Balmywood

Endpoint summary

Description of key information

- Skin irritation: irritating, based on in vitro skin irritation study (OECD 439, GLP, rel.1, K).

- Skin corrosion : not corrosive based on in vitro skin corrosion study (OECD 431, GLP, rel.1, K).

- Serious eye damage/ eye irritant: not irritant based on in vitro eye irritation study (OECD 492, GLP, rel.1, K).

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

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)

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 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

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%).

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

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”.

Reference 2

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 29 September 2021 to 24 November 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study performed in accordance with OECD guideline 431 and in compliance with GLP with the following deviations on composition and treatment duration: 1/ 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. 2/The epidermises were treated during 4 minutes instead of 3 minutes, as initially scheduled. As the exposure time is very slightly exceeded (+ 1 minute) and the viability obtained at this exposure time is close to that obtained after 1 hour of incubation (non-corrosive product), this deviation is considered as without impact on the conclusion of the study.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Dated to 18 June 2019.

Deviations:

yes

Remarks:

The epidermises were treated during 4 min instead of 3 min. As the exposure time is very slightly exceeded and the viability obtained at this exposure time is close to that obtained after 1 h of incubation, this deviation is considered as without impact.

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

other: reconstituted epidermis (epiCS cell system, Phenion-Henkel, Batch No. epiCS 21-43)

Cell source:

foreskin from multiple donors

Details on animal used as source of test system:

CELL SYSTEM
0.6 cm² reconstituted epidermis (epiCS, Phenion-Henkel, Batch No. epiCS 21-43).
The tissue integrity was checked using the OCT screening. It is considered valid.

BARRIER FUNCTION
The Exposure Time inducing 50% viability (ET50) was checked using Triton X-100 1% and should be included between 2 and 7 hours. As the ET50=2hours and 39min, 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 using a PCR analysis.
On cells from the donors, the absence of bacteria, fungus and mycoplasma (using an Elisa analysis) was verified.
> No contamination was detected on the tissues.

Vehicle:

unchanged (no vehicle)

Details on test system:

HUMAN SKIN MODEL
The 0.6 cm² reconstituted epidermis (epiCS, Phenion-Henkel, Batch No. epiCS 21-43) were received on 23 November 2021. 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 (Phenion-Henkel, Batch No.305-AK2236). The culture plates were incubated at 37°C, 5% CO2 for 20 hours and 55 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 solution with brown test item was observed after 57 minutes of incubation in the incubator, 5% CO2 in the dark.
> 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 50 µL of the test item to 2 mL of isopropanol (same conditions as in the main test). A brown solution was obtained after 2 hours and 01 minute of incubation at ambient temperature with gentle shaking. The mean of the corrected OD (blank subtracted) was 0.116 which is higher than 0.08 (value corresponding to approximately twice the OD of the extracting solvent).
>Therefore, the test item was identified as causing colour interference with the viability assay and four viable control tissues were added 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 4 minutes at room temperature and during 1 hour at 37±1°C, 5±1% CO2. The additonal living Human skin model surfaces (2 for each time) were treated in the same manner in order to generate non-specific Isopropanol interaction control.
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
4 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. 7380521). The rinsed tissues were checked for any coloration and noted to be yellow.

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 3 hours 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
- As the test item was identified as causing colour interference with the viability assay, four viable control tissues were added to the study. Therefore, the true viability is then calculated as follow:
viability % = (OD test item - Mean OD NSC living / 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 4 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 3 hours at 37±1°C, 5% CO2.

Number of replicates:

2 living human skin models for each time and 2 viable control tissues for each time in order to generate non-specific isopropanol interaction control.

Irritation / corrosion parameter:

other: Corrected mean percent viability (%)

Run / experiment:

at 4 minutes

Value:

105.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

other: Corrected mean percent viability (%)

Run / experiment:

at 60 minutes

Value:

101.2

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 corrected mean percent viability (%) of the treated tissues, after 4 minutes exposure, was 105.9%, versus 0.4% in the positive control.
- The corrected mean percent viability (%) of the treated tissues, after 60 minutes exposure, was 101.2%, versus 0.3% 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 0.623 for 4-minute of exposure and 0.769 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 0.3%, 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 2.3%, therefore, the criteria is met.

