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Administrative data

skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)

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)
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Test material form:
solid: particulate/powder
migrated information: powder
Details on test material:
Batch: C02421-1620
Purity: 98.6%

In vivo test system

Test animals

Details on test animals and environmental conditions:
- Source: Harlan Netherlands B.V. Postbus6174 NL -5960 AD Horst / The Netherlands
- Number of animals for the pre-test (non-GLP): 2 females
- Number of animals for the main study: 16 females
-Number of animals per group: 4 females (nulliparous and non-pregnant)
- Number of test groups: 3
- Number of control (vehicle) group: 1
- Age: 8-12 weeks (beginning of acclimatization)
- Body weight: 17.1g - 20.3g (beginning of acclimatization)
- Identification: Each cage by unique cage card, in every cage each animal by individual code marked at tail with a permanent pen
- Randomization: Randomly selected by computer algorithm at time of elivery.
- Acclimatization: Under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

- Conditions: Standard Laboratory Conditions. Air-conditioned with target ranges for room temperature 22 +/- 3 °C, relativehumidity 30-70 % and 10-15 air changes per hour. Room temperature and humidity were monitored continuously and values outside of these ranges occasionally occurred, usually following room cleaning. These transient variations are considered not to have any influence on the study and, therefore, these data are not reported but are retained at RCC. There was a 12 hour fluorescent light / 12 hour dark cycle with at least 8 hours music during the light period.
- Accommodation: In groups of four in Makrolon type-3 cages with standard softwood bedding (“Lignocel”, Schill AG, CH-4132 Muttenz).
- Diet: Pelleted standard Kiiba 34331 batch no. 84/02 mouse maintenance diet (Provimi Kliba AG, CH-4303 Kaiseraugst) available ad /~bifum. Results of analyses for contaminants are archived at RCC.
- Water: Community tap water from Itingen, available ad Iibitum. Results of representative bacteriological, chemical and contaminant analyses are archived at RCC.

Study design: in vivo (LLNA)

other: ethanol: water, 7:3 (v/v)
5%, 10% and 25%
No. of animals per dose:
Details on study design:
Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear lobe (left and right) with different test item concentrationsof 5%, 10% and 25% (w/v) in ethanol: water, 7:3 (v/v). The application volume, 25 µl, was spread over the entire dorsal surface of each ear lobe once daily for three consecutive days. A further group of mice was treated with an equivalent volume of the relevant vehicle alone (control animals). A hair dryer was used to dry the ear’s surface as quickly as possible to avoid loss of test item applied.

3H-methyl thymidine (3HTdR) was purchased from Amersham International (Amersham product code no. TRA 31O;specific activity, 2 Ci/mmol; concentration, 1 mCi/ml). Five days after the first topical application, all mice were administered with 250 PI of 83.50 pCi/ml 3HTdR (equal to 20.9 pCi 3HTdR) by intravenous injection via a tail vein.

Approximately five hours after treatment with 3HTdR all mice were euthanized by intraperitoneal injection of VETANARCOL VeterinarianAG, Zurich).The draining lymph nodes were rapidly excised and pooled for each experimental group (8 nodes per group). Single cell suspensions (phosphate buffered saline) of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 pm mesh size). After washing two times with phosphate buffered saline (approx. 10 ml) the lymph node cells were resuspended in 5 % trichloroacetic acid (approx. 3 ml) and incubated at approximately +4 “C for at least 18 hours for precipitation of macromolecules. The precipitates were then resuspended in 5 % trichloroacetic acid (1 ml) and transferred to glass scintillation vials with 10 ml of ‘Ultima Gold’ scintillation liquid and thoroughly mixed. The level of 3HTdR incorporation was then measured on a beta-scintillation counter. Similarly, background 3HTdR levels were also measured in two 1ml-aliquots of 5 % trichloroacetic acid.The Beta-scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM).

Results and discussion

In vivo (LLNA)

Remarks on result:
other: Group 2 (5%) : 1.3 Group 3 (10%) : 1.1 Group 4 (25%) : 1.3

Any other information on results incl. tables

No deaths occurred during the study period.

No test item-related clinical signs were observed in any animals of the control group, Group 2 (5 %) or Group 3 (1O%). On the third application day, a slight ear swelling was observed at both dosing sites in all mice of Group 4 (25 %), persisting for two days.

The body weight of the animals, recorded at the start of acclimatization period and prior to necropsy, was within the range commonly recorded for animals of this strain and age.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Migrated information Criteria used for interpretation of results: EU
In this study STIMULATION INDICES of 1.3, 1.1 and 1.3 were determined with the test item at concentrations of 5 %, 10 % and 25 % (w/v) in ethanol: water, 7:3 (v/v). A test item is regarded as a sensitizer in the LLNA if the exposure to one or more test concentrations resulted in 3-fold or greater increase in incorporation of 3HTdR compared with concurrent controls, as indicated by the STIMULATION INDEX (S.I.). The test item ucb 6474 was found tobe a non-sensitizer when tested at up to the highest achievable concentration of 25 %(w/v) in ethanol: water, 7:3 (v/v).