Disodium tetrachloropalladate

Endpoint summary

Description of key information

No irritation was observed following a 24-hr semi-occluded application of disodium tetrachloropalladate (500 mg) to the intact or abraded skin of six rabbits (Middleton, 1978a).

Instillation of disodium tetrachloropalladate (about 100 mg) into one eye of each of six white rabbits caused moderate irritation when the eyes were examined at up to 72 hr (Middleton, 1978b). However, recent and GLP-compliant in vitro skin irritation/corrosion data (OECD437) clearly demonstrated a corrosive potential of the substance. Therefore, the substance is being classified as Eye Damage cat 1.

No relevant respiratory tract data were identified.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

3 days; no data on actual dates

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Conducted according to US CFR (1973) guidelines, pre-GLP, but scientifically acceptable

Qualifier:

according to guideline

Guideline:

other: US Federal register Vol. 38, No. 187, section 1500:41 (1973)

Deviations:

no

GLP compliance:

no

Remarks:

pre-GLP

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: “recognised breeder”
- Age at study initiation: 14-16 weeks
- Weight at study initiation: mean 2.2 kg
- Housing: no data
- Diet (e.g. ad libitum): conventional; ad libitum
- Water (e.g. ad libitum): filtered water; ad libitum
- Acclimation period: no details

ENVIRONMENTAL CONDITIONS
- Temperature (°C): no data
- Humidity (%): no data
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): natural lighting
report states “environmentally controlled room”

Type of coverage:

semiocclusive

Preparation of test site:

other: shaved and (on one side) abraded

Vehicle:

unchanged (no vehicle)

Controls:

other: no concurrent control site

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 500 mg (no data if applied as the powder or made into a paste)

Duration of treatment / exposure:

24 hr

Observation period:

24 and 72 hr

Number of animals:

6 females

Details on study design:

TEST SITE
- Area of exposure: 6.25 cm2 each of shaved dorsal skin and shaved and abraded dorsal skin
- % coverage: about 5% each intact and abraded
- Type of wrap if used: gauze pad, secured by adhesive tape

REMOVAL OF TEST SUBSTANCE
- Washing (if done): no removal of test substance
- Time after start of exposure:

SCORING SYSTEM: assessed for primary irritation index at 24 and 72 hr using the Draize method of scoring. Scores for erythema/eschar formation and edema for both intact and abraded skin at both time points were totalled and divided by 24 to give the primary irritation index.
Primary irritation index Classification
0 non-irritant
> 0-2 mild irritant
> 2-5 moderate irritant
> 5-8 severe irritant

Irritation parameter:

primary dermal irritation index (PDII)

Basis:

mean

Time point:

other: Mean of 24 and 72 hr time points

Score:

0

Max. score:

8

Reversibility:

other: not relevant

Remarks on result:

no indication of irritation

Irritation parameter:

erythema score

Basis:

mean

Time point:

other: Mean of 24 and 72 hr time points

Score:

0

Max. score:

8

Reversibility:

other: not relevant

Remarks on result:

no indication of irritation

Irritation parameter:

edema score

Basis:

mean

Time point:

other: Mean of 24 and 72 hr time points

Score:

0

Max. score:

8

Reversibility:

other: not relevant

Remarks on result:

no indication of irritation

Irritant / corrosive response data:

No irritant effects were observed; no indication that corrosivity was assessed

Other effects:

No indication that systemic effects were assessed

Interpretation of results:

GHS criteria not met

Conclusions:

No irritation was observed following a 24-hr semi-occluded application of disodium tetrachloropalladate (500 mg) to the intact or abraded skin of six rabbits.

Executive summary:

In an early pre-GLP study, conducted according to US CFR guidelines, the irritant potential of disodium tetrachloropalladate, available as a brown powder, was assessed on the shaved intact and abraded skin of six New Zealand white rabbits.

