FINAL REPORT 

Study Title

ASSESSMENT OF CONTACT HYPERSENSITIVITY TO 

IN THE MOUSE (LOCAL LYMPH NODE ASSAY)

Author

Test Facility

 

Laboratory Project Identification

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1. CONTENTS

1. CONTENTS ...................................................................................................................................... 2

2. STATEMENT OF GLP COMPLIANCE............................................................................................. 3

3. QUALITY ASSURANCE STATEMENT ............................................................................................ 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 METHODS .......................................................................................................... 9

6.1. Test substance......................................................................................................................... 9

6.1.1. Test substance information............................................................................................. 9

6.1.2. Study specific test 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 protocol 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

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4. SUMMARY

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

Assay).

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 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.

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.

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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 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.

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5. INTRODUCTION

5.1. Preface

Sponsor

Study Monitor

(on behalf of the sponsor)

Test Facility

Study Director

Study Plan (In life 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 welfare 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 Lymph 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.

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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. 

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6. MATERIALS 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 nitrogen 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, Germany; 

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.

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

to each treatment. Homogeneity was obtained to visually acceptable 

levels.

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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). 

Source: 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 each 

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 home, 

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 water. 

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 

information 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). 

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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 highest 

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, height 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

Group1 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 

concentration, 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.

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

trichloroacetic acid (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 (PerkinElmer 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 

description of all other (local) effects was recorded.

Grading Irritation Reactions:

Erythema and eschar formation:

No erythema ...................................................................................................................................... 0 

Very slight erythema (barely perceptible) ............................................................................................ 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.

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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 (20011) and the Regulation (EC) No 1272/2008 of 

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

packaging 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 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.

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 macroscopic 

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

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7.2.4. Radioactivity measurements (Table 4)

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.

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.

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PRE-SCREEN TEST

Table 1: Body weights and skin reactions TS 1 (%) animal

Day 1 Day 2 Day 3 Day 4 Day 5 Day 6

bw

(g) 

2

erythema 3 erythema erythema erythema erythema erythema bw

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

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. 

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MAIN STUDY 

Table 3: Body weights and skin reactions group TS 1 (%) animal

Day 1 Day 2 Day 3 Day 4 Day 5 Day 6

bw

(g) 2

erythema 3 erythema erythema erythema erythema erythema bw

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

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 erythema 

1 = Very slight erythema (barely perceptible)

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MAIN STUDY

Table 4: Relative size lymph nodes, radioactivity counts (DPM) and Stimulation Index (SI) group TS 1 (%) 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 Mean

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.

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Appendix 1: RELIABILITY CHECK

ASSESSMENT OF CONTACT HYPERSENSITIVITY

TO ALPHA- HEXYLCINNAMALDEHYDE, TECHNICAL GRADE

IN THE MOUSE (LOCAL LYMPH NODE ASSAY)
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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 2011, 

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.

Final Report