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Toxicological information

Eye irritation

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Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2021-07-27 to 2021-10-06
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:
27 June 2018
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Sodium 1-[6-(morpholin-4-yl)pyrimidin-4-yl]-4-(1H-1,2,3-triazol-1-yl)-1H-pyrazol-5-olate
EC Number:
875-892-5
Cas Number:
1375799-59-9
Molecular formula:
C13 H14 N8 O2 . Na
IUPAC Name:
Sodium 1-[6-(morpholin-4-yl)pyrimidin-4-yl]-4-(1H-1,2,3-triazol-1-yl)-1H-pyrazol-5-olate

Test animals / tissue source

Species:
human
Details on test animals or tissues and environmental conditions:
- Justification of the test method (RhCE) and considerations regarding applicability: The EpiOcularTM EIT is an in vitro procedure allowing the identification of chemicals (substances and mixtures) not requiring classification and labelling for eye irritation or serious eye damage in accordance with UN GHS. It makes use of reconstructed human comea-like epithelium (RhCE) which closely mimics the histological, morphological, biochemical and physiological properties of the human corneal epithelium.
- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live: Reconstructed ocular tissue model containing normal human keratinocytes.
All cells used to produce EpiOcular™ are purchased or derived from tissue obtained by MatTek Corporation from accredited institutions. ln all cases, consent was obtained by these institutions from the donor or the donors legal nest of kin, for use of the cells or derivatives of the tissue for research purposes. HIV-1: negative; HepB: negative, HepC: negative; Bacteria, yeast and other fungi: negative.
- RhCE tissue or hCE cell construct used, including batch number: Kerationcyte strain: 4F1188, Lot Number: 34923
Analysis for tissue function ality and quality wil be decribed under 'Any other information on materials and methods incl. tables'

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 mg

Duration of treatment / exposure:
6 h treatment with 18 h post-treatment period
Duration of post- treatment incubation (in vitro):
18 h
Number of animals or in vitro replicates:
duplicates
Details on study design:
- Details of the test procedure used:
The EpiOcular™ RhCE tissue construct consists of 3 viable layers of cells and a non-keratinized surface as recommended by the test guidelines. The cell viability and barrier function as well as sterility of each batch of the RhCE tissue construct used is adequate, as has been demonstrated by the supplier. RhCE tissue viability in EpiOcular™ EIT is measured by enzymatic conversion of the vital dye MTT by the viable cells of the tissue into a blue MTT formazan salt that is quantitatively measured after extraction from tissues. The experiment was carried out on the EpiOcular™ RhCE tissue construct (about 0.6 cm² in size; MatTek Corporation, Slovakia).
The RhCE tissue equivalents were shipped in 24 well cell culture plates on semi solid agar’s
medium (Lot No.: 34923; Testing date: 03 Aug 2021).

- Doses of test chemical and control substances used:
Treatment: Before treatment 20 µL PBS were applied to all EpiOcular™ tissue surface for an incubation time of 30 ± 2 minutes under SCC. Following 50 mg of the neat test item, 50 µL negative control (deionized water) and positive control (neat methyl acetate), respectively, were applied to the EpiOcular™ tissue surface in duplicate.

- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods (where applicable):
The tissues were treated for 6 hours ± 15 min under SCC. After the end of treatment time the test item was removed by rinsing with PBS followed by a post-soak immersion period of 25 ± 2 min. in fresh medium. Post-treatment incubation: the tissues were incubated for 18 hours ± 15 min in fresh medium under SCC.

- Indication of controls used for direct MTT-reducers and/or colouring test chemicals (if applicable):
Optical properties of the test item or its chemical action on MTT may interfere with the measurement of MTT formazan leading to a false estimate of tissue viability.
The test item was therefore tested in advance for a potential direct influence on the test results not related to cytotoxic effects on tissue cells. For this pre-check the following parameters were tested:
1. Assessment ofpotential direct MTT-reduction of the test item. In case of a direct MTT-reduction of the test item a killed tissue control (inserts, which were killed by freezing) was used in the main assay
2. Assessment ofpotential interference of colored or staining test items, which become colored after application to the tissues, with OD read out
2.1. Assessment of the color reaction with water
2.2. Assessment of the color reaction with isopropanol
In case of an influence oftest item color on OD measurement, a color control was used in the
main assay. The evaluation criteria for the pre-check were:
- For MTT reduction (visual assessment): If the MTT solution color tums blue/purple, the test item is presumed to have reduced the MTT. A killed control must be conducted.
- For color reaction (measurement of the OD): If, after subtraction of the OD for water or isopropanol the OD of the test item solution is > 0.08 a Color Control must be conducted.
If a test item is identified as producing both, color interference and direct MTT reduction, a third set of controls (Non-specific killed control NS KC) is required, additionally from the PG KC and PG CC controls. This is usually the case with darkly coloured test items interfering
with the MTT assay (e.g. blue, purple, black) because their intrinsic colour impedes the assessment of their capacity to directly reduce MTT. Color Control and Killed Control were not required.