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 4 MINUTES EXPOSURE

 

	Skin	OD	Mean OD / disc (#)	Mean OD / product	Viability %	Mean viability %	SD viability	Viability difference between replicates %
Negative control	1	0.617 0.601 0.628	0.616	0.623	99.0	100.00	1.5	2.1
	2	0.626 0.630 0.631	0.629		101.0
Positive control	3	0.004 0.002 0.002	0.003	0.003	0.5	0.4	0.1	0.2
	4	0.002 0.002 0.002	0.002		0.3
Test item	7	0.660 0.660 0.657	0.659	0.665	105.9	106.7	1.2	1.8
	8	0.654 0.688 0.667	0.670		107.6
Test item NSC living control	9	0.006 0.006 0.007	0.007	0.006	1.1	0.9	0.3	0.5
	10	0.004 0.004 0.004	0.004		0.6
Test item corrected						105.9

 

 

 

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	11	0.677 0.659 0.649	0.662	0.769	86.1	100.00	19.6	27.7
	12	0.853 0.901 0.869	0.875		113.9
Positive control	13	0.002 0.002 0.002	0.002	0.002	0.3	0.3	0.1	0.1
	14	0.002 0.002 0.003	0.003		0.4
Test item	17	0.830 0.792 0.699	0.774	0.783	100.7	101.9	1.7	2.3
	18	0.828 0.805 0.741	0.792		103.1
Test item NSC living control	19	0.004 0.004 0.005	0.005	0.005	0.7	0.7	0.0	0.0
	20	0.005 0.005 0.004	0.005		0.7
Test item corrected						101.2

Note #: mean of 3 values OD: optical density

SPL: sample

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 BALMYWOOD ZA3155 after topical administration on in vitro human reconstituted epidermis (epiCS®, supplied by Phenion-Henkel).

The test item BALMYWOOD ZA3155 was applied as supplied, at the dose of 50 µL to 2 living Human skin model surfaces (epiCS®, supplied by Phenion-Henkel) for 4 minutes and 1 hour. The application was followed by a rinse with 20 mL of PBS. 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. Additionally, 4 lived Human skin model surfaces (epiCS®, supplied by Henkel) were treated in the same manner but they were incubated in culture medium instead of MTT solution in order to generate non-specific living color controls.

 

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

4 minutes and 1 hour after the test item application, the corrected mean percent viability of the epidermis skins treated with the test item were 105.9% and 101.2% (considered as 100%), versus 0.4% and 0.3%, 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 BALMYWOOD ZA3155 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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 26 January 2022 to 17 February 2022.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study performed according to OECD Guideline 492 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).

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Version / remarks:

Adopted 18 June 2019

Deviations:

no

Principles of method if other than guideline:

Not applicable.

GLP compliance:

yes (incl. QA statement)

Species:

human

Strain:

other: Keratinocyte strain No.4F1188

Details on test animals or tissues and environmental conditions:

CELL SYSTEM USED
0.60 cm² Reconstructed human Cornea-like Epithelia (EpiOcular™ OCL-200-EACH, supplied by MatTek Corporation, batch No. 34953) containing normal human keratinocytes.

ANALYSIS FOR POTENTIAL BIOLOGICAL CONTAMINANTS
The cells used to produce EpiOcular™ tissue are screened for potential biological contaminants.
HIV1 virus, hepatitis B virus and hepatitis C virus were checked using oligonucleotide-directed amplification and bacteria, yeast and other fungi were checked using long term antibiotic, antimycotic free culture.
> No contamination was detected for all contaminants.

ANALYSIS FOR TISSUE FUNCTIONALITY AND QUALITY
- The tissue viability was checked using a MTT QC assay for 1hour. The OD value (540-570nm) should be between 1.1 and 3. As the OD value = 1.783±0.055, the cell viability is confirmed.
- The barrier function represented by the Exposure Time inducing 50% viability (ET50) was checked using Triton X-100 and should be between 12.2 and 37.5 minutes. As the ET50= 28.17 minutes, the barrier function is considered valid.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

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

Duration of treatment / exposure:

30 minutes at 37°C, 5% CO2, 95% humidity (standard culture conditions)

Duration of post- treatment incubation (in vitro):

- Post-exposure immersion period: 12 minutes at room temperature - Post-exposure incubation period: 2 hours at standard culture conditions

Number of animals or in vitro replicates:

Test item, negative and positive controls were applied to the entire surface of 4 living RhCE tissue replicates including 2 RHE models for the non-specific colour (NSC) control.

