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### 2 - Bromo - 3,3,3 - Trifluoropropene Abiotic Degradation: Hydrolysis as a Function of pH

(Preliminary Test)

HLS study number: WA G 0007 Version ID: Final Issue date: 26 July 2012Huntingdon Life Sciences WA G0007

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## Sponsor and T

est Facility Sponsor American Pacific Corporation 3883 Ho ward Hughes Parkway Sui te 7 00 Las Vegas NV 89169 USA Test facility Hunt ingdon Life Sciences Eye Research Centre Eye Suffo lk IP23 7PX UKHuntingdon Life Sciences WA G0007

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## Ta ble of Content

s Sponsor and Test Facility ................................ ................................ ................................ ...... 2 Compliance with Good Laboratory Practice ................................ ................................ .......... 4 Quality Assurance Statement ................................ ................................ ................................ 5 Contributing Scientists ................................ ................................ ................................ .......... 6 Summary ................................ ................................ ................................ .............................. 7 1. Introduction ................................ ................................ ................................ .................. 8 1.1 Objective ................................ ................................ ................................ ........... 8 1.2 Regulatory compliance ................................ ................................ ..................... 8 1.3 Study schedule ................................ ................................ ................................ . 8 1.4 Maintenance of records ................................ ................................ .................... 8 2. Test Substance ................................ ................................ ................................ ............ 9 3. Abiotic Degradation: Hydrolysis as a Fun ction of pH (Preliminary Test) .................... 10 3.1 Method ................................ ................................ ................................ ........... 10 3.2 Definition and units ................................ ................................ ......................... 10 3.3 Procedure ................................ ................................ ................................ ....... 10 3.3.1 Preparation of buffer solutions ................................ ............................. 10 3.3.2 Preliminary test ................................ ................................ ................... 10 3.4 GC conditions ................................ ................................ ................................ . 11 3.5 Preparation of calibration ................................ ................................ ................ 12 3.6 Calculations ................................ ................................ ................................ .... 12 3.7 Results ................................ ................................ ................................ ........... 12 3.7.1 Linearity ................................ ................................ .............................. 12 3.7.2 Method verification ................................ ................................ .............. 12 3.7. 3 Preliminary test ................................ ................................ ................... 12 3.7.4 pH of test solutions and incubation temperature ................................ .. 13 3.8 Conclusion ................................ ................................ ................................ ...... 13 

## List of Annexes Annex 1 Eye Research Centre GLP Compliance Statement 2011 ............................. 23Huntingdon Life Sciences WA G0007

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## Contributing Scientist s 2 - Bromo - 3,3,3 - Trifluoropropene : Abiotic degradation: hydrolysis as a function of pH (preliminary test) Study management P Sy dney BSc MSc Study Di rector Product Chemistry Other responsible staff L Lawson MChem Scient ist Product ChemistryHuntingdon Life Sciences WA G0007

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## Summary A study w as perf

ormed to determine the rate of hydrolysis of 2 - Bromo - 3,3,3 - Trifluoropropene as a fu nct io n of p H. The test was co nducted in a ccordance wi th t he preliminary test as described in Co mmissio n Regulat io n (EC) No. 440/2008 (Method C.7) and the OECD Guidelines for Testing of Chemicals (Method 111). The preliminary

s tudy s howed that ate ach of

p H 4, 7 and 9 and 50  0.5oC, le ss than 1 0% hydro lysis had occurred af ter 5 days, equivalent to a half - life of greater than 1 year under environmental condit io ns (25°C). No further testing was considered necessary. 2 - Bromo - 3,3,3 - Trifluoropropene was determined to be hydrolyt ically stable under acid ic, neutral and basic condit io ns.Huntingdon Life Sciences WA G0007

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## 1. Introduction 1.1 Objective This study

w as designed to determine the rate of hydrolysis of 2 - Bromo - 3,3,3 - Trifluoropropene as a funct io n of p H. Determinat io n of

the rate of

hydrolysis of

a t est substance aids in predict ing it s e nvironmental f ate . 

### 1.2 Regulatory compliance The test was conducted in accordance wi th the preliminary test as described in Co mmissio n Regulation (EC) No. 440/2008 (Method C . 7) and the OECD Guidelin es fo r Testing of Chemicals (Method 111) . 

