Benzene-1,3-dicarbonitrile

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 November 2019 - 20 January 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

GLP study to current guideline

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 438 (Isolated Chicken Eye 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.48 (Isolated chicken eye test method for identifying occular corrosives and severe irritants)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Benzene-1,3-dicarbonitrile

EC Number:

210-933-7

EC Name:

Benzene-1,3-dicarbonitrile

Cas Number:

626-17-5

Molecular formula:

C8H4N2

IUPAC Name:

benzene-1,3-dicarbonitrile

Test material form:

solid: flakes

Details on test material:

Test substance name: Isophthalonitrile
Chemical name: 1,3-dicyanobenzene
CAS number: 626-17-5
EC number: 210-933-7
LOT number: JB1906150101
Purity: 99.8 % (w/w %)
Appearance: Off white solid flakes
Manufacture date: 15 June 2019
Expiry date:15 June 2020
Storage condition: Room temperature (15-25oC. below 70 RH%), protected from humidity

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:

Remarks:

COBB 500 (1) and ROSS 308 (2)

Details on test animals or tissues and environmental conditions:

Strain of chicken: COBB 500 (1) and ROSS 308 (2)
Source: TARAVIS KFT. 9600 Sárvár, Rábasömjéni út 129. Hungary

Head collection was performed by a slaughter house technician. Heads were removed immediately after sedation of the chickens (sedation was performed by electric current). The heads were transported to Toxi-Coop ZRT. at the earliest convenience for use approximately within 2 hours from collection. The ambient temperature was optimal (19.3ºC to 20.4ºC in the first and 19.2ºC to 20.4ºC additional experiment) during the transport. All eyes used in the assay were from the same groups of eyes collected on one specific day.
After collection, the heads were inspected for appropriate quality and wrapped with paper moistened with saline, then placed in a plastic box that can be closed (4-5 heads/box).

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

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

other: Fluorescein sodium salt

Amount / concentration applied:

The test item and positive control applied in an amount of 0.03 g/eye by powdering the entire surface of the cornea attempting to cover the cornea surface uniformly with the test substance or positive control.

Duration of treatment / exposure:

10 seconds

Observation period (in vivo):

Not applicable for In-Vitro study

Duration of post- treatment incubation (in vitro):

240 minutes

Number of animals or in vitro replicates:

Three test item treated eyes, three positive control treated eyes and one negative control treated eye which was treated with 30 µL saline solution were used in the first and additional experiment.

Details on study design:

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 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 eyeball was carefully removed from the orbit by holding the nictitating membrane with surgical forceps, while cutting the eye muscles with bent scissors without cutting off the optical nerve too short. The procedure avoided pressure on the eye 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 applied 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 saline solution dripping from a stainless steel tube, at a rate of approximately 3 to 4 drops/minutes. The door of the chamber was closed except for manipulations and examinations, to maintain temperature and humidity.
The appropriate numbers of eyes were selected and after being placed in the superfusion apparatus, the selected eyes 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 using the depth measuring device on the slit lamp microscope (Haag-Streit BQ 900) with the slit-width set at 9½, equalling 0.095 mm. 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 was conducted for approximately 45 to 60 minutes. The temperature was verified to be in the range of 32 ± 1.5 °C in all chambers during the acclimatization and treatment periods.

The baseline assessments
At the end of the acclimatization period, a zero reference measurement was recorded for cornea thickness and opacity to serve as a baseline (t=0) for each individual eye. The cornea thickness of the eyes should not change by more than ±5-7 % within approximately 45 to 60 minutes before the start of application. Changes in thickness were not observed in the eyes during the first and additional experiment. Following the equilibration period, the fluorescein retention was measured. Baseline values were required to evaluate any potential test item related effects after treatment. The location of any minor findings was marked on the record sheet, if applicable. None of the eyes were discarded as no eye was considered unsuitable after the baseline assessment.

