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Diss Factsheets

Administrative data

Description of key information

LLNA: sensitizing

Key value for chemical safety assessment

Skin sensitisation

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

The skin sensitizing potential of EEIM Acetate was assessed using the radioactive Murine Local Lymph Node Assay. The assay simulates the induction phase for skin sensitization in mice. It determines the response of cells in the auricular lymph nodes to repeated application of the test substance to the dorsal skin of the ears.
Groups of 5 female CBA/J mice each were treated with 10%, 25% and 50% w/w preparations of the test substance in 70% ethanol or with the vehicle alone. The high concentration was selected based on the presence of substance related mortality in a pre-test using the undiluted test substance.
The study used 3 test groups and 1 control group. Each test animal was treated with 25 µL per ear of the appropriate test-substance preparation, applied to the dorsal surfaces of both ears for three consecutive days. The control group was treated with 25 µL per ear of the vehicle alone.
Three days after the last application the mice were injected into the tail vein with 20 µCi of 3H-thymidine in 250 µL of sterile saline. About 5 hours after the 3H-thymidine injection, the mice were sacrificed and the auricular lymph nodes were removed. Lymph node response was evaluated by measuring 3H-thymidine incorporation (indicator of cell proliferation). Cell counts and weights of each animal’s pooled lymph nodes were also determined. In addition,a 0.8 cm diameter sample was punched out of the apical part of each ear and for each animal the weight of the pooled punches was determined in order to obtain an indication of possible skin irritation.No signs of systemic toxicity were noticed during general observation.
When applied as 25% and 50% preparations in 70% ethanol, the test substance induced a biologically relevant (increase above the cut off Stimulation Index of 3) and statistically significant increase of 3H-thymidine incorporation into the cells from the auricular lymph nodes. Concomitantly, the increase in the auricular lymph node cell counts was biologically relevant
(increase to 1.5 fold or above of control value = stimulation index (SI) ≥ 1.5) and statistically significant at these concentrations.
There was a relevant and statistically significant increase in lymph node weights at these concentrations, as well. In addition, a statistically significant increase in lymph node weights was noted at 10%.
The 10% and 25% test-substance preparations caused some and the 50% preparation a considerable increase in ear weights as indication of ear skin irritation. The increases were statistically significant at all concentrations. Compound residues were noted on the ear skin of the animals treated with the 50% concentration on day 5.
Because the lymph node response cannot be fully attributed to the ear skin irritation observed, it is concluded that EEIM Acetate exhibits a skin sensitizing potential in the Murine Local Lymph Node Assay under the test conditions chosen.
The threshold concentration for sensitization induction was >10% <25%. The EC 3 (estimated concentration that leads to the SI of 3.0) for 3 H-thymidine incorporation and the EC 1.5 (estimated concentration that leads to the SI of 1.5) for cell count was calculated by linear regression from the results of these concentrations to be 19.2% and 20.2%, respectively.

Justification for classification or non-classification

Based on the results of the skin sensitization testing, the test item is classified as skin sensitization cat. 1B (H317) according to Regulation (EC) No 1272/2008 (CLP).