35 1.316 0.593 2F 16 267 1.93 1.051 2.03 10.71 1.295 0.544 17 252 1.68 0.835 1.96 8.71 1.095 0.572 18 274 1.91 0.895 1.69 8.47 1.18 1 0.414 3F 19 275 1.73 0.940 1.78 10.68 1.089 0.469 20 301 1.98 1.044 2.03 12.43 1.314 0.418 21 274 1.94 1.164 1.93 11.43 1.492 0.620 4F 22 267 1.84 1.055 1.67 10.10 1.318 0.505 23 257 1.86 1.043 1.77 9.82 1.449 0.500 24 271 1 .90 0.988 1.71 10.31 1.216 0.448130Huntingdon Life SciencesWAG0013

APPENDIX 8 Request ID: 264438 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 -

t rifluoropropene Exposure (ppm) : 0 4721 8901 19098 Animal Number: 1 Sex: Male Dose Group: 1 Phase: Treatment Sacrifice Status: Final phase sacrifice Date of Death: 09 - Dec - 11 Day of Death: 15 Week of Death: 3 Subgroup: 1 Terminal Bodyweight: 455.2 g PATHOLOGY OBSERVATIONS MACROPATHOLOGY HISTOPATHOLOGY ***ANIMAL HAS NO MACROSCOPIC FINDINGS RECORDED*** Lungs + Bronchi Alveolit is, Focal, Minimal Per ivascular Inflammatory Cell Infiltrate, Minimal Arterial Mural Mineralisation, Focal, Minimal Tracheal Bifurc. Inflammatory Cell Infiltrate in Lamina Propria, Minimal The following examined tissues had no gross observations and no findings microscopically: Larynx, Nasal turbinates, Trachea131Huntingdon Life SciencesWAG0013

APPENDIX 8 - continued Request ID: 264438 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 4721 8901 19098 Animal Number: 2 Sex: Male Dose Group: 1 Phase: Treatment Sacrifice Status: Final phase sacrifice Date of Death: 09 - Dec - 11 Day of Death: 15 Week of Death: 3 Subgroup: 1 Terminal Bodyweight: 493.4 g PATHOLOGY OBSERVATIONS MACROPATHOLOGY HISTOPATHOLOGY Lungs + Bronchi Alveolar Macrophages, Focal, Minimal Skin Hair loss, Forelimb(s) Tracheal Bifurc. Inflammatory Cell Infiltrate in Lamina Propria, Minimal Trachea Inflammatory Cell Infiltrate - Lamina Propria, Focal, Minimal The following examined tissues had no gross observations and no findings microscopically: Larynx, Nasal turbinates132Huntingdon Life SciencesWAG0013

APPENDIX 8 - continued Request ID: 264438 Macropathology and histopathology - individual findings Gr oup : 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 4721 8901 19098 Animal Number: 3 Sex: Male Dose Group: 1 Phase: Treatment Sacrifice Status: Final phase sacrifice Date of Death: 09 - Dec - 11 Day of Death: 15 Week of Death: 3 Subgroup: 1 Terminal Bodyweight: 499.7 g PATHOLOGY OBSERVATIONS MACROPATHOLOGY HISTOPATHOLOGY Lungs + Bronchi Per ivascular Inflammatory Cell Infiltrate, Minimal Arterial Mural Mineralisation, Focal, Minimal Skin Hair loss, Forelimb(s) The following examined tissues had no gross obs ervations and no findings microscopically: Larynx, Nasal turbinates, Trachea, Tracheal Bifurc.133Huntingdon Life SciencesWAG0013

APPENDIX 8 - continued Request ID: 264438 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 4721 8901 19098 Animal Number: 4 Sex: Male Dose Group: 2 Phase: Treatment Sacrifice Status: Final phase sacrifice Date of Death: 25 - Nov - 11 Day of Death: 15 Week of Death: 3 Subgroup: 1 Terminal Bodyweight: 410.7 g PATHOLOGY OBSERVATIONS MACROPATHOLOGY HISTOPATHOLOGY Lary nx Epithelial Keratinisation - Ventrolateral, Minimal Epithelial Hyperplasia - Ar yt enoid, Minimal134Huntingdon Life SciencesWAG0013

APPENDIX 8 - continued Request ID: 264438 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 4721 8901 19098 Animal Number: 4 Sex: Male Dose Group: 2 Phase: Treatment Sacrifice Status: Final phase sacrifice Date of Death: 25 - Nov - 11 Day of Death: 15 Week of Death: 3 Subgroup: 1 Terminal Bodyweight: 410.7 g PATHOLOGY OBSERVATIONS MACROPATHOLOGY HISTOPATHOLOGY continued from previ ous page... Nasal turbinates Olfactory Epithelium - Erosion, Focal, Minimal Olfactory Epithelium - Degeneration/Atrophy/Disorganisation, Multifocal, Minimal Olfactory Epithelium - Squamous Metaplasia, Focal, Minimal Olfactory Epithelium - Respiratory Metaplasia With or Without Associated Inflammato

ry Cell Infiltrate, Focal, Minimal Olfactory Epithelium - Fibrosis With or Without Associated Cartilage Formation in Dorsal Lamina Propria, Focal, Minimal Olfactory Epithelium - Bowman's Gland Atrophy in Dorsal Lamina Propria, Slight Olfactory Nerve Fibres - Atr ophy, Slight Respiratory Epithelium - Disorganisation/Degeneration/ Regeneration, Focal, Minimal Debris a nd Inflammatory Cells in Airway, Multifocal, Minimal135Huntingdon Life SciencesWAG0013

APPENDIX 8 - continued Request ID: 264438 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 4721 8901 19098 Animal Number: 4 Sex: Male Dose Group: 2 Phase: Treatment Sacrifice Status: Final phase sacrifice Date of Death: 25 - Nov - 11 Day of Death: 15 Week of Death: 3 Subgroup: 1 Terminal Bo dyweight: 410.7 g PATHOLOGY OBSERVATIONS MACROPATHOLOGY HISTOPATHOLOGY continued from previous page... Spleen Capsule thickened, Ant erior pole, an area, 4x4mm. The following examined tissues had no gross observations and no findings microscopically: Trachea, Tracheal Bifurc.136Huntingdon Life SciencesWAG0013

APPENDIX 8 - continued Request ID: 264438 Macropathology and histopathology - individua l 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 4721 8901 19098 Animal Number: 5 Sex: Male Dose Group: 2 Phase: Treatment Sacrifice Statu s: Final phase sacrifice Date of Death: 25 - Nov - 11 Day of Death: 15 Week of Death: 3 Subgroup: 1 Terminal Bodyweight: 424.3 g PATHOLOGY OBSERVATIONS MACROPATHOLOGY HISTOPATHOLOGY Lungs + Bronchi Pale area(s), Right, caudal lobe, Punctate, One, subpleural. Lary nx Submucosal Foreign Body Granulomata - Ventral, Focal, Minimal Squamous Metaplasia - Ar yt enoid, S light137Huntingdon Life SciencesWAG0013

APPENDIX 8 - continued Request ID: 264438 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 4721 8901 19098 Animal Number: 5 Sex: Male Dose Group: 2 Phase: Treatment Sacrifice Status: Final phase sacrifice Date of Death: 25 - Nov - 11 Day of Death: 15 Week of Death: 3 Subgroup: 1 Terminal Bodyweight: 424.3 g PATHOLOGY OBSERVATIONS MACRO PATHOLOGY HIST OPATHOLOGY continued from previous page... Nasal turbinates Olfactory Epithelium - Degeneration/Atrophy/Disorganisati on, Focal, Minimal Olfactory Epithelium - Squamous Metaplasia, Multifocal, Minimal Olfactory Epithelium - Fibrosis With or Without Associated Cartilage Formation in Dorsal Lamina Propria, Focal, Minimal Olfactory Epithelium - Bowman's Gland Atrophy in Dorsal Lamina Propria, Minimal Olfactory Nerve Fibres - Atr ophy, Slight Respiratory Epithelium - Goblet Cell Hyperplasia, Minimal Transitional Epithelium - Disorganisation/Regeneration, Minimal Debris and Inflammatory Cells in Airway, Focal, Minimal Spleen Capsule thickened, A few, areas, up to 7x5mm.138Huntingdon Life SciencesWAG0013

APPENDIX 8 - continued Request ID: 264438 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 4721 8901 19098 Animal Number: 5 Sex: Male Dose Group: 2 Phase: Treatment Sacrifice Status: Final phase sacrifice Date of Death: 25 - Nov - 11 Day of Death: 15 Week of Death: 3 Subgroup: 1 Terminal Bodyweight: 424.3 g PATHOLOGY OBSERVATIONS MACROPATHOLOGY HISTOP ATHOLOGY continued from previous page... The following examined tissues had no gross observations and no findings microscopically: Trachea, Tracheal Bifurc.139Huntingdon Life SciencesWAG0013

APPENDIX 8 - continued Request ID: 264438 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 4721 8901 19098 Animal Number: 6 Sex: Male Dose Group: 2 Phase: Treatment Sacri fice Status: Final phase sacrifice Date of Death: 25 - Nov - 11 Day of Death: 15 Week of Death: 3 Subgroup: 1 Terminal Bodyweight: 459.5 g PATHOLOGY OBSERVATIONS MACROPATHOLOGY HISTOPATHOLOGY ***ANIMAL HAS NO MACROSCOPIC FINDINGS RECORDED*** Lary nx Epithelial Keratinisation - Ventrolateral, Minimal Squamous Metaplasia - Ar yt enoid, Minimal Nasal turbinates O lfactory Epithelium - Degeneration/Atrophy/Disorganisation, Focal, Minimal Olfactory Epithelium - Squamous Metaplasia, Focal, Minimal Olfactory Epithelium - Respiratory Metaplasia With or Without Associated Inflammato

ry Cell Infiltrat e, Focal, Minimal Olfactory Epithelium - Bowman's Gland Atrophy in Dorsal Lamina Propria, Minimal Olfactory Nerve Fibres - Atr ophy, Minimal Transitional Epithelium - Disorganisation/Regeneration, Minimal Nasolacrimal Ducts - Inflammation, Unilateral, Minimal140Huntingdon Life SciencesWAG0013

APPENDIX 8 - continued Request ID: 264438 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 - bromo - 3,3,3 -

trifluoropropene Exposure (ppm) : 0 4721 8901 19098 Animal Number: 6 Sex: Male Dose Group: 2 Phase: Treatment Sacrifice Status: Final phase sacrifice Date of Death: 25 - Nov - 11 Day of Death: 15 Week of Death: 3 Subgroup: 1 Terminal Bodyweight: 459.5 g PATHOLOGY OBSERVATIONS MACROPATHOLOGY HISTOPATHOLOGY continued from previous page... Tracheal Bifurc. Inflammatory Cel l Infiltrate in Lamina Propria, Minimal Trachea Inflammatory Cell Infiltrate - Lamina Propria, Focal, Minimal The following examined tissues had no gross observations and no findings microscopically:

