Alkali metal tetrakis (perfluoro-alcoholato)metallate

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

18.6.2019

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Test material form:

solid

Details on test material:

Test material was sublimed for purification.

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Details on animal used as source of test system:

EpiDerm™ tissues derived from MatTek Corporation, Lot# 30863, keratinocyte strain 00267

Justification for test system used:

The skin corrosion test refers to the production of irreversible tissue damage following the application of a test material on a reconstructed human skin model. It allows the identification of corrosive chemical substances and mixtures.
The test item is applied topically to a three-dimensional human skin model, comprising of non-transformed, human-derived epidermal keratinocytes, which have been cultured to form a multi-layered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers representing main lipid classes analogous to those found in vivo.
The principle of the human skin model assay is based on the hypothesis that corrosive chemicals are able to penetrate the stratum corneum by diffusion or erosion and are cytotoxic to the underlying cell layers.
Corrosive chemicals are identified by their ability to decrease cell viability. The viability is measured by enzymatic conversion of the vital dye MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide) into a blue formazan salt, that is quantitatively measured after extraction from tissues.

Vehicle:

water

Details on test system:

The test system is a commercially available EpiDermTM-Kit, procured by MatTek.
The EpiDermTM tissue consists of human-derived epidermal keratinocytes which have been cultured to form a multi-layered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDermTM tissues are cultured on specially prepared cell cultures inserts.
EpiDermTM tissues were procured from MatTek In Vitro Life Science Laboratories, Bratislava (Designation of the kit: EPI-200-SCT, Batch: 30863)

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

in the 3 minute exposure experiment two tissues were tested with 25.9 mg and 25.2 mg test substance; for the 1 hour exposure experiment also two tissues were exposed to 26.2 mg and 26.3 mg test substance, respectively.

Duration of treatment / exposure:

3 minutes and 1 hour exposure

Duration of post-treatment incubation (if applicable):

After the respective incubation time (“3 minutes” and “1 hour”) at 37 ± 1 °C and 5.0 ±1% CO2, the inserts were removed from the plates using sterile forceps. The inserts were thoroughly rinsed with DPBS, blotted with sterile cellulose tissue and set into the respective holding plate, using the wells containing assay medium. After transfer of all inserts, they were immediately moved to the wells containing MTT medium, blotting the bottom with cellulose tissue again before setting the insert into the MTT well. The tissues were incubated with MTT solution for 3 hours at 37 ±1 °C and 5.0 ±1% CO2.
After this time, the MTT solution was aspirated and replaced by DPBS. This was then aspirated, too, and replaced several times. At last, each insert was thoroughly dried and set into the empty, pre-warmed 24-well-plate. Into each well, 2 mL isopropanol were pipetted, taking care to reach the upper rim of the insert. The plate was then shaken for 2 hours at room temperature.

Number of replicates:

Two tissues per exposure time were tested

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Test item, 3 minutes incubation

Value:

109

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Positive control, 3 minutes incubation

Value:

20.7

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Test item, 1 hour incubation

Value:

79.2

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Results 4

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Positive control, 1 hour incubation

Value:

4.9

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Validity
The criterion for optical density of the negative control (≥ 0.8 and ≤ 2.8) was fulfilled: optical density was 1.6 (3 minutes) resp. 1.7 (1 hour).
The positive control showed clear corrosive effects. The criterion for the viability of the 1 hour experiment, expressed as % of the negative control (< 15%), was fulfilled, too. The mean value of relative tissue viability was 4.9%.
The values for negative control and for positive control were within the range of historical data of the test facility.
Therefore, the experiment is considered valid.

Any other information on results incl. tables

Measured Values

As blank, the optical density of isopropanol was measured in 12 wells of the 96-well-plate. The measured values and their mean are given as follows:

Absorbance: 0.035, 0.036, 0.035, 0.038, 0.035, 0.035, 0.035, 0.035, 0.035, 0.034, 0.034, 0.035, Mean of 12 values: 0.035

 

The absorbance values of negative control, test item and positive control are given in the following table:

Absorbance Values (OD 570 nm)

Incubation	Negative Control		Test Item		Positive Control
	Tissue 1	Tissue 2	Tissue 1	Tissue 2	Tissue 1	Tissue 2
3 min	1.575	1.686	1.753	1.798	0.354	0.376
	1.574	1.689	1.736	1.793	0.353	0.377
	1.567	1.668	1.747	1.794	0.351	0.379
1 h	1.760	1.623	1.397	1.302	0.118	0.114
	1.748	1.619	1.385	1.299	0.117	0.115
	1.750	1.616	1.381	1.294	0.118	0.114

 

From the measured absorbances, the mean absorbance of isopropanol was subtracted. The corrected mean and relative standard deviation (RSD) of the two tissues were also calculated.

Mean Absorbance Values of the 3 Minutes Experiment

Designation	Negative Control	Test Item	Positive Control
Mean – blank (tissue 1)	1.537	1.710	0.318
Mean – blank (tissue 2)	1.646	1.760	0.342
Mean of the two tissues	1.591	1.735	0.330
RSD	4.8%	2.0%	5.3%

 

Mean Absorbance Values of the 1 h Experiment

Designation	Negative Control	Test Item	Positive Control
Mean – blank (tissue 1)	1.718	1.353	0.083
Mean – blank (tissue 2)	1.584	1.263	0.079
Mean of the two tissues	1.651	1.308	0.081
RSD	5.7%	4.8%	2.9%

 

Comparison of Tissue Viability

For the test item and the positive control, the following percentage values of mean tissue viability were calculated in comparison to the mean of the negative controls:

% Tissue Viability

Test Item	Positive Control	Incubation
109.0%	20.7%	3 min
79.2%	4.9%	1 h

 

Corrosivity of the Test Item

The mean value of relative tissue viability of the test item was increased to 109.0% after 3 minutes treatment. This value is above the threshold for corrosivity (50%). After 1 hour treatment, the mean value of relative tissue viability of the test item was reduced to 79.2%, lying above the threshold for corrosivity (15%). Therefore, the test item is considered as non-corrosive to skin in this study.

Applicant's summary and conclusion

Interpretation of results:

other: substance is non-corrosive to skin

Conclusions:

The test item AM(pfa)4 is considered non-corrosive to skin.

Executive summary:

One valid experiment was performed.

Two tissues of the human skin model EpiDerm™ were treated with the test item for 3 minutes and 1 hour, respectively. The test item was applied to each tissue and spread to match the tissue size.

Demineralised water was used as negative control and 8 M KOH was used as positive control.

After treatment, the respective substance was rinsed from the tissues. Then, cell viability of the tissues was evaluated by addition of MTT, which can be reduced to a blue formazan. Formazan production was evaluated by measuring the optical density (OD) of the resulting solution.

After treatment with the negative control, the absorbance values were within the required acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 for both treatment intervals; thus, showing the quality of the tissues. The OD was 1.6 (3 minutes experiment) and 1.7 (1 hour experiment).

The positive control showed clear corrosive effects for both treatment intervals. The mean relative tissue viability value was reduced to 4.9% for the 1 hour treatment.

After 3 minutes treatment with the test item, the mean value of relative tissue viability was increased to 109.0 %. This value is above the threshold for corrosion potential (50%). After 1 hour treatment, the mean value of relative tissue viability was reduced to 79.2%. This value too is above the threshold for corrosion potential (15%).

 

Thus, under the conditions of this study, the test item AM(pfa)4 is considered non-corrosive to skin in the Reconstructed Human Epidermis (RHE) Test method.