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Description of key information

Skin sensitization (OECD TG 429): sensitizing

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25 August 2006 - 5 September 2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
2002
Deviations:
no
Remarks:
Remarks: No dose selection rationale is provided. Information on solubility of the substance in the solvent is not given.
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)
Specific details on test material used for the study:
- Name of test material (as cited in study report): Ylang Ylang extra EO Comores
- Physical state: liquid
-Colour: yellow
- Analytical purity: not specified (pure material)
- Storage condition of test material: ambient temperature in the dark
Species:
mouse
Strain:
other: CBA/Ca/Ola/Hsd
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan UK Limited, Shaw's Farm, Blackthorne, Bicester, Oxon, UK
- Age at study initiation: 8-12 weeks
- Housing: 4 mice/cage, suitable cages
- Diet: ad libitum, RM1 by Special Diet Services Limited, Witham, Essex, UK
- Water: ad libitum
- Acclimation period: 5 days prior dosing

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±3
- Humidity (%): 30-70
- Air changes (per hr): min 15
- Photoperiod (hrs dark / hrs light): 12
Vehicle:
other: 1:3 ethanol:diethylphthalate
Concentration:
0.5, 1, 2.5, 5 and 10% (w/v)
No. of animals per dose:
4
Details on study design:
MAIN STUDY

TREATMENT PREPARATION AND ADMINISTRATION:
25 µl of the substance was applied on the dorsal ear with a micro-pipette (injection), once for three consecutive days. Dose preparations were used within 24 hours. Three days after last treatment, 250 µl PBS 20 µCi/mmol specific activity 3H-methyl thymidine, were injected in the tail vein. Five hours thereafter the animals were sacrificed.

Cell suspension: mechanical disaggregation of pooled lymph nodes; properly prepared as described in the Guideline OECD 429.
Incorporation of 3H-methyl thymidine was measured by β-scintillation (disintegrations/min: DPM).

Criteria used to consider a positive response:
- Stimulation Index (SI) was calculated as Disintegrations per min (DPM) per concentration/DPM vehicle control. A positive response is indicated when the SI ≥ 3.
- The EC3 is the concentration for which a 3- fold increase in DPM is seen. The EC3 is calculated as follows: [(3-d)/(b-d)]* (a-c)+c, where a: concentration giving the SI immediately higher than 3, b: SI of a, c: concentration giving an SI immediately below 3, d: SI of c
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Positive control results:
The stimulation index at tested concentrations 5, 10 and 25% w/v were 1.7, 2.3, 6.4, respectively. This result confirms the validity of the test.
Key result
Parameter:
EC3
Remarks:
%
Value:
6.8
Parameter:
SI
Value:
1.5
Test group / Remarks:
0.5% concentration
Parameter:
SI
Value:
1.4
Test group / Remarks:
1% concentration
Parameter:
SI
Value:
2.1
Test group / Remarks:
2.5% concentration
Parameter:
SI
Value:
2.5
Test group / Remarks:
5% concentration
Parameter:
SI
Value:
3.9
Test group / Remarks:
10% concentration
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA
Ylang Ylang Extra EO comores induced a higher than 3-fold increase in lympocyte proliferation at the highest concentration tested (10% w/v).

DETAILS ON STIMULATION INDEX CALCULATION
Mean disintegrations per minute (DPM) values were:
- Vehicle control: 533
- 0.5%: 805
- 1%: 742
- 2.5%: 1099
- 5%: 1331
- 10%: 2090

EC3 CALCULATION
The calculated EC3 was 6.8%.

CLINICAL OBSERVATIONS:
No visual levels of skin irritancy were observed at any of the concentrations used on or around the esar area for the duration of the study.

BODY WEIGHTS
Body weights were recorded but showed no abnormalities.
Interpretation of results:
other: Skin Sensitiser 1B
Remarks:
Based on CLP criteria (Annex I of 1272/2008/EC)
Conclusions:
Ylang Ylang Extra EO comores induced a higher than 3-fold increase in lympocyte proliferation at the highest concentration tested (10% w/v). The calculated EC3 was 6.8%. Under the conditions of this assay, the test substance is needs to be classified as Skin Sensitiser Category 1B in accordance with Annex I of the CLP Regulation (1272/2008/EC).
Executive summary:

A Local Lymph Node Assay (LLNA) was performed according to the OECD guideline 429 (2002). Four mice per group were treated with 0%, 0.5%, 1%, 2.5%, 5%, and 10% w/v Ylang Ylang Extra EO Comores in 1:3 ethanol:diethyl phthalate (vehicle) for three consecutive days, followed by in vivo radiolabelling (by injection) and lymphocyte proliferation analysis with the liquid scintillation counter. The number of disintegrations per minute (dpm) was determined for each group for the pooled lymph nodes. Treatment with Ylang Ylang Extra EO Comores did induce significantly the lymph node proliferation in mice as compared to controls at the highest dose tested. A higher than 3-fold increase in lympocyte proliferation was found at the highest concentration tested (10% w/v). The calculated EC3 was 6.8%. Under the conditions of this assay, the test substance is needs to be classified as Skin Sensitiser Category 1B in accordance with Annex I of the CLP Regulation (1272/2008/EC).

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

A Local Lymph Node Assay (LLNA) was performed according to the OECD guideline 429 (2002). Four mice per group were treated with 0%, 0.5%, 1%, 2.5%, 5%, and 10% w/v Ylang Ylang Extra EO Comores in 1:3 ethanol:diethyl phthalate (vehicle) for three consecutive days, followed by in vivo radiolabelling (by injection) and lymphocyte proliferation analysis with the liquid scintillation counter. The number of disintegrations per minute (dpm) was determined for each group for the pooled lymph nodes. Treatment with Ylang Ylang Extra EO Comores did induce significantly the lymph node proliferation in mice as compared to controls at the highest dose tested. A higher than 3-fold increase in lympocyte proliferation was found at the highest concentration tested (10% w/v). The calculated EC3 was 6.8% and based on this the substance should be considered a skin sensitiser.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the available data, Ylang Ylang Ext, I and II should be classified for skin sensitisation (Skin Sens. 1B / H317) in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).