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### REPORT 2 - Bromo - 3,3,3 - trifluoropropene : Skin Irritation to the Rabbi t

HLS s tudy number: WA G0010 Version ID: Final Issue date: 27 September 2012Huntingdon Life Sciences WA G0010

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## Details of Sponsor and Te st Facilit y Sponsor American Pacific Corporation 3883 Howard Hughes Parkw ay Sui te 700 Las Vegas Nevada NV 89169 USA Test facility Huntingdon Life Sciences Eye Research Centre Eye Suffolk IP23 7PX UKHuntingdon Life Sciences WA G0010

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## Ta ble of Contents Details of Spons or and Test Facility

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

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

................................................................................................ 5 Contributing Scientist

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

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

.................................................................................................................. 8 2. Test substance

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

............................................................................................. 10 3.1 Animal management

....................................................................................... 10 3.2 Test substance preparation

............................................................................. 10 3.3 Treatment procedure

...................................................................................... 11 3.4 Serial observations

......................................................................................... 11 3.4.1 Clinical signs

....................................................................................... 11 3.4.2 Dermal responses

............................................................................... 11 3.4.3 Interpretation of responses

.................................................................. 12 3.4.4 Termination

......................................................................................... 13 3.5 Archives

.......................................................................................................... 14 3.6 Deviations from protocol

................................................................................. 14 4. Results

....................................................................................................................... 15 4.1 Clinical signs

................................................................................................... 15 4.2 Dermal responses

........................................................................................... 15 5. Conclusion

................................................................................................................. 16 

## List of T ables Table 1 Mean values for erythema and oedema

....................................................... 17 Table 2 Individual values for erythema and oedema

................................................. 18 

## L ist of An nexes Annex 1 We ight of evidence

...................................................................................... 19 Annex 2 GLP Compliance Statement

......................................................................... 20Huntingdon Life Sciences WAG0010 

## Compliance with Good Laboratory Practice 2-Bromo-3,3,3-trifluoropropene: Skin Irritation to the Rabbit The study described in this report was conducted in conlpliance with the following Good Laboratory Practice standards and I consider the data generated to be valid. The UK Good Laboratory Practice Regulations (Statutory Instrument 1999 No. 3106, as amended by Statutory Instrunlent 2004 No. 994). GECD Principles of Good Laboratory Practice (as revised in 1997), ENV/MCICHEM (98) 17. EC Commission Directive 2004/10/EC of 11 February 2004 (Official Journal No. L 50/44). These principles of Good Laboratory Practice are accepted by the regulatory authorities of the United States of America and Japan on the basis of intergovernmental agreements.

2:::l-**~~ &~.**  :z.p (1.= P BRees BSc (Hons) CBiol MSB Date Study Director Huntingdon Life Sciences

4Huntingdon Life Sciences WAG0010 

## Quality Assurance Statement 2-Bromo-3,3,3-trifluoropropene: Skin Irritation to the Rabbit The following inspections and audits have been carried out in relation to this study: Study Phase Date(s) of Inspection Date of Reporting to Study Director and Management Protocol Audit 07 Mar 2012 07 Mar 2012 Report Audit 19 Apr 2012-20 Apr 2012 20 Apr 2012

Process based inspections At or about the time this study was in progress il1spections of procedures employed on this type of study were carried out. These were conducted and reported to appropriate Company Management as indicated below: Process Based Inspections Date(s) of Inspection Date of Reporting to Management Dose administration & 28 Feb 2012 28 Feb 2012 Observations

- Dermal Study Management and 13 Jan 2012 16 Jan 2012 Conduct

In addition, an inspection of the facility where this study was conducted was carried out on an annual basis. These inspections were promptly reported to Company Management.



