Benzoic acid, 2-([1,1'-biphenyl]-4-ylcarbonyl)-

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From April 02, 2014 to May 26, 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

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:

yes

Remarks:

see 'Any other information on materials and methods'

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

2-{[1,1'-biphenyl]-4-carbonyl}benzoic acid

EC Number:

700-862-4

Cas Number:

42797-18-2

Molecular formula:

C20 H14 O3

IUPAC Name:

2-{[1,1'-biphenyl]-4-carbonyl}benzoic acid

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material (as cited in study report): 2-(4Phenylbenzoyl)benzoic acid
- Physical state: white powder
- Analytical purity: 99.9%
- Lot/batch No.: 5307
- Expiration date of the lot/batch: 01 May 2015
- Storage condition of test material: room temperature in the dark

Specific details on test material used for the study:

Batch No.: 5307; Purity: 99.9%; Appearance: white powder

Test animals / tissue source

Species:

cattle

Strain:

other:

Remarks:

in vitro bovine cornea study

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 refrigerated on arrival and used within 24 h of receipt.

Test system

Vehicle:

physiological saline

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

For the purpose of this study the test substance was prepared as a 20% w/v solution in 0.9% w/v sodium chloride solution. The test substance was formulated within two hours of being applied to the test system. It is assumed that the formulation was stable for this duration.

Duration of treatment / exposure:

240 min

Duration of post- treatment incubation (in vitro):

First incubation, with the test substance for 240 min. Subsequently a post treatment opacity reading was taken and each cornea was visually observed.
Second incubation: 1 mL of sodium fluorescein solution (5 mg/mL) at 32 ± 1ºC for 90 min.

Number of animals or in vitro replicates:

Three corneas were allocated to the negative control. Three corneas were also allocated to the test substance and three corneas to the positive control substance.

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 (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 Eagle’s minimum essential medium (MEM) and plugged. The holders were incubated at 32 ± 1ºC 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 (Appendix 1 in tab for other information). The average opacity for all corneas was calculated.
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 substance and three corneas to the positive control substance.

Treatment of Corneas
The MEM was removed from the anterior chamber of the BCOP holder and 0.75 mL of the test substance preparation or control substance were applied to the appropriate corneas. The holders were gently tilted back and forth to ensure a uniform application of the substance over the entire cornea. Each holder was incubated, anterior chamber uppermost, at 32 ± 1ºC for 240 minutes.
At the end of the exposure period the test substance preparation and control substances 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 ± 1ºC for 90 min.

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

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

The two endpoints opacity and permeability were combined in an empirically derived formula to generate an in vitro irritancy score (IVIS)

Value:

3.1

Negative controls validity:

valid

Remarks:

in vitro irritation score: 2.5

Positive controls validity:

valid

Remarks:

in vitro irritation score: 83.8

Remarks on result:

other: The result of this study has identified the test substance has not causing serious eye damage, but they do not permit conclusion that the test substance does not require classification for eye irritation.

Any other information on results incl. tables

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

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

Acceptance criteria:

The positive control In Vitro Irritancy Score was within the range of 73.8 – 103.6. The positive control acceptance criterion was therefore satisfied.

The negative control gave opacity of ≤4.7 and permeability ≤0.085. The negative control acceptance criteria were therefore satisfied.

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Conclusions:

Under the study conditions, although the test substance did not cause serious eye damage to the bovine corneas, based on the results no prediction no prediction could be made about eye irritation potential of the test substance

Executive summary:

An ex vivo study was conducted to determine the corneal damage potential of the test substance according to OECD Guideline 437, in compliance with GLP. In this in vitro method, damage is assessed by quantitative measurements of changes in corneal opacity and permeability. Bovine corneas were collected from slaughtered cattle which were between 12 and 60 months old. One valid experiment was performed with three replicates for negative control, positive control and the test substance. 0.75 mL of the test substance was applied at the concentration of 20 % w/v in 0.9 % w/v sodium chloride solute in a way that as much as possible of the corneas surface was covered. Subsequently, corneas were incubated for 240 min at 32 ± 1°C in a corneal holder. At the end of the exposure period, the test and control substances were removed from the anterior chamber and the corneas were 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. Following the opacity measurement, the permeability of the corneas to sodium fluorescein was evaluated. The medium was replaced with 1 mL of sodium fluorescein solution (5 mg/mL) and corneas were incubated again at 32 ± 1ºC for 90 min. After incubation 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 an Anthos 2001 microplate reader. The two endpoints opacity and permeability were combined in an empirically derived formula to generate an in vitro irritancy score (IVIS). The IVIS of the test substance was determined to be 3.1 which is well below the corrosive limit of 55 and above the non-corrosive limit of 3; therefore, no predictions could be made. Therefore, based on EU CLP criteria, no prediction could be made about eye irritation potential of the test substance. The IVIS of the positive control was within the range of 31.6 to 58.7, therefore acceptance criterion was satisfied. The negative control gave opacity of ≤3.0 and permeability ≤0.077, therefore the negative control acceptance criteria were satisfied. The quality criteria required for acceptance of results in the test were satisfied. Under the study conditions, although the test substance did not cause serious eye damage to the bovine corneas, based on the results no prediction no prediction could be made about eye irritation potential of the test substance (Warren, 2014).