Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20 January 2015 to 10 February 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP in accordance with recognised guideline.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Description: dark yellow viscous liquid
Purity: 100%
Expiry date: 12 June 2016
Storage conditions: room temperature in the dark

The integrity of supplied data relating to the identity, purity and stability of the test item is the responsibility of the Sponsor.
A Certificate of Analysis was supplied by the Sponsor.

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/CaCrl
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK), Kent, UK
- Weight at study initiation: 15 to 23 g
- Housing: individually housed in suspended solid floor polypropylene cages furnished with softwood woodflakes
- Diet (e.g. ad libitum): free access of food throughout the study (Rodent 2014C Teklad Global Diet, Harlan Laboratories U.K Ltd., Oxon, UK)
- Water (e.g. ad libitum): free access of tap water throughout the study
- Acclimation period: at least 5 days
- Age: 8 to 12 weeks

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.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity (%): 50 ± 20 %
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): 12 hours continuous light/dark.

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.

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Remarks:
α-Hexylcinnamaldehyde, tech., 85% at 25% v/v in acetone/olive oil 4:1
Concentration:
100% (neat) and as a solution at 1% and 10% w/w in acetone/olive 4:1
No. of animals per dose:
five
Details on study design:
Preliminary Screening Test
Using available information regarding the systemic toxicity/irritancy potential of the test item, a preliminary screening test was performed using one mouse.The mouse was treated by daily application of 25 µL of the undiluted test item to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). Themouse was observed twice daily on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. Local irritation was scored daily according to a scale of 0 to 4, see Appendix 3 as described in any other information. Any clinical signs of toxicity, if present, were also recorded. The body weight of the mouse was recorded on Day 1 (prior to dosing) and on Day 6.
The thickness of the ear was measured using a Mitutoyo 547-300S gauge (Mitutoyo Corporation), pre dose and 1 hour post dose on Day 1, 1 hour post dose on Days 2 and 3 and on Days 4, 5, 6. Any changes in the ear thickness were noted. Mean ear thickness changes were calculated between time periods Days 1 to 3 and Days 1 to 6. A mean ear thickness increase of equal to or greater than 25% was considered to indicate excessive irritation and limited biological relevance to the endpoint of sensitisation.

Main Test
Test Item Administration
Groups of five mice were treated with the test item at concentrations of 100%, 10% or 1% w/w in acetone/olive oil 4:1. The preliminary screening test suggested that the test item 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 item to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The test item formulation was 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.
The positive control animals were similarly treated to the test animals except that 25 µL of the positive control item, α-Hexylcinnamaldehyde, tech., 85%, at aconcentration of 25% v/v in acetone/olive oil 4:1 was applied to the dorsal surface of each ear.
The thickness of each ear was measured and recorded as described in the preliminary screening.

3H-Methyl Thymidine Administration
Five days following the first topical application of the test item, vehicle control or positive control item (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 study 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 re-suspended in 10 mL of PBS and re-pelleted. To precipitate out the radioactive material, the pellet was re-suspended 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, re-suspended 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).
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
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. Data was first assessed for suitability by analysis of normality and homogeneity of variance. If the assumptions that the data are both normally distributed and has homogeneity of variances, then parametric one way analysis of variance (ANOVA) and Dunnett's multiple comparison procedure were used to determine statistical significance. If the assumptions were not met, non-parametric Kruskal-Wallis Rank Sum and Marm-Whitney U test procedures were used.

Probability values (p) are presented as follows:

P<0.001 ***
P<0.01 **
P<0.05 *
P≥0.05 (not significant)

Results and discussion

Positive control results:
The positive control item, α-Hexylcinnamaldehyde, tech., 85%, gave a Stimulation Index of greater than 3 (4.46) when tested at a concentration of 25% v/v in acetone/olive oil 4:1.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: The Stimulation Index are given in Table 4 below.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: The radioactive disintegrations per minute per animal are given in Table 4 below.

Any other information on results incl. tables

Interpretation of Result

The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute per animal and as the ratio of 3HTdR incorporation into lymph node cells of test nodes relative to that recorded for the control nodes (Stimulation Index).

The test item will be regarded as a sensitizer if at least one concentration of the test item results in a threefold or greater increase in 3HTdR incorporation compared to control values. Any test item failing to produce a threefold or greater increase in 3HTdR incorporation will be regarded as a "non-sensitizer".

Preliminary Screening Test

Clinical observations, body weight and mortality data are given in Table 1 below and local skin irritation is given in Table 2 below. The ear thickness measurements and mean ear thickness changes are given in Table 3 below. 

Very slight erythema was noted on both ears on Day 3. No signs of systemic toxicity or irritation indicated by an equal to or greater than 25% increase in mean ear thickness were noted. 

Based on this information the undiluted test item and the test item at concentrations of 10% and 1% 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 radioactive disintegrations per minute per animal and the stimulation index are given in Table 4 below. 

