Androsta-1,4-diene-17-carbothioic acid, 6,9-difluoro-11-hydroxy-16-methyl-3-oxo-17-(1-oxopropoxy)-, (6.alpha.,11.beta.,16.alpha.,17.alpha.)-

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19th March 2015 - 29th December 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

equivalent or similar 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

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

(6ALPHA,11BETA,16ALPHA,17ALPHA)-6,9-DIFLUORO-11-HYDRO XY-16-METHYL-3-OXO-17- (1-OXOPROPOXY)ANDROSTA-1,4-DIENE-CARBOTHIOIC ACID

EC Number:

617-084-5

Cas Number:

80474-45-9

Molecular formula:

C24H30F2O5S

IUPAC Name:

(6ALPHA,11BETA,16ALPHA,17ALPHA)-6,9-DIFLUORO-11-HYDRO XY-16-METHYL-3-OXO-17- (1-OXOPROPOXY)ANDROSTA-1,4-DIENE-CARBOTHIOIC ACID

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

Identification: Thio Acid Propionate
Batch: 0000135240
Purity: 97.76%
Physical state/Appearance: Off white solid
Expiry Date: 26 July 2016
Storage Conditions: 4 o
C in the dark, over silica gel

Test animals / tissue source

Species:

cattle

Details on test animals or tissues and environmental conditions:

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

Hank's balanced salt solution

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

For the purpose of this study the test item was prepared as a 20% w/v solution in 0.9% w/v sodium chloride solution.

The negative control item, 0.9% w/v sodium chloride solution, was used as supplied.

The positive control item, Imidazole, was used as a 20% w/v solution in 0.9% w/v sodium chloride solution.

Duration of treatment / exposure:

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.

Observation period (in vivo):

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

Duration of post- treatment incubation (in vitro):

The anterior and posterior chambers of each BCOP holder were filled with complete Eagle’s Minimum Essential Medium (MEM) 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 animals or in vitro replicates:

Three corneas with opacity values close to the median value of all corneas were allocated to the negative control. Three corneas were also allocated to the test item and three corneas to the positive control item.

Details on study design:

The MEM was removed from the anterior chamber of the BCOP holder and 0.75 mL of the test item preparation or control items were 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, anterior chamber uppermost, at 32 ± 1 ºC for 240 minutes.

At the end of the exposure period the test item 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 without phenol red. The anterior chamber was refilled with fresh complete MEM without phenol red. A post-treatment opacity reading was taken and each cornea was visually observed.

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 ºC for 90 minutes.

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 492 nm (OD492) was measured using the Anthos 2001 microplate reader.

The corneas were retained after testing for possible conduct of histopathology. Each cornea was placed into a pre-labeled tissue cassette fitted with a histology sponge to protect the endothelial surface. The cassette was immersed in 10% neutral buffered formalin.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

Test Item

Value:

ca. 34.7

Negative controls validity:

valid

Remarks:

Irritation score: 1.8

Positive controls validity:

valid

Remarks:

Irritation Score: 78.1

Other effects / acceptance of results:

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 for this testing facility. Therefore the In Vitro Irritancy Score should fall
within the range of 66.9 to 101.4.

For an acceptable test the following negative control criteria should be achieved:
0.9% w/v sodium chloride solution 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 2014 for bovine corneas treated with the respective negative control. When testing solids the negative control limit for opacity should be ≤4.1 and for permeability ≤0.105.

In vivo

Irritant / corrosive response data:

The In Vitro irritancy scores are summarized as follows:

Test Item In Vitro Irritancy Score: 34.7
Negative Control In Vitro Irritancy Score: 1.8
Positive Control In Vitro Irritancy Score: 78.1

The positive control In Vitro Irritancy Score was within the range of 66.9 to 101.4. The positive control acceptance criterion was therefore satisfied. The negative control gave opacity of ≤4.1 and permeability ≤0.105. The negative control acceptance criteria were therefore satisfied.

No prediction of eye irritation can be made.

Any other information on results incl. tables

Individual and Mean Corneal Opacity and Permeability Measurements:

Treatment	Cornea Number	Opacity Pre-treatment	Opacity Post-treatment	Opacity Post-treatment - Pre-treatment	Corrected Value	Permeability (OD)	Permeability (OD) Corrected Value	In Vitro Irritancy Score
Negative Conrol	18	2	3	1		0.006
	23	2	4	2		0.006
	26	2	4	2		0.012
				1.7*		0.008♦		1.8
Positive Control	5	3	49	56	44.3	1.344	1.336
	19	3	65	62	60.3	1.873	1.865
	22	3	63	60	58.3	1.562	1.554
					54.3•		1.585•	78.1
Test Item	24	3	11	8	6.3	1.851	1.843
	27	2	9	7	5.3	2.276	2.268
	28	2	8	6	4.3	1.778	1.770
					5.3•		1.960•	34.7

OD = Optical density

* = Mean of the post-treatment − pre-treatment values

♦ = Mean permeability

• = Mean corrected value

Applicant's summary and conclusion

Interpretation of results:

other: No prediction of eye irritation can be made.

Conclusions:

No prediction of eye irritation can be made

Executive summary:

Introduction:

The purpose of this test was to identify test items that can induce serious eye damage and to identify test items not requiring classification for eye irritation or serious eye damage. The Bovine Corneal Opacity and Permeability (BCOP) test method is an organotypic model that provides short-term maintenance of normal physiological and biochemical function of the bovine cornea in vitro. In this test method, damage by the test item is assessed by quantitative measurements of changes in corneal opacity and permeability.

The test method can correctly identify test items (both chemicals and mixtures) inducing serious eye damage as well as those not requiring classification for eye irritation or serious eye damage, as defined by the United Nations (UN) Globally Harmonized System of Classification and Labelling of Items (GHS) and EU Classification, Labelling and Packaging (CLP) of chemicals (Regulation (EC) No 1272/2008), and it was therefore endorsed as scientifically valid for both purposes. Test items inducing serious eye damage are classified as UN GHS and EU CLP Category 1. Items not classified for eye irritation or serious eye damage are defined as those that do not meet the requirements for classification as UN GHS/ EU CLP Category 1 or 2 (2A or 2B), i.e. they are referred to as UN GHS/EU CLP No Category.

Method:

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).

Interpretation:

The test item is classified according to the prediction model below:

IVIS	UN GHS	European Regulation (EC) 1272/2008
≤ 3	No Category	Not classified for irritation
>3; ≤ 55	No prediction can be made	No prediction can be made
> 55	Category 1	Category 1 H318: Causes serious eye damage

Results:

The In Vitro irritancy scores are summarized as follows:

Treatment	In Vitro Irritancy Score
Test Item	34.7
Negative Control	1.8
Positive Control	78.1

Conclusion:

No prediction of eye irritation can be made.