Reaction mass of dieuropium tricarbonate and digadolinium tricarbonate and disamarium tricarbonate

Endpoint summary

Description of key information

Keratinosens (Michel, 2018)

Under the experimental conditions of this study, the test material, was positive in the KeratinoSens assay and therefore was considered to have potential to activate the Nrf2 transcription factor.

h-CLAT (Gerbeix, 2018)

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

Magnusson and Kligman (Tarcai, 2019)

Under the conditions of the test, challenge with the test material evoked no positive responses in the test animals previously sensitised with the test material or in the control group. The net response value represented an incidence rate of 0 % and the net score value of 0.00.

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:

28 August 2017 to 22 September 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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.

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.

Positive control results:

All acceptance criteria were reached in each run.

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

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

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.

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

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.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05 September 2017 to 22 September 2017

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Details on the study design:

VEHICLE AND NEGATIVE CONTROL
Based on solubility results, the selected vehicle was DMSO.
This vehicle was used as the negative control, and was applied to cells at a concentration of 1 % in culture medium.
Since several test materials were assayed concurrently, the results of the negative control were shared.

POSITIVE CONTROL
Cinnamic Aldehyde (CA)
Since several test materials were assayed concurrently, the results of the positive control were shared.
For each run, the positive control item was dissolved in DMSO to a final concentration of 200 mM. This solution was then further diluted to a final concentration of 6.4 mM. It was diluted in DMSO by serial dilutions in the Master plate 100x, using a dilution factor of 2, to obtain a total of five 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 FORMULATION
On the basis of solubility results, the test material was suspended in DMSO at 100 mM.
After preparation and in order to improve the solubility of the test material, the test material formulations were sonicated for 5 minutes. A homogeneous white suspension was obtained.
One formulation was prepared for each run. It was then 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 luciferase gene is the endpoint evaluated and reflects the activation by the test material of the Nrf2 transcription factor in this test.
• Supplier: this cell line was provided by Givaudan.
• Storage condition: at -80 °C.
• Mycoplasm: absence of mycoplasm was confirmed.

SPECIFIC EQUIPMENT
- 96-well plate Luminometer with injectors and optical density reader (Varioskan Flash).

MEDIA
- Maintenance medium No. 1: DMEM containing GlutaMAX™, 1000 mg/L D-Glucose, Sodium Pyruvate and supplemented with 9.1 % Foetal 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.

STUDY DESIGN
The test material was tested in two independent runs using cells from a different passage number. The plates were processed as described below.

SOLUBILITY ASSAY
A solubility assay was performed prior to the first treatment in order to select the vehicle (among DMSO, water for injections or treatment culture medium). Sonication for 5 minutes was used in order to improve the solubility of the test material.

During this solubility assay, the test material was first suspended in DMSO at 100 mM. This stock formulation was then diluted in treatment culture medium to the final concentration of 1000 µM. A visual inspection of the sample was performed to evaluate the presence or absence of precipitate/emulsion.

KERATINOSENS ASSAY
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 (three white plates and one 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 the 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 three white assay plates and 50 µL to the transparent plate for the 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 the 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 at room temperature and under orbital shaking, the plates containing the passive lysis buffer were then placed in the luminometer for reading using the following program:
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 the cytotoxicity - transparent plate
For the cell viability assay plate, the medium was replaced by 200 µL of treatment medium, a volume of 27 µL of a MTT solution at 5 mg/mL in D-PBS was then added to each well of the transparent 96-well plate. The plates were covered with a sealing membrane and returned at 37 °C in the incubator in 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 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.

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 was 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 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 wrong extrapolations.

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:
- The positive control results should be positive, thus the gene induction should be statistically significant 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 < 1000 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).

Key result

Run / experiment:

other: 1 and 2

Parameter:

other: IMAX

Value:

11.93

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Other effects / acceptance of results:

SOLUBILITY TEST
In the solubility test, the test material was found not soluble in DMSO, water for injections and culture medium at 200 mM even after a sonication step of 5 minutes.
Then a homogeneous suspension was obtained when the test material was suspended at 100 mM in DMSO followed by a sonication step of 5 minutes. Therefore, this vehicle (DMSO) was selected for the preparation of the test material formulations.
Moderate precipitate was observed once the test material stock formulation was diluted in the treatment culture medium to a final concentration of 1000 µM.

