2-Propenoic acid, 2-[[1,1,2-trifluoro-2-(1,1,2,2,3,3,3-heptafluoropropoxy)ethyl]thio]ethyl ester

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019-10-14 to 2019-11-15

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:

2020

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method: B.60 (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)

Version / remarks:

14 February 2017

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 luciferase KeratinoSens™ test method)

Version / remarks:

25 June 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:

2-{[1,1,2-trifluoro-2-(1,1,2,2,3,3,3-heptafluoropropoxy)ethyl]sulfanyl}ethyl prop-2-enoate

EC Number:

864-951-0

Cas Number:

2170099-74-6

Molecular formula:

C10H8F10O3S

IUPAC Name:

2-{[1,1,2-trifluoro-2-(1,1,2,2,3,3,3-heptafluoropropoxy)ethyl]sulfanyl}ethyl prop-2-enoate

Test material form:

liquid

In vitro test system

Details of test system:

Keratinoses transgenic cell line [442D]

Details on the study design:

PREPARATION OF TEST SOLUTIONS
- Preparation of the test chemical stock solution
The test item was dissolved in dimethyl sulfoxide (DMSO). A stock solution of 200 mM was prepared by pre-weighing the test item into 4 mL glass vessels.

- Preparation of the test chemical serial dilutions
Based on the stock Solution a set of twelve master Solutions in 100% DMSO were prepared. The stock solution (200 mM) of the test items were diluted eleven times using a constant dilution factor of 1:2 (100 µL stock Solution + 100 µL DMSO). Then the 100x concentrated master solutions were further diluted 1:25 in test item exposure medium (10 µL + 240 µL test item exposure medium) resulting in a 4% share of the solvent. These 4-fold concentrated test item solutions were finally diluted 1:4 when incubated with the cells (50 µL + 150 µL test item exposure medium). Based on this procedure the final concentration of the solvent was 1% (v/v) in all test item concentrations and controls.

- Preparation of the positive controls
Cinnamic aldehyde was dissolved in DMSO at a concentration of 6.4 mM and was further diluted four times with a constant dilution factor of 1:2 resulting in a concentration range of 0.4 mM - 6.4 mM. The following preparation of the positive control was carried out analogous to the preparation of the test items, resulting in a final concentration range of 4 µM - 64 µM. The final concentration of DMSO was 1% (v/v) for all wells.

- Preparation of the solvent and blank controls
DMSO at a final concentration of 1% (v/v) in test item exposure medium was used as negative control. Six wells filled with the negative control were included in every testing plate. The preparation of the negative control was carried out analogous to the test item. A blank well with no seeded cells was included in every plate to determine the background. The well was incubated with the negative control.


APPLICATION OF THE TEST CHEMICAL AND CONTROL SUBSTANCES
- Number of replicates: 3 (test item and positive control); 6 (solvent control)
- Number of repetitions: 2
- Test chemical concentrations: 0.98, 1.95, 3.9, 7.8, 15.6, 31.25, 62.5, 125, 250, 500, 1000 and 2000 µM
- Application procedure
- Exposure time: 48 hours
- Description on study acceptance criteria
The test met acceptance criteria if:
- the luciferase activity induction of the positive control is statistically significant above the threshold of 1.5 (using a t-test) in at least one of the tested concentrations
- the average induction in the three technical replicates for the positive control at a concentration of 64 µM is between 2 and 8
- the EC1.5 value of the positive control is within two Standard deviations of the historical mean (7 µM - 30 µM based on the Validation dataset)
- the average coefficient of Variation (CV) of the luminescence reading for the negative (solvent) control is <20% in each repetition which is consisting of 6 wells.

SEEDING AND INCUBATION
- Seeding conditions (passage number and seeding density)
A cell suspension of 8 x 10^4 cells/mL in assay medium was prepared. 125 µL of the cell suspension corresponding to 1 x 10^4 cells were dispensed in each well, except for the blank. To determine the luciferase activity cells were seeded in white 96-well plates (Hat bottom). In parallel cells were seeded in a transparent 96-well plate (flat bottom) for the determination of the cell viability.

- Incubation conditions: 37°C ± 1 °C and 5% CO2

- Washing conditions: After seeding, cells were grown for 24 ± 1h in assay medium at 37 ± 1 °C and 5% CO2. Thereafter, the assay medium was discarded and replaced by 150 µL test item exposure medium. 50 µL of the shortly before prepared 4x master concentrations were transferred to the luciferase and cell viability plates, resulting in an additional 1:4 dilution of the test item.

- Precipitation noted: No

LUCIFERASE ACTIVITY MEASUREMENTS
- Choice of luminometer: Multi-Mode Microplate Reader (Cytation 1, BioTek Instruments GmbH, Bad Friedrichshall, Germany).
- Plate used: 96 well plate
- Lysate preparation: After washing 20 µL of passive lysis buffer was added into each well and the plate was incubated for 20 min at room temperature in the absence of light.
Plates with the cell lysate were placed in the plate reader for luminescence measurement. Per well 50 µL of the luciferase substrate was injected by the injector of the plate reader. The plate reader waited for 1 s before assessing the luciferase activity for 2 s. This procedure was repeated for each individual well.

DATA EVALUATION
- Cytotoxicity assessment
2.7 mL of a MTT Solution (5 mg/mL in DPBS) was added to 20 mL test item exposure medium. For the cell viability assay plate, the medium was replaced with 200 µL of this fresh medium containing MTT. The plate was covered with a sealing tape and incubated for 4h at 37 ± 1°C and 5% CO2. Afterwards the medium was removed and replaced by 200 µL 10% SDS solution per well. The plate was covered with sealing tape and incubated in the incubator at 37 ± 1°C and 5% CO2 overnight. After the incubation period the plate was shaken for 10 min and the OD was measured at 600 nm.