-141Huntingdon Life SciencesWAG0013

APPENDIX 8 - con tinued Request ID: 264438 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 4721 8901 19098 Ani mal Number: 7 Sex: Male Dose Group: 3 Phase: Treatment Sacrifice Status: Final phase sacrifice Date of Death: 02 - Dec - 11 Day of Death: 15 Week of Death: 3 Subgroup: 1 Terminal Bodyweight: 500.7 g PATHOLOGY OBSERVATIONS MACROPATHOLOGY HISTOPATHOLOGY Lary nx Squamous Metaplasia - Ventral, Minimal Epithelial Keratinisation - Ventrolateral, Minimal Squamous Metap lasia - Ar yt enoid, Minimal142Huntingdon Life SciencesWAG0013

APPENDIX 8 - continued Request ID: 264438 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 4721 8901 19098 Animal Number: 7 Sex: Male Dose Group: 3 Phase: Treatment Sacrifice Status: Final phase sacrifice Date of Death: 02 - Dec - 11 Day of Death: 15 Week of Death: 3 Subgroup: 1 Terminal Bodyweight: 500.7 g PATHOLO GY OBSERVATIONS MACROPATHOLOGY HISTOPATHOLOGY continued from previous page... Nasal turbinates Olfactory Epithelium - Erosion, Foca l, Minimal Olfactory Epithelium - Degeneration/Atrophy/Disorganisation, Multifocal, Moderate Olfactory Epithelium - Squamous Metaplasia, Focal, Minimal Olfactory Epithelium - Respiratory Metaplasia With or Without Associate d Inflammato

ry Cell Infiltrate, Focal, Slight Olfactory Epithelium - Fibrosis With or Without Associated Cartilage Formation in Dorsal Lamina Propria, Moderate Olfactory Epithelium - Bowman's Gland Atrophy in Dorsal Lamina Propria, Sl ight Olfactory Nerve Fibres - Atr ophy, Slight Fusion of Dorsal Turbinate With Associated Epithelial Diverticula Formation, Multifocal, Moderate Respiratory Epithelium - Disorganisation/Degeneration/ Regeneration, Multifocal , Minimal Transitional Epithelium - Disorganisation/Regeneration, Minimal143Huntingdon Life SciencesWAG0013

APPENDIX 8 - continued Request ID: 264438 Macropathology and histopathology - individual findings Group : 1 2 3 4 Compound : Control 2 - br omo - 3,3,3 - trifluoroprop ene 2 - br omo - 3,3,3 -

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

trifluoropropene Exposure (ppm) : 0 4721 8901 19098 Animal Number: 7 Sex: Male Dose Group: 3 Phase: Treatment Sacrifice Status: Final phase sacrifice Date of Death: 02 - Dec - 11 Day of Death: 15 Week of Death: 3 Subgroup: 1 Terminal Bodyweight: 500.7 g PATHOLOGY OBSERVATIONS MACROPATHOLOGY HISTOPATHOLOGY continued from previous page... Skin Hair loss, Side(s) of face, diffuse. The following examined tissues had no gross observations and no findings microscopically: Trachea, Tracheal Bifurc.144Huntingdon Life SciencesWAG0013

APPENDIX 8 - continued Request ID: 264438 Macropathology and histopath ology - 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 4721 8901 19098 Animal Number: 8 Sex: Male Dose Group: 3 Phase: Treatment Sacrifice Status: Final phase sacrifice Date of Death: 02 - Dec - 11 Day of Death: 15 Week of Death: 3 Subgroup: 1 Terminal Bodyweight: 445.7 g PATHOLOGY OBSERVATIONS MACROPATHOLOGY HISTOPATHOLOGY ***ANIMAL HAS NO MACROSCOPIC FINDINGS RECORDED*** Lary nx Epithelial Keratinisation - Ventrolateral, Minimal Squamous Metaplasia - Ar yt enoid, Minimal145Huntingdon Life SciencesWAG0013

APPENDIX 8 - continue d Request ID: 264438 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 4721 8901 19098 Animal Number: 8 Sex: Male Dose Group: 3 Phase: Treatment Sacrifice Status: Final phase sacrifice Date of Death: 02 - Dec - 11 Day of Death: 15 Week of Death: 3 Subgroup: 1 Terminal Bodyweight: 445.7 g PATHOLOGY OBSERVATIONS MACROPATHOLOGY HISTOPATHOLOGY continued from previous page... Nasal turbinates Olfactory Epithelium - Degeneration/Atrophy/Disorganisation, Focal, Slight Olfa ctory Epithelium - Squamous Metaplasia, Focal, Minimal Olfactory Epithelium - Respiratory Metaplasia With or Without Associated Inflammato

ry Cell Infiltrate, Multifocal, Minimal Olfactory Epithelium - Fibrosis With or Without Associat ed Cartilage Formation in Dorsal Lamina Propria, Slight Olfactory Epithelium - Bowman's Gland Atrophy in Dorsal Lamina Propria, Slight Olfactory Nerve Fibres - Atr ophy, Slight Fusion of Dorsal Turbinate With Associated Epit helial Diverticula Formation, Focal, Moderate Respiratory Epithelium - Disorganisation/Degeneration/ Regeneration, Multifocal, Minimal Respiratory Epithelium - Goblet Cell Hyperplasia, Minimal Nasolacrimal Ducts - Inflammation, Minimal Debris and Inflammatory Cells in Airway, Multifocal, Minimal146Huntingdon Life SciencesWAG0013

APPENDIX 8 - continued Request ID: 264438 Macropathology and histopathology - individual findings Group : 1 2 3 4 Compound : Control 2 - br omo - 3,3,3 - trifluoroprop ene 2 - br omo - 3,3,3 -

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

trifluoropropene Exposure (ppm) : 0 4721 8901 19098 Animal Number: 8 Sex: Male Dose Group: 3 Phase: Treatment Sacrifice Status: Final phase sacrifice Date of Death: 02 - Dec - 11 Day of Death: 15 Week of Death: 3 Subgroup: 1 Terminal Bodyweight: 445.7 g PATHOLOGY OBSERVATIONS MACROPATHOLOGY HISTOPATHOLOGY continued from previous page... The following examined tissues had no gross observations and no findings microscopically: Trachea, Tracheal Bifurc.147Huntingdon Life SciencesWAG0013

APPENDIX 8 - continued Request ID: 264438 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 4721 8901 19098 Animal Number: 9 Sex: Male Dose Group: 3 Phase: Treatment Sacrifice Status: Final phase sacrifice Date of Death: 02 - Dec - 11 Day of Death: 15 Week of Death: 3 Subgroup: 1 Terminal Bodyweight: 461.8 g PATHOLOGY OBSERVATIONS MACROPATHOLOGY HISTOPATHOLOGY Lungs + Bronchi Dark area(s), Right, caudal lobe, One, subpleural, 1mm. Lary nx Submucosal Foreign Body Granulomata - Ventral, Focal, Minimal Squamous Metaplasia - Ar yt enoid, Minimal Epithelial Hyperplasia - Ar yt enoid, Minimal Inflammation, Multifocal, Minimal148Huntingdon Life SciencesWAG0013

APPENDIX 8 - continued Request ID: 264438 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 -

trifl uoropropene 2 - br omo - 3,3,3 -

trifluoropropene Exposure (ppm) : 0 4721 8901 19098 Animal Number: 9 Sex: Male Dose Group: 3 Phase: Treatment Sacrifice Status: Final phase sacrifice Date of Death: 02 - Dec - 11 Day of Death: 15 Week of Death: 3 Subgroup: 1 Terminal Bodyweight: 461.8 g PATHOLOGY OBSERVATIONS MACROPATHOLOGY HISTOPATHOLOGY continued from previous page... Nasal turbinates O lfactory Epithelium - Degeneration/Atrophy/Disorganisation, Multifocal, Minimal Olfactory Epithelium - Respiratory Metaplasia With or Without Associated Inflammato

ry Cell Infiltrate, Multifocal, Slight Olfactory Epithelium - Fibrosis With or Without Associated Cartilage Formation in Dorsal Lamina Propria, Moderate Olfactory Epithelium - Bowman's Gland Atrophy in Dorsal Lamina Propria, Slight Olfactory Nerve Fibres - Atr ophy, Slight Submucosal Foreign Bo dy Granulomata - Dor sal, Focal, Minimal Fusion of Dorsal Turbinate With Associated Epithelial Diverticula Formation, Focal, Moderate Respiratory Epithelium - Disorganisation/Degeneration/ Regeneration, Multifocal, Minimal Transitional Epithelium - Disorganisation/Regeneration, Minimal149Huntingdon Life SciencesWAG0013

APPENDIX 8 - continued Request ID: 264438 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 4721 8901 19098 Animal Number: 9 Sex: Male Dose Group: 3 Phase: Treatment Sacrifice Status: Final phase sacrifice Date of Death: 02 - Dec - 11 Day of Death: 15 Week of Death: 3 Subgroup: 1 T erminal Bodyweight: 461.8 g PATHOLOGY OBSERVATIONS MACROPATHOLOGY HISTOPATHOLOGY continued from previous page... Skin Scab(s), Side( s) of face, Left, One, 1mm. Hair loss, Side(s) of face, diffuse. The following examined tissues had no gross observations and no findings microscopically: Trachea, Tracheal Bifurc.150Huntingdon Life SciencesWAG0013

APPENDIX 8 - continued Request ID: 264438 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 4721 8901 19098 Animal Number: 10 Sex: Male Dose Group: 4 Phase: Treatment Sacrifice Status: Final phase sacrifice Date of Death: 09 - Dec - 11 Day of Death: 15 Week of Death: 3 Subgroup: 1 Terminal Bodyweight: 437.0 g PATHOLOGY OBSERVATIONS MACROPATHOLOGY HISTOPATHOLOGY Kidneys Pale area(s), Left, Cranial pole, One, subcapsular, 4x2mm, near to pelvic regio n. Lungs + Bronchi Arterial Mural Mineralisation, Focal, Minimal Lary nx Squamous Metaplasia - Ventral, Minimal Epithelial Keratinisation - Ventrolateral, Minimal Squamous Metaplasia - Ar yt enoid, Slight Inflammation, Multifocal, Minimal151Huntingdon Life SciencesWAG0013

