[No public or meaningful name is available]

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14.06.-05.08.2021

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:

2022

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 June 2019

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

bis(diethylamino)methylidene-dibutylazanium;chloride

Cas Number:

89450-30-6

Molecular formula:

C17H38N3Cl

IUPAC Name:

bis(diethylamino)methylidene-dibutylazanium;chloride

Test material form:

solid

Details on test material:

Batch No.: EJ/V35/21

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: The EpiDerm™ tissues were provided as kits (e.g. EPI-200-SIT, MatTek)

Source strain:

other: not applicable

Details on animal used as source of test system:

not applicable

Justification for test system used:

The EpiDerm™ Skin Model is a well-established organotypic, three-dimensional model of the human epidermis and is used for in vitro experiments since many years. It is known for its similarity to human skin.

Vehicle:

unchanged (no vehicle)

Remarks:

The test item was applied directly atop the EpiDerm™ tissue using an application spoon avoiding compression of the test item. To ensure good contact with the skin the test item was moistened with 25 μL H2O.

Details on test system:

Upon receipt of the EpiDerm™, the tissues were inspected visually and transferred into 6-well plates containing 0.9 mL pre-warmed assay medium per well. The 6-well plates were pre-incubated in a humidified incubator at 37 +/- 1 °C, 5.0% CO2 / 95% air for at least 1 h. Then the medium was replaced by 0.9 mL fresh assay medium and the surface was dried using a sterile cotton tip. The 6-well plate used for the 3 min experiment was placed back into the incubator. The other plate was used for the 60 min treatment. About 1 h before the end of the first treatment period the MTT solution was prepared and pre-warmed in the incubator.

60 min experiment: the tissues were treated with each dose group in duplicate, starting with the
negative control. Start time was recorded with dosing of the first tissue. A constant time interval of e.g. 20 sec. was kept between dosing. Then the 6-well plate was incubated at 37 +/- 1 °C, 5.0% CO2 / 95% air.

3 min experiment: the tissues were treated with each dose group in duplicate, starting with the
negative control. Start time was recorded with dosing of the first tissue. A constant time interval of 20 sec. was kept between dosing.
After 3 min of application, the first insert was removed from the 6-well plate with forceps. Using a wash bottle, the tissue was gently rinsed about 20 times with PBS (phosphate buffered saline) to remove any residual test item. Excess PBS was removed by gently shaking the insert and blotting bottom with blotting paper. The insert was placed in a prepared 24-well “holding plate“ containing 300 μL prewarmed assay medium per well. All inserts were treated in the same manner.
Then the inserts were dried again and transferred into a prepared 24-well “MTT assay plate“ containing 300 μL pre-warmed MTT solution. The plate was incubated for 3 h at 37 +/- 1 °C, 5.0% CO2 / 95% air.

60 min experiment: after 60 min application, the first insert was removed from the 6-well plate with forceps. Using a wash bottle, the tissue was gently rinsed about 20 times with PBS to remove any residual test item. Excess PBS was removed by gently shaking the insert and blotting bottom with blotting paper. The insert was placed in a prepared 24-well “holding plate“ containing 300 μL prewarmed assay medium per well. All inserts were treated in the same manner. Then the inserts were dried again and transferred into a prepared 24-well “MTT assay plate“ containing 300 μL pre-warmed MTT solution. The plate was incubated for 3 h at 37 +/- 1 °C, 5.0% CO2 / 95% air.

3 min and 60 min experiment: after the 3 h MTT incubation period the MTT solution was aspirated. The wells were refilled with PBS and the PBS was aspirated. The rinsing was repeated twice and the tissues were dried. Then the inserts were transferred into 12-well “extraction plates“. 2 mL of isopropanol were pipetted into each insert: thus, the insert was covered from both sides.
The extraction plates were sealed in zip-bags to inhibit isopropanol evaporation. Extraction was carried out either over night without shaking at room temperature or, alternatively, at least 2 h with shaking at room temperature.
After the extraction period the inserts were pierced with an injection needle to allow the extracts to run through the tissues into the corresponding wells. Then the inserts were discarded and the extraction plates were placed on a shaker for 15 min.
Per each tissue 3 x 200 μL aliquots of the extract were transferred into a 96-well plate and OD was
measured at 570 nm without reference wavelength in a plate spectrophotometer using isopropanol as
a blank.


MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 min, 60 min
- Spectrophotometer: plate spectrophotometer
- Wavelength: 570 nm

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 50%, or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15%.
- The test substance is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

25 mg + 25 μL H2O

Duration of treatment / exposure:

3 min, 60 min

Number of replicates:

2 tissues per time time point

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 minutes exposure

Value:

ca. 13

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minutes exposure

Value:

ca. 102.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

Test results are attached as pdf file

Applicant's summary and conclusion

Conclusions:

The test item showed corrosive effects. The mean relative tissue viability (% negative control) was reduced below 15% after 60 min treatment but not below 50% after 3 min treatment. The test item is therefore classified as “corrosive“ in accordance with a combination of optional UN GHS sub-categories 1B and 1C.

Executive summary:

The potential of the test item to induce skin corrosion was analysed by using the three-dimensional
human skin model EpiDerm™, comprising a reconstructed epidermis with a functional stratum
corneum.
In the present study Dibuthyltetraethylguanidinium chloride was applied topically to the EpiDerm™ tissue for 3 min and 60 min followed by immediate determination of cytotoxic effects via MTT reduction
assay.
Corrosivity potential of the test item was predicted from the relative mean tissue viabilities obtained
after both treatment periods had been compared to the corresponding negative control tissues.
The test item has no non-specific MTT-reducing or colouring potential, therefore no additional controls
were necessary.
The test item showed corrosive effects. The mean relative tissue viability (% negative control) was
reduced below 15% (13.0%) after 60 min treatment but not below 50% (102.3%) after 3 min treatment.
The controls confirmed the validity of the study. The mean OD570nm of the two negative control tissues
was ≥ 0.8 and ≤ 2.8 for each exposure period (1.760, 1.747). The mean relative tissue viability
(% negative control) of the positive control was ≤ 15% (6.1%) after 60 min treatment. The coefficient
of variation (CV) (in the range of 20 – 100% viability) of replicate tissues of all dose groups was ≤ 30%
(0.6% - 4.0%).