Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
August to November 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guideline
Qualifier:
according to
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:
28 July 2015
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): Solvent N1
- Physical state: liquid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch No.of test material: 24/02/2016
- Expiration date of the batch: July 2021
- Purity test date: 75.6 % (w/v) N1 SOLVENT

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Stability of the test substance: the substance is chemically stable at room temperature
- Storae conditions: In the refrigerator set at +5°C (for reducing the risk of evaporation)
- Correction factor: none

Test animals / tissue source

Species:
human
Strain:
other: not applicable.
Remarks:
Reconstructed human Cornea-like Epithelium
Details on test animals or tissues and environmental conditions:
Description: the EpiOcularTM model consists of an airlifted, living, multilayered ocular tissue construction (surface 0.60 cm2), reconstructed from normal (non-transformed) human-derived keratinocytes. This is a non-keratinized epithelium which models the cornea epithelium with progressively stratified, but not cornified cells. The cells are cultured in proprietary serum-free culture media, which induces corneal differentiation and the formation of the organotypic 3D cornea-like model. The 3D tissue consists of highly organized cell layers similar to that found in the cornea. The model features a normal ultra-structure and is functionally equivalent to human in vivo tissue.
Quality Control: the acceptability range for the barrier function as measured by the ET50 assay is established by the RhCE tissue construct developer/supplier to be between 12.2 and 37.5 min. Data demonstrating compliance with all production release criteria were provided by the RhCE tissue construct supplier and the information are documented in the certificate of analysis archived in the study files.
Selection: at receipt, the tissues were inspected for obvious defects as they could have been rejected based on blistering, excess fluid or air bubbles below the tissue insert. Cultures with air bubbles under the insert covering greater than 50% of the insert area were not used.
Expiry date: the EpiOcular tissues were used within 72 hours of their production.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
Undiluted test item was applied topically on each designated tissue, and gently spread onto the epithelium surfaces to ensure uniform covering of the tissues.
Duration of treatment / exposure:
Pre-incubation: 1 hour (+37°C, 5% CO2 in a humidified incubator). After the pre-incubation period, the assay medium was removed and replaced by 1 mL of fresh assay medium before incubation overnight (16-24h) at +37°C, 5% CO2 in a humidified incubator.
Exposure: The tissues were incubated at +37°C, 5% CO2 in a humidified incubator for 30 (± 2) minutes.
Observation period (in vivo):
N/A
Duration of post- treatment incubation (in vitro):
2 hours (± 15 minutes) at +37°C, 5% CO2 in a humidified incubator.
Number of animals or in vitro replicates:
Main test:
One 6-well plate was used for the test item-treated tissues.
Positive and negative controls were placed on separate 6-well plates (one plate for each).
Test item, negative and positive controls were applied on duplicate tissues.
Details on study design:
PRELIMINARY TESTS

1) Test for direct MTT reduction with the test item:
As a test item may directly reduce MTT, thus mimicking mitochondrial succinate dehydrogenase activity, it is necessary to test the ability of the test item to directly reduce MTT before performing the main test. This property of the test item would only be a problem if, at the time of the MTT viability test (after rinsing), there remained a sufficient amount of test item present on or in the tissue. In this case, the true metabolic MTT reduction and the false direct MTT reduction should be differentiated and quantified.
To identify any test substance interference with the MTT endpoint, the following preliminary test was performed:
. 50 μL of the test item were added to 1 mL of a 1.0 mg/mL freshly prepared MTT solution,
. a negative control was tested concurrently by adding 50 μL of sterile deionized water to 1 mL of a 1.0 mg/mL freshly prepared MTT solution,
. both mixtures were incubated in darkness at +37°C for 3 hours (± 10 minutes).
Then the color of the solutions obtained was evaluated.
If the MTT solution color turns blue/purple when compared to the negative control, the test item was presumed to reduce MTT.

2) Test for the detection of the coloring potential of the test item:
As a test item may be colored or become colored in contact with water and/or isopropanol, it is necessary to test its potential interference with the MTT determination in these two conditions.
The maximum amount of test item, 50 μL was added to 1 mL of water and incubated for at least 1 hour in the dark at +37°C, 5% CO2 and 2 mL of isopropanol, incubated in a 6-well plate and placed on an orbital plate shaker for 2 hours at room temperature. After that, the presence and intensity of the coloration were evaluated. If the solution changes color significantly, additional controls were performed on viable tissues in parallel to the main test. Otherwise, no additional tissue controls were used.

