Cedrus atlantica, ext.

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20-01-2016 to 08-02-2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

in accordance with GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

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:

Cedrus atlantica, ext.

EC Number:

295-985-9

EC Name:

Cedrus atlantica, ext.

Cas Number:

92201-55-3

Molecular formula:

Not applicable due to UVCB nature of the substance

IUPAC Name:

Essential oil of Cedarwood Atlas obtained from the wood of Cedrus atlantica by steam distillation

Test material form:

liquid

Details on test material:

Name of test material as cited in study report: Cedar atlantica oil

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature

Specific details on test material used for the study:

- pH (1% in water, indicative range): 7.31 - 7.08 (determined by WIL Research Europe)

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France
- Age at study initiation: approximately 10 weeks old
- Weight at study initiation: body weight variation was within +/-20% of the sex mean.
- Housing: sawdust as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG,
Rosenberg, Germany). Paper (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United
Kingdom) and shelters (disposable paper corner home, MCORN 404, Datesand Ltd, USA) were
supplied as cage-enrichment. On Day 6, the animals were group housed in Makrolon MII type
cages with a sheet of paper instead of sawdust and cage enrichment.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24 °C
- Humidity (%): relative humidity of 40 to 70%
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12- hour light/ 12-hour dark cycle

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

Prescreen study: 50 - 100%
Main study: 0 - 10- 25 - 50 % (v/v)

No. of animals per dose:

5

Details on study design:

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response: Increased T-lymphocyte proliferation in the test groups as compared to the concurrent vehicle control group.

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 response 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 (based on the following reference: Basketter DA, Lea LJ, Dickens A, Briggs, D, Pate I, Dearman RJ and Kimber I.A comparison of statistical approaches to the derivation of EC3 values from local lymph node assay dose responses. J Appl Toxicol 1999; 19:261-266

Positive control substance(s):

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

Results and discussion

Positive control results:

The six monthly reliability check with Hexylcinnamaldehyde, indicates that the Local Lymph Node Assay as performed at WIL Research Europe is an appropriate model for testing for contact hypersensitivity

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

1

Test group / Remarks:

Concentration 0%

Results 2

Parameter:

SI

Value:

5.3

Test group / Remarks:

Concentration 10%

Results 3

Parameter:

SI

Value:

5

Test group / Remarks:

Concentration 25%

Results 4

Parameter:

SI

Value:

4.5

Test group / Remarks:

Concentration 50%

Results 5

Parameter:

other: Mean disintegrations per minute (DPM)/animal

Value:

772

Test group / Remarks:

Concentration 0%

Results 6

Parameter:

other: Mean disintegrations per minute (DPM)/animal

Value:

4 096

Test group / Remarks:

Concentration 10%

Results 7

Parameter:

other: Mean disintegrations per minute (DPM)/animal

Value:

3 836

Test group / Remarks:

Concentration 25%

Results 8

Parameter:

other: Mean disintegrations per minute (DPM)/animal

Value:

3 492

Test group / Remarks:

Concentration 50%

Cellular proliferation data / Observations:

DETAILS ON STIMULATION INDEX CALCULATION
A Stimulation Index (SI) is calculated for each group using the individual SI values. The individual SI is the ratio of the DPM/animal compared to the DPM/vehicle control group mean.

EC3 CALCULATION
The calculated SI values did not show a dose-response relationship and therefore an exact EC3 value could not be calculated. The EC3 value was established to be between >0 and 10%.

CLINICAL OBSERVATIONS:
Systemic Toxicity: No mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the main study. Body weights and body weight gain of experimental animals remained in the same range as controls over the study period.
Macroscopic Examination of the Auricular Lymph Nodes and Surrounding Area: All auricular lymph nodes of the animals treated at 10% and control groups were considered normal in size. Most nodes of the animals treated at 25% and 50% were considered enlarged. No macroscopic abnormalities of the surrounding area were noted for any of the animals.

Any other information on results incl. tables

Tables and figures have been included in the attached document "LLNA tables and figures".

 

Preliminary irritation study:

The results of the epidermal exposures for the selection of highest test substance concentration to be tested in the main study are described in the table (see attachment). Based on signs of systemic toxicity found at 100% (e.g. hunched posture and piloerection were noted for the animals treated at 100% on days 3 and 4, and on the same days very slight erythema was noted for all animals), the highest test item concentration selected for the main study was a 50% concentration.

Main study:

Body weights and body weight gain of experimental animals remained in the same range as controls over the study period. The auricular lymph nodes of the control group and animals treated at 10% were considered normal in size however most nodes of the animals treated at 25% and 50% were considered enlarged. Very slight erythema was noted for most animals treated at 50% (Days 2-3) and three animals treated at 25% (Day 3). Scaliness or scabs were noted for most animals treated at 25% and 50% on Day 6.

Applicant's summary and conclusion

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

The results indicate that the test substance elicits a SI ≥ 3. The EC3 value (the estimated test substance concentration that will give a SI =3) was established to be between >0 and 10%. Under the conditions of this test, Cedarwood Atlas oil needs to be classified for skin sensitization (H317, Skin Sens 1) in accordance with the criteria outlined in Annex I of CLP (1272/2008/EC).

Executive summary:

The Local Lymph Node Assay (according to OECD 429) was conducted to determine the sensitising potential of Cedar atlantica oil in mice. Lymph node proliferation was determined after exposure to 0%, 10%, 25% or 50% Cedarwood Atlas oil in vehicle, using radioactivity counts (DPM). A pooled approach (per test group) was used. Stimulation indices were calculated.

 

Measured disintegrations per node (pooled) were 772, 4096, 3836 and 3492 for the 0%, 10%, 25% and 50% dosing groups, respectively. Corresponding stimulation indices (SI) were calculated to be 5.3, 5.0 and 4.5 for the 10%, 20% and 50% concentration, respectively. The EC3 value (the estimated test substance concentration that will give a SI =3) was established to be between >0 and 10%.