KERATINOSENS RUN
All acceptance criteria were met for the positive and negative controls in each run, both runs were therefore considered to be valid.
Both runs were performed using the following concentrations 0.49, 0.98, 1.95, 3.91, 7.81, 15.63, 31.25, 62.5, 125, 250, 500 and 1000 µM culture medium containing 1 % DMSO.
At these tested concentrations:
- Slight to strong precipitate was observed in test item-treated wells at concentrations ≥ 3.91 µM in the first run and ≥ 31.3 µM in the second run,
- No noteworthy decrease in cell viability was noted in either run (i.e. cell viability > 70 %).
- Statistically significant gene-fold inductions above the threshold of 1.5 compared to the negative control were noted at concentrations of 0.98, 7.81 and then ≥ 31.3 µM in the first run and ≥ 31.3 µM in the second run. An apparent dose-response relationship was noted up to 250 µM during both runs, followed by a decrease in gene-fold inductions values, related to the strong test material precipitate noted at the highest concentrations, which may have prevented the exposition of the cells to the test material,
- Moreover, the Imax values were > 1.5 in both runs (i.e. 11.93 and 13.37 in the first and second runs, respectively) and the calculated EC1.5 was 0.96 µM in the first run and 24.57 µM in the second run.
No geometric mean IC30 or IC50 was calculated since the cell viability was > 70 % in both runs.
The evaluation criteria for a positive response are met in both runs. The final outcome is therefore positive. This positive 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.

Table 1: Imax and EC1.5 Results

Test Material	Imax	EC1.5 (µm)	IC50 (µm)	IC30 (µm)
Run 1	11.93	0.96	-	-
Run 2	13.37	24.57	-	-
Mean	12.65	n.r.	n.r.	n.r.
Geometric Mean	n.r.	4.86	-	-
SD	1.02	16.70	-	-

- = no data available

n.r. = not requested by the OECD guideline

Interpretation of results:

other: Positive according to EU criteria

Conclusions:

Under the experimental conditions of this study, the test material was positive in the KeratinoSens assay and therefore was considered to have potential to activate the Nrf2 transcription factor.

Executive summary:

The objective of this study was to evaluate the potential of the test material, to activate the Nrf2 transcription factor. This test is a part of a tiered strategy for the evaluation of skin sensitisation potential. Thus, data generated with the present Test Guideline should be used to support the discrimination between skin sensitisers and non-sensitisers in the context of an integrated approach to testing and assessment. The test was performed in accordance with the standardised guideline OECD 422D, under GLP conditions.

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 were exposed to the vehicle control or to different concentrations of test material and of 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 was measured by flash luminescence. In parallel, the cytotoxicity was measured by a MTT reduction test and was taken into consideration in the interpretation of the sensitisation results. Two independent runs were performed as part of this study.

All acceptance criteria were met for the positive and negative controls in each run, both runs were therefore considered to be valid.Both runs were performed using the following concentrations 0.49, 0.98, 1.95, 3.91, 7.81, 15.63, 31.25, 62.5, 125, 250, 500 and 1 000 µM culture medium containing 1 % DMSO.

At these tested concentrations; slight to strong precipitate was observed in test material-treated wells at concentrations ≥ 3.91 µM in the first run and ≥ 31.3 µM in the second run; no noteworthy decrease in cell viability was noted in either run (i.e. cell viability > 70 %); statistically gene-fold inductions above the threshold of 1.5 compared to the negative control were noted at concentrations of 0.98, 7.81 and then ≥ 31.3 µM in the first run and ≥ 31.3 µM in the second run. An apparent dose‑response relationship was noted up to 250µM during both runs, followed by a decrease in gene-fold inductions values, related to the strong test material precipitate noted at the highest concentrations, which may have prevented the exposition of the cells to the test material;  moreover, the Imax values were > 1.5 in both runs (i.e. 11.93 and 13.37 in the first and second runs, respectively) and the calculated EC1.5 was 0.96 µM in the first run and 24.57 µM in the second run.

No geometric mean IC30 or IC50 was calculated since the cell viability was > 70 % in both runs.

The evaluation criteria for a positive response are met in both runs, the final outcome is therefore positive. This positive 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.

Under the experimental conditions of this study, the test material, was positive in the KeratinoSens assay and therefore was considered to have potential to activate the Nrf2 transcription factor.

Reference 3

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 March 2019 to 12 May 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Version / remarks:

2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

2003

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

Previous work undertaken by the Rare Earth Consortium to support a number of REACH registrations has shown that both the in vitro tests and in vivo LLNA studies have given false positive results for skin sensitisation for other rare earth substances, when compared with results from Magnusson and Kligman (M&K) studies. Given the contradictory result for this class of chemicals between the typical REACH studies and the M&K study, and bearing in mind the positive in vitro results on this substance and the known unreliability of the LLNA for certain metals, it is considered necessary to perform this study in order to obtain a definitive understanding the substances sensitisation potential.

