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

Endpoint summary

Description of key information

First of all, two in vitro skin sensitisation were performed on ethyl (3-benzoyl-2,4,6-trimethylbenzoyl)(phenyl)phosphinate. Based on the inconclusive results obtained (negative results in KeratinoSens Assay and positive results in hClat assay), an vivo study (LLNA) was performed and concluded that ethyl (3-benzoyl-2,4,6-trimethylbenzoyl)(phenyl)phosphinate is not skin sensitizer.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

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

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.

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

Positive control results:

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

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

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

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

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.

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

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.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 September 2019 to 08 November 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442E (In Vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT))

Version / remarks:

In Vitro Skin Sensitisation: In Vitro Skin Sensitisation Assays addressing the Key Event on activation of dendritic cells on the Adverse Outcome Pathway for Skin Sensitisation. Annex I: In Vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT), June 2018.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

human Cell Line Activation Test (h-CLAT)

Details of test system:

THP-1 cell line [442E]

Details on the study design:

CONTROLS
- Positive Controls (h-CLAT): DNCB (2,4-dinitrochlorobenzene) final concentration: 8 µg/mL, Purity = 99 %; NiSO4 (Nickel Sulphate) final concentration: 240 µg/mL, Purity: = 99 %
- Solvent Control for the Positive Control (h-CLAT): DMSO (Dimethyl sulfoxide, CAS No. 67-68-5) in culture medium (cRPMI), final concentration 0.2 %

SOLUBILITY ASSESSMENT
The solubility of the test material was assessed visually for each preparation (particles, drops, cloudiness, non-miscible phases, etc) and recorded in the study files. A preparation was deemed appropriate for cell treatment as long as it qualified as a solution or stable dispersion (homogenous emulsion/suspension).
Saline solution (0.9 % NaCl) and DMSO are the only vehicles allowed in this assay. The vehicle was chosen between these two in the order of preference, and in accordance with the steps described below.
First, the test material was suspended in saline solution at 100 mg/mL. Since the test material was not soluble at the concentration of 100 mg/mL in saline, it was tested at 500 mg/mL in DMSO. A orange solution was obtained after a 1-minute heating step at 50 °C.
Since a solution was obtained in DMSO, the formulation was then 500-fold diluted in RPMI-1640 culture medium. A visual inspection was performed immediately after dilution as well as after an overnight period. The presence or absence of precipitate/emulsion was recorded in the study files.
Vortexing was used to help solubilise the test material in vehicles.

POSITIVE CONTROL PREPARATION
The positive control DNCB was prepared at the concentration of 8 µg/mL in DMSO before treatment (final concentration in wells of 4 µg/mL) as follows:
- on the treatment day, the required quantity of DNCB was mixed with DMSO at the concentration of 2 mg/mL,
- this solution was then 250-fold diluted in cRPMI in order to obtain a 8 µg/mL DNCB stock solution.
The positive control NiSO4 was prepared at the concentration of 240 µg/mL in 0.9 % NaCl before treatment (final concentration in wells of 120 µg/mL) as follows:
- on the treatment day, the required quantity of NiSO4 was mixed with 0.9 % NaCl at the concentration of 12 mg/mL,
- this solution was then 50-fold diluted in cRPMI in order to obtain a 240 µg/mL NiSO4 stock solution.
Both positive control stock solutions were prepared within 4 hours of use, and kept at room temperature and protected from light until use.

VEHICLE CONTROL
As DMSO was the selected vehicle at completion of the solubility assay, a DMSO control formulation was included as the vehicle control, and consisted of DMSO dissolved at 0.2 % in cRPMI.

TEST MATERIAL PREPARATION
All test material preparations were prepared in glass vials. A fresh stock formulations of the test material were prepared for each run.
Test material formulations prepared in DMSO were 500 x concentrated; then 2 x concentrated formulations were prepared by 1:250 dilution in cRPMI. A DMSO vehicle control was also prepared (0.4% DMSO in cRPMI). The aforementioned mentioned dilutions of the test material and vehicle control were performed to ensure a constant percentage of the vehicle in the final volume of cell suspension in the well (i.e. 0.2 % for DMSO).
A visual assessment of the stock formulations was performed.
Precipitation in the treatment conditions (i.e. when diluted in cRPMI) was checked.
The test material formulations were kept at room temperature and protected from light until use, i.e. within 4 hours after preparation of the stock formulations. No control of concentration was performed during the study.

TEST SYSTEM
- Cells: THP-1 is an immortalised human monocytic leukemia cell line derived from an acute monocytic leukemia patient. The THP-1 cells were stored in a cryoprotective medium in a liquid nitrogen container. Cells were grown using general culture procedures. They were cultured in cRPMI medium and maintained in a humidified incubator set at 37 °C in 5 % CO2 and were not allowed to exceed a cell density of 1 x 10^6 cells/mL or more than 30 passages.
The culture medium (cRPMI) was composed of RPMI 1640 with 10 % Inactivated Fetal Bovine Serum (FBS), 0.05 mM 2 mercaptoethanol, penicillin and streptomycin.
During cell culturing, cell viability was checked using trypan blue.
- Cell culture for testing: For testing, THP-1 cells were seeded at a density between 0.1 – 0.2 x 10^6 cells/mL, and pre-cultured in culture flasks for 48 hours to 72 hours, respectively. Cells did not exceed a density of 1 x 10^6 to 1.2 x 10^6 cells/mL. On the day of testing, cells harvested from culture flasks were resuspended with fresh culture medium at 2 x 10^6 cells/mL. Then, 500 µL of cell suspension were distributed into each well of a 24 well flat bottom plate (i.e. 1 x 10^6 cells/well).
- Reactivity check: Two weeks after thawing, a reactivity check was performed to validate the cells of each working cell bank before testing. A reactivity check assay was performed by testing the cell response after contact with Lactic Acid, DNCB and NiSO4 .

