Pyrolytic lignin

Endpoint summary

Description of key information

Skin irritation

In a recent in vitro skin irritation study the test item showed a mean relative tissue viability of 57,6%. The test item is considered irritant to the skin if the mean relative tissue viability of three individual tissues is reduced ≤ 50% of the negative control.

Eye corrosion/irritation

The eye corrosion potential of the test item was investigated in the bovine corneal opacity and permeability (BCOP) assay. The mean in vitro irritation score (IVIS) was calculated as 3.11.

The eye irritation potential of the test item was investigated in a EpiOcularTM Eye irritation test (RhCE) model. The mean value of relative tissue viability was 49,5% which is below the threshold for eye irration potential (<= 60%).

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

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)

Qualifier:

according to guideline

Guideline:

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

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 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

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

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%

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%

 

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

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)

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)

Species:

other: EpiOcularTM tissue. Human-derived keratinocytes

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

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%

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%

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.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

November 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Version / remarks:

Feb 2017

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

Version / remarks:

Jun 2020

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

GLP compliance:

yes (incl. QA statement)

Species:

cattle

Strain:

other: Bos primigenius Taurus (fresh bovine corneas)

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Slaughterhouse Müller Fleisch GmbH
- Characteristics of donor animals (e.g. age, sex, weight): Age between 12 and 60 months old
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): T ransported in Hanks’ Balanced Salt Solution with 1% Penicillin-Streptomycin solution (Penicillin 100 U/mL, Streptomycin 100 μg/mL) in a suitable cooled container within 1 hour
- Selection and preparation of corneas: The corneas were excised, and incubated for 1h at 32 ± 1 °C in pre-warmed medium cMEM

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit):
- Concentration (if solution): 750 μL

VEHICLE
- Amount(s) applied (volume or weight with unit):
- Concentration (if solution):
- Lot/batch no. (if required):
- Purity:

Duration of treatment / exposure:

Exposure time: 10 minutes
Post-treatment incubation time: 2 hours

Details on study design:

NUMBER OF REPLICATES : 3

NEGATIVE CONTROL USED: HBSS, Hank's Balanced Salt solution diluted in water 1:10

POSITIVE CONTROL USED: Dimethylformamide (DMF), undiluted

APPLICATION DOSE AND EXPOSURE TIME: 750 µL. The exposure time was 10 minutes at 32°C.

TREATMENT METHOD: closed chamber for the negative and positive control / open chamber: test item

POST-INCUBATION PERIOD: 2 hours

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: 2 rinses. First with cMEM with phenol red and second with cMEM without phenol red.


METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: change of opacity was calculated by subtracting the intitial basal opacity from the post opacity reading
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate photometer. Corrected OD492 values were calculated

SCORING SYSTEM: In Vitro Irritancy Score (IVIS): The IVIS of each replicate of the negative control was calculated from the following equation: IVIS = opacity difference + (15 x corrected OD492 value)
The IVIS of each replicate of the positive control and of the test item were calculated from the following equation:
IVIS = (opacity difference – mean opacity difference of the negative control) + [15 x (OD492 – mean OD492 of the negative control)]

DECISION CRITERIA:
According to OECD Guideline no. 437 (Jun. 2020), a substance with an IVIS > 55 induces serious eye damage, that should be classified as UN GHS Category I.
Classification Scheme:
IVIS ≤ 3 --> no UN GHS category
IVIS  > 3 and ≤ 55 --> no prediction of UN GHS category can be made
IVIS > 55 --> UN GHS: Eye damage Category I

Irritation parameter:

in vitro irritation score

Run / experiment:

Three replicates

Value:

3.11

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS
- Visible damage on test system: No

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Mean IVIS of negative control is ≤3
- Acceptance criteria met for positive control: Mean IVIS of positive control is between 57.92 – 139.81

Opacity values of the test item:

Parameter	Test item
	Rep 1	Rep 2	Rep 3
opacity before exposure	3.14	2.82	2.45
opacity after exposure	6.71	7.45	6.66
Opacity difference	3.57	4.63	4.21
Opacity difference corrected	2.44	3.5	3.08
Mean opacity diff corrected	3.01

