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### REPORT

**Assessment of Skin Sensitization Potential using the**  **Local Ly mph Node Assay in the Mouse**  **(Pooled treatment group approach)** 

**HLS s tudy number:**  CEF0006 **Version ID:** Audited Draft 3 **Issue date:** 24 July 2013Huntingdon Life Sciences CEF0006

2 

## Sponsor **and Te**  **st Facility Details**  **Sponsor**

**Test facility** Huntingdon Life Sciences Huntingdon Research Centre Wo olley Road Alconbury Huntingdon Cambridgeshi re PE28 4HS UKHuntingdon Life Sciences CEF0006

3 

## Ta **ble 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 Principle of the local lymph node assay
............................................................ 9 1.3 Regulatory complianc e ..................................................................................... 9 1.4 Test system
...................................................................................................... 9 2. Experimental Procedure
............................................................................................. 10 2.1 Study schedule and structure
.......................................................................... 10 2.1.1 Time schedule
..................................................................................... 10 2.1.2 Identity of treatment groups
................................................................ . 10 2.2 Test substance and formulation
...................................................................... 11 2.2.1 Test substance
.................................................................................... 11 2.2.2 Vehicle selection
................................................................................. 11 2.2.3 Formulation
......................................................................................... 11 2.3 Animal management
....................................................................................... 12 2.3.1 Animal housing, diet and water supply
................................................ 12 2.4 Concentration selection
.................................................................................. 13 2.4.1 Preliminary investigations
.................................................................... 13 2.4.2 Main study
........................................................................................... 13 2.5 Administration of test substance
..................................................................... 13 2.5.1 Preliminary investigation
..................................................................... 13 2.5.2 Main phase
......................................................................................... 14 2.5.3 Administration of 3H- methyl Thymidine
............................................... 14 2.6 Observations
.................................................................................................. 14 2.6.1 Clinical signs
....................................................................................... 14 2.6.2 Dermal reactions
................................................................................. 14 2.6.3 Measurement of ear swelling
............................................................... 14 2.6.4 Bodyweight
......................................................................................... 15 2.7 Terminal studies
............................................................................................. 15 2.7.1 Termination
......................................................................................... 15 2.7.2 Preparation of sing le cell suspensions
................................................. 15 2.7.3 Determination of incorporated 3H - methyl Thymidine
........................... 15 2.8 Data treatment
................................................................................................ 15 2.8.1 Stimulation Index
................................................................................. 15 2.8.2 Estimated Concentration of Three
....................................................... 16 2.9 Computer systems
.......................................................................................... 16 2.10 Quality Assurance and archiving procedures
.................................................. 16 2.10.1 Quality Assurance
............................................................................... 16 2.10.2 Archives
.............................................................................................. 16 2.11 Deviations from protocol
................................................................................. 16 3. Results
....................................................................................................................... 17 3.1 Preliminary investigation
................................................................................. 17Huntingdon Life Sciences CEF0006

4 3.1.1 Mortality and clinical sign s ................................................................... 17 3.1.2 Dermal reactions
................................................................................. 17 3.1.3 Measurement of ear thickness
............................................................ 17 3.1.4 Bodyweight
......................................................................................... 17 3.2 Main phase
..................................................................................................... 17 3.2.1 Mortality and clinical signs
................................................................... 17 3.2.2 Dermal reactions
................................................................................. 18 3.2.3 Bodyweight
......................................................................................... 18 3.3 Stimulation Index and Estimated Concentration of Three
................................ 18 4. Conclusion
................................................................................................................. 19 5. References
................................................................................................................. 20 

## List of T **ables** Table 1 Group dpm/node and Stimulation Index
....................................................... 21 

## List of A**ppendi ces**  Appendix 1 Preliminary phase
– Measurement of ear thickness (mm)
............................ 22 Appendix 2 Preliminary and Main phases - Individual and group mean bodyweights and

individual bodyweight chan ge (g)
................................................................ . 23 

## List of **An nexes**  Annex 1 GLP Compliance Statement
......................................................................... 24Huntingdon Life Sciences CEF0006

5 

## Compliance with Good Laboratory **Practice Assessment of Skin Sensitization Potential using the Local**  **Lymph Node Assay in the Mouse (Pooled treatment group approach)** The study described in this report was conducted in co mp liance with the fo llo wing Good Laboratory

