1H-Imidazolium, 1,3-diethyl-, acetate (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:

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:

1,3-diethyl-1H-imidazol-3-ium acetate

EC Number:

692-759-5

Cas Number:

1040916-84-4

Molecular formula:

C7 H13 N2 . C2 H3 O2

IUPAC Name:

1,3-diethyl-1H-imidazol-3-ium acetate

Details on test material:

Name of the test substance used in the study report: EEIM Acetate
Purity: 97.4 ± 0.1 g/ 100 g

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

Acclimatization period at least 5 days before the first test substance application.
The single housed animals were identified by cage cards.
The animals were housed in fully air-conditioned rooms. Central air-conditioning guaranteed a range of 20 – 24°C for temperature and of 30 – 70% for relative humidity. The day/night rhythm was 12 h light and 12 h darkness.
Type of cage: Makrolon cage, type II.
Feeding: Kliba-Labordiaet (Maus / Ratte Haltung “GLP”), Provimi Kliba SA, Kaiseraugst, Basel, Switzerland, ad libitum
Drinking water: Tap water ad libitum

Study design: in vivo (LLNA)

Vehicle:

other: ethanol

Concentration:

Control group 1: 70% ethanol
Test group 2: test substance 10% in 70% ethanol (25 ul per ear)
Test group 3: test substance 25% in 70% ethanol (25 ul per ear)
Test group 4: test substance 50% in 70% ethanol (25 ul per ear)

No. of animals per dose:

5

Details on study design:

70% ethanol was used as the vehicle because good solubility of the preparation was achieved.
Control group 1 was used to rule out any influence of the vehicle on the parameters measured in the test.
The test substance preparation was produced on a weight per weight basis shortly before the application. After stirring with a magnetic stirrer the test substance was soluble in the vehicle.
Form of application: solution
_____
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 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.
The undiluted test substance caused mortality in both animals after the 2nd application. The 50% concentration caused considerably increased ear weights and lymph node weights as indication of ear skin irritation. Compound residues were observed on the ear skin on day 5. After application of the 25% test substance concentration the animals showed some increases in ear weights and lymph node weights. Beside slightly reduced mean body weights in the animals of both concentrations concentrations, no signs of systemic toxicity were noticed during general observation.
Therefore, the following dose levels were selected for the main study: 10%, 25% and 50%.

Positive control substance(s):

other: A concurrent positive control with a known sensitizer was not included in this study. Studies with alpha-Hexylcinnamaldehyde, techn. 85% are performed twice a year in order to show that the test system is able to detect sensitizing compounds.

Statistics:

yes

Results and discussion

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

1.2

Test group / Remarks:

10% in 70% ethanol

Results 2

Parameter:

SI

Value:

4.13

Test group / Remarks:

25% in 70% ethanol

Results 3

Parameter:

SI

Value:

4.61

Test group / Remarks:

50% in 70% ethanol

Results 4

Parameter:

EC3

Remarks:

(%)

Value:

19.2

Any other information on results incl. tables

Test Group	Treatment	3H-thymidine Incorporation Stimulation Index	Cell Count Stimulation Index	Lymph Node Weight Stimulation Index	Ear Weight Stimulation Index
1	vehicle 70% ethanol	1.00	1.00	1.00	1.00
2	10% in 70% ethanol	1.20	1.08	1.10     #	1.05   #
3	25% in 70% ethanol	4.13   ##	1.70   ##	1.63   ##	1.19   ##
4	50% in 70% ethanol	4.61   ##	2.16   ##	1.82   ##	1.39   ##

The statistical evaluations were performed using the WILCOXON-test ( # for p ≤ 0.05, ## for p ≤ 0.01 )

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.

The threshold concentration for sensitization induction was >10% < 25%. The EC 3 (estimated concentration that leads to the SI of 3.0) for 3H-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.

Applicant's summary and conclusion

Conclusions:

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.