1,2-dimethyl-3-propyl imidazolium bis((trifluoromethyl)sulfonyl)amide

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

31 AUG 2016 to 19 SEP 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

dated 22 july 2010

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

Council regulation No. 640/2012 of the 06 July 2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

GROUPE INTERMINISTERIEL DES PRODUITS CHIMIQUES, France

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

1,2-dimethyl-3-propyl imidazolium bis((trifluoromethyl)sulfonyl)amide

EC Number:

805-807-9

Cas Number:

169051-76-7

Molecular formula:

C10H15F6N3O4S2

IUPAC Name:

1,2-dimethyl-3-propyl imidazolium bis((trifluoromethyl)sulfonyl)amide

Test material form:

liquid

Details on test material:

Appearance : colorless oil
Composition: a base stock containing (C, 28.64; H, 3.61; F, 27.18; N, 10.02; O, 15.26; S, 15.29)
Molecular formula : C10H15F6N3O4S2
Molecular Weight : 419.12 g/mol
Purity >99% (purity determined by MNR)
Homogeneity : homogeneous

Specific details on test material used for the study:

Batch No.: L-16-0589
Manufacturing date: 2016-01-07
Expiry date: 2030-01-02
the test item was identified under the code number : PH-16/0440.
Storage: room temperature

In vivo test system

Test animals

Species:

mouse

Strain:

CBA:J

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
Source: CBA/JRj supplied by Elevage Janvier Labs (Le Genest Saint Isle, France)
Age at the study initiation: 8 or 9 weaks old. Weight at study initiation: 19.7 - 25.1 g
Housing : individually housed in suspended solid-flour polypropylene cages furnished with softwood woodflakes.
Diet: Teklad Global 16% Protein Rodent Diet (ENVIGO++++ - 2016), ad libitum- Water: tap water from public distribution system, ad libitum
Acclimation period: at least 5 days.

ENVIRONNEMENTAL CONDITIONS
Temperature (°C): 19 - 25, Humidity (%) 30 - 70, Air change (per h): at least 10, Photoperiod: (hrs dark / hrs light): 12 / 12.

Study design: in vivo (LLNA)

Vehicle:

dimethyl sulphoxide

Remarks:

This vehicle was chosen as it produced the most suitable formulation at the required concentration.

Concentration:

0, 25, 50 and 100%

No. of animals per dose:

4 (+1 additional animal/group as reserve)

Details on study design:

PRELIMINARY STUDY:
In the first pretest, one mortality occured at the concentration of 100% without any sign of systemic toxicity.
In the second pretest, no mortality and no signs of systemic toxicty were noted.
No cutaneous reactions were recorded at the concentration of 100%. Therefore this concentration was chosen as the highest concentration for the main study.


MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Lymphocyte cell counting
- Criteria used to consider a positive response: The test item will be regarded as a sensitiser if at least one concentration of the test item results is greater than 1.4 compared to control values (SI ≥ 1.4). Other relevant criteria such as dose-response and irritation level were also taken into account for the interpretation of the results. Any test item failing to produce a SI > 1.4 will be classified as a "non-sensitiser".

TREATMENT PREPARATION AND ADMINISTRATION:
Test item administration
Groups of four mice were treated with the test item undiluted (100%) and diluted at concentrations of 50% and 25% in dimethyl sulfoxide DMSO. The mice were treated by daily application of 25µl of the appropriate concentration of the test item to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The test item formulation was administrated using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette. A further group of four mice received the vehicle alone in the same manner.
Clinical observations
All animals were observed daily on Days 1, 2, 3, 4, 5 and 6.
The bodyweight of each mouse was recorded on Day 1 (prior to dosing) and day 6 (prior to termination).
Ear thickness measurements and recording of local reactions were performed in order to assess any possible irritant effect of the test item, as possible irritancy may be involved in false positive lymphoproliferative responses. On day 1 and on day 3 (before application) as well as on day 6 (after sacrifice) of each experiment, the thickness of the right ear of each animal of the vehicle control and treated groups was measured by a micrometer. Furthermore, on day 6, punch biopsies of 8 mm in diameter of the apical area of both ears were prepared and weighed in order to assess the irritation potential of the test item and the two lymph nodes per mouse were weighted. Any irritation reaction (erythema and oedema) was recorded in parallel. Any other observation (dryness, presence of residual test item…) was noted.
Terminal procedures
Termination: On day 6 (end of the test), the animals were anaesthetized with sodium pentobarbital and administration continued to fatal levels. The draining auricular lymph nodes from the four mice were excised.
Preparation of single cell Suspension: A single cell suspension of the lymph node cells of 4 mice of each group was prepared by gentle mechanical tissue disaggregation through a 200-meshcell strainers. 10 µL of this cell suspension was diluted in 10 mL of physiological saline solution before lymphocyte cells were counted using a cell counter (Beckman Coulter Z2). A size range of 5 - 15 µm was selected for counting which covers the average size of a lymphocyte of 8 µm.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Positive control results:

Three groups, each of four animals, were treated with 50µl (25µl per ear) of α-Hexylcinnamaldehyde, as a solution in acetone/olive oil (4:1; v/v) (AOO) at concentrations of 5%, 10% and 25% (v/v). A further control group of four animals was treated with acetone/olive oil (4:1; v/v) alone.