APPENDIX 8 - continue d Request ID: 264438 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 4721 8901 19098 Animal Number: 10 Sex: Male Dose Group: 4 Phase: Treatment Sacrifice Status: Final phase sacrifice Date of Death: 09 - Dec - 11 Day of Death: 15 Week of Death: 3 Subgroup: 1 Terminal Bodyweight: 437.0 g PATHOLOGY OBSERVATIONS MACROPATHOLOGY HISTOPATHOLOGY continued from previous page... Nasal turbinates Olfactory Epithelium - Erosion, Focal, Minimal Olfactory Epithelium - Degeneration/Atrophy/Disorganisation, Focal, Moderate Olfactory Epithelium - Respiratory Metaplasia With or Without Associated Inflammato

ry Cell Infiltrate, Slight Olfactory Epithelium - Fibrosis With or Without Associated Cartilage Formation in Dorsal Lamina Propria, Slight Olfactory Epithelium - Bowman's Gland Atrophy in Dorsal Lamina Propria, Moderate Olfactory Nerve Fibres - Atr ophy, Slight Submucosal Foreign Body Granulomata - Dor sal, Focal, Minimal Fusion of Dorsal Turbinate With Associated Epithelial Diverticula Formation, Multifocal, Slight Respiratory Epithelium - Squamous Metaplasia, Focal, Minimal Respiratory Epithelium - Disorganisation/Degeneration/ Regeneration, Min imal Transitional Epithelium - Disorganisation/Regeneration, Slight Nasolacrimal Ducts - Inflammation, Bilateral, Slight152Huntingdon Life SciencesWAG0013

APPENDIX 8 - continued Request ID: 264438 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 4721 8901 19098 Animal Number: 10 Sex: Male Dose Group: 4 Phase: Treatment Sacrifice Status: Final phase sa cr ifice Date of Death: 09 - Dec - 11 Day of Death: 15 Week of Death: 3 Subgroup: 1 Terminal Bodyweight: 437.0 g PATHOLOGY OBSERVATIONS MACROPATHOLOGY HISTOPATHOLOGY continued from previous page... Stomach Forestomach roughened, Epithelial aspect Tracheal Bifurc. Inflammatory Cell Infiltrate in Lamina Propria, Minimal Trachea Epithelial Degeneration/Regener atio n, Diffuse, Slight Inflammatory Cell Infiltrate - Lamina Propria, Diffuse, Minimal Thy mus Small The following examined tissues had no gross observations and no findings microscopically:

-153Huntingdon Life SciencesWAG0013

APPENDIX 8 - con tinued Request ID: 264438 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 4721 8901 19098 Ani mal Number: 11 Sex: Male Dose Group: 4 Phase: Treatment Sacrifice Status: Final phase sacrifice Date of Death: 09 - Dec - 11 Day of Death: 15 Week of Death: 3 Subgroup: 1 Terminal Bodyweight: 370.2 g PATHOLOGY OBSERVATIONS MACROPATHOLOGY HISTOPATHOLOGY Lungs + Bronchi Lungs + Bronchi Pale area(s), Left, Punctate, One, subpleural. Alveolitis, Focal, Minimal Alveolar Macrophages, Multifocal, Minimal Arterial Mural Mineralisation, Focal, Minimal Lary nx Epithelial Hyperplasia - Ventral, Minimal Epithelial Keratinisation - Ventrolateral, Minimal154Huntingdon Life SciencesWAG0013

APPENDIX 8 - continued Request ID : 264438 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 4721 8901 19098 Animal Number: 11 S ex: Male Dose Group: 4 Phase: Treatment Sacrifice Status: Final phase sacrifice Date of Death: 09 - Dec - 11 Day of Death: 15 Week of Death: 3 Subgroup: 1 Terminal Bodyweight: 370.2 g PATHOLOGY OBSERVATIONS MACROPATHOLOGY HISTOPATHOLOGY continued from previous page... Nasal turbinates Olfactory Epithelium - Necrosis, Focal, Minimal Olfactory Epithelium - Degeneration/Atroph y/ Disorganisation, Multifocal, Slight Olfactory Epithelium - Respiratory Metaplasia With or Without Associated Inflammato

ry Cell Infiltrate, Minimal Olfactory Epithelium - Fibrosis With or Without Associated Cartilage Formation in Dor sal Lamina Propria, Marked Olfactory Epithelium - Bowman's Gland Atrophy in Dorsal Lamina Propria, Marked Olfactory Nerve Fibres - Atr ophy, Slight Fusion of Dorsal Turbinate With Associated Epithelial Diverticula Formation, Multifocal, Moderate Respiratory Epithelium - Squamous Metaplasia, Focal, Minimal Respiratory Epithelium - Disorganisation/Degeneration/ Regeneration, Minimal Transitional Epithelium - Disorganisation/Regeneration, Minimal155Huntingdon Life SciencesWAG0013

APPENDIX 8 - continued Request ID: 264438 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 4721 8901 19098 Animal Number: 11 Sex: Male Dose Group: 4 Phase: Treatment Sacrifice Status: Final phase sacrifice Date of Death: 09 - Dec - 11 Day of Death: 15 Week of Death: 3 Subgroup: 1 Terminal Bodyweight: 370.2 g PATHOLOGY OBSERVATIONS MACROPATH OLOGY HISTOPAT HOLOGY continued from previous page... Stomach Forestomach depression(s), Epithelial aspect, Two, crater - like, up to 6m m. Trachea

Epithelial Degeneration/Regeneration, Diffuse, Minimal Thy mus Small The following examined tissues had no gross observations and no findings microscopically: Tracheal Bifurc.156Huntingdon Life SciencesWAG0013

APPENDIX 8 - con tinued Request ID: 264438 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 4721 8901 19098 Ani mal Number: 12 Sex: Male Dose Group: 4 Phase: Treatment Sacrifice Status: Final phase sacrifice Date of Death: 09 - Dec - 11 Day of Death: 15 Week of Death: 3 Subgroup: 1 Terminal Bodyweight: 427.3 g PATHOLOGY OBSERVATIONS MACROPATHOLOGY HISTOPATHOLOGY Kidneys Pelvic dilatation, Right Lary nx Squamous Metaplasia - Ventral, Minimal Epithelial Keratinisation - Ventrolateral, Minimal Squamous Metaplasia - Ar yt enoid, Minimal157Huntingdon Life SciencesWAG0013

APPENDIX 8 - continued Request ID: 264438 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 4721 8901 19098 Animal Number: 12 Sex: Male Dose Group: 4 Phase: Treatment Sacrifice Status: Final phase sacrifice Date of Death: 09 - Dec - 11 Day of Death: 15 Week of Death: 3 Subgroup: 1 Terminal Bodyweight: 427.3 g PATHOLOGY OBSERVATIONS MACROPATHOLOGY HISTOPATHOLOGY continued from previous page... Nasal turbinat es Olfactory Epithelium - Necrosis, Focal, Minimal Olfactory Epithelium - Erosion, Focal, Minimal Olfactory Epithelium - Degeneration/Atrophy/Disorganisation, Multifocal, Slight Olfactory Epithelium - Squamous Metaplasia, Focal, Minimal Olfactory Epithelium - Respiratory Metaplasia With or Without Associated Inflammato

ry Cell Infiltrate, Slight Olfactory Epithelium - Fibrosis With or Without Associated Cartilage Formation in Dorsal Lamina Propria, Marked Olfactory Epithelium - Bowman's Gland Atrophy in Dorsal Lamina Propria, Marked Olfactory Nerve Fibres - Atr ophy, Slight Fusion of Dorsal Turbinate With Associated Epithelial Diverticula Formation, Multifocal, M oderate Respiratory Epithelium - Squamous Metaplasia, Focal, Minimal Respiratory Epithelium - Disorganisation/Degeneration/ Regeneration, Minimal Transitional Epithelium - Disorganisation/Regeneration, Minimal Na solacrimal Ducts - Inflammation, Bilateral, Slight Debris and Inflammatory Cells in Airway, Focal, Slight158Huntingdon Life SciencesWAG0013

APPENDIX 8 - continued Request ID: 264438 Macropathology and histopathology - individual findings Group : 1 2 3 4 Compound : Con trol 2 - br omo - 3,3,3 - trifluoropropene 2 - br omo - 3,3,3 -

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

trifluoropropene Exposure (ppm) : 0 4721 8901 19098 Animal Number: 12 Sex: Male Dose Group: 4 Phase: Treatment Sacrifice Status: Final phase sacrifice Date of Death: 09 - Dec - 11 Day of Death: 15 Week of Death: 3 Subgroup: 1 Terminal Bodyweight: 427.3 g PATHOLOGY OBSERVATIONS MACROPATHOLOGY HISTOPATHOLOGY continue d f

rom previous page... Stomach Forestomach depression(s), Epithelial aspect, Punctate, One, crater - like. Tracheal Bifurc. Inflammatory Cell Infiltrate in Lamina Propria, Minimal Trachea Epithelial Degeneration/Regeneration, Diffuse, Slight Inflammatory Cell Infiltrate - Lamina Propria, Diffuse, Slight Thy mus Small The following examined tissues had no gross observations and no findings microscopically: Lungs + Bronchi159Huntingdon Life SciencesWAG0013

APPENDIX 8 - continued Request ID: 264438 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 4 721 8901 19098 Animal Number: 13 Sex: Female Dose Group: 1 Phase: Treatment Sacrifice Status: Final phase sacrifice Date of Death: 09 - Dec - 11 Day of Death: 15 Week of Death: 3 Subgroup: 1 Terminal Bodyweight: 319.3 g

PATHOLOGY OBSERVATIONS MACROPATHO LOGY 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.160Huntingdon Life SciencesWAG0013

APPENDIX 8 - continued Request ID: 264438 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 4721 8901 19098 Animal Number: 14 Sex: Female Dose Group: 1 Phase: Treatment Sacrifice Status: Final phase sacrifice Date of Death: 09 - Dec - 11 Day of Death: 15 Week of Death: 3 Subgroup: 1 Terminal Bodyweight: 322.8 g

PATHOLOGY OBSERVATIONS MACROPATHOLOGY HISTOPATHOLOGY ***ANIMAL HAS NO MACROSCOPIC FINDINGS RECORDED*** Lungs + Bronchi Alveolar Macrophages, Focal, Minimal Alveolar Eosinophilic Crystals with Associated Inflammatory Cell Infiltrate, Focal, Minimal Arterial Mural Mineralisation, Focal, Minimal The following examined tissues had no gross observations and no findings microscopically: Larynx, Nasal turbinates, Trachea, Tracheal Bifurc.161Huntingdon Life SciencesWAG0013

APPENDIX 8 - continued Request ID: 264438 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 4721 8901 19098 Animal Number: 15 Sex: Fem ale Dose Group: 1 Phase: Treatment Sacrifice Status: Final phase sacrifice Date of Death: 09 - Dec - 11 Day of Death: 15 Week of Death: 3 Subgroup: 1 Terminal Bodyweight: 301.3 g

PATHOLOGY OBSERVATIONS MACROPATHOLOGY HISTOPATHOLOGY ***ANIMAL HAS NO MACROSCOPIC FINDINGS RECORDED*** Lungs + Bronchi Alveolar Eosinophilic Crystals with Associated Inflammatory Cell Infiltrate, Focal, Min imal Arterial Mural Mineralisation, Focal, Minimal The following examined tissues had no gross observations and no findings microscopically: Larynx, Nasal turbinates, Trachea, Tracheal Bifurc.162Huntingdon Life SciencesWAG0013

