Huntingdon 

AGENT873 

AN INHALATION STUDY TO INVESTIGATE THE CARDIAC SENSmSATION 

POTENTIAL IN THE BEAGLE DOG 

'.I 

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I 

AAS 001/014630 

AGENT873 

AN INHALATION STUDY TO INVESTIGATE THE CARDIAC SENSITISATION 

POTENTIAL IN TilE BEAGLE DOG 

Sponsor 

BP Exploration Alaska, 

PO Box 196612, 

900 East Benson Boulevard, 

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 GOOD LABORATORY PRACTICE STANDARDS....................... 4 

QUALITY ASSUR.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 

DISCUSSION AND CONCLUSION......................................................................................... 17 

REFERENCES............................................................................................................................ 18 

: 2 : 
AAS 001/014630 

Page 

FIGURES 

1. Exposure system..................................................................................................................... 19 

TABLES 

1. Summary of cardiac responses to adrenaline administration during Agent 873 exposure .. 20 

2. Mean analysed concentrations of 2-bromo-3,3,3-trifluoropropene ...................................... 21 

APPENDICES 

1. Results of Stage 1 -individual responses to adrenaline alone.............................................. 22 

2. Results of Stage 2 - cardiac responses to adrenaline administration during 

exposure to air only................................................................................................................ 23 

3. Results of Stage 2 - cardiac responses to adrenaline administration during 

exposure to Agent 873 ........................................................................................................... 24 

4. Methods of sample 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 

I : 3 
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 of Good 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 of the test material, were not available to Huntingdon 

Life Sciences for compliance with the Good Laboratory Practice Regulations given above . 

....... ~ .................................... .. 

Paul J. Horrell, B.Sc. (Hons.), 

Study Director, 

Huntingdon Life Sciences Ltd. 

: 4: 

.......................... 

16 ~1'4'f18ttl... -~· ... • 6 •• ................................. ~ 

Date 
QUALITY ASSURANCE STATEMENT 

The following have been inspected or audited in relation to this study: 

Study Phases Inspected 

Protocol Review 

Study Based Inspections 

Study preparation ) 

Test item control 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 of Reporting 

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 of phases of this study were conducted and reported 

to the Study Director and Company Management 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 of study were carried out. These were 

promptly reported to appropriate Company Management 

Report Audit: This report has been audited by the Quality Assurance Department. This audit was 

conducted and reported to the Study Director 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.~~ ................................. . 

Amanda Thompson, M.R.Q.A., 

Group Manager, 

Department of Quality Assurance, 

Huntingdon Life Sciences Ltd. 

Date 

5 
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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 study was designed to assess the cardiac sensitisation potential of Agent 873 in beagle dogs. 

Adrenaline was administered by intravenous injection before and during inhalation of test 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 of the test substance. No such effect 

should be observed in the absence of the test substance. 

Agent 873 was administered at concentrations of 0.49, 1.00 , and 1.50% v/v in air. 

At 0.49% v/v Agent 873 there was no evidence of cardiac sensitisation. 

At 1.00% v/v Agent 873, 1 out of 6 dogs exposed gave a positive cardiac sensitisation response. 

At 1.50% v/v Agent 873, the single dog exposed gave a positive response. 

It is concluded :from the results of the study that for Agent 873 the No Adverse Effect Level is 0.49% 

v/v and the Low Adverse Effect Level is 1.00% v/v. 

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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 of 9 were used on this study. All of the six dogs had been used for 

previous studies of the 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 of the 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 of chloroform, 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 of adrenaline 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 of biological 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 extrapolating the 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 of the study all data pertaining to the study, including a copy of this fmal report, were 

transferred to Huntingdon Life Sciences Ltd Archives. The data will be retained in Archives for at least 

5 years 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 26 November 2001 respectively. 

The Stage II exposures were carried out during the period 13 August to 12 September 2001. 

8 
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Common name: 

Chemical name: 

Purity: 

Presentation: 

Storage conditions: 

Batch (lot): 

Expiry date: 

Date received: 

Supplier: 

AAS 001/014630 

TEST SUBSTANCES 

Agent 873 

2-bromo-3, 3, 3-triflouropropene 

Not given 

liquid 

Ambient temperature and humidity in the dark 

#83-70 

Not given. Assumed stable for the duration of the 

study 

15May2001 

Sponsor 

The complete description of the chemical and physical properties of the test substance, together with 

information regarding its purity, composition and stability are the responsibility of the Sponsor. 

: 9: 
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AAS 0011014630 

MATERIALS AND METHODS 

TEST ANIMALS 

Nine pure~bred male beagle dogs, approximately 6- 16 months old, were available for the study. 

