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Description of key information

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was conducted between 12 July 2010 and 11 August 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study is considered to be reliability 1 as it has been conducted according to OECD Test Guideline 429 using the Local Lymph Node Assay method and in compliance with GLP.
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymphnode assay (LLNA)
Test material information:
Composition 1
Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
Female CBA/Ca (CBA/CaOlaHsd) strain mice were supplied by Harlan Laboratories U.K. Ltd., Oxon, UK. On receipt the animals were randomly allocated to cages. The animals were nulliparous and non pregnant. After an acclimatisation period of at least five days the animals were selected at random and given a number unique within the study by indelible ink marking on the tail and a number written on a cage card. At the start of the study the animals were in the weight range of 15 to 23 g, and were eight to twelve weeks old.
The animals were individually housed in suspended solid floor polypropylene cages furnished with softwood woodflakes. Free access to mains tap water and food (2014 Teklad Global Rodent diet supplied by Harlan Laboratories U.K. Ltd., Oxon, UK) was allowed throughout the study.
The temperature and relative humidity were controlled to remain within target ranges of 19 to 25°C and 30 to 70%, respectively. Any occasional deviations from these targets were considered not to have affected the purpose or integrity of the study. The rate of air exchange was approximately fifteen changes per hour and the lighting was controlled by a time switch to give twelve hours continuous light (06.00 to 18.00) and twelve hours darkness.
The animals were provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
25 or 50% v/v in vehicle
No. of animals per dose:
Preliminary screening test: One
Main test: Groups of five
Details on study design:
For the purpose of the study, the test material was used undiluted and freshly prepared as a solution in acetone/olive oil 4:1. This vehicle was chosen as it produced the most suitable formulation at the required concentration.
The test material was formulated within two hours of being applied to the test system. It is assumed that the formulation was stable for this duration.

Preliminary Screening Test
Using available information regarding the irritancy potential of the test material, a preliminary screening test was performed using one mouse. The mouse was treated by daily application of 25 µl of the undiluted test material to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The mouse was observed twice daily on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. Any signs of toxicity or excessive local irritation noted during this period were recorded. The bodyweight was recorded on Day 1 (prior to dosing) and on Day 6.

Main Test
Test Material Administration
Groups of five mice were treated with the undiluted test material or the test material at concentrations of 50% or 25% v/v in acetone/olive oil 4:1. The preliminary screening test suggested that the test material would not produce systemic toxicity or excessive local irritation at the highest suitable concentration. The mice were treated by daily application of 25 µl of the appropriate concentration of the test material to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The undiluted test material and test material formulations were administered using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette.
A further group of five mice received the vehicle alone in the same manner.

3H-Methyl Thymidine Administration
Five days following the first topical application of the test material or vehicle (Day 6) all mice were injected via the tail vein with 250 µl of phosphate buffered saline (PBS) containing 3H methyl thymidine (3HTdR:80µCi/ml, specific activity 2.0 Ci/mmol, ARC UK Ltd) giving a total of 20 µCi to each mouse.

Observations
Clinical Observations:
All animals were observed twice daily on Days 1, 2 and 3 and on a daily basis on Days 4, 5 and 6. Any signs of toxicity or signs of ill health during the test were recorded.

Bodyweights:
The bodyweight of each mouse was recorded on Day 1 (prior to dosing) and Day 6 (prior to termination).

Terminal Procedures
Termination:
Five hours following the administration of 3HTdR all mice were killed by carbon dioxide asphyxiation followed by cervical separation. For each individual animal of each group the draining auricular lymph nodes were excised and processed. For each individual animal 1 ml of PBS was added to the lymph nodes.

Preparation of Single Cell Suspension:
A single cell suspension of the lymph node cells for each individual animal was prepared by gentle mechanical disaggregation through a 200 mesh stainless steel gauze. The lymph node cells were rinsed through the gauze with 4 ml of PBS into a petri dish labelled with the project number and dose concentration. The lymph node cells suspension was transferred to a centrifuge tube. The petri dish was washed with an additional 5 ml of PBS to remove all remaining lymph node cells and these were added to the centrifuge tube. The lymph node cells were pelleted at 1400 rpm (approximately 190 g) for ten minutes. The pellet was resuspended in 10 ml of PBS and re-pelleted. To precipitate out the radioactive material, the pellet was resuspended in 3 ml of 5% Trichloroacetic acid (TCA).

