Thermal cracking oil from blends of rubber, fuel oils and paraffin waxes, steam distillation concentrate

Endpoint summary

Description of key information

The skin corrosion potential of Thermal cracking oil from blends of rubber, fuel oils and paraffin waxes, steam distillation condensate was evaluated using OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method). The test was GLP compliant.
Mean tissue viability (as a percentage of the negative control) following a 3 and 60 minute exposure was 101.767% and 106.095% respectively. Thermal cracking oil from blends of rubber, fuel oils and paraffin waxes, steam distillation condensate was determined not to meet the criteria for classification according to CLP Regulation (EC) No. 1272/2008.

The in vitro skin irritation of Thermal cracking oil from blends of rubber, fuel oils and paraffin waxes, steam distillation condensate was determined in accordance with the OECD Guideline for Testing of Chemicals 439 during a GLP-compliant study.
Skin irritation of the test substance and controls was evaluated in triplicate.
The relative mean viability of the test item treated tissues was 105.4% after the 15-Minute exposure period and 42-Hours post-exposure incubation period.
The criteria required for acceptance of results in the test were satisfied.
In this study and under the experimental conditions reported, the test item was classified as non-irritant.

An in vitro Bovine Corneal Opacity and Permeability (BCOP) assay was performed in line with OECD Testing Guideline 437 (Bovine Corneal Opacity and Permeability Test Method). The study was GLP compliant. The undiluted test material was applied to three cattle corneas at a volume of 0.75 mL, after which each cornea was incubated at 32±1°C for 10 minutes. Thermal cracking oil from blends of rubber, fuel oils and paraffin waxes, steam distillation condensate, produced an IVIS of 3.8. According to UN GHS Classification for the test item, No Prediction of eye irritation can be made under the conditions of the test. Since the OECD 437 is designed to identify Eye damage 1 or not classified substances, a decision to classify the substance as eye irritant cat. 2 was taken.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17-22/02/2021

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 18 June 2019, corrected 26 June 2020

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

EC Number: 942-492-8
Batch: 02 November 2020
Purity: UVCB (Substance of Unknown or Variable
composition, Complex reaction products or Biological
materials)

Test system:

human skin model

Source species:

other: EPISKIN™ Reconstructed Human Epidermis Model Kit

Cell type:

other: adult human-derived epidermal keratinocytes

Cell source:

other: three-dimensional reconstructed human epidermis model consisting of adult human-derived epidermal keratinocytes seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen.

Justification for test system used:

Following a full validation study (ECVAM, 2009) the EpiSkinTM reconstructed human epidermis model showed evidence of being a reliable and relevant stand-alone test for predicting rabbit skin irritation when the endpoint is measured by MTT reduction and for being used as a replacement for the Draize Skin Irritation Test for the purpose of distinguishing between Irritating and Non-Irritating test items.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKINTM
- Tissue batch number(s): 21-EKIN-007
- Data received: 17/02/2021
- suggested expiration date: 22/02/2021
- Date of initiation of testing: 17/02/2021

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature / 15 minutes
- Temperature of post-treatment incubation (if applicable):The rinsed tissues were incubated at 37 °C, 5% CO2 in air for 42 hours.

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: each tissue was removed from the well using forceps and
rinsed using a wash bottle containing DPBS with Ca++ and Mg++. Rinsing was achieved by
filling and emptying each tissue insert for approximately 40 seconds using a constant soft
stream of DPBS to gently remove any residual test item.
- Observable damage in the tissue due to washing: not specified

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 2 mL of a 0.3 mg/mL MTT solution, freshly prepared in assay medium, was pipetted into the third column of 3 wells of the 12-well plates
- Incubation time: 3 hours at 37 °C, 5% CO2 in air
- Spectrophotometer: The optical density (OD570) was measured (quantitative viability
analysis) at 570 nm (without a reference filter) using the Labtech LT-4500 microplate reader.
- Wavelength: 570 nm


NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues / killed tissues
- Procedure used to prepare the killed tissues (if applicable):
- N. of replicates : 3
- Method of calculation used:


PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)

- The test substance is considered to be non-corrosive to skin if [complete, e.g. the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.]

Control samples:

yes, concurrent no treatment

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied:10 μL (26.3 μL/cm2)

VEHICLE
N/A

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 10 μL of DPBS served as the negative controls

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 10 μL of SDS 5% w/v served as the positive controls

Duration of treatment / exposure:

Triplicate tissues were treated with the test item for an exposure period of 15 minutes.
To
ensure satisfactory contact with the positive control item the SDS solution was spread over
the entire surface of the epidermis using a pipette tip (taking particular care to cover the
center). After a 7-Minute contact time the SDS solution was re-spread with a pipette tip to
maintain the distribution of the SDS for the remainder of the contact period (re-spreading is
not required for the negative control or test item). The plates were kept in the biological
safety cabinet at room temperature for 15 minutes.

