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Description of key information

In a dermal sensitization study conducted according to OECD 429 with Jasmine absolute (grandiflorium) in 1:3 ethanol:diethylphthalate, young adult female CBA/CaOlaHsd mice (4 per dose group) were tested at concentrations of 1, 2.5, 5, 10 or 25% (w/v) in a local lymph node assay (LLNA). The EC3 value (derived by linear interpolation) was calculated to be at a test item concentration of 5.9%. Based from the results of this study, the test item is a dermal sensitizer under "Category 1B" according to UN GHS.

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:
2006-01-04 to 2006-05-19
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
adopted 2002
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)
Specific details on test material used for the study:
- Source: Robertet
- Colour: Brown-red
- Physical state: Liquid
- Batch No.: 1488614
- CTL test substance reference number: Yl3546/001
- Purity: not specified
- Storage conditions: Ambient temperature in the dark
- Expiry: July 2006

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
All dose preparation were used within 24 hours of preparation. No correction was made for the purity of the active ingredient in the test substance.
Species:
mouse
Strain:
other: CBA/Ca/Ola/Hsd
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Interfauna UK Limited, Blackthorne, Bicester, Oxon, UK
- Females (if applicable) nulliparous and non-pregnant: not specified
- Age at study initiation: 8-12 weeks of age
- Housing: 4 per group
- Diet (e.g. ad libitum): Yes, Diet (RMl), supplied by Special Diet Services Limited, Witham, Essex, UK
- Water (e.g. ad libitum): Yes, mains water, supplied by an automatic system
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12
Vehicle:
other: 1:3 ethanol:diethylphthalate
Concentration:
25 µL of a 1, 2.5, 5, 10 or 25 % w/v preparation of the test substance in 1:3 EtOH:DEP was applied
No. of animals per dose:
4
Details on study design:
MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay
- Criteria used to consider a positive response: The criterion for a positive response is that one or more concentrations of the test substance should elicit a 3-fold or greater increase in isotope incorporation relative to the vehicle control group. Consequently, a test substance which does not fulfil the above criterion is designated as unlikely to be a skin sensitiser.

TREATMENT PREPARATION AND ADMINISTRATION:
Groups of four female mice were used for this study. Approximately 25 µL of a 1, 2.5, 5, 10 or 25% w/v preparation of the test substance in 1:3 EtOH:DEP was applied, using a variable volume micro-pipette, to the dorsal surface of each ear. A vehicle control group was similarly treated using 1:3 EtOH:DEP alone. The procedure was repeated daily for 3 consecutive days. Three days after the third application, all the animals were injected, via the tail vein, with approximately 250 µL of phosphate buffered saline (PBS) containing 20 µCi of a 2.0 Ci/mmol specific activity 3H-methyl thymidine. Approximately 5 hours later, the animals were humanely killed by inhalation of halothane vapour followed by cervical dislocation. The draining auricular lymph nodes were removed from each animal and, together with the nodes from the other animals in the group, were placed in a container of PBS. A single cell suspension was prepared by mechanical disaggregation of lymph nodes through a 200-mesh stainless steel gauze. The cell suspensions were then washed three times by centrifugation with approximately 10 mL of PBS. Approximately 3 mL of 5% w/v trichloroacetic acid (TCA) was added and, after overnight precipitation at 4 °C, the samples were pelleted by centrifugation and the supernatant was discarded. The cells were then resuspended in approximately 1 mL of TCA. The lymph node suspensions were transferred to scintillation vials and 10 mL of scintillant (Optiphase) was added prior to B-scintillation counting using a Packard Tri-Carb 3100TR Liquid Scintillation Counter.

Clinical Observations:
Animals were checked at least once daily for signs of systemic toxicity.

Bodyweights:
The bodyweight of each animal was recorded prior to dosing on day 1 and prior to injection of 3^H-methyl thymidine on day 6.

