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Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25 Jul - 24 Aug 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report date:
2019

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
adopted 28 Jul 2015
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
adopted 06 Jul 2012
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Schwabach, Germany

Test material

Constituent 1
Reference substance name:
Vinasses, residue of fermentation containing biomass of bakers yeast (Saccharomyces cerevisiae)
EC Number:
932-161-6
Molecular formula:
Not applicable (a generic molecular formula cannot be provided for this specific UVCB substance).
IUPAC Name:
Vinasses, residue of fermentation containing biomass of bakers yeast (Saccharomyces cerevisiae)
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: sterilization at 121 °C for 20 min to avoid contamination of the cell culture to avoid contamination of the cell culture

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: EpiDermTM reconstituted three-dimensional human epidermis (EPI-200)
Source strain:
not specified
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPI-200 SIT kit (MatTek Corporation, Bratislava, Slovakia)
- Tissue batch number: 28646

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1 °C for 35 min in the incubator followed by 25 min at room temperature under the sterile flow
- Temperature of post-treatment incubation: 37 °C for 42 h

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps:
The tissues were washed by filling and emptying the inserts 15 times with Dulbecco's phosphate buffered saline (DPBS) using a constant stream in about 1.5 cm distance from the tissue surface. This process also occured sequentially, e.g. in one-minute intervals. Subsequently, the inserts were completely submerged three times in 150 mL DPBS and shaken to remove rests of the test item. Finally, the inserts were rinsed once from the inside and the outside with sterile DPBS.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h ± 5 min at 37 ± 1 °C
- Spectrophotometer: plate spectrophotometer
- Wavelength: 570 nm without reference wavelength
- Filter bandpass: ± 30 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: The quality of the EpiDerm tissue was assessed via MTT cytotoxicity assay. The determined OD (540-570 nm) was 2.216 ± 0.075 and fits to the acceptance criteria of 1.0 - 3.0.
- Barrier function: The barrier function was evaluated by determination of the exposure time required to reduce tissue viability by 50% (ET-50) upon treatment with 100 µL of 1% Triton X-100. The ET-50 value was determined to be 6.01 h and fits to the acceptance criteria of 4.77 - 8.72 h.
- Contamination: The cells used to produce the EpiDermTM tissue were screened for the presence of viruses, bacteria, yeast and other fungi. No contamination was detected. Tissue sterility was confirmed by long-term incubation with antibiotic and antimycotic free culture.

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE:
Since the test item mixed with aqua dest. showed colouring detectable by unaided eye assessment and absorbed light in the range of 570 ± 30 nm, the test item was checked for its tissue colouring potential.
- Fresh tissues/killed tissues: Living tissues were treated with 30 µL of the test item (TVT). All steps were performed as in the main study except for the MTT staining (Incubation in medium without MTT).
- No. of replicates: 2 living tissues
- Method of calculation used: The non-specific colour of additional viable tissues (NSCliving) was calculated according to the following formular: NSCliving [%] = [ODTVT / ODNK] x 100
NK: negative control, TVT: additional test item treated living tissue without MTT staining.
If NSCliving is ≤ 5% relative to the negative control of living epidermis, no correction of the result is necessary. If NSCliving is > 5% relative to the negative control of living epidermis, the true MTT metabolic conversion (TODTT) was corrected according to the following formular: TODTT = OTM – ODTVT
TM: test item treated with living tissues
If NSCliving is > 30% relative to the negative control of living epidermis, the test item is considered as incompatible with the test method.
Since the test substance did not directly reduce MTT, an additional test with freeze-killed tissues to determine the non-specific reduction of MTT was not performed.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: single experiment

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin if the viability after 60 min exposure is less than or equal to 50%.
- The test substance is considered to be non-irritant to skin if the viability after 60 min exposure is greater than 50%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount applied: 30 µL (47 µL/cm2)

NEGATIVE CONTROL
- Amount applied: 30 µL (47 µL/cm2)

