Pyrolytic lignin

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

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

Test animals / tissue source

Species:

other: EpiOcularTM tissue. Human-derived keratinocytes

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Pyrolytic lignin
- Concentration (if solution): 50 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)

Duration of treatment / exposure:

1 hour main test
180 min MTT assay

Duration of post- treatment incubation (in vitro):

120 min

Details on study design:

NUMBER OF REPLICATES: 2

NEGATIVE CONTROL USED: Sterile demineralised water

POSITIVE CONTROL USED : Methyl acetate

APPLICATION DOSE AND EXPOSURE TIME: Test material 50 μL, 30 minutes exposure. MTT Solution 300 μL, 180 minutes exposure

REMOVAL OF TEST SUBSTANCE
- Dulbecco`s Phosphate Buffered Saline (DPBS) used for test item
- Number of washing steps after exposure period: 1 rinse

- POST-EXPOSURE INCUBATION: 120 minutes

METHODS FOR MEASURED ENDPOINTS:
- Optical density (OD): Measurement of MTT formazan precipitate extracted with isopropanol on the MTT assay after main test with a spectrophotometer at 570 nm.

DECISION CRITERIA:
% Viability > 60% - Non eye irritant - No UN GHS classification
% Viability ≤ 60 % - At least eye irritant - No prediction can be made for UN GHS classification

Results and discussion

In vitro

Results

Irritation parameter:

percent tissue viability 

Run / experiment:

Main test

Value:

49.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No

DEMONSTRATION OF TECHNICAL PROFICIENCY: The validity of the EpiOcularTM test at LAUS GmbH was demonstrated in a non-GLP pro-ficiency study. For this purpose 15 proficiency chemicals (indicated by the OECD 492 guideline) were tested. All of the 15 proficiency chemicals were correctly categorized. Therefore, the proficiency of the EpiOcularTM test was demonstrated

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Mean OD of negative control is >0.8 and <2.8
- Acceptance criteria met for positive control: % Mean relative viability of positive control is < 50% of negative control
- Acceptance criteria met for variability between replicate measurements: Variation within replicates is < 20%

Any other information on results incl. tables

Mean Absorbance Negative Control, Positive Control and Test Item

Designation	Negative Control	Positive Control	Test Item
Mean – blank (Tissue 1)	1.944	0.774	0.959
Mean – blank (Tissue 2)	1.997	0.619	0.992

% Viability Positive Control and Test Item

Designation	Positive Control	Test Item
% Viability (Tissue 1)	39.2%	48.6%
% Viability (Tissue 2)	31.4%	50.3%
% Viability Mean	35.3%	49.5%

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Remarks:

According to the OECD Guideline 492, the EpiOcularTM Eye Irritation Test does not allow discrimination between eye irritation/reversible effects on the eye (Category 2) and seri-ous eye damage/irreversible effects on the eye (Category 1)

Conclusions:

Under the conditions of the test, BTG Pyrolytic Lignin is considered either eye irritant or inducing serious eye damage in the EpiOcularTM Eye Irritation Test.

Executive summary:

The study was performed in order to evaluate the eye hazard potential of BTG Pyrolytic Lignin in a Reconstructed human Cornea-like Epithelium (RhCE) model in an in vitro study (e.g. EpiOcular^TM Eye Irritation Test). The EpiOcular^TM Eye Irritation Test (EIT) predicts the acute eye hazard potential of chemicals by measurement of tissue damage caused by cytotoxic effects in the reconstructed human cornea-like tissue model. The EpiOcular™ EIT can be used to identify chemicals that do not require classification for eye irritation or serious eye damage according to the UN GHS classification system. A limitation of this guideline is that it neither allows discrimination between eye irritation/reversible effects on the eye (Category 2) and serious eye damage/irreversible effects on the eye (Category 1), nor between eye irritants (optional Category 2A) and mild eye irritants (optional Category 2B).

The liquid test item was applied topically to a three-dimensional RhCE tissue construct in duplicate for an exposure time of 30 minutes.Eye hazard materials are identified by their ability to produce a decrease in cell viability as determined. The cell viability is measured by dehydrogenase conversion of MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide), 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 eye irritation potential. After treatment with the test item, the mean value of relative tissue viability was 49.5%. This value is below the threshold for eye irritation potential (≤ 60%) and therefore the test item is considered either eye irritant or inducing serious eye damage.