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### REPORT

2 - bromo - 3,3,3 - trifluoropropene : Activated Sludge Respiration Inhibition Test

HLS study number: WA G0020
Version ID: Final
Issue date: 21 October 2013Huntingdon Life Sciences WA G0020

2 

## Sponsor and T

est Facility Det ails Sponsor American Pacific Corporation 3883 Howard Hu ghes Parkway Suite 700 Las Vegas NV 89169 USA Test Facility In Life and Analysis Huntingdon Life Sciences Ltd Eye Research Centre Eye Suffolk IP23 7PX UKHuntingdon Life Sciences WA G0020

3 

## Ta ble of Content

s Sponsor a nd Test Facility Details

................................ ................................ .......................... 2 Compliance with Good Laboratory Practice

................................ ................................ .......... 4 Quality Assurance Statement

................................ ................................ ................................ 5 Contributing Scientists

................................ ................................ ................................ .......... 6 Summary

................................ ................................ ................................ .............................. 7 1. Introduction

................................ ................................ ................................ .................. 8 2. Test Substance

................................ ................................ ................................ ............ 9 3. Experimental Procedure

................................ ................................ ............................. 10 3.1 Dilution water

................................ ................................ ................................ .. 10 3.2 Preparation o f solutions of the test substance

................................ ................. 10 3.3 Test methods

................................ ................................ ................................ .. 10 3.4 Stability of test concentrations

................................ ................................ ........ 10 3.5 Toxicity Test

................................ ................................ ................................ ... 10 3.6 Deviations fro m Protocol

................................ ................................ ................. 10 4. Maintenance of Records

................................ ................................ ............................. 11 5. Results

................................ ................................ ................................ ....................... 12 6. Conclusion

................................ ................................ ................................ ................. 13 

## List of T ables

Table 1 Measured concentrations

................................ ................................ ............. 14 

## List of A ppendices
Appendix 1 Certificate of analysis

................................ ................................ ................... 15 Appendix 2 The determination of 2

- bromo
- 3,3,3

- trifluoropropene in water

...................... 16 Appendix 3 Compliance statement

................................ ................................ ................. 24HuntingdonLifeSciencesWAGOO2OCompliancewithGoodLaboratoryPractice2-bromo-3,3,34ritluoropropeneActivatedSludgeRespirationInhibitionTestThestudydescribedinthisreportwasconductedincompliancewiththefollowingGoodLaboratoryPracticeslandardsandIconsiderthedatageneratedtobevalid.Thisexcludestheanalyticalchemistrydatagenerated,which,whilstperformedtotheprinciplesofGLP,wasnotvalidated,andassuch,nocomplianceisclaimed.This wasnotvalidatedasduringthepreliminaryinvestigation,lossofthetestmaterialwassosignificant,thatifthetestsubstancewasusedinadefinitivetest,concentrationswouldnot havebeenmaintainedduringtheexposurephase.TheUKGoodLaboratoryPracticeRegulations(Statutory Instrument1999No.3106,asamendedbyStatutoryInstrument2004No.994).OECDPrinciplesofGoodLaboratoryPractice(asrevisedin1997),ENV/MC/CHEM(98)17.ECCommissionDirective2004/10/SCof11Februan’2004 (OfficialJournalNo.L50/44).TheseprinciplesofGoodLaboratoryPracticeareacceptedbytheregulatoryauthoritiesoftheUnitedStatesofAmericaandJapanonthebasisofintergovernmentalagreements.Z.iOycjherZ.cj3JordanDaviesBScMScDateStudyDirectorHuntingdonLifeSciences4QualityAssuranceStatementHuntingdonLifeSciencesWAGOO2O2-bromo-3,3,34rifluoropropene:ActivatedSludgeRespirationInhibitionTestThefollowing inspectionsandauditshavebeencarriedoutinrelationtothisstudy:StudyPhaseProtocolAuditProtocolAmendmentNo.1ProtocolAmendmentNo.2ProtocolAmendmentNo.3ReportAuditProtocolAmendment No.4Date(s)ofInspection10Aug201208May 201308May201329Jul201322Aug2013—23Aug201302October2013DateofReportingtoStudyDirectorandManagement10Aug201208May201308May201329Jul201323Aug201302October2013Processbasedinspections:At oraboutthetimethisstudywasinprogressinspectionsofprocedures employedon thistypeofstudywerecarriedout.Thesewereconductedandreportedtoappropriate Company Managementasindicatedbelow:ProcessBasedInspectionsDoseFormulationSamplingoftestmediaforchemical analysisEnvironmentalmeasurements&monitoring/AmmoniaanalysisSet-upoftestorreferencecompound/inoculummixtureStudymanagement&conductResultsProcessingChromatographySampleExtractionandCleanUpSamplePreparation(toincludesamplemanagement&tracking)Standardpreparation&Fortification proceduresDate(s)ofInspection15Jan201304Feb201307Mar201324Jan201326Mar 2013-27Mar201306Feb201312Feb201327Feb2013-28Feb201327Feb201305Aug2013-06Aug2013DateofReportingtoManagement16Jan201304Feb201307Mar201328Jan201327Mar201306Feb201312Feb201328Feb 201301Mar201307Aug2013wherethisstudywasconductedwerereportedtoCompany Management.wascarriedoutonanDateTPulhngerMRQAGroupManagerDepartmentofQualityAssuranceHuntingdonLifeSciences5Huntingdon Life Sciences WA G0020

