Salt reaction of cobalt(2+) and C2/C8/C20 carboxylates

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021-09-21 to 2021-12-20

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 14 June 2021

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Salt reaction of cobalt(2+) and C2/C8/C20 carboxylates

EC Number:

942-548-1

Molecular formula:

(CnH2n-102/Cn’H2n’-1O2) Co with n, n’ = 2 or 8 or 20

IUPAC Name:

Salt reaction of cobalt(2+) and C2/C8/C20 carboxylates

Test material form:

solid: pellets

In vitro test system

Test system:

human skin model

Test animals

Details on test animals or test system and environmental conditions:

Not applicable - Since this is an in-vitro study there is no information on test animals.

Test system

Type of coverage:

open

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

The test item was applied after being reduced to fine powder, at the dose of 16 mg, during 42 minutes at room temperature, to the epidermal surface of 5 living rhCE tissue replicates (including 2 for non-specific colour living control) and 4 killed RhCE tissues replicates (including 2 fo the non-specific MTT reduction control and 2 for the non-specific colour killed control). The Reconstructed Human epidermis had been previously moistened with 10 µL of distilled water.
Under the same experimental conditions, a positive control (16 µL 5% sodium dodecyl sulfate (SDS)) and a negative control (16 µL of distilled water - ADL Prochilab - batch n° 201117) were carried out. The 5% SDS solution was prepared by weighing 0.5 g of SDS (SIGMA - batch n° STBJ3028) in a 10 mL volumetric flask qsp 10 mL of distilled water. Then, the preparation was magnetically stirred, just before the treatment to obtain a colourless solution. To ensure good contact with the epidermis, during all the treatment period, the control items were recovered with a nylon mesh provided by Episkin SA.

Duration of treatment / exposure:

42 minutes

Observation period:

not applicable

Number of animals:

not applicable

Details on study design:

HUMAN SKIN MODEL
The 0.50 cm2 reconstructed epidermis (Episkin SA, RHE/S/17 batch n°21-RHE-169) were received on 26 October 2021. The 4 additional killed Human skin model surfaces (Episkin SA, RHE/S/17 - batch n°21-RHE-057 frozen on 20 April 2021) were defrozen the same day.
The inserts (filter + epidermis) was gently removed from the agarose while avoiding leaving agarose on the polycarbonate filter. The inserts were placed in 24 wells culture plate which had been previously filled with 300 µL of growth medium (Episkin SA, batch n° 21 SGM 118) fot 2 hours and 52 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 (Episkin SA, batch 21 SMM 042).

EVALUATION OF DIRECT INTERACTION WITH MTT
The direct interaction of MTT with the test item was checked by adding 16 mg of the test item to 300 µL of solution of MTT at 1 mg/L. A yellow solution with purple test item was observed after 3 hours of incubation between 37.2°C and 37.4°C, 5% CO2. > Therefore, due to its colour, the test item was identified as porducing interference with the MTT reduction reading. Two killed control tissue models were added to the study which underwent the entire testing procedure to generate a non-specific MTT reguction (NSMTT) control.

SPECTRAL ANALYSIS OF THE TEST ITEM IN ISOPROPANOL
The spectral properties at 570 nm of test item in isopropanol ware checked by adding 16 mg of the test item to 1.5 mL of isopropanol (same conditions as in the main test). A purple solution was obtained after 2 hours of incubation at ambient temperature with gentle shaking. The mean of the corrected OD (blank subtracted) was 0.446 which is higher than 0.08 (value corresponding to approximately twice the OD of the extracting solvent. > Therefore, the test item was identified as causing colour interference with the viability assay. Two viable control tissues were added to the study which underwent the entire testing procedure but were incubated in culture medium instead of MTT solution during the MTT incubbation step to generate a non-specific colour (NSC living) control.

> Therefore, as the test item was identified as interfering with MTT readin reduction and colour interference, two killed control tissues (NSC killed control) were added to the study, which underwent the entire testing procedure but were incubated in cluture medium instead of MTT solution during the incubation step.

GRADING OF REACTIONS
42 minutes after the test item application, the nylon mesh was removed and the human epidermies were washed with 25 x 1 mL of DPBS (Dutscher, batch n° 7380521). The rinsed tissues were checked for any coloration and noted to be white, comparable coloration to that of the negative control tissues.
The epidermises were incubated for 41 hours 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 responsibel 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 quantatively measured by Optical Density (OD) after extraction from tissues.
The measured OD are proportional to the number of living cells.

The skin samples were placed in 300 µL of a MTT solution at 1.0 mg/L for 3 hours at 37°C, 5%CO2; except for the NSC living and killed controls which were placed in the maintenance medium (Episkin SA, batch n° 21 SGM 118) instead of MTT solution.

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 extracts 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.

TREATMENT OF THE RESULTS
The results were expressed as a viability percentage compared with the negative control:
% viability = OD test item / OD negative control * 100

As the test item is identified as producing both direct MTT reduction and colour interference, the true viability was calculated as follows:
% true viability = [(OD of living tissues exposed to test item - OD of killed tissues exposed to test item (NSMTT) - OD of living tissues exposed to the test item incubated with medium without MTT (NSC living) + OD of killed tissues exposed to test item incubated with medium without MTT (NSC killed))/OD living tissues exposed to negative control] * 100

Results for test items producing non specifc MTT reduction (%NSMTT) and/or non-specific colour (%NSC living) ≥ 50% of the negative control should be taken with caution.

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.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean

Value:

104.9

Negative controls validity:

valid

Remarks:

mean viability (%) = 100

Positive controls validity:

valid

Remarks:

mean viability (%) = 1.6

Remarks on result:

other:

Remarks:

Time point: after 42 min of exposure and 42 hours post-treatment incubation.

Other effects / acceptance of results:

- SD value of the % viability ≤ 18%
- Negative control: OD values of the 3 replicates in the range of ≥0.8 and ≤ 3.0.
- The optical density was measured after 1:2 dilution of the formazan extracts in isopropanol; the acceptability criterai should be in the range of ≥0.4 and ≤ 1.5.
- Positive control: mean viability < 40%.

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	0.949	0.870	0.791	0.870	100.0
Positive control	42 min	0.016	0.013	0.013	0.014	1.6
Test item	42 min	0.958	0.892	0.910	0.920	105.7
Test item NSMTT	42 min	0.008	0.010		0.009	1.0
Test item NSC killed	42 min	0.005	0.002		0.004	0.4
Test item NSC living	42 min	0.003	0.001		0.002	0.2
Test item corrected						104.9

OD = Optical Density

The standard deviations between the % variabilities of the test item, the positive and negative controls were respectively 3.9 - 0.2 - 9.1%. The threshold of the "OECD TG 439 Guideline for the Testing of Chemicals 439: In vitro Skin Irritation: Reconstructed Human Epidermis Test Method” is 18%.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In accordance with the Regulation EC n° 1272/2008, the test item has to be considered as non-irritant to skin. It corresponds to UN GHS No Category. No hazard statement or signal word is required.