Reaction mass of dieuropium tricarbonate and digadolinium tricarbonate and disamarium tricarbonate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 August 2017 to 22 September 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD guideline No. 442E: "In vitro skin sensitization: human Cell Line Activation Test (h-CLAT)"

Version / remarks:

29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

other: HUMAN-CELL LINE ACTIVATION TEST

Justification for non-LLNA method:

Recommended test system in the international OECD guidelines.

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of dieuropium tricarbonate and digadolinium tricarbonate and disamarium tricarbonate

EC Number:

945-733-5

Molecular formula:

See remarks

IUPAC Name:

Reaction mass of dieuropium tricarbonate and digadolinium tricarbonate and disamarium tricarbonate

Test material form:

solid: particulate/powder

Details on test material:

- Appearance: Natural white powder
- Storage conditions: Room temperature (15-25 °C), protected from light and humidity

In vitro test system

Details on the study design:

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 was qualified as a solution or stable dispersion (homogenous emulsion/suspension).
- Saline (0.9 % NaCl) and DMSO are the only vehicles allowed in the 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 dissolved in saline at 100 mg/mL. As the test material was not soluble at the concentration of 100 mg/mL in 0.9 % NaCl, it was dissolved at 500 mg/mL in DMSO. As it was not soluble at 500 mg/mL in DMSO, the HSC was determined by testing lower concentrations in a common ratio of two (250 mg/mL → 125 mg/mL → 62.5 mg/mL → 31.25 mg/mL → 15.63 mg/mL → 7.81 mg/mL).
- First, only vortexing was used to help solubilise the test material. As only heterogeneous preparations were first obtained, the lowest concentrated preparation of 7.81 mg/mL was submitted to 10 minutes heating at 40 °C followed by 5 minute-sonication. The remaining preparation was a homogenous preparation another solubility assay was performed at 250 and 125 mg/mL using vortexing and 5 minutes sonication.
- Vortexing, sonication (5 minutes maximum) and heating were used at different occasions during the study to help solubilise the test material in vehicles as the Sponsor did not mention that such process can affect the test material stability.

PREPARATION OF THE TEST MATERIAL AND CONTROLS
- The positive control 2,4-Dinitrochlorobenzene (DNCB) was prepared at the concentration of 8 μg/mL in DMSO 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 Nickel Sulphate (NiSO4) was prepared at the concentration of 200 μg/mL in 0.9 % NaCl as follows: on the treatment day, the required quantity of NiSO4 was mixed with 0.9 % NaCl at the concentration of 10 mg/mL, this solution was then 50-fold diluted in cRPMI in order to obtain a 200 μg/mL NiSO4 stock solution.
- Both positive control stock solutions were prepared within 4 hours before use, and kept at room temperature and protected from light until use.

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

- All test material preparations were prepared in glass vials only.
- Fresh stock formulations of the test material were prepared for each run, using the vehicle and concentration identified in the Solubility assessment. These concentrations were the same for all runs.
- 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 above mentioned dilutions of the test material and vehicle controls were performed to insure a constant percentage of the vehicle in the final volume of cell suspension in the well (i.e. 0.2 % for DMSO).
- The aspect of the stock formulations was evaluated and recorded in the study files.
- The precipitation in the treatment conditions (i.e. when diluted in cRPMI) was checked and any observation was reported in the study files.
- 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
- The THP-1 is an immortalised human monocytic leukaemia cell line derived from an acute monocytic leukaemia patient. The THP-1 cell line is obtained from ATCC (Ref: TIB-202, American Type Culture Collection, Manassas, USA) by the intermediate of LGC Standards (Molsheim, France). The THP-1 cells are 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, 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 % FBS, 0.05 mM 2-mercaptoethanol and with 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 x 10^6 and 0.2 x 10^6 cells/mL, and pre-cultured in culture flasks for 48 hours to 72 hours, respectively. Cell did not exceed density of 1 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 cells suspension were distributed into 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 qualify the cells of each working cell bank before testing. A reactivity check assay was performed by testing cell response after contact with Lactic Acid, DNCB and NiSO4 under an internal Citoxlab France reference number. Results from negative and positive controls were obtained during each test.

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 result 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 tests.

