1H-Imidazole-4,5-dicarbonitrile, 2-(trifluoromethyl)-, lithium salt (1:1)

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 February 2014 -- 28 April 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

lithium(1+) 4,5-dicyano-2-(trifluoromethyl)-1H-imidazol-1-ide

EC Number:

691-963-1

Cas Number:

761441-54-7

Molecular formula:

C6 F3 N4. Li

IUPAC Name:

lithium(1+) 4,5-dicyano-2-(trifluoromethyl)-1H-imidazol-1-ide

Test material form:

other: white powder

In vivo test system

Test animals

Species:

mouse

Strain:

CBA:J

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: breeder: Janvier, Le Genest-Saint-Isle, France.
- Age/Mean body weight at study initiation: the animals of the preliminary test were approximately 8 to 11 weeks old and had a mean body weight of 21.8 g (range: 19.7 g to 24.2 g) and the animals of the main test were approximately 8 weeks old and had a mean body weight of 19.9 g (range: 18.6 g to 21.4 g).
- Fasting period before study: no
- Housing: polycarbonate cages
- Diet: SSNIFF R/M-H pelleted diet (free access)
- Water: tap water filtered with a 0.22 µm filter (free access)
- Acclimation period: 6 days before the beginning of the study

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): approximately 12 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 h/12 h

IN-LIFE DATES: 19 March 2014 to 28 April 2014

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

No. of animals per dose:

4 per dose.

Details on study design:

RANGE FINDING TESTS:
The maximum concentration tested in the main test was selected according to the criteria specified in the OECD Guidelines and on the basis of the data that was obtained in the preliminary test:
- the vehicle was selected on the basis of producing a homogeneous preparation suitable for application of the test item,
- the concentrations were selected from the concentration series 75%, 50%, 25%, 10%, 5%, 2.5%, 1%, 0.5%, etc,
- the maximum concentration of the test item was selected to avoid overt systemic toxicity and excessive local skin irritation the latter being defined by an increase in ear thickness = 25%.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: murine Local Lymph Node Assay
- Criteria used to consider a positive response: stimulation Index SI >= 3 and dose-relationship; additional consideration of ear thickness and radioactivity levels

TREATMENT PREPARATION AND ADMINISTRATION:
- Treatment preparation: The test item was prepared at the chosen concentrations in the vehicle.
The positive control was dissolved in AOO at the concentration of 25% (v/v).
- Administration:
On days 1, 2 and 3, a dose-volume of 25 µL of the control or dosage form preparations was applied to the dorsal surface of both ears, using an adjustable pipette fitted with a plastic tip.
In order to avoid licking and to ensure an optimized application of the test materials, the animals were placed under light isoflurane anesthezia during the administration.
No massage was performed but the tip was used to spread the preparation over the application sites. No rinsing was performed.

Positive control substance(s):

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

Results and discussion

Positive control results:

The threshold positive value of 3 for the SI was reached in the positive control group (see Executive summary).

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

1.23

Test group / Remarks:

0.5%

Results 2

Parameter:

SI

Value:

0.53

Test group / Remarks:

1%

Results 3

Parameter:

SI

Value:

0.79

Test group / Remarks:

2.5%

Results 4

Parameter:

SI

Value:

1.73

Test group / Remarks:

5%

Results 5

Parameter:

SI

Value:

2.02

Test group / Remarks:

10%

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test item was tested as a solution after preparation at concentrations of 0.5, 1, 2.5, 5 and 10% in dimethylformamide (DMF).
Under the experimental conditions of this study, the test item gave a negative result in the murine Local Lymph Node Assay, indicative of the absence of skin sensitization properties.

Therefore, the test item should not be classified as a skin sensitizer according to the criteria of CLP Regulation.

Executive summary:

The potential of Lithium 4,5-dicyano 2-(trifluomethyl) imidazolate to induce contact hypersensitivity was evaluated using the murine Local Lymph Node Assay (LLNA). This study was conducted in compliance with the OECD Guideline No. 429 and the Principles of Good Laboratory Practice. To assess the irritant potential of the test item (through ear thickness measurement), a first preliminary test was performed in order to define the test item concentrations to be used in the main test. Two groups of two female mice received the test item by topical route to the dorsal surface of both ears (one concentration per ear) on Days 1, 2 and 3 at concentrations of 5, 10, 25 or 50% under a dose-volume of 25 µL. Following unscheduled death or sacrifice of the four animals on Day 3, a second preliminary test was added with two groups of two females, at concentrations of 1, 2.5, 5 or 10% under a dose-volume of 25 µL. From Day 1 to Day 3 then on Day 6, the thickness of both ears of each animal was measured and the local reactions were recorded. Each animal was observed at least once a day for mortality and clinical signs. Body weight was recorded once during the acclimation period, and then on Days 1 and 6. On completion of the observation period, the surviving animals were sacrificed then discarded without macroscopic post-mortem examination. In the main test, five groups of four female mice received the test item by topical route to the dorsal surface of both ears on Days 1, 2 and 3 at concentrations of 0.5, 1.0, 2.5, 5.0 or 10.0% under a dose-volume of 25 µL. One negative control group of four females received the vehicle (dimethylformamide (DMF)) under the same experimental conditions. Additionally, one positive control group of four females received the positive control, a-hexylcinnamaldehyde (HCA), at 25% in a mixture Acetone/Olive Oil (4/1; v/v) under the same experimental conditions. From Day 1 to Day 3 then on Day 6, the thickness of the left ear of each animal was measured, except in animals of the positive control group, and the local reactions were recorded. Each animal was observed at least once a day for mortality and clinical signs. Body weight was recorded once during the acclimation period, and then on Days 1 and 6. After 2 days of resting, on Day 6, the animals received a single intravenous injection of tritiated methyl thymidine (3H-TdR). Approximately 5 hours later, the animals were sacrificed and the auricular lymph nodes were excised. The proliferation of lymphocytes in the lymph node draining the application site was measured by incorporation of3H-TdR.

The results are expressed as disintegrations per minute (dpm) per group and as dpm/node. The obtained values were used to calculate Stimulation Indices (SI).

As a solution was obtained at the concentration of 50% in dimethylformamide (DMF), DMF was selected for the preparation of the test item. In the first preliminary test, on Day 3, three females were found dead and one was prematurely sacrificed. No unscheduled deaths or clinical signs were observed in any animals during the second preliminary test. In the assessment of local reactions, the increase in ear thickness was lower than the limit of 25% for each tested concentration. The highest concentration retained for the main test was therefore 10%. In the main test, one control female was found dead on Day 2 without clinical signs. No other unscheduled deaths or clinical signs were observed in any animals. Body weight of animals was unaffected by the test item treatment. No local reactions were observed in any animals. The SI of the positive control was > 3 (SI = 9.30); this experiment was therefore considered valid. The observed SI values were 1.23, 0.53, 0.79, 1.73 and 2.02 at concentrations of 0.5%, 1.0%, 2.5%, 5.0% and 10.0% (w/v), respectively. No significant lymphoproliferation was noted with the test item at any tested concentrations as all SI values were below the cut-off value of 3.

Lithium 4,5-dicyano 2-(trifluomethyl) imidazolate was tested as a solution after preparation at concentrations of 0.5, 1, 2.5, 5 and 10% in dimethylformamide (DMF)and gave a negative result in the murine Local Lymph Node Assay, indicative of the absence of skin sensitization properties.

 

Lithium 4,5-dicyano 2-(trifluomethyl) imidazolate should not be classified as a skin sensitizer according to the criteria of CLP/GHS Regulations.