Registration Dossier

Toxicological information

Skin irritation / corrosion

Currently viewing:

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 February 2019 - 29 March 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:
yes
Remarks:
Corrections for color interference were performed although they were unnecessary. Study plan did not inform that the mean OD of negative controls were calculated after blank correction. Deviations had no affect on integrity or validity of the study.
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Specific details on test material used for the study:
- CAS No.: 68515-39-9 / 90193-82-1
- Batch No.: 20181013
- Expiration date of the lot/batch: 13 October 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

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 TISSUE
-Model used: EpiDerm™ (MatTek)
-Batch/lot no.: 82105
-Delivery date: 26 March 2019

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: The treatment time was 60 minutes in total. Within this period, the 6-well plates were put into the incubator for 35 minutes at 37 ± 1.5 °C, 5 ± 0.5% CO2. In the remaining period the plates were placed in a sterile bench at room temperature until the end of treatment.
- Temperature of post-treatment incubation: 37 ± 1.5 °C, 5 ± 0.5% CO2

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: gently rinsed with PBS for at least 15 times in order to remove any residual test material

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hour
- Spectrophotometer: Versamax® Molecular Devices, Softmax Pro Enterprise, version 4.7.1
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 3

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.
- Viability: 1.84 ± 0.034 (pass)
- Barrier function: 5.78 hours (pass)
- Contamination: sterile (pass)

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
The test item reduced MTT (pre-test for direct MTT reduction), but it did not change colour when mixed with deionised water (pre-test for colour interference). Consequently, additional tests with freeze-killed tissues were necessary. The optical pre-experiment (colour interference pre-experiment) to investigate the test item’s colour change potential in water did not lead to a change in colour.
Optical evaluation of the MTT-reducing capacity of the test item after 1 hour incubation with MTT-reagent showed purple colour.
- Control used: 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 study substance was classified as irritant by the SIT (tissue viability < 50 %), the correction procedures was not necessary. Although it is not necessary, the correction procedure was performed according to MatTek protocol.

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

PREDICTION MODEL / DECISION CRITERIA
For the current test, an irritation potential of a test item leading to EU classification H315 (according to regulation (EC) 1272/2008), and GHS category 2 according to UN GHS (published 2003, last (7th) revision 2017) is recommended if the mean relative tissue viability of three individual tissues is reduced ≤ 50% of the negative control.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
STUDY SUBSTANCE
- Amount applied: 30 μL (47 μL/cm2), applied to the epidermis surface ensuring uniform covering
- Concentration: purity 100%, used as supplied

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

POSITIVE CONTROL
- Amount applied: 30 μL
- Concentration: 5% w/v aqueous solution
Duration of treatment / exposure:
60 minutes
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
Triplicate

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Value:
13.72
Negative controls validity:
valid
Remarks:
The acceptance criterion for an acceptable test if the mean OD570 for the negative control treated tissues is ≥0.8 and ≤2.8, and the SD value of the percentage viability is ≤18%. The quality criteria required were satisfied.
Positive controls validity:
valid
Remarks:
The acceptance criterion for an acceptable test is ≤20% relative to the negative control treated tissues, and the standard deviation (SD) value of the percentage viability is ≤18%. The quality criteria required were satisfied.
Remarks on result:
positive indication of irritation

Applicant's summary and conclusion

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

This in vitro study was performed to assess the irritation potential of the study substance by means of the Human Skin Model Test.


The study substance reduced MTT (pre-test for direct MTT reduction), but it did not change colour when mixed with deionised water (pre-test for colour interference). Consequently, additional tests with freeze-killed tissues were necessary.


The study substance, the negative control (DPBS), and the positive control (5% SDS) were applied to triplicate tissue, respectively.


The study substance and the positive and negative controls were washed off the skin tissues after 60 minutes treatment. After further incubation for about 42 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.


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 showing the quality of the tissues.


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.


The standard deviations between the three tissue percentage viability values of each group (test item, positive and negative controls) in the main test were below 18 (threshold according OECD 439: ≤ 18), thus ensuring the validity of the study.


Compared to the negative control the mean relative viability was reduced to 13.72% after exposure of the skin tissues to the study substance. This value is below the threshold for irritancy of ≤ 50%. Therefore, the study substance is considered to possess an irritant potential.


In conclusion, it can be stated that in this study and under the experimental conditions reported, the study substance is an irritant to skin.