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:

Treatment Group

 

Concentration

Stimulation Index

 

Result

 

 

 

Test Item

1%v/v in

acetone/olive oil 4:1

0.75

Negative

10%v/v in

acetone/olive oil 4:1

1.30

Negative

100%

2.96

Negative

Positive Control Item

25%v/v in

acetone/olive oil 4:1

4.46

Positive

Clinical Observations and Mortality Data

Individual clinical observations and mortality data for test and control animals are given in Table 5 below and local skin irritation is given in Table 6. The ear thickness measurements and mean ear thickness changes are given in Table 7.

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

Body Weight

Individual body weights and body weight change for test and control animals are given in Table 8.

Body weight change of the test animals between Day 1 and Day 6 were comparable to that observed in the corresponding control group animals over the same period.

Table 1: Clinical Observations, Body Weight and Mortality Data-Preliminary Screening Test

 

 

 

Concentration

 

 

 

Animal Number

 

Body Weight (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

 

17.5

 

17.7

 

0

 

0

 

0

 

0

 

0

 

0

 

0

 

0

 

0

0= No mortality or signs of systemic toxicity

Table 2: Local Skin Irritation - Preliminary Screening Test

 

 

 

Concentration

 

 

 

Animal Number

 

Local Skin Irritation

Day1

Day2

Day3

Day4

Days

Day6

 

left

 

right

 

left

 

right

 

left

 

right

 

left

 

right

 

left

 

right

 

left

 

right

100%

 

S-1

0

0

0

0

1

1

0

0

0

0

0

0

Table 4 Individual Disintegrations per Minute and Stimulation Indices

Treatment Group

Animal Number

dpm/
Animal
a

Mean dpm/Animal
(Standard Deviation)

Stimulation Indexb

Result

Vehicle acetone/olive oil 4:1

1-1

2219.96

2067.31
(±228.28)

n/a

n/a

1-2

2381.08

1-3

1818.42

1-4

1935.27

1-5

1981.81

Test Item
1% v/v in
acetone/olive oil 4:1

2-1

1692.18

1554.53
(±401.18)

0.75

Negative

2-2

1087.73

2-3

1204.01

2-4

2057.28

2-5

1731.45

Test Item
10% v/v in
acetone/olive oil 4:1

3-1

2842.66

2682.81**
(±332.08)

1.30

Negative

3-2

3196.03

3-3

2483.30

3-4

2435.45

3-5

2456.62

Test Item
100% 

4-1

5421.72

6127.62**
(±745.04)

2.96

Negative

4-2

6858.88

4-3

6084.17

4-4

5366.83

4-5

6906.48

Positive Control Item

25% v/v in
acetone/olive oil 4:1

5-1

10234.21

9229.73**
(±1131.04)

4.46

Positive

5-2

10568.61

5-3

7995.21

5-4

8379.12

5-5

8972.50

dpm = Disintegrations per minute

n/a =  Not applicable

a =     Total number of lymph nodes per animal is 2

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

** =    Significantly different from control group p<0.01

Table 5 Individual Clinical Observations and Mortality Data

Treatment Group

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
acetone/olive oil 4:1

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

Test Item
1% v/v in
acetone/olive oil 4:1

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

Test Item
10% v/v in
acetone/olive oil 4:1

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

Test Item
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

Positive Control Item

25% v/v in
acetone/olive oil 4:1

5-1

0

0

0

0

0

0

0

0

0

5-2

0

0

0

0

0

0

0

0

0

5-3

0

0

0

0

0

0

0

0

0

5-4

0

0

0

0

0

0

0

0

0

5-5

0

0

0

0

0

0

0

0

0

0 =    No mortality or signs of systemic toxicity

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information
Conclusions:
The test item was considered to be a non-sensitiser under the conditions of the test.

α-Hexylcinnamaldehyde, tech., 85% gave a Stimulation Index of greater than 3 (4.46) when tested at a concentration of 25% v/v in acetone/olive oil 4: 1 thus demonstrating the sensitivity and reliability of the test system.
Executive summary:

Introduction

A study was performed in accordance with OECD Guideline 429 and EU Method B.42 (LLNA) to assess the skin sensitisation potential of the test item in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear.

Methods

Following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of 100%, this concentration was selected as the highest dose investigated in the main test of the Local Lymph Node Assay. Three groups, each of five animals, were treated with 50 µL (25 µL per ear) of the undiluted test item or the test item as a solution in acetone/olive oil 4: 1 at concentrations of 10% or 1% v/v. A further group of five animals was treated with acetone/olive oil 4: 1 alone. A concurrent positive control test, using a group of five animals, was also performed with the known sensitizer, α-Hexylcinnamaldehyde tech., 85%, at a concentration of 25% v/v in acetone/olive oil 4: 1.

Results

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:

Treatment Group

 

Concentration

Stimulation Index

 

Result

 

 

 

Test Item

1% v/v in

acetone/olive oil 4:1

0.75

Negative

10% v/v in

acetone/olive oil 4:1

1.30

Negative

100%

2.96

Negative

Positive Control Item

25% v/v in

acetone/olive oil 4:1

4.46

Positive

Conclusion

The test item was considered to be a non-sensitiser under the conditions of the test. The positive control, α-Hexylcinnamaldehyde, tech., 85% gave a Stimulation Index of greater than 3 (4.46) when tested at a concentration of 25% v/v in acetone/olive oil 4: 1 thus demonstrating the sensitivity and reliability of the test system.