Reaction mass of 2-aminoethanol and 6-[(p-tosyl)amino]hexanoic acid, compound with 2-aminoethanol (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:

From 31 January 2016 to 29 February 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study was conducted by a GLP accredited laboratory using OECD Testing Guideline 437. The study was conducted on the registered substance.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of 2-aminoethanol and 6-[(p- tosyl)amino] hexanoic acid, compound with 2- aminoethanol (1:1)

IUPAC Name:

Reaction mass of 2-aminoethanol and 6-[(p- tosyl)amino] hexanoic acid, compound with 2- aminoethanol (1:1)

Constituent 2

Reference substance name:

Reaction mass of 2-aminoethanol and 6-[(p-tosyl)amino]hexanoic acid, compound with 2-aminoethanol (1:1)

EC Number:

942-732-1

Molecular formula:

C13H19NO4S.C2H7NO/C2H7NO/H2O

IUPAC Name:

Reaction mass of 2-aminoethanol and 6-[(p-tosyl)amino]hexanoic acid, compound with 2-aminoethanol (1:1)

Test material form:

other: Liquid

Details on test material:

- Name of test material (as cited in study report): Reaction mass of 2-aminoethanol and 6-[(p-tosyl)amino] hexanoic acid, compound with 2-aminoethanol (1:1)- Expiration date of the lot/batch: 24 June 2016- Storage condition of test material: Room temperature in the dark

Test animals / tissue source

Species:

other: Bovine cornea

Strain:

other: Bovine cornea

Details on test animals or tissues and environmental conditions:

Not applicable

Test system

Vehicle:

physiological saline

Controls:

other:

Amount / concentration applied:

TEST MATERIAL- Concentration (if solution): 20%w/v solution in 0.9%w/v sodium chloride solution

Duration of treatment / exposure:

Each holder was incubated, anterior chamber uppermost, at 21 ± 1°C for 120 minutes.

Observation period (in vivo):

Not applicable

Number of animals or in vitro replicates:

Not applicable

Details on study design:

The negative control item, 0.9% w/v sodium chloride solution, was used as supplied.The positive control item, >99.8% Ethanol, was used as supplied.Preparation of CorneasAll eyes were macroscopically examined before and after dissection. Only corneas free of damage were used.The cornea from each selected eye was removed leaving a 2 to 3 mm rim of sclera to facilitate handling. The iris and lens were peeled away from the cornea. The isolated corneas were immersed (epithelial side uppermost) in a dish containing HBSS until they were mounted in Bovine Corneal Opacity and Permeability (BCOP) holders.The anterior and posterior chambers of each BCOP holder were filled with complete Eagle’s Minimum Essential Medium (EMEM) without phenol red and plugged. The holders were incubated at 32 ± 1 ºC for 60 minutes. At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.Selection of Corneas and Opacity ReadingThe medium from both chambers of each holder was replaced with fresh complete EMEM.A pre-treatment opacity reading was taken for each cornea using a calibrated opacitometer (Appendix 1). The average opacity for all corneas was calculated.Three corneas with opacity values close to the median value of all corneas were allocated to the negative control. Three corneas were also allocated to the test item and three corneas to the positive control item.Treatment of CorneasThe EMEM was removed from the anterior chamber of the BCOP holder and 0.75 mL of the test item preparation or control items were applied to the appropriate corneas. The holders were gently tilted back and forth to ensure a uniform application of the item over the entire cornea.Each holder was incubated, anterior chamber uppermost, at 32 ± 1 ºC for 240 minutes.At the end of the exposure period the test item and control items were removed from the anterior chamber and the cornea was rinsed three times with fresh complete EMEM containing phenol red before a final rinse with complete EMEM without phenol red. The anterior chamber was refilled with fresh complete EMEM without phenol red. A post-treatment opacity reading was taken and each cornea was visually observed.Application of Sodium FluoresceinFollowing the opacity measurement the permeability of the corneas to sodium fluorescein was evaluated. The medium from the anterior chamber was removed and replaced with 1 mL of sodium fluorescein solution (5 mg/mL). The dosing holes were plugged and the holders incubated, anterior chamber uppermost, at 32 ± 1 ºC for 90 minutes.Permeability DeterminationsAfter incubation the medium in the posterior chamber of each holder was decanted and retained.360 μL of medium representing each cornea was applied to a designated well on a 96-well plate and the optical density at 492 nm (OD492) was measured using the Anthos 2001 microplate reader.HistopathologyThe corneas were retained after testing for possible conduct of histopathology. Each cornea was placed into a pre-labeled tissue cassette fitted with a histology sponge to protect the endothelial surface. The cassette was immersed in 10% neutral buffered formalin.

Results and discussion

In vivo

Results

Irritation parameter:

other: In Vitro Irritancy Score (IVIS)

Basis:

mean

Time point:

other: 10 minutes

Score:

27.8

Reversibility:

other: not applicable

Irritant / corrosive response data:

In Vitro Irritancy Score = mean opacity value + (15 x mean permeability OD492 value)

Any other information on results incl. tables

The results of the BCOP on Reaction mass of 2-aminoethanol and 6-[(p-tosyl)amino] hexanoic acid, compound with 2-aminoethanol (1:1). The individual In Vitro Irritancy Scores (IVIS) are given below:

Substance	In-vitro Irritancy Scores (IVIS)
Negative control	0.5
Positive control	44.4
Test item	27.8

Applicant's summary and conclusion

Interpretation of results:

other: inconclusive

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

The test item produced an inconclusive IVIS of 27.8 when tested under a Bovine Corneal Opacity and Permeability (BCOP).

Executive summary:

The potential of the test item to be irritant to the eye was determined in accordance with the OECD Guideline for Testing of Chemicals 437. The purpose of this test was to identify test items that can induce serious eye damage and to identify test items not requiring classification for eye irritation or serious eye damage. The Bovine Corneal Opacity and Permeability (BCOP) test method is an organotypic model that provides short-term maintenance of normal physiological and biochemical function of the bovine cornea in vitro. In this test method, damage by the test item is assessed by quantitative measurements of changes in corneal opacity and permeability.

 

The test item was applied at a concentration of 20% w/v in 0.9% w/v sodium chloride solution for 120 minutes. Negative and positive control items were tested concurrently. The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate an In Vitro Irritancy Score (IVIS)

 

The test item produced an inconclusive IVIS of 27.8 when tested under a Bovine Corneal Opacity and Permeability (BCOP).