## '11

## ~\-~ 20(L T Whatling MRQA Date Group Manager Department of Quality Assurance Huntingdon Life Sciences

5Huntingdon Life Sciences WA G0010

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## Contributing Scientist 2-Bromo- 3,3,3 - trifluoropropene: Skin Irritation to the Rabbit 

### Study management P B Rees BSc (Hons) CBio l M SB Study Di rectorHuntingdon Life Sciences WA G0010

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## Summary A study was performed to assess the skin irritat io n potential o f 2- Bromo- 3,3,3

- trifluoropropene to the rabbit. The method fo llo wed was that described in: EEC Methods for the determination o f toxici ty and other healt h effects. Commissio n Regulat io n No. 440/2008. Part B, Method B.4 Acute Toxicit y: Dermal irri tation/ corrosi on. 30 May 2008

. OECD Guideline for Testing of Chemicals No.404 “Acute Dermal Irri tation/Corrosi on”. Adopted 24 April 2002. EPA Health Effects Test Guidelines OPPTS 870.2500 Acute Dermal Irr it ation EPA 712-C-98- 196. August 1998. Japanese Ministry

of Agriculture, Forestry

and Fisheries, Test Data for Registration of Agricultural Chemicals, Skin Irritation (2 -1- 4), 12 Nohsan No. 8147, Agricultural Producti on Bureau, November 24, 2000. Three rabbits received a single four hour, occlusive, dermal administration of approximately 0.5 m L o f th e test substance as supplied and were observed for four days. No dermal react io n was observed in any animal throughout the duration of the study

. The means of

scores for these reactions at approximately 24, 48 and 72 hours after administration, calculated separately f or each animal , are summarised below: Means of scores at approximately 24, 48 and 72 hours Animal number Erythema Oedema 5 0.0 0.0 78 0.0 0.0 79 0.0 0.0 EC trigger values*  2 .3  2 .3 *Classification Category 2 according to the Globally Harmonised System and European Commission Regulation 1272/2008 is triggered if means of scores for either effect are  2 .3 fortwo or three animals (or if effects persist to Day 14 in at least two animals). According to the criteria of the Globally Harmonised System and European Commissio n regulat io n 1272/2008 2- Bromo
- 3,3,3

- trifluoropropene did not requi re any cl assificat io n and labelling.Huntingdon Life Sciences WA G0010

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## 1. Introduction The study was designed to assess skin irritation potential o f 2- Bromo- 3,3,3

- trifluoropropene fo llo wing a single dermal applicat io n to rabbits. The test substance may come into contact wi th skin during handling or use. The study was conducted in co mp lianc e wi th the following guidelines: EEC Methods for the determination o f toxici ty and other healt h effects. Commissio n Regulat io n No. 440/2008. Part B, Method B.4 Acute Toxicit y: Dermal irri tation/ corrosi on. 30 May 2008

. OECD Guideline for Testing of Chem icals No.404 “Acute Dermal Irri tation/Corrosi on”. Adopted 24 April 2002. EPA Health Effects Test Guidelines OPPTS 870.2500 Acute Dermal Irritation EPA 712-C-98- 196. August 1998. Japanese Ministry

of Agriculture, Forestry

and Fisheries, Test Data for Registration of Agricultural Chemicals, Skin Irritation (2 -1- 4), 12 Nohsan No. 8147, Agricultural Producti on Bureau, November 24, 2000. The albino rabbit was chosen as it has been shown to be a suitable model for skin irritat io n studi es and i s the animal reco mmended in the test guidelines. The amount of

test substance administered was chosen in co mp liance with the guidelines. The protocol was approved by the Study Di rector and Hunt ingdon Life Sciences Management on 6 March 2012 and by the Sponsor on 3 Apri l 2012

. The experimental start date was 11 March 2012 and the experimental co mp letion date was 23 March 2012

.Huntingdon Life Sciences WA G00 10

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## 2. Te st substance Ident ification: 2- Bromo-3,3,3- trifluoropropene CAS Number: 1514-82-5 Act io n: Fi

re ext inguishing agent Descript io n: Clear liquid wi th slight yellow tint Storage condit io ns: Refrigerator (nominally 4  C) Batch number: Kg F080 Date of receipt: 12 September 2011 Expi ry date: 1 August 2012 Puri ty : 99.9% excluding stabilisers Stabilisers:Huntingdon Life Sciences WA G0010

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## 3. Experimental Procedure 3.1 Animal management Animals for this study

were selected from

a stock supply

of h eal thy adul t rabbi ts of th e New Zealand Whi

te strain. They

were in the weight range of 3.55 to 4.94 kg and 35 or 36 weeks of age, pri or to treatment (Day 1). All rabbits were acclimat is ed to the experimental environment for a period of ten weeks prior to the start of the study