Details on study design:

TISSUES CONSTRUCTS
- RhCE tissue construct used: 0.60 cm² Reconstructed human Cornea-like Epithelia (EpiOcular™ OCL-200-EACH, supplied by MatTek Corporation, batch No. 34953) were received on 15 February 2022.

- Pre-incubation of the tissues:
On 15 February 2022, the tissues in their well shipping container were equilibrated to room temperature for 15 minutes. Then the inserts (filter + epithelium) were gently removed from the agarose while avoiding leaving agarose on the polycarbonate filter. They were placed in 6 wells culture plate which had been previously filled with 1 mL of 37°C pre-warmed assay medium (MatTek Corporation, batch No. 021422MPA) and incubated for 22 hours and 02 minutes at standard culture conditions.

- Indication of controls used for direct MTT-reducers:
The direct interaction of MTT with the test item was checked by adding 50 µL of the test item to 1 mL of solution of MTT at 1 mg/mL (same conditions as in the main test). A yellow liquid 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 and there is no need to add non-specific MTT reduction (NSMTT) controls.

- Indication of controls used for colouring test chemicals:
The spectral properties at 570 nm of test item in isopropanol were checked by adding 50 µL of the test item to 2 mL of isopropanol (same conditions as in the main test). An orange solution was obtained after 2 hours of incubation at ambient temperature with gentle shaking. The mean of the corrected OD (blank subtracted) was 0.114 which is higher than 0.08 (value corresponding to approximately twice the OD of the extracting solvent).
> Therefore, the test item was identified as causing colour interference with the viability assay and two viable control tissues were added to the study which underwent the entire testing procedure but were incubated with culture medium instead of MTT solution during the MTT incubation step to generate a non-specific colour (NSC living) control.

MAIN TEST
- Pre-treatment:
After the overnight incubation, the tissues were pre-wetted with 20 µL of Ca2+Mg2+Free-DPBS (Dutscher - Batch No. 3730921). The tissues were incubated at standard culture conditions for 30 minutes.

- Treatment and post-treatment incubation of the tissues:
The test item was applied as supplied, at the dose of 50 μL, to the entire surface of 4 living RhCE tissue replicates including 2 RHE models for the non-specific colour (NSC) control, during 30 minutes at standard culture conditions.

In the same experimental conditions, a positive control (Methyl acetate - Sigma-Aldrich, batch No. BCBX8836) and a negative control (distilled water - ADL Prochilab - Batch No. 2011117) were carried out. The controls were applied, as supplied, at the dose of 50 μL, to the surface of 2 RhCE tissue replicates during 30 minutes at standard culture conditions.

After the treatment, the test item and control substances were carefully washed from the RhCE tissues by extensive rinsing with Ca2+Mg2+Free-DPBS (Dutscher - Batch No. 3730921). The rinsed tissues were checked for any coloration and noted to be white, comparable coloration to that of the negative control tissues, with residual brown coloration in the center.
This rinsing step was followed by a 12-minute post-exposure immersion period at room temperature in 5 mL of fresh medium to remove any test item absorbed into the tissue.
The RhCE constructs were then incubated for a 2 hours post-exposure incubation at standard culture conditions in 1 mL of fresh medium at 37°C, 5% CO2.

- MTT viability assay:
Following the exposure to the test item, viability measurements are performed immediately after the post-exposure incubation period of the rinsed tissues in fresh medium. This period allows both for recovery from weak cytotoxic effects and for appearance of clear cytotoxic effects.
The RhCE tissue viability was measured by enzymatic conversion of the vital dye MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Thiazolyl blue tetrazolium bromide; CAS No. 298-93-1] by the viable cells of the tissue into a blue MTT formazan salt that is quantitatively measured after extraction from tissues.
The RhCE constructs were placed in 300 µL of a MTT solution at 1.0 mg/mL for 3 hours at standard culture conditions. The two additional control tissues were incubated in assay medium (MatTek Corporation, batch No. 021422MPA) instead of MTT solution in order to generate NSC control.

