Diamminedichloropalladium

Endpoint summary

Description of key information

In an in vitro EpiSkin assay, conducted in accordance with OECD Test Guideline 439 and to GLP, diamminedichloropalladium was considered to be non-irritating to skin (Kiss, 2012b).










  
  
  
  


In an OECD Test Guideline 405 study, to GLP, diamminedichloropalladium (0.1 g, powdered) caused significant conjunctival and corneal irritant effects in the eyes of three New Zealand white rabbits which were not fully reversible within 3 weeks (Kiss, 2012d).















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 vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18-20 January 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OECD guideline study, to GLP.

Qualifier:

according to guideline

Guideline:

other: OECD Guidelines for Testing of Chemicals, Section 4, No. 439, “In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method” adopted 22 July 2010

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EpiSkin™ SOP, Version 1.8 (February 2009), ECVAM Skin Irritation Validation Study: Validation of the EpiSkin™ test method 15 min - 42 hours for the prediction of acute skin irritation of chemicals. Available at: [http://ecvam.jrc.ec.europa.eu]

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: IN VITRO SKIN IRRITATION: RECONSTRUCTED HUMAN EPIDERMIS MODEL TEST in relation to Regulation (EC) No 440/2008 (as amended) and Regulation (EC) No 1907/2006 on REACH (Annex III, B.46).

Deviations:

no

Principles of method if other than guideline:

The test is designed to predict and classify the skin irritant potential of chemicals according to chemical safety regulations, using the reconstructed human epidermis model EPISKIN-SM and parameters related to skin irritation.

EPISKIN-SM is a three-dimensional human skin model comprising a reconstructed epidermis with a functional stratum corneum. Its use for skin irritation testing involves topical application of test materials to the surface of the epidermis, and the subsequent assessment of their effects on cell viability. Cell viability determination is based on cellular mitochondrial dehydrogenase activity, measured by MTT reduction and conversion into a blue formazan salt that is quantitatively measured after extraction from tissues. The reduction of cell viability in treated tissues is compared to negative controls and expressed as a %. The % reduction in viability is used to predict the irritation potential.

The EPISKIN-SM has been found scientifically valid for reliably predicting no label and R38 (irritant) substances in respect to the previous EU classification scheme and has been confirmed in April 2009 by ESAC for use under the UN GHS system as "applicable to all authorities".

GLP compliance:

yes (incl. QA statement)

Species:

human

Strain:

other: Reconstructed human epidermis model (see details below)

Details on test animals or test system and environmental conditions:

EPISKIN-SM (Source: SkinEthic, France, Batch No.:12-EKIN-003, Expiry date: 23 January 2012) is a three-dimensional human epidermis model. Adult human-derived epidermal keratinocytes are seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after 13-day culture period comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.

Type of coverage:

other: Applied evenly to the epidermal surface following the application of 10 ul distilled water to this surface

Preparation of test site:

other: in vitro cell culture

Vehicle:

water

Remarks:

distilled

Controls:

other: negative control skin unit tested in triplicate

Amount / concentration applied:

20 mg to each of three test skin units.
10 µl PBS (phosphate buffered saline) was added to each of the three negative control skin units and 10 µl SDS (sodium dodecyl sulfate, 5% aqueous solution) was added to each of the three positive control skin units.
For additional control for staining effects of the test item, 10 µl distilled water was applied to the epidermal surface of a single skin unit to ensure good contact with the epidermis, then 20 mg of the test item was applied evenly to the epidermal surface.

Duration of treatment / exposure:

Exposure for 15 minutes (± 0.5 min) at room temperature (20-37°C).
Then incubated with fresh “maintenance medium” for 42 hours (± 1 hr) at 37°C.

Observation period:

Not applicable to this test system

Number of animals:

Not applicable to this test system

Details on study design:

EPISKIN-SM assay plate contained reconstructed epidermis units (area: 0.38 cm2); each was attached to the base of a tissue culture vessel and maintained on nutritive agar.

After test substance exposure and subsequent incubation, preparations for cell viability determination were: incubation with MTT solution (at 37 degrees C for 3 hours) followed by incubation with acidified isopropanol for formazan extraction (around two hours at room temperature with gentle agitation).

