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Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Jan- Mar 2021
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2021
Report date:
2021

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Qualifier:
according to guideline
Guideline:
other: EU-Method B.40 BIS. “in vitro skin corrosion: Human skin model test"
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
1,6-anhydro-β-D-glucose
EC Number:
207-855-0
EC Name:
1,6-anhydro-β-D-glucose
Cas Number:
498-07-7
Molecular formula:
C6H10O5
IUPAC Name:
(1R,2S,3S,4R,5R)-6,8-dioxabicyclo[3.2.1]octane-2,3,4-triol
Constituent 2
Chemical structure
Reference substance name:
Glycollaldehyde
EC Number:
205-484-9
EC Name:
Glycollaldehyde
Cas Number:
141-46-8
Molecular formula:
C2H4O2
IUPAC Name:
2-hydroxyacetaldehyde
Constituent 3
Reference substance name:
Organic acids
IUPAC Name:
Organic acids
Constituent 4
Reference substance name:
Ketones
IUPAC Name:
Ketones
Constituent 5
Reference substance name:
Phenols
IUPAC Name:
Phenols
Constituent 6
Reference substance name:
Water
EC Number:
231-791-2
EC Name:
Water
Cas Number:
7732-18-5
Molecular formula:
H2O
IUPAC Name:
water
Constituent 7
Reference substance name:
Oligomers of sugars and anhydrosugars
IUPAC Name:
Oligomers of sugars and anhydrosugars
Test material form:
liquid: viscous

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Vehicle:
unchanged (no vehicle)
Details on test system:
TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1°C


DYE BINDING METHOD
- Dye used in the dye-binding assay: [none / MTT / Sulforhodamine B / other:] MTT
- Spectrophotometer: Microtiter plate photometer
- Wavelength: 570 nm

NUMBER OF INDEPENDENT TESTING RUNS / EXPERIMENTS TO DERIVE FINAL PREDICTION:
One valid experiment and two additional pre-test

VALIDITY OF EXPERIMENT
- Mean OD of the tissue replicates treated with the negative control (H2O) should be ≥0.8 and ≤2.8 for every exposure time
- Mean viability of the tissue replicates exposed for 1 hour with the positive control (8N KOH), expressed as % of the negative control, should be ≤ 15%
- In the range 20 - 100% viability, the Coefficient of Variation (CV) between tissue replicates should be ≤ 30%

DECISION CRITERIA
- The test substance is considered to be corrosive to skin if:
% tissue viability after 3 min. incubation time is < 50% of negative control and % tissue viability after 1h. incubation time is irrelevant
% tissue viability after 3 min. incubation time is ≥ 50% of negative control and % tissue viability after 1h. incubation time is < 15% of negative control

- For substance considered corrosive:
% tissue viability after 3 min. incubation time is < 25% of negative control and % tissue viability after 1h. incubation time is irrelevant - GHS Skin Corrisive Sub-category 1A
% tissue viability after 3 min. incubation time is ≥ 25% of negative control and % tissue viability after 1h. incubation time is irrelevant - GHS Skin Corrisive Sub-category 1B or 1C

- The test substance is considered to be non-corrosive to skin if:
% tissue viability after 3 min. incubation time is ≥ 50% of negative control and % tissue viability after 1h. incubation time is ≥ 15% of negative control

Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Pyrolytic sugar
- Concentration (if solution): 50µL

MTT ASSAY
- 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT)
- Concentration (if solution): 1mL MTT solution (5 mg/mL MTT, in Dulbecco’s Phosphate-Buffered Saline (DPBS buffer))

NEGATIVE CONTROL
- Demineralised water
- Concentration (if solution): 50µL

POSITIVE CONTROL
- KOH solution in demineralised water
- Concentration (if solution): 50µL
Duration of treatment / exposure:
3 minutes with one human skin model
1 hour with a second human skin model
Number of replicates:
2 replicates per human skin model

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 min experiment of main test
Value:
93.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1 hour experiment of main test
Value:
72.8
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 min experiment corrected values of main test
Value:
16.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1 hour experiment corrected values of main test
Value:
6.6
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
CORRECTED VALUES OF MAIN TEST
As the test item had shown its ability to reduce MTT in the Direct-MTT reduction pre-test. Corrections were made on the final % tissue viability from the main test. The true tissue viability is calculated as the percentage tissue viability obtained with living tissues exposed to the MTT reducer, minus the percent not specific MTT reduction obtained with the killed tissues exposed to the same MTT reducer, calculated relative to the negative control run concurrently to the test being corrected.