Determination of 3HTdR Incorporation:
After approximately eighteen hours incubation at approximately 4°C, the precipitates were recovered by centrifugation at 2100 rpm (approximately 450 g) for ten minutes, resuspended in 1 ml of TCA and transferred to 10 ml of scintillation fluid (Optiphase 'Trisafe'). 3HTdR incorporation was measured by β scintillation counting. The "Poly QTM" vials containing the samples and scintillation fluid were placed in the sample changer of the scintillator and left for approximately twenty minutes. The purpose of this period of time in darkness was to reduce the risk of luminescence, which has been shown to affect the reliability of the results. After approximately twenty minutes, the vials were shaken vigorously. The number of radioactive disintegrations per minute was then measured using the Beckman LS6500 scintillation system (Beckman Instruments Inc, Fullerton, CA, USA).
Statistics:
Data was processed to give group mean values for disintegrations per minute and standard deviations where appropriate. Individual and group mean disintegrations per minute values were assessed for dose response relationships by analysis of homogeneity of variance followed by one way analysis of variance (ANOVA). In the event of a significant result from the ANOVA, pairwise comparisons were performed between control and treated groups. For homogenous datasets Dunnett’s Multiple Comparison test was used and for non homogenous datasets Dunnett’s T3 Multiple Comparison Method was used.
Probability values (p) are presented as follows:
P<0.001: ***
P<0.01: **
P<0.05: *
P>0.05: (not significant)
Positive control results:
A study was performed to assess the sensitivity of the strain of mouse used at these laboratories to a known sensitiser. The current positive control study was performed outside the recommended six months interval, but due to the historical data available, this deviation was considered not to affect the purpose or integrity of the study. The methodology for the LLNA is detailed in the OECD Guideline for the Testing of Chemicals, No. 429, and Method B.42 of Commission Regulation (EC) No. 440/2008. The study described in this document is based on these test methods but has been refined in order to reduce the number of animals required. The reduced LLNA (rLLNA) has been endorsed by the non Commission members of the European Centre for the Validation of Alternative Methods (ECVAM) Scientific Advisory Committee (ESAC) at its 26th meeting held on 26 – 27 April 2007 at ECVAM, Ispra, Italy.
Test Material: α Hexylcinnamaldehyde, tech., 85%
Project number: 41003764
Study dates: 26 August 2010 to 01 September 2010

A group of five animals was treated with 50 µl (25 µl per ear) of α Hexylcinnamaldehyde, tech., 85% as a solution in acetone/olive oil 4:1 at a concentration of 25% v/v. A further control group of five animals was treated with acetone/olive oil 4:1 alone.

The Stimulation Index expressed as the mean radioactive incorporation for the treatment group divided by the mean radioactive incorporation of the vehicle control group is as follows:

25 % (% v/v) in acetone / olive oil (4:1) gave a stimulation index of 7.25 (positive).

α Hexylcinnamaldehyde, tech., 85% was considered to be a sensitiser under the conditions of the test.
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
The Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group are as follows: 25 % (% v/v) in acetone/olive oil (4:1) gave a stimulation index of 1.77 (negative). 50 % (% v/v) in acetone/olive oil (4:1) gave a stimulation index of 2.92 (negative). 100 % (% v/v) in acetone/olive oil (4:1) gave a stimulation index of 4.68 (positive). The concentration of test material expected to cause a 3 fold increase in 3HTdR incorporation (EC3 value) was calculated to be 52%.

Preliminary screening test

No signs of systemic toxicity were noted.

Based on this information the undiluted test material and the test material at concentrations of 50% and25% v/v in acetone/olive oil 4:1 were selected for the main test.

Main test

Estimation of the proliferative response of lymph node cells.

The Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group are as follows:

Concentration (%v/v) in
acetone/olive oil 4:1

Stimulation Index

Result

25

1.77

Negative

50

2.92

Negative

100

4.68

Positive

Clinical observations and mortality data

There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test.

Body weight

Bodyweight changes of the test animals between Day 1 and Day 6 were comparable to those observed in the corresponding control group animals over the same period.