- Number of tissue replicates used per test chemical and controls (positive control, negative control): duplicates

- Wavelength and band pass (if applicable) used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer): 570 nm; spectrophotometer (EL808, Bio-Tek; 96 well format, 200 µL)

- Description of the method used to quantify MTT formazan, if applicable:
Immediately after the end of post-treatment incubation the MTT reduction assay was performed. For viability testing the inserts were placed in new plates containing MTT solution (1 mg/mL in Maintenance medium at 37°C, or Assay medium for the color control inserts).
The tissues were incubated for 180 ± 10 min. under SCC. The extraction of blue formazan was performed in isopropanol on a vertical shaker for 2-3 hours at room temperature. The concentration of formazan was measured by determination of the OD of the isopropanol-extracts in duplicate at 570 nm in an automatic reader of a spectrophotometer.

- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model:
For interpretation of cell viability results the cut-off value distinguishing classified (irritant)
from non-classified substances as given in OECD TG 492 was used:
- The test chemical is identified as not irritant and not requiring classification according to UN GHS (No Category) if the mean percent tissue viability after exposure and post-exposure incubation is more than (>) 60%.
- The test chemical is identified as irritant and potentially requiring classification according to UN GHS (Category 2 or Category 1) if the mean percent tissue viability after exposure and post exposure incubation is less than or equal (≤) to 60%.

- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria:
The following acceptance criteria determine the validity of an assay:
- mean OD 570 nm negative control (NC) is > 0.8 and < 2.8
- mean relative viability ofthe positive control (PC) is < 50 % (relative to negative control)
- the difference of viability between the two replicates is < 20 %.

- Complete supporting information for the specific RhCE tissue construct or hCE cells used: please refer to 'Any other infomation on materials and methods incl. tables'

Results and discussion

In vitro

Results
Irritation parameter:
mean percent tissue viability 
Run / experiment:
Mean
Value:
28
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

























































Summary of results



Compound



% measure Cell Viability



OD mean (n=4)



test item



27.46



0.479



test item KC



n.a.



n.a.



NC KC



n.a.



n.a.



Test item CC



n.a.



n.a.



Positive control



12.72



0.221



Negative control



100



1.739



% FINAL viability of the test item *



27.56



% FINAL viability of the test item rounded



28



*= % viability test item – (% viability test item KC - % viability NC KC) - % viability test item CC + viability NS KC



n.a. = not applicable, because no KC and NC KC control were required


Applicant's summary and conclusion

Interpretation of results:
other:
Remarks:
According to the Evaluation criteria of OECD test guideline 492 the test chemical is identified as irritant and potentially requiring classification according to UN GHS (Category 2 or Category 1) because the mean percent tissue viability after exposure and and post exposure incubation is less than or equal (≤) to 60%.
Conclusions:
In this study under the given conditions the test item showed irritant effects.
Executive summary:

In this study conducted according to OECD test guideline 492 (adopted June 27, 2018), the potential of the test item to induce eye irritation was analysed by using the three-dimensional human corneal epithelium model EpiOcular™, consisting of normal, human-derived epidermal keratinocytes mimicking characteristics of the corneal epithelium.


The test item was applied topically to the EpiOcular™ tissue for 6 h followed by 25 min post-soaking incubation after removal of the test item. After an 18 h post-treatment period cytotoxic effects were determined via MTT reduction assay.


Ocular irritation potential of the test item was predicted from the relative mean tissue viabilities compared to the negative control tissues concurrently treated with deionized water.


The test item showed no non-specific reduction of MTT and no relevant colouring after mixture with isopropanol and after mixture with deionized water. Therefore, NSCliving was not determined.


The test item showed irritant effects. The mean relative tissue viability (% negative control) was ≤ 60% (28 %).


The controls confirmed the validity of the study. The mean absolute OD570 of the two negative control tissues was > 0.8 and < 2.5 (1.739). The mean relative tissue viability (% negative control) of the positive control was < 50% (12.72%). The maximum inter tissue difference of replicate tissues of all dose groups was < 20%.


In this study under the given conditions the test item showed irritant effects.