4-[bis(phenylsubstituted)substituted]phthalic acid

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date 05 October 2017. Experimental completion date 05 October 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

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:

yes

Remarks:

The positive control group had an overall IVIS of 124.2, which was marginally higher than the criteria range set for an acceptable test. This deviation was considered to have not affected the integrity or validity of the study.

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:

yes

Remarks:

See entry for OECD Guideline 437

GLP compliance:

yes (incl. QA statement)

Test material

Reference

Name:

Unnamed

Type:

Constituent

Test material form:

solid: particulate/powder

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:

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

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

0.75 mL of test item preparation or control items were applied to the appropriate corneas.

Duration of treatment / exposure:

240 minutes

Number of animals or in vitro replicates:

Three corneas for test item.
Three corneas for positive control.
Three corneas for negative control.

Details on study design:

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 ºC 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 EMEM.

A pre-treatment opacity reading was taken for each cornea using a calibrated opacitometer. The average opacity for all corneas was calculated.
Calibtation of the opacitometer was considered to be acceptable.

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 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.

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.

Data Evlaution:
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 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.

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

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Value:

19.6

Negative controls validity:

valid

Remarks:

IVIS: 1.4

Positive controls validity:

valid

Remarks:

IVIS: 124.2

Remarks on result:

other:

Remarks:

No prediction on eye irritation can be made

Other effects / acceptance of results:

Corneal Opacity and Permeability Measurement:
Individual and mean corneal opacity measurements and individual and mean corneal permeability measurements are given in the table 'Individual and Mean Corneal Opacity and Permeability Measurements' (see any other information on results incl tables section).

Corneal Epithelium Condition:
The corneas treated with the test item were 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.

In Vitro Irritancy Score:
The In Vitro irritancy scores are summarised as follows:
Test Item: 19.6
Negative Control: 1.4
Positive Control: 124.2

Criteria for an Acceptable Test
The positive control In Vitro Irritancy Score was above the range of 65.1 to 123.3. The positive control acceptance criterion was therefore not satisfied. This is reported as a deviation (see below):

The negative control gave opacity of ≤2.4 and permeability ≤0.072. The negative control acceptance criteria were therefore satisfied.

The following deviation from the Study Plan occurred:
The positive control group had an overall IVIS of 124.2, which was marginally higher than the criteria range set for an acceptable test. As the score was only
marginally exceeded, it was decided that the result was acceptable as the positive control group still provided its intended function, which was to show the sensitivity
of the test system to a known ocular irritant.
This deviation was considered to have not affected the integrity or validity of the study.

Any other information on results incl. tables

Individual and Mean Corneal Opacity and Permeability Measurements:

Treatment	Cornea Number	Opacity				Permeability (OD)		In VitroIrritancy Score
		Pre-Treatment	Post-treatment	Post-treatment – Pre-treatment	Correct Value	-	Corrected Value
Negative Control	1	4	4	0	-	0.019	-	-
	2	3	6	3	-	0.027	-	-
	3	5	4	0	-	0.025	-	-
	-	-	-	1.0*	-	0.024^a	-	1.4
Positive Control	4	4	98	94	93.0	2.260	2.236	-
	5	3	93	90	89.0	2.910	2.886	-
	6	3	83	80	79.0	2.335	2.311	-
	-	-	-	-	87.0^b	-	2.478^b	124.2
Test Item	7	3	37	34	33.0	0.071	0.047	-
	8	3	16	13	12.0	0.027	0.003	-
	9	5	19	14	13.0	0.024	0.000	-
	-	-	-	-	19.3^b	-	0.017^b	19.6

OD = optical density

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

a = Mean permeability

b = Mean correct 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 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).

Data Interpretation:

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

IVIS	Classification
≤ 3	No category. Not requiring classification to UN GHS or EU CLP
> 3; ≤55	No prediction of eye irritation can be made
> 55	Category 1. UN GHS or EU CLP Causes serious eye damage

Results:

The In Vitro Irritancy scores are summarised as follows:

Treatment	In Vitro Irritancy Score
Test Item	19.6
Negative Control	1.4
Positive Control	124.2

Conclusion:

No prediction of eye irritation can be made.