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### HFCPE: Assessment of Skin Sens itization Potential using the Local Lymph Node Assay in the Mouse (Pooled treatment group approach)

HLS study number: VHJ0006
Version ID: Final
Issue date: 10 March 2010Huntingdon Life Sciences VHJ0006

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## Sponsor and Test Facility Details
Sponsor ZEON Corporation Shin Marunouchi Center Building 1-6-2 Marunouchi Chiyoda-ku Tokyo 100-8246 JAPAN Test facility Huntingdon Life Sciences Huntingdon Research Centre Woolley Road Alconbury Huntingdon Cambridgeshire PE28 4HS UKHuntingdon Life Sciences VHJ0006

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## Table of Contents
Sponsor and Test Facility Details ........................................................................................... 2 Compliance with Good Laboratory Practice ............................................................................ 5 Quality Assurance Statement ................................................................................................. 6 Contributing Scientist ........................................................................................................

...... 7 Summary

............................................................................................................................... . . 8 1. Introduction ..................................................................................................................

.. 9 1.1 Objective ............................................................................................................ 9 1.2 Regulatory compliance ....................................................................................... 9 1.3 Test system ........................................................................................................ 9 2. Experimental Procedure

............................................................................................... 10 2.1 Study schedule ................................................................................................. 10 2.1.1 Time schedule ...................................................................................... 10 2.2 Test substance and formulation ....................................................................... 10 2.2.1 Test substance ..................................................................................... 10 2.2.2 Test substance preparation .................................................................. 11 2.2.3 Formulation ........................................................................................... 11 2.3 Animal management ........................................................................................ 11 2.3.1 Animal housing, diet and water supply ................................................. 11 2.4 Positive control ................................................................................................. 12 2.5 Treatment procedure ........................................................................................ 13 2.5.1 Selection of dose levels ........................................................................ 13 2.6 Administration of test substance ...................................................................... 13 2.6.1 Preliminary investigation ....................................................................... 13 2.6.2 Main phase ........................................................................................... 13 2.6.3 Administration of 3 H-methyl Thymidine

................................................. 13 2.7 Observations .................................................................................................... 14 2.7.1 Clinical signs ......................................................................................... 14 2.7.2 Bodyweight ........................................................................................... 14 2.8 Terminal studies ............................................................................................... 14 2.8.1 Termination ........................................................................................... 14 2.8.2 Preparation of single cell suspensions ................................................. 14 2.8.3 Determination of incorporated 3 H-methyl Thymidine ............................ 14 2.9 Interpretation of results ..................................................................................... 14 2.10 Quality Assurance and archiving procedures ................................................... 15 2.10.1 Quality Assurance ................................................................................ 15 2.10.2 Archives ................................................................................................ 15 2.11 Deviations from protocol ................................................................................... 15 3. Results .......................................................................................................................

.. 16 3.1 Preliminary investigation .................................................................................. 16 3.1.1 Mortality and clinical signs .................................................................... 16 3.1.2 Bodyweight ........................................................................................... 16 3.2 Main phase ....................................................................................................... 16 3.2.1 Mortality and clinical signs .................................................................... 16 3.2.2 Bodyweight ........................................................................................... 16 3.3 Estimation of the proliferative response of lymph node cells ........................... 16 4. Conclusion ................................................................................................................... 17Huntingdon Life Sciences VHJ0006

4 5. References ................................................................................................................... 18 

## List of Tables

Table 1 Group dpm/node and test/control ratios ........................................................ 19 

## List of Appendices
Appendix 1 Individual and group mean bodyweights and individual bodyweight change (g)

...................................................................................................................... 20 Appendix 2 Summary of positive control data .................................................................. 21 

## List of Annexes

Annex 1 GLP Compliance Statements ........................................................................ 23Huntingdon Life Sciences VHJ0006

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## Contributing Scientist
HFCPE: Assessment of skin sensitiz ation potential usin g the local lymph node assay in the mouse (pool ed treatment group approach) 