STUDY DESIGN
The study was divided in two successive phases. First, a Dose-Range Finding assay (DRF) was performed to assess test material toxicity and, determine the CV75 (i.e. the test material concentration that results in 75 % cell viability compared to the vehicle control). Secondly, based on cytotoxicity data obtained from the DRF, a concentration series was tested in successive runs in the main test in order to evaluate the expression of CD86 and CD54.

- Dose-Range Finding assay (DRF)
Test material stock solutions were prepared at 8 different concentrations by 2-fold dilutions using the selected vehicle. These stock formulations were then diluted 250 fold (as DMSO was the selected vehicle) into cRPMI to obtain working solutions.
The working solutions were finally ready for treatment after adding 500 µL of working solutions to the volume of THP-1 cell suspension in the plate (500 µL) to achieve a further 2-fold dilution. In order to avoid evaporation of volatile chemicals and cross-contamination between wells, a sealer was placed on each 24-well plate just after treatment, before putting the plastic lids back on each plate.
The treated plates were then incubated for 24 hours ± 30 minutes in a humidified incubator set at 37 °C and 5 % CO2.
At the end of the treatment phase, an inspection under a light microscope was performed for each well. Then, cells were transferred into sample tubes and collected by centrifugation. The supernatants were discarded and the remaining cells were resuspended with 600 µL of FACS buffer. Finally, cells were resuspended in 200 µL FACS buffer and the plate positioned into the plate-reader of the flow cytometer. A volume of 10 µL of Propidium Iodide (PI) solution at 12.5 µg/mL was added automatically by the flow cytometer before acquisition of a sample to obtain a final PI concentration of 0.625 µg/mL per well.

- Main Test
The main test consisted of two separate validated runs.
Test material stock solutions were prepared at 8 different concentrations by 1.2-fold dilutions using the selected vehicle. The highest concentration corresponded to 1.2-fold the mean CV75.
All stock formulations were then 250-fold diluted into cRPMI to obtain working solutions.
In parallel, the working solutions of both positive controls (DNCB and NiSO4) and the vehicle control (DMSO) were prepared.
All working solutions were finally used for treatment after adding 500 µL of working solutions to the volume of THP-1 cell suspension in the plate (500 µL) to achieve a further 2-fold dilution. In order to avoid evaporation of volatile chemicals and cross-contamination between wells, a sealer was placed on each 24-well plate just after treatment, before putting the plastic lids back on each plate.
The treated plates were then incubated for 24 hours ± 30 minutes in a humidified incubator set at 37 °C and 5 % CO2.
At the end of the treatment phase, an inspection under a light microscope was performed for each well. Then, cells were transferred into sample tubes and collected by centrifugation, washed twice with 1 mL FACS buffer and blocked with 600 µL of blocking solution and incubated at 4 °C for 15 minutes (± 1 minute). After blocking, cells were split into three aliquots of 180 µL into a 96-well round bottom plate and centrifuged before staining with antibodies. A volume of 50 µL of FITC-labelled anti-CD86, anti-CD54 or mouse IgG1 (isotype) antibodies prepared in FACS buffer was added to each aliquot before incubation for 30 minutes (± 2 minutes) at 4 °C.
Finally, cells were washed with 150 µL FACS buffer twice and re-suspended in 200 µL FACS buffer. The plate was then positioned into the plate-reader of the flow cytometer. A volume of 10 µL of PI solution at 12.5 µg/mL was added automatically by the flow cytometer before acquisition of a sample to obtain a final PI concentration of 0.625 µg/mL per well.

FLOW CYTOMETRY ANALYSIS
- DRF assays: The PI uptake is analysed by flow cytometry using acquisition channel B3. A total of 10 000 living cells (PI negative) were acquired. In case of low viability (i.e. less than 10 000 living cells), a maximum of 100 µL per sample was acquired (corresponding to an acquisition time of 2 minutes).
- Main Test: The non-specific binding of IgG1 and the expression of CD86 and CD54 was analysed by flow cytometry with the acquisition channel B1 to obtain the Mean Fluorescence Intensity (MFI); whereas the viability (PI uptake) was analysed with the acquisition channel B3. A total of 10 000 living cells (PI negative) were acquired. When the viability was low (i.e. less than 10 000 living cells), a maximum of 100 µL per sample was acquired (corresponding to an acquisition time of 2 minutes).
Where viability is less than 50 %, the corresponding test material concentration is considered too high for interpretation because of the diffuse labelling cytoplasmic structures that are generated following cell membrane destruction.