- Dose-Range Finding assay (DRF)
The DRF consisted in two separated assays. Treatments of DRF assays were performed at the following concentrations: 1.95, 3.91, 7.81, 15.63, 31.25, 62.50, 125.00, 250.00 μg/mL.
Each assay was performed as described below:
Test material stock solutions were prepared at eight different concentrations by 2-fold dilutions using the selected vehicle. These stock formulations were then diluted 250-fold (as DMSO is the selected vehicle) into cRPMI to obtain working solutions.
The working solutions were finally used for exposure by 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, 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 was 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 tests
The main test consisted in two separated runs being performed as described below:
Test material stock solutions were prepared at eight different concentrations by 1.2-fold dilutions using the selected vehicle. The highest concentration corresponded to 1.2-fold the mean CV75 was obtained. The maximum concentration in the plates was 201.16 μg/mL.
All stock formulations were then 250-fold diluted into cRPMI to obtain working solutions.
In parallel, the working solutions of positive controls DNCB and NiSO4 and vehicle control were prepared as above.
All working solutions were finally used for exposure by 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.
During the main test, treatments were performed at the following concentrations: 56.14, 67.37, 80.84, 97.01, 116.41, 139.69, 167.63 and 201.16 µg/mL.
At the end of the treatment phase, 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 in 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 two times and resuspended 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 using flow cytometry with the acquisition channel B3. A total of 10 000 living cells (PI negative) are acquired. In case of low viability which does not allow obtaining 10 000 living cells, a total of 30 000 events is acquired. Alternatively, cells were acquired for a maximum of 1 minute after the initiation of the acquisition.
- Main test
The expression of IgG1, CD86 and CD54 was analysed by flow cytometry with the acquisition channel B1 in order 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 and did not allow obtaining 10 000 living cells, a total of 30 000 events was acquired. Alternatively, cells were acquired for a maximum of 1 minute after the initiation of the acquisition.
In case cell viability is less than 50 %, no MFI is presented in the study report and the corresponding test material concentration are 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 (75 % of cell viability, lying between a and c). CV75 is defined as the estimated concentration that is required to elicit 75 % cell viability. The CV75 value is calculated by log-linear interpolation utilizing the following equation:

Log CV75 = [(75 – c) x Log b – (75 – a) x Log d] / (a – c)

- 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

RFI = Relative Fluorescence Intensity
MFI = Mean Fluorescence Intensity


ACCEPTANCE CRITERIA
DRF
- Viability of control cells treated with cRPMI should be ≥ 90 %
- viability of control cells treated with 0.2 % DMSO should be ≥ 90 %.

Main test
The following applies for each run.

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 DMSO control, RFI values of both CD86 and CD54 should not exceed the positive criteria (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 (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 four out of eight concentrations should be > 50 %.

Main test interpretation
Individual run interpretation:
A run conclusion is positive if at least one of the conditions below is met:
- RFI of CD86 is ≥ 150 at any concentration leading to ≥ 50 % viability,
- RFI of CD54 is ≥ 200 at any concentration leading to ≥ 50 % viability.
- In other circumstances, the run is considered as negative.

Prediction model
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 will be 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.

CLASSIFICATION
Results from the present study can be used to support the discrimination between skin sensitisers (i.e. UN GHS Category 1) and non-sensitisers in the context of Integrated Approaches to Testing and Assessment (IATA). However, results obtained at completion of the study will not be usable on their own, neither to sub-categorise skin sensitisers into sub-categories 1A and 1B as defined by UN GHS, for authorities implementing these two optional sub-categories, nor to predict potency for safety assessment decisions.

Results and discussion

Positive control results:

All acceptance criteria were reached in each run.

In vitro / in chemico

Results

Results 1

Run / experiment:

other: DRF1

Parameter:

other: CV75 (µg/mL)

Value:

161.79

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Results 2

Run / experiment:

other: DRF 2

Parameter:

other: CV75 (µg/mL)

Value:

173.48

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Results 3

Key result

Run / experiment:

mean

Parameter:

other: CV75 (µg/mL)

Value:

167.63

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Other effects / acceptance of results:

SOLUBILITY ASSESSMENT
- The concentration at which the test material formulation retained vehicle and maximum stock concentration was at 125 mg/mL in DMSO with vortexing, 5 minutes sonication and 10 minutes magnetic stirring.
- Therefore, DMSO was selected as vehicle, and the following test material concentrations were tested in the DRF phase: 1.95, 3.91, 7.81, 15.63, 31.25, 62.50, 125.00, 250.00 μg/mL.

