2-ethoxyethyl ethyl carbonic acid ester

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 November 2018 to 29 November 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

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:

2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

2-ethoxyethyl ethyl carbonic acid ester

EC Number:

809-934-0

Cas Number:

627034-93-9

Molecular formula:

C7H14O4

IUPAC Name:

2-ethoxyethyl ethyl carbonic acid ester

Test material form:

liquid

Details on test material:

- Appearance: clear, colourless liquid
- Storage conditions: room temperature, in the dark

Test animals / tissue source

Species:

human

Strain:

not specified

Details on test animals or tissues and environmental conditions:

TEST SYSTEM
- EpiOcular™ kits and MTT-100 kits were purchased from MatTek Corporation (82105 Bratislava, Slovakia).
- The EpiOcular™ tissue consists of normal, human-derived epidermal keratinocytes which have been cultured to form a stratified squamous epithelium similar to that found in the human cornea. It consists of highly organised basal cells which progressively flatten out as the apical surface of the tissue is approached, analogous to the normal in vivo corneal epithelium. The EpiOcular™ tissues (surface 0.6 cm²) were cultured on specially prepared cell culture inserts.
- EpiOcular™ tissues were received at 2 - 8 °C on medium-supplemented agarose gels in a 24-well plate. On day of receipt (28 November 2018) of the EpiOcular™ tissues, the equilibration step (15 minutes at room temperature in the 24-well shipping container) started. 1.0 mL of the medium was aliquoted into the appropriate wells of pre-labelled 6-well plates.
- Each 24-well shipping container was removed from its plastic bag under sterile conditions and its surface disinfected by wiping with 70 % isopropanol- or ethanol-soaked tissue paper. The sterile gauze was removed and each tissue was inspected for air bubbles between the agarose gel and insert. Cultures with air bubbles under the insert covering greater than 50 % of the insert area were not used. The tissues were carefully removed from the 24-well shipping containers using sterile forceps. Any agarose adhering to the inserts was removed by gentle blotting on sterile filter paper or gauze. The insert was then transferred aseptically into the 6-well plates and pre-incubated at standard culture conditions for one hour in the Assay Medium. After one hour, the Assay Medium was replaced by 1 mL fresh Assay Medium at 37 °C and the EpiOcular™ tissues were incubated at standard culture conditions (37 ± 1.5 °C, 5 ± 0.5 % CO2) overnight (about 17 hours).

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 50 μL (83.3 μL/cm² according to guideline)

Duration of treatment / exposure:

30 minutes

Duration of post- treatment incubation (in vitro):

- 12 minute post-soak immersion incubation
- 120 minutes post-treatment incubation
- 180 minutes with MTT

Number of animals or in vitro replicates:

2

Details on study design:

ASSESSMENT OF DIRECT MTT REDUCTION BY THE TEST MATERIAL
- Test materials may have the ability to directly reduce MTT and to form a blue/purple reaction product which could have an impact on the quantitative MTT measurement. Therefore, it was necessary to assess this ability for the test material prior to conducting any assays with viable tissues. For this purpose approximately 50 μL of the test material were added to a 1 mL of a 1.0 mg/mL MTT solution (in DMEM) in a glass tube and the mixture was incubated in the dark at 37 ± 1.5 °C in a humidified atmosphere of 5 ± 0.5 % CO2 in air for three hours. A control (50 μL of deionised water in 1 mL of 1.0 mg/mL MTT solution) was performed concurrently. If the MTT solution colour turned blue/purple, the test material will be presumed to have reduced the MTT.
- Since the MTT solution colour did not turn blue/purple, the test material is not presumed to be a MTT reducer. An additional test with freeze-killed tissues did not have to be performed.

ASSESSMENT OF COLOURED OR STAINING MATERIALS
- Coloured test materials or test materials which become coloured after application to the tissues could interfere with the quantitative photometric MTT measurement if the colourant bound to the tissue and would be extracted together with MTT. Therefore, each test material had to be checked for its colouring properties
- Since the test material was non-coloured, additional tests had to be performed to assess, if it becomes coloured after contact with water or isopropanol. For this purpose, 50 μL of the test material were added to 2.0 mL of isopropanol (glass tube) and shaken for 2 to 3 hours at room temperature. 2.0 mL of isopropanol was used as control. Additionally 50 μL of the test material were added to 1.0 mL of deionised water (glass tube) and incubated at 37 ± 1.5 °C in a humidified atmosphere of 5 ± 0.5 % CO2 in air for at least 1 hour. 1 mL of deionised water was used as control.
- According to guideline the absorbance of all samples was measured in duplicates at 570 nm (OD570) with a plate reader (Versamax® Molecular Devices, 85737 Ismaning, Germany, Software Softmax Pro Enterprise, version 4.7.1). No reference wavelength measurement was used.
- Since the test material did not dye water or isopropanol, additional tests with viable tissues did not have to be performed.

