Trimethoxy(3,3,3-trifluoropropyl)silane

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date : 18 February 2021; Experimental completion date : 04 March 2021

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 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)

Version / remarks:

2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

ARE-Nrf2 luciferase KeratinoSens™ test method

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:

Test item: ST2092NM
Storage conditions: Room temperature (10 - 30˚C), in the dark
Purity: 100%
Appearance: Clear colorless liquid
Expiry date: 30 June 2021

In vitro test system

Details of test system:

Keratinoses transgenic cell line [442D]

Details on the study design:

Cell Culture:
The cells used in this assay were the transgenic cell line KeratinoSens™ with a stable insertion of the luciferase construct supplied by Givaudan (Dubendorf, Switzerland). The cells were routinely grown and subcultured in maintenance medium at 37°C ± 2°C in a humidified atmosphere containing 5% CO2 in air. Maintenance medium was 500 mL Dulbecco’s Modified Eagles Medium containing Glutamax (DMEM) (Gibco 21885), supplemented with 50 mL foetal bovine serum (FBS) (Gibco 10270) and 5.5 mL Geneticin (Gibco 10131).

Cell Culture from Frozen Stocks:
Vials of KeratinoSens™ cells, stored frozen in cryotubes at -196°C under liquid nitrogen, in DMEM containing 10% dimethyl sulfoxide and 20% FBS, were thawed rapidly at 37°C in a water-bath. The cells were then resuspended in 9 mL of pre-warmed maintenance medium without geneticin and pelleted by centrifugation at 125 g for 5 minutes. The cell pellet was resuspended in maintenance medium without geneticin in tissue culture flasks. The flasks were incubated until 80-90% confluent cell monolayers had been obtained. Geneticin-containing medium was used in subsequent passages. Establishing cell cultures from frozen stocks and subsequent passage was conducted prior to the start of this study.

Cell Passage:
Actively growing cell stocks were maintained and expanded by subculturing (passage). When the cells had reached 80 – 90% confluence, the medium from each flask was removed, the cells washed twice with Dulbecco’s phosphate buffered saline (DPBS) (Gibco 14190) and harvested using trypsin-EDTA solution. Cultures were incubated at 37 ± 2°C in a humidified atmosphere containing 5% CO2 in air until complete detachment and disaggregation of the cell monolayer had occurred. The cells were then resuspended in medium to neutralise the trypsin (cells from several flasks were pooled at this point). The cells were resuspended and distributed into flasks containing fresh maintenance medium. This passage procedure was repeated to provide a sufficient number of cells for a test and were passaged at least twice before using the cells in a test. The passages of KeratinoSens™ cells were limited to 25 passages from frozen stock.

Preparation of Test Cell Cultures:
The cells from flasks of actively growing cultures were detached and disaggregated as described above. The number of viable cells in the prepared cell suspension were determined by counting a trypan blue-stained cell preparation using an Improved Neubauer Haemocytometer. The cell suspension was diluted with maintenance medium without geneticin to give 1 x 10^5 viable cells/mL and 100 µL volumes pipetted into all wells except one well of sterile 96-well flat-bottomed microtitre plates. On each occasion four plates were prepared in parallel: three white plates for measuring luminescence and one transparent plate for measuring cell viability using the MTT assay. One well of each plate received 100 µL maintenance medium without geneticin with no cells. The plates were incubated for 24 ± 2 hours at 37 ± 2°C in a humidified atmosphere of 5% CO2 in air, to allow the cells to attach.

Test Item Solubility:
Prior to commencing testing, the solubility of the test item in DMSO at 200 mM was assessed. The test item, ST2092NM, was found to be soluble in DMSO at 200 mM, the highest concentration as recommended by the guideline this test follows. When dosed at 1% v/v into assay medium, it remained soluble to give a final in-assay concentration of 2000 µM.

Preparation of the Test Item:
A stock solution of the test item, ST2092NM, was prepared by weighing the test item into a tared glass container and diluting to 200 mM in DMSO.

Test Procedure:
Preparation of the 100x Solvent Plate:
A 100x solvent plate was set up by adding 200 µL of the stock solution of the test item to one well in column 12. 200 µL of the 6.4 mM stock solution of cinnamic aldehyde was added to the appropriate well in column 11. One well was left blank. All other wells contained 100 µL of the appropriate solvent.
Serial halving dilutions of the test item were prepared by transferring 100 µL from each dilution into 100 µL of solvent. Pipette tips were discarded after each transfer and then fresh pipette tips were used to mix each concentration prior to the next transfer.
The 6.4 mM stock solution of cinnamic aldehyde was diluted from column 11 to column 7 using the same procedure of serial halving dilutions.

Preparation of the Dilution Plate:
The 100x solvent plate was replicated into a fresh 96 well plate by adding 240 µL of assay medium to each well and then 10 µL solution per well from the 100x solvent plate was added to equivalent wells on the dilution plate. Assay medium was 495 mL DMEM (Gibco 21885), supplemented with 5.0 mL FBS.