Specific details on test material used for the study:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
Vehicle Selection (Preliminary Compatibility Test) and formulation:
- The vehicle used in the study was selected from available information provided by the Sponsor, from the results of solubility assays performed by the test facility and on tests to check the easy passage of dose formulation through a needle (for intradermal injections), prior to the preliminary test.
- The solubility of the test material was examined in a Preliminary Compatibility Test. The following vehicles were assessed: Physiological saline solution (saline) and 1 % Methylcellulose (1 % MC). A 1:1 ratio stable emulsion of Freund's complete adjuvant mixed with the chosen solvent and easy passage through a needle was also tested for intradermal treatment.
- Formulation with saline: The highest achievable concentration in saline was 50 % (w/v), which appeared to be a suspension. The 10 % (w/v) concentration was a very fluid suspension. The easy passage of a 10 % (w/v) concentration in saline through a 26G needle was considered possible based on the formulation. The formulation of 5 % (w/v) test material in FCA:saline (1:1) mixture blocked the passage thorough the needle. The maximum appropriate concentration in FCA:saline was 2.5 % (w/v) with continuous mixing.
- Formulation with 1 % methylcellulose: The highest achievable concentration in 1 % MC was 100 % (w/v), which appeared to be a creamy material. The easy passage of a 10 % (w/v) concentration in 1 % MC through a 26G needle was possible. The passage of 5 % (w/v) test material in FCA:1 % MC (1:1) mixture was also possible. Taking into account the test material characteristics, the preliminary test results and the study requirements, 1 % MC was selected as the vehicle for the main test.

Formulation:
- The formulations were freshly formulated at appropriate concentrations in the vehicle on the day of administration. The stability and homogeneity were assessed visually; chemical analysis was not performed.
- The formulations were used within approximately 4 hours after mixing the test material with the vehicle and the test material preparation was performed with approved procedures and documented in detail. The concentrations were applied as suspensions.
- No correction for purity of the test material was applied.
- The dose levels and the vehicle selection for the main test were based on the results of a Preliminary Dose Range Finding Study. The test material was weighed and formulations were prepared daily on a weight:volume basis (as % (w/v)) with usage of mortar and pestle.

Vehicle:
- The selection of the vehicle for the main test was based on trial formulations and preliminary test with the test material. It is preferred to use aqueous vehicles whenever possible, therefore physiological saline solution (saline) should be the first choice whenever possible (as distilled water is not compatible with intradermal treatments). During the tests, saline was not suitable as a vehicle for the study due to the settlement of the test material, however 1 % MC was appropriate as a vehicle.
- Vehicle: 1 % methylcellulose

Species:

guinea pig

Strain:

other: LAL/HA/BR

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Females nulliparous and non-pregnant: Not specified
- Age at study initiation: Young adult, ~ 8 weeks old (main test)
- Weight at study initiation: 400 – 468 g
- Housing: Animals were housed in Macrolon cages size IV, with up to 3 animals/cage to allow socialisation
- Diet: Ad libitum
- Water: Ad libitum
- Acclimation period: 29 days before main test and at least 35 days before preliminary test under laboratory conditions
- Indication of any skin lesions: Health inspection was performed after arrival of the animals. Only healthy animals were used for the test. The health status was certified by the staff Veterinarian.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.1 – 22.9 °C
- Humidity (%): 22 – 59 %
- Air changes (per hr): 15 - 20 air exchange/hour.
- Photoperiod (hrs dark / hrs light): Light 12 hours daily from 6 a.m. to 6 p.m.

Route:

intradermal

Vehicle:

other: 1 % methylcellulose

Concentration / amount:

1 % (w/v)

Day(s)/duration:

24 ± 2 h

Route:

epicutaneous, occlusive

Vehicle:

other: 1 % methyl cellulose

Concentration / amount:

100 % (w/v)

Day(s)/duration:

72 ± 2 h

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

other: 1 % methyl cellulose

Concentration / amount:

Left side: 25 % (w/v)
Right side: 12.5 % (w/v)

Day(s)/duration:

24 ± 2 h

No. of animals per dose:

10

Details on study design:

RANGE FINDING TESTS:
- The dose levels for the main test are selected based on the results of the Preliminary Test.
- The concentration of test material to be used for each induction exposure should be well tolerated systemically and should be the highest to cause no more than mild-to-moderate skin irritation. The concentration used for the challenge exposure should be the highest non-irritant dose.
- A day prior to the test, the hair was removed from the right and/or left sides of the animals (approximately 5 x 5 cm). The hair removal was performed carefully to ensure animals are closely shaven.
- A series of test material concentrations were tested to identify the primary irritation following intradermal injection and topical application: 5, 2.5, 1, 0.5 % (w/v) in 1 % MC and 2.5, 1 % (w/v) in saline were used for intradermal injection; 100, 75, 50 and 25 % (w/v) concentrations in 1 % MC for topical application. Local effects were examined and scored approximately 1, 24, 48 and 72 hours after the treatment (in case of intradermal treatment) or after patch removal (in case of topical application). Skin effects were scored for erythema and oedema, any other observations of changes to the skin was recorded if present.

