Rhamnolipids: fermentation products of glucose with Pseudomonas putida, Pseudomonadaceae

Endpoint summary

Description of key information

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 test substance Rhamnolipids compared 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 to the skin.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

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

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(26 July 2013)

Deviations:

no

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 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

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

	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%.

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2014-06-26 to 2014-07-11

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

pH of the preparation : 7.0
Concentration of the tracer : 79% of Rhamnolipids
Dilution rate : 1.2

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: bovine eyes (from cattle less than 12 months old) collected at the slaughterhouses
of La Talaudiere
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): at room temperature, and used 4 hours maximum after killing the animals.

Vehicle:

water

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): neat solid - (previously powdered) 750 ± 75 mg, preparation 750 ± 8 μL

- Concentration (if solution): 10% (w/w) in distilled water

Duration of treatment / exposure:

30 ± 5 minutes

Duration of post- treatment incubation (in vitro):

10 ± 1 minutes

Number of animals or in vitro replicates:

3

Details on study design:

QUALITY CHECK OF THE ISOLATED CORNEAS
- measurement of the initial opacity (OPTO) of each cornea using an opacitometer OPKlT, which determines a change in light transmission between an "empty" holder (containing nutritive medium only) and a "treated" holder, and displays a numerical value of opacity

NUMBER OF REPLICATES: 3

NEGATIVE CONTROL USED: nutritive medium

POSITIVE CONTROL USED: Cetyl Trimethylarnmonium Bromide (CTAB - CAS N° 57-09-0) to 0.5% (W/W) in sterile water (CAS N° 7732-18-5), heated to 32 ± 1°C, during 10 minutes ± 2 minutes, until complete dissolution

APPLICATION DOSE AND EXPOSURE TIME: - tested as supplied and extemporaneous dilution to 10% (W/W) in distilled water exposure time 30 min and 10 min

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: 3 times for each control and 4 times for the test item as supplied and
3 times for the diluted test item with nutritive medium at 32 ± 1 "C with a 50 mL syringe
- POST-EXPOSURE INCUBATION: yes after rinsing incubation of the holders in a water-bath at about 32 ± 1 "C, in a horizontal position, for 2 hours ± 10 minutes.


METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: - replacing of the nutritive medium in the posterior compartrnent using a 50 mL syringe
- wiping of each holder
- measurement of the opacity OPT2 (= OPT "2 hours") of each cornea versus an "empty" holder (containing nutritive medium only).
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader (OD490)
- Others (e.g, pertinent visual observations, histopathology): observation of the cornea condition: epithelium detachment, visible modification of the cornea (edema, colouring ... ).

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
Corneal score = (OPT2 - OPTO) + (15 x O.D.)
Classsification table given below

DECISION CRITERIA: please specify if the decision criteria as indicated in the TG was used: no

Irritation parameter:

in vitro irritation score

Remarks:

30 min

Run / experiment:

neat substance

Value:

96.7

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Irritation parameter:

in vitro irritation score

Remarks:

10 min

Run / experiment:

neat substance

Value:

52.7

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Irritation parameter:

in vitro irritation score

Remarks:

30 min

Run / experiment:

10 % (w/w) dilution

Value:

104.4

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Irritation parameter:

in vitro irritation score

Remarks:

10 min

Run / experiment:

10% (w/w) dilution

Value:

90.4

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

- the results of the positive control (C.T.A.B. to 0.5%) are in conformity with the awaited data
- the results of the calibrators of the opacitometer are in conformity with the awaited data
- the results of the opacity and the O.D. of the negative control (nutritive medium) are in conformity with the criteria of validity of the test
- the results of the O.D. of fluorescein (5 μg/mL) are in conformity with the criteria of validity of the test

Other effects:

Detachment oft the epithelium and edema in all cornea treated with the testsubstance and the positive control samples (contact timepoints 10 min and 30 min)

Results after 30 minutes contact timepoint

Treatment	Mean Opacity	Mean Permeability	Mean In vitro Irritation Score
Negative control nutritive medium	-2.7 ± 0.6	0.013 ± 0.004
Positive Control CTAB 0.5%	34.3 ± 1.5	4.230 ± 2.03	97.8 ± 2.9
Test substance as supplied	3.0 ± 0.6	6.244 ± 0.068	96.7 ± 0.6
Test substance diluted to 10%	2.0 ± 1.2	6.830 ± 0.217	104.4± 2.2

 

