Registration Dossier

Toxicological information

Eye irritation

Currently viewing:

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Experimental study based on OECD guideline 437

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report Date:
2019

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 438 (Isolated Chicken Eye 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:
9 October 2017
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Benzylidenchlorid
No more data available

Test animals / tissue source

Species:
pig
Strain:
not specified
Details on test animals or tissues and environmental conditions:
The assay uses isolated corneas obtained as a by-product from animals freshly slaughtered at the abattoir A. Moksel AG, Buchloe, Germany.
On the test day, fresh eyes were collected from the slaughterhouse and were transported in HBSS containing Pen/Strep on ice to the laboratories. Immediately after arrival of the eyes, cornea preparation was initiated.
The eyes were carefully examined for defects and any defective eyes were discarded.
The tissue surrounding the eyeball was carefully pulled away and the cornea was excised leaving a 2 to 3 mm rim of sclera. The isolated corneas were stored in a petri dish containing HBSS. Before the corneas were mounted in corneal holders (Duratec GmbH) with the endothelial side against the O-ring of the posterior chamber, they had been visually examined for defects and any defective cornea had been discarded. The anterior chamber was then positioned on top of the cornea and tightened with screws. The chambers of the corneal holder were then filled with RPMI 1640 medium (without phenol red) containing 1% FBS and 2 mM L-glutamine (complete RPMI 1640 medium). The posterior chamber was always filled first. The corneas were incubated for one hour at 32 ± 1 °C.

Test system

Vehicle:
Hank's balanced salt solution
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
750 µL for 10 min incubation

3 corneas for the test item
3 corneas as negative controls treated with physiological saline 0.9% NaCl
3 corneas as positive controls treated with ethanol 100%
Duration of treatment / exposure:
After the equilibration period, the medium was removed from both chambers and replaced with fresh complete RPMI 1640 medium. An initial measurement was performed on each of the corneas using the opacitometer. Three corneas with illuminance readings approximately equivalent to the median illuminance of all corneas were selected as negative-control corneas. The illuminance of each cornea was read and recorded. Only corneas that had an initial illuminance reading I > I0/1.1651 lux were used for the assay. The medium was removed from the anterior chamber and replaced with the test item or control.
750 μL of the test substance or the control substance was introduced into the anterior chamber. After 10 minutes incubation at 32 ± 1 °C either the test substance or the control substance was removed and the epithelium washed at least three times with MEM (containing phenol red). Once the medium was free of test substance, the cornea was finally rinsed with complete RPMI 1640 medium (without phenol red). The anterior chamber was refilled with complete RPMI 1640 medium and an illuminance measurement was performed after 2 hours incubation at 32 ± 1 °C. Also, each cornea was observed visually and pertinent observations were recorded.
After the illuminance measurement was performed, the medium was removed from both chambers of the holder. The posterior chamber was refilled with fresh complete RPMI 1640 medium. 1 mL of a 4 mg/mL sodium fluorescein solution was added to the anterior chamber and the corneas were incubated for 90 minutes at 32 ± 1 °C. Then the medium from the posterior chamber was removed and its optical density at 490 nm (OD490) was determined, using a spectrophotometer (Jenway 6405 UV/VIS).
Duration of post- treatment incubation (in vitro):
2h
Number of animals or in vitro replicates:
3

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Run / experiment:
1
Value:
ca. 6.44
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
not determinable because of methodological limitations

In vivo

Irritant / corrosive response data:
The eye irritancy potential of Benzal chloride was investigated in the bovine corneal opacity and permeability assay.
The test item was tested as provided by the sponsor.
All 3 corneas treated with Benzal chloride showed moderate opacity of the tissue.
The following mean in vitro irritation score was calculated:
6.44

Any other information on results incl. tables

The eye irritancy potential of Benzal chloride was investigated in the bovine corneal opacity and permeability assay.

The test item was tested as provided by the sponsor.

All 3 corneas treated with Benzal chloride showed moderate opacity of the tissue.

The following mean in vitro irritation score was calculated:

6.44

No prediction can be made regarding the classification of the test substance Benzal chloride according to the evaluation criteria. Further testing in another suitable method is required.

The in vitro irritation score obtained with the positive control fell within the two standard deviations of the current historical mean and therefore this assay is considered to be valid.

The negative control responses resulted in opacity and permeability values that are less than the established upper limits for background bovine corneas treated with the respective negative control.

Applicant's summary and conclusion

Interpretation of results:
other: Slightly irritating
Conclusions:
No prediction can be made regarding the classification of the test substance Benzal chloride according to the evaluation criteria. Further testing in another suitable method is required. Based on these results and the one of other studies show a slightly irritating potential for benzal chloride.
Executive summary:

The eye irritancy potential of Benzal chloride was investigated in the bovine corneal opacity and permeability assay.

Preparation of the test item: tested as provided by the sponsor

Visual Observation after treatment: All 3 corneas treated with Benzal chloride showed moderate opacity of the tissue.

Mean in vitro irritation score: 6.44

The in vitro irritation score obtained with the positive control fell within the two standard deviations of the current historical mean and therefore this assay is considered to be valid.

The negative control responses resulted in opacity and permeability values that are less than the established upper limits for background bovine corneas treated with the respective negative control.