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Working for a better future

HFCPE: Assessment of Skin Sensitization 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 2010 
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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 

 UK 
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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 ................................................................................................................... 17
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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 ........................................................................ 23


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Contributing Scientist 

HFCPE: Assessment of skin sensitization potential using the local lymph 

node assay in the mouse (pooled treatment group approach) 

Study management 

Rachel Clarke BSc (Hons) 

Study Director 
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Summary 

The study was performed to assess the skin sensitization potential of HFCPE using the 

murine local lymph node assay (LLNA). 

In order to find a suitable level for the main phase of the study, 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 was too volatile to be administered 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 the lymph node cells (LNC) from 

the draining auricular lymph nodes was assessed five days following the initial application, 

by measurement of the incorporation of 3H-methyl Thymidine (3HTdR) 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 3HTdR incorporation into LNC of test 

nodes relative to that recorded for control nodes (test/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 3HTdR incorporation compared to control values. 

Results 

In this assay the test/control 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 substance 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. 
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1. Introduction 

1.1 Objective 

The local lymph node assay (LLNA) was designed to assess skin sensitization potential 

(delayed type hypersensitivity) of HFCPE (used in electronic applications) in the mouse 

following topical application to the dorsal surface of the ear. In this study lymphocyte 

proliferation was assessed during the sensitizing phase of the response. Lymphocyte 

proliferation in lymph nodes draining the site of application of the test substance was 

quantified by measuring the incorporation of radiolabelled Thymidine. 

1.2 Regulatory compliance 

The study was designed to meet the requirements of the following guidelines: 

OECD Guideline for Testing of Chemicals No. 429 “Skin Sensitization: Local 

Lymph Node Assay”. (24 April 2002). 

EEC Methods for the determination of toxicity. 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. 

1.3 Test system 

The mouse was chosen as the test species as it has been shown to be a suitable model. 
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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: Electronic 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 characterisation of the test substance and the 

documentation of the methods of synthesis, fabrication or derivation and stability. 
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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 investigation 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 purity of the test substance or test substance 

formulations were not undertaken as part of this study. 

Detailed records of test substance usage were maintained. The amount of test substance 

necessary to prepare the formulations 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 prepared on the day of dosing at the required 

concentrations. 

The absorption of the test substance was not determined. 

2.3 Animal management 

Seventeen healthy female CBA/Ca mice were obtained from Harlan UK Ltd. One of these 

animals arrived on 19 November 2009 and was used for the preliminary investigation. The 

remainder arrived on 03 December 2009 and were used for the main phase of the study. 

All preliminary and main phase animals were in the weight range 16.1 to 22.2 g and 

approximately eight to twelve weeks of age prior 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 code and identified uniquely within the study by 

tail marking. Each cage label was colour-coded 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 facility (Building F21, Room 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 treatment groups. They 

were housed individually in polycarbonate cages with woodflake bedding. The mice were 

also given Nestlets for environmental enrichment. Certificates of analysis for woodflake 

bedding and Nestlets were lodged in Huntingdon 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 temperature 
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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 air changes in the animal rooms. Temperature 

and humidity were monitored daily and records are 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 public 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 was 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 any 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 were received routinely from the water supplier. 

Since the results of these various analyses did not provide evidence of contamination that 

might have prejudiced the study, 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 checked periodically at Huntingdon Life Sciences 

Ltd. with hexyl cinnamic aldehyde (HCA), a known moderate sensitizer (Appendix 2). 
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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 neat liquid test substance, however, the test 

substance is a volatile liquid and it was considered not appropriate to perform dosing due to 

the rapid evaporation. 

Based on this information the following concentrations were selected for the main phase of 

the study: 

10, 25 and 50% v/v 

Prior to dose administration the Study Director authorised the choice 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 μl 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 using an automatic micropipette. The test substance was spread 

over the entire dorsal surface of the ear using the tip of the pipette.

