Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26-02-2015 to 19-05-2015
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 442B (Skin Sensitization: Local Lymph Node Assay: BrdU-ELISA)
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
Test item Disperse Blue CVG

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
Age and weight range at order: 7 to 8 weeks old, 24 to 28 grams
Breeder: Charles River France Laboratories, Iffa Credo, Domaine des Oncins B.P. 0109, F 69592 L’ARBRESLE CEDEX, France.
Weight range at arrival: 20 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):
yes
Remarks:
alpha-hexylcinnamaldehyde

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
5%, 10%, 25% (Test Item); 25% (Positive Control)
No. of animals per dose:
4 females
Details on study design:
RANGE FINDING TEST:
- 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
Statistics:
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) s applied.

Results and discussion

Positive control results:
Stimulation Index of 2.47 was calculated. As it was greater than 2, the study was regarded as valid.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: No increase in cell proliferation of draining lymph nodes was observed in any treatment group. The calculated Stimulation Indices (SI) were 0.82, 0.78 and 0.98, respectively at low, mid- and high dose levels.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: BrdU Labelling index/group (OD, Optical Density) Group 1: 0.239 Group 2: 0.197 Group 3: 0.187 Group 4: 0.234 Group 5 (Positive Control): 0.590

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 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). An increase of ear punch weight was noted in animals treated with the test item at dose levels of 25 and 10 %. This increase was not dose-related and therefore did not affect the choice of dose levels.

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 in mid- and high dose animals from Day 3 up to Day 4 or 6. 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.

No increase in cell proliferation of draining lymph nodes was observed in any treatment group. The calculated Stimulation Indices (SI) were 0.82, 0.78 and 0.98, respectively at low, mid- and high dose levels.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Remarks:
Migrated information
Conclusions:
No increase in cell proliferation of draining lymph nodes was observed in any treatment group. The calculated Stimulation Indices were 0.82, 0.78 and 0.98 at low, mid- and high dose levels, respectively.
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 significant 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.

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 3 up to Day 6.

No increase in cell proliferation of draining lymph nodes was observed in any treatment group. The calculated Stimulation Indices (SI) were 0.82, 0.78 and 0.98 at low, mid- and high dose levels, respectively.