Balmywood

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 26 January 2022 to 10 March 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study performed according to OECD Guideline No.439 and under GLP compliance (the exact composition of the test item cannot be exactly determined because the element is an UVCB substance. This deviation to GLP is under the responsibility of the Sponsor and is considered as without impact on the conclusion of the study)

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

Version / remarks:

adopted 14 June 2021

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Light fraction extract obtained from the labdanum gum issued from the exudate of Cistus ladaniferus (Cistaceae) by maceration with essential oil of Cedarwood obtained from the wood of Juniperus Mexicana (Cupressaceae)

Molecular formula:

not applicable for NCS substances

IUPAC Name:

Light fraction extract obtained from the labdanum gum issued from the exudate of Cistus ladaniferus (Cistaceae) by maceration with essential oil of Cedarwood obtained from the wood of Juniperus Mexicana (Cupressaceae)

Test material form:

liquid

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

other: reconstructed epidermises (SkinEthic RHE, RHE/S/17 Batch No. 22-RHE-042)

Cell source:

foreskin from multiple donors

Details on animal used as source of test system:

0.5 cm² reconstructed epidermis of normal human keratinocytes. Cells are grown on inert polycarbonates filters in chemically defined medium, for 17 days.
The epidermises was prepared and packaged using aseptic techniques and store in an incubator at 37°C, 5% CO2 with saturated humidity.

HISTOLOGY
Satisfactory for multi-layered, highly differentiated epidermis consisting of organized basal, spinous and granular layers, and a multilayered stratum corneum.
Number of cell layer ≥4: 5 cell layers

CELL VIABILITY
The OD = 1.0 with the CV = 12.9%. As the OD > 7, the cell viability is confirmed.

BARRIER FUNCTION
The Exposure Time inducing 50% viability (ET50) was checked using Triton X-100 1% and should be between 4 and 10 hours. As the ET50=5.6hours, the barrier function is considered valid.

BIOLOGICAL SAFETY
On blood of the donors, the absence of HIV1 and HIV2 antibodies, hepatitis C antibodies and hepatitis B antigen HBs was verified.
On cells from the donors, the absence of bacteria, fungus and mycoplasma was verified.

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. 22-RHE-042) were received on 08 March 2022. 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 24 wells culture plate which had been previously filled with 300 μL of growth medium (Episkin SA, batch No. 22 SGM 024) for 3 hours and 40 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. No. 22 SMM 011).

EVALUATION OF DIRECT INTERACTION WITH MTT
The direct interaction of MTT with the test item was checked by adding 16 µL of the test item to 300 µL of the solution of MTT at 1 mg/mL (same conditions as in the main test). A yellow solution with brown to black product was observed after 3 hours of incubation between 36.5°C and 36.9°C, 5% CO2.
> Therefore, there is no direct interaction between the test item and MTT.

SPECTRAL ANALYSIS OF THE TEST ITEM IN ISOPROPANOL
The spectral properties at 570 nm of the test item in isopropanol were checked by adding 16 µL of the test item to 1.5 mL of isopropanol (same conditions as in the main test). A yellow liquid was obtained after 2 hours of incubation at ambient temperature with gentle shaking. The mean of the corrected OD (blank subtracted) was 0.051which is lower than 0.08 (value corresponding to approximately twice the OD of the extracting solvent).
>Therefore, the test item will not interfere with the viability 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 16 μL to the epidermal surface of 3 living human skin models during 42 minutes at room temperature. To ensure a good contact with the epidermises, during all the treatment period, the test item was covered with a nylon mesh provided by Episkin SA.

- In the same experimental conditions, a positive control (16 μL of 5% sodium dodecyl sulfate - SDS) and a negative control (16 μL of distilled water – ADL Prochilab - Batch No. 211021) were carried out. The 5% SDS solution was prepared by weighing 0.5001 g of SDS (SIGMA Batch No. STBJ3028) in a 10 mL volumetric flask q.s. 10 mL of distilled water. Then, the preparation was magnetically stirred, just before the treatment to obtain a colourless solution.
To ensure a good contact with the epidermises, during all the treatment period, the control items were covered with a nylon mesh provided by Episkin SA.

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 (Dutscher - Batch No. 3730921). The rinsed tissues were checked for any coloration: slight brown coloration was noted on the epidermises treated with the test item after the rinse. They were incubated for a 42 hours 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 the 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 that is quantitatively measured by Optical Density (OD) after extraction from tissues. The measured OD were proportional to the number of living cells.

- The skin samples 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 2 hours under gentle agitation in the dark, and the concentration of formazan was measured by determining the OD (Optical Density) at 570 nm, just after dilution of the extracts (1:2 in isopropanol).

- The OD of MTT extract 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 V1.05.11 supplied by BioTek.

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 were used to calculate a percentage of viability relative to the negative control, which was arbitrarily set at 100%. The cut-off value of percentage cell viability distinguishing irritant from non-classified test items associated with the SkinEthic RHE model is given below:

- The test item is considered as non-irritant to skin in accordance with UN GHS No Category, if the mean percent viability after 42 minutes exposure and 42 hours of post-treatment incubation is > 50%.