POSITIVE CONTROL
- Amount applied: 30 µL (47 µL/cm2)
- Concentration (if solution): 5% (v/v) in deionised water
Duration of treatment / exposure:
35 min at 37 ± 1 °C and 25 min at room temperature
Duration of post-treatment incubation (if applicable):
42 h
Number of replicates:
triplicates for each treatment and control group

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Remarks:
mean value of 3 tissues
Run / experiment:
60 min exposure
Value:
85.2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
OTHER EFFECTS:
- Direct MTT reduction: The test substance was not considered to be a MTT reducer. Thus, an additional test with freeze-killed tissues was not performed.
- Colour interference: The substance showed colouring upon mixing with water and absorbed light in the relevant range of 570 ± 30 nm, therefore the non-specific color of additional viable tissue was determined. Because the non-specific colour of additional viable tissues was ≤ 5% (NSCliving: 0.3%) relative to the negative control of living epidermis, no correction of the results was necessary.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD 570 nm of the tissue replicates treated with the negative control is ≥ 0.8 and ≤ 2.8. The mean OD 570 nm is 1.744 and matches the required acceptability criterion.
- Acceptance criteria met for positive control: Mean relative tissue viability is ≤ 20%. Treatment with the positive control for 1 h induced a decrease in relative absorbance to 4.7% as compared to the negative control.
- Acceptance criteria met for variability between replicate measurements: The relative standard deviation of 3 identical replicates is < 18%. The relative standard deviations between the % variability values of the test item, the positive and negative controls are 5.8% at the highest.

Any other information on results incl. tables

Table 1: Results of the test item

Name

Negative Control

Positive Control

Test Item

Tissue

1

2

3

1

2

3

1

2

3

 

Absolute OD570

1.765

1.845

1.763

0.119

0.126

0.125

1.395

1.577

1.607

1.773

1.807

1.778

0.123

0.133

0.134

1.442

1.530

1.628

Mean Absolute OD570

1.789

0.127

1.530

 

OD570 (Blank Corrected)

1.720

1.800

1.718

0.074

0.081

0.081

1.350

1.532

1.562

1.729

1.762

1.734

0.078

0.089

0.089

1.398

1.485

1.583

Mean OD570 of the Duplicates(Blank Corrected)

1.725

1.781

1.726

0.076

0.085

0.085

1.374

1.509

1.573

Total Mean OD570 of the 3 Replicate Tissues (Blank Corrected)

1.744*

0.082

1.485

SD of Mean OD570 of the Duplicates (BlankCorrected)

0.032

0.005

0.101

Relative Tissue Viability [%]

98.9

102.1

99.0

4.4

4.9

4.9

78.8

86.5

90.2

Mean Relative Tissue Viability [%]

100.0

4.7

85.2

SD of Relative Tissue Viability [%]

1.8

0.3

5.8

CV [% Viabilities]

1.8

6.2

6.8

* Blank-corrected mean OD 570 nm of the negative control corresponds to 100% absolute tissue viability.

Table 2: Results of the NSCliving control

NSCliving

TVT

Negative Control

Tissue

1

2

1

2

3

 

Absolute OD570

0.049

0.048

1.765

1.845

1.763

0.052

0.050

1.773

1.807

1.778

 

OD570 (Blank Corrected)

0.004

0.003

1.720

1.800

1.718

0.007

0.005

1.729

1.762

1.734

Mean OD570 of the Duplicates (Blank Corrected)

0.006

0.004

1.725

1.781

1.726

Total Mean OD570 of the 2 or 3 Replicate Tissues (Blank Corrected)

0.005

1.744

SD of Mean OD570 of the Duplicates (Blank Corrected)

0.001

0.032

NSCliving [%]

0.3

-

Relative Tissue Viability [%]

 

-

98.9

102.1

99.0

Mean Relative Tissue Viability [%]

 

-

100.0

SD of Relative Tissue Viability [%]

-

1.8

CV [% Viabilities]

-

1.8

Applicant's summary and conclusion

Interpretation of results:
other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No. 1272/2008.
Conclusions:
CLP: not irritating

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