PERFORMANCE OF THE EXPERIMENT
- After the overnight incubation, the tissues were pre-wetted with 20 μL of Ca++Mg++free-DPBS. The tissues were incubated at standard culture conditions (37 ± 1.5 °C, 5 ± 0.5 % CO2) for 30 minutes.
- After the 30 minute Ca++Mg++free-DPBS pre-treatment, the test and control materials were tested by applying 50 μL topically on the EpiOcular™ tissues. The tissues were incubated at standard culture conditions for 30 minutes.
- At the end of the 30 minutes treatment time, the test material was removed by extensively rinsing the tissues with Ca++Mg++-free DPBS (brought to room temperature). Three clean beakers containing a minimum of 100 mL each of Ca++Mg++-free DPBS were used per test material. Each test material utilised a different set of three beakers. The inserts containing the tissues were lifted out of the medium by grasping the upper edge of the plastic "collar" with fine forceps. To assure throughput, the tissues were rinsed two at a time by holding replicate inserts together by their collars using forceps. The test or control materials were decanted from the tissue surface onto a clean absorbent material and the cultures dipped into the first beaker of DPBS, swirled in a circular motion in the liquid for approximately 2 seconds, lifted out so that the inserts were mostly filled with DPBS, and the liquid was decanted back into the container. This process was performed two additional times in the first beaker. The culture was then rinsed in the second and third beaker of DPBS three times each in the same fashion. Finally, any remaining liquid was decanted onto the absorbent material by rotating the insert to approximately a 45° angle (open end down) and touching the upper lip to the absorbent material (to break the surface tension).
- After rinsing, the tissues were immediately transferred to and immersed in 5 mL of previously warmed Assay Medium (room temperature) in a pre-labelled 12-well plate for a 12 minute immersion incubation (post-soak) at room temperature. This incubation in Assay Medium was intended to remove any test material absorbed into the tissue.
- At the end of the post-soak immersion, each insert was removed from the Assay Medium, the medium was decanted off the tissue, and the inserts were blotted on absorbent material, and transferred to the appropriate well of the pre-labelled 6-well plate containing 1 mL of warm Assay Medium. The tissues were incubated for 120 minutes at 37 ± 1.5 °C in a humidified atmosphere of 5 ± 0.5 % CO2 (post-treatment incubation).

MTT ASSAY
- At the end of the post-treatment incubation, each insert was removed from the 6-well plate and gently blotted on absorbent material. The tissues were placed into the 24-well plate containing 0.3 mL of MTT solution. Once all the tissues were placed into the 24-well plate, the plate was incubated for 180 minutes at standard culture conditions.
- Since the test material was colourless inserts were removed from the 24-well plate after 180 minutes; the bottom of the insert was blotted on absorbent material, and then transferred to a pre-labelled 24-well plate containing 2 mL isopropanol in each well so that isopropanol was flowing into the insert. The plates were sealed with parafilm and a standard plate sealer, and were immediately extracted. To extract the MTT, the plates were placed on an orbital plate shaker and shaken for 2 to 3 hours at room temperature. The tissues were pierced. The corresponding negative, positive, and additional viable tissues (without MTT addition) were treated identically with piercing.
- The extract solution was mixed and two 200 μL aliquots were transferred to the appropriate wells of a pre-labelled 96-well plate.
- The absorbance at 570 nm (OD570) of each well was measured with a plate reader (Versamax® Molecular Devices, 85737 Ismaning, Germany, Software Softmax Pro Enterprise, version 4.7.1). No reference wavelength measurement was used.

DATA EVALUATION
1) The mean OD value of the blank control wells (ODBlk) for each experiment was calculated.
2) The mean value of the two replicates for each tissue was calculated.
3) The mean ODBlk from each mean OD value of the same experiment was subtracted (blank corrected values).
4) The mean value of the two relating tissues for each control (negative control (NC) and positive control (PC) and test material (TM) was calculated (ODTM, ODNC, ODPC).
5) The mean OD value of the negative control corresponds to 100 % viability.
Corrected negative control OD = Negative Control OD - ODBlk = 100 % Viability

Calculations for Viability Tests only
1) The percent viability of each of the two relating tissues for each control and test material relative to the negative control (= 100%) was calculated:
Viability (%) = 100. [(ODTM/ODPC/ODNC) / meanODNC]
2) The difference of the viability between duplicate tissues was calculated. If the difference is > 20 % the test is considered as non-qualified.
3) The mean test material viability (TM viability) was calculated and the test material was classified according to the prediction model.

