L-lysine, compound with S-(carboxymethyl)-L-cysteine (1:1)

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From February 6 to 21, 2014

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Principles of method if other than guideline:

In the test, a cell model of dendritic cells was used to evaluate to immunological reactivity of typical immunity cells, specialised as antigen presenting cells, exposed for prolonged time (48h) to the tested product at different concentrations.

GLP compliance:

no

Remarks:

the available study was originally run not for REACH purposes; however, it is scientifically valid and well documented.

Type of study:

activation of dendritic cells

Test material

Test material information

Constituent 1

Reference substance name:

L-lysine, S-carboxymethyl-L-cysteine salt

IUPAC Name:

L-lysine, S-carboxymethyl-L-cysteine salt

In vitro test system

Details on the study design:

PRINCIPLE OF THE TEST
In the skin, the dendritic cells specialized for the first contact as antigen presenting cells, are named Langerhans Cells.
Cells used are monocytes-derived immature dendritic cells from human healthy blood volunteers, as prototypic blood-derived immunologically active cells. These cells play a central role in the skin immune response towards topical products. Human primary dendritic cells are an in vitro model of Langerhans cells. The expression of two co-stimulatory molecules, CD80 and CD86 is checked, using bacterial lipopolysaccharides (LPS, generic stimulators of immune response) and nickel sulphate as two positive controls, nickel being a well known contact sensitising agent, both in in vitro and in vivo models.
When the lymphocytes T’ TCR recognize the antigen (signal 1) on the Antigen-Presenting Cell (APC), additional molecules (called co-stimulatory) on the APC’s membrane are necessary to obtain a complete functional immune response (signal 2).
The signal 2 is very important to define the kind of immune response that is going to be activated (umoral, cellular, etc.)
The costimulatory molecules CD80 and CD86 (also calle B7.1 and B7.2) are necessary to obtain an efficient antigen presentation by the T cell receptor (TCR) and hence to obtain a correct immune response.
Both these molecules are membrane glycoprotein expressed on the surface of different antigen-presenting cells (dendritic cells, Langerhans cells, monocytes/macrophages, keratinocytes) and they recognise a further molecule, a glycoprotein called CD28 on the T lymphocyte membrane.
The switching on of the ligand/receptor system CD28/B7 avoids the T cell apoptosis and sustains their proliferation and differentiation and the production of many cytokines.
In the first phase of the physiological immune response, B7.2 is expressed as default and it modulates both the Th1 and Th2 responses. As the immune response goes on, also B7.1 is up-regulated and the costimulatory signal increase, with an expansion of T cells and the production of different cytokines. B7.1 is also preferentially up-regulated during the acute phase of auto- immune response.
The increasing level of expression of CD80 and CD86 on the dendritic cells is a signal of activation of the immune response derived from the exposition to a potentially sensitising contact antigen. Functionally, the expression of co-stimulatory molecules on the dendritic cells means activation of the immunological response in terms of capability to present the antigen in the typical tissues (skin in our case), where, in vivo, the immune protective response is triggered.

SAMPLE PREPARATION
The sample was dissolved in ethanol and then diluted in the cell culture medium at different concentrations.
The product underwent a preliminary cytotoxicity screening on the cells to decide the best concentration to test it without cytotoxic effects on the cells, in order to avoid false results. Cell medium exposed to the same experimental conditions were used as a negative control.

CELL MODEL
The test is carried out on a primary cell line of dendritic cells derived from human monocytes of healthy volunteers peripheral blood . Cells are kept in RPMI 1640, FCS (10 %), GM-CSF (50 ng/ml) e IL-4 (1000 IU/ml) added.

MTT PRELIMINARY ASSAY EXECUTION
The MTT assay is simple, accurate and yields reproducible results. The key component is (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) or MTT. This product is of yellowish colour in solution. Mitochondrial dehydrogenases of viable cells
cleave the tetrazolium ring, leading to the formation of purple crystals which are insoluble in aqueous solutions. The crystals are re-dissolved in acidified isopropanol and the resulting purple solution is measured spectrophotometrically. An increase or decrease in cell number results in a concomitant change in the amount of formazan formed, indicating the degree of cytotoxicity caused by the test material.
After exposure of the cells to the test material, the cells are washed with PBS and exposed to the MTT-medium at 37 °C. At the end of the incubation period, the MTT-medium is removed and the cells receive the MTT solubilization solution. The plate is shaken on a rotatory plate shaker for 20-30 minutes, ensuring that all the crystals have dissolved from the cells and have formed a homogeneous solution.
The absorbance is measured as described with background elimination.
The results are expressed in terms of viability:
% viability = 100 × OD treated cultures / OD untreated control cultures

TREATMENT AND EXPOSURE
Following the preliminary cytototoxicity investigation, the optimal testing concentrations were defined. The sample was diluted in the cell medium at the wished final concentrations and put in contact with the dendritic cells in vitro. The exposure has been carried out for 48 h at 37 °C with 5 % CO2.