APPENDIX 8 - continued Request ID: 264438 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 4721 8901 19098 Animal Number: 1 6 Sex: Female Dose Group: 2 Phase: Treatment Sacrifice Status: Final phase sacrifice Date of Death: 25 - Nov - 11 Day of Death: 15 Week of Death: 3 Subgroup: 1 Terminal Bodyweight: 267.0 g

PATHOLOGY OBSERVATIONS MACROPATHOLOGY HISTOPATHOLOGY ***ANIMAL HAS NO MACROSCOPIC FINDINGS RECORDED*** Lary nx Epithelial Keratinisation - Ventrolateral, Minimal Squamous Metaplasia - A ry tenoid, Minimal163Huntingdon Life SciencesWAG0013

APPENDIX 8 - continued Request ID: 264438 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 4721 8901 19098 Animal Number: 16 Sex: Female Dose Group: 2 Phase: Treatment Sacrifice Status: Final phase sacrifice Date of Death: 25 - Nov - 11 Day of Death: 15 Week of Death: 3 Subgroup: 1 Terminal Bodyweight: 267.0 g

PATHOLOGY OBS ERVATIONS MACROPATHOLOGY HISTOPATHOLOGY continued from previous page... Nasal turbinates Olfactory Epithelium - Squamous Metaplasia , Multifocal, Minimal Olfactory Epithelium - Respiratory Metaplasia With or Without Associated Inflammato

ry Cell Infiltrate, Multifocal, Minimal Olfactory Epithelium - Fibrosis With or Without Associated Cartilage Formation in Dorsal Lamina Propria, Minimal Olfactory Epithelium - Bowman's Gland Atrophy in Dorsal Lamina Propria, Moderate Olfactory Nerve Fibres - Atr ophy, Minimal Respiratory Epithelium - Disorganisation/Degeneration/ Regeneration, Focal, Minimal Nasolacrimal Ducts - Inflammation, Unilateral, Minimal The following examined tissues had no gross observations and no findings microscopically: Trachea, Tracheal Bifurc.164Huntingdon Life SciencesWAG0013

APPENDIX 8 - continued Request ID: 264438 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 4721 8901 19098 Animal Number: 17 Sex: Female Dose Group: 2 Phase: Treatment Sacrifice Status: Final phase sacrifice Date of Death: 25 - Nov - 11 Day of Death: 15 Week of Death: 3 Subgroup: 1 Terminal Bodyweight: 252.2 g

PATHOLOGY OBSERVATIONS MACROPATHOLOGY HISTOPATHOLOGY LN Bronchial Enlarged, 3mm. Lary nx Epithelial Keratinisation - Ventrolateral, Minimal165Huntingdon Life SciencesWAG0013

APPENDIX 8 - continued Request ID: 264438 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 4721 8901 19098 Animal Number: 17 Sex: Female Dose Group: 2 Phase: Treatment Sacrifice Status: Final phase sacrifice Date of Death: 25 - Nov - 11 Day of Death: 15 Week of Death: 3 Subgroup: 1 Terminal Bodyweight: 252.2 g

PATHOLOGY OBSERVATIONS MACROPATHOLOGY HISTOPATHOLOGY continued from previous page... Nasal turbinates Olfactory Epithelium - Erosion, Focal, Minimal Olfactory Epithelium - Degeneration/Atrophy/Disorganisation , Focal, Slight Olfactory Epithelium - Squamous Metaplasia, Multifocal, Minimal Olfactory Epithelium - Respiratory Metaplasia With or Without Associated Inflammato

ry Cell Infiltrate, Multifocal, Minimal Olfactory Epithelium - Fibrosis With or Without Associated Cartilage Formation in Dorsal Lamina Propria, Minimal Olfactory Epithelium - Bowman's Gland Atrophy in Dorsal Lamina Propria, Moderate Olfactory Nerve Fibres - Atr ophy, Minimal Respirato ry Epithelium - Goblet Cell Hyperplasia, Minimal Transitional Epithelium - Disorganisation/Regeneration, Minimal166Huntingdon Life SciencesWAG0013

APPENDIX 8 - continued Request ID: 264438 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 4721 8901 19098 Animal Number: 17 Sex: Female Dose Group: 2 Phase: Treatment Sacrifice Status: Final phase sacrifice Date o f Death: 25 - Nov - 11 Day of Death: 15 Week of Death: 3 Subgroup: 1 Terminal Bodyweight: 252.2 g

PATHOLOGY OBSERVATIONS MACROPATHOLOGY HISTOPATHOLO GY continued from previous page... The following examined tissues had no gross observations and no findings microscopically: Trachea, Tracheal Bifurc.167Huntingdon Life SciencesWAG0013

APPENDIX 8 - continued Request ID: 264438 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 4721 8901 19098 Animal Number: 18 Sex: Female Dose Group: 2 Phase: Treatment Sacrific e Status: Final phase sacrifice Date of Death: 25 - Nov - 11 Day of Death: 15 Week of Death: 3 Subgroup: 1 Terminal Bodyweight: 273.6 g

PATHOLOGY OBSERVATIONS MACROPATHOLOGY HISTOPATHOLOGY LN Mandibular Enlarged, Left, One, 13mm. Lungs + Bronchi Pale area(s), Punctate, A few, subpleural. Lary nx Squamous Metaplasia - Ar yt enoid, Minimal168Huntingdon Life SciencesWAG0013

APPENDIX 8 - continued Request ID: 264438 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 4721 8901 19098 An imal Number: 18 Sex: Female Dose Group: 2 Phase: Treatment Sacrifice Status: Final phase sacrifice Date of Death: 25 - Nov - 11 Day of Death: 15 Week of Death: 3 Subgroup: 1 Terminal Bodyweight: 273.6 g

PATHOLOGY OBSERVATIONS MACROPATHOLOGY HISTOPATHOLOGY continued from previous page... Nasal turbinates Olfactory Epithelium - Degeneration/Atrophy/Disorganisation, Focal, Minimal Olfactory Epithelium - Squamous Metaplasia, Multifocal, Minimal Olfactory Epithelium - Respiratory Metaplasia With or Without Associated Inflammato

ry Cell Infiltrate, Multifocal, Minimal Olfactory Epithelium - Fibrosis With or Without Associated Cartilage Formation in Dorsal Lamina Propria, Minimal Olfactory Epithelium - Bowman's Gland Atrophy in Dorsal Lamina Propria, Moderate Olfactory Nerve Fibres - Atr ophy, Slight Fusion of Dorsal Turbinate Wi th Associated Epithelial Diverticula Formation, Focal, Minimal Transitional Epithelium - Disorganisation/Regeneration, Minimal169Huntingdon Life SciencesWAG0013

APPENDIX 8 - continued Request ID: 264438 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 4721 8901 19098 Animal Number: 18 Sex: Female Dose Group: 2 Phase: Treatment Sacrifice Status: Final phase sacri fice Date of Death: 25 - Nov - 11 Day of Death: 15 Week of Death: 3 Subgroup: 1 Terminal Bodyweight: 273.6 g

PATHOLOGY OBSERVATIONS MACROPATHOLOGY HISTOPATHOLOGY continued from previous page... The following examined tissues had no gross observations and no findings microscopically: Trachea, Tracheal Bifurc.170Huntingdon Life SciencesWAG0013

APPENDIX 8 - continued Request ID: 264438 Macropathology and histopatho logy - 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 4721 8901 19098 Animal Number: 19 Sex: Female Dose Group: 3 Phase: Treatm ent Sacrifice Status: Final phase sacrifice Date of Death: 02 - Dec - 11 Day of Death: 15 Week of Death: 3 Subgroup: 1 Terminal Bodyweight: 275.2 g

PATHOLOGY OBSERVATIONS MACROPATHOLOGY HISTOPATHOLOGY ***ANIMAL HAS NO MACROSCOPIC FINDINGS RECORDED*** Lary nx Squamous Metaplasia - Ventral, Minimal Epithelial Keratinisation - Ventrolateral, Minimal Squamous Metapla sia - Ar yt enoid, Minimal171Huntingdon Life SciencesWAG0013

APPENDIX 8 - continued Request ID: 264438 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 -

trifluoropr opene Exposure (ppm) : 0 4721 8901 19098 Animal Number: 19 Sex: Female Dose Group: 3 Phase: Treatment Sacrifice Status: Final phase sacrifice Date of Death: 02 - Dec - 11 Day of Death: 15 Week of Death: 3 Subgroup: 1 Terminal Bodyweight: 275.2 g

PATHOL OGY OBSERVATIONS MACROPATHOLOGY HISTOPATHOLOGY continued from previous page... Nasal turbinates Olfactory Epithelium - Degeneration /Atrophy/Disorganisation, Multifocal, Slight Olfactory Epithelium - Squamous Metaplasia, Multifocal, Minimal Olfactory Epithelium - Respiratory Metaplasia With or Without Associated Inflammato

ry Cell Infiltrate, Focal, Minimal Olfactory Epithelium - Fibrosis With or Without Associated Cartilage Formation in Dorsal Lamina Propria, Moderate Olfactory Epithelium - Bowman's Gland Atrophy in Dorsal Lamina Propria, Moderate Olfactory Nerve Fibres - Atr ophy, S light Fusion of Dorsal Turbinate With Associated Epithelial Diverticula Formation, Multifocal, Moderate Respiratory Epithelium - Disorganisation/Degeneration/ Regeneration, Focal, Minimal Debris and Inflammatory Cells in Airway, Multifocal, Minimal172Huntingdon Life SciencesWAG0013

APPENDIX 8 - continued Request ID: 264438 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 -

trifluorop ropene Exposure (ppm) : 0 4721 8901 19098 Animal Number: 19 Sex: Female Dose Group: 3 Phase: Treatment Sacrifice Status: Final phase sacrifice Date of Death: 02 - Dec - 11 Day of Death: 15 Week of Death: 3 Subgroup: 1 Terminal Bodyweight: 275.2 g

PATHO LOGY OBSERVATIONS MACROPATHOLOGY HISTOPATHOLOGY continued from previous page... Trachea Inflammatory Cell Infiltrate - Lamina Propria, Diffuse, Minimal The following examined tissues had no gross observations and no findings microscopically: Tracheal Bifurc.173Huntingdon Life SciencesWAG0013

APPENDIX 8 - continued Request ID: 264438 Macropathology and histopathology - individual findings Grou p : 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 4721 8901 19098 Animal Number: 20 Sex: Female Dose Group: 3 Phase: Treatment Sacrifice Status: Final phase sacrifice Date of Death: 02 - Dec - 11 Day of Death: 15 Week of Death: 3 Subgroup: 1 Terminal Bodyweight: 300.7 g

PATHOLOGY OBSERVATIONS MACROPATHOLOGY HISTOPATHOLOGY ***ANIMAL HAS NO MACROSCOPIC FINDINGS RECORDED*** Lary nx Squamous Metaplasia - Ventral, Minimal Squamous Metaplasia - Ar yt enoid, Minimal174Huntingdon Life SciencesWAG0013