. The rabbits w ere housed individually in plastic cages with perforated flo ors. Each rabbit was offered 125 g of a standard laboratory rabbi t diet per day ; drinking water was provided ad libitum . The batch of diet used for the study was analysed for nutrients, possible contaminants and micro

- organi sms likely to be present in the diet and which, if in excess of specified amounts, might have an undesirable effect on the test system . The animals were given a dietary

supplement of hay

. During the acclimat is ation and study period the animals were given small so ftwhi te untreated wood blocks for environmental enrichment. Results of routine physical and chemical examinat ion of drinking water, as conducted by the supplier are made available to Huntingdon Life Sciences Limited. Animal room environmental controls were set to maintain temperature within the range 16 to 20°C, and relat ive humidit y wi thin 40 to 70%. These environmental parameters were recorded and the permanent record archived wit h other departmental raw data. Ligh ting was controlled by means o f a time switch to give 12 hours of artificial light (06:00 to 18:00 GMT) in each 24 hour period. Each animal was ident ified by a numbered tag placed through the edge of one ear. This ident ificat io n was unique wit hin the Department throughout the duration of the study . Each cage was ident ified by a co lo ured label displaying the study number and animal number. 

### 3.2 Test substance preparation 2- Bromo-3,3,3- trifluoropropene was administered as supplied by the Sponsor. The absorption of

2- Bromo- 3,3,3

- trifluoropropene was not determined. The ident it y, strength and purit y of the test substance received, its stabilit y under the storage condi tions and the condit io ns of administration were the responsibilit y of the Sponsor.Huntingdon Life Sciences WA G0010

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### 3.3 Treatment proced ure On the day

b ef ore applicat io n of the test substance, hair was remo ved with clippers fro m th e dorso

- lu mbar region of

each rabbit exposing an appropriate sized area of skin. Approximately 0.5 ml of

the test substance was applied under a 2

- ply 25 mm x 25 mm porous gauze pad secured with ‘blenderm’ surgical tape to intact skin sites on three animals. An addi tional si te was similarly treated with the exception of test substance and acted as a control . Each treatment site was covered with a square piece of aluminium fo il (approximately 55 mm x 55 mm) all four sides of which were secured to the skin by impermeable surgical tape (Blenderm). A single animal (number 5 ) received three exposures of three minutes, one or four hours duration in a step

- wise manner and acted as a preliminary screen. In the absence of a severe effect on removal o f th e dressings the next exposure was init iated. In the absence of a severe response in this animal, two further animals were committed to the study; each animal received a single four hour exposure. For exposures of one hour or more each treatment site was covered with cotton wool and "Tubigrip" elast icated bandage dressing for the duration of the exposure period. The animals were returned to their cages immediately af ter tre atment. Atthe end of the exposure period the semi- occlusive dressing and gauze pad were removed and the treatment site was washed with lukewarm water (30

- 40°C) to remove any residual test substance. The treated area was blotted dry with absorbent paper. 

### 3.4 Serial observations 3.4.1 Clinical signs All animals were observed daily f or signs of ill healt h or toxicit y. 3.4.2 Dermal responses Examinat io n o f th e treated skin was made on removal of the dressings (for 3 minute or one hour exposures) and approximately 1, 24, 48 and 72 hours later. Only the data for the four hour exposure are reported, the data from the three minute and one hour exposures are held in th e archives. Local dermal i rri tation was assessed using the prescribed numerical system: Ery th ema and eschar for mat io n: No ery thema 0 Very slight ery

th ema (barely percept ible) 1 Well

- defined ery

th ema 2 Moderate to severe ery

th ema 3 Severe ery

th ema (beet redness) or eschar format io n (injuries in depth) prevent ing grading of eryt hema 4Huntingdon Life Sciences WA G0010

12 Oedema format io n: No oedema 0 Very slight oedema (barely

percept ible) 1 Sl ight oedema (edges o f area well

- defined by defini te raising) 2 Moderate oedema (raised approximately 1 millimetre) 3 Severe oedema (raised more than 1 millimetre and extending beyo nd the area of exposure) 4
3.4.3 Interpretation of responses Primary Irritation Index A primary

irri tation index (PII) was calculated from the ery th ema and oedema scores according to the fo llo wing form ula as described in Technical Report No. 66 “Skin irritation and Corrosi on: Reference chemicals data bank” (March 1995) ECETOC, Brussels.

