Essential oil of Matricaria recutita (Asteraceae) obtained from flower tops by steam distillation of European origin

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 - February 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date: 27 April 2017

Test material

Test material information

Constituent 1

Reference substance name:

(R*,R*)-α,4-dimethyl-α-(4-methyl-3-pentenyl)cyclohex-3-ene-1-methanol

EC Number:

208-205-9

EC Name:

(R*,R*)-α,4-dimethyl-α-(4-methyl-3-pentenyl)cyclohex-3-ene-1-methanol

Cas Number:

515-69-5

Molecular formula:

C15H26O

IUPAC Name:

6-methyl-2-(4-methylcyclohex-3-en-1-yl)hept-5-en-2-ol

Constituent 2

Reference substance name:

[3S-[3α,6α(R*)]-tetrahydro-2,2,6-trimethyl-6-(4-methyl-3-cyclohexen-1-yl)-2H-pyran-3-ol

EC Number:

245-086-2

EC Name:

[3S-[3α,6α(R*)]-tetrahydro-2,2,6-trimethyl-6-(4-methyl-3-cyclohexen-1-yl)-2H-pyran-3-ol

Cas Number:

22567-36-8

Molecular formula:

C15H26O2

IUPAC Name:

2,2,6-trimethyl-6-(4-methylcyclohex-3-en-1-yl)tetrahydro-2H-pyran-3-ol

Constituent 3

Reference substance name:

[2S-[2α,5β(R*)]]-tetrahydro-α,α,5-trimethyl-5-(4-methyl-3-cyclohexen-1-yl)furan-2-methanol

EC Number:

247-509-6

EC Name:

[2S-[2α,5β(R*)]]-tetrahydro-α,α,5-trimethyl-5-(4-methyl-3-cyclohexen-1-yl)furan-2-methanol

Cas Number:

26184-88-3

Molecular formula:

C15H26O2

IUPAC Name:

2-[5-methyl-5-(4-methylcyclohex-3-en-1-yl)tetrahydrofuran-2-yl]propan-2-ol

Constituent 4

Reference substance name:

7,11-dimethyl-3-methylenedodeca-1,6,10-triene

EC Number:

278-628-1

EC Name:

7,11-dimethyl-3-methylenedodeca-1,6,10-triene

Cas Number:

77129-48-7

Molecular formula:

C15H24

IUPAC Name:

7,11-dimethyl-3-methylidenedodeca-1,6,10-triene

Constituent 5

Reference substance name:

7-ethyl-1,4-dimethylazulene

EC Number:

208-449-6

EC Name:

7-ethyl-1,4-dimethylazulene

Cas Number:

529-05-5

Molecular formula:

C14H16

IUPAC Name:

7-ethyl-1,4-dimethylazulene

Constituent 6

Reference substance name:

[S-(R*,R*)]-dihydro-2,2,6-trimethyl-6-(4-methyl-3-cyclohexen-1-yl)-2H-pyran-3(4H)-one

EC Number:

245-087-8

EC Name:

[S-(R*,R*)]-dihydro-2,2,6-trimethyl-6-(4-methyl-3-cyclohexen-1-yl)-2H-pyran-3(4H)-one

Cas Number:

22567-38-0

Molecular formula:

C15H24O2

IUPAC Name:

2,2,6-trimethyl-6-(4-methylcyclohex-3-en-1-yl)dihydro-2H-pyran-3(4H)-one

Constituent 7

Reference substance name:

2,6,10-trimethyldodeca-2,6,9,11-tetraene

EC Number:

207-948-6

EC Name:

2,6,10-trimethyldodeca-2,6,9,11-tetraene

Cas Number:

502-61-4

Molecular formula:

C15H24

IUPAC Name:

(3E,6E)-3,7,11-trimethyldodeca-1,3,6,10-tetraene

Constituent 8

Reference substance name:

(Z)-en-yn-dicycloether

Cas Number:

4575-53-5

Molecular formula:

C13H14O2

IUPAC Name:

(Z)-en-yn-dicycloether

Test material form:

liquid

Details on test material:

Colour: deep blue
CAS No.: 8002-66-2
Storage: room temperature

Specific details on test material used for the study:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- the test material was used as supplied

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 was applied, as supplied, at the dose of 50 µL to 2 living and 4 killed Human skin model surfaces during 3 minutes at room temperature and during 1 hour at 37°C ± 1°C, 5% ± 1% CO2.
Additionally, 2 living and 4 killed Human skin model surfaces (epiCS®, CellSystems®) were treated in the same manner during both 3 minutes and 1 hour, but they were incubated in MTT assay medium instead of MTT solution in order to generate non-specific living and killed colour controls.
- In the same experimental conditions, a positive control and a negative control were carried out.


