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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
07-04-2015 to 21-04-2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
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

Constituent 1
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): Acid Red EAY 9656

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, 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: 18 to 19 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:
Test Item: 50% 25% 10%

Positive Control: 25%
No. of animals per dose:
4 females
Details on study design:
RANGE FINDING TESTS:
- Irritation: Animals treated for three consecutive days (Days 1, 2, 3) with 25 µL/ear/day ofthe vehicle or test item formulations at 50, 25, 10, 5 and
2.5%:
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 STUDY
- 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: 10%, 25% and 50%(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 rispective vehicle group.




























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:
In the group treated with the positive control item, a Stimulation Index of 5.27 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: see Remark
Remarks:
An increase in cell proliferation of draining lymph nodes, statistically significant at the 2 higher concentrations, was observed in all dose groups, with a Stimulation Index of 1.86, 2.11 and 2.07 in low, medium and high dose (10 %, 25 % and 50 %) respectively.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: BrdU Labelling index/group (OD, Optical Density): Group 1: 0.132 Group 2: 0.245 Group 3: 0.278 Group 4: 0.273 Group 5 (Positive Control): 0.694

Any other information on results incl. tables

Preliminary test:

Five concentrations (50, 25, 10, 5 and 2.5% w/w) of the test item were selected to 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 only finding, observed in two animals on Day 3, was slight red coloration of the ears. This finding was also noted on Day 4 and 5 but only in 1 animal. This sign was due to the coloration of the test item. The evaluation of visible reactions showed no erythema at any of the concentrations investigated (50, 25, 10, 5 and 2.5% 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 found at any dose level investigated. Based on the results described above, the highest concentration selected for the main assay was 50% w/w.

Main assay:

No mortality or clinical signs were recorded in animals treated at all dose levels investigated (50, 25 and 10% w/w). Red coloration of the treated site was observed from Day 2 up to Day 4 in all animals of medium and high dose groups. This discoloration 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.

An increase in cell proliferation of draining lymph nodes, statistically significant at the 2 higher concentrations, was observed in all dose groups, with a Stimulation Index of 1.86, 2.11 and 2.07 in low, medium and high dose (10 %, 25% and 50 %) respectively.

Applicant's summary and conclusion

Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Remarks:
Migrated information
Conclusions:
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:

Five concentrations [50, 25, 10, 5 and 2.5 % w/w in acetone:olive oil 4:1 (v/v)] were tested in the preliminary phase, in order to identify a non toxic and minimally irritant concentration and avoid false positive results. No signs of toxicity (significant clinical signs or body weight losses) were observed at the tested concentrations. According to the results of the irritation screening, the concentration of 50 % w/w was judged to be not irritant. In the main assay, the test item was topically administered at the concentrations of 50, 25 and 10 % w/w, in acetone:olive oil 4:1 (v/v). No mortality or clinical signs were recorded in any animal. Changes in body weight observed during the study were within the expected range for this strain and age of animals. An increase in cell proliferation of draining lymph nodes, statistically significant at the 2 higher concentrations, was observed in the all dose group, with a Stimulation Index of 1.86, 2.11 and 2.07 in low, medium and high dose (10 %, 25 % and 50 %) respectively. 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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