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Skin sensitisation

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skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 December 2010 to 21 December 2010
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Test material form:
Details on test material:
- Storage: room temperature 15-25 °C, continuously protected from all light

In vivo test system

Test animals

CBA/J Rj mice
Details on test animals and environmental conditions:
- Females (if applicable) nulliparous and non-pregnant: yes
- Microbiological status of animals, when known: SPF
- Age at study initiation: 8 – 9 weeks old
- Weight at study initiation: 19.0 – 21.8 grams (the weight variation in animals involved in the study did not exceed ± 20% of the mean weight)
- Housing: Group caging in Type II polypropylene/ polycarbonate cages. Bedding was available to animals during the study and they were provided with glass tunnel-tubes.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 5 days

- Temperature: 22 ± 3°C
- Humidity: 30 - 70%
- Air changes: 15- 2 0 air exchange/hour
- Photoperiod: 12 hours daily, from 6.00 a.m. to 6.00 p.m.

Study design: in vivo (LLNA)

acetone/olive oil (4:1 v/v)
10, 5 and 2.5 (w/v) %
No. of animals per dose:
4 animals per dose
Details on study design:
- Compound solubility: -During the Preliminary Compatibility Test, based on the solubility of the test material acetone/olive oil 4:1 (v/v) mixture (AOO) was chosen as vehicle for the experiment. The maximum attainable concentration was 10 (w/v) %. The test material was weighed and formulations prepared daily on a weight: volume basis.
- A Preliminary Irritation/Toxicity Test was performed on CBA/J Rj mice using two doses, at test material concentrations of 10 and 5 (w/v) %, respectively. This preliminary experiment was conducted in a similar experimental manner to the main study, but it was terminated on Day 6. An assessment of ear swelling was made by measuring thickness on Days 1, 3 and 6, and the weight of an ear punch on Day 6.
- During the Preliminary Irritation / Toxicity Test no mortality, systemic toxicity or local irritation were observed (score of erythema was 0 during the observation time). There was no local swelling on Day 3 and on Day 6 in either group. The ear punch weights were in the normal range. No treatment related effect on body weights was observed. The observations recorded in this preliminary test suggested that the application of the material at 10 and 5% were acceptable for the LLNA main study.

- Topical application: During the assay each mouse was topically dosed on the dorsal surface of each ear with 25 μL of the appropriate formulation applied using a pipette. Each animal was dosed once a day for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6.
- Injection of Tritiated Thymidine (3HTdR): On Day 6, animals were taken to the radioactive suite and each mouse was intravenously injected via the tail vein with 250 μL of sterile PBS (phosphate buffered saline) containing approximately 20 μCi of 3HTdR using a gauge 25G1" hypodermic needle with 1 mL sterile syringe. Once injected, the mice were left for 5 hours (± 30 minutes).
- Removal and Preparation of Draining Auricular Lymph Nodes: Five hours (± 30 minutes) after intravenous injection the mice were euthanised by asphyxiation with ascending doses of carbon dioxide (deep anaesthesia was confirmed before making incision, death was confirmed before discarding carcasses). The draining auricular lymph nodes were excised by making a small incision on the skin between the jaw and sternum, pulling the skin gently back towards the ears and exposing the lymph nodes. The nodes were then removed using forceps. The carcasses were discarded after cervical dislocation or after cutting through major cervical blood vessels. Once removed, the nodes of mice were collected in separate Petri dishes containing a small amount (1-2 mL) of PBS to keep the nodes wet before processing.
- Preparation of Single Cell Suspension of Lymph Node Cells: A single cell suspension (SCS) of lymph node cells (LNCs) was prepared and collected in disposable tubes by gentle mechanical disaggregating of the lymph nodes through a cell strainer using the plunger of a disposable syringe. The cell strainer was washed with PBS (up to 10 mL). Pooled LNCs were pelleted with a relative centrifugal force (RCF) of 190 x g (approximately) for 10 minutes at 4°C. After centrifugation supernatants were discarded. Pellets were gently resuspended and 10 mL of PBS was added to the tubes. The washing step was repeated twice.
- Determination of Incorporated 3HTdR: After the final wash, supernatants were removed leaving a small volume (< 0.5 mL) of supernatant above each pellet. Each pellet was gently agitated before suspending the LNCs in 3 mL of 5% TCA (trichloroacetic acid) for precipitation of macromolecules. After incubation with 5% TCA at 2 - 8°C overnight (approximately 18 hours) precipitate was recovered by centrifugation at 190 x g for 10 minutes, supernatants were removed and pellets were suspended in 1 mL of 5% TCA and dispersed using ultrasonic water bath. Each precipitate was transferred to a suitable sized scintillation vial with 10 mL of scintillation liquid and thoroughly mixed. The vials were loaded to a β-scintillation counter and 3HTdR incorporation was measured for up to 10 minutes per sample. The β-counter expresses the 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM) for each animal, and the results were pooled. Similarly, background 3HTdR levels were also measured in two 1 mL aliquots of 5% TCA.