CALCULATIONS
- Estimation of the CV75 value: The percentage of living cells (PI negative cells) is used as the value for cell viability. The CV75 value is derived from the dose-response curve.
- Main Test: Based on the Mean Fluorescence Intensity (MFI), the Relative Fluorescence Intensity (RFI) of CD86 and CD54 were calculated according to the following equation:

RFI = [(MFI of test material-treated (CD86 or CD54) - MFI of test material-treated IgG1) / (MFI of control-treated (CD86 or CD54) - MFI of control-treated IgG1)] x 100

Where:
RFI = Relative Fluorescence Intensity
MFI = Mean Fluorescence Intensity

Moreover, for the test chemicals predicted as positive with the h-CLAT, two Effective Concentrations (EC) values, the EC150 for CD86 and EC200 for CD54, i.e. the concentration at which the test chemicals induced a RFI of 150 or 200, will be determined.

They can be calculated by the following equations:

EC150 (for CD86) = Bconcentration + [(150 - BRFI) / (ARFI - BRFI) × (Aconcentration - Bconcentration)].
EC200 (for CD54) = Bconcentration + [(200 - BRFI) / (ARFI- BRFI) × (Aconcentration - Bconcentration)].
Aconcentration is the lowest concentration in µg/mL with RFI > 150 (CD86) or 200 (CD54).
Bconcentration is the highest concentration in µg/mL with RFI < 150 (CD86) or 200 (CD54).
ARFI is the RFI at the lowest concentration with RFI > 150 (CD86) or 200 (CD54).
BRFI is the RFI at the highest concentration with RFI < 150 (CD86) or 200 (CD54).

ACCEPTABILITY CRITERIA
- DRF: Viability of control cells treated with cRPMI (and DMSO if applicable) should be = 90 %.
- Main Test:
Controls acceptance criteria:
- Viability of cells treated with cRPMI and DMSO controls should be = 90 %,
- in cRPMI and DMSO control wells, MFI ratio of both CD86 and CD54 to isotype control should be > 105 %,
- in the cRPMI [except the cRPMI(1)] and DMSO controls, RFI values of both CD86 and CD54 should not exceed the positive criteria (which are CD86 RFI = 150 % and CD54 RFI = 200 %),
- in the positive controls (DNCB and NiSO4), RFI values of both CD86 and CD54 should meet positive criteria (i.e. CD86 RFI = 150 and CD54 RFI = 200) and cell viability should be more than 50 %.
Test material acceptance criteria:
- For a test material noted as cytotoxic in the DRF phase, and resulting in a negative outcome in the main test, cell viability at 1.2 x CV75 should be < 90 % in each run,
- cell viability of at least 4 out of 8 concentrations should be > 50 %.

CRITERIA FOR A POSITIVE RESULT
A run conclusion is positive if at least one of the conditions below is met:
- RFI of CD86 is = 150 in at least one tested concentration leading to = 50 % viability,
- RFI of CD54 is = 200 in at least one tested concentration leading to = 50 % viability.
In other circumstances, the run is considered as negative.

RESULTS EVALUATION
Based on the individual run conclusions, a final prediction is made as follows:
- if the first two runs are both positive for CD86 and/or are both positive for CD54, the h-CLAT prediction is considered positive and a third run does not need to be conducted,
- if the first two runs are negative for both markers, the h-CLAT prediction is considered negative (with due consideration of the highest-tested dose conditions) without the need for a third run,
- if however, the first two runs are not concordant for at least one of the markers (CD54 or CD86), a third run is needed and the final prediction is based on the majority result of the three individual runs (i.e. 2 out of 3). In this respect, it should be noted that if two independent runs are conducted and one is only positive for CD86 (hereinafter referred to as P1) and the other is only positive for CD54 (hereinafter referred to as P2), a third run is required. If this third run is negative for both markers (hereinafter referred to as N), the h-CLAT prediction is considered negative. On the other hand, if the third run is positive for either marker (P1 or P2) or for both markers (hereinafter referred to as P12), the h-CLAT prediction is considered positive.

Vehicle / solvent control:

DMSO

Negative control:

not applicable

Positive control:

dinitrochlorobenzene (DNCB) [442E]

Positive control results:

Results with the two positive control materials (DNCB and NiSO4) indicated that these were correctly classified in a maximum of three validated runs using working cell bank 160718A.

Key result

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

RFI CD54>150 [442E]

Value:

203

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: (6.23 µg/mL)

Key result

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

RFI CD54>150 [442E]

Value:

207

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: (7.48 - 15.51 µg/mL)

Other effects / acceptance of results:

SOLUBILITY ASSESSMENT
A 100-fold dilution in culture medium was performed, test material precipitate was observed, immediately as well as after an overnight period of incubation.
A 0.9 % NaCl solution (concentration: 100 mg/mL) resulted in a heterogeneous suspension of test material.
A DMSO soludion (concentration: 500 mg/mL) resulted in a homogeneous soultion of test material.
Based on these results, 500 mg/mL in DMSO was retained as the highest stock concentration to be tested in the DRF assays.

DRF RESULTS
The following test material concentrations were tested in both DRF assays: 7.81, 15.63, 31.25, 62.50, 125, 250, 500 and 1 000 µg/mL.
At these tested concentrations:
- at post-treatment observation, slight to moderate emulsion was noted at concentration = 250 µg/mL in both DRF assays,
- during both DRF assays, flow cytometry measurement after PI staining revealed a cell viability decrease below 75 % at concentrations = 31.25 µg/mL. The corresponding CV75 values were 18.59 µg/mL and 18.63 µg/mL in the DRF1 and DRF2, respectively.
Based on the results from both DRF runs, the mean CV75 was 18.61 µg/mL, and the highest concentration tested in the main test was therefore 22.33 µg/mL (i.e. 1.2-fold the mean CV75).