APPENDIX 8 - continued Request ID: 264438 Macropathology and h istopathology - 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 4721 8901 19098 Animal Number: 20 Sex: Female Dose Group: 3 Phas e: Treatment Sacrifice Status: Final phase sacrifice Date of Death: 02 - Dec - 11 Day of Death: 15 Week of Death: 3 Subgroup: 1 Terminal Bodyweight: 300.7 g

PATHOLOGY OBSERVATIONS MACROPATHOLOGY HISTOPATHOLOGY continued from previous page... Nasal turbinates Olfactory Epithelium - Degeneration/Atrophy/Disorganisation, Multifocal, Slight Olfactory Epithelium - Squamous Metapla sia, Focal, Minimal Olfactory Epithelium - Respiratory Metaplasia With or Without Associated Inflammato

ry Cell Infiltrate, Multifocal, Minimal Olfactory Epithelium - Fibrosis With or Without Associated Cartilage Formation in Dorsal Lamina Propria, Moderate Olfactory Epithelium - Bowman's Gland Atrophy in Dorsal Lamina Propria, Moderate Olfactory Nerve Fibres - Atr ophy, Slight Fusion of Dorsal Turbinate With Associated Epithelial Diverticula Formation, Multifocal, Moderate Transitional Epithelium - Disorganisation/Regeneration, Minimal175Huntingdon Life SciencesWAG0013

APPENDIX 8 - continued Request ID: 264438 Macropathology and histopathology - individual findings Group : 1 2 3 4 Compound : Control 2 - br omo - 3,3,3 - tri fluoropropene 2 - br omo - 3,3,3 -

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

trifluoropropene Exposure (ppm) : 0 4721 8901 19098 Animal Number: 20 Sex: Female Dose Group: 3 Phase: Treatment Sacrifice Status: Final phase sacrifice Date of Death: 02 - Dec - 11 Day of Death : 15 Week of Death: 3 Subgroup: 1 Terminal Bodyweight: 300.7 g

PATHOLOGY OBSERVATIONS MACROPATHOLOGY HISTOPATHOLOGY continued from previous page ... The following examined tissues had no gross observations and no findings microscopically: Trachea, Tracheal Bifurc.176Huntingdon Life SciencesWAG0013

APPENDIX 8 - continued Request ID: 264438 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 4721 8901 19098 Animal Number: 21 Sex: Female Dose Group: 3 Phase: Treatment Sacrifice Status: Final phase sacrifice Date of Death: 02 - Dec - 11 Day of Death: 15 Week of Death: 3 Subgroup: 1 Terminal Bodyweight: 274.4 g

PATHOLOGY OBSERVATIONS MACROPATHOLOGY HIST OPATHOLOGY ***ANIMAL HAS NO MACROSCOPIC FINDINGS RECORDED*** Lary nx Squamous Metaplasia - Ventral, Minimal Epithelial Keratinisation - Ventrolateral, Minimal Squamous Metaplasia - Ar yt enoid, Minimal177Huntingdon Life SciencesWAG0013

APPENDIX 8 - continued Request ID: 264438 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 4721 8901 19098 Animal Number: 21 Sex: Female Dose Group: 3 Phase: Treatment Sacrifice Status: Final phase sacrifice Date of Death: 02 - Dec - 11 Day of Death: 15 Week of Death: 3 Subgroup: 1 Terminal Bodyweight: 274.4 g

PATHOLOGY OBSERVATIONS MACROPATHOLOGY HISTOPATHOLOGY continued from previous page... Nasal turbinates Olfactory Epithelium - Necrosis, Focal, Minimal Olfactory Epithelium - Erosion, Focal, Minimal Olfactory Epithelium - Degeneration/Atrophy/Disorganisation, Multifocal, Slight Olfactory Epithelium - Squamous Metaplasia, Multifocal, Minimal Olfactory Epithelium - Respiratory Metaplasia With or Without Associated Inflammato

ry Cell Infiltrate, Multifocal, Minimal Olfactory Epithelium - Fibrosis With or Without Associated Cartilage Formation in Dorsal Lamina Propria, Moderate Olfactory Epithelium - Bowman's Gland Atrophy in Dorsal Lamina Propria, Moderate Olfactory Nerve Fibres - Atr ophy, Moderate Fusion of Dorsal Turbinate With Associated Epithelial Diverticula Formation, Multifocal, Moderate Respiratory Epithelium - Disorganisation/Degeneration/ Regeneration, Multifocal, Slight Respiratory Epithelium - Goblet Cell Hyperplasia, Minimal Transitional Epithelium - Disorganisation/Regeneration, Minimal Debris and Inflammatory Cells in Airway, Focal, Minimal178Huntingdon Life SciencesWAG0013

APPENDIX 8 - continued Request ID: 264438 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 4721 8901 19098 Animal Number: 21 Sex: Female Dose Group: 3 Phase: Treatment Sacrifice Status: Final phase sacrifice Date of Death: 02 - Dec - 11 Day of Death: 15 Week of Death: 3 Subgroup: 1 Terminal Bodyweight: 274.4 g

PATHOLOGY OBSERVATIONS MACROPATHOLOGY HISTOPATHOLOGY continued from previous page... Tracheal Bifurc. Inflammatory Cell Infiltrate in Lamina Propria, Minimal Trach ea Epithelial Degeneration/Regeneration, Minimal The following examined tissues had no gross observations and no findings microscopically:

-179Huntingdon Life SciencesWAG0013

APPENDIX 8 - continued Request ID: 264438 Macropathology and histopathology - in dividual 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 4721 8901 19098 Animal Number: 22 Sex: Female Dose Group: 4 Phase: Treatment Sacri fice Status: Final phase sacrifice Date of Death: 09 - Dec - 11 Day of Death: 15 Week of Death: 3 Subgroup: 1 Terminal Bodyweight: 266.8 g

PATHOLOGY OBSERVATIONS MACROPATHOLOGY HISTOPATHOLOGY Lungs + Bronchi Alveolar Macrophages, Focal, Minimal Lary nx Squamous Metaplasia - Ventral, Minimal Epithelial Keratinisation - Ventrolateral, Minimal Squamous Metaplasia - Ar yt enoid, Minimal180Huntingdon Life SciencesWAG0013

APPENDIX 8 - continued Request ID: 264438 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 -

tri fluoropropene Exposure (ppm) : 0 4721 8901 19098 Animal Number: 22 Sex: Female Dose Group: 4 Phase: Treatment Sacrifice Status: Final phase sacrifice Date of Death: 09 - Dec - 11 Day of Death: 15 Week of Death: 3 Subgroup: 1 Terminal Bodyweight: 266.8 g

PATHOLOGY OBSERVATIONS MACROPATHOLOGY HISTOPATHOLOGY continued from previous page... Nasal turbinates Olfactory Epithelium - Dege neration/Atrophy/Disorganisation, Multifocal, Slight Olfactory Epithelium - Fibrosis With or Without Associated Cartilage Formation in Dorsal Lamina Propria, Moderate Olfactory Epithelium - Bowman's Gland Atrophy in Dorsal Lamina Prop ria, Moderate Olfactory Nerve Fibres - Atr ophy, Moderate Fusion of Dorsal Turbinate With Associated Epithelial Diverticula Formation, Multifocal, Moderate Respiratory Epithelium - Squamous Metaplasia, Focal, Minimal Respiratory Epithelium - Disorganisation/Degeneration/ Regeneration, Minimal Transitional Epithelium - Disorganisation/Regeneration, Minimal Nasolacrimal Ducts - Inflammation, Bilateral, Minimal181Huntingdon Life SciencesWAG0013

APPENDIX 8 - continued Reques t ID: 264438 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 4721 8901 19098 Animal Number: 22 Sex: Female Dose Group: 4 Phase: Treatment Sacrifice Status: Final phase sacrifice Date of Death: 09 - Dec - 11 Day of Death: 15 Week of Death: 3 Subgroup: 1 Terminal Bodyweight: 266.8 g

PATHOLOGY OBSERVATIONS MACROPATHOLOGY HISTOPATHOLOGY continued from previous page... Stomach Forestomach roughened, Epithelial aspect, an area, 6mm. Thy mus Small The following examined tissues had no gross observations and no findings microscopically: Trachea, Tracheal Bifurc.182Huntingdon Life SciencesWAG0013

APPENDIX 8 - continued Request ID: 264438 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 4721 8901 19098 Animal Number: 23 Sex: Female Dose Group: 4 Phase: Treatment Sacrifice Status: Final phase sacrifice Date of Death: 09 - Dec - 11 Day of Death: 15 Week of Death: 3 Subgroup: 1 Terminal Bodyweight: 256.7 g

PATHOLOGY OBSERVATIONS MACROPATHOLOGY HISTOPATHOLOGY Lungs + Br onchi Alveolar Macrophages, Diffuse, Minimal Arterial Mural Mineralisation, Focal, Minimal Lary nx Squamous Metaplasia - Ventral, Minimal Epithelial Keratinisation - Ventrolateral, Minimal Squamous Metaplasia - Ar yt enoid, Minimal Epithelial Hyperplasia - Ar yt enoid, Minimal Inflammation, Multifocal, Slight183Huntingdon Life SciencesWAG0013

APPENDIX 8 - continued Request ID: 264438 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 4721 8901 19098 Animal Number: 23 Sex: Female Dose Group: 4 Phase: Treatment Sacrifice Status: Final phase sacrifice Date of Death: 09 - Dec - 11 Day of Death: 15 Week of Death: 3 Subgroup: 1 Terminal Bodyweight: 256.7 g

PATHOLOGY OBSERVATIONS MACROPATHOLOGY HIST OPATHOLOGY continued from previous page... Nasal turbinates Olfactory Epithelium - Erosion, Focal, Minimal Olfactory Epithelium - Degeneration/Atrophy/Disorganisation, Multifocal, Moderate Olfactory Epithelium - Squamous Metaplasia, Focal, Minimal Olfactory Epithelium - Respiratory Metaplasia With or Without Associated Inflammato

ry Cell Infiltrate, Focal, Minimal Olfactory Epithelium - Fibrosis With or Without Associated Cartilage Formation in Dorsal Lamina Propria, Moderate Olfactory Epithelium - Bowman's Gland Atrophy in Dorsal Lamina Propria, Moderate Olfactory Nerve Fibres - Atr ophy, Slight Fusion of Dorsal Turbinate With Associated Epithelial Diverticula Formation , Multifocal, Moderate Respiratory Epithelium - Disorganisation/Degeneration/ Regeneration, Minimal Transitional Epithelium - Disorganisation/Regeneration, Slight184Huntingdon Life SciencesWAG0013

APPENDIX 8 - continued Request ID: 264438 Macropathology and hi stopathology - 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 4721 8901 19098 Animal Number: 23 Sex: Female Dose Group: 4 Phase : Treatment Sacrifice Status: Final phase sacrifice Date of Death: 09 - Dec - 11 Day of Death: 15 Week of Death: 3 Subgroup: 1 Terminal Bodyweight: 256.7 g

