[No public or meaningful name is available]

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 03 February 2022 to 23 February 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study performed according to OECD Guideline No.431 and under GLP compliance

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Dated to 18 June 2019.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

litsea cubeba (fruit) extract – terpenes fraction

Molecular formula:

not applicable

IUPAC Name:

litsea cubeba (fruit) extract – terpenes fraction

Test material form:

liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No. of test material: 0000765018
- Date received : 25 January 2022
- Expiration date of the lot/batch: 15 December 2022
- Manufacture date : 15 December 2021

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

FORM AS APPLIED IN THE TEST
The test item was used as supplied in the study.

In vitro test system

Test system:

human skin model

Source species:

other: reconstituted epidermis (epiCS, Phenion)

Cell type:

other: epiCS, Cell Systems

Vehicle:

unchanged (no vehicle)

Details on test system:

HUMAN SKIN MODEL
The 0.6 cm² reconstituted epidermis (epiCS, Phenion - Batch No.epiCS 22-05) were received on 22 February 2022. The insert (filter + epidermis) was gently removed from the agarose while avoiding leaving agarose on the polycarbonate filter. The inserts were placed in a 6 wells culture plate which had been previously filled with 1 mL of culture medium. The culture plates were incubated at 37°C, 5% CO2 for 20 hours and 48 minutes before treatment.

EVALUATION OF DIRECT INTERACTION WITH MTT
The direct interaction of MTT with the test item was checked by adding 50 µL of the test item to 1mL of the solution of MTT at 1 mg/mL (same conditions as in the main test). A yellow liquid was observed after 1 hour of incubation between 36.5°C and 37.9°C, 5% CO2 in the dark.
> Therefore, there is no direct interaction between the test item and MTT.

SPECTRAL ANALYSIS OF THE TEST ITEM
- IN WATER:
The coloration potential of the test item in water was checked by adding 50 µL of the test item to 300 µL of distilled water. A colourless liquid was obtained after 1 hour of incubation between 36.5°C and 36.9°C, 5% CO2 in the dark.
- IN ISOPROPANOL:
The coloration potential of the test item in isopropanol was checked by adding 50 µL of the test item to 2 mL of isopropanol. A colourless liquid was obtained after 2 hours of incubation at room temperature under gentle agitation.
>No significant coloration appeared. Therefore, the test item will not interfere with the MTT assay and there is no need to add non-specific coloration controls to the study.

TREATMENT
The test item was applied as supplied at the dose of 50 µL to the epidermal surface of the 2 living human skin models during 3 minutes at room temperature and during 1 hour at 37±1°C, 5±1% CO2.
In the same experimental conditions, a positive control (50µL of 8N KOH - Fisher Scientific, Batch No. A0412420) and a negative control (50µL of distilled water - ADL Prochilab - Batch No. 201117) were carried out.

REMOVAL OF TEST MATERIAL AND CONTROLS
3 minutes and 1 hour after the test item application the human epidermis were washed 20 times with 1 mL of DPBS (DPBS – Dutscher, Batch No. 3730921).

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 into 300 µL of MTT solution, at the concentration of 1 mg/mL, for 2 hours and 45 minutes at 37±1°C, 5% CO2. The precipitated blue formazan product was then extracted using 2 mL of isopropanol for 2 hours under agitation in the dark, and the concentration of formazan was measured by determining the Optical Density (OD) at 570 nm, just after dilution of the extraction in isopropanol (1:3).
The absorbance was measured in triplicate of MTT extract.
The measured absorbances were proportional to the number of living cells.

The measurement of OD was performed using the Elx800 absorbance microplate reader (controlled and calibrated every year 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 CALCULATION:
- The results were expressed as a viability percentage compared with the negative control:
viability % = (mean OD test item / mean OD negative control) * 100
- Data from individual replicate tissues (OD values and calculated percent tissue viability data for the test item and controls), mean percent tissue viability and standard deviation for each individual test item and control were reported in Table 7.3.1/1.

PREDICTION MODEL / DECISION CRITERIA:
The OD values obtained for each test sample are used to calculate a percentage of viability relative to the negative control, which is arbitrarily set at 100%. The cut-off values for the prediction of corrosion associated with the epiCS® models are as follows:

Viability measured after exposure time points (t=3 and 60 minutes)

STEP 1 (corrosive or not corrosive)
< 50% after 3 min exposure ==> Corrosive: Corresponding hazard statement “H314: Causes severe skin burns and eye damage” and signal word “Danger”
≥ 50% after 3 min exposure AND < 15% after 60 min exposure ==> Corrosive: Corresponding hazard statement “H314: Causes severe skin burns and eye damage” and signal word “Danger”
≥ 50% after 3 min exposure AND ≥ 15% after 60 min exposure ==> Non-corrosive

STEP 2 (subcategories for items identified as corrosive in step 1)
< 15% after 3 min exposure ==> Optional Sub-category 1A*
≥ 15% after 3 min exposure ==> A combination of optional Sub-categories 1B-and-1C

* According to the data generated in view of assessing the usefulness of the RhE test methods for supporting subcategorisation, it was shown that around 33% of the Sub-category 1A results of the epiCS® test methods may actually constitute Sub-category 1B or Sub-category 1C substances/mixtures (i.e. over-classifications).
It must be noted that a limitation of this Test Guideline is that it does not allow discriminating between skin corrosive sub-categories 1B and 1C.

