5-(3-Phenylpropioloyl)-1,3-isobenzofurandione

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 February 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

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying Ocular Corrosives and Severe Irritants)

Version / remarks:

OECD Guidelines for Testing of Chemicals 438 (26th July 2013)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU method B.48 (Isolated chicken eye test method for identifying occular corrosives and severe irritants)

Version / remarks:

EU Commission Regulation (EC) No 1152/2010 (8th December 2010) amending, Regulation (EC) No 440/2008: Method B 48.

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2400 (Acute Eye Irritation)

Version / remarks:

OPPTS 870.2400 (EPA 712-C-98-195) August 1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

5-(3-phenylprop-2-ynoyl)-1,3-dihydro-2-benzofuran-1,3-dione

EC Number:

935-606-2

Cas Number:

1329658-14-1

Molecular formula:

C17H8O4

IUPAC Name:

5-(3-phenylprop-2-ynoyl)-1,3-dihydro-2-benzofuran-1,3-dione

Test material form:

solid: particulate/powder

Details on test material:

Short Name: PETA
Long Name: NNEXAMITE™ A56 (PETA)
Chemical Name: Phenylacetylene modified trimellitic anhydride
Lot Number: NEX-X61-A02
CAS Number: 1329658-14-1
Description: Yellow powder
Purity: >99 %
Molecular weight: 276.24
Manufacture date: 04 September 2013
Expiry date: 01 May 2014
Storage conditions: Room temperature (15-25 °C), protected from humidity
Safety Precautions: Routine safety precautions (gloves, goggles, face mask, lab coat) for unknown materials were applied to assure personnel health and safety

Specific details on test material used for the study:

No further details specified in the study report.

Test animals / tissue source

Species:

chicken

Strain:

other: COBB 500

Details on test animals or tissues and environmental conditions:

Strain of chicken: COBB 500
Source: TARAVIS KFT. 9600 Sárvár, Rábasömjéni út. 129.
Chicken heads were collected after slaughter in a commercial abattoir from chickens which are used for human consumption. Heads were collected by a slaughter house technician and heads transported to CiToxLAB Hungary Ltd. at ambient temperature at the earliest convenience.
After collection, the heads were inspected for appropriate quality and wrapped with tissue paper moistened with saline, then placed in a plastic box which was closed (4-5 heads per box). The heads were transported to CiToxLAB Hungary Ltd. and received into the laboratory for use within 2 hours of collection.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

The test item was applied in an amount of 30 mg onto the entire surface of the cornea.

Duration of treatment / exposure:

An exposure period of 10 seconds from the end of the application the cornea surface.

Duration of post- treatment incubation (in vitro):

The control eyes and test eyes were evaluated pre-treatment and at approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse.

Number of animals or in vitro replicates:

3 replicates per test group

Details on study design:

SELECTION AND PREPARATION OF EYES FOR THE TEST
Eyes selection
After removing the head from the plastic box, it was put on soft paper. The eyelids were carefully cut away with scissors, avoiding damaging the cornea. One small drop of fluorescein solution 2 % (w/v) was applied onto the cornea surface for a few seconds and subsequently rinsed off with 20 mL isotonic saline. Then the fluorescein-treated cornea was examined with a hand-held slit lamp or slit lamp microscope, with the eye in the head, to ensure that the cornea was not damaged. If the cornea was in good condition, the eyeball was carefully removed from the orbit.

Preparation of eyes
The eye ball was carefully removed from the orbit by holding the nictitating membrane with surgical forceps, while cutting the eye muscles with bent scissors. Care was taken to remove the eyeball from the orbit without cutting off the optical nerve too short. The procedure avoided pressure on the eye while removing the eyeball from the orbit, in order to prevent distortion of the cornea and subsequent corneal opacity. Once removed from the orbit, the eye was placed onto damp paper and the nictitating membrane was cut away with other connective tissue. The prepared eyes were kept on the wet papers in a closed box so that the appropriate humidity was maintained.