Practi ce Standards and, with th e exceptio n noted below ; I consider the data generated to be valid. The UK Good Laboratory Practice Regulat io ns (Statutory Instrument 1999 No. 3106, as amended by Statutory Instrument 2004 No. 994). EC Commissio n Di

rective 2004/10/EC of 11 February 2004 ( Official Journal No. L 50/44). OECD Principles of Good Laboratory Practice (as revised in 1997), ENV/MC/CHEM(98)17. These principles of Good Laboratory Practice are accepted by

the regulatory

authori ti es of th e United States of America and Japan on the bas is of intergovernmental agreements. Chemical analysis of formulated test articles for determination o f stabilit y, ho mo geneit y and concentration was not undertaken for this study
.

Clare Chesher Study Di rector Hunt ingdon Life Sciences DateHuntingdon Life Sciences CEF0006

6 

## Quality **As surance St** **atement**

**Assessment of Skin Sensitization Potential using the Local Lymph Node Assay in the Mouse (Pooled treatment group app**  **roach)** The fo llo wing inspect io ns and audits have been carried out in relation to this study: **Study Phase Date of Inspection**  **Date of Reporting to Study**  **Director and Management Protocol Audit**  01 May 2013 01 May 2013 **Report Audit** 27 June 2013
- 28 June 2013 28 Jun 2013 **Process based inspections:** At or about th e time this study was in progress inspect io ns of procedures emplo yed on this t ype 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** Study management and conduct 08 Apr 2013
- 09 Apr 2013 10 Apr 2013 Preparati on and administration of radi olabel 15 Apr 2013 15 Apr 2013 Terminal procedures 15 Apr 2013 15 Apr 2013 Form ulat io n procedures 02 May 2013 02 May 2013 Body weight 23 May 2013
- 28 May

2013 28 May

2013 Dose administration 30 May 2013
- 05 June 2013 05 Jun 2013 Assay Procedures 05 Jun 2013 05 Jun 2013 In addit io n, an inspect io n of the facilit y where thi s study was conducted was carried out on an annual b asis. These inspect io ns were promptly reported to Company Management.

Kevin Oldham MRQA Lead Auditor Department of Qualit y Assurance Hunt ingdon Life Sciences DateHuntingdon Life Sciences CEF0006

7 

## Contributing Scientist

**Assessment of Skin Sensitization Potential using the Local Lymph Node Assay in the Mouse (Pooled treatment group approach)**  

### Study management Clare J Chesher BSc (Hons) MSc Study Di rectorHuntingdon Life Sciences CEF00 06

8 

## Summary The study was performed to assess the skin sensit ization potential of using the lo cal lymph node assay

(LLNA). Preliminary invest igat io ns were performed at 25 and 50 % w/v wi th 2 mice per concentration to establish the highest concentration o f test substance which did not lead to systemic toxici ty or excessive loc al

i rri tation. The results of preliminary invest igat io ns indicated th at 50% w/v would be a sui

table high concentration for use on the main study
. The study com prised three treat ed groups, each comprising four fe male mice receiving at concentrations of 10, 25 or 50 % w/v. Similarly const it uted groups received the vehicle Acetone: olive oil (4:1 v/v) or posit ive control substance (2 5% v/v hexyl cinnamic aldehyde). T he mice were treated by daily applicat io n of 25  L of the appropri ate concentration or contro l (vehicle or posit ive), to the dorsal surface of both ears for three consecutive days. T he proliferat ive response of the lymph node cells (LNC) fro m th e draining auricular lymph nodes was assessed five days fo llo wing the init ial applicat io n, by measuremen t of the incorporation of 3 H- met hyl Thymidine ( 3 HTdR) by 
- scint illat io n counting of LNC suspensions. The response was expressed as radioactive disintegrat io ns per minute per lymph node (dpm/node) and as the ratio of 3 HTdR incorporation into LNC of test nodes relat ive to that recorded for con trol nodes (test/control

ratio) , termed as Stimulat io n Index (SI)
. The test substance is regarded as a sensit izer if at least one concentration of the chemical has a SI of three or more. 

### Results The SI obtained for 10, 25 and 50 % w/v were 1.1, 1.0 and 1.5 respectively which indicates th at did not show th e potenti al to induce skin sensit iz ation. The SI for the posit ive control substance hexyl cinnamic aldehyde was 5.6 , whi ch demonstrates the validit y of thi s stud y. 

