4,4'-oxydianiline

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 February 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

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)

Principles of method if other than guideline:

A study was performed to assess the ocular irritancy potential of the test material to the isolated bovine cornea. The neat test material was applied for 240 minutes. Negative and positive control materials 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 (MS).

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

4,4'-oxydianiline

EC Number:

202-977-0

EC Name:

4,4'-oxydianiline

Cas Number:

101-80-4

Molecular formula:

C12H12N2O

IUPAC Name:

4,4'-oxydianiline

Test material form:

solid: crystalline

Remarks:

white

Specific details on test material used for the study:

4,4'-oxydianiline
4,4'-diaminodiphenyl ether
CAS 101-80-4

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

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 corneas were prepared immediately on arrival.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

not required

Amount / concentration applied:

350 mg of the neat material was applied directly to cornea.

Duration of treatment / exposure:

240 minutes

Observation period (in vivo):

None - observed at end of treatment process.

Number of animals or in vitro replicates:

None - in vitro test. 3 bovine eyes were used for the test.

Details on study design:

4.1 – Pre-Test Procedures

4.1.1 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 corneas were prepared immediately on arrival.

4.1.2 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 at least 60 minutes. At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.

4.1.3 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 material. Three corneas were also numerically allocated to the negative control material and three corneas to the positive control material.

4.2 Treatment of Corneas

The MEM was removed from the anterior chamber of the BCOP holder. The window-locking ring and glass window from the anterior chamber were removed and the neat test material was evenly sprinkled over the cornea. 350 mg of the neat test material was found to adequately cover the cornea surface. After dosing, the glass window and locking ring were replaced and each holder was incubated, anterior chamber uppermost, at 32 +/- 1°C for 240 minutes.

At the end of the exposure period the window-locking ring and glass window were removed to allow the test material preparation or control material to be removed from the anterior chamber. 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.

4.3 Application of Sodium Fluorescein and Permeability Determinations

4.3.1 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 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 +/- 5 minutes.

4.3.2 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 1.500 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.

4.4 Histopathology

The corneas were retained after testing for possible conduct of histopathology. Each cornea was placed into a pre-labelled 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

Value:

1.6

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

EVALUATION OF RESULTS

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 from each 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 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 material induced a response through only one of the two endpoints.

Visual Observation

The condition of the cornea was visually assessed immediately after rinsing and at the final opacity measurement.

DATA INTERPRETATION

A test material that induces an in vitro irritancy score >55.1 is defined as an ocular corrosive or severe irritant.

In Vitro Irritancy Score

The results are summarised as follows:

Treatment In Vitro Irritancy Score

Test Material 1.6
Negative Control 1.8
Positive Control 95.4

CONCLUSION

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

Any other information on results incl. tables

Individual and Mean Corneal Opacity and Permeability Results

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	2	4	2	-	0.025	-	-
	2	1	2	1	-	0.042	-	-
	3	2	3	1	-	0.025	-	-
Mean		-	-	1.3*	-	0.031	-	1.8
Positive Control	7	2	75	73	71.7	2.285	2.254	-
	8	1	62	61	59.7	1.925	1.894	-
	9	1	62	61	59.7	2.220	2.189	-
Mean	-	-	-	-	63.7.	-	2.113*	95.4
Test Material	4	1		2	0.7	0.027	0.0	-
	5	2	5	3	1.7	0.059	0.026	-
	6	2	5	3	1.7	0.067	0.036	-
Mean	-	-	-	-	1.4.		0.021*	1.6

OD = Optical density

* = Mean of the post treatment - pre-treatment values                • = Mean corrected value Corneal Epithilium Opacity

Treatment	Cornea Number	Observation
		Immediately after Rinsing
Negative Control	1	clear
	2	clear
	3	clear
Positive Control	7	cloudy
	8	cloudy
	9	cloudy
Test Material	4	clear
	5	clear
	6	clear

Applicant's summary and conclusion

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

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

Executive summary:

A study was performed to assess the ocular corrosivity potential of the test material to the isolated bovine cornea. This study was conducted according to the OECD guideline 437 "Bovine Corneal opacity and Permeability" (BCOP).

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