Eyes examination and acclimatization time
The prepared eye was placed in a steel clamp with the cornea positioned vertically with the eye in the correct relative position (same position as in the chicken head). Again avoiding too much pressure on the eye by the clamp. Because of the relatively firm sclera of the chicken eyeball, only slight pressure was needed to fix the eye properly. The clamp with the eyeball was transferred to a chamber of the superfusion apparatus. The clamp holding the eye was positioned in such a way that the entire cornea was supplied with isotonic saline dripping from a stainless steel tube, at a rate of approximately 3-5 drops/minute. The door of the chamber was closed except for manipulations and examinations, to maintain temperature and humidity.
The appropriate number of eyes was selected and after being placed in the superfusion apparatus, were examined again with the slit lamp microscope to ensure that they were in good condition. The focus was adjusted to see clearly the isotonic saline which was flowing on the cornea surface. Eyes with a high baseline fluorescein staining (i.e., > 0.5) or corneal opacity score (i.e., > 0.5) were rejected. The cornea thickness was measured, any eye with cornea thickness deviating more than 10 % from the mean value for all eyes, or eyes that showed any other signs of damage, were rejected and replaced. If the selected eyes were appropriate for the test, acclimatization started and it was conducted for approximately 45 to 60 minutes. The chambers of the superfusion apparatus were at controlled temperature (32±1.5°C) during the acclimatization and treatment periods.

Identification
The eyes were identified by chamber number, marked on the door of each chamber.

THE BASE LINE ASSESSMENTS
The base line assessments
At the end of the equilibration period, a zero reference measurement was recorded for cornea thickness and opacity to serve as a base line (t=0) for each individual eye. The cornea thickness of the eyes should not change by more than 5% or -7% between the -45 min and the zero time. No changes in thickness were observed in the eyes. Following the equilibration period, the fluorescein retention was measured. Base line values were required to evaluate any potential test item related effect after treatment. All eyes were considered to be suitable for the assay.

TEST PROCEDURE
Treatment
After the zero reference measurements, the eye (in its retainer) was removed from the chamber, and placed on a layer of tissue with the cornea facing upwards. The eyes were held in horizontal position, while the test item was applied onto the cornea. The test item was applied in an amount of 30 mg onto the entire surface of the cornea attempting to cover the cornea surface uniformly with the test substance, taking care not to damage or touch the cornea.
The positive control eyes were treated in a similar way with 30 mg powdered Imidazole. The negative control eye was treated with 30μL of 0.9% w/v physiological saline (Salsol solution).
One eye was treated with isotonic saline, three eyes with the test item and another three with Imidazole in accordance with the current OECD guideline.

Test item removal
The time of application was observed and recorded, then after an exposure period of 10 seconds from the end of the application the cornea surface was rinsed thoroughly with 20 mL isotonic saline at ambient temperature, taking care not to damage the cornea but attempting to remove all residual test item if possible.
The test item and the Imidazole were adhered to the corneal surface after the post-treatment rinse. Gentle rinsing with 20 mL saline was performed and the rate of saline-drops was increased at each observation time point.
The test item and the Imidazole treated cornea surfaces were not cleared 240 minutes after the post-treatment rinse.

OBSERVATION
Observation and assessment of corneal effects
The control eyes and test eyes were evaluated pre-treatment and at approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse. Minor variations within approximately ±5 minutes were considered acceptable.
Corneal thickness and corneal opacity were measured at all time points. Fluorescein retention was measured on two occasions, at base line (t=0) and approximately 30 minutes after the post-treatment rinse. Haag-Streit Bern 900 slit-lamp microscope was used for the measurements.

Results and discussion

In vitro

Results

Irritation parameter:

percent corneal swelling

Run / experiment:

Test Item

Value:

1.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Based on the in vitro eye irritation assay in the isolated chicken eyes test with NEXAMITETM A56 (PETA), the test item is not classified as a severe irritant and not classified as non-irritant. No conclusion of in vivo significance can be made from the adherence of the test item to the cornea, since in vivo eye lids will probably clear the surface, but abrasion may occur. It is concluded that further information is required for classification.

Validity of the test: The results from all eyes used met the quality control standards. The negative control and positive control results were in line with historic data. This experiment was considered to be valid.

Any other information on results incl. tables

Test Item

Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	1.8%	I
Mean maximum corneal swelling at up to 240 min	1.8%	I
Mean maximum corneal opacity	0.33	I
Mean fluorescein retention	1.67	III
Other observations	The test Item was adhered to all corneal surfaces after the post-treatment rinse. The cornea surfaces were not cleared 240 minutes after the post-treatment rinse.
Overall ICE Class	2xI 1xIII

 

Positive Control

Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	3.5%	I
Mean maximum corneal swelling at up to 240 min	7.1%	II
Mean maximum corneal opacity	3.83	IV
Mean fluorescein retention	3.00	IV
Other observations	The Imidazole was adhered to all corneal surfaces after the post-treatment rinse. The cornea surfaces were not cleared 240 minutes after the post-treatment rinse.
Overall ICE Class	1xII 2xIV

 

Negative Control

Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	0.0%	I
Mean maximum corneal swelling at up to 240 min	0.0%	I
Mean maximum corneal opacity	0.00	I
Mean fluorescein retention	0.00	I
Other observations	None
Overall ICE Class	3xI