### Conclusion

is not regarded as a potential skin sensitizer.Huntingdon Life Sciences CEF0006

9 

## 1. **Introduction 1.1**  **Objective** The obj ect ive o f thi s study was to assess the skin sensit iz ation potenti al of 

### 1.2 **Principle of the local lymph node assay** The theory

behind the lo cal

lymph node assay (LLNA) is that a sensit izing substance induces proliferat io n of lymphocy tes in the lymph nodes draining the site of test substance exposure. The lymphocy

te proliferat io n is proportional to the dose and potency o f th e sensit izing substance and is quantified by measuring the incorporation of radio labelled Thymidine. Assessment of sensit izat io n potential is made by comparing the mean proliferation in each test group to the mean proliferat io n in the vehicle control group, with the ratio between tr eated and control groups termed the Stimulat io n Index (SI). A test substance is regarded as a sensit izer if the SI is 3 or more in any o f th e concentrations tested. 

### 1.3 **Regulatory compliance** The study was designed to meet the requirements of the fo llo wing guidelines: OECD Guideline for Testing of Chemicals No. 429 “Skin Sensit ization: Local Lymph Node Assay”. 22 July 2010
. EEC Methods for the determination o f toxi ci ty and other healt h effects. Commissio n Regulat io n No. 440/2008. Part B, Method B.42. Skin se nsi ti sat io n: Local Lymph Node Assay. 30 May 2008. As amended by Regulat io n No. 640/2012 06 Jul 2012. 

### 1.4 **Test system** The fe male CBA/Ca mouse is chosen as the test species as it has been shown to be a suitable mo del f or thi s study

and is the species/strain/s ex recommended by the test guidelines.Huntingdon Life Sciences CEF0006

10 

## 2. **Experiment al Procedure**  **2.1 Study schedule**  **and structure 2.1.1**  **Time schedule** Study init iat io n: (p rotocol signed by Study Di rector) 29 April 2013 Experimental start date: (Ini ti al animal

allocation) 02 May 2013 Treatment commenced: 08 May 2013 Com plet io n of live phase : 20 May 2013 Experimental co mp let io n date: (date of scint illat io n counting) 21 May 2013 Study com plet io n: When Study Di rector signs final

report **2.1.2 Identity of treatment groups**  Groups o f mice were treated as follo ws: **Group Treatment**  **Concentration Animal numbers**  1# 25%w/v D1
- D2 2# 50%w/v D3
- D4 3 AOO 0 D5
- D8 4 10% w/v D9
- D12 5 25% w/v D13
- D16 6 50% w/v D17
- D20 7 HCA 25% w/v D21
- D24 AOO Acetone:olive oil (4:1 v /v) (vehicle control) # Preliminary investigations HCA Hexy

l cinnamic aldehy

de (positive control)Huntingdon Life Sciences CEF0006

11 

### 2.2 **Test substance and formulation 2.2.1**  **Test substance** Ident ification: Chemical name: Intended use: Appearance: White crystalline powder Storage condit io ns: Room tem perature in the dark Supplier: Sponsor Batch or L ot number: 12342 Expi ry date: W ill be documented in the raw data and in th e final report Puri ty W ill be documented in the raw data and in th e final report Sam ple receipt: 4 March 2013 The Sponsor was responsible for the characterisat ion of the test substance and the documentation of

the methods of synt hesis, fabrication or derivat io n and stabilit y. **2.2.2 Vehicl**  **e selection** A vehicle trial perf

ormed wi th showed that it formed a thick off whi te suspensi on at 50 % w/v in acetone:olive oil (4:1 v/v)
. It was not possible to formulate at 100% as the materi al was too bul

ky . A cetone:olive oil (4:1 v/v) is th e pr ef erred vehicle for the local lymph node assay and as the test materia l was sui ta ble for dosing in acetone:olive o il (4:1 v/v) at 50% no other vehicles required testing. **2.2.3 Formulation**  The test substance, was prepared for administration as a series o f graded concentrations in the vehicle, by direct dilut io n. The test substance was used as supplied and all formulat io ns were prepared on the day of dosing at the requi

red concentration s. The absorption of the test substance was not determined. Determinat i on of

the ho mo geneit y, stabili

ty and puri ty of the test substance or test substance fo rm ulat io ns were not undertaken as part of this study
.Huntingdon Life Sciences CEF0006

12 Detailed records of test substance usage were maintained. The amount of test substance necessary to prepare the formu lat io ns and the amount actually used were determined on each occasion. The difference between these amounts was checked before the formulat io ns were di spensed. 

