Santalum austro-caledonicum, ext.

Endpoint summary

Description of key information

In an in vitro skin irritation and skin corrosion study (OECD 439 & OECD 431) test item was irritant to skin (GLP, Rel. K1)

In an in vitro eye irritation study (OECD 492) test item was considered non irritant to the eyes (GLP, Rel K1)

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

February 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted in compliance with OECD Guideline No. 431 without any deviation.

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Version / remarks:

13 April 2004

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

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

Test system:

human skin model

Source species:

human

Cell type:

other: reconstituted epidermis

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: 0.38 cm2 reconstituted epidermis (Episkin®)
- Tissue batch number(s): Episkin® - Batch No. 09-EKIN-006
- Delivery date: 10 February 2009
- The insert (filter + epidermis) was gently removed from the agarose while avoiding leaving agarose on the polycarbonate filter. The insert was placed in a 12 wells culture plated which has been previously filled with 2.2 mL of assay medium (Skinethic 09-ESSC-006).
- Date of initiation of testing: 10 February 2009

EXPOSURE
- The test item has been applied to the epidermal surface of 2 human skin model, during 3 minutes and during 1 hour.

REMOVAL OF TEST MATERIAL AND CONTROLS
- 3 minutes and 1 hour after the test item application, the human epidermis was washed with PBS (3 x 0.5 mL).

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
The cell viability was quantified by measurement of the cell succinate dehydrogenase activity. This enzyme was responsible for the MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue; CAS No. 298-93-1)] reduction into blue formazan crystal. The skin sample was placed in MTT solution of 0.5 mg/mL concentration for 3 hours at 37 °C. The precipitated blue formazan product is then extracted using acidic isopropanol between 2 and 4 hours under agitation in the dark, and the concentration of formazan was measured by determining the Optical Density (OD) at 570 nm.
The absorbance was measured in triplicate of MTT extract. The measured absorbances are proportional to the number of live cells.

NUMBER OF REPLICATE TISSUES:
Duplicate skin tissues for test item, negative and positive controls

VIABILITY
Viability = (OD test item / OD negative control) x 100
For each tissue, OD values and calculated percentage cell viability data for the test item, positive and negative controls, should be reported in tabular form, including data from replicate repeat experiments as appropriate, mean and individual values.

PREDICTION MODEL / DECISION CRITERIA
The optical density (OD) values obtained for each test sample were used to calculate a percentage of viability relative to the negative control, which was arbitrarily set at 100%. The cut-off values for the prediction of corrosion associated with the EPISKlN models were as follows:
The item is to be classified as non-corrosive: If the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%
The item is to be classified as corrosive R35: Causes severe burns: If the viability after 3 minutes exposure is strictly less than 50%,
The item is to be classified as corrosive R34: Causes burus: If the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is strictly less than 15%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 µL
- Concentration (if solution): Undiluted

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 10 µL distilled water

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 10 µL 8N KOH

TREATMENT
- The test item has been applied, as supplied, at the dose of 10 µL, to the epidermal surface of 2 human skin model, during 3 minutes and during 1 hour. In the same experimental conditions, the positive and negative controls have been carried out.

Duration of treatment / exposure:

Test item has been applied to the epidermal surface of human skin models during 3 minutes and during 1 hour.

Number of replicates:

Duplicate skin tissues for test item, negative and positive controls

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Main test (3 minutes)

Value:

99

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

34.7% of viability

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Main test (1 hour)

Value:

106

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

10.9% of viability

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

VIABILITY
- 3 minutes and 1 hour after the test item application, the viability of the human skin model has been 99% and 106% (considering as 100%) respectively.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes; 3 minutes and 1 hour after the negative control application, the viability of the human skin model has been 100%.
- Acceptance criteria met for positive control: Yes; 3 minutes and 1 hour after the positive control application, the viability of the human skin model has been 34.7% and 10.9%, respectively.