6 

## Contributing Scientist s 2 - bromo - 3,3,3 - trifluoropropene : Activated Sludge Respiration Inhibition Test 

### Study management Aquatic Ecotoxicology and Biodegradation Jordan Davies BSc MSc Study Di rector (repl acement) Robert A Dickinso n BSc Study Di rector (ori ginal) 

### Expert department Environmenta l Analysis Si mo n Tate Higher National Certificate (Applied Chemistry) Senior Study

ManagerHuntingdon Life Sciences WA G0020

7 

## Summary A preliminary invest igat io n was conducted to assess if test concentrati ons of 2

- bro mo - 3,3,3

- trifluoropropene coul d be adequately m aintained under the condit io ns of the act ivated sludg e respi rat io n inhibit io n test ( OCSPP 850.3300: Modi fied activated sludge , respi rati on inhibi

ti on test, January

2012 )

. Sam ples o f 2

- brom o - 3,3,3

- trifluoropropene were prepared in reverse osmosis water (500 mL) a t nominal concentrat io ns o f 10 and 10 00 mg/L. The test substance was added direct ly to the water in bottl es ( 500 m

L ) housed in a Coordinated E nviro nmental Services (CES) lt d autom ated r espi

rometer. Mixtures were aerated at ca .1 L/min ute and st ir red for three hours. Sam ples of the mixt ures were taken before the instrument was init iated (0 hours) and after th ree hours of aerati on . Results of the analy

ti cal m easurements show that at the start of the test , recoveries of 2

- bro mo - 3,3,3

- trifluoropropene were between 42% and 71% of no minal. A t the end of the incubat io n phase measured test le vels had decreased to <1 % of no minal. As the analy ti cal method was not fu lly validated, no claim o f com pliance for GLP i s made for th ese resul ts. This was not validated as during the preliminary investigation, loss of the test material was so si gnificant, that if the test substance was used in a definit ive test, concentrations would not have been maintained during the exposure phase. These results indicate that under condit io ns of the test it is n ot possible to m aintain test concentrations of 2

- brom o - 3,3,3

- trifluoropropene and therefore, it would not be feasible to conduct a toxi ci

ty assay using th e activated sludge respi rat io n inhibit io n test as the test substance would be lost through vo lat ilit y . Due to the vo lat ile nature of the test co mpound and the losses observed during the preliminary investigations it would not be expected that sewage micro

- organi sms woul d be impacted by this com pound.Huntingdon Life Sciences WA G0020

8 

## 1. Introduction The obj ect ive o f thi s study was to assess th e effects of 2

- brom o - 3,3,3

- trifluoropropene on sewage micro

- organi sms by m easuring the rate of oxy gen uptake of act ivated sludge at 20  2  C in i ts presence at a range of concentrations. Th is ty pe of test i s m ost appropri ate for pure substances, mixtur es or commercial fo rm ulat io ns that are soluble in water

. As t he test substance was known to be volat ile, th e study i ncluded an analyt ical

trial to assess the feasibilit y of using th e activated sludge respi rat io n inhibit io n test to determine the toxicit y of th e test substance to activated sludge. The analyt ical

m ethod used during the trial was not fully v alidated. Thi s woul d have been com pleted if the tri al

had indicated that further testing was feasible. Since further testing was not practicable, the assay was not validated, so no claim of compliance to GLP is made fo r th e analy ti cal resul ts al th ough the work followed the principles of GLP. The invest igation was designed to meet the requirements of OCSPP 850.3300: Modified activated sludge, respirat io n inh ibit io n test, January 2012. The protocol was approved by the Study Di rector , by Huntin gdon Life Sciences Management and by the Sponsor on 14 August 2012

