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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:
25 January to 06 February 2012.
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
GLP study conducted in compliance with OECD Guideline No. 429 without any deviation. The substance is adequately identified, but some data on composition is missing. Therefore validation applies with restrictions.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
22 July 2010.
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. QA statement)
Remarks:
Inspected on 2010-12-07 / Signed on 2011-03-07.
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Chemical structure
Reference substance name:
2,6-dimethyl-4-(2,2,3-trimethylcyclopent-3-en-1-yl)cyclohex-2-en-1-ol [ISOMER 1]
Molecular formula:
C16 H26 O
IUPAC Name:
2,6-dimethyl-4-(2,2,3-trimethylcyclopent-3-en-1-yl)cyclohex-2-en-1-ol [ISOMER 1]
Test material form:
other: See specific details on each Endpoint Study Records
Details on test material:
- Storage condition of test material: Room temperature
Specific details on test material used for the study:
- Physical state: Colourless gel
- Date received: 13 January 2012

In vivo test system

Test animals

Species:
mouse
Strain:
CBA:J
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Elevage Janvier (F-53941 Le Genest Saint Isle)
- Age at study initiation: 9-10 weeks
- Weight at study initiation: 20.6-23.8 g
- Housing: Animals were individually housed in suspended solid-floor polypropylene cages furnished with softwood woodflakes.
- Diet: food (M20, SDS), ad libitum
- Water: tap water, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19-25 °C
- Humidity: 30-70 %
- Air changes: Approximately 15 changes/h
- Photoperiod: 12 h dark / 12 h light

IN-LIFE DATES: From 25 January to 06 February, 2012.

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
Preliminary screening test: 100%
Main test: 10% (v/v), 25% (v/v) and 50% (w/w) in the vehicle Acetone/olive oil (4/1, v/v)
No. of animals per dose:
4
Details on study design:
RANGE FINDING TESTS:
- As no available information was available regarding irritant potential or systemic toxicity of the test item in the mouse, a preliminary screening test was performed using one mouse. The mouse was treated by daily application of 25 μL of the test item at 100% to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The mouse was observed daily from day 1. Any signs of toxicity or excessive local irritation noted during this period were recorded. Ear thickness was recorded on day 1, day 3 and on day 6. The bodyweight of the mouse was recorded on Day 1 (prior to dosing) and Day 6.
- Irritation: No mortality and no signs of systemic toxicity were noted. Significant increase in ear thickness and in ear weight was noted in animal treated at 100%.
Therefore, the concentration of 50% was chosen as the highest concentration for the main study.

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay
- Criteria used to consider a positive response: The test item will be regarded as a sensitiser if at least one concentration of the test item results is greater than 1.4 compared to control values. Other relevant criteria such as dose-response and irritation level were also taken into account for the interpretation of the results. Any test item failing to produce a SI < 1.4 will be classified as a "non-sensitiser".

TREATMENT PREPARATION AND ADMINISTRATION:
25 μL of control or test substance was applied topically on the dorsal surface of both ears using a micropipette daily for three consecutive days (Days 1-3). On day 6 (end of the test), the animals were anaesthetised with sodium pentobarbital and administration continued to fatal levels. The draining auricular lymph nodes from the four mice were excised and pooled for each experimental group. A single cell suspension of the lymph node cells of 4 mice of each group was prepared by gentle mechanical tissue disaggregation through a 200-mesh cell strainers in 4 mL of PBS (Ca2+ / Mg2+ - free) containing 0.5% BSA into a well of a multi-well 6. 10 μL of this cell suspension was diluted in 10 mL of physiological saline solution (NaCl 0.9%). The lymphocyte cells were counting using a cell counter (Beckman Coulter Z2). For the run, the lower size selected was 5 μm and the upper size selected was 15 μm (the average size of a lymphocyte is 8 μm).
The proliferation response of lymph node cells was expressed as the number of lymphocytes per lymph node and as the ratio of lymphocytes into lymph node cells of test nodes relative to that recorded for the control nodes (Stimulation Index).
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
None.

Results and discussion

Positive control results:
Three groups, each of four animals, were treated with 50 μL (25 μL per ear) of α- Hexylcinnamaldehyde, as a solution in acetone/olive oil (4:1, v/v) at concentrations of 2.5%, 5% and 10% (v/v). With EC1.4 = 6.75, α-Hexylcinnamaldehyde is considered to be a sensitiser under the conditions of the test.

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
0.85
Test group / Remarks:
10% (v/v) in Acetone/Olive oil (4:1)
Key result
Parameter:
SI
Value:
1.19
Test group / Remarks:
25% (v/v) in Acetone/Olive oil (4:1)
Key result
Parameter:
SI
Value:
3.1
Test group / Remarks:
50% (v/v) in Acetone/Olive oil (4:1)
Key result
Parameter:
other: EC1.4
Remarks:
%
Value:
27.75
Test group / Remarks:
Test item
Cellular proliferation data / Observations:
- A stimulation index of more than 1.4 was recorded for one concentration of the test item (50% (v/v) in acetone/olive oil).
The Stimulation Index (SI) calculated by pooled approach was respectively 0.85, 1.19 and 3.1 for the treated group at 10%, 25% and 50%. The EC1.4 value determined by linear interpolation of points on the dose-response curve was 27.75%.

LOCAL IRRITATION
- No cutaneous reaction was noted in the treated animals.
- No significant increase in ear thickness and in ear weight was noted in animals treated at 10%, 25% and 50%. Therefore, the test item must be considered as not excessively irritant at the three concentrations.

CLINICAL OBSERVATIONS
- No mortality and no signs of systemic toxicity were noted in the test and controls animals during the test.

BODY WEIGHTS
- Bodyweight changes of the test animals between day 1 and day 6 were comparable to those observed in the corresponding control group animals over the same period.

Any other information on results incl. tables

None.

Applicant's summary and conclusion

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
Under these experimental conditions, the test material is classified as skin sensitiser in category 1B according to the annex I of the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.
Executive summary:

In a local lymph node assay performed according to OECD Guideline 429 and in compliance with GLP, three groups of CBA/J (CBA/J@Rj) strain mice (4 females/dose) were treated for three consecutive days (D1, D2, D3) with 50 μL (25 μL per ear) of the test item at concentrations of 10%, 25% and 50% (v/v) in Acetone/Olive oil (4:1). A further group of four animals was treated with Acetone/Olive oil (4:1). At D6, the proliferation of lymphocytes in the draining auricular lymph nodes was determined by cell counting. The test concentrations for the main study were determined from a preliminary study tested at 100 %. Based on the results of preliminary study, the dose levels selected for the main test were 10%, 25% and 50% (v/v) in Acetone/Olive oil (4:1).

 

No mortality and no signs of systemic toxicity were noted in the test and controls animals during the test. No significant increase in ear thickness and in ear weight was noted in animals treated at 10%, 25% and 50%. Therefore, the test item must be considered as not excessively irritant at the three concentrations.

A stimulation index of more than 1.4 was recorded for one concentration of the test item (50% (v/v) in acetone/olive oil). The Stimulation Index (SI) calculated by pooled approach was respectively 0.85, 1.19 and 3.10 for the treated group at 10%, 25% and 50%. The EC1.4 value determined by linear interpolation of points on the dose-response curve was 27.75%.

 

Under these experimental conditions, the test material is classified as skin sensitiser in category 1B according to the annex I of the Regulation (EC) No. 1272 /2008 (CLP) and to the GHS.

This study is considered as acceptable and satisfies the requirement for skin sensitisation endpoint.