Cedrus atlantica, ext.

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18-01-2016 to 25-01-2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

GLP compliance:

yes

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

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Adult donors

Source strain:

other: Strains no: 09-KERA-009, 08-KERA-008, 08-KERA-001, 10-KERA-005

Details on animal used as source of test system:

Not relevant

Justification for test system used:

In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimise the need of in vivo testing. One of the validated in vitro skin irritation tests is the EPISKIN test, which is recommended in international guidelines (e.g. OECD and EC).

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN Small ModelTM (EPISKIN-SMTM, 0.38 cm2, Batch no.: 16-EKIN-003)TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37.0 ± 1.0°C (actual range 36.2 - 36.7°C), with the exception of the test item incubation for 15 minutes at room temperature
- Temperature of post-treatment incubation (if applicable): 37.0 ± 1.0°C (actual range 36.2 - 36.7°C)

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: Tissues were washed with phosphate buffered saline to remove residual test item.
- Observable damage in the tissue due to washing: Not reported

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/ml- Incubation time: 3 hours at 37°C
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 3

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin if the relative mean tissue viability of three viability tissues after 15 minutes exposure and 42 hours post incubation is less than or equal to 50% of the mean viability of the negative controls.
- The test substance is considered to be non-irritant to skin if the relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is > 50% of the mean viability of the negative controls.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 µl

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 25 µl PBS

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 25 µl SDS
- Concentration (if solution): 5% in PBS

Duration of treatment / exposure:

15 ± 0.5 minutes at room temperature

Duration of post-treatment incubation (if applicable):

42 hours at 37°C

Number of replicates:

3

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean (percentage of control)

Value:

63

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

Mean tissue viability (percentage of control): 17%

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

- OTHER EFFECTS:
Cedar atlantica oil was checked for colour interference in aqueous conditions and possible direct MTT reduction by adding the test item to MTT medium. Because no colour changes were observed it was concluded that Cedar atlantica oil did not interact with the MTT endpoint.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The absolute mean OD570 (optical density at 570 nm) of the three tissues of the negative control should reasonably be within the laboratory historical control data range and the Standard Deviation value (SD) of the % viability should be ≤18. The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range.
- Acceptance criteria met for positive control: The mean relative tissue viability of the positive control should be ≤50% relative to the negative control and the Standard Deviation value (SD) of the % viability should be ≤18.The positive control had a mean cell viability after 15 ± 0.5 minutes exposure of 17%.
- Acceptance criteria met for variability between replicate measurements: The SD calculated from individual % tissue viabilities of the three identically treated replicates should be ≤18.The standard deviation value of the percentage viability of three tissues treated identically was less than 10%, indicating that the test system functioned properly.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

After treatment with the test item Cedarwood Atlas oil the relative cell viability values (compared to the negative control tissues) decreased to 63%. This value is above the threshold for irritancy of 50%. Therefore it can be concluded that Cedarwood Atlas oil is not irritant to skin and does not need to be classified for skin irritation in accordance with the criteria outlined in Annex I of CLP (1272/2008/EC).

Executive summary:

This in vitro study was performed to assess the irritation potential of Cedarwood Atlas oil by means of the Human Skin Model Test (OECD TG 439). Three tissues of the human skin model EpiSkin™ were treated with the test item, the negative or the positive control for 15 minutes. 25 µL of the test item was applied to each tissue, spread to match the tissue size. The test item and the positive and negative controls were washed off the skin tissues after 15 minutes treatment. After further incubation for about 42 hours the tissues were treated with the MTT [(3-4,5-dimethyl thiazole 2-yl) 2,5-diphenyl-tetrazoliumbromide] solution for 3 hours following approximately 71 hours extraction of the colorant from the cells. The amount of extracted colorant was determined photometrically at 570 nm. 25 µL of either the negative control (PBS) or the positive control (SDS) were applied to each tissue.

After treatment with the test item Cedarwood Atlas oil the relative cell viability values (compared to the negative control tissues) decreased to 63%. This value is above the threshold for irritancy of 50% as reported in the OECD guideline. Therefore it can be concluded that Cedarwood Atlas oil is not irritant to skin and does not need to be classified for skin irritation in accordance with the criteria outlined in Annex I of CLP (1272/2008/EC).