All dogs were obtained from Interfauna UK Ltd, Abbots Ripton Road, Wyton, Huntingdon, England. 

At the start of the Stage II procedures (Air only) the dogs weighed between 9.5 and 19.5 kg. 

The dogs were each identified by a unique number tattooed on the 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 of the left hindleg. 

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 of at 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 bag containing air only. Details of training are stored with 

the raw data for this study but are not commented on further in this report. 

ACCOMMODATION AND HUSBANDRY 

The dogs were housed in pens within a purpose designed dog holding facility (Building J24, Unit 25). 

The pens were designed in accordance with the requirements of the United Kingdom Home Office Code 

of Practice for Housing and Care of Animals used in Scientific Procedures. The pens had a floor area of 

4.5 square metres and could accommodate up to 2 dogs of the same sex and group. The pens could be 

subdivided into equal areas by means of a moveable partition so that each day the dogs could be 

segregated for assessment of individual clinical signs and food consumption. Both parts of the pen were 

fitted with an automatic valve for the supply of drinking water. During week days the dogs were 

segregated from approximately 0800 to 1500 hours. 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 controlled to give 12 hours of light and 12 hours of dark in each 24-hour period. Air extraction, via 

a balanced system, provided approximately 10 - 12 air changes per hour. Actual minimum and 

maximum temperature and relative humidity values were 17/23°C and 40/90% respectively. 

The exposure sessions were carried out in the Department of Dog Toxicology (Building J24 

Room 1005). The dogs were transported from the holding unit to the experimental Wlit as required. 

10 
AAS 001/014630 

The dogs were provided with 500 - 600 g of dry diet daily (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, of any non-nutrient substance likeJy to influence the outcome of this study. The results of 

chemical analysis during the study are lodged in the Huntingdon Life Sciences Archives. 

As part of the routine husbandry, food consumption records and weekly bodyweights were recorded and 

veterinary records were maintained. These have been archived and are not commented on further in this 

report 

On completion of the study the surviving dogs were retained for possible use in further studies of the 

same type. 

EXPOSURE SYSTEM (Figure 1) 

The dogs were exposed to the test gas by a snout-only system. 

A Halls face mask (Phoenix Medical Ltd) was used. The mask consisted of a 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 of the 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 could thus support itself standing or be supported by the 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 

exhaled to waste. During the first experimental session the gas bag was filled with room air only. 

For gas exposures, the reservoir gas bag contained known concentrations of the 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 of air 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 

throughout the exposure period. 

The test atmosphere in the reservoir gas bag was sampled before and after exposure for each dog. 

Samples were analysed using infra-red spectrophotometry. The procedures are detailed in Appendix 4. 

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AAS 0011014630 

MEASUREMENT OF THE ELECTROCARDIOGRAM (ECG) 

The standard lead 11 electrocardiograph was used throughout the 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. 

EXPERIMENTAL PROCEDURE 

The experimental procedure was based on published 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 breathing room 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 of 0.1 mJJsecond. Appropriate dilutions were 

used such that the volume of adrenaline solution used was consistently 0.1 m1lkg bodyweight. 

The ECG was established (as described previously) and the timing commenced as shown below: 

Time (minutes) 

0 

2 

7 

12 

17 

Event 

Start ECG recording 

FastadRmatineQDl)cballengea~rered 

Gas reservoir connected to air supply line 

Second adRmatine QD2) challenge ~ered 

Gas reservoir disconnected 

Stop ECG recording 

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

DESIGN section of this report. 

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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 of the 9 dogs using a range 

of adrenaline doses. 

The response to administration of adrenaline is dependant on the dose given and the responsiveness of 

each individual dog. Typically the response consists of a 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 ventricular tachycardia may occur. 

The purpose of Stage 1 of the 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 of the previously used dogs and was established 

for each of the naive dogs in the following manner. 

The initial dose adrenaline for each dog was 4 J.Lg/kg. If the response to the first adrenaline challenge 

was too large, as defined by an excessive number of ectopic 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 half or 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, if too 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 a judgement in the selection of an 

appropriate adrenaline dose in the best interests of the dog and the study integrity. 

When an appropriate dose had been selected, that dose was used for the exposures in Stage 2. 

13 
AAS 001/014630 

Each of the dogs allocated to the study was tested as indicated in the EXPERIMENTAL 

PROCEDURE, except that no test gas was used and the 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 of the results of 

Stage 1 of the study and the mtionale for the selection of the dogs for Stage 2 of the study are given in 

the RESULTS section of this report. 