Results after 10 minutes contact timepoint

Treatment	Mean Opacity	Mean Permeability	Mean In vitro Irritation Score
Negative control nutritive medium	0.7±1.2	0.027± 0.
Positive Control CTAB 0.5%	16.0± 1.5	0.978± 0.072	30.7± 1.4
Test substance as supplied	1.3± 0.0	3.425± 0.130	52.7 ± 1.9
Test substance diluted to 10%	4.3± 1.0	5.740± 0.077	90.4 ± 0.2

 

Interpretation of results:

other: expert statement

Remarks:

severely irritating / corrosive

Conclusions:

From the results obtained under the experimental conditions adopted, the test item, applied as supplied and diluted to 10% (WIW) in distilled water is classified "IRRITANT TO SEVERELY IRRITANT" for the isolated bovine cornea, after 30 and 10 minutes of contact.

Executive summary:

This in vitro study was performed to assess the corneal irritation and damage potential of Rhamnolipid by means of the BCOP assay using fresh bovine corneas comparable to OECD guideline 437.

The corneas were incubated with the test substance 30 ± 5 minutes or 10 ± 1 minutes at 32±1°C. The test was performed in triplicates. After rinsing the corneas at least 3 times, opacity and permeability were determined. The in vitro irritancy score (IVIS) were calculated as mean opacity value + (15 x mean OD490 value); a substance that induces an IVIS ≥ 55.1 is defined as a corrosive or severe irritant.

The positive and negative controls induced the appropriate responses.

The corneas treated with the neat Rhamnolipids showed opacity values ranging from 2.7 to 3.7 and and permeability values ranging from 6.188 to 6.324 after 30 min contact time and opacity values of 1.3 and permeability values ranging from 3.285 to 3.541 after 10 min contact time. The mean in vitro irritancy score was 96.7 after 30 minutes of treatment and 52.7 after 10 minutes of treatment.

The corneas treated with the 10% dilution Rhamnolipids showed opacity values ranging from 7.7 to 2.7 and and permeability values ranging from 6.635 to 7.076 after 30 min contact time and opacity values of 3.3 to 5.3 and permeability values ranging from 3.5.657 to 5.809 after 10 min contact time. The mean in vitro irritancy score was 104.4 after 30 minutes of treatment and 90.4 after 10 minutes of treatment.

Since the mean in vitro irritancy scores after 30 min of contact were above 55.1 and after 10 min of contact point were above 10, the test substance Rhamnolipids is considered to be severely irritating/ corrosive in the Bovine Corneal Opacity and Permeability test under the experimental conditions described in this report.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-19-03 to 2015-08-24

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

adopted 26 July 2013

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Substance was tested at different pH values and various concentrations:
pH 5.8; 100%, 50% and 10%
pH 7.0; 100%, 50% and 10%

Species:

chicken

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: ROSS, spring chickens from poultry slaughterhouse v.d. Bor, Nijkerkerveen, the Netherlands
- Age at study initiation: Approximately 7 weeks old
- Weight at study initiation: body weight range approximately 1.5 - 2.5 kg

Heads of the animals were cut off immediately after sedation of the animals by electric shock and incision of the neck for bleeding, and before they reached the next station on the process line. The heads were placed in small plastic boxes on a bedding of paper tissues moistened with isotonic saline. Next, they were transported to the testing facility. During transportation, the heads were kept at ambient temperature.

Within 2 hours after kill, eyes were carefully dissected and placed in a superfusion apparatus. Eyes with a corneal thickness deviating more than 10% of the average corneal thickness of the eyes, eyes that showed opacity (score higher than 0.5), were unacceptably stained with fluorescein (score higher than 0.5), or showed any other signs of damage were rejected and were replaced.

After an equilibration period of 45-60 minutes, the corneal thickness of the eyes was measured once more to determine the zero reference value for corneal swelling calculations.

Vehicle:

unchanged (no vehicle)

Remarks:

liquids used as supplied by the sponsor

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 mg for solid test substance; 30 µL for liquid preparations

Duration of treatment / exposure:

10 s

Observation period (in vivo):

eyes were examined at approximately 0, 30, 75, 120, 180 and 240 minutes after treatment

Number of animals or in vitro replicates:

negative control (physiological saline, 30 µL): 2
positive control (NaOH, 30 mg): 3
test group: 3 per concentration / pH

Details on study design:

REMOVAL OF TEST SUBSTANCE - Washing (if done): 20 mL saline
- Time after start of exposure: 10 s
SCORING SYSTEM: according to ICE classification criteria OECD TG 438
TOOL USED TO ASSESS SCORE: slit lamp microscope / fluorescein

