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Description of key information

In an in vitro reconstructed human epidermis (EpiSkin) assay, conducted in accordance with OECD Test Guideline 439 and to GLP, palladium di(4-oxopent-2-en-2-oate) was considered to be non-irritating to skin (Kiss, 2012a).

In an in vitro reconstructed human epidermis (EpiSkin) assay, conducted in accordance with OECD Test Guideline 439 and to GLP, palladium di(4-oxopent-2-en-2-oate) was considered to be non-irritating to skin (Kiss, 2012a).

 

In an OECD Test Guideline 405 study, to GLP, palladium di(4-oxopent-2-en-2-oate) was shown to cause significant conjunctival and corneal irritant effects in the eyes of New Zealand white rabbits within 1 hr of instillation, which were not fully reversible within the 3-week observation period (Kiss, 2012b).

No relevant respiratory tract irritation data were identified.


In an OECD Test Guideline 405 study, to GLP, palladium di(4-oxopent-2-en-2-oate) was shown to cause significant conjunctival and corneal irritant effects in the eyes of New Zealand white rabbits within 1 hr of instillation, which were not fully reversible within the 3-week observation period (Kiss, 2012b).
No relevant respiratory tract irritation 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". It is approved by international regulatory agencies as a replacement for the identification of irritants/corrosives in the in vivo rabbit skin assay (OECD 404).
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 (± 1h) 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 / corrosion parameter:
% tissue viability
Value:
94
Negative controls validity:
valid
Remarks on result:
no indication of irritation
Remarks:
Score is a percentage (%) of negative control. Mean relative viability <=50% the test substance is considered to be irritant to skin.
Other effects / acceptance of results:
Mean cell viability (as adjusted for intrinsic colour) was 94% of the negative control (range 90%-97%).

For individual results of each test substance, positive control and negative control test unit, see 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, palladium di(4-oxopent-2-en-2-oate) was considered to be non-irritating to skin.
Executive summary:

Palladium di(4-oxopent-2-en-2-oate) was tested for skin irritation potential in an in vitro reconstructed human epidermis model (EPISKIN assay) conducted in accordance with OECD Test Guideline 439, and to GLP.

 

EPISKIN 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 %. 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.

 

Following a 15-minute exposure to the test substance, the test system skin cell viability was calculated to be greater than 50% (the average was 94% and the range was 90-97%), and it was therefore considered to be non-irritating to skin.

 

Under the conditions of this assay, palladium di(4-oxopent-2-en-2-oate) 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: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 March-17 April 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD guideline study, to GLP, with minor humidity deviations.
Qualifier:
according to guideline
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Deviations:
no
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:
no
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:
no
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: 3119 – 3200 g
- Body weight range at the end of the in-life phase: 3658 – 3752 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: 20 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 ± 3°C
- Humidity (%): 24 – 61 %
- 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 24, 48 and 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 24, 48 and 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:
3.78
Max. score:
4
Reversibility:
not fully reversible within: 3 weeks
Remarks on result:
other: In one animal, chemosis persisted until study termination 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: Discharge persisted in one animal until study termination at 3 weeks
Irritation parameter:
other: Conjunctival redness
Basis:
mean
Time point:
other: Mean of 24, 48 and 72-hour timepoints
Score:
2.56
Max. score:
3
Reversibility:
not fully reversible within: 3 weeks
Remarks on result:
other: Redness persisted in two animals until study termination 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:
1.33
Max. score:
4
Reversibility:
not fully reversible within: 3 weeks
Remarks on result:
other: Corneal opacity persisted in one animal until study termination 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 2), discharge (score 3) and chemosis (score 2) were found in all animals. Two rabbits showed corneal opacity (score 1, area 4). The initial test in one rabbit showed no corneal effect at 1 hour.

At 24 hours after treatment: Conjunctival redness (score 2 or 3), 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.

At 48 hours after treatment: Conjunctival redness (score 2 or 3), discharge (score 3) and chemosis (score 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 72 hours after treatment: Conjunctival redness (score 2 or 3), discharge (score 3) and chemosis (score 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 1 week after treatment: Conjunctival redness (score 2 or 3), discharge (score 1, 2 or 3) and chemosis (score 1 or 2) 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 one animal.

At 2 weeks after treatment: Conjunctival redness (score 1) and/or discharge (score 1) was found in two animals. Conjunctival chemosis (score 1) was observed in all animals. Corneal opacity (score 2, area 4) and surface damage on the conjunctivae and on the nictitating membrane was noted in one animal.

