Ferrocholinate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20. Feb. 2020 (Study Plan dated); 24. Feb. 2020 (Experimental Starting Date); 03. Mar. 2020 (Experimental Completion Date)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Reference 2

Reference Type:

other: Standard Operating Procedure (SOP)

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Adopted 25. June 2018.

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: OECD. (2012). The Adverse Outcome Pathway for Skin Sensitisation Initiated by Covalent Binding, Part 1: Scientific Evidence. Series on Testing and Assessment No. 168, Paris.

Version / remarks:

2012

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Bauch, C., Kolle, S. N., & Ramirez, T. (2012). Putting the parts together: combining in vitro methods to test for skin sensitizing potentials. Regulation of Toxicology and Pharmacology, 63, 489–504.

Version / remarks:

2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of dendritic cells

Test material

Test material information

Constituent 1

Reference substance name:

Ferrocholinate

EC Number:

215-649-7

EC Name:

Ferrocholinate

Cas Number:

1336-80-7

Molecular formula:

C11H24FeNO11

IUPAC Name:

2-hydroxyethyl(trimethyl)azanium;iron(3+);2-oxidopropane-1,2,3-tricarboxylate;trihydrate

Test material form:

solid: particulate/powder

Details on test material:

- Appearance: Yellow-green powder
- Purity: 12.2 % Ferric
- Homogeneity: Homogeneous
- Molecular Weight: 941.56 g/mol

Specific details on test material used for the study:

PROPERTIES OF TEST MATERIAL
LogPow: < 0.3 (GLP study 19100101G930 performed at LAUS GmbH).

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
First, a stock solution (nominal concentration: 100 mg/mL) of the test item in RPMI 1640 was prepared and used to prepare a geometric series of solutions (factor 2 for pre-test; factor 1.2 for runs). Afterwards all concentrations were further diluted (1:50 fold) in complete culture medium (working solutions). Another 1:2 dilution was achieved by adding 1 part of each test item concentration and 1 part of the cell suspension to the treatment plate. In the end, the total dilution factor was 1:100. The stock solutions as well as the dilutions were freshly prepared on the day of treatment.

In vitro test system

Details on the study design:

1. Reasons for the Choice of the THP-1 Cell Line
The OECD 442E indicates that the human monocytic leukaemia cell line, THP-1 should be used for the h-CLAT.

2. Cell Cultures
THP-1 cells are stored in liquid nitrogen in a cell bank to allow a continuous stock of cells, which guarantees similar parameters of the experiment and reproducible characteristics of the cells.
The THP-1 cells are routinely seeded every 2-3 days at the density of 0.1 – 0.2 * 106 cells/mL. They were maintained at densities from 0.1 to 1.0 * 106 cells/mL. Prior to using them for testing, the cells were qualified by conducting a reactivity check.
For the pre-test cells of passage 20 were used. For the two runs cells of passage 23 were used. After thawing the cells were cultivated in RPMI 1640 complete culture medium in cell culture flasks at 37 ± 1 °C in a humidified atmosphere with 5.0 ± 0.5 % CO2.

3. Reactivity Check
Four weeks after thawing, a reactivity check of the cells was performed. For that, the two positive controls 2,4-dinitrochlorobenzene (DNCB) (CAS n. 97-00-7, ≥ 99% purity, test concentration: 4 µg/mL) and nickel sulfate (NiSO4) (CAS n. 10101-97-0, ≥ 99% purity, test concentration: 200 µg/mL) as well as the negative control, lactic acid (LA) (CAS n. 50-21-5, ≥ 85% purity, test concentration: 1000 µg/mL) were used. These substances as well as all additional information are given by the OECD 442E. The experimental procedure was identical to the two runs in this study.
The two positive controls produced a clearly positive result of the two surface markers CD86 and CD54. The negative control produced a clearly negative result of both surface markers. Therefore, the cells were found to be suitable for the experiment.
For the pre-test as well as the two runs only cells which have successfully passed the reactivity check were used.
 

Results and discussion

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

other: Both independent runs of the experiment at any tested concentration

Parameter:

other: Relative Fluorescense Intensity (RFI) of CD86 (%)

Value:

150

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

The test item is negative in the h-CLAT.

Results 2

Key result

Run / experiment:

other: Both independent runs of the experiment at any tested concentration

Parameter:

other: Relative Fluorescense Intensity (RFI) of CD54 (%)

Value:

200

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

The test item is negative in the h-CLAT.

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
The assay is considered acceptable if it meets the following criteria:
- The cell viabilities of medium and solvent/vehicle controls are higher than 90%.
- In the solvent/vehicle control, RFI values of both CD86 and CD54 do not exceed the positive crite-ria (CD86 RFI ≥ 150 % and CD54 RFI ≥ 200 %).
- For both medium and solvent controls, the MFI ratio of both CD86 and CD54 to isotype control should be > 105%.
- In the positive control, RFI values of both CD86 and CD54 should meet the positive criteria (CD86 RFI ≥ 150 % and CD54 RFI ≥ 200 %) and cell viability should be more than 50 %.
- For the test item, the cell viability should be more than 50 % in at least four tested concentrations in each run.
Negative results are acceptable only for test items exhibiting a cell viability of less than 90 % at the highest concentration tested. If the cell viability at 1.2 × CV75 is equal or above 90 % the negative result should be discarded. In such a case another Pre-test has to be performed to refine the dose selection. It should be noted that when 5000 µg/mL in PBS or medium, 1000 µg/mL in DMSO or the highest soluble concentration is used as the maximal test concentration of a test item, a negative result is acceptable even if the cell viability is above 90 %.

All validity criteria were met. Therefore, the study is considered as valid.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Remarks:

A final conclusion on skin sensitisation hazard (sensitiser vs non-sensitiser) or potency (Cat 1A or 1B according to CLP) cannot be made as this method is not a stand-alone assay.

Conclusions:

Under the experimental conditions of this study, the test item Ferric Choline Citrate was negative in the h-CLAT and is therefore considered not to have the potential to activate dendritic cells and therefore to up-regulate the cell surface marker (CD86 and CD54) expression of THP-1 cells.