Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Remarks:
in the Bovine Corneal Opacity and Permeability
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 guidelineopen allclose all
Qualifier:
according to
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. Oct. 2017: “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”
Deviations:
not specified
Qualifier:
according to
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Version / remarks:
adopted 14. Feb. 2017
Deviations:
not specified
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
- Name of test material: 4,4'-bis(2-methoxystyryl)-1,1'-biphenyl
- IUPAC name: 1-[(E)-2-(2-methoxyphenyl)ethenyl]-4-{4-[(E)-2-(2-methoxyphenyl)ethenyl]phenyl}benzene
- Molecular formula: C30H26O2
- Molecular weight: 418.53 g/mol
- Smiles notation: COC1=CC=CC=C1/C=C/C2=CC=C(C3=CC=C(/C=C/C4=CC=CC=C4OC)C=C3)C=C2
- InChl: 1S/C30H26O2/c1-31-29-9-5-3-7-27(29)21-15-23-11-17-25(18-12-23)26-19-13-24(14-20-26)16-22-28-8-4-6-10-30(28)32-2/h3-22H,1-2H3/b21-15+,22-16+
- Substance type: Organic
- Physical state: Solid

Test animals / tissue source

Species:
other: Bovine
Strain:
other: Bos primigenius Taurus (fresh bovine corneas)
Details on test animals or tissues and environmental conditions:
Fresh bovine eyes were obtained from the slaughterhouse Müller Fleisch GmbH, Enzstr. 2-4, 75217 Birkenfeld, Germany, on the day of the test. The cattle were between 12 and 60 months old. The eyes were transported to the test facility in Hanks’ Balanced Salt Solution with 1% Penicillin-Streptomycin solution (Penicillin 100 U/mL, Streptomycin 100 µg/mL) in a suitable cooled container within 1 hour.

Test system

Vehicle:
Hank's balanced salt solution
Controls:
yes
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit):
Test item: 750 µL
Positive control: 750 µL
Negative control: 750 µL

- Concentration
Test item: 20% suspension in HBSS
Positive control: imidazole, 20 % solution in HBSS
Negative control: HBSS
Duration of treatment / exposure:
4h at 32 ± 1 °C
Number of animals or in vitro replicates:
3
Details on study design:
-SELECTION AND PREPARATION OF CORNEAS : The fresh bovine eyes were obtained from cattle which were between 12 and 60 months old. The eyes were transported to the test facility in Hanks’ Balanced Salt Solution(HBSS) with 1% Penicillin-Streptomycin solution (Penicillin 100 U/mL, Streptomycin 100 µg/mL) in a suitable cooled container within 1 hour.
After having carefully cleaned and sterilised the cornea holders, they were kept in the incubation chamber at 32 ± 1 °C.
On the day of the assay, the MEM without phenol red was supplemented with sodium bicarbonate, L-glutamine and 1% fetal calf serum (= complete MEM) and stored in a water bath at 32 ± 1 °C.
The same was performed with the MEM with phenol red, but without addition of sodium bicarbonate.

-QUALITY CHECK OF THE ISOLATED CORNEAS : The corneas were examined and only corneas which were free from damages were used

-NUMBER OF REPLICATES : 3

-NEGATIVE CONTROL USED : Yes (HBSS)

-POSITIVE CONTROL USED : Yes (imidazole, 20 % solution in HBSS)

-APPLICATION DOSE AND EXPOSURE TIME : 750 µL, 4h

-TREATMENT METHOD: Open chamber

-POST-INCUBATION PERIOD: no

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: Rinsing the anterior chamber with cMEM with phenol red and final rinsing with cMEM without phenol red.
- POST-EXPOSURE INCUBATION: 90 minutes at 32 ± 1 °C.

-METHODS FOR MEASURED ENDPOINTS:
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of [UV/VIS spectrophotometry / microtiter plate reader] (OD490)

-SCORING SYSTEM: In Vitro Irritancy Score (IVIS) :
The IVIS of each replicate of the negative control: IVIS = opacity difference + (15 x corrected OD492 value).
The IVIS of each replicate of the positive control and of the test item: IVIS = (opacity difference – mean opacity difference of the negative control) + [15 x (OD492 – mean OD492 of the negative control)].

-DECISION CRITERIA: According to OECD Guideline no. 437 (Oct. 2017).

Results and discussion

In vitro

Results
Irritation parameter:
cornea opacity score
Remarks:
Bovine Corneal
Value:
ca. 0.54
Vehicle controls validity:
not specified
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Under the conditions of this study, the test item 4,4'-bis(2-methoxystyryl)-1,1'-biphenyl (EC:254-935-6) showed no effects on the cornea of the bovine eye. The calculated mean IVIS (In Vitro Irritancy Score) was 0.54.
According to OECD Guideline no. 437 (Oct. 2017), a substance with an IVIS ≤ 3 re-quires no classification for eye irritation or serious eye damage.
Executive summary:

This in vitro study was performed to assess corneal damage potential of 4,4'-bis(2-methoxystyryl)-1,1'-biphenylby quantitative measurements of changes in opacity and permeability in a bovine cornea.

The test item 4,4'-bis(2-methoxystyryl)-1,1'-biphenyl was brought onto the cornea of a bovine eye which previously had been incubated with cMEM without phenol red at 32 ± 1 °C for 1 hour and whose opacity had been determined. The test item was incubated on the cornea for 4 hoursat32 ± 1 °C. After removal of the test item, opacity and permeability values were measured.

 

The test item was tested as 20% suspension in HBSS.

Under the conditions of this test, the test item 4,4'-bis(2-methoxystyryl)-1,1'-biphenyl showed no effects on the cornea of the bovine eye. The calculated mean IVIS was 0.54.

According to OECD Guideline no. 437 (Oct. 2017), a substance with an IVIS ≤ 3 requires no classification for eye irritation or serious eye damage.

 

The negative control (HBSS) and the positive control (20% imidazole solution) have met the validity criteria.

 

No observations were made which might cause doubts concerning the validity of the study outcome. The test is considered valid.