At time t=0, i.e. immediately after the zero reference measurement, the following procedure was applied for each test eye:
The clamp holding the test eye was placed on paper tissues outside the chamber with the cornea facing upwards. Next, three corneas were treated with 30 mg test item. After an exposure period of 10 seconds, the corneal surface was rinsed thoroughly with 20 mL of isotonic saline at ambient temperature. After rinsing, each eye in the holder was returned to its chamber. The eyes were examined at 0, 30, 75, 120, 180 and 240 minutes after treatment. All examinations were performed with a slit-lamp microscope. Fluorescein retention was scored only at 30 minutes after treatment.
After the final examination, the test and control eyes were preserved in a neutral aqueous phosphate-buffered solution of 4% formaldehyde.
The tissues selected were embedded in paraffin wax, sectioned at 5 μm and stained with Periodic Acid-Schiff for histopathology examination. Ocular effects were evaluated using the endpoints of corneal thickness (swelling), corneal opacity and fluorescein retention.

Irritation parameter:

percent corneal swelling

Run / experiment:

100% pH 5.8

Value:

33

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Irritation parameter:

cornea opacity score

Run / experiment:

100% pH 5.8

Value:

3.5

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Irritation parameter:

fluorescein retention score

Run / experiment:

100% pH 5.8

Value:

2.8

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Irritation parameter:

percent corneal swelling

Run / experiment:

100 % pH 7

Value:

16

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Irritation parameter:

cornea opacity score

Run / experiment:

100 % pH 7

Value:

2.3

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Irritation parameter:

fluorescein retention score

Run / experiment:

100 % pH 7

Value:

2.2

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Irritation parameter:

percent corneal swelling

Run / experiment:

50% pH 5.8

Value:

28

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Irritation parameter:

cornea opacity score

Run / experiment:

50% pH 5.8

Value:

3

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Irritation parameter:

fluorescein retention score

Run / experiment:

50% pH 5.8

Value:

3

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
The test item at 50% / pH 5.8 and 100% / pH 5.8 caused corneal effects leading to Category 1 classification, consisting of moderate or severe corneal swelling (28 and 33%), severe or severe to very severe opacity (mean score of 3.0 or 3.5) and severe or moderate to severe fluorescein retention (mean score of 2.8 or 3.0). In addition, loosening (veil-like) of epithelium was observed.

Microscopic examination of the corneas treated with teh test item at 100% / pH 5.8 revealed very slight, slight or severe erosion and very slight vacuolation (one cornea top region; one cornea low region) of the epithelium. In addition, the epithelium top ceillayer was hypertrophic in one cornea.
Microscopic examination of the corneas treated with the test item at 50% / pH 5.8 revealed slight or moderate erosion and slight vacuolation (one cornea mid region) of the epithelium. In addition, the epithelium top cell layer was hypertrophic all corneas.
The AlSE histopathology criteria for upgrading to category 1 were met, but did not need to be applied, because it was already classified as category 1 on the basis of the slit-Iamp observations.

The test item at 100% / pH 7.0 caused corneal effects leading to Category 2/2A classification, consisting of slight corneal swelling (16%), moderate or moderate to severe opacity (mean score of 2.3) and moderate or moderate to severe fluorescein retention (mean score of 2.2). In addition, loosening (veil-like) of epithelium was observed.
Microscopic examination ofthe corneas treated with the test item at 100% / pH 7.0 revealed moderate or severe erosion, slight necrosis (one cornea) and very slight (one cornea low region) or slight (one cornea mid region) vacuolation of the epithelium.
According to the AlSE histopathology criteria, upgrading to category 1 on basis of the histopathology of the corneas is required.


DEMONSTRATION OF TECHNICAL PROFICIENCY:

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

The negative control eyes did not show any corneal effect and demonstrated that the general conditions during the tests were adequate. The positive control NaOH caused (very) severe corneal effects and demonstrated the ICE test valid to detect severe eye irritants. Microscopic examination of the corneas treated with the negative control (saline) did not reveal any abnormalities. The positive control NaOH caused very slight, moderate or severe erosion and slight necrosis of the epithelium, and endothelial necrosis (one cornea).

