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Description of key information

Concentration (% v/v) in acetone/olive oil 4:1  Stimulation Index Result 
 25  1.16 Negative
 50  1.77 Negative 
 100  4.00  Positive

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:
07 March 2013 - 21 March 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in accordance with OECD test guidelines and in compliance with GLP.
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
not specified
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
not specified
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)
Specific details on test material used for the study:
Description: Brown Liquid
Batch: 12D25
Purity: 94.0%
Expiry Date: 30 January 2014
Storage Conditions: Room temperature in the dark
Species:
mouse
Strain:
CBA/Ca
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories UK Ltd, Oxon, UK
- Age at study initiation: Eight to twelve weeks old
- Weight at study initiation: 15 to 23 g
- Housing: Animals were randomly allocated to cages
- Diet (e.g. ad libitum): Ad libitum
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: At least five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25 °C
- Humidity (%): 30 to 70%
- Air changes (per hr): Approximately fifteen changes per hour
- Photoperiod (hrs dark / hrs light): Twelve hours continuous light (06.00 to 18.00) and twelve hours darkness

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
100% (v/v) in acetone/olive oil 4:1
No. of animals per dose:
4
Details on study design:
RANGE FINDING TESTS:
- Lymph node proliferation response: One mouse was treated by daily application of 25 ?L of the undiluted test item to the dorsal surface of each ear
for three consecutive days (Days 1, 2, 3). The mouse was observed twice daily on Days 1, 2 and 3 and once daily on Days 4, 5 and 6.
A mean ear thickness increase of equal to or greater than 25% was considered to indicate excessive irritation and limited biological relevance to the endpoint of
sensitisation.

MAIN STUDY:
ANIMAL ASSIGNMENT AND TREATMENT: Groups of four mice were treated with the undiluted test item or the test item at concentrations of 50% or 25% v/v in acetone/olive oil 4: 1. The preliminary screening test suggested that the test item would not produce systemic toxicity or excessive local skin irritation at the highest suitable concentration. The mice were treated by daily application of 25 ?L of the appropriate concentration of the test item to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3).
- Name of test method: Local Lymph Node Assay.
- Criteria used to consider a positive response: The test item will be regarded as a sensitizer if at least one concentration of the test item results
in a threefold or greater increase in 3HTdR incorporation compared to control values.

Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Positive control results:
The Stimulation Index expressed as the mean radioactive incorporation for the treatment group divided by the mean radioactive incorporation of the vehicle control group is as follows:
Concentration (% v/v) in acetone/olive oil (4:1): 25
Stimulation Index: 7.33
Result: Positive
Parameter:
SI
Remarks on result:
other: vehicle: n/a 25% (v/v): 1.16 50% (v/v): 1.77 100% (v/v): 4.00
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: vehicle: 19354.91 25% (v/v): 22450.71 50% (v/v): 34320.62 100% (v/v): 77510.55

The Stimulation Index expressed as the mean radioactive incorporation for each treatment group

divided by the mean radioactive incorporation of the vehicle control group are as follows:

 Concentration (% v/v) in acetone/olive oil 4:1  Stimulation Index Result 
 25  1.16 Negative
 50  1.77 Negative 
 100  4.00  Positive
Interpretation of results:
sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test item was considered to be a sensitizer under the conditions of the test.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

A study was performed to assess the skin sensitization potential of the test item in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear. The study was performed in compliance with the OECD Guidelines for Testing of Chemicals No. 429 "Skin Sensitization: Local Lymph Node Assay" (adopted 22 July 2010) and Method B42 Skin Sensitization (Local Lymph Node Assay) of Commission Regulation (EC) No. 440/2008.

The Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group are as follows:

 Concentration (% v/v) in acetone/olive oil 4:1  Stimulation Index  Result
 25  1.16  Negative
 50  1.77  Negative
 100  4.00  Positive

The concentration of test item expected to cause a 3 fold increase in 3HTdR incorporation (EC3 value) was calculated to be 78%.

The test item was considered to be a sensitizer under the conditions of the test.


Migrated from Short description of key information:
Skin Sensitization: Local Lymph Node Assay in female (CBA/Ca) mice - sensitizer under the conditions of the test.

Justification for selection of skin sensitisation endpoint:
Mice are the preferred species of choice since quantitative methods have been developed for the measurement of skin sensitization responses in the mouse, and are specified in the appropriate test guidelines.

Justification for classification or non-classification

Substances showing a low to moderate frequency of occurrence in humans and/or a low to moderate potency in animals can be presumed to have the potential to produce sensitisation in humans. Severity of reaction may also be considered.

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