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Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16Nov2001 to 09Apr2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report Date:
2002

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
See individual study reports for purity information and CofA where applicable
Specific details on test material used for the study:
supplier: Ciba Specialty Chemicals
. batch number:013673A1
. description: violet powder
. purity: 94.1%

In vivo test system

Test animals

Species:
mouse
Strain:
CBA:J
Sex:
female
Details on test animals and environmental conditions:
Age/weight: on the first day of treatment, the animals were approximately 9 weeks old and had a mean body weight ± standard deviation of 20.8±1.6g.
Acclimation: at least 5 days before the beginning of the study.
Allocation: on day 1, animals were assigned to the treatment groups by hand procedure.
Identification : individually by a number on the tail.

The conditions in the animal room were set as follows:
. temperature: 22 ± 2°C
. relative humidity: 30 to 70%
. light/dark cycle: 12 h/12 h
. ventilation: approximately 12 cycles/hour of filtered, non-recycled air.
The temperature and relative humidity were under continuous control and recording. The records were checked daily and filed. In addition to these daily checks, the housing conditions and corresponding instrumentation and equipment are verified and calibrated at regular intervals. The animals were housed individually in disposable crystal polystyrene cages (22.00 cm x 8.50 cm x 8.00 cm). Each cage contained autoclaved sawdust (SICSA, Alfortville, France). Sawdust is analysed by the supplier for composition and contaminant levels.

Study design: in vivo (LLNA)

Vehicle:
other: Ethanol/water (50:50)
Concentration:
0.25-5%
No. of animals per dose:
4
Details on study design:
Compound solubility:
Due to the unsatisfactory solubility of the test item in the first recommended vehicle (acetone/olive oil (4/1, v/v)) and in dimethylformamide (DMF), a mixture ethanol/water (50/50 (v/v)) was chosen among the other proposed vehicles; a homogeneous preparation was obtained at the maximal concentration of 5%.

MAIN STUDY
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 site. No rinsing was performed between each application.

Clinical signs, morbidity and mortality
The animals were observed at least once a day during the study for clinical signs, signs of morbidity or mortality.

Bodyweight
The animals were weighed individually on the first day of the study (day 1) and on the day of sacrifice (day 6).

Ear thickness measurements and recording of local reactions
On days 1, 2 and 3 (before application) and on day 6 (after sacrifice), the thickness of the left ear of each animal of the vehicle control and treated groups was measured using a micrometer. No measurement of ear thickness was carried out for animals of the reference treated group. Any irritation reaction (erythema and oedema) was recorded in parallel. Any other observation (coloration, presence of residual test item,...) was noted.

Intravenous injection of 3H-TdR and sampling of auricular lymph nodes
Lymph node cell proliferative responses were measured as described by Kimber, I. and Dearman R.J. (1991). On day 6, all animals of all groups received a single intravenous injection of 250 µL of 0.9% NaCl containing 20 µCi of 3H-TdR, via the tail vein. Approximately 5 hours later, the animals were killed by cervical dislocation and the auricular lymph nodes were excised. The lymph nodes were pooled for each experimental group. A single cell suspension of auricular lymph node cells (ALNC) was prepared by mechanical disaggregation in Petri dishes with the plunger of a syringe.

Preparation of auricular lymph node cell suspensions and determination of proliferation
Cell suspensions were washed with 15 mL of 0.9% NaCl and pellets obtained were re-suspended in 0.9% NaCl for numeration of lymphocytes (cellularity) and determination of their viability by exclusion of Trypan blue. Each cell suspension was then centrifuged and pellets were precipitated with 3 mL of 5% (w/v) trichloroacetic acid (TCA) in purified water at +4°C overnight. After a last centrifugation, the pellets were precipitated with 1 mL of 5% TCA.
Three mL of Ultima Gold'* scintillation fluid (Packard) were added in order to measure incorporation of 3H-TdR using P-scintillation counting. The results were expressed as disintegrations/mn (dpm) per group. Stimulation Indices (SI) were calculated.

