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Toxicological information

Skin sensitisation

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Administrative data

skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010-08-02 to 2010-12-09
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and Guideline Study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guidelineopen allclose all
according to guideline
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
July 22, 2010
according to guideline
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
30 May 2008
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Test material form:
other: colourless liquid

In vivo test system

Test animals

Details on test animals and environmental conditions:
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sulzfeld, Germany
- Age at study initiation: 8 – 12 weeks
- Weight at study initiation: 18.8 g – 24.1 g
- Housing: The animals were housed in fully air-conditioned rooms.Type of cage: Makrolon cage, type II Enrichment: PLEXX mouse tunnel (red, transparent) and nest building material Nestlets NES 3600 (PLEXX b.v.; AB Elst, Netherlands). No. of animals per cage: 1
- Diet: Kliba-Labordiät (Maus / Ratte Haltung “GLP”), Provimi Kliba SA, Kaiseraugst, Basel, Switzerland, ad libitum
- Water: Tap water ad libitum
- Acclimation period: at least 5 days before the first test substance application

- Temperature (°C): 20 – 24
- Humidity (%): 30 – 70
- Air changes (per hr): Central air-conditioning
- Photoperiod (hrs dark / hrs light): 12 h / 12 h (6.00 a.m. – 6.00 p.m. / 6.00 p.m. – 6.00 a.m.)

IN-LIFE DATES: From: 2010-07-21 To: 2010-08-09

Study design: in vivo (LLNA)

other: Methyl ethyl ketone
0, 0.75, 2.5, 7.5 % (w/w)
No. of animals per dose:
5 animals per dose
Details on study design:
- Compound solubility: MEK was used as the vehicle because good solubility of the preparation was achieved.
- Irritation: At the tested concentration of 50% the animals showed severely increased lymph node weights and slightly increased ear weights.
- Lymph node proliferation response: After application of a 5% test-substance preparation the animals did not show any signs of local irritation as confirmed by the ear weight measurements, but considerable increase in lymph node weights was observed. Signs of systemic toxicity were not observed during the pre-tests.

- Criteria used to consider a positive response: In order to reveal a possible induction of sensitization, the response in the draining lymph node after epicutaneous application of several concentrations of the test substance to the skin of the dorsal surface of both ears is determined by measuring 3H-thymidine incorporation into the lymph node cells. Additional parameters used to characterize the response are, lymph node cell count and, to a certain extent, lymph node weight. Because irritation by the test substance may also induce lymph node responses, the weights of ear punches taken from the area of test-substance application are determined as an indicator for inflammatory ear swelling due to any irritant action of the test substance.

The study comprised three treatment groups and a vehicle control group. Each group consisted of 5 mice.
Randomization: Prior to first application, the animals were distributed to the individual groups, received their animal numbers and were allocated to the respective cages according to the randomization instructions of „Nijenhuis, A. and Wilf, H.S.: Combinatorial Algorithms, Academic Press, New York, San Francisco, London, 1978, pp. 62 – 64“.
Body weight determination: Individual body weights on day 0 prior to the first application and on day 5 prior to the sacrifice of the animals.
Signs and symptoms: No detailed clinical examination of the individual animals was performed but any obvious signs of systemic toxicity and/or local inflammation at the application sites were noted.
Mortality: A check for moribund and dead animals was made twice each workday (beginning and end) and once on Saturdays, Sundays and on public holidays.
Form of application: Epicutaneous application is simulating dermal contact with the compound which is possible to occur under practical use conditions.
Application volume: 25 μL per ear
Site of application: Dorsal surface of both ears
Frequency of application: 3 consecutive applications (day 0 – day 2) to the same application site
The animals of control group 1 and test groups 2-4 were treated with vehicle or test substance preparation
³H-thymidine injection: On study day five (about 66 to 72 hours after the last application of test substance to the ears) the mice were injected into a tail vein with 20 μCi of 3H-thymidine in 250 μL of sterile saline.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Stimulation Index (SI): A value calculated to assess the skin sensitization potential of a test substance that is the ratio of the proliferation in treated groups to that in the concurrent vehicle control group.
EC3 = estimated concentration needed to produce a stimulation index of 3;
EC1.5 = estimated concentration needed to produce a stimulation index of 1.5

Results and discussion

Positive control results:
The sensitivity of mice (CBA/J, Charles River Laboratories, Research Models and Services, Germany GmbH, Sulzfeld, Germany) and the reliability of experimental techniques were assessed regularly using a known sensitizer as recommended by the test guidelines.
Positive results were consistently obtained over the years using several variations of the methods and different vehicles.
A list of tests including stimulation indices (fold of change as compared to the vehicle control) for 3H-thymidine incorporation and cell count is presented.

In vivo (LLNA)

Resultsopen allclose all
Remarks on result:
other: Test Group / Treatment / Stimulation Index 1 * / vehicle MEK / 1.00 2 / 0.75% in MEK / 1.66 3 / 2.5% in MEK / 2.20 4 / 7.5% in MEK / 5.52 * Calculation on basis of 4 animals, as one animal died during 3H-thymidine injection
other: disintegrations per minute (DPM)
Remarks on result:
other: see Remark
Test Group / Treatment / [DPM/Lymph Node Pair] / Stimulation Index 1 * / vehicle MEK / 580.0 / 1.00 2 / 0.75% in MEK / 962.1 / 1.66 3 / 2.5% in MEK / 1,273.6 / 2.20 4 / 7.5% in MEK / 3,202.2 / 5.52 - test group x / test group 1 (vehicle control) * Calculation on basis of 4 animals, as one animal died during 3H-thymidine injection.

Applicant's summary and conclusion