Summary results of the slit-Iamp examination

Test material	Maximum mean score for:			Irritation categories^1	Irritation Index^2	Classifications (EU-CLP^3/UN-GHS^4)
	Swelling	Opacity	Fluorescin retention
test item(pH 5.8)_100%	33	3.5^5	2.8	IV;IV;IV	159	1/1
test item (pH 5.8)_50%	28	3.0^5	3.0	III;IV;IV	148	1/1
test item (pH 5.8)_10%	6	1.3^5	2.0	II;II;III	72	2/2B
test item (pH 7.0)_100%	16	2.3^5	2.2	II;III;III	106	1^7/1^7
test item (pH 7.0)_50%	8	1.3^5	2.0	II;II;III	74	2/2B
test item (pH 7.0)_10%	8	1.5^5	2.0	II;II;III	78	2/2B
NaOH (positive control)	46	4.0^6	3.0	IV;IV;IV	186	1/1

 

1 I= no effect; II = slight effect; III = moderate effect; IV = severe effect.

2 Irritation Index = maximum mean corneal swelling + maximum mean opacity (x 20) + mean fluorescein score (x 20)

3 EU-CLP: NC = not classified; Category 2 = Irritating to eyes; Category 1 = irreversible effects on the eye/serious damage to the eye. Regulation (EC)

No 1272/2808 of the European Parliament and of the Council of 16 December 2008 on classification, labelling and packaging of substances and mixtures, amending and repealing Oirectives 67/548/EEC and 1999/45/EC, and amending Regulation (EC) No 1907/2806.

4 UN-GHS: NC = not classified; Category 2B = mild irritant, causes eye irritation; Category 2A = irritant, causes eye irritation; Category 1 = irreversible effects on the eye/serious damage to the eye. United Nations-Economic Commission for Europe (UN/ECE) (2003). Globally Harmonised System of

Classification and Labelling of Chemicals (GHS). UN, New York and Geneva, 2007.

5 Loosening (veil-like) of epithelium

6 Severe loosening of epithelium

7 According to the AlSE histopathology criteria, upgrading to category 1 on basis of the histopathology of the corneas is required.

 

 

Individual histopathological findings

Test material	Eye no	Epithelium						Stroma			Endothelium
		Erosion	Necrosis	Vacuolation			Notes	Disorder of fibers	Pyknotic nuclei
				top	mid	low			outer region (adjacent to epithelium)	inner region (adjacent to endothelium)
test item 100% pH 5.8	4	3	-	-	-	-	-	-	-	-	-
	6	1	-	-	-	½	-	-	-	-	-
	8	½	-	½	-	-	‡	-	-	-	-
test item 100% pH 7.0	5	3	-	-	-	-	-	-	-	-	-
	7	2	1	-	1	-	-	-	-	-	-
	9	2	-	-	-	½	-	-	-	-	-
NaOH (positive control)	1	3	1	-	-	-	-	-	-	-	P
	2	2	1	-	-	-	-	-	-	-	-
	3	½	1	-	-	-	-	-	-	-	-
Saline (negative control)	10	-	-	-	-	-	-	-	-	-	-

- = not observed; P = present; 1/2 = very slight; 1 = slight; 2 = moderate; 3 = severe;

† = scored in the top/mid/low section of the epithelium; ‡ = epithelium top cell layer hypertrophic

 

Test material	Eye no	Epithelium						Stroma			Endothelium
		Erosion	Necrosis	Vacuolation			Notes	Disorder of fibers	Pyknotic nuclei
				Top	mid	low			outer region (adjacent to epithelium)	inner region (adjacent to endothelium)
test item 50% pH 5.8	21	2	-	-	-	-	‡	-	-	-	-
	25	1	-	-	-	-	‡	-	-	-	-
	29	2	-	-	1	-	‡	-	-	-	-
test item 50% pH 7.0	22	½	-	1	-	-	‡	-	-	-	-
	26	½	-	1	-	-	‡	-	-	-	-
	30	½	-	-	-	-	-	-	-	-	-
test item 10% pH 5.8	23	½	-	-	-	-	‡	-	-	-	-
	27	½	1	-	-	-	‡	-	-	-	-
	31	½	1	½	-	-	‡	-	-	-	-
test item 10% pH 7.0	24	½	½	½	-	-	‡	-	-	-	-
	28	½	1	-	-	-		-	-	-	-
	32	½	½	-	-	-	‡	-	-	-	-
Saline (negative control)	33	-	-	-	-	-	-	-	-	-	-

- = not observed; P = present; 1/2 = very slight; 1 = slight; 2 = moderate; 3 = severe;

† = scored in the top/mid/low section of the epithelium; ‡ = epithelium top cell layer hypertrophic

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Conclusions:

Based on the results from this Isolated Chicken Eye test, the testsubstance Rhamnolipids applied neat (100%) is considered "Irreversible effects on the eye/serious damage to the eye" (Category 1) under the experimental conditions described in this report (pH 7 and pH 5.8).

