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Description of key information

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.

Sandalwood, ext. was also assessed for corrsivity/irritancy to eyes in an OECD 437 study. The corrected mean opacity score found was 3. The corrected mean optical density (permeability) score was 0.033. The in vitro irritancy score (IVIS) was calculated as 3.5. TFS plantation-grown Indian sandalwood (santalum album) oil, Batch# APIS0-130608SD/SA therefore was not corrosive or a severe irritant in this test.

Furthermore, two in vivo studies have been cited in a review article, indicating an irritant effect to skin (rabbit and mice), but due to lack of access to the study reports, this information is only used as supportive information, as validity cannot be assessed appropriately (Klimisch 4).

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
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
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
GLP compliance:
yes (incl. QA statement)
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
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
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
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%.

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.

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.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye 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
Qualifier:
according to guideline
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Batch# APISO-130608SD/SA
Species:
cattle
Details on test animals or tissues and environmental conditions:
The bovine eyes were received from Spear Products on 01 Aug 2013 and transported to MB Research in Hank's Balanced Salt Solution with Pennstrep in a refrigerated container.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
Liquids were tested undiluted. Volume applied: 0.75 ml of each liquid (ethanol, MEM or liquid test article)
Duration of treatment / exposure:
10 (±1) minute exposure
Duration of post- treatment incubation (in vitro):
2 hours
Number of animals or in vitro replicates:
3 replicates for each treatment (ethanol, MEM or liquid test article)
Details on study design:
Test System
The bovine eyes were received from Spear Products on 01 Aug 2013 and transported to MB Research in Hank's Balanced Salt Solution with Pennstrep in a refrigerated container.
Pretest Procedure
Fresh assay solutions were prepared prior to use. MEM solution was prepared by stirring together one jar of MEM powder (sufficient to make one liter of solution), 2.2 g Sodium Bicarbonate, 0.292 g L-Glutamine, 10 ml of Fetal Bovine Serum (FBS) and brought to a final volume of 1000 mL with distilled water. The MEM solution was kept in a 32 °C (±1 °C) incubator for the duration of testing. Hanks Balanced Salt Solution (HBSS) was prepared by stirring together one jar of HBSS powder (sufficient to make one liter), 0.35 g Sodium Bicarbonate and brought to a final volume of 1000 mL with distilled water. HBSS was maintained at room temperature.
In addition, MEM solution with Phenol Red was prepared by stirring together 9.3 g MEM with Phenol Red (sufficient to make one liter), 2.2 g Sodium Bicarbonate, 0.292 g L-Glutamine, 10 mL Fetal bovine Serum and brought to a final volume of 1000 mL with distilled water. The MEM solution with Phenol Red was kept in a 32 °C (±1 °C) incubator for the duration of testing.
The eyes were examined prior to use on the day of dosing. Any eye with a cornea exhibiting evidence of vascularization, pigmentation, opacity or scratches was discarded. Corneas from eyes that were free of defects were dissected from the surrounding tissues. A 2 - 3 mm rim of sclera was left attached to each cornea. The corneas were then placed in a container of fresh HBSS.
The dissected corneas were mounted in specially designed holders that were separated into anterior and posterior chambers and filled separately. Each cornea was mounted allowing the epithelium of the cornea to project into the anterior chamber. The posterior chamber was filled with MEM solution ensuring contact with the endothelium. The anterior chamber was filled with MEM solution, ensuring contact with the epithelium. Each cornea was visually inspected again to ensure there were no defects.
The entire holder was incubated at 32 °C (±1 °C) and allowed to equilibrate for at least one hour but not longer than two hours.
A pre-exposure determination of opacity was made for each cornea by measuring each against the blank supplied with the opacitometer.
Following the equilibration, fresh pre-warmed MEM was added to both chambers and baseline opacity readings were taken for each cornea. Any corneas that showed macroscopic tissue damage (e.g., scratches, pigmentation, neovascularization) or an opacity unit of greater than 7, was discarded. The mean opacity of all equilibrated corneas was calculated. Three corneas with opacity values close to the median value for all corneas were selected as negative (or solvent) control corneas. The remaining corneas were then distributed into treatment and positive control groups.
A pre-exposure determination of opacity was made for each cornea by measuring each against the blank supplied with the opacitometer.
Study Procedure
Liquids were tested undiluted. Following the pretest observations, the MEM solution was removed from the anterior chamber. A volume of 0.75 mL of the ethanol, MEM or liquid test article was applied to the epithelium of each of the three positive controls, three negative controls, or three test article-treated corneas in a manner, which ensured the entire cornea was covered.
All holders and corneas were placed in a horizontal position (anterior side up) in the 32 °C (±1 °C) incubator. After 10 (±1) minute, the test article, ethanol or MEM solution in the controls were removed from the epithelium of the cornea and anterior chamber of the holder by washing with MEM solution containing phenol red. The anterior and posterior chambers of the holders were refilled with fresh MEM solution. Opacity measurements were made following the 10-minute exposure and MEM solution refill.
All corneas were incubated at 32 °C (±1 °C) for an additional two hours at which time the MEM solution in the anterior and posterior chambers was removed and the holders refilled with fresh MEM solution. A measurement of opacity was taken with each treated cornea compared to the blank supplied with the OP-KIT. This is the reading that was used in the final in-vitro calculations.
Immediately following the 2-hour opacity measurement, the MEM solution was removed from the anterior chamber and replaced with 1.0 mL of 0.4% sodium fluorescein solution (in Dulbecco's Phosphate Buffered Saline) for liquid test articles and corresponding controls. Each holder was returned to the 32 °C (±1 °C) incubator in a horizontal position (anterior side up) ensuring contact of the fluorescein with the cornea.
After 90 minutes, the fluid from the posterior chamber was removed and the amount of dye, which passed through the cornea, was measured as the optical density at 490 nm by a plate reader or spectrophotometer. A 1:1000 dilution of the fluorescein was prepared and measured in the plate reader or spectrophotometer as a measure of consistency.
Analysis of Data
Individual corrected opacity scores were calculated by subtracting the pretest score from the two-hour score. Corrected mean opacity scores were calculated by averaging the individual two-hour corrected opacity scores for a given dose group and subtracting the mean opacity score for the negative control group. A corrected mean opacity score was not calculated for the negative control, rather only the mean of the individual two-hour corrected opacity scores (with no subtraction of mean opacity score for negative control).
Individual corrected optical densities were calculated by subtracting the mean optical density for the negative control group from the individual optical density values. Individual corrected optical densities were not calculated for the negative control group. Corrected mean optical densities were calculated by averaging the individual corrected optical density values for a given dose group. A corrected mean optical density was not calculated for the negative control, rather only the mean of the individual optical densities.
The In Vitro Irritancy Score (IVIS) for the test article and positive control were calculated by adding the corrected mean opacity score to fifteen times the corrected mean optical density as shown by the equation below:
in Vitro Score = Corrected Mean Opacity Score + 15(Corrected Mean Optical Density Score)
OECD Guideline #437 defines a substance, which produces an In Vitro score of ≥55.1 as a corrosive or severe irritant. IVIS < 55.1 is not a corrosive or severe irritant and additional testing should be conducted for classification and labelling purposes.
Irritation parameter:
cornea opacity score
Run / experiment:
mean value, test item
Value:
3
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
cornea opacity score
Run / experiment:
mean value, positive control (Ethanol)
Value:
21.3
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
cornea opacity score
Run / experiment:
mean value, negative control (MEM)
Value:
-1
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
in vitro irritation score
Run / experiment:
calculated value, test item
Value:
3.5
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
in vitro irritation score
Run / experiment:
calculated value, positive control (Ethanol)
Value:
28.6
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
in vitro irritation score
Run / experiment:
calculated score, negative control (MEM)
Value:
-0.7
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
The corrected mean optical density (permeability) score for TEST ARTICLE was 0.033.
The corrected mean optical density (permeability) score for NEGATIVE CONTROL was 0.020
The corrected mean optical density (permeability) score for POSITIVE CONTROL was 0.486
Interpretation of results:
GHS criteria not met
Conclusions:
The corrected mean opacity score was 3. The corrected mean optical density (permeability) score was 0.033. The in vitro irritancy score (IVIS) was calculated as 3.5. TFS plantation-grown Indian sandalwood (santalum album) oil, Batch# APIS0-130608SD/SA is not corrosive or a severe irritant.
Executive summary:

