Zinc EDDHA

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 FEB 2021 - 2 JUN 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Version / remarks:

adopted 18. Jun. 2019

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt Rheinland-Pfalz 15 May 2018

Test material

Test material information

1

Reference substance name:

Acetic acid, oxo-, sodium salt, reaction products with ethylenediamine and phenol, zinc sodium salts

Molecular formula:

non specified (UVCB substance)

IUPAC Name:

Acetic acid, oxo-, sodium salt, reaction products with ethylenediamine and phenol, zinc sodium salts

Test material form:

solid: particulate/powder

Test animals / tissue source

Species:

human

Details on test animals or tissues and environmental conditions:

- Justification of the test method (e.g. ICE, EIT, RhCE) and considerations regarding applicability: This study was performed in order to evaluate the eye hazard potential of ManganeseEDDHA sodium salts in a Reconstructed human Cornea-like Epithelium (RhCE) model in an in vitro study (e.g. EpiOcularTM Eye Irritation Test). The EpiOcular™ Eye Irritation Test (EIT) predicts the acute eye hazard potential of chemicals by measurement of tissue damage caused by cytotoxic effects in the reconstructed
human cornea-like tissue model. Within a testing strategy, the EpiOcular™ EIT can be used as a replacement of the in vivo Draize Eye Irritation Test. It is utilized for the classification and labelling of chemicals concerning their eye hazard potential. The EpiOcular™ EIT can be used to identify chemicals that do not require classification for eye irritation or serious eye damage according to the UN GHS classification system. A limitation of this guideline is that it neither allows discrimination between eye irritation/reversible effects on the eye (Category 2) and serious eye damage/irreversible effects on the eye (Category 1), nor between eye irritants (optional Category 2A) and mild eye irritants (optional Category 2B). For these purposes, further testing with other suitable test methods is required The solid test item was applied topically to a three-dimensional RhCE tissue construct in duplicate for an exposure time of 6 hours. Eye hazard materials are identified by their ability to produce a decrease in cell viability as determined. The 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 eye irritation potential. The test substance falls within the applicaility domain of this model.
- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live: No bacteria, yeast and other fungi were detected, Tissue viability and Barrier function were within the acceptance criteria
- Cell line used, its source, passage number and confluence of cells used for testing: Keratinocyte strain 4F1188
- RhCE tissue or hCE cell construct used, including batch number: EpiOcular™ Tissue (Lot No. 34901)

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50.5mg

Duration of treatment / exposure:

6 h

Duration of post- treatment incubation (in vitro):

18 h

Number of animals or in vitro replicates:

2

Details on study design:

- Details of the test procedure used :
The solid test item was applied topically to a three-dimensional RhCE tissue construct in duplicate for an exposure time of 6 hours. Eye hazard materials are identified by their ability to produce a decrease in cell viability as determined. The 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 eye irritation potential
- Doses of test chemical and control substances used :
test item: 50 mg, positive and negative control: 50 µL
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods (where applicable) : 6 h exposure 37 ± 1 °C, 18 h post-exposure incubation at 37 ± 1 °C
- Number of tissue replicates used per test chemical and controls (positive control, negative control) : 2
- For hCE cells: number of runs and of hCE models used within each run : 1 run, 2 tissues per test item/controls
- Wavelength and band pass (if applicable) used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer) : 570 nm
- Description of the method used to quantify MTT formazan, if applicable : The inserts were removed from the 6-well plate and discarded. 1 mL isopropanol was added and the content of each well was thoroughly mixed in order to achieve homogenisation.
From each well, two replicates with 200 µL solution (each) were pipetted into a 96-wellplate. The plate was read in a plate spectrophotometer at 570 nm. In addition, eight wells of the 96-well-plate were filled with 200 µL isopropanol each, serving as blank
- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model : according to OECD TG 492
- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria :
negative control OD: historical range 1.047 - 2.340 (mean 1.660) - study 1.618
positive control viability: historical range 20.4 - 47.8% (mean 34.1%) - study 26.4%
- Complete supporting information for the specific RhCE tissue construct or hCE cells used :
EpiOcularTM tissues were procured from MatTek In Vitro Life Science Laboratories, Mlynské Nivy 73, 82105 Bratislava, Slovakia.
Designation of the kit: OCL-200-EIT
Day of delivery: 16. Mar. 2021
Batch no.: 34901
- Demonstration of proficiency in performing the test method before routine use by testing of the proficiency chemicals : All 15 proficiency chemicals of the OECD TG 492 were correctly classified.
- Acceptable variability between tissue replicates for positive and negative controls : <20%
- Acceptable variability between tissue replicates for the test chemical: <20%

