(2S,3S)-2,3-Bis[(4-methylbenzoyl)oxy]butanedioic acid — methyl {(4S)-8-fluoro-2-[4-(3methoxyphenyl)piperazin-1-yl]-3-[2-methoxy-5-(trifluoromethyl)phenyl]-3,4-dihydroquinazolin-4yl}acetate — ethyl acetate (1:1:1)

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17-06-2014 to 19-08 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

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

Test material information

Constituent 1

Reference substance name:

(2S,3S)-2,3-Bis[(4-methylbenzoyl)oxy]butanedioic acid — methyl {(4S)-8-fluoro-2-[4-(3methoxyphenyl)piperazin-1-yl]-3-[2-methoxy-5-(trifluoromethyl)phenyl]-3,4-dihydroquinazolin-4yl}acetate — ethyl acetate (1:1:1)

EC Number:

947-432-4

Cas Number:

2241754-97-0

Molecular formula:

C30H30F4N4O4.C20H18O8.C4H8O2

IUPAC Name:

(2S,3S)-2,3-Bis[(4-methylbenzoyl)oxy]butanedioic acid — methyl {(4S)-8-fluoro-2-[4-(3methoxyphenyl)piperazin-1-yl]-3-[2-methoxy-5-(trifluoromethyl)phenyl]-3,4-dihydroquinazolin-4yl}acetate — ethyl acetate (1:1:1)

Test material form:

solid

Details on test material:

lot L--005457795-001 G001

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 23 Jun 2014 and 26 Jun 2014 and transported to
MB Research in Hank's Balanced Salt Solution (HBSS) in a refrigerated container.

Test system

Vehicle:

other: Minimal Eagle's Medium

Amount / concentration applied:

2.0 g of test article were brought to a total volume of 10 ml with MEM and mixed prior to dosing. (opaque off-white suspension)

Details on study design:

Pretest Procedures
Fresh assay solutions were prepared prior to use. Minimum Essential Media (MEM) solution was
prepared by combining together one jar of MEM powder (sufficient to make one liter), 2.2 g Sodium
Bicarbonate, 0.292 g L-Glutamine, 10 ml of Fetal Bovine Serum (FBS) and distilled water was added to a
total volume of 1000 ml. The MEM solution was kept in an incubator for the duration of testing. HBSS
was prepared by combining together HBSS powder (sufficient to make one liter) and 0.35 g Sodium
Bicarbonate; the solution was brought to a final volume of 1000 ml with distilled water. HBSS was
maintained at room temperature.
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.
The dissected corneas were mounted in specially designed holders that were separated into anterior and
posterior chambers and filled separately. Each cornea was mounted allowing the epithelium of the cornea
to project into the anterior chamber. The posterior chamber was filled with (MEM) solution ensuring
contact with the endothelium. The anterior chamber was filled with MEM solution, ensuring contact with
the epithelium. Each cornea was visually inspected again to ensure there were no defects.
The entire holder with the cornea was then placed in a 32°C incubator and allowed to equilibrate for at
least one hour, but not longer than two hours.
Following the equilibration, the holders containing the corneas were removed from the incubator. The
MEM solution was removed from both chambers and the chambers refilled with fresh MEM solution. At
this time, five corneas were selected for dosing with the test article and two were selected as controls.
A pre-exposure determination of opacity was made for each control by measuring each against the blanks
supplied by the opacitometer. A pre-exposure determination of opacity was made for each test cornea by
measuring against each control cornea (a total of 10 determinations).