### 1.3 S tudy schedule Study init iat io n d ate : 06 December 2011 Experimental start d at e : 20 January 2012 Experimental c om

plet io n d ate : 12 March 2012 Study c om

plet io n d ate : 26 July 2012 

### 1.4 Maintenance of records Primary data fro m the test , and a copy o f the final report, are stored in the archives of Hunt ingdon Life Sciences.Huntingdon Life Sciences WA G0007

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## 2. Te st Substance Ident ity: 2 - Bromo-3,3,3 - trifluoropropene Chemical structure: F F F CH 2

Br CAS number: 1514 -82 -5 Appearance: Clear liquid wi th a slight yellow tint Storage co ndit io ns: Room tem perature Lot number: Kg F080 Puri ty : 99.9% excluding stabilisers Stabilisers: Expi ry date: 01 August 2012 Date received: 12 September 2011Huntingdon Life Sciences WA G0007

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## 3. 

## Ab iotic Degradation: Hydrolysis as a Function of pH (Preliminary T est) (EC Method C . 7, OECD Method 1 11 ) 3.1 Method The hydro lysis of

the test substance was investigated according to the pre liminary test as described in E C Method C . 7 and OECD Method 111. 

### 3.2 Definition and units Hy drolysis refers to a reaction of a c hemical RX wi

th w ater. Thi s react io n may be represented by

the net exchange of the X with OH:

RX + HOH → ROH + HX The concentrations can be expressed in units such as milligrams per litre or moles per litre. 

### 3.3 Procedure Tests were carried out using reagent grade chemicals, purified sterile water and sterilised glassware. 

### 3.3.1 Preparation of buffer solutio ns Buffer solut io ns were prepared as fo llo ws: pH 4: 0.2M aqueous potassi um dihydrogen orthophosphate (55 ml) was mixed wi th 0.07M aqueous disodi

um hydrogen orthophosphate dodecahy drate (125 ml) and purified water (320 ml). The pH was adjusted to 4.0  0.0 5 wi th orthophosphoric acid. pH 7: 0.2M aqueous potassium dihydrogen orthophosphate (125 ml) was mixed wi th 1M sodi um hydroxide (15 ml) and purified water (360 ml). The pH was adj usted to 7.0  0.05 wi th 1M hydrochloric acid. pH 9: 0.1M boric acid in 0.1 M aqueous potassi um c hloride (250 ml) was mixed wi th 1M sodi um hydroxide (10.5 ml) and purified water (240 ml). The pH was adjusted to 9.0  0.05 wi th 1M hydrochloric acid. 

### 3.3.2 Preliminary test The fo llo wing procedur e was carried out at each of pH 4, 7 and 9: Aliquots (1 00 μ l) o f a stock sol ut io n of 2 - Bromo - 3,3,3 - Trifluoropropene in methano l (approximately 2.5 g/l) were added to separate 10 ml Wheaton via ls containing buffer sol ut io n ( 9.9 ml), which had been purged with nitrogen and pre - equilibrated at test t emperature (50 ± 0.5°C).Huntingdon Life Sciences WA G0007

11 The samples, of

nominal concentration 25 mg/ l, were placed in a 50°C waterbath in the dark unt il sampling was required (af ter a short incubat ion period of a pproximately 5 minutes, and then after 2.4 , 48, 120 and 24 0 hours). Ate a ch sam

pling time, two sam

ples were removed fro m the bath . After cooling, each via l was opened and the ent ir e vo lu me was quant it atively transferred to a separate vessel wi th a portion (10 ml) o f hexane. After shaking fo r 90 seconds, the upper hexane phase w as analysed by gas chro mat ography (GC) . Sam ple pH and incubat io n temperature were monit ored over the period of the test. Method verificat io n samples were prepared at room t emp erature ate ach pH value, the samples being fortified and processed as above. 

### 3.4 G C conditions Instrument: Ag ilent 5890 Series Gas Chro mat ograph Col umn: DB - 5 (30 m x 0.32 mm internal diameter x 1.00 m film thickness) Carrier gas: Helium Flow: 2.0 ml/minute at 35°C Oven tem perature Ini ti al : 35°C (5 minutes) Ram p: 5°C /minute Final: 80°C (2 minutes) Inject io n temperature: 175 °C Inject io n technique: Split (1:25) Inject io n vo lu me: 1 μl Detector: ECD Detector gas flows Anode purge (N 2 ) : 10 ml/minute Make - up (N 2 ) 80 ml/minute Detector tem

perature: 320  C Retention t ime: Approximately 2. 7 minutesHuntingdon Life Sciences WA G0007

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### 3.5 Preparation of calibration A stock calibrat io n sol

ut io n of a pproximate concentration 500 mg/l was prepared by adding test substance ( 30 μl ; densit y = 1.653 g/ml, Hunt ingdon Life Sciences Study Number WAG0005) to a 100 ml vo lu metric flask containing hexane and dissolving in and dilut ing to vo lu me with the same . Calibrat io n so lut io ns in the range 5 to 50 mg/ l were prepared by dilut io ns of the stock sol ut io n wi th hexane. 

