estr-4-ene-3,11,17-trione cyclic 3-(1,2-ethanediylmercaptole)

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: Study performed at a registered GLP site and reported a high standard andis in compliance with recognised OECD standards with no deviations.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

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)

Deviations:

no

Principles of method if other than guideline:

No analysis was carried out to determine the homogeneity, concentration or stability of the test item formulation. The test item was formulated within two hours of being applied to the test system; it is assumed that the formulation was stable for this duration. This is an exception with regard to GLP, but is considered not to affect the purpose or integrity of the study.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

11-KN

IUPAC Name:

11-KN

Constituent 2

Reference substance name:

estr-4-ene-3,11,17-trione cyclic 3-(1,2-ethanediylmercaptole)

Molecular formula:

C20H26O2S2

IUPAC Name:

estr-4-ene-3,11,17-trione cyclic 3-(1,2-ethanediylmercaptole)

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): 11-KN
- Physical state: White powder
- Lot/batch No.: CGG238K1
- Expiration date of the lot/batch: 12 February 2014
- Storage condition of test material: room temperature in the dark

Test animals / tissue source

Species:

other: Bovine Eyes

Strain:

not specified

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
Source of Bovine Eyes
Eyes from adult cattle were obtained from a local abattoir as a by-product from freshly slaughtered animals. The eyes were excised by an abattoir employee and placed in Hanks' Balanced Salt Solution (HBSS), supplemented with Penicillin/Streptomycin, and transported to the laboratory on ice packs. The eyes were refrigerated on arrival and used within 24 hours of receipt.

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 (epithelial side uppermost) 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 minimum essential medium (MEM) 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.

Selection of Corneas and Opacity Reading
The medium from both chambers of each holder was replaced with fresh complete MEM.
A pre-treatment opacity reading was taken for each cornea using a calibrated opacitometer. The average opacity for all corneas was calculated.
Three corneas were numerically allocated to the test item. Three corneas were also numerically allocated to the negative control item and three corneas to the positive control item.

Test system

Vehicle:

other: 20% w/v dilution in 0.9% w/v sodium chloride solution.

Controls:

other: Negative Control and Positive Control (20% w/v Imidazole)

Amount / concentration applied:

TEST MATERIAL
- Concentration : 20% w/v

VEHICLE
- Concentration : 0.9% w/v sodium chloride solution.

Duration of treatment / exposure:

240 minutes Treatment

Observation period (in vivo):

Initial post treatment observations to assess Opacity.
Further 90 minutes for Permeability assessement

Number of animals or in vitro replicates:

number of corneas - 9

Details on study design:

REMOVAL OF TEST SUBSTANCE
Treatment of Corneas
The MEM was removed from the anterior chamber of the BCOP holder and the test item preparation or control items were applied to the cornea. The holders were gently tilted back and forth to ensure a uniform application of the item over the entire cornea. Each holder was incubated, anterior chamber uppermost, at 32 ±1°C for 240 minutes.
At the end of the exposure period the test item preparation and control items were removed from the anterior chamber and the cornea was rinsed three times with fresh complete MEM containing phenol red before a final rinse with complete MEM. The anterior and posterior chambers were refilled with fresh complete MEM. A post-treatment opacity reading was taken and each cornea was visually observed.
Application of Sodium Fluorescein
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

Permeability Determinations
After incubation the medium in the posterior chamber of each holder was decanted and retained.
360 µL of medium representing each cornea was applied to a designated well on a 96-well plate and the optical density at 492nm (OD492) was measured.
If values greater than 1500 OD₄₉₂ were obtained a 1 in 5 dilution of the medium in complete MEM was performed and the measurement repeated. The modified value was multiplied by 5 to reflect the 1 in 5 dilution.


SCORING SYSTEM:
Results from the two test method endpoints, opacity and permeability, were combined in an empirically derived formula to generate an In Vitro Irritancy Score.
Opacity Measurement
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.
Permeability Measurement
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.
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 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.

DATA INTERPRETATION
A test item that induces an In Vitro Irritancy Score 255.1 is defined as an ocular corrosive or severe irritant.
Criterion for an Acceptable Test
For an acceptable test the following positive control criterion must be achieved:
200% 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 for this testing facility. Therefore the In Vitro Irritancy Score should fall within the range of 55.8 to 126.1.

Results and discussion

In vivo

Results

Irritation parameter:

other: In Vitro Irritancy Score

Basis:

mean

Score:

5.7

Irritant / corrosive response data:

Treatment                    In Vitro Irritancy Score
Test Item                              5.7
Negative Control                3.3
Positive Control                  94.7

Individual and mean corneal opacity measurements and individual and mean corneal permeability measurements are given in the table below entitled Individual and Mean Corneal Opacity and Permeability Measurements.

Criterion for an Acceptable Test
The positive control In Vitro Irritancy Score was within the range 55.8 to 126.1. The positive control acceptance criterion was therefore satisfied.

Other effects:

Corneal Epithelium Condition
The condition of each cornea post treatment is given in the table below entitled Corneal Epithelium Condition Post Treatment.
The corneas treated with the test item were slightly cloudy 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.

Any other information on results incl. tables

Individual and Mean Corneal Opacity and Permeability Measurements

Treatment	Cornea Number	Opacity				Permeability (OD)		In vitro Irritancy Score
		Pre-treatment	Post-treatment	Post-treatment - Pre-treatment	Corrected Value		Corrected Value
Negative Control	1	1	4	3		0.015
	2	3	6	3		0.011
	3	2	5	3		0.024
	mean			3.0		0.017		3.3
Positive Control	4	3	68	65	62.0	2.48	2.463
	5	2	48	46	43.0	2.315	2.298
	6	3	73	70	67.0	2.72	2.703
	mean				57.3		2.488	94.7
Test Item	7	2	12	10	7.0	0.069	0.052
	8	2	10	8	5.0	0.031	0.014
	9	2	9	7	4.0	0.025	0.008
	mean				5.3		0.025	5.7

Corneal Epithelium Condition Post Treatment

Treatment	Cornea Number	Observation Post Treatment
Negative control	1	clear
	2	clear
	3	clear
Positive Control	4	cloudy
	5	cloudy
	6	cloudy
Test Item	7	slightly cloudy
	8	slightly cloudy
	9	slightly cloudy

Applicant's summary and conclusion

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The test item was considered not to be an ocular corrosive or severe irritant.

Executive summary:

The test item was applied at a concentration of 20% w/v in 0.9% w/v sodium chloride solution for 240 minutes. Negative and positive control items 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).

A test item that induces an In Vitro Irritancy Score ≥55.1 is defined as an ocular corrosive or severe irritant.

The in vitro Irritancy scores are summarised as follows:

Treatment                    In Vitro Irritancy Score

Test Item                            5.7

Negative Control                3.3

Positive Control                  94.7

Therefore the test item was considered not to be an ocular corrosive or severe irritant.