2-{[(2-{3-[(4-{Ethyl[3-(vinylsulfonyl)phenyl]amino}-6-fluoro-heteromonocycle-2-yl)amino]-2- (hydroxy-O)phenyl}hydrazono-N2)(phenyl)methyl]diazenyl-N1}benzoato(6-)-O]cuprate(4-) sodium salt, polysulfo

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011-09-07

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliance

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD 437

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Test animals / tissue source

Species:

other: cattle

Details on test animals or tissues and environmental conditions:

the BCOP test method uses isolated corneas from the eyes of freshly slaughtered cattle. Eyes are collected in the slaughterhouse, immersed completely in Hanks' Balanced Salt Solution (HBSS) and transported to the laboratory in such a manner as to minimize deterioration and/or bacterial contamination.

Test system

Vehicle:

physiological saline

Controls:

yes, concurrent vehicle

Amount / concentration applied:

The test item as dissolved with physiological saline to gain a 20% concentration

Duration of treatment / exposure:

4 hours +/- 5 minutes at 32 +/-1°C

Observation period (in vivo):

after the incubation period the test substance or the control substance was removed and the epithelium washed at least three times with MEM. The anterior chamber was refilled with complete RPMI and an opacity measurement was performed. After the opacity measurement the medium was reomved from both chambers of the holder. The posterior chamber was refilled with fresh complete RPMI. 1mL of a 5 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 ant its optical density at 490 nm was determined using a spectrophotometer.

Number of animals or in vitro replicates:

3 corneas for the test item
3 corneas for the negative control treated with physiological saline
3 corneas as positive control treated with imidazole 20% in physiological saline

Details on study design:

Preparation of the Corneas:
The assay uses isolated corneas obtained as a by-product from an abattoir from freshly slaughtered animals.
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. Corneas were then mounted in corneal holders (MC2, Clermont, France) with the endothelial side against the O-ring of the posterior chamber. 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 in a water bath.
Treatment of the Corneas:
After the incubation period, the medium was removed from both chambers and replaced with fresh Complete RPMI. An initial opacity measurement was performed on each of the corneas using an opacitometer (MC2, Clermont, France). Three corneas with opacity readings approximately equivalent to the median opacity of all corneas were selected as negative-control corneas. The opacity of each cornea was read against an air-filled chamber and recorded. Corneas that have an initial opacity reading above 7 units were not dosed. The medium was removed from the anterior chamber and replaced with the test item or control.
750 L of the test item preparation or the control substance was introduced into the anterior chamber. After 4 hours ± 5 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 opacity measurement was performed.
After the opacity measurement the medium was removed from both chambers of the holder. The posterior chamber was refilled with fresh complete RPMI. 1 mL of a 5 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.
Test Groups:
3 corneas for the test item
3 corneas as negative controls treated with physiological saline 0.9% NaCl
3 corneas as positive control treated with imidazole 20% in physiological saline 0.9% NaCl
The BCOP assay is considered to be valid if the in vitro score obtained with the positive control falls within the two standard deviations of the current historical mean.
Evaluation of Results:
The change in opacity for each cornea was calculated by subtracting the initial opacity reading from the final opacity reading. These values were corrected by subtracting from each the average change in opacity observed for the negative-control corneas. The mean opacity value for each treatment was calculated by averaging the corrected opacity values of each cornea for a given treatment.
The mean OD490 for the blank wells were calculated. The mean blank OD490 was subtracted from the OD490 of each well (corrected OD490). Any dilutions that were made to bring the OD490 values into the linear range of the spectrophotometer (OD490 should be less than 1.500), were taken into account by multiplying the OD490 value of the dilution by the dilution factor. The final-corrected OD490 of the test article and the positive control were calculated by subtracting the average corrected OD490 of the negative control corneas from the corrected OD490 value of each treated cornea:
Final-corrected OD490 = (OD490 – mean blank OD490) – average-corrected negative control OD490
The mean OD490 value of each treatment group was calculated by averaging the final corrected OD490 values of the treated corneas for that treatment condition.
The following formula was used to determine the in vitro score:
In vitro score = mean opacity value + (15 x mean OD490 value)

Results and discussion

In vivo

Results

Irritation parameter:

other: in vitro score

Basis:

other: corneas: mean value

Time point:

other: 4 hours

Score:

26.16

Remarks on result:

other: score is calculated only for the mean values; as in vitro method no reversibilty

Irritant / corrosive response data:

The eye irritancy potential of FAT 40853/A TE was investigated in the bovine corneal opacity and permeability assay.
The test item was dissolved with physiological saline 0.9% NaCl to gain a 20% concentration.
The following mean in vitro score was calculated:
26.16
Therefore the test item was classified as moderate irritant.
The in vitro 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.

Applicant's summary and conclusion

Interpretation of results:

moderately irritating

Remarks:

Migrated information Criteria used for interpretation of results: OECD GHS

Conclusions:

According to the evaluation criteria the test item FAT 40853/A TE is classified as moderate eye irritant.

Executive summary:

The eye irritancy potential ofFAT 40853/A TEwas investigated in the bovine corneal opacity and permeability assay.

 

Preparation of the test item:                     dissolvedwith physiological saline 0.9% NaCl to gain a 20% concentration

Mean in vitro score:                                26.16

Classification: moderate irritant

The in vitroscore 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.

 Conclusion:

According to the evaluation criteria the test itemFAT 40853/A TEis classified as moderate eye irritant.