Sandalwood, 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:

2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

5-(2,3-dimethyltricyclo[2.2.1.02,6]hept-3-yl)-2-methylpent-2-en-1-ol, stereoisomer

EC Number:

204-102-8

EC Name:

5-(2,3-dimethyltricyclo[2.2.1.02,6]hept-3-yl)-2-methylpent-2-en-1-ol, stereoisomer

Cas Number:

115-71-9

Molecular formula:

C15H24O

IUPAC Name:

5-(2,3-dimethyltricyclo[2.2.1.0~2,6~]hept-3-yl)-2-methylpent-2-en-1-ol

Constituent 2

Reference substance name:

[1S-[1α,2α(Z),4α]]-2-methyl-5-(2-methyl-3-methylenebicyclo[2.2.1]hept-2-yl)-2-penten-1-ol

EC Number:

201-027-2

EC Name:

[1S-[1α,2α(Z),4α]]-2-methyl-5-(2-methyl-3-methylenebicyclo[2.2.1]hept-2-yl)-2-penten-1-ol

Cas Number:

77-42-9

Molecular formula:

C15H24O

IUPAC Name:

2-methyl-5-(2-methyl-3-methylenebicyclo[2.2.1]hept-2-yl)pent-2-en-1-ol

Constituent 3

Reference substance name:

(Z)-5-[(6R)-4,6-dimethyl-6-bicyclo[3.1.1]hept-3-enyl]-2-methylpent-2-en-1-ol

Cas Number:

176777-61-0

Molecular formula:

C15H24O

IUPAC Name:

(Z)-5-[(6R)-4,6-dimethyl-6-bicyclo[3.1.1]hept-3-enyl]-2-methylpent-2-en-1-ol

Constituent 4

Reference substance name:

(E)-(R)-(+)-5-(2,3-dimethyltricyclo[2.2.1.0^2,6]hept-3-yl)-2-methyl-2-penten-1-ol

Cas Number:

14490-17-6

Molecular formula:

C15H24O

IUPAC Name:

(E)-(R)-(+)-5-(2,3-dimethyltricyclo[2.2.1.0^2,6]hept-3-yl)-2-methyl-2-penten-1-ol

Constituent 5

Reference substance name:

2-​Hepten-​1-​ol, 2-​methyl-​6-​(4-​methylphenyl)​-​, (2Z)​-

Cas Number:

122442-36-8

Molecular formula:

C15H22O

IUPAC Name:

2-​Hepten-​1-​ol, 2-​methyl-​6-​(4-​methylphenyl)​-​, (2Z)​-

Test material form:

liquid

In vitro test system

Test system:

human skin model

Source species:

human

Vehicle:

unchanged (no vehicle)

Details on test system:

