Rhamnolipids: fermentation products of glucose with Pseudomonas putida, Pseudomonadaceae

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014-06-23 to 2014-07-17

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

(26 July 2013)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

(06-Jul-2012)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: no data

Justification for test system used:

recommended by guideline

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Episkin Small Model
This skin model consists of normal (non-cancerous), adult human-derived epidermal keratinocytes (NHEK) which have been cultured to form a multilayered, highly differentiated model of the human epidermis. The NHEK are cultured on chemically modified, collagen-coated cell culture inserts. A highly differentiated and stratified epidermis model is obtained after 13-day culture period comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.
- Tissue batch number(s): Lot: 14-EKIN-024
- certification date: 2014-07-01
- 'Expiration date: 2014-07-07

- Date of initiation of testing: 2014-06-26

TEMPERATURE USED FOR TEST SYSTEM
Upon receipt of the EPISKIN-SM™, the tissues were transferred into 12-well plates containing 2 mL prewarmed maintenance medium per well
- Conditions used during pre incubation: 37 ± 1°C, 5.0% CO2 for at least 24 h
After this pre-incubation the tissues were treated with each dose group in triplicate, starting with the negative control. Start time was recorded with dosing of the first tissue.
- Temperature used during treatment / exposure: room temperature for 15 ± 0.5 min
Then the tissues were incubated at room temperature for 15 ± 0.5 min. Afterwards, the tissues were washed with PBS to remove any residual test item. Excess PBS was removed by blotting bottom with blotting paper. The inserts were placed in a prepared 12-well plate containing 2 mL prewarmed fresh maintenance medium and post-incubated
- Temperature of post-treatment incubation (if applicable): 37 ± 1°C 5.0% CO2 for 42 ± 1 h

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: tissues were washed with PBS, Excess PBS was removed by blotting bottom with blotting paper. The inserts were placed in a prepared 12-well plate containing 2 mL prewarmed fresh maintenance medium
- Observable damage in the tissue due to washing: not recorded
- Modifications to validated SOP:

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration:
MTT stock solution: 3 mg/mL MTT (Applichem; Lot 2G002451) in PBS (Gibco; Lot 1563913)
MTT medium: MTT stock solution diluted 1 + 9 with OMEM-based medium (final concentration 0.3 mg/mL)
- Incubation time: 3 h ± 5 min. at 37 ± 1°C, 5.0% CO2
After this incubation period the plates were placed for 15 ± 2 min. on a plate shaker. Then the inserts were transferred in a prepared 12-well plate containing 2 mL prewarmed MTT medium and further incubated for 3 h ± 5 min. at 37 ± 1°C, 5.0% CO2. After the 3 h MTT incubation period the tissues were placed on blotting paper to dry the tissues. Afterwards a total biopsy of the epidermis by using the special biopsy punch was performed and the epidermis was separated from the collagen matrix with the aid of forceps. Both parts (epidermis and collagen matrix) were transferred into suitable tubes and 500 μL of acidic isopropanol were added. Extraction was carried out protected from light over the weekend at 2 - 8°C. At the end of the formazan extraction period the tubes were mixed by vortexing until solution colour became homogeneous.
If any visible cell/tissue fragments were in suspension, the tubes were centrifuged at 500 rpm to eliminate the fragments and avoid further possible interference with the absorbance readings.
Per each tissue 2 x 200 μL aliquots of the extract were transferred into a 96-well plate and OD was measured at 550 nm without reference wavelength in a plate spectrophotometer.

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
-Morphology:
Histology scoring (HES stained vertical paraffin sections, n = 6):
specification ~ 19.5
result: 22.2 ± 0.3, CV = 1.2%
Well-differentiated epidermis consisting of a basal layer, several spinous and granular layers and a thick stratum corneum.
Barrier function:
IC50 determination (SOS concentration, MTT test, n = 14):
specification ~ 1.5 mg/mL
result: 2.3 mg/mL

NUMBER OF REPLICATE TISSUES: 3


NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION:

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test item is considered to be irritant to skin in accordance with regulation EC 1272/2008 and UN GHS Category 2, if the tissue viability after 15 min of exposure and 42 h of post-incubation is less or equal to 50%.
- The test substance may be considered as non-irritant to skin in accordance with UN GHS No Category if the tissue viability after exposure and posttreatment incubation is higher than 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 mg (26.3 mg/cm3)

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 10 μL PBS
- Concentration (if solution):

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 10 μL SDS
- Concentration (if solution): 5% solution

Duration of treatment / exposure:

15 ± 0.5 min

Duration of post-treatment incubation (if applicable):

42 ± 1 h

Number of replicates:

3

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

test substance

Value:

92.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

OD550: 0.898

Positive controls validity:

valid

Remarks:

23.2 % tissue viability

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: The mixture of 10 mg test item per 2 mL MTT medium showed no reduction of MTT compared to the solvent. The mixture did not turn blue/purple.
- Colour interference with MTT: The mixture of 10 mg of the test item per 90 μI aqua dest. showed no colouring detectable by unaided eye-assessment.

DEMONSTRATION OF TECHNICAL PROFICIENCY:

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

Any other information on results incl. tables

	Negative Control			Positive Control			Test Item
Tissue	1	2	4	1	2	3	1	2	3
Mean OD550 of the duplicates  (blank corrected)	0.850	0.893	0.823	0.191	0.183	0.221	0.810	0.739	0.833
Total mean OD550 of 3 replicates  (blank corrected)	0.855*			0.199			0.794
SDOD550	0.032			0.018			0.044
Relative tissue viabilities %	99.3	104.4	96.3	22.4	21.4	25.9	94.7	86.5	97.4
Mean relative tissue viability	100.0			23.2**			92.9
SD tissue viability %***	4.1			2.3			5.7

 

*Corrected mean OD550of the negative control corresponds to 100% absolute tissue viability.

**Mean relative tissue viability of the three positive control tissues is≤40%.

***The standard deviation (SD) obtained from the three concurrently tested tissues is < 18%.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test item Rhamnolipids was not irritating in the in vitro skin irritation test under the experimental conditions described in this report.

Executive summary:

In a dermal irritation study performed in accordance with OECD Guideline 439 (In Vitro Skin Irritation) (adopted 26 July 2013), the test item Rhamnolipids (79% a.i.) was applied to the three-dimensional human epidermis model tissue for an exposure period of 15 minutes in triplicates. 5 μL of deionised water were topically applied to the epidermal surface in order to improve further contact between the solid and the epidermis. Each approximately 10 mg of the test item were applied to the wetted tissues.

After 15 minutes exposure at room temperature, the tissues were washed with phosphate buffered saline to remove any residual test material. Subsequently the tissue constructs were incubated for 42 h at 37°C. Cytotoxicity (irritancy) was expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.

The positive (5% SDS) and negative (PBS) control gave responses that were within the acceptance criteria and as such demonstrated the validity of the study.

The relative mean tissue viability obtained after 15 minutes treatment with the testsubstanceRhamnolipidscompared to the negative control tissues was 92.9%. Since the mean relative tissue viability for the test substance was above 50%, the test item is identified to be not irritating.

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