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Working for a better future

REPORT

 Assessment of Skin Sensitization Potential using the 

Local Lymph Node Assay in the Mouse (Pooled treatment group 

approach)

HLS study number: CEF0006

Version ID: Audited Draft 3

Issue date: 24 July 2013
Huntingdon Life Sciences

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Sponsor and Test Facility Details

Sponsor

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 Principle of the local lymph node assay ............................................................ 9

1.3 Regulatory compliance ..................................................................................... 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 single 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................................................................................. 17
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3.1.1 Mortality and clinical signs................................................................... 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 Tables

Table 1 Group dpm/node and Stimulation Index .......................................................21

List of Appendices

Appendix 1 Preliminary phase – Measurement of ear thickness (mm)............................22

Appendix 2 Preliminary and Main phases - Individual and group mean bodyweights and 

individual bodyweight change (g).................................................................23

List of Annexes

Annex 1 GLP Compliance Statement.........................................................................24
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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 compliance with the following Good 

Laboratory Practice Standards and, with the exception noted below; I consider the data 

generated to be valid.

The UK Good Laboratory Practice Regulations (Statutory Instrument 1999 

No. 3106, as amended by Statutory Instrument 2004 No. 994).

EC Commission Directive 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 authorities of 

the United States of America and Japan on the basis of intergovernmental agreements.

Chemical analysis of formulated test articles for determination of stability, homogeneity and 

concentration was not undertaken for this study.

Clare Chesher

Study Director

Huntingdon Life Sciences

Date
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Quality Assurance Statement

 Assessment of Skin Sensitization Potential using the Local 

Lymph Node Assay in the Mouse (Pooled treatment group approach)

The following inspections 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 the time this study was in progress inspections of 

procedures employed on this type of study were carried out. These were conducted and 

reported to appropriate Company Management as indicated below:

Process Based Inspections Date(s) of Inspection Date of Reporting to 

Management

Study management and conduct 08 Apr 2013-09 Apr 2013 10 Apr 2013

Preparation and administration 

of radiolabel 

15 Apr 2013 15 Apr 2013

Terminal procedures 15 Apr 2013 15 Apr 2013

Formulation procedures 02 May 2013 02 May 2013

Bodyweight 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 addition, an inspection of the facility where this study was conducted was carried out on an 

annual basis. These inspections were promptly reported to Company Management.

Kevin Oldham MRQA

Lead Auditor

Department of Quality Assurance

Huntingdon Life Sciences

Date
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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 Director
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Summary

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

local lymph node assay (LLNA).

Preliminary investigations were performed at 25 and 50% w/v with 2 mice per concentration 

to establish the highest concentration of test substance which did not lead to systemic toxicity

or excessive local irritation. The results of preliminary investigations indicated that 50% w/v

would be a suitable high concentration for use on the main study.

The study comprised three treated groups, each comprising four female mice receiving at 

concentrations of 10, 25 or 50% w/v. Similarly constituted groups received the vehicle 

Acetone: olive oil (4:1 v/v) or positive control substance (25% v/v hexyl cinnamic aldehyde). 

The mice were treated by daily application of 25 L of the appropriate concentration or 

control (vehicle or positive), 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 3H-methyl Thymidine (

3HTdR) by -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), termed as Stimulation Index 

(SI).

The test substance is regarded as a sensitizer 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 

that did not show the potential to induce skin sensitization.

The SI for the positive control substance hexyl cinnamic aldehyde was 5.6, which 

demonstrates the validity of this study.

Conclusion

is not regarded as a potential skin sensitizer. 
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1. Introduction

1.1 Objective

The objective of this study was to assess the skin sensitization potential of

1.2 Principle of the local lymph node assay 

The theory behind the local lymph node assay (LLNA) is that a sensitizing substance induces 

proliferation of lymphocytes in the lymph nodes draining the site of test substance exposure. 

The lymphocyte proliferation is proportional to the dose and potency of the sensitizing 

substance and is quantified by measuring the incorporation of radiolabelled Thymidine. 