MAIN TEST:

Pre-incubation of the tissues:
As the tissues were shipped before the day prior the treatment, tissues were stored between +2°C and +8°C, prior their pre-incubation. The tissues were equilibrated (in the 24-well shipping container) to room temperature for at least 15 minutes. The underside of each tissue was inspected for air bubbles between the agarose gel and the insert. Tissues with air bubbles under the insert covering greater than 50% of the insert area were not used. Any unusual observation was noted separately. The plastic bag containing the 24-well plate shipping container was disinfected by wiping with 70% ethanol-soaked tissue paper. Each 24-well shipping container was then removed from its plastic bag under sterile conditions. A volume of 1 mL assay medium pre-warmed (+37°C) was added to 2 wells per pre-labeled 6-well plate. 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 plate and pre-incubated at +37°C, 5% CO2 in a humidified incubator for 1 hour. After the pre-incubation period, the assay medium was removed and replaced by 1 mL of fresh assay medium before incubation overnight (16-24h) at +37°C, 5% CO2 in a humidified incubator.
Each 6-well plate was labeled with the test item or control codes.

Treatment of tissues:
Following the pre-incubation period, the tissues were pre-wetted with 20 μL of D-PBS. Each insert was tapped on the walI of the plate to ensure that the entire tissue surface was wetted with D-PBS. The tissues were then incubated at +37°C, 5% CO2 in a humidified incubator for 30 (± 2) minutes.
The test item, negative and positive controls were applied topically on each designated tissue, and gently spread onto the epithelium surfaces to ensure uniform covering of the tissues. Inserts were then tapped on the wall of the plate to ensure that the items were applied evenly to the surface of each tissue.
All treated plates (including those treated with positive and negative controls) were covered by a plate sealer in order to avoid any evaporation/volatility of test item.
Then, all tissues (test item, negative and positive controls) were incubated at +37°C, 5% CO2 in a humidified incubator for 30 minutes (± 2 minutes).
The tissues were processed (treatment and rinsing) in the same order and at regular time-intervals to ensure each tissue receives an equal exposure period.

Rinsing of tissues:
At the end of the treatment period, each tissue was removed from the well of the treatment plate and rinsed to gently remove any residual test or control items. A set of three clean beakers containing a minimum of 100 mL each of D-PBS was used per test or control item. The tissues were rinsed two at time by holding replicate inserts together. The test or control items were firstly decanted from the tissue surface onto a clean absorbent paper. The tissues were then dipped into the first beaker of D-PBS, swirled in a circular motion during approximately 2 seconds, lifted out and decanted back into the beaker. This process was performed three times per beaker. Any remaining liquid was decanted onto an absorbent paper.

Post-soak and post-incubation:
The rinsed tissues were transferred to new wells of a pre-labeled 12-well plate containing 5 mL of assay medium pre-warmed at room temperature. This incubation in assay medium was intended to remove any test article from the tissue.
Each tissue was incubated for 12 minutes (± 2 minutes) at room temperature to remove any liquid test item or negative and positive controls from the tissue.
At the end of the Post-soak immersion, each insert was blotted on absorbent material and transferred to appropriate well of the pre-labeled 6-well plate containing 1 mL of assay medium. The tissues were then incubated for 2 hours (± 15 minutes) at +37°C, 5% CO2 in a humidified incubator for test item, negative and positive controls.

MTT viability assay:
Following the post-treatment incubation, a volume of 0.3 mL of a freshly prepared MTT solution (1.0 mg/mL) was added into new wells of pre-labeled 24-well plates.
At the end of the post-treatment incubation, each insert was removed from the 6-well plate and gently blotted on absorbent paper. The tissues were then transferred to the MTT pre-filled wells and incubated for 3 hours (± 10 minutes) at +37°C, 5% CO2 in a humidified incubator.
At the end of the 3-hour incubation period, the underside of each tissue was blotted on absorbent paper to dry. Each tissue was examined with the naked eye and the degree of MTT staining was evaluated.
For the test item, negative and positive control-treated tissues, the inserts were transferred to new wells of the 24-well plate containing 2 mL of isopropanol per well so that isopropanol was flowing into the insert on the tissue surface.
Plates were surrounded with parafilm to prevent evaporation. Formazan extraction was performed overnight at +2-8°C and protected from light.