### 3.6 Calculations The concentration of 2 - Bromo - 3,3,3 - Trifluoropropene in the analysed so lut io n (C A) was calculated fro m standards introduced before and af ter samples (bracket ing standards) by the fo llo wing equat io n:

C A (m g/l) = standards bracketing of are a peak mean l) (mg ion concentrat standard x are a peak sam ple 

### 3.7 Results 3.7.1 Linearity Under the condi

tions de scribed, the calibrat io n of 2 - Bromo - 3,3,3 - Trifluoropropene was found to be linear over the concentration range 0 to 50 mg/l, wi th a regression

coefficient of 0.9992 (Table 1, Figure 1). An example standard chromatogram is shown in Figure 2. It was consider ed that the analyt ical method was sufficient ly sensi tive to quantify test substance concentrations down to 10% or less of the init ial concentration. 

### 3.7.2 Method verification An overall mean recovery value of 9 1 % was determined, verifying the efficiency of s ampl e processing . 

### 3.7.3 Preliminary test The preliminary study

s howed that ate ach of

p H 4, 7 and 9 and 50  0.5oC, le ss than 10% hydro lysis had occurred aft er 120 hours (5 days), equivalent to a half - life of greater than 1 year under environmental condit io ns (25°C ). No further testing was considered necessary . Table 2 summarises the resul ts fro m the preliminary

test, whilst Table 3 presents the primar y data. Example chromatograms are shown in Figures 3 to 8 . Although slow hydro lysis was evident, the t 240h chro mat ograms did not show a ddi tional degradate peaks. It is p ossible that any degradation products may have been mo re water sol uble than the parent com pound and did not parti

tion into the extraction so lvent (hexane ).Huntingdon Life Sciences WA G0007

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### 3.7.4 pH of test solutions and incubation temperature The pH val

ues of t est sol ut io ns and incubation tem perature over the period of the test are presented in Table 4 . The resul ts show that there were no significant changes in pH and tem perature wi

th time. 

### 3.8 Conclusion 2 - Bromo - 3,3,3 - Trifluoropropene was determined to be hydrolyt ically stable under acidic, neutral and basic condit io ns.Huntingdon Life Sciences WA G0007

14 Table 1 Standard calibration for 2 - Bromo - 3,3,3 -Trifluoropropene

Standard concentration (mg/l) Peak area

49.54 2270800 39.63 1833100 29.72 1392800 1 9.82 991250 9.908 536170 4.954 291960 Linear regression: y = 44990 x + 58040 (including x=0, y

=0) r = 0.9992 x = concentration y = peak areaHuntingdon Life SciencesWAG000715 Table 2 Preliminary test results for hydrolysis of 2 - Bromo - 3,3,3 -Tr ifluoropropene C t ( m g/l) pH t 0h t 2.4h t 48 h t 120h t 24 0h

Measured Mean Measured Mean Measured Mean Measured Mean Measured Mean 4 22.9, 21.8 22.3 20.1, 21.3 20.7 20.2, 20.5 20.3 19.8, 18.2 19.0 18.0, 18.3 18.1 7 21.1, 22 .0 21.6 19.7, 19.8 19.7 20.3, 18.1 19.2 17.8, 19.9 18.9 18.1, 18.2 18.1 9 22.9, 22.0 22.5 21.5, 21.4 21.4 19.9, 19.2 19.5 20.2, 20.0 20.1 17.8, 19.7 18.7 wh ere Ct is the concentration of 2 - Br omo - 3,3,3 - Trifluoropropene in solution at time th (in hours) At pH 4, ln Ct = 3.05 - 0.000714t; t 0 = 21.2 mg/l, t 120 = 19.5 mg/l; Hydrolysis at t 120 = 8% At pH 7, ln Ct = 3.01 - 0.000505t; t 0 = 20.3 mg/l, t 120 = 19.1 mg/l; Hydrolysis at t 120 = 6% At pH 9, ln Ct = 3.06 - 0.000587t; t 0 = 21. 4 mg/l, t 120 = 19.9 mg/l; Hydrolysis at t 120 = 7%Huntingdon Life Sciences WA G0007