DRF RESULTS
The following results were obtained in the first DRF test (i.e. DRF 1):
- at post-treatment observation, precipitate was noted at concentration ≥ 62.50 μg/mL
- flow cytometry measurement after Propidium Iodide staining revealed a cell viability decrease below 75 % at the highest tested concentration of 250 μg/mL. The corresponding CV75 value was 161.79 μg/mL.
The following results were obtained in the second DRF test (i.e. DRF 2):
- at post-treatment observation, precipitate was noted at concentration ≥ 31.25 μg/mL,
- flow cytometry measurement after Propidium Iodide staining revealed a cell viability decrease below 75 % at the highest tested concentration of 250 μg/mL. The corresponding CV75 value was 173.48 μg/mL.
Based on the results from both DRF runs, the mean CV75 was 167.63 μg/mL, and the highest concentration tested in the main test was therefore 201.16 μg/mL (i.e. 1.2-fold the mean CV75).

MAIN TEST: INDIVIDUAL RUN RESULTS
- All acceptance criteria were reached in each run.
- Run A:
Post-treatment observations: Precipitate was noted in wells treated at all tested concentration.
Run outcome: RFI CD54 exceeded the positivity threshold at the concentration of 56.14 μg/mL and concentration ≥ 97.01 μg/mL. The run was therefore considered positive.
- Run B:
Post-treatment observations: Precipitate was noted in wells treated at all tested concentration.
Run outcome: RFI CD86 and/or RFI CD54 exceeded the positivity threshold at the concentrations of 80.84 μg/mL. The run was therefore considered positive.

Any other information on results incl. tables

Table 1: Summary of Results

Concentration (µg/mL)	RFI for CD86		RFI for CD54		Viability (%)		Run Conclusion		General Conclusion
	A	B	A	B	A	B	A	B
56.14	104	108	235	94	90.7	91.8	P2	P2	Positive
67.37	95	109	166	108	91.5	91.3
80.84	105	112	193	204	87.5	89.3
97.01	80	100	387	130	80.0	88.3
116.41	106	104	375	115	79.8	91.1
139.69	105	101	339	140	77.7	88.3
167.63	101	87	456	108	71.4	88.4
201.16	112	89	246	96	82.6	84.2

RFI = Relative Fluorescence Index

N = run with negative outcome

P1 = run with positive outcome for CD86

P2 = run with positive outcome for CD54

P12 = run with positive outcome for CD86 and CD54

Applicant's summary and conclusion

Interpretation of results:

other: Positive in the h-CLAT

Conclusions:

Under the conditions of this study, the test material was positive in the h-CLAT.

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 cell surface markers expression in THP-1 cells using the h-CLAT test method.

A solubility assessment was first performed in 0.9 % NaCl and then DMSO to select the vehicle and highest concentration to be used for test material preparations.

Following the solubility assays, the cytotoxic potential was assessed in a Dose-Range Finding assay in order 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 two successive runs. In each run, the test material formulations were applied to THP-1 cells and cultured in a 24-well plate for 24 h ± 30 minutes at 37 °C, 5 % CO2 in a humidified incubator. A set of control wells was also added in each plate to guarantee the validity of each run. At the end of the incubation period, cells from each well were distributed to three wells of 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. Then, 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 Fluoresence Intensity (MFI) obtained for each test sample was corrected by the isotype control IgG1 MFI value to obtain the corrected MFI. 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).

The test material was found soluble in DMSO at the concentration of 125 mg/mL. The test material induced a decrease in cell viability < 75 % in both DRF runs and the calculated mean CV75 value was: 167.63 μg/mL. The highest concentration tested in the main test was therefore 201.16 μg/mL (i.e. 1.2-fold the mean CV75). Positive outcomes for CD54 were observed in Runs A and B.

Under the conditions of this study, the test material was positive in the h-CLAT.