PII=  Ery

thema at 24/48/72 hours +  Oedema at 24/48/72 hours

3 x number of animals The maximum

possible score was 8.0. The PII was then used to classify the test substance as fo llo ws: Primary Ir ritation Index Classification

0 no n - irritant >0

- 2.0 mildly irritating 2.1

- 5.0 moderate irritant 5.1

- 6.0 moderate to severe irritant > 6.0 severe irritant
This classificat io n system is a modificat io n based on “Appraisal of the Safet y of C hemicals in Foods Drugs and Cosmet ics” published by the Associat io n of Food and Drug Officials o f th e United States, 1959.Huntingdon Life Sciences WAG0010

13 Globally Harmonised System (United Nations) European Commission regulation 1272/2008 contains the following criteria for classification of irritants and corrosives to skin. These we re derived from the Globally Harmonised System (United Nations 2005 and more recent revisions). Corrosion: a corrosive substance is a substance th at produces destructi on of skin tissue, namely, visible necrosis through the epidermis and into the dermis, in at least one tested animal after exposure up to four hours duration. Corrosive reactions ar e typified by ulcers, bleeding, bloody scabs and, by the end of observation at 14 days, by discolouration due to blanching of the skin, complete areas of alopecia and scars. Corrosive substances are assigned to Category 1; three subcategories are provided within the corrosive category: Subcategory 1A: where responses ar e noted following up to 3 minutes exposure and up to 1 hour observation Subcategory 1B: where responses are described following exposure between 3 minutes and 1 hour and observations up to 14 days Subcategory 1C: where responses occur after exposures between 1 hour and 4 hours and observations up to 14 days Irritation: an irritant substance is a substance thatproduces reversible damage to the skin following an exposure of up to four hours. Irritant substances are assi gned to Category 2; the following thresholds apply: Mean value of >2.3 - <4.0 for erythema/eschar or for oedema in at least 2 of 3 tested animals from gradings at 24, 48 and 72 hours after patch removal or, if reactions are delayed, from grades on 3 consecutive days after the onset of skin reactions. Inflammation that persists to th e end of the observation period normally 14 days in at least 2 anim als, particularly taking in to account alopecia (limited area), hype rkeratosis, hyperplasia, and scaling. In some cases where there is pronounced variability of response among animals, with very definite positive effects related to chemical exposure in a single animal butless than the criteria above. 3.4.4 Termination Following completion of the observation period the animals were humanely killed by an intravenous injection of sodium pentobarbital.Huntingdon Life Sciences WA G0010

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### 3.5 Archives All raw data arising from the performance of this study at Huntingdon Life Sciences is the property of the Sponsor and will be lodged together with a copy of the final report in the Hunt ingdon Life Sciences Archive. Such records will be retaine d f or a minimum

of one year from the date on which the Study Di rector signs the final

report. At the end of the retention period the Sponsor will be contacted and advice sought on the return, disposal or further retention of the records. Hunt ingdon Life S ciences will retain the Qualit y Assurance records relevant to this study and a copy of the final report in i

ts archive indefinitely. 

### 3.6 Deviations from protocol The study protocol indicated that the test substance should be stored r efr igerated (no minally 4  C) , desiccated and under a nitrogen blanket, however, the material was stored refrigerated without neither desiccant nor nitrogen blanket. The Sponsor advised that to evaluate the material, a couple of tests should be performed: 1. Appearance: th e materi al should be a clear liquid with a slight yellow colour and no particulates. 2. pH: th e pH of an aqueous 50% v/v preparation should be determined. Shake a 1:1 mixture of the test material and dist illed water for 30 seconds, then, allow the two layers to separate (about 2 minutes) and using a calibrated pH meter measure the pH of the aqueous layer. If there is no chang e in the appearance and the pH value is above 5 then the material would be considered to have been unaffected. The appearance was considered to have been unchanged and the pH value for the working container for this study

was f ound to be 6.60. Accordingly, it is considered that the incorrect storage condit io ns did not affect the integrit y of the test substance. There w as no other deviat io n fro m protocol

.Huntingdon Life Sciences WA G0010

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## 4. Results 4.1 Clinical signs There was no sign of toxicit y or ill h eal th in any rabbi t during the observat io n period. 