Assessment of sensitization potential is made by comparing the mean proliferation in each 

test group to the mean proliferation in the vehicle control group, with the ratio between 

treated and control groups termed the Stimulation Index (SI). A test substance is regarded as 

a sensitizer if the SI is 3 or more in any of the concentrations tested.

1.3 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”. 22 July 2010.

EEC Methods for the determination of toxicity and other health effects. 

Commission Regulation No. 440/2008. Part B, Method B.42. Skin sensitisation: 

Local Lymph Node Assay. 30 May 2008. As amended by Regulation No. 640/2012 

06 Jul 2012.

1.4 Test system

The female CBA/Ca mouse is chosen as the test species as it has been shown to be a suitable 

model for this study and is the species/strain/sex recommended by the test guidelines.
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2. Experimental Procedure

2.1 Study schedule and structure

2.1.1 Time schedule

Study initiation:

(protocol signed by Study Director)

29 April 2013

Experimental start date:

(Initial animal allocation)

02 May 2013

Treatment commenced: 08 May 2013

Completion of live phase: 20 May 2013

Experimental completion date:

(date of scintillation counting)

21 May 2013

Study completion: When Study Director signs final report

2.1.2 Identity of treatment groups

Groups of mice were treated as follows:

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 Hexyl cinnamic aldehyde (positive control)
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2.2 Test substance and formulation

2.2.1 Test substance

Identification:

Chemical name:

Intended use:

Appearance: White crystalline powder

Storage conditions: Room temperature in the dark

Supplier: Sponsor

Batch or Lot number: 12342

Expiry date: Will be documented in the raw data and in 

the final report

Purity Will be documented in the raw data and in 

the final report

Sample receipt: 4 March 2013

The Sponsor was responsible for the characterisation of the test substance and the 

documentation of the methods of synthesis, fabrication or derivation and stability.

2.2.2 Vehicle selection

A vehicle trial performed with showed that it formed a thick off white suspension

at 50% w/v in acetone:olive oil (4:1 v/v). It was not possible to formulate at 100% as the 

material was too bulky. Acetone:olive oil (4:1 v/v) is the preferred vehicle for the local 

lymph node assay and as the test material was suitable for dosing in acetone:olive oil 

(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 of graded 

concentrations in the vehicle, by direct dilution.

The test substance was used as supplied and all formulations were prepared on the day of

dosing at the required concentrations.

The absorption of the test substance was not determined.

Determination of the homogeneity, stability and purity of the test substance or test substance 

formulations were not undertaken as part of this study.
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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.

2.3 Animal management

Healthy female CBA/Ca mice (a total of 24 females) were obtained from Charles 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 acclimatised to the experimental environment for

6 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, test substance concentration and animal mark(s).

2.3.1 Animal housing, diet and water supply

Animals were housed inside a barriered rodent facility (Building F21, Room 061/062). 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 animals were allocated without conscious bias to cages within the treatment groups. 

They were housed two animals per cage, in solid bottomed polycarbonate cages with a 

stainless steel mesh lid. Each cage contained a quantity of autoclaved wood flake bedding, 

additionally Nestlets and a plastic shelter were included 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 

and relative humidity controls were set to 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.
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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 Concentration selection

2.4.1 Preliminary investigations 

Preliminary investigations were performed to ensure the highest concentration to be used on 

the main study did not result in systemic toxicity or excessive local irritation.

An assessment of local irritation was performed and an erythema score greater or equal to 3 

and/or an increase in ear thickness of greater than 25% at Day 3 or 6 compared with predose 

would be regarded as irritation which precludes the use of that concentration on the main 

study.

2.4.2 Main study 

The results of the preliminary investigations 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 regulatory guidelines and 

the concentrations administered on the main study were:

10, 25 and 50% w/v in Acetone: olive oil (4:1 v/v)

The positive control group received 25% v/v hexylcinnamic aldehyde in Acetone: olive oil 

(4:1 v/v) based on previous experience at this laboratory. 