MAIN TEST
The two runs were performed at the following final concentrations: 6.23, 7.48, 8.97, 10.77, 12.92, 15.51, 18.61 and 22.33 µg/mL.
At these tested concentrations:
- at post-treatment observation, no abnormalities and no precipitate/emulsion was noted in test material treated wells at the end of treatment period, at any tested concentrations in either run,
- RFI CD54 exceeded the positivity threshold only at the lowest tested concentration in the Run A, and at concentrations = 7.48 µg/mL and up to 18.61 µg/mL in the Run B, whereas RFI CD86 did not exceed the positivity threshold at any tested concentrations in either run.
Both runs were therefore considered positive.

All acceptance criteria were fulfilled in each validated run.

Summary Results

Test Material Conc. (µg/mL)	RFI for CD86		RFI for CD54		Viability (%)		Run Conclusion		General Conclusion
	A	B	A	B	A	B	A	B
6.23	73	58	203*	197	93	94	Run with positive outcome for CD54	Run with positive outcome for CD54	Positive
7.48	74	61	189	217*	92	93
8.97	80	75	150	210*	93	93
10.77	79	62	172	227*	93	91
12.92	90	83	150	217*	92	90
15.51	79	57	156	207*	92	88
18.61	75	69	142	167	92	89
22.33	129	62	106	123	85	84

 * Positive result

RFI: Relative Fluorescence Index

Interpretation of results:

other: Positive results

Conclusions:

Under the experimental conditions of this study, the test material was found to be positive in the h-CLAT test method.

Executive summary:

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

The objective of the study was to determine the ability of the test material to induce an increase in expression of cell surface markers in THP-1 cells using the h-CLAT test method.

The study was initiated with a solubility assessment in saline solution (0.9 % NaCl) and dimethylsulfoxide (DMSO) to select an appropriate vehicle and the highest concentration to be used for test material formulations. The test material was found to be soluble in DMSO at the concentration of 500 mg/mL.

Following the solubility assays, the cytotoxic potential was assessed in a Dose-Range Finding assay to select sub-toxic concentrations for testing in the main test.

The skin sensitising potential of the test material was then tested in the main test in 2 successive validated runs.

In each run, test material formulations were applied to THP-1 cells and cultured in a 24-well plate for 24h ± 30 minutes at 37 °C and 5 % CO2 in a humidified incubator. A set of control wells was also added in each plate to assure the validity of each run. At the end of the incubation period, cells from each well were distributed into three wells of a 96-well plate: the first well was labelled with IgG1-FITC antibodies, the second one was labelled with CD86-FITC antibodies and the third one was labelled with CD54-FITC antibodies. Just before flow cytometry analysis of CD86 and CD54 expression, all cells were dyed with Propidium Iodide for viability discrimination.

For each run, the Mean Fluorescence Intensity (MFI) from each test sample was corrected by the isotype control IgG1 MFI value to obtain the corrected MFI. The corrected MFI value from the corresponding vehicle control was set to 100 % CD54 and CD86 expression by default. Then, corrected MFI values from each test sample were compared to the corresponding vehicle control to obtain the Relative Fluorescence Index for CD86 and CD54 expression for each tested concentration (RFI CD86 and RFI CD54).

During both DRF assays, the test material induced a decrease in cell viability < 75 % and the calculated mean CV75 value was 18.61 µg/mL. The highest concentration tested in the main test was therefore 22.33 µg/mL (i.e. 1.2-fold the mean CV75).

Under the experimental conditions of this study, the test material was found to be positive in the h-CLAT test method.

Reference 3

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05 February 2020 to 18 February 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

22 July 2010

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

6 July 2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA

Remarks:

CBA/CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Females nulliparous and non-pregnant: Yes
- Age at study initiation: 10 weeks old (age-matched, within one week). In the Preliminary Experiment mice of 9 weeks of age were used.
- Weight at study initiation: 19.4 - 22.6 g (The weight variation in animals in the study did not exceed ± 20 % of the mean weight). In the Preliminary Experiment mice of 21.1 - 23.3 g were used.
- Housing: Group housing in Type II polypropylene / polycarbonate cages. Additional enrichment (tunnel, bedding and certified nest building material) was available for the animals during the study.
- Diet: Ad libitum
- Water: Tap water, ad libitum.
- Acclimation period: 20 days
- Indication of any skin lesions: Only healthy animals were used for the study. Health status was certified by the veterinarian.

ENVIRONMENTAL CONDITIONS
- Temperature: 19.8 - 24.3 °C
- Humidity: 22 - 74 %. Due to technical reasons, the relative humidity values (minimum of 22 % and maximum of 74 %) outside the expected range of 30 - 70 % were recorded occasionally times during the study. The observed differences in the environmental parameters were considered not to adversely affect the results or integrity of the study.
- Air changes: 15 - 20 air exchanges/hour
- Photoperiod: 12 hours dark / 12 hours light , from 6.00 a.m. to 6.00 p.m.

Vehicle:

dimethylformamide

Concentration:

Test material treatment groups: 5, 10 and 25 % w/v in DMF.

