1H-Imidazolium, 3-ethyl-1-methyl-, ethyl sulfate (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: Guideline study (429)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

-temperary fluctuations of relative humidity above level of 70%. Based on laboratory historical data, these fluctuations were considered not to have affected the study integrity

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Details on test material:

- Physical apprearance: Light yellow viscous liquid
- Batch: 99/484
- Storage: RT in the dark
- Stability under in vehicle (Dimethyl foramamide): at least 96 h
- Specific gravity: 1.23
- Purity: 99% (HPLC)
- Epiry date: 2005 (allocated by NOTOX, 1 year after receipt of the test substance)

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA strain, inbred, SPF-Quality

Sex:

female

Details on test animals and environmental conditions:

- Source: Charles River France, L'Arbresle Cedex, France
- Age at study initiation: approx 10 wks
- Weight at study initiation: +/- 20% of the sex mean
- Housing: individually
- Diet : standard pelleted laboratory animal diet (code VRF 1, Altromin, Lage, Germany)
- Water: Free access to tap-water
- Acclimation period: =>5 days
- Preparation of test formulations: the test substance formulations (wlw) were prepared within 4 hours prior to each treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.4 - 21.9
- Humidity (%): 41 - 75
- Air changes (per hr): approx. 15
- Photoperiod (hrs dark / hrs light): 12

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Remarks:

(selected based on trial formulations performed at NOTOX)

Concentration:

25%, 50% in vehicle and 100% (undiluted)

No. of animals per dose:

5 females

Details on study design:

RANGE FINDING TESTS:
- Concentrations: 50%, 100% (undiluted)
- # of animals: 1 per dose (total of 2)
- Age of animals: 5-14 weeks old
- Duration of treatment: Each animal was treated with one concentration on three consecutive days. Approximately 4 hours after the last exposure, the ear was cleaned of residual test substance with water and the irritation was assessed.
- Procedure: Identical to those used during days 1 to 3 of the main study unless where specified. Bodyweights were determined on day 3. No necropsy was performed after termination.


MAIN STUDY
INDUCTION
- Frequency of application: once daily for 3 days (days 1, 2 and 3)
- Site of application: ears
- Route of application: epidermal
- Volume applied: 25 µl/ear
- Concentrations: 25%, 50% in vehicle and 100% (undiluted)

INJECTION OF 3H-methyl thymidine
- Day of injection: 3 days after last treatment (day 6)
- Site of injection: tail vein
- Vehicle: PBS
- Volume injected: 0.25 ml
- Specific radioactivity: 20 µCi/injection

SACRIFICE
- Time schedule: 5 hours after injection of 3H-methyl thymidine
- Method: injection of phenobarbital

TISSUE PROCESSING AND MEASUREMENTS
- Lymph node processed: auricular lymph nodes
- Pooling of lymph nodes: yes
- Measurement of radioactivity: Packard scintillation counter (1900TR) (Counting time was to a statistical precision of ± 0.2% or a maximum of 5 minutes whichever comes first)

CRITERIA FOR POSTIVE RESULT
The stimulation index (SI) is the ratio of the disintergrations per minute (DPM) per group compared to DPM/vehicle control group. If the results indicate a stimulation index (SI) greater or equal to 3, the test substance may be regarded as a skin sensitiser, based on the test guideline and recommendations done by ICCVAM (NIH publication; No 99-4494, February 1999). If possible, an EC3 value (the estimated test substance concentration that will give a SI of 3 was determined, using linear interpolation.


OTHER
The test animals were checked twice daily for mortality/viability and at least once a day for toxicity signs. Bodyweight of the test animals was taken on day 1, prior to treatment and on days 6. On day 3 (3-4 hours after treatment), the skin reactions were assessed. If possible, skin reactions were graded following the draize numerical scoring system. Furthermore descriptions of all other (local) effects were recorded.

Positive control substance(s):

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

other: Concentrations: 5, 10 and 25 % in acetone: olive oil (4:1)

Results and discussion

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: TEST GROUPS - control (dimethylformamide): 1 - 25 %: 1.1 - 50%: 1.0 - 100 %:0.7 POSITIVE CONTROL - control (acetone/olive oil): 1 - 5%: 1 - 10%: 3.2 - 25%: 7.1

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: TEST GROUP (Mean DPM/Animal) - Control (dimethylformamide): 373 - 25 %: 411 - 50%: 385 - 100 %: 243 POSITIVE CONTROL (DPM/animal) (see attachment 1) - control (acetone/olive oil): 241 ± 75 - 5%: 235 ± 53 - 10%: 766 ± 225 - 25%: 1708 ± 509

Any other information on results incl. tables

OBSERVATIONS (MAIN STUDY)

No mortality occurred and no symptoms of systemic toxicity were observed in the animals of the main study.

The majority of the nodes of the experimental and control groups were considered normal in size (visual inspection). The nodes of two animals treated at 25 and 100% were decreased in size. No other macroscopic abnormalities of the nodes were noted (see attachment # 1).

The simulation index was in the range of 0.7 to 1.1 % for all concentrations tested. Theris no indication that the substance could elicid an SI > 3. Based on the results the test substance should be regarded as not sensitizing..

RESULTS (RELIABILITY CHECK WITH POSITIVE CONTROL SUBSTANCE

The SI values calculated for the posive control at concentrations of 5, 10 and 25% were 1.0, 3.2 and 7.1 respectively. An EC3 value of 9.5% was calculated for the positive control using linear interpolation. The calculated EC3 value was found to be in the acceptable range of 2 and 20%. The results of the 6 monthly HCA reliability checks of the recent years were 8 .8, 5 .5, 7.3 and 10.3%. The study results are valid therefore.

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information