Pyrolytic sugar

Endpoint summary

Description of key information

Skin irritation:

The skin irritation potential was investigated in a Reconstructed human Epidermis (RhE) study (OECD 431). After the treatment with the test item, the mean value of relative tissue viability was reduced to 30.4%. This value is below the threshold for skin irritation potential (50%).

Next, the skin corrosion potential was investigated in a Reconstructed human Epidermis (RhE) study (OECD 439). The result of this test was inconclusive; 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. In order to reach a conclusion for the skin irritation endpoint, the test results on the fast pyrolyis bio-oil , from which the Pyrolytic sugar is extracted, were evaluated. The same MTT observation were noted in the OECD 439 of the fast pyrolyiss bio-oil (FBPO) (CAS 1207435-39-9 EC 692-061-0)

. For this substance, the skin corrosion potential on this substance was further evaluated in a Corrositex test according to OECD 435 and concluded to be non-corrosive. Based on these findings in FBPO , it is considered that pyrolytic sugar is also non-corrosive but irritating to the skin.

Eye corrosion:

The test item BTG Pyrolytic Sugar is found to induce serious eye damage in the Bovine Corneal Opacity and Permeability (BCOP) Test.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

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)

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 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

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.

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%

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 EpiDerm^TM 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.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Oct 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Commission Regulation (EU) No. 640/2012 amending EU method B.46 , adopted Jul. 2012

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Jun 2020

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Remarks:

EpiDerm tissue

Source species:

human

Cell type:

other: epidermal keratinocytes

Details on test system:

The test system is a commercially available EpiDermTM-Kit, procured by MatTek.
The EpiDermTM tissue consists of human-derived epidermal keratinocytes which have been cultured to form a multi-layered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers representing main
lipid classes analogous to those found in vivo. The EpiDermTM tissues are cultured on spe- cially prepared cell culture inserts.

EpiDermTM tissues were procured from MatTek In Vitro Life Science Laboratories, Bratislava.

Designation of the kit: EPI-218-SIT
Day of delivery: 06. Oct. 2020
Batch no.: 30896


PRE-TESTS:
1) Nylon mesh compatibility:
The test item was tested for possible reaction with the nylon mesh, which is used to ensure sufficient
contact with the tissue surface. 30 μL of the test item were pipetted onto a nylon mesh on a microsc ope slide.
No reaction with the nylon mesh was visible after an exposure time of 1 hour.
2) Assessment of coloured or staining test items:
It was tested whether the test item develops a colour without MTT addition. 30 μL test item were given
in a test tube with 0.3 mL H2O demin. and incubated at 37 ± 1°C and 5.0 ± 1% CO2 and ≥ 95% relati ve humidity for 1 hour. The resulting solution showed a significant change in colour, therefore binding capacity should to be tested, but due to an irritant result of the main test no additional test and no data correction is necessary.
3) Assessment of direct reduction of MTT by the test item:
The test item was tested for the ability of direct MTT reduction. To test for this ability, 30 μL test item
were added to 1 mL of MTT solution and the mixture was incubated in the dark at 37 ± 1°C and 5.0 ±
1% CO2 and ≥ 95% relative humidity for 1 hour. Untreated MTT medium was used as control.
The MTT solution showed a significant change in colour within 1 hour.
Therefore, direct MTT reduction by the test item had taken place and an additional test should be performed, but due to an irritant result in the main test no additional test and no data correction is necessary.

PRE-INCUBATION OF TISSUES:
All working steps were performed under sterile conditions. For each treatment group (negative control,
test item and positive control) a 6-well-plate was prepared with 0.9 mL assay medium in 3 of the 6 ells (upper row). The tissues were inspected for viability. Then, the tissues were transferred into the
wells, which contain medium by using sterile forceps and placed into the incubator at 37 ± 1°C and 5
± 1% CO2 and ≥ 95% relative humidity for 1 hour.
After 1 hour pre-incubation, the other 3 wells of each plate (lower row) were filled with fresh assay
medium (0.9 mL). Every tissue was transferred into a well of the lower row. All 6-well-plates were set
into the incubator at 37 ± 1°C and 5.0 ± 1% CO2 and ≥ 95% relative humidity for 19 hours 25 minutes.

