## FINAL REPORT

Study Title



## ASSESSMENT OF CONTAC T HYPERSENSITIVITY T O

IN THE MOUSE (LOCAL LYMPH NODE ASSAY)

Author

Test Facility

Laboratory Project Identification- Page 2 - 1. CONTENTS

1. CONTENTS

...................................................................................................................................... 2

2. STATEMENT OF GLP COM PLIANCE

............................................................................................. 3

3. QUALITY ASSURANCE ST ATEMENT

............................................................................................ 4

4. SUMMARY

........................................................................................................................................ 5

5. INTRODUCTION

.............................................................................................................................. 7

5.1. Preface

..................................................................................................................................... 7

5.2. Aims of the study

...................................................................................................................... 7

5.3. Guidelines

................................................................................................................................ 7

5.4. Storage and retention of records and materials

....................................................................... 8

6. MATERIALS AND METHOD S

.......................................................................................................... 9

6.1. Test substance

......................................................................................................................... 9

6.1.1. Test substance information

............................................................................................. 9

6.1.2. Study specific t est substance information

...................................................................... 9

6.1.3. Test substance preparation

............................................................................................ 9

6.2. Test system

............................................................................................................................ 10

6.3. Animal husbandry

.................................................................................................................. 10

6.4. Weight of evidence analysis

.................................................................................................. 10

6.5. Pre

- screen test

....................................................................................................................... 11

6.6. Main study

.............................................................................................................................. 11

6.6.1. Allocation

...................................................................................................................... 11

6.6.2. Induction

- Days 1, 2 and 3

........................................................................................... 11

6.6.3. Excision of the nodes

- Day 6

....................................................................................... 11

6.6.4. Tissue processing for radioactivity

- Day 6

................................................................... 12

6.6.5. Radioactivity measurements

- Day 7

............................................................................ 12

6.7. Observations

.......................................................................................................................... 12

6.8. Electronic data capture

.......................................................................................................... 12

6.9. Interpretation

.......................................................................................................................... 13

6.10. List of deviations

.................................................................................................................... 13

6.10.1. List of protoc ol deviations

........................................................................................ 13

6.10.2. List of standard operating procedures deviations

.................................................... 13

7. RESULTS

....................................................................................................................................... 13

7.1. Pre

- screen test (Table 1 and Table 2)

................................................................................... 13

7.2. Main study

.............................................................................................................................. 13

7.2.1. Skin reactions / Irritation (Table 3)

................................................................................ 13

7.2.2. Systemic toxicity (Body weights, see Table 3)

............................................................. 13

7.2.3. Macroscopy of the auricular lymph nodes and surrounding area (Table 4)

................. 13

7.2.4. Radioactivity measurements (Table 4)

......................................................................... 14

8. CONCLUSION

................................................................................................................................ 14

9. REFERENCES

............................................................................................................................... 14

TABLES

Table 1: Body weights and skin reactions

.............................................................................................. 15

Table 2: Ear thickness measurements

................................................................................................... 15

Table 3: Body weights and skin reactions

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

Table 4: Relative size lymph nodes, radioactivity counts (DPM) and Stimulation Index (SI)

................. 17

FIGURES

Figure 1: Dose

- response curve

.............................................................................................................. 17

APPENDICES

Appendix 1: RELIABILITY CHECK- Page 5 - 4. SUMMARY

Assessment of Contact Hypersensitivity to in the Mouse (Local Lymph Node

A ssay).

The study was carried out based on the guidelines described in:

OECD, Section 4, Health Effects, No.429 (2010),

EC, No 440/2008; B42: "Skin Sensitization: Local Lymph Node Assay"

EPA, OPPTS 870.2600 (2003) “Skin Sensitization”.

Test substance concentrations selected for the main study were based on the results of a pre

- screen

test. The two animals treated at 100% were found dead or sacrificed for humane reasons on Day 2.

The two animals treated at 50% showed signs of systemic toxicity (hunched p osture). It was

concluded that these concentrations could not be tolerated well and did not comply with the selection

criteria, and therefore the highest test substance concentration selected for the main study was a 25%

concentration.

