Registration Dossier

Administrative data

skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant guideline study, available as unpublished report, no restrictions, fully adequate for assessment.

Data source

Reference Type:
study report
Report Date:

Materials and methods

Test guidelineopen allclose all
according to
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
according to
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
according to
EPA OPPTS 870.2600 (Skin Sensitisation)
GLP compliance:
yes (incl. certificate)
RCC Ltd, CH-4452 ltingen, Switzerland
Type of study:
mouse local lymphnode assay (LLNA)

Test material

Test material form:
solid: particulate/powder
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): FAT 40826/A
- Lot/batch No.: TZ 5604 BOP 01/06
- Expiration date: 1 February 2011
- Purity: Content of organic part (Na-salt): approx. 78%. Oligomers: 13%. Main component: approx. 48%.
- Stability of test item: Stable under storage conditions
- Storage conditions: At room temperature (range of 20 ± 5 °C) light protected. Store in a desiccator.

In vivo test system

Test animals

Details on test animals and environmental conditions:
- Source: RCC Ltd., Laboratory Animal Service, CH-4414 Füllinsdorf/Switzerland
- Age at acclimatization: 8 - 12 weeks
- Weight at study initiation: 16 g-24 g (ordered)
- Housing: lndividual in Makrolon type-2 cages with standard softwood bedding ("Lignocel", Schill AG, CH-4132 Muttenz).
- Diet: Pelleted standard Kliba 3433, batch no. 001/06 mouse maintenance diet (Provimi Kliba AG, CH-4303 Kaiseraugst), ad libitum.
- Water: Community tap water from ltingen, available ad libitum.
- Acclimation period: Under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

- Temperature (°C): 22±3
- Humidity (%): 30-70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

5, 10, 25% (w/v)
No. of animals per dose:
- 4 females/dose
- Number of animals for the pre-test (non-GLP): 3 females
Details on study design:
In non-GLP solubility pre-test, the test item was tested in different vehicles: acetone/olive oil (4/1, v/v), ethanol/water (7/3, v/v), DMF and propylene glycol. DMF was found to be a suitable vehicle and was selected and used in the main test. 25 % was the highest technically achievable concentration in the chosen vehicle. A non-GLP local toxicity pre-test was performed for determination of concentrations for the main test. Three single animals were each treated with one of three different concentrations: 5 %, 10 % and 25 %, on both ears on three consecutive days. No clinical signs were observed at these concentrations one day after each topical application. A yellow-orange stain was found at all the dosing sites one day after the second and the third application. 25 % was the highest dosing concentration in the main tests.

Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear lobe (left and right) with the test item at concentrations of 5 %, 10 % and 25% in DMF. The application volume, 25 µl, was spread over the entire dorsal surface (diameter: approx. 8 mm) 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).

Five days after the first topical application, all mice were administered with 250 µL of PBS containing 79.38 µCi/mL 3HTdR (equal to 19.8 µCi 3HTdR) by intravenous injection via a tail vein.

Approximately five hours after treatment with ³HTdR all mice were euthanized by inhalation of CO2 (dry ice). 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 µm mesh size). After washing twice 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 'Irga-Safe plus' scintillation liquid and thoroughly mixed. The level of ³HTdR incorporation was then measured on a β-scintillation counter. Similarly, background ³HTdR levels were also measured in two 1 mL-aliquots of 5 % trichloroacetic acid. The β-scintillation counter expresses ³HTdR incorporation as the number of radioactive disintegrations per minute (DPM).

The proliferative response of lymph node cells is expressed as the number of radioactive disintegrations per minute per lymph node (DPM/node) and as the ratio of ³HTdR incorporated into lymph node cells of test group relative to that recorded for control group (STIMULATION INDEX) (S.I.). Before DPM/node values were determined, mean scintillation-background DPM was subtracted from test and control raw data.
A test item is regarded as a sensitizer in the LLNA if the following criteria are fulfilled:
- First, 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.
- Second, the data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentration) for either local toxicity or immunological suppression.
Positive control substance(s):
other: alpha-hexylcinnamaldehyde in acetone/olive oil (4/1, v/v) using CBA/Ca mice (RCC Study Number A51085) from 11-JAN-2006 to 25-JAN-2006.

Results and discussion

Positive control results:
In this study (RCC Study number A51085) STIMULATION INDICES of 1.8, 2.9 and 6.2 were determined with the test item at concentrations of 5 %, 10 % and 25 % (w/v), respectively, in acetone:olive oil, 4:1 (v/v). The positive control item ALPHA-HEXYLCINNAMALDEHYDE showed an allergenic potential, and an EC3 value of 10.5 % (w/v) was derived.

In vivo (LLNA)

Resultsopen allclose all
Remarks on result:
other: Test substance concentration 5% (w/v): 16.7 Test substance concentration 10% (w/v): 13.9 Test substance concentration 25% (w/v): 18.6
other: disintegrations per minute (DPM)
Remarks on result:
other: see Remark
Control group: 181 DPM/node Test substance concentration 5% (w/v): 3021 DPM/node Test substance concentration 10% (w/v): 2525 DPM/node Test substance concentration 25% (w/v): 3371 DPM/node Since the lymph nodes of the animals of a dose group were pooled, DPM/node was determined by dividing the measured value by the number of lymph nodes pooled.

Any other information on results incl. tables

- Viability/mortality: No death occurred during the study period.

- Clinical signs: Neither clinical / local signs nor other findings were observed in any animals of the control group or in the 5% and 10% test substance group. One day after the second topical application, a slight lid adhesion was observed at both ears in all mice of Group 4 (25 %), persisting for a total of two days.

- Body weights: The body weight of the animals, recorded prior to the first application and prior to necropsy, was within the range commonly recorded for animals of this strain and age.

- Size of the draining lymph nodes of the treated groups was 2 -5 fold larger compared to the control group.

Applicant's summary and conclusion

Interpretation of results:
Migrated information Criteria used for interpretation of results: EU
The test substance was found to be a sensitizer.
Executive summary:

In a GLP compliant Local Lymph Node Assay, tested according to OECD guideline 429, 4 female CBA mice per dose were treated daily with the test item at concentrations of 5, 10, and 25% in DMF by topical application to the dorsum of each ear lobe (left and right) for three consecutive days. 25% was the highest technically applicable concentration in the vehicle. A control group of four mice was treated with the vehicle 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 thereafter the mice were sacrificed and the draining auricular lymph nodes were excised and pooled per group. The proliferative capacity of pooled lymph node cells was determined by the incorporation of ³H-methyl thymidine. Stimulation Indices of 16.7, 13.9 and 18.6 were determined with the test item at concentrations of 5, 10, and 25% in DMF, respectively. Based on the described study and under the conditions reported, it is concluded that the test substance is a sensitizer.