TREATMENT:
- One plate (3 tissues) was used as negative control; each tissue was treated with 30 μL DPBS
buffer, a nylon mesh was added in order to ensure sufficient contact with the tissue surface.
- One plate was used as positive control; each tissue was treated with 30 μL 5% SDS-solution, a
nylon mesh was added in order to ensure sufficient contact with the tissue surface.
- One plate was used for treatment with the test item: 30 μL test item was applied and a nylon mesh w as added in order to ensure sufficient contact with the tissue surface.
Tissues were dosed in 1-minute-intervals. 25 min after dosing the last tissue, all plates were transferred into
the incubator for 35 minutes at 37 ± 1°C and 5.0 ± 1% CO2 and ≥ 95% relative humidity. 1 hour after
the first application, the inserts were removed from the plates using sterile forceps and rinsed imm ediately in 1-minute-intervals. After rinsing thoroughly with DPBS, each tissue was blotted with sterile
cellulose tissue and then transferred into a new 6-well-plate with fresh assay medium (0.9 mL). The
surface of the inserts was then carefully dried with a sterile cotton tipped swab. Then, the tissues were set in the incubator for 23 hours at 37 ± 1°C and 5.0 ± 1% CO2 and ≥ 95% relati ve humidity.

MEDIUM RENEWAL:
After post-incubation, the tissues were removed from the incubator and shaken for 5 minutes (120
rpm). 0.9 mL assay medium were filled in the lower row of the 6-well-plate. Then the inserts were transferred into the lower row of the 6-well-plate and set into the incubator for 19 hours and 30 minutes for post-incubation at 37 ± 1°C and 5.0 ± 1% CO2 and ≥ 95% relative humidity.

MTT ASSAY:
After a total incubation time of 42 hours and 30 minutes, a 24-well-plate was prepared with 300 μL freshly prepared MTT-solution (1 mg/ml) in each well. The tissues were blotted on the bottom and then
transferred into the 24-well-plate. Then the 24-well-plate was set into the incubator for 3 hours and
2 minutes at 37 ± 1°C and 5.0 ± 1% CO2 and ≥ 95% relative humidity. After this time, the MTT-solution was aspirated and replaced by DPBS buffer. This was then aspirated, too, and replaced several times. At last, each insert was thoroughly dried and set into the empty, pre-warmed 24-well-plate. Into each well, 2 mL isopropanol were pipetted, taking care to reach the upper rim of the insert. The plate was then shaken (120 rpm) for 2 hours at room temperature.
After 2 hours, the inserts were pierced with an injection needle, taking care that all colour was extracted. The inserts were then discarded and the content of each well was thoroughly mixed in order to achieve homogenisation. From each well, two replicates with 200 μL solution (each) were pipetted into a 96-well- plate which was read in a plate spectrophotometer at 570 nm. In addition, eight wells of the 96-well-plate were filled with 200 μl isopropanol each, serving as blank.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30μL
NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30μL DPBS buffer
POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30μL 5% SDS-solution

Duration of treatment / exposure:

1h

Duration of post-treatment incubation (if applicable):

23 hours

Number of replicates:

2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean of three replicates

Value:

30.4

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

DEMONSTRATION OF TECHNICAL PROFICIENCY:
The validity of the skin irritation study was demonstrated in a 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:
- Optical density of negative control: 1.4 -- acceptance criterion: ≥ 0.8 and ≤ 2.8 --> accepted
- % tissue viability of positive control SDS: 11.0% -- acceptance criterion: ≤ 20% of negative control --> accepted
- SD of mean viability of the tissue replicates (%): 9.0% (negative control), 3.5% (positive control),
5.0% (test item) -- acceptance criterion ≤ 18% --> accepted

The values for negative control and for positive control were within the range of historical data of the test facility.

Interpretation of results:

other: the study alone does not allow conclusion on the classification

Conclusions:

The test item is considered as a least irritant to skin the Reconstructed human Epidermis (RhE) Test Method

Executive summary:

The skin irritation potential of the test item was assessed according to the Reconstructed human Epidermis (RhE) test method following OECD guideline 439.

Three tissues of the human skin model EpiDermTM were treated with the test item for 60 minutes. The test item was applied directly to each tissue and spread to match the tissue size (0.63 cm2; as indicated by the supplier).

DPBS-buffer was used as negative control and 5% SDS solution was used as positive control.

After treatment with the negative control, the mean absorbance value was within the required acceptability criterion of 0.8 ≤ mean OD ≤ 2.8, OD was 1.4.

The positive control showed clear irritating effects. The mean value of relative tissue viability was reduced to 11.0 % (required: ≤20%).

The variation within the tissue replicates of negative control, positive control and test item was acceptable (required: ≤ 18%).

After the treatment with the test item, the mean value of relative tissue viability was reduced to 30.4%. This value is below the threshold for skin irritation potential (50%). Test items that induce values below the threshold of 50% are considered at least irritant to skin.

The test item Pyrolytic Sugar is considered irritant to skin in the Reconstructed human Epidermis (RhE) Test Method. The study does not allow to determine the skin corrosion properties of the substance.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Nov 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

GLP compliance:

yes (incl. QA statement)

Species:

cattle

Strain:

other: Bos rimigenius Taurus (fresh bovine corneas) p

Details on test animals or tissues and environmental conditions:

Fresh bovine eyes were obtained from the slaughterhouse Müller Fleisch GmbH, Industri- estraße 42, 75217 Birkenfeld, Germany, on the day of the test. The cattle were between 12 and 60 months old. The eyes were transported to the test facility 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.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

Test material:
-amount applied: 750 μL
- concentration: neat

Duration of treatment / exposure:

Exposure time: 10 minutes at 32 ± 1 °C.
After thorough rinsing the anterior chambers with cMEM with phenol red and final rinsing with cMEM without phenol red, the anterior chambers were filled with cMEM without phenol red and the cornea holders were stored for additional 2 hours at 32 ± 1 °C (post-incubation).
After post-incubation time the final opacity value of each cornea was recorded.

Number of animals or in vitro replicates:

3 replicates

Details on study design:

Preparation of Test System
After having carefully cleaned and sterilized the cornea holders, they were kept in the incu bation chamber at 32 ± 1 °C.
After the arrival of the corneas, they were examined and only corneas which were free from damages were used.
The corneas were excised with a scalpel and cut from the globe with a 2-3 mm ring of sclera around the outside. Each cornea was transferred to a cornea holder in which pre-warmed cMEM (32 ± 1 °C) without phenol red was filled. The holders were then incubated for 1 hour in the incubation chamber at 32 ± 1 °C. The formation of bubbles was prevented.
After the initial incubation, the medium was completely changed and the baseline opacity for each cornea was recorded. The baseline opacity was measured by placing the cornea holder in an opacitometer and recording the illuminance (unit: LUX).
None of the corneas showed an opacity greater than seven opacity units; therefore, all corneas were used.

Preparations
On the day of the assay, the MEM without phenol red was supplemented with sodium bicarbonate, L-glutamine and 1% fetal calf serum (= complete MEM) and stored in a water bath at 32 ± 1 °C.
The same was performed with the MEM with phenol red, but without addition of sodium bi- carbonate.

Application
For each treatment group (negative control solution, test item or positive control), three replicates were used. After removal of the pre-incubation medium (cMEM without phenol red), 750 μL of negative control solution, 750 μL of test item or 750 μL of positive control were applied to each replicate to the epithelial side of the cornea.
According to the characteristics of the controls, the following treatment procedure was performed:
Closed Chamber Method:
The respective substance (negative control solution, test item solution or positive control) was applied by pipetting 750 μL of the appropriate liquid through the refill hole in the anterior holder on the cornea. The controls and the test item were given on the epithelium that the cornea was evenly covered.
According to the characteristics of the test item, the following treatment procedure was per- formed:

Open Chamber Method:
In order to apply the test item, the window-locking ring and glass window of the anterior chamber of each cornea holder was unscrewed to remove the glass disc.
750 μL of the test item was applied directly onto the cornea. After dosing, the glass windows were replaced on the anterior chambers to recreate a closed system.

Permeability test
After the recording of the final opacity values, the cMEM without phenol red was removed from both chambers of each cornea holder. The posterior chamber, which interfaces with the endothelial side of the cornea was filled with fresh cMEM. Then 1 mL sodium fluorescein solution was added to the front chamber of each cornea holder for the detection of permea- bility of the corneas.
For the controls and the test item a sodium fluorescein solution with a concentration of 4 mg/mL was used.
The chambers were then closed again and incubated for 90 minutes at 32 ± 1 °C in a hori- zontal position. After incubation, the content of each posterior chamber was thoroughly mixed and pipetted in a 96-well plate. Then, its optical density at 492 nm was measured with the microtiter plate photometer.

