Ferrocholinate

Endpoint summary

Description of key information

Experimental studies:

Under the experimental conditions of the in vitro human Cell Line Activation Test (h-CLAT) 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. Remarks: This test is not currently suitable on their own for predicting skin sensitising potency.

Under the experimental conditions of the ARE-Nrf2 Luciferase LuSens Test Method, the test item, Ferric Choline Citrate, was positive in the LuSens assay and is therefore considered to have the potential to activate the Nrf2 transcription factor (sensitizing potential). A categorization in the sub-categories 1 A and 1 B is not possible.

QSAR studies:

The value obtained for Skin Sensitsation in vitro (EC3, Keratinocyte activation human cells) of the prediction was 63.7 mg/L.

The value obtained for Skin Sensitisation in vivo (EC3, LLNA mouse) of the prediction was 14.7 % (Moderate Sensitiser).

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:

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

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

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

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

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.

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.
 

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.

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.

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.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06. Mar. 2020 (Study Plan dated); 09. Mar. 2020 (Experimental Starting Date); 14. May 2020 (Experimental Completion Date)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)

Version / remarks:

“In Vitro Skin Sensitisation assays addressing the AOP Key Event on Keratinocyte activation”. Adapted: 25 June 2018.

Deviations:

yes

Remarks:

In repetition I, II, III and IV the viability values of the positive control were slightly below 70 %. The concentration was adopted during the experimental phase. The deviation is considered as uncritical.

Qualifier:

according to guideline

Guideline:

other: OECD Series on Testing and Assessment No. 2013, “PERFORMANCE STANDARDS FOR ASSESSMENT OF PROPOSED SIMILAR OR MODIFIED IN VITRO SKIN SENSITISATION ARE-NRF2 LUCIFERASE TEST METHODS” ENV/JM/MONO(2015)6.

Version / remarks:

Aadapted 22 May 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EU-Method B.60 (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)

Version / remarks:

Adapted 14. Feb. 2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Specific details on test material used for the study:

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

Positive control results:

p-Phenylenediamine (CAS no. 106-50-3) was used as positive control (80 µM, 60 µM, 25 µM) and induced a distinct increase in luciferase induction above 2.5 fold in comparison to the solvent control in all repetitions. This luciferase induction is well within the historical data range of the positive control.
In repetition I, II, III and IV the viability values of the positive control were slightly below 70 %. This effect is a result of a new batch of the positive control. For that reason, the concentration was adopted during the experimental phase. Since no cytotoxic effect of the positive control was ob-served in repetition V and the results of the test item concentrations are in accordance to the ones of repetition I to IV the deviation is considered as uncritical. Therefore, the study is valid.

Key result

Run / experiment:

other: Repetition I: 401.9 µg/mL to 1000 µg/mL concentration

Parameter:

other: Fold in luciferase induction

Value:

1.5

Vehicle controls validity:

not specified

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Key result

Run / experiment:

other: Repetition II: 334.9 µg/mL to 1000 µg/mL concentration

Parameter:

other:

Remarks:

Fold in luciferase induction

Value:

1.5

Vehicle controls validity:

not specified

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Key result

Run / experiment:

other: Repetition III: 18.11 µg/mL and 26.08 µg/mL, 77.89 µg/mL and 112.16 µg/mL as well as 232.57 to 1000 µg/mL concentrations

Parameter:

other: Fold in luciferase induction

Value:

1.5

Vehicle controls validity:

not specified

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Key result

Run / experiment:

other: Repetition IV: 18.11 µg/mL to 1000 µg/mL concentration

Parameter:

other: Fold in luciferase induction

Value:

1.5

Vehicle controls validity:

not specified

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Key result

Run / experiment:

other: Repetition V: 18.11 µg/mL to 1000 µg/mL concentration

Parameter:

other: Fold in luciferase induction

Value:

1.5

Vehicle controls validity:

not specified

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Other effects / acceptance of results:

In total five repetitions were performed. Repetition III and IV were performed because 1) in repetition I and II no clear dose-dependent effect (biphasic dose response curve) of the test item concentrations was detected and 2) one of the validity criteria was not met (cytotoxic effect of the positive control). Repetition V was performed because again one of the validity criteria was not met (slight cytotoxic effect of the positive control) in repetition III and IV.