. The experim ental

start and com

plet io n date w as on 31 January 2013

.Huntingdon Life Sciences WA G0020

9 

## 2. Te st Subst ance
Nam e: 2

- brom o- 3,3,3

- trifluoropropene Chemical Name: 2

- brom o- 3,3,3

- trifluoroprop -1

- ene CAS Number: 1514 -82 -5 Use: Fi re ext inguishing agent Molecular Formula: CF 3 CBr=CH 2 Molecular Weight: 174.948 Physical State: Cl ear liqui d wi th a slight y ell ow tint Source: American Pa cific Corp. Batch Number: WN CC

- BTP111128 Puri ty : >99.6 % (as received with stabilizers)

Stabilisers:

S pecific gravit y: 1.6 5 Boiling point: 34 oC at 760 mmHg Water sol ubili ty : 1.0 g/L at 20oC Storage: Stored free of air and water at room temperature Re

- assay date: 12 January 2014 Sam ple received: 2 March 2012
The Sponsor’s Certificate of analysis is given in Appendix 1

.Huntingdon Life Sciences WA G0020

10 

## 3. Experiment al Procedure 3.1 Dilution w ater The dilut io n water used to prepare solut io ns of test mixtures was reverse osmosis (RO) water. 

### 3.2 Preparation of solutions of the test substance Inform at io n from the Sponsor indicated that the test su bstance was a vo lat ile liquid. Therefore 2

- brom o - 3,3,3

- trifluoropropene (3 .1 μL and 313 μL cal culated based on a specific gravit y of 1.6 ) was added direct ly to bottles (500 mL) containing 5 00 mL of RO water to give final, no minal test concentration s of 1 0 mg/L and 1000 mg/L

. The mixtures were shaken unt il the test substance was no lo nger visible. 

### 3.3 Test m ethods In thi s test, a Co

- ordi

nated Environmental Services (CES) Ltd automated respirometer and associ ated software was used to incubate and aerate th e t est mixtures in the analyt ical tri al

. Mixtures were incubated in 500 mL bottles fitted with a sinter to deliver air and a m agnet ic sti rrer bar for mixing

. Aliquots of RO water (500 mL) were added to duplicate bottles at each concentration

. The test subs tance was then added as a vo lu me ( 3.1 and 313 μL ) to give the final no minal concentrati ons of 10 and 1000 as m g/L respectively. Sam ples were taken immediately after dosing for chemical analysis . The instrum ent was then init iated and aerati on ( ca . 1 L/min ) and st ir ri ng was em pl oy ed on the mixtures fo r 3 hours. At the end of the incubation period , th e mixtures were re - sam pled for chemical analysis

. 

### 3.4 Stability of test concentrations The test concentrations of 2

- brom o - 3,3,3

- trifluoropropene were quantified us ing gas chro mat ography wi

th Mass Spectrometric detecti on ( GC

- MS , see Appendix 2 )

. Two samples ( 2 0 mL) of media were taken from the test vessels at 0 and 3 hours

. Each sample was added to a polypropylene tube (50 mL) conta ining 5 mL of ethyl acetate and ca . 5 g sodium chloride. On each occasio n, one of the samples was analysed and the other was stored in a freezer in case further analysis was required. 

### 3.5 Toxicity Test Not performed as the results of the analyt ical tri al indic ated that i

t was not possible to maintain exposure concentrations of the test substance due to its vo lat ilit y. 

### 3.6 Deviations from Protocol There were no deviat io ns from the protocol

.Huntingdon Life Sciences WA G0020

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## 4. Ma intenance of Records This report has been compiled fro m ori ginal data, w hich will be stored, together with a copy of the final report, a copy

of the protocol

and am endm ents in Hunt ingdon Life Sciences’ Archives, Huntingdon Life Sciences Ltd, Ey e, Suffol k, UK. Records will be retained for a minimum period of at least one year f rom the date of i ssue of th e final report. At the end of the one y ear retenti on peri od the Sponsor will be contacted and advice sought on the future requirements. Under no circumstances will any i tem be di scarded wi th out the Sponsor’s knowledge. Hunt ingd on Life Sciences will retain the Qualit y Assurance records relevant to this study f or twenty y ears, and a copy

of the final report in its archives indefinitely

.Huntingdon Life Sciences WA G0020

12 

## 5. Result s Results of the analy

ti cal chemistry

are given in Table 1 and methods are given in Appen dix 2. A nalyt ical measurements show that at the start of the test recoveries of 2