- The test item is identified as requiring classification and labelling according to UN GHS (Category 2), 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”. In accordance with the Regulation (CE) No.1272/2008 and with a classification non-corrosive on a skin corrosion test, the item has to be classified in Category 2 “Irritant”. The corresponding hazard statement is “H315: Causes skin irritation” with the signal word “Warning”.

- The test item is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1), 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. In accordance with the Regulation (CE) No.1272/2008 and in absence of information on a skin corrosion test, the item has to be classified in Category 2 “Irritant” or in Category 1 “Corrosive”. The corresponding hazard statement is respectively, “H315: Causes skin irritation” with the signal word “Warning” or “H314: Causes severe skin burns and eye damage” with the signal word “Danger”.

ACCEPTABILITY CRITERIA
The results of the assay are considered acceptable if the following assay acceptance criteria are achieved:

- Positive Control
The assay establishes the acceptance criterion for an acceptable test if the relative mean tissue viability for the positive control treated tissues was <40% relative to the negative control treated tissues, and the standard deviation value of the percentage viability is lower or equal to 18%.

- Negative Control
The assay establishes the acceptance criterion for an acceptable test if the mean OD for the negative control treated tissues was higher or equal to 0.8 and lower or equal to 3, and the standard deviation value of the percentage viability is lower or equal to 18%.

- Test Item
The assay establishes the acceptance criterion for an acceptable test if the standard deviation calculated from individual percentage tissue viabilities of the three identically treated tissues is lower or equal to 18%.

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):

42 hours post-incubation period in fresh medium at 37°C, 5% CO2

Number of replicates:

3 living human skin models and controls

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

At 42 minutes exposure

Value:

6.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

DEMONSTRATION OF TECHNICAL PROFICIENCY: Proficiency chemicals were tested according to the OECD TG 439.

MTT VIABILITY ASSAY RESULTS
- The mean percent viability (%) of the treated tissues was 6.9%, versus 1.2% in the positive control (5% SDS).

ACCEPTANCE OF RESULTS:
These results are in accordance with the acceptability criteria:
- The OD of the negative control was ≥ 0.8 and ≤ 3, and the standard deviation value of the percentage viability is ≤ 18% (mean OD of the negative control = 0.856 and SD = 7.8%);
- The relative mean tissue viability for the positive control treated tissues was <40% relative to the negative control treated tissues, and the standard deviation value of the percentage viability is ≤ 18% (mean viability of the positive control = 1.2% and SD = 0.1%);
- The standard deviation calculated from individual percentage tissue viabilities of the three identically treated tissues is ≤ 18% (SD = 0.3%).

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 OF OD AFTER 42 MINUTES EXPOSURE

	Skin	OD	Mean OD / disc (#)	Mean OD / product	Viability %	Mean viability %	SD viability	Conclusion
Negative control	1	0.868 0.872 0.884	0.875	0.856	102.3	100.00	7.8	No category
	2	0.887 0.916 0.930	0.911		106.5
	3	0.754 0.776 0.811	0.781		91.3
Positive control	4	0.010 0.010 0.011	0.011	0.010	1.3	1.2	0.1	Category 2 'irritant'
	5	0.010 0.010 0.010	0.010		1.2
	6	0.010 0.010 0.010	0.010		1.2
Test item	7	0.063 0.061 0.059	0.061	0.059	7.1	6.9	0.3	Category 2 'irritant'
	8	0.059 0.058 0.058	0.059		6.9
	9	0.055 0.055 0.057	0.056		6.5

Note #: mean of 3 values

OD: optical density

SPL: sample

SD: Standard deviation

Applicant's summary and conclusion

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

The mean percent viability of the treated tissues was 6.9%, versus 1.2% in the positive control (5% Sodium Dodecyl Sulfate).
In accordance with the Regulation (CE) No.1272/2008 and with a classification non-corrosive on a skin corrosion test, the item has to be classified in Category 2 “Irritant”. The corresponding hazard statement is “H315: Causes skin irritation” with the signal word “Warning”.

Executive summary:

An in vitro skin irritation test using the Reconstructed Human Epidermis (SkinEthic RHE® model) was performed according to the OECD Guideline 439 and in compliance with GLP to predict the acute skin irritation potential of the test item.

The test item was applied as supplied, at the dose of 16 μL, to 3 living Reconstructed Human epidermis (SkinEthic RHE® model) for 42 minutes at room temperature. The application was followed by a rinse with 25 mL of PBS and a 42 hours incubation period at 37°C, 5% CO2. In the same experimental conditions, a positive control (16 μL of 5% sodium dodecyl sulfate - SDS) and a negative control (16 μL of distilled water) were carried out. 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 quality criteria required for acceptance of results in the test were satisfied.
The mean percent viability of the treated tissues was 6.9% versus 1.2% in the positive control (5% Sodium Dodecyl Sulfate).

In accordance with the Regulation (CE) No.1272/2008 and with a classification non-corrosive on a skin corrosion test, the item has to be classified in Category 2 “Irritant”. The corresponding hazard statement is “H315: Causes skin irritation” with the signal word “Warning”.