PATHOLOGY OBSERVATIONS MACROPATHOLOGY HISTOPATHOLOGY continued from previous page... Tracheal Bifurc. Inflammatory Cell Infiltrate in Lamina Propria, Minimal Trachea Epithelial Degeneration/Regeneration, Diffuse, Minimal Inflammatory Cell Infiltrate - Lamina Propria, Diffuse, Slight Thy mus Small The following examined tissues had no gross observations and no findings microscopically:

-185Huntingdon Life SciencesWAG0013

APPENDIX 8 - continued Request ID: 2 64438 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 4721 8901 19098 Animal Number: 24 Sex: Female Dose Group: 4 Phase: Treatment Sacrifice Status: Final phase sacrifice Date of Death: 09 - Dec - 11 Day of Death: 15 Week of Death: 3 Subgroup: 1 Terminal Bodyweight: 271.3 g

PATHOLOGY OBSERVATIONS MACROPATHOLOGY HISTOPATHOLOGY Lungs + Bronchi Alveolar Macrophages, Multifocal, Minimal Lary nx Squamous Metaplasia - Ventral, Minimal Epithelial Keratini sation - Ventrolateral, Minimal Squamous Metaplasia - Ar yt enoid, Minimal Epithelial Hyperplasia - Ar yt enoid, Minimal186Huntingdon Life SciencesWAG0013

APPENDIX 8 - continued Request ID: 264438 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 4721 8901 19098 Animal Number: 24 Sex: Female Dose Group: 4 Phase: Treatment Sacrifice Status: Final phase sacr ifice Date of Death: 09 - Dec - 11 Day of Death: 15 Week of Death: 3 Subgroup: 1 Terminal Bodyweight: 271.3 g

PATHOLOGY OBSERVATIONS MACROPATHOLOGY HISTOPATHOLOGY continued from previous page... Nasal turbinates Olfactory Epithelium - Degeneration/Atrophy/Disorganisation, Multifocal, Moderate Olfactory Epithelium - Squamous Metaplasia, Focal, Minimal Olfactory Epit

helium - Respiratory Metaplasia With or Without Associated Inflammato

ry Cell Infiltrate, Focal, Minimal Olfactory Epithelium - Fibrosis With or Without Associated Cartilage Formation in Dorsal Lamina Propria, Moderate Olfactory Epithe lium - Bowman's Gland Atrophy in Dorsal Lamina Propria, Moderate Olfactory Nerve Fibres - Atr ophy, Slight Fusion of Dorsal Turbinate With Associated Epithelial Diverticula Formation, Multifocal, Moderate Respiratory Epithel ium - Disorganisation/Degeneration/ Regeneration, Slight Transitional Epithelium - Disorganisation/Regeneration, Minimal Nasolacrimal Ducts - Epithelial Degeneration, Bilateral, Minimal Nasolacrimal Ducts - Inflammation, Un ilateral, Minimal Debris and Inflammatory Cells in Airway, Focal, Minimal187Huntingdon Life SciencesWAG0013

APPENDIX 8 - continued Request ID: 264438 Macropathology and histopathology - individual findings Group : 1 2 3 4 Compound : Control 2 - br omo - 3,3,3 - trifluoroprop ene 2 - br omo - 3,3,3 -

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

trifluoropropene Exposure (ppm) : 0 4721 8901 19098 Animal Number: 24 Sex: Female Dose Group: 4 Phase: Treatment Sacrifice Status: Final phase sacrifice Date of Death: 09 - Dec - 11 Day of Death: 15 Week of Death: 3 Subgroup: 1 Terminal Bodyweight: 271.3 g

PATHOLOGY OBSERVATIONS MACROPATHOLOGY HISTOPATHOLOGY continued from previous page... Stom ach Forestomach roughened, Epithelial aspect Trachea Epithelial Degeneration/Regeneration, Diffuse, Minimal Thy mus Small The following examined tissues had no gross observations and no findings microscopical ly : Tracheal Bifurc.Huntingdon Life Sciences WA G0013

188 

### Annex 1 Administration of 2 -bromo- 3,3,3 - trifluoropropene to ratsHuntingdon Life Sciences WA G0013

189 

### Working for a better future



## Aerosol Technology R eport

HLS s tudy number: WA G0013 Author A Allen Version ID: Audited Draft 1 Issue date:Huntingdon Life Sciences WAG0013

190 

# Table of Contents Signature Page ................................................................................................................ ... 191 1. Pre-study Atmosphere Characterization ..................................................................... 192 2. Test Article Handling and Processing for Inhalation Generation ................................. 192 3. Exposure System ........................................................................................................ 192 4. Administration ............................................................................................................. 19 3 5. Atmosphere Analysis .................................................................................................. 193 5.1 Nominal Concentration ......................................................................................... 193 5.2 Nominal Concentration ......................................................................................... 194 5.3 Chamber Temperature ......................................................................................... 194 5.4 Chamber Humidity ................................................................................................ 194 6. Data and Calculations ................................................................................................. 195 7. Results and Discussion ............................................................................................... 195 

### 7.1 Aerosol Concent ration s ............................................................................. 195 

# List of Tables Table 1 Chamber atmosphere concentration of 2-bromo-3,3,3-trifluoropropene - individual exposure values for Group 1 ....................................................... 196 Table 2 Chamber atmosphere concentration of 2-bromo-3,3,3-trifluoropropene – daily means and individual exposure values for Group 2 .................................... 197 Table 3 Chamber atmosphere concentration of 2-bromo-3,3,3-trifluoropropene - daily means and individual exposure values for Group 3 .................................... 198 Table 4 Chamber atmosphere concentration of 2-bromo-3,3,3-trifluoropropene - daily means and individual exposure values for Group 4 .................................... 199 Table 5 Chamber Temperatures - daily mean .......................................................... 200 Table 6 Chamber Humidity - daily mean .................................................................. 201 Table 7 Nominal Chamber atmosphere concentration of 2-bromo-3,3,3-trifluoropropene - individual exposure values for Group 2 ... 202 Table 8 Nominal Chamber atmosphere concentration of 2-bromo-3,3,3-trifluoropropene - individual exposure values for Group 3 ... 203 Table 9 Nominal Chamber atmosphere concentration of 2-bromo-3,3,3-trifluoropropene - individual exposure values for Group 4 ... 204 

# List of Figures Figure 1 Schematic of Inhalation Exposure System .................................................. 205Huntingdon Life Sciences WA G0013

192 

# 1. Pre - study

A tmosphere Ch aracterization Inhalat io n exposure system characterized including determinat io ns of: Achieved vapour concentration; Spati al 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 vaporised using warmed air th rough a sintered glass vaporiser. 

# 3. Exposure Sy

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

rected flow Whole body

exposure chamber: 0.75m 3 

### chamber of

s tainless

– steel

and glass construction Animal Restraint: Wire mesh modular caging Aerosol Generator: Sint ered glass vaporiser for Groups 2 to 4 Liquid reservoi

r and feed rate used to alter concentration Group 2

– 29L/min Groups 3 and 4

– 49L/min Diluen t Ai rflow: From in- house co mpressed air system

– breathing qualit y Group 1

– 149L/min Group 2

– 120L/min Groups 3 and 4

– 100L/min Extract Ai

rflow: Drawn by a fan extract system Drawn fro m base of exposure system Controlled by a gate clamp situated at the rear of the chamber Airflow Monitoring: In - line flowmeters monitored continuously for generation and diluent ai rflows Chamber pressure different ial mo nit ored by Magnahelic set at 1 to 4mm/H2O to ensure a slight negative pressure within the chamber~150L/min .Huntingdon Life Sciences WA G0013

193 

# 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 

# 5. At mosphere A nalysis 5.1 Nominal Concentration Aerosol sa mples 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 spectrophotometerHuntingdon Life Sciences WA G0013

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## Flow diagram of the analytical method for the a nalysis 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 measure on the IR spectrophotometer Calibration Prepare a ser ies of calibration standards by direct addition of a suitable volume of test item directly into gas bags containing a suitable volume of air

 Select and prepare the appropriate calibration range for the expected concentration of the test atmosphere sample s to be analysed

 Prepare a set of calibration standards for low range analysis at nominal concentrations of 1167, 2334, 3501, 4668, 7002 and 9336 ppm

 Prepare a set of calibration standards for inter range analysis at nominal concentrations of 2334, 350 1, 4668, 9336, 14004 and 18672 ppm

 Prepare a set of calibration standards for high range analysis at nominal concentrations of 4668, 9336, 14004, 18672, 23340 and 28008 ppm

 Measure the required calibration range standards using the IR spectrophotometer fo r calibration 

### IR conditions Instrument: MIRAN SapphIRe model ML Path length: Short (0.5m) Wa velength: 10.699 μm (low calibration range)

12.026 μm (inter and high calibration range) The IR spectrophotometer was calibrated 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 . 

## 5.2 Nominal Concentration Moni tored and summarised inTables 7 t o 9. 

## 5.3 Chamber Temperature Moni tored using a liquid in glass thermometer, daily m eans presented in Table 5. 

## 5.4 Chamber Humidity Moni tored using wet and dry

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

tables, daily means presente d inTable 6.Huntingdon Life Sciences WA G0013

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# 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 r ounding errors in the last significant figure. All data not presented in this report is retained in the study data files and archives. 

