Registration Dossier

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:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
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.