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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:
weight of evidence
Study period:
2015-05-29 to 2015-09-17
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline and GLP study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

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:
2013
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
2009
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
other: Epi-200- SIT Kit Components, MTT-100 Assay Kit Components (human derived epidermal keratinocytes)
Strain:
other: not applicable
Details on test animals and environmental conditions:
Epi-200 SIT kits and MTT-100 assays diluent were purchased from MatTek Corporation (82105 Bratislava, Slovakia). The EpiDerm™ tissue consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multilayered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDerm™ tissues (surface 0.6 cm²) are cultured on specially prepared cell culture inserts. EpiDerm™ tissues were shipped with cool packs on medium-supplemented agarose gels in a 24-well plate and reached Envigo CRS on June 30, 2015. On day of receipt the pre-incubation phase of the EpiDerm™ tissues started.

Test system

Type of coverage:
other: not applicable
Preparation of test site:
other: not applicable
Vehicle:
unchanged (no vehicle)
Controls:
other: No control animals because Human skin model. Negative and positive control were used in this test.
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 30 µL
Duration of treatment / exposure:
60 min
Observation period:
not applicable
Number of animals:
Human in vitro skin model, 3 tissues per dose
Details on study design:
Treatment
Time: 60 min
Incubator: Within this period the 6-well plates were put into the incubator for 35 min at 37 +/-1.5 °C, 5+/- 0.5 % CO2.
Rinsing: After the end of the treatment interval the inserts were removed immediately with DPBS (Dulbecco’s Phosphate Buffered Saline) at least 15 times in order to remove any residual test material.
Incubation time: 42 hours

MTT Assay
A volume of 300 µL of the MTT solution (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl-tetrazoliumbromide) was added to each well and the plates were kept in an incubator (37 +/- 1 °C, 5 +/- 0.5% CO2) until further use.
Incubation period: 3 hours
Time: After the 42 hours incubation period was completed for all tissues and exposure groups, culture inserts were transferred from the holding plates to the MTT plates.
Rinsing: After 3 h incubation, wells were rinsed three times with DPBS (Dulbecco's Phosphate Buffered Saline) and were transferred onto new 24-well plates.
Extraction time: About 69 hours and 55 min at room temperature the formazan salt was extracted.
Extractant solution: Isopropanol

OD reading: OD was read in a microplate reader (Versamax Molecular Devices, Softmax Pro, version 4.7.1) with 570 +/- 1 nm filter. Mean values were calculated from the 3 wells per tissue.

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
other: other: Relative absorbance value compared to the relative absorbance value of the negative control
Value:
4.9
Remarks on result:
other:
Remarks:
Basis: other: test item, mean of 3 tissues. Time point: 60 min treatment. Max. score: 100.0. Reversibility: other: not examined. (migrated information)
Irritation / corrosion parameter:
other: other: Relative absorbance value compared to the relative absorbance value of the negative control
Value:
6.5
Remarks on result:
other:
Remarks:
Basis: other: positive control, mean of 3 tissues. Time point: 60 min treatment. Max. score: 100.0. Reversibility: other: not examined. (migrated information)

In vivo

Irritant / corrosive response data:
The mean relative absorbance value of the test item, corresponding to the cell viability, decreased to 4.9% (threshold for irritancy: ≤ 50%), consequently the test item was irritant to skin.

Applicant's summary and conclusion

Interpretation of results:
irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test item is irritating to skin according to EU CLP and UN GHS.
Executive summary:

This in vitro study was performed to assess the irritation potential of the test item by means of the Human Skin Model Test. The test item did not reduce MTT (test for direct MTT (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl-tetrazoliumbromide) reduction), and it did not change colour when mixed with deionised water (test for colour interference). Also its intrinsic colour was not intensive. Consequently, additional tests with freeze-killed or viable tissues were not necessary. Each three tissues of the human skin model EpiDerm™ were treated with the test item, the negative or the positive control for 60 minutes. 30 μL of the test item were applied to each tissue, and spread to match the surface of the tissue. 30 μL of either the negative control (DPBS, Dulbecco's Phosphate Buffered Saline) or the positive control (5% SLS) were applied to each tissue. After treatment with the negative control the absorbance values were well in the required range of the acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 for the 60 minutes treatment interval, thus assuring the quality of the tissues. Treatment with the positive control induced a sufficient decrease in the relative absorbance as compared to the negative control for the 60 minutes treatment interval, and thus assuring the validity of the test system. After treatment with the test item the mean relative absorbance value decreased to 4.9% compared to the relative absorbance value of the negative control. This value is below the threshold for irritancy of ≤ 50%. Therefore, the test item is considered to possess an irritant potential. In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item is irritant to skin.