Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 - 25 February 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study has been performed according to OECD and EC guidelines and according to GLP principles.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2008

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymphnode assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Aquapel® 203
- Substance type: yellow fatty liquid
- Physical state: liquid
- Analytical purity: 89.9%
- Lot/batch No.: G12FY067
- Expiration date of the lot/batch: 12 June 2008
- Stability under storage conditions: stable
- Storage condition of test material: at room temperature in the dark

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, L’Arbresle Cedex, France
- Age at study initiation: approx. 9 weeks old
- Weight at study initiation: 20-24 g
- Housing: Individual housing in labeled Macrolon cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.4 - 23.7
- Humidity (%): 40 - 67
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12


Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
0, 10, 25 and 50%
No. of animals per dose:
5
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: The test substance formulations (w/w) were prepared within 4 hours prior to each treatment. No adjustment was made for specific gravity of the vehicle. Homogeneity was obtained to visually acceptable levels.
- Irritation: well defined or severe erythema at 50 and 100% concentration. Based on the results, the highest test substance concentration selected for the main study was a 50% concentration.


MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA
- Criteria used to consider a positive response:
DPM values are presented for each animal and for each dose group. A Stimulation Index (SI) is calculated for each group. The SI is the ratio of the DPM/group compared to DPM/vehicle control group.
If the results indicate a SI >= 3, the test substance may be regarded as a skin sensitiser, based on the test guideline and recommendations done by ICCVAM.


TREATMENT PREPARATION AND ADMINISTRATION:
Induction - Days 1, 2 and 3
The dorsal surface of both ears was epidermally treated (25 μl/ear) with the test substance concentration, at approximately the same time per day. The concentrations were mixed thoroughly using a vortex mixer immediately prior to dosing.
The control animals were treated the same as the experimental animals, except that, instead of the test substance, the vehicle alone was administered.

Treatment - Day 6
All animals:
Each animal was injected via the tail vein with 0.25 mL of sterile phosphate buffered saline (PBS) containing 20 μCi of 3H-methyl thymidine.
After approximately five hours, all animals were killed by intraperitoneal injection with pentobarbital Euthesate® (0.2 ml/animal). The draining (auricular) lymph node of each ear was excised.
The relative size of the nodes (as compared to normal) was estimated by visual examination and abnormalities of the nodes and surrounding area were recorded. The nodes were pooled for each animal in approximately 3 mL PBS.

Tissue processing for radioactivity - Day 6
A single cell suspension of lymph node cells (LNC) was prepared in PBS by gentle separation through stainless steel gauze (diameter 125 μm). LNC were washed twice with an excess of PBS by centrifugation at 200g for 10 minutes at 4º C. To precipitate the DNA, the LNC were exposed to 5% trichloroacetic acid (TCA) at 4º C during the night.

Radioactivity measurements - Day 7
Precipitates were recovered by centrifugation, resuspended in 1 mL TCA and transferred to 10 mL of Ultima Gold cocktail as the scintillation fluid. Radioactive measurements were performed using a Packard scintillation counter (2800TR). Counting time was to a statistical precision of ± 0.2% or a maximum of 5 minutes whichever comes first. The scintillation counter was programmed to automatically subtract background and convert Counts Per Minute (CPM) to Disintegrations Per Minute (DPM).
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
The EC3 value (the estimated test substance concentration that will give a SI =3) was determined, using linear interpolation .

Results and discussion

Positive control results:
The SI values calculated for the substance concentrations 5, 10 and 25% were 1.0, 2.0 and 5.7 respectively. An EC3 value of 14.1% was calculated using linear interpolation.

The calculated EC3 value was found to be in the acceptable range of 2 and 20%. The results of the 6 monthly HCA reliability checks of the recent years were 10.3, 9.5, 13.1 and 15.6%.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: The SI values calculated for the substance concentrations 10, 25 and 50% were 5.6, 13.0 and 11.0 respectively. These results show that the test substance elicits an SI >= 3. The EC3 value was established to be between 0 and 10%.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Mean DPM/animal values for the experimental groups treated with test substance concentrations 10, 25 and 50% were 2916, 6700 and 5652 respectively. The mean DPM/animal value for the vehicle control group was 516.

Any other information on results incl. tables

These results indicate that the test substance could elicit a SI>=3. These data did not show a clear dose-response and no reliable EC3 value could be calculated. It was possible to strengthen the outcome of the study by adding lower concentrations. Since the SI values clearly exceeded 3 and since extension of the study would not alter the classification, this was considered not appropriate for ethical reasons.

Applicant's summary and conclusion

Interpretation of results:
sensitising
Remarks:
Migrated information
Conclusions:
Based on the results:
- according to the recommendations made in the test guidelines, Aquapel® 203 would be regarded as skin sensitizer.
- according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations (2004), Aquapel® 203 should be classified as skin sensitizer (Category 1).
- according to the EC criteria for classification and labeling requirements for dangerous substances and preparations (Council Directive 67/548/EEC), Aquapel® 203 should be labeled as: may cause sensitization by skin contact (R 43).