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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:
The study was conducted between 9 October 2002 and 14 October 2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report Date:
2003

Materials and methods

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

Test material

Reference
Name:
Unnamed
Test material form:
liquid

In vivo test system

Test animals

Species:
mouse
Strain:
CBA:J
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France.
- Females nulliparous and non-pregnant: yes
- Age at study initiation: approximately 9 weeks
- Weight at study initiation: 20.1 ± 1.2 g (mean body weight ± standard deviation)
- Housing: individually, disposable crystal polystyrene cages (22.00 cm x 8.50 cm x 8.00 cm).
- Diet: A04 C pelleted diet (UAR, Villemoisson, Epinay-sur-Orge, France), ad libitum.
- Water: tap water (filtered using a 0.22 micron filter), ad libitum.
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): : 22 ± 2
- Humidity (%): 30 to 70
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Remarks:
acetone, batch No. 0126152 and olive oil, batch No. 050K6072
Concentration:
0, 5, 10, 25, 50, and 100 (v/v%)
No. of animals per dose:
4
Details on study design:
MAIN TEST
- Administration of the dosage forms: 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 between each application.

- Clinical examinations:
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.
Body weight: 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.

- Proliferations assay:
Intravenous injection of 3H-TdR and sampling of auricular lymph nodes: Lymph node cell proliferative responses were measured as described by Kimber and Dearman (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 (specific activity of 25 Ci/mmol), 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.

Preparation of auricular lymph node cell suspensions and determination of proliferation: For each experimental group, a single cell suspension of auricular lymph node cells (ALNC) was prepared by mechanical dissagregation in Petri dishes with the plunger of a syringe. 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 GoldxR scintillation fluid (Packard) were added in order to measure incorporation of 3H-TdR using β-scintillation counting. The results were expressed as disintegrations/mn (dpm) per group. Stimulation Indices (SI) were calculated according to the following formula:

SI = dpm of treated group / dpm of control group

The same calculation was made for the positive control group.

Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)

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 = 5.65) were noted.

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
EC3
Remarks:
%
Value:
38
Test group / Remarks:
Based on interpolation between the 25 and 50% dose (no dose-response relationship was established)
Parameter:
SI
Value:
0.69
Test group / Remarks:
5% test substance
Parameter:
SI
Value:
0.91
Test group / Remarks:
10% test substance
Parameter:
SI
Value:
1.04
Test group / Remarks:
25% test substance
Parameter:
SI
Value:
4.95
Test group / Remarks:
50% test substance
Parameter:
SI
Value:
2.23
Test group / Remarks:
100% test substance
Cellular proliferation data / Observations:
CHOICE OF THE VEHICLE
The test item was freely soluble in the first recommended vehicle, acetone/olive oil (4/1, v/v). A homogeneous dosage form preparation was obtained whatever the proportion.

CELLULAR PROLIFERATION DATA
Grouped disintegrations per minute (dpm):
- Vehicle group: 856.44
- 5% test group: 592.18
- 10% test group: 781.60
- 25% test group: 893.85
- 50% test group: 4239.23
- 100% test group: 239.13

The quantity of cells obtained in each group was satisfactory and the cellularity correlated with incorporation of 3H-TdR. The cell viability was higher than 80% in each group. 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 = 5.65) were noted. The study was therefore considered valid.

DETAILS ON STIMULATION INDEX CALCULATION
The stimulation index was calculated by dividing the dpm/group for the test groups through the dpm/group for the vehicle group.
In the treated groups, a positive lymphoproliferative response (SI > 3) was noted at the concentration of 50%, without clear evidence of a dose-response relationship.

EC3 CALCULATION
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, excluding the result at the 100% concentration.

CLINICAL OBSERVATIONS
No clinical signs and no mortality were observed during the study. No cutaneous reactions and noteworthy increase in ear thickness were observed at any tested concentrations.

BODY WEIGHTS
The body weight gain of the treated animals was similar to that of controls.

Applicant's summary and conclusion

Interpretation of results:
other: Skin Sensitiser 1B
Remarks:
in accordance with EU CLP (EC 1272/2008 and its updates)
Conclusions:
A positive lymphoproliferative response (SI > 3) was noted at the concentration of 50%, without clear evidence of a dose-response relationship. Based interpolation between the 25 and 50% dose, the test item was found to have an EC3 value of 38% and is considered to be a sensitiser under the conditions of the test.
Executive summary:

The skin sensitisation potential of the Geranium oil has been tested according to the OECD TG 429 (Local Lymph Node Assay) guideline. At 5, 10, 25, 50, and 100% the substance showed SI values of 0.69, 0.91, 1.04, 4.95 and 2.23, respectively. No cutaneous reactions and noteworthy increase in ear thickness were observed at any tested concentrations. In the treated groups, a positive lymphoproliferative response (SI > 3) was noted at the concentration of 50%, without clear evidence of a dose-response relationship. Based interpolation between the 25 and 50% dose, the test item was found to have an EC3 value of 38% and is considered to be a sensitiser under the conditions of the test.