### 4.2 Dermal responses No dermal react io n was observed in any animal throughout the duration of the study

.Huntingdon Life Sciences WA G0010

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## 5. Conclusion According to the crit eri a of the Globally Harmonised System and European Commissio n regulat io n 1272/2008 2

- Bromo
- 3,3,3

- trifluoropropene did not produce irritation or corrosivit y and did not requi

re any classificat io n and labelling.Huntingdon Life Sciences WA G0010

17 Table 1 Mean values for erythema and oedema 24, 48 and 72 hours after removal of dressings * Animal number and sex Ery thema Oedema Test Control Test Control 5 F 0.0 0.0 0.0 0.0 78 F 0.0 0.0 0.0 0.0 79 F 0.0 0.0 0.0 0.0

* 4 hour exposure F FemaleHuntingdon Life SciencesWAG001018 Table 2 Individual values for erythema and oedema

Test site: 0.5 mL 2- Bromo - 3,3,3 - trifluoropropene 

# } semi occluded for four hours Control site: No treatment
Animal number and sex Type of

Response Score after removal of dressings 1 h our 24 hours 48 hours 72 hours Test site Control site Test site Control site Test site Control site Test site Control site Ery thema 0 0 0 0 0 0 0 0 5 F # Oedema 0 0 0 0 0 0 0 0

Ery thema 0 0 0 0 0 0 0 0 7 8 F Oedema 0 0 0 0 0 0 0 0

Ery thema 0 0 0 0 0 0 0 0 7 9 F Oedema 0 0 0 0 0 0 0 0 F Female # Sentinel animalHuntingdon Life Sciences WAG0010

19 Annex 1 Weight of evidence Prior to undertaking in-vivo irritation testing the Study Di rector conducted a weight-of-theevidence analysis to ensure that the in-vivo testing was sufficiently justified. The following factors have been take n into consideration: 1. Nature of test substance. A clear liquid with a slight ye llow tint for use as a fire extinguishing agent. The sponsor advises the following: Chemical name: 2-Bromo- 3,3,3-trifluoro-1-propene Molecular weight: 174.95 Purity: 99.9% excluding stabilisers Boiling point: 34 ° C 2. Evaluation of existing human and anim al data (includingdermal toxicity) Internet searches indicated that the materialis extremely flammable and harmful or an irritant. These indications were clarified with “the toxicological properties of this product have not been fully determined” and ar e considered to be precautionary. 3. Analysis of structure acti vity relationships (SAR). None available. 4. Physicochemical properties. Physicochemical properties (HLS study WAG0005) indicated that the test material is not flammable and the partition coefficient is 2.7. pH recorded at HLS as a 10% aqueous preparation: 5.2 5. Results from in-vitro or ex-vivo tests. Due to the low evaporation point and the small amount of test material used in the EpiSkin assay, the test material was considered unsuitable for testing by this method. The Physicochemical properties show a lack of autoflammability and flash point, however, data available on the InterNet quotes a flash point in the region of -23 ° C to -25 ° C. These MSDS’s also state that the “chemical, physicaland toxicological prope rties have not been thoroughly investigated” so it is considered that the extremely flammable and -25 ° C flash point labels are precautionary based on its volatility; any irritati on label is likewise considered to be precautionary. It is generally accepted that compounds with a partition coefficient in the range of 1 to 4 (optimally between 1 and 2) and a molecular weight of less than 500 may be absorbed, at least in part, via the dermal route. The partition coefficient of 2-Bromo-3,3,3-trifluoro-1- propene is 2.7 and the molecular weight is 174.95; accordingly, it was considered likely that this material will cross the skin barrier. The low boiling point (below body temperature) indicates that the material w ill not be in contact with the body for a long period of time, although steps (airtight admi nistration) were taken to maximise exposure time. The Study Director was satisfied that, in the ab sence of obtainable data, there was sufficient justification to conduct an in-vi vo rabbit skin irri tation study.Huntingdon Life Sciences WA G0010

20 Annex 2 GLP Compliance Statement