Fenuron

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

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)

Version / remarks:

adopted 09 October 2017

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Version / remarks:

February 14, 2017

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

1

Reference substance name:

Fenuron

EC Number:

202-941-4

EC Name:

Fenuron

Cas Number:

101-42-8

Molecular formula:

C9H12N2O

IUPAC Name:

fenuron

Test material form:

other: white to off-white powder

Details on test material:

- Particle size distribution: 100 % particle size < 12 µm (method: Laser Diffraction; Certificate of Analysis)
Particle size parameter determined with a Malvern Mastersizer 2000 (Non-GLP determination)
D10% = 1.18 µm
D50% = 7.72 µm
D90% = 16.00 µm
D: Diameter; 10, 50, 90: percentage cumulative
- Mass median aerodynamic diameter (MMAD): 1.595 µm
- Geometric standard deviation (GSD): calculated as 2.09

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Isochem Kautschuk GmbH, Batch no. 0010416
- Expiration date of the lot/batch: April 2019
- Purity test date: 5 October 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material : at room temperature (+10°C to +25°C) in a tightly closed container in a dry, cool and well-ventilated place, avoiding exposure to sunlight and moisture

OTHER SPECIFICS: IsoQure UR 300

Test animals / tissue source

Species:

cattle

Strain:

other: Bovine eyes

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Hubert Bahlmann GmbH & Co. Versandschlachterei Spezialmischfutterwerk KG, 49699 Lindern, Germany
- Characteristics of donor animals (e.g. age, sex, weight): 6 to 12 months old
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): On collection the eyes were completely submerged in Hanks’ Balanced Salt Solution (HBSS) containing penicillin at 100 IU/mL and streptomycin at 100 µg/mL .
- Indication of any existing defects or lesions in ocular tissue samples: Only corneas from eyes free of defects were used.
- Indication of any antibiotics used: penicillin at 100 IU/mL and streptomycin at 100 µg/mL .

Test system

Vehicle:

physiological saline

Remarks:

0.9% sodium chloride solution

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 µL
- Concentration (if solution): 20% (w/v)

VEHICLE / NEGATIVE CONTROL
- Amount(s) applied (volume or weight with unit): 750 µL
- Concentration (if solution):0.9% sodium chloride solution
- Lot/batch no. (if required): Batch no. 173568002; B. Braun Melsungen AG, 34212 Melsungen, Germany

Duration of treatment / exposure:

240 min

Number of animals or in vitro replicates:

Three corneas were used for each treatment group (test item, negative control and positive control).

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
Upon arrival at the laboratory, the eyes were examined for defects such as but not limited to increased opacity, scratches, and neovascularisation. Only corneas from eyes free of defects were used. Corneas that had opacity greater than seven opacity units or equivalent for the opacitometer and cornea holders used after an initial one hour equilibration period had to be discarded.
The open-chamber method was used. The corneas were dissected with a 2 to 3 mm rim of sclera and mounted in corneal holders with anterior (epithelium) and posterior (endothelium) chambers. Beginning with the posterior chambers, the chambers were filled to excess with pre-warmed Eagle’s Minimum Essential Medium (EMEM) , while preventing bubble formation. The corneal holder was equilibrated at 32 °C ± 1 °C for at least one hour.

QUALITY CHECK OF THE ISOLATED CORNEAS
After the equilibration period, fresh pre-warmed EMEM was added to both chambers and baseline opacity readings were taken for each cornea. Corneas exhibiting macroscopic tissue damage (e.g. scratches, pigmentation, neovascularisation) or an opacity >7 opacity units were discarded. The mean opacity of all equilibrated corneas was calculated by use of an opacitometer. A minimum of three corneas with opacity values close to the median value for all corneas were selected as negative control corneas. The remaining corneas were then distributed into treatment, solvent and positive control groups.

