(Z)-α-[[2-(tert-butoxy)-1,1-dimethyl-2-oxoethoxy]imino]-2-(tritylamino)thiazol-4-acetic acid

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 December 2017 to 14 December 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

(Z)-α-[[2-(tert-butoxy)-1,1-dimethyl-2-oxoethoxy]imino]-2-(tritylamino)thiazol-4-acetic acid

EC Number:

272-056-6

EC Name:

(Z)-α-[[2-(tert-butoxy)-1,1-dimethyl-2-oxoethoxy]imino]-2-(tritylamino)thiazol-4-acetic acid

Cas Number:

68672-66-2

Molecular formula:

C32H33N3O5S

IUPAC Name:

(2Z)-2-({[1-(tert-butoxy)-2-methyl-1-oxopropan-2-yl]oxy}imino)-2-{2-[(triphenylmethyl)amino]-1,3-thiazol-4-yl}acetic acid

Test material form:

solid: particulate/powder

Details on test material:

white powder

Specific details on test material used for the study:

Batch: 14TATT-S01025C
Purity: 100.1% w/w (HPLC Analysis)
Physical state/Appearance: White powder
Expiry Date: 24 December 2017
Storage Conditions: Room temperature in the dark over silica gel

Test animals / tissue source

Species:

cattle

Strain:

not specified

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:

not specified

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

20% w/v in sodium chloride 0.9% w/v

Duration of treatment / exposure:

240 minutes

Duration of post- treatment incubation (in vitro):

240 minutes

Number of animals or in vitro replicates:

3

Details on study design:

SELECTION AND PREPARATION OF CORNEAS :
Selection of Corneas and Opacity Reading
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. Three corneas were randomly allocated to the negative control. Three corneas were also allocated to the test item and three corneas to the positive control item.

Treatment of Corneas
The EMEM 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 3 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. The negative and positive control data was shared with Envigo - study number VB92TC.

NUMBER OF REPLICATES : 3

NEGATIVE CONTROL USED : Sodium chloride 0.9% w/v

POSITIVE CONTROL USED : Imidazole

APPLICATION DOSE AND EXPOSURE TIME : The test item was applied at a concentration of 20% w/v in sodium chloride 0.9% w/v for 240 minutes.

TREATMENT METHOD: chamber

POST-INCUBATION PERIOD: yes/no. 240 minutes.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period:
- POST-EXPOSURE INCUBATION:

METHODS FOR MEASURED ENDPOINTS:
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 ºC for 90 minutes.

Permeability Determinations
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-labeled 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.

Histopathology
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. No histopathology was required for this study.

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 OD 492 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 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.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
IVIS                                                 UN GHS                             EU CLP (Regulation (EC) No 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

Visual Observation
The condition of the cornea was visually assessed post treatment.

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 2016 for this testing facility. Therefore the In Vitro Irritancy Score should fall within the range of 65.1 to 123.3. 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 2016 for bovine corneas treated with the respective negative control. When testing solids the negative control limit for opacity should be ≤2.4 and for permeability ≤0.072.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

in vitro irritation score

Run / experiment:

Test Item

Value:

ca. 0.1

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 2

Irritation parameter:

in vitro irritation score

Run / experiment:

Negative Control

Value:

ca. 1.4

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 3

Irritation parameter:

in vitro irritation score

Run / experiment:

Positive Control

Value:

ca. 110.8

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

No category. Not requiring classification to UN GHS or EU CLP.

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 sodium chloride 0.9% w/v 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).  The negative and positive control data was shared with Envigo - study number VB92TC.

Data Interpretation

The test item is classified according to the prediction model as follows:

IVIS                                                 UN GHS                            EU CLP (Regulation (EC) No 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: