Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
May 2019
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:
June 2018
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
2-[5-fluoro-1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]-5-[(E)-phenyldiazenyl]pyrimidine-4,6-diamine
EC Number:
811-933-5
Cas Number:
1350653-29-0
Molecular formula:
C23 H17 F2 N9
IUPAC Name:
2-[5-fluoro-1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]-5-[(E)-phenyldiazenyl]pyrimidine-4,6-diamine
Test material form:
solid

Test animals / tissue source

Species:
other: reconstructed human cornea-like epithelium (RhCE)
Strain:
other: not applicable
Details on test animals or tissues and environmental conditions:
JUSTIFICATION OF THE TEST METHOD AND CONSIDERATIONS REGARDING APPLICABILITY :
The EpiOcular™ eye irritation test (EIT) follows international OECD test guidelines. The EIT measures the ocular irritation potential of a test item by determination of cytotoxic effects on a reconstructed human cornea epithelium (RhCE) tissue model to discriminate chemicals not requiring classification for eye irritancy (UN GHS No Category) from those requiring classification. The EpiOcular™ EIT is not intended to differentiate between UN GHS Category 1 (serious eye damage) and UN GHS Category 2 (eye irritation).

DESCRIPTION OF THE CELL SYSTEM USED, INCL. CERTIFICATE OF AUTHENTICITY AND THE MYCOPLASMA STATUS OF THE CELL LINE :
The EpiOcular™ RhCE tissue construct consists of 3 viable layers of cells and a non-keratinized surface as recommended by the test guidelines. The model is standardized and commercially available. 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 (MatTek Corporation, Slovakia).

ENVIRONMENTAL CONDITIONS :
The environmental conditions in the incubator were standardized as follows:
Incubator temperature: 37 +/- 2° C
CO2 gas concentration: 5 %
Humidity: 95 %
All incubation steps were performed in a CO2 atmosphere incubator (Heraeus, Osterode, Germany).

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 per tissue insert in duplicate
- Concentration (if solution): neat test item

POSITIVE CONTROL
- Amount(s) applied (volume or weight with unit): 50 μLper tissue insert in duplicate

Duration of treatment / exposure:
6 hours
Duration of post- treatment incubation (in vitro):
18 hours
Number of animals or in vitro replicates:
tissue inserts were used in duplicate for test item, negative and positive control
Details on study design:
RhCE TISSUE CONSTRUCT USED, INCLUDING BATCH NUMBER :
EpiOcular RhCE tissue supplied by MatTek Corporation,

PRE-CHECK FOR POTENTIAL OPTICAL INTERFERENCES OF THE TEST ITEM :
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 of potential 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 of potential 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 of test 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 turns 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.


DESCRIPTION OF THE METHOD USED TO QUANTIFY MTT FORMAZAN :
For viability testing the inserts were placed in new plates containing MTT solution (1 mg/ml in Maintenance medium at 37°C). The tissues were incubated for 180 ± 10 min. under standard culture conditions. 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 (EL808, Bio-Tek; 96 well format, 200 μl). Data acquisition and evaluation were performed with the software "Gen5" (Bio-Tek).

ASSAY ACCEPTANCE CRITERIA :
The following acceptance criteria determine the validity of an assay:
- mean OD 570 nm negative control (NC) is > 0.8 and < 2.5
- mean relative viability of the positive control (PC) is < 50 % (relative to negative control)
- the difference of viability between the two replicates is < 20 %.

Results and discussion

In vitro

Results
Irritation parameter:
other: final cell viability (%)
Value:
ca. 69
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:
- OTHER EFFECTS:
- Visible damage on test system: No
- Direct-MTT reduction: No - according to the results of the pre-check
- Potential interference of colored or staining test items: Yes (color reaction in water) - according to the results of the pre-check

DEMONSTRATION OF TECHNICAL PROFICIENCY:
- Reliability of the test was previously confirmed by interlaboratory validation

ACCEPTANCE OF RESULTS :
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes

Applicant's summary and conclusion

Interpretation of results:
other: negative
Executive summary:

An in vitro study for assessing ocular irritation properties of the test item was performed using the reconstructed human cornea-like epithelium (RhCE) cell model EpiOcularTM. The EpiOcularTM Eye Irritation Test (EIT) was conducted in accordance with OECD 492. The solid test item was applied topically to the RhCE tissue surface in duplicate for 6 hours, followed by an 18 hour post-treatment incubation period. Cell viability was measured in a spectrophotometer by assessing the extent of MTT (methylthiazole tetrazolium) reduction. The optical density value obtained for the test item was used to calculate the percentage of viability relative to the negative control, which was set at 100 %. The results of the concurrent negative control (NC, deionized water) and positive control (PC, neat methyl acetate) demonstrated the viability (NC) and sensitivity (PC) of the tissue model. As the final mean percent tissue viability recorded for the test item is above 60% (69%) relative to the negative control, the test item was characterized as having no eye irritating properties.