STAGE2 

Each of the dogs selected for Stage 2 was exposed to the test gas according to the EXPERIMENTAL 

PROCEDURE. The dogs were scheduled to be exposed according to the following schedule, 

Exposure session 

1 

2 

3 

4 

5 

6 

7 

Agent 873 concentration (% v/v in 

air) 

Air only 

1 

3 

5 

7 

10 

15 

At least one calendar day was planned between each exposure session to allow the dogs to recover. 

JNTERPRETATION OF RESULTS 

The study was designed to provide infonnation as to any dose level that gave rise to clear signs of test 

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 

exposure to the test gas. 

Interpretation of the results was made for each dog, taking into account the response of each dog during 

Stage 1 of this study. 

DEVIATIONS FROM PROTOCOL 

The correct name of the dog breeder is Harlan UK Ltd, not Harlan UK U.K. Ltd as stated in section 

4.1.1 of the Protocol and Protocol Amendment 1. 

The date of arrival, at Huntingdon Life Sciences, for Dogs 1061, 1063, 1065 and 1067 was 

incorrectly stated, in Protocol Amendment 1, as 10/07/00. The correct da.te of arrival was 10/07/01. 

Dog 1061 was removed from the study, prior to the start of Stage 1 procedures due to ill health, and 

was replaced with Dog 1315. 

These deviations from Protocol have had no impact on the integrity of the 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 of any test gas are summarised in Appendix 1. 

On the basis of these results the dose of adrenaline 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 would not tolerate the challenge process. 

STAGE 2- EXPOSURE TO TEST ATMOSPHERE 

Six dogs were selected from the 9 available. 

The dogs selected represented a range of responses 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 
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AAS 0011014630 

The results are presented as follows: 

Table 1 Summary of cardiac 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 air progressing 

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 half the concentration used in the 

firSt exposure was conducted in order to either further refine the Low Adverse Effect Level or establish 

a No Adverse Effect Level. If the 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 agreement between achieved and target concentrations of test material. 

The results of exposure are presented below: 

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 of multifocal 

ventricular ectopic activity (MVEA) approximately 6 seconds after administration of the 

second adrenaline challenge. 

At the 1.50% level 1 dog was exposed. Clinical signs included deep slow breathing, agitation and 

limb tremors. The animal responded positively with an episode of M:VEA approximately 13 

seconds after administration of the second adrenaline challenge. 

At the 0.49% level 6 dogs were exposed. Clinical signs included agitation, deep slow breathing 

and muscle tremors. There were no positive cardiac responses. 

16 : 
AAS 001/014630 

DISCUSSION AND CONCLUSION 

Agent 873 is being developed by the Sponsor for 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/v Agent 873 in air. 

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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 of the cardiac sensitisation potential of some 

halogenated and non-halogenated hydrocarbons. 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 of a range of halogenated materials. Poster Presentation at Society ofToxicology 1994 Annual 

Meeting, Dallas, Texas. 

LEVY, A.G. (1913), The exciting causes of ventricular fibrillation in animals under chloroform 

anaesthesia Heart, 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 
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FIGUREl 

Exposure system 

w 

v 

GB Gas bag reservoir 

M Mask 

V N on-rebreatldng valve 

W Waste 

To breathe the air Vis open to ambient 

To dose GB is connected to V 

19 : 

AAS 001/014630 

GB 
1 

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TABLE I 

. Summary of cardiac responses to adrenaline administration 

during Agent 873 exposure 

AAS 001/014630 

Dog Adrenaline dose Concentration in 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 Not performed 

AP Assumed positive due to positive response at lower concentration 

MVEA Multifocal ventricular ectopic activity 

20 

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AAS 0011014630 

TABLE2 

Mean analysed concentrations of 2-bromo·3,3,3-trifluoropropene 

T arget 0 . 5° *:1 v v I 

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 arget 10o/c . oVV I 

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 1 arget . So/c oVV I 

Dog identification Mean exposure concentration 

(ppm) I (% v/v) 

1003 14988 1.50 

: 21 
AAS 001/014630 

APPENDIX I 

Results of Stage 1 - individual responses to adrenaline alone 

Dog Adrenaline Description of cardiac response following 

number dose adrenaline challenge* 

* 

L 

H 

A 

NP 

(!lg/kg) 

Number of ectopic 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 of ectopic 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 "Description of cardiac response" 

Adrenaline dose considered too low 

Adrenaline dose considered too high 

Adrenaline dose considered acceptable 

Not performed due to excessive response after first challenge 

: 22 
AAS 0011014630 

APPENDIX2 

Results of Stage 2 - Cardiac responses to adrenaline administration 

, during exposure to air only 

Dog Adrenaline Description of cardiac response 

number dose following adrenaline challenge* 

* 

(N) 

(~glkg) Number of ectopic 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 in height ofT -wave) is not mentioned in the "Description of cardiac 

response". 

