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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:
2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Acceptable, well-documented study report equivalent or similar to OECD guideline (439): GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report Date:
2019

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Specific details on test material used for the study:
- CAS No.: 84852-02-8
- Batch No.: 20181120
- Expiration date of the lot/batch: 20 November 2021
- Purity: 100%, based on above name treat as a UVCB mixture
- Physical state/Appearance: clear liquid
- Storage conditions: room temperature
- Treatment of study substance prior to testing: used as supplied
- Stability in Solvent: Stable in water (not quantified)

In vitro test system

Test system:
human skin model
Remarks:
EpiDerm™ human reconstructed epidermis model
Source species:
human
Cell type:
other: epidermal keratinocytes
Cell source:
other: adult human
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Epi-200 SIT kits, EpiDerm™(MatTek Corporation, Slovakia)
- Lot No.: 28689
- Delivery date: March 26, 2019
- Date of initiation of testing: March 26, 2019

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1.5 °C
- Temperature of post-treatment incubation (if applicable): 37 ± 1.5 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: gently rinsed with PBS for at least 15 times; after rinsing, the inserts were submerged in PBS at least three times, then the inserts were once again rinsed with sterile PBS from the inside and the outside

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: Versamax®, Molecular Devices
- Wavelength: 570 nm
The test item and the positive and negative controls were washed off the skin tissues after 60 minutes treatment. After further incubation for about 41.5 hours, the tissues were treated with the MTT solution for 3 hours followed by 2.5 hours of extraction of the colorant from the cells. The amount of extracted colorant was determined photometrically at 570 nm.

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
Historical data and the quality certificate of the supplier of the test kit demonstrating its robustness are annexed to the report, including quality control data (determined by MatTek Corporation, 82105 Bratislava, Slovakia) of the respective EpiDerm™ lot. According to the OECD TG 439, the acceptance limit of the ET50 should be between 4.0 hours and 8.7 hours after treatment with 1% Triton X-100 (QC batch release criteria).
- Viability: 1.84 ± 0.034 (pass)
- Barrier function: 5.78 hours (pass)
- Contamination: sterile (pass)

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Freeze-killed tissues
- No. of replicates: 2
- Method of calculation used:
True viability = Viability of treated tissue – Interference from test chemical =
ODtvt – ODkt where ODkt = (mean ODtkt – mean ODukt)
tvt = treated viable tissue kt = killed tissues tkt = treated killed tissue ukt = untreated killed tissue (NC treated tissue)
Since the interference by the test item is < 30% of the negative control value, the net OD of the test item treated killed control was subtracted from the mean OD of the test item treated viable tissues to obtain the true amount of MTT reduction that reflected metabolic conversion only.

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

PREDICTION MODEL / DECISION CRITERIA
- The study substance is considered to be irritant to skin if the mean percent tissue viability after exposure and post-treatment incubation is less than or equal to 50%.
- The study substance is considered to be non-irritant to skin if the mean percent tissue viability after exposure and post-treatment incubation is greater than 50%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
STUDY SUBSTANCE
- Amount applied: 30 μL (47 μL/cm2 according to guideline) of the undiluted test item was dispensed directly atop the EpiDerm™ tissues and spread to match the surface of the tissues for a complete treatment time of 60 minutes.
- Concentration: purity 100%, used as supplied

NEGATIVE CONTROL
- Amount applied: 30 μL
- Concentration: as supplied

POSITIVE CONTROL
- Amount applied: 30 μL
- Concentration: 5% solution in deionised water
Duration of treatment / exposure:
60 minutes
Duration of post-treatment incubation (if applicable):
41.5 hours
Number of replicates:
Triplicate

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Value:
3.81
Negative controls validity:
valid
Remarks:
After treatment with the negative control, the absorbance values were well within the required acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 for the 60 minutes treatment interval thus assuring the quality of the tissues.
Positive controls validity:
valid
Remarks:
Treatment with the positive control induced a decrease in the relative absorbance compared with the negative control to 4.02% thus ensuring the validity of the test system.
Remarks on result:
positive indication of irritation

Applicant's summary and conclusion

Interpretation of results:
Category 2 (irritant) based on GHS criteria
Conclusions:
In conclusion, it can be stated that in this study and under the experimental conditions reported, the study substance is irritant to skin according to UN GHS and EU CLP regulation.
Executive summary:

INTRODUCTION


The purpose of this test was to evaluate the skin irritation potential of the study substance using the EpiDermTM reconstructed human epidermis model after a treatment period of 60 minutes followed by a post-exposure incubation period of 41.5 hours. The principle of the assay was based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the study substance by means of the colorimetric MTT reduction assay. Cell viability is measured by dehydrogenase conversion of MTT [3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl-tetrazoliumbromide], in cell mitochondria, into a blue formazan salt that is quantitatively measured after extraction from tissues. The percent reduction of cell viability in comparison of untreated negative controls is used to predict skin irritation potential.


METHOD


Triplicate tissues of the human skin model EpiDerm™ were treated with the study substance, the negative control (DPBS) or the positive control (5% SDS) for 60 minutes. At the end of the exposure period each tissue was rinsed before incubating for 41.5 hours. The study substance was found to directly reduce MTT and therefore additional non-viable (freeze-killed) tissues were incorporated into the testing for correction purposes. At the end of the post-exposure incubation period each tissue was taken for MTT-loading. 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, the optical density was measured at 570 nm.


RESULTS


The relative mean relative viability of the study substance treated tissues was 3.81% after the 60-Minute exposure period and 41.5-Hours post-exposure incubation period. This value is below the threshold for irritancy of ≤ 50%. Therefore, the study substance is considered to possess an irritant potential.


Quality criteria: The quality criteria required for acceptance of results in the test were satisfied.


CONCLUSION


In this study and under the experimental conditions reported, the study substance is irritant to skin. The following classification criteria apply:


 


Category 2 Irritant based on GHS Criteria