4-aminobenzonitrile

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-07-17

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

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)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

4-aminobenzonitrile

EC Number:

212-850-1

EC Name:

4-aminobenzonitrile

Cas Number:

873-74-5

Molecular formula:

C7H6N2

IUPAC Name:

4-aminobenzonitrile

Test material form:

solid: flakes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: no data
- Expiration date of the lot/batch: no data
- Purity test date: no data
- Other: physical state: solid; form: white to yellow brown flakes

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room tempearture
- Stability under test conditions: no data
- Solubility and stability of the test substance in the solvent/vehicle: no data

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Final preparation of a solid: the test item was tested as a 20% suspension (w/v) in saline. The test item could not be suspended or solved homogeneously, therefore, each 0.75 mL of the prepared stock was distributed to each cornea.

FORM AS APPLIED IN THE TEST (if different from that of starting material): 20% suspension (w/v) in saline

Test animals / tissue source

Species:

other: bovine eyes

Strain:

other: not applicable

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: freshly isolated bovine corneas (at least 9 month to 60 month old donor cattle), Schlachthof Bensheim, 64625 Bensheim, Germany
- Freshly isolated bovine eyes were collected from the abattoir. The isolated eyes were transported to the laboratory in a cool box containing HBSS (Gibco, Ref. 14025-050) cooled in the refrigerator prior to use. Since the transport period is less than one hour, the described cooling procedure for the bovine eyes is sufficient. The corneae were isolated on the same day after delivery of the eyes.

Test system

Vehicle:

physiological saline

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.75 mL
- Concentration (if solution): 20% suspension (w/v) in saline

VEHICLE/NEGATIVE CONTROL
- Amount(s) applied (volume or weight with unit): 0.75 mL

POSITIVE CONTROL
- Amount(s) applied (volume or weight with unit): 0.75 mL
- Concentration (if solution): 10% (w/v) in 0.9% natriumchloride (saline)

Duration of treatment / exposure:

240 minutes

Duration of post- treatment incubation (in vitro):

After 240 minutes of treatment, opacity was measured. The permeability measurement of the corneas was performed after the incubation period of 90 minutes ± 10 minutes following the opacity measurement.

Number of animals or in vitro replicates:

Sets of three corneae were used for treatment with the test item and the negative and positive control

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
- All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity and scratches were discarded. The cornea was carefully removed from the eye using scalpel and rounded scissors. A rim of about 2 mm of tissue (sclera) was left for stability and handling of the isolated cornea. The corneae were directly used in the BCOP test on the same day. Each isolated cornea was mounted in a specially designed cornea holder according to the description given in OECD guideline 437, that consists of anterior and posterior compartments, which interface with the epithelial and endothelial sides of the cornea, respectively. The endothelial side of the cornea was positioned against the sealing ring (O-ring) of the posterior part of the holder. The cornea was gently flattened over the O-ring but stretching was avoided. The anterior part of the holder was positioned on top of the cornea and fixed in place with screws. Both compartments of the holder were filled with incubation medium. The posterior compartment was filled first to return the cornea to its natural convex position. Care was taken to assure no air bubbles were present within the compartments. For equilibration, the corneae in the holder were incubated in a vertical position for about one hour at 32 ± 1 °C in a water-bath.

TREATMENT METHOD: [closed chamber / open chamber]
- The anterior compartment received the test item suspension or negative or positive control at a volume of 0.75 mL ensuring sufficient coverage of the epithelial surface of the cornea (closed chamber method; open chamber method is only used for testing of very viscous substances). The corneae were incubated in a horizontal position at 32 ± 1 °C in the water-bath. The incubation time lasted 240 minutes.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: the test item or control items, respectively, were rinsed off from the application side with saline, and fresh incubation medium was added into the anterior compartment and opacity was measured (t240).

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The opacitometer determines changes in the light transmission passing through the corneae, and displays a numerical opacity value. This value was recorded in a table. The opacitometer (OP_KiT opacitometer (Electro Design, 63-Riom, France)) was calibrated as described in the manual and the opacity of each of the corneae was determined by reading each holder placed in the photoreceptor compartment for treated cornea.
For equilibration and prior to application of the test item or controls, the corneae in the holder was incubated in a vertical position for about one hour at 32 ± 1 °C in a water-bath. At the end of the incubation period, the basal opacity was determined (t0). Each corneae with a value of the basal opacity > 7 was discarded. After exposure of the corneae to the test groups and after rinsing the opacity value was determined again (t240).
The change of opacity value of each treated cornea or positive and negative control corneae was calculated by subtracting the initial basal opacity from the post treatment opacity reading (t240 – t0), for each individual cornea. The average change in opacity of the negative control corneae was calculated and this value was subtracted from the change in opacity of each treated cornea or positive control to obtain a corrected opacity.