Based on the results from this Isolated Chicken Eye test, the testsubstance Rhamnolipids applied as a 50% dilution at pH 5.8 is considered "Irreversible effects on the eye/serious damage to the eye" (Category 1) under the experimental conditions described in this report.

Executive summary:

In an Isolated Chicken Eye (ICE) test according to OECD Guideline 438 (adopted 26 July 2013), the eye irritation potential of the testsubstance Rhamnolipids was assessed at different concentrations and pH vaues. In addition, the test included a negative control (saline) and a positive control (NaOH). Chicken eyes were obtained from slaughter animals used for human consumption.

The isolated chicken eyes were exposed to a single application of 30 mg for 10 seconds followed by a 20 mL saline rinse. Three main parameters were measured to disclose possible adverse eye effects: corneal thickness (expressed as corneal swelling), corneal opacity and fluorescein retention of damaged epithelial cells. In addition, histopathology of the corneas was performed.

 

The test item at 50% / pH 5.8 and 100% / pH 5.8 caused corneal effects leading to Category 1 classification, consisting of moderate or severe corneal swelling (28 and 33%), severe or severe to very severe opacity (mean score of 3.0 or 3.5) and severe or moderate to severe fluorescein retention (mean score of 2.8 or 3.0). In addition, loosening (veil-like) of epithelium was observed.

Microscopic examination of the corneas treated with the test item at 100% / pH 5.8 revealed very slight, slight or severe erosion and very slight vacuolation (one cornea top region; one cornea low region) of the epithelium. In addition, the epithelium top ceillayer was hypertrophic in one cornea.

Microscopic examination of the corneas treated with the test item at 50% / pH 5.8 revealed slight or moderate erosion and slight vacuolation (one cornea mid region) of the epithelium. In addition, the epithelium top cell layer was hypertrophic all corneas.

The AlSE histopathology criteria for upgrading to category 1 were met, but did not need to be applied, because it was already classified as category 1 on the basis of the slit-Iamp observations.

The test item at 100% / pH 7.0 caused corneal effects leading to Category 2/2A classification, consisting of slight corneal swelling (16%), moderate or moderate to severe opacity (mean score of 2.3) and moderate or moderate to severe fluorescein retention (mean score of 2.2). In addition, loosening (veil-like) of epithelium was observed.

Microscopic examination of the corneas treated with the test item at 100% / pH 7.0 revealed moderate or severe erosion, slight necrosis (one cornea) and very slight (one cornea low region) or slight (one cornea mid region) vacuolation of the epithelium.

According to the AlSE histopathology criteria, upgrading to category 1 on basis of the histopathology of the corneas is required.

 

The negative control eye did not show any corneal effect and demonstrated that the general conditions during the tests were adequate.

The positive control NaOH caused severe corneal effects and demonstrated the ICE test valid to detect severe eye irritants.

Reference 3

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-19-03 to 2015-08-24

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

(adopted 26 July 2013)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Substance was tested at different pH values and various concentrations:
pH 5.8; 100%, 50% and 10%
pH 7.0; 100%, 50% and 10%

Species:

chicken

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: ROSS, spring chickens from poultry slaughterhouse v.d. Bor, Nijkerkerveen, the Netherlands
- Age at study initiation: Approximately 7 weeks old
- Weight at study initiation: body weight range approximately 1.5 2.5 kg

Heads of the animals were cut off immediately after sedation of the animals by electric shock and incision of the neck for bleeding, and before they reached the next station on the process line. The heads were placed in small plastic boxes on a bedding of paper tissues moistened with isotonic saline. Next, they were transported to the testing facility. During transportation, the heads were kept at ambient temperature.

Within 2 hours after kill, eyes were carefully dissected and placed in a superfusion apparatus. Eyes with a corneal thickness deviating more than 10% of the average corneal thickness of the eyes, eyes that showed opacity (score higher than 0.5), were unacceptably stained with fluorescein (score higher than 0.5), or showed any other signs of damage were rejected and were replaced.

After an equilibration period of 45-60 minutes, the corneal thickness of the eyes was measured once more to determine the zero reference value for corneal swelling calculations.