Negative response 

: 23 
AAS 001/014630 

APPENDIX3 

Results of Stage 2 - cardiac responses to adrenaline administration 

during exposure to Agent 873 

Gas Dog Adrenaline Description of cardiac response 

concentration number dose following adrenaline challenge* 

(%v/v) (Jlglkg) Number of ectopic Comment 

beats (response) 

First Second 

challenge challenge 

1.00 1001 12 10 1 Agitation, arched back, pawing at mask, 

vocalising, deep slow breathing, struggling (N) 

1131 12 0 MVEA Agitation, arched back, deep slow breathing, 

(VEA) struggling, pawing at mask, back legs tense (P) 

1003 8 8 8 Agitation, arched back, muscle tremors, deep 

slow breathing (N) 

1063 2 0 3 Arched back, deep slow breathing, agitation, 

struggling (N) 

1133 2 2 8 Agitation, arched back, deep slow breathing (N) 

1065 1 1 0 Agitation, pawing at mask, muscle tremors, 

struggling (N) 

1.50 1003 8 1 MVEA Deep slow breathing, arched back , agitation, 

limb tremors_JP) 

0.49 1001 12 0 1 Agitation, deep slow breathing, arched back, 

muscle tremors, shallow breathing (N) 

1131 12 1 1 Agitation , deep slow breathing, salivation, 

muscle tremors (N) 

1003 8 0 2 Agitation, deep slow breathing, muscle tremors, 

shallow breathing 

(N) 

1063 2 0 0 Agitation, arched back, 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 of ectopic 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 "Description of cardiac response" 

(N) 

(P) 

(UA) 

VEA 

MVEA 

Negative response 

Positive response 

Unable to assess due to struggling disturbing ECG recording. 

Ventricular ectopic activity 

Multi focal ventricular ectopic activity 

24 
AAS 0011014630 

APPENDIX4 

Meth~ds of sample collection and analysis for l-bromo-3,3,3-triftuoropropene .·" 

SAMPLE COLLECTION 

Chamber concentration 

Samples of the test atmosphere were drawn directly from the 200 litre gas-sampling bag containing 

the test atmosphere into the cell of the 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 of sample analysis is detailed, together with a 

summary of the method validation, in the Inhalation Analytical Procedure at the end of this 

Appendix. 

Preparation of the test atmospheres 

The test atmospheres were prepared in gas sampling bags (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 of the bag were mixed thoroughly prior to use. 

CALCULATIONS 

The method for calculating the approximate 2-bromo-3,3,3-trifluropropene concentration from the 

volumes used to prepare each bag is as follows: 

where V 

Va = 

Concentration (ppm)= V x 106 

Va+V 

volume of2-bromo-3,3,3-trifluropropene vapour added to the bag (litres); 

volume of air added to the bag (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 of numbers, much of the 

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 and recalculation may lead to small apparent discrepancies with other data in the report. 

: 25 
t 

! 

1 

I 

l 

l 

i 

i 

t 

l 

AAS 001/014630 

APPENDIX4 

(Method of sample collection and analysts for 2·bromo-3,3,3-trifluoropropene - continued) 

Compound specific Inhalation Analytical Procedure for 2-Bromo-3,3,3-trifluoropropene 

The analysis of 2-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 an Inhalation 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 supplementary document. 

I EFFECTIVE FROM: I 16 August 2001 

Test substance 

2-Bromo-3,3,3-trifluoropropene, has the following formula H2=CBrCF3 

Appearance 

Storage 

Reagents 

N/A 

Equipment 

Infra-Red analyser 

Sample pump 

Gas sample bags 

Syringes 

Syringe valves 

Integrator 

Balance and data printer 

Clear liquid 

Room temperature 

Miran lA serial no. 1A2123 

Metal Bellows Corporation 

SKC "Tedlar" 3 or 5 litre capacity 

Hamilton series 500 (SOOml capacity) 

Hamilton series 1001 (l.Oml capacity) 

Mininert push-valve with luer fitting 

Thermoquest Chromjet 4400 

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

Preparation of calibration standards 

Draw the required amount of 2-Bromo-3,3,3-trifluoropropene into a syringe (of appropriate 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 of the 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 x l,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 of 2-Bromo-3,3,3-trifluoropropene (ml) 

mass of 2-Bromo-3,3,3-trifluoropropene (mg) 

molecular weight of 2-Bromo-3,3,3-trifluoropropene 

(174.95 glmol) 

0.08205 L atm./mol.K 

temperature CCC) + 273 

barometric pressure (mm Hg) 

volume of air added to the calibration bag 

The range of calibration concentrations used in the measurement of 2-Bromo-3,3,3-trifluoropropene 

is detailed in the study specific supplement. 

