Rhamnolipids: fermentation products of glucose with Pseudomonas putida, Pseudomonadaceae

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2015-02-18 to 2015-02-27

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:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

other: U-SENS (former MUSST)

Version / remarks:

MUSST SOP ECVAM version 03/26/2013

Deviations:

not applicable

Principles of method if other than guideline:

- Principle of test:
Cell activation by contact sensitizers is determined by the overexpression of CD86 on U937 cell surface, which is a ligand required for T-cell activation. CD86 expression is measured using fluorescent labeled anti-CD86 monoclonal antibody after a 45 hours treatment by flow cytometry.

Cell culture
U937, a human myelomonocytic cell line, was obtained from the America, Type Culture Collection (ATCC, Ref: CRL-1593.2). On the day of experiment, cells were seeded in 96-well culture plates at a density of 0.5 x 106 cells/mL with or without a test items (doses ranging from 1 to 200 μg/mL) for 45±3 hours to determine a dose-response.

Cell treatment
Treatments were performed at the following concentration for the first run: 1, 10,20,50, 100 and 200 μg/mL. For the next runs, the adjustment of the dose could be necessary to determine a sub-toxic dose-response of the test item and the calculation of the EC150. Test item is tested up to five validated runs. The sensitizing potential of the test item is determined after a minimum of two concordant runs.
Determination of CD86 Expression
Cell staining was performed using mouse mAb FITC-anti-human-CD86. Briefly, cells were washed in cold PBS (containing 5% FBS), stained with antibodies in the dark at 4°C for 30 min, washed twice with cold PBS (containing 5% FBS) and once with PBS without FBS and resuspended in PBS. Cells were then analyzed using FACS DIVA Software based on a collection of 10,000 cells with a FACS CANTO II flow cytometer. Necrotic cells were gated out after staining with 50 μg/mL Propidium Iodide solution. The percentage of CD86 positive cells was determined according to the IgGl isotype control (% cCD86).

Automatized test system using MAIA (Methode Alternative In vitro Automatisée) was used for cell treatment and cell staining.

GLP compliance:

no

Remarks:

adequate for C&L; sufficient documentation is provided to assess the adequacy of the study; the data are valid for the endpoint investigated and the study is performed using an acceptable level of quality assurance

Type of study:

activation of dendritic cells

Test material

Test material information

Constituent 1

Reference substance name:

Rhamnolipids: fermentation products of glucose with Pseudomonas bacteria

EC Number:

943-175-7

IUPAC Name:

Rhamnolipids: fermentation products of glucose with Pseudomonas bacteria

Test material form:

solid: particulate/powder

In vitro test system

Details on the study design:

Skin sensitisation (In vitro test system) - Details on study design:

No solubility assessment was performed because the solvent RPMI was weil known for this test item

Lactic acid (LA 200 μg/mL as known non sensitizer) treated cells were used as negative control 2,4,6- Trinitrobenzenesulfonic acid (TNBS 50 μg/mL, as well known skin sensitizer) treated cells were used as positive control.

A set of control wells corresponding to untreated (RPMI control) and treated with either the vehicle (DMSO), negative and positive controls were also added in each plate to guarantee the validity of each run.

Cell culture
U937, a human myelomonocytic cell line, was obtained from the ATCC (Ref: CRL-1593.2). On the day of experiment, cells were seeded in 96-well culture plates at a density of 0.5E06 cells/mL with or without a test items (doses ranging from 1 to 200 μg/mL) for 45±3 hours to determine a dose-response.

Cell treatment
Test item was tested in two validated runs.
Concentration for the first run: 1, 10, 20, 50, 100 and 200 μg/mL.
Concentration for the second run: 10, 20, 50, 100, 160 and 200 μg/mL.