### 2.3 **Animal management** H eal thy female CBA/Ca mice (a total of 24 fe males) we re obtained fro m Charl es River UK. The mice were in the weight range 17.3 to 21.9 g and approximately eight to twelve weeks of age prior to dosing on Day 1. They were acclimat ised to the exper imental environment for 6 days pri or to the start of the study
. Each animal was assig ned an alpha -numeric code and ident ified uniquely wit hin the study by tail marking. Each cage label was co lo ur- co ded and was ident ified uniquely wit h the study number, test substance concentration and animal mark (s)
. **2.3.1 Animal housing, diet and water supply**  Animals were housed inside a barriered rodent facilit y (Building F21 , Room 061/062 ). The facilit y was designed and operated to minimise the entry of external biological and chemical agents and to minimise the transference of such agents between rooms. T he animals were allocated without conscious bias to cages within the treatment groups. They were housed two animals per cage , in so lid bottomed polycarbonate cages with a stainless steel mesh lid. Each cage contained a quant it y of autoclaved wood flake b edding , addi tionally Nest let s and a plast ic shelter were included fo r envi ronmental

enrichment. Certificates of analysis for woodflake bedding and Nest let s were lodged in Hunt ingdon Life Sci ences Ltd. Archives. The animal room was kept at positive pressur e wi th respect to the outside by its own supply of f iltered fresh air, which was passed to atmosphere and not re
- circulated. The temperature and relative humidit y control s were set to maintain the range of 19 to 23  C and 40 to 70% respectively . Any minor deviat io ns fro m th ese ranges would not have had an adverse effect on the animals and would not affect the integrit y or validit y of the study . Artificial light ing was controlled to give a cy cle of 12 hours continuous light and 12 hours continuous dark per 24 hours. Peri odic checks were made on the number of air changes in the animal rooms. Temperature and humidit y were moni

tored daily and records are archived with the departmental raw data
. Alarms were activated if there was any failure of the vent ilation system , or tem perature limits were exceeded. A stand
- by electrici ty supply was available to be automat ically brought into operati on should the public supply fail. The animals were allowed free access to a standard rodent diet (Rat and Mouse No. 1 Maint enance Diet). This diet contained no added ant ibiotic or other chemotherapeut ic or prophylact ic agent. Potable water taken fro m th e public supply was f reely available via polycarbonate bottles fi tted wi th sipper tubes.Huntingdon Life Sciences CEF0006

13 Each batch of diet was analysed ro ut inely by the supplier for various nutrit io nal com ponents and chemical and microbio lo gical contaminants. Supplier’s analy ti cal certificates were scrutinised and approved before any b atch of di et was released for use. The qualit y of the water supply

i s g overned by

regulat io ns published by the Department f

or Envi ronment, Food and Rural Affairs. Certificates of analysis were received routinely fro m th e 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 **C oncentration selection**  **2.4.1 Preliminary investigations**  Preliminary invest igat io ns were performed to ensure the highest concentration to be used on th e main study

did not resul

t in systemic toxicit y or excessive local i rri tation. An assessment of local irritati on was performed and an ery th ema score greater or equal to 3 and/or an increase in ear thickness of greater than 25% at Day 3 or 6 com

pared wi th predose would be regarded as i

rri tation which precludes the use of that concentration on the main study
. **2.4.2 Main s**  **tudy** The results of the preliminary invest igat io ns indicated that 50 % w/v was a suitable high concentration for administration in the main phase of the study . The low and intermediate concentrations were selected from the concentration series given in re gulatory guidelines and th e concentrations administered on the main study were: 10, 25 and 50% w/v in Acetone: olive oil (4:1 v/v) The posi tive control

group received 25% v/v hexylcinnamic aldehyde in Acetone: olive oil (4:1 v/v) based on previous experie nce at this laboratory
. Pri or to dose administration the Study Di rector authori sed the cho ic e of vehicle and dose levels in the raw data. 