Vehicle:

not specified

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 mg for solid testsubstance; 30 µL for liquid preparations

Duration of treatment / exposure:

10 s

Observation period (in vivo):

eyes were examined at approximately 0, 30, 75, 120, 180 and 240 minutes after treatment

Number of animals or in vitro replicates:

negative control (physiological saline, 30 µL): 2
positive control (NaOH, 30 mg): 3
test group: 3

Details on study design:

REMOVAL OF TEST SUBSTANCE - Washing (if done): 20 mL saline
- Time after start of exposure: 10 s
SCORING SYSTEM: according to ICE classification criteria OECD TG 438
TOOL USED TO ASSESS SCORE: slit lamp microscope / fluorescein

At time t=0, i.e. immediately after the zero reference measurement, the following procedure was applied for each test eye:
The clamp holding the test eye was placed on paper tissues outside the chamber with the cornea facing upwards. Next, three corneas were treated with 30 mg test item. After an exposure period of 10 seconds, the corneal surface was rinsed thoroughly with 20 mL of isotonic saline at ambient temperature. After rinsing, each eye in the holder was returned to its chamber. The eyes were examined at 0, 30, 75, 120, 180 and 240 minutes after treatment. All examinations were performed with a slit-lamp microscope. Fluorescein retention was scored only at 30 minutes after treatment.
After the final examination, the test and control eyes were preserved in a neutral aqueous phosphate-buffered solution of 4% formaldehyde.
The tissues selected were embedded in paraffin wax, sectioned at 5 μm and stained with Periodic Acid-Schiff for histopathology examination. Ocular effects were evaluated using the endpoints of corneal thickness (swelling), corneal opacity and fluorescein retention.

Irritation parameter:

percent corneal swelling

Run / experiment:

50% pH 7

Value:

8

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Irritation parameter:

cornea opacity score

Run / experiment:

50% pH 7

Value:

1.3

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Irritation parameter:

fluorescein retention score

Run / experiment:

50% pH 7

Value:

2

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Irritation parameter:

percent corneal swelling

Run / experiment:

10% pH 7

Value:

8

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Irritation parameter:

cornea opacity score

Run / experiment:

10% pH 7

Value:

1.5

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Irritation parameter:

fluorescein retention score

Run / experiment:

10% pH 7

Value:

2

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Irritation parameter:

percent corneal swelling

Run / experiment:

10% pH 5.8

Value:

6

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Irritation parameter:

cornea opacity score

Run / experiment:

10% pH 5.8

Value:

1.3

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Irritation parameter:

fluorescein retention score

Run / experiment:

10% pH 5.8

Value:

2

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:

The test item at 50% / pH 7.0 and 10% / pH 7.0 caused corneal effects leading to Category 2/2B classification, consisting of slight corneal swelling (8%), slight or slight to moderate opacity (range mean score of 1.3 or 1.5) and moderate fluorescein retention (mean score of 2.0).

Microscopic examination of the corneas treated with the test item at 50% / pH 7.0 revealed very slight erosion and slight vacuolation (two corneas top region) of the epithelium. In addition, the epithelium top cell layer was hypertrophic in two corneas. Microscopic examination of the corneas treated with the test utem at 10% / pH 7.0 revealed very slight erosion, very slight or slight necrosis and very slight vacuolation (one cornea top region) of the epithelium. In addition, the epithelium top cell layer was hypertrophic in two corneas. According to the AlSE histopathology criteria, upgrading to category 1 on basis of the histopathology of the corneas is not required.

The test item at 10% / pH 5.8 caused corneal effects leading to Category 2/2B classification, consisting of slight corneal swelling (6%), slight or slight to moderate opacity (mean score of 1.3) and moderate fluorescein retention (mean score of 2.0). In addition, loosening (veil-like) of epithelium was observed. Microscopic examination of the corneas revealed very slight erosion, slight necrosis (two corneas) and very slight vacuolation (one cornea top region) of the epithelium.. In addition, the epithelium top cell layer was hypertrophic in all corneas. According to the AlSE histopathology criteria, upgrading to category 1 on basis of the histopathology of the corneas is not required.


DEMONSTRATION OF TECHNICAL PROFICIENCY:

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


The negative control eyes did not show any corneal effect and demonstrated that the general conditions during the tests were adequate. The positive control NaOH caused (very) severe corneal effects and demonstrated the ICE test valid to detect severe eye irritants. Microscopic examination of the corneas treated with the negative control (saline) did not reveal any abnormalities. The positive control NaOH caused very slight, moderate or severe erosion and slight necrosis of the epithelium, and endothelial necrosis (one cornea).

