Rhamnolipids: fermentation products of glucose with Pseudomonas putida, Pseudomonadaceae

Endpoint summary

Description of key information

negative in DPRA, KeratinoSens and MUSST

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:

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

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

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).

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).

Parameter:

other: EC150 [µg/ml]

Value:

200

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

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

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.

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.

Reference 2

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2014-09-09 to 2014-09-10

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD draft proposal for a new test guideline: in chemico skin sensitization: Direct Peptide Reactivity Assay (DPRA).

Version / remarks:

DB-ALM Protocol W154, Direct Peptide Reactivity Assay (DPRA) for Skin Sensitization Testing. Gerberick G.F. et al. (2004)

Deviations:

not applicable

GLP compliance:

yes

Type of study:

direct peptide reactivity assay (DPRA)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- analytical purity: 79%
- correction factor: 1.26

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: solubility testing
- Based on solubility results, the vehicle was a 1:1 mixture of RO water:acetonitrile

FORM AS APPLIED IN THE TEST (if different from that of starting material)
test item was dissolved in the selected vehicle (1:1 mixture of RO water:acetonitrile) at 100 mM taking into account the correction factor. This formulation had the aspect of a yellowish liquid.

OTHER SPECIFICS:
Each formulation was immediately used for the incubation with peptides.

Details on the study design:

Skin sensitisation (In chemico test system) - Details on study design:
Cysteine peptide: AC-RFAACAA-COOH Molecular weight 750.88 g/mol
Lysine peptide: AC-RFAAKAA-COOH Molecular weight 775.91 g/mol

Number of replicates: triplicate; co-elution control samples: one sample per peptide

- Co-elution control with cysteine peptide: 50 μL of test item formulation was incubated with 750 μL of cysteine peptide dilution buffer (without cysteine peptide) and 200 μL of acetonitrile.
- Co-elution control with lysine peptide: 250 μL of test item formulation was incubated with 750 μL of lysine peptide dilution buffer (without lysine peptide).

- Reference control A and B samples: Acetonitrile was added to a volume of peptide solution (cysteine or lysine) to achieve a nominal concentration of 0.500 mM.

- Reference control C prepared with cysteine peptide: 50 μL of each vehicle (RO water:acetonitrile and acetonitrile) was incubated with 750 μL of cysteine peptide solution (at 0.667 mM in phosphate buffer at pH 7.5) and 200 μL of acetonitrile.
- Reference control C prepared with lysine peptide: 250 μL of each vehicle (RO water:acetonitrile and acetonitrile) was incubated with 750 μL of lysine peptide solution (at 0.667 mM in ammonium acetate buffer at pH 10.2).

- Reactivity of cinnamaldehyde with cysteine peptide: 50 μL of cinnamaldehyde at 100 mM in acetonitrile was incubated with 750 μL of cysteine peptide solution (at 0.667 mM in phosphate buffer at pH 7.5) and 200 μL of acetonitrile.
- Reactivity of cinnamaldehyde with lysine peptide: 250 μL of cinnamaldehyde at 100 mM in acetonitrile was incubated with 750 μL of lysine peptide solution (at 0.667 mM in ammonium acetate at pH 10.2).
- Reactivity of test item with cysteine peptide: 50 μL of test item formulation was incubated with 750 μL of cysteine peptide solution (at 0.667 mM in phosphate buffer at pH 7.5) and 200 μL of acetonitrile.
- Reactivity of test item with lysine peptide: 250 μL of test item formulation was incubated with 750 μL of lysine peptide solution (at 0.667 mM in ammonium acetate at pH 10.2).
- Reactivity of cinnamaldehyde with lysine peptide: 250 μL of cinnamaldehyde at 100 mM in acetonitrile was incubated with 750 μL of lysine peptide solution (at 0.667 mM in ammonium acetate at pH 10.2).
All samples (co-elution controls, reference controls, test item and positive control samples) were then incubated during 24 (± 2) hours at 25°C and protected from light before injection onto the HPLC/UV system.
At the end of the incubation period, a visual inspection of the samples was performed prior to HPLC analysis.
As precipitates were observed in all test item samples and in the co-elution sample prepared with the lysine dilution buffer, these vials were centrifuged at 400 g for aperiod of 5 minutes at room temperature to force precipitate to the bottom of the viaI. Positive control samples, all reference control sampies for cysteine peptide and the reference control for lysine peptide prepared in acetonitrile were also centrifuged at the same conditions to force precipitate to the bottom of the vials. Only supernatants were then injected onto the HPLC/UV system.
For the other samples, the vials were directly transferred onto the HPLC/UV system.

