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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:
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:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
Version 439, adopted 28. July 2015
Deviations:
not specified
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
adopted 06. Jul. 2012
Deviations:
not specified
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Name of test material: tert-butylbenzene
- IUPAC name: tert-Butylbenzene
- Molecular formula: C10H14
- Molecular weight: 134.22 g/mol
- Substance type: Organic
- Physical state: Liquid

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
other: human-derived epidermal keratinocytes
Cell source:
other: EpiDermTM tissues were procured from MatTek In Vitro Life Science Laboratories, Brati-slava.
Source strain:
not specified
Details on animal used as source of test system:
The test system is a commercially available EpiDermTM-Kit, procured by MatTek.
The EpiDermTM tissues are cultured on specially prepared cell culture inserts.
Origin: EpiDermTM tissues were procured from MatTek In Vitro Life Science Laboratories, Brati-slava.
Designation of the kit: EPI-200-SIT
Day of delivery: 23. Oct. 2018
Batch no.: 28665
Justification for test system used:
The test consists of a topical exposure of the neat test item to a human reconstructed epi-dermis model followed by a cell viability test. 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 skin irritation potential.
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 (rinsing thoroughly with DPBS)
- Observable damage in the tissue due to washing: Blotted with sterile cellulose tissue
DYE BINDING METHOD
- Dye used in the dye-binding assay: 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (=MTT)
- Spectrophotometer: Plate spectrophotometer
- Wavelength: 570 nm
NUMBER OF INDEPENDENT TESTING RUNS / EXPERIMENTS TO DERIVE FINAL PREDICTION: 3 (Pre - Tests, Pre-Incubation of Tissues and Treatment)
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): The test item was applied directly to each tissue and spread to match the tissue size (0.63 cm2; as indicated by the supplier). 30 µL

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL
- Concentration: DPBS buffer

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL
- Concentration: 5% SDS solution
Duration of treatment / exposure:
24 hours at 37 ± 1°C and 5.0 ± 0.5% CO2
Number of replicates:
3

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Value:
34.3
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: 34.3%+-0.3%

Applicant's summary and conclusion

Interpretation of results:
Category 2 (irritant) based on GHS criteria
Conclusions:
According to the Reconstructed human Epidermis (RhE) Test Method. After the treatment with the test item, the mean value of relative tissue viability was re-duced to 34.3%. This value is below the threshold for skin irritation potential (50%). The test item tert-Butylbenzene that induce values below the threshold of 50% are considered at least irritant to skin.
Executive summary:

Three experiments were performed.

The first experiment was not valid, because the standard deviation of the three replicates of the test item was too high. This is why a second experiment was performed.

The second experiment was not valid, because the standard deviation of the three replicates of the negative control was too high. The first two experiments are not reported in this report, but the raw data are kept in the GLP-archive of the test facility.

The third experiment was valid.

 

The test itemtert-Butylbenzeneis considered as at least irritant to skin.

After the treatment, the mean value of relative tissue viability was reduced to 34.3%. This value is below 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.