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

Endpoint summary

Description of key information

- In an in vitro skin irritation study performed in accordance with OECD draft proposal for a new guideline “In vitro Skin Irritation: Reconstructed Human Epidermis (RhE) Test Method”, GLP, Reliability 1, the read-across substance showed a mean relative tissue viability of 113.2 % and was considered as not irritating to skin.

- In an ex vivo eye irritation guideline study (bovine corneal opacity and permeability assay) according to OECD 437, GLP, Reliability 1, the mean in vitro irritation score was 10. According to the guideline, no prediction can be made using this value.

- In an in vitro eye irritation guideline study (EpiOcular™ Cornea Epithelial Model) according to OECD 492 under GLP conditions, the mean tissue viability was 11% in a first experiment and 21% in a second experiment. According to the guideline, no prediction can be made using this values. 

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:

2010-05-04 to 2010-05-14

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

according to an OECD draft guideline

Qualifier:

according to guideline

Guideline:

other: OECD Guideline for the testing of chemicals draft proposal for a new guideline In vitro Skin Irritation: Reconstructed Human Epidermis (RhE) Test Method

Deviations:

yes

Remarks:

re-spreading of sodium dodecyl sulphate was avoided.

GLP compliance:

yes

Test system:

human skin model

Remarks:

EPISKIN reconstructed human epidermis

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

skin obtained from plastic surgery from multiple donors

Details on animal used as source of test system:

not applicable

Justification for test system used:

The Episkin® Skin Irritation Test is accepted as a reliable and relevant stand-alone replacement test for in vivo skin irritation testing. This test uses a reconstructed human epidermis model which consists of normal human epidermal keratinocytes and therefore represents in vitro the target organ of the species of interest and closely mimics the biochemical and physiological properties of the upper parts of the human, i.e. the epidermis. An ECVAM-validated protocol was followed.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN® reconstructed human epidermis
- Tissue batch number: 10-EKIN-016
- Production date: 2010-05-04
TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 °C
REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 10x, 2.5 mL PBS
- Observable damage in the tissue due to washing: not reported
MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL, freshly prepared
- Incubation time: 3 h
- Wavelength: 570 mnm
- spectrophotometer: BMG Labtech PC Logiciel BMG Optima Mars V2.00
NUMBER OF REPLICATE TISSUES: 3
SCORING SYSTEM
A test compound was classified as irritant if tissue viability was equal or below 50 %.
A test compound was classified as non-irritant if tissue viability was above 50 %.
For further details, please see section "any other information on materials and methods incl. tables".

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

Amount of test item applied: 10 µL, neat
For further details, please see section "any other information on materials and methods incl. tables".

Duration of treatment / exposure:

15 minutes, observation period 42 h

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

TEST ITEM (mean of 3 replicates)

Value:

ca. 113.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

positive control (mean of 3 replicates)

Value:

ca. 29

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

not applicable

Remarks on result:

positive indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

negative control (mean of 3 replicates)

Value:

100

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: not reported
- Direct MTT reduction: no
- Colour interference with MTT: no
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes

Experiment validation

The results of the MTT assay for negative and positive controls are shown in Table 1. Optical density for negative control (PBS) was above 0.6 and the standard deviation value of the percentage of viability was lower than 18 %. The tissue viability for the positive control (5 % SDS) was below 40 %, with the standard deviation value lower than 18 %. Therefore, the acceptance criteria were met for this experiment.

MTT-interaction

No interaction with MTT: no risk of false negatives.

The coloring potential of test-substances

There was no change to the colour of the tissues and no perturbation the OD measurement.

Tissue viability

For each treated tissue, the tissue viability was expressed as a percentage of the mean negative control. The tissue viability data obtained with the MTT assay for test substances are presented in Table 1. In every case, the relative viabilities of tissues exposed to test substances were above 50 % for neat concentration.

Table 1: summary results and classification

Substance	OD mean	OD s.d	Viability mean (%)	Viability s.d	Classification
Negative control (PBS)	0.866	0.070	100.0	8.1	Non-irritant
Positive control (SDS 5%)	0.251	0.008	29.0	1.0	irritant
Test item	0.980	0.072	113.2	8.3	Non-irritant

 

Interpretation of results:

GHS criteria not met

Conclusions:

The substance is not irritating in the in vitro skin irritation test under the experimental conditions described in this report.

Executive summary:

In an in vitro skin irritation study performed in accordance with OECD draft proposal for a new guideline “In vitro Skin Irritation: Reconstructed Human Epidermis (RhE) Test Method”, the test substance “Sophorolipids: fermentation products of glucose and fatty acids, C18-unsatd., esters with glycerol with yeast Candida Bombicola, partially hydrolysed” was applied neat to reconstructed human epidermis for an exposure period of 15 minutes. The number of replicate tissues was three. After 15 minutes exposure the tissues were washed with phosphate buffered saline. Subsequently the tissue constructs were incubated for 42 h at 37 °C. Afterwards, tissues were placed in MTT working solution and incubated at 37 °C, 5 % CO₂, for approximately 3 h. MTT was reduced by viable cells into blue formazan crystals which were extracted and transferred for 570-nm optical density reading in duplicate. Relative cell viability for each test compound was calculated as the OD570 ratio to negative control.

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 compared to the negative control tissues was 113.2 %. Since the mean relative tissue viability for the test substance was above 50 %, the test substance “Sophorolipids: fermentation products of glucose and fatty acids, C18-unsatd., esters with glycerol with yeast Candida Bombicola, partially hydrolysed” 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:

key study

Study period:

2022-03-04 to 2022-03-08

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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

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.