EpiDermTm Tissue Samples:
EpiDermTm tissues, Lot 18581 Kit M, were received from MatTek on 23 Jul 2013. Upon receipt, tissues were incubated (37 °C ±1 °C, 5% CO2 ±1% CO2) with assay medium (MatTek) for a one-hour equilibration. The tissues were then moved to new wells with fresh medium for an additional overnight equilibration, for 18 ±3 hour. Equilibration medium was replaced with fresh medium before dosing.
Test Article Reduction of MTT:
100 pL of the test article were mixed with 1 mL of MTT solution (1 mg/mL Methyl thiazole tetrazolium diluted in Dulbecco's Modified Eagle's Medium (DMEM)). A Negative Control, 100 pL of Phosphate Buffered Saline, was tested concurrently. The solutions were incubated at room temperature in the dark for 60 minutes. After incubation, the solutions were visually inspected for purple coloration, which indicates that the test article reduced MTT. Since tissue viability is based on MIT reduction, direct reduction by a test article can exaggerate viability, making a test article seem less irritating than it really is. The test article did not reduce MTT and the assay continued as per the protocol.
Mesh Compatibility:
Pre-cut nylon meshes supplied with the tissues were placed on a slide and 30 pL of the test article or PBS Negative Control was applied. After 60 minutes exposure, the mesh was checked microscopically. No interaction between the test article or PBS and the mesh was observed so the test article was dosed using the mesh as a spreading aid.
Dosing:
30 pL of the test article or control articles were applied to the EpiDermTM tissue. The nylon mesh was then placed on top to facilitate even distribution of the test material. A Negative Control (phosphate buffered saline (PBS)) and a Positive Control (5% sodium dodecyl sulfate (SDS) solution, MatTek) were tested concurrently. Each treatment with test article or control was conducted in triplicate. The exposure period for the test article and controls was 60 minutes. The dosed tissues were placed in an incubator at 37 °C ±1 °C, 5% CO2 ±1% CO2 for 35 ±1 minutes, then returned to the sterile hood for the remainder of the 60 minute exposure period.
After dosing and incubation, the tissues were rinsed with PBS, blotted to remove the test substance and dry the tissue, and transferred to fresh medium. The rinsed EpiDermTM tissues were returned to the incubator for 24 ±2 hours. Medium was changed at 24 ±2 hours. Tissues were returned to the incubator for an additional 18 ±2 hours.
Tissue Viability (MTT Reduction):
At the end of the incubation period, each EpiDermTM tissue was rinsed with phosphate buffered saline (PBS) and transferred to a 24-well plate containing 300 pL of MTT solution (1 mg/mL MTT in DMEM). The tissues were then returned to the incubator for a three-hour MTT incubation period. Following the MTT incubation period, each EpiDermTM tissue was rinsed with PBS and then treated with 2.0 mL of extractant solution (isopropanol) per well for at least 2 hours, with shaking, at room temperature. Two aliquots of the extracted MTT formazan were measured at 540 nm using a plate reader (pQuant Plate Reader, Bio-Tek Instruments, Winooski, VT).
Analysis of Data:
The mean absorbance value for each time point was calculated from the optical density (OD) of the duplicate samples and expressed as percent viability for each sample using the following formula:
% viability = 100 * (OD sample/OD Negative Control)

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

30 µL of the test article or control articles were applied to the EpiDermTM tissue.

Duration of treatment / exposure:

60 minutes

Duration of post-treatment incubation (if applicable):

24 ±2 hours.
Medium was changed at 24 ±2 hours. Tissues were returned to the incubator for an additional 18 ±2 hours.

Number of replicates:

Each treatment with test article or control was conducted in triplicate

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean value, test article

Value:

6.5

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean value, positive control

Value:

4.6

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean value, negative control

Value:

100

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

The assay meets the acceptance criterion if the mean OD540 of the Negative Control tissues is ≥1.0 and ≤2.5, and the mean viability of Positive Control tissues, expressed as percentage of the Negative Control tissues, is ≤20%. In addition, the standard deviation (SD) calculated from individual percent tissue viabilities of the three identically-treated replicates is <18%.

Any other information on results incl. tables

Quality Controls:

The mean OD540 of the Negative Control tissues is 1.793, and the mean viability of Positive Control tissues is 4.6%. The standard deviation (SD) calculated from individual percent tissue viabilities of the three identically-treated replicates is 8.66% for the Negative Control and 0.21 % for the Positive Control. All Controls pass the acceptance criteria.

Applicant's summary and conclusion

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

The test article provided by MT Romance Australia Pty Ltd was tested using the MatTek EpiDermTM' Skin Irritation Test (SIT).
The mean viability of the test article tissues is 6.5% (SD 0.31).
The test article is irritant.

Executive summary:

Sandalwood, ext. was assessed in a skin irritation test according to OECD Guideline for the Testing of Chemicals No. 439 — In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method.

The mean viability of the test article tissues is 6.5% (SD 0.31), compared to 100% (negative control) and 4.6% positive control (5% sodium dodecyl sulfate solution). Thus, the test article is irritant.