In the main study, three experimental groups of five female CBA/J mice were treated with test

substance concentrations of 5, 10 or 25% w/w on three consecutive days, by open application on the

ears. Five vehicle control animals were similarly treated, but with vehicle alone (Acetone/Olive oil (4:1

v/v)).

Three days after the last exposure, all animals were injected with 3 H- methyl thymidine and after five

hours the draining (auricular) lymph nodes were excised and pooled for each animal.

After precipitating the DNA of the lymph node cells, radioactivity measurements were performed. The

activity was expressed as the number of Disintegrations Per Minute (DPM) and a stimulation index (SI)

was subsequently calculated for each group.

RESULTS

The slight irritation of the ears as shown by the animals treated at 25% was considered not to have a

toxicologically significant effect on the activity of the nodes. No irritation of the ears was observed in

any of the animals treated with vehicle or at test substance concentration of 10% or 5%.

No mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the

main study. Body weights and body weight gain of the experimental animals remained in the same

range as controls over the study period, except for the slight body weight loss noted in two animals

treated at 25% and one at 5%. This body weight loss was considered not toxicologically significant.

The auricular lymph nodes of the animals of the experimental and control groups were considered

normal in size, except for the enlarged nodes found in two animals treated at 25%. No macroscopic

abnormalities of the surrounding area were noted in any of the animals.

Mean DPM/animal values for the experimental groups treated with test substance concentrations 5, 10

and 25% were 139, 98 and 132 DPM respectively. The mean DPM/animal value for the vehicle control

group was 97 DPM.- Page 6 - CONCLUSION

The SI values calculated for the substance concentrations 5, 10 and 25% were 1.4, 1.0 and 1.4

respectively.

Since there was no indication that the test substance elicits an SI ≥ 3 when tested up to the highest

tolerable concentration of 25%, was considered not to be a skin sensitizer. It was

established that the EC3 value (the estimated test substance conc entration that will give a SI =3) (if

any) exceeds 25%.

Based on these results, would not be regarded as a skin sensitizer according to

the recommendations made in the test guidelines. The test substance does not have to be classified

and has no obligatory labelling requirement for sensitization by skin contact according to the Globally

Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2011)

and the Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and

mixtures.- Page 7 - 5. INTRODUCTION

5.1. Preface

Sponsor

Study Monitor

(on behalf of the sponsor)

Test Facility

Study Director

Study Plan (In li fe phase) Start : 30 November 2011

Completion : 12 December 2011

5.2. Aims of the study

The purpose of this study was to evaluate whether the test substance induces contact hypersensitivity

in mice after three epidermal induction exposures of the animals under the conditions described in this

report. This study should provide a rational basis for risk assessment in man. Compared to

sensitization tests using Guinea pigs, the Local Lymph Node Assay (LLNA) provides certain

advantages with regard to animal w elfare and scientific aspects.

5.3. Guidelines

As required by the Dutch Act on Animal Experimentation (February 1997), this type of protocol was

reviewed and agreed by the Laboratory Animal Welfare Officer and the Ethical Committee of

. The study procedures described in this report were based on the following

guidelines:

Organization for Economic Co

- operation and Development (OECD), OECD Guidelines for Testing of

Chemicals, Section 4, Health Effects, No.429, "Skin Sensitization: Local L ymph Node Assay", Paris

Cedex, July 2010.

Commission Regulation (EC) No 440/2008 Part B: Methods for the Determination of Toxicity and other

Health Effects; B42: "Skin Sensitization: Local Lymph Node Assay". Official Journal of the European

Union No. L142, May 2008, including most recent amendments.