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:

During 3 minutes at room temperature and during 1 hour at 37±1°C, 5±1% CO2.

Duration of post-treatment incubation (if applicable):

The skin sample was placed in MTT solution of 1 mg/mL concentration for 2 hours and 45 minutes at 37±1°C, 5% CO2.

Number of replicates:

2 living human skin models for each time

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

at 3 minutes

Value:

83.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

at 60 minutes

Value:

46.3

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:

MTT VIABILITY ASSAY RESULTS
- The mean percent viability of the treated tissues, after 3 minutes exposure, was 83.9%, versus 10.9% in the positive control.
- The mean percent viability of the treated tissues, after 1 hour exposure, was 46.3 %, versus 0.7% in the positive control.

ACCEPTANCE CRITERIA
- Negative control: the mean OD of the tissue replicates should be ≥ 0.8 and ≤ 2.8 for epiCS® model, for every exposure time. As the extract was diluted at 1:3 just before the OD measure, the acceptability criteria should be in the range ≥ 0.3 and ≤ 0.9 for the negative control. As the mean OD of the negative control is 0.673 for 3-minute of exposure and 0.667 for 1-hour of exposure, therefore, the criteria is met.
- Positive control: the mean viability of the tissue replicates exposed for 1 hour with the positive control (8N KOH), expressed as % of the negative control, should be ≤ 15%. As the mean viability is 0.7%, therefore, the criteria is met.
- Test item: in the range 20-100% viability, and for ODs ≥ 0.3, difference of viability between the two tissue replicates should not exceed 30%. As the maximum difference of viability between the two tissue replicates is 14.6%, therefore, the criteria is met.

Any other information on results incl. tables

Table 7.3.1/1: Main test - Individual and mean OD values and tissue viabilities for the test item, the negative and positive controls

 

INDIVIDUAL AND AVERAGE VALUES AFTER 3 MINUTES EXPOSURE

 

	Skin	OD	Mean OD / disc (#)	Mean OD / product	Viability %	Mean viability %	SD viability	Viability difference between replicates %
Negative control	1	0.748 0.723 0.694	0.722	0.673	107.3	100.00	10.3	14.6
	2	0.630 0.609 0.632	0.624		92.7
Positive control	3	0.082 0.085 0.084	0.084	0.074	12.5	10.9	2.2	3.1
	4	0.061 0.063 0.065	0.063		9.4
Test item	5	0.555 0.540 0.538	0.544	0.565	80.8	83.8	4.3	6.1
	6	0.577 0.579 0.600	0.585		86.9

 

 

 

INDIVIDUAL AND AVERAGE VALUES AFTER 1 HOUR EXPOSURE

	Skin	OD	Mean OD / disc (#)	Mean OD / product	Viability %	Mean viability %	SD viability	Viability difference between replicates %
Negative control	9	0.706 0.690 0.693	0.696	0.667	104.3	100.00	6.1	8.7
	10	0.638 0.619 0.658	0.638		95.7
Positive control	11	0.006 0.006 0.007	0.006	0.005	0.9	0.7	0.2	0.3
	12	0.004 0.004 0.003	0.004		0.6
Test item	13	0.315 0.324 0.317	0.319	0.309	47.8	46.3	2.1	3.0
	14	0.299 0.299 0.299	0.299		44.8

Note #: mean of 3 values OD: optical density

SPL: sample

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In accordance with the CLP 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 test according to the OECD Guideline OECD 431 and in compliance with GLP was performed. The aim of the study was to evaluate the possible corrosive effects of the test item LITSEA CUBEBA TERPENES L62840 after topical administration on in vitro human reconstituted epidermis (epiCS®, supplied by Phenion).

The test item LITSEA CUBEBA TERPENES L62840 was applied as supplied, at the dose of 50 μL to 2 living Human skin model surfaces for each time (epiCS®, supplied by Phenion) for 3 minutes at room temperature and for 1 hour at 37°C ± 1°C, 5% ± 1% CO2. In the same experimental conditions, a positive control (50 µL of 8N KOH) and a negative control (50 µL of distilled water) were carried out. The application was 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.

 

Quality criteria: The quality criteria required for acceptance of results in the test were satisfied.

3 minutes and 1 hour after the test item application, the mean corrected percent viability of the epidermis skins treated with the test item were 83.9% and 46.3%, versus 10.9% and 0.7%, respectively, with the positive control item (potassium hydroxide 8N).

 

In accordance with the Regulation (EC) No. 1272/2008, the results obtained under these experimental conditions enable to conclude that the test item LITSEA CUBEBA TERPENES L62840 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.