Prediction Model
- If the test material-treated tissue viability is > 60 % relative to the negative control treated tissue viability, the test material is identified as not requiring classification and labelling according to UN GHS (No Category).
- If the test material-treated tissue viability is ≤ 60 % relative to negative control treated tissue viability, no prediction can be made from this result in isolation and requires additional information for classification purposes.
- A single test composed of at least two tissue replicates should be sufficient for a test chemical, when the result is unequivocal. However, in cases of borderline results, such as non-concordant replicate measurements and/or mean percent tissue viability equal to 60 ± 5 %, a second test should be considered, as well as a third one in case of discordant results between the first two tests.

Acceptability of the Assay
The results are acceptable according to MatTek Protocol, if:
1) The negative control OD is > 0.8 and < 2.5,
2) The mean relative viability of the positive control is below 50 % of the negative control viability.
3) The difference of viability between the two relating tissues of a single test material is < 20 % in the same run (for positive and negative control tissues and tissues of test materials). This applies also to the freeze-killed tissues (materials and negative control) and the additional viable tissues (without MTT addition) which are calculated as percent values related to the viability of the relating negative control.
The positive and negative control data shall fall within the historical control data.

Results and discussion

In vitro

Results

Irritation parameter:

other: Mean absorbance value

Run / experiment:

Test material mean

Value:

81.32

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

DIRECT MTT REDUCTION
- The optical pre-experiment (colour interference pre-experiment) to investigate the test material’s colour change potential in water or isopropanol did not lead to a change in colour. Therefore, an additional test with viable tissues without MTT addition was not necessary.
- Optical evaluation of the MTT-reducing capacity of the test material with MTT-reagent did not show blue/purple colour. Therefore, an additional test with freeze-killed tissues was not necessary.

MAIN EXPERIMENT
- The mean absorbance value of the test material, corresponding to the relative cell viability, decreased to 81.32 % (threshold: ≤ 60 %), consequently the test material is considered as “No Category” according to UN GHS.
- Concerning acceptance criteria:
The negative control OD is > 0.8 and < 2.5 (2.167 and 2.243).
The tissue viability of the positive control is below 50 % of the negative control viability (42.88 %).
- The difference of viability between the two relating tissues of a single material is < 20 % (values between 1.46 p.p and 7.56 p.p) in the same run (for positive and negative control tissues and tissues of single test materials).

Any other information on results incl. tables

Table 1: Results of the Main Experiment

Test Group	Tissue No.	Well 1 [OD570]	Well 2 [OD570]	Mean [OD570] (Well 1 and well 2)	Mean [OD570] blank corr. (Well 1 and well 2)	Mean [OD570] of T1 and T2	Tissue viabil.* [%]	rel. viabil. of T1 and T2**	Diff. of viabil. between T1 and T2 [p.p.]
Blank		0.035	0.035	0.035
Negative Control	1	2.243	2.192	2.218	2.182	2.158	100.0	101.2	2.30
	2	2.169	2.167	2.168	2.133			98.8
Positive Control	1	1.062	1.021	1.042	1.007	0.925	42.88	46.7	7.56
	2	0.892	0.865	0.879	0.844			39.1
Test Material	1	1.828	1.783	1.806	1.770	1.755	81.32	82.1	1.46
	2	1.804	1.744	1.774	1.739			80.6

* Tissue viability = (100 × (meanOD of T1&T2)(test material/ positve control/ negative control)/ (meanOD of T1&T2)(negative control)

** Relative Tissue viability = (100 × (meanOD blank corrected)(test material/ positve control/ negative control)/ (meanOD of T1&T2)(negative control)

Applicant's summary and conclusion

Interpretation of results:

other: Not classified in accordance with EU Criteria

Conclusions:

Under the conditions of this study, it can be stated that the test material does not need to be classified for eye irritation.

Executive summary:

The eye irritation potential of the test material was investigated in accordance with the standardised guideline OECD 492, under GLP conditions.

This in vitro study was performed to assess the eye irritation potential of the test material by means of the Human Cornea Model Test. The test material did not prove to be an MTT reducer in the MTT pre-test. Also, its intrinsic colour was not intensive and it did not prove to dye water or isopropanol in the colour interference pre-test. Therefore, additional tests with freeze-killed or viable tissues did not have to be performed.

Each 50 μL of the test material, the negative control (deionised water) or the positive control (methyl acetate) were applied to each of duplicate tissues for 30 minutes.

After treatment with the negative control, the absorbance values were well within the required acceptability criterion of OD > 0.8 and < 2.5, thus showing the quality of the tissues.

Treatment with the positive control induced a decrease below 50 % viability compared with the negative control value in the relative absorbance, thus ensuring the validity of the test system.

The difference of relative viability between the two relating tissues was < 20 % in the same run (for test material tissues, positive and negative control tissues).

Irritating effects were not observed following incubation with the test material. Compared with the value of the negative control, the mean absorption value corresponding to the tissue viability did not decrease below 60 % (determined value for the test material: 81.32 %).

Under the conditions of this study, it can be stated that the test material does not need to be classified for eye irritation.