For cell viability measurements, the OD (Absorbance / Optical Density) of the samples in a spectrophotometer was read at 540 nm using acidified isopropanol solution blank (6×200 µL). (The validity of the microplate reader was verified with a standard verification plate daily before use. The standard plate was calibrated yearly by the manufacturer.)

For each treated tissue, OD (as adjusted for colouring potential of the test substance) was calculated and the tissue viability was expressed as a % relative to negative control.

Criteria for classification as irritant/non-irritant: If the resulting mean relative viability (as adjusted for intrinsic colour) is less than or equal to 50% of the negative control, the test substance is considered to be irritant to skin.

Irritation parameter:

other:

Basis:

mean

Remarks:

Cell/tissue viability

Time point:

other: 42 hours

Score:

56

Reversibility:

other: Not applicable

Remarks on result:

other: Score is a percentage (%) of negative control. Mean relative viability <=50% the test substance is considered to be irritant to skin.

Irritant / corrosive response data:

Mean cell viability (as adjusted for intrinsic colour) was 56% of the negative control (range 54%-58%).

Other effects:

No data

The results of the optical density (OD) measured at 540 nm of each replicate and the calculated % viability of the cells are presented in the attached document.

Interpretation of results:

not classified

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

In an in vitro reconstructed human epidermis (EpiSkin) assay, conducted in accordance with OECD Test Guideline 439 and to GLP, diamminedichloropalladium was considered to be non-irritating.

Executive summary:

Diamminedichloropalladium was tested for skin irritation potential in an in vitro reconstructed human epidermis (EpiSkin) assay conducted in accordance with OECD Test Guideline 439, and to GLP.

Following exposure to diamminedichloropalladium, the test system skin cell viability was calculated to be greater than 50% (the average was 56% and the range was 54-58%). As such, it was considered to be non-irritating to skin.

As such, diamminedichloropalladium does not require classification for skin irritation 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, other

Remarks:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 March-16 April 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OECD guideline study, to GLP. Minor humidity deviations were not expected to have any impact on the study outcome.

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

yes

Remarks:

Minor humidity deviations were expected to have no impact on the study outcome

Qualifier:

according to guideline

Guideline:

EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)

Deviations:

yes

Remarks:

Minor humidity deviations were expected to have no impact on the study outcom

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2400 (Acute Eye Irritation)

Deviations:

yes

Remarks:

Minor humidity deviations were expected to have no impact on the study outcom

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: S&K-LAP Kft., 2173 Kartal, Császár road 135, Hungary
- Age at study initiation: ~12 weeks
- Body weight range at the beginning of the in-life phase: 3188 – 3218 g
- Body weight range at the end of the in-life phase: 3768 – 3880 g
- Housing: individually in AAALAC approved metal wire rabbit cages. Cages were of an open wire structure and cages were placed together to allow some social interaction with rabbit(s) in adjoining cages
- Diet (e.g. ad libitum): Purina and UNI diet (Lot number: 00701211, 0030 03 12 and 0060 04 12) for rabbits produced by AGRIBRANDS Europe Hungary PLC, H-5300 Karcag, Madarasi road, Hungary, ad libitum.
- Water (e.g. ad libitum): municipal tap water, as for human consumption, ad libitum
- Acclimation period: 19 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 ± 3°C
- Humidity (%): 24 – 61 % [slightly outside of recommending range]
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12/12

Vehicle:

unchanged (no vehicle)

Controls:

not required

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 g

Duration of treatment / exposure:

Following instillation into the conjunctival sac of one eye, the eyelids were held closed for several seconds. Washing with physiological saline was performed at 72 hours.

Observation period (in vivo):

3 weeks (The eyes were examined at 1, 24, 48, 72 hours, 1, 2 and 3 weeks after treatment).

Number of animals or in vitro replicates:

3 (males)

Details on study design:

Washing with physiological saline was performed at 72 hours.

The eyes were examined at 1, 24, 48, 72 hours, 1, 2 and 3 weeks after treatment. The nature, severity and duration of all lesions observed were described.

