Reaction mass of 4,4'-[2,2,2-trifluoro-1-(trifluoromethyl)ethylidene]diphenol and benzyltriphenylphosphonium, salt with 4,4'-[2,2,2-trifluoro-1-(trifluoromethyl) ethylidene]bis[phenol] (1:1)

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 January 2018 to 31 January 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Benzyltriphenylphosphonium, salt with 4,4'- [2,2,2- trifluoro-1- (trifluoromethyl)ethylidene]bis[phenol] (1:1)

Cas Number:

75768-65-9

IUPAC Name:

Benzyltriphenylphosphonium, salt with 4,4'- [2,2,2- trifluoro-1- (trifluoromethyl)ethylidene]bis[phenol] (1:1)

Test material form:

solid

Details on test material:

Purity: 98 %

In vitro test system

Test system:

human skin model

Source species:

other: EpiDerm™ tissues

Cell type:

other: EpiDerm™ tissues

Cell source:

other: EpiDerm™ tissues

Source strain:

not specified

Vehicle:

unchanged (no vehicle)

Details on test system:

Receipt of the EpiDerm™ Skin Model
Upon receipt of the EpiDerm™ Skin Bioassay Kit, the solutions were stored as indicated by the manufacturer. The EpiDerm™ tissues were stored at 2-8ºC until used. On the day of dosing, an appropriate volume of EpiDerm™ assay medium was removed and warmed to approximately 37ºC. Nine-tenths (0.9) mL of assay medium were aliquotted into the wells of each 6-well plate. The six-well plates were labeled to indicate test article and exposure time. The EpiDerm™ tissues were inspected for air bubbles between the agarose gel and cell culture insert prior to opening the sealed package. Tissues with air bubbles covering greater than 50% of the cell culture insert area were not used. The 24-well shipping containers were removed from the plastic bag and their surfaces were disinfected with 70% ethanol. The EpiDerm™ tissues were transferred aseptically into the 6-well plates. The EpiDerm™ tissues were then incubated in the dark at 37±1ºC in a humidified atmosphere of 5±1% CO2 in air (standard culture conditions) for at least one hour. The medium was then aspirated and 0.9 mL of fresh medium were added to each assay well below the EpiDerm™ tissues. The plates were returned to the incubator until treatment was initiated. Upon opening the bag, any remaining unused tissues were briefly gassed with an atmosphere of 5% CO2/95% air and placed back at 2-8ºC for later use.

Preparation and Delivery of Test Article
The tissues designated to the negative control were treated with 50 μL of sterile, deionized water. The tissues designated to the positive control, 8N KOH (Sigma), were tested using the same method. All exposure conditions were documented in the study workbook.
The solid test article was placed directly onto the tissue using a 25 mg sharp spoon (Aesculap, Cat. No. FK 623R). The sharp spoon was filled with the test article, and leveled by gently stroking away excess test article using a rod-shaped instrument. Care was taken to avoid packing the material into the spoon. The contents of the spoon was poured over the tissue surface. Each EpiDerm™ tissue treated with the solid test article received 25 μL of sterile, deionized water applied directly onto the test article. The test article was gently mixed, and spread over the tissue surface using a bulb-headed rod if needed.

Assessment of Direct Test Article Reduction of MTT
The test article was added to a 1.0 mg/mL MTT (Sigma) solution in warm Dulbecco’s Modified Eagle’s Medium (DMEM) containing 2 mM L-glutamine (MTT Addition Medium) to assess its ability to directly reduce MTT. Approximately 25 mg of the test article were added to 1 mL of the MTT solution, and the mixture was incubated at standard culture conditions for at least one hour. A negative control, 50 μL of sterile, deionized water (Quality Biological), was tested concurrently. If the MTT solution color turned blue/purple, the test article was presumed to have reduced the MTT.
The test article was not observed to directly reduce MTT in the absence of viable cells.
The positive control, 8N potassium hydroxide (8N KOH), is known to directly reduce MTT in the absence of viable cells. Therefore, a killed control experiment was performed concurrently in the definitive assay to determine the extent of the direct MTT reduction (if any) by the positive control in non-viable freeze-killed tissues.

Assessment of Colored or Staining Materials
Approximately one leveled spoonful (approximately 25 mg) (solid test articles) were added to 2.0 mL isopropanol in 6-well plates and placed on an orbital place shaker for 2-3 hours at room temperature. After shaking, 200 μL aliquots of the isopropanol solutions and two blank samples of isopropanol were transferred to a 96-well plate and the absorbance was measured with a plate reader at the MTT measurement wavelength (550 nm).
Some test articles may precipitate or become cloudy after addition to the isopropanol and interfere with the optical density (OD) measurement. In such cases, after the 2-3 hour shaking period, the test article-isopropanol mixture was transferred into centrifuge tubes and centrifuged (e.g., 100 x g, for 5 minutes at room temperature) prior to transfer to the 96-well plates for the absorbance determination. The same centrifugation procedure may be performed after the isopropanol extraction period in the definitive assay.
The absorbance of the test article samples was determined by subtracting the mean isopropanol blank value from the absorbance of the test article samples. If the OD550 of the test article sample is > 0.08, the material has to be considered as possibly interacting with the MTT measurement.
The test article was not considered to have probable photometric MTT interference.

pH Determination
The pH of the test article was not measured as the test article is a solid.

