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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:
04-March-2015 to 20-May-2015
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: 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
migrated information: powder
Details on test material:
Test item: Disperse Blue 1092

In vivo test system

Test animals

Details on test animals and environmental conditions:
Age and weight range at order: 7 to 8 weeks old, 21 to 25 grams
Breeder: Charles River France Laboratories, Iffa Credo, Domaine des Oncins B.P. 0109, F 69592 L’ARBRESLE CEDEX, France.
Weight range at arrival: 19 to 21 grams
Acclimatisation period: At least 5 days
Animals per cage:1/cage during the study; up to 5 during acclimatisation
Housing: Polysulphone solid bottomed cages measuring 35.5 \times 23.5 \times 19 cm with nesting material
Cage control: Daily inspected and changed as necessary (at least twice/week)
Water Drinking water supplied to each cage via a water bottle
Water supply Ad libitum
Diet 4 RF 21 (Mucedola S.r.l., Via G. Galilei, 4, 20019, Settimo Milanese (MI) Italy)
Diet supply Ad libitum throughout the study
Room lighting Artificial (fluorescent tubes), daily light/dark cycle of 12/12 hours
Air changes Approximately 15 to 20 air changes per hour
Temperature range 22 °C \pm 2 °C
Relative humidity range 55 % \pm 15 %

Study design: in vivo (non-LLNA)

Positive control substance(s):

Study design: in vivo (LLNA)

acetone/olive oil (4:1 v/v)
Test Item: 5%, 10%, 25%
Positive Control:25%
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.

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. Data were found to be inhomogeneous and 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 4.07 was calculated. As it was greater than 2, the study was regarded as valid.

In vivo (LLNA)

Resultsopen allclose all
Test group / Remarks:
Test group / Remarks:
Test group / Remarks:
other: disintegrations per minute (DPM)
Remarks on result:
other: BrdU Labelling index/group (OD, Optical Density): Group 1: 0.137 Group 2: 0.242 Group 3: 0.278 Group 4: 0.349 Group 5 Positive Control) : 0.349

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 selected to be used in the preliminary phase. No systemic signs of toxicity (clinical signs or toxicologically relevant body weight losses) were observed at any of the tested concentrations. The only abnormality observed was blue coloration of the treated site from Day 2 up to Day 4 in animals treated with the test item at the concentration of 25, 10, 5 and 1 %. This finding was due to the coloration of the substance. 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 Days 3 and 6 compared to Day 1). The evaluation of ear punch weight indicated that no significant increase was found at any dose level investigated. The increase observed at 10% concentration which was greater than 25% (35%) was considered to be incidental, as no other dose-related increased were seen. Based on the results described above, the highest concentration selected for the main assay was 25% w/w.

Main assay:

No mortality was recorded in animals treated at all dose levels investigated (25, 10 and 5% w/w). Blue coloration of the ears was detected from Day 2 up to Day Day 4 or 5, in animals from all treated groups. This finding was due to the coloration of the test item. Changes in body weight observed during the study were within the expected range for this strain and age of animals.

Dose-related increases in cell proliferation of draining lymph nodes were observed in all treated groups when compared to controls, achieving statistical significance only in the mid-dose group (the high dose group was not statistical significant since the values were inhomogeneus). The calculated Stimulation Indices (SI) were 1.77, 2.03 and 2.55 at low, mid- and high dose levels, respectively.

Applicant's summary and conclusion

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
The results obtained in this study indicate that the test item may elicit a sensitisation response in mice following dermal exposure, since in all dose groups the Stimulation Index was greater than 1.6. Therefore, the test item should be classified as a sensitiser
Executive summary:

Preliminary test:

Five concentrations were investigated 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. No signs of systemic toxicity (clinical signs or body weight losses) were observed at the tested concentrations.

According to the results of the irritation screening, the concentration of 25% w/w was judged to be not irritant. Blue coloration of the treated site was also noted. 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 was recorded in any animal. Body weight changes were considered not remarkable. Blue coloration of the ear was observed from Day 2 up to Day 4 or 5. Statistically significant and dose-related increases in cell proliferation of draining lymph nodes were observed in the three treatment groups, being the calculated Stimulation Indices (SI) 1.77, 2.03 and 2.55, respectively at low-, mid- and high-dose levels (5%, 10% and 25%).

The results obtained in this study indicate that the test item may elicit a sensitisation response in mice following dermal exposure, since in all dose groups the Stimulation Index was greater than 1.6. Therefore, the test item should be classified as a sensitiser.

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