ethyl (3-benzoyl-2,4,6-trimethylbenzoyl)(phenyl)phosphinate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 September 2019 to 18 October 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

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:

yes

Remarks:

See 'Principles of method if other than guideline'.

Principles of method if other than guideline:

The OECD Test Guideline 442D mentions that test materials prepared in sterile water or culture medium have to be filtered after preparation for sterilisation prior treatment. Filtration of such preparations can be possible when a test material formulation is a solution. However, in case the test material formulation is a suspension, filtration process can retain test material particles in the filter and considerably decrease test material concentration in the filtered formulation. Therefore, in view of the above and considering that pre treatment filtration procedure is not of common practice in other in vitro cell-based assays, and that water and culture media used in the assay are already sterile, test material formulations were not filtered prior treatment.

GLP compliance:

yes (incl. QA statement)

Type of study:

ARE-Nrf2 luciferase KeratinoSens™ test method

Justification for non-LLNA method:

The KeratinoSens™ test is a method for which validation studies have been completed followed by an independent peer review conducted by the European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM). The KeratinoSens™ test method was considered scientifically valid to be used as part of an IATA (Integrated Approach to Testing and Assessment), to support the discrimination between skin sensitisers and non-sensitisers for the purpose of hazard classification and labelling.

Test material

Test material information

Constituent 1

Reference substance name:

ethyl (3-benzoyl-2,4,6-trimethylbenzoyl)(phenyl)phosphinate

EC Number:

806-801-9

Cas Number:

1539267-56-5

Molecular formula:

C25H25O4P

IUPAC Name:

ethyl (3-benzoyl-2,4,6-trimethylbenzoyl)(phenyl)phosphinate

Test material form:

solid: particulate/powder

Details on test material:

- Appearance: White to off white.
- Storage conditions: Controlled room temperature (15 - 25 °C, =70 % relative humidity), protected from light.

In vitro test system

Details of test system:

Keratinoses transgenic cell line [442D]

Details on the study design:

CONTROLS
- Negative Control: 1 % DMSO in culture medium.
- Positive Control: Cinnamic Aldehyde in DMSO.

POSITIVE CONTROL FORMULATIONS
For each run, the positive control item was dissolved in DMSO to a final concentration of 200 mM. This solution was further diluted to a final concentration of 6.4 mM. Then, it was diluted in DMSO by serial dilutions in the Master plate 100x, using a dilution factor of 2, to obtain a total of 5 concentrations. Subsequently, each formulation of the Master plate 100x was diluted 25-fold in treatment medium in another 96-well plate called "Master plate 4x". The final tested concentrations ranged from 4 to 64 µM. All these formulations were prepared within 4 hours before use, then kept at room temperature and protected from light until use.

TEST MATERIAL FORMULATIONS
On the basis of solubility results, the test material was solubilised in DMSO at 200 mM (stock formulation). One stock formulation was prepared for each run. The stock formulation was diluted in DMSO by serial dilutions, using a dilution factor of 2, to obtain a total of 12 concentrations in a 96-well plate; this 96 well plate was called "Master plate 100x". Subsequently, each formulation of the Master plate 100x was 25-fold diluted in treatment medium in another 96 well plate called "Master plate 4x" taking care to adjust all wells to the same DMSO level.
All formulations were prepared within 4 hours before use, and kept at room temperature and protected from light until use.

TEST SYSTEM
- KeratinoSens cells: the cell line KeratinoSens is stably transfected with a modified plasmid. This plasmid contains an ARE sequence from the AKR1C2 gene and a SV40 promotor which are inserted upstream of a luciferase gene. The resulting plasmid was transfected into HaCaT keratinocytes and clones with a stable insertion selected in the presence of Geneticin / G-418. Induction of the luciferase gene is the endpoint evaluated and reflects the activation by the test material of the Nrf2 transcription factor in this test.
- Storage conditions: -80 °C
The absence of mycoplasm was confirmed.
- Media:
Maintenance medium No. 1: DMEM containing GlutaMAXTM, 1 000 mg/L D-Glucose, Sodium Pyruvate and supplemented with 9.1 % Fetal Calf Serum (FCS) and 500 µg/mL G-418.
Maintenance medium No. 2: DMEM with 9.1 % FCS without G-418.
Treatment medium: DMEM with 1 % FCS without G-418.
Freezing medium: DMEM with 20 % FCS and 10% DMSO.

