Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Test according to internationla guidelines, but the result of the test item is considered not fully according to validiay criteria due to the fact that the physicochemical properties of the test item do not allow to remove the test item completely from the cornea. The results, however, suggfest that the test item does not cause serious eye damage.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
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
Qualifier:
according to
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Qualifier:
according to
Guideline:
other: OECD No.160. Guidance document on the bovine corneal opacity and permeability (BCOP) and isolated chicken eye (ICE) test methods: collection of tissues for histological evaluation and collection of data on non-severe irritants; 25. Oct.2011
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
Identification: V516690
Appearance: Brown powder
Batch: V516690/AY
Purity/Composition: LC-MSpurity (Area% UV-VIS): 95.8% (Reference: PA064492)
Test item storage: At room temperature
Stable under storage conditions until 17 July 2016 (expiry date)
Molecular formula: C49H49ClN2O6S2
MW: 861.52

Test animals / tissue source

Species:
other: Bos primigenius Taurus
Strain:
other: 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 Hank’s balanced salt solu tion (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 h.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes
Amount / concentration applied:
The test item was applied directly on the cornea
Tissue 1: 292.7 mg
Tissue 2: 282.8 mg
Tissue 3: 305.5 mg
Duration of treatment / exposure:
Incubation time; 4h and 5 minutes
Observation period (in vivo):
4 hours and 5 minutes at 32 +/-1°C
Number of animals or in vitro replicates:
3 tissue samples
Details on study design:
After having carefully cleaned and sterilised the cornea holders, they were kept in the in- cubation chamber at 32 ± 1 °C.
On the day of the assay, the MEM without phenol red was supplemented with sodium bi- carbonate, 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 (32 ± 1 °C) without phenol red was filled. The holders were then incubated for 1 h in the incubation chamber at 32 ± 1 °C.

Results and discussion

In vivo

Resultsopen allclose all
Irritation parameter:
other: IVIS (In vitro Irritancy Score of the BCOP test)
Basis:
mean
Time point:
other: 4 hours and 5 minutes
Score:
3.47
Max. score:
100
Remarks on result:
other: Relative Standard Deviation of IVIS was 64.8%

Any other information on results incl. tables

Table 9.2-a IVIS

Test Group

IVIS

Mean IVIS

Relative Standard Deviation IVIS

Negative Control 0.9% NaCl

0.78

0.58

69.7%

0.84

0.11

Test Item V516690

1.28

3.47

64.8%

5.78

3.36

Positive Control 20% imidazole solution

73.64

79.23

12.7%

73.18

90.88

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.

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

Parameter

Criterion

Found

Assessment

IVIS of negative control 0.9% NaCl

≤3

0.58

ok

IVIS of positive control 20% imidazole solution

33.87 – 134.19

79.23

ok


Applicant's summary and conclusion

Conclusions:
This in vitro study was performed to assess corneal damage potential of V516690 by quantitative measurements of changes in opacity and permeability in a bovine cornea. The test item V516690 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 and 5 min. at 32 ± 1 °C. After removal of the test item, opacity and permeability values were measured. The negative control (physiological sodium chloride solution) and the positive control (undiluted dimethylformamide) have met the validity criteria. The test item was tested pure. Under the conditions of this study, the test item V516690 showed effects on the cornea of the bovine eye. The calculated IVIS (in vitro irritancy score) is 3.47. As the test substance could not be completely removed from the cornea, the opacity value in the photometrical measurement was too high. This is why it is possible, that the IVIS value of the test item was wrongly too high and the result of the study cannot be assessed. But it can be stated that the test item does not cause serious eye damage. The result of the test item is considered not valid due to the fact that the physicochemical properties of the test item do not allow to remove the test item completely from the cornea.