Reaction mass of 1,1-bis(phenethyloxy)ethane and [2-(1-ethoxyethoxy)ethyl]benzene

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

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

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

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of 1,1-bis(phenethyloxy)ethane and [2-(1-ethoxyethoxy)ethyl]benzene

EC Number:

907-481-4

Molecular formula:

C18H22O2 + C12H18O2

IUPAC Name:

Reaction mass of 1,1-bis(phenethyloxy)ethane and [2-(1-ethoxyethoxy)ethyl]benzene

Test material form:

liquid

Specific details on test material used for the study:

- Physical Appearance: Colourless liquid
- Date of Expiry: 12/07/2018
- Storage Condition: Ambient (21 to 29°C)

Test animals / tissue source

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: slaughter house.
- Number of animals: 10
- Characteristics of donor animals: Age of cattle : 3.8 to 4.5 years, 5 males and 5 females
- Storage, temperature and transport conditions of ocular tissue: The head of the cattle were not rinsed with detergent. Eyes were enucleated as soon as possible after death and were immersed in Hank’s Balanced Salt Solution (HBSS) with antibiotics (Penicillin and Streptomycin), placed in a suitable container and transported to the test facility in cool packs.
- indication of any existing defects or lesions in ocular tissue samples: Upon arrival at the test facility, eyes were examined for defects including opacity, scratches and no vascularization. Only corneas free of such defects were used in the experiment. Horizontal diameter and central corneal thickness (CCT) was measured. Cornea with horizontal diameter <28.5 mm and CCT <900 μm were selected.

Test system

Details on study design:

PROCEDURE
- Preparation of Eyes: Before the start of the experiment, the opacity of empty cornea holders filled with pre-warmed EMEM was measured and the mean opacity value obtained was considered as I0. Corneas free of defects were dissected with 2 to 3 mm rim of sclera remaining. Isolated corneas were mounted in designated corneal holders 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 the chambers were joined together by tightening the chamber screws. The corneal holders were equilibrated for at least 1 hour by allowing the corneas to equilibrate and to achieve normal metabolic activity with pre-warmed and red-free EMEM supplemented with 1% Fetal
- Bovine Serum and 1% antibiotics - Penicillin and Streptomycin in an incubator at 32±1°C. Fresh pre-warmed and red-free EMEM was added to both chambers of the cornea holders after completion of the equilibrium period and baseline opacity was recorded for each cornea. The opacity of each cornea was calculated by using the formula I0/I and the opacity value was calculated for initial readouts (Before Treatment) by using the formula [(I0/I-b)/a] where a=0.0251, b=0.9894. Corneas lesser than 7 opacity units, after an initial 1-hour equilibration period, were used for the experiment.
- Application of the Test item: The medium from the anterior chamber was removed and the test item was added by following the Close Chamber Method. A quantity of 750 μL of test item was introduced into the anterior chamber through the dosing hole of the corneal holder and the holes were sealed during exposure and the holders were incubated horizontally at 32±1°C. The corneas were exposed to the test item for approximately 10 minutes. Similar procedure was followed for the negative and positive controls. Three corneas per group were used.
- Post Exposure: 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 till no visual evidence of the test item was observed. Finally, the corneas were rinsed with EMEM without phenol red. The anterior chamber was then refilled with fresh EMEM without phenol red and incubated at 32±1°C for 2 hours. Post exposure corneas were observed visually for tissue peeling, residual test chemical, and non-uniform opacity patterns.
- Measurements of End Points: After a post-incubation period of 2 hours, the opacity was measured with the aid of an opacitometer. The permeability was determined by adding 1 mL of 4 mg/mL of sodium fluorescein to the anterior chamber of the cornea holder, while the posterior chamber was filled with fresh MEM. The holders were incubated in a horizontal position for 90 min at 32±1°C. The permeability was measured with the aid of a spectrophotometry as optical density at 490 nm.
- Histopathological Evaluation: Corneas were preserved in 10% neutral buffered formalin for further histopathological examination. All the corneas including the negative control, positive control and test item were processed, embedded in paraffin, cut at a thickness of 4 to 6 micrometers and stained with hematoxylin and eosin and subjected to histopathological evaluation.

DATA EVALUATION
- Data Derivation: The opacity and mean permeability values were corrected for background opacity and permeability at OD490 values using the following formula: True value of opacity of positive control/test item = Change in opacity value of positive control/test item - mean change in opacity value of negative control. True value of optical density at 490 nm of positive control/test item = OD of positive control/test item - OD of negative control. The mean opacity and permeability at OD490 values for each treatment group was combined and in vitro Irritancy Score (IVIS) for each group was derived using following formula. IVIS= Mean Opacity value + (15 × Mean permeability OD490 value).
- Decision Criteria: Test item with a IVIS score ≤ 3 would be considered as UN GHS no category. Test item with a IVIS score >3 and ≤ 55 would be considered as: “no prediction can be made”, subsequently testing with any other adequate method remains at the discretion of the sponsor. Test item with a IVIS score > 55 would be considered as severe irritant causing serious eye damage and classified as UN GHS category 1 without further testing.
- Study Acceptance Criteria: The study was accepted: As the negative control response resulted in opacity and permeability values less than the established upper limits of background opacity and permeability values for bovine corneas treated with the respective negative control. As the positive control gave an IVIS that falls within two standard deviations of the historical mean.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Value:

0.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- In vitro Irritancy Score (IVIS): The mean corrected opacity and mean corrected permeability values of the test item are 0.59 and -0.007, respectively. The in vitro Irritancy Score (IVIS) of the test item is 0.5 which is considered as UN GHS no category. Treatment with the positive control resulted in mean corrected opacity and mean corrected permeability values of 92.79 and 0.829, respectively. The in vitro Irritancy Score (IVIS) of the positive control was determined to be 105.2, indicating corrosivity or severe irritancy to Bovine corneas.
- Histopathological Evaluation: In the histopathological examination, the corneas treated with the negative control and test item did not show any abnormalities. In the positive control treated corneas, histopathology data showed moderate diffuse cytoplasmic and nuclear vacuolization in the wing and basal layers of the epithelium. In all the corneas, multifocal minimal expansion of the superficial collagen was observed in stroma. Endothelium was apparently normal in all three corneas.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met