1H-Imidazolium, 3-ethyl-1-methyl-, 1,1,1-trifluoromethanesulfonate (1:1)

Skin sensitisation

Administrative data

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:

other: GLP and guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Commission Regulation (EC) No 440/2008

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

1-ethyl-3-methyl-1H-imidazol-3-ium trifluoromethanesulfonate

EC Number:

680-002-1

Cas Number:

145022-44-2

Molecular formula:

C6 H11 N2. C F3 O3 S

IUPAC Name:

1-ethyl-3-methyl-1H-imidazol-3-ium trifluoromethanesulfonate

Details on test material:

- Name of test material (as cited in study report): EMIM Triflat
- Test-substance No.: 09/0070-2
- Purity: The test substance has been characterized by analyses
- Lot/batch No.: Los-Nr. 0000000001
- Storage Stability: The stability under storage conditions over the study period was guaranteed by the sponsor, and the sponsor holds this
responsibility

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/J

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sulzfeld, Germany
- Age at study initiation: 8 - 12 weks
- Weight at study initiation: 18.9 - 24.2 g
- Housing: single housed
- Diet (e.g. ad libitum): Kliba-Labordiaet (Maus / Ratte Haltung “GLP”), Provimi Kliba SA, Kaiseraugst, Basel, Switzerland, ad libitum.
- Water (e.g. ad libitum): Tap water ad libitum
- Acclimation period: at least 5 days before the first application


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 – 24°C
- Humidity (%): 30 - 70
- Photoperiod (hrs dark / hrs light): 12h/12h

Study design: in vivo (LLNA)

Vehicle:

other: diluted or test substance solutions in ethanol

Concentration:

A solubility experiment was performed according to the recommendations given by OECD 429. The highest test-substance concentration, which can
be technically used, was the undiluted test substance. The test-substance solutions (25 resp. 50%) were formulated in 70% ethanol.

No. of animals per dose:

5

Details on study design:

To determine the highest test-substance concentration that does not induce local signs of skin irritation and/or systemic toxicity, a pre-test (experimental conduct in accordance with GLP but without a GLP status) was performed. Two mice per dose group were treated with test-substance concentrations of 25% and 50% or with the undiluted test substance on three consecutive days.
In the pre-test clinical signs were recorded after each application as well as on day 5. Signs of local irritation were recorded on day 1, 2 and 5. Furthermore, the ears were punched after sacrifice at the apical area using a biopsy punch (Ø 0.8 cm) and were immediately pooled per animal and weighed using an analytical balance. Additionally, the weight of the pooled lymph nodes from both sides was determined for each animal.
At all tested concentrations the animals did not show relevant signs of local irritation as confirmed by the ear weight measurements. Some increase in lymph node weights were observed after application of the undiluted test substance.
Beside slightly reduced mean body weights in all dose groups, no signs of systemic toxicity were noticed during general observation in the pre-test. Therefore, the following dose levels were selected for the main study: 25%, 50% and the undiluted test substance.

Statistics:

Mean values and standard deviations of the measured parameters were calculated for the test and control groups from the individual values. The stimulation indices of 3H-thymidine incorporation, cell count, lymph node weight and ear weight measurements were calculated as the ratio of the test group mean values for these parameters divided by those of the vehicle control group.
Further statistical analyses were performed for:
3H-thymidine incorporation, cell count, lymph node weight and ear weight :WILCOXON - Test

Results and discussion

Positive control results:

A concurrent positive control (reliability check) with a known sensitizer was not included in this study.
Studies using the positive control substance Alpha-Hexylcinnamaldehyde, techn. 85% are performed twice a year in the laboratory in order to show that the test system is able to detect sensitizing compounds under the test conditions chosen.

In vivo (LLNA)

Results

Parameter:

SI

Remarks on result:

other: see Remark

Remarks:

When applied undiluted or as 50% and 25% solutions in 70% ethanol, the test substance did not induce a biologically relevant (no increase above the cut off Stimulation Index of 3) increase of 3H-thymidine incorporation into the cells from the auricular lymph nodes or in lymph node cell counts (no increase to 1.5 fold or above of control value = stimulation index (SI) ≥ 1.5).

Any other information on results incl. tables

No signs of systemic toxicity were noticed during general observation.

In addition, there were no relevant increases in lymph node weights or ear weights. Some statistically significant increases in 3H-thymidine incorporation, cell counts, lymph node weights and ear weight occurred without biological relevance or concentration relation.

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information

Conclusions:

Thus it is concluded that EMIM Triflat does not exhibit a skin sensitizing potential in the Murine Local Lymph Node Assay under the test conditions chosen.

Executive summary:

The skin sensitizing potential of EMIM Triflat 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 the undiluted test substance, 50% and 25% w/w preparations of the test substance in 70% ethanol or with the vehicle alone.

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 undiluted or as 50% and 25% solutions in 70% ethanol, the test substance did not induce a biologically relevant (no increase above the cut off Stimulation Index of 3) increase of 3H-thymidine incorporation into the cells from the auricular lymph nodes or in lymph node cell counts (no increase to 1.5 fold or above of control value = stimulation index (SI) ≥ 1.5). In addition, there were no relevant increases in lymph node weights or ear weights. Some statistically significant increases in 3H-thymidine incorporation, cell counts, lymph node weights and ear weight occurred without biological relevance or concentration relation.

Thus it is concluded that EMIM Triflat does not exhibit a skin sensitizing potential in the Murine Local Lymph Node Assay under the test conditions chosen.