Environmental Protection Agency (EPA): Health Effects Test Guidelines OPPTS 870.2600. “Skin

Sensitization”, March 2003.- Page 8 - 5.4. Storage and retention of records and materials

Records and materials pertaining to the study including protocol, raw data and the final report are

retained in the archives for a period of at least 2 years after finalization of the report. After this

period, the sponsor will be contacted to determine how the records and materials should be handled.

will retain information concerning decisions made.

will retain a test substance sample until the expiry date, but no longer than 10 years after

finalization of the report. After this period the sample will be destroyed.- Page 9 - 6. MATERIA LS AND METHODS

6.1. Test substance

6.1.1. Test substance information

Identification

Structure

Molecular formula

Molecular weight

CAS Number

Description

Batch

Purity

Test substance storage

Stability under storage conditions

Expiry date 17 October 2012

6.1.2. Study specific test substance information

Volatile No

Test substance handling Flush container with nitr ogen after handling

pH (1% in water, indicative value)

Specific Gravity / Density

Stability at higher temperatures Yes, maximum temperature: 130 ° C

maximum duration: 24 hours under nitrogen seal

Stability in vehicle:

• Acetone At least 24 hours

• Olive oil At least 24 hours

Solubility in vehicle:

• Acetone Not indicated

• Olive oil Not indicated

6.1.3. Test substance preparation

Vehicle Acetone/Olive oil (4:1 v/v) (Acetone p.a.: Merck, Darmstadt, German y;

Olive oil: Sigma

- Aldrich, Steinheim, Germany).

Rationale The vehicle was selected based on trial formulations performed at

and on test substance data supplied by the sponsor.

P reparation The test substance formulations (w/w) were prepared within 4 hours prior

to each treatment. Homogeneity was obtained to visually acceptable

levels.- Page 10

- 6.2. Test system

Species Mouse, CBA/J strain, inbred, SPF

- Quality.

Recognized by the international guidelines as the recommended test

system (e.g. OECD, EC, EPA).

S ource: Janvier, Le Genest

- Saint

- Isle, France

Number of animals 20 females (nulliparous and non

- pregnant), five females per group.

Age and body weight Young adult animals (approx. 10 weeks old) were selected. Body weight

variation was within +/

- 20% of the sex mean.

Identification Tail mark with marker pen.

Health inspection A health inspection was performed prior to treatment, to ensure that the

animals were in a good state of health. Special attention was paid to the

ears, which were intact and free from any abnormality.

Reliability check The results of a reliability test with three concentrations of

Hexylcinnamaldehyde in Acetone/Olive oil (4:1 v/v), performed not more

than 6 months previously and using the same materials, animal supplier,

animal strain and essential procedures is summarized in the appendix of

this report. For both scientific and animal welfare reasons, no concurrent

positive control group was added to the study.

An extensive data base is available with reliability checks performed e ach

half year during at least the recent 9 years showing reproducible and

consistent positive results.

6.3. Animal husbandry

Conditions

Animals were housed in a controlled environment, in which optimal conditions were considered to be

approximately 15 air changes per hour, a temperature of 21.0 ± 3.0oC (actual range: 18.1

– 22.6oC), a

relative humidity of 40

- 70% (actual range: 46

- 68%) and 12 hours artificial fluorescent light and 12

hours darkness per day.

Accommodation

Animals were group housed in labeled Makrolon cages (MIII type; height 18 cm) containing sterilised

sawdust as bedding material ( Litalabo, S.P.P.S., Argenteuil, France ). Paper ( Enviro

- dri, Wm. Lillico &

Son (Wonham Mill Ltd), Surrey, United Kingdom) and shelters (disposable paper corner hom e,

MCORN 404, Datesand Ltd, USA) were supplied as cage

- enrichment. The acclimatization period was

at least 5 days before the start of treatment under laboratory conditions. On Day 6, the animals were

group housed in Makrolon MII type cages with a sheet of paper instead of sawdust and cage

enrichment. Certificates of analysis were examined and then retained in the archives.

Diet

Free access to pelleted rodent diet (SM R/M

- Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).

Water

Free access to tap w ater.

Results of analysis for diet (nutrients and contaminants), sawdust, paper, shelters and water were

assessed and did not reveal any findings that were considered to have affected the study integrity. All

certificates and results of analysis are retained in the archives.