NUMBER OF REPLICATES
3

NEGATIVE CONTROL USED
0.9% sodium chloride solution

SOLVENT CONTROL USED = NEGATIVE CONTROL = VEHICLE CONTROL

POSITIVE CONTROL USED:
20% Imidazole (CAS no. 288-32-4) in 0.9% sodium chloride solution

APPLICATION DOSE AND EXPOSURE TIME
750 µL / 240 minutes

TREATMENT METHOD: open chamber

POST-INCUBATION PERIOD: no

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: The epithelium was washed with EMEM containing phenol red at least three times. Washing was repeated until no test item or discolouration (yellow or purple) of phenol red was visible. The corneas were rinsed a final time with EMEM only to remove any remaining phenol red from the chamber. The chamber was then filled with EMEM without phenol red.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Corneal opacity was determined by the amount of light transmission through the cornea measured quantitatively with the aid of an opacitometer resulting in opacity values measured on a continuous scale.
- Corneal permeability: To determine the corneal permeability 1 mL sodium fluorescein solution (5 mg/mL in 0.9% sodium chloride solution) was added to the anterior chamber (epithelial surface) while the posterior chamber (endothelial surface) was refilled with fresh EMEM. The holder was incubated in a horizontal position at 32 ± 1 °C for 90 ± 5 minutes. The amount of sodium fluorescein that crossed from the anterior to the posterior chamber was measured quantitatively using a microplate reader (Tecan Sunrise Magellan Version 7.2 ). Measurements at 490 nm were recorded as optical density (OD490). The fluorescein permeability values were determined using OD490 values based upon a visible light spectrophotometer (Tecan Sunrise) using a standard 1 cm path length.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
IVIS = mean opacity value + (15 x mean permeability OD490 value)

DECISION CRITERIA:
IVIS UN GHS
≤ 3 No Category
> 3 and ≤ 55 No prediction can be made
> 55 Category 1

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

in vitro irritation score

Remarks:

mean test substance

Run / experiment:

240 min exposure time

Value:

0.285

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: test material

Remarks:

standard deviation ± 0.362

Results 2

Irritation parameter:

in vitro irritation score

Remarks:

cornea 7

Run / experiment:

240 min exposure time

Value:

0.41

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: test material

Results 3

Irritation parameter:

in vitro irritation score

Remarks:

cornea 8

Run / experiment:

240 min exposure time

Value:

0.569

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: test material

Results 4

Irritation parameter:

in vitro irritation score

Remarks:

cornea 9

Run / experiment:

240 min exposure time

Value:

-0.123

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: test material

Results 5

Irritation parameter:

in vitro irritation score

Remarks:

mean negative control

Run / experiment:

240 min exposure time

Value:

0.442

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: vehicle 0.9% NaCl

Remarks:

stadard deviation ± 0.189

Results 6

Irritation parameter:

in vitro irritation score

Remarks:

mean positive control

Run / experiment:

240 min exposure time

Value:

89.075

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Positive control 20% Imidazole

Remarks:

