# Huntingdon

AGENT873 ANINHALATION STUDY TOINVESTIGATE THE CARDIAC SENSmSATION POTENTIALINTHEBEAGLE DOG

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# I

AAS 001/014630

AGENT873 ANINHALATION STUDY TO INVESTIGATE THE CARDIAC SENSITISATION POTENTIALINTilE BEAGLE DOG

Sponsor 

### BP Exploration Alaska, PO Box 196612, 900 

### EastBensonBoulevard, Anchorage, 

### AK 99519-6612, USA

Page 1

### of33 Research Laboratory 

### Huntingdon Life Sciences Ltd., Woolley Road, Alconbury, Huntingdon, Cambridgeshire, PE284HS, ENGLAND. 

### Report issued: 16th 

### September 2002

## AAS 001/014630 CONTENTS 

### Page 

## COMPLIANCE wnH

## GOODLABORATORYPRACTICE STANDARDS....................... 4 

## QUALITYASSUR.ANCE STA TEI.\.1ENT
................................................................................. 5 

## CONTRIBUTIN"G SClENTISTS................................................................................................ 6**S~Y.................................................................................................................................** 7 

## INTRODUCTION....................................................................................................................... 8 

## TEST SUBSTAN"CES................................................................................................................. 9 

## MATERIALS AND 1\ffiffiODS................................................................................................ 10**EXPE~ALDESIGN** 

## ...................................................................................................... 13, 

## RESULTS.................................................................................................................................... 15 

## DISCUSSIONAND CONCLUSION......................................................................................... 17 

## REFERENCES............................................................................................................................ 18

: 2 :

### AAS 001/014630 Page 

### FIGURES 1. 

### Exposure system..................................................................................................................... 19


### TABLES 1. 

### Summary ofcardiac responses to adrenaline administration during Agent 873 exposure .. 20 2. Mean analysed concentrations of2-bromo-3,3,3-trifluoropropene
...................................... 21
APPENDICES 1. Results ofStage 1

### -individualresponses to adrenaline alone.............................................. 22 2. Results ofStage 2 - cardiac responses to adrenaline administration during exposure to 

### air only................................................................................................................ 23 3. 

### Results ofStage 2

- 

### cardiac responses to adrenaline administration during exposure to Agent 873
........................................................................................................... 24 4. Methods ofsample collection and analysis for 2-bromo-3,3,3-trifluoropropene................. 25 5. Gas bag concentrations of2-bromo-3,3,3-trifluoropropene- individual sample values...... 32 HUNTINGDON RESEARCH CENTRE GLP COMPLIANCE STATEMENT 2001............ 33



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### AAS 001/014630 COMPLIANCE WITH GOOD LABORATORY PRACTICE STANDARDS

The study described in this report was conducted in compliance with the following Good Laboratory Practice standards and I 

### consider the data generated to be valid: The United Kingdom Good Laboratory Practice Regulations, 1999, (Statutory Instrument No. 3106). OECD Principles ofGood Laboratory Practice (as revised in 1997), ENV/MC/CHEM (98) 17. EC Council Directive, 1999/11/EC of8 March 1999, (Official Journal No. L 77/8). Information regarding the purity and expiry date ofthe test material, were not available to Huntingdon Life Sciences for compliance with the Good Laboratory Practice Regulations given above
.



# .......

# ~...................................... PaulJ. 

### Horrell, B.Sc. (Hons.), StudyDirector, Huntingdon Life Sciences Ltd.

: 4: 

## 16**~1'4'f18ttl... ~** 

..........................**-~·** ... • 6 •• ................................. 

### DateQUALITYASSURANCE STATEMENT

The following have been inspected or audited inrelation to this study: Study Phases Inspected ProtocolReview Study Based Inspections Studypreparation ) Test itemcontrol and disposition ) Exposure and generation of test atmospheres ) Atmosphere sampling ) Clinical signs examination ) Report audit Date oflnspection 09 July 2001

23 August 2001

04 September 2002 AAS 001/014630

Date ofReporting 12 July 2001

28 August 2001

05 September 2002 Protocol: An audit of the protocol for this study was conducted and reported to the Study Director and Company Management as indicated above. Study based inspections: Inspections and audits ofphases ofthis study were conducted and reported to the StudyDirector and CompanyManagement as indicated above. Process based inspections: At or about the time this study was in progress inspections and audits of other routine and repetitive procedures employed on this type ofstudy were carried out. These were promptly reportedto appropriate Company Management Report Audit: This report has been audited by the Quality Assurance Department. This audit was conducted and reported to the StudyDirector and Company Management as indicated above. The methods, procedures and observations were found to be accurately described and the reported results to reflect the raw data.**6.~~**

### .................................. AmandaThompson, M.R.Q.A., Group Manager, Department ofQuality Assurance, Huntingdon Life Sciences Ltd. Date

5

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# I STUDY MANAGEMENT Paul J. Horrell, B.Sc. (Hons.), Study Director.