To verify if the biphasic response of the test item in repetition I and II whether it is specific to the test item or to an experimental artefact, different dilution steps of the test item were used in the following repetitions.

Since the biphasic effect was reproducible and seems to be specific to the test chemical, the positive result of repetition I and II was confirmed.

In repetition I and II, the positive control p-Phenylenediamine (80 µM) induced a cytotoxic effect but still a clear fold induction above 2.5 fold in comparison to the solvent control. Both repetitions were repeated with a lower concentration of the positive control (60 µM). However, in repetition III and IV, the viability values of the positive control were still slightly below the threshold of 70 %. For that reason, a fifth repetition was performed with an even lower concentration of the positive control (25 µm) to confirm the validity of the results of repetition I to IV.

Since all acceptability criteria were met in repetition V and all results of the test item concentrations were in accordance to those ones of repetition I, II, III and IV, also the first four repetitions were declared as valid and were used for the final evaluation.

12 concentrations of the test item were evaluated. The exposure time was 48 h. The following nominal concentrations of the test item were investigated in repetition I and II: 34.6 µg/mL, 161.5 µg/mL, 193.8 µg/mL, 232.6 µg/mL, 279.1 µg/mL, 334.9 µg/mL, 401.9 µg/mL, 482.3 µg/mL, 578.7 µg/mL, 694.4 µg/mL, 833.3 µg/mL, 1000 µg/mL

The following nominal concentrations of the test item were investigated in repetition III, IV and V: 18.11 µg/mL, 26.08 µg/mL, 37.56 µg/mL, 54.09 µg/mL, 77.89 µg/mL, 112.16 µg/mL, 161.51 µg/mL, 232.57 µg/mL, 334.90 µg/mL, 482.25 µg/mL, 694.44 µg/mL, 1000.00 µg/mL

None of the real treatment concentrations in all repetitions deviated more than 10 % from the nominal concentration. Precipitation of the test item was not visible up to the highest concentration in the repetitions.

p-Phenylenediamine (80 µM, 60 µM, 25 µM) was used as positive control and induced a distinct increase in luciferase induction above 2.5 fold in comparison to the solvent control in all repetitions. This luciferase induction is well within the historical data range of the positive control.

DL-lactic acid (5000 µM) was used as negative control. The induction of the luciferase was < 1.5 fold in comparison to the solvent control and well within the historical data range of the negative control.

The induction of the luciferase of the growth control (Medium no. 3) was < 1.5 fold.

Despite the uncritical cytotoxic effect of the positive control in the repetitions I to IV all acceptability criteria of the assay were met. Therefore, the study is valid.

No cytotoxic effect was observed in all tested test item concentrations. Therefore, all tested concentrations could be evaluated for luciferase induction.

Repetition I: 401.9 µg/mL to 1000 µg/mL
Repetition II: 334.9 µg/mL to 1000 µg/mL
Repetition III: 18.11 µg/mL and 26.08 µg/mL, 77.89 µg/mL and 112.16 µg/mL as well as 232.57 to 1000 µg/mL
Repetition IV: 18.11 µg/mL to 1000 µg/mL
Repetition V: 18.11 µg/mL to 1000 µg/mL

Therefore, all repetitions are clearly positive.

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

Under the experimental conditions of the ARE-Nrf2 Luciferase LuSens Test Method, the test item, Ferric Choline Citrate, was positive in the LuSens assay and is therefore considered to have the potential to activate the Nrf2 transcription factor (sensitizing potential). A categorization in the sub-categories 1 A and 1 B is not possible.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available