### Study management Rachel Clarke BSc (Hons) Study DirectorHuntingdon Life Sciences VHJ0006

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## Summary
The study was performed to assess the skin sensitization po tential of HFCPE using the murine local lymph node assay (LLNA).
In order to find a suitable leve l for the main phase of the st udy, a preliminary investigation was conducted. One female (no. B1) was dosed at 50% v/v in AOO (4:1 v/v acetone:olive oil) and those results indicated that 50% v/v was a suitable high dose level for the main phase of the study. The neat liquid test substance wa s too volatile to be admi nistered as supplied. In the main phase of the study, groups of four mice were treated as follows: Group Treatment Concentration Animal numbers
2 AOO 0 B2-B5 3 HFCPE 10% v/v B6-B9 4 HFCPE 25% v/v B10-B13 5 HFCPE 50% v/v B14-B17 AOO acetone:olive oil (4:1 v/v) (vehicle control) In each phase of the study, the mice were treated by daily application of 25 μl of each of one of these three concentrations, or control (vehicle ), to the dorsal surface of both ears for three consecutive days. In the main phase of the study, the proliferative response of th e lymph node cells (LNC) from the draining auricular lymph node s was assessed five days follo wing the initial application, by measurement of the incorporation of 3 H-methyl Thymidine ( 3 HTdR) by β -scintillation counting of LNC suspensions. The response was expressed as radioactiv e disintegrations per minute per lymph node (dpm/node) and as the ratio of 3 HTdR incorporation into LNC of test nodes relative to that recorded for control nodes (tes t/control ratio). The test substance is regarded as a sensitizer if at least one concentration of the chemical results in a three-fold greater increase in 3 HTdR incorporation compared to control values. 

### Results In this assay the test/contro l ratios obtained for 10, 25 or 50% v/v were 0.8, 0.6 or 0.8 respectively which indicates that HFCPE did not show the potential to induce skin sensitization (delayed contact hypersensitivity). Responses to the positive control substa nce hexyl cinnamic aldehyde (HCA), in contemporaneous studies ( Appendix 2 ) demonstrate the reliability and sensitivity of this assay to detect skin sensitization potential in this laboratory. 

### Conclusion HFCPE is not regarded as a potential skin sensitizer.Huntingdon Life Sciences VHJ0006

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## 1. Introduction 1.1 Objective The local lymph node assay (LLNA) was designe d to assess skin sensitization potential (delayed type hypersensitivity) of HFCPE (use d in electronic applic ations) in the mouse following topical application to the dorsal surface of the ear. In this study lymphocyte proliferation was assessed during the sensit izing phase of the response. Lymphocyte proliferation in lymph nodes draining the site of application of the test substance was quantified by measuring the incorpor ation of radiolab elled Thymidine. 

### 1.2 Regulatory compliance The study was designed to meet the re quirements of the following guidelines: OECD Guideline for Testing of Chemi cals No. 429 “Skin Sensitization: Local Lymph Node Assay”. (24 April 2002).
EEC Methods for the determination of t oxicity. Annex to Commission Directive 2004/73/EC (Official Journal No. L 152, 30.4.2004), Part B, Method B.42. Skin sensitisation: Local Lymph Node Assay. 

### 1.3 Test system The mouse was chosen as the test species as it has been shown to be a suitable model.Huntingdon Life Sciences VHJ0006

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## 2. Experimental Procedure 2.1 Study schedule
2.1.1 Time schedule Study initiation: (Protocol signed by Study Director) 19 November 2009

Experimental start date: (Initial animal allocation) 19 November 2009

Day of initial treatment: 24 November 2009 Completion of live phase: 14 December 2009 Experimental completion date: (Date of scintillation count) 15 December 2009

Study completion: 10 March 2010 

### 2.2 Test substance and formulation
2.2.1 Test substance
Identification: HFCPE Chemical name: 1,3,3,4,4,5,5-Heptafluorocyclopentene
Intended use: Electr onic applications Appearance: Colourless liquid Storage conditions: Room temperature Supplier: Sponsor Batch/lot number: 20090601 Expiry date: End of June 2010 Purity/composition: 99.3% Date of receipt: 22 June 2009 The Sponsor was responsible for the charac terisation of the test substance and the documentation of the methods of synthesis, fabrication or derivation and stability.Huntingdon Life Sciences VHJ0006