### TOXICOLOGY CONTRIBUTING SCIENTISTS

Colin J. Hardy B.Sc., Ph.D., M.I.Biol., C.Biol., Dip.R.C.Path (Toxicology), Principal Toxicologist,



### ANALYSIS Ian S. Gilkison, M.A., Ph.D., Section Head, Aerosol Technology and Analysis.

: 6 : AAS 001/014630

# 1
AAS 001/014630 SUMMARY

This studywas designed to assess the cardiac sensitisationpotential ofAgent 873 inbeagle dogs. Adrenaline was administered by intravenous injection before and during inhalation oftest substance. The effect of the adrenaline on the ECG pattern was examined. Positive evidence of cardiac sensitisation would be observed as the presence of multiple multifocal ectopic beats or ventricular fibrillation following adrenaline administration during inhalation ofthe test substance. No such effect should

### be observed inthe absence ofthetest substance. Agent 873 was administered atconcentrations of0.49, 1.00 , and 1.50%v/v in air. At0.49% v/vAgent 873 there

### was noevidence ofcardiac sensitisation. At 1.00% v/v Agent 873, 1 outof6 dogs exposed gave a positive cardiac sensitisationresponse. At 1.50% v/v Agent 873, the single dog exposedgave a positive response. It is concluded :from the results ofthe study that for Agent 873 the No Adverse Effect Level is 0.49% v/v and the LowAdverseEffectLevel is 1.00% v/v.

: 7 :

# 1

I AAS 001/014630 

### INTRODUCTION

The potential of Agent 873 to cause cardiac sensitisation by inhalation in the beagle dog was determined. Six dogs from an available batch of9 were used on this study. All ofthe six dogs had been used for previous studies ofthe same type. Cardiac sensitisation to adrenaline is a phenomenon associated with the inhalation of a number of unsubstituted and halogenated hydrocarbons (Becket al, 

### 1973; Clark, 1973, 1982; Hardy, 1994). After inhalation ofthe sensitising agent, challenge with adrenaline causes cardiac arrhythmias. This type of response was first demonstrated by Levy (Levy, 1913) when he induced ventricular fibrillation in cats during inhalation ofchloroform, by injecting adrenaline. There are no regulatory guidelines for this type of study, therefore design was in accordance with accepted pharmacological principles and methods (Reinhardt, 1971, 1973). The technique used involved the intravenous injection ofadrenaline before and during the inhalation of the test gas. The effect of adrenaline on the electrocardiogram was examined in both cases and compared to 

### assess any positive response to the test gas. It should be noted that the mammalian response to exogenous adrenaline can be dependant upon a number ofbiological factors alone or in combination for example plasma hormone levels and temperament. This stresses the importance of acclimatisation and training. The beagle dog was chosen for three reasons. Firstly available cardiac sensitisation data pertains to beagles and therefore provides comparative data. Secondly, a docile and accommodating species is required to accept training and venepuncture. Thirdly, a non-rodent species provides more suitable electrocardiographic data when extrapolatingthe data to man. The protocol was approved by the Study Director and Huntingdon Life Sciences Management on 28 June 2001 and by the Sponsor on 20 July 2001. 

### On completion ofthe study all data pertaining to the study, including a copy ofthis fmal report, were transferred to Huntingdon Life Sciences Ltd Archives. The data will be retained in Archives for at least 5years from the completion date for the study. Huntingdon Life Sciences will retain the Quality Assurance records relevant to this study and a copy of the fmal report indefinitely. The experimental start and finish dates for this study were 30 July and 26November 2001 respectively. The Stage II exposures were carried out during the period 13 August to 12 September 2001.

8

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Commonname:
Chemicalname: 

### Purity:
Presentation: Storage conditions:
Batch(lot): Expirydate:

Datereceived: Supplier: AAS 001/014630 

### TEST SUBSTANCES
Agent 873 2-bromo-3, 3, 3-triflouropropene
Notgiven liquid
Ambient temperature andhumidity 

### in the

### dark
#83-70
Not given. Assumed stable for the duration ofthe study 15May2001 Sponsor
The complete description ofthe chemical and physical properties ofthe test substance, together with informationregarding its purity, composition and stability are the responsibility ofthe Sponsor.



### : 9:

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I AAS 0011014630 MATERIALSANDMETHODS

TEST ANIMALS Nine**pure~bred** male beagle dogs, approximately 6- 16 months old, were available for the study. All dogs were obtained from InterfaunaUKLtd, Abbots RiptonRoad, Wyton, Huntingdon, England. Atthe 

### start ofthe Stage IIprocedures (Air only) the dogs weighedbetween9.5 and 19.5 kg. The dogs were eachidentified by a uniquenumber tattooed onthe 

### ear pinna by the supplier. Permanent study numbers were assigned to all the dogs. The individual permanent study number was tattooed on 

### the inside aspect ofthe lefthindleg.

ACCLIMATISATION The dogs were acclimatised to laboratory conditions and handling procedures. Each dog was further trained in order to acclimatise it to the exposure apparatus. The training consisted ofat least 5 sessions during which the dogs were restrained in a canvas sling whilst breathing through a face mask and non-return valve connected to a reservoir gas bagcontaining air only. Details oftraining are storedwith theraw data for this studybut arenot commentedon further inthisreport.

ACCOMMODATIONAND HUSBANDRY The dogs were housed in pens within a purpose designed dog holding facility (Building J24, Unit 25). Thepenswere designedin accordancewith therequirements oftheUnitedKingdomHome Office 

### Code ofPractice for Housing andCare ofAnimals usedin Scientific Procedures. The penshad a floor area of 4.5 square metres and could accommodate up to 2 dogs ofthe same sex and group. Thepens could be subdivided into equal 

### areas by means of 

### a moveable partition so that each day the dogs could be segregatedfor assessment ofindividual clinical signs and food consumption. Bothparts ofthe pen were fitted with an automatic valve for the supply of drinking 

### water. During week days the dogs were segregated from approximately 0800 to 1500hours. At weekends dogs were paired up at approximately 1200 hours. Animal room temperature controls were set to maintain, as far as possible, a temperature range of 15

