O-(ethoxycarbonyl)-N-(1-methyl-2-oxo-2-phenylethylidene)hydroxylamine

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 June 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:

2017

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)

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

O-(ethoxycarbonyl)-N-(1-methyl-2-oxo-2-phenylethylidene)hydroxylamine

EC Number:

265-967-5

EC Name:

O-(ethoxycarbonyl)-N-(1-methyl-2-oxo-2-phenylethylidene)hydroxylamine

Cas Number:

65894-76-0

Molecular formula:

C12H13NO4

IUPAC Name:

O-(ethoxycarbonyl)-N-(1-methyl-2-oxo-2-phenylethylidene)hydroxylamine

Test material form:

solid: particulate/powder

Details on test material:

- Appearance: Off white powder

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
The test item was formulated within 2 hours of being applied to the test system. It is assumed that the formulation was stable for this duration.

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: A local abattoir
- Characteristics of donor animals: Eyes from adult cattle (typically 12 to 60 months old) were obtained from a local abattoir as a by-product from freshly slaughtered animals.
- Storage, temperature and transport conditions of ocular: They were transported to the test facility over ice packs on the same day of slaughter. The corneas were prepared immediately on arrival.
- Indication of any antibiotics used: The eyes were excised by an abattoir employee after slaughter, and were placed in Hanks’ Balanced Salt Solution (HBSS) supplemented with antibiotics (penicillin at 100 IU/mL and streptomycin at 100 µg/mL).

Test system

Vehicle:

other: sodium chloride 0.9 % w/v

Controls:

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 0.75 mL
- Concentration: 20 % w/v suspension

VEHICLE
- Amount(s) applied: 0.75 mL
- Concentration: 0.9 % w/v
- Lot/batch no.: 3012488
- Purity: 0.9 %

Duration of treatment / exposure:

240 minutes

Duration of post- treatment incubation (in vitro):

90 minutes with Sodium Fluorescein

Number of animals or in vitro replicates:

The test was performed in triplicate.

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
- All 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 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 °C for 60 minutes. At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.

NUMBER OF REPLICATES: 3

SOLVENT CONTROL USED: Yes, sodium chloride 0.9 % w/v

POSITIVE CONTROL USED: Yes, Imidazole as a 20 % w/v solution in sodium chloride 0.9 % w/v.

APPLICATION DOSE AND EXPOSURE TIME: 0.75 mL for 240 minutes

TREATMENT METHOD:
- The 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. The average opacity for all corneas was calculated.
- The EMEM was removed from the anterior chamber of the BCOP holder and 0.75 mL of the test material preparation or control materials were applied to the appropriate corneas. The holders were gently tilted back and forth to ensure a uniform application of the material over the entire cornea. Each holder was incubated, anterior chamber uppermost, at 32 ± 1 ºC for 240 minutes.

REMOVAL OF TEST SUBSTANCE
- At the end of the exposure period the test material and control materials 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.

POST-INCUBATION PERIOD: Yes, following 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 °C for 90 minutes.
- After incubation the medium in the posterior chamber of each holder was decanted and retained. 360 µL of media representing each cornea was dispensed into the appropriate wells of a pre-labelled 96 well plate. The optical density was measured (quantitative viability analysis) at 492 nm (without a reference filter) using the Labtech LT-4500 microplate reader.

DATA EVALUATION FOR MEASURED ENDPOINTS
- Corneal opacity: The change in opacity for each cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final opacity reading. These values were then corrected by subtracting the average change in opacity observed for the negative control corneas. The mean opacity value of each treatment group was then calculated by averaging the corrected opacity values of each cornea for that treatment group.
- Corneal permeability: The corrected OD492 was calculated by subtracting the mean OD492 of the negative control corneas from the OD492 value of each treated cornea. The OD492 value of each treatment group was calculated by averaging the corrected OD492 values of the treated corneas for the treatment group.
- Others: Visual observations were performed post treatment to assess the condition of the cornea.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS).
Results from the two test method endpoints, opacity and permeability, were combined in an empirically derived formula to generate an In Vitro Irritancy Score. The following formula was used to determine the In Vitro Irritancy Score:

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

Additionally, the opacity and permeability values were evaluated independently to determine whether the test material induced a response through only one of the two endpoints.

DECISION CRITERIA:
The test material was classified according to the following prediction model:
- IVIS ≤ 3: No category. Not requiring classification to UN GHS or EU CLP.
- IVIS > 3; ≤ 55: No prediction of eye irritation can be made
- IVIS > 55: Category 1. UN GHS or EU CLP Causes serious eye damage

ACCEPTABILITY CRITERIA
For an acceptable test the following positive control criterion should be achieved:
- 20 % w/v Imidazole was used for positive control purposes. The test was acceptable if the positive control produced an In Vitro Irritancy Score which fell within two standard deviations of the historical mean during 2015 for this testing facility. Therefore the In Vitro Irritancy Score should fall within the range of 50.8 to 100.4.
For an acceptable test the following negative control criteria should be achieved:
- Sodium chloride 0.9 % w/v was used for negative control purposes. The test was acceptable if the negative control produced an In Vitro Irritancy Score which is less than or equal to the upper limit for background opacity and permeability values during 2015 for bovine corneas treated with the respective negative control. When testing solids the negative control limit for opacity should be ≤ 5.4 and for permeability ≤ 0.070.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

Mean

Value:

0.9

Vehicle controls validity:

valid

Remarks:

IVIS score 2.0

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks:

IVIS score 95.9

Other effects / acceptance of results:

CORNEAL EPITHELIUM CONDITION
- The corneas treated with the test material were clear post treatment.
- The corneas treated with the negative control were clear post treatment.
- The corneas treated with the positive control were cloudy post treatment.

ACCEPTANCE OF RESULTS:
- The positive control In Vitro Irritancy Score was within the range of 50.8 to 100.4. The positive control acceptance criterion was therefore satisfied.
- The negative control gave opacity of ≤ 5.4 and permeability ≤ 0.070. The negative control acceptance criteria were therefore satisfied.

Any other information on results incl. tables

Table 1: Summary of opacity, permeability and in vitro scores

Treatment	Mean Opacity	Mean Permeability	Mean In vitro Irritation Score*
Negative control	1.7	0.025	2.0
Positive control	65.3	2.035	95.9
Test material	0.6	0.022	0.9

*Calculated using the negative control mean opacity and mean permeability values for the positive control and test material.

In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value)

Applicant's summary and conclusion

Interpretation of results:

other: Not classified in accordance with EU criteria

Conclusions:

Under the conditions of this study, as the test material induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.

Executive summary:

The hazard potential of the test material to the eye was evaluated in vitro in accordance with the standardised guidelines OECD 437 and EU Method B.47 under GLP conditions using the Bovine Corneal Opacity and Permeability test (BCOP test).

The test material was applied at a concentration of 20 % w/v in sodium chloride 0.9 % w/v for 240 minutes. Negative and positive control materials 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 material did not induce ocular irritation through both endpoints (opacity and permeability), resulting in a mean in vitro irritancy score of 0.9 after 240 minutes of treatment.

The IVIS for the negative control was 2; the IVIS for the positive control was 95.9.

The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control was 95.9 and within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

Under the conditions of this study, as the test material induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.