Table 7.3.1/1: Assessment of the skin corrosion

Items	Skin	Absorbances #	Mean	Viability%	Conclusion
Individual and average values of OD after 3 minutes exposure
Negative control	1	0.471	0.464	100.0	Non Corrosive
	2	0.457
Positive control	3	0.158	0.161	34.7	Corrosive
	4	0.164
Test item	5	0.427	0.460	99.0	Non Corrosive
	6	0.492
Individual and average values of OD after 1 hour exposure
Negative control	1	0.514	0.523	100.0	Non Corrosive
	2	0.531
Positive control	3	0.055	0.057	10.9	Corrosive
	4	0.059
Test item	5	0.538	0.555	106.2	Non Corrosive
	6	0.572

#: mean of 3 values

OD: optical density

Note:

30 minutes exposure: If the viability obtained for the test substance is greater than 50%, then it is non-corrosive.

1 hour exposure: If the viability obtained for the test substance is greater than15%, then it is non-corrosive.

Interpretation of results:

other: the test item must not be classified in category 1"Corrosive".

Conclusions:

Under the experimental conditions adopted and in accordance with the Globally Harmonized System (Regulation (EC) No.1272/2008), the test item must not be classified in category 1"Corrosive".

Executive summary:

An in vitro skin corrosion study was performed according to OECD Guideline 431 and in compliance with GLP to evaluate the possible corrosive effects of the test item after topical administration on in vitro human reconstituted epidermis (Episkin^®model).

 

The test item was applied, as supplied, at the dose of 10 µL, to 2 Human skin model surfaces (Episkin^®), during 3 minutes and 1 hour (n=4).

 

3 minutes and 1 hour after the test item application, the viability of the human skin model has been 99% and 106% (considering as 100%) respectively, versus 34.7% and 10.9% respectively in the positive control (8NKOH).

 

Under the experimental conditions adopted and in accordance with the Globally Harmonized System (Regulation (EC) No.1272/2008), the test item must not be classified in category 1"Corrosive".

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 February to 02 March 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted in compliance with OECD Guideline No. 439 without any deviation.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

28 July 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

27 April 2017

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Robertet / 2582250
- Appearance: Colorless to pale yellow liquid
- Manufacturing date: 04 November 2016
- Date received: 27 January 2017
- Expiration date of the lot/batch: 04 November 2018
- Purity test date: 10 February 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, darkness

Test system:

human skin model

Source species:

other:

Cell type:

other: reconstructed epidermis

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- The 0.50 cm² reconstructed epidermises (Episkin SA, RHE/S/17 Batch No. 17-RHE-023) were received on 28 February 2017. The same day, the insert (filter + epidermis) was gently removed from the agarose while avoiding leaving agarose on the polycarbonate filter. The inserts were placed in 6 wells culture plate which had been previously filled with 300 µL of growth medium (Episkin SA, batch No. 17 MPE 018) during 2 hours and 20 minutes. Then just before treatment, the inserts were placed in 24 wells culture plate which had been previously filled with 300 µL of maintenance medium (Episkin SA, batch No. 17 MA 012).
The two killed Reconstructed Human epidermis control tissue models, supplied by Episkin (Batch No. 17-RHE-021) were defrost on 21 February 2017, re-freeze on 21 February 2017 and defrost to be used on 28 February 2017.

TREATMENT
- The test item was applied, as supplied, at the dose of 16 µL, to the epidermal surface of 3 living human skin models and 2 killed Reconstructed Human epidermis (for non-specific MTT reduction control) during 42 minutes at room temperature. To ensure a good contact with the epidermises, during all the treatment period, the test item was recovered with a nylon mesh provided by Episkin SA.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: Test item was applied for 42 minutes at room temperature
- Temperature of post-treatment incubation: 41 hours and 50 minutes post-treatment incubation period in fresh medium at 37 °C, 5% CO2

