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Toxicological information

Skin sensitisation

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Administrative data

skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
02 - 05 Apr 2008
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
24 April 2002
GLP compliance:
Type of study:
mouse local lymph node assay (LLNA): BrdU-ELISA

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:

In vivo test system

Test animals

other: CBA/J Rj.
Details on test animals and environmental conditions:
- Source: Janvier S.A.S., Le Gnest st Isle, France
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: 20.8 - 24.0 g
- Housing: individually in cages of standard dimensions on sawdust bedding
- Diet: RM1(E)-SQC SDS/DIETEX feed (quality controlled / radiation sterilised)
- Water: ad libitum
- Acclimation period: minimum of 5 days

- Temperature (°C): 19 - 23
- Humidity (%): 45 - 65
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 / 12

Study design: in vivo (LLNA)

other: for 25, 50 and 75% solutions: sterile water
25, 50, 75% (v/v) and 100%
No. of animals per dose:
2 (preliminary study), 4 (main study)
Details on study design:
A preliminary study was carried out to evaluate the maximum homogeneous concentration causing slight to moderate irritation by cutaneous application without causing toxic effects for the main study. Four concentrations of 25, 50 and 75% (v/v) in sterile water and 100% (undiluted) were tested, each in at least two different animals (two concentrations per mouse, one concentration per ear).
- Compound solubility: The test susbtance was soluble at 75% (v/v) in sterile water.
- Irritation: The application of the test substance at 25%, 50%, 75% (v/v) in sterile water and 100% did not induce colouring of the application site. Grading of all skin reactions was therefore possible. No sign of irritation was observed in animals.
- Systemic toxicity: No mortality was observed during the preliminary study.
- Ear thickness measurements: No increase of ear thickness was noted at all tested concentrations.

Since undiluted test substance caused no irritation in preliminary study, same doses were chosen.
- Name of test method: ELISA BrdU
- Criteria used to consider a positive response: The test substance can be regarded as a sensitiser if at least one test concentration produces a test/control ratio equal to or greater than 3.0 (Stimulation Index SI > 3) [1]. However, the magnitude of the stimulation index was not the only factor used in determining the biological significance of a skin sensitisation response. Other relevant criteria such as cellularity and ear thickness were taken into account for the interpretation of the results.

TREATMENT PREPARATION AND ADMINISTRATION: A volume of 25 μL of test substance, vehicle or positive control was applied to the dorsum of both ears of all animals daily for three consecutive days. Two days after the third application on Day 5, an intraperitoneal injection of 250 µL of 0.9% sodium chloride containing 2.5 mg of BrdU was made. Approximately 5 h after BrdU injection, the mice were sacrificed and draining auricular lymph nodes from each mouse ear were excised. A single cell suspension was prepared by separation through a 200 µm-mesh nylon filter. Cell suspensions were re-suspended in 2 mL of a solution containing phosphate buffer solution and 1% of antibiotics and antimycotics for counting of lymphocytes (cellularity). 25 μL of cell suspension was seeded in 96 well tissue culture plate. On the next day (Day 7) BrdU was measured by ELISA using a commercial kit.
Positive control substance(s):
other: Dinitrochlorbenzol (DNCB) 0.5% in acetone
Results of Stimulation Index and ear thickness were given as means ± SEM (Standard Error of the Mean). The effects of the test item on Stimulation Index and ear thickness were compared with that of the control group using an analysis of variance with a Dunnett´s test in case of significance (P ≤ 0.05). Body weight changes were given as mean ± SEM (Standard Error of the Mean) and were compared with the control group using an analysis of variance with Dunett´s test in case of significance (P ≤ 0.05).

Results and discussion

Positive control results:
In the positive control animals were treated with DNCB 0.5% discrete erythema was noted on both ears on Day 4 and Day 6. An increase in ear thickness (+10%) was noted compared to Day 1. This increase was statistically significant in comparison with ear thickness of animals treated with vehicle positive control (acetone). Cellularity index was 21.88 with a total amount of viable cells of 14.1x10E6 compared to 1.21x10E6 with the vehicle control group and the significant stimulation index was 4.3.

In vivo (LLNA)

Resultsopen allclose all
Test group / Remarks:
50% (v/v)
Test group / Remarks:
75% (v/v)
Test group / Remarks:
100% (v/v)
Cellular proliferation data / Observations:
Erythema: The test substance at 50%, 75% and 100% did not provoked irritation.
Ear thickness: No significant increase was noted in animals treated with the test substance at 50%, 75% and 100% during the study except in animals treated with the test substacne at 75% on Day 2 in comparison with vehicle group but there were only an increase of 5% in comparison with Day 1. Therefore it was not a relevant significant increase.

CELLULAR PROLIFERATION DATA: Groups treated with vehicle, test substance at 50%, 75% and 100% , acetone and DNCB at 0.5% showed a cell viability of 73%, 68%, 73%, 64%, 71%, and 86% respectively. The total amount of viable cells for each groups were of 1.21x10E6 ; 1.24x10E6 ; 1.19x10E6 ; 0.86x10E6 , 0.58x10E6 and 14.1x10E6 cells per node, respectively. Cellularity index were of 1.15; 0.98 and 1.06 and stimulation index were 1.1, 0.9 and 1.1with the test substance at 50%, 75%, and 100%, respectively.

DETAILS ON STIMULATION INDEX CALCULATION: Stimulation Index (SI), expressed as the ratio of mean OD of the test nodes relative to that recorded for control (vehicle) nodes and Cellularity Index expressed as the ratio of mean amount of cells (x 10E6 cells) in the treated group and the mean amount of cells (x 10E6 cells) in the vehicle group.

EC3 CALCULATION: No EC3 value could be calculated.

CLINICAL OBSERVATIONS: No clinical signs or mortalities were observed during the main study.

BODY WEIGHTS: The body weight change of the treated animals with test substance was similar to that of the vehicle control animals.

Applicant's summary and conclusion

Interpretation of results:
other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008
Under the conditions of the local lymph node assay, the test substance at concentrations of 50, 75 and 100% did not produce a stimulation index equal to or greater than 3 and no EC3 value was calculated. Therefore, the test substance was considered to be not sensitising.