(Z)-4-[C11-13 (branched) alkylamino]-4-oxo-2-butenoic acid

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

(Z)-4-[C11-13 (branched) alkylamino]-4-oxo-2-butenoic acid

EC Number:

701-116-0

Cas Number:

2156592-45-7

Molecular formula:

C17H31NO3

IUPAC Name:

(Z)-4-[C11-13 (branched) alkylamino]-4-oxo-2-butenoic acid

Test animals / tissue source

Species:

other: in vitro system, isolated bovine cornea

Strain:

other: not applicable, in vitro system

Details on test animals or tissues and environmental conditions:

An in vitro system (isolated bovine cornea) was applied to analyse the irritating potential of the test item for the eye. The bovine eyes were obtained as a by-producr of freshly slaughtered cattle (age of animals was minimum 12 months and maximum 60 months).
Corneas free of defects (opacity, scratches, pigmentation etc.) were dissected with a 2 to 3 mm rim of sclera. Isolated corneas were mounted in specially designed corneal holders that consists of anterior and posterior chambers. Both chambers were filled to excess with prewarmed Eagles’s MEM (without phenol red) and then equilibrated in a vertical position at about 32 °C for at least 1 hour. After the equilibration period the medium in both chambers was replaced with fresh prewarmed medium and initial corneal opacity readings were taken for each cornea with an opacitometer. Any corneas that show macroscopic tissue damage or an opacity value < 507 opacity units were discarded. Corneas with opacity values close to the median value of all corneas were selected as negative control (NC). The remaining corneas were then distributed into treatment and positive control groups.
Each corneal holder was uniquely identified with a number on the chambers.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

other: 3 corneas were used as PC and NC, respectively

Amount / concentration applied:

Before application of the test item the medium in the anterior chamber was removed using a syringe.
750 μL of the undiluted liquid test substance was applied directly to the epithelial surface of the cornea using a pipette (open chamber method).
Control tissues were concurrently applied into the anterior chamber with 750 μL of de-ionized water (NC) or with 750 μL of 1% (w/v) solution of sodium hydroxide in deionized water (PC) using a pipette.

Duration of treatment / exposure:

The corneas were incubated in a horizontal position at about 32 °C for approximately 10 minutes (liquids and surfactants). The NC and PC were then removed from the anterior chamber using a syringe and the epithelium was washed at least 3 times with Eagle’s MEM (containing phenol red) and once with Eagle’s MEM (without phenol red). Both chambers were then refilled with fresh Eagle’s MEM (without phenol red). Because the test substance could not be removed using a syringe, the epithelium was rinsed with the open chamber method.

Observation period (in vivo):

The corneas were incubated for further 2 hours at about 32 °C. After the incubation period the medium was removed and both chambers were then refilled with fresh Eagle’s MEM.

Number of animals or in vitro replicates:

not applicable; 3 corneas were used for each treatment group (PC, NC, test item)

Details on study design:

Determination of permeability
- For determination of permeability the medium in the anterior chamber was replaced by 1 mL sodium fluorescein solution (4 mg/mL for liquid test substances and surfactants) and incubated for 90 ± 5 min in a horizontal position at about 32 °C. The amount of sodium fluorescein that permeated through the corneas into the posterior chamber was measured spectrophotometrically. Three aliquots per cornea were transferred to a 96-well microtiter plate and the optical density (OD490) was determined.
Measurement of final corneal opacity
- Before measurement, each cornea was observed visually and observations were recorded. Final corneal opacity readings were taken for each cornea with an opacitometer.

The mean corneal opacity and permeability values of each treatment group were used to calculate an In Vitro Irritancy Score (IVIS). An IVIS >55 indicates the risk of serious damage to the eyes, wehereas an IVIA < 55 predicts that the test item does not cause any risk of serious eye damage.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

cornea opacity score

Run / experiment:

test substance

Value:

7.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: No indication for serious eye damage

Results 2

Irritation parameter:

cornea opacity score

Run / experiment:

negative control

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not measured/tested

Results 3

Irritation parameter:

cornea opacity score

Run / experiment:

positive control

Value:

88

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not measured/tested

Applicant's summary and conclusion

Interpretation of results:

other: no serious eye damage

Remarks:

Criteria used for interpretation of results: EU