6.4. Weight of evidence analysis

In the interest of animal welfare and to minimize any testing likely to produce severe responses in

animals, a weight of evidence analysis was performed prior to start of this study. All available

in formation was evaluated (e.g. existing human and animal data, literature, substance data supplied

by the sponsor, analysis of structure activity relationships (SAR), physicochemical properties and

reactivity (pH, buffering capacity).- Page 11

- 6.5. Pre

- screen test

A pre

- screen test was conducted in order to select the highest test substance concentration to be

used in the main study. In principle, this highest concentration should cause no systemic toxicity, may

give well

- defined irritation at the most (maximum grade 2 (see section 6.7 ) and/or an increase in ear

thickness < 25%) and is the highest possible concentration that can technically be applied.

Two test substance concentrations were tested; a 100% and 50% concentration. The highes t

concentration was the maximum concentration as required in the test guidelines (undiluted for liquids).

The test system, procedures and techniques were identical to those used in the main study except that

assessment of lymph node proliferation and necropsy were not performed. Two young adult animals

per concentration were selected (in the range of 8 to14 weeks old). Each animal was treated with one

concentration on three consecutive days. Animals were group housed in labeled Makrolon cages (MII

type, h eight 14 cm). Ear thickness measurements were conducted using a digital thickness gauge

prior to dosing on Days 1 and 3, and on Day 6. The surviving animals were sacrificed after the final

observation.

6.6. Main study

Three groups of five animals were treated with one test substance concentration per group. The

highest test substance concentration was selected from the pre

- screen test.

One group of five animals was treated with vehicle.

6.6.1. Allocation

Group 1 animal numbers induction (test substance; % w/w)

1 01

- 05 0 (Acetone/Olive oil (4:1 v/v))

2 06

- 10 5

3 11

- 15 10

4 16

- 20 25

1 . five females each group

6.6.2. Induction

- Days 1, 2 and 3

The dorsal surface of both ears was topically treated (25 μL/ear) with the test substance

concentratio n, at approximately the same time on each day. The concentrations were stirred with a

magnetic stirrer immediately prior to dosing.

The control animals were treated in the same way as the experimental animals, except that the vehicle

was administered instead of the test substance.

6.6.3. Excision of the nodes

- Day 6

Each animal was injected via the tail vein with 0.25 mL of sterile phosphate buffered saline (PBS)

(Merck, Darmstadt, Germany) containing 20 μCi of 3 H- methyl thymidine (PerkinElmer Life and

Analytical Sciences, Boston, MA, US ).

After approximately five hours, all animals were killed by intraperitoneal injection (0.2 mL/animal)

with Euthasol® 20% (AST Farma BV, Oudewater, The Netherlands). The draining (auricular) lymph

node of each ear was excised. The relative size of the nodes (as compared to normal) was estimated

by visual examination and abnormalities of the nodes and surrounding area were recorded. The nodes

were pooled for each animal in approximately 3 mL PBS.- Page 12

- 6.6.4. Tissue processing for radioactivity

- Day 6

A single cell suspension of lymph node cells (LNC) was prepared in PBS by gentle separation through

stainless steel gauze (diameter 125 μm). LNC were washed twice with an excess of PBS by

centrifugation at 200 g for 10 minutes at 4 o C. To precipitate the DNA, the LNC were exposed to 5%

trichloroacetic a cid (TCA) ( Merck, Darmstadt, Germany) and stored in the refrigerator until the next

day.

6.6.5. Radioactivity measurements

- Day 7

Precipitates were recovered by centrifugation, resuspended in 1 mL TCA and transferred to 10 mL of

Ultima Gold cocktail ( PerkinElme r Life and Analytical Sciences, Boston, MA, US) as the scintillation

fluid. Radioactive measurements were performed using a Packard scintillation counter (2800TR).

Counting time was to a statistical precision of ± 0.2% or a maximum of 5 minutes whichever came first.

The scintillation counter was programmed to automatically subtract background and convert Counts

Per Minute (CPM) to Disintegrations Per Minute (DPM).

6.7. Observations

Mortality/Viability Twice daily.

Body weights On Day 1 (pre

- dose) and Day 6 (prior to necropsy).

Clinical signs Once daily on Days 1

- 6 (on Days 1

- 3 between 3 and 4 hours after

dosing).