SCORING SYSTEM: The eye irritation scores were evaluated according to the scoring system by Draize (1977) and OECD 405 (24 April 2002).

TOOL USED TO ASSESS SCORE: none reported; presumably subjective observation by an experienced evaluator

Irritation parameter:

chemosis score

Remarks:

Conjunctival chemosis

Basis:

mean

Remarks:

of all 3 animals

Time point:

other: Mean of 24, 48 and 72-hour timepoints

Score:

3.11

Max. score:

4

Reversibility:

not fully reversible within: 3 weeks

Remarks on result:

other: An effect remained evident in all animals at 3 weeks

Irritation parameter:

other: Conjunctival discharge

Basis:

mean

Time point:

other: Mean of 24, 48 and 72-hour timepoints

Score:

3

Max. score:

3

Reversibility:

not fully reversible within: 3 weeks

Remarks on result:

other: An effect remained evident in all animals at 3 weeks

Irritation parameter:

other: Conjunctival redness

Basis:

mean

Time point:

other: Mean of 24, 48 and 72-hour timepoints

Score:

2

Max. score:

3

Reversibility:

not fully reversible within: 3 weeks

Remarks on result:

other: An effect remained evident in all animals at 3 weeks

Irritation parameter:

cornea opacity score

Remarks:

opacity, degree of density

Basis:

mean

Time point:

other: Mean of 24, 48 and 72-hour timepoints

Score:

1.45

Max. score:

4

Reversibility:

not fully reversible within: 3 weeks

Remarks on result:

other: An effect remained evident in all animals at 3 weeks

Irritation parameter:

iris score

Basis:

mean

Time point:

other: Mean of 24, 48 and 72-hour timepoints

Score:

0

Max. score:

2

Reversibility:

other: Not applicable

Remarks on result:

other: The iris was not affected in any animal at any time point

Irritant / corrosive response data:

Initial Pain Reaction (IPR) (score 2) was observed in all animals.

One hour after the application: Conjunctival redness (score 1 or 2), discharge
(score 3) and chemosis (score 1 or 3) were found in all animals. All rabbits showed corneal opacity (score 1, area 3 or 4).

At 24 hours after treatment: Conjunctival redness (score 2), discharge (score 3) and chemosis (score 3) were found in all animals. Additionally, corneal opacity (score 1, area 4) was seen in all animals.

At 48 hours after treatment: Conjunctival redness (score 2), discharge (score 3) and chemosis (score 3) were found in all animals. Additionally, corneal opacity (score 1 or 2, area 3 or 4) was seen in all animals.

At 72 hours after treatment: Conjunctival redness (score 2), discharge (score 3) and chemosis (score 3 or 4) were found in all animals. Additionally, corneal opacity (score 1 or 2, area 3 or 4) was seen in all animals. Surface damage on the conjunctivae and on the nictitating membrane was noted in all animals.

At 1 week after treatment: Conjunctival redness (score 2), discharge (score 3) and chemosis (score 3 or 4) were found in all animals. Additionally, corneal opacity (score 1 or 2, area 4) was seen in all animals. Surface damage on the conjunctivae and on the nictitating membrane was noted in all animals.

At 2 weeks after treatment: Conjunctival redness (score 2), discharge (score 1 or 3) and chemosis (score 2 or 3) were found in all animals. Additionally, corneal opacity (score 1, 2 or 3, area 2, 3 or 4) was seen in all animals. Surface damage on the conjunctivae and on the nictitating membrane was noted in all animals.

At 3 weeks after treatment: Conjunctival redness (score 2), discharge (score 1, 2 or 3) and chemosis (score 2) were found in all animals. Additionally, corneal opacity (score 1 or 2, area 2 or 4) was seen in all animals. Surface damage on the conjunctivae and on the nictitating membrane was noted in all animals.

The individual mean scores for each animal (considering readings at 24, 48 and 72 hours after the treatment) were as follows:
chemosis : 3.00, 3.33, 3.00
discharge : 3.00, 3.00, 3.00
redness : 2.00, 2.00, 2.00
cornea opacity : 1.00, 1.67, 1.67
iris : 0.00, 0.00, 0.00

Surface damage on the conjunctivae and on the nictitating membrane was seen in all animals at all timepoints from 72 hours (inclusive).

