ethyl 2-[[(1R,2S,5R)-5-methyl-2-propan-2-ylcyclohexanecarbonyl]amino]acetate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01 August 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

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 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)

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

ethyl 2-{[(1R,2S,5R)-5-methyl-2-(propan-2-yl)cyclohexyl]formamido}acetate

EC Number:

695-735-2

Cas Number:

68489-14-5

Molecular formula:

C15H27NO3

IUPAC Name:

ethyl 2-{[(1R,2S,5R)-5-methyl-2-(propan-2-yl)cyclohexyl]formamido}acetate

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and batch No.of test material: WS-5, 83100003
- Expiration date of the lot/batch: 12 September 2018
- Purity test date: 99.76%
- Physical state/Appearance: white solid

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature in the dark

Test animals / tissue source

Species:

other: Eyes from adult cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Source of Bovine Eyes
- Source: adult cattle
- Age: typically 12 to 60 months old

Conditions:
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). They were transported to the test facility over ice packs on the same day of slaughter. The corneas were prepared immediately on arrival.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amounts applied: Approximately 0.53 g of the solid test item, WS-5

Duration of treatment / exposure:

240 minutes

Duration of post- treatment incubation (in vitro):

The condition of the cornea was visually assessed post- treatment

Number of animals or in vitro replicates:

3 in vitro replicates

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.

QUALITY CHECK OF THE ISOLATED CORNEAS : 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.

NUMBER OF REPLICATES : 3 replicates

NEGATIVE CONTROL USED : Three corneas with opacity values close to the median value of all corneas were allocated to the negative control. Sodium chloride 0.9% w/v was used for negative control purposes.

SOLVENT CONTROL USED (if applicable) - no

POSITIVE CONTROL USED : Three corneas were allocated to the positive control item. 20% w/v Imidazole was used for positive control purposes.

APPLICATION DOSE AND EXPOSURE TIME : Approximately 0.53 g of the solid test item, WS-5 was found to adequately cover the corneal surface. 0.75 ml of each negative and positive control item was 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 uppermost, at 32 ± 1 ºC for 240 minutes.

TREATMENT METHOD: anterior chamber

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: pre treatment opacity reading was taken for each cornea using a calibrated opacitometer. The average opacity for all corneas was calculated. 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 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. The optical density was measured (quantitative viability analysis) at 492 nm (without a reference filter) using the Labtech LT-4500 microplate reader. 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.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS) . 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 item induced a response through only one of the two endpoints.

DECISION 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:

3

Value:

ca. 0

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control gave opacity of ≤5.4 and permeability ≤0.070. The negative control acceptance criteria were therefore satisfied.
- Acceptance criteria met for positive control: 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.

Any other information on results incl. tables

Table 1. The results of the In Vitro irritancy scores (IVI):

Treatment	In Vitro Irritancy Score
Test Item	0.0
Negative Control	2.4
Positive Control	73.9

Applicant's summary and conclusion

Interpretation of results:

other: Not requiring classification according to EU CLP

Conclusions:

Based on available data on eye irritaiton performed according the OECD TG 437, WS-5 is not classified for eye irritation according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.

Executive summary:

The purpose of The Bovine Corneal Opacity and Permeability (BCOP) testing performed according to OECD TG 437, was to identify, whether WS-5 can induce a serious eye damage. In this test method, damage by the test item is assessed by quantitative measurements of changes in corneal opacity and permeability.

Eyes from adult cattle (typically 12 to 60 months old) were obtained from a local abattoir as a by-product from freshly slaughtered animals.

All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used.

The corneas treated with the test item were clear post treatment. The corneas treated with the negative control item were clear post treatment. The corneas treated with the positive control item were cloudy post treatment.

Results from the two test method endpoints, opacity and permeability, were combined in an empirically derived formula to generate anIn Vitro Irritancy Score. Additionally, the opacity and permeability values were evaluated independently to determine whether the test item induced a response through only one of the two endpoints.

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.

Based on the In Vitro irritancy score (IVIS), no category is applied for WS-5. Therefore, WS-5 is not classified as eye irritant according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.