Escherichia coli dehalogenase catalyst

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

31st January 2018

Reliability:

1 (reliable without restriction)

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Unknown 6

IUPAC Name:

Unknown 6

Constituent 2

Reference substance name:

Unknown 7

IUPAC Name:

Unknown 7

Constituent 3

Reference substance name:

Unknown 5

IUPAC Name:

Unknown 5

Constituent 4

Reference substance name:

Unknown 4

IUPAC Name:

Unknown 4

Constituent 5

Reference substance name:

Unknown 3

IUPAC Name:

Unknown 3

Constituent 6

Reference substance name:

Unknown 2

IUPAC Name:

Unknown 2

Constituent 7

Reference substance name:

Unknown 1

IUPAC Name:

Unknown 1

Test material form:

solid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 11879017
- Expiration date of the lot/batch: August 2018

RADIOLABELLING INFORMATION : Not applicable.

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Not specified
- Stability under test conditions: Stable
- Solubility and stability of the test substance in the solvent/vehicle: Test item suspended in saline 0.9% NaCl to give a 20% concentration. Test item stable in the test medium.

Test animals / tissue source

Species:

cattle

Strain:

not specified

Test system

Vehicle:

physiological saline

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Concentration (if solution): 20% solution of test item in physiological saline 0.9% NaCl

Duration of treatment / exposure:

4 hours ± 5 minutes at 32 ±1oC

Observation period (in vivo):

Not applicable

Duration of post- treatment incubation (in vitro):

90 minutes at 32 ±1oC

Number of animals or in vitro replicates:

3 replicates in each test group.

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
The corneas were excised leaving 2 – 3 mm of sclera and stored in HBSS solution. A visual inspection of the excised corneas was performed before the corneas were mounted in corneal holders, any defective corneas were discarded. The corneal holders were filled with RPMI (without phenol red) containing 1% FBS and 2mM L-glutamine (complete RPMI). The corneas were incubated for one hour at 32 ±1oC.

QUALITY CHECK OF THE ISOLATED CORNEAS
After equilibration of the corneas, the medium was removed from both chambers and replaced with fresh RPMI. Initial measurement of the opacity using the opacitometer was performed, only corneas with initial illuminance readings I > I0/1.1651 lux were used in the assay.

NUMBER OF REPLICATES
Three replicates were used for each of the test item, negative control and positive controls.

NEGATIVE CONTROL USED
The three corneas with illuminance reading closest to the median value across all corneas were selected as negative control corneas.

POSITIVE CONTROL USED
Imidazole 20% in physiological saline 0.9% NaCl.

APPLICATION DOSE AND EXPOSURE TIME
750µl of test item (20% in physiological saline 0.9% NaCl) or control substance was introduced into the anterior chamber. After an incubation period of 4 hours ±5 minutes at 32 ±1oC the test item of control substance was removed.

TREATMENT METHOD: closed chamber

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: After removal of the test item or control substance, the epithelium was washed a minimum of three times with MEM (containing phenol red). Once the medium was free of test substance a final rinse with complete RPMI (without phenol red) was performed. The chambers were refilled with complete RPMI and illuminance measurement recorded.

POST-INCUBATION PERIOD: 90 minutes at 4 hours ± 5 minutes at 32 ±1oC

POST-EXPOSURE INCUBATION: Post illuminance measurement the medium was removed from both chambers of the holder. The posterior chamber was filled with fresh complete RPMI, the anterior chamber was filled with 1ml of a 5 mg/ml sodium fluorescein solution and the cornea incubated for 90 minutes at 4 hours ± 5 minutes at 32 ±1oC.
After incubation, the medium from the posterior chamber was removed and the optical density of this solution, at 490 nm, determined using a spectrophotometer.
METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Corneal opacity was measured using a BASF-OP3.0, Duratec GmbH opacitometer.
- Corneal permeability: Passage of sodium fluorescein dye measured with the aid of UV/VIS spectrophotometry
SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
DECISION CRITERIA: The decision criteria were in accordance with OECD Guideline 437 and GHS. IVIS values ≤ 3, No GHS Category; IVIS values > 3 but ≤55, No prediction can be made with respect to eye irritancy; IVIS values >55, Serious eye damage, Category 1.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

Mean value reported

Value:

ca. 4.73

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable because of methodological limitations

Any other information on results incl. tables

Please refer to document attached below (BCOP Study 179041 Results Tables) .

Applicant's summary and conclusion

Interpretation of results:

Category 2 (irritating to eyes) based on GHS criteria

Remarks:

Classified using the precautionary principle on the basis that the in vitro skin irritation study results are not yet available.

Conclusions:

A mean in vitro irritation score of 4.73 was obtained, this value falls between the cut-off values for no classification required and Category 1: Causes severe eye damage. As a result, no prediction with respect to eye irritancy can be made.

Executive summary:

The eye irritancy potential of Escherichia coli dehalogenase catalyst was investigated in a bovine corneal opacity and permeability assay. The assay was performed to GLP and in accordance with OECD Guideline 437.

The test item was applied as a suspension in physiological saline 0.9% NaCl at a concentration of 20%. Triplicate assays were run for the test item, positive and negative controls. A mean in vitro irritation score of 4.73 was obtained, this value falls between the cut-off values for no classification required and Category 1: Causes severe eye damage. As a result, no prediction with respect to eye irritancy can be made.