Duration of post-treatment incubation (if applicable):

At the end of the exposure period, each tissue was removed from the well using forceps and
rinsed using a wash bottle containing DPBS with Ca++ and Mg++. Rinsing was achieved by
filling and emptying each tissue insert for approximately 40 seconds using a constant soft
stream of DPBS to gently remove any residual test item. The rinsed tissues were transferred to the second column of 3 wells containing 2 mL of maintenance medium in each well. The
rinsed tissues were incubated at 37 °C, 5% CO2 in air for 42 hours.

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Value:

>= 105.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: none
- Direct-MTT reduction: The MTT solution containing the test item did not turn blue or purple which indicated that the test item did not directly reduce MTT.
- Colour interference with MTT: The solution containing the test item was colorless. It was therefore unnecessary to run color correction tissues.

DEMONSTRATION OF TECHNICAL PROFICIENCY:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD570 for the negative control treated tissues was 0.918 and the standard deviation value of the viability was 6.4%. The negative control acceptance criteria were therefore satisfied.
- Acceptance criteria met for positive control: The relative mean tissue viability for the positive control treated tissues was 4.4% relative to
the negative control treated tissues and the standard deviation value of the viability was 2.3%.
The positive control acceptance criteria were therefore satisfied.
- Acceptance criteria met for variability between replicate measurements: The standard deviation calculated from individual tissue viabilities of the three identically test
item treated tissues was 4.8%. The test item acceptance criterion was therefore satisfied.
- Range of historical values if different from the ones specified in the test guideline: N/A

Interpretation of results:

GHS criteria not met

Conclusions:

In this study and under the experimental conditions reported, the test item was classified as
non-irritant

Executive summary:

A GLP-compliant skin irritation study in vitro was carried out for the test item using EPISKINTM reconstructed human epidermis model after a treatment period of 15 minutes followed by a post-exposure incubation period of 42 hours. The experiment was performed in line with OECD Guideline 439 and EU Method B46.

Triplicate tissues were treated with the test item for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 hours. At the end of the post-exposure incubation period each tissue was taken for MTT-loading. The maintenance medium from beneath each tissue was transferred to pre-labeled micro tubes and stored in a freezer for possible inflammatory mediator determination. After MTT-loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT-loaded tissues. At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 μL samples were transferred to the appropriate wells of a pre-labeled 96-well plate. The optical density was measured at 570 nm.

The relative mean viability of the test item treated tissues was 105.4% after the 15-Minute exposure period and 42-Hours post-exposure incubation period.
The criteria required for acceptance of results in the test were satisfied.

In this study and under the experimental conditions reported, the test item was classified as non-irritant.

Reference 2

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 April -18 April 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Deviations:

no

Principles of method if other than guideline:

Due to the harmful nature and volatility of the test item, the dosing for the 1hr timepoint was carried out in a ducted Class II B2 cabinet that used 100% extraction to kepp both user protected and environment sterile. This was at room temperature under ambient conditions and not at 37°C, 5% CO2 and equal or greater than 95% Relative Humidity as prescribed by the guidelines. This did not affect the study due to the short timepoint and also due to the fact that the medium used in the assay was pre-warmed to 37°C, the negative control and positive control acceptance criteria were met demonstrating that the integrity of the test remained and therefore the data was deemed acceptable for use.

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Human-derived epidermal keratinocytes

Source strain:

other: Keratinocyte Strain

Details on animal used as source of test system:

Not applicable.

Justification for test system used:

The EpiDerm(tm) skin model and assay for skin corrosion testing is endorsed by OECD TG 431.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ Reconstructed Human Epidermis (EPI-200 MatTek Corporation)
- Lot number: 28692
- Receipt date: 16 APR 19
- Date of initiation of testing: 16 APR 19

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37°C, 5% CO2 and equal or greater than 95% Relative Humidity (for 3 minute exposure only)
-- Temperature of post-treatment incubation (if applicable): 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: Not addressed.
- Observable damage in the tissue due to washing: Not addressed.
- Modifications to validated SOP: Due to the harmful nature and volatility of the test item, the dosing for the 1hr timepoint was carried out in a ducted Class II B2 cabinet that used 100% extraction. This was at room temperature under ambient conditions and not at 37°C, 5% CO2 and equal or greater than 95% Relative Humidity as prescribed by the guidelines.This was deemed not to have affected the study, and the data was deemed acceptable for use. This is due to: the short timepoint; the fact that the medium used was pre-warmed to 37°C; the control substances met acceptance criteria.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: Not addressed
- Incubation time: Not addressed
- Spectrophotometer: BMG LabTech FluoStar Optima.
- Wavelength: 570 nm
- Filter: (no reference filter).
- Linear OD range of spectrophotometer: Not stated.