The Stimulation Index expressed as a cell count for each treatment group divided by mean cell count of the vehicle control group are: 1.67; 3.32; 3.86 for the treated groups at 5%, 10% and 25% respectively. The EC1.4 value is 4.19%

In conclusion, in view of the results, the test item α-Hexylcinnamaldehyde has to be classified as a sensitizer in category 1, Sub-category 1B, in accordance withthe regulation EC No. 1272/2008.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

2.06

Test group / Remarks:

25%

Remarks on result:

other: Positive

Results 2

Key result

Parameter:

SI

Value:

2.16

Test group / Remarks:

50%

Remarks on result:

other: Positive

Results 3

Key result

Parameter:

SI

Value:

1.49

Test group / Remarks:

100%

Remarks on result:

other: Positive

Results 4

Parameter:

other: EC1.4

Remarks on result:

not determinable

Remarks:

can not be determined and was lower than 25%

Cellular proliferation data / Observations:

The lowest SI value at the tested concentration 100% may be explained by a better bioavailability of the test item at lowest concentrations.

Any other information on results incl. tables

PRELIMINARY SCREENING TEST

In the first pretest, one mortality occured at concentration of 100% without any sign of systemic toxicity.
In the second pretest, no mortality and no sign of systémic toxicity were noted.

No cutaneous reactions were recorded at the concentration of 100%. Therefore this concentration was chosen as the highest concentration for the main study.

MAIN TEST

Bodyweight changes of the test animals between Day 1 and Day 6 were comparable to those observed in the corresponding control group animals over the same period.

A simulation index of more than 1.4 was recorded whatever the concentration.

The stimulation index (SI) calculated by pooled approach was 2.06, 2.16 and 1.49 for the treated groups at 25%, 50% and 100%, respectively. The EC_1.4can not be determined and was lower than 25%.
Furthermore, the lowest SI value at the tested concentration 100%, may be explained by a better bioavailability of the test item at lowest concentrations.

No cutaneous reaction was observed except splight dryness of the skin noted in animak treated at 25% on day 6.

No significant increase in ear thickness and in ear weight was noted in animals treated at 25%, 50% and 100%. Therefore, the test item has to be considered as not excessively irritant at these concentrations.

Applicant's summary and conclusion

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

In view of these results, under these experimental conditions, the test item 1-Propyl-2,3-Dimethylimidazolium bis(trifluoromethanesulfonyl)imide has to be classified as a sensitizer, in Category 1, in accordance with the criteria for classification, packaging and labelling of dangerous substances and mixturees of the Regulation No. 1272/2008. The signal word "warning" and hazard statement H317 "May cause an allergic skin reaction" are required.

Executive summary:

The test was performed to assess the skin sensitization potential of the test item 1-Propyl-2,3-Dimethylimidazolium bis(trifluoromethanesulfonyl)imide in the CBA/J strain mouse following topical application to the dorsal surface of the ear.

Three groups of four animals were treated for the three consecutive days (D1, D2, D3) with 50µl (25µl per ear) of the test item undiluted at 100% and diluted in DMSO at concentrations of 25% and 50%. A further group of four animals was treated with DMSO.

On D6, the proliferation of lymphocytes in the draining auricular lymph nodes was determined by cell counting. The experimental protocol was established according to the O.E.C.D. Test Guideline No. 429 dated 22 july 2010 and the test method B.42 of the Council Regulation No. 640/2012 of 06 july 2012.

No mortality and no sign of systemic toxicity were noted in the treated and control animals during the test.

No cutaneous reaction was observed except slight dryness of the skin noted in one animal treated at 25% on day 6.

No significant increase in ear thickness and in ear weight was noted in animals treated at 25%, 50% and 100%. Therefore, the test item has to be considered as not excessively irritant at these concentrations.

The stimulation index (SI) calculated by pooled approach was 2.06, 2.16 and 1.49 for the treated groups at 25%, 50% and 100%, respectively. The EC_1.4 can not be determined and was lower than 25%.

Furthermore, the lowest SI value at the tested concentration 100%, may be explained by a better bioavailability of the test item at lowest concentrations.

In view of these results, under these experimental conditions, the test item 1-Propyl-2,3-Dimethylimidazolium bis(trifluoromethanesulfonyl)imide has to be classified as a sensitizer, in Category 1, in accordance with the criteria for classification, packaging and labelling of dangerous substances and mixturees of the Regulation No. 1272/2008.

The signal word "warning" and hazard statement H317 "May cause an allergic skin reaction" are required.