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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions

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
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
- Source: Harlan Laboratories B.V. Postbus 6174 5960 AD Horst / The Netherlands
- Age at study initiation: 10-12 weeks
- Weight at study initiation: 21-22g
- Housing: groups of five
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 5d
- Fasting: 16h before administration

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2
- Humidity (%): 45-65
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:
dimethyl sulphoxide
Concentration:
2.5, 5 and 10%
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 could be technically used was a 25 % solution 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. vortexing, sonicating, warming to 37°C).
- Irritation: Two mice were treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 10 and 25% (w/w) once daily each on three consecutive days. At the tested concentrations the animals did not show any signs of systemic toxicity. On day 6, the animal treated with 25% test item concentration showed an increase in ear thickness of 30% compared to pre-treatment value.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node assay
- Criteria used to consider a positive response:
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:
The test item in the main study was assayed at 2.5, 5, and 10% (w/w) in DMSO. The different test item concentrations were prepared serially. The preparations were made freshly before each dosing occasion.

Topical Application
Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear with different test item concentrations of 2.5, 5, and 10% (w/w) in dimethylsulfoxide. The application volume, 25 ul/ear/day, was spread over the entire dorsal surface ( ~8 mm) of each ear once daily for three consecutive days. A further group of mice was treated with an equivalent volume of the relevant vehicle alone (control animals).

Administration of 3H-Methyl Thymidine
3H-methyl thymidine (3HTdR) was purchased from Hartmann Analytic, 38124 Braunschweig, Germany (specific activity, 2 Ci/mmol; concentration, 1 mCi/mL). Five days after the first topical application (day 6) 250 WL of phosphate-buffered saline (PBS) containing 19.5 WCi of 3HTdR (equivalent to 3HTdR 78 WCi/mL) were injected into each test and control mouse via the tail vein.
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). The Dean-Dixon-Test was used for identification of possible outliers (performed with Microsoft Excel 2003).
However, both biological and statistical significance were considered together.

Results and discussion

Positive control results:
substance: \-Hexylcinnamaldehyde
Vehicle: acetone:olive oil (4+1 v/v)
concentration: 0, 5, 10, 25%
DPM/lymph node: 382.2; 224.6; 577.4; 1424.4
S.I.: 1; 0.88; 1.51; 3.73

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Value:
1
Test group / Remarks:
vehicle
Parameter:
SI
Value:
1.67
Test group / Remarks:
2.5 % group
Parameter:
SI
Value:
1.16
Test group / Remarks:
5 % group
Parameter:
SI
Value:
1.95
Test group / Remarks:
10 % group
Parameter:
other: disintegrations per minute (DPM)
Value:
826.1
Test group / Remarks:
vehicle group
Parameter:
other: disintegrations per minute (DPM)
Value:
1 379.3
Test group / Remarks:
2.5 % group
Parameter:
other: disintegrations per minute (DPM)
Value:
959.9
Test group / Remarks:
5 % group
Parameter:
other: disintegrations per minute (DPM)
Value:
1 607.1
Test group / Remarks:
10 % group

Any other information on results incl. tables

Viability / Mortality

No deaths occurred during the study period.

Clinical Signs

No symptoms of local toxicity at the ears of the animals and no systemic findings were observed during the study period. Eventual erythema of the ear skin could not be evaluated due to the colour of the test item.

Body Weights

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:
GHS criteria not met