Registration Dossier

Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
Qualifier:
according to
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 material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

In vitro test system

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

Results and discussion

In vitro

Resultsopen allclose all
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

Any other information on results incl. tables

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

 

Applicant's summary and conclusion

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.