Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
01 - 23 August 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well conducted and well described study in accordance with GLP and OECD Guideline 429 without any deviation.
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

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
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
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:
- Name of test material: MEXORYL SCK
- Physical state: Beige powder
- Analytical purity: saponines content (determined by HPLC assay) 57.0 % w/w
- Lot/batch No.: R0069579A 008 X 001
- Expiration date of the lot/batch: September 2012
- Storage condition of test material: At room temperature away from light

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/J
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Animal breeding facility, JRF.
- Age at study initiation: 9-10 weeks
- Weight at study initiation: 18-23.5 g
- Housing: Animals were housed individually in solid floor polypropylene cages. On Day 5, post administration of the radiolabelled material, the animals were transferred to the metal metabolic cages.
- Diet: Teklad certified Global High Fiber Rat/Mice feed (manufactured by Harlan, Nederland), ad libitum
- Water: UV sterilised water (Reverse Osmosis water filtration system), ad libitum
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19-22 °C
- Humidity: 64-65 %
- Photoperiod: 12 h dark / 12 h light

Study design: in vivo (LLNA)

Vehicle:
other: 1 % pluronic L-92
Concentration:
Preliminary study: 5, 10, 25 and 50 % (w/v) in 1 % pluronic L-92
Main study: 2.5, 5, 10, 25 and 50 % (w/v) in 1 % pluronic L-92
No. of animals per dose:
Preliminary study: 2 females/dose
Main study: 4 females/dose
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: Test item was found to be soluble in 1 % pluronic L-92 and homogenous solution was obtained up to the maximum concentration of 50 % (w/v)
- Irritation: No sign of toxicity at the concentrations of 5, 10, 25 and 50 % (w/v). There was no noteworthy increase observed in ear thickness measurement. Therefore, the concentrations selected for the main study were 2.5, 5, 10, 25 and 50 % (w/v).

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Individual approach, using tritiated (3H)-methyl thymidine, according to the OECD 429 test guideline.
- Additional investigation: Measurement of ear thickness - Ear thickness of individual animal was measured using a digital micrometer on the day of the commencement (Day 0) and on Days 1, 2 and 5, altogether with recording of local reactions if any before each application. Group mean ear thickness was calculated.
- Criteria used to consider a positive response: Test item was regarded as skin sensitizer when the Stimulation Index (SI) for a dose group is ≥ 3 together with consideration of a dose-response relationship.

TREATMENT PREPARATION AND ADMINISTRATION:
- Fresh dose solutions were prepared every day prior to application. Test item dosage forms were used within 1 hour after preparation.
- 25 µL of test material was applied to the dorsum surface of each ear of each mouse for three consecutive days (Days 0, 1 and 2). On day 5 an injection of 250 µL phosphate buffered saline (PBS) containing 20 µCi of 3H-methyl thymidine (3H-TdR) or 250 µl PBS containing 2 µCi of (3H)-methyl thymidine was made into the tail vein of each experimental mouse. Five hours later, the draining Auricular lymph node of each ear was excised into PBS. A single cell suspension of lymph node cells was prepared from each mouse. Cells were precipitated with 5% trichloroacetic acid at 4 ± 1 °C for 18 hours. Then precipitate was transferred to a scintillation vials with 10 mL scintillation fluid, vials were loaded into a β- scintillation counter and after approximately 30 minutes 3H-TdR incorporation was measured as disintegrations per minute (DPM) for each group and expressed DPM/group and DPM/node.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Statistical analysis was carried for the assessment of the dose response relationship and pair wise comparison between the treatment and vehicle control group. The body weight data was subjected to Bartlett's test to meet the homogeneity of variance before conducting ANOVA followed by Dunnett's t test. Where the data do not meet the homogeneity of variance Student's t test was performed to calculate significance.

Results and discussion

Positive control results:
Stimulation index for positive control group treated with 25 % (v/v) hexyl cinnamic aldehyde was found to be 3.77 and classified as skin sensitizer.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: Stimulation index for 2.5, 5, 10, 25 and 50 % (w/v) were 1.33, 1.78, 2.02, 2.13 and 3.54, respectively. EC3 value calculated for the test item was found to be 40.43 %.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: DPM/group for vehicle, 2.5, 5, 10, 25 and 50 % (w/v) were 2477.87, 3290.39, 4402.91, 5006.32, 5289.28 and 8782.63, respectively.

Any other information on results incl. tables

None

Applicant's summary and conclusion

Interpretation of results:
sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Under the test conditions, test item, MEXORYL SCK is classified as a skin sensitiser according to the annex VI of the Regulation EC No. 1272/2008 (CLP) and of the Directive 67/548/EEC.
Executive summary:
In a local lymph node assay performed according to OECD Guideline 429 and in compliance with GLP, groups of CBA/J mice (4 females/dose) were treated with test item, MEXORYL SCK at concentrations of 2.5, 5, 10, 25 and 50 % (w/v) in 1 % pluronic L-92 to the dorsum of ears (25 µL/ear) on three consecutive days. Four vehicle control animals were treated with vehicle alone (1 % pluronic L-92) and four positive controls were treated with Hexylcinnamaldehyde (25 %). Three days after the last exposure, all animals were injected with 3H–methyl thymidine and after five hours the draining (auricular) lymph nodes were excised. After precipitating the DNA of the lymph node cells, radioactivity measurements were performed. The activity was expressed as the number of Disintegrations Per Minute (DPM) and a stimulation index (SI) was subsequently calculated for each group. Animals were observed for mortality, clinical signs and body weight during the study. The test concentrations for the main study were determined from a preliminary irritation study at 5, 10, 25 and 50 % (w/v) in 1 % pluronic L-92 using two females/dose. 

 

No mortality and no clinical signs were observed during the observation period. Mean body weights of treatment group animals was comparable to that of the vehicle control. There was no noteworthy increase observed in ear thickness measurement on Days 0, 1, 2 and 5 in all groups. DPM/group for vehicle, 2.5, 5, 10, 25 and 50 % (w/v) were 2477.87, 3290.39, 4402.91, 5006.32, 5289.28 and 8782.63, respectively. Stimulation index for 2.5, 5, 10, 25 and 50 % (w/v) were 1.33, 1.78, 2.02, 2.13 and 3.54, respectively. EC3 value calculated for the test item was found to be 40.43 %. Stimulation index for positive control group treated with 25 % (v/v) hexyl cinnamic aldehyde was found to be 3.77 and classified as skin sensitizer; this experiment was therefore considered valid. Since Stimulation index obtained for 50 % (w/v) tested dose concentrations of test item revealed three-fold increase over the vehicle control, associated with dose-response relationship, the test item may be categorized as a weak sensitizer.

 

Under the test conditions, test item, MEXORYL SCK is classified as a skin sensitiser according to the annex VI of the Regulation EC No. 1272/2008 (CLP) and of the Directive 67/548/EEC.