[No public or meaningful name is available]

Endpoint summary

Description of key information

- Skin corrosive Category 1 (OECD 439, K, Rel.1: positive ; OECD 431, K, Rel.1: positive)

- Eye corrsive Category 1 (OECD 492, GLP, K, Rel.1: positive)

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

February - May 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

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)

Test system:

human skin model

Source species:

other: Reconstructed Human Epidermis Model

Cell type:

non-transformed keratinocytes

Cell source:

other: Not applicable

Source strain:

other: Not applicable

Justification for test system used:

Following the REACH bottom-up strategy, as the result of the OECD test guideline No. 439 was positive, the epiCS® Reconstructed Human Epidermis Model method was used to assess skin corrosion as recommended in the OECD test guideline No. 431.

Vehicle:

unchanged (no vehicle)

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 2 killed Human skin model surfaces (epiCS®, CellSystems®) during 3 minutes and 1 hour.

REMOVAL OF TEST MATERIAL AND CONTROLS
- 3 minutes and 1 hour after the test item application, the human epidermis was washed with 20 mL of PBS.

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 at 37°C ± 1°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.

VIABILITY
Viability = (OD test item / OD negative control) x 100

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:

2 living skin model surfaces (epiCS®, CellSystems®) for test item; 2 lived skins each for positive and negative controls

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Experiment 1 at 3 minutes

Value:

47.94

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Experiment 1 at 1 hour

Value:

3.63

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:

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 47.94 and 3.63%, respectively, versus 6.08% and 0.39%, respectively, with the positive control item (potassium hydroxide 8N).

Table 7.3.1/1: Skin corrosion assay: Results

 

	Skin	OD	Mean OD / disc (#)	Mean OD / product	Viability %	Mean viability %	Viability difference between replicates %
Treatment: 3 min
Negative control	1	0.669	0.686	0.0691	99.35	100	1.3
		0.688
		0.701
	2	0.715	0.695		100.65
		0.652
		0.717
Positive control	3	0.025	0.025	0.042	3.62	6.08	4.9
		0.025
		0.024
	4	0.059	0.059		8.54
		0.058
		0.059
Test item	7	0.232	0.233	0.331	33.74	47.94	28.4
		0.231
		0.235
	8	0.440	0.429		62.13
		0.428
		0.419
Treatment: 1 hour
Negative control	11	0.856	0.842	0.771	109.28	100	18.6
		0.384
		0.835
	12	0.710	0.699		90.72
		0.676
		0.711
Positive control	13	0.004	0.004	0.003	0.52	0.39	0.3
		0.004
		0.004
	14	0.002	0.002		0.26
		0.002
		0.002
Test item	17	0.026	0.026	0.028	3.37	3.63	0.5
		0.027
		0.024
	18	0.030	0.030		3.89
		0.029
		0.029

#: mean of 3 values

OD: optical density

Interpretation of results:

Category 1 (corrosive) based on GHS criteria

Conclusions:

In accordance with the Regulation (EC) No. 1272/2008, the results obtained under these experimental conditions enable to conclude that the test item has to be classified in Category 1B/1C “Corrosive”.
The corresponding hazard statement is “H314: Causes severe skin burns and eye damage” with the signal word “Danger

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 47.94% and 3.63% versus 6.08% and 0.39%, 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 has to be classified in Category 1 “Corrosive”. The corresponding hazard statement is “H314: Causes severe skin burns and eye damage” with the signal word “Danger

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

January - March 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Used as supplied

Test system:

human skin model

Source species:

other: reconstructed epidermises

Cell type:

non-transformed keratinocytes

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- 0.50 cm² reconstructed epidermis (Episkin SA, RHE/S/17) were received, and on 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 1 mL of growth medium (Episkin SA) during 2 hours and 25 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).

TREATMENT
- The test item was applied as supplied, at the approximate dose of 16 µL, on the epidermal surface of 3 living human skin models during 42 minutes at room temperature.
- In the same experimental conditions, a positive control (5% SDS) and a negative control (DPBS) were carried out. The 5% SDS solution was prepared by weighing 0.5 g of SDS in a 10 mL volumetric flask qsp 10 mL of distilled water. Then, the preparation was magnetically stirred, just before the treatment. 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.

REMOVAL OF TEST MATERIAL AND CONTROLS
- 42 minutes after the test item application, 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 brown instead of being whitish as for the coloration of the negative control tissues. Residual test item with brown coloration was noted on all Reconstructed Human epidermis after the rinse. They were incubated for a 42 hours and 05 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 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. 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.

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 values for the prediction of irritation associated with the RHE models were as follows:
- The test item is considered to be non-irritant to skin if the tissue viability after 42 minutes of exposure and 42 hours of post-treatment incubation is >50%.
- The test item is considered to be irritant to skin if the tissue viability after 42 minutes of exposure and 42 hours of post-treatment incubation is ≤ 50% and the result of a skin corrosion test is "non-corrosive". In accordance with Regulation EC No. 1272/2008, the test 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 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. 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".

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 at 37°C, 5% CO2

Number of replicates:

3 living human skin models

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

main test (mean value)

Value:

1.2

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:

The mean percent viability of the treated tissues was 1.2%, versus 1.0% in the positive control (5% Sodium Dodecyl Sulfate ).

