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

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2022-03-04 to 2022-03-08

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Version / remarks:

Adopted June 18, 2019

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

941-810-2

IUPAC Name:

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

Test material form:

solid

Details on test material:

59.0% dry matter

Test animals / tissue source

Species:

other: Reconstructed Human Cornea-like Epithelium (RhCE)

Details on test animals or tissues and environmental conditions:

The EpiOcular tissue construct is a nonkeratinized epithelium prepared from normal human keratinocytes. It models the cornea epithelium with progressively stratified, but not cornified cells. A cell suspension is seeded into the insert in specialized medium. After an initial period of submerged culture, the medium is removed from the top of the tissue so that the epithelial surface is in direct contact with the air. This allows the test material to be directly applied to the epithelial surface in a fashion similar to how the corneal epithelium would be exposed in vivo.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
The viscous liquid test material was applied undiluted (50 µL) directly on top of the tissue.

Duration of treatment / exposure:

30 minutes

Observation period (in vivo):

120 minutes

Number of animals or in vitro replicates:

The test was performed on a total of 2 tissues per test material together with a negative control and positive control. Two tissues were treated with 50 µL Milli-Q water (negative control) and 2 tissues with 50 µL Methyl Acetate (positive control) respectively.

Details on study design:

REMOVAL OF TEST SUBSTANCE
After an initial period of submerged culture, the medium is removed from the top of the tissue so that the epithelial surface is in direct contact with the air. This allows the test material to be directly applied to the epithelial surface in a fashion similar to how the corneal epithelium would be exposed in vivo.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

mean percent tissue viability 

Run / experiment:

mean of 2 replicates

Value:

ca. 11

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Difference between two tissues (percentage): 3.4. Acceptability criteria fulfilled.

Results 2

Irritation parameter:

mean percent tissue viability 

Run / experiment:

mean of 2 replicates

Value:

ca. 21

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Difference between two tissues (percentage): 29. Since the difference was above the maximum of 20%, the experiment was repeated.

Other effects / acceptance of results:

OTHER EFFECTS:
- non
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for solvent control: yes
- Acceptance criteria met for positive control: yes

Any other information on results incl. tables

Interference of the Test Material with the MTT Endpoint:
The test material was checked for possible direct MTT reduction by adding the test material to MTT medium. Because no color changes were observed it was concluded that the test material did not interact with the MTT endpoint.
The test material was checked for color interference in aqueous conditions. Addition of the test material to Milli-Q and isopropanol resulted after subtraction of the blank in an OD of
-0.0002 and 0.0049, respectively. Therefore it was concluded that the test material did not induce color interference.

Main Assay:
The mean absorption at 570 nm measured after treatment with the test material and controls are presented in Table 1.
The individual OD570 measurements are presented in Table 3.
Table 2 shows the mean tissue viability obtained after 30 ± 2 minutes treatment with the test material compared to the negative control tissues. Eye hazard potential is expressed as the remaining cell viability after exposure to the test material. The relative mean tissue viability obtained after 30 ± 2 minutes treatment with the test material compared to the negative control tissues was 21%. The difference between the percentage of viability of two tissues treated with the test material was 29%. Since the difference was above the maximum of 20%, the experiment was repeated.
In the repeat experiment, the relative mean tissue viability obtained after 30 ± 2 minutes treatment with the test material compared to the negative control tissues was 11%. The difference between the percentage of viability of two tissues treated identically was 9.2%, indicating that the test system functioned properly.
The positive control had a mean cell viability of 20% and 37% after 30 ± 2 minutes exposure, in the initial and repeat experiment, respectively. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range (See Table 4).
Since the mean relative tissue viability for the test material in both experiments was below 60% after 30 ± 2 minutes treatment it is considered to be potentially irritant or corrosive to the eye.

Mean Absorption in the EpiOcular™ Test with PC-2021-999

First experiment

	A (OD570)	B (OD570)	Mean (OD570)		SD
Negative control	1.204	1.121	1.163	±	0.058
Test material	0.080	0.418	0.249	±	0.239
Positive control	0.244	0.228	0.236	±	0.011

OD = optical density

SD = Standard deviation

Duplicate exposures are indicated by A and B.

In this table the values are corrected for background absorption (0.041). Isopropanol was used to measure the background absorption.

Second experiment

	A (OD570)	B (OD570)	Mean (OD570)		SD
Negative control	1.754	1.923	1.838	±	0.119
Test material	0.237	0.175	0.206	±	0.044
Positive control	0.729	0.616	0.672	±	0.080

OD = optical density

SD = Standard deviation

Duplicate exposures are indicated by A and B.

In this table the values are corrected for background absorption (0.041). Isopropanol was used to measure the background absorption.

Mean Tissue Viability in the EpiOcular™ Test with PC-2021-999

First experiment

	Mean tissue viability (percentage of control)	Difference between two tissues  (percentage)
Negative control	100	7.1
Test material	21	29
Positive control	20	1.4

 

 

 

Second experiment

	Mean tissue viability (percentage of control)	Difference between two tissues  (percentage)
Negative control	100	9.2
Test material	11	3.4
Positive control	37	6.2

 

 

Applicant's summary and conclusion

Interpretation of results:

other: Expert judgement: not Category 1

Conclusions:

In conclusion, the test material is identified as no prediction can be made regarding the classification in the EpiOcular™ test under the experimental conditions described in this report.

Executive summary:

In an in vitro eye irritation guideline study (EpiOcular™ Cornea Epithelial Model) according to OECD 492 under GLP conditions, undiluted “Sophorolipids: fermentation products of glucose and fatty acids, C18-unsatd., esters with glycerol with yeast Candida Bombicola” was applied to the models for 30 min at 37 °C. The test was performed in duplicates.

Physiological saline was used as negative control, Methyl Acetate as positive control. Both controls confirmed the validity of the study.

In this study, the mean tissue viability was 21% in the first experiment, and 11% in the second experiment. According to the guideline, no prediction can be made using this values. For precautionary measures, “Sophorolipids: fermentation products of glucose and fatty acids, C18-unsatd., esters with glycerol with yeast Candida Bombicola” is classified voluntarily as irritating to eyes (GHS Category 2).