Determination of CD86 Expression
Cell staining was performed using mouse mAb FITC-anti-human-CD86. Briefly, cells were washed in cold PBS (containing 5% FBS), stained with antibodies in the dark at 4°C for 30 min, washed twice with cold PBS (containing 5% FBS) and once with PBS without FBS and resuspended in PBS. Cells were then analyzed using FACS DIVA Software based on a collection of 10,000 cells with a FACS CANTO II flow cytometer. Necrotic cells were gated out after staining with 50 μg/mL Propidium Iodide solution. The percentage of CD86 positive cells was determined according to the IgG1 isotype control (%cCD86).

Results and discussion

Positive control results:

2,4,6- Trinitrobenzenesulfonic acid (TNBS 50 μg/mL, as well known skin sensitizer) treated cells were used as positive control. In both runs, all positive control wells gave positive results (CD86 SI ≥ 150).

In vitro / in chemico

Results

Results 1

Parameter:

other: EC150 [µg/ml]

Value:

200

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Results 2

Parameter:

other: CV70 [µg/mL]

Value:

200

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Other effects / acceptance of results:

DEMONSTRATION OF TECHNICAL PROFICIENCY:
If one or more of the following acceptance criteria is not met, the run is invalidated and had to be repeated:
- At least 2 RPMI controls out of 3 presents a % IgG1 between 0.6% and 1.5%.
- The mean viability of RPMI cells have to be >90%.
- The mean corrected %CD86+ values of RPMI control wells have to be ≥ 2 and ≤ 25%.
- At least 2 out of 3 wells of the positive control TNBS have to be positives (CD86 SI ≥ 150).
- At least 2 out of 3 wells of the negative control LA have to be negatives (CD86 SI ≤ 150).
- The mean viability of the vehicle control DMSO has to be ≥ 90%.The mean CD86 SI of the vehicle control DMSO has to be ≤ 250. If not a marginal value can be discarded.


ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Other acceptance criteria met: yes

Any other information on results incl. tables

Summary results

	Dose (µg/ml)	Individual runs				Mean and SD				FL3 (lgG1)		FL1 (lgG1)
	testsubstance	% vialbility (mean)		CD86-lgG1(S.I.)		Viability		CD86-lgG1		X Geo	Mean S.I.	X Geo	Mean S.I.
		A	B	A	B	Mean	SD	Mean	SD	A	B	A	B
	1.00	95		76		95	0	76	0	165		108
	10.00	96	96	66	70	96	0	68	2	110	93	96	95
	20.00	96	96	61	70	96	0	65	4	96	97	96	94
	50.00	95	96	77	61	96	1	69	8	92	95	95	95
	100.00	95	96	90	72	95	0	81	9	105	87	101	99
	160.00		93		78	93	0	78	0		82		109
	200.00	67	76		99	72	5	99	0		84		95
CV70 / EC 150		189	NA	NA	NA	NA		NA
Run conclusion				NS	NS

NA: Not applicable NS: Non-Sensitizer

EC150: calculated value corresponding to the concentration beyond which the RM leads 150% of expression of CD86 with regard to the control.

CV70: calculated value corresponding to the concentration beyond which the moleeule is considered as being cytotoxic.

Applicant's summary and conclusion

Interpretation of results:

other: not sensitising

Remarks:

expert statement

Conclusions:

Under the experimental conditions of this study, the test item, Rhamnolipids is predicted to be a non sensitizer (no EC150) in the MUSST assay without cytotoxicity.

Executive summary:

The purpose of this study was to evaluate the capability of Rhamnolipids (79% a.i.) to induce the surface marker CD86 expression in the human myeloid U937 cell line. This study was conducted in compliance with the MUSST SOP ECVAM version 03/26/2013.

Cell activation by contact sensitizers is determined by the overexpression of CD86 on U937 cell surface, which is a ligand required for T-cell activation. CD86 expression is measured using fluorescent labeled anti-CD86 monoclonal antibody after a 45 hours treatment by flow cytometry.

All validity criteria were fulfilled. An EC150 could not be determined up to the highest tested concentration of 200 µg/L. Rhamnolipids is predicted a non-sensitizer in the MUSST assay without cytotoxicity.