Positive control study:
Approximately 25 µL of 5%. 10% or 25% w/v preparation of hexylcinnamaldehyde in acetone in olive oil (4:1) was applied, and a vehicle control group was similarly treated using acetone in olive oil (4:1) alone.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
The EC3 value was derived by interpolating between two points on the Stimulation Index (SI) axis, one immediately above and the other immediately below the SI value of 3 (vehicle-treated control values [SI=1] not being used for the latter). Where the data points lying immediately above and below the SI value of three have the co-ordinates a (the concentration giving the SI immediately above 3), b (the SI of a), c (the concentration giving the SI immediately below 3) and d (the SI of c), the EC3 value was calculated using the following equation: EC3= [(3-d)/(b-d)] x (a-c) + c. The quantity applied per square centimetre was derived from this value, assuming that the area of the mouse ear is 1cm2 and that 1 ul is equivalent to 1mg.
Positive control results:
The application of hexylcinnamaldehyde at concentrations of 5%, 10% and 25% w/v in acetone in olive oil (4:1) resulted in a greater than 3-fold increase in isotope incorporation at the 10 and 25 %w/v concentrations. Therefore, hexylcinnamaldehyde was shown to be a skin sensitiser, confirming the validity of the protocol used for the study.
Key result
Parameter:
EC3
Value:
5.9
Key result
Parameter:
SI
Value:
11.8
Test group / Remarks:
25%
Key result
Parameter:
SI
Value:
7.4
Test group / Remarks:
10%
Key result
Parameter:
SI
Value:
2
Test group / Remarks:
5%
Key result
Parameter:
SI
Value:
1.8
Test group / Remarks:
2.5%
Key result
Parameter:
SI
Value:
1.2
Test group / Remarks:
1%
Cellular proliferation data / Observations:
Evaluation of test substance:
The application of the test substance at concentrations of 1, 2.5, 5,10 and 25% in 1:3 EtOH:DEP resulted in an isotope incorporation which was greater than 3-fold at the 10 and 25% w/v concentrations. Consequently, the test substance is likely to be a skon sensitiser under the conditions of the test. The concentration given rise to a 3-fold increase in lymphocyte proliferation (EC3) was calculated to be 5.9% w/v. For detailed results please refer to table 1 in box "Any other information on results incl. tables".

Table 1: Skin sensitizing potential of the test item

Concentration of test substance (% w/v)

Number of lymph nodes assayed

Disintegrations per minute (dpm)

Dpm per lymph node

Test: control ratio (SI)

0 (vehicle only)

8

4785

598

N/A

1

8

5546

693

1.2

2.5

8

8817

1102

1.8

5

8

9653

1207

2.0

10

8

35190

4399

7.4

25

8

56341

7043

11.8

EC3

Calculated to be 5.9 % (1475 µg/cm2)

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
In conclusion, in a mouse local lymph node assay, the test item is considered to be a skin sensitizer (Skin Sens. 1B) with an EC3 value of 5.9%.
Executive summary:

In a dermal sensitization study conducted according to OECD 429 with Jasmine absolute (grandiflorium) in 1:3 ethanol:diethylphthalate, young adult female CBA/CaOlaHsd mice (4 per dose group) were tested at concentrations of 1, 2.5, 5, 10 or 25% (w/v) in a local lymph node assay (LLNA). The EC3 value (derived by linear interpolation) was calculated to be at a test item concentration of 5.9%. Based from the results of this study, the test item is a dermal sensitizer under "Category 1B" according to UN GHS.

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

In a dermal sensitization study conducted according to OECD 429 with Jasmine absolute (grandiflorium) in 1:3 ethanol:diethylphthalate, young adult female CBA/CaOlaHsd mice (4 per dose group) were tested at concentrations of 1, 2.5, 5, 10 or 25% (w/v) in a local lymph node assay (LLNA). The EC3 value (derived by linear interpolation) was calculated to be at a test item concentration of 5.9%. Based from the results of this study, the test item is a dermal sensitizer under "Category 1B" according to UN GHS.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the result from a dermal sensitization study conducted according to OECD 429, the target substance needs to be classified as Skin Sens 1B, H317 in accordance with CLP Regulation 1272/2008.