- 25°C. Relative humidity was recorded continuously but was not controlled. Fluorescent lighting was controlledto give 12hours oflight and 12 hours ofdark in each24-hourperiod. Airextraction, via a balanced system, provided approximately 10

- 12 air changes per hour. Actual minimum and maximumtemperature andrelative humidityvalues were 17/23°C and40/90%respectively. The exposure sessions were carried out in the Department of Dog Toxicology (Building J24 Room 1005). The dogs were transported from theholding

### unit to the experimental

### Wlit

## asrequired.

10AAS 001/014630 Thedogs wereprovided with 500

- 600 g ofdry dietdaily (Diet A, Special Diets Services Ltd) and fresh tap water daily. There was no infonnation available to indicate to the Study Director the presence, in food or water, ofany non-nutrient substance likeJy toinfluence the outcome ofthis study. Theresults of chemical analysis duringthe study are lodged in the Huntingdon Life Sciences Archives. Aspart oftheroutine husbandry, food consumptionrecords and weekly bodyweights wererecorded and veterinaryrecords were maintained. These have been archived and arenot commented on further inthis 

### report On completion ofthe study the surviving dogs were retained for possible use in further studies ofthe sametype.

EXPOSURE SYSTEM (Figure 1) Thedogs were exposed to the test gasby a snout-only system. A Halls face mask (Phoenix Medical Ltd) was used. The mask consisted ofa rubber cone, one end of which 

### was connected 

### to the air supply line, while the other end was placed over the dog's snout. To ensure a reasonably airtight fit around the dog's snout, a latex sheet with a hole was placed over the snout end ofthe mask and the dog's snout protruded through the sheet into the mask The mask was held in place by a leather muzzle. The dog was restrained in a canvas sling through which the limbs extended,the dog couldthus supportitselfstanding orbe supportedbythe sling. The dog face mask was connected 

### via a non-return valve to a reservoir gas bag (fedlar® SKC Inc, USA) of 200 litres capacity. The dogs inhaled from the gas bag through the non-return valve and exhaledto waste. Duringthe first experimental sessionthe

### gas bagwas filled withroomaironly. For 

### gas exposures, the reservoir 

### gas bag contained known concentrations ofthe test material. The test atmosphere was produced by injecting volumes of the test material through a port into a gas bag containing a known volume ofair which had been metered into the bag through a wet-type 

### gas meter (Alexander Wright and Co (Westminster) Ltd). The liquid test substance was then allowed to vaporise completely. During the gas exposure period the dog was permitted to breathe through the non-return valve from the test gas bag and exhale to waste. In this way the concentration in the gas bag remained constant throughoutthe exposureperiod. The test atmosphere in the reservoir gas bag was sampled before and after exposure for each dog. Samples were analysed using infra-red spectrophotometry. Theprocedures are detailed inAppendix4.



### 11

# I AAS 0011014630 MEASUREMENT OFTHEELECTROCARDIOGRAM (ECG) 

### The standard lead 11 electrocardiograph was used throughoutthe study. Appropriate areas on the dog's limbs were shaved and electrode gel applied. Standard ECG limb leads were then connected to the prepared areas on the dog with blunt clips. The electrocardiograph (Electromed multi-trace 11) 

### was calibrated with 1mV peaks. 

### The electrocardiogram was 

### then checked for clarity before dosing commenced.

EXPERIMENTALPROCEDURE 

### The experimental procedure was based onpublished techniques (Reinhardt, 1971, 1973). The gas bags were prepared before each exposure session and the target concentration was established before any dogs were exposed. Once the level was established, the dog was placed in 

### the sling and fitted with the mask At this stage the dog was breathingroom air. Adrenaline solutions were prepared immediately prior to each exposure session from a stock solution (Adrenaline 1: 

### 1000, Batch 026007, Phoenix Pharmaceuticals Ltd., Gloucester, England and Batch 184575, Martindale Pharmaceuticals, Romford, Essex, England) and sterile pyrogen-free water (Fonnulation Department). Injections were given at a rate of0.1 mJJsecond. Appropriate dilutions were used such 

### that the volume ofadrenaline solution used was consistently 0.1 m1lkg

### bodyweight. The ECG was established (as describedpreviously) and the timing commenced as shownbelow: Time 

### (minutes) 0 2 7 

### 12 17 Event StartECGrecording**FastadRmatineQDl)cballengea~rered** Gas reservoir connected to airsupply line SecondadRmatine QD2) challenge**~ered** Gas reservoir disconnected Stop ECGrecording 

### The test animals were exposed to various test gas concentrations as detailed in the EXPERIMENTAL DESIGN 

### section ofthis report.



### 12 :

# I AAS 0011014630 

### EXPERIMENTAL DESIGN

The study was performed in 2 stages:



### STAGEl In

### this stage an individual response to adrenaline alone was selected for each ofthe 9 dogs using a range ofadrenaline doses. The response to administration ofadrenaline is dependant on the dose given and the responsiveness of each individual dog. Typically the response consists ofa transient increase in heart rate followed by a reflex slowing of the heart rate and an increase in the height of the T-wave, occasionally multiple unifocal ventriculartachycardia may occur. The purpose ofStage 1 ofthe study was to establish an adrenaline dose at which there was a clear but 

### minimal effect on the ECG ideally with a few ectopic beats. The actual dose level at which the various responses OCCUlTed was dependent on the individual dog. A suitable adrenaline dose had been selected for each ofthe previously used dogs and was established for eachofthe naive dogs in the following manner. The initial dose adrenaline for each dog was 4 

