isliFe

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 May 2019 - 06 June 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Test material form:

solid

Specific details on test material used for the study:

Batch no : TROD7BB06

In vitro test system

Test system:

human skin model

Remarks:

Episkin SA

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

foreskin from multiple donors

Vehicle:

unchanged (no vehicle)

Details on test system:

The 0.50 cm² reconstructed epidermises (Episkin SA, RHE/S/17 Batch No. 19-RHE-096) were received on 04 June 2019. The 4 additional killed Human skin model surfaces were defrozen the day of the treatment. 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 300 μL of growth medium (Episkin SA, batch No. 19 SGM 065) during 2 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 (EpiskinSA, batch No. 19 SMM 022).

42 minutes after the test item application, the human epidermises were washed with 25 x 1 mL of DPBS (Dutscher - Batch No. 3630319). The rinsed tissues were checked for any coloration: after the rinse, the surface of the epidermises was slightly red with a darker outline. The 4 additional control tissues were darker.
The epidermises were incubated for a 41 hours and 40 minutes post-treatment incubation-period in fresh medium at 37°C, 5% CO2. Then, the epidermises were put in contact with the MTT solution. 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 samples were placed in 300 μL of a MTT solution at 1.0 mg/mL for 3 hours at 37°C, 5% CO2. except for the 2 living and 2 killed Human skin model surfaces for the non-specific colour control which were placed in the maintenance medium (Episkin SA, batch No. 19 SMM 022).
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 at 570 nm, just after dilution of the extracts 1:2 in isopropanol.
The OD was measured in triplicate of MTT extract.
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.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

The test item was applied as supplied, to the epidermal surface of 3 living Reconstructed Human epidermis, at the dose of 16 mg per 0.50 cm² of human skin model, during 42 minutes at room temperature.

Duration of treatment / exposure:

42 minutes

Duration of post-treatment incubation (if applicable):

41 hours and 40 minutes

Number of replicates:

3 living Reconstructed Human Epidermis

Two killed Human skin tissues were treated in order to generate non-specific MTT reduction control and 2 additional killed and 2 additional living control tissues were treated to generate non-specific living and killed colour controls.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean

Value:

86.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

mean tissue viability 1.3% for 5% sodium dodecy sulfate solution

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In accordance with the Regulation EC No. 1272/2008, the test item Acetic acid, oxo-, sodium salt, reaction products with ethanolamine and phenol, sodium hydroxide and ferric chloride (MEAHA Fe) has to be considered as Non-irritant to skin. It corresponds to UN GHS No Category.
No hazard statement or signal word is required.

Executive summary:

In accordance with the Regulation EC No. 1272/2008, the test item Acetic acid, oxo-, sodium salt, reaction products with ethanolamine and phenol, sodium hydroxide and ferric chloride (MEAHA Fe) has to be considered as Non-irritant to skin. It corresponds to UN GHS No Category.

No hazard statement or signal word is required.