1-ethyl-3-methyl-3H-imidazolium dicyanidoamide(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:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

3-ethyl-1-methyl-1H-Imidazolium salt with N-cyanocyanamide (1:1)

EC Number:

609-330-5

Cas Number:

370865-89-7

Molecular formula:

C6 H11 N2 .C2 N3

IUPAC Name:

3-ethyl-1-methyl-1H-Imidazolium salt with N-cyanocyanamide (1:1)

Specific details on test material used for the study:

- Name of test material (as cited in study report)t: EMIM Dicyanamid
- Purity: 97.4 g/100 g

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Weight at study initiation: 0: 18.9 g – 21.8 g
- Housing: One animal per cage (type: Makrolon cage, type II).
- Diet: Kliba-Labordiaet (Maus / Ratte Haltung “GLP”), Provimi Kliba SA, Kaiseraugst, Basel, Switzerland, ad libitum
- Water: Tap water ad libitum
- Acclimation period: at least 5 days before the first test substance application
- Other: The single housed animals were identified by cage cards.

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

Study design: in vivo (LLNA)

Vehicle:

other: 70% ethanol was used as the vehicle because good solubility of the preparation was achieved

Concentration:

Control group 1: 25 µL of 70% ethanol
Test group 2: 25 µL of 10% in 70% ethanol
Test group 3: 25 µL of 25% in 70% ethanol
Test group 4: 50% in 70% ethanol

No. of animals per dose:

5

Details on study design:

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 preparations/solutions at different concentrations were formulated in 70% ethanol. 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. The test-substance preparation was produced on a weight per weight basis shortly before the application by stirring with a magnetic stirrer. After stirring with a magnetic stirrer the test substance was soluble in the vehicle. Form of application: solution

Positive control substance(s):

other: Alpha-Hexylcinnamaldehyde, techn. 85%

Results and discussion

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

EC3

Value:

26

Remarks on result:

other: 3H-thymidine incorporation

Results 2

Parameter:

SI

Value:

2.27

Test group / Remarks:

10% in 70% ethanol

Remarks on result:

other: 3H-thymidine incorporation

Results 3

Parameter:

SI

Value:

2.98

Test group / Remarks:

25% in 70% ethanol

Remarks on result:

other: 3H-thymidine incorporation

Results 4

Parameter:

SI

Value:

3.53

Test group / Remarks:

50% in 70% ethanol

Remarks on result:

other: 3H-thymidine incorporation

Cellular proliferation data / Observations:

Beside slightly reduced mean body weights in test groups 3 and 4, no signs of systemic toxicity were noticed during general observation. When applied as 10%, 25% and 50% solution in 70% ethanol, the test substance induced a statistically significant increase of 3H-thymidine incorporation into the cells from the auricular lymph nodes, which was above the cut-off value for skin sensitization (increase above the cut off stimulation index (SI) of 3) at the 50% concentration. The 25% test substance preparation caused an increase of 3H-thymidine incorporation at the border of this value. Concomitantly, all concentrations induced statistically significant responses in the auricular lymph node cell counts, which failed the border of biological relevance (increase to 1.5fold or above of control value = stimulation index (SI) ≥ 1.5) at the 25% and 50% concentrations. In addition, both concentrations caused about the same statistically significant increase in lymph node weights. The ear weights of all concentrations showed some statistically significant increase as indication of slight ear skin irritation, but without a clear concentration response relation. Because the lymph node response cannot be fully attributed to the ear skin irritation observed, it is concluded that EMIM Dicyanamid exhibits a skin sensitizing potential in the Murine Local Lymph Node Assay under the test conditions chosen The threshold concentration for sensitization induction was ca. 25% for 3H-thymidine incorporation. Due to the difference in concentration-effect relationships of 3H-thymidine incorporation and cell count, no congruent estimated concentrations were obtained. The EC 3 (estimated concentration that leads to the SI of 3.0) for 3H-thymidine incorporation was calculated by linear regression from the results of the 25% and 50% concentrations to be 26%, while the SI of 1.5 for cell count was >50%.

Applicant's summary and conclusion

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria