Pyrolytic lignin

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Oct 2020- Mar2021

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Low molecular weight organic substances from fast pyrolysis bio-oil

IUPAC Name:

Low molecular weight organic substances from fast pyrolysis bio-oil

Constituent 2

Reference substance name:

Monomers from fast pyrolysis bio-oil

IUPAC Name:

Monomers from fast pyrolysis bio-oil

Constituent 3

Reference substance name:

Dimers from fast pyrolysis bio-oil

IUPAC Name:

Dimers from fast pyrolysis bio-oil

Constituent 4

Reference substance name:

Trimers from fast pyrolysis bio-oil

IUPAC Name:

Trimers from fast pyrolysis bio-oil

Constituent 5

Reference substance name:

Higher oligomers from fast pyrolysis bio-oil

IUPAC Name:

Higher oligomers from fast pyrolysis bio-oil

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

Test material form:

liquid: viscous

Remarks:

Brown /black highly vicous liquid

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
- Temperature of post-treatment incubation (if applicable): 37 ± 1°C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: 1
- Observable damage in the tissue due to washing: No

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

EXPERIMENTS TO DERIVE FINAL PREDICTION:
One valid experiment and three additional pre-tests

DECISION CRITERIA
% Tissue viability is ≤ 50 % of negative control - Corrosive/Irritant to skin - UN GHS Category 1 or 2
% Tissue viability is > 50% of negative control - Non-irritant to skin - No Category for Skin Irritation

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Pyrolytic lignin
- Concentration (if solution): 30 µL

MTT ASSAY
- 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT)
- Concentration (if solution): 300 µL (1 mg/ml MTT in Dulbecco’s Phosphate-Buffered Saline (DPBS buffer) and 8mL of medium)

NEGATIVE CONTROL
- Dulbecco’s Phosphate Buffered Saline (DPBS buffer without CaCl2 and without MgCl2)
- Concentration (if solution): 30 µL

POSITIVE CONTROL
- Solution of demineralised water containing 5% Sodium dodecyl sulphate (SDS)
- Concentration (if solution): 30 µL

Duration of treatment / exposure:

1 hour main test

Duration of post-treatment incubation (if applicable):

43 hours in total (25 hours after DPBS rinse and assay medium application and 18 hours after medium renewal) on main test

Number of replicates:

3 replicates for main test

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Main test

Value:

61.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Main test (Corrected Values)

Value:

57.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No

PRE-TEST
- Direct-MTT reduction: Yes
The test item was tested for the ability of direct MTT reduction. The MTT solution turns blue/purple within 1 hour. Therefore, direct MTT reduction by the test item had taken place and an additional test was necessary.

- Additional Test Direct Reduction of MTT with freeze killed Tissues: Yes
Two freeze-killed tissues treated with the MTT reducing test item and two untreated killed tissues that showed the small amount of MTT reduction due to residual NADH and associated enzymes within the killed tissues. Therefore, direct MTT reduction had taken place and data correction with the main test was necessary.

- Colour interference with MTT: No
It was tested whether the test item develops a colour without MTT addition as the solution showed no significant change in colour, no binding capacity had to be tested.

DEMONSTRATION OF TECHNICAL PROFICIENCY: The validity of the skin irritation study at LAUS GmbH was demonstrated in a non-GLP proficiency study. For this purpose, 10 proficiency chemicals (indicated by the OECD 439 guideline) were tested. All of the 10 proficiency chemicals were correctly categorized. Therefore, the proficiency of the skin irritation study was demonstrated.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Negative control Optical Density (OD )≥ 0.8 and ≤ 2.8
- Acceptance criteria met for positive control: % tissue viability of positive control SDS ≤ 20% of negative control
- Acceptance criteria met for variability between replicate measurements: SD of mean viability of the tissue replicates ≤ 18%

Any other information on results incl. tables

Mean Absorbance Values (main test)

Designation	Negative Control	Test Item	Positive Control
Mean – blank (tissue 1)	1.264	0.753	0.066
Mean – blank (tissue 2)	1.337	0.778	0.073
Mean – blank (tissue 3)	1.456	0.968	0.075
Mean of the three tissues	1.352	0.833	0.071

% Tissue Viability (main test)

Designation	Test ItemBTG Pyrolytic Lignin	Positive Control
% Tissue viability (tissue 1)	55.7%	4.9%
% Tissue viability (tissue 2)	57.5%	5.4%
% Tissue viability (tissue 3)	71.6%	5.5%
% Tissue viability (mean)	61.6%	5.3%
± SD of mean tissue viability (%)	8.7%	0.3%

Mean Absorbance Values (corrected with freeze killed control)

Designation	Negative Control	Test Item
Mean – blank (tissue 1)	1.264	0.699
Mean – blank (tissue 2)	1.337	0.724
Mean – blank (tissue 3)	1.456	0.914
Mean of the three tissues	1.352	0.779

% Tissue Viability (corrected with freeze killed control)

Designation	Test Item
% Tissue viability (tissue 1)	51.7%
% Tissue viability (tissue 2)	53.6%
% Tissue viability (tissue 3)	67.6%
% Tissue viability (mean)	57.6%
± SD of mean tissue viability (%)	8.7%

 

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test item Pyrolytic Lignin is considered non-irritant to skin in the Recon-structed human Epidermis (RhE) Test Method.

Executive summary:

This in vitro study was performed in order to evaluate the potential of Pyrolytic Lignin to evoke skin irritation in a reconstructed human epidermis (RhE) test method. The test consists of a topical exposure of the neat test item to a human reconstructed epidermis model followed by a cell viability test. Cell viability is measured by dehydrogenase conversion of MTT 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, present in cell mitochondria, into a blue formazan salt that is quantitatively measured after extraction from tissues. The percentage reduction of cell viability in comparison of untreated negative controls is used to predict the skin irritation potential. The study was conducted following the OECD Guideline 439 and the EU method B.46. A valid experiment (main test) with additional test were performed. The additional tests included an assessment of coloured or staining test item to test whether the test item develops a colour without MTT addition, that showed no significant change in colour; an assessment to test the potetntial of the test item of direct reduction of MTT. As the test item was capable of reducting MTT and additional direct reduction of MTT tes was performed with freeze killed tissues. Because direct MTT reduction took place, data correction for the results of the main test was necessary.

In the main test, tissues of the human skin model EpiDerm^TM were treated with the test item for 60 minutes. After the treatment with the test item, the mean value of relative tissue viability was reduced to 57.6% after correction. This value is above the threshold for skin irritation potential (50%). Therefore, the test item BTG Pyrolytic Lignin is considered non-irritant to skin.