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Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 July 2017 - 22 August 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guidelineopen allclose all
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)
Version / remarks:
2013
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Version / remarks:
2017
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Tetrachloro-μ-hydroxy(μ-methacrylato-O:O')dichromium
EC Number:
239-147-2
EC Name:
Tetrachloro-μ-hydroxy(μ-methacrylato-O:O')dichromium
Cas Number:
15096-41-0
Molecular formula:
C10H23Cl4Cr2O5
IUPAC Name:
tetrachloro-μ-hydroxy(μ-methacrylato-O:O')dichromium
Test material form:
liquid

Test animals / tissue source

Species:
other: isolated bovine cornea
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Odenwaldschlachthof Brensbach, 64395 Brensbach, Germany
- Number of animals: not specified
- Characteristics of donor animals: 14 - 196 months old
- Storage, temperature and transport conditions of ocular tissue: The eyes were kept and transported in transport medium cooled on ice.
- Time interval prior to initiating testing: The corneas were prepared immediately after delivery of the eyes to the laboratory.
- Indication of any existing defects or lesions in ocular tissue samples: Eyes presenting defects such as vascularization, pigmentation, opacity and scratches were discarded.
- Indication of any antibiotics used: Streptomycin and Penicillin was added for the transport (5 mL/500 mL HBSS).

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 0.75 mL
Duration of treatment / exposure:
10 minutes
Duration of post- treatment incubation (in vitro):
120 minutes
Number of animals or in vitro replicates:
3 corneas per group
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
The corneas were carefully removed from the eyes using scalpel and rounded scissors. A rim of about 2 to 3 mm of tissue (sclera) was left for stability and handling of the isolated cornea. All corneas used in the study were collected in incubation medium (pre-warmed at 32 ± 1°C) and the corneal diameter of each cornea was measured and recorded. Each cornea was mounted in a cornea holder (CiToxLAB, Veszprem, Hungary) with the endothelial side against the sealing ring (O-ring) of the posterior part of the holder. The cornea was gently flattened over the O-ring without stretching the cornea. Afterwards, the anterior part of the holder was positioned on top of the cornea and fixed in place with screws. Both compartments of the holder were filled with incubation medium. The posterior compartment was filled first to return the cornea to its natural convex form.

QUALITY CHECK OF THE ISOLATED CORNEAS
All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity and scratches were discarded. At the end of the incubation period, the basal opacity was determined with a calibrated opacitometer (BASF-OP2.0, BASF SE, Ludwigshafen, Germany). The light transmission through the corneas, given as lux value, was recorded in a table and thereafter converted into an opacity value (baseline opacity values). Any corneas that showed macroscopic tissue damage (e.g. scratches, pigmentation, neovascularization) or an opacity > 7 opacity units were discarded.

NUMBER OF REPLICATES: 3

NEGATIVE CONTROL USED: Yes

POSITIVE CONTROL USED: Yes

APPLICATION DOSE AND EXPOSURE TIME: 0.75 mL and 10 minutes

POST-INCUBATION PERIOD: 120 minutes

REMOVAL OF TEST SUBSTANCE:
After the incubation period the corneal surface was washed three times with wash medium. Incubation medium was used as final rinse to ensure removal of the phenol red from the anterior chamber prior to the opacity measurement.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The light transmission through the corneas was determined with a calibrated opacitometer (BASF-OP2.0, BASF SE, Ludwigshafen, Germany).
- Corneal permeability: The amount of fluorescein that crossed the cornea was measured spectrophotometrically with a microplate reader (ELx800, BioTek Instruments GmbH, Bad Friedrichshall, Germany)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA:
IVIS ≤ 3 No Category (according to GHS)
IVIS > 3; ≤ 55 No prediction can be made
IVIS > 55 Serious eye damage according, Category 1 (according to GHS)

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
in vitro irritation score
Run / experiment:
1
Value:
156.7
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
in vitro irritation score
Run / experiment:
2
Value:
146
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
in vitro irritation score
Run / experiment:
3
Value:
132.4
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: none

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes

Any other information on results incl. tables

Study Acceptance Criteria

After treatment with the negative control (0.9 % sodium chloride solution) the calculated IVIS was 1.2 and, thus, within three standard deviations of the current historical mean of the negative control (IVIS: -1.1 - 5.3). After treatment with the positive control (N,N-dimethylformamide) the calculated IVIS was 95.2 and, thus, also within two standard deviations of the current historical mean of the positive control (IVIS 76.1 - 120.0). Therefore, the study fulfilled the acceptance criteria. The resulting classification of the test item in this study is unequivocal and no borderline results were obtained. Therefore, a single testing run composed of three corneas per group was considered sufficient.

Results

 

Opacity

Permeability

IVIS

per cornea

per group (mean value)

Standard Deviation

Negative Control

1.0

0.009

1.135

1.2

0.6

1.08

0.005

1.875

0.6

0.007

0.705

Positive control

80.5

0.236

84.040

95.2

11.0

86.1

0.615

95.325

87.2

1.259

106.085

Test Substance

156.7

0.000

156.700

145.0

12.2

145.6

0.027

146.005

132.3

0.008

132.420

Applicant's summary and conclusion

Interpretation of results:
Category 1 (irreversible effects on the eye) based on GHS criteria
Conclusions:
Based on the results of the OECD 437 study, the test item induced serious eye damage (UN GHS: Category 1).
Executive summary:

The objective of the present study was to examine the potential of the test substance to induce serious eye damage in the BCOP assay. To determine the eye hazard potential the induced opacity and increased permeability was investigated in isolated bovine corneas after exposure to the test item according to OECD Guideline 437. As negative control 0.9 % sodium chloride solution and as positive control N,N-dimethylformamide was used. Three corneas were used per group (negative control, positive control or test item group). After a first opacity measurement of the untreated bovine corneas, 750 µL of the test item, positive or negative control were applied on the corneas and incubated for 10 minutes. After the incubation phase the test item, the positive, and the negative control were rinsed from the corneas and the opacity was measured again. After the opacity measurements, the permeability of the corneas was determined by application of a fluorescein solution for 90 minutes. The amount of fluorescein solution that crossed the cornea was measured spectrophotometrically. The opacity and permeability assessments were combined to determine an In Vitro Irritancy Score (IVIS).

After treatment with the negative control (0.9 % sodium chloride solution) the calculated IVIS was 1.2 (study acceptance criteria range: -1.1 - 5.3). Treatment with the positive control (N,N-dimethylformamide) revealed an IVIS of 95.2 (study acceptance criteria range: 76.1 - 120.0). Therefore, the study fulfilled the acceptance criteria.

The IVIS obtained after treatment with the test item was 145.0 and, thus, higher than 55, i.e. according to OECD 437 the test item induced serious eye damage (UN GHS: Category 1).