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Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19. Mar. 2018 to 23. Mar. 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
OECD Guideline for the Testing of Chemicals, Version 439, adopted 28. July 2015, “In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method”
Deviations:
no
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 Regulation (EC) No. 761/2009, Annex III, EU method B.46 “IN VITRO SKIN IRRITATION: RECONSTRUCTED HUMAN EPIDERMIS MODEL TEST”, adopted 06. Jul. 2012
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Reference substance name:
Petroleum resins
EC Number:
265-116-8
EC Name:
Petroleum resins
Cas Number:
64742-16-1
Molecular formula:
UVCB substance containing saturated aliphatic hydrocarbon and aromatic hydrocarbons
IUPAC Name:
naphthalene
Test material form:
liquid
Details on test material:
Reference Name: Petroleum Resins (Kendex 0897)
Sponsor Information: CAS Number 64742-16-1
Label Identification: LANXESS Lot # E267112117, 18 Jan 18, Kendex 0897
Quantity & Date Received: 24 Jan 18; 76.1 g (GW)
Physical Description: Brown liquid
Storage: Room temperature
Purity: See Certificate of Analysis
Stability: Not provided to testing facility
Specific details on test material used for the study:
No further details specified in the study report

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: not specified
Source strain:
not specified
Details on animal used as source of test system:
Specification
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 specially prepared cell culture inserts.

Origin
EpiDermTM tissues were procured from MatTek In Vitro Life Science Laboratories, Bratislava.
Designation of the kit: EPI-200-SIT
Day of delivery: 20. Mar. 2018
Batch no.: 25887
Justification for test system used:
In accordance with test guidelines
Vehicle:
unchanged (no vehicle)
Details on test system:
Pre - Tests
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 microscope slide.
No reaction with the nylon mesh was visible after an exposure time of 1 hour.

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 ± 0.5% CO2 for 1 hour.
The resulting solution was colourless, therefore no binding capacity had to be tested.

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 ± 0.5% CO2 for 1 hour. Untreated MTT medium was used as control.
The MTT solution did not change its colour within 1 hour. Therefore, direct MTT reduction by the test item had not taken place and no data correction was 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 wells (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 ± 0.5% CO2 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 ± 0.5% CO2 for 18 hours 55 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% SDSsolution, 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 were applied, and a nylon mesh was added in order to ensure sufficient contact with the tissue surface.
Tissues were dosed in 1-minute-intervals. After dosing the last tissue, all plates were transferred into the incubator for 35 minutes at 37 ± 1°C and 5.0 ± 0.5% CO2.
1 hour after the first application, the inserts were removed from the plates using sterile forceps and rinsed immediately in 1-minute-intervals.
After rinsing, each tissue was blotted with sterile cellulose tissue and then transferred into a new 6-well-plate with fresh assay medium (0.9 mL).
Then, the tissues were set in the incubator for 23 hours 25 minutes at 37 ± 1°C and 5.0 ± 0.5% CO2.

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-wellplate.
Then the inserts were transferred into the lower row of the 6-well-plate and set into the incubator for 19 hours 15 minutes for post-incubation at 37 ± 1°C and 5.0 ± 0.5% CO2.

MTT Assay
After a total incubation time of 42 hours 40 minutes, a 24-well-plate was prepared with 300 μL freshly prepared MTT-solution 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 at 37 ± 1°C and 5.0 ± 0.5% CO2.
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-wellplate which was read in a plate spectrophotometer at 570 nm.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
30 μL test item was applied
Duration of treatment / exposure:
1 hour
Duration of post-treatment incubation (if applicable):
Then, the tissues were set in the incubator for 23 hours 25 minutes at 37 ± 1°C and 5.0 ± 0.5% CO2.
Number of replicates:
3 tissues per test system (test item, negative control, positive control)

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Test Item
Value:
108.4
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
The mean value of relative tissue viability of the test item was increased to 108.4% after the treatment. This value is above the threshold for skin irritation (50%). Therefore, the test item is considered as non-irritant to skin. All validity criteria were met.
Values for negative control and for positive control were within the range of historical data of the test facility.
Therefore, the experiment is considered valid.

