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Description of key information

Topical application with 5, 25 and 50 % v/v test substance elicited a stimulation index (SI) of 2.13, 7.25 and 9.05, respectively in a local lymph node assay (OECD 429), thus the material is a sensitiser.

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
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)
Deviations:
yes
Remarks:
, none that affected the integrity of the study
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
other: CBA/Ca
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan UK
- Age at study initiation: 11 to 12 weeks
- Weight at study initiation: 20.7 to 24.5 grams
- Housing: housed individually in solid floor standard polysulfone cages (Size: Approximately L 360 x B 205 x H 140 mm), with stainless steel top grill
- Diet (e.g. ad libitum): Ssniff mice pellet food, ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 days before the start of treatment


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 23 deg C
- Humidity (%): 57 to 66 %
- Air changes (per hr): 14.2 air changes/hr
- Photoperiod (hrs dark / hrs light): 12 hrs light/ 12 hrs dark


IN-LIFE DATES: From: 2009-08-03 To: 2009-08-18
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
5, 25, 50, 75 % in vehicle and 100 % of the test substance
No. of animals per dose:
6
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: The test item was miscible at all the tested concentrations in Acetone:olive oil
- Irritation: erythema was observed at 75 and 100 % concentrations
- Lymph node proliferation response: screen looked at irritation only

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: randomly distributed to different groups by body weight stratification method.
- Criteria used to consider a positive response: Any test material that produces a SI , 3 in the LLNA is normally considered "positive" for dermal sensitisation potential

TREATMENT PREPARATION AND ADMINISTRATION:
The test substance was combined with Acetone Olive oil to obtain concentrations of 5, 25 and 50 % v/v. Test item solution was prepared daily just prior to dosing.
The application of the test item (25 µL/ear) was made on the dorsum of both ears in a manner to prevent test item loss. Six female
mice/group received vehicle (Acetone : Olive oil) or positive control (30 % α-hexylcinnamaldehyde) or 5, 25 and 50 % test substance once daily for three consecutive days. Ears were inspected prior to application on test item solution, and erythema was evaluated on days 2,3 and 6. All mice were weighed on days 1 and 6.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Means and SD were generated for body weight data (absolute and gain) and LLNA response (dpm and SI values). The body weight and dpm data
were analysed by one-way analysis of variance. When the differences are indicated by the ANOVA, a comparison of treated vs. control groups was done using a Dunnett's t-test (Ps0.05). Statistically significant differences (P≤ 0.05).
Positive control results:
30 % HCA in Acetone: olive oil elicited a stimulation index (SI) of 8.48, in comparison with the vehicle-treated mice
Key result
Parameter:
SI
Value:
2.13
Test group / Remarks:
5 % v/v test substance
Key result
Parameter:
SI
Value:
7.25
Test group / Remarks:
25 % v/v test substance
Key result
Parameter:
SI
Value:
9.05
Test group / Remarks:
50 % v/v test substance
Key result
Parameter:
EC3
Value:
8.4
Cellular proliferation data / Observations:
DPM/Mouse:
- Vehicle (AOO): 1000.50; 378.07
- 30 % HCA in AOO: 8482.50; 2857.66
- 5 % test substance in AOO: 2131.33; 568.45
- 25 % test substance in AOO: 7251.83; 1066.03
- 50 % test substance in AOO: 9054.67; 2509.62

The analysed radioactivity of 3H-TdR working solution was 83.72 uCi/mL against the nominal concentration of 80 uCi/mL.

Interpretation of results:
other: Classified as a sensitiser as Category 1B in accordance with EU criteria
Conclusions:
Topical application with 5, 25 and 50 % v/v test substance elicited a stimulation index (SI) of 2.13, 7.25 and 9.05, respectively. The calculated EC3 value was 8.40 %
Executive summary:

The Local Lymph Node Assay (LLNA) was conducted to evaluate the potential of the test substance to cause contact sensitization by measuring lymphocyte proliferative response from auricular lymph nodes following topical application of the test item to the female CBA/Ca mouse ear.

Screening Study: Three daily topical application of 5, 25, 50 and 75 % v/v in Acetone Olive oil and 100 % of the test substance were given to one animal at each dose level. Erythema was observed at 75 and 100 % concentration. The mice exposed to 50, 75 and 100 % concentration lost body weights. Results from this study were used to determine the dosing concentration in the LLNA study.

LLNA study: Six female CBA/Ca mice group received vehicle (Acetone Olive oil [AOO]) or 30 % α-hexylcinnarnaldehyde (HCA: positive control in AOO) or 5, 25 and 50 % of the test substance on days 1 to 3. On day 6, uptake of 3H-methyl thymidine into the auricular lymph nodes draining the site of test item application was measured five hours post administration. Proper conduct of the LLNA was confirmed via a positive response using 30 % α-hexylcinnamaldehyde, contact sensitiser, which elicited proliferation with a Stimulation Index (SI) value of 8.48, in comparison to vehicle-treated mice. The test substance did not elicit erythema. The test substance at the dose concentration of 50 % v/v decreased the body weight gain. The SI values for 5, 25 and 50 % of test substance were 2.13, 7.25 and 9.05, respectively. The EC3 value was 8.40 %.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

A local lymph node assay is available for the test susbtance. The assay was conducted to evaluate the potential of the test substance to cause contact sensitisation by measuring lymphocyte proliferative response from auricular lymph nodes following topical application of the test item to the female CBA/Ca mouse ear.

The test substance did not elicit erythema. The test item at the dose concentration of 50 % v/v decreased the body weight gain. The SI values for 5, 25 and 50 % of test substance were 2.13, 7.25 and 9.05, respectively. The EC3 value was 8.40 %.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance requires classification with respect to skin sensitsation as Category 1B ( H317: May cause an allergic skin reaction).