Test procedure

Treatment
After the zero reference measurements in each experiment, one out of three test item treated eyes was taken out of its chamber and placed on a layer of tissue with the cornea facing upwards. The eye was held in horizontal position over a container to catch waste, while the test item was applied onto the centre of the cornea. The test item was applied in an amount of 0.03 g by attempting to cover the cornea surface uniformly with the test substance, while taking care not to damage or touch the cornea with the application equipment. This procedure was repeated for each test item treated eye.
The three positive control eyes were treated in a similar way with 0.03 g Imidazole.
One negative control eye was treated with saline solution. The saline solution was applied in a volume of 30 µL from micropipette, in such a way that the entire surface of the cornea was covered with negative control, taking care not to damage or touch the cornea with the application equipment.

Test item removal
The time of application was monitored, then after an exposure period of 10 seconds from the end of the application the cornea surface was rinsed thoroughly with 20 mL saline solution at ambient temperature, while taking care not to damage the cornea but attempting to remove all the residual test item if possible. The eye in the holder was then returned to its chamber. The time while the eye was out of the chamber was limited to the minimum.

The Imidazole was stuck on the corneas’ surface in all eyes at 30 minutes after the post-treatment rinse. The gentle rinsing with 20 mL saline was performed in all Imidazole treated eyes after the 30, 75, 120 and 180 minutes of observation. The Imidazole treated cornea surfaces were not totally cleared at 240 minutes after the post-treatment rinse in the first and additional experiment.

Observation and assessment of corneal effects
The control 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 ±5 minutes were considered acceptable.

The cornea thickness and cornea opacity were measured at all time points. Fluorescein retention was determined at baseline (t=0) and 30 minutes after the post-treatment rinse.

Retention of corneas
At the end of the procedures, the corneas were carefully removed from the eyes and placed individually into labeled containers of preservative fluid (4% formaldehyde) for potential histopathology and stored.

Histopathology
A histopathology of the corneas was not performed. Corneas are discarded 2 months after the final report.

Demonstration of Proficiency
Prior to routine use of the method Toxi-Coop ZRT. demonstrated the technical proficiency in a separate study (study no.: 392.549.3229) using the ten Proficiency Chemicals according to OECD Test Guideline No. 438.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

cornea opacity score

Run / experiment:

Mean maximum corneal opacity

Value:

> 0.5 - < 1

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 2

Irritation parameter:

percent corneal swelling

Run / experiment:

Mean maximum corneal swelling at up to 75 min

Value:

> 3 - < 5

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 3

Irritation parameter:

percent corneal swelling

Run / experiment:

Mean maximum corneal swelling at up to 240 min

Value:

> 4 - < 5

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 4

Irritation parameter:

fluorescein retention score

Run / experiment:

Mean fluorescein retention

Value:

> 0.2 - < 0.5

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The negative control NaCl (9g/L saline) had no significant effects on the chicken eye in this study.
Positive and negative control values were within the corresponding historical control data ranges

Any other information on results incl. tables

The mean values of the treated eyes for maximum corneal thickness change, corneal opacity, fluorescein retention and other observation (morphological effect etc.) are given below.

 

Test Item: Isophthalonitrile

 

Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	3%	I
Mean maximum corneal swelling at up to 240 min	4%	I
Mean maximum corneal opacity	0.8	II
Mean fluorescein retention	0.2	I
Other Observations	None
Overall ICE Class	2xI, 1xII

 

Positive Control: Imidazole

 

Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	31%	IV
Mean maximum corneal swelling at up to 240 min	36%	IV
Mean maximum corneal opacity	3.8	IV
Mean fluorescein retention	3.0	IV
Other Observations	Corneal opacity score 4 was observed in three eyes eye at 30 minutes after the post-treatment rinse.
Overall ICE Class	3xIV

 

The positive control Acetic acid 10% (v/v) solution was classed as corrosive/severely irritating, UN GHS Classification: Category 1.