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.

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.

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

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

 

 

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).

 

Reference 2

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

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

26 June 2020

Deviations:

no

GLP compliance:

yes

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Source: Bovine eyes from young cattle were obtained from the slaughterhouse (Vitelco, 's Hertogenbosch, The Netherlands), where the eyes were excised by a slaughterhouse employee as soon as possible after slaughter.
Transport: Eyes were collected and transported in physiological saline in a suitable container under cooled conditions and tested the day of arrival in the laboratory.

Vehicle:

physiological saline

Controls:

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

TEST MATERIAL
No correction was made for the purity/composition of the test material.
The test material was tested neat.

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
After the incubation the solutions were removed and the epithelium was washed with MEM with phenol red (Earle’s Minimum Essential Medium, Life Technologies) and thereafter with cMEM. Possible pH effects of the test material on the corneas were recorded.

Irritation parameter:

in vitro irritation score

Run / experiment:

mean of 3 replicates

Value:

ca. 10

Vehicle controls validity:

valid

Negative controls validity:

valid

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

RESULTS
The test material was tested neat.
Table 1 summarizes the opacity, permeability and in vitro irritancy scores of Sophorolipids lactone intermediate and the controls. 

Table 1          
Summary of Opacity, Permeability and In Vitro Scores

Treatment	Mean Opacity ^1	Mean Permeability ^1	Mean In vitro Irritation Score ^1, 2
Negative control	0.3	-0.001	0.3
Positive control (Ethanol)	22	1.962	51
Test material	2.0	0.544	10

¹      Calculated using the negative control corrected mean opacity and mean permeability values for the positive control and test material.

²      In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD₄₉₀ value).

The individual in vitro irritancy scores for the negative controls ranged from 0.26 to 0.31. The corneas treated with the negative control material were translucent after the 10 minutes of treatment. The individual positive control in vitro irritancy scores ranged from 35 to 65 (Table 5). The corneas treated with the positive control material were turbid after the 10 minutes of treatment. No pH effects of the negative and positive control were observed on the rinsing medium.
The corneas treated with the test material showed opacity values ranging from 1.2 to 3.0 and permeability values ranging from 0.342 to 0.710. The corneas were translucent after the 10 minutes of treatment with the test material. A pH effect of the test material was observed on the rinsing medium, the corneas were rinsed until no color change of the medium was observed. Hence, the in vitro irritancy scores ranged from 8.2 to 12 after 10 minutes of treatment with the test material.

DISCUSSION
The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. Altough the negative control treated corneas were translucent, all results are within the acceptability range therefore this has no impact on the study result. The mean in vitro irritancy score of the positive control (Ethanol) was 51 and within two standard deviations of the current historical positive control mean (Table 6). It was therefore concluded that the test conditions were adequate and that the test system functioned properly.
The test material induced ocular irritation through one endpoint (permeability), resulting in a mean in vitro irritancy score of 10 after 10 minutes of treatment.

CONCLUSION
In conclusion, since Sophorolipids lactone intermediate induced an IVIS > 3 ≤ 55, no prediction on the classification can be made.

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: 10. In conclusion, since Sophorolipids lactone intermediate induced an IVIS > 3 ≤ 55, no prediction on the classification can be made. 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, undiluted “Sophorolipids: fermentation products of glucose and fatty acids, C18-unsatd., esters with glycerol with yeast Candida Bombicola” 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, ethanol as positive control. Both controls confirmed the validity of the study.

In this study, the mean in vitro irritation score was 10. In conclusion, since Sophorolipids lactone intermediate induced an IVIS > 3 ≤ 55, no prediction on the classification can be made. 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).

 

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation

In an in vitro skin irritation study performed in accordance with OECD draft proposal for a new guideline “In vitro Skin Irritation: Reconstructed Human Epidermis (RhE) Test Method”, the read-across substance “Sophorolipids: fermentation products of glucose and fatty acids, C18-unsatd., esters with glycerol with yeast Candida Bombicola, partially hydrolysed” was applied neat to reconstructed human epidermis for an exposure period of 15 minutes. The number of replicate tissues was three. After 15 minutes exposure the tissues were washed with phosphate buffered saline. Subsequently the tissue constructs were incubated for 42 h at 37 °C. Afterwards, tissues were placed in MTT working solution and incubated at 37 °C, 5 % CO₂, for approximately 3 h. MTT was reduced by viable cells into blue formazan crystals which were extracted and transferred for 570-nm optical density reading in duplicate. Relative cell viability for each test compound was calculated as the OD570 ratio to negative control.

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 compared to the negative control tissues was 113.2 %. Since the mean relative tissue viability for the test substance was above 50 %, the read-across substance “Sophorolipids: fermentation products of glucose and fatty acids, C18-unsatd., esters with glycerol with yeast Candida Bombicola, partially hydrolysed” is identified to be not irritating.

 

Eye irritation

In an ex vivo eye irritation guideline study (bovine corneal opacity and permeability assay) according to OECD 437 under GLP conditions, undiluted “Sophorolipids: fermentation products of glucose and fatty acids, C18-unsatd., esters with glycerol with yeast Candida Bombicola” was applied on corneas for 10 min. The test was performed in triplicates. In conclusion, since Sophorolipids lactone intermediate induced an IVIS > 3 ≤ 55, no prediction on the classification can be made. 

 

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. 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).

 

Justification for classification or non-classification

Justification for classification as "irritating to eyes": Study data do not allow a prediction. For precautionary measures, the test substance “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).