Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2011-2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and OECD 429 compliant study with well characterized test material

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Substance type: orange-red powder
- Physical state: solid
- Analytical purity: 97.7 area%
- Purity test date: 2011
- Lot/batch No.: 10-0189
- Expiration date of the lot/batch: Unlimited stability
- Stability under test conditions: stable
- Storage condition of test material: Room temperature
-

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River UK
- Age at study initiation: pre-test: 9-10 weeks (beginning of treatment), main study: 8-9 weeks (beginning of treatment)
- Weight at study initiation: 14.3 - 20.4 g
- Housing: group
- Diet: Pelleted standard diet, ad libitum
- Water: ad libitum
- Acclimation period: at least five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 + 2°C
- Humidity (%): 45-108%
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2011-08-24 To: 2011-09-14

Study design: in vivo (LLNA)

Vehicle:
dimethyl sulphoxide
Concentration:
2.5, 5%, and 10% (w/w).
No. of animals per dose:
5
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: A solubility experiment was performed according to the recommendations given by OECD 429. The highest test item concentration, which can be technically used was a 10% (w/w) suspension in dimethylsulfoxide. At higher concentrations, an applicable formulation of the test item was not achieved, neither by the use of other vehicles nor by using additional methods to formulate the test item (e.g. grinding of the test item in a mortar, vortexing, sonicating, warming to 37°C).


MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA
- Criteria used to consider a positive response: A test item is regarded as a sensitiser in the LLNA if the following criteria are fulfilled:
•First, that exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the Stimulation Index.
•Second, that the data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.


TREATMENT PREPARATION AND ADMINISTRATION: Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear with test item concentrations of 2.5, 5 and 10% (w/w) in dimethylsulfoxide. The application volume, 25 µL/ear/day, was spread over the entire dorsal surface (diameter ca 8 mm) of each ear once daily for three consecutive days. A further group of mice (control animals) was treated with an equivalent volume of the relevant vehicle alone.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
The mean values and standard deviations were calculated in the body weight tables and for the DPM values (group mean DPM ± standard deviation) as well as for the ear weights, lymph node weights and lymph node cell counts.
A statistical analysis was conducted on the DPM values, the ear weights, the lymph node weights and the lymph node cell count to assess whether the difference was statistically significant between test item groups and negative control group.
For all statistical calculations SigmaStat for Windows (Version 2.0) was used. A One-Way-Analysis-of-Variance was used as statistical method. In case of significant results of the One-Way-ANOVA, multiple comparisons were performed with the Dunnett test. Statistical significance was set at the five per cent level (p < 0.05). The Dean-Dixon-Test and Grubb’s test were used for identification of possible outliers (performed with Microsoft Excel 2003).
However, both biological and statistical significance were considered together.

Results and discussion

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: The EC3 value could not be calculated, since all S.I.s are below the threshold value of 3. For details see table below.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see table

Any other information on results incl. tables

Test item concentration
% (w/w)

Group Calculation

Mean DPM
  per animala)

SDb)

S.I.

Vehicle
(dimethylsulfoxide)

968.1

490.1

1.00

2.5 %
test substance

1083.5

117.9

1.12

5 %
test substance

1011.9

294.2

1.05

10 %
test substance

1091.9

373.1

1.13

a) Mean DPM/animal was determined by dividing the sum of the measured values from lymph nodes of all animals within a group by the number of animals in that group (5 animals per group)

b) SD = Standard Deviation

No deaths occurred during the study period.

No symptoms of local toxicity at the ears of the animals and no systemic findings were observed during the study period. An erythema of the ear skin could not be determined due to intense coloration of the ear skin caused by the test item. The body weight of the animals, recorded prior to the first application and prior to treatment with 3HTdR, was within the range commonly recorded for animals of this strain and age.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information
Executive summary:

The substance did not cause sensitization in the LLNA at doses of 2.5, 5%, and 10% (w/w).