Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 February 2012 - 10 March 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

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
Principles of method if other than guideline:
There was an exception with regard to GLP: No analysis was carried out to determine the homogeneity, concentration or stability of the test item formulation. The test item was formulated within two hours of being applied to the test system; it is assumed that the formulation was stable for this duration. This exception is considered not to affect the purpose or integrity of the study.
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/Ca
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories UK Limited, Bicester, Oxon, UK
- Age at study initiation: 8 - 12 weeks old
- Weight at study initiation: 15 - 23 g
- Housing: individually housed in suspended solid-floor polypropylene cages furnished with softwood woodflakes
- Diet (e.g. ad libitum):2014C Teklad Global Rodent diet supplied by Harlan Laboratories UK Ltd., Oxon, UK ad libitum
- Water (e.g. ad libitum): mains tap water ad libitum
- Acclimation period: at least five days
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.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): target range of 19 to 25°C
- Humidity (%): target range of 30 to 70%
Any occasional deviations from these targets were considered not to have affected the purpose or integrity of the study.
- Air changes (per hr): approximately fifteen changes per hour
- Photoperiod (hrs dark / hrs light): twelve hours continuous light (06.00 to 18.00) and twelve hours darkness

IN-LIFE DATES: From: 10 February 2012 - 8 March 2012

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Remarks:
This vehicle was chosen as it produced the highest concentration that was suitable for dosing.
Concentration:
10 %, 5 % and 2.5 % w/w in acetone/olive oil 4:1
No. of animals per dose:
5
Details on study design:
PRELIMINARY SCREENING TEST
Using available information regarding the systemic toxicity of the test item, a preliminary screening test was performed using three mice, one mouse per test item concentration. The mice were treated by daily application of 25 µL of the test item at concentrations of 50 %, 25 % or 10 % w/w in acetone/olive oil 4:1, to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The mice were observed twice daily on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. Local irritation was scored daily and clinical signs of toxicity, if present, were also recorded. The bodyweight of each mouse was recorded on Day 1 (prior to dosing) and on Day 6.

MAIN TEST
Test Item Administration:
Groups of five mice were treated with the test item at concentrations of 10 %, 5 % or 2.5 % 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.

3H-Methyl Thymidine Administration:
Five days following the first topical application of the test item 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. 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 x gravity) 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 x gravity) 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 Q" 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:
Statistical Analysis
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.

Interpretation of Results
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 sensitiser 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 classified as a "non-sensitiser".

The EC3 value was also calculated. The EC3 value is the concentration of test item expected to cause a 3 fold increase in 3HTdR incorporation. The equation used for the calculation of EC3 is:

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

Results and discussion

Positive control results:
Project number: 41103434
Study dates: 05 October 2011 - 11 October 2011

Methods. A group of five animals was treated with 50 µL (25 µL per ear) of α-Hexylcinnamaldehyde 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.

Results. 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:

Concentration (% v/v) in Stimulation Index Result
acetone/olive oil 4:1
25 4.05 Positive

Conclusion: α-Hexylcinnamaldehyde was considered to be a sensitiser under the conditions of the test.

In vivo (LLNA)

Resultsopen allclose all
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: Concentration (% w/w) in Stimulation Index Result acetone/olive oil 4:1 2.5 2.33 Negative 5 2.40 Negative 10 4.79 Positive
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: The radioactive disintegrations per minute per animal and the stimulation index are given in Table 1.

Any other information on results incl. tables

Table 1 Individual Disintegrations per Minute and Stimulation Indices

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

 

Animal Number

 

dpm/Animal

Mean dpm/Animal and Standard Deviation

 

Stimulation Index

 

Result

 

 

Vehicle

1-1

1156.42

 

 

1315.45 ± 322.55

 

 

n/a

 

 

n/a

1-2

1269.53

1-3

1091.83

1-4

1178.36

1-5

1881.10

 

 

2.5

2-1

3278.14

 

 

3060.13 ± 1063.78

 

 

2.33

 

 

Negative

2-2

3548.73

2-3

4472.14

2-4

2034.45

2-5

1967.21

 

 

5

3-1

2664.51

 

 

3155.04** ± 477.66

 

 

2.40

 

 

Negative

3-2

3258.18

3-3

3812.63

3-4

2709.74

3-5

3330.13

 

 

10

4-1

4542.83

 

 

6305.34* ± 2422.81

 

 

4.79

 

 

Positive

4-2

9364.26

4-3

4870.27

4-4

8489.79

4-5

4259.55

dpm = Disintegrations per minute

Total number of lymph nodes per animal is 2

Stimulation Index of 3.0 or greater indicates a positive result

n/a = not applicable

** Significantly different from control group p<0.01

* Significantly different from control group p<0.05

                              

                                                                               

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.

 

Bodyweight

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 = 10

b = 4.79

c = 5

d = 2.40

EC3 = 6

The concentration of test item expected to cause a 3 fold increase in 3HTdR incorporation was calculated to be 6 %.

Applicant's summary and conclusion

Interpretation of results:
sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test item was considered to be a sensitiser under the conditions of the test.
Executive summary:

The test item was considered to be a sensitiser under the conditions of the test. The test was conducted in accordance with OECD Guideline for the Testing of Chemicals No. 429 "Skin Sensitisation: Local Lymph Node Assay" (adopted 22 July 2010) and Method B42 Skin Sensitisation (Local Lymph Node Assay) of Commission Regulation (EC) No. 440/2008.