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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:
From 04 December 2014 To 16 February 2015
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 442B (Skin Sensitization: Local Lymph Node Assay: BrdU-ELISA)
GLP compliance:
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Test material form:
solid: particulate/powder
Details on test material:
Test item: Disperse Yellow DYLA 1306

In vivo test system

Test animals

Details on test animals and environmental conditions:
- Source: Charles River France Laboratories, Domaine des Oncins B.P. 0109, F 69592 L’ARBRESLE CEDEX, France.
- Age at arrival: 7 to 8 weeks old
- Weight at arrival: 18.7 to 19.8 grams
- Housing: Polysulphone solid bottomed cages measuring 35.5 x 23.5 x 19 cm with nesting material
- Diet (e.g. ad libitum): Ad libitum 4 RF 21 (Mucedola S.r.l., Via G. Galilei, 4, 20019, Settimo Milanese (MI) Italy)
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: At least 5 days

- Temperature (°C): 22 °C+/-2 °C
- Humidity (%): 55%+/-15%
- Air changes (per hr): Approximately 15 to 20 air changes per hour
- Photoperiod (hrs dark / hrs light): Artificial (fluorescent tubes), daily light/dark cycle of 12/12 hours

IN-LIFE DATES: From: 28 January 2015 To: 16 February 2015

Study design: in vivo (LLNA)

acetone/olive oil (4:1 v/v)
25% 10% 5% (Test Item) 25% (Positive Control)
No. of animals per dose:
4 females
Details on study design:
- Irritation: Animals treated for three consecutive days (Days 1, 2, 3) with 25 µL/ear/day ofthe vehicle or test item formulations at 1, 2.5, 5, 10, 25%:
The treated sites of all animals were examined daily, ear thickness measured by a suitable micrometer on Day 1 (before dosing),
on Day 3 (before dosing) and on Day 6. After sacrifice, regularly shaped biopsies obtained from both ears and weighed together.
Main test:
- No. of exposures:3
- Test groups: 3 with test item, 1 with positive control
- Control groups: 2 (test item negative control and vehicle of positive control )
- Site: Ears, 25 µL/ear/day
- Frequency of applications: once daily
- Duration: 3 days
- Concentrations: 5%, 10%, 25% (Test Item); 25% (Positive control)
- Day 5: intraperitoneal injection of 0.5 mL/animal of a solution of BrdU at a concentration of 10 mg/mL in physiological saline
- Day 6 : Sacrifice, the auricular lymph nodes were excised, pooled on individual basis and individually collected in a solution of 2 % BSA-PBS [2 % bovine serum albumine (BSA) in phosphate buffered saline, PBS]. Cell suspensions were prepared for the evaluation of proliferation .
BrdU was measured by ELISA using a commercial kit (Roche Applied Science, Mannheim, Germany, Catalogue Number 11 647 229 001, batch no. 10493100).
Absorbance (OD) was detected at 450 nm (with reference wavelength: 690 nm).

The BrdU labelling index was calculated for each mouse and a group mean was subsequently calculated. Results for each treatment group were expressed as the mean Stimulation Index (SI). The SI was derived by dividing the mean BrdU labelling index/mouse within each test item group and the positive control groups by the mean labelling indices for the respective vehicle group.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Differences between each treated group and the concurrent negative control group (individual BrdU labelling indices) were assessed by Dunnett’s
test. The homogeneity of the data was verified by Bartlett’s test before Dunnett’s test. If data were found to be inhomogeneous a Modified t test (Cochran and Cox) was applied.

Results and discussion

Positive control results:
In the group treated with the positive control item, a Stimulation Index of 3.10 was calculated. As it was greater than 2, the study was regarded as

In vivo (LLNA)

Resultsopen allclose all
Test group / Remarks:
Test group / Remarks:
Test group / Remarks:

Any other information on results incl. tables

Preliminary phase:

Five concentrations (25, 10, 5, 2.5 and 1% w/w) of the test item were selectedto be used in the preliminary phase.

No signs of toxicity (clinical signs or toxicologically relevant body weight losses) were observed at any of the tested concentrations.

The evaluation of visible reactions showed no erythema at any of the concentrations investigated (25, 10, 5, 2.5 and 1% w/w).

The evaluation of ear thickness indicated that no increase was induced by treatment (values of Day 6 compared to Day 1).

The evaluation of ear punch weight indicated that no increase was observed in the treated animals, when compared to the animals treated with the vehicle.

Based on the results described above, although administered with difficulty due to the viscosity of the obtained formulation, the highest concentration selected for the main assay was 25% w/w.

Main assay:

No mortality and no clinical signs were recorded in animals treated at all dose levels investigated (25, 10 and 5% w/w). Red staining of the ears, due to the coloration of the test item, was noted in the animals treated with the test item during the observation period.

Changes in body weight observed during the study were within the expected range for this strain and age of animals.

No significant increase in cell proliferation of draining lymph nodes was observed in any treatment group. The calculated Stimulation Indices (SI) were 0.71, 0.76 and 0.59, respectively at low, mid- and high dose levels.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
No significant increase in cell proliferation of draining lymph nodes was observed in any treatment group. The calculated Stimulation Indices (SI) were 0.71, 0.76 and 0.59, respectively at low-, mid- and high-dose levels.
Executive summary:

Preliminary test

Five concentrations were tested in the preliminary test [25, 10, 5, 2.5 and 1% w/w in acetone:olive oil 4:1 (v/v)] in order to identify a non toxic and minimally irritant concentration and avoid false positive results. Concentration of 25% w/w was administered with difficulty due to the viscosity of the formulation. No signs of toxicity (significant clinical signs or body weight losses) were observed at the administered concentrations. According to the results obtained, the concentration of 25% w/w was judged to be not irritant.

Main assay

In the main assay, the test item was topically administered at the concentrations of 25, 10 and 5% w/w in acetone:olive oil 4:1 (v/v). No mortality nor signs of systemical toxicity were recorded in any animal. Body weight changes were considered not remarkable. No increase in cell proliferation of draining lymph nodes was observed in any treatment group. The calculated Stimulation Indices were 0.71, 0.76 and 0.59 at low, mid- and high dose levels, respectively.