 

In a 24 hr semi-occluded patch test, 500 mg of the test substance did not cause any irritation to either intact or abraded skin when assessed immediately on removal of the patch and 48 hr later. Residual test material was not washed off the skin during this period. As such, no classification for skin irritancy is required according to EU CLP criteria (EC 1272/2008).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

5 Jan - 12 Jan 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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)

Version / remarks:

version adopted 26 June 2020

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Pd content 36.11%

Species:

cattle

Details on test animals or tissues and environmental conditions:

Bovine eyes from young cattle were obtained from the
slaughterhouse (Vitelco, -'s Hertogenbosch, The Netherlands),
where the eyes were excised by a slaughterhouse employee as
soon as possible after slaughter.
Eyes were collected and transported in physiological saline in a
suitable container under cooled conditions and tested the day of
arrival in the laboratory.

Vehicle:

physiological saline

Controls:

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

No correction was made for the purity/composition of the test item.
A solubility test in physiological saline was performed based on visual assessment. A 20% (w/v) (dark brown) suspension of the test item was prepared in physiological saline. The stock solution was treated with ultrasonic waves to obtain a homogeneous suspension.

The medium from the anterior compartment was removed and 750 µL of either the negative control, positive control (20% (w/v) Imidazole solution) or 20% (w/v) suspension of the test
item was introduced onto the epithelium of the cornea.

Duration of treatment / exposure:

Corneas were incubated in a horizontal position for 240 +/- 10 minutes
at 32 +/- 1°C. After the incubation the solutions and the test item suspension were removed and
the epithelium was washed at least three times with MEM with phenol red (Eagle’s Minimum
Essential Medium Life Technologies). Possible pH effects of the test item on the corneas
were recorded. Each cornea was inspected visually for dissimilar opacity patterns. The
medium in the posterior compartment was removed and both compartments were refilled with
fresh cMEM and the opacity determinations were performed.

Number of animals or in vitro replicates:

3 replicates per treatment (vehicle control, test item and positive control)

Details on study design:

Preparation of Corneas
The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and
neovascularization by removing them from the physiological saline and holding them in the
light. Those exhibiting defects were discarded.
The isolated corneas were stored in a petri dish with cMEM (Eagle’s Minimum Essential
Medium (Life Technologies, Bleiswijk, The Netherlands) containing 1% (v/v) L-glutamine
(Life Technologies) and 1% (v/v) Fetal Bovine Serum (Life Technologies)). The isolated
corneas were mounted in a corneal holder (one cornea per holder) of Duratec Analysentechnik GmbH (Hockenheim, Germany) with the endothelial side against the O-ring
of the posterior half of the holder. The anterior half of the holder was positioned on top of the
cornea and tightened with screws. The compartments of the corneal holder were filled with
cMEM of 32 +/- 1°C. The corneas were incubated for the minimum of 1 hour at 32 +/- 1°C.

Cornea Selection and Opacity Reading
After the incubation period, the medium was removed from both compartments and replaced
with fresh cMEM. Opacity determinations were performed on each of the corneas using an
opacitometer (BASF-OP3.0, Duratec GmbH). The opacity of each cornea was read against a
cMEM filled chamber, and the initial opacity reading thus determined was recorded. Corneas
that had an initial opacity reading higher than 7 were not used. Three corneas were selected at
random for each treatment group.

Treatment of Corneas and Opacity Measurements
The medium from the anterior compartment was removed and 750 µL of either the negative
control, positive control (20% (w/v) Imidazole solution) or 20% (w/v) suspension of the test
item was introduced onto the epithelium of the cornea. The holder was slightly rotated, with
the corneas maintained in a horizontal position, to ensure uniform distribution of the solutions
over the entire cornea. Corneas were incubated in a horizontal position for 240 +/- 10 minutes
at 32 +/- 1°C. After the incubation the solutions and the test item suspension were removed and
the epithelium was washed at least three times with MEM with phenol red (Eagle’s Minimum
Essential Medium Life Technologies). Possible pH effects of the test item on the corneas
were recorded. Each cornea was inspected visually for dissimilar opacity patterns. The
medium in the posterior compartment was removed and both compartments were refilled with
fresh cMEM and the opacity determinations were performed.

