Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental starting date: 19 August 2015 and Experimental completion date: 19 August 2015.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Deviations:
yes
Remarks:
The measurement of the opacity was performed with a photometer (570 nm) instead of an opacitometer. This can be seen as uncritical, because the opacity can be calculated from the absorbances. Deviation was signed and assessed by the S.D. on 20 August 2015
Qualifier:
according to guideline
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Deviations:
yes
Remarks:
The measurement of the opacity was performed with a photometer (570 nm) instead of an opacitometer. This can be seen as uncritical, because the opacity can be calculated from the absorbances. Deviation was signed and assessed by the S.D. on 20 August 2015
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
2,2'-{octahydro-1H-4,7-methanoindene-2,5-diylbis[(2,1-phenylene)oxymethylene]}bis(oxirane)
EC Number:
943-519-6
Cas Number:
2149603-92-7
Molecular formula:
C28H32O4
IUPAC Name:
2,2'-{octahydro-1H-4,7-methanoindene-2,5-diylbis[(2,1-phenylene)oxymethylene]}bis(oxirane)
Test material form:
not specified

Test animals / tissue source

Species:
other: Bos primigenius Taurus (fresh bovine cornea)
Strain:
not specified
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 Hank’s balanced salt solution (supplemented with 0.01% streptomycin and 0.01% penicillin). Then, the corneas were dissected and incubated in medium at 32 ± 1 °C in an incubation chamber for 1 hour.

Test system

Vehicle:
unchanged (no vehicle)
Remarks:
The test item is a solid substance. It was tested directly, without dilution or preparation of a solution.
Controls:
yes
Amount / concentration applied:
Tissue 1: 469.7 mg of the test item
Tissue 2: 394,3 mg of the test item
Tissue 3: 455.7 mg of the test item
Duration of treatment / exposure:
4 hours
Observation period (in vivo):
Not applicable
Number of animals or in vitro replicates:
Not applicable
Details on study design:
Conduct of the study:
Preparations:
After having carefully cleaned and sterilized 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.
After the arrival of the corneas, they were examined and only corneas which were free from damages were used. The corneas were excised with a scalpel and cut from the globe with a 2-3 mm ring of sclera around the outside. Each cornea was transferred to a cornea holder in which pre-warmed cMEM without phenol red was filled. The holders were then incubated for 1 h in the incubation chamber at 32 ± 1 °C.

Method Description
After the initial incubation, the medium was changed and the baseline opacity for each cornea was recorded. None of the corneas showed tissue damage; therefore, all corneas were used.
The baseline opacity was measured by placing the holder with the cornea in a spectral photometer and recording the absorbance at 570 nm.
For each treatment group (negative control solution, test item and positive control solution), three replicates were used. After removal of the pre-incubation medium, 750 μL negative control solution resp. positive control solution were applied to each replicate.
According to the characteristics of the test item, the following treatment procedure was performed:

Open Chamber Method
The “open chamber-method” is used for solids. In order to apply the test item, the nut was unscrewed to remove the glass disc. The test item could be applied directly on the cornea now.

The test item was given on the epithelium in such a manner that as much as possible of the cornea was covered with the test item.
Exposition time on the corneas was 4 h at 32 ± 1 °C. After thorough rinsing with cMEM with phenol red and final rinsing with cMEM without phenol red, both chambers were filled with cMEM without phenol red, and the final opacity value of each cornea was recorded at once (again by measurement at 570 nm). The cMEM without phenol red was then removed from the front chamber, and 1 mL sodium fluorescein solution (concentration:
5 mg/mL) was added to the front chamber.
The chambers were then closed again and incubated for 90 min at 32 ± 1 °C. After incubation, the content of the posterior chamber was thoroughly mixed. Then, the permeability of the cornea was measured as optical density of the liquid at 490 nm.

SCORING SYSTEM: Classification scheme.

IVIS<=3: No cetgory
IVIS >3 and <=55: No prediction can be made
IVIS>55: Eye damage category 1

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Value:
< 3
Remarks on result:
no indication of irritation

Any other information on results incl. tables

The calculated IVIS for each replicate and the corresponding means are presented in the following table:

 TEST GROUP IVIS  MEAN IVIS  Relative standard deviation IVIS 
   1.84    
 Negative control 0.9% NaCl  1.51  1.43  31.6%
   0.95    
   -0.20    
 Test item  -0.92  -0.61  60.3%
   -0.70    
   73.79    
 Positive control imidazole 20%  56.25  62.88  15.1%
   58.60    

VALIDITY:

According to the guideline, the test is considered as valid if the positive control causes an IVIS that falls within two standard deviations of the current historical mean. The negative control has to show an IVIS ≤ 3.

The validity criteria and findings are given in the following table:

 Parameter  Criterion  Found  Assessment
 IVIS of negative control 0.9% NaCl  <=3  1.43  OK
 IVIS of positive control 20% imidazole solution  33.19 -135.95 62.88   OK

Values for negative and positive controls were within the range of historical data of the test facility. Therefore, the test system was acceptable.

COMPARISON WITH HISTORICAL DATA:

In the following table, the means of the negative control and positive control of all experiments which were performed with this positive control up to 16. Jun. 2015 are stated and compared with the values which were found in this study.

 Parameter  IVIS negative control  IVIS positive control
 Substance  0.9% sodium chloride solution  20% imidazole solution
 Mean  1.18 84.57 
 Standard deviation  0.93 25.69 
 Range (min-max)  0.13 -6.86 32.62 -161.29 
 Range (validity)  <=3 33.19 -135.95 
 Study 15061202G850  1.43 62.88 

Applicant's summary and conclusion

Interpretation of results:
study cannot be used for classification
Remarks:
Migrated information
Conclusions:
Under the conditions of this study, the test item 2,5-bis[(glycidyloxy)phenyl]octahydro-4,7- methano-5h-indene (TK30250) showed no effects on the cornea of the bovine eye. The calculated IVIS (in vitro irritancy score) is -0.61.
According to OECD Guideline no. 437 (Jul. 2013), a substance with an IVIS ≤ 3 requires no classification for eye irritation or serious eye damage.
Executive summary:

This in vitro study was performed to assess corneal damage potential of 2,5- bis[(glycidyloxy)phenyl]octahydro-4,7-methano-5h-indene (TK30250) by quantitative measurements of changes in opacity and permeability in a bovine cornea.

The test item 2,5-bis[(glycidyloxy)phenyl]octahydro-4,7-methano-5h-indene (TK30250) 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 h and whose opacity had been determined.

The test item was incubated on the cornea for 4 h at 32 ± 1 °C. After removal of the test item, opacity and permeability values were measured.

The test item was tested pure.

Under the conditions of this test, the test item 2,5-bis[(glycidyloxy)phenyl]octahydro-4,7- methano-5h-indene (TK30250) showed no effects on the cornea of the bovine eye. The calculated IVIS (in vitro irritancy score) is -0.61.

According to OECD Guideline no. 437 (Jul. 2013), a substance with an IVIS ≤ 3 requires no classification for eye irritation or serious eye damage.

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

Physiological sodium chloride solution was used as negative control. The negative control showed no irritating effect on the cornea and the calculated IVIS (in vitro irritancy score) is 1.43.

20% Imidazole solution was used as positive control. The positive control induced serious eye damage on the cornea and falls within two standard deviations of the current historical mean. The calculated IVIS (in vitro irritancy score) is 62.88.

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