Sophorolipids: fermentation products of glucose and fatty acids, C18 (unsaturated), glycerol esters with yeast Candida Bombicola, partially hydrolysed

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010-11-29 to 2010-11-30

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

7 September 2009

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

(8Z)-16-{[(2R,4S,5S)-6-[(acetyloxy)methyl]-3-{[(2S,4S,5S)-6-[(acetyloxy)methyl]-3,4,5-trihydroxyoxan-2-yl]oxy}-4,5-dihydroxyoxan-2-yl]oxy}heptadec-8-enoic acid; [(1S,4S,5S,8R,17Z,27S,31R)-28-[(acetyloxy)methyl]-4,5,30,31-tetrahydroxy-10-methyl-25-oxo-2,7,9,26,29-pentaoxatricyclo[25.2.2.0³,⁸]hentriacont-17-en-6-yl]methyl acetate

EC Number:

941-809-7

IUPAC Name:

(8Z)-16-{[(2R,4S,5S)-6-[(acetyloxy)methyl]-3-{[(2S,4S,5S)-6-[(acetyloxy)methyl]-3,4,5-trihydroxyoxan-2-yl]oxy}-4,5-dihydroxyoxan-2-yl]oxy}heptadec-8-enoic acid; [(1S,4S,5S,8R,17Z,27S,31R)-28-[(acetyloxy)methyl]-4,5,30,31-tetrahydroxy-10-methyl-25-oxo-2,7,9,26,29-pentaoxatricyclo[25.2.2.0³,⁸]hentriacont-17-en-6-yl]methyl acetate

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

- Source: slaughterhouse, not specified
- Calf eyes were excised as soon as possible after slaughter. Care was taken to avoid damaging the cornea during the enucleation procedure. Eyes were collected in a plastic container containing 1 L of sterile Hanks's Balanced Salt Solution (HBSS). Medium storage and transportation of eyes to the laboratory was performed at room temperature. The eyes were used within 3 hours after slaughter.

Test system

Vehicle:

physiological saline

Controls:

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 0.75 mL
- Concentration: 10 % solution of test item prepared in 0.9 % sodium chloride
For further details, please refer to section "any other information on materials and methods incl. tables".

Duration of treatment / exposure:

10 ± 1 minutes

Observation period (in vivo):

120 (+/-) 10 minutes

Number of animals or in vitro replicates:

triplicates

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): at least three times(until the medium was clear with approximately 4 ml of EMEM solution
- Time after start of exposure: 130 minutes
For further details, please refer to section "any other information on materials and methods incl. tables".

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

mean of 3 replicates

Value:

ca. 32.3

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: no abnormalities.
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for solvent control: yes
- Acceptance criteria met for positive control: yes

Any other information on results incl. tables

RESULTS

For each exposure condition, three corneas were visually inspected and showed no abnormalities. Three corneas were treated with 0.9 % sodium chloride as solvent/vehicle control. The IVIS of 0.9 % sodium chloride was -0.2 ± 0.4 (-0.6 to 0.2) with a mean opacity value of -0.2 ± 0.4 (-0.6 to 0.2) and a mean permeability value of 0.001 ± 0.001 (0.001 to 0.002).

Three corneas were selected for treatment with the positive control 100 % N,N-dimethylformamide. Treatment resulted in a mean IVIS of 100.5 ± 9.9. The corrected mean value of the opacity was 81.9 ± 6.0, ranging from 76.4 to 88.3. The corrected mean value of the permeability was 1.240 ± 0.340, ranging from 0.890 to 1.570. Before starting the permeability test, the dye solution sodium fluorescein was checked for its quality. The dye solution is valid for use, if a dilution of the stock solution containing 10 µg/mL gave the correct concentration on the calibration curve (acceptable range OD490: 1.75 ± 0.2). The average concentration of 2 measurements calculated by the spectrophotometer was 1.7797 µg/mL (OD 1.7692 and OD 1.7902) and thus valid for use. This means that the test conditions were optimal to determine the irritating potential of test item.

Three corneas were selected for treatment with the test item. Treatment resulted in a mean IVIS of 32.3 ± 5.8 after 10 minutes treatment, ranging from 28.2 to 38.9. The net value of the opacity score ranged from 2.6 to 4.4, the mean value was 3.8 ± 1.0. The mean corrected permeability value of the corneas was 1.900 ± 0.372, ranging from 1.583 to 2.310.

Treatment	Mean opacity	Mean permeability	Mean in vitro irritation score
Negative control	0.2 (+/-) 0.4	0.001  (+/-) 0.001	- 0.2  (+/-) 0.4
Positive control	81.9 (+/-) 6.0	1.240  (+/-) 0.340	100.5  (+/-) 9.9
Test substance	3.8  (+/-) 1.0	1.9  (+/-) 0.372	32.3  (+/-) 5.8

Applicant's summary and conclusion

Interpretation of results:

other: Expert judgement: not Category 1

Conclusions:

The eye irritancy potential of the test substance was investigated in the bovine corneal opacity and permeability assay. The following mean in vitro irritation score was calculated: 32.3 ± 5.8. No prediction can be made regarding the classification of the test substance to the evaluation criteria. For precautionary reasons, the test substance is classified as Category 2.

Executive summary:

In an ex vivo eye irritation guideline study (bovine corneal opacity and permeability assay) according to OECD 437 under GLP conditions, 0.75 mL of a 10 % solution of “Sophorolipids: fermentation products of glucose and fatty acids, C18-unsatd., esters with glycerol with yeast Candida Bombicola, partially hydrolysed” in physiological saline was applied on corneas for 10 min at 32 °C. The test was performed in triplicates.

Physiological saline was used as negative control, N,N-dimethylformamide as positive control. Both controls confirmed the validity of the study.

In this study, the mean in vitro irritation score was 32.3 ± 5.8. According to the guideline, no prediction can be made using this value. For precautionary measures, “Sophorolipids: fermentation products of glucose and fatty acids, C18-unsatd., esters with glycerol with yeast Candida Bombicola, partially hydrolysed” is classified voluntarily as irritating to eyes (GHS Category 2).