- bro mo - 3,3,3

- trifluoropropene were between 54 % and 71% of nominal at 10 mg/L and between 42% and 48% of no minal at 1000 m

g/L. At the end of the incubat io n phase , test l evels had decrease d to <1 % of no minal in all mixtures. These results indicate that under condit io ns of the test it is not possible to maintain test concentrations of 2

- brom o - 3,3,3

- trifluoropropene and therefore, it would not be feasible to conduct a toxi ci

ty as say using the activated sludge respi rat io n inhibit io n test as 2

- brom o 3,3,3

- trifluoropropene woul d be l ost through vol atilit y. Therefore no further testing was carried out and the results of the preliminary i nvesti gati on were reported.Huntingdon Life Sciences WA G0020

13 

## 6. Conclusion
After 3 hours of aeration and st ir ri ng, analyt ical measurements of test samples s howed that <1 % of the no minal test concentrations of 2

- bro mo - 3,3,3

- trifluoropropene remained. These results indicate that under condit io ns of the test it is not possible to maintain t est concentrations of 2

- brom o - 3,3,3

- trifluoropropene and therefore, it would not be feasible to conduct a toxi ci

ty assay using the activated sludge respi rat io n inhibit io n test as the test substance would be lost through vo lat ilit y. Due to the vo lat ile natu re of the test compound and the losses observed during the preliminary invest igat io ns it woul d not be expected that sewage micro

- organisms would be impacted by

this com pound.H untingdon Life Sciences WA G0020

14 Table 1 Measured concentrations
Nominal 2
- bromo
- 3,3,3
- trifluoropropene conc. (mg/L) Measured 2
- bromo
- 3,3,3
- trifluoropropene concentrations (mg/L)

0 hours %N 3 hours %N

10 7.06 71 nd

- 10 5.43 54 n d

- 1000 420 42 <0.5 <1 1000 483 48 1.57 <1
nd Not detected above the limit of detection of the analy tical method (<LOD 0.005 mg/L).H untingdon Life Sciences WA G0020

15 Appendix 1 Certificate of analysisH untingdon Life Sciences WA G0020

16 Appendix 2 The determination of 2 - bromo - 3,3,3 - trifluoropropene in water 

## 1. Method 1.1 General principle
Sam ples were extr acted with ethyl acetate, prior to quantitation by GC

- MS

.

Materials Grade Acetone AR Ethyl acetate HPLC Hexane Gl ass dist illed
1.1.1 Test article fortifying and calibration solutions An appropriate amount of 2

- brom o - 3,3,3

- trifluoropropene was accura tely weighed (corrected fo r puri ty ) and di ssolved in acetone to give a 1 mg/mL stock standard solut io n. Appropriate dilut io ns of the stock standard solution were made with hexane to give fortificat io n standard sol ut io ns. An appropriate fortificat io n stan dard soluti on was progressively diluted wi

th ethyl acetate in order to produce a calibrat io n line in the range 1 to 50 ng/mL. 

### 1.2 Procedure An aliquot of water (20 mL) was taken and fortified if required. An aliquot of ethyl acetate (5 mL) was added and th e samples were shaken vigorously. The phases were centrifuged at 3500 rpm f or 3 minutes and an aliquot of the ethyl acetate layer was diluted by a f actor of 20 wi th ethyl acetate, prior to analysis by GC

- MS. Any f urther dilut io ns, where required, were perform ed using ethyl acetate.H untingdon Life Sciences WA G0020

17 1.2.1 Instrumentation GC - MS conditions Instrum ent: Voy ager Data system: MassLab v1.3 Ioni sat io n mo de: CI

– Ion m oni toring details: m/z 79 Col umn: Rtx

- 1 (60 m x 0.32 mm x 5μm film thickness) Tem perature programme: 40°C hel d fo r 4 min th en ramped at 30°C/min to 250°C and held for 3 min Injector temperature: 200°C Inject io n vo lu me: 2 μL Retention t ime: approx. 5.9 minutes LOD: 1 ng/mL (equivalent to 0.00 5 mg/L in aqueous media) 1.2.2 Calculation of results Test samples were quant ified using the fo llo wing equati on: Residue found (mg/L) = 1000 1 x D x M 1 x x Where x (resi due concentrati on in final so lut io n) was calculated using the linear regression: y = m x + c where x (concentration in ng/mL) = y  c m c = int ercept m = sl ope y = peak area of sample M = matri

x concentration (mL/mL) D = dilut io n factorH untingdon Life Sciences WA G0020

18 

## 2. Results and Discussion 2.1 Calibration The chro mat ographi c response to 2

- bromo

- 3,3,3

- trifluoropropene was shown to be linear over the range of concentr ati ons 1 to 50 ng/m L. Typical calibration data is presented in Table 1 and Figure 1. Ty pical chrom atogram s of calibrat io n standards are presented in Figures 2 and 3. 