Opacity Measurement
The opacity of a cornea was measured by the diminution of light passing through the cornea.
The light was measured as illuminance (I = luminous flux per area, unit: lux) by a light meter.
The change in opacity for each individual cornea (including the negative control) was
calculated by subtracting the initial opacity reading from the final post-treatment reading. The
corrected opacity for each treated cornea with the test item or positive control was calculated
by subtracting the average change in opacity of the negative control corneas from the change
in opacity of each test item or positive control treated cornea.
The mean opacity value of each treatment group was calculated by averaging the corrected
opacity values of the treated corneas for each treatment group.

Application of Sodium Fluorescein
Following the final opacity measurement, permeability of the cornea to Na-fluorescein
(Sigma-Aldrich, Germany) was evaluated.
The medium of both compartments (anterior compartment first) was removed. The posterior
compartment was refilled with fresh cMEM. The anterior compartment was filled with mL of 5 mg Na-fluorescein/mL cMEM solution (Sigma-Aldrich Chemie GmbH, Germany).
The holders were slightly rotated, with the corneas maintained in a horizontal position, to
ensure uniform distribution of the sodium-fluorescein solution over the entire cornea. Corneas
were incubated in a horizontal position for 90 +/- 5 minutes at 32 +/- 1°C.

Permeability Determinations
After the incubation period, the medium in the posterior compartment of each holder was
removed and placed into a sampling tube labelled according to holder number. 360 µL of the
medium from each sampling tube was transferred to a 96-well plate. The optical density at
490 nm (OD490) of each sampling tube was measured in triplicate using a microplate reader
(TECAN Infinite® M200 Pro Plate Reader). Any OD490 that was 1.500 or higher was diluted
to bring the OD490 into the acceptable range (linearity up to OD490 of 1.500 was verified
before the start of the experiment). OD490 values of less than 1.500 were used in the
permeability calculation.
The mean OD490 for each treatment was calculated using cMEM corrected OD490 values. If a
dilution has been performed, the OD490 of each reading of the positive control and the test
item was corrected for the mean negative control OD490 before the dilution factor was applied
to the reading.

Irritation parameter:

in vitro irritation score

Run / experiment:

run 1, 240h treatment

Value:

424

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

The negative control responses for opacity and permeability were less than the upper limits of
the laboratory historical range indicating that the negative control did not induce irritancy on
the corneas.
The positive control (20% (w/v) Imidazole) showed clear opacity and increased permeability
of the corneas. The mean in vitro irritancy score of the positive control (20% (w/v) Imidazole)
was 121 and within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and
that the test system functioned properly. The mean IVIS score of 121 for the positive control
was greater than the cut-off value of >55 and corresponds to a classification as serious eye
damaging (EU CLP/GHS Category 1).
The test item induced serious eye damage through a single endpoint (opacity), resulting in a
mean in vitro irritancy score of 424 after 240 minutes of treatment, corresponding to a
classification as serious eye damaging (EU CLP/GHS Category 1).

The individual in vitro irritancy scores for the negative controls ranged from 0.6 to 1.5. The corneas treated with the negative control item were clear after the 240 minutes of treatment. 

The individual positive control in vitro irritancy scores ranged from 117 to 124. The corneas treated with the positive control were turbid after the 240 minutes of
treatment.

Any possible pH effect of the test item on the corneas could not be observed on the rinsing medium following incubation, due to the dark brown color of the test item suspension. The corneas treated with the test item showed opacity values ranging from 269 to 510 and permeability values ranging from -0.029 to -0.021. The corneas were turbid after the 240 minutes of treatment with the test item. Hence, the in vitro irritancy scores ranged from 269 to 510 after 240 minutes of treatment with the test item.

Summary Table of Opacity, Permeability and In Vitro Scores

treatment	mean opacity	mean permeability	mean in vitro irritation score(1,2)
negative control (vehicle only)	0.6	0.029	1.0
positive control	95	1.699	121
test item	425	-0.026	424

1 Calculated using the negative control corrected mean opacity and mean permeability values for the positive
control and test item.
2 In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value).