Irritation Once daily on Days 1

- 6 (on Days 1

- 3 within 1 hour after dosing)

according to the following numerical scoring system. Furthermore, a

descr iption of all other (local) effects was recorded.

Grading Irritation Reactions:

Erythema and eschar formation:

No erythema .............................................................................................................................

......... 0

Very slight erythema (barely perce ptible) ........................................................................................

.... 1

Well

- defined erythema .............................................................................................................

.......... 2

Moderate to severe erythema (beet redness) to slight eschar formation (injuries in depth) ..................

... 3

Severe erythema (beet redness) to eschar formation preventing grading of erythema ..................

......... 4

6.8. Electronic data capture

Observations/measurements in the study were recorded electronically using the following

programmes: REES Centron Environmental Monitoring system version SQL 2.0 (REES scientific,

Trenton, NJ, USA); Quantasmart 2.03 (PerkinElmer Life Sciences, Boston, MA, USA): LSC software.- Page 13

- 6.9. Interpretation

DPM values are presented for each animal and for each dose group. A Stimulation Index (SI) is

calculated for each group. The SI is the ratio of the DPM/group compared to DPM/vehicle control

group.

If the results indicate a SI ≥ 3, the test substance may be regarded as a skin sensitizer.

The results were evaluated according to the Globally Harmonized System of Classification and

Labelling of Chemicals (GHS) of the United Nations (200 11 ) and the Regulation (EC) No 1272/2008 of

the European Parliament and of the Council of 16 December 2008 on classification, labelling and

p ackaging of substances and mixtures.

Consideration was given to the EC3 value (the estimated test substance concentration that will give a

SI =3) ( Ref. 1 )

.

6.10. List of deviations

6.10.1. List of protocol deviations

There were no deviations from the protocol.

6.10.2. List of standard operating procedures deviations

There were no deviations from standard operating procedures during the study.

7. RESULTS

7.1. Pre

- screen test ( Table 1 and Table 2 )

The two animals treated at 100% were found dead or sacrificed for humane reasons on Day 2. The

two animals treated at 50% showed signs of systemic toxicity (hunched posture). It was concluded that

these concentrations could not be tolerated well and did not comply with the selection criteria, and

therefore the highest test substance concentration selected for the main study was a 25%

concentration.

7.2. Main study

7.2.1. Skin reactions / Irritation ( Table 3 )

The slight irritation of the ears as shown by the animals treated at 25% was considered not to have a

toxicologically significant effect on the activity of the nodes. No irritation of the ears was observed in

any of the animals treated with vehicle or at test substance concentration of 10% or 5%.

7.2.2. Systemic toxicity (Body weights, see Table 3 )

No mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the

main study. Body weights and body weight gain of the experimental a nimals remained in the same

range as controls over the study period, except for the slight body weight loss noted in two animals

treated at 25% and one at 5%. This body weight loss was considered not toxicologically significant.

7.2.3. Macroscopy of the auricular lymph nodes and surrounding area ( Table 4 )

The auricular lymph nodes of the animals of the experimental and control groups were considered

normal in size, except for the enlarged nodes found in two animals treated at 25%. No mac roscopic

abnormalities of the surrounding area were noted in any of the animals.- Page 14

- 7.2.4. Radioactivity measurements ( Table 4 )

Mean DPM/animal values for the experimental groups treated with test substance concentrations 5, 10

and 25% wer e 139, 98 and 132 DPM respectively. The mean DPM/animal value for the vehicle control

group was 97 DPM.

8. CONCLUSION

The SI values calculated for the substance concentrations 5, 10 and 25% were 1.4, 1.0 and 1.4

respectively ( Table 4 and Figure 1 )

.

Since there was no indication that the test substance elicits an SI ≥ 3 when tested up to the highest

tolerable concentration of 25%, was considered not to be a skin sensitizer. It was

established that the EC3 value (the estimated test substance concentration that will give a SI =3) (if

any) exceeds 25%

.

Based on these results, would not be regarded as a skin sensitizer according to

the recommendations made in the test guidelines. The test substance does not have to be classified

and has no obligatory labelling requirement for sensitization by skin contact according to the Globally

Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2011)

and the Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and

mixtures.