- Intradermal treatment: For the intradermal application, 0.1 mL per concentration was injected intradermally into the hair free skin of the animals. One or two formulations were injected on the right and left side of the animals. The concentrations for intradermal treatment were also tested in a 1:1 mixture (v/v) of Freund's Complete Adjuvant (FCA) and saline. Each concentration was injected in duplicate. Two animals were used per concentration. Since some concentrations proved to be not suitable for treatment as a single injection as required, but could be administered in several parts, the results of these scorings are not described below.
The injection of 5 % (w/v) concentration in 1 % MC or FCA:1 % MC (1:1) was not possible as a whole or not possible at all. This concentration was not suitable for treatment.
The injection of 2.5 % (w/v) concentration in 1 % MC was feasible however the injection of 2.5 % (w/v) in FCA:1 % MC (1:1) was only possible in multiple administrations, therefore this concentration was not suitable for treatment.
The injection of 1 and 0.5 % (w/v) concentration in 1 % MC and FCA:1 % MC (1:1) were feasible. The formulation without FCA caused erythema score 1 (except 0.5 % at 1 hour, score 0), and score 1 or 2 with FCA.
The 2.5 and 1 % (w/v) concentrations were also tested in saline or FCA:saline (1:1) and found to be inappropriate for treatment. The injection of 0.1 mL in one injection cannot be guaranteed. Furthermore, it was recognised that the test material tends to settle in the syringe after a relatively short period of time , therefore formulation with saline was excluded from intradermal treatment.
Based on the preliminary test results and considering the requirement of the intradermal induction phase, concentration of 1 % (w/v) in 1 % MC and FCA:1 % MC (1:1) were selected for the main test.
- Topical treatment: The volume for the dermal application was 0.5 mL. A closed patch exposure was performed by means of an occlusive bandage using similar treatment procedures as for the main test. The time of exposure for the dermal application was approximately 48 hours. In the preliminary tests one concentration was used on the right side and another concentration on the left side of animals if appropriate. Two animals per concentration were used in the preliminary test.
At a concentration of 100 % (w/v) in 1 % MC, slight erythema (score 1) was noted during the whole observation period. This concentration was chosen for topical induction exposure.
At concentration of 75 % (w/v) in 1 % MC, slight erythema (score 1) was observed at 1 hour in one animal and at 1, 24 hours in the other animal. During the rest of the observation period the animals were symptom-free.
At concentration of 50 % (w/v) in 1 % MC, slight erythema (score 1) was observed at 1, 24 hours in one animal and other animal was symptom-free. During the rest of the observation period the animals were symptom-free. In order to choose the appropriate concentration for challenge phase this concentration level was tested one more time. One of the animals was symptom-free during the second test, in the other animal slight erythema was observed at 1 hour. Although the 1-hour observation is not part of the sensitisation evaluation, the evaluated irritation response at 50 % across the preliminary testing indicates that it cannot be used as a challenge concentration as it does have irritation potential.
At a concentration of 25 % (w/v) in 1 % MC, the animals were symptom-free, therefore this concentration was selected for challenge exposure.
- On the basis of results of the Preliminary Dose Range Finding Tests, the following treatments were selected for the main test:
• Intradermal induction: 1 % (w/v) test material formulated in 1 % MC and FCA:1 % MC (1:1) in the test group; and 1 % MC and FCA:1 % MC (1:1) in the control group for intradermal injections.
• Topical induction: 100 % (w/v) test material in 1 % MC in test group and 1 % MC only in control group for dermal treatment. Since erythema was observed in the preliminary test, application of 0.5 mL of 10 % sodium dodecyl sulphate (SDS) was not selected prior to topical induction.
• Challenge phase: All animals of the treatment and control groups: 25 % (w/v) test material in 1 % MC on the left side as a challenge exposure (as this concentration was the highest which caused no irritation) and 12.5 % (w/v) in 1 % MC on the right side (50 % of the maximum challenge concentration (safeguard dose), in order to facilitate the decision between a true sensitiser or an irritation response).


MAIN STUDY
Induction involved two main procedures: Intradermal induction (Main test I) and topical induction exposure (Main test II) with closed patch technique. The results of the intradermal and the topical induction treatments were observed and recorded.
INDUCTION EXPOSURE 1: INTERADERMAL INDUCTION
On the day before treatment, an area approximately 5 x 5 cm on the scapular region of the animals was clipped free of hair and was carefully shaved. Three pairs of intradermal injections (0.1 mL/site) were made in the clipped region as follows (the first listed nearest the head):
Test group:
• 2 injections of Freund's Complete Adjuvant and 1 % MC in a 1:1 (v/v) mixture,
• 2 injections of 1 % (w/v) test material in 1% MC,
• 2 injections of 1 % (w/v) test material, formulated in a 1:1 mixture (v/v) of Freund's Complete Adjuvant and 1% MC.
Control group:
• 2 injections of Freund's Complete Adjuvant and 1 % MC in a 1:1 (v/v) mixture,
• 2 injections of 1 % MC,
• 2 injections of 1 % MC, formulated in a 1:1 mixture (v/v) of Freund's Complete Adjuvant and 1% MC.
- No. of exposures: 1
- Control group: 1 % MC. Control animals were treated similarly to test animals, except that during the induction phase, the test material was omitted.
- Site: Scapular region