Storage of standards 

Type 

Standard gas bags (5,000- 20,000 ppm) 

Storage conditions 

ambient 

: 27 

Maximum storage period 

1 Day 
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 of the 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 of sample through the chamber for up to 10 seconds, then switching back to room air. The 

integrator measures peak height which is proportional to absorbance of the 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 

of best 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 present using the equation below: 

Concentration (ppm)= (A-I) 

s 

Where A = 

s = 

I = 

Absorbance of the sample 

Slope of calibration line derived from calibration data 

Intercept of calibration line derived from calibration data 

Infra-red spectrophotometer conditions 

IR Spectrophotometer 

Cell type 

Operating wavelength 

Slit width 

Detector range 

Meter response settings 

Quality assurance measures 

Wilkes Miran lA-CVF 

1 em path 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 of the instrument will be performed with the appropriate standards on each day of use. 

The highest calibration standard will be compared against a standard of similar concentration 

prepared independently. The ratio of response factors will be acceptable if within 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 of the quality check standards must lie within 5% of the nominal value. 

Samples must lie within the range of acceptable quality check standards for the result to be regarded 

as valid. The LOQ for the run will be regarded as the concentration of the lowest acceptable quality 

check standard. 

: 28 : 
AAS 001/014630 

APPENDIX4 

(Method of sample collection and analysis for 2-bromo-3,3,3-tritluoropropene- continued) 

Summary of method 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 with no weighting of the 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 of Quantification (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 of concentration 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% of their nominal 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 the range of 5,000 to 20,000 ppm. 

Details given in this supplement supersede those in the compound specific lAP. 

I EFFECTIVE DATE: l16 August 2001 

Analytical standard 

Name 

Batch number 

Purity 

Expiry date 

Supplier 

Preparation of calibration standards 

2-Bromo-3,3,3-trifluoropropene 

83-70 

98% 

08/04 

BP Exploration 

Prepare calibration standards in the nominal range 5,000 to 20,000 ppm. 

Instrument conditions 

Detector range 

Attenuation 

0.25A 

1024 

I EFFECTIVE DATE : I 23 August 2001 

Instrument conditions 

Attenuation 2048 

I EFFECTIVE DATE : I 28 Aupst 2001 

Instrument conditions 

Detector range 

Attenuation 

lA 

512 

: 30 : 
I 

I 

·. 

! 

¥ 

AAS 001/014630 

APPENDIX4 

(Method of sample collection and analysis for 2-bromo-3,3,3-triOuoropropene - continued) 

I EFFECTIVE DATE : lu September 2001 

Instrument conditions 

Detector range 

Attenuation 

Additional Method Validation 

0.2SA 

1024 

The raw data for this method validation is located in study AAS/00 1. 

Precision data for repeatability (peak height) showed coefficients of variation for 

2-Bromo-3,3,3-trifluoropropene of less than 1% using standard concentrations of9,000 to 3,000 ppm. 

Least squares regression analysis with no weighting of the peak height response against concentration 

of standard (9,000 to 3,000 ppm) produced a correlation coefficient of 0.997 and-relative errors less 

than 4.5%. The Limit of Quantification (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 275 and 91 ppm respectively (calculated statistically using the standard deviation obtained for 

a standard of concentration 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 of 2-bromo-3,3,3-tritluoropropene - individual sample values 

T arget concentrat10n 0 5'Y< oV I 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 

T arget concentrat10n 1 0~ oV.V I 

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 arge t concentra f ton 15~ oVV I 

SampleiD Gas Measured concentration 

bag I 

No. (ppm) I (% v/v) 

Pre dog 1003 1 14950 1.49 ! 

Post dog 1003 1 15026 1.50 I 

32 
AAS 001/014630 

HUNTINGDON RESEARCH CENTRE GLP COMPLIANCE STATEMENT 2001 

THE DEPARTMENT OF HEALTH OFTBE GOVERNMENT 

OF THE UNITED KINGDOM 

GOOD LABOR.Al'ORY PRACDCE 

LA:BOifATOitY 

DATE OF INSPECnON 

tsa JauuyHOt 

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