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Description of key information

The key skin sensitisation study, conducted according to an appropriate OECD test guideline (local lymph node assay) and in compliance with GLP, reported the submission substance to be not sensitising (RCC, 2004).

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21 April 2004 to 05 May 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
2002
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature, protected from light
- Stability under test conditions: stable under test conditions
- Solubility and stability of the test substance in the solvent/vehicle: not applicable
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not applicable

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was placed in a volumetric flask on a tared Mettler balance and the vehicle was quantitatively added. The weight/volume dilutions were prepared individually using a magnetic stirrer as homogeniser. The test formulations were made freshly before each dosing occasion and no more than 4 hours prior to application to the ears.
- Preliminary purification step (if any): none
- Final dilution of a dissolved solid, stock liquid or gel: not applicable
- Final preparation of a solid: not applicable
Species:
mouse
Strain:
CBA/Ca
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan, the Netherlands
- Females (if applicable) nulliparous and non-pregnant: yes
- Microbiological status of animals, when known: not specified
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 16-24 g
- Housing: Individually in Makrolon type-2 cages with standard softwood bedding
- Diet: pelleted standard mouse maintenance diet, ad libitum
- Water: community tap water, ad libitum
- Acclimation period: Under test conditions after health examination. Only animals without any visible signs of illness were used for the study.
- Indication of any skin lesions: none reported

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22+/-3°C
- Humidity (%): 30-70%
- Air changes (per hour): 10-15
- Photoperiod (hours dark / hours light): 12/12
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
25 and 50% in acetone/olive oil, 4:1 (v/v) and 100% (undiluted)
No. of animals per dose:
4
Details on study design:
PRE-SCREEN TESTS:
- Compound solubility: yes. A non-GLP solubility test was performed where a suitable vehicle was chosen.
- Irritation: yes. A non-GLP pre-test was performed in two mice. The test item was tested at four different concentrations 10, 25, 50 in acetone/olive oil and 100%. It was determined that 100% was the highest technically applicable concentration whilst avoiding systemic toxicity.
- Systemic toxicity: yes
- Ear thickness measurements: no
- Erythema scores: no

MAIN STUDY
Each test group was treated by topic application to the dorsal surface of each ear lobe with different test item concentrations of 25, 50 % in acetone/olive oil and 100% undiluted. The application volume was 25 µl. It was spread over the entire dorsal surface of each ear lobe once daily for three consecutive days. A control group was treated with the vehicle only. A hair dryer was passed briefly over the ear's surface to prevent the loss of any of the test item applied.
250µl of 81.5 µCi/ml 3H-Methyl-thymidine was administered to all animals by intravenous injection via a tail vein five days after the first topical application.

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Skin Sensitisation: Local Lymph Node Assay
- Criteria used to consider a positive response: First, 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, the data are compatible with a conventional dose response, although allowance must be made for either local toxicity or immunological suppression.

TREATMENT PREPARATION AND ADMINISTRATION: Dose formulations were prepared at least 4 hours prior to application. The test material was applied topically to the dorsal surface of each ear.
Statistics:
Not used
Key result
Parameter:
SI
Value:
1
Test group / Remarks:
25% in acetone/olive oil
Key result
Parameter:
SI
Value:
2
Test group / Remarks:
50% in acetone/olive oil
Key result
Parameter:
SI
Value:
2.4
Test group / Remarks:
100%
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA: Background 3HTdR levels were measured in two 1ml-aliquots of 5% trichloroacetic acid. The β-scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute. The proliferative capacity of pooled lymph node cells was determined by the incorporation of 3HTdRmeasured on a β-scintillation counter.

DETAILS ON STIMULATION INDEX CALCULATION: Not specified

EC3 CALCULATION: Calculation of EC3 value was not done because no test concentration produced a stimulation index of 3 or higher.

CLINICAL OBSERVATIONS: No deaths occurred during the study. No clinical signs were observed in any of the animals of the control group or 25% treatment group. On the second application day, a slight ear swelling was observed at both dosing sites in all mice of 100% group, persisted for a total of four days. On the third application day, a slight ear erythema was observed at both dosing sites in all mice of 50% and 100% groups, persisting for a total of 3 days.

BODY WEIGHTS: The recorded body weights of the animals were within the range commonly recorded for animals of this strain and age.
Interpretation of results:
GHS criteria not met
Conclusions:
The Local Lymph Node Assay, conducted according to an appropriate OECD test guideline and in compliance with GLP, reported the test substance to be not a skin sensitiser.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

The key skin sensitisation study, conducted according to an appropriate OECD test guideline (local lymph node assay) and in compliance with GLP, reported the submission substance to be not sensitising (RCC, 2004).

Test substance (25%, 50%) in acetone/olive oil and 100 % undiluted test substance was applied topically to the dorsal area of the ears of 4 female mice per dose for three consecutive days. A control group of four mice was treated the same way but with vehicle only. Five days after the first topical application the mice were injected intravenously into a tail vein with radiolabelled thymidine (3H-methyl thymidine).

Approximately, five hours after injection the mice were sacrificed. The draining auricular lymph nodes excised and pooled per group. The proliferative capacity of pooled lymph node cells was determined by the incorporation of 3H-methyl thymidine measured in a β scintillation counter.

No deaths occurred during the study. No clinical signs were observed in any of the animals of the control group or 25% treatment group. On the second application day, a slight ear swelling was observed at both dosing sites in all mice of 100% group, persisted for a total of four days. On the third application day, a slight ear erythema was observed at both dosing sites in all mice of 50% and 100% groups, persisting for a total of 3 days.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the available data for the submission substance, no classification is required for sensitisation according to Regulation (EC) No 1272/2008.