METHOD
The test material was tested in two independent validated runs using cells of a different passage number.
- Solubility assay: A solubility assay was performed prior the first treatment to select the most appropriate vehicle (between DMSO, water for injections or treatment culture medium). When a solution or a stable dispersion was obtained in these vehicles, the formulations were 100-fold diluted in culture medium. Then, a visual inspection of the samples was performed immediately as well as after an overnight period of incubation at 37 °C to evaluate the presence or absence of precipitate/emulsion.

Main Test:
- Cell seeding for testing: Cells were grown using general culture procedures up to 80 - 90 % confluence. The day prior to treatment, cells were washed twice with D-PBS containing 0.05 % EDTA, harvested, re-suspended in Maintenance medium No. 2 and counted using Trypan Blue dye. Cell concentration was adjusted to a density of 8 x 10^4 cells/mL. Cells were then distributed into four 96-well plates (3 white plates and 1 transparent plate), by adding 125 µL (representing 1 x 10^4 cells) per well taking care to avoid sedimentation of the cells during seeding. After seeding, the cells were grown for 24 (± 1) hours in 96-well microtiter plates prior to test material addition.
- Treatment: After the 24-hour growing period, the medium was removed by aspiration and replaced by 150 µL of treatment medium. From the Master plate 4x, a volume of 50 µL was added to each well of the 3 white assay plates and 50 µL to the transparent plate for cytotoxicity evaluation. All plates were covered by a sealing membrane to avoid evaporation of volatile test materials and to avoid cross-contamination between wells. The plates were then incubated for 48 (± 2) hours at 37 °C, 5 % CO2, 90 % humidity.

ENDPOINT MEASUREMENTS
- Microscopic observation to evaluate the presence or absence of precipitate - transparent plate: After 48 (± 2) hours incubation period, the presence or absence of precipitate/emulsion was determined in each well by microscopic inspection.
- Luminescence flash signal to evaluate induction signal - white plates: After incubation, the supernatants from the white assay plates were discarded. The cells were washed once with D-PBS. A volume of 20 µL of passive lysis buffer was added to each well and the cells were incubated for 20 (± 2) minutes preferentially (not exceeding 30 minutes) at room temperature under orbital shaking. The plates containing the passive lysis buffer were then placed in the luminometer for reading using the following program:
A volume of 50 µL of the luciferase substrate was added to each well,
1 second after this addition, the luciferase signal was integrated for 2 seconds.
- Absorbance signal to evaluate cytotoxicity - transparent plate: For the cell viability assay plate (transparent plate), the medium was removed by aspiration and was replaced by 200 µL of treatment medium. A volume of 27 µL of MTT solution at 5 mg/mL in D-PBS was then added to each well. The plates were covered with a sealing membrane and returned at 37 °C in the incubator in a humidified atmosphere for 4 hours (± 10 minutes). At the end of the incubation period, the medium was removed and a volume of 200 µL of a 10 % SDS solution was added to each well. The plates were covered with a sealing membrane and placed at 37 °C in the incubator in a humidified atmosphere for an overnight period to extract the formazan from cells. After the overnight incubation, the absorption of each well was determined at 600 nm using the plate reader.

DATA ANALYSIS
Data evaluation was performed using a validated Excel sheet. The generated raw data (luminescence data for the luciferase activity and absorbance data for the MTT test) were pasted into an Excel template, and all data processing performed automatically.

For the MTT and the luciferase data, the background value recorded in the empty well without cells (blank) was subtracted.
For the MTT data, the % viability was calculated for each well in the test plate in relation to average of the six negative control wells.
For the luciferase data, the average value of the six negative control wells was set to 1, and for each well in the plate, the fold induction was calculated in relation to this value.