Optical Density measurements:
At the end of the formazan extraction period, tissues (test item, negative and positive control-treated tissues) were pierced.
The extract solution was mixed using a pipette and two 200 μL aliquots were transferred to the appropriate wells of a pre-labeled 96-well plate.
One 96-well plate was used for the negative and positive controls (placed at opposite end of the plate) and a separate 96-well plate was used for test item-treated tissues.
For each 96-well plate, the average Optical Density value (OD) of 4 wells containing 200 μL of isopropanol only was used as the blank.
The OD was measured at 570 nm using a plate reader.

Data analysis:
As the test item was found in the preliminary test not to have any coloring potential and any direct MTT reducing properties, no additional controls were run during the main test. Therefore, the mean blank OD value (mean ODblank) was calculated from the four replicates.
Then, the mean ODblank was subtracted from each OD value and the corrected mean OD values (mean cOD) of the two aliquots were calculated for each tissue.
The corrected mean OD of the two negative controls-treated tissues (mean cODNC) was set to 100% viability and was used as a reference.
For the tissues treated with the test item or negative or positive controls, the relative viabilities for each tissue were expressed as percentages of the reference viability and were calculated as follows:
TI relative viability (%) = (cOD TI or NC or PC / mean cOD NC) x 100
with:
cOD TI = corrected OD of each tissue treated with test item.
cOD NC = corrected OD of each tissue treated with negative control.
cOD PC = corrected OD of each tissue treated with positive control.

Acceptance criteria
The results of the study were considered acceptable if the following criteria are fully met:
. the mean cOD of the negative controls is between 0.8 and 2.5,
. relative mean viability of the positive control is < 50% of the relative mean viability of the negative control,
. the difference of viability between the two tissue replicate is < 20%.

Results and discussion

In vitro

Results
Irritation parameter:
other: relative mean viability (%)
Run / experiment:
Main test / Mean value of the two treated tissues. Difference of 2% between duplicate tissues.
Value:
99
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: FULFILLED.
Mean cOD of the negative control = 1.763 (SD = 0.066). (Acceptance criteria: Mean cOD of the negative control between 0.8 and 2.5)

- Acceptance criteria met for positive control: FULFILLED.
Relative mean viability of the positive control = 26% (SD = 5%). (Acceptance criteria: Relative mean viability of the positive control < 50% of the relative mean viability of the negative control)

. Acceptance criteria: difference of viability between the two tissue replicate is < 20%. FULFILLED
Difference of viability between the two tissue replicate:
2% for test item treated tissues
5% for negative control tissues
7% for positive control tissues

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Under the experimental conditions of this study, the test item, N1 SOLVENT, is considered to be non-irritant to Reconstructed human Cornea-like Epithelium.
According to the results of this study, the classification of the test item should be: "No Category" (GHS 2015 and Regulation (EC) No. 1272/2008).
Executive summary:

The study was conducted according to OECD Guideline No. 492 and in compliance with the principles of Good Laboratory Practice. No deviation from OECD Guideline No. 492 occurred.

Preliminary tests were performed to detect the ability of the test item to directly reduce MTT as well as its coloring potential.

Following the preliminary tests, the eye irritation potential of the test item was assessed in the main test. The test item and both negative and positive controls were applied topically on duplicate tissues and incubated at +37°C for 30 minutes.

As the test item is a volatile liquid, measures for avoiding losses due to evaporation and ensuring appropriate exposure of tissues were applied during the test. Indeed, all treated plates (including those treated with positive and negative controls) were covered by a plate sealer in order to avoid any evaporation/volatility.

At the end of the treatment period, each tissue was rinsed with D-PBS, incubated for 12 minutes at room temperature, blotted on absorbent material, and then incubated for another 2 hours at 37°C, 5% CO2 in a humidified incubator. The cell viability was then assessed by means of the colorimetric MTT reduction assay.

Mean viability values were calculated for each tissue and expressed as a percentage of the mean viability of the negative control tissues which was set at 100% (reference viability).

In the preliminary tests, the test item was found not to have direct MTT reducing properties or coloring potential.

In the main test, all the acceptance criteria were fulfilled. The study is therefore considered valid.

The relative mean viability of the tissues treated with the test item was 99% with a difference of 2% between duplicate tissues. As the mean viability was > 60% after the MTT reduction, the results met the criteria for a non-irritant response.