Analytical Column: Zorbax SB C18, 100 x 2.1 mm, 3.5 μm, (Waters), In-line filter C18, 4.0 x 2.0 mm (Phenomenex)
Mobile phase (gradient mode): Mobile phase A: acetonitrile + 0.085% TFA, Mobile phase B: milli-Q water + 0.1% TFA
Flow: 350 μL/minute
UV Wavelenght: 220 nm
Injection volume: 7 μL
Retention times: Cysteine-peptide: approx. 10 minutes; Lysine-peptide: approx. 6.3 minutes
Total analysis time: 20 min

Positive control results:

Mean depletion rate (%) of cinnamaldehyde: 69.17

% depletion cystein peptide: mean 83.39; SD 0.69; % CV 0.8
% depletion lysine peptide: mean 55.16; SD 0.85; % CV 1.5

Key result

Parameter:

other: mean percent cysteine and lysine depletions

Value:

4.13

Negative controls validity:

not applicable

Positive controls validity:

not applicable

Remarks on result:

other: no/minimal peptide reactivity

Run / experiment:

other: cysteine peptide

Parameter:

other: mean percent depletion values

Value:

6.59

Negative controls validity:

valid

Positive controls validity:

valid

Run / experiment:

other: lysine peptide

Parameter:

other: mean percent depletion values

Value:

1.68

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system:
precipitates were observed at the end of the incubation with the peptides

DEMONSTRATION OF TECHNICAL PROFICIENCY:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control:
- Acceptance criteria met for positive control:
- Acceptance criteria met for variability between replicate measurements:
- Range of historical values if different from the ones specified in the test guideline:

Several vehicles were tested during the solubility assay. The test item was found not soluble at 100 mM in acetonitrile even after sonication. A solution was obtained at 100 mM with RO water after sonification and in a 1:1 mixture of RO water:acetonitrile. Therefore, since the formulation prepared in a 1:1 mixture of RO water:acetonitrile has a better aspect than that prepared in RO water and sonicated, the vehicle retained was 1:1 mixture of RO water:acetonitrile.

The acceptance criteria for the calibration curve samples, the reference and positive controls as well as for the study samples were satisfied. The study was therefore considered to be valid.

 

Analysis of the chromatograms of the co-elution samples indicated that the test item did not co-elute with either the lysine or the cysteine peptides. As a result, the mean percent depletion values were calculated for each peptide.

The mean of the percent cysteine and percent lysine depletions was equal to 4.13%. Accordingly, the test item was considered to have no/minimal peptide reactivity. Therefore, the DPRA prediction would be considered as negative and the test item may have no potential to cause skin sensitization.

 

Since precipitates were observed at the end of the incubation with the peptides, the peptide depletion may be underestimated.

Interpretation of results:

other: not sensitising

Remarks:

expert statement

Conclusions:

Under the experimental conditions of this study, the test item Rhamnolipids was considered to have no/minimal peptide reactivity and therefore, has no potential to cause skin sensitization.

Executive summary:

In an in chemico skin sensitisation study performed according to the OECD draft proposal for a new test guideline 442C (Direct Peptide Reactivity Assay) the reactivity of the test item Rhamnolipids was evaluated by monitoring peptide depletion following a 24-hour contact between the test item and synthetic cysteine and lysine peptides.