INDUCTION EXPOSURE 2: TOPICAL INDUCTION
- The same scapular region which received the intradermal injections, was used for dermal induction exposure.
- The scapular area (approximately 5 x 5 cm) was clipped free of hair one day prior to the dermal treatment (Day 6).
- Seven days after intradermal induction a 2.5 x 2.5 cm sterile gauze patch (approximately 4 layers of porous gauze pads) was saturated with approximately 0.5 mL of the test material (at 100 % (w/v) concentration, the highest dose found to cause no more than mild-to-moderate skin irritation in the preliminary dose range finding test) and placed over the injection sites (scapular region). The control group was treated with 0.5 mL vehicle (1 % MC) with the same method.
- The gauze patches were kept in contact with the skin by a patch with a surrounding adhesive hypoallergenic plaster. The treated areas were covered for approximately 48 hours with a fully occlusive foil (Closed Patch Test). After the patch removal, any remaining test material was removed with a wet gauze swab.
- Following the dermal induction treatment, the animals were left untreated for 14 days prior to challenge applications.
- No. of exposures: 1
- Control group: 1 % MC. Control animals were treated similarly to test animals, except that during the induction phase, the test material was omitted.
- Site: Scapular region
- Exposure period: 48 h

B. CHALLENGE EXPOSURE
- Two weeks after the topical induction application, the animals were exposed to a dermal challenge dose. Approximately 24 hours before the treatment, the hair was removed from an area of approximately 5 x 5 cm on the left and right sides of each animal.
- For the challenge, a 2.5 x 2.5 cm patch of sterile gauze was saturated with approximately 0.5 mL of the test material at 25 % (w/v) concentration (the highest dose found to cause no skin irritation in the preliminary dose range finding test) and applied to the left side of all animals (both the test and the control).
- The right shaved side area of all animals was treated with the safeguard dose (12.5 % (w/v) concentration of the test material).
- Treatment was performed as described above (Closed Patch Test). The time of the exposure was approximately 24 hours. After the patch removal, any remaining test material was removed with a wet gauze swab.
- No. of exposures: 1
- Day(s) of challenge: 21
- Exposure period: 24 h
- Site: Left and right sides
- Concentrations:
Left side: 25 % (w/v) in 1 % MC
Right side: 12.5 % (w/v) in 1 % MC
- Evaluation (hr after challenge): 24 ± 2 h and 48 ± 2 h after the patch removal.


OBSERVATION AND SCORING
- Detailed clinical observations were made on all animals outside the home cage in a standard arena at randomisation (Day -1), on the day of treatment (Day 0, prior to treatment) and at least weekly thereafter and also on the last day of experiment.
- The intradermal and dermal irritation scores (in cases of induction exposures) were evaluated according to the scoring system by Draize (1959)
Erythema and eschar formation:
0: No erythema
1: Very slight erythema (barely perceptible)
2: Well defined erythema
3: Moderate to severe erythema
4: Severe erythema (beef redness) to slight eschar formation (injuries in depth)

Oedema formation:
0: No oedema
1: Very slight oedema (barely perceptible)
2: Slight oedema (edges of area well defined by definite raising)
3: Moderate oedema (raised approx. 1 mm)
4: Severe oedema (raised more than 1 mm and extending beyond area of exposure)

After the challenge exposure, each animal was examined and scored approximately at 24 and 48 hours after the end of the exposure period.
Grading was performed according to the following system:
0 = no visible change
1 = discrete or patchy erythema
2 = moderate and confluent erythema
3 = intense erythema and swelling


CLASSIFICATION OF SENSITISER
- The results obtained for test animals at the challenge application were compared with those simultaneously obtained in control animals.
- A test animal was considered to show evidence of contact hypersensitivity if the observed dermal reaction at challenge is more marked and/or persistent than dermal reaction seen in animals of the control group. If dermal scores of ≥ 1 were noted in control animals and the dermal reactions of the test group animals were not clearly different from the reaction in the animals of the control group, the test results were classified as "inconclusive".
- The percentage of animals showing positive reaction was calculated in both (test and control) groups. As a result of these, the percentage value of the control animals that responded were subtracted from the percentage of the test animals responding positively to the challenge. The net response value is the percent sensitisation. 
- The evaluation of skin sensitisation was performed in accordance with Commission Regulation (EC) No 1272/2008 of 16 December 2008 and Globally Harmonized System of Classification and Labelling of Chemicals (GHS) version 7, 2017.
- Category 1:
Substances shall be classified as skin sensitisers (Category 1) where data are not sufficient for sub-categorisation. Allergic response of at least 30 % of the animals is considered as positive.
 - Subcategory 1 A:
≥ 30 % responding at ≤ 0.1 % intradermal induction dose, or
≥ 60 % responding at > 0.1 % to ≤ intradermal induction dose.
- Subcategory 1 B:
≥ 30 % to < 60 % responding at > 0.1% to ≤ 1% intradermal induction dose, or
≥ 30 % respo nding at > 1% intradermal induction dose.
- If at least 30 % of the animals show allergic response, after the Magnusson and Kligman procedure, the test material has to be classified as a "sensitiser".
- In case the results are clearly positive (at least 30 % considered as a positive response) then the test material has to be considered to be positive and no further testing is required.
- In the case where no animals (0/10) or only one animal with a questionable reaction (no more than score 1) were observed, the test material has to be considered negative and no further testing is required. This is a standard practice, taking into account animal welfare grounds. It is implausible that adding a second group of 10 animals would result in a positive rate of 60 %.