Prior to dose administration the Study Director authorised the choice of vehicle and dose 

levels in the raw data.

2.5 Administration of test substance

2.5.1 Preliminary investigation

Two female mice (per concentration) received a daily application 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 

automatic micropipette and was spread over the entire dorsal surface of the ear using the tip 

of the pipette.
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2.5.2 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 L 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 and 

was spread over the entire dorsal surface of the ear using the tip of the pipette. Further 

groups of four mice received the vehicle alone or the positive control substance in the same 

manner.

2.5.3 Administration of 3H-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 L of phosphate buffered 

saline containing 3H-methyl Thymidine

a

(

3HTdR: 80 Ci/mL) giving a nominal 20 Ci 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.

2.6 Observations

2.6.1 Clinical signs

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

for signs of irritation.

2.6.2 Dermal reactions

In the preliminary phase of the study, an assessment of erythema was made predose on

Day 1, 2 and 3 and daily thereafter using the following numerical scoring system:

Erythema and eschar formation:

No erythema 0

Very slight erythema (barely perceptible) 1

Well-defined erythema 2

Moderate to severe erythema 3

Severe erythema (beet redness) to eschar formation (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 

mouse on Days 1 (predose), 3 (predose) and 6 (preterminal). Measurements were taken in 

triplicate 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 occasion was the mean of the three measurements.

 

a

Specific activity 2.0 Ci/mmol, concentration 1.0 mCi/mL
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2.6.4 Bodyweight

The weight of each mouse was recorded on arrival (data not reported), Day 1 (before dosing) 

and prior to termination (Day 6).

2.7 Terminal studies

2.7.1 Termination

The mice in the preliminary investigations were humanely killed by carbon dioxide 

asphyxiation on Day 6 of the study. The carcasses were discarded and no further 

investigations were carried out with these animals.

In the main phase of the study, five hours following the administration of 3HTdR on Day 6 all 

mice were humanely killed by carbon dioxide asphyxiation. The draining auricular lymph 

nodes were excised and pooled for each experimental group in 1.0 mL of phosphate buffered 

saline (PBS). The carcasses were then discarded and no further investigations were carried 

out. The following procedures were carried out with the lymph nodes from these animals 

only.

2.7.2 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 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 

the final wash.

2.7.3 Determination of incorporated 3H-methyl Thymidine

After overnight incubation (minimum of 18 hours) 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. The 3HTdR incorporation was measured by 

-scintillation counting. The proliferative response of LNC was expressed as radioactive 

disintegrations per minute per lymph node (dpm/node).

2.8 Data treatment

2.8.1 Stimulation Index 

Results for each treatment group were expressed as the Stimulation Index (SI). This was 

derived by dividing the mean dpm/mouse for each treated group and the positive control 

group by the mean dpm/mouse in the vehicle control group. 

If the SI is 3 or more, the test substance is regarded as a skin sensitizer.

The positive control group is expected to give an SI of 3 or more to demonstrate the validity 

of the study.
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2.8.2 Estimated Concentration of Three

The Estimated Concentration of Three (EC3) is the concentration of test substance which 

would result in a SI of 3 and, where data permits, this value was calculated (Ryan 2007).

As all concentrations tested resulted 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 quantify data include: 

System name* System function

Pristima Pharmacy data management

* All version numbers of the systems are maintained by Huntingdon Life Sciences

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 will be retained indefinitely and all Quality Assurance inspection 

records for a period of 20 years. All other appropriate specimens and records will be retained 

for a minimum period of one year from the date of issue of the final report. At the end of this 

retention period the Sponsor will be contacted and advice sought on future archiving 

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 were observed during this study.

Greasy fur or dose residue on the head was noted following each dosing occasion, this was 

related to unoccluded dermal administration of a liquid formulation and not an effect of the 

test substance.

3.1.2 Dermal reactions

No erythema was observed on the ears of any mouse on Days 1 to 6.

3.1.3 Measurement of ear thickness

Appendix 1

There was no evidence of an effect of treatment on ear thickness.