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: 2.04±0.538 = PASS.
- Barrier function: 8.1 hrs = PASS.
- Morphology: 13 layers present & 98.1 µm = PASS .
- Contamination: No contamination reported = PASS.
- Reproducibility: All acceptance criteria PASS: Mean OD570 of negative control tissues were greater than or equal to 0.8 (2.131 and 2.142); Mean positive control viability was <15% after 1hr (9.328%); Coefficients of variation were stated to be acceptable.

NUMBER OF REPLICATE TISSUES: 3 for test substance, positive control and negative control. 2 for colourant control.

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Other: NO interference with MTT was found, but a formation of coloured formation in water wass observed.
- No. of replicates: 2
- Method of calculation used: Substraction of colourant control value from from viability.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1.

PREDICTION MODEL / DECISION CRITERIA
- A tesst item is classified "corrosive" if the relative tissue viability after 3 minute treatment with a test material is decreased below 50%. In addition, those materials classified "non-corrosive" after 3 minute treatment (viability more than or equal to 50%) are classified "corrosive" if the relative tissue viability after 1 hour treatment with a test material is decreased below 15%.
- The test substance is considered to be non-corrosive if the viability after 3 minute exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 µl
- Concentration (if solution): Neat

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 µl
- Concentration (if solution): Neat

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 µl
- Concentration (if solution): 8N

Duration of treatment / exposure:

3 mins and 60 mins

Number of replicates:

3 for test substance, positive control and negative control. 2 for colourant control.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minute dose: Mean of Viability (%)

Value:

101.767

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 minute dose: Mean of Viability (%)

Value:

106.095

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: Not addressed.
- Direct-MTT reduction: None stated.
- Colour interference with MTT: No interference with MTT. However coloured solution was formed in water and as such was controlled by the use of a colourant control.

DEMONSTRATION OF TECHNICAL PROFICIENCY: OECD TG 431

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Mean OD570 of negative control tissues must be greater ≥0.8. Results for 3 mins = 2.131 and for 1hr = 2.142 therefore passed
- Acceptance criteria met for positive control: The mean of the positive control relative percentage viability, after 1hour exposure must be < 15% of the mean of the negative control. The result was a pass at 9.328%.
- Acceptance criteria met for variability between replicate measurements: should not exceed 0.3 (30%). Results= Pass.the CV value of the PC after 1hr was above 30% (56.079%). However, as the percentage of viability was below 20% (9.328%), acceptance criterion 3 is met.

Interpretation of results:

GHS criteria not met

Conclusions:

In the reported study, the mean tissue viability (as a percentage of the negative control) following a 3 and 60 minute exposure was 101.767% and 106.095% respectively. Thermal cracking oil from blends of rubber, fuel oils and paraffin waxes, steam distillation condensate was determined not to meet the criteria for classification according to CLP Regulation (EC) No. 1272/2008.

Executive summary:

In order to determine the skin corrosion potential of Thermal cracking oil from blends of rubber, fuel oils and paraffin waxes, steam distillation condensate, a GLP complaint study was carried out in accordance with OECD guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method).Thermal cracking oil from blends of rubber, fuel oils and paraffin waxes, steam distillation condensate was applied as single topical application of 50µl of the neat test item, negative controls of tissue grade sterile water and positive controls of potassium hydroxide were also prepared and applied at 50 µl for 3 and 60±1 minute to the surface of the EpiDerm™ skin models at 37 °C, 5 % CO2, 95 % RH (relative humidity), prior to the MTT endpoint: three tissues per condition (n=3).Prior to the assay, the test item was checked for interference (water colouration or MTT interference) and was found not to interfere with MTT but was found to form a coloured solution in water therefore additional colourant controls were included in the study. Corrosion is directly related to cytotoxicity in the EpiDerm™ tissue. Cytotoxicity is determined by the reduction of MTT (3-[4,5dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to formazan by viable cells in the test item treated tissues relative to the corresponding negative control. The results are then used to make a prediction of the corrosivity potential of the test item. All controls were valid and demonstrated the reliability of the test system. Although the colourant control values were minimal, they were removed from the overall viability. Mean tissue viability (as a percentage of the negative control), was 101.767% and 106.095% after 3 and 60 minutes of exposure, respectively. Therefore, Thermal cracking oil from blends of rubber, fuel oils and paraffin waxes, steam distillation condensate does not meet the criteria for classification according to CLP Regulation (EC) No. 1272/2008.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not 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:

21 April 2021

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

Update 26/06/2020

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Batch 02 November 2020

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Eyes from adult cattle (typically 12 to 60 months old) were obtained from a local abattoir as a
by-product from freshly slaughtered animals. The eyes were excised by an abattoir employee
after slaughter, and were placed in Hanks’ Balanced Salt Solution (HBSS) supplemented with
antibiotics (penicillin at 100 IU/mL and streptomycin at 100 μg/mL). They were transported
to the test facility over ice packs on the same day of slaughter. The corneas were refrigerated
on arrival and used within 24 hours of receipt.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied (volume or weight with unit): 0.75 ml neat substance

Duration of treatment / exposure:

10 min

Duration of post- treatment incubation (in vitro):

120 minutes

Number of animals or in vitro replicates:

Three corneas were randomly allocated to the negative control. Three corneas were also
allocated to the test item and three corneas to the positive control item.

Details on study design:

NUMBER OF REPLICATES: 3

NEGATIVE CONTROL USED: Sodium chloride 0.9% w/v

SOLVENT CONTROL USED (if applicable): no

POSITIVE CONTROL USED: Neat Ethanol

APPLICATION DOSE AND EXPOSURE TIME: 0.75 ml / 10 min

TREATMENT METHOD: [closed chamber / open chamber]

POST-INCUBATION PERIOD: yes. If YES please specify duration: 120 min

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: rinsed 3 times with fresh complete EMEM containing phenol red before a final rinse with complete EMEM without phenol red.
- POST-EXPOSURE INCUBATION: The holders were incubated, anterior chamber facing forward, at 32 ± 1 ºC for 120 minutes.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The change in opacity for each cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final opacity reading. These values were then corrected by subtracting the average change in opacity observed for the negative control corneas. The mean opacity value of each treatment group was then calculated by averaging the corrected opacity values of each cornea for that treatment group
- Corneal permeability: 360 μL of media representing each cornea was dispensed into the appropriate wells of a pre-labeled 96-well plate. The optical density was measured (quantitative viability analysis) at 492 nm (without a reference filter) using the Labtech LT-4500 microplate reader.


SCORING SYSTEM: In Vitro Irritancy Score (IVIS): mean opacity value + (15 x mean permeability OD492 value)

DECISION CRITERIA: please specify if the decision criteria as indicated in the TG was used: YES

Irritation parameter:

in vitro irritation score

Value:

3.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

Value 0.3

Positive controls validity:

valid

Remarks:

Value 38.2

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: The corneas treated with the test item were clear post treatment and post incubation.


ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control values were within accepted limits.
- Acceptance criteria met for positive control: The positive control In Vitro Irritancy Score was within the acceptance range.

Interpretation of results:

study cannot be used for classification

Conclusions:

According to UN GHS Classification for the test item Thermal cracking oil from blends of
rubber, fuel oils and paraffin waxes, steam distillation condensate, No stand-alone prediction
of eye irritation can be made under the conditions of the test.

Executive summary:

An in vitro Bovine Corneal Opacity and Permeability (BCOP) assay was performed in line with OECD Testing Guideline 437 (Bovine Corneal Opacity and Permeability Test Method). The study was GLPcompliant.
The undiluted test material was applied to three cattle corneas obtained from an abattoir at a volume of 0.75 mL, after which each cornea was incubated at 32±1°C for 10 minutes.

Opacity and permeability of corneas were measured following exposition.
Negative and positive controls were performed and considered as valid.

An In Vitro Irritation Score (IVIS) of 3.8 was calculated for the test item from corneal opacity and permeability measurements. According to UN GHS Classification for the test item Thermal cracking oil from blends of rubber, fuel oils and paraffin waxes, steam distillation condensate, No stand-alone prediction of eye irritation can be made under the conditions of the test.

Since the OECD 437 is designed to identify Eye damage 1 or not classified substances, a decision to classify the substance as eye irritant cat. 2 was taken.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Since the OECD 437 is designed to identify Eye damage 1 or not classified substances, a decision to classify the substance as eye irritant cat. 2 was taken.