Other effects:

There were no clinical signs observed that could be related to treatment.

Interpretation of results:

Category 1 (irreversible effects on the eye)

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

In a OECD Test Guideline 405 study, to GLP, diamminedichloropalladium (0.1 g, powdered) caused significant conjunctival and corneal irritant effects in the eyes of three New Zealand white rabbits which were not fully reversible within 3 weeks.

Executive summary:

In an in vivo rabbit eye irritation study, conducted in accordance with OECD Test Guideline 405 and to GLP, diamminedichloropalladium (0.1 g, powdered) was instilled into the right eye of each of three New Zealand white rabbits. The eyelids were held closed for several seconds and washing with physiological saline was performed at 72 hours. The contralateral eye remained untreated and was used for control purposes.

No clinical signs of systemic toxic effects were reported. Diamminedichloropalladium was shown to cause significant conjunctival and corneal irritant effects (scores of 1 to 4 of 4) within one hour of application that were not fully reversible within 3 weeks (Kiss, 2012d). According to EU CLP criteria (EC 1272/2008), diamminedichloropalladium should be classified as Category 1 (irreversible effects on the eye).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

No relevant irritation/corrosion human data were identified.

Diamminedichloropalladium was tested for skin irritation potential in an in vitro reconstructed human epidermis (EpiSkin) assay conducted in accordance with OECD Test Guideline 439, and to GLP. Following exposure to diamminedichloropalladium, the test system skin cell viability was calculated to be greater than 50% (the average was 56% and the range was 54-58%). As such, it was considered to be non-irritating to skin (Kiss, 2012b). In support,neatdiamminedichloropalladium was not irritant in a limited (non-GLP) study involving 24-hr occluded application to the intact and abraded skin of six rabbits (Campbell et al., 1975). As such,diamminedichloropalladium does not require classification for skin irritation according to EU CLP criteria (EC 1272/2008).

Diamminedichloropalladium was tested in an in vitro eye irritation study using isolated chicken eyes, conducted according to OECD Test Guideline 438 and to GLP. After reference measurements, 30 mg of test material was applied to cover the entire surface of the cornea. After 10 seconds, the surface was rinsed with saline and observations of corneal thickness and corneal opacity were made over a 240-minute post-exposure period; fluorescein retention was measured at 30 minutes. Positive control eyes were treated in a similar way with 30 mg imidazole and a single negative control eye was treated with 30 µL of isotonic saline. Results suggested that diamminedichloropalladium was not severely irritating or corrosive. However, this test substance remained adhered to the corneal surface after the post-treatment rinse. No conclusion of the in vivo significance can be made from this observation since in vivo eye lids will probably clear the surface, although abrasion may occur. An in vivo study is required for precise classification (Kiss, 2012c).

In an in vivo rabbit eye irritation study, conducted in accordance with OECD Test Guideline 405 and to GLP, diamminedichloropalladium (0.1 g, powdered) was instilled into the right eye of each of three New Zealand white rabbits. The eyelids were held closed for several seconds and washing with physiological saline was performed at 72 hours. The contralateral eye remained untreated and was used for control purposes. No clinical signs of systemic toxic effects were reported. Diamminedichloropalladium was shown to cause significant conjunctival and corneal irritant effects within one hour of application that were not fully reversible within 3 weeks (Kiss, 2012d). According to EU CLP criteria (EC 1272/2008), diamminedichloropalladium should be classified for eye damage as Category 1 (irreversible effects on the eye).

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:
OECD guideline study, to GLP. Both skin irritation studies were negative.

Justification for selection of eye irritation endpoint:
In vivo OECD Guideline GLP study, providing the most severe effect.

Justification for classification or non-classification

Based on the results of the available reliable skin and eye irritation studies, diamminedichloropalladium should not be classified for skin irritation, but should be classified for ‘irreversible effects on the eye’ (category 1), according to EU CLP criteria (EC 1272/2008).