9. REFERENCES

Ref. 1 Basketter DA, Lea LJ, Dickens A, Briggs, D, Pate I, Dearman RJ and Kimber I. A

comparison of statistical approaches to the derivation of EC3 values from local lymph node

assay dose responses. J Appl Toxicol 1999;19 :261

- 266.- Page 15

- PRE

- SCREEN TEST

Table 1 : Body weights and skin reactionsTS1(%)animal Day 1 Day 2 Day 3 Day 4 Day 5 Day 6

bw

(g)

2 erythema 3 erythema e rythema erythema erythema erythema bw

(g) left right left right left right left right left right left right

50 1 20 0 0 1 1 1 1 6 1 1 0 0 0 0 21

2 23 0 0 1 1 1 1 6 1 1 0 0 0 0 22

100 3 22 0 0 n.d. 4 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d.

4 21 0 0 n.d. 5 n.d. n.d. n.d. n .d. n.d. n.d. n.d. n.d. n.d. n.d.

1

. TS = test substance (% w/w).

2

. Body weight (grams).

3 Grading erythema and eschar formation (Left = dorsal surface of left ear; right = dorsal surface of right ear) :

0 = No erythema

1 = Very slight erythema (barely perceptible)

4

. Animal sacrificed for humane reasons on Day 2 after showing flat posture, lethargy, ptosis and slow respiration.

5

. Animal showed lethargy on Day 1 and was found dead on Day 2.

6

. Animal showed hunched posture.

n.d . not determined, animals found dead or sacrificed on day 2.

Table 2 : Ear thickness measurements

TS 1 (%) Animal Day 1 Day 3 Day 6

left right left right left right

(mm) (mm) (mm) % 2 (mm) % (mm) % (mm) %

50 1 0.230 0.230 0.225 -2 0.230 0 0.230 0 0.230 0

2 0.230 0.235 0.225 -2 0.225 -4 0.230 0 0.230 -2

100 3 0.230 0.230 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d.

4 0.230 0.235 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d.

Left (mm) = thickness of left ear in millimeters; right (mm) = thickness of right ear in millimeters.

1

. TS = test substance (% w/w).

2

. Percentual increase compared to Day 1 pre

- dose value.

n.d. not determined, animals found dead or sacrificed on day 2.- Page 16

- M AIN STUD Y

Table 3 : Body weights and skin reactionsgroupTS1(%)animal Day 1 Day 2 Day 3 Day 4 Day 5 Day 6

bw

(g) 2 erythema 3 erythema erythema erythema erythema erythema bw

(g) left right left right left right left right left right left right

1 0 1 23 0 0 0 0 0 0 0 0 0 0 0 0 24

2 22 0 0 0 0 0 0 0 0 0 0 0 0 23

3 24 0 0 0 0 0 0 0 0 0 0 0 0 24

4 22 0 0 0 0 0 0 0 0 0 0 0 0 24

5 24 0 0 0 0 0 0 0 0 0 0 0 0 25

2 5 6 23 0 0 0 0 0 0 0 0 0 0 0 0 23

7 22 0 0 0 0 0 0 0 0 0 0 0 0 22

8 22 0 0 0 0 0 0 0 0 0 0 0 0 23

9 23 0 0 0 0 0 0 0 0 0 0 0 0 22

10 22 0 0 0 0 0 0 0 0 0 0 0 0 24

3 10 11 23 0 0 0 0 0 0 0 0 0 0 0 0 25

12 22 0 0 0 0 0 0 0 0 0 0 0 0 23

13 22 0 0 0 0 0 0 0 0 0 0 0 0 23

14 22 0 0 0 0 0 0 0 0 0 0 0 0 22

15 23 0 0 0 0 0 0 0 0 0 0 0 0 24

4 25 16 24 0 0 0 0 1 1 0 0 0 0 0 0 23

17 23 0 0 0 0 1 1 0 0 0 0 0 0 24

18 24 0 0 0 0 1 1 0 0 0 0 0 0 25

19 22 0 0 0 0 1 1 0 0 0 0 0 0 23

20 25 0 0 0 0 1 1 0 0 0 0 0 0 24

1

. TS = test substance (% w/w).