MEASUREMENT OF BODY WEIGHT
- Body weight was recorded with a precision of 1 g at randomisation (Day -1), on the day of treatment (Day 0, prior to treatment), then at least weekly, including Day 24 prior to euthanasia. The mean values and the standard deviations were calculated and reported.


OBSERVATIONS
- Mortality/Clinical signs: Daily during the test. As part of this observation, detailed clinical observation was made at randomisation, at start of the experiment and then weekly during the test including the last day.
Skin reactions were observed and recorded as follows:
• Preliminary test: 1 (± 5 min), 24 (± 2), 48 (± 2) and 72 (± 2) hours after treatment and/or patch removal.
• Intradermal induction exposure: 24 (± 2) hours after treatment.
• Dermal induction exposure: 1 (± 5 min), 24 (± 2), 48 (± 2) and 72 (± 2) hours after the patch removal.
• Challenge exposure: 24 (± 2) and 48 (± 2) hours after the patch removal.

Challenge controls:

Control animals were treated similarly to test animals, except that during the induction phase, the test material was omitted.

Positive control substance(s):

yes

Remarks:

The sensitivity and reliability of the experimental procedure is assessed twice a year by use of reference materials which are known to have mild-to-moderate skin sensitisation properties such as 2-mercaptobenzothiazole.

Positive control results:

The selection of dose levels was made on the basis of the previous reliability study.

Intradermal induction exposure: 1 % (w/v)
Dermal induction exposure: 75 % (w/v)
Challenge treatment: 50 % (w/v)

Challenge with 2-mercaptobenzothiazole elicited discrete and moderate erythema (score 1 and 2) on the skin surface of previously sensitised guinea pigs. The mean of the scores were 1.00 (90 % of animals) at the 24-hour observation and 0.80 (80 % of animals) at the 48-hour observation. In the control group the mean of the scores was 0.00.

On the basis of the results of the present study, 2-mercaptobenzothiazole was classified as a skin sensitiser. This demonstrates that the Magnusson and Kligman method in this laboratory is considered to be reliable.

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

25 % (w/v) formulated in 1 % MC and 12.5% (w/v) formulated in 1% MC

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

No signs of systemic or local toxicity were observed.

Remarks on result:

no indication of skin sensitisation

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

25% (w/v) formulated in 1% MC and 12.5 % (w/v) formulated in 1 % MC

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

No signs of systemic or local toxicity were observed.

Remarks on result:

no indication of skin sensitisation

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

25% (w/v) formulated in 1% MC and 12.5% (w/v) formulated in 1% MC

No. with + reactions:

0

Total no. in group:

5

Remarks on result:

no indication of skin sensitisation

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

25% (w/v) formulated in 1% MC and 12.5% (w/v) formulated in 1% MC

No. with + reactions:

0

Total no. in group:

5

Remarks on result:

no indication of skin sensitisation

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Dose level:

50% (w/v) 2-mercaptobenzothiazole

No. with + reactions:

9

Total no. in group:

10

Clinical observations:

Challenge with the test item 2-mercaptobenzothiazole elicited discrete and moderate erythema (score 1 and 2) on the skin surface of previously sensitised guinea pigs

Remarks on result:

positive indication of skin sensitisation

Reading:

2nd reading

Hours after challenge:

48

Group:

positive control

Dose level:

50% (w/v) 2-Mercaptobenzothiazole

No. with + reactions:

8

Total no. in group:

10

Clinical observations:

Challenge with the test item 2-mercaptobenzothiazole elicited discrete and moderate erythema (score 1 and 2) on the skin surface of previously sensitised guinea pigs

Remarks on result:

positive indication of skin sensitisation

Main Test Results

- Ten test animals were subjected to sensitisation procedures in a two-stage process, i.e. an intradermal injection and a topical application. The test material was used at a concentration of 1 % (w/v) for intradermal injections and at a concentration of 100 % (w/v) for topical (dermal) sensitisation treatment. Two weeks after the last induction exposure, a challenge dose at a concentration of 25 % (w/v) formulated in 1 % MC was administered on the left side of animals. The right side of animals was treated with safeguard dose 12.5 % (w/v). Challenge was performed by dermal application of the test material.

- Five control guinea pigs were simultaneously exposed to 1 % MC only during the sensitisation phase I (intradermal treatment) and during the sensitisation phase II (dermal treatment). Control animals were treated with the test material at a concentration of 25 % (w/v) formulated in 1 % MC on the left side during challenge. The right side was treated with a safeguard dose 12.5 % (w/v).