### 2. 2 Limit of detection (LOD) The limit of detection of the method is defined as the va lue of the lowest calibrat io n standard chro mat ographed that gave a peak height to baseline no ise ratio  3. For this study the limit of detecti on was 1 ng/mL (equivalent to 0.005 mg/L in water). 

### 2. 3 Samples Sam ples o f water from

the test were analysed in a sui tably sized batch alo ng with a control sample fortified with 2

- bro mo - 3,3,3

- trifluoropropene which acted as a procedural recovery sample. The p rocedural recover y perf orm ed on control

HPLC water w as in the range 70 to 110% confirming the validit y of t he met hodol ogy on th e day

of analysis. Ty

pical chro mat ogram s of untreated, un treated fortified with 2

- brom o - 3,3,3

- trifluoropropene and treated extracts are presented in Figures 4 to 7.H untingdon Life Sciences WA G0020

19 Table 1 Ty pical standard calibration for 2 - bromo - 3,3,3 - trifluoroprope ne by

GC - MS Standard concentration (ng/mL) Peak area

0 0 1 104.415 2 182.686 4 273.549 8 622.550 10 773.833 20 1422.779 30 2103.317 40 2721.646 50 3404.771

Calibratio n curve: y = 67.5580 x + 46.2481 (batch 1 ) r = 0.999 6 Where x = conc entration y = peak area r = regression coefficientH untingdon Life Sciences WA G0020

20 Figure 1 Ty pical standard chemical calibration for

2 - bromo - 3,3,3 - trifluoropropene by GC - MSH untingdon Life Sciences WA G0020

2 1 Figure 2 Ty pical chromatogram of a 1 ng/mL

2 - bromo - 3,3,3 - trifluoropropene calibration standard

ID E:\STUDYDATA\WAG_0020\Voyager_W AG_0020.PRO\Data\020B107.RAW

4.00 6.00 8.00 10.00 12.00 Time 57 100

% 020B107 Sm (Mn, 2x3) SIR of 1 Channel CI79 5.50e4 Area

5.99 104.415

F igure 3 Ty pical chromatogram of a 50 ng/mL

2 - bromo - 3,3,3 - trifluoropropene calibration standard

ID E:\STUDYDATA\WAG_0020\Voyager_W AG_0020.PRO\Data\020B117.RAW

4.00 6.00 8.00 10.00 12.00 Time 57 100

% 020B117 Sm (Mn, 2x3) SIR of 1 Channel CI79 5.50e4 Area 5.99 3404.771H untingdon Life Sciences WA G0020

22 Figure 4 Chromatogram of a sample of untreated HPLC water (analytical

identification H 2 O Con)

ID E:\STUDYDATA\WAG_0020\Voyager_W AG_0020.PRO\Data\020B112.RAW

4.00 6.00 8.00 10.00 12.00 Time 57 100

% 020B112 Sm (Mn, 2x3) SIR of 1 Channel CI79 5.50e4

8.33

Figure 5 Chromatogram of a sample of untreated HPLC w ater fortified

with 2 - bromo - 3,3,3 - trifluoropropene at 10 mg/L (analytical identification H 2 O F10)

ID E:\STUDYDATA\WAG_0020\Voyager_W AG_0020.PRO\Data\020B114.RAW

4.00 6.00 8.00 10.00 12.00 Time 57 100

% 020B114 Sm (Mn, 2x3) SIR of 1 Channel CI79 5.50e4 Area

5.96 1160.464H untingdon Life Sciences WA G0020

23 Figure 6 Chromatogram of a te st sample at 10 mg/L of BTFP at 0 hours

(analytical identification 13/WAG/A0317 D*100)

ID E:\STUDYDATA\WAG_0020\Voyager_W AG_0020.PRO\Data\020B102.RAW

4.00 6.00 8.00 10.00 12.00 Time 57 100

% 020B102 Sm (Mn, 2x3) SIR of 1 Channel CI79 5.50e4 Area

5.97 999.715

Figure 7 Chromatogram of a test sample at 10 mg/L of BTFP at 3 hours

(analytical identification 13/WAG/A0321)

ID E:\STUDYDATA\WAG_0020\Voyager_W AG_0020.PRO\Data\020B120.RAW

4.00 6.00 8.00 10.00 12.00 Time 57 100

% 020B120 Sm (Mn, 2x3) SIR of 1 Channel CI79 5.50e4

8.36H untingdon Life Sciences WA G0020

24 Appendix 3 Compliance statement