No. of animals per dose:

4 animals per dose/ treatment group.

Details on study design:

ANIMAL IDENTIFICATION AND RANDOMISATION
A unique number written on the tail with a permanent marker identified each animal. The animal number was assigned on the basis of the Test Facility's master file. The cages were marked with identity cards with information including study code, cage number, and dose group, sex and individual animal number. The animals were randomised and allocated to the experimental groups. The randomisation was checked by computer software using the body weight to verify the homogeneity and variability between the groups.

PRE-SCREEN TESTS:
- Compound solubility: The solubility of the test material was examined in a short Preliminary Compatibility Test. The following standard OECD vehicles were assessed: AOO (acetone:olive oil 4:1 (v:v) mixture), N,N-dimethylformamide (DMF), methyl ethyl ketone (MEK), propylene glycol (PG), dimethyl sulfoxide (DMSO) and 1 % aqueous Pluronic® PE9200 (1 % Pluronic). The best vehicle taking into account the test material characteristics, its usage and the requirements of the relevant OECD guideline was considered to be N,N-dimethylformamide (DMF). The 50 % (w/v) formulation was the highest concentration which was suitable for the preliminary test. The 50 % (w/v) and the 25 % (w/v) formulations appeared to be solutions by visual examination.
The test material was weighed and formulations prepared the day before the treatment on a weight: volume basis (as % (w/v)).
- Preliminary Irritation/ Toxicity Test: The preliminary experiment was conducted in a similar experimental manner to the main study, but it was terminated on Day 6 and the radioactive proliferation assay was not performed. During the test, 2 animals/dose received test material concentrations of 50 % and 25 % (w/v) in DMF.
All mice were observed daily for any clinical signs of systemic toxicity or local irritation at the application site. Both ears of each mouse were observed for erythema. Ear thickness was also measured using a thickness gauge on Day 1 (pre-dose), Day 3 (approximately 48 hours after the first dose) and Day 6. Additional quantification of the ear thickness was performed by ear punch weight determination after the euthanasia of the experimental animals.
During the Preliminary Irritation / Toxicity Test no mortality or signs of systemic toxicity were observed. Minimal amount of test material residue was observed on the ears of all animals in the 50 % (w/v) and 25 % (w/v) dose groups on Days 2 - 3. Extensive alopecia was observed for both animals of the 50 % (w/v) dose group on Days 2 - 6 and for both animals of the 25 % (w/v) dose group on Days 4 - 6 or Day 6. Alopecia around the ears was observed for both animals of the 25 % (w/v) dose group on Days 3 - 5 or Day 3. No marked body weight loss (> 5 % reduction in body weight) was observed in any animals.
The detected ear thickness values clearly indicated excessive local irritation (= 25 %) for one animal (both ears) and the other animal (right ear) of the 50 % (w/v) on Day 6.
It was considered that the ear thickness increase was not caused by residual test material on the ears, because minimal amount of test material residue was detected only on Days 2 - 3, hence this result indicated ear swelling.
The ear thickness values of the animals of the 25 % (w/v) dose group was within the acceptable range.
The ear punch weights were within the acceptable range in all animals.
The draining auricular lymph nodes of the animals were visually examined and were considered normal in all animals of the 50 % (w/v) and 25 % (w/v) dose groups.
Based on these results, 25 % (w/v) dose was selected as top dose for the main test.

MAIN STUDY
> Topical application: animals were topically dosed with 25 µL of the appropriate formulation using a pipette on the dorsal surface of each ear. Each animal was dosed once a day for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6.
The principles and criteria summarised in the OECD Humane Endpoints Guidance Document No. 19 was taken into consideration.
- Clinical Observations: During the study (Day 1 to Day 6) each animal was observed daily for any clinical signs, including local irritation and systemic toxicity. Clinical observations were performed twice a day (before and after treatments) on Days 1, 2 and 3 and once daily on Days 4, 5 and 6.
Both ears of each mouse were observed for erythema and scored. Individual records were maintained.
- Measurement of Body Weight: Individual body weights were recorded on Day 1 (beginning of the test) and on Day 6 (prior to 3HTdR injection) with a precision of 0.1 g.

