[No public or meaningful name is available]

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Tributyl(ethyl)phosphonium diphenyl phosphate

Cas Number:

2409816-82-4

Molecular formula:

C26H42O4P2

IUPAC Name:

Tributyl(ethyl)phosphonium diphenyl phosphate

Test material form:

liquid

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: not specified

Source strain:

not specified

Details on animal used as source of 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 representing main
lipid classes analogous to those found in vivo. The EpiDermTM tissues are cultured on specially prepared cell culture inserts.

EpiDermTM tissues were procured from MatTek In Vitro Life Science Laboratories, Bratis-lava.

Vehicle:

not specified

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

30 µL

Duration of treatment / exposure:

24 hours and 35 minutes

Duration of post-treatment incubation (if applicable):

19 hours

Number of replicates:

two

Test system

Details on study design:

7.1 Non-GLP pre - tests
7.1.1 Non- GLP pre-test: Nylon mesh compatibility

It was tested whether the test item reacts with the nylon mesh (EPI-MESH). The mesh was brought onto a slide, then, 30 µL test item were applied.
After 1 hour, the mesh was evaluated microscopically.

No reaction with the mesh was occurred, the nylon mesh was used, to ensure spreading.

7.1.2 Non- GLP pre-test: Assessment of Coloured or Staining Test Items
In a non-GLP pre-test, the light yellow test item was tested for color formation without MTT addition. To test for this ability, 30 µL test item and 300 µL demin. H2O were given in a test tube and the mixture was incubated in the dark at 37 ± 1 °C, 5 ± 1% CO2 and ≥ 95% rela-tive humidityfor 1 hour.

The color of the solution was not significantly changed; therefore, the binding capacity was not tested.

7.1.3 Non- GLP pre-test: Assessment of Direct Reduction of MTT by the Test Item
In a non-GLP pre-test, the test item was tested for the ability of direct MTT reduction. To test for this ability, 30 µL test item were added to 1 mL of MTT solution and the mixture was incubated in the dark at 37 ± 1 °C, 5 ± 1% CO2 and ≥ 95% relative humidityfor 1 hour. Untreated MTT solution was used as control.

The color of the MTT solution did not turn to blue/purple, therefore, the test item was not presumed to have reduced the MTT and no additional test on freeze killed tissues was performed.

7.2 Pre-Incubation of Tissues
All working steps were performed under sterile conditions. For each treatment group (neg-ative control, test item and positive control) a 6-well-platewas prepared with 0.9 mL assay medium in 3 of the 6 wells (upper row). The tissues were inspected for viability. Then, the tissues were transferred into the wells, which contain mediumby using sterile forceps and placed into the incubator at 37 ± 1°C and 5 ± 1% CO2and ≥ 95% relative humidity for 1 hour.
After 1 hour pre-incubation, the other 3 wells of each plate (lower row) were filled with fresh assay medium (0.9 mL). Every tissue was transferred into a well of the lower row. All 6-well-plates were set into the incubator at 37 ± 1°C and 5.0 ± 1% CO2 and ≥ 95% relative humidity for 20 hours.

7.3 Treatment
One plate (3 tissues) was used as negative control; each tissue was treated with 30 µL DPBS buffer, a nylon mesh was added in order to ensure sufficient contact with the tissue surface.
One plate(3 tissues) was used as positive control; each tissue was treated with 30 µL 5% SDS-solution, a nylon mesh was added in order to ensure sufficient contact with the tissue surface.
One plate (3 tissues) was used for treatment with the test item:
30 µL test item were applied, and a nylon mesh was added in order to ensure sufficient contact with the tissue surface.
Tissues were dosed in 1-minute-intervals. After dosing the last tissue, all plates were transferred into the incubator for 35 minutes at 37 ± 1°C and 5.0 ± 1% CO2 and ≥ 95% rel-ative humidity.
1 hour after the first application, the inserts were removed from the plates using sterile forceps and rinsed immediately in 1-minute-intervals.
After rinsing thoroughly with DPBS, each tissue was blotted with sterile cellulose tissue and then transferred into a new 6-well-plate with fresh assay medium (0.9 mL).The surface of theinserts was then carefully dried with a sterile cotton tipped swab.
Then, the tissues were set in the incubator for 24 hours at 37 ± 1°C and 5.0 ± 1% CO2 and ≥ 95% relative humidity.

7.4 Medium Renewal
After post-incubation, the tissues were removed from the incubator and shaken for 5 minutes (120 rpm). 0.9 mL assay medium were filled in the lower row of the 6-well-plate. Then the inserts were transferred into the lower row of the 6-well-plate and set into the incubator for 19 hours for post-incubation at 37 ± 1°C and 5.0 ± 1% CO2 and ≥ 95% rela-tive humidity.

7.5 MTT Assay
After a total incubation time of 43 hoursa 24-well-plate was prepared with 300 µL freshly prepared MTT-solution (1 mg/mL) in each well. The tissues were blotted on the bottom and then transferred into the 24-well-plate. Then the 24-well-plate was set into the incubator for 3 hours at 37 ± 1°C and 5.0 ± 1% CO2and ≥ 95% relative humidity.
After this time, the MTT-solution was aspirated and replaced by DPBS buffer. 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 (120 rpm) for 2 hours at room temperature.
After 2 hours, the inserts were pierced with an injection needle, taking care that all colour was extracted. The inserts were then discarded and the content of each well was tho-roughly mixed in order to achieve homogenisation.
From each well, two replicates with 200 µL solution (each) were pipetted into a 96-well-plate which was read in a plate spectrophotometer at 570 nm. In addition, eight wells of the 96-well-plate were filled with 200 µl isopropanol each, serving as blank.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean

Value:

2.1

Vehicle controls validity:

not specified

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

2.2%

Other effects / acceptance of results:

Three tissues of the human skin model EpiDermTMwere treated with the test itemfor 60 minutes.

After the treatment with the test item, the mean value of relative tissue viability was re-duced to 2.1%. This value is below the threshold for skin irritation potential (50%). Test items that induce values below the threshold of 50% are considered at least irritant to skin.
The optical density of the negative control was well within the required acceptability crite-rion of 0.8≤ mean OD ≤ 2.8.
The positive control has met the acceptance criterion too, for thus ensuring the validity of the test system.

For these reasons, the result of the test is considered valid.

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Conclusions:

The test item tributyl(ethyl)phosphonium diphenyl phosphate is considered at least irritant to skin in the Reconstructed human Epidermis (RhE) Test Method.