Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-07-10 to 2017-08-15
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 guideline
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
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
2-hydroxy-5-sulfobenzoic acid dihydrate
Cas Number:
5965-83-3
Molecular formula:
C7H6O6S*2H20
IUPAC Name:
2-hydroxy-5-sulfobenzoic acid dihydrate
Test material form:
solid

Test animals / tissue source

Species:
cattle
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Odenwaldschlachthof Brensbach, 64395 Brensbach, Germany
- Characteristics of donor animals: 11 - 135 months old
- Storage, temperature and transport conditions of ocular tissue: The eyes were kept and transported in transport medium (Hanks´ Balanced Salt Solution) cooled on ice.

Test system

Vehicle:
other: 0.9% sodium chloride solution
Controls:
yes, concurrent vehicle
yes, concurrent positive control
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 750 µL ( = 150 mg)
- Concentration: 20%

Duration of treatment / exposure:
4 h
Number of animals or in vitro replicates:
3 replicates
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
Freshly isolated bovine eyes of cattle were collected from the slaughterhouse. Excess tissue was removed from the eyes. The corneas were prepared immediately after delivery of the eyes to the laboratory. All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity or scratches were discarded. 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
For equilibration, the corneas in the holder were incubated (BSS 160, Grumbach Brutgerate GmbH, Asslar, Germany) in a vertical position at 32 ± 1 °C for about one hour. At the end of the incubation period, the incubation medium was replaced by fresh pre-warmed (32 ± 1°C) incubation medium in both compartments. The baseline 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. Three corneas were selected as negative control corneas. The remaining corneas were distributed into treatment and positive control group.

NUMBER OF REPLICATES
3

NEGATIVE CONTROL USED
0.9% sodium chloride solution

POSITIVE CONTROL USED
Imidazole

APPLICATION DOSE AND EXPOSURE TIME
750 µL, 20%, 4 h

TREATMENT METHOD
closed chamber

POST-INCUBATION PERIOD
no

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: 3 times with EMEM liquid medium with Earle's salts and phenol red (MEM Earle's)

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: calibrated opacitometer (BASF-OP2.0, BASF SE, Ludwigshafen, Germany)
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader (OD490)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: see "Any other information on materials and methods"

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Value:
184
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: No

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

Any other information on results incl. tables

Table 1: Summary of the results

 

Opacity

Permeability

IVIS

Per cornea

Per Group (Mean Value)

S.D.

Negative

control

0.0

0.001

0.015

0.4

0.9

1.4

0.000

1.400

-0.3

-0.001

-0.315

Positive

control

88.4

2.480

125.600

120.4

6.2

79.5

2.841

122.115

78.2

2.351

113.465

Test item

165.5

0.002

165.530

184.0

39.2

229.0

0.001

229.015

157.5

-0.002

157.470

Applicant's summary and conclusion

Interpretation of results:
Category 1 (irreversible effects on the eye) based on GHS criteria
Conclusions:
In an in vitro eye irritation study according to OECD 437 (BCOP), an IVIS of 184.0 was determined. The test item is considered to induce serious eye damage (UN GHS Category 1).
Executive summary:

The potential of the test substance to cause ocular irritation or serious damage to the eyes was assessed by a single topical application of 750 μL of a 20% (w/v) test item preparation to the epithelial surface of isolated bovine corneas according to OECD Guideline 437. Three corneas were treated with the test item preparation for an exposure period of 4 hours. In addition to the test substance a negative control (NC; 0.9 % sodium chloride solution) and a positive control (PC; 20% imidazole) were applied to three corneas each. Corneal opacity was measured quantitatively as the amount of light transmitted through the cornea. Permeability was measured quantitatively as the amount of sodium fluorescein dye that passes across the full thickness of the cornea. Both measurements were used to calculate an In Vitro Irritancy Score of the test substance. The test substance induced an IVIS score of 184.0. Based on the results for BCOP test and considering the decision criteria, the test substance does induce serious eye damage (UN GHS Category 1).

Furthermore, it was assumed, that the anhydrate form has the same corrosive potential to the eye and thus the result of the test item is also applicable