Lecithins, hydrogenated

Endpoint summary

Description of key information

A theorical AOP (adverse outcome pathway) approach was considered on the test substance but shows its limits when it comes to complexe substances:

The substance is a UVCB with logPow < 0,3 and water solubility < 5 mg/L (20°C, pH 6).

 

-the key event n°1, DPRA test (OECD 442C) was unrealizable due to the fact that this test is unrealizable due to the fact that the substance is a UVCB and that this test is not applicable to UVCB.

-the key event n°2, Keratinosens test (OECD 442D) was unrealizable because it was impossible to obtain a solution homogeneous and stable enough.

-the key event n°3,h-Clat test (OECD 442E) was successfully performed and concluded that this substance isn’t a skin sensitizer.

 

No other test with other key event was available. The AOP approach can’t be concluded.

It was then decided to use literature data to complete the results of the h-Clat test.

Data in literature (including CIR report which is written by experts) show that neither the hydrogenated lecithins neither lecithins are considered as sensitizers.

Please see justification report in section 13.2.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD 442E: Human Cell Line Activation Test (h-CLAT)

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of dendritic cells

Details on the study design:

An alternative method has been developed for the evaluation of the skin sensitisation potential by measuring phenotypic changes, such as CD86 and CD54 expression on dendritic cells. The human leukemia cell line THP-1 is used as surrogate for human myeloid dendritic cells, since these cells show also enhanced CD86 and/or CD54 expression when treated with sensitisers.
The purpose of the Human Cell Line Activation Test (h-CLAT) is to assess the skin sensitising potential of the test item in an appropriate solvent (DMSO, saline or culture medium) when administered to THP-1 cells for 24 hours. The test item concentrations for the main experiment (h-CLAT) of test item are determined by two XTT tests.

Cytotoxic effects were not observed following incubation with the test item up to the highest tested concentration (625 µg/mL). Due the lack of cytotoxicity, a CV75 value could not be calculated. In addition, due to precipitations observed in the five highest test item concentrations of the first and in the three highest test item concentrations of the second XTT test, the lowest concentration with precipitations (156.3 µg/mL) of the second XTT test was used as highest concentration for the h-CLAT runs.
The following concentrations of the test item were tested in the main experiments (h-CLAT):
43.6, 52.3, 62.8, 75.4, 90.5, 108.5, 130.3 and 156.3 µg/mL.

--> Treatment of the cells
Each volume (500 µL) of the dilutions of the test item, medium control, positive and DMSO control was added to the cells. The treated THP-1 cells were incubated for 24 ± 0.5 hours. At the end of the incubation period, the cell cultures were microscopically evaluated for morphological alterations.
Each concentration of the test item, medium control, positive and DMSO control was prepared in triplicates for the different staining (with FITC-labelled anti-CD86, CD54 antibody or mouse IgG1).

-->Staining of the Cells
The triplicates of each test item-treated and not test item-treated cells were pooled and equally distributed into three sample tubes, collected by centrifugation (approx. 250  g, 5 min) and then washed twice with approx. 2 mL of FACS buffer (PBS with 0.1% (w/v) BSA). Thereafter, the cells were centrifuged, re-suspended and blocked with 600 µL of blocking solution at 2 8 °C (on ice) for approx. 15 min. After blocking, the cells were centrifuged and the cell pellets were re-suspended in 100 µL FACS buffer. The cells were stained with FITC-labelled anti-CD86, CD54 antibody or mouse IgG1 (isotype control).
All solutions were kept light protected at 2 - 8 °C or on ice during the staining and analysis procedures.
The cells with the different antibodies or the IgG1 were mixed and incubated light protected for 30 ± 5 min. at 2 - 8 °C (on ice).

-->Sample Preparation for Measurement
After staining with the antibodies, the cells were washed twice (2-8 °C) with 2 mL FACS buffer and re-suspended in a final volume of 2 mL/tube FACS buffer. At least 10 minutes before the flow cytometry acquisition, 5 µL of a 7-AAD solution were added.

-->Flow Cytometry Acquisition
Before using the flow cytometer (FACSCalibur, Becton Dickinson GmbH), the device was calibrated with appropriate beads in accordance with the manufacturer’s instructions.
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.

Positive control results:

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%.

Parameter:

other: RFI (relative fluorescence intensity) CD86

Remarks:

used as an indicator of CD86 and CD54 expression

Value:

116.2

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

The RFI value that fulfill the positive criteria is set to >=150% for CD86 cells

Parameter:

other: RFI (relative fluorescence intensity) CD54

Remarks:

used as an indicator of CD86 and CD54 expression

Value:

103.6

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

The RFI value that fulfill the positive criteria is set to >=200% for CD54 cells

Other effects / acceptance of results:

Due to precipitations observed in the three highest tested test item concentrations (108.5, 130.3 and 156.3 µg/mL) of both independent runs, these concentrations were excluded from the evaluation. The RFI of CD86 and CD54 was not equal or greater than 150% and 200%, respectively at any dose in both independent runs.

Interpretation of results:

GHS criteria not met

Executive summary:

The skin sensitization test was assessed with the OECD 442E guideline (h-CLAT).

The test item did not activate THP-1 cells up to a concentration of 90.5µg/mL and is therefore not considered as a skin sensitizer.

This test is part of an adverse outcome pathway and should be used in combinaison with other tests included in this AOP (OECD 442C and 442D).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available