PROLIFERATION ASSAY
- Injection of Tritiated Thymidine (3HTdR): On Day 6, animals were taken to the radioactive suite and each mouse was intravenously injected via the tail vein with 250 µL of sterile PBS (phosphate buffered saline) containing approximately 20 µCi of 3HTdR using a gauge 25G x 1" hypodermic needle with 1 mL sterile syringe. Once injected, the mice were left for 5 hours (± 30 minutes).
- Removal and Preparation of Draining Auricular Lymph Nodes: Five hours (± 30 minutes) after intravenous injection the mice were euthanised by asphyxiation with ascending doses of carbon dioxide (deep anaesthesia was confirmed before making incision, death was confirmed before discarding carcasses).
The draining auricular lymph nodes were excised by making a small incision on the skin between the jaw and sternum, pulling the skin gently back towards the ears and exposing the lymph nodes. The nodes were then removed using forceps. The carcasses were discarded after cervical dislocation or after cutting through major cervical blood vessels.
Once removed, the nodes of mice from each test group was pooled and collected in separate Petri dishes containing a small amount (1 - 2 mL) of PBS to keep the nodes wet before processing.
- Preparation of Single Cell Suspension of Lymph Node Cells: A single cell suspension (SCS) of pooled lymph node cells (LNCs) was prepared and collected in disposable tubes by gentle mechanical disaggregating of the lymph nodes through a cell strainer using the plunger of a disposable syringe. The cell strainer was washed with PBS (up to 10 mL). Pooled LNCs were pelleted with a relative centrifugal force (RCF) of 190 x g (approximately) for 10 minutes at 4 °C. After centrifugation supernatants were discarded. Pellets were gently resuspended and 10 mL of PBS was added to the tubes. The washing step was repeated twice. This procedure was repeated for each group of pooled lymph nodes.
- Determination of Incorporated 3HTdR: After the final washing step, supernatants were removed. Pellets were gently resuspended and 3 mL of 5 % (w/v) TCA solution was added to the tubes for precipitation of macromolecules.
After overnight incubation (approximately 18 hours) at 2 - 8 °C, precipitates were centrifuged (approximately 190 x g for 10 minutes at 4 °C), and supernatants were removed. Pellets were resuspended in 1 mL of 5 % (w/v) TCA solution and dispersed by using an ultrasonic bath. Samples were transferred into a suitable sized scintillation vial filled with 10 mL of scintillation liquid and thoroughly mixed. The vials were loaded into a ß-scintillation counter and 3HTdR incorporation was measured (10-minute measurement).
The ß-counter expresses the 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM). Background level was also measured in duplicates by adding 1 mL of 5 % (w/v) TCA solution into a scintillation vial filled with 10 mL of scintillation liquid.

EVALUATION OF RESULTS
The number of radioactive disintegrations per minute (DPM) was measured for each pooled group of nodes. The measured DPM values were corrected with the background DPM value (“DPM”). The average of the two measured DPM values of 5 % (w/v) TCA solutions was used as background DPM value.
Stimulation index (SI = DPN value of a treated group divided by the DPN value of the negative control group) for each treatment group was also calculated. A stimulation index of 3 or greater is an indication of a positive result.

> Interpretation of Results: A test material is regarded as a sensitiser if both of the following criteria are fulfilled:
- That exposure to at least one concentration of the test material resulted in an incorporation of 3HTdR at least 3-fold or greater than recorded in control mice, as indicated by the stimulation index.
- The data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.

> Acceptability of the test: The Local Lymph Node Assay is considered valid if it meets the following criteria:
- the DPN value of the negative (vehicle) control group falls within the range of historical laboratory control data,
- the positive control substance produces a significant lymphoproliferative response increases (SI > 3),
- each treated and control group includes at least 4 animals,
- the test material does not cause serious systemic or local toxicity.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Positive control results:

The result of the positive control substance a-Hexylcinnamaldehyde (HCA) dissolved in the same vehicle was used to demonstrate the appropriate performance of the assay. The positive control substance was examined at a concentration of 25 % (w/v) in the relevant vehicle (DMF) using CBA/CaOlaHsd mice.
No mortality, cutaneous reactions or signs of toxicity were observed for the positive control substance in the study. A significant lymphoproliferative response (stimulation index value of 4.1) was noted for HCA in the main experiment. The results of the positive control group demonstrated the appropriate performance of the assay.
Furthermore, the DPN values observed for the vehicle and positive control substance in this experiment were within the historical control range. Each treated and control group included 4 animals.

Key result

Parameter:

SI

Value:

1

Test group / Remarks:

25 % (w/v) test material in DMF

Key result

Parameter:

SI

Value:

0.9

Test group / Remarks:

10 % (w/v) test material in DMF

Key result

Parameter:

SI

Value:

1.3

Test group / Remarks:

5 % (w/v) test material in DMF

Parameter:

SI

Value:

1

Test group / Remarks:

Negative control (DMF)

Parameter:

SI

Value:

4.1

Test group / Remarks:

Positive control (25 % (w/v) HCA in DMF)

Cellular proliferation data / Observations:

PROLIFERATION ASSAY
The appearance of the lymph nodes was normal in the negative control group and in all test material treated groups. Larger than normal lymph nodes were observed in the positive control group.
The stimulation index values were 1.0, 0.9 and 1.3 at concentrations of 25 % (w/v), 10 % (w/v) and 5 % (w/v), respectively.

CLINICAL OBSERVATIONS:
No mortality or systemic toxicity was observed during the main study. No test material residue was observed on the ears of the animals. Alopecia around the ears was observed in all animals at 25 % (w/v) on Days 2 - 3 and in one animal on Day 4. Extensive alopecia around the ears was observed in three animals at 25 % (w/v) dose group on Days 4 - 6 and in one animal on Day 5 - 6.

BODY WEIGHTS
Marked body weight loss (> 5 % reduction in body weight) was observed in one animal of the 25 % (w/v) group and in one animal of the positive control group, however these changes were considered as individual variability.
No evidence of test material related effects observed on the mean body weight changes in the main study.