standard deviation ± 3.632

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control:
The negative or solvent/vehicle control responses should result in opacity and permeability values, that are less than the established upper limits for background opacity and permeability values for bovine corneas treated with the respective negative or solvent/vehicle control. In cases of borderline results in the first testing run, a second testing run should be considered (but not necessarily required), as well as a third one in case of discordant mean IVIS results between the first two testing runs. A result in the first testing run is considered borderline if the predictions from the 3 corneas were non-concordant, such that:
•2 of the 3 corneas gave discordant predictions from the mean of all 3 corneas, OR,
•1 of the 3 corneas gave discordant prediction from the mean of all 3 corneas AND the discordant result was > 10 IVIS units from the cut-off threshold of 55.
•If the repeat testing run corroborates the prediction of the initial testing run (based upon the mean IVIS value), then a final decision can be taken without further testing. If the repeat testing run results in a non-concordant prediction from the initial testing run (based upon the mean IVIS value), then a third and final testing run should be conducted to resolve equivocal predictions, and to classify the test chemical. It may be permissible to waive further testing for classification and labelling in the event any testing run results in a UN GHS Category 1 prediction.
The calculated IVIS value of 0.442 ± 0.189 was well below the cut-off value of 3 (UN GHS no category).
- Acceptance criteria met for positive control: A test is considered acceptable if the positive control gives an IVIS that falls within two standard deviations of the current historical mean, which was updated at least every three months. . The calculated IVIS value of 89.075 ± 3.632 was within two standard deviations of the current historical mean and well above the cut-off value of 55. Hence, the acceptance criteria for the test were fulfilled.
- Range of historical values if different from the ones specified in the test guideline:
IVIS: lower and upper limits of acceptance according to OECD Guideline No. 437;
Opacity: upper limit of acceptance according to OECD Guideline No. 437;
Permeability: upper limit of acceptance according to OECD Guideline No. 437.
Historical Control Data (GLP studies of the years 2015 - 2017 (n = 11). Background data are not audited by LPT-QAU.):
-NaCl 0.9%:
*IVIS
Mean: 0.414
Standard deviation: 0.796
Lower limit of acceptance (mean-2*SD ): -1.178
Upper limit of acceptance (mean-2*SD): 2.005
*Opacity
Mean: 0.108
Standard deviation: 0.768
Lower limit of acceptance (mean-2*SD ): -1.428
Upper limit of acceptance (mean-2*SD): 1.643
*Permeability
Mean: 0.024
Standard deviation: 0.012
Lower limit of acceptance (mean-2*SD ): -0.001
Upper limit of acceptance (mean-2*SD): 0.048

-Imidazol 20%
*IVIS
Mean: 84.593
Standard deviation: 12.257
Lower limit of acceptance (mean-2*SD ): 60.079
Upper limit of acceptance (mean-2*SD): 109.108
*Opacity
Mean: 60.730
Standard deviation: 10.816
Lower limit of acceptance (mean-2*SD ): 39.098
Upper limit of acceptance (mean-2*SD): 82.363
*Permeability
Mean: 1.589
Standard deviation: 0.485
Lower limit of acceptance (mean-2*SD ): 0.619
Upper limit of acceptance (mean-2*SD): 2.558

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the present test conditions IsoQure UR 300 tested in the in vitro BCOP test method, had an IVIS value of 0.285, which is below the cut-off value of 3 (UN GHS no category) and consequently it is not classified for irritation or serious eye damage according to UN GHS classification.

Executive summary:

The purpose of this study was to determine a possible potency of IsoQure UR 300 of being ocular corrosive and severe irritant employing an in vitro system.The Bovine Corneal Opacity and Permeability Assay (BCOP) test method is an organotypic model that provides short-term maintenance of normal physiological and biochemical function of the bovine cornea in vitro. Three corneas were used for each treatment group (test item, solvent control and positive control). The solid test item was suspended in a 0.9% sodium chloride solution with a final concentration of 20% IsoQure UR 300 as recommended in the test guideline 437 for non-surfactant solids. 0.9% NaCl solution was used as the solvent control and 20% Imidazole in 0.9% NaCl solution as the positive control item. The test item and the controls were applied to the epithelial surface of the cornea by addition to the anterior chamber of the corneal holder. The exposure time for the test item and the controls was 240 minutes. The optical density (OD) was measured at a wavelength of 490 nm. The acceptance criteria of validity were fulfilled in this test. Following treatment with IsoQure UR 300 a mean opacity of 0.305 ± 0.354 and a mean permeability value of <0.001 compared to the negative control were determined. The calculated IVIS of 0.285 ± 0.362 is below the cut-off value of 3 (UN GHS no category). Hence, the test item did not show severely irritant or corrosive properties and consequently it is not classified for irritation or serious eye damage according to UN GHS classification.