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Eye irritation

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Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
20 Dec 2016 to 23 Nov 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

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:
26 July 2013
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
BASF SE, Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany

Test material

Constituent 1
Chemical structure
Reference substance name:
4,5,6,7-tetrahydro-1H-benzotriazole
EC Number:
229-858-6
EC Name:
4,5,6,7-tetrahydro-1H-benzotriazole
Cas Number:
6789-99-7
Molecular formula:
C6H9N3
IUPAC Name:
4,5,6,7-tetrahydro-1H-1,2,3-benzotriazole
Details on test material:
Physical state, appearance: solid, white
Storage conditions: room temperature

Test animals / tissue source

Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Schlachthof Mannheim, Schlachthofstr. 21, 68165 Mannheim, Germany
- Characteristics of donor animals: age min. 12 months, max. 60 months
- indication of any existing defects or lesions in ocular tissue samples: Only Corneas free of defects were used

Test system

Vehicle:
water
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Concentration (if solution):
Treatment: 750 µL, 20 % (w/v) test substance in water
Negative control: 750 µL de-ionized water
Positive control: 750 µL, 20 % (w/v) imidazole/de-ionized water
Duration of treatment / exposure:
Corneal opacity: 4 h at 32 °C
Duration of post- treatment incubation (in vitro):
Permeability: replacement of the test substance with 1 mL sodium fluorescin solution (5 mg/mL) and 90 ±5 min. incubation at 32 °C
Number of animals or in vitro replicates:
3 corneas in each group (Treatment, NC, PC)
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
Corneas free of defects (opacity, scratches, pigmentation etc.) were dissected with a 2 to 3 mm rim of sclera. Isolated corneas were mounted in cornea holders that consists of anterior and posterior chambers. Both chambers were filled to excess with pre-warmed Eagle’s MEM (without phenol red) and then equilibrated in a vertical position at about 32 °C for at least 1 hour.

QUALITY CHECK OF THE ISOLATED CORNEAS
After the equilibration period the medium in both chambers was replaced with fresh prewarmed medium and initial corneal opacity readings were taken for each cornea with an opacitometer. Any corneas that showed macroscopic tissue damage or an opacity value < 548 opacity units were discarded.

NUMBER OF REPLICATES
3

NEGATIVE CONTROL USED
Yes, 750 µL de-ionized water

POSITIVE CONTROL USED
Yes, 20% (w/v) solution of imidazole in de-ionized water

POST-INCUBATION PERIOD
no

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: at least 3 times with Eagle’s MEM (containing phenol red) and once with Eagle’s MEM (without phenol red).

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: corneal opacity readings were taken for each cornea with an opacitometer
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of a 96-well microtiter plate reader (OD490)

SCORING SYSTEM:
In Vitro Irritancy Score (IVIS)
Evaluation of the test results based on to the criteria given in OECD 437 (July 2013).

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
in vitro irritation score
Remarks:
1st run
Run / experiment:
BCOP Test
Value:
60.7
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
in vitro irritation score
Remarks:
2nd run
Run / experiment:
BCOP Test
Value:
55.4
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: Not reported

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: 1st test run: mean values for opacity, permeability and IVIS of the test substance, the NC and the PC

Test substance identification Mean Opacity Value Mean Permeability Value Mean  In VitroIrritancy
Score
test article 1.7 3.935 60.7
NC 6.2 0.003 6.3
PC 89.6 2.484 126.9

Table 2: 2nd test run: mean values for opacity, permeability and IVIS of the test substance, the NC and the PC

Test substance identification Mean Opacity Value Mean Permeability Value Mean  In VitroIrritancy
Score
test article 0.0 3.692 55.4
NC 14.1 0.003 14.1
PC 112.0 3.222 160.3

Applicant's summary and conclusion

Interpretation of results:
study cannot be used for classification
Conclusions:
The mean IVIS of both test runs was calculated to be 58.1. Of the six corneas tested, only one cornea produced an IVIS value above the cut-off for severe eye damage, five corneas showed borderline results.
The overall borderline results of the BCOP Tests do not clearly allow for the evaluation of risk of serious eye damage. On basis of the results of this study, a potential of the test substance to bear a risk of serious eye damage cannot be excluded. According to OECD criteria, the substance is considered to cause serious eye damage. Classification with Category 1 is therefore recommended.
Executive summary:

The potential of the test item to cause ocular irritation or serious damage to the eyes was assessed by a single topical application of 750 μL of a 20% testsubstance preparation in deionized water to the epithelial surface of isolated bovine corneas. Three corneas were treated with the test-substance preparation for an exposure period of 4 hours. In addition to the test substance, a negative control (NC; deionized water) and a positive control (PC; 20% imidazole in deionized water) were applied to three corneas each. Corneal opacity was quantitatively measured as the amount of light transmitted through the cornea. Permeability was quantitatively measured 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. Two test runs of the BCOP assay were performed. Due to high variability in single IVIS values of corneas treated with the test substance (IVIS values for single corneas: 57.1, 48.7 and 76.4) and the borderline result (mean IVIS = 60.7), a 2nd test run was performed to clarify the result. Slight compound residues remained on the test-substance treated corneas after the washing procedure of the 2nd test run. The IVIS values for the single corneas of the 2nd test run were: 57.9, 53.6 and 54.6. The mean IVIS of both test runs was calculated to be 58.1. Of the six corneas tested, only one cornea produced an IVIS value above the cut-off for severe eye damage, five corneas showed borderline results.

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