Reaction mass of morpholine and 6-[(p-tosyl)amino]hexanoic acid, compound with morpholine (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 10 October 2017 to 26 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:

2018

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 i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

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:

morpholin-4-ium morpholine 6-(4-methylbenzenesulfonamido)hexanoate

EC Number:

944-090-8

Molecular formula:

C13H19NO4S.C4H9NO/C4H9NO/H2O

IUPAC Name:

morpholin-4-ium morpholine 6-(4-methylbenzenesulfonamido)hexanoate

Test material form:

liquid

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Corneas from bovine eyes were obtained from a local abattoir. The eyes were removed after slaughter, completely immersed in Hanks’ Balanced Salt Solution (containing penicillin at 100 IU/mL and streptomycin at 100 µg/mL) in a suitably sized container and transported on the same day to the testing facility.

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): 750 µL

Duration of treatment / exposure:

Ten minutes

Duration of post- treatment incubation (in vitro):

Two hours

Number of animals or in vitro replicates:

Three corneas (triplicate)

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
Upon arrival at the test facility, the corneas were excised from the eyes and loaded onto the specifically designed holders . Both chambers of each holder were filled with pre-warmed Minimal Essential Medium (MEM), with the posterior chamber filled first, ensuring that no bubbles were formed. The holders were incubated at 32±1°C for at least 1 hour. After the incubation, the media was removed from both the anterior and posterior chambers. Fresh media was added to the posterior chamber first and then the anterior chamber (this media replacement order ensured the cornea retained its natural curvature as much as possible). The opacity of each cornea was measured using an opacitometer. Any corneas found to have scratches or increased neovascularization or an opacity of >7 opacity units when examined prior to treatment were discarded.

QUALITY CHECK OF THE ISOLATED CORNEAS
On arrival at the test facility the eyes were carefully examined for defects including increased opacity, scratches and neovascularisation. Only corneas free from such defects were used.

NUMBER OF REPLICATES
Three.

NEGATIVE CONTROL USED
0.9% sodium chloride solution

POSITIVE CONTROL USED
Dimethylformamide

APPLICATION DOSE AND EXPOSURE TIME
A volume of 750 µL of the test article (or enough test article to completely cover the cornea) was applied to each of three corneas followed by a ten minute incubation at 32±1°C

POST-INCUBATION PERIOD: Two hours

REMOVAL OF TEST SUBSTANCE
After the incubation, each cornea was washed with media containing phenol red (as a pH indicator) until this indicator showed no pH effect occurring (and demonstrating that the test article had been removed successfully). The corneas were then washed once in media without phenol red.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: opacitometer
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of spectrophotometer (OD490)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA:
The test article is concluded as inducing serious eye damage if the IVIS is >55.
The test article is concluded as not requiring classification for eye irritation if the IVIS is ≤3.
No prediction can be made if the IVIS is >3 but ≤55.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Value:

20.69

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: The corneas treated with the test article were noted to be cloudy and slightly wrinkled following treatment

DEMONSTRATION OF TECHNICAL PROFICIENCY:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Negative control yields opacity and permeability values that are less than the established upper limits for these endpoints for bovine corneas as treated at the testing facility
- Acceptance criteria met for positive control: Positive control yields an IVIS within 2 standard deviations of the historical control mean.

Any other information on results incl. tables

See attached brackground documents.

Applicant's summary and conclusion

Interpretation of results:

other: Inconclusive

Conclusions:

The test article, Reaction mass of morpholine and 6 [(p tosyl)amino]hexanoic acid, compound with morpholine (1:1) (EC 944 090 8), produced an IVIS score of 20.69 and therefore no prediction can be made in respect of it’s potential to cause eye irritation as the IVIS was >3 but <55.

Executive summary:

An in vitro Bovine Corneal Opacity and Permeability (BCOP) assay was performed in line with OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method).

 

The undiluted test material was applied to three cattle corneas obtained from an abattoir at a volume of 750 μL, after which each cornea was incubated at 32 ± 1 °C for 10 minutes and then washed with phenol red-containing media followed by a media without phenol red. Corneal opacity was measured after a 2 hour period of horizontal incubation. For permeability, the corneas were incubated in the vertical position for 1 hour and 25 minutes at 32 ± 1°C within a sodium fluorescein solution. Thereafter, three 350 μL aliquots of this media (per cornea) were analysed for optical density at 490 nanometers (OD490). A positive (dimethylformamide) and negative (0.9% sodium chloride solution) control was applied using the same procedure to additional groups of corneas.

 

The mean corrected opacity reading for the test article, positive control, and negative control was 16.0, 62.7, and 0.0, respectively. The mean group corrected optical density for the test article was 0.180 and the mean group corrected optical density for the positive and negative control was 0.243 and 0.000, respectively. An In Vitro Irritation Score (IVIS) of 20.69 was calculated for Reaction mass of morpholine and 6-[(p-tosyl)amino]hexanoic acid, compound with morpholine (1:1) from corneal opacity and permeability measurements. No prediction can be made if the IVIS is >3 but ≤ 55 and, therefore, the study is inconclusive.