Santalum austro-caledonicum, ext.

Skin irritation / corrosion

Administrative data

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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

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

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

Cas Number:

88034-74-6

Molecular formula:

C15H24O

IUPAC Name:

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

Constituent 4

Reference substance name:

(2E)-2-methyl-6-[(1S)-4-methylcyclohex-3-en-1-yl]hepta-2,6-dien-1-ol

Cas Number:

942226-77-9

Molecular formula:

C15H24O

IUPAC Name:

(2E)-2-methyl-6-[(1S)-4-methylcyclohex-3-en-1-yl]hepta-2,6-dien-1-ol

Constituent 5

Reference substance name:

(Z)-2-Methyl-5-((1R,2R,4S)-2-methyl-3-methylenebicyclo[2.2.1]heptan-2-yl)pent-2-en-1-ol

Cas Number:

79081-90-6

Molecular formula:

C15H24O

IUPAC Name:

(Z)-2-Methyl-5-((1R,2R,4S)-2-methyl-3-methylenebicyclo[2.2.1]heptan-2-yl)pent-2-en-1-ol

Test material form:

liquid

Details on test material:

Manufacturing date: 30/05/2008
Expiry date: 30/05/2010

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

In vitro test system

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

Results and discussion

In vitro

Results

Results 1

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

Results 2

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.

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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".