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

percent tissue viability 

Run / experiment:

Tissue 1

Value:

43.9

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation parameter:

percent tissue viability 

Run / experiment:

Tissue 2

Value:

40.4

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
The pre-test showed, that the test item was intensely coloured (OD > 0.08), but the mean viability of the tissues treated with test item was 42.2% (≤ 60%) and therefore the additional test was not necessary.

DEMONSTRATION OF TECHNICAL PROFICIENCY: demonstrated on the 15 proficiency chemicals indicated in the OECD TG 492

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: demanded OD > 0.8 < 2.8 - found OD 1.618
- Acceptance criteria met for positive control: demanded < 50 % of negative control - found 26.4 %

Any other information on results incl. tables

Table 1: Absorbance Values Negative Control, Positive Control and Test Item (OD at 570 nm)

Designation	Measurement	Negative Control	Positive Control	Test Item
Tissue 1	1	1.740	0.442	0.744
	2	1.750	0.443	0.745
Tissue 2	1	1.561	0.479	0.687
	2	1.558	0.482	0.688

Table 2: Mean Absorbance Negative Control, Positive Control and Test Item

Designation	Negative Control	Positive Control	Test Item
Mean – blank (Tissue 1)	1.711	0.409	0.711
Mean – blank (Tissue 2)	1.526	0.447	0.654

Table 3: % Viability Positive Control and Test Item

Designation	Positive Control	Test Item
% Viability (Tissue 1)	25.2%	43.9%
% Viability (Tissue 2)	27.6%	40.4%
% Viability Mean	26.4%	42.2%

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Conclusions:

After treatment with the test item, the mean value of relative tissue viability was 42.2%. This value is below the threshold for eye irritation potential (≤ 60%). Test items that induce values below the threshold are considered either eye irritant or inducing serious eye damage.

Executive summary:

The registered substance was tested for eye irritation in a GLP compliant in vitro study according to OECD Guideline 492. The test item was applied to a three-dimensional human cornea tissue model in duplicate for an exposure time of 6 hours. The solid test item was applied to two tissue replicates. After treatment, the respective substance was rinsed from the tissue; then, cell viability of the tissues was evaluated by addition of MTT, which can be reduced to formazan. The formazan production was evaluated by measuring the optical density (OD) of the resulting solution. Demineralised water was used as negative control and methyl acetate was used as positive control. The controls showed the following results: After treatment with the negative control, the absorbance values were within the required acceptability criterion of mean OD > 0.8 and < 2.8, OD was 1.618. The positive control showed clear eye irritating effects, the mean value of the relative tissue viability was 26.4% (< 50%). The variation within tissue replicates of the controls and the test item was acceptable (< 20%). After treatment with the test item, the mean value of relative tissue viability was 42.2%. This value is below the threshold for eye irritation potential (≤ 60%). Test items that induce values below the threshold are considered either eye irritant or inducing serious eye damage. According to the OECD Guideline 492, the EpiOcular^TM Eye Irritation Test does not allow discrimination between eye irritation/reversible effects on the eye (Category 2) and serious eye damage/irreversible effects on the eye (Category 1). In this case no prediction can be made and further testing with other suitable test methods is required. Under the conditions of the test, Zinc-EDDHA sodium salts is considered either eye irritant or inducing serious eye damage in the EpiOcular^TM Eye Irritation Test.