- Clinical Observations: During the study (Day 1 to Day 6) each animal was observed daily for any clinical signs, including local irritation and systemic toxicity. Clinical observations were performed twice a day (before and after treatments) on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. Individual records were maintained.
- Measurement of Body Weight: Individual body weights were recorded on Day 1 (beginning of the test) and on Day 6 (prior to 3HTdR injection) with a precision of ± 0.1 g.

- DPM was measured for each group. The measured DPM values were corrected with the background DPM value (“DPM”). The average of the two measured DPM values of 5 (w/v) % TCA solutions was used as the background DPM value. The results were expressed as “DPN” (DPM divided by the number of lymph nodes) following the industry standard for data presentation. Stimulation index (SI = DPN value of a treated group divided by the DPN value of the negative control group) for each treatment group was also calculated. A stimulation index of 3 or greater is an indication of a positive result.
- Interpretation of Results: The test material is regarded as a sensitiser if both of the following criteria are fulfilled:
(1) That exposure to at least one concentration of the test material resulted in an incorporation of 3HTdR at least 3-fold or greater than recorded in control mice, as indicated by the stimulation index.
(2) The data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Positive control results:
- The positive control group animals were treated with 25 (w/v) % HCA solution in a relevant vehicle (AOO) concurrent to the test material groups. The positive control substance was chosen according to the OECD guideline. No mortality, cutaneous reactions or signs of toxicity were observed in the positive control group. A significant lymphoproliferative response (stimulation index value of 4.2) was noted for α-hexylcinnamaldehyde in this experiment. The results of the positive control group demonstrated the appropriate performance of the assay.

In vivo (LLNA)

Resultsopen allclose all
Key result
Test group / Remarks:
10 (w/v) %
Key result
Test group / Remarks:
5 (w/v) %
Key result
Test group / Remarks:
2.5 (w/v) %
Cellular proliferation data / Observations:
- No mortality or signs of systemic toxicity were observed during the study.

- No treatment related effects were observed on animal body weights.

- Appearance of the lymph nodes was normal in the negative control group and in the test material treated groups. Larger than normal lymph nodes was observed in the positive control group.

- The test material was a light yellow solid, which was dissolved in acetone/olive oil 4:1 (v/v) mixture (AOO). Since there were no confounding effects of irritation or systemic toxicity at the applied concentrations, the proliferation values obtained are considered to reflect the real potential of the test material to cause lymphoproliferation in the Local Lymph Node Assay. The lack of any positive result under these exaggerated test conditions is considered to be good evidence that the test material is not a sensitiser.

- The results of the positive control group demonstrated the appropriate performance of the assay.

Any other information on results incl. tables

Table 1: DPM, DPN and Stimulation Index Values for all Groups

Test Group

Measured DPM/group

Group DPM

No. of Nodes


Stimulation Index Values


(5 (w/v) % TCA )






Negative control







Test material

10 (w/v) % in AOO






Test material

5 (w/v) % in AOO






Test material

2.5 (w/v) % in AOO






Positive control

25 % HCA in AOO






Applicant's summary and conclusion

Interpretation of results:
other: Not classified in accordance with EU Criteria.
Under the conditions of this study, the test material was shown to have no sensitisation potential.
Executive summary:

The skin sensitisation potential of the test material was investigated in accordance with the standardised guideline OECD 429, under GLP conditions.

Based on the results of the Preliminary Compatibility Test and on the recommendations of the OECD Guideline, the test material was dissolved in acetone/olive oil 4:1 (v/v) mixture (AOO). The maximum attainable concentration was 10 (w/v) %.

The Preliminary Irritation/Toxicity Test was performed in CBA/J Rj mice using two doses (test material concentrations of 10 and 5 (w/v) %) in the selected vehicle. The applicability and biocompatibility of the test material on the ears of animals at the maximum concentration of test material of 10 (w/v) % was acceptable.

In the main assay, twenty female CBA/J Rj mice were allocated to five groups of four animals each: three groups received the appropriate formulation of the test material at concentrations of 10, 5 and 2.5 (w/v) %, the negative control group received AOO and the positive control group received 25% α-hexylcinnamaldehyde (HCA) in AOO. The solutions of the test material were applied on the dorsal surface of ears of experimental animals (25 μL/ear) for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6. On Day 6, the cell proliferation in the local lymph nodes was measured by incorporation of tritiated methyl thymidine (3HTdR) and the values obtained were used to calculate stimulation indices (SI).

No mortality, systemic toxicity or local irritation was observed during the study. No treatment related effects were observed on animal body weights in any treated groups. Stimulation index values of the test material were 0.5, 0.7 and 0.8 at treatment concentrations of 10, 5 and 2.5 (w/v) %, respectively. α-hexylcinnamaldehyde (25 (w/v) % dissolved in AOO) was used as a positive control to demonstrate the appropriate performance of the assay. A significant lymphoproliferative response (stimulation index value of 4.2) was noted for the positive control chemical and this result confirmed the validity of the assay.

Under the conditions of this study, the test material was shown to have no sensitisation potential.