OTHER EFFECTS
- Visible damage on test system: No

PRE TESTS
- Direct-MTT reduction: Yes
The MTT solution showed a significant change in colour within 1 hour. Therefore, a functional test with freeze killed tissues that possess no metabolic activity was performed.
Two freeze-killed tissues were treated with the MTT reducing test item for 3 minutes and 1 hour and two untreated killed tissues for 3 minutes and 1 hour.


- Colour interference with MTT: No
It was tested whether the test item develops a colour without MTT addition. The resulting suspension was coloured red brown and thereforethe binding capacity of the test item to the tissue was tested. As the stained control result was ≤ 5 % of the viable negative control, data correction procedure is not necessary.

Any other information on results incl. tables

Mean Absorbance Values of the 3 Minutes Experiment of the main test

Designation

Negative Control

Test Item

Positive Control

Mean – blank

(tissue 1)

1.648

1.478

0.362

Mean – blank

(tissue 2)

1.724

1.676

0.376

Mean of the

two tissues

1.686

1.577

0.369

RSD

3.2%

8.9%

2.6%

Mean Absorbance Values of the 1 h Experiment of the main

Designation

Negative Control

Test Item

Positive Control

Mean – blank

(tissue 1)

1.536

1.182

0.108

Mean – blank

(tissue 2)

1.816

1.259

0.079

Mean of the

two tissues

1.676

1.220

0.093

RSD

11.8%

4.5%

22.0%

% Tissue Viability of the Main test

Incubation

Test Item

Positive Control

3 min

93.5%

21.9%

1 h

72.8%

5.6%

 

Mean Absorbance Values of the 3 Minutes Experiment of the Direct Reduction of MTT test

Designation

Negative Control

Test Item

Mean – blank

(tissue 1)

0.080

1.342

Mean – blank

(tissue 2)

0.083

1.418

Mean of the

two tissues

0.082

1.380

RSD

2.6%

3.9%

Mean Absorbance Values of the 1 h Experiment of the Direct Reduction of MTT test

Designation

Negative Control

Test Item

Mean – blank

(tissue 1)

0.079

1.310

Mean – blank

(tissue 2)

0.082

1.071

Mean of the

two tissues

0.081

1.190

RSD

2.9%

14.2%

Corrected Absorbance Values (OD 570 nm) and viabilty

Designation

3 Minutes

Experiment

1 hour

Experiment

Mean absorbance test item additional test

1.380

1.190

Mean absorbance negative control additional test

0.082

0.081

Mean difference absorbance

1.298

1.110

% Viability mean additional test in comparison to the negative control of the main test

77.0%

66.2%

 

Corrected absorbance in the additional test

0.279

0.110

Corrected% Viability

16.5%

6.6%

Applicant's summary and conclusion

Interpretation of results:
other: the study alone does not allow conclusion on the classification
Conclusions:
It is not possible to categorize the test item Pyrolytic Sugar as corrosive or not with the performance described in this study
Executive summary:

The study was conducted to determine the skin corrosion potential of Pyrolytic Sugar in the Reconstructed Human Epidermis (RHE). The study was conducted following the OECD Guideline 431 and EU Method B.40-BIS. One valid experiment with additional tests was performed. As the test item showed intense coloring in the pre-test, there was the risk to influence the photometric measurement and a false negative result. Therefore, an additional test for intensely coloured test items was performed. But the result of the additional test showed, that the test item colour did not critically influence the result of the study. In the pre-test the test item showed MTT-reducing properties. The probability to influence the photometric measurement and of a false negative result had to be excluded. Therefore, an additional test using freeze-killed tissues was performed. The result of the additional test showed that MTT reduction by the test item did influence the measurement and a correction of the result of the main test was necessary.

In the main test two tissues of the human skin model EpiDermTM were treated with the test itemfor 3 minutes and 1 hour, the, cell viability of the tissues was evaluated by addition of MTT, which can be reduced to a blue formazan. Formazan production was evaluated by measuring the optical density (OD) of the resulting solution. After 3 minutes treatment with the test item, the mean value of relative tissue viability was reduced to 93.5% in the main test and 16.5% after correction. After 1 hour treatment, the mean value of relative tissue viability was reduced to 72.8% in the main test and 6.6% after correction. As the correction was greater than 30% of the negative control value (77% in the 3 min. exp. and 66.2% in the 1 hour exp.), it is not possible to obtain a reliable result without performing further tests to consider whether it is tissue damage and therefore corrosive properties or MTT reduction by the test item itself.