 

Historical Control data (n=17, data from 2013):

Negative Control: Physiological saline (Salsol solution, NaCl 0.9% w/v)

Observation	Min. Value	Max. Value
Maximum corneal swelling at up to 75 min	0.0%	1.2%
Maximum corneal swelling at up to 240 min	0.0%	1.2%
Maximum corneal opacity change	0.00	0.00
Fluorescein retention	0.00	0.00

Positive Control: (Imidazole)

Observation	Min. Value	Max. Value
Maximum corneal swelling at up to 75 min	1.1%	5.9%
Maximum corneal swelling at up to 240 min	4.6%	10.6%
Maximum corneal opacity change	3.50	4.00
Fluorescein retention	2.00	3.00

 

Table of individual date (NEXAMITE™ A56 (PETA))

Chamber number ↓	Corneal thickness (instrument units)										Corneal opacity score							Fluorescein retention
Relative observation time (min) →	-45	0	Change	30	75	Max change up to 75	120	180	240	Max change up to 240	0	30	75	120	180	240	Max ∆ Opac	0	30	∆ Flu ret
1	76	76	0.0%	78	78	2.6%	78	78	78	2.6%	0	0	0	0	1	1	1.0	0	2	2.0
2	76	76	0.0%	77	77	1.3%	77	77	77	1.3%	0	0	0	0	0	0	0.0	0	1	1.0
3	76	76	0.0%	77	77	1.3%	77	77	77	1.3%	0	0	0	0	0	0	0.0	0	2	2.0
Mean values:						1.8%				1.8%							0.33			1.67

Comment: The Test Item was adhered to all corneal surfaces after the post-treatment rinse. The cornea surfaces was not cleared 240 minutes after the post-treatment rinse.

 

Table of individual date (Imidazole)

Chamber number ↓	Corneal thickness (instrument units)										Corneal opacity score							Fluorescein retention
Relative observation time (min) →	-45	0	Change	30	75	Max change up to 75	120	180	240	Max change up to 240	0	30	75	120	180	240	Max ∆ Opac	0	30	∆ Flu ret
4	75	75	0.0%	76	78	4.0%	79	80	80	6.7%	0	4	4	4	4	4	4.0	0	3	3.0
5	76	76	0.0%	78	78	2.6%	80	81	82	7.9%	0.5	4	4	4	4	4	3.5	0	3	3.0
6	75	75	0.0%	76	78	4.0%	78	79	80	6.7%	0	4	4	4	4	4	4.0	0	3	3.0
Mean values:						3.5%				7.1%							3.83			3.00

Comment: The Imidazole adhered to all corneal surfaces after the post-treatment rinse. The cornea surfaces was not cleared 240 minutes after the post-treatment rinse.

 

Table of individual date (Saline (Salsol solution, NaCl 0.9% w/v))

Chamber number ↓	Corneal thickness (instrument units)										Corneal opacity score							Fluorescein retention
Relative observation time (min) →	-45	0	Change	30	75	Max change up to 75	120	180	240	Max change up to 240	0	30	75	120	180	240	Max ∆ Opac	0	30	∆ Flu ret
7	75	75	0.0%	75	75	0.0%	75	75	75	0.0%	0	0	0	0	0	0	0.00	0	0	0.00

 

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Conclusions:

Based on the in vitro eye irritation in the isolated chicken eyes test with NEXAMITETM A56 (PETA), the test item is not classified as a severe irritant and not classified as non-irritant. It is concluded that further information is required for definitive classification. On the basis of worst case hazard assessment, the substance is classified as eye irritation Category 2.

Executive summary:

An in vitro eye irritation study of the test item NEXAMITE™ A56 (PETA) was performed in isolated chicken’s eyes. The irritation effects of the test item were evaluated according to the OECD No.: 438 (26th July 2013).

 

After the zero reference measurements, the eye was held in horizontal position and 30 mg of NEXAMITETM A56 (PETA) was applied onto the centre of the cornea such that the entire surface of the cornea was covered. After 10 seconds, the surface was rinsed with saline. The positive control eyes were treated with 30 mg Imidazole. The negative control eye was treated with 30 μL of Saline (Salsol solution, NaCl 0.9% w/v).

 

Based on this in vitro eye irritation in the isolated chicken eyes test with NEXAMITE™ A56 (PETA), the test item is not classified as a severe irritant and not classified as non-irritant. It is concluded that further information is required for definitive classification. On the basis of worst case hazard assessment, the substance is classified as eye irritation Category 2.