Trimethoxy(3,3,3-trifluoropropyl)silane

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date: 02 June 2020, Experimental completion date: 04 June 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

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 June 2019)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Trimethoxy(3,3,3-trifluoropropyl)silane

EC Number:

207-059-3

EC Name:

Trimethoxy(3,3,3-trifluoropropyl)silane

Cas Number:

429-60-7

Molecular formula:

C6H13F3O3Si

IUPAC Name:

trimethoxy(3,3,3-trifluoropropyl)silane

Test material form:

liquid

Specific details on test material used for the study:

Identification: ST2092NM
Batch: 802496
Purity: 100%
Physical state/Appearance: Clear colorless liquid
Expiry Date: 31 October 2020
Storage Conditions: Room temperature in the dark

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: derived from human skin

Justification for test system used:

The purpose of this test was to evaluate the corrosivity potential of the test item using the EpiDerm™ Human Skin Model after treatment periods of 3 and 60 minutes.
Corrosion is directly related to cytotoxicity in the EpiDerm™ tissue. Cytotoxicity is determined by the reduction of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to formazan by viable cells in the test item treated tissues relative to the corresponding negative control. Viable cells are able to reduce MTT to formazan whereas non-viable cells cannot. The results are used to make a prediction of the corrosivity potential of the test item (increased cytotoxicity is indicative of corrosion potential).
This model incorporates several features, which make it advantageous in the study of potential dermal corrosivity. The target cells are epithelial, derived from human skin, and formed into a stratified, cornified epithelium. Test items are applied to the culture surface, at the air interface, so that undiluted and/or end use dilutions can be tested directly.

Vehicle:

unchanged (no vehicle)

Details on test system:

TEST SYSTEM:
EpiDerm™ Reconstructed Human Epidermis Model Kit:
Supplier: MatTek In Vitro Life Sciences Laboratories
Date received: 02 June 2020
EpiDermTM Tissues (0.63cm2) lot number: 30870
Assay Medium lot number : 052820MJC
Upon receipt of the EpidermTM tissues, the sealed 24 well plate was stored in a refrigerator until use.

PRE-TEST PROCEDURES:
ASSESSMENT OF DIRECT TEST ITEM REDUCTION OF MTT:
MTT Dye Metabolism, Cell Viability Assay:
The MTT assay, a colorimetric method of determining cell viability, is based on reduction of the yellow tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue/purple formazan salt by mitochondrial succinate dehydrogenase in viable cells.
One limitation of the assay is possible interference of the test item with MTT. A test item may directly reduce MTT, thus mimicking dehydrogenase activity of the cellular mitochondria. This property of the test item is only a problem if at the time of the MTT test (after rinsing) there is still a sufficient amount of the test item present on or in the tissues. In this case, the true metabolic MTT reduction and the false direct MTT reduction can be differentiated and quantified.

Test for Direct MTT Reduction:
As specified, a test item may interfere with the MTT endpoint, if it was able to directly reduce MTT and at the same time was present on or in the tissues when the MTT viability test was performed. To identify this possible interference, the test item was checked for the ability to directly reduce MTT according to the procedure below:
50 µL of the test item was added to 1 mL of a freshly prepared 1.0 mg/mL MTT solution. The solution was incubated in the dark at 37 °C, 5% CO2 in air for 60 minutes. Untreated MTT solution was tested concurrently to act as a control.
If the MTT solution containing the test item turned blue/purple relative to the control, the test item was presumed to have reduced the MTT.
The MTT solution containing the test item turned black. Therefore, although the solution did not turn blue/purple, the darkening to black was taken as an indication that the test item could directly reduce the MTT. There was a possibility that if the test item could not be totally rinsed off the tissues, any residual test item present on or in the tissue may directly reduce MTT and could have given rise to a false negative result. Therefore, the determination of skin corrosion potential was performed in parallel on viable and non-viable, freeze-killed, tissues.
This step was a functional check which employs freeze killed tissues that possess no metabolic activity but may absorb and bind the test item in the same way as viable tissues.
Freeze killed tissues were prepared prior to the study by placing untreated EPIDERMTM tissues in an empty 12 well plate and storing in a freezer (35 to 10 °C) for a minimum of 24 hours. Before use each tissue was thawed by placing in 0.9 mL of assay medium for approximately 1 hour in an incubator at 37 °C, 5% CO2.
In addition to the normal test procedure, the test item was applied to two freeze killed tissues per exposure period. In addition, two freeze killed tissues per exposure period remained untreated. The untreated freeze killed control normally show a small amount of MTT reduction due to residual reducing enzymes within the killed tissues.

