Aluminate(2),[[N,N'-1,2-ethanediylbis[N-(carboxymethyl)glycinato]](4-)-N,N',O,O',ON]hydroxy-,disodium

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2017-2018

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 442E; In vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT), July 2016

Deviations:

yes

Remarks:

Yes. The cytotoxicity measurement and estimation of the CV75 value of the dose finding assay is performed by XTT test instead of flow cytometry.

GLP compliance:

yes

Type of study:

activation of dendritic cells

Test material

Test material information

Constituent 1

Test material form:

solid: flakes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: #070601
- Expiration date of the lot/batch: 06/06/2019
- Purity test date: 92.7%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature, moisture protected
- Solubility and stability of the test substance in the solvent/vehicle: soluble and stable in water or saline

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: dissolution in saline
- Final dilution of a dissolved solid,: 39.1, 78.1, 156.3, 312.5, 625, 1250, 2500 and 5000 μg/mL for the cytotoxicity test (XTT test) and 848, 1018, 1222, 1466, 1759, 2111, 2533 and 3040 for the main experiment

FORM AS APPLIED IN THE TEST: liquid (powder dissolved in saline)

In vitro test system

Details on the study design:

TEST SYSTEM: THP-1 cells,
SOURCE SPECIES: human
CELL TYPE: Leukemic monocyte
SOURCE: ATCC, #TIB-202
JUSTIFICATION OF THE TEST SYSTEM USED:
THP-1 cells are used as surrogate for human myeloid dendritic cells and show enhanced CD86 and/or CD54 expression when treated with sensitisers.

VEHICLE:
Saline solution (0.9% NaCl).
JUSTIFICATION OF THE CHOICE OF THE VEHICLE: test item is soluble in saline solution and insoluble in DMSO.
CULTURE MEDIUM: RPMI-1640 supplemented with 10 % FBS (v/v), 0.05 mM 2-mercaptoethanol, 4.5 g/L glucose, 1% (v/v) sodium pyruvate, 1% (v/v) L-glutamine and appropriate antibiotics (100 U/mL of penicillin and 100 μg/mL of streptomycin)

CONTROLS
Solvent control: saline (0.9% NaCl), diluted 1:100 in culture medium
Medium control: culture medium
Positive control: DNCB
Concentration of the positive control: 2 and 3 µg/ml
Solvent of the positive control: DMSO 0.2% in culture medium
Solvent control of the positive control: DMSO 0.2% in culture medium

PREPARATION OF THE CELLS
THP-1 Cell Cultures: aliquots of cells in freezing medium at 1 × 10E6 to 2 × 10E6 cells/mL
Thawed stock cultures are propagated at 37 ± 1.5 °C in plastic flasks. The cells are sub-cultured twice weekly. The cell density should not exceed 1 × 10E6 cells/mL. The THP-1 cell suspension is incubated at 37 ± 1.5 °C and 5.0 ± 0.5 % carbon dioxide atmosphere.
Prior to using a THP-1 cell batch for testing, the cells were qualified by conducting a reactivity check.
The passage numbers of the used THP-1 cells were 20 and 8 in the XTT assays and 12 and 13 in the h-CLAT for runs 1 and 2, respectively.

Preparation and Seeding of THP-1 Cells: seeded at a density between 0.2 × 10E6 cells/mL and 0.5 × 10E6 cells/mL + pre-cultured in culture flasks for 48 or 72 hours.

XTT experiments: a volume of 100 μL with a cell density of 0.9 - 1 × 10E6 THP-1 cells/mL was seeded in each well of a 96-well flat bottom plate.

Main experiment: cells were resuspended at 2 × 10E6 cells/mL. For the main experiment (h-CLAT) 0.9 - 1 × 10E6 cells/well in a volume of 500 μL were seeded in a 24-well plate before the treatment.