Skin Corrosion Assay
The test and control articles were tested by treating four EpiDerm™ tissues per material. Two tissues were used to assess viability after the 3-minute exposure, and two were used to assess viability after the 60-minute exposure. Twenty-five mg of solid (powdered) test article were applied topically on the EpiDerm™ tissue. Each EpiDerm™ tissue treated with a solid test article also received 25 μL of sterile, deionized water applied directly onto the test article. Fifty (50) microliters of the negative and positive controls were applied topically on the EpiDerm™ tissue. The three-minute exposure time began as soon as the material was spread onto the tissue. This short exposure time precluded treating more than a small number of tissues at once. The cultures exposed for 3 minutes were held at room temperature during dosing, while the cultures exposed for the 60 minutes were incubated at standard culture conditions until the completion of the exposure time.
A 1.0 mg/mL solution of MTT in warm MTT Addition Medium was prepared no more than 2 hours before use. Three hundred (300) μL of MTT reagent solution were added to designated wells in a pre-labeled 24-well plate. The plate was held in the incubator until tissues were added. After the appropriate exposure time, the EpiDerm™ tissues were extensively rinsed with warm (approximately 37ºC) Calcium and Magnesium-Free Dulbecco's Phosphate Buffered Saline (Ca++Mg++-Free DPBS) and the wash medium was decanted. The EpiDerm™ tissues were transferred to the appropriate wells after rinsing. The plates were incubated at standard culture conditions for 3 ± 0.1 hours.
After the incubation period with MTT solution, the EpiDerm™ tissues were blotted on absorbent paper, cleared of excess liquid, and transferred to a pre-labeled 24-well plate containing 2.0 mL of isopropanol in each designated well. The plates were covered with paraffin film and stored in the refrigerator (2-8ºC) until the last exposure time was harvested. Then the plates were shaken for 2 - 3 hours at room temperature.
At the end of the extraction period, the liquid within the cell culture inserts was decanted into the well from which the cell culture insert was taken. The extract solution was mixed and 200 μL were transferred to the appropriate wells of a 96-well plate. Two hundred (200) μL of isopropanol were placed in the two wells designated as the blanks. The absorbance at 550 nm (OD550) of each well was measured with a Molecular Devices Vmax plate reader.

Killed Controls (KC)
To evaluate whether residual positive control was binding to the tissue and leading to a false MTT reduction signal, a functional check (using freeze-killed control tissue) was performed. Freeze-killed tissues were received from MatTek Corporation, and were stored in the freezer until use.
For the positive control, 8N KOH, duplicate killed tissues were treated in the normal fashion for 3 and 60 minutes. The rinsing, MTT exposure, and solvent extraction procedures were performed exactly as described for the viable tissues. Duplicate killed-control tissues were treated with the negative control for 3 and 60 minutes. A small amount of MTT reduction is expected from the residual NADH and associated enzymes within the killed tissue. This background reduction of MTT will be compared to the MTT reduction observed in the positive control treated killed-control tissues using calculations described in the Presentation of Data.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

25 mg of solid (powdered) test article were applied topically on the EpiDerm(TM) tissue;

Duration of treatment / exposure:

3-minute exposure
60-minute exposure

Number of replicates:

2 replicates were used to assess viability after 3-minute exposure
2 replicates were used to assess viability after 60-minute exposure

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3-minute exposure

Value:

108

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Non-corrosive

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60-minute exposure

Value:

81.3

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Non-corrosive

Results 3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Positive control 3-minutes exposure

Value:

21

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Corrosive 1A

Results 4

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Positive control 60-minutes exposure

Value:

10.4

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Corrosive 1A

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test article was not determined to be a colorant (was not considered to have potential interference with the MTT measurement) or a direct MTT reducer.

Executive summary:

The purpose of this study was to assess the potential skin corrosivity of the test article supplied by Blue Frog Scientific, in the EpiDerm Kit (MatTek Corporation). The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) conversion assay, which measures the NAD(P)H-dependent microsomal enzyme reduction of MTT (and to a lesser extent, the succinate dehydrogenase reduction of MTT) to a blue formazan precipitate, was used to assess cellular metabolism after test article exposure1. The protocol is consistent with the OECD Test Guideline 431 “In Vitro Skin Corrosion: Human Skin Model Test”.

The method utilizes a 3-minute exposure for a corrosive classification and a 60-minute confirmatory exposure for materials found to be non-corrosive at the 3-minute exposure. Viable cells reduce the yellow, soluble, oxidized form of the MTT to the blue-black, insoluble, reduced form. The reduced dye is extracted from the tissue with isopropanol, and the amount of reduced dye is determined spectrophotometrically. The relative viability of the treated tissues is calculated as a percentage of the negative control viability from the absorbance data by dividing the corrected test article-treated tissue absorbance by the corrected control tissue absorbance, and multiplying by 100. Test materials which reduce tissue viability to < 50% within 3 minutes are considered corrosive by this method. In addition, test materials which result in tissue viability of ≥ 50% after a 3-minute exposure, but result in tissue viability of < 15% after a 60-minute exposure are also classified corrosive. Test materials which result in tissue viabilities of ≥ 50% after a 3-minute exposure and ≥15% after a 60-minute exposure are classified non-corrosive. Furthermore, sub-classification of corrosive materials is possible using the 3 minute exposure time as follows: a sub-category classification of 1A is assigned if the viability is < 25%, and 1B/1C if the viability is ≥ 25%.

Viability of 108% for 3-minutes exposure and 81.3% for 60 -minutes exposure were obtained for the test substance, therefore the test substance was not determined to be a colorant (was not considered to have potential interference with the MTT measurement) or a direct MTT reducer. Based on these findings the substance should not be classified as skin corrosive.