L-Leucine, 4-fluoro-N-[(1S)-2,2,2-trifluoro-1-[4'-(methylsulfonyl)[1,1'-biphenyl]-4-yl]ethyl]-, compd. with N-cyclohexylcyclohexanamine (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:

10 July - 13 July, 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study was conducted in accordance with GLP. The study material is well characterized

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: Gautheron, P., Dukic, M., Alix, D. and Sina, J.F. (1992) Bovine Corneal Opacity and Permeability Test: An in vitro Assay of Occular Irritancy. Fund. Appl. Toxicol. 18:442-449

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Sina, J.F., (1994) Validation of the Bovine Corneal Opacity – Permiability Assay as a Predictor of Ocular Irritation Potential. In Vitro Toxicology 7(3):283−29

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Sina, J.F., Galer, D.M., Sussman, R.G., Gautheron, P.D., Sargent, E.V., Leong, B., Shah, P.V., Curren, R.D. and Miller, K. (1995). A Collaborative Evaluation of Seven Alternatives to the Draize Eye Irritation Test using Pharmaceutical Intermediates.

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: INVITTOX Protocol 124. 1999. Bovine Corneal Opacity and Permeability (BCOP) Assay – SOP of Microbiological Associates Ltd. UK

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

N-cyclohexylcyclohexanaminium (2S)-4-fluoro-4-methyl-2-{[(1S)-2,2,2-trifluoro-1-{4'-methanesulfonyl-[1,1'-biphenyl]-4-yl}ethyl]amino}pentanoate

EC Number:

700-778-8

Cas Number:

1064076-86-3

Molecular formula:

C21H23F4NO4S.C12H23N

IUPAC Name:

N-cyclohexylcyclohexanaminium (2S)-4-fluoro-4-methyl-2-{[(1S)-2,2,2-trifluoro-1-{4'-methanesulfonyl-[1,1'-biphenyl]-4-yl}ethyl]amino}pentanoate

Test animals / tissue source

Species:

other: Bovine eyes from local abattoir delived to lab within 4hrs of sacrifice.

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Bovine eyes from local abattoir delived to lab within 4hrs of sacrifice and were kept moist and transported chilled.

Test system

Vehicle:

other: Modified Minimum Essential Medium (MEM)

Controls:

other: Modified Minimum Essential Medium (MEM) used as a negative control. A 20% solution of Imidazole in MEM was used as a positive control.

Amount / concentration applied:

20% solution of test substance in MEM

Duration of treatment / exposure:

4 ± 0.15 hrs
32 ± 2°C

Observation period (in vivo):

Opacity and Permeability readings were taken immediately after the exposure period.

Number of animals or in vitro replicates:

3 eyes used for the negative control, 2 used for the positive control and 3 eyes used for the test solution.

Details on study design:

EXPERIMENTAL DESIGN
1 Eyes were carefully examined for defects such as neovascularization, pigmentation, opacity, or
scratches (a slit lamp may be used to facilitate this evaluation).
2 Preparation of Corneas:
2.1 The back of the eye was grabbed with a small piece of gauze while slicing through the
middle of the eye. Halfway through the slice, the front half of eye was stabilized by grabbing
the cut edge with forceps in order to prevent the cornea from getting scratched or damaged.
2.2 The front half of the eye was placed, cornea side down into Petri dish filled with
Phosphate Buffered Saline (PBS). The lens and uveal tissues (i.e. iris, ciliary body) were peeled
away leaving only the cornea and sclera.
2.3 While holding the eye with forceps at the cut edge, excess tissue was cut leaving a
2-3 mm rim of sclera around the cornea.
2.4 Corneas were mounted in holders with the endothelial side applied to the o-ring of the
posterior part of the holder.
2.5 The anterior part of the holder was applied to the cornea and held in place with 3
screws. The compartments were filled (posterior part first) with MEM medium and the
corneas were incubated for 60 ± 10 minutes in a water-bath at 32 ± 2 °C.
2.6 At the end of the incubation period, the media were completely removed from both
compartments using a blunt needle attached to a 5 cc syringe.
2.7 The baseline opacity (OI) was measured.
2.8 The average OI was calculated for all of the corneas.
2.9 Three corneas with opacity values close to the average value for all corneas were selected
as the negative control corneas.
2.10 Corneas that had an initial opacity reading greater than 10 were discarded.
2.11 The remaining corneas were distributed into the test groups and the positive control
groups.
3 Treatment:
3.1 Using a blunt needle attached to a 5 cc syringe, the media was completely removed
from the anterior chamber.
3.2 The anterior compartment was then filled with 0.75 mL of the test substance, positive
control or the negative control. All holes were capped.
3.3 The test substance was applied using a glass syringe.
3.4 The holders were rotated so that the cornea was in a horizontal position with the anterior
compartment on top.
3.5 Incubation was carried out at 32 ± 2 °C in a water-bath for 4 ± 0.15 hours.
3.6 After incubation, the test substance was removed.
3.7 The epithelium was washed at least 3 times with MEM containing phenol red (to monitor
the pH) using a gentle swirling motion. A final wash was done in MEM without phenol red.
3.8 The final post-treatment reading (OF) was measured.

Results and discussion

In vivo

Results

Irritation parameter:

overall irritation score

Basis:

mean

Score:

0.006

Irritant / corrosive response data:

The numerical results and calculations of the study are presented in Table I. Based on the
measurements obtained, the in vitro score for the test article was 0.058, classifying it as a mild
irritant. This score was lower than that of the negative control.

Any other information on results incl. tables

Based on the result of this calculation, the predicted ocular irritancy was classified as follows:

In vitro Score

Irritancy/Classification

0-3.0 Non-irritant

3.1-25.0 Mild irritant

25.1-55.0 Moderate irritant

55.1-80.0 Severe irritant

>= 80.1 Very Severe irritant

TABLE I

Opacity and Permeability (Mean +- SD)

Parameter (n) Negative Control (MEM) (n) Positive Control (Imidazole 20%) (n) L-001054748 -001X001 (20%)

Opacity (Initial) (3) 2.33 ± 1.58 (2) 5.50 ± 3.54 (3) 0.67 ± 1.15

Opacity (Final) (3) 3.67 ± 3.79 (2) 83.00 ± 22.63 (3) 2.67 ± 1.16

delta Opacity(3)1.33 ± 4.16 (2) 77.50 ± 19.01 (3) 2.00 ± 2.003

Corrected delta Opacity (3) N/A (2) 76.17 ± 19.10 (3 )1.67 ± 1.41

Permeability (3) 0.108 ± 0.124 (2) 1.760 ± 0.392 (3) -0.0002 ± 0.003

Corrected Permeability (3) N/A (2) 1.643 ± 0.381 (3) -0.109 ± 0.003

In vitro Score negative control: 2.96 postive control 100.93 test substance -0.962

Classification Negative Non Irritant Postive control Very Severe test substance N/A

Applicant's summary and conclusion

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: other:

Conclusions:

Based on the available data, the test substance does not appear to be a greater corneal irritant than does the negative
control.

Executive summary:

A 20% solution of the test substance L-001054748-001X001, was evaluated for its ocular

irritancy potential using corneas collected from bovine eyes. Opacity in treated corneas was

noted as slightly greater than that of negative control eyes, but within the variability of the

respective means. The permeability reading of the test substance was actually less than that of

the negative control but not different than the variability of the means. Based on the available

data, the test substance does not appear to be a greater corneal irritant than does the negative

control.