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Description of key information

Sensitisation (OECD TG 429): sensitising

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
No data available
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The study was conducted according to OECD guideline 429 and under GLP conditions.
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Principles of method if other than guideline:
Not relevant
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Interfauna UK Limited, Blackthorne, Bicester, Oxon, UK
- Age at study initiation: Young adults (no specific data available)
Weight at study initiation: 17.1-20.3 g (1 animal with weight of 10.5 g, was possibly mis-weighed)
- Housing: Maximum of 4 mice per cage, under standard laboratory conditions
- Diet: Ad libitum, RM1 diet
- Water: Ad libitum, mains water
- Acclimation period: At least 5 days prior to start of dosing (under experimental conditions)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22+/-3
- Humidity (%): 30-70
- Air changes (per hr): min. 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: No data available
Vehicle:
other: 1:3 ethanol:diethyl phthalate
Concentration:
2.5%, 5%, 10%, 25%, 50% w/v and concurrent vehicle
No. of animals per dose:
4
Details on study design:
RANGE FINDING TESTS: No data available

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: OECD guideline 429 was followed.
- Criteria used to consider a positive response: Increased T-lymphocyte proliferation in the test groups as compared to the concurrent vehicle control group.

TREATMENT PREPARATION AND ADMINISTRATION:
All dose preparations were used within 24 hours of preparation. No correction was made based on the purity of the active ingredient in the test substance (purity not determined). Stability and achieved concentration were not determined. 25 uL of vehicle or Litsea cubeba oil in vehicle at a concentration of 2.5% -5% -10% - 25% or 50% w/v was applied to the dorsal surface of each ear during 3 consecutive days. Three days after the last application, the mice were injected with approx. 250 µl of PBS containing 3H-methyl thymidine (20 µCi).

ANALYSIS OF LYMPHOCYTE PROLIFERATION
Five hours after injecting radiolabelled thymidine animals were sacrificed (halothane inhalation). The lymph nodes of the mice in the same group were pooled, washed with PBS and resuspended in TCA before counted by liquid scintillation.

EXAMINATIONS
- Signs of systemic toxicity: checked at least once daily
- Bodyweights: recorded prior to dosing (day 1) and prior to injection of 3H-thymidine (day 6)

DATA EVALUATION
The disintegrations per minute (dpm) value was determined for each test group. This information was used to calculate the Stimulation Index (SI) for each of the test groups (disintegrations per minute of treatment group / disintegrations per minute of control group). A positive respons is indicated when one or more test groups shows a SI of 3. The EC3 value is the concentration at which a 3-fold increase of lymph node proliferation is observed. Interpolation is used to determine the EC3 value between two test concentrations.

POSITIVE CONTROL
Male mice were treated with 25 uL of acetone or 1%, 3% or 10% hexyl cinnamic aldehyde in acetone, using the same method as for the test groups.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
- Calculation of the Stimulation Index per treatment group
- Determination of the EC3 value (by interpolation)
Positive control results:
A dose-related response (increase of Stimulation Index; or SI) was observed for the three treatment groups as compared to the control group. The SI was >3 for the 2 highest concentrations applied, indicating that the study protocol is valid for determination of the substance potential to sensitize in mice.
Parameter:
SI
Remarks on result:
other: - 2.5% treatment group: 2.09 - 5% treatment group: 2.35 - 10% treatment group: 3.31 - 25% treatment group: 7.96 - 50% treatment group: 16.03
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: - Control group: 4866 dpm - 2.5% treatment group: 10143 dpm - 5% treatment group: 11431 dpm - 10% treatment group: 16087 dpm - 25% treatment group: 38734 dpm - 50% treatment group: 77974 dpm
Key result
Parameter:
EC3
Remarks:
% (w/v)
Value:
8.4

A dose-related increase in Stimulation Index was observed for the groups treated with Litsea cubeba oil. The EC3 value was determined to be 8.4% w/v.

Interpretation of results:
other: Sensitising
Remarks:
in accordance with the criteria outline in Annex I of 1272/2008/EC (CLP)
Conclusions:
In this study, treatment with Litsea cubeba oil was found to significantly induce lymph node proliferation in mice as compared to controls. The Stimulation Index showed a dose-related increase and the EC3 value was determined to be 8.4% w/v (based on interpolation between the 5% and 10% w/v dose). Based on this EC3 value, the substance should be classified as skin sensitiser (Cat. 1B) in accordance with the criteria outline in Annex I of 1272/2008/EC (CLP).
Executive summary:

This Local Lymph Node Assay (LLNA) was performed according to the methods outlined in OECD guideline 429. Four mice per group were treated with 0%, 2.5%, 5%, 10%, 25% or 50% Litsea Cubeba oil 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 Litsea Cubeba oil was found to significantly induce lymph node proliferation in mice as compared to controls. The Stimulation Index (SI) was calculated for the treatment groups (dpm treatment group / dpm control group) and a dose-related increase in SI was observed for the animal groups treated with Litsea cubeba oil. The EC3 value was calculated to be 8.4% w/v based on interpolation between the 5% and 10% w/v dose.

Based on the EC3 value of 8.4% that was found in this study, the substance should be classified as skin sensitiser (Cat. 1B) in accordance with the criteria outline in Annex I of 1272/2008/EC (CLP).

Endpoint:
skin sensitisation: in vitro
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
an in vitro skin sensitisation study does not need to be conducted because adequate data from an in vivo skin sensitisation study are available
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

A Local Lymph Node Assay (LLNA) was performed according to the methods outlined in OECD guideline 429. Treatment with Litsea Cubeba oil was found to significantly induce lymph node proliferation in mice as compared to controls. A dose-related increase in Stimulation Index (SI) was observed for the animal groups treated with Litsea cubeba oil and the EC3 value was determined to be 8.4% w/v (based on interpolation between the 5% and 10% w/v dose).

Justification for classification or non-classification

The available information indicates that Lisea cubeba oil has the potency to cause skin sensitisation. Based on the derived EC3 value of 8.4%, the substance should be classified for skin sensitisation (Skin Sens. 1B / H317) in accordance with the criteria outlined in Annex I of 1272/2008/EC (CLP).