### 2.5 **Administration of test substance 2.5.1**  **Preliminary investigation**  Two fe male mice (per concentration) received a daily appli cation of

25  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 using an automat ic micropipette and was spread over th e enti re dorsal surface of the ear using the tip of the pi pette.Huntingdon Life Sciences CEF0006

14 **2.5.2 Main**  **phase** Groups of fo ur mice were treated at one of three concentrations of the test substance. The mice were treated by daily applicat io n of 25  L of the appropri ate concentration of t he 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 and was spread over the ent ir e dorsal surface o f th e ear using the ti p of the pi pette. Furth er groups of four mice received the vehicle alone or the posit ive control substance in the same manner. **2.5.3 Administration of**  **3H- methyl Thymidine**  In the main phase of the study, five days fo llo wing the fi rst topi cal applicat io n of tes t substance (Day

6) all mice were injected via the tail vein wit h 250  L of phosphate buffered saline containing 3 H- met hyl Thymidin e a ( 3 HTdR: 80  Ci /mL) giving a no minal 20  Ci to each mouse. The inject io n int o the tail vein was carried out using a plast ic syringe and needle af ter the mouse had been heated in a warming chamber. 

### 2.6 **Observations 2.6.1**  **Clinical signs** All animals were observed daily f or signs of ill healt h or toxicit y. The ears were examined fo r signs of

i rri tation. **2.6.2 Dermal re**  **actions** In the preliminar y phase of the study, an assessment of ery th ema was made predose o n Day 1 , 2 and 3 and daily th ereafter using the follo wing numerical scoring system: Ery th ema and eschar format io n: No ery thema 0 Very slight ery

th ema (barely percept ible) 1 Well
- defined e ry

th ema 2 Moderate to severe ery

th ema 3 Severe ery

th ema (beet redness) to eschar format io n (injuries in depth) 4 **2.6.3 Measurement of ear swelling**  On the preliminary

phase of the study, ear thickness was measured for both ears of each mo use on Days 1 (predo se), 3 (predose) and 6 (preterminal). Measurements were taken in tri plicate by placing a thickness gauge in the centre of each ear. The gauge was completely removed from

the ear and replaced for each measurement. The reported ear thickness on each occas io n was the mean of the three measurements.

a Specific activity 2.0 Ci/mmol, concentration 1.0 mCi/mLHuntingdon Life Sciences CEF0006

15 **2.6.4 Bodyweight**  The weight of each mouse was recorded on arrival (data not reported), Day 1 ( before dosing) and prior to terminat io n (Day 6). 

### 2.7 **Terminal studies 2.7.1**  **Termination** The mice in the preliminary invest igat io ns wer e humanely killed by carbon dioxide asphyxiation on Day

6 of the study
. The carcasses were discarded and no further investigat io ns were c arried out with these animals. In the main phase of the study, five hours fo llo wing the administration of 3 HTdR on Day 6 all mice were humanely killed by carbon dioxide asphyxiat io n . T he draining auricular lymph nodes were excised and poo led fo r each experimental group in 1.0 mL of phosphate buffered saline (PBS) . The carcasses were then discarded and no further invest igat io ns were carried out. The fo llo wing procedures were carried out with the lymph nodes from these animals only
. **2.7.2 Preparation of single cell suspensions**  A single cell suspensio n of lymph node cells (LNC) was prepared by gent le mechanical di saggregation th

rough a stainless steel gauze (200 mesh size). The pooled LNC were then washed by adding 10 mL PBS , 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 th e final wash. **2.7.3 Determination of incorporated**  **3H- methyl Thymidine**  After overnight incubat io n (minimum o f 18 hours) wit h 5% TCA at 4  C, the precipitate was recovered by

centrifugati on and resuspended in 1 mL 5% TCA and transferred to 10 mL Ul tima gold scint ill at io n fluid on Day 7. The 3 HTdR incorporation was measured by 
- scintillat io n count ing. The proliferat ive response of LNC was expressed as radioactive di sintegrat io ns per minute per lymph node (dpm/node). 