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

 

	Skin tissues	OD	Mean OD/disc (#)	Mean OD/product	Viability %	Mean viability %	Standard deviation (SD)
Negative control	1	0.973	1.047	1.002	104.5	100	5.3
		1.074
		1.095
	2	0.938	1.016		101.4
		1.047
		1.064
	3	0.873	0.943		94.1
		1.047
		1.064
Positive control	1	0.009	0.010	0.010	1.0	1.0	0.1
		0.011
		0.011
	2	0.010	0.011		1.1
		0.012
		0.012
	3	0.010	0.010		1.0
		0.010
		0.010
Test item	1	0.009	0.009	0.012	0.9	1.2	0.2
		0.010
		0.010
	2	0.013	0.013		1.3
		0.014
		0.014
	3	0.013	0.013		1.3
		0.013
		0.014

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

OD: optical density

Interpretation of results:

study cannot be used for classification

Conclusions:

In accordance with Regulation EC No. 1272/2008 and in absence of information on skin corrosion, test item has to be classified in Category 2 “Irritating to skin” or in Category 1 “Corrosive”. The hazard statement “H315: Causes skin irritation” with the signal word “Warning” or “H314: Causes severe skin burns and eye damage” with the signal word “Danger” are required.

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) during 42 minutes, followed by a rinse with 25 mL of PBS and a 42 hours 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 viability of the treated tissues was1.2 %, versus 1.0 % in the positive control (5% Sodium Dodecyl Sulfate).

 

Under the test conditions and in accordance with Regulation EC No. 1272/2008 and in absence of information on skin corrosion, the test item has to be classified in Category 2 "Irritating to skin" or in Category 1 "Corrosive". The hazard statement "H315: Causes skin irritation" with the signal word "Warning" or "H314: Causes severe skin burns and eye damage" with the signal word "Danger" are required.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (corrosive)

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:

January - May 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline 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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

other: Reconstructed human Cornea-like Epithelia

Details on test animals or tissues and environmental conditions:

- Description of the cell system used: 0.60 cm² Reconstructed human Cornea-like Epithelia [EpiOcular(TM) OCL-212-ver2.0, supplied by MatTek Corporation]

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Concentration: Undiluted 50 µL

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 on duplicate tissues.

Details on study design:

MAIN TEST
- Pre-incubation of the tissues:
On the day of receipt, the tissues in their well shipping container were equilibrated at 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 and incubated during 20 hours and 34 minutes at standard culture conditions.
After the overnight incubation, the tissues were pre-wetted with 20 μL of Ca2+Mg2+Free-DPBS. 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 2 living PBS 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 used. The controls were applied, as supplied, at the dose of 50 µL, to the surface of 2 viable 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. The rinsed tissues were checked for any colouration and noted for comparable colour with the negative control. 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-hour 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 by Optical Density (OD) after extraction from tissues. The measured OD was proportional to the number of living cells.
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 precipitated blue formazan product was then extracted from the tissues by placing each insert in 2 mL of isopropanol during 17 hours and 40 minutes at 6±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 measurement of formazan extracts at 570 nm was measured 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.

Irritation parameter:

other: % tissu viability

Run / experiment:

main experiment (mean value)

Value:

2.64

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:

The mean percent tissue viability of the RhCE replicates treated with the test item was 2.64% versus 17.72% in the positive control (Methyl acetate).

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

 

	Tissue	OD	Mean OD/disc (#)	Mean OD/product	Viability %	Mean viability %	Difference of viability %
Negative control	1	1.128	1.054	1.041	101.25	100.00	2.50
		1.062
		0.971
	2	1.018	1.028		98.75
		1.054
		1.012
Positive control	3	0.204	0.193	0.185	18.54	17.72	1.63
		0.184
		0.189
	4	0.172	0.176		16.91
		0.175
		0.180
Test item	9	0.027	0.029	0.028	2.79	2.64	0.29
		0.029
		0.029
	10	0.027	0.026		2.50
		0.027
		0.024

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

OD: optical density

Interpretation of results:

study cannot be used for classification

Conclusions:

In accordance with the Regulation EC No. 1272/2008, the test item has to be identified as potentially requiring classification and labeling according to UN GHS Category 2 or Category 1.

Executive summary:

In an OECD 492 study, the test item was applied, as supplied, at the dose of 50 µL, to 2 living DPBS pre-treated RhCE (EpiOcular^TMtissue model) during 30 minutes at 37°C, 5% CO₂, 95% humidity. 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.

 

 The mean percent tissue viability of the RhCE replicates treated with the test item was2.64%versus 17.72% in the positive control (Methyl acetate).

 

 

In accordance with the Regulation EC No. 1272/2008, the test item has to be identified as potentially requiring classification and labeling according to UN GHS Category 2 or Category 1.

 

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin corrosion:

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 47.94% and 3.63% versus 6.08% and 0.39%, respectively, with the positive control item (potassium hydroxide 8N).

Eye irritation:

In an OECD 492 study, the test item was applied, as supplied, at the dose of 50 µL, to 2 living DPBS pre-treated RhCE (EpiOcular^TMtissue model) during 30 minutes at 37°C, 5% CO₂, 95% humidity. 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.

 

 The mean percent tissue viability of the RhCE replicates treated with the test item was2.64%versus 17.72% in the positive control (Methyl acetate).

 

Justification for classification or non-classification

Self - classification :

According to results from OECD 439 and OECD 431 study, the registered substance is classified as category 1 for skin irritation (H314) according to Regulation EC N° 1272/2008 (CLP) and GHS.

According to the results of OECD 492 and OECD 431, the registered substance should be classified as category 1 for eye irritation (H318) according to Regulation EC N° 1272/2008 (CLP) and GHS.