The test item was dissolved at 100 mM in a 1:1 mixture of water:acetonitrile. The positive control was cinnamaldehyde, and for each peptide, the analytical batch included co-elution control samples and three reference control samples.

Reactivity (%depletion) was determined following 24-hour contact between test item and peptide in acetonitrile at the ratios 1:10 cysteine:test item and 1:50 lysine:test item by liquid chromatography with UV-detection.

Peptide reactivity was reported as percent depletion based on the peptide peak area of the replicate injection and the mean peptide peak area in the three relevant reference control C samples. The acceptance criteria for the calibration curve samples, the reference and positive controls as well as for the study samples were satisfied and thus demonstrated the validity of the study.

 

Analysis of the chromatograms of the co-elution samples indicated that the test item did not co-elute with either the lysine or the cysteine peptides. As a result, the mean percent depletion values were calculated for each peptide. For the cysteine peptide, the mean depletion value was 6.59% and for the lysine peptide, the mean depletion value was 1.68%.

Since the mean of the percent cysteine and percent lysine depletions was equal to 4.13%, the test item was considered to have no/minimal peptide reactivity. Therefore, the DPRA prediction would be considered as negative and the test item may have no potential to cause skin sensitisation.

 

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2014-08-05

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:

OECD Draft Guideline, In Vitro Skin Sensitization: ARE-Nrf2 Luciferase Test method; 16 May 2014.

Deviations:

no

GLP compliance:

yes

Type of study:

activation of keratinocytes

Specific details on test material used for the study:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: solubility testing
- Final dilution of a dissolved solid, stock liquid or gel:
- correction factor: 1.26 to account for purity < 100%
On the basis of solubility results, the test item was dissolved in DMSO at 200 mM for each run. This formulation was then diluted in DMSO by serial dilutions, using a dilution factor of 2, to obtain a total of 12 concentrations in a 96-well plate; this 96-well plate was called "Master plate 100x".
Subsequently, each formulation of the Master plate 100x was 25-fold diluted in treatment medium in another 96-well plate called Master plate 4x. This plate was prepared just before each treatment taking care to adjust all wells to the same DMSO level.

FORM AS APPLIED IN THE TEST (if different from that of starting material)
solution in DMSO

OTHER SPECIFICS:
All formulations were prepared within 4 hours before use, and kept at room temperature and protected from light until use.

Details on the study design:

Skin sensitisation (In vitro test system) - Details on study design:
KeratinoSens cells: Supplier: Givaudan, batch used (A1) was validated by the Supplier, Mycoplasm: absence of mycoplasm was confirmed on February 2014.

Solubility test was performed prior the first treatment. In the solubility test, the test item was found soluble in DMSO at 200 mM.

Cell seeding for testing
Cells were grown using general culture procedures and the day prior to treatment, cells were washed twice with D-PBS containing 0.05% EDTA, harvested, re-suspended in maintenance medium No.2 and adjusted to a density of 8 x 104 cells/mL, cells were then distributed into four 96-well plates (three white plates and one transparent plate), by adding 125 μL (representing 1 x 104 cells) per weil,
after seeding, the cells were grown for 24 (± 1) hours in the 96-well microtiter plates prior to test item
addition.

Positive control
The positive control was first dissolved in DMSO at 200 mM and then to 6.4 mM. Four serial dilutions were then prepared from the formulation at 6.4 mM in DMSO in the "Master plate 100x" and using a dilution factor of 2. Subsequently, each formulation of the Master plate 100x was 25-fold diluted in treatment medium in another 96-well plate called Master plate 4x.
These formulations were prepared within 4 hours before use and kept at room temperature and protected from light.