 

Skin Effects During Induction Phase

- At the 24-hour observation after intradermal induction very slight or well defined erythema were observed in the test group and no reaction was observed in the control group. No oedema was noted in the control or test group.

- At the 1 and 24-hour observations after patch removal of topical induction treatment, very slight erythema was observed in some of the animals. No erythema was observed at 48 and 72-hours observations. In the control group no reaction was observed. No oedema was noted in the control or test group.

 

Skin Effects After the Challenge Exposure

- Test group: After the challenge with the test material at a concentration of 25 % (w/v) formulated in 1 % MC, no positive response was observed in the treated animals on the left flank. The mean of the scores was 0.00 according to the 24 and 48-hour results. The right shaved side of test animals was treated with safeguard dose (12.5 % (w/v)) and no reaction was noted.

- Control group: After the challenge with the test material at a concentration of 25 % (w/v) formulated in 1 % MC, no visible changes were found at the 24 and 48-hour examinations on the left flank. The right shaved side of control animals were treated with safeguard dose (12.5 % (w/v)) and no reaction was noted.

 

Clinical Observations / Mortality

- No signs of systemic or local toxicity were observed.

- No mortality was observed during the study.

 

Body Weight

- There were no notable differences between the test animal group and the control group.

Interpretation of results:

other: Not classified according to EU criteria.

Conclusions:

Under the conditions of the test, challenge with test material evoked no positive responses in the test animals previously sensitised with the test material or in the control group. The net response value represented an incidence rate of 0 % and the net score value of 0.00.

Executive summary:

A skin sensitisation study was performed in the guinea pig according to the Magnusson and Kligman method, using a maximisation method with Freund's Complete Adjuvant to evaluate the sensitisation potential of the test material. The study was performed according to OECD Guideline No. 406 (adopted in 1992) and in compliance with GLP guidelines.

Prior to the study the Sponsor confirmed that the chemical nature of the test material is not compatible with the available in vitro or in vivo alternative tests.

Previous work undertaken by the Rare Earth Consortium to support a number of REACH registrations has shown that both the in vitro tests and in vivo LLNA studies have given false positive results for skin sensitisation for other rare earth substances, when compared with results from Magnusson and Kligman (M&K) studies. Given the contradictory result for this class of chemicals between the typical REACH studies and the M&K study, and bearing in mind the positive in vitro results on this substance and the known unreliability of the LLNA for certain metals, it is considered necessary to perform this study in order to obtain a definitive understanding the substances sensitisation potential.

Based on the results of the preliminary tests, ten test animals were subjected to sensitisation procedures in a two-stage process, named induction phase: i.e. an intradermal treatment and a 48-hour topical application (dermal treatment under an occlusive dressing). The test material was used at a concentration of 1 % (w/v) suspended in 1 % methyl cellulose (abbreviation: 1 % MC) for intradermal injections and at a concentration of 100 % (w/v) for topical sensitisation treatment. Five control guinea pigs were simultaneously exposed to 1 % MC during the sensitisation phase.

Two weeks after the last induction exposure, a challenge dose at a concentration of 25 % (w/v) in 1 % MC was administered on the left side of all animals. The right side of the animals was treated with safeguard dose (12.5 % (w/v)). Challenge was performed by dermal application of the test material for 24 hours with a fully occlusive foil (Closed Patch Test). Skin reactions were measured 24 and 48 hours after patch removal.

Induction: At the 24-hour observation after intradermal induction very slight or well defined erythema were observed in the test group and no reaction was observed in the control group. No oedema was noted in the control or test group.

At the 1 and 24-hour observations after patch removal of topical induction treatment, very slight erythema was observed in some of the animals. No erythema was observed at 48 and 72-hours observations. In the control group no reaction was observed. No oedema was noted in the control or test group.

Challenge: No test material related signs of erythema, local reactions or systemic toxicity were observed in any animals after the challenge.

Incidence rate: No signs of contact sensitisation were detected after the challenge exposure in guinea pigs previously exposed to the test material during the experiment.

Intensity of sensitisation response: In the control and treatment groups, the mean of the score was 0.00 according to the 24 and 48-hour results after the challenge exposure.

In conclusion, under the conditions of the present assay the test material was shown to have no sensitisation potential and is classified as a non-sensitiser, according to current GHS criteria and EU-regulations.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

Magnusson and Kligman (Tarcai, 2019)

A skin sensitisation study was performed in the guinea pig according to the Magnusson and Kligman method, using a maximisation method with Freund's Complete Adjuvant to evaluate the sensitisation potential of test material. The study was performed according to OECD Guideline No. 406 (adopted in 1992) and in compliance with GLP guidelines. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Prior to the study the Sponsor confirmed that the chemical nature of the test material is not compatible with the available in vitro or in vivo alternative tests.