For wells in which a statistically significant gene-induction (using a student test, also called T-test) over the 1.5 threshold was found, the following parameters were calculated from the processed raw data:
Imax: Maximal induction factor of luciferase activity compared to the negative control over the complete dose-response range measured,
EC1.5: Concentration at which a 1.5-fold luciferase gene induction is obtained,
IC50 and IC30: Concentrations effecting a reduction of cellular viability by 50 % and 30 %,
Indication whether a significant 1.5-fold gene induction occurred below the IC30.

The data were plotted in graphs and the Imax and the EC1.5 values were visually checked since uneven dose response curves or large variation may lead to incorrect extrapolation.
Also, the individual and overall geometric means (IC50 and IC30) were calculated, when applicable.

ACCEPTANCE CRITERIA
Each run was considered valid if the following criteria were met:
- At least 2 consecutive concentrations should have a viability = 70 %,
- The positive control results should be positive, thus the gene induction should be statistically significant and above the threshold of 1.5 in at least one of the tested concentrations,
- The average EC1.5 value for the positive control should be within two standard deviations of the historical mean. In addition, the average induction (Imax) in the three replicate plates for the positive control at 64 µM should be between 2 and 8. If the latter criterion was not fulfilled, the dose-response of Cinnamic Aldehyde was carefully checked, and the run was accepted if there was a clear dose response with increasing luciferase activity at increasing concentrations for the positive control,
- The average coefficient of variation of the luminescence reading in the negative control wells of the triplicate plates should be < 20 %.

EVALUATION CRITERIA
The results of each run are analysed individually and if the test material is classified as positive in two runs, the final outcome is considered positive. If the test material is classified as negative in two runs, the final outcome is negative. In case, the first two runs were not concordant, a third run was performed and the final outcome was that of the two concordant runs.

The test material is considered as positive if the following four conditions are all met in two of two or in two of three runs, otherwise the KeratinoSens prediction is considered as negative:
- the Imax is > 1.5-fold and statistically significantly different as compared to the negative control (as determined by a two-tailed, unpaired Student’s T-test),
- at the lowest concentration with a gene induction > 1.5-fold (i.e. at the EC1.5 determining value), the cell viability is > 70 %,
- the EC1.5 value is < 1 000 µM (or < 200 µg/mL for test material without MW),
- there is an apparent overall dose-response for luciferase induction (or a reproducible biphasic response).

Vehicle / solvent control:

DMSO

Results and discussion

Positive control results:

The cell viability for the positive control (Run 1 and Run 2) were all > 70 % (96 - 117 %), indicating a positive result.

In vitro / in chemico

Results

Results 1

Group:

test chemical

Run / experiment:

mean

Parameter:

IC50 [442D]

Value:

45.34 µM

Cell viability:

yes at 62.5 µM

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Group:

test chemical

Run / experiment:

mean

Parameter:

IC30 [442D]

Value:

38.41 µM

Cell viability:

yes at 62.5 µM

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

Key result

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

Imax [442D]

Value:

1.24 %

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Results 4

Key result

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

Imax [442D]

Value:

1.33 %

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

SOLUBILITY ASSAY
In the solubility assay, and at all tested concentrations (i.e. at 100 and 200 mM), the test material was found soluble in DMSO (yellow solution) whereas white heterogeneous suspensions were obtained when tested in water for injections and culture medium, despite a 5-minute of vortexing.
Once the test material stock formulations in DMSO were diluted in the treatment culture medium to final concentrations of 2 000 µM or 1 000 µM, a strong precipitate was immediately observed. This strong precipitate was not observed anymore after an overnight period of incubation at 37 °C for the formulation at 1 000 µM, and a slight emulsion was noted for the formulation at 2 000 µM.
On the basis of these results, the test material stock formulation to be used in the main test was therefore selected to be 200 mM in DMSO.