Assessment of Color Interference with the MTT Endpoint:
A test item may interfere with the MTT endpoint if it is colored or if it becomes colored when in wet or aqueous conditions. The MTT assay is affected only if the test item is present in the tissues when the MTT viability assay is performed.
50 µL of test item was added to 300 µL of sterile water. The solution was incubated in the dark at 37 °C, 5% CO2 in air for 60 minutes. A visual assessment of the color was then made.

MAIN TEST:
Pre-Incubation:
The assay medium was brought to room temperature before use. 0.9 mL of this assay medium was pipetted into the appropriate wells of two pre-labeled 6-well plates for both the 3 Minute and 60 Minute exposure periods. EpiDerm™ tissues were transferred into the 6 well plates containing the assay medium. The 6 well plates containing the EpiDerm™ samples were pre-incubated (37 °C, 5% CO2) for approximately 1 hour before dosing.

Application of Test Item and Rinsing:
Before pre-incubation was complete, a 24 well plate was prepared for use as a “holding plate” for both the 3 Minute and 60 Minute exposure periods. This plate was used to maintain the viability of the tissue inserts between rinsing following chemical exposure and MTT-loading. Another 24 well plate was prepared for the MTT-loading. 300 µL of either pre warmed assay medium (holding plate) or MTT medium (MTT-loading plate) was dispensed into each well. The two plates were placed into the incubator until required.
After pre incubation of the EpiDerm™ tissues, the medium was aspirated and replaced with 0.9 mL of fresh assay medium. The 6-well plate for the 3 Minute exposure period was returned to the incubator, while the other was being dosed for the 60 Minute exposure. For the 60 Minute exposure period, 50 µL of sterile distilled water (negative control) was added to the first two tissues. The tissues were dosed at regular intervals to allow for the time taken to rinse each tissue following exposure and to ensure that each tissue gets an equal exposure time. 50 µL of the test item and 50 µL of 8.0 N Potassium Hydroxide (positive control) were also applied to the corresponding tissues in turn. The plate was returned to the incubator (37 °C, 5% CO2) for the 60 Minute exposure period.
When dosing for the 60 Minute exposure period was complete, the same procedure was repeated for the 3 Minute exposure period. Because the exposure time was so short, the tissues were dosed at regular intervals to ensure that each tissue received an equal exposure time and to allow for the time taken to rinse each tissue following exposure. Rinsing was achieved by filling and emptying each tissue under a constant soft stream of Dulbecco’s Phosphate Buffered Saline (DPBS) (without Ca++ Mg++) for approximately 40 seconds, to gently remove any residual test item. Excess DPBS was removed by blotting the bottom of the tissue insert with tissue paper. Each tissue was placed into the prepared holding plate until all tissues were rinsed. They were then blotted and transferred to the 24 well plate prepared for MTT-loading. The plate was incubated (37 °C, 5% CO2) for 3 hours. Once the 60 Minute exposure period was complete, the same rinsing and MTT-loading procedure was repeated.
After the 3 Hour MTT incubation was complete, the tissue inserts were blotted and transferred to 24 well plates for formazan (reduced MTT) extraction. The formazan was extracted from the top and bottom of the tissue by completely immersing the tissue insert in 2 mL of isopropanol. The plate was covered with plate sealer, to prevent isopropanol evaporation, and stood overnight at room temperature, to allow extraction to proceed.

Absorbance/Optical Density Measurements:
After extraction, each tissue was pierced with a pipette fitted with a 1000 µL tip and the extraction solution was forced vigorously up and down to form a homogenous solution. 3 x 200 µL aliquots of the extract were transferred to the appropriate wells of a pre labeled 96 well plate. 200 µL of isopropanol alone was added to the three wells designated as blanks. Absorbency at 570 nm (OD570) of each well was measured using the Labtech LT 4500 microplate reader and LT-com analysis software.

DATA EVALUATION:
Quantitative MTT Assessment (percentage tissue viability):
The corrosivity potential of the test item was predicted from the relative mean tissue viabilities obtained after the 3 and 60 Minute exposure periods, compared to the mean of the negative control tissues (n=2) treated with sterile distilled water. The relative mean viabilities were calculated in the following way:
Relative mean viability (%) = mean OD570 of test item / mean OD570 of negative control x 100

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:

50 µL of the test item, positive and negative controls were applied to tissues.

Duration of treatment / exposure:

3 and 60 minute exposure periods.

Duration of post-treatment incubation (if applicable):

3 hours

Number of replicates:

Dulpicate tissues were treated.