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 and 3 minutes 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
As the test item is identified as producing both direct MTT reduction and colour interference:
True viability % = [(OD of living tissues exposed to test item - OD of killed tissues exposed to test item - OD of living tissues exposed to test item incubated with medium without MTT + OD of killed tissues exposed to test item incubated with medium without MTT) / OD of living tissues exposed to negative control] x 100

Results for test items producing non specific MTT reduction (%NSMTT) and/or non specific colour (%NSCliving) ≥ 50% of the negative control should be taken with caution.

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:

mean at 3 minutes

Value:

128.16

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean at 1 hour

Value:

100.07

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 128.16% and 100.07%, respectively.

Any other information on results incl. tables

Table 7.3.1/1: Skin corrosion assay: Results at 3 minutes

	Skin	OD	Mean OD / disc(#)	Mean OD / product	Viability %	Meanviability %	Viability difference between replicates %
Negativecontrol	1	0.524 0.591 0.618	0.578	0.570	101.40	100.00	2.8
	2	0.608 0.558 0.521	0.562		98.60
Positive control	3	0.053 0.045 0.040	0.046	0.116	8.07	20.26	24.4
	4	0.215 0.178 0.162	0.185		32.46
Test item PH-17/0609	5	0.832 0.690 0.686	0.736	0.745	129.12	130.70	3.2
	6	0.672 0.923 0.668	0.754		132.28
Test item PH-17/0609NSMTT	7	0.020 0.018 0.016	0.018	0.016	3.16	2.72	0.9
	8	0.013 0.013 0.013	0.013		2.28
Test item PH-17/0609 NSC livingcontrol	11	0.004 0.004 0.004	0.004	0.004	0.70	0.70	0.0
	12	0.005 0.004 0.003	0.004		0.70
Test item PH-17/0609 NSC killedcontrol	9	0.003 0.003 0.003	0.003	0.005	0.53	0.88	0.7
	10	0.008 0.007 0.006	0.007		1.23
Test item PH-17/0609 corrected						128.16

Table 7.3.1/1: Skin corrosion assay: Results at 1 hour

	Skin	OD	Mean OD / disc(#)	Mean OD / product	Viability %	Mean viability %	Viability difference between replicates %
Negativecontrol	1 (Plate 2)	0.672 0.802 0.841	0.772	0.688	112.21	100.00	24.4
	2 (Plate 2)	0.603 0.622 0.587	0.604		87.79
Positivecontrol	3 (Plate 2)	0.002 0.001 0.000	0.001	0.000	0.15	0.00	0.3
	4 (Plate 2)	-0.001 -0.001 -0.001	-0.001		-0.15
Test itemPH-17/0609	5 (Plate 2)	0.852 0.722 0.706	0.760	0.716	110.47	104.07	12.8
	6 (Plate 2)	0.720 0.648 0.647	0.672		97.67
Test itemPH-17/0609NSMTT	7 (Plate 2)	0.012 0.009 0.009	0.010	0.020	1.45	2.83	2.8
	8 (Plate 2)	0.032 0.027 0.028	0.029		4.22
Test itemPH-17/0609 NSC livingcontrol	11 (Plate 2)	0.009 0.008 0.008	0.009	0.010	1.31	1.38	0.1
	12 (Plate 2)	0.011 0.008 0.010	0.010		1.45
Test itemPH-17/0609 NSC killedcontrol	9 (Plate 2)	0.000 0.000 0.000	0.000	0.002	0.00	0.22	0.4
	10 (Plate 2)	0.004 0.002 0.001	0.003		0.44
Test itemPH-17/0609 corrected						100.07

#: mean of 3 values

OD: optical density

 

 

Applicant's summary and conclusion

Interpretation of results:

other: test item does not have to be classified in Category 1 “Corrosive”

Conclusions:

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.

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 were 128.16% and 100.7% 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.