# 7. Results and Discussion 7.1 Aerosol Concentrations Individual exposure concentrations and daily m eans are presented in Tables 1 to 4 and summarized below: Group Concentration (ppm)

Target Mean 1 0 2 5000 4721 3 10000 8901 4 20000 19098 

### Addit io nal samples were taken for confirmat io n of atypical resul ts and a ft er minor feed rate adj ustments. All groups were relatively close to target.Huntingdon Life Sciences WA G0013

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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 (p pm)

Sample 1 Sample 2 Sample 3 1 ND ND ND 2 ND ND ND 3 ND ND ND 4 1 ND ND ND 5 ND ND ND 6 1 ND ND ND 7 ND ND ND 8 1 ND ND ND 9 ND ND ND 1 0 1 ND ND ND ND No test item detected 1 Only one standard curve was used for calibration due to insufficient volume remaining for measurementHuntingdon Life Sciences WA G0013

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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 No. Chamber concentration (ppm)

Sample 1 Sa mple 2 Sample 3 Sample 4 Sample 5 Sample 6 Daily mean 1 5296 5108 4751 4588 4936 2 5581 4383 4267 5123 4423 4912 4782 3 1 5546 4760 4878 5061 4 4062 5661 4859 3554 4534 5 6113 5613 4530 3170 2946 4474 6 6611 4160 4732 5758 5315 7 5 789 4101 4851 4914 8 1 5978 5008 4357 3851 4736 4786 9 1 4 380 4 298 4 013 4 230 1 0 4523 4021 3981 4175 Mean 2 4 721 sd 3 64 CV (%) 7. 7 1 Only one standard curve was used for calibration due to insufficient volume remaining for measurement 2 Mean calculated from the individual mean values sd Standard deviation CV (%) Coefficient of variation (sd × 100/mean)Huntingdon Life Sciences WA G0013

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### Table 3 Chamber atmosphere concentration of

2-bromo- 3,3,3 - trifluoropropene - d aily means and individual exposure values for Group 3 Exposure No. Chamber concentration (ppm)

Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Sample 6 Daily mean 1 9817 6681 10288 10080 9217 2 6902 4723 11694 7773 3 1 11319 7964 7288 15143 7056 10692 9910 4 1 7524 10077 8648 8750 5 7860 9833 9563 9085 6 9304 11418 9570 10097 7 2 8247 8593 8017 8 1 7130 7928 7977 7678 9 1 9170 8419 7304 8298 1 0 8947 9003 9939 9296 Mean 3 8901 sd 858 CV (%) 9.6 1 Only one standard curve was used for calibration due to insufficient volume remaining for measurement 2 Results from Exposure 7 have been excluded from the mean, due to the standard measurements falling outside acceptance criteria 3 Mean calculated from the individual mean values sd Standard deviation CV (%) Coefficient of variation (sd × 100/mean)Huntingdon Life Sciences WA G0013

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### Table 4 Chamber atmosphere concentration of

2-bromo- 3,3,3 - trifluoropropene - daily means and individual expos ure values for Group 4 Exposure No. Aerosol concentration (ppm)

Sample 1 Sample 2 Sample 3 Sample 4 Daily mean 1 20053 17539 19272 18955 2 1 18656 17536 18093 3 2 18487 18472 18193 18384 4 2 10617 20131 18076 19121 16986 5 20434 19500 20019 19984 6 2 18547 20381 17974 18967 7 16028 18501 19457 17995 8 2 23547 17121 19444 20037 9 20840 23887 20573 21767 1 0 2 13683 25746 20232 15580 18810 Mean 3 19098 sd 1374 CV (%) 7.2 1 Results from Exposure 2 have be en excluded from the mean, due to the standard measurements falling outside acceptance criteria 2 Only one standard curve was used for calibration due to insufficient volume remaining for measurement 3 Mean calculated from the individual mean values sd Standar d deviation CV (%) Coefficient of variation (sd × 100/mean)Huntingdon Life Sciences WA G0013

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### Table 5 Chamber Temperatures - daily mean Exposure No. Chamber Temperature (°C )

Group 1 Group 2 Group 3 Group 4 1 21.0 20.7 20.5 20.8 2 21.0 20.8 20.6 20.4 3 2 0.6 20.5 20.4 20.0 4 21.0 20.7 20.5 20.2 5 21.0 20.5 20.6 20.1 6 20.9 20.6 20.7 20.0 7 21.0 20.6 20.7 20.0 8 21.0 20.5 20.0 20.0 9 21.0 20.5 20.2 20.2 1 0 21.1 20.6 20.4 20.4Huntingdon Life Sciences WA G0013

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### Table 6 Chamber Humidity - daily mean Expo su re No. Chamber Temperature ( %RH )

Group 1 Group 2 Group 3 Group 4 1 28 34 28 31 2 29 25 25 32 3 29 29 26 32 4 33 27 27 34 5 26 26 26 32 6 22 30 29 35 7 23 28 26 32 8 24 28 28 32 9 23 30 28 31 1 0 22 29 25 31Huntingdon Life Sciences WA G0013

202 

### Table 7 Nominal Chamber atmosphere concentration of

2-bromo- 3,3,3 - trifluoropropene - individual exposure values for Group 2 Day 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 % 1 764 1980 271.3 4936 4999 98.7 2 769 2020 275.1 4782 5068 94.4 3 766 2060 281.4 5061 5184 97.6 4 786 1610 214.5 4534 3956 115 5 757 1760 243.3 4474 4485 99.8 6 763 2050 281.2 5315 5180 103 7 761 2050 281.9 4914 5194 94.6 8 764 19 60 268.4 4786 4946 96.8 9 770 1700 231.0 4230 4259 99.3 10 770 1550 210.7 4175 3887 107 1 Analysed/Nominal (A/N) ratio (%) = Analysed concentration (ppm) / Nominal Concentration (ppm)*100 

#   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 (0.08206Latm/mol°K) MW =Molecular weight (174.95g/mol) F =Chamber airflow (150L/min) D =Duration 6 Hours (360mins)Huntingdon Life Sciences WA G0013

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### Table 8 Nominal Chamber atmosphere concentration of

2-bromo- 3,3,3 - trifluoropropene - individual exposure values for Group 3 Day Barometric Pressure P (mmHg) Test

Compound usage TCU (g) Volume of

Test Compound V (L) Analysed

Conc.(ppm) Nominal

Conc.(ppm) A/N 2

ratio %

1 763 3470 475.9 9217 8736 106 2 761 3330 457.9 7773 8409 92.4 3 764 3470 475.0 9910 8720 114 4 770 3440 467.4 8750 8582 102 5 770 3490 474.3 9085 8708 104 6 767 3660 499.5 10097 9166 110 7 768 2980 406.2 7467 1 8 753 3250 450.9 7678 8280 92.7 9 765 3350 457.6 8298 8403 98.7 10 756 3630 502.2 9296 9214 101 1 No value due to analysed value excluded 2 Analysed/Nominal (A/N) ratio (%) = Analysed concentration (ppm) / Nominal Concentration (ppm)*100 

#   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 (0.08206Latm/mol°K) MW =Molecular weight (174.95g/mol) F =Chamber airflow (150L/min) D =Duration 6 Hours (360mins)Huntingdon Life Sciences WA G0013

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### Table 9 Nominal Chamber atmosphere concentration of

2-bromo- 3,3,3 - trifluoropropene - individual exposure values for Group 4 Day Barometric Pressure P (mmHg) Test

Compound usage TCU (g) Volume of

Test Compound V (L) Analysed

Conc.(ppm) Nominal

Conc.(ppm) A/N 2

ratio %

1 767 7120 972.4 18955 17689 107 2 768 6270 853.9 15568 1 3 753 6580 912.7 18384 16621 111 4 765 6100 833.3 16986 151 97 112 5 756 6780 937.0 19984 17055 117 6 761 7060 969.0 18967 17628 108 7 754 7340 1016.9 17995 18484 97.4 8 754 7030 974.0 20037 17717 113 9 753 7400 1027.1 21767 18666 117 10 754 7220 1001.5 18810 18208 103 1 No value due to analysed valu e excluded 2 Analysed/Nominal (A/N) ratio (%) = Analysed concentration (ppm) / Nominal Concentration (ppm)*100



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

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



#   P(mmHg)

760 (g/mol) MW 273 C) T( R (g) TCU )( 

  LVHuntingdon Life Sciences WA G0013

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### Figure 1 Schematic of Inhalation Exposure System

A B

D

F C E

F

H I

J K Front view Side view

L Generation Trolley

G



### Key A Who le body

chamber G Load cells B 76.2mm test material delivery tubing H Air - line co il C El utri ator I Sint ered glass vaporiser D Secondary

generation trolley J Water bath E Magnahelix K Generat io n flo wmeters F Pressure Vessels L Control valvesHuntingdon Life Sciences WA G0013

206 

### Annex 2 Pathology reportHuntingdon Life Sciences WAG0013

Issue No: 2 Page 2 of 10 

# 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 inhalati on toxicity study.

Group Treatment Achieved exposure level (ppm) Number of animals Male Female 1 Control 0 3 3 2 2-bromo-3,3,3-trifluoropropene 4721 3 3 3 2-bromo-3,3,3-trifluoropropene 8901 3 3 4 2-bromo-3,3,3-trifluoropropene 19098 3 3 

# 2. Results 2.1 Decedents There were no unscheduled deaths during the 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 the following intergroup differences: Thymus A reduction in the size of the thymus was noted in animals treated with 19098 ppm.

Summary of findings in the thymus for animals killed after 2 weeks of treatment Group/sex 1M 2M 3M 4M 1F 2F 3F 4F Achieved Exposure Level (ppm) 0 4721 8901 19098 0 4721 8901 19098 Small 0 0 0 3 0 0 0 3 Number of animals examined 3 3 3 3 3 3 3 3Huntingdon Life Sciences WAG0013

Issue No: 2 Page 3 of 10 

### Stomach Depressions or roughening of the stomach was observed in animals treated with 19098 ppm.

Summary of findings in the stomach for animals killed after 2 weeks of treatment Group/sex 1M 2M 3M 4M 1F 2F 3F 4F Achieved Exposure Level (ppm) 0 4721 8901 19098 0 4721 8901 19098 Forestomach Depression(s) 0 0 0 2 0 0 0 0 Forestomach Roughened 0 0 0 1 0 0 0 2 Number of animals examined 3 3 3 3 3 3 3 3 

### The nature and incidence of all other findings were consis tent with the commonly seen background of macroscopic changes inrats at these laboratories. 

## 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 in the nasal turbinates, larynx and trachea. Nasal turbinates In the olfactory epithelium of male and female animals exposed to concentrations of 4721, 8901 or 19098 ppm, the test article-related findings seen included the following. Minimal to marked fibrosis w ith or without associated cartilage formation in the dorsal lamina propria. Minimal to marked atrophy of the Bowman’s glands in the dorsal lamina propria. Minimal to moderate atrophy of olfactory nerve fibres. Minimal epithelial necrosis was seen in tw o male animals exposed to 19098 ppm and one female exposed to 8901 ppm. Minimal erosion of the epithelium. Minimal to moderate degeneration/atr ophy/disorganisation of the epithelium. Minimal squamous metaplasia of the epithelium. Minimal or slight respiratory metaplasia of the epithelium with or without associated inflammatory cell infiltrate.Huntingdon Life Sciences WAG0013

Issue No: 2 Page 4 of 10 

### In the turbinates, minimal to moderate fusion of dorsal turbinates with associated epithelial diverticula formation was seen in male and female animals exposed to 4721, 8901 or 19098 ppm. In the respiratory epitheliu m of male and female animals exposed to 4721, 8901 or 19098 ppm, the test article-related findings seen included the following. Minimal or slight disorgan isation/degeneration/regene ration of the epithelium. Minimal squamous metaplasia of the epithelium was only seen in animals exposed to 19098 ppm. In the transitional epithelium, minimal or slight disorganisatio n/regeneration of the epithelium was seen in male and female animals exposed to 4721, 8901 or 19098 ppm. In the nasolacrimal ducts, mini mal epithelial degeneration was seen in one female exposed to 19098 ppm and minimal or slight inflammation wa s seen in animals exposed to 4721, 8901 or 19098 ppm. In the airway, minimal or slight debris and inflammatory cells were also seen in animals exposed to 4721, 8901 or 19098 ppm.Huntingdon Life Sciences WAG0013

