Phosphoric acid, mixed esters with alcohols, C7-9-iso-, C8-rich and alcohols, C11-14-iso-, C13-rich, ethoxylated, potassium salt

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July-September 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 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:

Phosphoric acid, mixed esters with alcohols, C7-9-iso-, C8-rich and alcohols, C11-14-iso-, C13-rich, ethoxylated, potassium salt

EC Number:

947-738-8

Molecular formula:

Molecular formulas of exemplary components: C33H68KO10P C49H100KO16P C24H49K2O10P C24H50KO10P C16H34KO4P C8H17K2O4P C8H18KO4P

IUPAC Name:

Phosphoric acid, mixed esters with alcohols, C7-9-iso-, C8-rich and alcohols, C11-14-iso-, C13-rich, ethoxylated, potassium salt

Test material form:

liquid

Test animals / tissue source

Species:

cattle

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.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

750 uL of the test substance or the control substances

Duration of treatment / exposure:

10 minutes

Observation period (in vivo):

N/A

Duration of post- treatment incubation (in vitro):

2 hours

Number of animals or in vitro replicates:

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%

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
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 (without phenol red) containing 1% FBS and 2 mM L-glutamine (complete RPMI). The posterior chamber was always filled first. The corneas were incubated for one hour at 32 +/- 1 °C.


TREATMENT METHOD
After the equilibration period, the medium was removed from both chambers and replaced with fresh complete RPMI. 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 uL of the test substance or the control substance was introduced into the anterior chamber. As the viscosity of the test item was relatively high, it was applied directly onto the cornea by removing the window-locking ring and glass window prior to treatment. 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 (without phenol red). The anterior chamber was refilled with complete RPMI 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.
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).

DECISION CRITERIA: as per OECD TG 437

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

in vitro irritation score

Run / experiment:

mean value

Value:

46.54

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation parameter:

cornea opacity score

Remarks:

corrected opacity value

Run / experiment:

mean value

Value:

10.47

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

Irritation parameter:

other: permeability

Remarks:

corrected OD490 value

Run / experiment:

mean value

Value:

2.405

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

All 3 corneas treated with test item showed slight opacity of the tissue.

No prediction can be made regarding the classification of the test substance Strodex PK-90 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: No prediction can be made

Remarks:

3 < IVIS <= 55

Conclusions:

No prediction can be made regarding the classification of the test substance according to the evaluation criteria.