[No public or meaningful name is available]

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 May 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

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 9 October 2017

Deviations:

no

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:

2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Hess. Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany

Test material

Test material information

1

Reference substance name:

Reaction mass of dipotassium 2-(3,4-dimethyl-1H-pyrazol-1-yl) succinate and dipotassium 2-(4,5-dimethy-1H-pyrazol-1-yl) succinate

Molecular formula:

C9H10K2N2O4

IUPAC Name:

Reaction mass of dipotassium 2-(3,4-dimethyl-1H-pyrazol-1-yl) succinate and dipotassium 2-(4,5-dimethy-1H-pyrazol-1-yl) succinate

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

TEST METHOD
The bovine corneal opacity and permeability (BCOP) test is an in vitro test method used for identifying i) chemicals inducing serious eye damage and ii) chemicals not requiring classification for eye irritation or serious eye damage. The potential of a test substance to cause ocular corrosivity or severe irritancy is measured by its ability to induce opacity and increased permeability in an isolated bovine cornea. The opacity and permeability assessments of the cornea are combined to derive an in vitro irritancy score (IVIS), which is used to classify the irritancy level of the test substance.

SOURCE OF COLLECTED EYES
- Source: AB Schlachthof GmbH & Co. KG, Aschaffenburg, Germany
- Characteristics of donor animals: 14 months old
- Storage, temperature and transport conditions of ocular tissue: The isolated bovine eyes were stored in HBSS (Hank’s Buffered Salt Solution) containing 1% [v/v] penicillin/streptomycin.
- Time interval prior to initiating testing: On the test day, fresh eyes were collected from the slaughter house. The corneae were isolated on the same day after delivery of the eyes.
- indication of any existing defects or lesions in ocular tissue samples: All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity and scratches were discarded.
- Indication of any antibiotics used: 1% [v/v] penicillin/streptomycin (100 units/mL penicillin and 100 µg/mL streptomycin) containing HBSS was used for storage in the slaughter-house and for transport.

Test system

Vehicle:

physiological saline

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 750 µL
- Concentration: 20%

VEHICLE
- Substance: Physiological saline
- Amount applied: 750 µL
- Concentration: 0.9% Natrium chloride in deionised water

POSITIVE SUBSTANCE
- Substance: Benzalkonium chloride
- Concentration: 10% Benzalkonium chloride in 0.9% Natrium chloride solution in deionised water
- Amount applied: 750 µL

Duration of treatment / exposure:

4 h at 32 ± 1 °C

Duration of post- treatment incubation (in vitro):

90 ± 5 min at 32 ± 1 °C

Number of animals or in vitro replicates:

triplicates each for treatment and control groups

Details on study design:

SELECTION AND PREPARATION OF CORNEAS :
- Dissection of the eyes and treatment: Corneas were 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 corneas.
- Description of the cornea holder: The cornea holders consist of an anterior and a posterior compartment, which interface with the epithelial and endothelial sides of the cornea. 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.
- Test medium and temperature conditions used in the cornea holder: The incubation medium consisted of Minimum Essential Medium (MEM) supplemented with 1.1 g sodium bicarbonate, 5 mL L-glutamine, 5 mL penicillin/streptomycin per 500 mL medium (final concentration of 100 units penicillin per mL medium, and 100 µg streptomycin per mL medium). Immediately before starting the test, MEM was supplemented with 1% fetal calf serum (FCS) and prior to use it was pre-warmed to 32 ± 1 °C.
- Equilibration time: 1 h at 32 ± 1 °C
- Quality check of the equilibrated corneas: Only corneaes with an initial basal opacity value < 7 were used

TREATMENT METHOD: closed chamber

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: Corneas were washed at least 3 times with Minimum Essential Medium (MEM) containing phenol red (or more if phenol red was still discoloured (yellow or purple), or the test item was still visible). Once the medium was free of the test item the corneaes were given a final rinse with MEM containing all supplements without phenol red.
- POST-EXPOSURE INCUBATION: 90 ± 5 min at 32 ± 1 °C

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Corneal opacity was determined by the amount of light transmission through the cornea via an opacitometer (OP KiT opacitometer (Electro Design, 63-Riom, France)). The change of the opacity value of each treated cornea or of the positive and negative control corneae is 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 is calculated and this value is subtracted from the change in opacity of each treated cornea or positive control to obtain a corrected opacity.
- Corneal permeability: The passage of sodium fluorescein dye was measured with the aid of a microtiter plate reader (Versamax® Molecular Devices) at 490 nm (OD490). The corrected OD490 value of each cornea treated with positive control or test item is calculated by subtracting the average negative control cornea value from the original permeability value for each cornea.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
- Calculation IVIS of the negative control: IVIS = opacity value + (15 x OD490 value), Calculation IVIScorrected of the positive control and the test item: IVIScorrected = (opacity value – mean of opacity of negative control) + 15 x (permeability value – mean permeability of the negative control)

DECISION CRITERIA:
Test substance with an IVIS ≥ 55 was regarded as severe irritant/corrosive and labelled Category 1.
Test substance with an IVIS ≤ 3 was regarded as non-irritant and labelled in no category.
Test substance with an IVIS > 3 ≤ 55 was regarded as no prediction can be made.

ACCEPTABILITY CRITERIA
The test will be acceptable if
- the positive control gives an IVIS that falls within two standard deviations of the current historical mean (updated every three months), and if
- 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.
A single testing run composed of at least three corneae should be sufficient for a test chemical when the resulting classification is unequivocal. In cases of borderline results in the first testing run, a second testing run will be considered, 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 corneae are non-concordant, such that:
- 2 of the 3 corneae give discordant predictions from the mean of all corneae, or,
- 1 of the 3 corneae gives a discordant prediction from the mean of all 3 corneae, and the discordant result is >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.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Remarks:

mean out of all 3 eyes

Run / experiment:

4 h

Value:

3.4

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: Test substance

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No macroscopically visible defects were observed.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, the negative control gives a mean IVIS of 0.77, the opacity (mean 0.00) and permeability (mean 0.051) values are less than the established upper limits for background opacity and permeability values for bovine corneae treated with the respective negative control
- Acceptance criteria met for positive control: yes, the positive control gives a mean IVIS of 91.16, that falls within two standard deviations of the current historical mean

Any other information on results incl. tables

Table 2: Results after 240 minutes treatment time

Test Group	Opacity value = Difference (t240-t0) of Opacity		Permeability at 490 nm (OD490)		IVIS	Mean IVIS
		Mean		Mean
Negative Control	0	0.00	0.054	0.051	0.81	0.77
	0		0.050		0.75
	0		0.050		0.75
Positive Control	74.00*		0.404*		80.06	91.16
	99.00*		0.243*		102.64
	83.00*		0.520*		90.80
Test Item	3.00*		-0.004*		3.00	3.40
	3.00*		0.013*		3.19
	4.00*		-0.003*		4.00

*corrected values

Applicant's summary and conclusion

Interpretation of results:

other: non-corrosive

Conclusions:

Under the conditions of the Bovine Corneal Opacity and Permeability Test (BCOP) the test substance was not identified to induce serious eye damage, but the test substance could also not be identied to not require classification for eye irritation or serious eye damge. Thus, the test substance is considered to be non-corrosive, but no prediction on the irritation potential can be made and further evaluation and/or data generation is required.