Registration Dossier

Administrative data

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 test 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 32.3 ± 5.8. According to the guideline, no prediction can be made using this value.

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
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
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
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.
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".
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

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
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).

 

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 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.

Eye irritation

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, potassium hexafluorophosphate 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).