Summary results of the slit-Iamp examination

Test material	Maximum mean score for:			Irritation categories^1	Irritation Index^2	Classifications (EU-CLP^3/UN-GHS^4)
	Swelling	Opacity	Fluorescin retention
test item (pH 5.8)_100%	33	3.5^5	2.8	IV;IV;IV	159	1/1
test item (pH 5.8)_50%	28	3.0^5	3.0	III;IV;IV	148	1/1
test item (pH 5.8)_10%	6	1.3^5	2.0	II;II;III	72	2/2B
test item (pH 7.0)_100%	16	2.3^5	2.2	II;III;III	106	1^7/1^7
test item(pH 7.0)_50%	8	1.3^5	2.0	II;II;III	74	2/2B
test item (pH 7.0)_10%	8	1.5^5	2.0	II;II;III	78	2/2B
NaOH (positive control)	46	4.0^6	3.0	IV;IV;IV	186	1/1

 

1 I= no effect; II = slight effect; III = moderate effect; IV = severe effect.

2 Irritation Index = maximum mean corneal swelling + maximum mean opacity (x 20) + mean fluorescein score (x 20)

3 EU-CLP: NC = not classified; Category 2 = Irritating to eyes; Category 1 = irreversible effects on the eye/serious damage to the eye. Regulation (EC)

No 1272/2808 of the European Parliament and of the Council of 16 December 2008 on classification, labelling and packaging of substances and mixtures, amending and repealing Oirectives 67/548/EEC and 1999/45/EC, and amending Regulation (EC) No 1907/2806.

4 UN-GHS: NC = not classified; Category 2B = mild irritant, causes eye irritation; Category 2A = irritant, causes eye irritation; Category 1 = irreversible effects on the eye/serious damage to the eye. United Nations-Economic Commission for Europe (UN/ECE) (2003). Globally Harmonised System of

Classification and Labelling of Chemicals (GHS). UN, New York and Geneva, 2007.

5 Loosening (veil-like) of epithelium

6 Severe loosening of epithelium

7 According to the AlSE histopathology criteria, upgrading to category 1 on basis of the histopathology of the corneas is required.

 

 

Individual histopathological findings

Test material	Eye no	Epithelium						Stroma			Endothelium
		Erosion	Necrosis	Vacuolation			Notes	Disorder of fibers	Pyknotic nuclei
				top	mid	low			outer region (adjacent to epithelium)	inner region (adjacent to endothelium)
test item 100% pH 5.8	4	3	-	-	-	-	-	-	-	-	-
	6	1	-	-	-	½	-	-	-	-	-
	8	½	-	½	-	-	‡	-	-	-	-
test item 100% pH 7.0	5	3	-	-	-	-	-	-	-	-	-
	7	2	1	-	1	-	-	-	-	-	-
	9	2	-	-	-	½	-	-	-	-	-
NaOH (positive control)	1	3	1	-	-	-	-	-	-	-	P
	2	2	1	-	-	-	-	-	-	-	-
	3	½	1	-	-	-	-	-	-	-	-
Saline (negative control)	10	-	-	-	-	-	-	-	-	-	-

- = not observed; P = present; 1/2 = very slight; 1 = slight; 2 = moderate; 3 = severe;

† = scored in the top/mid/low section of the epithelium; ‡ = epithelium top cell layer hypertrophic

 

Test material	Eye no	Epithelium						Stroma			Endothelium
		Erosion	Necrosis	Vacuolation			Notes	Disorder of fibers	Pyknotic nuclei
				Top	mid	low			outer region (adjacent to epithelium)	inner region (adjacent to endothelium)
test item 50% pH 5.8	21	2	-	-	-	-	‡	-	-	-	-
	25	1	-	-	-	-	‡	-	-	-	-
	29	2	-	-	1	-	‡	-	-	-	-
test item 50% pH 7.0	22	½	-	1	-	-	‡	-	-	-	-
	26	½	-	1	-	-	‡	-	-	-	-
	30	½	-	-	-	-	-	-	-	-	-
test item 10% pH 5.8	23	½	-	-	-	-	‡	-	-	-	-
	27	½	1	-	-	-	‡	-	-	-	-
	31	½	1	½	-	-	‡	-	-	-	-
test item 10% pH 7.0	24	½	½	½	-	-	‡	-	-	-	-
	28	½	1	-	-	-		-	-	-	-
	32	½	½	-	-	-	‡	-	-	-	-
Saline (negative control)	33	-	-	-	-	-	-	-	-	-	-

- = not observed; P = present; 1/2 = very slight; 1 = slight; 2 = moderate; 3 = severe;

† = scored in the top/mid/low section of the epithelium; ‡ = epithelium top cell layer hypertrophic

Interpretation of results:

Category 2 (irritating to eyes) based on GHS criteria

Conclusions:

Based on the results from this Isolated Chicken Eye test, the testsubstance Rhamnolipids applied at a 50% dilution at pH 7 and at a 10% dilution at pH 7 and 5.8 is considered to be irritating to the eyes (Category 2 (EU-CLP classification); Category 2B:"Mild irritant/causes eye irritation" (UN-GHS classification)) under the experimental conditions described in this report.

