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

Skin irritation / corrosion

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

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)

Test material

Test material information

Constituent 1

Reference substance name:

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

Molecular formula:

Substance is a UVCB and cannot be defined by molecular and structural information

IUPAC Name:

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

Test material form:

liquid

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)

In vitro test system

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

Results and discussion

In vitro

Results

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

Applicant's summary and conclusion

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.