Cyclopropanecarboxylic acid, 1-amino-2-ethenyl-, ethyl ester, (1R,2S)-, sulfate (2:1)

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2011-11-22

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

This study does not comply with the GLP requirements nor Quality Assurance Procedures. It was carried out in conformity with the EEC validation study with some modifications, but given the impossibility to check these pieces of information, the OECD Guideline N°437 was chosen as a reference to assess the reliability of this study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

equivalent or similar to guideline

Guideline:

other: EEC validation study (contract ref. B91/B4-3081/013188, 1991; Gautheron et al., 1994) with some modifications (Vanparys et al., 1993)

Deviations:

not specified

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

no

GLP compliance:

no

Test material

Test material information

Constituent 1

Reference substance name:

bis(ethyl (1R,2S)-1-amino-2-ethenylcyclopropane-1-carboxylate); sulfuric acid

EC Number:

689-958-4

Cas Number:

1173807-85-6

Molecular formula:

C8H13NO2.0.5H2SO4

IUPAC Name:

bis(ethyl (1R,2S)-1-amino-2-ethenylcyclopropane-1-carboxylate); sulfuric acid

Test material form:

not specified

Details on test material:

- Name of test material (as cited in study report): JNJ-31052047-ABI
- Physical state: solid
- Lot/batch No.: 00549620
- Purity : 100.1% w/w
- Purity test date : 6 July 2011
- Storage condition of test material: Stored at room temperature in a closed and labeled container

Test animals / tissue source

Species:

other: bovine

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Bovine (<12 months) eyes were excised by a slaughterhouse veterinarian and collected in a plastic jar containing one liter of Hank’s Balanced Salt Solution (HBSS, Sigma). Medium storage and transportation of eyes to the laboratory was performed at room temperature. The eyes were used within 24 hours after killing the animals.

Corneas Preparation and Selection :
The intact corneas were selected, dissected with a 2-3 mm rim of sclera for easier handling and stored in a petri-dish containing Hank’s Balanced Salt Solution (HBSS) until use. Corneas were mounted in holders, the endothelial side being applied on the O-ring of the posterior part of the cornea holder. The anterior part of the cornea holder was applied onto the posterior part and held in place with 3 screws. The compartments were filled with Minimal Essential Medium (MEM, Sigma) and the corneas were then incubated for one hour in a water-bath at 32°C.
After measuring opacity and permeability, corneas were rejected for use if they showed a background opacity grade greater than three.

Test system

Vehicle:

other: 0.9% (w/v) NaCl in water

Controls:

yes

Amount / concentration applied:

TEST MATERIAL
- Concentration (if solution): a 20 % (w/w) formulation (e.g. suspension)

Duration of treatment / exposure:

Corneas were incubated in a horizontal position for 4 hours (240 min) at 32°C in a water-bath, according to the experimental protocol for solid test compounds.

Observation period (in vivo):

Background opacity : After incubation of the corneas for one hour in a water-bath at 32°C, the MEMsolution was removed and both compartments were refilled with fresh medium. After careful removal of the air-bubbles, background opacity of all corneas was measured with an opacitometer (OP-KIT, Electro Design, Riom, France) at time zero.
Opacity and permeability were measured with the opacitometer and the spectrophotometer respectively at time t240.

Number of animals or in vitro replicates:

3 corneas per control, 3 corneas per test item

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing and opacity measurement: After incubation, the vehicle control, the positive control, and the JNJ-31052047-ABI formulation were removed from the anterior compartment and the epithelium was washed at least 3 times with approximately 4 ml of MEM-solution until the medium was clear. The medium was then removed from the posterior compartment and both compartments were finally refilled with MEM-solution until the medium was clear. The medium was then removed from the posterior compartment and both compartments were finally refilled with MEM-solution. Then opacity was measured with the opacitometer.
- Permeability determination: The permeability of the corneas was evaluated immediately after measuring the opacity. The medium was removed from the anterior compartment and replaced by 1 ml of a 0.5% sodium-fluorescein solution (sodium fluorescein (Sigma) was disolved in Dulbecco’s phosphate buffered saline). Corneas were incubated in a horizontal position for 90 minutes at 32°C in a water-bath. After incubation, medium from the posterior chamber was removed, and its optical density (OD) was determined spectrophotometrically at 490 nm. The results on the printout of the spectrophotometer were entered into a specially prepared spreadsheet. The mean permeability value obtained for the vehicle control (n=3) is subtracted from the individual test item permeability value.

- Time after start of exposure: 240 min (corresponding to the incubation period)

SCORING SYSTEM:
An in vitro score for the corneal injury was calculated with a formula, which combines results from both opacity and permeability: In vitro score = opacity value + 15 times OD value for permeability. Individual in vitro scores were used to calculate the mean in vitro score.
In vitro scores were sorted into five different classes:
 ≤ 3.0 = non eye irritant
 from 3.1 to 25.0 = mild eye irritant
 from 25.1 to 55.0 = moderate eye irritant
 from 55.1 to 80.0 = severe eye irritant
 ≥ 80.1 = very severe eye irritant.

TOOLS USED TO ASSESS SCORE:
The opacity was measured with an opacitometer (OP-KIT, Electro Design, Riom, France), and permeability was assed by optical density measurement with a spectrophotomer at 490nm.

Results and discussion

In vivo

Results

Irritation parameter:

other: opacity and permeability of the cornea

Basis:

mean

Time point:

other: 240 min

Score:

42.3

Max. score:

80

Reversibility:

other: reversibility was not checked on this study type

Irritant / corrosive response data:

When tested as a 20 % (w/w) suspension, JNJ-31052047-ABI induced a moderate increase in opacity (41.0 ± 11.1) and no increase in permeability (0.089 ± 0.043). Consequently, an in vitro score of 42.3 +/- 11.3 was calculated which classified the test item as a moderate eye irritant when tested in vitro under the conditions described in this report.
With the negative control (0.9 % NaCl solution) neither an increase of opacity nor permeability of the corneae could be observed. The in vitro score was calculated as 0.3.
The positive control (20% w/w imidazole) showed clear opacity and distinctive permeability of the corneae and therefore, is classified as very severe eye irritant. The in vitro score was calculated as 217.6.

Any other information on results incl. tables

Background opacity measurement

For all corneas examined, background opacity ranged between 0 and 3, indicating that no corneas had to be excluded from the experimental test design.

In vitro scores of control corneas

In this study, the vehicle control induced a mean opacity, permeability and an in vitro score which fell within our laboratory range of historical control data.

The corneas treated with the positive control imidazole (20 % (w/w)) showed a marked increase in opacity and permeability. Imidazole was classified as a very severe eye irritant. Based on these findings, it was concluded that the assay acceptance criteria for the evaluation of the in vitro eye irritating potential of JNJ-31052047-ABI were met.

Applicant's summary and conclusion

Interpretation of results:

moderately irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

In conclusion it can be stated that under the experimental conditions reported, the test item T0033325 is considered to be a moderately irritating substance.