[No public or meaningful name is available]

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

December 21, 2021 to February 10,2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

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:

18 Jun 2019

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Phosphonic acid, (4-morpholinylmethylene)bis-, tetraethyl ester

Cas Number:

59646-46-7

Molecular formula:

C13H29NO7P2

IUPAC Name:

Phosphonic acid, (4-morpholinylmethylene)bis-, tetraethyl ester

Test material form:

liquid: viscous

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

50 µL (83.3 µL/cm2) of the test item were dispensed directly atop the EpiOcular™ tissue.

Number of animals or in vitro replicates:

The test was performed on a total of 2 tissues per dose group.

Details on study design:

Pre-Experiments
To check the non-specific MTT-reducing capability of the test item 50 µL of the test item were mixed per 1 mL MTT medium and incubated for 3 h in a humidified incubator at 37 ± 2 °C, 5.0% CO2 / 95% humidity.
As the mixture turned blue/purple, the test item was presumed to have reduced MTT. The part of absorption due to the non-specific reduction of MTT (NSMTT) was determined by using killed tissues if the mean relative tissue viability of the test item treated tissues (TM) was above the 60% threshold value. For quantitative correction of results, two killed tissues were treated with 50 µL of the test item (KT) and one tissue was treated with 50 µl of the negative control (Aqua dest.; KU), respectively. All steps were performed exactly as described in the chapter below. NSMTT was calculated relative to the negative control of living tissues (NC) according to the following formula:
NSMTT [%] = [(ODKT - ODKU)/ODNC] * 100
If the viability difference of the two identically treated killed test item treated tissues (KT) was > 20% the killed control was considered as non-qualified.
If NSMTT was ≤ 60% relative to the negative control of living tissues it was used for quantitative correction of the results by calculation of the killed control corrected viability (KCCV) which was considered for the classification of the test item according to the following formula:
KCCV [%] = viabilityTM – NSMTT
If NSMTT was > 60% relative to the negative control of living tissues the results obtained should be taken with caution as this is the cut-off used to distinguish classified from not classified test items.
If uncorrected ODKT of the tissue extracts fell outside the linear range of the spectrophotometer the test item was considered as incompatible with the test method.

To check the colouring potential of the test item 50 µL of the test item were mixed per 1 mL Aqua dest. and per 2 mL isopropanol each in a 6-well plate. The water solution was incubated for at least 1 h in a humidified incubator at 37 ± 2 °C, 5.0% CO2 / 95% humidity. The isopropanol solution was shaken on a plate shaker for 2 to 3 h. After the respective incubation period, 2 x 200 µL aliquots per test solution were transferred into a 96-well plate, using 200 µL Aqua dest. and isopropanol as respective blanks and OD was measured in a range of 570 ± 30 nm without reference wavelength in a plate spectrophotometer.
The mixture showed an ODnet < 0.08. Thus, the additional test with viable tissues and the quantitative corrections were not necessary.
The test item showed non-specific reduction of MTT but no relevant colouring potential after mixture with aqua dest. and with isopropanol. Therefore, no additional controls for correction of possible false-negative results were necessary.

Experimental Procedure
Upon receipt of the EpiOcular™, the tissues were equilibrated in the 24-well shipment plate to room temperature for about 15 min. Then, the EpiOcular™ tissues were transferred into 6-well plates containing 1 mL pre-warmed assay medium per well and incubated for 1 h in a humidified incubator at 37 ± 2 °C, 5.0% CO2 / 95% humidity. Then the inserts were transferred into new 6-well plates containing 1 mL fresh assay medium per well and pre-incubated in a humidified incubator at 37 ± 2 °C, 5.0% CO2 / 95% humidity for 16 - 24 h.
After the overnight incubation the tissues were pre-treated with 20 µL of DPBS-buffer and incubated for 30 ± 2 min in a humidified incubator at 37 ± 2 °C, 5.0% CO2 / 95% humidity to mimic the wet conditions of the human eye.
 
