Melaleuca viridiflora extract

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 September – 04 October 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study conducted in compliance with OECD guideline 429.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Melaleuca viridiflora extract

EC Number:

310-217-5

EC Name:

Melaleuca viridiflora extract

Cas Number:

132940-73-9

IUPAC Name:

Essential oil of Melaleuca viridiflora (Myrtaceae) obtained from twigs and leaves by steam distillation

Test material form:

other: liquid

Details on test material:

- Name of test material (as cited in study report): NIAOULI Essential Oil (Melaleuca quinquenervia) 021261
- Physical state: Colourless liquid
- Analytical purity: Considered as 100 %
- Lot/batch No.: 1855343
- Date received: 06 September 2010
- Expiry date: 23 January 2011
- Storage condition of test material: Room temperature

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/J

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Elevage Janvier, France
- Age at study initiation: 8 weeks
- Weight at study initiation: 19.3-22.1 g
- Housing: Individually housed in suspended solid-floor polypropylene cages
- Diet (e.g. ad libitum): Diet (M20, SDS), ad libitum
- Water (e.g. ad libitum): Tap water, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25 °C
- Humidity (%): 30-70 %
- Air changes (per hour): 15/hour
- Photoperiod (hours dark / hours light): 12 hours dark / 12 hours light

Study design: in vivo (LLNA)

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

0, 5, 10, 25, 50 and 100 % (v/v) in acetone/olive oil (4:1, v/v)

No. of animals per dose:

Four females

Details on study design:

RANGE FINDING TESTS:
- Method: In the preliminary test, a mouse was treated with daily application of 25 µL of the test item (undiluted) to the dorsal surface of each ear for three consecutive days (Days 1, 2 and 3). Any signs of toxicity or excessive local irritation were recorded daily on Days 1, 2, 3, 4, 5 and 6. Ear thickness was recorded on Days 1, 3 and 6. Bodyweight of the mouse was recorded on Day 1 (prior to dosing) and on Day 6.
- Results: No systemic toxicity or excessive irritation was noted at 100 %. Therefore, the concentration of 100 % 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: Test item would be regarded as a sensitiser if at least one concentration of the test item produce a Stimulation Index > 1.4 (SI = cell count of treated group / cell count of control group).

TREATMENT PREPARATION AND ADMINISTRATION: 25 µL of test material at concentrations of 0 (vehicle control), 5, 10, 25, 50 and 100 % (v/v) in acetone/olive oil (4:1, v/v) were applied to the dorsal surface of each ear on Days 1, 2 and 3. On Day 6, all mice were euthanized with sodium pentobarbital and the draining auricular lymph node 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. A volume of 10 μL of this cell suspension was diluted in 10 mL of physiological saline solution (NaCl 0.9 %). The lymphocyte cells were counted using a cell counter (Beckman Coulter Z2).

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

Not applicable

Results and discussion

Positive control results:

Stimulation Index = 2.06

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: see Remark

Remarks:

- Stimulation Index (SI) calculated by pooled approach was 1.18, 0.96, 1.01, 1.52 and 2.35 for the treated groups at 5, 10, 25, 50 and 100 %, respectively. - EC1.4 value determined by linear interpolation of points on the dose-response curve was 44.12 %.

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: Not applicable

Any other information on results incl. tables

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

- Body weight: Bodyweight changes of the test animals were comparable to those observed in the corresponding control group animals, throughout the study.

- Skin irritation and ear thickness: Slight dryness to dryness was noted on the treatment site on Day 6 in all animals treated at 100 %. No significant increase in ear thickness and in ear weight was recorded at the concentrations up to 100 %.

Applicant's summary and conclusion

Interpretation of results:

sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Under the test conditions, NIAOULI Essential Oil (Melaleuca quinquenervia) 021261 is classified as ‘R43 May cause sensitisation by skin contact’, according to the criteria of Annex VI to the Directive 67/548/EEC and ‘Category 1’ according to the Regulation (EC) No 1272/2008 (CLP).

Executive summary:

In a local lymph node assay performed in CBA/J strain mice according to OECD guideline 429 and in compliance with GLP, six groups of mice (4 females/dose) were applied with 25 µL of the test item, NIAOULI Essential Oil (Melaleuca quinquenervia) 021261, at concentrations of 0 (vehicle control), 5, 10, 25, 50 and 100 % (v/v) in acetone/olive oil (4:1, v/v) (AOO) to the dorsal surface of each ear for three consecutive days. A group of 4 mice was treated with a positive control (alpha-hexylcinnamaldehyde) diluted at 25 % (v/v) in AOO. On Day 6, all animals were sacrificed and the proliferation of lymphocytes in the draining auricular lymph nodes was determined by cell counting.

No mortality or clinical sign of toxicity was noted at any dose level. No significant increase in ear thickness and in ear weight was recorded at any dose level. Stimulation Index (SI) calculated by pooled approach was 1.18, 0.96, 1.01, 1.52 and 2.35 for the treated groups at 5, 10, 25, 50 and 100 %, respectively, and EC1.4 value was determined to be 44.12 %. The SI value for the positive control was 2.06.

Under the test conditions, NIAOULI Essential Oil (Melaleuca quinquenervia) 021261 is classified as ‘R43 May cause sensitisation by skin contact’, according to the criteria of Annex VI to the Directive 67/548/EEC and ‘Category 1’ according to the Regulation (EC) No 1272/2008 (CLP).