### 2.8 **Data treatment 2.8.1**  **Stimulation Index** Results for eac h treatment group were expressed as the Stimulat io n Index (SI). This was derived by dividing the mean dpm /mouse f or each treated group and the posit ive control group by the mean dpm

/mouse in the vehicle control group. If the SI is 3 or more, the test sub stance is regarded as a skin sensit izer. The posi tive control

group is expected to give an SI of 3 or more to demonstrate the validit y of the study
.Huntingdon Life Sciences CEF0006

16 **2.8.2 Estimated Concentration of Three**  The Estimated Concentration of Three ( EC3 ) is the concentration of test su bstance which would resul

t in a SI of 3 and, where data permits, this value was calculated ( Ryan 2007 )
. As all concentrations tested result ed in an SI of less than 3 then the EC3 was reported as greater than the highest concentration tested 50% w/v
. 

### 2.9 **Computer systems** The computer systems that were used on this study to acquire and quant ify data include: **System name*** **System function** Pristima Pharmacy data management
* All versio

n numbers of the systems are maintained by Huntingdon Life Sciences 

### 2.10 **Quality A** **ssurance and archiving procedures 2.10.1**  **Quality A** **ssurance** Details o f th e Qualit y Assurance inspections and audits are presented on the Qualit y Assurance Statement. **2.10.2 Archives**  Fo llo wing com

plet io n of this study

all raw data were store d in the archives of Hunt ingdon Life Sciences. A copy of the final report will

b e retained indefinit ely and all Qualit y Assurance inspection records for a period of 20 years. All other appropriate specimens and records will be retained fo r a minimu m peri od of one year from th e date of issue of the final report. At the end of this retention peri od the Sponsor will be contacted and advice sought on future archiving requi rements. Under no circumstances will any item be discarded without the Sponsor’s knowledge. 

### 2.11 **Deviations from protocol** There wer e no deviat io ns from protocol
.Huntingdon Life Sciences CEF0006

17 

## 3. **Result s**  **3.1 Preliminary**  **investigation 3.1.1**  **Mortality and clinical signs** There were no deaths and no signs of ill health or toxici ty were observed during this study
. Greasy f ur or dose re sidue on the head was noted following each dosing occasion, this was related to unoccluded dermal administration of a liquid f orm ulation and not an effect of the test substance. **3.1.2 Dermal re**  **actions** No eryt hema wa s observed on the ears of any mo use on Days 1 t o 6. **3.1.3 Measurement of ear thickness**  Appendix 1 There was no evidence o f an effect of treatment on ear thickness. **3.1.4 Bodyweight**  Appendix 2 There was no indicat io n of an overt effect of treatment on body weight gain. A lo ss in body

weight was noted for both mice re ceiving 25% w/v over the study period, however, a small loss in body weight i s not uncommo n in yo ung laboratory mice and this is not considered to be an effect of treatment. On the basis of the results fro m th e preliminary investigation , 50 % w/v was conside red a sui table high concentration f or administration in the ma in 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 toxici ty were observed in treated animals during this study
. Greasy f ur and/or dose residue on the head was noted fo llo wing each dosing occasion, this was related to unoccluded dermal administration of a liquid formulat io n/ vehicle and not an effect of the test substance. Parti ally closed eyelids were observed for all mice rec eiving the HCA posit ive control fo llo wing dosing on Day

2 only.Huntingdon Life Sciences CEF0006

18 **3.2.2 Dermal re**  **actions** No signs of dermal irri

tation were seen o n th e ear during the study
. **3.2.3 Bodyweight**  Appendix 2 There was no indicat io n of an effect of treatment on bodyweight gain. A lo ss in body

weight was noted for one mou se receiving 50% w/v (No. D19 ) over the study peri od, however, a small lo ss in body weight is not uncommo n in yo ung laboratory mice and is not considered to be an effect of treatment. 

### 3.3 **Stimulation Index and Estimated Concentration**  **of Three** Table 1 The SI (test/control ratios) obtained for 10, 25 and 50 % w/v were 1.1, 1.0 and 1.5 respectively
. As a SI of 3 or more was not recorded for any of the concentrations tested, is not considered to have the potential to cause skin sensit izat io n. The SI for the posit ive control substance hexyl cinnamic aldehyde (HCA) was 5.6 which demonstrates the validit y of thi s study
.Huntingdon Life Sciences CEF0006