The precipitated blue formazan product was then extracted from the tissues by placing each insert in 2 mL of isopropanol for 16 hours and 40 minutes at 7+/-3°C in the dark.
The concentration of formazan was measured by determining the OD at 570 nm, just after dilution of the extractions in isopropanol (1:2).

The OD at 570 nm was measured in triplicate samples of formazan extracts.
The measured OD are proportional to the number of living cells.
The measurement of OD was performed 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.

Irritation parameter:

other: % mean corrected percent tissue viability

Run / experiment:

After 30 minutes exposure

Value:

104

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
As the test item was identified as causing colour interference with the viability assay, the true tissue viability is calculated as the percent tissue viability obtained with living tissues exposed to the interfering test chemical and incubated with the MTT solution (%Viability test) minus the percent non-specific colour obtained with living tissues exposed to the interfering test chemical and incubated with medium without MTT, run concurrently to the test being corrected (%NSC living).

> Therefore, the corrected mean percent viability of the RhCE replicates treated with the test item BALMYWOOD ZA3155 was 104.0% (considered as 100%) versus 48.4% in the positive control (Methyl acetate).

DEMONSTRATION OF TECHNICAL PROFICIENCY: Proficiency chemicals were tested according to the OECD TG 492.

ACCEPTANCE OF RESULTS:
These results are in accordance with the acceptability criteria:
- The OD of the negative control was > 0.8 and < 2.8. As the extract was diluted at 50% just before the OD measurement, the acceptability criteria should be in the range > 0.4 and < 1.4 for the negative control (mean OD of the negative control = 0.793);
- The relative mean tissue viability for the positive control treated tissues was <50% relative to the negative control treated tissues (mean viability of the positive control = 48.4%);
- The difference of viability between two tissue replicates is < 20% (difference of viability between two tissue replicates is 7.9% and 0.4% for living tissues exposed to the test item and NSC living tissues exposed to the test item and then incubated in medium instead of MTT, respectively).

Table 7.3.2/1: Main test - Individual and mean OD values and tissue viabilities for the test item, the negative and positive controls after 30 minutes exposure

 

Tissue	OD	Mean OD/ disc (#)	Mean OD / Product	Viability (%)	Mean viability (%)	SD viability	Viability difference between replicates (%)	Conclusion
Negative Control	0.743 0.816 0.800	0.786	0.793	99.1	100.00	1.2	1.8	No category
	0.769 0.853 0.778	0.800		100.9
Positive Control	0.350 0.366 0.366	0.361	0.384	45.5	48.4	4.1	5.8	UN GHS Category 2 or 1
	0.394 0.417 0.409	0.407		51.3
Test Item	0.782 0.828 0.787	0.799	0.831	100.8	104.7	5.6	7.9	No category
	0.843 0.898 0.846	0.862		108.7
Test item NSC living	0.009 0.007 0.006	0.007	0.006	0.9	0.7	0.3	0.4
	0.004 0.004 0.004	0.004		0.5
Test item corrected					104.0

#: mean of 3 values

OD: optical density

SPL: sample

Interpretation of results:

GHS criteria not met

Conclusions:

The corrected mean percent viability of the RhCE replicates treated with the test item BALMYWOOD ZA3155 was 104.0% (considered as 100%) versus 48.4% in the positive control (Methyl acetate).
Under the experimental conditions adopted and in accordance with the Regulation EC No. 1272/2008, the test item does not require classification for eye irritation or serious eye damage. It corresponds to the UN GHS No Category. No hazard statement and the signal word are required.

Executive summary:

An OECD 492 study was performed to evaluate the eye hazard potential of test item after topical administration on in vitro reconstructed human cornea-like epithelium tissues (EpiOcular™ tissue model).

 

The test item BALMYWOOD ZA3155 was applied as supplied, at the dose of 50 µL, to 2 living DPBS pre-treated RhCE (EpiOcular™ tissue model) during 30 minutes at 37°C, 5% CO2, 95% humidity (standard culture conditions). In the same experimental conditions, a positive control (Methyl acetate) and a negative control (distilled water) were carried out. The controls were applied, as supplied, at the dose of 50 µL, to the surface of 2 RhCE tissue replicates during 30 minutes at standard culture conditions.

