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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:
2022-11-02 to 2022-11-18
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2023

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:
14 June 2021
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
06 July 2012
Qualifier:
according to guideline
Guideline:
other: UN GHS (published 2003, last (8th) revision 2019)
Version / remarks:
2019
Qualifier:
according to guideline
Guideline:
other: MatTek Corporation Protocol: In vitro EpiDermTM Skin Irritation Test (EPI-200-SIT) For use with MatTek Corporation’s Reconstructed Human Epidermal Model EpiDerm (EPI-200-SIT); Version 12/04/2020.
Version / remarks:
12/04/2020
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
2‐aminoacetate, hydron, zinc(2+) sulfate hydrate
Molecular formula:
C2H7NO7SZn
IUPAC Name:
2‐aminoacetate, hydron, zinc(2+) sulfate hydrate
Test material form:
solid

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
foreskin from a single donor
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Epi-200-SIT kits and MTT-100 assays are purchased from MatTek Corporation (82105 Bratislava, Slovakia).
- Tissue batch number(s): lot nr: 36184, keratinocyte strain: 00267
- Shipping date: 2022-11-16
- Delivery date: 2022-11-16
- Date of initiation of testing: 2022-11-16
TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: Each incubation of the tissues was performed under 37 ± 1.5°C and 5 ± 0.5% CO2 in DMEM Medium.
- Temperature of post-treatment incubation: The post-treatment incubation was under standard incubation conditions (37 ± 1.5°C and 5 ± 0.5% CO2).
REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: At the end of the treatment interval the tissues were gently rinsed with PBS several times in order to remove any residual test material.
- Observable damage in the tissue due to washing: Optical evaluation of the test item treated tissues revealed no visible damage.
- Modifications to validated SOP: no
MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: On the day of the experiment a MTT solution of 1 mg/mL in DMEM was prepared, 300µL MTT was used.
- Incubation time: The MTT incubation time was 3 hour ± 5 min.
- Spectrophotometer: The microplate reader Versamax® Molecular Devices was used.
- Wavelength: The optical density at OD570nm was determined.
- Filter: The absorbance values were determined using the software SoftMax Pro Enterprise (version 4.7.1).
FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: MTT QC, 4 hours, n=3, Viability should be between 1.0 -3.0, viability = 1.499 +/- 0.046, Pass
- Barrier function: ET-50 assay, 100 µL 1% Triton X-100, 4 time points, n=3, MTT assay, ET-50 schould be between 4.77-8.72 hours, ET-50 = 8.05 hours, Pass.
- Contamination: no contamination detected
- Reproducibility: yes
NUMBER OF REPLICATE TISSUES:
Each group (negative control, positive control, test item) was tested in triplicates.
CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
The test item did not prove to be a MTT reducer in the MTT interference pre-experiment.
NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION:
1 main experiment was used for prediction.
PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive to skin if the viability after 1 hour exposure is less than or equal to 50 %.
- The test substance is considered to be non-corrosive to skin if the viability after 1 hour exposure is greater than 50%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
DPBS (MatTek)
Duration of treatment / exposure:
Pre-experiment (colour interference test): The treatment in the colour interference test was 3 hours with deionised water under standard conditions, and in parallel 3 hours with isopropanol at room temperature.
Pre-experiment (MTT-interference test): in the MTT interference test treatment was 3 hours under standard conditions.
Main-experiment: The test item and the controls, respectively, were tested in triplicate tissues with an exposure time of 60 minutes. Within this period the 6-well plates were placed in the incubator for 35 minutes at standard incubation conditions. In the remaining period the plates were placed in a sterile bench at room temperature until the end of treatment.
Duration of post-treatment incubation (if applicable):
At the end of the treatment interval the tissues were gently rinsed with PBS several times in order to remove any residual test material. Afterwards, the tissues were incubated in 0.9 mL of fresh assay medium for 24 ± 2 hours. After this incubation period the medium was changed, and the tissues were incubated for another 18 ± 2 hours at standard incubation conditions. The complete incubation time was 42 ± 4 hours.
After the 42 hour incubation period was completed, the tissues were transferred to the MTT-plates. After a 3 hour ± 5 minutes incubation period at standard conditions the tissues were rinsed with PBS and carefully dried with blotting paper.
The tissues were transferred into new 24-well plates containing 2 mL of extractant solution (isopropanol) in each well ensuring that the tissues were completely covered. The 24-well plates were sealed to minimise isopropanol evaporation. The formazan salt was extracted within 2-4 hours while shaking at room temperature.
At the end of the extraction period the inserts were pierced with an injection needle to allow the extract to run into the well from which the insert was taken, and the insert were discarded.
Per each tissue, 3 x 200 µL aliquots of the formazan solution were transferred into a 96-well, flat bottom, microtiter plate. 200 µL of isopropanol were added to the wells designated as blanks for 96-well plate.
Number of replicates:
negative control: 3
positive control: 3
test material: 3