Calculation of EC3 value

EC3 = c + [[(3 - d)/(b - d)] x (a - c)]a

Where:

a = 100

b = 4.68

c = 50

d = 2.92

EC3= 50 + [[(3-2.92)/(4.68-2.92)] x (100-50)] = 52

The concentration of test material expected to cause a 3 fold increase in3HTdR incorporation (EC3value) was calculated to be 52%. a= lowest concentration giving stimulation index >3

b = actual stimulation index caused by ‘a’

c = highest concentration failing to produce a stimulation index of 3

d = actual stimulation index caused by ‘c’ Clinical observations, bodyweight and mortality data - preliminary screening test

Concentration

Animal Number

Bodyweight (g)

Day

1

2

3

4

5

6

Day 1

Day 6

Pre-Dose

Post

Dose

Pre-Dose

Post

Dose

Pre-Dose

Post

Dose

100%

S-1

19

19

0

0

0

0

0

0

0

0

0

0 = No signs of systemic toxicity

Individual disintegrations per minute and stimulation indices

Concentration
(% v/v) in
acetone/olive oil 4:1

Animal Number

dpm/
Animala

Mean dpm/Animal
(Standard Deviation)

Stimulation Indexb

Result

Vehicle

1-1

521.79

1149.36
(±630.41)

N/A

N/A

1-2

711.54

1-3

1177.44

1-4

1645.40

1-5

1690.62

25

2-1

2433.12

2033.61
(±935.56)

1.77

Negative

2-2

3346.22

2-3

1038.24

2-4

2105.88

2-5

1244.58

50

3-1

2982.24

3357.72*
(±730.21)

2.92

Negative

3-2

3456.29

3-3

2308.90

3-4

4061.16

3-5

3980.02

100

4-1

4009.95

5383.10***
(±1755.99)

4.68

Positive

4-2

4581.20

4-3

4927.86

4-4

4946.26

4-5

8450.25

dpm = Disintegrations per minute

a = Total number of lymph nodes per animal is 2

b = Stimulation Index of 3.0 or greater indicates a positive result

N/A=  Not applicable

*=      Significantly different from control group p<0.05

Individual clinical observations and mortality data

Concentration
(% v/v) in
acetone/olive oil 4:1

Animal Number

Day 1

Day 2

Day 3

Day 4

Day 5

Day 6

Pre-Dose

Post Dose

Pre-Dose

Post Dose

Pre-Dose

Post Dose

Vehicle

1-1

0

0

0

0

0

0

0

0

0

1-2

0

0

0

0

0

0

0

0

0

1-3

0

0

0

0

0

0

0

0

0

1-4

0

0

0

0

0

0

0

0

0

1-5

0

0

0

0

0

0

0

0

0

25

2-1

0

0

0

0

0

0

0

0

0

2-2

0

0

0

0

0

0

0

0

0

2-3

0

0

0

0

0

0

0

0

0

2-4

0

0

0

0

0

0

0

0

0

2-5

0

0

0

0

0

0

0

0

0

50

3-1

0

0

0

0

0

0

0

0

0

3-2

0

0

0

0

0

0

0

0

0

3-3

0

0

0

0

0

0

0

0

0

3-4

0

0

0

0

0

0

0

0

0

3-5

0

0

0

0

0

0

0

0

0

100

4-1

0

0

0

0

0

0

0

0

0

4-2

0

0

0

0

0

0

0

0

0

4-3

0

0

0

0

0

0

0

0

0

4-4

0

0

0

0

0

0

0

0

0

4-5

0

0

0

0

0

0

0

0

0

0=      No signs of systemic toxicity

***=   Significantly different from control group p<0.001

Individual Bodyweights and Bodyweight Changes

Concentration
(% v/v) in
acetone/olive oil 4:1

Animal Number

Bodyweight (g)

Bodyweight Change (g)