- Prediction model used
A KeratinoSens™ prediction was considered positive if the following conditions were met in at least two independently prepared test repetitions:
- Imax is > 1.5-fold increased and statistically significant (p<0.05) compared to the negative control
- cell viability is >70% at the lowest concentration with an induction of luciferase activity > 1.5-fold (i.e. at the EC1.5 determining concentration)
- EC1.5 value is <1000 µM
- an apparent overall dose-response for luciferase induction


If in a given repetition, all of the three first conditions are met but a clear dose-dependent increase in luciferase induction cannot be observed, then the result of that repetition should be considered inconclusive and further testing may be required. In addition, a negative result obtained with test chemicals tested at a maximal test concentration <1000 µM (or 200 µg/mL for test chemicals with no defined MW) and which do not reach cytotoxicity (<70% viability) at the maximal tested concentration should also be considered as inconclusive. A negative result for test items with a log Kow >7 should be interpreted with care due to the applicability of the test method.

Vehicle / solvent control:

DMSO

Negative control:

not applicable

Positive control:

cinnamic aldehyde [442D]

Results and discussion

Positive control results:

The average luciferase activity induction obtained with the positive control cinnamic aldehyde at the highest tested concentration 64 μM was 2.83 at Run 1 and 2.49 at Run 2.

In vitro / in chemico

Results

Results 1

Key result

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

EC 1.5 [442D]

Value:

7.81 µM

Cell viability:

108.78 %

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Results 2

Key result

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

Imax [442D]

Value:

7.82

Cell viability:

108.78 %

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Results 3

Key result

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

EC 1.5 [442D]

Value:

9.91 µM

Cell viability:

134.27 %

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Results 4

Key result

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

Imax [442D]

Value:

6.59

Cell viability:

134.27 %

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Outcome of the prediction model:

positive [in vitro/in chemico]

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for solvent control: yes, the average coefficient of Variation (CV) of the luminescence reading is <20% in each repetition which is consisting of 6 wells (Run 1: 19.2 %; Run 2: 13.1 %)
- Acceptance criteria met for positive control: yes, the luciferase activity induction is statistically significant above the threshold of 1.5 (using a t-test) in at least one of the tested concentrations and the average induction in the three technical replicates for the positive control at a concentration of 64 µM is between 2 and 8 (Run 1: 2.83; Run 2: 1.52).
The EC1.5 value of the positive control is within two standard deviations of the historical mean; 7 µM - 30 µM based on the Validation dataset. (Run 1: 30 µM; Run 2: 16 µM).

Applicant's summary and conclusion

Interpretation of results:

other: positive for the second key event of the skin sensitisation Adverse Outcome Pathway

Conclusions:

The test item induced the luciferase activity in the transgenic KeratinoSens™ cell line under the test conditions of the study. Therefore, the test item is considered positive for the second key event of the skin sensitisation Adverse Outcome Pathway (AOP).

Executive summary:

This in vitro Skin Sensitisation Test ARE-Nrf2 Luciferase Test Method (KeratinoSens) was performed according to OECD 442D. It is used to assess the inflammatory responses in the keratinocytes as changes in gene expression associated with specific cell signalling pathways such as the antioxidant/electrophile response element (ARE)-dependent pathways (second key event of a skin sensitization Adverse Outcome Pathway) of the test item. In the present study the test substance was dissolved in DMSO. Based on a molecular weight of 398.22 g/mol a stock Solution of 200 mM was prepared. The cells were treated with different concentrations (0.98, 1.95, 3.9, 7.8, 15.6, 31.25, 62.5, 125, 250, 500, 1000 and 2000 µM) of the test item and the luciferase gene induction was measured using light producing luciferase substrate. Cell viability was determined by enzymatic conversion of vital dye MTT into a blue formazan salt and measurement of the formazan salt after extraction from cells.

Test chemicals are considered positive in the KeratinoSens™ test method if they induce a statistically significant induction of the luciferase activity above a given threshold (>1.5 fold), below a defined concentration which does not significantly affect cell viability (< 1000 uM) and at a concentration at which the cellular viability is above 70%. For this purpose, the maximal fold induction of the luciferase activity over solvent control (Imax) was determined. Furthermore, since cells were exposed to series of concentrations of the test chemical, the concentration needed for a statistically significant induction of luciferase activity above the threshold (EC1.5 value) was interpolated from the dose-response curve obtained from the series of tested concentrations of the test chemical. Finally, parallel cytotoxicity measurements were conducted to assess whether luciferase induction occurs at sub-cytotoxic concentrations.

In the first run, a max luciferase activity induction (Imax) of 7.82 was determined. The lowest tested concentration with a luciferase induction >1.5 (2.10) was found to be 15.63 µM, the corresponding cell viability was >70% (108.78%). The calculated EC1.5 value was 7.81 µM. In the second run, a max luciferase activity induction (Imax) of 6.59 was determined. The lowest tested concentration with a luciferase induction >1.5 (1.80) was found to be 15.63 µM, the corresponding cell viability was >70% (134.27%). The calculated EC1.5 value was 9.91 µM. A dose response for luciferase activity induction was observed for each individual run as well as for an overall luciferase activity induction. Therefore, the test item is considered positive for the second key event of the skin sensitisation Adverse Outcome Pathway (AOP).