REMOVAL OF TEST MATERIAL AND CONTROLS
- 42 minutes after the test item application, the nylon mesh was removed and the human epidermises were washed with 25 x 1 mL of DPBS. The rinsed tissues were checked for any coloration and noted to be whitish, comparable coloration to that of the negative control tissues. They were incubated for a 41 hours and 50 minutes post-treatment incubation period in fresh medium at 37°C, 5% CO2. Then, the epidermises were put in contact with the MTT solution.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- The cell viability was quantified by measurement of the cell succinate dehydrogenase activity. This enzyme is responsible for the MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue; CAS No. 298-93-1)] reduction into blue formazan crystal that is quantitatively measured by Optical Density (OD) after extraction from tissues. The measured OD are proportional to the number of living cells.
- The skin samples (included the 2 killed Reconstructed Human epidermis ) were placed in 300 µL of a MTT solution at 1.0 mg/mL for 2 hours and 55 minutes at 37°C, 5% CO2. The precipitated blue formazan product was then extracted using isopropanol during 2 hours under gentle agitation in the dark, and the concentration of formazan was determined by measuring the OD at 570 nm, just after dilution of the extracts 1:2 in isopropanol.
- The OD was measured in triplicate of MTT extract. The measurement of OD at 570 nm of formazan extracts was performed in triplicate samples using the ELx800 absorbance microplate reader (controlled every year and calibrated if necessary) supplied by BioTek and the validated software Gen5 ELISA V1.05.11 supplied by BioTek.
- The linearity range of optical density measured is validated for an optical density between 0 and 2.0.

VIABILITY:
Viability % = (mean OD test item / mean OD negative control) x100
As the test item was a direct MTT-reducer, true tissue viability was calculated as follows:
True Viability % = [(mean OD test item – mean OD non-specific MTT killed control)/ mean OD negative control] x 100
For each tissue, OD values and calculated percentage cell viability data for the test item, positive and negative controls, were reported in tabular form, including data from replicate repeat experiments as appropriate, mean and individual values.

NUMBER OF REPLICATE TISSUES:
3 living human skin models and 2 killed Reconstructed Human epidermis (for non-specific MTT reduction control)

PREDICTION MODEL / DECISION CRITERIA
- The OD values obtained for each test sample were used to calculate a percentage of viability relative to the negative control, which was arbitrarily set at 100%. The cut-off values for the prediction of irritation associated with the RHE models were as follows:
The test item is considered as non-irritant to skin: if the mean percent viability after 42 minutes exposure and 42 hours of post-treatment incubation is > 50%.
The test item is considered to be irritant to skin: if the mean percent tissue viability after 42 minutes exposure and 42 hours of post-treatment incubation is ≤ 50% and the result of a skin corrosion test is “non-corrosive”.
The test item is considered to be irritant or corrosive to skin: if the mean percent tissue viability after 42 minutes exposure and 42 hours of post-treatment incubation is ≤ 50% and in absence of information on a skin corrosion test.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 16 µL
- Concentration (if solution): Undiluted

Duration of treatment / exposure:

42 minutes at room temperature.

Duration of post-treatment incubation (if applicable):

41 hours and 50 minutes post-incubation period at 37 °C, 5% CO2

Number of replicates:

3 living human skin models and 2 killed Reconstructed Human epidermis (for non-specific MTT reduction control)

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean

Run / experiment:

Main test (42 minutes)

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

VIABILITY
- The mean corrected percent viability of the treated tissues was -2.7% (considered as 0%), versus 1.3% in the positive control (5% Sodium Dodecyl Sulfate).

ACCEPTANCE OF RESULTS:
- The mean percent tissue viabilities obtained with the negative control and positive controls are within the range of historical data and therefore validate the experiment.