### J.Lg/kg. Ifthe response to 

### the first adrenaline challenge was too large, as defined by an excessive number ofectopic beats (more than 

### approximately 

### 1 0) or following both injections there were no ectopic beats, the experimental session did not proceed further and the dog was allowed to rest for at least 20 minutes. The procedure was then repeated with an adrenaline dose halfor double that previously given (except that the highest dose was 12 J.Lg/kg, ie: 

### if there 

### was no response at 8 J.Lg/kg, this was followed by 12 J.Lg/kg). It 

### was recognised that there were occasions when a judgement needed to be applied, for example, if there were 15

- 20 ectopic beats at 8 Jig/kg but at 4 

### J.Lg/kg there were none, then 8 Jlg/kg was likely to be acceptable as a suitable dose. Each previously used dog was re-tested at the established dose and where necessary a new adrenaline dose 

### was established. If

### no ectopic beats were observed for a particular dog the adrenaline dose was increased until a response was observed. Conversely, iftoo many ectopic beats were observed the dog 

### was allowed to recover and the adrenaline dose 

### was reduced until an acceptable dose was found. A maximum dose of 12 J.Lg/kg was used. If

### necessary, the Study Director and monitoring toxicologist applied ajudgement in the selection ofan appropriate adrenaline dose in the bestinterests ofthe dog andthe study integrity. When an appropriate dose had been selected, that dose was used for the exposures in Stage 2.

13AAS 001/014630 Each of the dogs allocated to the study 

### was tested as indicated in the EXPERIMENTAL PROCEDURE, exceptthatnotest gas was used andthe dogs received air only for the 17 minutes. On completion of Stage 1, 6 of the dogs were allocated for use in Stage 2. Details ofthe results of Stage 

### 1ofthe study and the mtionale for the selection ofthe dogs for Stage 

### 2 ofthe study are given in the RESULTS section ofthis report.

STAGE2 Each ofthe dogs selected for Stage 2 was exposed to the test 

### gas according to the EXPERIMENTAL PROCEDURE. Thedogs were scheduled to beexposed according to the following schedule, Exposure session 1 

### 2 3 4 5 6 7 Agent 873 concentration (% v/v in air) Aironly 1 3 5 7 10 15 Atleastone calendardaywas plannedbetween eachexposure sessionto allowthe dogs to recover.

JNTERPRETATION OF RESULTS The study was designed to provide infonnation as to any dose level that gave rise to clear signs oftest gas related cardiac sensitisation. The criterion for a positive effect was the appearance of a burst of multifocal ventricular ectopic activity (MVEA) or ventricular fibrillation (VF) at any time during exposuretothe test gas. Interpretation ofthe results was made for each dog, taking into account the response ofeach dog during Stage 1ofthis study.

DEVIATIONS FROMPROTOCOL The correct name ofthe dog breeder is Harlan UK Ltd, not Harlan UK U.K. Ltd as stated in section 4.1.1 ofthe Protocol and Protocol Amendment 1. The date of arrival, at Huntingdon Life Sciences, for Dogs 1061, 1063, 1065 and 1067 was incorrectly stated, inProtocol Amendment 1, as 10/07/00. The correct da.te ofarrival was 10/07/01. Dog 1061 was removed from the study, prior to the start ofStage 1 procedures due to 

### ill health, and was replaced withDog 1315. These deviations fromProtocol have hadno impact onthe integrity ofthe study.
14 :

### AAS 001/014630 RESULTS

STAGE 1- CHALLENGES WITH ADRENALINE ALONE 

### The results of 

### the experimental sessions in which the dogs were challenged with adrenaline in 

### the absence ofany test gas are summarised in Appendix 1. On 

### the basis ofthese results the dose ofadrenaline selected for each dog was: 1 

### )lgfkg: 2 

### )lgfkg: 8 

### )lgfkg: 

### 12 Jlg/kg: 

### Dog: Dogs: Dog: Dogs: 1065 1315,1063, l067and 1133 1003 1001 and 1131 Dog 1307 wouldnot tolerate the challengeprocess.

STAGE 2- EXPOSURE TOTESTATMOSPHERE Six 

### dogs were selected from the 9 

### available. The dogs selectedrepresented a range ofresponses to adrenaline. The following dogs were selected: Dog number 1001 1003 1063 1065 1131 1133 Adrenaline dose (Jlg/kg) 12 8 2 1 12 2



### 15 

# t

# I AAS 0011014630 Theresults are presented as follows: Table 1 Summary ofcardiac responses to adrenaline administration during Agent 873 exposure. Appendix 2 - Results of Stage 2 - Cardiac responses to adrenaline administration during exposure to air only. Appendix 3

- Results of Stage 2 - Cardiac responses to adrenaline administration during exposure to Agent 873. The achieved exposure concentrations for each dog are presented as follows: Table 2 Mean analysed concentrations of2-bromo-3,3,3-trifluoropropene Appendix 5 - 

### Gas bag concentrations of 2-bromo-3,3,3-trifluoropropene- individual sample values The original exposure regimen required exposure at an initial concentration of 1% v/v in airprogressing 

### to 15% v/v in air. However, due to a positive response seen during the first exposure the regimen was revised. Following the positive response at 1.00% v/v the sensitisation potential was confirmed by exposing a single animal to 1.5% v/v. Subsequently an exposure at halfthe concentration used in the firSt exposure was conducted in order 

### to either further refine the Low Adverse Effect Level orestablish a No Adverse Effect Level. 

### Ifthe latter occurred no further exposures would be required. The revised target concentrations and actual gas bag concentrations adchived for 2-bromo-3,3,3-trifluoropropene are summarised below: Target concentration (Oft• v/v) 1.0 1.5 0.5 Mean achieved concentration (% vtv)

