[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:

2018-04-03 to 2018-05-05

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:

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)

Test material

Test material information

Constituent 1

Reference substance name:

Ammonium bis(2-ethylhexyl) phosphate

EC Number:

225-615-3

EC Name:

Ammonium bis(2-ethylhexyl) phosphate

Cas Number:

4971-47-5

Molecular formula:

C16H35O4P.H3N

IUPAC Name:

ammonium bis(2-ethylhexyl) phosphate

Test animals / tissue source

Species:

cattle

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: slaughter house (Chowdeshwari Chicken Center, Tumkur)
- Storage, temperature and transport conditions of ocular tissue: Eyes were enucleated as soon as possible after death and immersed in the Hank's Balanced Salt Solution (HBSS) with 10% antibiotics (Penicillin and Streptomycin) in a suitable container and were transported to the test facility by placing in cool packs.
- indication of any existing defects or lesions in ocular tissue samples: Upon arrival to the test facility, eyes were examined for defects including opacity, scratches and neovascularization. Only corneas free of such defects were used in the experiment.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 750 µL

Duration of treatment / exposure:

10 minutes

Duration of post- treatment incubation (in vitro):

2 h

Number of animals or in vitro replicates:

3

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
Before the start of the experiment, opacity of empty cornea holders filled with MEM media were measured and the mean opacity value of the empty corneal holders obtained was considered as l0. Corneas free of defects were dissected with a 2 to 3 mm rim of sclera. Isolated corneas were mounted in designated corneal holder's by placing the endothelial side of the cornea against the O-ring of the posterior chamber. The anterior chamber was placed over the cornea and both Chambers were joined together by tightening the chamber screws then the posterior and anterior chambers were filled with MEM without phenol red (Minimum Essential Medium supplemented with 1% Fetal Bovine Serum and 3% Penicillin and Streptomycin). The corneal holders were equilibrated at 32±1°C for one hour to allow the corneas to equilibrate with the medium and to achieve normal metabolic activity, MEM was replaced with fresh pre-warmed MEM without phenol red in both chambers of cornea holders after completion of equilibrium period. An opacity determination was performed on each of the corneas using an Opacitometer (BASF Opacitometer 2013-19). The opacity of each cornea was read against a MEM filled chamber, and the initial opacity reading thus determined was recorded us baseline opacity.
Opacity of each cornea was calculated by using a formula I0/I and opacity value was calculated for initial readouts (before treatment) by using the formula [(I0/I-b)/a] where a=0.0251, b=0.9894 (Opacitometer specific empirically determined variables) I0 is the mean opacity value obtained for the empty corneal holders without corneas and with MEM, I is the individual opacity value of cornea. Corneas showing opacity greater than 7 opacity units after an initial 3-hour equilibration period were not used for the experiment,

TREATMENT METHOD: closed chamber

POST-EXPOSURE PERIOD: yes. 2 h

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: After the exposure period, the test item, negative and positive controls were removed from the anterior chamber and the epithelium was washed with EMEM containing phenol red until no visual evidence of the test item was observed. Finally the corneas were rinsed with MEM without phenol red.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Opacity was measured with the aid of an opacitometer. Opacity was then calculated using the formula mentioned in section 7,1.
The change in opacity for each individual cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final post-treatment reading. The corrected opacity for each treated cornea and positive control was calculated by subtracting the average change in opacity of die negative control corneas from the change in opacity of each test item treated and positive control cornea. The mean opacity value of each treatment group was then calculated by averaging the corrected opacity values of the treated corneas for each treatment group.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader (OD490)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: decision criteria as indicated in the TG was used.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

in vitro irritation score

Run / experiment:

test item

Value:

82.3

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation parameter:

in vitro irritation score

Run / experiment:

negative control

Vehicle controls validity:

not examined

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: IVIS not applicable for negative control

Results 3

Irritation parameter:

in vitro irritation score

Run / experiment:

positive control

Value:

116.8

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

not applicable

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: none

DEMONSTRATION OF TECHNICAL PROFICIENCY:
Prior to routine use the technical proficiency of the tost method was established by using proficiency chemicals under Bioneeds Study No.: BIO-TX 421, according to OECD Test Guideline No. 437. Proficiency chemicals are periodically tested in order to ensure the accuracy and reliability of the test method overtime (once in three years).

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes

Any other information on results incl. tables

Table 1 Summary of in vitro irritancy score (IVIS)

Group & Treatment	Mean change in opacity value	Mean corrected opacity value	Mean Corrected permeability value	IVIS value
Negative control	1.3	-	-	-
Positive control	93.6	92.30 ± 6.37	1.635 ± 0.009	116.8
Test item	60.6	59.30 ± 3.75	1.536 ± 0.103	82.3

 

Applicant's summary and conclusion

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Conclusions:

Based on the results obtained in the Bovine Corneal Opacity and Permeability Test according to OECD 437, the test item, induced an IVIS of 82.3 at 10 minutes of treatment. As the test item induced an IVIS >55, it is considered as severe irritant/causing serious eye damage to bovine cornea and classified as UN GHS category 1.

Executive summary:

The test item was evaluated for ocular corrosion or severe irritancy as per the OECD guideline for the testing of chemicals No. 437 "Bovine Corneal Opacity and Permeability Test Method for Identifying Chemicals Inducing Serious Eye Damage and Chemicals not requiring Classification for Eye Irritation or Serious Eye Damage", adopted October 2017.

Eyes of cattle were collected from a slaughter house by immersing them in the Hank's Balanced Salt Solution (HBSS) with antibiotics (penicillin and streptomycin) in a suitable container and transported to the test facility by placing on cool packs. Only eye balls free of defects were selected for the experiment. Empty cornea holder's opacity with pre-warmed Eagle's Minimum Essential Medium was measured and the mean opacity value obtained was determined as I0. Cornea holders with selected Corneas were equilibrated at 32±3 °C for 1 hour with Eagle's Minimum Essential Medium with 1% Fetal Bovine Scrum supplemented with 1% antibiotics and baseline opacity was recoiled for each cornea. Corneas with opacity units less than 7 were selected and used for the study and distributed for the treatment groups.

A volume of 750 µL of test item, negative (distilled water) and positive control (Ethanol) was introduced into anterior chamber in triplicates to die designated cornea holders and incubated at 32±1 °C for 10 minutes. Treated corneas were washed till no visual evidence of test item observed with EMEM containing phenol red and finally with EMEM without phenol red. The anterior chamber was then refilled with fresh EMEM without phenol red. Opacity was measured with the aid of opacitometer and permeability was determined spectrophotometrically at 490 nm (OD490) using 4 mg/mL sodium fluorescein, post incubation of 90 min at 32±1 °C.

The test item resulted in the mean corrected opacity and mean corrected permeability values of test item are 59.30 and 1.536, respectively. The in vitro Irritancy Score (IVIS) of test item resulted in 82.3, whereas the positive control resulted in mean corrected opacity and mean corrected permeability values of 92,30 and 1,635, respectively where the in vitro Irritancy Score (IVIS) of 116.8, indicating corrosion or severe irritancy to Bovine cornea.