Sage, Salvia officinalis, 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:

December 2017 - april 2018

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:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

1-isopropyl-4-methylbicyclo[3.1.0]hexan-3-one

EC Number:

208-912-2

EC Name:

1-isopropyl-4-methylbicyclo[3.1.0]hexan-3-one

Cas Number:

546-80-5

Molecular formula:

C10H16O

IUPAC Name:

(1S,4R,5R)-4-methyl-1-propan-2-ylbicyclo[3.1.0]hexan-3-one

Constituent 2

Reference substance name:

DL-bornan-2-one

EC Number:

244-350-4

EC Name:

DL-bornan-2-one

Cas Number:

21368-68-3

Molecular formula:

C10H16O

IUPAC Name:

1,7,7-trimethylbicyclo[2.2.1]heptan-2-one

Constituent 3

Reference substance name:

Cineole

EC Number:

207-431-5

EC Name:

Cineole

Cas Number:

470-82-6

Molecular formula:

C10H18O

IUPAC Name:

1,3,3-trimethyl-2-oxabicyclo[2.2.2]octane

Constituent 4

Reference substance name:

(1S, 4S, 5R)-4-Methyl-1-(1-methylethyl)bicyclo[3.1.0]hexan-3-one

Cas Number:

471-15-8

Molecular formula:

C10H16O

IUPAC Name:

(1S, 4S, 5R)-4-Methyl-1-(1-methylethyl)bicyclo[3.1.0]hexan-3-one

Constituent 5

Reference substance name:

Camphene

EC Number:

201-234-8

EC Name:

Camphene

Cas Number:

79-92-5

Molecular formula:

C10H16

IUPAC Name:

2,2-diméthyl-3-méthylène-bicyclo[2,2,1]heptane

Constituent 6

Reference substance name:

Humulene

EC Number:

229-816-7

EC Name:

Humulene

Cas Number:

6753-98-6

Molecular formula:

C15H24

IUPAC Name:

(1E,4E,8E)-2,6,6,9-tetramethylcycloundeca-1,4,8-triene

Constituent 7

Reference substance name:

Pin-2(3)-ene

EC Number:

201-291-9

EC Name:

Pin-2(3)-ene

Cas Number:

80-56-8

Molecular formula:

C10H16

IUPAC Name:

2,6,6-trimethylbicyclo[3.1.1]hept-2-ene

Constituent 8

Reference substance name:

Caryophyllene

EC Number:

201-746-1

EC Name:

Caryophyllene

Cas Number:

87-44-5

Molecular formula:

C15H24

IUPAC Name:

(1R,4E,9S)-4,11,11-trimethyl-8-methylidenebicyclo[7.2.0]undec-4-ene

Constituent 9

Reference substance name:

(1S-endo)-1,7,7-trimethylbicyclo[2.2.1]heptan-2-ol

EC Number:

207-353-1

EC Name:

(1S-endo)-1,7,7-trimethylbicyclo[2.2.1]heptan-2-ol

Cas Number:

464-45-9

Molecular formula:

C10H18O

IUPAC Name:

endo-1,7,7-trimethylbicyclo[2.2.1]heptan-2-ol

Constituent 10

Reference substance name:

Pin-2(10)-ene

EC Number:

204-872-5

EC Name:

Pin-2(10)-ene

Cas Number:

127-91-3

Molecular formula:

C10H16

IUPAC Name:

6,6-dimethyl-2-methylenebicyclo[3.1.1]heptane

Test material form:

liquid

Details on test material:

-Name: SAGE DALMATIAN OIL
-IUPAC Name: Essential oil of Salvia officinalis (Lamiaceae) obtained from leaves, flowers and stalks by steam distillation.
-CAS: 84082-79-1
-EINECS: 282-025-9
-Batch no.: BU201707
-Purity: 100 % wt – UVCB substance
-Appearance: pale to slightly yellow
-Expiry date: 03/07/2019
-Storage: Sample should be stored tightly closed, below ambient temperature, preferably under inert gas atmosphere in dark, lightproofed bottles.