Interpretation of results:

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

Conclusions:

Disodium tetrachloropalladate induced an IVIS ≥ 55 in a BCOP assay (according to OECD437). It is concluded that Disodium tetrachloropalladate induces serious eye damage in the Bovine Corneal Opacity and Permeability test under the experimental conditions described in this report and should be classified category 1 according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations.

Executive summary:

The objective of this study was to evaluate the eye hazard potential of Disodium
tetrachloropalladate as measured by its ability to induce opacity and increase permeability in an isolated bovine cornea using the Bovine Corneal Opacity and Permeability test (BCOP test).
This report describes the potency of chemicals to induce serious eye damage using isolated bovine corneas. The eye damage of the test item was tested through topical application for approximately 240 minutes.
The study procedures described in this report were based on the most recent OECD guideline. 
The test item was available as a brown-red hygroscopic solid and was applied as a 20% (w/v) suspension (750 μL) directly on top of the corneas.
The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (20% (w/v) Imidazole) was 121 and within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system
functioned properly.
The test item induced serious eye damage through a single endpoint (opacity), resulting in a mean in vitro irritancy score of 424 after 4 hours of treatment.
In conclusion, since Disodium tetrachloropalladate induced an IVIS ≥ 55, it is concluded that Disodium tetrachloropalladate induces serious eye damage in the Bovine Corneal Opacity and Permeability test under the experimental conditions described in this report and should be classified category 1 according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

No relevant human irritation/corrosion data were identified. No in vitro skin or eye irritation studies were identified, or are needed, as reliable in vivo studies are already available.

 

In an early pre-GLP study, conducted according to US CFR guidelines, the irritant potential of disodium tetrachloropalladate, available as a brown powder, was assessed on the shaved intact and abraded skin of six New Zealand white rabbits. In a 24-hr semi-occluded patch test, 500 mg of the test substance did not cause any irritation to either intact or abraded skin when assessed immediately on removal of the patch and 48 hr later. Residual test material was not washed off the skin during this period(Middleton, 1978a). As such, no classification for skin irritancy is required according to EU CLP criteria (EC 1272/2008).

 

In an early pre-GLP study, conducted according to US CFR guidelines, the eye irritancy potential of disodium tetrachloropalladate was assessed following instillation of about 100 mg to one conjunctival sac of each of six White Russian (albino) rabbits. Moderate irritation to the eyes (including effects on the cornea, conjunctivae, and iris) of each of the animals was observed, when assessed at 24, 48 and 72 hr, which had not reversed during the short (3-day) observation period. The primary irritation index was calculated to be 48.1 (out of a possible 110) for the mean of the three time points. Disodium tetrachloropalladate was moderately irritating when the powdered material was applied to the eyes of rabbits(Middleton, 1978b). Classification as an eye irritant (category 2) appears appropriate according to EU CLP criteria (EC 1272/2008). However, in a recent and GLP-compliant in vitro skin irritation/corrosion study according to OECD437 (Gijsbrechts 2021), disodium tetrachloropalladate clearly exceeded the threshold value for classification as causing irreversible damage to the eye. Therefore, the substance is precautionarily classified as Eye Damage cat1.

 

No respiratory tract data were identified. A new study was not conducted as it is not a REACH Standard Information Requirement. Further, the compound is not expected to reach the lungs in appreciable quantities (based on respiratory tract deposition modelling data). Thus, inhalation will not be a significant route of exposure.

Justification for selection of skin irritation / corrosion endpoint:
Conducted according to US CFR (1973) guidelines, pre-GLP, but scientifically acceptable, and the only skin irritation study available

Justification for selection of eye irritation endpoint:
Conducted according to OECD437, GLP compliant, and considered superior for classification&labelling purposes to the pre-GLP in vivo study performed according to US CFR (1973), pre-GLP.

Justification for classification or non-classification

Based on the results of the available reliable skin and eye irritation studies, disodium tetrachloropalladate should not be classified for skin irritation, but should be classified for eye damage (Category 1), according to EU CLP criteria (EC 1272/2008).