Afterwards, the tissues were treated with each dose group in duplicate, starting with the negative and positive control. Then the 6-well plate(s) were incubated for 30 ± 2 min at 37 ± 2 °C, 5.0% CO2 / 95% humidity. At the end of the exposure period, the test item and control substances were removed by extensively rinsing the tissue with DPBS. Excess DPBS was removed by decanting the insert and blotting bottom with blotting paper. After rinsing, the inserts were transferred to and immersed in a prepared 12-well “post-soak plate“, containing 5 mL fresh pre-warmed assay medium per well and incubated for 12 ± 2 min at room temperature. Afterwards, the inserts were removed from the assay medium, the medium was decanted off the tissue and the tissues were blotted on blotting paper. The inserts were transferred to a new 6-well plate (post-treatment plate) containing 1 mL pre-warmed assay medium. The tissues were incubated for 120 ± 15 min at 37 ± 2 °C, 5.0% CO2 / 95% humidity.
After this incubation period excess medium was removed by blotting bottom on absorbent paper before the inserts were transferred in a prepared 24-well “MTT assay plate” containing 0.3 mL pre-warmed MTT medium and further incubated for 3 h ± 15 min at 37 ± 2 °C, 5.0% CO2 / 95% humidity.
After the 3 h MTT incubation period the inserts were removed, the bottom of the inserts blotted on blotting paper, and then transferred into new 6-well “extraction plates“, containing 2 mL of isopropanol to extract only the bottom of the tissues. The extraction plates were sealed to inhibit isopropanol evaporation. Extraction was carried out after storage overnight in the dark at 2 - 8 °C. At the end of the extraction period the tissues were not pierced to avoid contamination of the extract with remaining test item.
Then the inserts were discarded and the extracts were mixed three times using a pipette. If any visible cell/tissue fragments were in suspension, extracts were centrifuged to eliminate the fragments and avoid further possible interference with the absorbance readings.
For each tissue 2 x 200 µL aliquots of the extract were transferred into a 96-well plate and OD was measured at 570 nm using a filter band pass of maximum ± 30 nm in a plate spectrophotometer using isopropanol as a blank.

Data Analysis
Ocular irritation potential of the test item was predicted from the relative mean tissue viabilities obtained after treatment compared to the negative control tissues concurrently treated with Aqua dest. The test item is considered to be irritant to the eye but it cannot be differentiated between UN GHS “Category 1” or “Category 2”, if the relative tissue viability is less or equal to 60%. The test item is considered to be non-irritant in accordance with UN GHS “No Category” if relative tissue viability is higher than 60%

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

mean percent tissue viability 

Run / experiment:

Experiment 2

Value:

74.5

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation parameter:

mean percent tissue viability 

Run / experiment:

Experiment 1

Value:

65.6

Negative controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In this study under the given conditions the test item showed no irritant effects in both experiments. The test item is classified as “non-irritant“ in accordance with UN GHS “No Category”

Executive summary:

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.

In the present study was applied topically to the EpiOcular tissue for 30 min followed by 12 min post-soaking incubation after removal of the test item. After a 120 min 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 Aqua dest.

The test item showed non-specific MTT-reducing potential. Therefore, additional killed tissue controls were treated with the test item to determine the non-specific reduction of MTT (NSMTT) and the results were corrected to the true MTT metabolic conversion (TOD_TT). The test item showed no water-colouring potential.

In the first experiment, the test item showed no irritant effects. The mean relative tissue viability (% negative control) was > 60% (65.6%).

Since the result is very close to what is considered to be the borderline range (mean percent tissue viability equal to 60±5%), a second experiment was conducted to verify the result.

In the second experiment, the test item showed no irritant effects. The mean relative tissue viability (% negative control) was > 60% (74.5% NSMTT- corrected).

As the test item showed no irritant effects in both experiments, the test item can be considered as non-irritant.

The controls confirmed the validity of the study. The mean absolute OD₅₇₀ of the two negative control tissues was > 0.8 and < 2.8 (1.893 [exp. 1] and 2.072 [exp. 2]). The mean relative tissue viability (% negative control) of the positive control was < 50% (43.5% [exp. 1 and 2]). The maximum inter tissue difference of replicate tissues of all dose groups was < 20% (10.6% [exp. 1] and 3.9% [exp. 2]).