2-Propenoic acid, 2-hydroxyethyl ester, reaction products with O,O-bis(dialkyalkyl) hydrogen phosphorodithioate and dimethyl phosphonate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 August 2019 to 26 September 2019

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

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

June 2018

Deviations:

yes

Remarks:

deviations with respect to incubation time and cell confluency with no impact on results or integrity of the study (see below)

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Test material

Reference

Name:

Unnamed

Type:

Constituent

Test material form:

liquid

Details on test material:

- Appearance/physical state: Light yellow liquid
- Storage conditions: Ambient temperature

In vitro test system

Details on the study design:

PURPOSE OF STUDY
- The purpose of this study was to support a predictive, adverse-outcome-pathway evaluation of whether the test item is likely to be a skin sensitizer using the ARE-Nrf2 Luciferase Test (KeratinoSens).
- The KeratinoSens in vitro skin sensitisation assay uses an immortalised adherent cell line derived from HaCaT human keratinocytes stably transfected with a selectable plasmid. The cell line contains the luciferase gene under the transcriptional control of a constitutive promoter fused with an ARE element from a gene that is known to be up-regulated by contact sensitisers. The luciferase signal reflects the activation by sensitisers of endogenous Nrf2 dependent genes, and the dependence of the luciferase signal in the recombinant cell line on Nrf2 has been demonstrated. This allows quantitative measurement (by luminescence detection) of luciferase gene induction, using well established light producing luciferase substrates, as an indicator of the activity of the Nrf2 transcription factor in cells following exposure to electrophilic test chemicals.

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) (10500) 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 sulphoxide 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 to 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 the study.

CELL PASSAGE
- Actively growing cell stocks were maintained and expanded by subculturing (passage).
- When the cells had reached 80 to 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 10E+05 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.

PREPARATION OF POSITIVE CONTROL
- Cinnamic aldehyde (Sigma, 239968, lot: STBG0250V, expiry: July 2020) was prepared by weighing between 20 to 40 mg into a tared glass container and diluted to a final concentration of 200 mM in DMSO using the formula V = 5 * [(p / 100) * w) / MW] - (w / 1000) where V = volume of DMSO in mL to be added; p = purity of chemical in %; MW = molecular weight of the chemical in g/moL; w = exact weight of the chemical added to the vial in mg.
- The 200 mM cinnamic aldehyde solution was further diluted to a final concentration of 6.4 mM by adding 32 µL of the 200 mM solution to 968 µL of DMSO.
- Preparation of the positive control was shared with other studies performed in the same assay.

TEST ITEM SOLUBILITY
- Prior to commencing testing, the solubility of the test item in assay medium containing 1 % DMSO at 200 mM was assessed.
- The test item was found to be soluble in DMSO at 200 mM, the highest concentration as recommended by the guideline this test follows.

PREPARATION OF THE TEST ITEM
- A stock solution of the test item was prepared by weighing the test item into a tared glass container and diluting to 200 mM in DMSO.

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.
- A 200 µL volume of the 6.4 mM stock solution of cinnamic aldehyde was added to the appropriate well in column 11 as per the example plate layout.
- 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 (see table below).

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.
- Assay medium (150 µL) was added to every well of the 96 well plates.
- A 50 µL volume 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.
- Fresh assay medium (100 µL) was added to each well. MTT solution (10 µL of 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.
- DMSO (50 µL) 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).
- The 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.
- Frozen reconstituted reagent was used for tests 1 and 2 and 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.
- Fresh assay medium (100 µL) 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.

ASSESSMENT OF RESULTS
- The following parameters were calculated in the KeratinoSens test method:
[i] the maximal average fold induction of luciferase activity (Imax) value observed at any
concentration of the tested chemical and positive control.
[ii] the EC1.5 value representing the concentration for which induction of luciferase activity is above the 1.5 fold threshold (i.e. 50% enhanced luciferase activity) was obtained.
[iii] the IC50 and IC30 concentration values for 50% and 30% reduction of cellular viability.
- Fold luciferase activity induction was calculated by Equation 1 (see attached document), and the overall maximal fold induction (Imax) was calculated as the average of the individual repetitions.
- EC1.5 was calculated by linear interpolation according to Equation 2 (see attached document), and the overall EC1.5 was calculated as the geometric mean of the individual repetitions.
- Viability was calculated by Equation 3 (see attached document).
- IC50 and IC30 were calculated by linear interpolation according to Equation 4 (see attached document), and the overall IC50 and IC30 were calculated as the geometric mean of the individual repetitions.
- For each concentration showing ≥ 1.5 fold luciferase activity induction, statistical significance was calculated (by a two-tailed Student's t-test), comparing the luminescence values for the three replicate samples with the luminescence values in the solvent (negative) control wells to determine whether the luciferase activity induction was statistically significant (p < 0.05). The lowest concentration with ≥ 1.5 fold luciferase activity induction was the value determining the EC1.5 value. It was checked in each case whether this value was below the IC30 value, indicating that there was less than 30% reduction in cellular viability at the EC1.5 determining concentration. Furthermore, it was checked that no significant cytotoxic effects occur at the lowest concentration leading to ≥1.5 fold luciferase induction.
- The data was visually checked with the help of graphs. Where a statistically non-significant induction above 1.5 fold was observed followed by a higher concentration with a statistically significant induction, results from this test were considered as valid and positive only if the statistically significant induction above the threshold of 1.5 was obtained for a non-cytotoxic concentration.