Negative Control: NaCl (9 g/L saline)

 

Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	0%	I
Mean maximum corneal swelling at up to 240 min	0%	I
Mean maximum corneal opacity	0.0	I
Mean fluorescein retention	0.0	I
Other Observations	None
Overall ICE Class	3xI

 

The negative control NaCl (9g/L saline) had no significant effects on the chicken eye in this study.

 

Additional experiment:

 

Test Item: Isophthalonitrile

 

Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	5%	I
Mean maximum corneal swelling at up to 240 min	5%	I
Mean maximum corneal opacity	1.0	II
Mean fluorescein retention	0.5	I
Other Observations	None
Overall ICE Class	2xI, 1xII

  

Positive Control: Imidazole

 

Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	30%	IV
Mean maximum corneal swelling at up to 240 min	36%	IV
Mean maximum corneal opacity	3.7	IV
Mean fluorescein retention	2.7	IV
Other Observations	Corneal opacity score 4 was observed in two eyes and score 3 was seen in one eye at 30 minutes after the post-treatment rinse.
Overall ICE Class	3xIV

The positive control Acetic acid 10% (v/v) solution was classed as corrosive/severely irritating, UN GHS Classification: Category 1.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In this ICET, Isophthalonitrile did not cause ocular corrosion or severe irritation in the enucleated chicken eyes in either cases. The overall ICE class was 2xI, 1xII in the first and the additional experiment.
The positive control was classed as corrosive/severely irritating, UN GHS Classification: Category 1 and the negative control had no significant effects on the chicken eye in this study. Furthermore, the three endpoints of the positive and the negative control were in the historical control range. So the positive and negative controls showed the expected results. The experiment was considered to be valid in both occasions.

In this in vitro eye irritation study, using the Isolated Chicken Eye model with Isophthalonitrile, no ocular corrosion or severe irritation potential was observed. The overall ICE class was 2xI, 1xII in the first and the additional experiment.
According to the guideline OECD 438, Isophthalonitrile is categorized as “No Category”.

Executive summary:

The purpose of this Isolated Chicken Eye Test (ICET) was to evaluate the potential ocular corrosivity and irritancy of the test item Isophthalonitrile by its ability to induce toxicity in enucleated chicken eyes. The test compound was applied in a single dose (30 mg/eye) onto the cornea of isolated chicken eyes in order to potentially classify the test compound as either 1: causing "serious eye damage" (category 1 of the Globally Harmonised System for the Classification and Labelling of Chemicals (GHS)), or 2: not requiring classification for eye irritation or serious eye damage according to the GHS. Tested corneas were evaluated pre-treatment and at approximately 30, 75, 120, 180, and 240 minutes after the post-treatment rinse. The endpoints evaluated were corneal opacity, swelling, fluorescein retention, and morphological effects. All of the endpoints, with the exception of fluorescein retention (which was determined only at pre-treatment and 30 minutes after test substance exposure) were determined at each of the above time points.

 

According to the first experiment results the test item showed negative outcome (GHS NC). As it was a solid test item, according to the OECD 438 additional experiment was necessary to confirm or discard the negative outcome.

 

The Imidazole (positive control) and test item Isophthalonitrile was ground before use in the study. The test item and positive control applied in an amount of 0.03 g/eye by powdering the entire surface of the cornea attempting to cover the cornea surface uniformly with the test substance or positive control. Three test item treated eyes, three positive control treated eyes and one negative control treated eye which was treated with 30 µL saline solution were used in the first and additional experiment.

After an exposure period of 10 seconds from the end of the application the cornea surface was rinsed thoroughly with ~20 mL saline solution at ambient temperature and this procedure was repeated for each eye.

 

The Imidazole was stuck on the corneas’ surface in all eyes at 30 minutes after the post-treatment rinse in the first and additional experiment. The Imidazole treated cornea surfaces were not totally cleared at 240 minutes after the post-treatment rinse in the first and additional experiment.