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Remarks:

Test Item

Run / experiment:

3 minute exposure / mean viabilities

Value:

95.3

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: non-corrosive

Results 2

Irritation / corrosion parameter:

% tissue viability

Remarks:

Test item

Run / experiment:

60 minutes exposure / mean viabilities

Value:

98.1

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: non-corrosive

Other effects / acceptance of results:

Direct MTT Reduction:
The MTT solution containing the test item turned black. Therefore, although the solution did not turn blue/purple, the darkening to black was taken as an indication that the test item could directly reduce the MTT and thus MTT corrective procedures were performed using non-viable, freeze-killed, tissues. However, the results obtained showed that no or negligible interference due to direct reduction of MTT occurred. It was therefore considered unnecessary to use the results of the freeze killed tissues for quantitative correction of results or for reporting purposes.

Assessment of Color Interference with the MTT endpoint:
The solution containing the test item did not become colored. This was taken to indicate the test item did not have the potential to cause color interference.

Test Item, Positive Control Item and Negative Control Item:
Mean OD570 values and viabilities for the negative control, positive control and test item are given in any other information on results incl. tables section.

Acceptance Criteria:
The mean OD570 for the negative control treated tissues was 1.814 for the 3 Minute exposure period and 1.950 for the 60 Minute exposure period. The negative control acceptance criteria were therefore satisfied.
The relative mean tissue viability for the positive control treated tissues was 2.6% relative to the negative control following the 60 Minute exposure period. The positive control acceptance criterion was therefore satisfied.
In the range 20 to 100% viability the Coefficient of Variation between the two tissue replicates of each treatment group did not exceed 30%. The acceptance criterion was therefore satisfied.

Any other information on results incl. tables

Test Item, Positive Control Item and Negative Control Item:

Mean OD₅₇₀values and viabilities for the negative control, positive control and test item are given below.

Mean OD₅₇₀Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item

Tissue	Exposure Period	MeanOD570of individual tissues	Mean OD570of duplicate tissues	Standard Deviation	Coefficient of Variation (%)	Relative Mean Viability (%)
Negative Control	3 Minutes	1.670	1.814	0.203	11.2	100*
		1.957
	60 Minutes	1.883	1.950	0.094	4.8
		2.016
Positive Control	3 Minutes	0.066	0.062	0.006	Na	3.4
		0.057
	60 Minutes	0.060	0.051	0.013	Na	2.6
		0.041
Test Item	3 Minutes	1.722	1.729	0.010	0.6	95.3
		1.736
	60 Minutes	1.896	1.912	0.023	1.2	98.1
		1.928

OD= Optical density

*=          The mean percentage viability of the negative control tissue is set at 100%

OD = Optical density

*=          The mean percentage viability of the negative control tissue is set at 100%

Applicant's summary and conclusion

Interpretation of results:

other: Non-corrosive prediction according to EU CLP

Conclusions:

The test item was considered to be non-corrosive to the skin.

Executive summary:

Introduction

The purpose of this test was to evaluate the corrosivity potential of the test item using the EpiDerm™ Human Skin Model after treatment periods of 3 and 60 minutes.

Corrosion is directly related to cytotoxicity in the EpiDerm™ tissue. Cytotoxicity is determined by the reduction of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to formazan by viable cells in the test item treated tissues relative to the corresponding negative control. Viable cells are able toreduce MTT to formazanwhereas non-viable cells cannot. The results are used to make a prediction of the corrosivity potential of the test item (increased cytotoxicity is indicative of corrosion potential).

Methods

Duplicate tissues were treated with the test item for exposure periods of 3 and 60 minutes. Negative and positive control groups were treated for each exposure period. As the test item had been shown to directly reduce MTT additional non-viable, freeze-killed, tissues were incorporated into the testing for correction purposes. At the end of the exposure period the test item was rinsed from each tissue before each tissue was taken for MTT‑loading. After MTT-loading each tissue was placed in 2 mL of isopropanol for MTT extraction.

At the end of the formazan extraction period each well was mixed thoroughly and triplicate 200 mL samples were transferred to the appropriate wells of a pre-labeled 96‑well plate. The optical density (OD) was measured at 570 nm (OD₅₇₀).

Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

Results

The results obtained showed that no or negligible interference due to direct reduction of MTT occurred in the main test. It was therefore considered unnecessary to use the results of the freeze-killed tissues for quantitative correction of results.

The relative mean viabilities for each treatment group were as follows:

Exposure Period	Percentage Viability
	Negative Control	Positive Control	Test Item
3 minute	100*	3.4	95.3
60 minute	100*	2.6	98.1

*The mean viability of the negative control tissues is set at 100%

Acceptance criteria: The criteria required for acceptance of results in the test were satisfied.

Conclusion

In this study and under the experimental conditions reported:

The test item was considered to be non-corrosive to the skin.