Study Procedure
Following the pretest observations, the MEM solution was removed from the anterior chamber and 0.75 ml
of the test article mixture was applied to the epithelium of each of the five treated corneas.
The holders and corneas were then placed in the 32°C incubator in a horizontal position to ensure contact
of the test article with the corneas. After four hours, 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°C incubator in a horizontal position
insuring 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

Run / experiment:

one

Value:

ca. -1.22

Vehicle controls validity:

valid

Negative controls validity:

not valid

Positive controls validity:

not specified

Remarks on result:

no indication of irritation

Any other information on results incl. tables

cornea#	pretest	4hrs	OD scores
C3	0	2	0.149*
C4	0	3	0.086*
mean	0	2.5	*
corrected Mean control opacity score1	2.5		*

1Corrected Mean Control Opacity Score = 4 hour mean score minus pretest mean score

* = The permeability scores of the control corneas, C3 and C4, used for the BCQP testing on 23 Jun 2014, significantly exceeded

the expected OD of untreated and intact corneas. Additionally, the C3 and C4 permeability scores exceeded those of the test

article corneas. The BCOP was repeated on 26 Jun 2014. The conclusion was based on the BCOP performed on 26 Jun 2014.

RESULTS for 23 Jun 2014

INDIVIDUAL CONTROL SCORES FOR BCOP

Cornea #	pretest scores		4 hour scores		OD scores
1	C3 -1	C4 -2	C3 -2	C4 -3	0.013
2	C3 -1	C4 -2	C3 -1	C4 -1	0.054
3	C3 -3	C4 -3	C3 -2	C4 -3	0.035
4	C3 -2	C4 -3	C3 -3	C4 -3	0.019
5	C3 -0	C4 -0	C3 -0	C4 -0	0.019

RESULTS for 26 Jun 2014

INDIVIDUAL CONTROL SCORES FOR BCOP

cornea#	pretest	4hrs	OD scores
C3	2	3	0.03
C4	0	4	0.055
meaN	1	3.5	0.043
corrected Mean control opacity score1	2.5

INDIVIDUAL TEST SCORES

rnea #	pretest scores		4 hour scores		OD scores
1	C3 -2	C4 0	C3 0	C4 0	0.091
2	C3 0	C4 2	C3 3	C4 3	0.065
3	C3 -2	C4 0	C3 1	C4 1	0.054
4	C3 -2	C4 0	C3 0	C4 0	0.025
5	C3 -1	C4 0	C3 -1	C4 -1	0.042

CALCULATED SCORES

CORNEA #	CORRECTED OPACITY   SCORES		CORRECTED OD
	4 HOUR SCORES
1	2	0	0.048
2	3	1	0.022
3	3	1	0.011
4	2	0	-0.018
5	0	-1	-0.001
CORRECTED MEAN OPTICAL DENSITY			0.012
CORRECTED MEAN OPACITY SCORE 2			-1.4

1Corrected Mean Control Opacity Score = 4 hour mean score minus pretest mean score

2Corrected Mean Opacity Score = mean treated opacity score minus corrected mean control opacity score

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The corrected mean opacity score was -1.4 The corrected mean optical density
(permeability) score was 0.012. The in vitro score was calculated as -1.22 and is classified as a non-irritant (Southee,
1998).

Executive summary:

Objective: To determine the potential for ocular irritation using an alternative to the Draize methodology.

This protocol is based on the methodology described in "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. 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 lED ISP GB

and included an analysis based on OECD Guideline for the Testing of Chemicals #437, adopted

September 7, 2009

Method Synopsis: Ten corneas were dosed with 0.75 ml of a 20% suspension of

L-005457795-001G001. Five corneas were dosed on 23 Ju12014. Opacity measurements and sodium

fluorescein permeability were determined. The permeability scores of the control corneas, C3 and C4,

used for the BCOP testing on 23 Jun 2014. significantly exceeded the expected OD490

of untreated and intact corneas. Additionally, the C3 and C4 permeability scores exceeded those of the test article

corneas. As a result, the BCOP was repeated on 26 Jun 2014. The conclusion was based on the BCOP

performed on 26 Jun 2014.

Summary: The corrected mean opacity score was -1.4 The corrected mean optical density

(permeability) score was 0.012.

Conclusion: The in vitro score was calculated as -1.22 and is classified as a non-irritant (Southee,

1998).