Executive summary:

In an Isolated Chicken Eye (ICE) test according to OECD Guideline 438 (adopted 26 July 2013), the eye irritation potential of the testsubstance Rhamnolipids was assessed at different concentrations and pH vaues. In addition, the test included a negative control (saline) and a positive control (NaOH). Chicken eyes were obtained from slaughter animals used for human consumption.

The isolated chicken eyes were exposed to a single application of 30 mg for 10 seconds followed by a 20 mL saline rinse. Three main parameters were measured to disclose possible adverse eye effects: corneal thickness (expressed as corneal swelling), corneal opacity and fluorescein retention of damaged epithelial cells. In addition, histopathology of the corneas was performed.

 

The test item at 50% / pH 7.0 and 10% / pH 7.0 caused corneal effects leading to Category 2/2B classification, consisting of slight corneal swelling (8%), slight or slight to moderate opacity (range mean score of 1.3 or 1.5) and moderate fluorescein retention (mean score of 2.0).

Microscopic examination of the corneas treated with the test item at 50% / pH 7.0 revealed very slight erosion and slight vacuolation (two corneas top region) of the epithelium. In addition, the epithelium top cell layer was hypertrophic in two corneas.

Microscopic examination of the corneas treated with the test item at 10% / pH 7.0 revealed very slight erosion, very slight or slight necrosis and very slight vacuolation (one cornea top region) of the epithelium. In addition, the epithelium top cell layer was hypertrophic in two corneas. According to the AlSE histopathology criteria, upgrading to category 1 on basis of the histopathology of the corneas is not required.

 

The test item at 10% / pH 5.8 caused corneal effects leading to Category 2/2B classification, consisting of slight corneal swelling (6%), slight or slight to moderate opacity (mean score of 1.3) and moderate fluorescein retention (mean score of 2.0). In addition, loosening (veil-like) of epithelium was observed.

Microscopic examination of the corneas revealed very slight erosion, slight necrosis (two corneas) and very slight vacuolation (one cornea top region) of the epithelium.. In addition, the epithelium top cell layer was hypertrophic in all corneas. According to the AlSE histopathology criteria, upgrading to category 1 on basis of the histopathology of the corneas is not required.

 

The negative control eye did not show any corneal effect and demonstrated that the general conditions during the tests were adequate.

The positive control NaOH caused severe corneal effects and demonstrated the ICE test valid to detect severe eye irritants.

 

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

An in vitro study was performed to assess the corneal irritation and damage potential of Rhamnolipid by means of the BCOP assay using fresh bovine corneas comparable to OECD guideline 437.

The corneas treated with the neat Rhamnolipids showed opacity values ranging from 2.7 to 3.7 and permeability values ranging from 6.188 to 6.324 after 30 min contact time and opacity values of 1.3 and permeability values ranging from 3.285 to 3.541 after 10 min contact time. The mean in vitro irritancy score was 96.7 after 30 minutes of treatment and 52.7 after 10 minutes of treatment.

The corneas treated with the 10% dilution Rhamnolipids showed opacity values ranging from 7.7 to 2.7 and permeability values ranging from 6.635 to 7.076 after 30 min contact time and opacity values of 3.3 to 5.3 and permeability values ranging from 3.5.657 to 5.809 after 10 min contact time. The mean in vitro irritancy score was 104.4 after 30 minutes of treatment and 90.4 after 10 minutes of treatment.

Since the mean in vitro irritancy scores after 30 min of contact were above 55.1 and after 10 min of contact point were above 10, the test substance Rhamnolipids is considered to be severely irritating/ corrosive > 10 % dilution in the Bovine Corneal Opacity and Permeability test under the experimental conditions described in this report.  It could be concluded that the corrosive effects of the source substances are dilution- and pH-dependent

Justification for classification or non-classification

The available data for skin irritation do not meet the classification criteria according to Regulation (EC) 1272/2008 and are therefore conclusive but not sufficient for classification.

The available data for eye damage / corrosion meet the criteria for classification to Regulation (EC) 1272/2008. The registered substance is classified as Eye Damage 1, H318 at a concentration > 10%, and as Eye irritation 2, H319 at a concentration < 10%.