Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22Apr2015
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:
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)
Qualifier:
according to
Guideline:
other: OECD (2014a) Draft Proposal for a New Test Guideline (EpiOcular™)
Version / remarks:
OECD (2014a) Draft
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
See individual study reports for purity information and CofA where applicable
Specific details on test material used for the study:
Test substance No.: 14/0424-1
Batch identification: 0008205061
CAS No.: 97404-02-9
Physical state / color: Solid I red

Test animals / tissue source

Species:
other: Both Cattle (BCOP) and human cornea model (Epiücular) were tsted in this study
Strain:
not specified
Details on test animals or tissues and environmental conditions:
BCOP: isolated corneas from the eyes of freshly slaughtered cattle
Human: three dimensional human cornea model Epiocular™

Test system

Vehicle:
other: Undiluted, 5%, and 50% (w/v) suspension in de-ionized water
Controls:
other: BCOP: Historical Positive and Negative Control. EpiOcular: Historical Positive, Negative Control and Viability, concurrent Kllled Control for MTT-reduction, Killed Control for Color Control
Amount / concentration applied:
BCOP: 750 μL of 50% and 5% test-substance preparations in deionized water
Epiocular: 50 μL bulk volume (about 12 mg) of the undiluted test substance or 50 μL of a 5% test-substance
Duration of treatment / exposure:
BCOP: 10 minutes
Epiocular: 6 hours
Duration of post- treatment incubation (in vitro):
BCOP: 2-hours post-incubation period.
Epiocular: 18-hours post-incubation period
Number of animals or in vitro replicates:
BCOP: Three
Epiocular: Two
Details on study design:
BCOP:
Washing performed after 10 mins, fluid not stated.

After determination of opacity and permeability, the corneas were fixed in 4% formaldehyde for at least 24 h and transferred to the laboratory of General Pathology for further histotechnical processing and examination by light microscopy.

Histopathological findings were summarized in a histopathological score of irritation (HSI) as follows:
0 = no findings
I = minimal
II = mild
III = moderate
IV= severe

Epiocular:
Washing performed after 6 hours, fluid not stated.

After application of the test material to the surface of the Epiocular™ tissue the induced cytotoxicity (= loss of viability) is measured by a colorimetric assay. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity. The mitochondrial dehydrogenase reduces the yellow colored water-soluble 3-[4,5-dimethylthiazol-2-yl]-2,5- diphenyltetrazolium bromide (MTT) to the insoluble blue colored formazan. After isopropanol extraction of the formazan from the tissues, the optical density of the extract is determined spectrophotometrically. Optical density of the extracts of test-substance treated tissues is compared to values from negative control tissues and expressed as relative tissue viability.

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
in vitro irritation score
Run / experiment:
BCOP, 50% test substance
Value:
1
Vehicle controls validity:
valid
Remarks:
vehicle control was negative control (water)
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
in vitro irritation score
Run / experiment:
BCOP, 5% test substance
Value:
1.5
Vehicle controls validity:
valid
Remarks:
vehicle control was negative control (water)
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
other: Mean Viability
Run / experiment:
Epiocular, Undiluted test substance
Value:
13.7
Vehicle controls validity:
valid
Remarks:
vehicle control was negative control (water)
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
other: Mean Viability
Run / experiment:
Epiocular, 5% test substance
Value:
44.5
Vehicle controls validity:
valid
Remarks:
vehicle control was negative control (water)
Negative controls validity:
valid
Positive controls validity:
valid

Applicant's summary and conclusion

Interpretation of results:
Category 1 (irreversible effects on the eye) based on GHS criteria
Conclusions:
Applying the criteria of the OECD 437 for the assessment of the BCOP test results, the IVIS scores of the test substance of 1.0 (50% test substance preparation) or 1.5 (5% test substance preparation) would classify it as a substance that does not cause eye irritation or serious eye damage. However, the Guideline also states, that due to limited accuracy of the BCOP test to correctly identify substances that do not require classification for eye irritation or serious eye damage, this test method should not be the first choice to initiate a bottom-up approach. Based on this statement and the experience of the test facility, test substances leading to IVIS <3 are generally examined in the EpiOcular test as well. In the current case, the EpiOcular test was positive and the test substance was assessed to be irritating.

Based on the results for BCOP and EpiOcular Test and applying the evaluation criteria described in section 3, the test item shows an eye irritation potential in the in vitro eye irritation test strategy under the test conditions chosen .
Executive summary:

Applying the criteria of the OECD 437 for the assessment of the BCOP test results, the IVIS scores of the test substance of 1.0 (50% test substance preparation) or 1.5 (5% test substance preparation) would classify it as a substance that does not cause eye irritation or serious eye damage. However, the Guideline also states, that due to limited accuracy of the BCOP test to correctly identify substances that do not require classification for eye irritation or serious eye damage, this test method should not be the first choice to initiate a bottom-up approach. Based on this statement and the experience of the test facility, test substances leading to IVIS<3 are generally examined in the EpiOcular test as well. In the current case, the EpiOcular test was positive and the test substance was assessed to be irritating.

 

Based on the results for BCOP and EpiOcular Test and applying the evaluation criteria described in section 3,the test item shows an eye irritation potential in thein vitro eye irritation test strategy under the test conditions chosen.