Treatment of Cultured Plates:
Approximately 24 hours after the test cell culture plates were established, the medium was removed from the wells by careful inversion of the plates and blotting onto sterile paper towel. 150 µL of assay medium was added to every well of the 96 well plates. 50 µL from each well of the dilution plate was transferred to equivalent wells in the 96 well plates. Three white plates were dosed for measuring luminescence and one transparent plate for measuring cell viability using the MTT assay.
The plates were then covered with a plate seal and placed in the incubator at 37 ± 2°C, in a humidified atmosphere of 5% CO2 in air for 48 ± 2 hours.

Cell Viability Measurement:
After incubation, the transparent plate was removed from the incubator and the plate seal discarded. The cell culture medium was removed by careful inversion of the plate and blotted onto sterile paper towel to remove residual culture medium. 100 µL fresh assay medium was added to each well. 10 µL of MTT solution (5 mg/mL in PBS) was added to each well of the 96-well plate. The plate was incubated at 37 ± 2°C in a humidified atmosphere of 5% CO2 in air for 4 hours ± 10 minutes. The medium was then removed by careful inversion of the plate and blotted onto sterile paper towel to remove residual culture medium. 50 µL of DMSO was added to each well. The plate was then placed in the incubator at 37 ± 2°C, in a humidified atmosphere of 5% CO2 in air, protected from light, for at least 10 minutes. The absorbance value of each well was read using a plate reader with a 540 nm filter.

Luciferase Measurement:
Luciferase was measured using the Steady Glo® Luciferase Assay system kit supplied by Promega (E2550). Steady-Glo® luciferase reagent was prepared by transferring the contents of one bottle of Steady-Glo® buffer to one bottle of Steady-Glo® substrate. The reagent was mixed by inversion until the substrate had dissolved. The reconstituted reagent was either used on the same day it was prepared or, if frozen reconstituted reagent was used, it was thawed to room temperature before use.
After incubation the medium was removed from the wells of the triplicate white plates by careful inversion of the plates and blotting on sterile absorbent paper. 100 µL of fresh assay medium was added to each well before 100 µL of Steady-Glo® luciferase reagent was added to each well of the plate. The plates were shaken on a plate shaker for at least 5 minutes until the cells had lysed. Luminescence (emitted light) was measured using a SpectraMax L luminometer. Each plate was read for total photon count with an integration time of 1 second. The plates were dark adapted for 1 minute prior to measurement.

Number of Tests Required:
Two independent tests each containing three replicates (i.e. n=6) were required to make a conclusion. Each independent test was performed on a different day with fresh stock solutions of the chemicals and independently harvested cells.

Vehicle / solvent control:

DMSO

Positive control:

cinnamic aldehyde [442D]

Results and discussion

Positive control results:

The results for the positive control, cinnamic aldehyde, are shown in Table 3, Table 4. The luciferase activity induction obtained with the positive control, cinnamic aldehyde, was statistically significant above the threshold of 1.5 in at least one of the tested concentrations (4 to 64 μM) in both tests.
The EC1.5 values of the positive control, cinnamic aldehyde, were 9.15 μM and 10.21 μM for test 1 and 2, respectively, which lay within the historical control range for this laboratory (see Table 5). The average induction in the three replicates for cinnamic aldehyde at 64 µM were 5.36 and 5.64 for test 1 and 2, respectively, which met the acceptance criterion of between 2 and 8.

In vitro / in chemico

Results

Results 1

Key result

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

EC 1.5 [442D]

Value:

1 978.01 µM

Cell viability:

>70%

Vehicle controls validity:

valid

Positive controls validity:

valid

Results 2

Key result

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

EC 1.5 [442D]

Value:

1 581.23 µM

Cell viability:

>70%

Vehicle controls validity:

valid

Positive controls validity:

valid

Results 3

Key result

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

Imax [442D]

Value:

1.51

Cell viability:

>70%

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: unit not applicable for imax

Results 4

Key result

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

Imax [442D]

Value:

1.94

Cell viability:

>70%

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: unit not applicable for Imax

Outcome of the prediction model:

negative [in vitro/in chemico]

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative/solvent control: yes; The average coefficient of variation of the luminescence reading for the negative solvent control (DMSO) was 16.4% and 9.2% for test 1 and 2, respectively, which met the acceptance criterion of below 20%.
- Acceptance criteria met for positive control: yes

Any other information on results incl. tables

The Imax for ST2092NM was 1.51 in test 1 and 1.94 in test 2. The Imax for both tests was >1.5-fold and statistically significant compared to the DMSO control. The EC1.5 for ST2092NM was 1978.01 µM and 1581.23 µM for tests 1 and 2, respectively. As the EC1.5 value was >1000 µM this was classified as a negative response. The cellular viability did not fall below 91.30% in test 1 and 90.18% in test 2 and therefore the IC30 and IC50 could not be calculated.