3.1.4 Bodyweight

Appendix 2

There was no indication of an overt effect of treatment on bodyweight gain.

A loss in bodyweight was noted for both mice receiving 25% w/v over the study period, 

however, a small loss in bodyweight is not uncommon in young laboratory mice and this is 

not considered to be an effect of treatment.

On the basis of the results from the preliminary investigation, 50% w/v was considered a 

suitable high concentration for administration in 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 were observed in 

treated animals during this study.

Greasy fur and/or dose residue on the head was noted following each dosing occasion, this 

was related to unoccluded dermal administration of a liquid formulation/vehicle and not an 

effect of the test substance.

Partially closed eyelids were observed for all mice receiving the HCA positive control 

following dosing on Day 2 only.
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3.2.2 Dermal reactions

No signs of dermal irritation were seen on the ear during the study.

3.2.3 Bodyweight

Appendix 2

There was no indication of an effect of treatment on bodyweight gain.

A loss in bodyweight was noted for one mouse receiving 50% w/v (No. D19) over the study 

period, however, a small loss in bodyweight is not uncommon in young 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 sensitization.

The SI for the positive control substance hexyl cinnamic aldehyde (HCA) was 5.6 which 

demonstrates the validity of this study.
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4. Conclusion

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

Ryan CA, Chaney JG, Gerberick GF, Kern PS, Dearman RJ, Kimber I, Basketter, DA (2007). 

Extrapolating local lymph node assay EC3 values to estimate relative sensitizing potency. 

Cutaneous and Ocular Toxicology, 26: 135-145.
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Table 1 Group dpm/node and Stimulation 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 control)

HCA Hexyl cinnamic aldehyde (positive control)
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Appendix 1 Preliminary phase – Measurement of ear thickness (mm)

Concentration 

(% w/v)

Animal 

No.

Days

1 (Predose) 3 (Predose) 6 (Preterminal)

25

D1

L 0.21 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

50

D3

L 0.21 0.21 0.20

% difference from Day 1 - 0 -5

R 0.21 0.20 0.21

% difference from Day 1 - -5 0

D4

L 0.20 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 Right ear

- Not applicable
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Appendix 2 Preliminary and Main phases - Individual and group mean 

bodyweights and individual bodyweight change (g)

Group Animal number Day 1* Day 6

(Termination)

Individual 

bodyweight change

1

25% w/v

D1 21.9 21.0 (0.9)

D2 19.3 18.7 (0.6)

Mean 20.6 19.9 -

2

50% w/v

D3 17.8 18.2 0.4

D4 17.8 18.0 0.2

Mean 17.8 18.1 -

3

Vehicle 

(AOO)

D5 19.4 19.8 0.4

D6 20.1 20.7 0.6

D7 19.6 19.7 0.1

D8 18.5 19.1 0.6

Mean 19.4 19.8 -

4

10% w/v

D9 20.5 21.1 0.6

D10 20.5 21.4 0.9

D11 19.8 20.2 0.4

D12 18.0 18.9 0.9

Mean 19.7 20.4 -

5

25% w/v

D13 19.9 21.1 1.2

D14 19.2 19.6 0.4

D15 19.1 19.6 0.5

D16 17.3 18.1 0.8

Mean 18.9 19.6 -

6

50% w/v

D17 20.3 20.5 0.2

D18 20.1 20.1 0.0

D19 19.9 19.8 (0.1)

D20 19.5 20.1 0.6

Mean 20.0 20.1 -

7

HCA

D21 18.0 18.8 0.8

D22 19.4 19.6 0.2

D23 18.0 18.8 0.8

D24 17.4 18.0 0.6

Mean 18.2 18.8 -

AOO Acetone:olive oil (4:1 v/v) (vehicle control)

HCA Hexyl cinnamic aldehyde (positive control)

( ) Loss in bodyweight

- Not applicable

* Prior to dosing
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Annex 1 GLP Compliance Statement

HUNTINGDON RESEARCH CENTRE GLP COMPLIANCE STATEMENT 2012