Irritation parameter:

in vitro irritation score

Run / experiment:

Three replicates

Value:

57.59

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

cornea opacity score

Run / experiment:

mean of three replicates

Value:

40.61

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

other: permeability

Run / experiment:

three replicates

Value:

1.131

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

According to the guideline, the test is considered as valid if the positive control causes an IVIS that falls within two standard deviations of the current historical mean.
The mean IVIS of the negative control has to show an IVIS ≤ 3.

The validity criteria
Mean IVIS of negative control HBSS, criterion: ≤3, result: 1.24 -->ok
Mean IVIS of positive control DMF undiluted, criterion: between 57.92 and 139.81, result: 107.83 -->ok

All validity criteria were met. The values for the negative and positive control were within the range of historical data of the test facility (see Annex 2, page 22). Therefore, the test system was acceptable.

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Conclusions:

The test item Pyrolytic Sugar is found to induce serious eye damage in the Bovine Corneal Opacity and Permeability (BCOP) Test.

Executive summary:

The test was performed in accordance to the OECD TG 437 and in compliance to GLP.

Bovine corneas were used. They were collected from slaughtered cattle that were between 12 and 60 months old.

The test item Pyrolytic Sugar was applied onto the cornea of a bovine eye which had been previously incubated with cMEM without phenol red at 32 ± 1 °C for 1 hour and whose opacity had been measured.

The test item was incubated on the cornea for 10 minutes at 32 ± 1 °C. After removal of the test item and 2 hours post-incubation, opacity and permeability values were measured.

Hank’s Balanced Salt Solution (HBSS) was used as negative control. The negative control showed no irritating effect on the cornea and the calculated mean IVIS (In Vitro Irritancy Score) was 1.24.

Dimethylformamide (DMF) undiluted was used as positive control. The positive control induced serious eye damage on the cornea and was within two standard deviations of the current historical mean. The calculated mean IVIS was 107.83.

Under the conditions of this study, the test item Pyrolytic Sugar induced serious eye damage on the cornea of the bovine eye. The calculated mean IVIS was 57.59.

According to OECD Guideline no. 437 (Jun. 2020), a substance with an IVIS > 55 in- duces serious eye damage, that should be classified as UN GHS Category I.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Justification for classification or non-classification

Skin irritation:

In function of the determination of the skin irritation potential of the substance, the following information was taken into account:

A bottom up approach was applied. First the skin irritation potential was investigated in a Reconstructed human Epidermis (RhE) study (OECD 431).

Three tissues of the human skin model EpiDermTM were treated with the test item for 60 minutes. The test item was applied directly to each tissue and spread to match the tissue size (0.63 cm2; as indicated by the supplier).

After the treatment with the test item, the mean value of relative tissue viability was reduced to 30.4%. This value is below the threshold for skin irritation potential (50%). Test items that induce values below the threshold of 50% are considered at least irritant to skin. To evaluate the skin corrosion potential of the substance, a Reconstructed Human Epidermis (RhE) study (OECD 439) was performed.

In the main test two tissues of the human skin model EpiDerm^TM were treated with the test item for 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 of 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.

During the performance of the same test on the fast pyrolyis bio-oil, from which the Pyrolytic sugar is extracted, the MTT interaction was also observed and no conclusion on skin corrosivity could be made. The skin corrosion potential on this substance was further evaluated in a Corrositex test according to OECD 435.

According to the corrositex skin corrosion scale stated in the INVITTOX protocol no 116 “CORROSITEX according to the CTM Continuous Time Monitor Assay”, dated February 2001, and the OECD guideline 435 “In Vitro Membrane Barrier Test Method for Skin Corrosion”, dated 16 July 2006, the test substance Pyrolysis oil (20 wt.%) is considered to be non-corrosive.

Therefore, the three studies were taken into account to determine the skin irritation potential of the substance (skin irritation positive, inconclusive skin corrosion study and conclusive non corrosive study on the related substance). This results in a positive classification for skin irritation. In conclusion the substance should be considered as to be a Skin irritant Category 2 according to Regulation No 1272/2008.

 

Eye irritation:

In a recent in vitro eye corrosion study (BCOP) the test item BTG Pyrolytic Sugar induced serious eye damage on the cornea of the bovine eye. The calculated mean IVIS was 57.59.

Thus, based on the available data on eye irritation, the test item should be considered as Eye damage Category 1 according to Regulation (EC) 1272/2008 (CLP).