Test item
All concentrations take into account the rhamnolipid sample composition of 79% by applying a correction factor of 1.26.
On the basis of solubility results, the test item was dissolved in DMSO at 200 mM for each run. This formulation was then diluted in DMSO by serial dilutions, using a dilution factor of 2, to obtain a total of 12 concentrations in a 96-well plate; this 96-well plate was called "Master plate 100 x".
Subsequently, each formulation of the Master plate 100x was 25-fold diluted in treatment medium in another 96-well plate called Master plate 4x. This plate was prepared just before each treatment taking care to adjust all wells to the same DMSO level.
All formulations were prepared within 4 hours before use, and kept at room temperature and protected from light until use.

Treatment
After the 24-hour growing period, the medium was removed by aspiration and replaced by 150 μL of
treatment medium, from the Master plate 4x, a volume of 50 μL was added to each weil of the three white assay plates and 50 μL to the transparent plate for the cytotoxicity evaluation, all plates were covered bya sealing membrane to avoid evaporation of volatile test items and to avoid cross-contamination between wells, the plates were then incubated for 48 (± 2) hours at 3rC, 5% CO2, 90% humidity.

The runs 1 and 2 were performed using the following final concentrations: 0.977, 1.953, 3.906, 7.81, 15.63, 31.25, 62.5, 125, 250, 500, 1000 and 2000 μM in culture medium containing 1% DMSO.

Positive control results:

Cinnamic aldehyde was used as a positive control in concentrations of 4, 8, 16, 32 and 64 µM. The mean viability was above 100 % for all tested concentrations.
The luciferase activity induction obtained with the positive control, cinnamic aldehyde, was above the threshold of 1.5 in the two highest tested concentrations (mean 2.3 and 4.9 at 32 and 64 µM).
The EC1.5 value (geometric mean) was 14.9 (SD 5.6)
The average induction (lmax) for the positive control at 64 μM was 4.9 (SD 0.2)

Key result

Run / experiment:

other: Run 1

Parameter:

other: I max

Value:

2.5

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: Run 2

Parameter:

other: I max

Value:

1.3

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

mean

Parameter:

other: I max

Value:

1.9

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: Geometric mean

Parameter:

other: EC 1.5 [µM]

Value:

42.2

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Parameter:

other: cell viability at EC1.5 [%]

Value:

70

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: no

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

In the solubility test, the test item was found soluble in DMSO at 200 mM.

Results:

 

Test item	I max	EC 1.5
Run 1	2.5	42.2
Run 2	1.3	-
Mean	1.9	-
Geometric mean	-	42.2
SD	0.9	-

 

 

Viability of culture treated with the test item

 

Test item	0.977	1.953	3.906	7.81	15.63	31.25	62.5	125	250	500	1000	2000
Run 1	102	104	100	89	83	60	0	0	0	0	0	0
Run 2	94	89	86	81	74	60	27	0	0	-1	-1	6
Mean viability	98	96	93	85	78	63	13	0	0	-1	-1	3
Geometric mean viability	97	96	93	85	78	63	n.c.	n.c.	n.c.	n.c.	n.c.	0
SD	6	11	9	6	6	4	19	1	0	1	1	4

n.c.: not calclulated

 

Interpretation of results:

other: not sensitising

Remarks:

expert statement

Conclusions:

Under the experimental conditions of this study, the test item Rhamnolipids was considered to be negative in the KeratinoSens assay and therefore has no potential to induce genes that are regulated via a reaction with Nrf2 pathway.

Executive summary:

The in vitro the skin sensitization potential of Rhamnolipids (79% a.i.) was tested in the KeratinoSens assay detecting the electrophilic properties via a reaction with the nuclear factor Nrf2 pathway according to OECD Draft Guideline, In Vitro Skin Sensitization: ARE-Nrf2 Luciferase Test method; 16 May 2014.