The exposure period was followed by extensive rinsing with DPBS at room temperature, a 12 minutes post-exposure immersion period at room temperature and a 2 hours post-exposure incubation at standard culture conditions. The tissue viability was measured by performing an MTT assay. Additionally, 2 RhCE (EpiOcular™ tissue model) were treated in the same manner but were incubated in culture medium instead of MTT solution in-order-to generate non-specific living color controls.

 

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

The mean corrected percent tissue viability of the RhCE replicates treated with the test item BALMYWOOD ZA3155 was 104.0% (considered as 100%) versus 48.4% in the positive control (Methyl acetate).

  

In conclusion, under the experimental conditions adopted and in accordance with the Regulation EC No. 1272/2008, the test item BALMYWOOD ZA3155 does not require classification for eye irritation or serious eye damage. It corresponds to the UN GHS No Category. No hazard statement and the signal word are required.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In vitro studies

Skin corrosion (OECD 431, GLP, rel.1, RS, K)

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 BALMYWOOD ZA3155 after topical administration on in vitro human reconstituted epidermis (epiCS®, supplied by Phenion-Henkel).

The test item BALMYWOOD ZA3155 was applied as supplied, at the dose of 50 µL to 2 living Human skin model surfaces (epiCS®, supplied by Phenion-Henkel) for 4 minutes and 1 hour. The application was followed by a rinse with 20 mL of PBS. 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. Additionally, 4 lived Human skin model surfaces (epiCS®, supplied by Henkel) were treated in the same manner but they were incubated in culture medium instead of MTT solution in order to generate non-specific living color controls.

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

4 minutes and 1 hour after the test item application, the corrected mean percent viability of the epidermis skins treated with the test item were 105.9% and 101.2% (considered as 100%), versus 0.4% and 0.3%, 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 BALMYWOOD ZA3155 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.

 

Skin irritation (OECD 439, GLP, rel.1, RS, K)

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. 

Quality criteria: 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”.

 

Eye irritation (OECD 492, GLP, rel.1, RS, K)

An OECD 492 study was performed to evaluate the eye hazard potential of test item after topical administration on in vitro reconstructed human cornea-like epithelium tissues (EpiOcular™ tissue model).

The test item BALMYWOOD ZA3155 was applied as supplied, at the dose of 50 µL, to 2 living DPBS pre-treated RhCE (EpiOcular™ tissue model) during 30 minutes at 37°C, 5% CO2, 95% humidity (standard culture conditions). In the same experimental conditions, a positive control (Methyl acetate) and a negative control (distilled water) were carried out. The controls were applied, as supplied, at the dose of 50 µL, to the surface of 2 RhCE tissue replicates during 30 minutes at standard culture conditions.

The exposure period was followed by extensive rinsing with DPBS at room temperature, a 12 minutes post-exposure immersion period at room temperature and a 2 hours post-exposure incubation at standard culture conditions. The tissue viability was measured by performing an MTT assay. Additionally, 2 RhCE (EpiOcular™ tissue model) were treated in the same manner but were incubated in culture medium instead of MTT solution in-order-to generate non-specific living color controls.

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

The mean corrected percent tissue viability of the RhCE replicates treated with the test item BALMYWOOD ZA3155 was 104.0% (considered as 100%) versus 48.4% in the positive control (Methyl acetate).

In conclusion, under the experimental conditions adopted and in accordance with the Regulation EC No. 1272/2008, the test item BALMYWOOD ZA3155 does not require classification for eye irritation or serious eye damage. It corresponds to the UN GHS No Category. No hazard statement and the signal word are required.

Justification for classification or non-classification

Harmonized classification:

The registered substance has no harmonized classification according to the Regulation (EC) No. 1272/2008.

 

Self-classification:

Based on an in vitro skin irritation study (OECD 439), the registered substance is classified as skin irritant: Skin Irritant Category 2 (H315: Causes skin irritation) according to the criteria of the Regulation (EC) No. 1272/2008 (CLP). Moreover, an assessment of the skin corrosivity has been performed. An in vitro skin corrosion study (OECD 431) has allowed to show that the test item is not considered as a skin corrosive.

Concerning the eye irritation/corrosion, an in vitro eye irritationstudy (OECD 492) has allowed to show that the test item is not considered as irritant to the eyes. No classification is required.