Chromium tris((2-ethylhexanoate)

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-01-10 to 2017-03-31

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

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)

Version / remarks:

dated 29 July 2016

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Test method B.40bis Council regulation n°440/2008

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 23 October 2015

Test material

Test material information

Constituent 1

Reference substance name:

Chromium tris((2-ethylhexanoate)

EC Number:

222-357-3

EC Name:

Chromium tris((2-ethylhexanoate)

Cas Number:

3444-17-5

Molecular formula:

Cr(C8H16O2)3

IUPAC Name:

chromium(3+); 2-ethylhexanoate

Test material form:

other: Solid/paste

In vitro test system

Test system:

human skin model

Vehicle:

unchanged (no vehicle)

Details on test system:

The 0.6 cm² reconstructed epidermis (epiCS, Cell Systems - batch n° 100-AF2446-1) were received on 17 January 2017. The insert (filter + epidermis) was gently removed from the agarose while avoiding leaving agarose on the polycarbonate filter. The insert was placed in a 6 wells culture plate which had been previously filled with 1 mL of culture medium (Cell Systems batch n° 305-AG0224). The culture dishes were incubated at 37 +/- 2°C, 5% CO2 during 20 hours and 45 minutes before treatment. Just before the treatment, the culture medium was replaced by a new culture medium (Cell Systems, batch n° 305-AG0224).

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

The test item was applied, as supplied,after being applied on a nylon mesh, at the dose of 25 mg,during 3 minutes and during 1 hour at room temperature, to the epidermal surface of 4 living human skin models, including two for the non-specific colour (NSC) control.
In the same experimental conditions, a positive control (8N KOH - Sigma batch n° SLB3295V) and a negative control (DPBS - PAN BIOTECH GmbH, batch 9510916) were carried out.

Duration of treatment / exposure:

3 minutes and 1 hour

Number of replicates:

4 living human skin models (2 living for non-specific colour living control)

Test system

Details on study design:

3 minutes and 1 hour after the test item applicationn, the human epidermis was washed 20 times with 1 mL of DPBS (PAN BIOTECH GmbH, batch n° 9510916). The rinsed tissues were checked for coloration and presented green spots.
The cell viability was quatified 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. The skin sample was placed in MTT solution of 1 mg/L concentration, except for the two tissues for the non-specific control which were placed in MTT assay medium (Cell Systems batch n° 303-AG0224), for 3 hours between 36.8°C and 37.8°C, 5% CO2. The precipitated blue formazan product was then extracted using isopropanol during 2 hours under agitation in the dark, and the concentration of formazan was measured by determining the Optical Density (OD) at 570 nm, just after dilution of the extraction in isopropanol (1:3).
The absorbance was measured in triplicate of MTT extract.
The measured absorbances were proportional to the number of living cells.

The measurement of OD was performed using the Elx800 absorbance microplate reader (controlled and calibrated every year if necessary) supplied by BioTek and the validated software Gen5 ELISA V1.05.11 supplied by BioTek.

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minutes after the test item application

Value:

78.19

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1 hour after the test item application

Value:

84.55

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

The results are expressed as a viability percentage compared with the negative control:
viability % = OD test item / OD negative control x 100
Because the test item was potentially causing colour interferences, true viability was calculated as follows:
true viability % = [(OD of living tissues exposed to test item - OD of living tissues exposed to test item incubated with medium without MTT) / OD of living tissues exposed to negative control] x 100

Any other information on results incl. tables

The mean viability of epidermis skins treated with the positive control item (potassium hydroxide 8N) was 58.81% and 0.74% respectively 3 minutes and 1 hour after the application.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In accordance with Regulation EC 1272/2008, the results obtained under these experimental conditions unable to conclude that the test item does not have to be classified in category 1 'Corrosive'.
The hazard statement 'H314: Causes severe skin burns and eye damage' with the signal word 'Danger' are not required.