In vitro test system

Test system:

human skin model

Source species:

other: reconstituted epidermis

Cell type:

other: reconstituted epidermis (epiCS®, CellSystems®)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: 0.60 cm2 reconstituted epidermis (epiCS®)

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 20 times with 20 mL of DPBS.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
The cell viability is quantified by measurement of the cellular mitochondrial dehydrogenases activity. These enzymes are responsible for the MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue; EINECS number 206-069-5, CAS number 298-93-1)] reduction into blue formazan in the viable cells. The skin sample is placed in MTT solution of appropriate concentration (e.g. 0.3 or 1 mg/mL) for 3 hours between 36.4°C and 37.9°C. The precipitated blue formazan product is then extracted using a solvent (e.g. isopropanol), and the concentration of formazan is measured by determining the Optical Density (OD) at a wavelength between 540 and 600 nm (preferably 570 nm). The measured absorbances are proportional to the number of living cells.
The measurement of OD was performed using the ELx800 absorbance microplate reader supplied by BioTek and the validated software Gens ELISA V1.05.11 supplied by BioTek.

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.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

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

Duration of treatment / exposure:

3 minutes and 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:

100

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

main test (1 hour)

Value:

39.83

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

VIABILITY
- 3 minutes and 1 hour after the test item application, the mean percent viability of the epidermis skins treated with the test item were 106.40% considered as 100% and 39.83%, 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; 1 hour after the positive control application, the viability of the human skin model has been 0.00%.

Any other information on results incl. tables

Table 7.3.1/1: Skin corrosion assay: Results

 

	Skin	OD	Mean OD / disc (#)	Mean OD / product	Viability %	Mean viability %	Viability difference between replicates %
Treatment: 3 min
Negative control	1	0.524	0.578	0.570	101.40	100	2.8
		0.591
		0.618
	2	0.608	0.562		98.60
		0.558
		0.521
Positive control	3	0.053	0.046	0.116	8.07	20.26	24.4
		0.045
		0.040
	4	0.215	0.185		32.46
		0.178
		0.162
Test item	13	0.649	0.597	0.607	104.74	106.40	3.3
		0.590
		0.162
	14	0.550	0.616		108.07
		0.616
		0.683
Treatment: 1 hour
Negative control	1	0.672	0.772	0.688	112.21	100	24.4
		0.802
		0.841
	2	0.603	0.604		87.79
		0.622
		0.587
Positive control	3	0.002	0.001	0.000	0.15	0.00	0.3
		0.001
		0.000
	4	-0.001	- 0.001		-0.15
		-0.001
		-0.001
Test item	13	0.302	0.268	0.274	38.95	39.83	1.7
		0.245
		0.255
	14	0.234	0.280		40.70
		0.292
		0.312

#: 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: not classified as skin corrosive

Conclusions:

In accordance with the Regulation (EC) No. 1272/2008, the results obtained under these experimental conditions enable to conclude that the test item does not have to be classified in Category 1 “Corrosive”.
The hazard statement “H314: Causes severe skin burns and eye damage” with the signal word “Danger” are not required.

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 (epiCS®, CellSystems®).

The test item was applied as supplied, at the dose of 50 μL, to 2 living Human skin model surfaces (epiCS®, CellSystems®) during 3 minutes and 1 hour, followed by a rinse with 20 mL of DPBS. 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.

3 minutes and 1 hour after the test item application, the mean percent viability of the epidermis skins treated with the test item were100% and 39.83% versus 20.26% and 0.00%, respectively, with the positive control item (potassium hydroxide 8N).

Under the test conditions, in accordance with the Regulation (EC) No. 1272/2008, the results obtained enable to conclude that the test item does not have to be classified in Category 1 “Corrosive”. No hazard statement or signal word are required.