- Corneal permeability: Following to the opacity readings, the permeability was measured as an indication of the integrity of the epithelial cell sheets. After the final opacity measurement was performed, the incubation medium was removed from the anterior and posterior compartment and replaced by 1 mL incubation medium in the posterior compartment and by 1 mL of a 0.5% (w/v) sodium fluorescein solution in HBSS in the anterior compartment. Corneae were incubated again in a horizontal position for 90 ± 10 minutes in a water-bath at 32 ± 1 °C. Incubation medium from the posterior compartment were removed, well mixed and transferred into a 96 well plate and the optical density at 490 nm (OD490) was determined with a spectrophotometer (Versamax® Molecular Devices). The absorbance values were determined using the software SoftMax Pro Enterprise (version 4.7.1).
The corrected OD490 value of each cornea treated with positive control and test item was calculated by subtracting the average negative control cornea value from the original permeability value for each cornea.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
- The following formula was used to determine the IVIS of the negative control: IVIS = opacity value + (15 x OD490 value)
The following formula was used to determine the IVIS of the positive control and the test item: IVIS = (opacity value – opacity value mean negative control) + (15 x corrected OD490 value)
The mean IVIS value of each treated group was calculated from the IVIS values.

DECISION CRITERIA:
- Depending on the score obtained, the test item was classified into the following category according to OECD guideline 437:
IVIS In vitro Irritancy Score (according to OECD 437)
<= 3 No Category (according to GHS)
> 3; <= 55 No prediction can be made
> 55 Serious eye damage according to CLP/EPA/GHS (Cat 1)

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Remarks:

mean

Run / experiment:

1

Value:

7.47

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: increase in corneal opacity

Other effects / acceptance of results:

Mean in vitro irritancy score:
- negative control: 1.04
- positive control: 128.43

ACCEPTANCE OF RESULTS:
The test is acceptable because
- the positive control gives an IVIS that falls within two standard deviations of the current historical mean (updated every three months), and because
- the negative control responses result in opacity and permeability values that are less than the established upper limits for background opacity and permeability values for bovine corneae treated with the respective negative control.

DISCUSSION:
After a first opacity measurement of the fresh bovine corneae (t0), the 20% (w/v) suspension in saline of the test item 4-Aminobenzonitrile, the positive, and the negative controls were applied to corneae and incubated for 240 minutes at 32 ± 1 °C. After the incubation phase the test item, the positive, and the negative controls were each rinsed from the corneae and opacity was measured again (t240).
After the opacity measurements permeability of the corneae was determined by measuring spectrophotometrically the transfer of sodium fluorescein after incubation in a horizontal position for 90 minutes at 32 ± 1 °C.
With the negative control (saline) neither an increase of opacity nor permeability of the corneae could be observed (mean IVIS = 1.04).
The positive control (10% (w/v) Benzalkonium chloride in saline) showed clear opacity and distinctive permeability of the corneae (mean IVIS = 128.43) corresponding to a classification as serious eye damaging (CLP/EPA/GHS (Cat 1)). The mean IVIS of the positive control (mean IVIS = 128.43) was in the range of the historical data (mean IVIS 116.24 ± 2 * 9.31).
Relative to the negative control, the test item 4-Aminobenzonitrile caused an increase of the corneal opacity but not of the permeability. The calculated mean IVIS was 7.47 (threshold for serious eye damage: IVIS ≥ 55). According to OECD 437 no prediction for the damage hazard of the test item to the eye can be made.

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Conclusions:

In conclusion, according to the current study and under the experimental conditions reported, 4-Aminobenzonitrile is not serious eye damaging (CLP/EPA/GHS (Cat 1) but a prediction for the damage hazard cannot be made (GHS). However based on the results neither classification nor an in vivo test is indicated.

Based on the criteria of the CLP regulation, no decision can be made on the classification for eye irritation or serious eye damage.

Executive summary:

An in vitro study was performed to assess the corneal damage potential of 4 -Aminobenzonitrile by means of the BCOP assay according to OECD 437 using fresh bovine corneae.

After a first opacity measurement of the fresh bovine corneae (t0), the 20% (w/v) suspension in saline of the test item 4-Aminobenzonitrile, the positive, and the negative controls were applied to corneae and incubated for 240 minutes at 32 ± 1 °C. After the incubation phase the test item, the positive, and the negative controls were each rinsed from the corneae and opacity was measured again (t240).

After the opacity measurements permeability of the corneae was determined by measuring spectrophotometrically the transfer of sodium fluorescein after incubation in a horizontal position for 90 minutes at 32 ± 1 °C.

With the negative control (saline) neither an increase of opacity nor permeability of the corneae could be observed (mean IVIS = 1.04).

The positive control (10% (w/v) Benzalkonium chloride in saline) showed clear opacity and distinctive permeability of the corneae (mean IVIS = 128.43) corresponding to a classification as serious eye damaging (CLP/EPA/GHS (Cat 1)). The mean IVIS of the positive control (mean IVIS = 128.43) was in the range of the historical data (mean IVIS 116.24 ± 2 * 9.31).

Relative to the negative control, the test item 4-Aminobenzonitrile caused an increase of the corneal opacity but not of the permeability. The calculated mean IVIS was 7.47 (threshold for serious eye damage: IVIS ≥ 55). According to OECD 437 no prediction for the damage hazard of 4 -Aminobenzonitrile to the eye can be made.

Based on the results no classification should be made. But neither the performance of an in vivo study is indicated. Because of the result is very close to "evidence for not irritating" and the substance is handled according to strictly controlled conditions no additional animal study should be performed.