Table 7.3.1/1: Assessment of the skin irritation - individual and average values of OD after 42 minutes exposure

 

Items	Skin	OD	Mean OD/disc#	Mean OD/product	Viability%	Mean viability%	SD	Conclusion
Negative control	1	1.094	1.109	1.113	99.6	100.0	0.7	-
		1.161
		1.073
	2	1.098	1.108		99.6
		1.137
		1.090
	3	1.057	1.122		100.8
		1.182
		1.126
Positive control	4	0.015	0.015	0.014	1.3	1.3	0.1	Irritant
		0.015
		0.015
	5	0.014	0.014		1.3
		0.014
		0.014
	6	0.012	0.013		1.2
		0.014
		0.012
Test item	7	0.047	0.049	0.045	4.4	4.1	0.4	-
		0.051
		0.049
	8	0.040	0.040		3.6
		0.040
		0.041
	9	0.048	0.047		4.2
		0.047
		0.047
Test item Killed tissues	13	0.067	0.068	0.075	6.1	6.7	0.9
		0.069
		0.067
	14	0.082	0.082		7.4
		0.080
		0.083
Test item corrected	-					-2.7	-	Corrosive or irritant

 

≠: mean of 3 values (triplicate of the same extract)

OD: optical density

Acceptance criteria: SD ≤ 18%

Interpretation of results:

other: Category 2 “Irritant” or Category 1 “Corrosive” based on GHS criteria

Conclusions:

Under these experimental conditions and in accordance with the Regulation (CE) No.1272/2008 and in absence of information on a skin corrosion, the test item has to be classified in Category 2 “Irritant” or in Category 1 “Corrosive”.

Executive summary:

An in vitro skin irritation study was performed according to OECD Guideline 439 and in compliance with GLP to evaluate the possible irritating effects of the test item Sandalwood Austrocaledonien Oil after topical application on in vitro human reconstructed epidermis (SkinEthic RHE^®model).

 

The test item was applied, as supplied, at the dose of 16 µL, to 3 living and 2 killed Reconstructed Human epidermis (SkinEthic RHE^® model) during 42 minutes, followed by a rinse with 25 mL of DPBS and a41 hours and 50 minutes post-incubation period at 37 °C, 5% CO₂. Cell viability was then measured by enzymatic conversion of the vital dye MTT into a blue formazan salt that was quantitatively measured after extraction from tissues.

 

The mean percent tissue viabilities obtained with the negative control and positive controls are within the range of historical data and therefore validate the experiment.

 

The mean corrected percent viability of the treated tissues was 0%, versus 1.3% in the positive control (5% Sodium Dodecyl Sulfate).

 

Under these experimental conditions and in accordance with the Regulation (CE) No.1272/2008 and in absence of information on a skin corrosion, the test item has to be classified in Category 2 “Irritant” or in Category 1 “Corrosive”.

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:

15 February to 09 March 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study performed according to OECD Guideline 492 without any deviation.

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Version / remarks:

adopted 28 July 2015

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

27 April 2017

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Robertet / 2582250
- Appearance: Colorless to pale yellow liquid
- Manufacturing date: 04 November 2016
- Date received: 27 January 2017
- Expiration date of the lot/batch: 04 November 2018
- Purity test date: 10 February 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, darkness

Species:

other: reconstructed human cornea-like epithelium tissues

Details on test animals or tissues and environmental conditions:

- Description of the cell system used:
Reconstructed human Cornea-like Epithelium (RhCE) tissue constructs: The 0.60 cm² Reconstructed human Cornea-like Epithelia (EpiOcularTM OCL-212-ver2.0, supplied by MatTek Corporation, batch No. 23770) were received on 08 March 2017.
The killed Reconstructed human Cornea-like Epithelia (EpiOcularTM OCL-212-ver2.0, supplied by MatTek Corporation, batch No. 23706) were defrost on 03 March 2017, re-freeze on 03 March 2017 and defrost to be used on 08 March 2017.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 μL
- Concentration (if solution): Undiluted

Duration of treatment / exposure:

30 minutes at 37 °C, 5% CO2, 95% humidity (standard culture conditions).

Duration of post- treatment incubation (in vitro):

After 30 minutes of exposure, extensive rinsing with DPBS at room temperature, a 12 minutes post-exposure immersion period at room temperature and a 2 hours and 03 minutes post-exposure incubation at standard culture conditions.