KERATINOSENS RUN
All acceptance criteria were fulfilled in each run, both runs were therefore considered as validated.
The first run was performed using the following concentrations: 0.98, 1.95, 3.91, 7.81, 15.6, 31.3, 62.5, 125, 250, 500, 1 000 and 2 000 µM in culture medium containing 1 % DMSO.
Based on the cytotoxicity observed on the first run, a lower range of concentrations was tested on the second run, as follows: 0.12; 0.24; 0.49; 0.98; 1.95; 3.91; 7.81; 15.6; 31.3; 62.5; 125 and 250 µM in culture medium containing 1 % DMSO.
At these tested concentrations:
- slight to strong test material precipitate was observed in treated wells at concentrations = 250 µM at the end of the 48-hour treatment period in the first run, at a slight one only at the highest tested concentration (i.e. 250 µM) in the second run,
- in both runs, a high decrease in cell viability (i.e. cell viability < 70 %) was noted at concentrations = 62.5 µM,
- no statistically significant gene-fold induction above the threshold of 1.5 was noted in comparison to the negative control at any tested concentrations, in either run,
- moreover, and for both runs, the Imax values were < 1.5 (i.e. 1.24 and 1.33 in the first and second runs, respectively) and thus no EC1.5 was calculated.
The geometric means IC30 and IC50 of the two validated runs were calculated to be 38.41 and 45.34 µM, respectively.
The evaluation criteria for a negative response are therefore met in both runs.

Any other information on results incl. tables

Imax, IC30, IC50 and EC1.5 Values, Mean and SD Values Obtained After Treatment with the Test Material.

Test Material	Imax	EC1.5 (µm)	IC50 (µm)	IC30 (µm)
Run 1	1.24	-	44.92	37.84
Run 2	1.33	-	45.75	38.99
Mean	1.28	-	n.r.	n.r.
Geometric mean	n.r.	n.c.	45.34	38.41
SD	0.66	n.c.	0.59	0.81

-: No data available

n.c.: Not calculated

n.r.: Not required by OECD Guideline

Applicant's summary and conclusion

Interpretation of results:

other: Negative results

Conclusions:

Under the experimental conditions of this study, the test material was negative in the KeratinoSens assay and therefore was considered to have no potential to activate the Nrf2 transcription factor.

Executive summary:

The skin sensitisation potential of the test material was investigated in accordance with the standardised guideline OECD 442D, under GLP conditions.

This in vitro test uses the KeratinoSens cell line, an immortalised and genetically modified Human adherent HaCaT keratinocyte cell line. The KeratinoSens cell line is stably transfected with a plasmid containing a luciferase gene under the transcriptional control of the SV40 origin of replication promoter. This promoter is fused with an ARE sequence. Sensitisers with electrophilic properties provoke the dissociation of Keap-1 from the transcription factor Nrf2. The free Nrf2 binds to the ARE sequence contained in the plasmid and therefore induces transcription of firefly luciferase.

During the study, the KeratinoSens cells were first plated on 96-well plates and grown for 24 hours at 37 °C. Then the medium was removed and the cells exposed to the vehicle control or to different concentrations of test material and positive controls. The treated plates were then incubated for 48 hours at 37 °C. At the end of the treatment, cells were washed and the luciferase production measured by flash luminescence. In parallel, cytotoxicity was measured by a MTT reduction, this was taken into consideration in the interpretation of the sensitisation results. Two independent validated runs were performed as part of this study.

This study was performed at test concentrations ranging from 0.98 to 2 000 µM in the first run and from 0.12 to 250 µM in the second one, in culture medium containing 1 % DMSO.

At these tested concentrations, no statistically significant gene-fold induction above the threshold of 1.5 was noted in comparison to the negative control at any tested concentration in either run.

All acceptance criteria were met in both runs; the study was therefore considered as validated.

The evaluation criteria for a negative response are met in both runs, the final outcome is therefore negative. It can therefore be concluded that the test material has no potential to activate the Nrf2 transcription factor. This negative result can be used to support the discrimination between skin sensitisers and non sensitisers in the context of an integrated approach to testing and assessment. It cannot be used on its own to conclude on a skin sensitisation potential.