CYTOTOXICITY ASSAY
Dose finding assay: XTT test
Number of test: 2
Volume of test item and each controls applied: 100 µl
Number of replicates: 7
Incubation time: 24 hours

XTT concentration: XTT buffer solution and substrate solution mixed right before application at a ratio 1:100
Volume added: 50µl
Spectrophotometer:
Wavelenght: 450 nm (reference wavelength 690 nm)
Determination of the absorbance value: SoftMax Pro Enterprise (version 4.7.1)

ACCEPTABILITY OF THE CITOTOXICITY ASSAY:
• mean absorbance of the medium control is ≥ 0.5
• mean viability of the solvent control is ≥ 90% in comparison to the medium control

MAIN TEST:
Number of replicate :2
Dose of the test item: 1.2 × CV75
Range of concentrations: 7 dilutions were prepared by serial 1:1.2 dilution in saline. All concentrations were further diluted 1:50 in culture medium.
Concentrations applied:
Volume of each test item : 500 µl
Volume of cells: 500 µl
Time of incubation: 24 hours
Staining of the cells: with FITC-labelled anti-CD86, CD54 antibody or mouse IgG1 (isotype control).
Flow cytometry acquisition: The expression of cell surface antigens (CD54, CD86) was analysed by flow cytometry using the software Cellquest Pro 6.0. The FITC acquisition channel (FL-1) was set for the optimal detection of the FITC fluorescence signal, and the 7-AAD acquisition channel (FL-3) was set for the optimal detection of DNA-bound 7-AAD fluorescence signal.

ACCEPTANCE CRITERIA FOR THE MAIN TEST
The following acceptance criteria should be met when using the h-CLAT method:
• Cell viability of medium control is adjusted to 100% and the cell viability of the DMSO control should be more than 90% in comparison to the medium control.
• In the solvent/vehicle control (i.e. DMSO), RFI values compared to the medium control of both CD86 and CD54 should not exceed the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%).
• For both medium and solvent/vehicle controls (i.e. DMSO), the MFI ratio of CD86 and CD54 to isotype control should be > 105%.
• In the positive control (DNCB), RFI values of both CD86 and CD54 should meet the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%) and the cell viability should be > 50% in at least one concentration of the two tested positive control concentrations.
• For the test chemical, the cell viability should be more than 50% in at least four tested concentrations in each run.

PREDICTION MODEL
For CD86/CD54 expression measurement, each test item is tested in at least two independent runs to derive a single prediction (POSITIVE or NEGATIVE). An h-CLAT prediction is considered POSITIVE if at least one of the following conditions is met in 2 of 2 or in at least 2 of 3 independent runs (OECD 442E guideline):
− The RFI of CD86 is ≥ 150% at any tested concentration (with cell viability ≥ 50%);
− The RFI of CD54 is ≥ 200% at any tested concentration (with cell viability ≥ 50%).
Otherwise, the h-CLAT prediction is considered NEGATIVE

Results and discussion

Positive control results:

The RFI values of the positive controls (DNCB) for CD54 and CD86 exceeded the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%) and the cell viability was >50%.

In vitro / in chemico

Results

Results 1

Run / experiment:

other: mean of 2 runs (µg/ml)

Parameter:

other: mean CV75 value

Remarks:

XTT tests

Value:

2 533.35

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

not examined

Results 2

Key result

Run / experiment:

other: 1

Parameter:

other: RFI(%) CD54

Remarks:

for all tested concentrations

Value:

200

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Results 3

Run / experiment:

other: 2

Parameter:

other: RFI (%) CD54

Remarks:

not for all tested concentrations

Value:

200

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Results 4

Key result

Run / experiment:

other: 1

Parameter:

other: RFI (%) CD86

Remarks:

for all tested concentrations

Value:

150

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Results 5

Key result

Run / experiment:

other: 2

Parameter:

other: RFI (%) CD86

Remarks:

for all tested concentrations

Value:

150

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Other effects / acceptance of results:

DEMONSTRATION OF TECHNICAL PROFICIENCY:Results of the h-CLAT proficviency of the lab are provided on a list of defined chemicals as described into the guideline.

