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Administrative data

Description of key information

Key value for chemical safety assessment

Skin sensitisation

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

GPMT:

In order to assess the cutaneous allergenic potential of the test substance, the Maximization-Test was performed in 15 (10 test and 5 control) female albino guinea pigs, in accordance with OECD Guideline No. 406, the Directive 96/54/EEC, B.6 and GLP (RCC, 2000). The intradermal induction of sensitization in the test group was performed in the nuchal region with a 1 % dilution of the test article in PEG 400 and in an emulsion of Freund's Complete Adjuvant (FCA) / physiological saline. The epidermal induction of sensitization was conducted for 48 hours under occlusion with the test article at 50 % in PEG 400 one week after the intradermal induction and following pretreatment of the test areas with 10 % Sodium-Lauryl-Sulfate (SLS) approximately 23.5 hours prior to application of the test article. The animals of the control group were intradermally induced with PEG 400 and FCA/physiological saline and epidermally induced with PEG 400 under occlusion following pretreatment with 10 % SLS. Two weeks after epidermal induction the control and test animals were challenged by epidermal application of the test article at 50 % in PEG 400 and PEG 400 alone under occlusive dressing. Cutaneous reactions were evaluated at 24 and 48 hours after removal of the dressing.

No toxic symptoms were evident in the guinea pigs of the control or test group. No deaths occurred. Five out of 10 test animals showed delayed skin reactions after the first challenge treatment with the test substance at 50 % (w/w) in PEG 400. No skin effect was observed in the control group.

Discrete/patchy erythema was observed in 5 test animals during the challenge procedure. As they did not fade but were reproducible in all five animals 24 hours later, the skin reactions should be regarded as a response to an allergenic potential of the test article.

LLNA:

1st study:

In order to study the possible allergenic potential of the test substance five groups of four female mice were each treated with the test item at concentrations of 1 %, 2.5 %, 5 %, 10 % and 25 % (w/v) in acetone:olive oil, 4:1 (v/v) by topical application to the dorsum of each ear lobe (left and right) on three consecutive days (RCC, 2000). The study was conducted according to OECD 429 guideline and GLP. A control group of four mice was treated with the vehicle (acetone:olive oil, 4:1 (v/v)) only. Five days after the first topical application the mice were injected intravenously into a tail vein with radio-labelled thymidine (3H-methyl thymidine). Approximately five hours after intravenous injection, the mice were sacrificed, the draining auricular lymph nodes excised and pooled per group. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes which were washed subsequently

and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of 3H-methyl thymidine measured in a beta-scintillation counter. No test item-related clinical signs were observed.

All treated animals survived the scheduled study period.

A test item is regarded as a sensitizer in the LLNA if the exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the STIMULATION INDEX (S.I.).

In this study STIMULATION INDICES of 1.7, 1.4, 1.3, 1.3 and 1.0 were determined with the test item at concentrations of 1 %, 2.5 %, 5 %, 10 % and 25 % (w/v) in acetone:olive oil, 4:1 (v/v).

A test item is regarded as a sensitizer in the LLNA if the exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the STIMULATION INDEX.

Based on these criteria, the test item was found to be a non-sensitizer when tested up to 25 % (w/v) in acetone:olive oil, 4:1 (v/v).

2nd study:

The aim of this study was to evaluate the potential of the test item to induce delayed contact hypersensitivity using the murine Local Lymph Node Assay (LLNA).

Evaluation of local irritation was also carried out in parallel.

This study was conducted in compliance with the principles of Good Laboratory Practice Regulations and according to OECD 429 guideline.

A preliminary test was first performed in order to defme the concentrations of test item to be used in the main test.

In the main test, twenty-eight female CBA/J mice were allocated to seven groups:

five treated groups of four animals receiving the test item at the concentration of 1,2.5,5,10 or 25%,

one negative control group of four animals receiving the vehicle (dimethylformamide = DMF), one positive control group of four animals receiving the reference item, a-hexylcinnamaldehyde (HCA), a moderate sensitizer, at the concentration of 25%.

During the induction phase, the test item, vehicle or reference item was applied over the ears (25 microliter per ear) for 3 consecutive days (days 1, 2 and 3). After 2 days of resting, the proliferation of lymphocytes in the lymph node draining the application site was measured by incorporation of tritiated methyl thymidine (day 6). The obtained values were used to calculate stimulation indices (SI).

The irritant potential of the test item was assessed in parallel by measurement of ear thickness on days 1,2, 3 and 6.

Due to the unsatisfactory solubility of the test item in the first recommended vehicle (acetone/olive oil (4/1, v/v)), dimethylformamide was chosen from the other proposed vehicles.

A homogeneous dosage form preparation was obtained at the maximum concentration of 25%.

Consequently, the concentrations selected for the preliminary test were 2.5, 5, 10 and 25%.

Since the test item was non-irritant in the preliminary test, the highest concentration retained for the main test was the maximal practicable concentration (25%). No deaths occurred during the study.

Hypoactivity was noted on day 6 in 1/4 females given the concentration of 1 %.

No cutaneous reactions and no increase in ear thickness were observed in the animals of the treated groups.

Proliferation assav

No noteworthy lymphoproliferation was noted at any tested concentration (highest S.I: 1.21, not dose-dependant), while significant lymphoproliferation was observed with HCA at 25% (S.I. 5.95).

Under otir experimental conditions, the test item did not induce delayed contact hypersensitivity in the murine Local Lymph Node Assay.


Migrated from Short description of key information:
GPMT: skin sensitizer (RCC, 2000)
LLNA: not sensitizing (RCC, 2000 and CIT, 2006)

Justification for classification or non-classification

Two LLNA's did not indicate a skin sensitization potential, a GPMT gave a positive result. Taken together all available data, a skin sensitization potential at concentrations above 25 % can not be excluded. The test substance therefore has to be classified as skin sensitizer according to DSD-DPD and CLP.