Complexation products of bismuth(3+) neodecanoate with propane-1,2-diol, propoxylated and 2,2',2'',2'''-ethylenedinitrilotetraethanol

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-01-16 to 2018-04-06

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

Version / remarks:

adopted 28 July 2015

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: ECVAM international validation study on in vitro tests for acute skin irritation (Altern Lab Anim. 2007 Dec; 35 (6):559-601)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 27 April 2017

Test material

Test material information

Constituent 1

Reference substance name:

Complexation products of bismuth(3+) neodecanoate with propane-1,2-diol, propoxylated and 2,2',2'',2'''-ethylenedinitrilotetraethanol

EC Number:

948-264-4

IUPAC Name:

Complexation products of bismuth(3+) neodecanoate with propane-1,2-diol, propoxylated and 2,2',2'',2'''-ethylenedinitrilotetraethanol

Test material form:

liquid

In vitro test system

Test system:

human skin model

Vehicle:

unchanged (no vehicle)

Details on test system:

The 0.5 cm² reconstructed epidermises (Episkin SA, RHE/S/17 - batch n° 18-RHE-006) were received on 30 January 2018. 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 a 6 wells culture plate which had been previously filled with 1 mL of growth medium (EpiSkin SA, batch n° 18 SGM 007) during 2 hours and 34 minutes. Then just before treatment, the inserts were placed in 24 wells culture plate whic had been previously filled with 300 µL of maintenance medium (EpiSkin, batch n° 18 SMM 004).

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

The test item was applied at the dose of 16 µL to the epidermal surface of 3 living skin models during 42 minutes at room temperature. To ensure good contact with the epidermises, during all the treatment period, the test item was recovered with a nylon mesh provided by Episkin SA.
In the same experimental conditions, a positive control (5% SDS), and a negative control ( DPBS -Dutscher - batch n°3149117) were carried out. The 5% SDS solution was prepared by weighing 0.5 g of SDS (SIGMA - batch n° STBG6142V) in a 10 mL volumetric flask qsp 10 mL of distilled water. Then, the preparation was magnetically stirred, just before the treatment. To ensure good contact with the epidermises, during all the treatment period, the control items were recovered with a nylon mesh provided by Episkin SA.

Duration of treatment / exposure:

42 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3 living skin models

Test system

Vehicle:

unchanged (no vehicle)

Details on study design:

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 n° 3149117). The rinsed tissues were checked for any coloration and noted to be whitish, comparable comparison to that of the negative control tissues.

They were incubated for 42 hours post-treatment incubation period in fresh medium between 36.1°c and 38.0°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, Thiazol blue; CAS n° 298-93-1)] reduction into blue formazan crystal that is quantitatively measured by Optical Density (OD) after extraction of tissues.
The skin samples were placed in 300 µL of a MTT solution at 1.0 mg/L for 3 hours between 37.0°C and 38.0°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 at 570 nm, just after dilution of the extracts 1:2 in isopropanol.
The OD was measured in triplicate of MTT extract.

The measurement of OD at 570 nm of formazan was performed in triplicate samples using the ELx800 absorbance microplte reader (controlled every year and calibrated if necessary) supplied by BioTek and the validated software Gen5 ELISA V1.05.11 supplied by BioTek.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Value:

44.3

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

mean viability (%) = 1.6 (5% Sodium dodecyl sulfate)

Other effects / acceptance of results:

The results were expressed as a viability percentage compared with the negative control:
% viability = ODtest item / OD negative control * 100

Any other information on results incl. tables

Results after treatment with test item and the controls

Dose group	Treatment	Mean OD Skin 1*	Mean OD Skin 2*	Mean OD Skin 3*	Mean OD Product	Mean Viability %
Negative control	42 min	0.680	0.742	0.703	0.708	100.0
Positive control	42 min	0.013	0.010	0.010	0.011	1.6
Test item	42 min	0.228	0.377	0.337	0.314	44.3

OD = Optical Density

EVALUATION AND INTERPRETATION OF THE RESULTS

The OD values obtained for each test sample are used to calculate a percentage of viability relative to the negative control, which was arbitrarely 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 of 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 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 the Regulation EC n° 1272/2008 and with a classification non-corrosive on a skin corrosion test, 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 identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1): if the mean percent viability after 42 minutes of 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 EC n° 1272/2008 and in absence of information on a skin corrosion test, the test 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'.

Applicant's summary and conclusion

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

In accordance with the Regulation EC n° 1272/2008 and considering the results obtained during the in vitro skin corrosion study (study n° HSMC-PH-17/0679, test item classified as non corrosive), the test item has to be classified in Category 2 'Irritating to skin'. The hazard statement 'H315: Causes skin irritation' with the signal word 'Warning' are required.