The KeratinoSens cells were plated on 96-well plates and grown for 24 hours at 37°C. Then the medium was removed and the cells were exposed to the negative control and to several concentrations of test item and of positive controls. The treated plates were then incubated for 48 hours at 37°C. At the end of the treatment, cells were washed and the luciferase production was measured by flash luminescence. In parallel, the cytotoxicity was measured by a MTT reduction and was taken into consideration in the interpretation of the sensitization results. Two independent runs were performed. All concentrations take into account the rhamnolipid sample composition of 79% by applying a correction factor of 1.26. These runs were performed using the following final concentrations: 0.977, 1.953, 3.906, 7.81, 15.63, 31.25, 62.5, 125, 250, 500, 1000 and 2000 μM in culture medium containing 1% DMSO.

All acceptance criteria were fulfilled and the study was therefore considered to be valid. An important decrease in cell viability (viability < 70%) was noted from concentrations ≥ 31.25 μM. No statistically significant gene induction was noted in comparison to the negative control at the non-cytotoxic concentrations. A gene induction above 1.5 was noted at 62.5 μM in the first run but this concentration presented 100% toxicity. Therefore it was not considered as biologically significant. Up to the highest non cytotoxic concentration (15.63 μM), none of the evaluation criteria for a positive prediction were met; the test item was therefore considered to have a negative KeratinoSens prediction.

Under the experimental conditions of this study, Rhamnolipids was considered to be negative in the KeratinoSens assay and therefore has no potential to induce genes that are regulated via a reaction with Nrf2 pathway. This negative result is used to support the discrimination between skin sensitizers and non-sensitizers in the context of an integrated approach to testing and assessment.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

An in chemico and two in vitro tests are available for the assessment of sensitisation potential of Rhamnolipids. All tests were consistently negative. Overall, Rhamnolipids is not a dermal sensitizer.

 

In an in chemico skin sensitisation study performed according to the OECD draft proposal for a new test guideline 442C (Direct Peptide Reactivity Assay) the reactivity of the test item Rhamnolipids was evaluated by monitoring peptide depletion following a 24-hour contact between the test item and synthetic cysteine and lysine peptides.

Analysis of the chromatograms of the co-elution samples indicated that the test item did not co-elute with either the lysine or the cysteine peptides. As a result, the mean percent depletion values were calculated for each peptide. For the cysteine peptide, the mean depletion value was 6.59% and for the lysine peptide, the mean depletion value was 1.68%.

Since the mean of the percent cysteine and percent lysine depletions was equal to 4.13%, the test item was considered to have no/minimal peptide reactivity. Therefore, the DPRA prediction would be considered as negative and the test item may have no potential to cause skin sensitisation.

 

The in vitro the skin sensitization potential of Rhamnolipids (79% a.i.) was tested in the KeratinoSens assay detecting the electrophilic properties via a reaction with the nuclear factor Nrf2 pathway according to OECD Draft Guideline, In Vitro Skin Sensitization: ARE-Nrf2 Luciferase Test method; 16 May 2014.

All acceptance criteria were fulfilled and the study was therefore considered to be valid. An important decrease in cell viability (viability < 70%) was noted from concentrations ≥ 31.25 μM. No statistically significant gene induction was noted in comparison to the negative control at the non-cytotoxic concentrations. A gene induction above 1.5 was noted at 62.5 μM in the first run but this concentration presented 100% toxicity. Therefore it was not considered as biologically significant. Up to the highest non cytotoxic concentration (15.63 μM), none of the evaluation criteria for a positive prediction were met; the test item was therefore considered to have a negative KeratinoSens prediction.

Under the experimental conditions of this study, Rhamnolipids was considered to be negative in the KeratinoSens assay and therefore has no potential to induce genes that are regulated via a reaction with Nrf2 pathway. This negative result is used to support the discrimination between skin sensitizers and non-sensitizers in the context of an integrated approach to testing and assessment.

 

The capability of Rhamnolipids (79% a.i.) to induce the surface marker CD86 expression in the human myeloid U937 cell line was evaluated in a study 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.