ACCEPTANCE OF RESULTS:
Cytotoxicity test: The RFI values of the positive controls (DNCB) for CD54 and CD86 exceeded the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%) and the cell viability was >50%

- Acceptance criteria met for negative control: RFI values compared to the medium control of both CD54 and CD86 did not exceed the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%).

- Acceptance criteria met for positive control:The RFI values of the positive controls (DNCB) for CD54 and CD86 exceeded the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%) and the cell viability was >50%

Any other information on results incl. tables

	run 1			run2
conc. (µg/ml)	RFI (%) CD54	RFI (%) CD86	cell viability (%)	RFI (%) CD54	RFI (%) CD86	cell viability (%)
848	234.8	224.1	90.4	173.0	186.1	90.5
1018	247.0	274.6	87.0	186.5	216.4	89.1
1222	283.3	323.7	81.6	209.0	229.9	89.9
1466	287.9	268.9	80.3	242.7	361.3	85.9
1759	253.0	304.8	66.2	205.6	319.7	82.0
2111	307.6	324.6	74.0	240.4	441.6	73.5
2533	322.7	379.8	62.2	277.5	491.6	64.0
3040	359.1*	545.2 *	42.2	321.3*	773.7*	47.5

*: as cell vaibility below 50%, these results are excluded from the evaluation

Applicant's summary and conclusion

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

The test item X300 activated THP-1 cells under the test conditions of this study. Therefore the test item is considered positive for the third key event of the skin sensitisation Adverse Outcome Pathway (AOP).

Executive summary:

The test item X300 was tested for its potential to induce skin sensitization in a strategy of assay. It was tested according to OECD guideline 442E, In Vitro Skin Sensitisation assays addressing the Key Event on activation of dendritic cells on the Adverse Outcome Pathway for Skin Sensitisation, with the human cell Line Activation Test or h-CLAT method.

The test item X300 was dissolved in saline and further diluted in culture medium when administered to THP-1 cells for 24 ± 0.5 hours. The highest test item concentration for the main experiment (h-CLAT) of X300 was previously determined by two XTT tests (deviation from the guideline but XTT tests are recognized as tests to assess the cytotoxicity).

Cytotoxic effects were observed following incubation with the test item starting with the concentration of 2500 μg/mL up to the highest tested concentration (5000 μg/mL) in the first XTT test and with the highest tested concentration (5000 μg/mL) in the second XTT test (threshold of cytotoxicity: < 75%). The mean CV75 value of both XTT tests was calculated as 2533.35 μg/mL.

The changes of surface markers expression (CD54, CD86) are measured by flow cytometry following cell staining with fluorochrome-tagged antibodies. The relative fluorescence or luminescence intensity of the treated cells compared to solvent/vehicle control are calculated and used in the prediction model, to support the discrimination between sensitisers and non-sensitisers.

The following concentrations of the test item (dissolved in saline) were tested in the main experiment (h-CLAT):

848, 1018, 1222, 1466, 1759, 2111, 2533 and 3040 µg/ml.

The test item X300 was tested in 2 independent runs. The highest tested test item concentration (3040 μg/mL) of both h-CLAT runs was excluded from the evaluation, since the cell viability was below 50%. The RFI of CD86 was greater than 150% in all concentrations of both runs. The RFI of CD54 was greater than 200% in all concentrations of the first run and in the most concentrations of the second run. Therefore, the h-CLAT prediction is considered positive for the tested test item in this h-CLAT. A median EC150 and EC200 value could not be calculated.

In the DMSO control, RFI values compared to the medium control of both CD54 and CD86 did not exceed the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%). The RFI values of the positive controls (DNCB) for CD54 and CD86 exceeded the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%) and the cell viability was >50%.

In conclusion, the test item X300 activated THP-1 cells under the test conditions of this study. Therefore the test item is considered positive for the third key event of the skin sensitisation Adverse Outcome Pathway (AOP).