Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
June 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

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:
Test item: Disperse Red DYGJ 0702
Chemical name: ethyl 3-{6-[2-(2-cyano-4-nitrophenyl)diazen-1-yl]-7-hydroxy-4-oxo-1,2,3,4-tetrahydroquinolin-1-yl}propanoate
CAS no.: 167652-69-5
Batch No.: 20150119
Content: 70.6% (w/w)
Empirical formula: C21H19N5O6
Molecular weigh: 437.41 g/mol
Physical state: Red powder
Description at first use: Red powder
Certificate No.: PS 20150081
Storage conditions: Room temperature
Expiry date: January 2020

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- 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 to 20 grams
- Housing: Polysulphone solid bottomed cages measuring 35.5 x 23.5 x 19 cm with nesting material
- Animals per cage: 1/cage during the study; up to 5 during acclimatisation
- Diet (e.g. ad libitum): ad libitum4 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

ENVIRONMENTAL CONDITIONS
- 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: 10-22 June 2015

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
25, 10 and 5% w/w (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 25, 10, 5, 2.5 and 1% w/w
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: 1
- Site: Ears, 25 µL/ear/day
- Frequency of applications: once daily
- Duration: 3 days
- Concentrations: 25, 10 and 5% w/w (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. 10872700).
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)
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. 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 3.68 was calculated. As it was greater than 2, the study was regarded as
valid.

In vivo (LLNA)

Results
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 were 1.23, 1.32 and 1.17 at low, mid- and high dose levels, respectively.

Any other information on results incl. tables

Preliminary test

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 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 the 25% and 10% concentrations were administered with difficulty, due to the viscosity of the obtained formulations, the concentrations selected for the main assay were 25%, 10% and 5% w/w. 5.2

Main Assay

In vivo phase

Neither mortality nor clinical signs were recorded in animals treated at all dose levels investigated (25, 10 and 5% w/w). Changes in body weight observed during the study were within the expected range for this strain and age of animals. 5.3

Evaluation of cell proliferation

No significant increase in cell proliferation of draining lymph nodes was observed in any treatment group. The calculated stimulation indices (SI) were 1.23, 1.32 and 1.17, respectively at low, mid- and high dose levels.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
No increase in cell proliferation of draining lymph nodes was observed in any treatment group. The calculated Stimulation Indices were 1.23, 1.32 and 1.17 at low, mid- and high dose levels, respectively
Executive summary:

Preliminary phase

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. Concentrations of 25% and 10% w/w were 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 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.

No increase in cell proliferation of draining lymph nodes was observed in any treatment group. The calculated Stimulation Indices were 1.23, 1.32 and 1.17 at low, medium and high dose levels, respectively.