Permeability values of the test item:

Parameter	Test item
	rep 1	rep 2	rep 3
Meas 1	0.046	0.052	0.053
Meas 2	0.043	0.053	0.053
Meas 3	0.044	0.052	0.055
Meas blank 1	0.0097	0.0157	0.0167
Meas blank 2	0.0067	0.0167	0.0167
Meas blank 3	0.0077	0.0157	0.0187
Mean of each rep blank	0.0080	0.0160	0.0174
corrected	0.0009	0.0089	0.0102
Corrected mean of 3 replicates	0.0067

IVIS values of the test item:

test group	IVIS	mean IVIS	rel std dev
Negative control	0.75	1.24	0.3534
	1.35
	1.61
Test item	2.45	3.11	0.1937
	3.64
	3.23
Positive control	107.3	107.83	0.0754
	116.21
	99.98

Interpretation of results:

study cannot be used for classification

Conclusions:

Under the conditions of the study, the test item, Pyrolytic Lignin, showed effects on the cornea of the bovine eye. However, according to OECD Guideline no. 437 (Jun. 2020), a substance with an IVIS > 3 and ≤ 55 induces effects on the cornea, that cannot be classified in an UN GHS Category for eye damage with the BCOP study only. In this case no prediction can be made and further testing must be considered for correct classification of the test item.

Executive summary:

This in vitro study was performed to assess the corneal damage potential of Pyrolytic Lignin by quantitative measurements of changes in opacity and permeability in a bovine cornea. The study was conducted in compliance with the OECD Guideline for the Testing of Chemicals, Method No. 437, the OECD Guideline for the Testing of Chemicals, Series on Testing and Assessment No.160 and the Commission Regulation (EU) Method B.47. The BCOP test method is an organotypic model that provides short-term maintenance of normal physiological and biochemical function of the bovine corneain vitro. In this test method, damage by the test item is assessed by quantitative measurements of changes in corneal opacity and permeability. Both measurements are used to calculate an“In VitroIrritancy Score (IVIS)”, which is used to classify the test item in the UN Globally Harmonised System (GHS).

The test item Pyrolytic Lignin was applied onto the cornea of a bovine eye and was incubated on the cornea for 10 minutes at 32 ± 1 °C. After removal of the test item and 2 hours of post-incubation, opacity and permeability values were measured. Under the conditions of this study, the test item BTG Pyrolytic Lignin showed effects on the cornea of the bovine eyeas the calculated mean IVIS was 3.11.

 

According to OECD Guideline no. 437 (Jun. 2020), a substance with an IVIS > 3 and ≤ 55 induces effects on the cornea, that cannot be classified in an UN GHS Category for eye damage with the BCOP study only. In this case no prediction can be made and further testing must be considered for correct classification of the test item.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Justification for classification or non-classification

Skin irritation

In a recent in vitro skin irritation study the test item showed a mean relative tissue viability of 57,6%. The test item is considered irritant to the skin if the mean relative tissue viability of three individual tissues is reduced ≤ 50% of the negative control.

Thus, based on the available data on skin irritation, the test item does not meet the criteria for classification according to Regulation (EC) 1272/2008 (CLP).

Eye corrosion/irritation

The top down approach was applied for the evaluation of the eye irritation potential of the substance. The eye corrosion potential of the test item was investigated in the bovine corneal opacity and permeability (BCOP) assay. The mean in vitro irritation score (IVIS) was calculated as 3.11. Substances with an IVIS >3 and <= 55 cannot be classifed in a CLP category for eye damage with the BCOP study only. Therefore an eye irritation study was perfomed.

The eye irritation potential of the test item was investigated in an EpiOcularTM Eye irritation test (RhCE) model. The mean value of relative tissue viability was 49,5% which is below the threshold for eye irritation potential (<= 60%).

The weight of evidence analyse of the results of both performed in vitro studies, concludes that the substance is to be considered as Eye irritant category 2.

Thus, based on the available data on eye irritation, the test item meets the criteria for classification as Eye irritant Category 2 according to Regulation (EC) 1272/2008 (CLP).