1.00 1.50 0.49 There was good agreementbetweenachieved and target concentrations oftest material. Theresults ofexposure arepresentedbelow: At the 1.00% level 6 dogs were exposed. Clinical signs included agitation, deep slow breathing, muscle tremors and struggling. One animal responded positively with an episode ofmultifocal ventricular ectopic activity (MVEA) approximately 6 seconds after administration of the second adrenaline challenge. Atthe 1.50% level 1dog was exposed. Clinical signs included deep slowbreathing, agitation and limb tremors. The animal responded positively with an episode of M:VEA approximately 13 seconds after administration ofthe second adrenaline challenge. At the 0.49% level 6 dogs were exposed. Clinical signs included agitation, deep slow breathing andmuscle tremors. There were nopositive cardiac responses.



### 16 :AAS 001/014630 

### DISCUSSION AND CONCLUSION
Agent 873 is beingdevelopedbythe Sponsorfor fire suppressant use. 

### In this study the 

### Low Adverse Effect Level was 1.00% v/v and the No Adverse Effect Level was 0.49%v/vAgent 873 in air.



### 17 :

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I AAS 001/014630 

### REFERENCES

BECK PHILLIPPA, S., CLARK., D.G. AND TINSTON, D.J. (1973). The phannacological actions of bromochlorodifluormethane (BCF) 

### Tax. andApp. Pharmacal., 24, 1 • 10. CLARK, D.G. and TINSTON, D.J. (1973). Correlation ofthe cardiac sensitisation potential ofsome halogenated andnon-halogenatedhydrocarbons.

### Brit.j. Pharmacal., 49, 355 • 357. CLARK, D.G. 

### and TINSTON, D.J. (1982). Acute inhalation toxicity of some halogenated and non-halogenated hydrocarbons

### Human Taxical., 1, 239- 247. HARDY, C.J., KIERAN, P.C., SHARMAN, 

### I.J. (1994). Assessment of the cardiac sensitisation potential ofa range ofhalogenatedmaterials. PosterPresentation at Society ofToxicology 1994

### Annual Meeting, Dallas, Texas. LEVY, A.G. (1913), The exciting causes of ventricular fibrillation in animals under chloroform anaesthesiaHeart, 4, 319-378. RElNHARDT

### et al (1971). Cardiac arrhythmias and aerosol sniffing

### Arch. Env. Hlth., 22,265-279. REINHARDT et 

### al (1973). Epinephrine induced cardiac arrhythmia potential of some conunon industrial solvents.!. 

### Occup. Med., 15, (12) 953-955.

18

# I

I FIGUREl Exposure system



## w

v

GB Gas bagreservoir 

### M Mask V Non-rebreatldngvalve W 

### Waste To breathe the air

### Vis opento ambient To dose GB is 

### connectedto V

19 : AAS 001/014630



### GB

# 1

I TABLE I

. Summaryofcardiac responses to adrenaline administration during Agent 873 exposure AAS 001/014630

Dog Adrenaline dose Concentrationin air_(% v/v) number (112/k!) 0.49 1.00 1.50

1001 

### 12 N N NP 1003 8 N N P(MVEA) 1063 

### 2 N N NP 1065 1 UA N NP 1131 

### 12 N P(MVEA) AP 1133 2 N N NP Number positive 016 116 2/2 N Negative response P Positive response UA Unable to assess due to struggling disturbing ECG recording NP Notperformed AP Assumed positive due to positive response at lower concentration MVEA Multifocal ventricular ectopic activity

20 I

### AAS 0011014630 TABLE2 

### Mean analysed concentrations of2-bromo·3,3,3-trifluoropropene T 0 5° I

arget . *:1 v v Dog identification Mean exposure concentration (ppm) 

### I (% v/v) 

### 1131 4932 0.493 1001 4911 0.491 1003 4945 0.494 1063 4928 0.493 1133 4866 0.487 1065 4866 0.487 Mean 

### 4908 0.491 T 10o/c I

arget . oVV Dog identification Mean exposure concentration (ppm) 

### I (%v/v) 

### 1001 10200 1.02 1131 10180 1.02 1003 9946 0.99 1063 9876 0.99 1133 9929 0.99 1065 9950 0.99 Mean 

### 10014 1.00 T 1So/c I

arget . oVV Dog identification Meanexposure concentration (ppm) 

### I (% v/v) 

### 1003 14988 1.50



### : 21

### AAS 001/014630 APPENDIX I 

### Results ofStage 1 - individual responses to adrenaline alone Dog Adrenaline Description ofcardiac response following number dose adrenaline challenge*



## *

L H A NP (!lg/kg) Number ofectopic beats Connnent (response) First Second challenge challenge 

### 1001 4 

### 0 0 L 8 

### 0 0 L 

### 12 1 11 A 

### 1003 4 0 0 L 8 

### 0 2 A 1315 

### 4 23 NP H 

### 2 0 

### 0 A 

### 4 19 29 H 

### 1063 4 0 15 H 

### 2 0 8 A 

### 1065 4 20 NP H 

### 2 14 16 H 1 13 9 A 

### 1067 4 11 

### 40 H 

### 2 0 0 A 

### 4 28 NP H 1131 

### 12 0 1 A 1133 

### 2 0 18 A 1 0 0 L In this appendix, mention is made only ofectopic beats or unexpected responses. The expected response to intravenous injection ofadrenaline (initial tachycardia followed by bradycardia and increase in height of T-wave) is notmentioned inthe "Descriptionofcardiac response" Adrenaline dose consideredtoo low Adrenaline dose considered too high Adrenaline dose considered acceptable Notperformed due to excessive response after first challenge