11 2.2.2 Test substance preparation
A vehicle trial was conducted and HFCPE showed that it was miscible with acetone:olive oil (4:1 v/v) forming an off white emulsion at 100% w/v and 50% w/v. 2.2.3 Formulation The dose levels for the preliminary inves tigation were chosen based on the physical properties of the test substance e.g. solubility, viscosity. The doses for the main phase of the study were based on the results of the preliminary investigations. Determination of the homogeneity, stability and pur ity of the test substance or test substance formulations were not undert aken as part of this study. Detailed records of test substance usage were maintained. The amount of test substance necessary to prepare the formul ations and the amount actually used were determined on each occasion. The difference between these amounts was checked before the formulations were dispensed. The test substance formulations were prep ared on the day of dosing at the required concentrations. The absorption of the test s ubstance was not determined. 

### 2.3 Animal management Seventeen healthy female CBA/Ca mice were obt ained from Harlan UK Ltd. One of these animals arrived on 19 November 2009 and was us ed for the preliminar y investigation. The remainder arrived on 03 December 2009 and were used for the main phase of the study. All preliminary and main phase animals we re in the weight range 16.1 to 22.2 g and approximately eight to twelve weeks of age pr ior to dosing on Day 1. All the mice were acclimatised to the experimental environment for at least 5 days prior to the start of the study. Each animal was assigned an alpha-numeric c ode and identified unique ly within the study by tail marking. Each cage label was colour-code d and was identified uniquely with the study number, dose level and animal mark. 2.3.1 Animal housing, diet and water supply
Animals were housed inside a barriered rodent f acility (Building F21, Ro om 12). The facility was designed and operated to minimise the entry of external biological and chemical agents and to minimise the transference of such agents between rooms.
The mice were allocated without conscious bias to cages within the tr eatment groups. They were housed individually in polycarbonate cages with woodf lake bedding. The mice were also given Nestlets for environmental enrichme nt. Certificates of analysis for woodflake bedding and Nestlets were lodged in H untingdon Life Sciences Ltd. Archives. The animal room was kept at positive pressure with respect to the outside by its own supply of filtered fresh air, which was passed to atmosphere and not re-circulated. The temperatureHuntingdon Life Sciences VHJ0006

12 and relative humidity controls were set to generally maintain the range of 19 to 23 ° C and 40 to 70% respectively. Any minor deviations from these ranges would not have had an adverse effect on the animals and would not affect the integrity or validity of the study. Artificial lighting was controlled to give a cycle of 12 hours continuous light and 12 hours continuous dark per 24 hours.
Periodic checks were made on the number of ai r changes in the animal rooms. Temperature and humidity were monitored daily and records ar e archived with the departmental raw data. Alarms were activated if there was any failure of the ventilation system, or temperature limits were exceeded. A stand-by electricity supply was available to be automatically brought into operation should the pub lic supply fail. The animals were allowed free access to a standard rodent diet (Rat and Mouse No. 1 Maintenance Diet). This diet contained no added antibiotic or other chemotherapeutic or prophylactic agent. Potable water taken from the public supply wa s freely available via polycarbonate bottles fitted with sipper tubes. Each batch of diet was analysed routinely by the supplier for various nutritional components and chemical and microbiological contaminants . Supplier’s analytical certificates were scrutinised and approved before a ny batch of diet was released for use. The quality of the water supply is governed by regulations published by the Department for Environment, Food and Rural Affairs. Certificates of analysis we re received routinely from the water supplier. Since the results of these vari ous analyses did not provide ev idence of contamination that might have prejudiced the st udy, they are not presented. No other specific contaminants that were likely to have been present in the diet or water were analysed, as none that may have interfered with or prejudiced the outcome of the study was known. 