 

Table 1: Results for ST2092NM– Test 1

Test item conc. (µM)	0.98	1.95	3.91	7.81	15.63	31.25	62.5	125	250	500	1000	2000
Mean fold induction	0.86	0.89	0.80	0.82	0.98	1.01	0.86	0.84	0.90	0.87	0.95	1.51
Statistically significant	N/A	N/A	N/A	N/A	N/A	N/A	N/A	N/A	N/A	N/A	N/A	Yes
Viability (%)	100.09	91.30	98.20	92.03	103.96	92.77	104.69	97.89	99.36	103.75	102.91	96.32
Imax	1.51
EC1.5 (µM)	1978.01
IC30 (µM)	N/A
IC50 (µM)	N/A

Determination criteria for the skin sensitisation potential of the test item	Result
Is the Imax >1.5 fold and statistically significant	Yes
Is the cellular viability >70% at the EC1.5 determining concentration	Yes
Is the EC1.5 value < 1000 µM	No
Is there an apparent overall dose-response for luciferase induction	No
KeratinoSens™ prediction	Negative

 

Table 2: Results for ST2092NM – Test 2

Test item conc. (µM)	0.98	1.95	3.91	7.81	15.63	31.25	62.5	125	250	500	1000	2000
Mean fold induction	0.80	0.87	0.81	0.84	0.91	1.05	0.96	0.86	0.90	1.06	0.89	1.94
Statistically significant	N/A	N/A	N/A	N/A	N/A	N/A	N/A	N/A	N/A	N/A	N/A	Yes
Viability (%)	90.18	92.34	92.51	92.86	93.98	94.67	113.33	100.63	106.68	105.47	111.78	95.02
Imax	1.94
EC1.5 (µM)	1581.23
IC30 (µM)	N/A
IC50 (µM)	N/A

 

Determination criteria for the skin sensitisation potential of the test item	Result
Is the Imax >1.5 fold and statistically significant	Yes
Is the cellular viability >70% at the EC1.5 determining concentration	Yes
Is the EC1.5 value < 1000 µM	No
Is there an apparent overall dose-response for luciferase induction	No
KeratinoSens™ prediction	Negative

 

Table 3: Results for Cinnamic Aldehyde – Test 1

Positive control conc. (µM)	4	8	16	32	64
Mean fold induction	1.21	1.43	1.93	2.61	5.36
Statistically significant	N/A	N/A	Yes	Yes	Yes
Viability (%)	106.68	116.40	120.38	137.01	132.30
Imax	5.36
EC1.5 (µM)	9.15
IC30 (µM)	N/A
IC50 (µM)	N/A

 

Test Acceptance Criteria	Result
Luciferase activity induction obtained with the positive control statistically significant above the threshold of 1.5 in at least one of the test concentrations	Yes	Pass
Average induction of positive control at 64 µM between 2 – 8	Yes (5.36)	Pass
EC1.5 of positive control within two standard deviations of the historical mean (-2.72 to 29.00)	Yes (9.15)	Pass
CV% of blank values < 20%	Yes (16.4%)	Pass

 

Table 4: Results for Cinnamic Aldehyde – Test 2

Positive control conc. (µM)	4	8	16	32	64
Mean fold induction	1.17	1.37	1.85	2.66	5.64
Statistically significant	N/A	N/A	Yes	Yes	Yes
Viability (%)	108.15	104.87	114.63	119.81	110.31
Imax	5.64
EC1.5 (µM)	10.21
IC30 (µM)	N/A
IC50 (µM)	N/A

 

 

Test Acceptance Criteria	Result
Luciferase activity induction obtained with the positive control statistically significant above the threshold of 1.5 in at least one of the test concentrations	Yes	Pass
Average induction of positive control at 64 µM between 2 – 8	Yes (5.64)	Pass
EC1.5 of positive control within two standard deviations of the historical mean (-2.72 to 29.00)	Yes (10.21)	Pass
CV% of blank values < 20%	Yes (9.2%)	Pass

 

Table 5: Historical Control Data for Cinnamic Aldehyde

	Mean	Standard Deviation	Laboratory Historical Data Range (Mean +/- 2xSD)
EC1.5 (µM)	13.14	7.93	-2.72 to 29.00

 

Applicant's summary and conclusion

Interpretation of results:

other: Test item is not a skin sensitizer.

Conclusions:

It was concluded that the test item gave a negative response in the ARE-Nrf2 Luciferase Test (KeratinoSens™), supporting the prediction that the test item is not a skin sensitizer.

Executive summary:

The purpose of this study was to support a predictive, adverse-outcome-pathway evaluation of whether the test item, ST2092NM, is likely to be a skin sensitizer using the ARE-Nrf2 Luciferase Test (KeratinoSens™).

The Imax for ST2092NM was 1.51 in test 1 and 1.94 in test 2. The Imax for both tests was >1.5 fold and statistically significant compared to the DMSO control. The EC1.5 was 1978.01 µM and 1581.23 µM for Tests 1 and 2, respectively. The cellular viability did not fall below 91.30% in test 1 and 90.18% in test 2 and therefore the IC30 and IC50 could not be calculated. Data showed no overall dose-response for luciferase induction.

All acceptance criteria for the positive control, cinnamic aldehyde, were met.

It was concluded that the test item gave a negative response in the ARE-Nrf2 Luciferase Test (KeratinoSens™), supporting the prediction that the test item is not a skin sensitizer.