### : 22

### AAS 0011014630 

### APPENDIX2 Results ofStage 2 - Cardiac responses to adrenaline administration

, during exposure to 

### air only Dog Adrenaline Description ofcardiac response 

### number dose following adrenaline challenge*



## *
(N)**(~glkg)** Number ofectopic beats Comment First Second (response) challenge challenge 1001 12 0 13 (N) 1003 8 1 4 (N) 1131 12 0 0 (N) 1133 2 0 3 (N) 1063 2 0 0 (N) 1065 1 0 0 _(N) In this appendix, mention is made only of ectopic beats or unexpected responses. The expected response to intravenous injection of adrenaline (initial tachycardia followed by bradycardia and increase inheight ofT -wave) is not mentioned inthe "Description ofcardiac response". Negative response

: 23AAS 001/014630 APPENDIX3 Results ofStage 2 - cardiac responses to adrenaline administration during exposure to Agent 873 Gas Dog Adrenaline Descriptionofcardiac response concentration number dose following adrenaline challenge* (%v/v) (Jlglkg) Number ofectopic Comment beats (response) First Second challenge challenge 1.00 1001 12 10 1 Agitation, arched back, pawing atmask, vocalising, deep slowbreathing, struggling (N) 1131 12 0 MVEA Agitation, arched back, deep slowbreathing, (VEA) struggling, pawing at mask, back legs tense (P) 1003 8 8 8 Agitation, archedback, muscle tremors, deep slowbreathing (N) 1063 2 0 3 Arched back, deep slow breathing, agitation, struggling (N) 1133 2 2 8 Agitation, archedback, deep slow breathing (N) 1065 1 1 0 Agitation, pawing at mask, muscletremors, struggling (N) 1.50 1003 8 1 MVEA Deep slowbreathing, arched back , agitation, limb tremors_JP) 0.49 1001 12 0 1 Agitation, deep slow breathing, archedback, muscle tremors, shallow breathing (N) 1131 12 1 1 Agitation , deep slowbreathing, salivation, muscle tremors (N) 1003 8 0 2 Agitation, deep slow breathing, muscle tremors, shallow breathing (N) 1063 2 0 0 Agitation, archedback, muscle tremors, shallow breathing, struggling (N) 1133 2 0 3 Arched back, deep slow breathing, agitation (N) 1065 1 0 UA Violent strugg}ing(UA) 

## * In this appendix, mentton is made only ofectopic beats or unexpected responses. The expected response to intravenous injection of adrenaline (initial tachycardia followed by bradycardia and increase in height of T-wave) is not mentioned in the "Descriptionofcardiac response" (N) (P) (UA) VEA MVEA Negative response Positive response Unable to assess due to struggling disturbing ECG recording. Ventricularectopic activity Multifocal ventricular ectopic activity



### 24AAS 0011014630 APPENDIX4**Meth~ds** ofsample collection and analysis for l-bromo-3,3,3-triftuoropropene .·"
SAMPLE COLLECTION Chamber concentration Samples ofthe test atmosphere were drawn directly from the 200 litre gas-sampling bag containing the test atmosphere into the cell ofthe infra-red (IR) spectrometer to exhaust using a Metal Bellows sample pump. The absorbance at 8.6 J.lm was measured relative to room air and compared to that of standards prepared in gas-sampling bags. The method ofsample analysis is detailed, together with a summary of the method validation, in the Inhalation Analytical Procedure at the end of this Appendix. Preparation ofthe test atmospheres Thetest atmospheres were prepared in gas samplingbags (ca. 200 litre capacity). A known volume of air was metered into the bag and the required volume of2-bromo-3,3,3-trifluropropene vapour added. The contents ofthe bag weremixed thoroughly prior to use.

CALCULATIONS The method for calculating the approximate 2-bromo-3,3,3-trifluropropene concentration from the volumes used to prepare eachbag is as follows:

where V 

### Va = Concentration

## (ppm)= V 

## x106 Va+V
volume of2-bromo-3,3,3-trifluropropene vapour added to the bag (litres); volume ofair added to thebag (litres). The exact concentration of 2-bromo-3,3,3-trifluropropene present in each test atmosphere was determined analytically by 1R analysis.
In order to minimise the cumulative errors that result from repeated rounding ofnumbers, much ofthe data in this report has been calculated continuously using unrounded numbers and only rounded for printing. Consequently, these rounded numbers may include rounding errors in the last significant figure andrecalculation may lead to small apparent discrepancies with other data inthe

### report.

: 25

# t ! 1 

## I l 

## l i 

# i 

# t 

# l AAS 001/014630 APPENDIX4 (Method ofsample collection and analysts for 2·bromo-3,3,3-trifluoropropene

- continued)

Compound specificInhalation Analytical Procedurefor 2-Bromo-3,3,3-trifluoropropene The analysis of2-Bromo-3,3,3-trifluoropropene In air sample substrate The method outlined in this document has been validated and is considered fit for the purpose of monitoring conditions in anInhalation Toxicology study. This document details the basic procedures for the analysis of 2-Bromo-3,3,3-trifluoropropene sampled directly from a test chamber. The test atmosphere and gas calibration standards are quantified by IR. Study specific amendments and additions will be detailed within a supplementarydocument. 