Issue No: 2 Page 5 of 10 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 4721 8901 19098 0 4721 8901 19098 Olfactory epithelium Fibrosis with or without associated cartilage formation in dorsal lamina propria Minimal Slight Moderate

Marked 0 0 0 0 2 0 0 0 0 1 2 0 0 1 0 2 0 0 0 0 3 0 0 0 0 0 3 0 0 0 3 0 Total 0 2 3 3 0 3 3 3 Bowman’s gland atrophy in dorsal lamina propria Minimal Slight Moderate

Marked 0 0 0 0 2 1 0 0 0 3 0 0 0 0 1 2 0 0 0 0 0 0 3 0 0 0 3 0 0 0 3 0 Total 0 3 3 3 0 3 3 3 Olfactory nerve fibres - atrophy Minimal Slight Moderate 0 0 0 1 2 0 0 3 0 0 3 0 0 0 0 2 1 0 0 2 1 0 2 1 To tal 0 3 3 3 0 3 3 3 Necrosis Minimal 0 0 0 2 0 0 1 0 Total 0 0 0 2 0 0 1 0 Erosion Minimal 0 1 1 2 0 1 1 1 Total 0 1 1 2 0 1 1 1 Degeneration/atrophy/disorganisation Minimal Slight Moderate 0 0 0 3 0 0 1 1 1 0 2 1 0 0 0 1 1 0 0 3 0 0 1 2 Total 0 3 3 3 0 2 3 3 Squamous metaplasia Minimal 0 3 2 1 0 3 3 2 Total 0 3 2 1 0 3 3 2 Respiratory metaplasia with or without associated inflammatory cell infiltrate Minimal Slight 0 0 2 0 1 2 1 2 0 0 3 0 3 0 2 0 Total 0 2 3 3 0 3 3 2Huntingdon Life Sciences WAG0013

Issue No: 2 Page 6 of 10 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 4721 8901 19098 0 4721 8901 19098 Turbinates Fusion of dorsal turbinate with associated epithelial diverticula formation Minimal Slight Moderate 0 0 0 0 0 0 0 0 3 0 1 2 0 0 0 1 0 0 0 0 3 0 0 3 Total 0 0 3 3 0 1 3 3 Respiratory epithelium Disorganisation/Degeneration/ Regeneration Minimal Slight 0 0 1 0 3 0 3 0 0 0 1 0 1 1 2 1 Total 0 1 3 3 0 1 2 3 Squamous metaplasia Minimal 0 0 0 3 0 0 0 1 Total 0 0 0 3 0 0 0 1 Transitional epithelium Disorganisation/Regeneration Minimal Slight 0 0 2 0 2 0 2 1 0 0 2 0 2 0 2 1 Total 0 2 2 3 0 2 2 3 Nasolacrimal ducts Epithelial degeneration Minimal 0 0 0 0 0 0 0 1 Total 0 0 0 0 0 0 0 1 Inflammation Minimal Slight 0 0 1 0 1 0 0 2 0 0 1 0 0 0 2 0 To tal 0 1 1 2 0 1 0 2 Airway Debris and inflammatory cells Minimal Slight 0 0 2 0 1 0 0 1 0 0 0. 0 2 0 1 0 Total 0 2 1 1 0 0 2 1 Number of tissues examined 3 3 3 3 3 3 3 3Huntingdon Life Sciences WAG0013

Issue No: 2 Page 7 of 10 

### Larynx In the ventral larynx, minimal squamous metapl asia was seen in male and female animals exposed to 8901 or 19098 ppm and minimal epithelial hyperplasia was seen in one male exposed to 19098 ppm. In the ventrolateral larynx, minimal epithelial keratinisation was seen in male and female animals of all groups exposed to 2-bromo-3,3,3-trifluoropropene. In the arytenoids, minimal or slight squamous metaplasia and minimal epithelial hyperplasia were seen in animals of all test article groups. Minimal or slight inflammation was seen in one male and one female exposed to 19098 ppm and one male exposed to 8901 ppm.

Summary of treatment related findings in the lary nx for animals killed after 2 weeks of treatment Group/sex 1M 2M 3M 4M 1F 2F 3F 4F Achieved Exposure Level (ppm) 0 4721 8901 19098 0 4721 8901 19098 Squamous metaplasia - ventral Minimal 0 0 1 2 0 0 3 3 Total 0 0 1 2 0 0 3 3 Epithelial hyperplasia - ventral Minimal 0 0 0 1 0 0 0 0 Total 0 0 0 1 0 0 0 0 Epithelial keratinisation - ventrolateral Minimal 0 2 2 3 0 2 2 3 Total 0 2 2 3 0 2 2 3 Squamous metaplasia - arytenoid Minimal Slight 0 0 1 1 3 0 1 1 0 0 2 0 3 0 3 0 Total 0 2 3 2 0 2 3 3 Epithelial hyperplasia - arytenoid Minimal 0 1 1 0 0 0 0 2 Total 0 1 1 0 0 0 0 2 Inflammation Minimal Slight 0 0 0 0 1 0 1 0 0 0 0 0 0 0 0 1 Total 0 0 1 1 0 0 0 1 Number of tissues examined 3 3 3 3 3 3 3 3Huntingdon Life Sciences WAG0013

Issue No: 2 Page 8 of 10 

### Trachea Minimal or slight epithelial de generation/regeneration was seen in male and female animals exposed to 19098 ppm and one female expos ed to 8901 ppm. Minimal or slight inflammatory cell infiltrate in the lamina propriawas seen at a higher incidence and/or degree in animals exposed to 19098 ppm, than in control animals.

Summary of treatment related findings in the trac hea for animals killed afte r 2 weeks of treatment Group/sex 1M 2M 3M 4M 1F 2F 3F 4F Achieved Exposure Level (ppm) 0 4721 8901 19098 0 4721 8901 19098 Epithelial degeneration/regeneration Minimal Slight 0 0 0 0 0 0 1 2 0 0 0 0 1 0 2 0 Total 0 0 0 3 0 0 1 2 Inflammatory cell infiltrate – lamina propria Minimal Slight 1 0 1 0 0 0 1 1 0 0 0 0 1 0 0 1 Total 1 1 0 2 0 0 1 1 Number of tissues examined 3 3 3 3 3 3 3 3Huntingdon Life Sciences WAG0013

Issue No: 2 Page 9 of 10 

# 3. Discussion In the nasal turbinates, the findings related to treatment were generally seen in the tissues of the dorsal half of the nasal cavity, with the se verity of findings increasing in a rostral to caudal direction and the olf actory epithelium being the most extensively affected. In the region of the olfactory epithelium, fi brosis with or withoutassociated cartilage formation, atrophy of the Bowman’s glands and atrophy of olfactory nerve fibres were seen in the dorsal lamina propria and erosion of the epithelium wereseen in animals of all groups exposed to 2-bromo-3,3,3-trifluoropropene. Necros is of the olfactory epithelium was seen in animals exposed to 8901 or 19098 ppm. The fibros is with or without associated cartilage formation is considered a protective response to irritation by the test substance, which would have caused the necrosis and erosion, with th e atrophy of the Bowman’s glands and olfactory nerves being either a direct effect of the irritation or a consequence of the epithelial degeneration. The fusion of dorsal turbinates with associated epithelial diverticula formation seen in animals of all test article groups, was consid ered a result of damage to the epithelium overlying the turbinates and dorsa l aspect of the nasal cavity with subsequent fusion of the turbinates and formation of epithelial diverticula. In the various epithelia of the nasal cavity (olfactory, respiratory and transitional) of animals of all test article groups similar findings were seen. These included metaplasia and combinations of disorganisation, atrophy, dege neration and regeneration. The metaplasia was an adaptive change to produce a type of epithelium which was more resistant to the irritation. The combinations of disorganisation, atrophy and degeneration were due to the irritation of the epithelium and the regenerati on was part of the ti ssue repair process. In the nasolacrimal ducts, inflammation was s een in animals of all test article groups. Although epithelial degeneration was seen in on ly one female exposed to 19098 ppm, both the inflammation and epithelial de generation are considered due to the irritancy of the test substance. In the airway, debris and inflammatory cells were seen in of all testarticle groups and are considered a consequence of the other changes in the nasal cavity. In general, the changes seen in the nasal turbinates of animals exposed to 8901 or 19098 ppm were of a similar severity and incidence and of a higher severity than those seen for animals exposed to 4721 ppm. In consideration of the epithelial changes the olfactory epithelium was the most extensively affected at all three exposure levels. In the larynx, epithelial metaplasia, hyperplasia and keratinisation were seen in animals of all test article groups. As with th e nasal epithelia, thes e changes were an adaptive response to produce an epithelium more resist ant to irritation. Inflammati on was seen in one male and one female exposed to 19098 ppm and one male exposed to 8901 ppm and was considered a consequence of irritancy. In general, the findings in the larynx were of a similar incidence and degree in all groups exposed to 2-bromo-3,3,3-trifluoropropene.Huntingdon Life Sciences WAG0013

Issue No: 2 Page 10 of 10 

### In the trachea, epithelial dege neration/regeneration was seen in male and female animals exposed to 19098 ppm and one female exposed to 8901 ppm. As in the nasal cavity, the degeneration was due to the irritation of the epithelium and the regeneration was part of the tissue repair process. Inflammatory cell infiltrate in the lami na propria was seen at a higher incidence and/or degree in animals exposed to 19098 ppm, compared with control animals, which again was considered a consequence of irritancy. Overall, the most marked changes were in th e nasal cavity, with less severe (minimal or slight) changes in the larynx and trachea. For the nasal cavity, ingeneral the changes seen in animals exposed to 8901 or 19098 ppm were of a si milar severity and incidence. As rodents are obligate nose-breathers, this distribution of findings is considered accounted for by the nasal cavity being the first part of the respiratory tract to have contact with th e test article. In addition, the nasal epithelium, and especially th e olfactory epithelium which is anatomically confined to the dorsal half of the nasal cav ity, is a site of metabolic enzymes and so metabolism of the test substance may have c ontributed to the severity of changes seen. At necropsy, a reduction in the size of the thymus and depressions or roughening of the stomach were noted in animals exposed to 19098 pp m. These tissues were not required to be examined microscopically. 

# 4. Conclusion Changes related to treatment with 2-bromo-3,3,3 -trifluoropropene, were seen in the nasal turbinates, larynx and trachea following i nhalation administration for 2 weeks.Huntingdon Life Sciences WA G001 3

217 

### Annex 3 Certificate of AnalysisHuntingdon Life Sciences WA G001 3

218Huntingdon Life Sciences WA G0013

219 

### Annex 4 GLP compliance statement sHuntingdon Life Sciences WA G0013

220 

### EYE RESEARCH CENTRE GLP COMPLIANCE STATEMENT 2011Huntingdon Life Sciences WA G0013

221 

### HUNTINGDON RESEARCH CENTRE GLP COMPLIANCE STATEMENT 2011