Santalum austro-caledonicum, ext.

Endpoint summary

Description of key information

Skin sensitisation: skin sensitiser, LLNA study (OECD 429, GLP, K1).

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:

29 October to 15 December 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted in compliance with OECD Guideline No. 429 without any deviation.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

24 April 2002

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:

16 July 2008

Type of study:

mouse local lymph node assay (LLNA)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Robertet / 1733624
- Appearance: Yellow liquid
- Production date: 30 May 2008
- Date received: 28 October 2008
- Expiration date of the lot/batch: 30 May 2010

STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature

Species:

mouse

Strain:

other: CBA:J CBA/J@Rj

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Elevage Janvier (F-53941 Le Genest Saint Isle).
- Females nulliparous and non-pregnant: Yes
- Age at study initiation: 8 weeks
- Weight at study initiation: 19-23.8 g
- Housing: Animals were individually housed in suspended solid-floor polypropylene cages furnished with softwood woodflakes.
- Diet: Food, ad libitum
- Water: Drinking water (tap water from public distribution system), ad libitum
- Acclimation period: 5 days

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

IN-LIFE DATES: 29 October to 15 December 2008

Vehicle:

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

Concentration:

Preliminary screening test: undiluted test item (100%)
Main test: 5%, 10%, 25% (v/v) in Acetone/Olive oil (4:1) and 100% (first step); 2.5% (v/v) in Acetone/Olive oil (4:1) (second step)

No. of animals per dose:

Preliminary screening test: One animal/dose
Main test: 4 animals/dose

Details on study design:

PRELIMINARY STUDY
- As no toxicological information was available regarding the systemic toxicity/irritancy potential of the test item, a preliminary screening test was performed using one mouse. The mouse was treated by daily application of 25 µ of the undiluted test item to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The mouse was observed daily on Days 1, 2, 3, 4, 5 and 6. Any signs of toxicity or excessive local irritation noted during this period were recorded. The bodyweight of the mouse was recorded on Day1 (prior to dosing) and on Day 6.
- Irritation: No signs of systemic toxicity were noted. Based on this information the dose levels selected for the main test were 2.5%, 5%, 10%, 25% (v/v) in Acetone/Olive oil (4:1) and 100%. At the request of the sponsor the dose of 50% was not tested.

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

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) at concentrations of 5%, 10% and 25% (v/v). With EC1.4 = 4.27, α-Hexylcinnamaldehyde is considered to be a sensitiser under the conditions of the test.

Key result

Parameter:

SI

Value:

1.16

Test group / Remarks:

2.5%

Key result

Parameter:

SI

Value:

1.53

Test group / Remarks:

5%

Key result

Parameter:

SI

Value:

2.01

Test group / Remarks:

10%

Key result

Parameter:

SI

Value:

3.68

Test group / Remarks:

25%

Key result

Parameter:

SI

Value:

4.67

Test group / Remarks:

100%

Key result

Parameter:

other: EC1.4

Value:

4.12

Cellular proliferation data / Observations:

ESTIMATION OF THE PROLIFERATIVE RESPONSE OF LYMPH NODE CELLS
- A stimulation index of more than 1.4 was recorded for four concentrations of the test item (5%, 10%, 25% (v/v) in Acetone/Olive oil (4:1) and 100%).
- The Stimulation Index (SI) calculated by pooled approach was respectively 1.16, 1.53, 2.01, 3.68 and 4.67 for the treated group at 2.5%, 5%, 10%, 25% and 100%.
- The EC1.4 value determined by linear interpolation of points on the dose-response curve was 4.12.

LOCAL IRRITATION
- Slight dryness to dryness was registered on day 5 and day 6, in the treated group at 10%, 25% and 100%.
- A slight increase in ear thickness (+24.8% and +14.3%) and in ear weight (+13.0% and + 14.3%) was recorded at 25% and 100% respectively.
- Therefore, the test item must be considered "slightly irritant" at the concentrations of 25% and 100% and "non irritant" at the concentrations of 2.5%, 5% and 10%.

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.

None

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Conclusions:

Under these experimental conditions and in accordance with the Globally Harmonized System (COM (2007)355 final), the test item must be classified in category 1 "Skin sensitisation".

Executive summary:

In a local lymph node assay performed according to OECD Guideline 429 and in compliance with GLP, four groups, each of four female CBA:J CBA/J@Rj mice, were treated for three consecutive days (D1, D2, D3) with 50 µL (25 µL per ear) of the undiluted test item and the test item as a solution in Acetone/Olive oil (4:1) at concentrations of 5%, 10% and 25% (v/v) in a first step and at concentration of 2.5% (v/v) in a second step. A further group of four animals was treated with Acetone/Olive oil (4:1). On D5, 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 2.5%, 5%, 10%, 25% (v/v) in Acetone/Olive oil (4:1) and 100%.

 

No mortality and no signs of systemic toxicity were noted in the test and controls animals during the test. Slight dryness to dryness was registered on day 5 and day 6, in the treated group at 10%, 25% and 100%. A slight increase in ear thickness (+24.8% and +14.3%) and in ear weight (+13.0% and +14.3%) was recorded at 25% and 100% respectively. Therefore, the test item must be considered "slightly irritant" at the concentrations of 25% and 100% and "non irritant" at the concentrations of 2.5%, 5% and 10%.

 

The Stimulation Index (SI) calculated by pooled approach was respectively 1.16, 1.53, 2.01, 3.68 and 4.67 for the treated group at 2.5%, 5%, 10%, 25% and 100%. The EC1.4 value determined by linear interpolation of points on the dose-response curve was 4.12.

 

Under these experimental conditions and in accordance with the Globally Harmonized System , the test item must be classified in category 1 "Skin sensitisation".

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information:

In a local lymph node assay performed according to OECD Guideline 429 and in compliance with GLP,four groups, each of four femaleCBA:J CBA/J@Rj mice, were treated for three consecutive days (D1, D2, D3) with 50 µL(25 µLper ear) of the undiluted test item and the test item as a solution in Acetone/Olive oil (4:1) at concentrations of 5%, 10% and 25% (v/v) in a first step and at concentration of 2.5% (v/v) in a secondstep. A further group of four animals was treated with Acetone/Olive oil (4:1). On D5, 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 2.5%, 5%, 10%, 25% (v/v) in Acetone/Olive oil (4:1) and 100%.

 

No mortality and no signs of systemic toxicity were noted in the test and controls animals during thetest. Slight dryness to dryness was registered on day 5 and day 6, in the treated group at 10%, 25% and 100%. A slight increase in ear thickness (+24.8% and +14.3%) and in ear weight (+13.0% and +14.3%) was recorded at 25% and 100% respectively. Therefore, the test item must be considered "slightly irritant" at the concentrations of 25% and 100% and "non irritant" at the concentrations of 2.5%, 5% and 10%.

 

The Stimulation Index (SI) calculated by pooled approach was respectively 1.16, 1.53, 2.01, 3.68 and 4.67 for the treated group at 2.5%, 5%, 10%, 25% and 100%. The EC1.4 value determined by linear interpolation of points on the dose-response curve was 4.12.

 

Under these experimental conditions and in accordance with the Globally Harmonized System , the test item must be classified in category 1 "Skin sensitisation".

Justification for classification or non-classification

Harmonized classification:

The registered substance has no harmonized classification according to the Regulation (EC) No. 1272/2008.

 

Self-classification:

Based on the available information the substance is classified as:

- Skin Sens. 1B, H317 (May cause an allergic skin reaction) according to the criteria of the Annex VI of the Regulation (EC) No. 1272/2008 (CLP).

No information was available regarding respiratory sensitisation.