Objective: To determine the potential for ocular irritation using an alternative to the Draize methodology. This protocol is based on the methodology described in the current OECD Guideline for the Testing of Chemicals #437.

Method Synopsis: Three bovine corneas per group were dosed with 0.75 ml of TFS plantation-grown Indian sandalwood (santalum album) oil, Batch# APISO-130608SD/SA, Minimal Essential Media (MEM) (negative control), or 100% Ethanol (positive control). Following a two-hour exposure for each group of dosed corneas, opacity measurements and sodium fluorescein permeability were determined.

Summary/Conclusion:

Test Article:The corrected mean opacity score was 3. The corrected mean optical density (permeability) score was 0.033. The in vitro irritancy score (IVIS) was calculated as 3.5. TFS plantation-grown Indian sandalwood (santalum album) oil, Batch# APIS0-130608SD/SA is not corrosive or a severe irritant.

Negative Control: The corrected mean opacity score was -1. The corrected mean optical density (permeability) score was 0.020. The IVIS was calculated as -0.70.

Positive Control: The corrected mean opacity score was 21.3. The corrected mean optical density (permeability) score was 0.486. The IVIS was calculated as 28.6.

All Controls were within normal limits.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

Sandalwood, ext. was tested for skin corrosion/irritation as well as for damage to eyes. Whereas the in vitro study for skin irritation showed a skin irritation potential, the study for eye irritation potential was negative. However, as some positive reactions were seen in human skin (patch tests 4 positives out of 203) as a pre-cautionary measure the substance is classified also for irritation to eyes (categroy 2). Thus, the substance sandalwood, ext. is considered a skin irritant, category 2 (H315) and an eye irritant (categroy 2, H319) according to GHS and/or CLP (Regulation EC No 1272/2008). Data on respiratory irritation is lacking.

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