Palladium dihydroxide

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 Nov 2020-17 Dec 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Version / remarks:

9.12.2010

Test guideline 2

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:

Palladium dihydroxide

EC Number:

235-219-2

EC Name:

Palladium dihydroxide

Cas Number:

12135-22-7

Molecular formula:

H2O2Pd

IUPAC Name:

palladium(2+) dihydroxide

Details on test material:

Brown solid, purity 65.3% (as Palladium), stored at room temperature in the dark

Test animals / tissue source

Species:

cattle

Details on test animals or tissues and environmental conditions:

Test System: Freshly isolated bovine cornea (14 month old donor cattle)
Source: AB Schlachthof GmbH & Co. KG, 63739 Aschaffenburg, Germany

Collection of Bovine Eyes
Freshly isolated bovine eyes of donor cattle were collected from the abattoir. Excess tissue was removed from the excised eyes. The isolated eyes were stored in HBSS (Hank’s Balanced Salt Solution) containing 1% (v/v) penicillin/streptomycin (100 units/mL penicillin and 100 μg/mL streptomycin) in the cooled slaughter-house and during transportation on the same morning to the laboratory using a Styrofoam box. The corneas were isolated on the same day after delivery of the eyes.

Preparation of Corneas
All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity and scratches were discarded. The cornea was carefully removed from the eye using scalpel and rounded scissors. A rim of about 2 mm of tissue (sclera) was left for stability and handling of the isolated cornea. The corneas were directly used in the BCOP test on the same day.
Each isolated cornea was mounted in a specially designed cornea holder according to the description given in OECD guideline 437, which consists of anterior and posterior compartments, which interface with the epithelial and the endothelial sides of the cornea, respectively. The endothelial side of the cornea was positioned against the sealing ring (O-ring) of the posterior part of the holder. The cornea was gently flattened over the O-ring but stretching was avoided. The anterior part of the holder was positioned on top of the cornea and fixed in place with screws. Both compartments of the holder were filled with incubation medium. The posterior compartment was filled first to return the cornea to its natural convex position. Care was taken to assure that no air bubbles were present within the compartments.
For equilibration, the corneas in the holder were incubated in a vertical position for about one hour at 32 ± 1 °C in a water-bath. At the end of the incubation period, the medium was changed before basal opacity measurement (t0). Only corneas with a value of the basal opacity < 7 were used. Sets of three corneas were used for treatment with the test item and for the negative and positive controls, respectively.

Test system

Vehicle:

physiological saline

Remarks:

The test item was ground in a mortar with a pistil to improve its consistency and tested as a 20% suspension (w/v) in saline using sonication for 10 minutes.

Controls:

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

The anterior compartment received the test item suspension or the negative or positive controls at a volume of 0.75 mL each on the surface of the corneas via open chamber method, respectively.

Duration of treatment / exposure:

The corneas were incubated in a horizontal position at 32 ± 1 °C in the water-bath for 240 minutes.

Duration of post- treatment incubation (in vitro):

After exposure, the test item or the control items, respectively, were each rinsed off from the according application sides with EMEM containing phenol red for at least three times or more until phenol red was still discoloured (yellow or purple), or the test item was still visible. Since the test item proved difficult to remove by the rinsing method, the front cover of the holder was opened and the cornea was carefully washed using a gentle stream of incubation medium. Once the medium was free of the test item the corneas were given a final rinse with cMEM without phenol red.

Number of animals or in vitro replicates:

3 corneas per group (pos control, neg control, test item) were used.

Details on study design:

Opacity Measurement
The opacitometer determines changes in the light transmission passing through the corneas and displays a numerical opacity value.

Permeability Determination
Following to the opacity readings, the permeability endpoint was measured as an indication of the integrity of the epithelial cell sheets. After the final opacity measurement was performed, the incubation medium was removed from both chambers. The posterior chamber was filled with fresh cMEM first. Then the anterior compartment was filled with 1 mL of a 0.5% (w/v) sodium fluorescein solution in HBSS. Corneas were incubated again in a horizontal position for 90 ± 5 minutes in a water-bath at 32 ± 1 °C. Incubation medium from the posterior compartment was removed, well mixed and transferred into a 96 well plate. The optical density was measured with a microplate reader (Versamax® Molecular Devices) at 490 nm (OD490). The absorbance values were determined using the software SoftMax Pro Enterprise (version 4.7.1).

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Value:

5.87

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: No prediction can be made

Applicant's summary and conclusion

Interpretation of results:

other: No prediction can be made

Conclusions:

According to the current study (in accordance to OECD437) and under the experimental conditions reported, a prediction for the eye damage hazard cannot be made (GHS) for Palladium dihydroxide.

Executive summary:

This in vitro study was performed to assess the corneal irritation and damage potential of Palladium dihydroxide by means of the BCOP assay using fresh bovine corneas.
Sets of three corneas were used for treatment with the test item (Palladium dihydroxide as a 20% (w/v) suspension in saline (0.9% (w/v) NaCl in deionised water)) and for the negative and positive controls. Prior to treatment, opacity was measured in the fresh bovine corneas following equilibration (t0). 0.75 mL of the test item, positive or negative controls were each applied to different corneas fixed in an incubation chamber. The corneas were then incubated in a horizontal position for 240 minutes at 32 ± 1 °C. The posterior chamber contained incubation medium. After the incubation phase, the test item, the positive, and the negative controls were each rinsed from the corneas and opacity was measured again (t240).
After the opacity measurements, the posterior chamber was re-filled with fresh cMEM and then the anterior compartment was filled with 1 mL of a 0.5% (w/v) sodium fluorescein solution in HBSS. Corneas were incubated in a horizontal position for 90 minutes at 32 ± 1 °C and then the incubation medium from the posterior chamber was removed and transferred to a 96 well-plate. Permeability of the corneas was determined by assessing the transfer of sodium fluorescein after incubation, measured spectrophotometrically.
With the negative control (physiological saline), neither an increase of opacity nor permeability of the corneas was observed.
The positive control (10% (w/v) benzalkonium chloride in saline) showed clear opacity and increased permeability of the cornea, corresponding to a classification as serious eye damage (EU CLP/GHS Category 1).
Relative to the negative control, the test item Palladium dihydroxide caused an increase of the corneal opacity and permeability and the calculated mean in vitro irritancy score was 5.87. Since this value is between the cut-off values of 3 (threshold for no category) and 55 (threshold for serious eye damage) no prediction for corneal irritation and damage potential can be made according to OECD 437 (see table in chapter 3.9.3).
In conclusion, according to the current study and under the experimental conditions reported, a prediction for the damage hazard cannot be made (GHS) for Palladium dihydroxide.