### 2.4 Positive control The sensitivity of the mouse strain used is ch ecked periodically at Huntingdon Life Sciences Ltd. with hexyl cinnamic aldehyde (HCA ), a known moderate sensitizer ( Appendix 2 )

.Huntingdon Life Sciences VHJ0006

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### 2.5 Treatment procedure 2.5.1 Selection of dose levels Based on the toxicity information from the acute oral toxicity study, 50% v/v was considered a suitable dose level in the preliminary investigation. The results of the preliminary investigation indicated that 50% v/v was well tolerated. There was consideration to perform a further preliminary investigation with the n eat liquid test substan ce, however, the test substance is a volatile liquid and it was consid ered not appropriate to perform dosing due to the rapid evaporation. Based on this information the following concentra tions were selected for the main phase of the study: 10, 25 and 50% v/v Prior to dose administration the Study Direct or authorised the choi ce of vehicle and dose levels in the raw data. 

### 2.6 Administration of test substance 2.6.1 Preliminary investigation
One female was treated with one concentration of the test substance. The mouse was treated by daily application of 25 μ l of appropriate concentration of the test substance to the dorsal surface of each ear for three consecutive days (Days 1-3). The test substance was applied to the dorsal surface of each ear usi ng an automatic micropipette. The test substance was spread over the entire dorsal su rface of the ear using th e tip of the pipette. 2.6.2 Main phase Groups of four mice were treated at one of three concentrations of the test substance. The mice were treated by da ily application of 25 μ l of the appropriate conc entration of the test substance to the dorsal surface of each ear for th ree consecutive days (Days 1-3). The test substance was applied to the dorsal surface of each ear using an automatic micropipette. The test substance was spread over the entire dorsal surface of the ear using the tip of the pipette. 2.6.3 Administration of 3 H-methyl Thymidine In the main phase of the study, five days follo wing the first topical application of test substance (Day 6) all mice were in jected via the tail vein with 250 μ l of phosphate buffered saline containing 3 H-methyl Thymidine a ( 3 HTdR: 80 μ Ci/mL) giving a nominal 20 μ Ci to each mouse. The injection into the tail vein wa s carried out using a plastic syringe and needle after the mouse had been heated in a warming chamber.

a Specific activity 2.0 Ci/mmol, concentration 1.0 mCi/mLHuntingdon Life Sciences VHJ0006

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### 2.7 Observations 2.7.1 Clinical signs All animals were observed daily for signs of il l health or toxicity. The ears were also examined for signs of irritation. 2.7.2 Bodyweight The weight of the mouse in the preliminary inves tigation was recorded on arrival, Day 1 (first day of dosing) and prior to termination (Day 4). These data are not reported. The weight of each mouse in the main phase of the study was recorded on arrival (these data are not reported), Day 1 (first day of dosi ng) and prior to te rmination (Day 6). 

### 2.8 Terminal studies
2.8.1 Termination The mouse in the preliminary investiga tion was humanely killed by carbon dioxide asphyxiation on Day 4 of the study. No further i nvestigations were carried out with this animal. In the main phase of the study, five hours following the administration of 3 HTdR on Day 6 all mice were humanely killed by carbon dioxide asphyxiation and the drai ning auricular lymph nodes excised and pooled for each experimental group. 1.0 mL of phosphate buffered saline was added to the pooled lymph nodes for each group. The animals were then discarded and no further investigations were carried out. The following proce dures were carried out with the lymph nodes from these animals only. 2.8.2 Preparation of single cell suspensions A single cell suspension of ly mph node cells (LNC) was prepared by gentle mechanical disaggregation through a stainle ss steel gauze (200 mesh size). The pooled LNC were then washed by adding 10 mL phosphate buffered sali ne, pelleted at 190 x g for 10 minutes and resuspended. The cells were washed twice ag ain and resuspended in 3 mL trichloroacetic acid (TCA: 5%) following the final wash. 2.8.3 Determination of incorporated 3 H-methyl Thymidine After overnight incubati on with 5% TCA at 4 ° C, the precipitate was recovered by centrifugation and resuspended in 1 mL 5% TCA and transferred to 10 mL Ultima gold scintillation fluid on Day 7. 3 HTdR incorporation was measured by β -scintillation counting. The proliferative response of LNC was expressed as radioac tive disintegrations per minute per lymph node (dpm/node) and as the ratio of 3 HTdR incorporation into LNC of test nodes relative to that recorded for control nodes (test/control ratio). 