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
The test item was tested neat.
In the pre-experiments (colour inteference test and MTT inteference test) 50 ± 2 mg of the test item was applied onto the surface of the tissue.
In the main-experiment 25 ± 2 mg (39.7 mg/cm2 according to guideline) of the test item was applied onto the surface of the tissue.
Duration of treatment / exposure:
Pre-experiment (colour interference test): 3 hours with deionised water under standard conditions, and in parallel 3 hours with isopropanol at room temperature.
Pre-experiment (MTT-interference test): 3 hours under standard conditions.
Main-experiment: Exposure time of 60 minutes in total. The plates were placed for 35 minutes in the incubator at standard incubation conditions. In the remaining period the plates were placed in a sterile bench at room temperature until the end of treatment.
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing: At the end of the treatment interval the tissues were gently rinsed with PBS several times in order to remove any residual test material.
- Time after start of exposure: 1 hour
OBSERVATION TIME POINTS
60 minutes
SCORING SYSTEM:
- Method of calculation:
1)The mean OD value of the three wells for each tissue and the blank control (ODBlk) was calculated (Mean [OD570] (well 1, well 2 and well 3).
2) The mean ODBlk was subtracted from each mean OD value of the three wells.
Mean [OD570] blank corr. (well 1, well 2 and well 3)). These values were used for all further calculations below.
3) The mean OD of the three relating tissues for each test group (negative control (NC), positive control (PC)) and the test item (TI) were calculated with the blank corrected mean OD (Mean [OD570] of T1, T2 and T3).
4) The percent viability of each test group relative to the negative control (= 100%) was calculated:
Viability (%)=100 ×〖mean OD〗_(TI⁄PC/NC)/〖mean OD〗_NC
5) The viability of each test group was calculated for each tissue replicate.
6) The standard deviation between the OD values within one test group was calculated (not reported). In addition, the standard deviation between the viability values within one test group was calculated.

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1
Value:
57.88
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation

Any other information on results incl. tables

Pre-experiment:


 


Assesment of Colour Interference:












































Treatment Group



OD 570 nm
Well 1



OD 570 nm
Well 2



Mean OD of
2 Wells



Mean OD


of 2 Wells blank


corrected



Evaluation Mean OD570 (blank corrected)
> 0.08



Blank
Aqua Deion.



0.037



0.036



0.037



 



Test Item + Aqua Deion.



0.042



0.041



0.042



0.005



no



Blank Isopropanol



0.037



0.038



0.037



 



Test Item+


Isopropanol



0.043



0.044



0.044



0.007



no



The mean OD of the test item in deionised water or isopropanol was < 0.08 and therefore, an additional test with viable tissues without MTT addition was not necessary in the main experiment.


 


 


Assesment of MTT Interference:


In the pre-experiment for MTT interference the test item did not reduce MTT to Formazan salt after optical evaluation. Therefore, an additional test with freeze-killed tissues was not necessary for the quantitative correction of the test item viability.


 


 


Main-experiment:


Results after treatment with Zinc Monoglycinate Sulfate Hydrate and the controls




























































































































Treatment Group



Tissue No.



OD 570 nm



Mean OD of
3 Wells



Mean OD


of 3 Wells blank


corrected



Mean


OD


of 3 tissues



Rel. Viability [%] Tissue
1, 2 + 3



Standard Deviation



Mean Rel. Viability


[%]



Well 1



Well 2



Well 3



Blank



 



0.040



0.040



0.040



0.040



 



Negative Control



1



1.737



1.700



1.669



1.702



1.662



1.631



101.942



1.7



100.0



2



1.647



1.649



1.655



1.650



1.611



98.784



3



1.648



1.670



1.658



1.658



1.619



99.274



Positive Control



1



0.134



0.123



0.117



0.125



0.085



0.073



5.217



0.7



4.47



2



0.106



0.104



0.102



0.104



0.064



3.941



3



0.109



0.110



0.108



0.109



0.069



4.238



Test Item



1



0.967



0.954



0.949



0.957



0.917



0.944



56.227



1.7



57.88



2



0.989



0.978



0.983



0.983



0.944



57.865



3



0.999



1.016



1.018



1.011



0.971



59.553


Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
In conclusion, it can be stated that in this study and under the experimental conditions reported the test item is non-irritant to skin according to UN GHS and EU CLP regulation.
Executive summary:

This in vitro study was performed to assess the skin irritation potential of Zinc Monoglycinate Sulfate Hydrate by means of the Human Skin Model Test.


The test item did not prove to be a MTT reducer in the MTT interference pre-experiment. The OD of the test item in deionised water or isopropanol at 570 nm after blank correction was < 0.08. Therefore, additional tests with freeze-killed tissues or viable tissues (without MTT addition) did not have to be performed.


Three tissues each of the human skin model EpiDerm were treated with the test item, the negative control (DPBS) or the positive control (5% SDS) for 60 minutes.


After treatment with the negative control the absorbance values were well within 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 viability as compared to the negative control for the 60 minutes treatment interval, thus assuring the validity of the test system.


After treatment with the test item Zinc Monoglycinate Sulfate Hydrate the mean relative viability value was 57.88% compared to the relative absorbance value of the negative control. This value is above the threshold for irritancy of ≤ 50%.


Therefore, the test item is not considered to possess an irritant potential. In conclusion, it can be stated that in this study and under the experimental conditions reported, Zinc Monoglycinate Sulfate Hydrate is non-irritant to skin according to UN GHS and EU CLP regulation.