Day 1

Day 6

Vehicle

1-1

19

20

1

1-2

18

19

1

1-3

18

18

0

1-4

20

19

-1

1-5

18

18

0

25

2-1

21

22

1

2-2

18

19

1

2-3

19

20

1

2-4

19

19

0

2-5

18

19

1

50

3-1

21

22

1

3-2

19

21

2

3-3

21

22

1

3-4

19

21

2

3-5

20

19

-1

100

4-1

19

20

1

4-2

19

19

0

4-3

17

18

1

4-4

18

18

0

4-5

18

18

0

Summary of positive control data for the local lymph node assay

Project Number

Start Date

Finish Date

Test Material

Concentration

Vehicle

Stimulation Indexa

Classificationb

0039/1117

11/11/09

17/11/09

α‑Hexylcinnamaldehyde, tech., 85%

50% v/v

cottonseed oil

6.40

Positive

0039/1118

11/11/09

17/11/09

α‑Hexylcinnamaldehyde, tech., 85%

15% v/v

butanone

7.45

Positive

0039/1119

11/11/09

17/11/09

α‑Hexylcinnamaldehyde, tech., 85%

15% v/v

dimethyl sulphoxide

6.07

Positive

0039/1120

05/11/09

11/11/09

α‑Hexylcinnamaldehyde, tech., 85%

15% v/v

acetone/olive oil 4:1

3.12

Positive

0039/1138

12/02/10

18/02/10

α‑Hexylcinnamaldehyde, tech., 85%

15% v/v

ethanol/distilled water 7:3

9.61

Positive

0039/1139

17/02/10

23/02/10

α‑Hexylcinnamaldehyde, tech., 85%

15% v/v

acetone

5.55

Positive

0039/1151

11/06/10

17/06/10

α‑Hexylcinnamaldehyde, tech., 85%

25% v/v

1% pluronic L92
in distilled water

6.77

Positive

0039/1152

11/06/10

17/06/10

α‑Hexylcinnamaldehyde, tech., 85%

15% v/v

dimethyl formamide

4.77

Positive

0039/1153

09/06/10

15/06/10

α‑Hexylcinnamaldehyde, tech., 85%

15% v/v

butanone

6.63

Positive

0039/1154

09/06/10

15/06/10

α‑Hexylcinnamaldehyde, tech., 85%

15% v/v

dimethyl sulphoxide

3.25

Positive

0039/1156

09/06/10

15/06/10

Phenylacetaldehyde (90%)

2.5% v/v

propylene glycol

18.43

Positive

0039/1157

09/06/10

15/06/10

α‑Hexylcinnamaldehyde, tech., 85%

50% v/v

cottonseed oil

5.93

Positive

41003764

26/08/10

01/09/10

α‑Hexylcinnamaldehyde, tech., 85%

25% v/v

acetone/olive oil 4:1

7.25

Positive

a=      Ratio of test to control lymphocyte proliferation

b=      Stimulation index greater than 3.0 indicates a positive result

Vehicle Determination Record

Vehicle

Concentration

Method of Preparation

Description of Formulation

Suitability*

acetone/olive oil (4:1)

50%
0.5 ml test material + 0.5 ml vehicle

Vortex mixer

solution

suitable for dosing

* = Suitable for dosing if formulation is a solution or fine homogenous suspension which can be administered via a micropipette

Interpretation of results:
sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group are as follows:
25 % (% v/v) in acetone/olive oil (4:1) gave a stimulation index of 1.77 (negative).
50 % (% v/v) in acetone/olive oil (4:1) gave a stimulation index of 2.92 (negative).
100 % (% v/v) in acetone/olive oil (4:1) gave a stimulation index of 4.68 (positive).

The concentration of test material expected to cause a 3 fold increase in 3HTdR incorporation (EC3 value) was calculated to be 52%.
The test material was considered to be a sensitiser under the conditions of the test.
Executive summary:

The sensitisation potential of the test substance, TM 10-202, was assessed as positive according to OECD Test Guideline 429 using the Local Lymph Node method.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:
Migrated from Short description of key information:
The sensitisation potential of the test substance, TM 10-202, was assessed as positive according to OECD Test Guideline 429 using the Local Lymph Node method.

Justification for selection of skin sensitisation endpoint:
The study was conducted according in vivo to an appropriate test species according to internationally recognised guidelines.

Justification for classification or non-classification

For classification of a substance as a skin sensitiser, evidence from appropriate animal studies is considered. In the case of the local lymph node assay, the proliferation response of lymph node cells is expressed as the number of radioactive disintegrations per minute per animal and as the ratio of3HRdR incorporation into lymph node cells of test nodes relative to that recorded for the control nodes (Stimulation index). A substance is regarded as a sensitiser if at least one concentration of the test item results in a three-fold or greater increase in3HTdR incorporation compared to control values. A test substance that fails to produce a three-fold or greater increase in3HTdR incorporation is classified as a non-sensitiser. Chemicals for which the lowest concentration tested resulted in a stimulation index of greater than three, an EC3 value can be extrapolated from the two lowest doses. 

One of the three concentrations tested for the test substance TM 10-202 gave a stimulation index of greater than 3 and therefore TM 10-202 is considered to be a sensitiser. The concentration of test material expected to cause a 3 fold increase in 3HTdR incorporation (EC3 value) was calculated to be 52%.

The test material was considered to be a sensitiser under the conditions of the test.