# IEFFECTIVE FROM: 

# I 16 August 2001

Test substance 2-Bromo-3,3,3-trifluoropropene, has thefollowing formula H2=CBrCF3 Appearance Storage Reagents N/A Equipment Infra-Red analyser Sample pump Gas samplebags Syringes Syringe valves Integrator Balanceand dataprinter Clearliquid Roomtemperature

Miran 

### lA serialno. 1A2123 Metal Bellows Corporation SKC "Tedlar" 3 or 5 litre capacity Hamilton series 500 

### (SOOml capacity) Hamilton series 1001 

### (l.Oml capacity) Mininertpush-valve with luer fitting Thermoquest Chromjet4400 Sartorius R200D with YDP-02 

### A 3 way valve was arranged to allow the flow of atmosphere through the flowcell to be rapidly switched between room air (used as the baseline measurement) and either chamber atmosphere or a standard gas bag. Consumables N/A
: 26 :

### AAS 001/014630 

### APPENDIX4 

### (Method ofsample collection and analysis for 2-bromo-3,3,3-triftuoropropene

- continued)
Preparation ofcalibration standards Draw the required amount of2-Bromo-3,3,3-trifluoropropene into a syringe (ofappropriate volume) fitted with a mininert syringe valve, taking care to remove any air bubbles from the syringe. Close the valve and accurately weigh (to 0.1 mg) the syringe and contents. After weighing, dispense the contents ofthe syringe into a Tedlar gas sampling bag (containing a known volume of air) via the injection port. Close the syringe valve and reweigh the syringe. From the weight of 2-Bromo-3,3,3-trifluoropropene dispensed the standard concentration is calculated using the following equations:

where Concentration

### (ppm)= V 

### xl,OOO,OOOppm Va+V



## v 

# w M 

### Gas constant Temperature (K) 

### Atm. Pressure 

# v. V = 

### W 

### x R 

### x T 

### x 760

### mm Hg

= 

## =

= = = 

## = M Atm
gaseous volume of2-Bromo-3,3,3-trifluoropropene (ml) 

### mass of2-Bromo-3,3,3-trifluoropropene (mg) molecular weight of2-Bromo-3,3,3-trifluoropropene (174.95 glmol) 0.08205 L 

### atm./mol.K temperature CCC) 

### +273 barometric pressure (mmHg) 

### volume ofair added to the calibration bag The range ofcalibration concentrations used in the measurement of 2-Bromo-3,3,3-trifluoropropene is detailed in the study specific supplement. 

### Storage ofstandards Type Standard gas bags (5,000- 20,000ppm) Storageconditions ambient



### : 27 Maximum storageperiod 1Day

# I

AAS 001/014630 

### APPENDIX4 (Method of**~ample** coDection and analysis for 2-bromo-3,3,3-trifluoropropene

- continued)

Calibration and quantification The instrument is allowed to warm-up for at least 

### IS minutes) then zeroed on room air. The wavelength ofthe instrument is fixed and room air is pumped through the flowcell immediately prior to measurement. Relative absorbance measurements are made by starting the integrator, switching the flow ofsample through the chamber for up to 10 seconds, then switching back to room air. The integrator measures peakheight which is proportional to absorbance ofthe sample. Calibrate by measuring replicates of each calibration standard) as detailed in the study specific supplement. Measure the absorbance (peak height) for each calibration standard and derive the line ofbest fit using an unweighted least squares linear method. For each sample, measure the absorbance at 8.6 J.l.m and determine the concentration of 2-Bromo-3,3,3-trifluoropropene presentusing the equation below: Concentration (ppm)= (A- I) 

## s Where 

### A = 

# s = I 

## = Absorbance ofthe sample Slope ofcalibration line derived from calibration data Intercept ofcalibrationline derived from calibration data Infra-red spectrophotometer conditions IRSpectrophotometer Cell type Operating wavelength Slitwidth Detectorrange Meterresponse settings Quality assurance measures Wilkes Miran lA-CVF 1empath length cell fitted with zinc selenide windows 8.6 J.l.ffi lmm 0.25 A for concentrations between 5,000 and 20,000 ppm 1 second

Calibration ofthe instrument will be performed with the appropriate standards oneach day ofuse. The highest calibration standard will be compared against a standard of similar concentration prepared independently. The ratio ofresponse factors will be acceptable ifwithin the range 0.95 to 1.05. A quality check standard must follow every 

### 6 test atmosphere samples for the analysis to be regarded as valid. The results ofthe quality check standards must lie within 

### 5% ofthenominal value. 

### Samples must lie within 

### the range ofacceptable quality check standards for the result to be regarded as valid. The LOQ for the run will be regarded as the concentration ofthe lowest acceptable quality check standard. : 28 :

### AAS 001/014630 APPENDIX4 (Method ofsample collection and analysis for 2-bromo-3,3,3-tritluoropropene- continued)
Summaryofmethod validation The raw data for the method validation is located in study AAS/00 1. Test blanks (room air, drawn 

### through the flow cell} produced no quantifiable peaks at the measurement wavelength. Precision data for repeatability (peak height) showed coefficients of variation for 2-Bromo-3,3,3-trifluoropropene of less 

### than 

### 5% using standard concentrations of 20,000 to 6,000ppm. Least squares regression analysis withno weighting ofthe peak height response against concentration of standard (20,000 to 6,000 ppm) produced a correlation coefficient of 0.9995 