16 Table 3 Measurement of 2 - Bromo - 3,3,3 -Trifluoropropene in hydrolysis

test solutions Sample Peak area C A (mg/l) 19.82 mg/l std 959030 - pH 4/t 0 A 1125100 22.89 pH 4/t 0 B 1069100 21.75 pH 7/t 0 A 10372 00 21.10 pH 7/t 0 B 1083100 22.03 19.82 mg/l std 989660 - pH 9/t 0 A 1139100 22.94 pH 9/t 0 B 1094000 22.03 pH 4/t 2.4 A 998570 20.11 pH 4/t 2.4 B 1056100 21.27 19.82 mg/l std 978910 - pH 7/t 2.4 A 981540 19.67 pH 7/t 2.4 B 986880 19.78 pH 9/t 2.4 A 1073 000 21.51 pH 9/t 2.4 B 1066200 21.37 19.82 mg/l std 998910 - pH 4/t48 A 1011300 20.15 pH 4/t48 B 1026100 20.45 pH 7/t48 A 1016500 20.26 pH 7/t48 B 908840 18.11 19.82 mg/l std 990150 - pH 9/t48 A 1015700 19.87 pH 9/t48 B 981250 19.20 pH 4/t 120 A 10 10500 19.77 pH 4/t 120 B 928340 18.16 19.82 mg/l std 1036200 - pH 7/t 120 A 951140 17.84 pH 7/t 120 B 1059300 19.86 pH 9/t 120 A 1076300 20.18 pH 9/t 120 B 1065900 19.99 19.82 mg/l std 1077700 - 19.82 mg/l std 975780 - pH 4/t 240 A 893770 17.95 pH 4/t 240 B 910070 18.28 pH 7/t 240 A 899690 18.07 pH 7/t 240 B 906750 18.21 19.82 mg/l std 997760 - pH 9/t 240 A 892030 17.76 pH 9/t 240 B 989290 19.70 19.82 mg/l std 993090 -Huntingdon Life Sciences WA G0007

17 Table 4 pH change and temperature during preliminary hydrolysis tests Nominal pH Initial pH Final pH Initial temperature (°C) Final temperature

(°C) 4 4.0 4.1 50 50 7 7.1 7.1 50 50 9 9. 0 9. 0 50 50Huntingdon Life Sciences WA G0007

18 Figure 1 Standard calibration for 2 - Bromo - 3,3,3 -Trifluoropropene

0.0E+00 5.0E+05 1.0E+06 1.5E+06 2.0E+06 2.5E+06

0 5 10 15 20 25 30 35 40 45 50Peak area

Concentration (mg/l)Huntingdon Life Sciences WA G0007

19 Figure 2 Example standard chromatogram (19.82 mg/l)

min

0 2 4 6 8 10 12 14 counts

0 100000 200000 300000 400000 ECD1 B, (0HY_00F\010F1001.D)2.693Huntingdon Life Sciences WA G0007

20 Figure 3 Example pH 4 t 0h chromatogram

min

0 2 4 6 8 10 12 14 counts

0 100000 200000 300000 400000 500000 ECD1 B, (0HY_00F\011F1101.D)2.692

Figure 4 Example pH 4 t 240 h chromatogram

min

0 2 4 6 8 10 12 14 counts

0 100000 200000 300000 400000 ECD1 B, (3HY_00F\011F1101.D)2.706Huntingdon Life Sciences WA G0007

21 Figure 5 Example pH 7 t 0h chromatogram

min

0 2 4 6 8 10 12 14 counts

0 100000 200000 300000 400000 500000 ECD1 B, (0HY_00F\013F1301.D)2.693

Figure 6 Example pH 7 t 24 0h chromatogram

min

0 2 4 6 8 10 12 14 counts

0 100000 200000 300000 400000 ECD1 B, (3HY_00F\013F1301.D)2.705Huntingdon Life Sciences WA G0007

22 Figure 7 Example pH 9 t 0h chromatogram

min

0 2 4 6 8 10 12 14 counts

0 100000 200000 300000 400000 500000 ECD1 B, (0HY_00F\016F1601.D)2.693

Figure 8 Example pH 9 t 24 0h chromatogram

min

0 2 4 6 8 10 12 14 counts

0 50000 100000 150000 200000 250000 300000 350000 400000 450000 ECD1 B, (3HY_00F\016F1601.D)2.705Huntingdon Life Sciences WA G0007

23 Annex 1 Eye Research Centre GLP Compliance Statement 201 1