Any other information on results incl. tables

Measured Values

As blank, the optical density of isopropanol was measured in 8 wells of the 96-well-plate.

The measured values and their mean are given in the following table:

Absorbance values blank isopropanol (OD 570 nm)

Replicate

1

2

3

4

5

6

7

8

Mean

Absorbance

0.039

0.038

0.040

0.040

0.039

0.038

0.040

0.039

0.039

 

The absorbance values of negative control, test item and positive control are given in the following table:

Absorbance Values negative control, test item and positive control (OD 570 nm)

Designation

Measurement

Negative Control

Petroleum Resins (Kendex 0897)

Positive Control

Tissue 1

1

1.314

1.269

0.072

2

1.484

1.344

0.072

Tissue 2

1

1.512

1.645

0.071

2

1.477

1.607

0.073

Tissue 3

1

1.187

1.470

0.071

2

1.230

1.540

0.071

 

From the measured absorbances, the mean of each tissue was calculated, subtracting the mean absorbance of isopropanol as given in table 9.1-a. The mean of the three tissues was also calculated.

Mean Absorbance Values

Designation

Negative Control

Petroleum Resins (Kendex 0897)

Positive Control

Mean – blank (tissue 1)

1.360

1.268

0.033

Mean – blank (tissue 2)

1.456

1.587

0.033

Mean – blank (tissue 3)

1.170

1.466

0.032

Mean of the three tissues

1.329

1.440

0.033

 

Comparison of Tissue Viability

For the test item and the positive control, the following percentage values of tissue viability were calculated in comparison to the negative control:

% Tissue Viability

Designation

Petroleum Resins (Kendex 0897)

Positive Control

% Tissue viability (tissue 1)

95.4%

2.5%

% Tissue viability (tissue 2)

119.4%

2.5%

% Tissue viability (tissue 3)

110.3%

2.4%

% Tissue viability (mean)

108.4%

2.5%

± SD of mean tissue viability (%)

12.1%

0.0%

 

Validity and Acceptability

Validity criteria and results are stated in the following table:

Validity

Criterion

Demanded

Found

OD of negative control

≥ 0.8 and ≤ 2.8

1.3

% tissue viability of positive control SDS

≤ 20% of negative control

2.5%

SD of mean viability of the tissue replicates (%)

≤ 18%

11.0% (negative control)

0.0% (positive control)

12.1% (test item)

 

Historical Data

Parameter

Negative Control

(OD)

Positive Control

(% OD compared to Negative Control)

Substance

DPBS buffer

Sodium Dodecyl Sulphate Solution 5%

Mean

1.829

4.6%

Standard deviation

0.319

3.2%

Range

0.476 – 2.471

1.7 – 17.1%

Study 18012205G840

1.329

2.5%

 

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The test item Petroleum Resins (Kendex 0897) is considered as non-irritant to skin.
After the treatment, the mean value of relative tissue viability was increased to 108.4%.
This value is above the threshold for skin irritation (50%).
The optical density of the negative control was well within the required acceptability criterion of 0.8 ≤ mean OD ≤ 2.8.
The positive control has met the acceptance criterion too, for thus ensuring the validity of the test system.
Variation within replicates was within the accepted range for negative control, positive control and test item (required: ≤ 18%).
For these reasons, the result of the test is considered valid.
Executive summary:

Title of Study: Determination of Skin Irritation Potential of Petroleum Resins (Kendex 0897) in the Reconstructed human Epidermis (RhE) Test Method following EU-Method B.46 and OECD 439

 

Findings and Results:

One valid experiment was performed.

Three tissues of the human skin model EpiDermTM were treated with Petroleum Resins (Kendex 0897) 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 was1.3. The positive controlshowed clear irritating effects. The mean value of relative tissue viability was reduced to2.5% (required:20%).

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

 

After the treatment with the test item, the mean value of relative tissue viability was increased to 108.4%. This value is above the threshold for skin irritation potential (50%).

Test items that induce values above the threshold of 50% are considered non-irritant to skin.

 

Therefore, Petroleum Resins (Kendex 0897) is considered non-irritant to skin in the Reconstructed human Epidermis (RhE) Test Method.