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Diss Factsheets

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
4th October 2017 to 4th October 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
other: In Vitro classification based on Southee JA., 1998. Evaluation of the Prevalidation Process, Part 2, final report, Volume 2, The Bovine Corneal Opacity and Permeability (BCOP) Assay. European Community Contract No. 11279-95-10F IED ISP GB
Qualifier:
according to guideline
Guideline:
other: "Bovine Corneal Opacity and Permeability Test: An In Vitro Assay of Ocular Irritancy, (1992)"; Gautheron, Pierre; Dukic, Martine; Alix, Danielle and Sina, Joseph F.; Fundamental and Applied Toxicology 18, 442-449.
Qualifier:
according to guideline
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
5-[4-Methoxy-2-(propan-2-yl)phenoxy]pyrimidine-2,4-diamine
Cas Number:
865304-71-8
Molecular formula:
C14H18N4O2
IUPAC Name:
5-[4-Methoxy-2-(propan-2-yl)phenoxy]pyrimidine-2,4-diamine
Test material form:
solid: crystalline
Details on test material:

Off-white

Test animals / tissue source

Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
The bovine eyes were received from Spear Products on 04 Oct 2017 and transported to MB Research in Hank’s Balanced Salt Solution (HBSS) in a refrigerated container.
The eyes were examined and any eye with a cornea exhibiting evidence of vascularization, pigmentation, opacity or scratches was discarded.
Corneas from eyes that were free of defects were dissected from the surrounding tissues. A 2-3 mm rim of sclera was left attached to each cornea. The corneas were then placed in a container of fresh HBSS.

Test system

Vehicle:
other: Minimum Essential Media (MEM)
Controls:
yes, concurrent no treatment
Amount / concentration applied:
2.000 g of test article was brought to a total volume of 10.0 ml with Minimal Essential Medium (MEM)
0.75 ml of the test article mixture was applied to the epithelium of each of the five treated corneas.
Duration of treatment / exposure:
Four hours (±10 minutes)
Number of animals or in vitro replicates:
Five corneas were incubated in the presence of the test article. Two corneas were incubated untreated.
Details on study design:
After application of the test item, the holders and corneas were then placed in the 32 (±2)°C incubator in a horizontal position to ensure contact of the test article with the corneas. After four hours (±10 minutes), the test article (or MEM solution in the controls) was removed from the epithelium of the cornea and anterior chamber of the holder by washing with MEM solution. The anterior and posterior chambers of the holders were then refilled with fresh MEM solution and opacity measurements were made taken with each treated cornea compared to each of the two control corneas. Opacity measurement of the cornea was made using an OP-KIT opacitometer produced by Electro-Design Corporation of Riom, France.

Immediately following the four hour opacity measurement, the MEM solution was removed from the anterior chamber and replaced with 1.0 ml of 0.5% sodium fluorescein solution in Dulbecco's Phosphate Buffered Saline (DPBS). Each holder was then returned to the 32 (±2)°C incubator in a horizontal position ensuring contact of the fluorescein with the cornea.
After 90 minutes, the fluid from the posterior chamber was removed and the amount of dye that passed through the cornea was measured as the optical density at 490 nm by spectrophotometric analysis

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Value:
-0.3
Negative controls validity:
valid
Remarks on result:
not determinable

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The in vitro score was calculated as -0.30 and is classified as a non-irritant. (Southee, 1998).
Executive summary:

Five corneas were dosed with 0.75 ml of a 20% formulation of L-001739764-000M020. Opacity measurements and sodium fluorescein permeability were determined.


The corrected mean opacity score was -0.6. The corrected mean optical density (permeability) score was 0.020.


The in vitro score was calculated as -0.30 and is classified as a non-irritant. (Southee, 1998).