19 

## 4. **Conclusion**

is not regarded as a potential skin sensitizer.Huntingdon Life Sciences CEF0006

20 

## 5. **References** Ryan CA, Chaney JG, Gerberick GF, Kern PS, Dearman RJ, Kimber I, Basketter, DA (2007). Extrapolat ing lo cal

lymph node assay

EC3 val ues to estimate relat ive sensit izing potency
. Cutaneous and O cular Toxicology , **26** : 135
- 145.Huntingdon Life Sciences CEF0006

21 **Table 1**  **Group dpm/node and Stimulati**  **on Index Group**  **Concentration dpm**  **Number of lymph**  **nodes per animal**  **dpm/node Stimulation**  **Index† Result** 

**+ = positive**

**- = negative**  3 AOO 357.10 8.0 44.64 n/a n/a 4 10% w/v 395.20 8.0 49.40 1.1
- 5 25% w

/v 364.80 8.0 45.60 1.0
- 6 50 % w/v 520.50 8.0 65.06 1.5
- 7 HCA 25% v/v 1985.10 8.0 248.14 5.6 + † Stimulation Index of 3 or more indicates a positive result dpm Disintegrations per minute less background count of 35.80 AOO Acetone:olive oil (4:1 v/v) (vehicle contro l) HCA Hexy

l cinnamic aldehy

de (positive control)Huntingdon Life Sciences CEF0006

22 **Appendix 1**  **Preliminary phase –**  **Measurement of ear thickness (m**  **m) Concentration**  **( % w/v**  **) Animal**  **No. Days**  **1 (Predose) 3 (Predose)**  **6 (Preterminal)**

25 D1 L 0.2 1 0.21 0.20

% difference from Day 1
- 0
- 5

R 0.21 0.20 0.21 % difference from Day 1
- - 5 0
D2 L 0.21 0.20 0.20

% difference from Day 1
- - 5
- 5

R 0.21 0.20 0.20 % difference from Day 1
- - 5
- 5

5 0 D3 L 0.21 0.21 0.2 0

% difference from Day 1
- 0
- 5

R 0.21 0.20 0.21 % difference from Day 1
- - 5 0
D4 L 0.2 0 0.21 0.20

% difference from Day 1
- +5 0

R 0.21 0.21 0.20 % difference from Day 1
- 0
- 5 The values above represent the mean of three readings, the individual readings are retained in the raw data. L Left ear R R

ight ear

- Not applicableHuntingdon Life Sciences CEF0006

23 **Appendix 2**  **Preliminary and Main phases -**  **Individual and group mean**

**bodyweights and individual bodyweight change (g) Grou** **p Animal number**  **Day 1*** **Day 6 (**  **Termination )**  **Individual**

**bodyweight change** 1 25 % w/v D 1 21.9 21.0 (0.9)

D 2 19.3 18.7 (0.6) Mean 20.6 19.9
- 2 50 % w/v D 3 17.8 18.2 0.4

D 4 17.8 18.0 0.2 Mean 17.8 18.1
- 3 Vehicle (AO O) D 5 19.4 19.8 0.4

D 6 20.1 20.7 0.6 D 7 19.6 19.7 0.1 D 8 18.5 19.1 0.6 Mean 19.4 19.8
- 4 10% w /v D 9 20.5 21.1 0.6

D 10 20.5 21.4 0.9 D 11 19.8 20.2 0.4 D 12 18.0 18.9 0.9 Mean 19.7 20.4
- 5 25% w /v D 13 19.9 21.1 1.2

D 14 19.2 19.6 0.4 D 15 19.1 19.6 0. 5 D 16 17.3 18.1 0.8 Mean 18.9 19.6
- 6 50% w /v D 17 20.3 20.5 0.2

D 18 20.1 20.1 0 .0 D 19 19.9 19.8 (0.1) D 20 19.5 20.1 0.6 Mean 20.0 20.1
-
7 HCA D 21 18.0 18.8 0.8

D 22 19.4 19.6 0. 2 D 23 18.0 18.8 0.8 D 24 17.4 18.0 0.6 Mean 18.2 18.8
- AOO Acetone:olive oil (4:1 v/v) (vehicle control) HCA Hexy

l cinnamic aldehy

de (positive control) ( ) Loss in bodyweight

- Not applicable * Prio

r to dosingHuntingdon Life Sciences CEF0006

24 **Annex 1 GLP Compliance Statement**  **HUNTINGDON RESEARCH CENTRE GLP COMPLIANCE STATEMENT 2012**