2

. Body weight (grams).

3 Grading erythema and eschar formation (Left = dorsal surface of left ear; right = dorsal surface of right ear) :

0 = No eryt hema

1 = Very slight erythema (barely perceptible)- Page 17

- MAIN STUDY

Table 4 : Relative size lymph nodes, radioactivity counts (DPM) and Stimulation Index (SI)groupTS1(%)animal Size nodes 2

DPM 3 /

animal mean

DPM ± SEM 4 mean

SI ± SEM

left right

1 0 1 n n 70

97 ± 15 1.0 ± 0.2 2 n n 130

3 n n 137

4 n n 89

5 n n 61

2 5 6 n n 173

139 ± 18 1.4 ± 0.3 7 n n 188

8 n n 86

9 n n 119

10 n n 131

3 10 11 n n 125

98 ± 9 1.0 ± 0.2 12 n n 72

13 n n 82

14 n n 106

15 n n 105

4 25 16 n n 93

132 ± 27 1.4 ± 0.3 17 + + 199

18 n n 96

19 n n 78

20 + n 196

1

. TS = test substance (% w/w).

2

. Relative size auricular lymph nodes (

- , -- or --- : degree of reduction, +,++ or +++: degree of enlargement, n: considered to be

normal).

3

. DPM = Disintegrations per minute

4

. SEM = Standard Error of the M ean

Figure 1 : Dose

- response curve

An individual Stimulation Index (SI) is calculated for each animal. The individual SI

is the ratio of the DPM/animal compared to the DPM/vehicle control group.

A mean SI is calculated for each group using the individual SI values.- Page 18

- Appendix 1 : RELIABILITY CHECK

ASSESSMENT OF CONTACT HYPERSENSITIVITY

TO ALPHA

- HEXYLCINNAMALDEHYDE, TECHNICAL GRADE

IN THE MOUSE (LOCAL LYMPH NODE ASSAY)- Page 19

- SUMMARY RELIABILITY CHECK

A reliability check is carried out at regular intervals to check the sensitivity of the test system and the

reliability of the experimental techniques as used by In this study, performed in March 20 11,

females of the CBA/J mouse strain (Janvier, Le Genest

- Saint

- Isle, France) were checked for

sensitivity to Hexylcinnamaldehyde. The females were approx. 10 weeks old at commencement of the

study. The study was based on the OECD Guideline No. 429, EC No 440/2008, Part B.42 and EPA,

OPPTS 870.2600 “Skin Sensitization”. Alpha

- hexylcinnamaldehyde, technical grade (CAS no. 101

- 86

- 0) was fabricated under lot no. 04012JE (Sigma

- Aldrich, Steinheim, Germany). Concentrations used

for this study were 5, 10 and 25% in Acetone/Olive oil (4:1 v/v).

group test substance 1

(% w/w) mean

DPM ± SEM 2 mean

SI ± SEM 2

2 5 673 ± 87 1.7 ± 0.3

3 10 599 ± 79 1.5 ± 0.3

4 25 2577 ± 290 6.6 ± 1.0

1 0 (vehicle) 390 ± 38 1.0 ± 0.1

1

. Vehicle: Acetone/Olive oil (4:1 (v/v).

Dose

- response curve

CONCLUSION

The SI values calculated for the substance concentrations 5, 10 and 25% were 1.7, 1.5 and 6.6

respectively.

An EC3 value of 14.4% was calculated using linear interpolation.

The calculated EC3 value was found to be in the acceptable range of 4.8 and 19.5%. The results of

the 6 monthly HCA reliability checks of the recent years were 13.8, 13.9, 16.0, 11.9, 16.9 and 10.7%.

Based on these results it was concluded that the Local Lymph Node Assay as performed at is

suitable for testing for contact hypersensitivity.

The raw data, protocol and report from this study are kept in the archives. The test described

above was performed in accordance with Standard Operating Procedures and the report was

audited by the QA

- unit.