Summary of Preliminary Test Data

Preliminary Concentrations	Physical Formulation	Clinical Observations	Body Weight	Erythema	Ear Thickness	Ear Biopsy Weight
100 % (w/v)	U	NM	NM	NM	NM	NM
50 % (w/v)	A	A	A	A	U	A
25 % (w/v)	A	A	A	A	A	A

Notes: A = Acceptable, U = Unacceptable, NM = Not measured

DPM, DPN and Stimulation Index Values for all Groups

Test Group Name	Measured DPM / Group	Total DPM	Number of Lymph Nodes	DPN	Stimulation Index
Background (5 % (w/v) TCA)	37 35	-	-	-	-
Negative control (DMF)	4430	4394.0	8	549.3	1.0
25 % (w/v) test material in DMF	4614	4578.0	8	572.3	1.0
10 % (w/v) test material in DMF	3834	3798.0	8	474.8	0.9
5 % (w/v) test material in DMF	5943	5907.0	8	738.4	1.3
Positive control (25% (w/v) HCA inDMF)	18221	18185.0	8	2273.1	4.1

Notes:

1. Total DPM (Disintegrations Per Minute) = Measured DPM – Background DPM

2. DPN (Disintegrations Per Node) = Mean total DPM divided by the number of lymph nodes.

3. Stimulation Index = DPN of a treated group divided by DPN of the appropriate control group.

Historical Control Data of the Positive and Negative Controls for CBA/CaOlaHsdmice(2014 - 2018)

	Vehicles
	Acetone: Olive oil 4:1 (AOO)			1 % Pluronic PE9200 in water (1 % Plu)
	DPN Values		SI Value	DPN Values		SI Value
	Control	HCA 25 %	HCA 25 %	Control	HCA 25 %	HCA 25 %
Average	472.7	3851.3	9.0	198.7	1988.1	11.2
Range: min	35.8	890.3	3.3	23.0	154.0	3.0
Range: max	1990.1	10336.0	20.2	680.8	6755.8	33.6
Number of cases	92	88	86	234	226	218
	Vehicles
	N,N-Dimethylformamide (DMF)			Dimethyl Sulfoxide (DMSO)
	DPN Values		SI Value	DPN Values		SI Value
	Control	HCA 25 %	HCA 25 %	Control	HCA 25 %	HCA 25 %
Average	256.1	2738.9	11.3	466.0	3014.4	7.2
Range: min	62.0	1201.3	4.9	218.3	1461.3	3.1
Range:max	649.6	5817.9	21.3	934.6	4877.5	14.5
Number of cases	68	68	68	22	22	21
	Vehicles
	Propylene Glycol (PG)			Methyl Ethyl Ketone (MEK)
	DPN Values		SI Value	DPN Values		SI Value
	Control	HCA 25 %	HCA 25 %	Control	HCA 25 %	HCA 25 %
Average	245.1	2278.6	9.4	264.5	4129.5	16.9
Range: min	63.3	817.3	5.8	80.5	1562.5	8.8
Range:max	506.0	4978.0	14.4	516.2	8682.5	36.3
Number of cases	18	18	18	18	19	19

HCA 25 % = alpha-Hexylcinnamaldehyde 25 % (w/v)

SI (Stimulation Index) = DPN of a treated group divided by DPN of the appropriate control group.

DPN (Disintegrations Per Node) = DPM (Disintegrations Per Minute) divided by the number of lymph nodes.

In case of individual approach, SI values were calculated from the mean DPN values of the group.

Interpretation of results:

GHS criteria not met

Remarks:

Not skin sensitizer

Conclusions:

Under the conditions of the present assay, the test material was shown to have no sensitisation potential (non-sensitiser) in the Local Lymph Node Assay.

Executive summary:

The skin sensitisation potential of the test material was investigated in a study which was conducted in accordance with the standardised guidelines OECD 429 and EU Method B.42, under GLP conditions.

Based on the results of the Preliminary Compatibility Test, the test material characteristics and on the recommendations of the OECD Guideline, the best vehicle for the test material was N,N-dimethylformamide (DMF). The 50 % (w/v) formulation was the highest concentration which was suitable for the preliminary test. The formulations at 25 % (w/v), 10 % (w/v) and 5 % (w/v) in DMF were also suitable for treatment. The formulations at 50 %, 25 %, 10 % and 5 % (w/v) appeared to be solutions by visual examination.

The Preliminary Irritation/Toxicity Test was performed in CBA/CaOlaHsd mice using two doses (2 animals/dose) 50 % (w/v) and 25 % (w/v) in DMF. Based on the observations recorded in the preliminary test, the 25 % (w/v) dose was selected as top dose for the main test.

In the main assay, twenty female CBA/CaOlaHsd mice were allocated to five groups of four animals each:

- three groups received test material (formulated in DMF) at 25 % (w/v), 10 % (w/v) and 5 % (w/v) concentrations,

- the negative control group received the vehicle (DMF) only,

- the positive control group received 25 % (w/v) HCA (dissolved in DMF).

During the main assay, the test material formulations were applied on the dorsal surface of ears of experimental animals (25 µL/ear) for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6. The cell proliferation in the local lymph nodes was assessed by measuring the incorporation of tritiated methyl thymidine (3HTdR) and the values obtained were used to calculate stimulation indices (SI) in comparison with the control group.

Under the conditions of the study, no mortality or systemic toxicity was observed during the main assay. No test material residue was observed on the ears of the animals. Alopecia around the ears was observed in all animals at 25 % (w/v) on Days 2 - 3. Extensive alopecia around the ears was observed in all animals at 25 % (w/v) dose group on Days 4 - 6. No test material related effects were observed on the mean body weight changes in the main study.

