Disodium [5-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-4-hydroxybenzene-1,3-disulphonato(4-)]cuprate(2-)

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-06-20 to 2017-08-16

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

according to OECD and GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD Guideline for the Testing of Chemicals, no. 437 (adopted: 26 July 2013)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Munich, Germany

Test material

Test material information

Constituent 1

Reference substance name:

Disodium [5-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-4-hydroxybenzene-1,3-disulphonato(4-)]cuprate(2-)

EC Number:

276-541-3

EC Name:

Disodium [5-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-4-hydroxybenzene-1,3-disulphonato(4-)]cuprate(2-)

Cas Number:

72252-61-0

Molecular formula:

C16H10CuN4O8S2.2Na

IUPAC Name:

disodium [5-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-4-hydroxybenzene-1,3-disulphonato(4-)]cuprate(2-)

Test material form:

solid: nanoform

Test system

Vehicle:

physiological saline

Amount / concentration applied:

The test item was suspended with physiological saline 0.9% NaCl (AlleMan Pharma, lot no. 609709, expiry date: 21/06/2017) to give a 20% concentration. 750 µL of the test item preparation or the control substance was introduced into the anterior chamber (closed-chamber method).

Duration of treatment / exposure:

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).

Duration of post- treatment incubation (in vitro):

1 mL of a 5 mg/mL sodium fluorescein solution was added to the anterior chamber and the corneae were incubated for 90 minutes at 32 ± 1 °C.

Number of animals or in vitro replicates:

3 corneae per group

Details on study design:

Test System

Preparation of the Corneae:
The assay uses isolated corneae 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 corneae were stored in a petri dish containing HBSS. Before the corneae 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 corneae were incubated for one hour at 32 ± 1 °C.

Calibration of the Opacitometer:
The opacitometer (BASF-OP3.0, Duratec GmbH) was switched on at least 15 min before starting the calibration procedure. The filter holder was placed into the opacitometer and the readout was adjusted to 1000 lux ± 10 lux using the “Calibrate”-turning knob. For calibration the glass filter F2 was introduced into the filter holder. The readout lay in the range between 540-560 lux. To test the linearity of the measurement, two additional calibration filters, glass filter F3 and glass filter F4, were measured. For these glass filters, the opacitometer displayed values between 300-310 lux and between 95-105 lux. The calibration procedure was performed before the test and is documented in the raw data.

Treatment of the Corneae:
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 corneae using the opacitometer. Three corneae with illuminance readings approximately equivalent to the median illuminance of all corneae were selected as negative-control corneae. The illuminance of each cornea was read and recorded. Only corneae 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 item preparation or the control substance was introduced into the anterior chamber (closed-chamber method). 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 illuminance measurement was performed. 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 5 mg/mL sodium fluorescein solution was added to the anterior chamber and the corneae 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).

Test Groups:
3 corneae for the test item
3 corneae as negative controls treated with physiological saline 0.9% NaCl
3 corneae as positive controls treated with imidazole 20% in physiological saline 0.9% NaCl
The BCOP assay is considered to be valid if the in vitro irritation score obtained with the positive control falls within the two standard deviations of the current historical mean.
The negative control responses should result in opacity and permeability values that are less than the established upper limits for background bovine corneae treated with the respective negative control.


Evaluation of Results:
TThe following formula was used to calculate the opacity, whereas the values a and b are equipment-specific variables empirically determined by the manufacturer:
Opacity = (I0/I - b)/a

with a = 0.025 and b = 0.9894

The value I0 = I(zero) is the illuminance through a holder without cornea, but with windows and liquid. This value is determined by taking the mean for a set of cornea holders and is reevaluated periodically.
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 cuvettes was calculated. The mean blank OD490 was subtracted from the OD490 of each cuvette (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 irritation score (IVIS):
IVIS = mean opacity value + (15 x mean permeability OD490 value)
An identification of test substances that should be classified as irritating to eyes (UN GHS Category 2 or Category 2A) or test substances that should be classified as mildly irritating to eyes (UN GHS Category 2B) cannot be made.
For this purpose further testing with another suitable method is required

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Value:

ca. 68.76

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

The eye irritancy potential of the test item was investigated in the bovine corneal opacity and permeability assay.
The test item was suspended with physiological saline 0.9% NaCl to give a 20% concentration.
All 3 corneae treated with the test item showed an orange discoloration and opacification of the tissue.
The following mean in vitro irritation score was calculated:
68.76

Applicant's summary and conclusion

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Conclusions:

According to the bovine corneal opacity and permeability assay, since the IVIS was > 55, the test item can be considered as inducing eye irritation or serious eye damage.

Executive summary:

 

Summary Results

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

Preparation of the test item:                   The test item was suspended with physiological saline 0.9% NaCl to give a 20% concentration.

Visual Observation after treatment:       All 3 corneae treated with the test item showed an orange discoloration and opacification of the tissue.

Mean in vitro irritation score:                   68.76

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 corneae treated with the respective negative control.

Conclusion

According to the bovine corneal opacity and permeability assay, since the IVIS was > 55, the test item can be considered as inducing eye irritation or serious eye damage.

Thus, the test item has to be classified as eye irritant cat. 1, H318 Causes serious eye damage.