SENSITISATION ASSAY EXECUTION
After the incubation with the tested substance and the controls, cells are collected, checked under the microscope for their vitality by staining with Trypan Blue dye and counting in a cell counter chamber, washed in PBS and then marked with a fluoresceinated anti-B7.1 or B7.2 antibody.
After washing, to eliminate the excess antibody, the MFI (Mean Fluorescence Intensity) linked to the cells was evaluated by means of a flux cytofluorimeter.
This value is proportional to the expression of costimulatory molecules.
The MFI of the non-treated cells and of cells after reaction with a monoclonal isotype-matched antibody was used as an internal control (basal fluorescence).

POSITIVE CONTROLS
- nickel sulfate crystals dissolved in PBS (well-known skin sensitiser both in vitro and in vivo)
- bacterial lipopolysaccharides (LPS, generic stimulators of immune response)

Results and discussion

Positive control results:

sample CD80 (MFI*) CD86 (MFI*)
nickel sulfate 20 µg/ml 28.06 38.56
nickel sulfate 10 µg/ml 20.04 25.82
nickel sulfate 4 µg/ml 14.23 5.36
LPS 1 µg/ml 30.81 74.08

*MFI = mean fluorescence intensity - it is the geometric average of the fluorescence intensity of the cells decorated with fluoresceinated antibody and it is proportional to the number of stained molecules per cell.

Nickel, a prototypic sensitising substance, showed:
- high increase of both the markers, with a predominance of CD80;
- direct correlation between concentration and intensity of the response;
- relevant effects even at very low doses. Also LPS increases the expression of both the investigated markers.

The tested dose of 4 mg/ml of nickel sulphate (NiSO4×6H2O) corresponds to more or less 1 ppm of nickel, dosage that is around the minimal sensitising threshold in already sensitised individuals with irritated skin. At this dose, Ni is already able to cause an increase from 2 times upwards of the investigated molecules. The concentration that is able to cause an allergic reaction in most of the sensitive subjects is anyway around higher value, over the 100 ppm of nickel sulphate in contact with safe and intact skin.

In vitro / in chemico

Results

Results 1

Run / experiment:

other: 10 mg/ml test substance

Parameter:

other: CD80 (MFI)

Remarks:

MFI = mean fluorescence intensity

Value:

-0.51

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Results 2

Run / experiment:

other: 2 mg/ml test substance

Parameter:

other: CD80 (MFI)

Remarks:

MFI = mean fluorescence intensity

Value:

-0.45

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Results 3

Run / experiment:

other: 10 mg/ml test substance

Parameter:

other: CD86 (MFI)

Remarks:

MFI = mean fluorescence intensity

Value:

-1.45

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Results 4

Run / experiment:

other: 2 mg/ml test substance

Parameter:

other: CD86 (MFI)

Remarks:

MFI = mean fluorescence intensity

Value:

-1.62

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

The sample did not cause any increase in the expression of both the investigated markers.
The sample did not cause any cytotoxic effect on the cells used for the test (IC50= 50 mg/ml).
No signal related to apoptosis on the treated cells was seen.

Applicant's summary and conclusion

Interpretation of results:

other: not sensitising (data used as part of a weight of evidence evaluation)

Conclusions:

No sensitising potential was seen in the in vitro test on human dendritic cells.

Executive summary:

Method

An in vitro test was conducted on human dendritic cells to analyse the sensitising potential of the substance. Dendritic cells were derived from human healthy blood volunteers.

Potential effects of the test substance were related to the expression of markers CD80 and CD86.

A preliminary MTT assay was run to evaluate the cytotoxicity of the substance and decide the test concentrations for the sensitisation assay. The sensitisation assay was tun at 37 °C with 5 % CO2 for 48 h using test concentrations of 10 and 2 mg/ml.

Bacterial lypopolysaccharides (LPS) and nickel sulphate were used as positive controls.

Results

The sample was not cytotoxic to the cells used for the test (IC50 = 50 mg/ml).

Positive controls were valid. The sample did not cause any increase in the expression of both markers; no apoptosis of treated cells was seen.