Number of animals or in vitro replicates:

Test item, negative and positive controls were applied on duplicate tissues (2 living RhCE tissue replicates and 2 killed DPBS pre-treated Reconstructed human Cornea-like Epithelia tissue replicates).

Details on study design:

- RhCE tissue construct used, including batch number: The 0.60 cm² Reconstructed human Cornea-like Epithelia (EpiOcularTM OCL-212-ver2.0, supplied by MatTek Corporation, batch No. 23770) were received on 08 March 2017. The killed Reconstructed human Cornea-like Epithelia (EpiOcularTM OCL-212-ver2.0, supplied by MatTek Corporation, batch No. 23706) were defrost on 03 March 2017, re-freeze on 03 March 2017 and defrost to be used on 08 March 2017.
The same day, the 12 tissues in their 24-well shipping container were equilibrated at room temperature for 15 minutes. Then the inserts (filter + epithelium) were gently removed from the agarose while avoiding leaving agarose on the polycarbonate filter. They were placed in 6 wells culture plate which had been previously filled with 1 mL of 37 °C pre-warmed assay medium (MatTek Corporation, batch No. 030617MGKA) and incubated during 19 hours and 55 minutes at standard culture conditions.

- Evaluation of direct interaction with MTT: The direct interaction of MTT with the test item was checked by adding 16 µL of the test item to 300 µL of solution of MTT at 1 mg/mL. A yellow solution with purple spots on the surface was observed after 3 hours of incubation between 36.3 °C and 37.6 °C, 5% CO2. Therefore, the test item was identified as a direct MTT reducer and two killed control tissue models were added to the study which underwent the entire testing procedure to generate a non-specific MTT reduction control.

- Doses of test chemical and control substances used: 50 μL of test substance, negative or positive controls used.

- Pre-treatment: After the overnight incubation, the tissues were pre-wetted with 20 μL of Ca2+Mg2+Free-DPBS. The tissues were incubated at standard culture conditions for 30 minutes.

Treatment of tissues
Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods:
- The test item was applied, as supplied, at the dose of 50 μL, to the entire surface of 2 living RhCE tissue replicates and 2 killed DPBS pre-treated Reconstructed human Cornea-like Epithelia tissue replicates during 30 minutes at standard culture conditions. In the same experimental conditions, the positive and negative controls were carried out.
- After the treatment, the test item and control substances were carefully washed from the RhCE tissues by extensive rinsing with Ca2+Mg2+Free-DPBS. The rinsed tissues were checked for any coloration and noted to be of comparable colour with the negative control treated tissues (whitish). This rinsing step was followed by a 12-minute post-exposure immersion period at room temperature in 5 mL of fresh medium to remove any test item absorbed into the tissue. The RhCE constructs were then incubated for a 2 hours and 03 minutes post-exposure incubation at standard culture conditions in 1 mL of fresh medium at 37 °C, 5% CO2.

- Indication of controls used for direct MTT-reducers and/or colouring test chemicals (if applicable):
The spectral properties at 570 nm of test item in isopropanol were checked by adding 16 µL of the test item to 1.5 mL of isopropanol (same conditions as in the main test). A yellowish solution was obtained after 2 hours of incubation at ambient temperature with gentle shaking. The mean of the corrected OD (Optical density) (blank subtracted) was 0.000 which is less than 0.08 (value corresponding to approximately twice the OD of the extracting solvent). Therefore the test item was considered not to interfere with the MTT assay and there was no need to add non-specific coloration controls to the study.

- Cell viability measurements: Following the exposure to the test item, viability measurements are performed immediately after the post-exposure incubation period of the rinsed tissues in fresh medium. This period allows both for recovery from weak cytotoxic effects and for appearance of clear cytotoxic effects. The RhCE tissue viability was measured by enzymatic conversion of the vital dye MTT by the viable cells of the tissue into a blue MTT formazan salt that is quantitatively measured after extraction from tissues.