Interpretation of results
The test item was considered as a skin sensitizer when the SI for a dose group is > 3. Other relevant criteria such as cellularity, radioactivity levels and ear thickness were also taken into account for the interpretation of results.

Determination of the EC3 value
Calculation of the EC3 value (theoretical concentration of the test item resulting in a SI value of 3) was performed on the basis of a dose-effect response.
Statistics:
Stimulation Indices (SI) were calculated.

Interpretation of results
The test item was considered as a skin sensitizer when the SI for a dose group is > 3. Other relevant criteria such as cellularity, radioactivity levels and ear thickness were also taken into account for the interpretation of results.

Determination of the EC3 value
Calculation of the EC3 value (theoretical concentration of the test item resulting in a SI value of 3) was performed on the basis of a dose-effect response.

Results and discussion

Positive control results:
In the positive control group given HCA at the concentration of 25%, an increase in cellularity and a stimulation index exceeding the threshold value of 3 (SI = 13.56) were noted. The study was therefore considered valid.

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
1.55
Test group / Remarks:
0.25% dose group
Key result
Parameter:
SI
Value:
2.26
Test group / Remarks:
0.50% dose group
Key result
Parameter:
SI
Value:
3.26
Test group / Remarks:
1 % dose group
Key result
Parameter:
SI
Value:
2.52
Test group / Remarks:
2.5% dose group
Key result
Parameter:
SI
Value:
3.88
Test group / Remarks:
5% dose group
Key result
Parameter:
EC3
Value:
3.12
Test group / Remarks:
Curve calcualted from 0.25, 0.5, 2.5 and 5% results
Cellular proliferation data / Observations:
The quantity of cells obtained in each group was satisfactory and the cell viability was higher than 80% in each group.

Applicant's summary and conclusion

Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Conclusions:
Under our experimental conditions, the test item induces delayed contact hypersensitivity in the murine Local Lymph Node Assay.
Executive summary:

The aim of this study was to evaluate the potential of the test item to induce delayed contact hypersensitivity using the murine Local Lymph Node Assay (LLNA). This study was conducted in compliance with the principles of Good Laboratory Practice Regulations.

Methods

Twenty-eight female CBA/J mice were allocated to seven groups of four animals each: five treated groups receiving the test item MIP 3100 at the concentrations of 0.25, 0.5, 1, 2.5 and 5%, one negative control group receiving the vehicle (a mixture of ethanol/water (50/50, v/v)), one positive control group receiving the reference item, a-hexylcinnamaldehyde (HCA), a moderate sensitizer, at the concentration of 25%.

The test item, vehicle or reference item were applied over the ears (25µL per ear) for three consecutive days (days 1, 2 and 3). After 2 days of resting, the proliferation of the lymph node cells in the lymph node draining the application site was measured by incorporation of tritiated methyl thymidine (day 6). The obtained values were used to calculate stimulation indices (SI). The irritant potential of the test item was assessed in parallel by measurement of ear thickness on days 1,2,3 and 6.

Results

Due to the unsatisfactory solubility of the test item in the first recommended vehicle (acetone/olive oil (4/1, v/v)), a mixture ethanol/water (50/50 (v/v)) was chosen among the other proposed vehicles; a homogeneous preparation was obtained at the maximal concentration of 5%.

Systemic clinical signs and mortality

No mortality and no clinical signs were observed during the study.

 

Local irritation

No increase in ear thickness was observed in the animals of the treated groups. A red coloration of the skin which could have masked a possible discrete to moderate erythema was noted on the ears of all treated animals from day 2 up to the end of the study (day 6).

 

Proliferation assay

A dose-related increase in the stimulation index was noted and the threshold positive value of 3 was exceeded at the concentrations of 1 and 5%

 

In the absence of local irritation, the positive lymphoproliferative responses observed at the concentrations of 1 and 5% were attributed to delayed contact hypersensitivity. The extrapolated EC3 value for the test item was 3.12%

Conclusion

Under our experimental conditions, the test item induces delayed contact hypersensitivity in the murine Local Lymph Node Assay.