Previous work undertaken by the Rare Earth Consortium to support a number of REACH registrations has shown that both the in vitro tests and in vivo LLNA studies have given false positive results for skin sensitisation for other rare earth substances, when compared with results from Magnusson and Kligman (M&K) studies. Given the contradictory result for this class of chemicals between the typical REACH studies and the M&K study, and bearing in mind the positive in vitro results on this substance and the known unreliability of the LLNA for certain metals, it is considered necessary to perform this study in order to obtain a definitive understanding the substances sensitisation potential.

Based on the results of the preliminary tests, ten test animals were subjected to sensitisation procedures in a two-stage process, named induction phase: i.e. an intradermal treatment and a 48-hour topical application (dermal treatment under an occlusive dressing). The test material was used at a concentration of 1 % (w/v) suspended in 1 % Methyl cellulose (abbreviation: 1 % MC) for intradermal injections and at a concentration of 100 % (w/v) for topical sensitisation treatment. Five control guinea pigs were simultaneously exposed to 1 % MC during the sensitisation phase.

Two weeks after the last induction exposure, a challenge dose at a concentration of 25% (w/v) in 1 % MC wasadministeredon the left side of all animals.The right side of the animals was treated with safeguard dose (12.5 % (w/v)).Challenge was performed by dermal application of the test materialfor 24 hours with a fully occlusive foil (Closed Patch Test). Skin reactions were measured 24 and 48 hours after patch removal.

Induction:At the 24-hour observation after intradermal induction very slight or well defined erythema were observed in the test group and no reaction was observed in the control group. No oedema was noted in the control or test group.

At the 1 and 24-hour observations after patch removal of topical induction treatment, very slight erythema was observed in some of the animals. No erythema was observed at 48 and 72-hours observations. In the control group no reaction was observed. No oedema was noted in the control or test group.

Challenge:No test material related signs of erythema, local reactions or systemic toxicity were observed in any animals after the challenge.

Incidence rate: No signs of contact sensitisation were detected after the challenge exposure in guinea pigs previously exposed to the test material during the experiment.

Intensity of sensitisation response: In the control and treatment groups, the mean of the score was 0.00 according to the 24 and 48-hour results after the challenge exposure.

In conclusion, under the conditions of the present assay the test material was shown to have no sensitisation potential and is classified as a non-sensitiser, according to current GHS criteria and EU-regulations.

Keratinosens (Michel, 2018)

The objective of this study was to evaluatethe potential of the testmaterial, toactivate the Nrf2 transcription factor. This test is a part of a tiered strategy for the evaluation of skin sensitisation potential. Thus, data generated with the present Test Guideline should be used to support the discrimination between skin sensitizers and non-sensitizers in the context of an integrated approach to testing and assessment.

The skin sensitisation potential of the test material was investigated in accordance with the standardised guideline OECD 442d, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

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 were exposed to the vehicle control or to different concentrations of test item and of 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 was measured by flash luminescence.In parallel, the cytotoxicity was measured by a MTT reduction test and was taken into consideration in the interpretation of the sensitisation results. Two independent runs were performed as part of this study.

All acceptance criteria were met for the positive and negative controls in each run, both runs were therefore considered to be valid.

Both runs were performed using the following concentrations 0.49, 0.98, 1.95, 3.91, 7.81, 15.63, 31.25, 62.5, 125, 250, 500 and 1 000 µM culture medium containing 1 % DMSO.

At these tested concentrations; slight to strong precipitate was observed in test item-treated wells atconcentrations ≥ 3.91 µM in the first run and ≥ 31.3 µM in the second run; no noteworthy decrease in cell viability was noted in either run (i.e. cell viability > 70 %) statistically gene-fold inductions above the threshold of 1.5comparedto the negative controlwere noted at concentrations of 0.98, 7.81 and then ≥ 31.3 µM in the first run and ≥ 31.3 µM in the second run. An apparent dose-response relationship was noted up to 250 µM during both runs, followed by a decrease in gene-fold inductions values, related to the strong test item precipitate noted at the highest concentrations,which may have prevented the exposition of the cells to the test item; moreover, the Imax values were > 1.5 in both runs (i.e. 11.93 and 13.37 in the first and second runs, respectively) and the calculated EC1.5 was 0.96 µM in the first run and 24.57 µM in the second run.

No geometric mean IC30 or IC50 was calculated since the cell viability was > 70 % in both runs.

The evaluation criteria for a positive response are met in both runs, the final outcome is therefore positive. This positive result can be used to support the discrimination between skin sensitizers and non-sensitizers 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.

Under the experimental conditions of this study, the test material, was positive in the KeratinoSens assay and therefore was considered to have potential to activate the Nrf2 transcription factor.

h-CLAT (Gerbeix, 2018)

The skin sensitisation potential of the test material was investigated in accordance with the standardised guideline OECD 442E, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

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 formulation 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.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance is not classified as a skin sensitiser.