- Optical Density measurements: The RhCE constructs were placed in 300 µL of a MTT solution at 1.0 mg/mL for 3 hours and 05 minutes at standard culture conditions. The precipitated blue formazan product was then extracted from the tissues by placing each insert in 2 mL of isopropanol during 17 hours and 28 minutes at 6±3 °C in the dark. The concentration of formazan was measured by determining the OD at 570 nm, just after dilution of the extractions in isopropanol (1:2). The OD at 570 nm was measured in triplicate samples of formazan extracts. The measured OD are proportional to the number of living cells. The measurement of OD was performed using the ELx800 absorbance microplate reader (controlled every year and calibrated if necessary) supplied by BioTek and the validated software Gen5 ELISA V1.05.11 supplied by BioTek.

- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model: The OD values obtained with the replicate tissue extracts for each test item were used to calculate the mean percent tissue viability normalized to the negative control, which was set to 100%. The percentage tissue viability cut-off value distinguishing classified from non-classified test items is 60%. Results are interpreted as follows:
The test item is identified as not requiring classification and labelling according to UN GHS No Category: if the mean percent tissue viability after exposure and post-exposure incubation is > 60%. In this case, no further testing in other test methods is required.
The test item is identified as potentially requiring classification and labelling according to UN GHS (Category 2 or Category 1): if the mean percent tissue viability after exposure and post-exposure incubation is ≤ 60%.
When the final mean percent tissue viability is ≤ 60%, further testing with other test methods will be required because the RhCE test method shows a certain number of false positive results and cannot resolve between UN GHS Categories 1 and 2.

- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria: The mean percent tissue viabilities obtained with the positive control and negative controls are within the range of historical data and therefore validate the experiment.

Irritation parameter:

other: % mean viability of the tissues

Run / experiment:

Main test - 30 minutes

Value:

73.93

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

MEAN PERCENT TISSUE VIABILITIES
- The mean corrected percent tissue viability of the RhCE replicates treated with the test item was 73.93 %, versus 39.16% in the positive control (Methyl acetate).

ACCEPTANCE OF RESULTS:
- The mean percent tissue viabilities obtained with the positive control and negative controls are within the range of historical data and therefore validate the experiment.

Table 7.3.2/1: Assessment of the eye irritation potential individual and average values of OD after 30 minutes exposure

 

Items	Tissues	OD	Mean OD/disc#	Mean OD / product	Viability%	Mean viability%	Difference of viability%	Conclusion
Negative control	1	0.947	0.912	0.959	95.10	100.00	9.80	-
		0.902
		0.887
	2	1.162	1.006		104.90
		0.966
		0.891
Positive control	3	0.318	0.366	0.376	38.16	39.16	1.98	UN GHS category 2 or 1
		0.372
		0.408
	4	0.382	0.385		40.15
		0.394
		0.379
Test item	5	0.806	0.768	0.767	80.083	79.98	0.21	-
		0.758
		0.739
	6	0.791	0.766		79.875
		0.753
		0.753
Test item killed control	9	0.054	0.057	0.058	5.944	6.05	0.21
		0.055
		0.061
	10	0.061	0.059		6.152
		0.058
		0.057
Test item corrected	-					73.93	-	No category

#: mean of 3 values (triplicate of the same extract)

OD: optical density

Interpretation of results:

GHS criteria not met

Conclusions:

Under the experimental conditions adopted and in accordance with the Regulation EC No. 1272/2008, the test item does not require classification for eye irritation or serious eye damage. It corresponds to the UN GHS No Category.

Executive summary:

An in vitro eye irritation test, reconstructed human cornea-like epithelium tissues (EpiOcular^TM tissue model) was performed according to the OECD Guideline 492 and in compliance with GLP to evaluate the eye hazard potential of the test item Sandalwood Austrocaledonien Oil.