### 2.9 Interpretation of results The test substance is regarded as a sensitizer if at l east one concentration of the test substance results in a three-fold greater increase in 3 HTdR incorporation compared to control values.Huntingdon Life Sciences VHJ0006

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### 2.10 Quality Assurance and archiving procedures 2.10.1 Quality Assurance Details of the Quality Assurance inspections and audits are presented on the Quality Assurance Statement. 2.10.2 Archives Following completion of this study all raw da ta were stored in the archives of Huntingdon Life Sciences. A copy of the final report and all Quality A ssurance inspection reco rds will be retained indefinitely. All other appropriate records will be retained for a minimum period of one year from the date of issue of the final report. At the end of the one y ear retention period the Sponsor will be contacted and advice s ought on the above requirements. Under no circumstances will any item be discar ded without the Sponsor’s knowledge. 

### 2.11 Deviations from protocol There were no deviations from protocol.Huntingdon Life Sciences VHJ0006

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## 3. Results 3.1 Preliminary investigation 3.1.1 Mortality and clinical signs There were no deaths and no signs of ill health or toxicity observed during this study. No signs of irritation were seen over the dosed ar ea during the study. 3.1.2 Bodyweight A bodyweight increase was recorded for the mous e over the period of the preliminary phase. On the basis of the results from the preliminary investigation, 50% v/v was considered a suitable high dose level for the main phase of the study. 

### 3.2 Main phase 3.2.1 Mortality and clinical signs There were no deaths and no signs of ill hea lth or toxicity observed during this study, however, the following obs ervations were noted: Greasy fur on the cranial region was observed po st-dose from Day 1 in the control and all treated groups. This sign had resolv ed completely in all animals by Day 5. 3.2.2 Bodyweight Appendix 1 Bodyweight stasis was recorded for two females (nos. B14 and B17) treated at 50% v/v. All remaining animals gained bodyweight during the study. 

### 3.3 Estimation of the proliferati ve response of lymph node cells
Table 1
The test/control ratios obtained for 10, 25 or 50% v/v HFCPE were 0.8, 0.6 or 0.8 respectively. As a test/cont rol ratio of 3 or more was not recorded for any of the concentrations tested, HFCPE is not consider ed to have the potential to cause skin sensitization (delayed contact hypersensitivity). Responses to the positive control substa nce hexyl cinnamic aldehyde (HCA), in contemporaneous studies ( Appendix 2 ) demonstrate the reliability and sensitivity of this assay to detect skin sensitization potential in this laboratory.Huntingdon Life Sciences VHJ0006

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## 4. Conclusion
HFCPE is not regarded as a potential skin sensitizer.Huntingdon Life Sciences VHJ0006

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## 5. References
OECD Guideline for Testing of Chemicals N o. 429 "Skin Sensitization: Local Lymph Node Assay". (24 April 2002).
EEC Methods for the determination of toxic ity. Annex to Commission Directive 2004/73/EC (Official Journal No. L152, 30.4.2004), Part B, Method B.42. Skin Sensitisation: Local Lymph Node Assay.Huntingdon Life Sciences VHJ0006

19 Table 1 Group dpm/node and test/control ratios Group Concentration % v/v dpm Number of lymph nodes

per group dpm/node Test/control ratio† Result + = positive - = negative
2 AOO 5242.1 8.0 655.3 n/a n/a 3 10 4142.7 8.0 517.8 0.8 - 4 25 3187.8 8.0 398.5 0.6 - 5 50 4129.2 8.0 516.1 0.8 - † Test/control of 3 or greater indicates a positive result n/a Not applicable dpm Disintegrations per minute (less background count of 55.7 dpm) AOO Acetone:olive oil (4:1 v/v) (vehicle control)Huntingdon Life Sciences VHJ0006

20 Appendix 1 Individual and group m ean bodyweights and individual bodyweight change (g) Group Animal number Day 1* (09 December 2009) Termination (14 December 2009) Individual bodyweight change