2.6.2 Main phase 

Groups of four mice were treated at one of three concentrations of the test substance. The 

mice were treated by daily application of 25 μl of the 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 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 following the first topical application of test 

substance (Day 6) all mice were injected via the tail vein with 250 μl of phosphate buffered 

saline containing 3

H-methyl Thymidinea (3HTdR: 80 μCi/mL) giving a nominal 20 μCi to 

each mouse. The injection into the tail vein was 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/mL 
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2.7 Observations 

2.7.1 Clinical signs 

All animals were observed daily for signs of ill health or toxicity. The ears were also 

examined for signs of irritation. 

2.7.2 Bodyweight 

The weight of the mouse in the preliminary investigation 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 dosing) and prior to termination (Day 6). 

2.8 Terminal studies 

2.8.1 Termination 

The mouse in the preliminary investigation was humanely killed by carbon dioxide 

asphyxiation on Day 4 of the study. No further investigations 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 draining 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 procedures were carried out with 

the lymph nodes from these animals only. 

2.8.2 Preparation of single cell suspensions 

A single cell suspension of lymph node cells (LNC) was prepared by gentle mechanical 

disaggregation through a stainless steel gauze (200 mesh size). The pooled LNC were then 

washed by adding 10 mL phosphate buffered saline, pelleted at 190 x g for 10 minutes and 

resuspended. The cells were washed twice again 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 incubation 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 radioactive disintegrations per minute 

per lymph node (dpm/node) and as the ratio of 3HTdR 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 least one concentration of the test substance 

results in a three-fold greater increase in 3HTdR incorporation compared to control values. 
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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 data were stored in the archives of 

Huntingdon Life Sciences. 

A copy of the final report and all Quality Assurance inspection records 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 year retention period the 

Sponsor will be contacted and advice sought on the above requirements. Under no 

circumstances will any item be discarded without the Sponsor’s knowledge. 

2.11 Deviations from protocol 

There were no deviations from protocol. 
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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 area during the study. 

3.1.2 Bodyweight 

A bodyweight increase was recorded for the mouse 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 health or toxicity observed during this study, 

however, the following observations were noted: 

Greasy fur on the cranial region was observed post-dose from Day 1 in the control and all 

treated groups. This sign had resolved 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 proliferative 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/control ratio of 3 or more was not recorded for any of the 

concentrations tested, HFCPE is not considered to have the potential to cause skin 

sensitization (delayed contact hypersensitivity). 

Responses to the positive control substance 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. 
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4. Conclusion 

HFCPE is not regarded as a potential skin sensitizer. 
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5. References 

OECD Guideline for Testing of Chemicals No. 429 "Skin Sensitization: Local Lymph Node 

Assay". (24 April 2002). 

EEC Methods for the determination of toxicity. 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. 
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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) 
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Appendix 1 Individual and group mean 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 dosing 
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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 sensitivity and reliability of the experimental 

technique used at Huntingdon Life Sciences to detect skin sensitization potential. The study 

was performed using the murine local lymph node assay (OECD 429 individual animal 

approach) and a known moderate sensitizer – hexyl cinnamic aldehyde (HCA). 

This study was conducted between the 14 and 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. England (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 initial application, by measurement of the 

incorporation of 3

H-methyl Thymidine (3HTdR) 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 considered to be valid if the results from the HCA group have a 

three-fold greater increase in 3HTdR incorporation compared to control values. 

In this assay the test/control ratio obtained for HCA at 25% was 6.0. This indicates that HCA 

demonstrates the potential to induce skin sensitization (delayed contact hypersensitivity) and 

confirms the sensitivity of the technique to detect sensitization potential. 
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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 

Group/treatment Animal number dpm 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 deviation 
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Annex 1 GLP Compliance Statements 
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HUNTINGDON RESEARCH CENTRE GLP COMPLIANCE STATEMENT 2008 
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HUNTINGDON RESEARCH CENTRE GLP COMPLIANCE STATEMENT 2009