 

The test item was applied, as supplied, at the dose of 50 µL, to 2 living and 2 killed DPBS pre-treated RhCE (EpiOcular^TM tissue model) during 30 minutes at 37 °C, 5% CO₂, 95% humidity (standard culture conditions). The exposure period was followed by extensive rinsing with DPBS at room temperature, a 12 minutes post-exposure immersion period at room temperature and a 2 hours and 03 minutes post-exposure incubation at standard culture conditions. The tissue viability was measured by performing an MTT assay.

 

The mean corrected percent tissue viability of the RhCE replicates treated with the test item was 73.93 %, versus 39.16% in the positive control (Methyl acetate). The mean percent tissue viabilities obtained with the positive control and negative controls are within the range of historical data and therefore validate the experiment.

 

In conclusion, under the experimental conditions adopted and in accordance with the Regulation EC No. 1272/2008, the test item does not require classification for eye irritation or serious eye damage. It corresponds to the UN GHS No Category.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin corrosion:

An in vitro skin corrosion study was performed according to OECD Guideline 431 and in compliance with GLP to evaluate the possible corrosive effects of the test item after topical administration on in vitro human reconstituted epidermis (Episkin^®model).

 

The test item was applied, as supplied, at the dose of 10 µL, to 2 Human skin model surfaces (Episkin^®), during 3 minutes and 1 hour (n=4).

 

3 minutes and 1 hour after the test item application, the viability of the human skin model has been 99% and 106% (considering as 100%) respectively, versus 34.7% and 10.9% respectively in the positive control (8NKOH).

 

Under the experimental conditions adopted and in accordance with the Globally Harmonized System (Regulation (EC) No.1272/2008), the test item must not be classified in category 1"Corrosive".

Skin irritation:

An in vitro skin irritation study was performed according to OECD Guideline 439 and in compliance with GLP to evaluate the possible irritating effects of the test item Sandalwood Austrocaledonien Oil after topical application onin vitrohuman reconstructed epidermis (SkinEthic RHE^®model).

 

The test item was applied, as supplied, at the dose of 16 µL, to 3 living and 2 killed Reconstructed Human epidermis (SkinEthic RHE^®model) during 42 minutes, followed by a rinse with 25 mL of DPBS and a41 hours and 50 minutespost-incubation period at 37 °C, 5% CO₂. Cell viability was then measured by enzymatic conversion of the vital dye MTT into a blue formazan salt that was quantitatively measured after extraction from tissues.

 

The mean percent tissue viabilities obtained with the negative control and positive controls are within the range of historical data and therefore validate the experiment.

 

The mean corrected percent viability of the treated tissues was 0%, versus 1.3% in the positive control (5% Sodium Dodecyl Sulfate).

 

Eye irritation:

An in vitro eye irritation test,reconstructed human cornea-like epithelium tissues (EpiOcular^TMtissue model)was performed according to the OECD Guideline 492 and in compliance with GLPto evaluate the eye hazard potential of the test item Sandalwood Austrocaledonien Oil.

 

The test item was applied, as supplied, at the dose of 50 µL, to 2 living and 2 killed DPBS pre-treated RhCE (EpiOcular^TMtissue model) during 30 minutes at 37 °C, 5% CO₂, 95% humidity (standard culture conditions). The exposure period was followed by extensive rinsing with DPBS at room temperature, a 12 minutes post-exposure immersion period at room temperature and a 2 hours and 03 minutes post-exposure incubation at standard culture conditions. The tissue viability was measured by performing an MTT assay.

 

The mean corrected percent tissue viability of the RhCE replicates treated with the test item was 73.93 %, versus 39.16% in the positive control (Methyl acetate). The mean percent tissue viabilities obtained with the positive control and negative controls are within the range of historical data and therefore validate the experiment.

 

Justification for classification or non-classification

Self-classification:

Based on the available information (OECD 492 test), the substance is not classified for eye irritation/corrosion according to the Regulation (EC) No. 1272/2008.

Based on the available information (OECD 439, and OECD 431 tests), the substance has to be classified as Category 2 irritant to the skin (H315) according to CLP regulation 1272/2008 (CE).

No data was available regarding respiratory irritation.