INTERPRETATION OF RESULTS AND PREDICTION MODEL
- A KeratinoSens prediction is considered positive if the following 4 conditions are all met in 2 of 2 or in the same 2 of 3 repetitions, otherwise the KeratinoSens prediction is considered negative:
[i] the Imax is ≥1.5 fold and statistically significantly different as compared to the solvent vehicle control (as determined by a two-tailed, unpaired Student's T-test).
[ii] the cellular viability is >70% at the lowest concentration with induction of luciferase activity ≥ 1.5 fold (i.e. at the EC1.5 determining concentration).
[iii] the EC1.5 value is < 1000 µM.
[iv] there is an apparent overall dose-response for luciferase induction (or a biphasic response).
- If in a given test, all of the first three conditions listed above are met, but a clear dose response for the 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 concentrations < 1000 µM and that do not reach cytotoxicity (< 70% viability) at the maximal tested concentration should also be considered as inconclusive.

TEST ACCEPTANCE CRITERIA
- In order for an assay to be accepted the following criteria must be met:
[i] The luciferase activity induction obtained with the positive control, cinnamic aldehyde, should be statistically significant above the threshold of 1.5 (e.g. using a t-test) in at least one of the tested concentrations (4 to 64 µM).
[ii] The EC1.5 value of the positive control should be within two standard deviations of the historical mean of the testing facility. In addition, the average induction in the three replicates for cinnamic aldehyde at 64 µM should be between 2 and 8. If the latter criterion is not fulfilled, the dose response of cinnamic aldehyde should be carefully checked, and tests may be accepted only if there is a clear dose-response with increasing luciferase activity induction at increasing concentrations for the positive control.
[iii] The average coefficient of variation of the luminescence reading for the negative solvent control (i.e. DMSO) should be below 20% in each repetition which consists of 6 wells tested in triplicate. If the variability is higher, results should be discarded.

MAJOR COMPUTERISED SYSTEMS
- Pharmacy test item management: Xybion Pristima
- Plate reader: Wallac EnVision model 2103
- Luminometer: Molecular Devices Spectramax L
- Statistical software: SAS 9.4, SAS Institute Inc. 2002
- All versions of the systems are maintained by Covance.

Results and discussion

Positive control results:

- The results for the positive control, cinnamic aldehyde, are shown in Table 2, Table 4, Figure 3 and Figure 4 (attached).
- 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 11.13 μM and 3.88 μM for test 1 and 2, respectively, which lay within the historical control range for this laboratory (see Table 5, attached).
- The average induction in the three replicates for cinnamic aldehyde at 64 μM were 3.65 and 2.18 for test 1 and 2, respectively, which met the acceptance criterion of between 2 and 8.

In vitro / in chemico

Results

Results 1

Run / experiment:

other: Test 1

Parameter:

other: Imax

Value:

3.35

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: > 1.5 fold and statistically significant

Results 2

Run / experiment:

other: Test 2

Parameter:

other: Imax

Value:

5.98

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: > 1.5 fold and statistically significant

Other effects / acceptance of results:

RESULTS
- The results for the test item are shown in Table 1 (attached) and Table 3 (attached).
- The Imax for the test item was 3.35 in test 1 and 5.98 in test 2. The Imax for both tests was > 1.5 fold and statistically significant compared to the DMSO control.
- The EC1.5 was 5.22 µM and 3.88 µM for tests 1 and 2, respectively.
- The IC30 value was 15.43 µM in test 1 and 47.20 µM in test 2 and the IC50 values were 20.04 µM and 57.74 µM in tests 1 and 2, respectively.
- Figure 1 (attached) and Figure 2 (attached) showed an overall dose-response for luciferase induction.
- The average coefficient of variation of the luminescence reading for the negative solvent control (DMSO) was 15.0 % and 16.8 % for test 1 and 2, respectively, which met the acceptance criterion of below 20 %.

Applicant's summary and conclusion

Interpretation of results:

other: substance predicted likely to be a skin sensitiser

Conclusions:

It was concluded that the test item gave a positive response in the ARE-Nrf2 Luciferase Test (KeratinoSens), supporting the prediction that the test item is likely to be a skin sensitiser.

Executive summary:

GUIDELINE

The test was conducted in accordance with OECD Guideline for the Testing of Chemicals, In Vitro Skin Sensitisation Assays Addressing the AOP Key Event on Keratinocyte Activation, Guideline 442D, June 2018 and DB-ALM Protocol no 155: KeratinoSens, March 2018.

 

METHODS

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

 

RESULTS

The Imax for the test item was 3.35 in test 1 and 5.98 in test 2. The Imax for both tests was > 1.5 fold and statistically significant compared to the DMSO control. The EC1.5 was 5.22µM and 3.88µM for tests 1 and 2, respectively. The IC30 value was 15.43 µM in test 1 and 47.20 µM in test 2 and the IC50 values were 20.04 µM and 57.74 µM in tests 1 and 2, respectively. Graphs showed an overall dose-response for luciferase induction. All acceptance criteria for the positive control, cinnamic aldehyde, were met.

 

CONCLUSION

It was concluded that the test item gave a positive response in the ARE-Nrf2 Luciferase Test (KeratinoSens), supporting the prediction that the test item is likely to be a skin sensitiser.