### and relative errors less than 

### 3%. The Limit ofQuantification (LOQ) for 2-Bromo-3,3,3-trifluoropropene will 

### be set by the lowest acceptable check standard, however, the LOQ and Limit of Detection (LOD) are potentially 

### as low as 2,943 and 971 

### ppm respectively (calculated statistically using the standard deviation obtained for a standard ofconcentration 5,963 ppm). Standards of 2-Bromo-3,3,3-trifluoropropene in air of 20,000 to 6,000 ppm stored at ambient temperature for 1 day and subsequently analysed against fresh standards showed concentrations within 

### 5% oftheirnominal concentrations.

: 29:AAS 0011014630 

### APPENDIX4 (Method**~f** 

### sample collection and analysis for 2-bromo-3,3,3-triftuoropropene- continued)

AAS/001

- STUDY SPECIFIC SUPPLEMENT to the Inhalation analytical procedure for 2-Bromo-3,3,3-triftuoropropene This supplement details additions and amendments to the procedure to be used for the IR assay of 2-Bromo-3,3,3-trifluoropropene obtained from air samples collected on the above study. The assay, incorporating the additions and amendments, is suitable for the analysis of 2-Bromo-3,3,3-trifluoropropene, in air, at concentrations within therange of5,000 to 20,000 ppm. Details given inthis supplement supersede those in the compound specific

### lAP. 

# IEFFECTIVE DATE: l16August 2001

Analytical standard Name Batchnumber Purity Expirydate Supplier 

### Preparation ofcalibration standards2-Bromo-3,3,3-trifluoropropene 83-70 

### 98% 08/04 BP Exploration
Prepare calibration standards inthe nominal range 5,000 to 20,000 ppm. 

### Instrument conditions Detectorrange Attenuation 0.25A 1024 

# IEFFECTIVE DATE : 

# I23 August 2001

Instrument conditions Attenuation 2048 

# IEFFECTIVE DATE : 

# I28 Aupst 2001

Instrument conditions Detectorrange Attenuation lA 512

: 30 :

# I

I

·. 

# !
¥ AAS 001/014630 APPENDIX4 (Method ofsample collection and analysis for 2-bromo-3,3,3-triOuoropropene

- continued) 

# IEFFECTIVEDATE : 

# lu September 2001

Instrumentconditions Detectorrange Attenuation AdditionalMethod Validation 0.2SA 1024

Theraw data for this method validation is located in study AAS/001. Precision data for repeatability (peak height) showed coefficients of variation for 2-Bromo-3,3,3-trifluoropropene ofless than 1% using standard concentrations of9,000 to 3,000 ppm. Least squares regression analysis withno weighting ofthe peakheight response against concentration ofstandard (9,000 to 3,000 ppm) produced a correlation coefficient of0.997 and-relative errors less 

### than 4.5%. The Limit ofQuantification (LOQ) for 2-Bromo-3,3,3-trifluoropropene will be setby the lowest acceptable check standard, however, the LOQ and Limit ofDetection (LOD) are potentially as low as 275 and 91 ppmrespectively (calculated statistically using the standard deviation obtained for a standard ofconcentration 3,172 ppm). Standards of 2-Bromo-3,3,3-trifluoropropene in air of 9,000 to 3,000 ppm are stable on the day of preparation (when analysed against alternate standards they showed concentrations within 

### 5% of their nominal concentrations).

: 31

### AAS 0011014630 APPENDIXS Gas bag c?ncentrations of2-bromo-3,3,3-tritluoropropene

- individual sample values


### Target concentrat10n 

### 0 

### 5'Y< I oV v SampleiD Gas Measured concentration bag No. (ppm) 

## I (% 

### v/v) Pre dog 1131 1 4943 0.494 Post dog 1131, Pre dog 1001 1 4921 0.492 Post dog 1001 1 4900 0.490 Pre dog 1003 2 4948 0.495 Post dog 1003, Pre dog 1063 2 4941 0.494 Post dog 1063 2 4914 0.491 Pre dog 1133 3 4873 0.487 i Post dog 1065 3 4859 0.486 

### J
Target concentrat10n 

### 1**0~** 

### I oV.V SampleiD Gas Measured concentration bag No. (ppm) 

## I (% v/v) Pre dog 1001 1 10194 1.02 Post dog 1001, Pre dog 1131 1 10206 1.02 Post dog 1131 1 10154 1.02 Pre dog 1003 2 9983 1.00 Post dog 1003, Pre dog 1063 2 9910 0.99 Post dog 1 063 2 9842 0.98 Pre dog 1133 3 9929 0.99 Post dog 1133, Pre dog 1065 3 9930 0.99 Post dog 1065 3 9969 1.00 

### T t

arge concentra ton oVV f**15~** 

### I SampleiD Gas Measured concentration 

## I bag No. (ppm) 

## I (% v/v) Pre dog 1003 1 14950 1.49 

### ! Post dog 1003 1 15026 1.50 I



### 32AAS 001/014630 

### HUNTINGDON RESEARCHCENTRE GLP COMPLIANCE STATEMENT 2001

THEDEPARTMENTOFHEALTH OFTBEGOVERNMENT OFTHEUNITED KINGDOM GOODLABOR.Al'ORYPRACDCE

LA:BOifATOitY

DATE OF INSPECnON tsaJauuyHOt TEST TYPE

A .-a!**~011** for**~** wllb tho Prilldplu of Good Laboratory Pradice wu carried out 11 the obove lt.borak>ry u part of UK GLP Complilnco Pro...,.....,. At lbe lime of the**~on** 110 deoial!ola

# w- !OIIIId of aufllcieQI mapill«k to affect lbe 1raiUIII)' of--.:llnicollllllcllec pe.rfom1e<! at tbe&e facllillec.

33