[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:

February - May 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

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 material

Test material information

Constituent 1

Test material form:

liquid

Details on test material:

Storage: room temperature
Form: liquid
Colour: colorless to yellowish

In vitro test system

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

Results and discussion

In vitro

Results

Results 1

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

Results 2

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).

Any other information on results incl. tables

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

Applicant's summary and conclusion

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