The stimulation index values were 1.0, 0.9 and 1.3 at concentrations of 25 % (w/v), 10 % (w/v) and 5 % (w/v), respectively.

The result of the positive control substance (25 % (w/v) HCA) dissolved in the same vehicle was used to demonstrate the appropriate performance of the assay. A lymphoproliferative response (SI = 4.1) in line with historical control data was noted for the positive control chemical, this result confirmed the validity of the assay.

In conclusion, the test material was shown to have no sensitisation potential in this Local Lymph Node Assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

KeratinoSens Assay

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.

h-CLAT Assay

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

The objective of the study was to determine the ability of the test material to induce an increase in expression of cell surface markers in THP-1 cells using the h-CLAT test method.

The study was initiated with a solubility assessment in saline solution (0.9 % NaCl) and dimethylsulfoxide (DMSO) to select an appropriate vehicle and the highest concentration to be used for test material formulations. The test material was found to be soluble in DMSO at the concentration of 500 mg/mL.

Following the solubility assays, the cytotoxic potential was assessed in a Dose-Range Finding assay to select sub-toxic concentrations for testing in the main test.

The skin sensitising potential of the test material was then tested in the main test in 2 successive validated runs.

In each run, test material formulations were applied to THP-1 cells and cultured in a 24-well plate for 24h ± 30 minutes at 37 °C and 5 % CO2 in a humidified incubator. A set of control wells was also added in each plate to assure the validity of each run. At the end of the incubation period, cells from each well were distributed into three wells of a 96-well plate: the first well was labelled with IgG1-FITC antibodies, the second one was labelled with CD86-FITC antibodies and the third one was labelled with CD54-FITC antibodies. Just before flow cytometry analysis of CD86 and CD54 expression, all cells were dyed with Propidium Iodide for viability discrimination.

For each run, the Mean Fluorescence Intensity (MFI) from each test sample was corrected by the isotype control IgG1 MFI value to obtain the corrected MFI. The corrected MFI value from the corresponding vehicle control was set to 100 % CD54 and CD86 expression by default. Then, corrected MFI values from each test sample were compared to the corresponding vehicle control to obtain the Relative Fluorescence Index for CD86 and CD54 expression for each tested concentration (RFI CD86 and RFI CD54).

During both DRF assays, the test material induced a decrease in cell viability < 75 % and the calculated mean CV75 value was 18.61 µg/mL. The highest concentration tested in the main test was therefore 22.33 µg/mL (i.e. 1.2-fold the mean CV75).

Under the experimental conditions of this study, the test material was found to be positive in the h-CLAT test method.

Local Lymph Node Assay

The skin sensitisation potential of the test material was investigated in a study which was conducted in accordance with the standardised guidelines OECD 429 and EU Method B.42, under GLP conditions.

Based on the results of the Preliminary Compatibility Test, the test material characteristics and on the recommendations of the OECD Guideline, the best vehicle for the test material was N,N-dimethylformamide (DMF). The 50 % (w/v) formulation was the highest concentration which was suitable for the preliminary test. The formulations at 25 % (w/v), 10 % (w/v) and 5 % (w/v) in DMF were also suitable for treatment. The formulations at 50 %, 25 %, 10 % and 5 % (w/v) appeared to be solutions by visual examination.

The Preliminary Irritation/Toxicity Test was performed in CBA/CaOlaHsd mice using two doses (2 animals/dose) 50 % (w/v) and 25 % (w/v) in DMF. Based on the observations recorded in the preliminary test, the 25 % (w/v) dose was selected as top dose for the main test.

In the main assay, twenty female CBA/CaOlaHsd mice were allocated to five groups of four animals each:

- three groups received test material (formulated in DMF) at 25 % (w/v), 10 % (w/v) and 5 % (w/v) concentrations,

- the negative control group received the vehicle (DMF) only,

- the positive control group received 25 % (w/v) HCA (dissolved in DMF).

During the main assay, the test material formulations were applied on the dorsal surface of ears of experimental animals (25 µL/ear) for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6. The cell proliferation in the local lymph nodes was assessed by measuring the incorporation of tritiated methyl thymidine (3HTdR) and the values obtained were used to calculate stimulation indices (SI) in comparison with the control group.

Under the conditions of the study, no mortality or systemic toxicity was observed during the main assay. No test material residue was observed on the ears of the animals. Alopecia around the ears was observed in all animals at 25 % (w/v) on Days 2-3. Extensive alopecia around the ears was observed in all animals at 25 % (w/v) dose group on Days 4 - 6. No test material related effects were observed on the mean body weight changes in the main study.

The stimulation index values were 1.0, 0.9 and 1.3 at concentrations of 25 % (w/v), 10 % (w/v) and 5 % (w/v), respectively.

The result of the positive control substance (25 % (w/v) HCA) dissolved in the same vehicle was used to demonstrate the appropriate performance of the assay. A lymphoproliferative response (SI = 4.1) in line with historical control data was noted for the positive control chemical, this result confirmed the validity of the assay.

In conclusion, the test material was shown to have no sensitisation potential in this Local Lymph Node Assay.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the available data, no classification of ethyl (3-benzoyl-2,4,6-trimethylbenzoyl)(phenyl)phosphinate is required for skin sensitisation according to the Regulation (EC) No 1272/2008.