2
AOO B2 16.1 17.6 1.5 B3 17.9 19.1 1.2 B4 19.7 20.4 0.7 B5 16.4 17.2 0.8 Mean 17.5 18.6 -

3
10% v/v B6 17.9 18.6 0.7 B7 17.4 18.4 1.0 B8 19.6 20.6 1.0 B9 18.5 19.4 0.9 Mean 18.4 19.3 -

4
25% v/v B10 18.6 18.7 0.1 B11 19.8 19.9 0.1 B12 17.4 18.4 1.0 B13 18.9 20.1 1.2 Mean 18.7 19.3 -

5
50% v/v B14 19.7 19.7 0.0 B15 17.9 18.7 0.8 B16 19.2 19.5 0.3 B17 22.2 22.2 0.0 Mean 19.8 20.0 - AOO Acetone:olive oil (4:1 v/v) (vehicle control) - Not applicable * Prior to dosingHuntingdon Life Sciences VHJ0006

21 Appendix 2 Summary of positive control data Positive control study with hexyl cinnamic aldehyde using the local lymph node assay (Study No. HLS0732) A study was performed to confirm the sensitiv ity and reliability of the experimental technique used at Huntingdon Life Sciences to detect skin sensitization potential. The study was performed using the murine local ly mph node assay (OECD 429 individual animal approach) and a known moderate sensiti zer – hexyl cinnami c aldehyde (HCA). This study was conducted between the 14 a nd 20 October 2009 using ten mice of the CBA/Ca strain supplied by Harlan UK Ltd., Bicester, Oxon, England. The HCA was supplied by Sigma Aldrich Chemical Co. E ngland (Lot No. 02002DH, expiry 17 October 2009). The dose level selected was based on previous experience with HCA at this laboratory and groups of five mice were treated as follows: Group Treatment Concentration Animal numbers
1 AOO 0 B1 – B5 2 HCA 25% v/v B6 – B10 AOO acetone:olive oil (4:1 v/ v) (vehicle control) HCA hexyl cinnamic aldehyde (positive control) The mice were treated by daily application of 25 μl of HCA, or vehicle control to the dorsal surface of both ears for three consecutive days. The proliferative response of the lymph node cells (LNC) from the draining auricular lymph nodes was assessed five days following the in itial application, by measurement of the incorporation of 3 H-methyl Thymidine ( 3 HTdR) by β -scintillation counting of LNC suspensions. The response was expressed as radioactive disintegrations per minute per lymph node (dpm/node) and as the ratio of 3 HTdR incorporation into LNC of test nodes relative to that recorded for control nodes (test/control ratio). The positive control study is cons idered to be valid if the re sults from the HCA group have a three-fold greater increase in 3 HTdR incorporation compared to control values. In this assay the test/control ratio obtained fo r HCA at 25% was 6.0. This indicates that HCA demonstrates the potential to i nduce skin sensitization (delayed contact hypersensitivity) and confirms the sensitivity of the techni que to detect sensitization potential.Huntingdon Life Sciences VHJ0006

22 Group dpm/node and test/control ratios Group Concentration % v/v Group mean dpm/node test/control ratio† Classification + = positive - = negative 1 Control, AOO 460.53 n/a n/a 2 25 2757.13 6.0 + AOO Acetone:olive oil (4:1 v/v) † Test/control ratio of greater than 3.0 indicates a positive result dpm Disintegrations per minute (Less mean background count of 53.55 dpm) n/a Not applicable
Individual dpm values
Grou p /treatment Animal number d p m count* B1 456.4 1 B2 997.1 B3 763.9 B4 1338.4 AOO B5 1317.3 Mean 974.6 SD 375.24

B6 2463.7 2 B7 7196.7 B8 7278.0 B9 1471.0 HCA 25% v/v B10 9429.7 Mean 5567.8 SD 3424.60

* Includes background count of 53.55 dpm SD Standard deviationHuntingdon Life Sciences VHJ0006

23 Annex 1 GLP Compliance StatementsHuntingdon Life Sciences VHJ0006

24 HUNTINGDON RESEARCH CENTRE GLP COMPLIANCE STATEMENT 2008Huntingdon Life Sciences VHJ0006

25 HUNTINGDON RESEARCH CENTRE GLP COMPLIANCE STATEMENT 2009