Tagetes minuta, ext.

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01-16 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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 28 July 2015

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

dated 23 July 2009

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

23 October 2015

Test material

Test material information

Constituent 1

Reference substance name:

(Z)-3,7-dimethylocta-1,3,6,-triene

EC Number:

222-081-3

EC Name:

(Z)-3,7-dimethylocta-1,3,6,-triene

Cas Number:

3338-55-4

Molecular formula:

C10H16

IUPAC Name:

3,7-dimethylocta-1,3,6-triene

Constituent 2

Reference substance name:

(5Z)-2,6-dimethylocta-2,5,7-trien-4-one (5E)-2,6-Dimethyl-2,5,7-octatrien-4-oneChl

Cas Number:

502-33-0

Molecular formula:

C10H14O

IUPAC Name:

(5Z)-2,6-dimethylocta-2,5,7-trien-4-one (5E)-2,6-Dimethyl-2,5,7-octatrien-4-oneChl

Constituent 3

Reference substance name:

(R)-p-mentha-1,8-diene

EC Number:

227-813-5

EC Name:

(R)-p-mentha-1,8-diene

Cas Number:

5989-27-5

Molecular formula:

C10H16

IUPAC Name:

(4R)-1-methyl-4-(prop-1-en-2-yl)cyclohexene

Constituent 4

Reference substance name:

2,6-dimethyloct-7-en-4-one

EC Number:

217-532-6

EC Name:

2,6-dimethyloct-7-en-4-one

Cas Number:

1879-00-1

Molecular formula:

C10H18O

IUPAC Name:

2,6-dimethyloct-7-en-4-one

Constituent 5

Reference substance name:

2,6-dimethylocta-5,7-dien-4-one

EC Number:

245-971-3

EC Name:

2,6-dimethylocta-5,7-dien-4-one

Cas Number:

23985-25-3

Molecular formula:

C10H16O

IUPAC Name:

2,6-dimethylocta-5,7-dien-4-one

Test material form:

liquid

Details on test material:

-Name: Tagete Essential Oil
-Other identifiers: Tagete Minuta Essential Oil
-Batch no.: 2563305
-Appearance: mobile liquid with characteristic odour
-CAS No.: 91770-75-1 / 8016-84-0
-EINECS-No.: 294-862-7
-Purity: 100% wt (UVCB substance)
-Homogeneity: 100%
-Production date: 07 Sep. 2016
-Expiry date: 07. Mar. 2018
-Storage Room Temprature (20 ± 5°C); Keep away from light/humidity and under inert gas

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Robertet / 2563305
- Appearance: Yellow to orange liquid
- Manufacturing date: 07 September 2016
- Date received: 27 January 2017
- Expiration date of the lot/batch: 07 March 2018
- Purity test date: 03 April 2017

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

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

other: living human skin models and killed reconstructed human 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-031) were received on 14 March 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 021) during 2 hours and 30 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 017).
The two killed Reconstructed Human epidermis control tissue models, supplied by Episkin (Batch No. 17-RHE-023) frozen on 28 February 2017 were defrost to be used on 16 March 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 15 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 slightly yellow instead of being whitish as for the coloration of the negative control tissues. They were incubated for a 41 hours and 15 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 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 3 hours at 37 °C, 5% CO2. The precipitated blue formazan product
was then extracted using isopropanol during 1 hour and 57 minutes 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 Vl.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 15 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)

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean

Run / experiment:

Main test (42 minutes)

Value:

-1.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Remarks:

mean corrected percent viability of the treated tissues was -1.2% (considered as 0%)

Other effects / acceptance of results:

VIABILITY
- The mean corrected percent viability of the treated tissues was -1.2% (considered as 0%), versus 0.9% 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.

Any other information on results incl. tables

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	1.195	1.272	1.090	116.7	100.0	16.4	-
		1.294
		1.327
	2	1.070	1.084		99.4
		1.095
		1.088
	3	0.936	0.915		83.9
		0.933
		0.878
Positive control	4	0.008	0.008	0.010	0.7	0.9	0.2	Irritant
		0.009
		0.008
	5	0.010	0.010		0.9
		0.010
		0.011
	6	0.012	0.012		1.1
		0.012
		0.013
Test item	7	0.018	0.019	0.018	1.7	1.7	0.1	-
		0.020
		0.020
	8	0.019	0.018		1.7
		0.019
		0.018
	9	0.017	0.017		1.6
		0.017
		0.017
Test item Killed tissues	10	0.031	0.031	0.031	2.8	2.8	0.0
		0.032
		0.032
	11	0.031	0.031		2.8
		0.030
		0.032
Test item corrected	-					-1.2	-	Corrosive or irritant

 

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

OD: optical density

 

Acceptability criteria:

• SD≤18%

• Negative control: OD value of the 3 replicates in the range ≥0.8 and ≤ 3.0.

The optical density was measured after a 1:2 dilution of the formazan extracts in isopropanol: the acceptability criteria should be in the range ≥0.4 and ≤1.5 for the negative control.

Applicant's summary and conclusion

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 EC 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 Tagete essential oil 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 a 41 hours and 15 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 -1.2% (considered as 0%), versus 0.9% in the positive control (5% Sodium Dodecyl Sulfate).

 

Under these experimental conditions and in accordance with the Regulation EC 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”.