At 3 weeks after treatment: Conjunctival redness (score 1) was found in two animals. Conjunctival chemosis (score 1) and discharge (score 2) was observed in one animal. One rabbit showed corneal opacity (score 2, area 2).

During the study, the control eye of each animal was symptom-free.

The individual mean scores for each animal (considering readings at 24, 48 and 72 hours after the treatment) were as follows:
chemosis : 3.67, 3.67, 4.00
discharge : 3.00, 3.00, 3.00
redness : 2.67, 2.00, 3.00
cornea opacity : 1.00, 1.00, 2.00
iris : 0.00, 0.00, 0.00

Surface damage on the conjunctivae and on the nictitating membrane was seen in all animals at 48 and 72 hours, and in one animal at 1 and 2 weeks.
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 an OECD Test Guideline 405 study, to GLP, palladium di(4-oxopent-2-en-2-oate) was shown to cause significant conjunctival and corneal irritant effects in the eyes of New Zealand white rabbits within 1 hr of instillation, which were not fully reversible within the 3-week observation period.
Executive summary:

In an in vivo eye irritation study carried out in accordance with OECD Test Guideline 405, and to GLP, 0.1 g of powdered palladium di(4-oxopent-2-en-2-oate) was instilled into the conjunctival sac of one eye of 3 male New Zealand White rabbits. Following instillation the eyelids were held closed for several seconds. Washing with physiological saline was performed at 24, 48 and 72 hr. The eyes were examined at 1, 24, 48, 72 hr, 1, 2 and 3 weeks after treatment, and scored according to the Draize system.

Significant conjunctival redness, discharge, and chemosis were seen in all animals, and corneal opacity in 2 rabbits, within 1 hr of instillation. These were not fully reversible within the 3-week observation period. According to EU CLP criteria (EC 1272/2008), palladium di(4 -oxopent-2 -en-2 -oate) 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 human irritation/corrosion data were identified.

Palladium di(4-oxopent-2-en-2-oate) was tested for skin irritation potential in an in vitro reconstructed human epidermis model (EpiSkin assay) conducted in accordance with OECD Test Guideline 439, and to GLP. EpiSkin 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 %. 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. Following a 15-minute exposure to the test substance, the test system skin cell viability was calculated to be greater than 50% (the average was 94% and the range was 90-97%), and it was therefore considered to be non-irritating to skin (Kiss, 2012a). Under the conditions of this assay, palladium di(4-oxopent-2-en-2-oate) does not require classification for skin irritation according to EU CLP criteria (EC 1272/2008).

 

In an in vivo eye irritation study carried out in accordance with OECD Test Guideline 405, and to GLP, 0.1 g of powdered palladium di(4-oxopent-2-en-2-oate) was instilled into the conjunctival sac of one eye of 3 male New Zealand White rabbits. Following instillation the eyelids were held closed for several seconds. Washing with physiological saline was performed at 24, 48 and 72 hours. The eyes were examined at 1, 24, 48, 72 hours, 1, 2 and 3 weeks after treatment, and scored according to the Draize system. Significant conjunctival redness, discharge, and chemosis were seen in all animals, and corneal opacity in 2 rabbits, within 1 hr of instillation. These were not fully reversible within the 3‑week observation period. According to EU CLP criteria (EC 1272/2008), palladium di(4-oxopent-2-en-2-oate) should be classified as Category 1 (irreversible effects on the eye) (Kiss, 2012b).

 

In contrast, in two in vitro assays, palladium di(4-oxopent-2-en-2-oate) was negative for eye irritation. In the first in vitro eye irritation study in isolated chicken eyes, performed in accordance with OECD Test Guideline 438 and to GLP, palladium di(4-oxopent-2-en-2-oate) appeared to be not severely irritating/corrosive and would not require classification as a severe eye irritant. However, the test substance remained adhered to the corneal surface after the post-treatment rinse. As such, an in vivo study is required to determine classification (Kiss, 2012c). It was also negative in an in vitro chorioallantoic membrane (CAM) assay using incubated hens eggs. As no irritation of the mucous membranes was seen, palladium di(4-oxopent-2-en-2-oate) in this study would not have predicted eye irritation in vivo (Albrecht, 2006).

No respiratory tract irritation 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, and the only skin irritation study available.

Justification for selection of eye irritation endpoint:
OECD guideline study, to GLP. This in vivo study shows the most severe effect.

Justification for classification or non-classification

Based on the results of the available reliable in vitro skin irritation study, there is no requirement to classify palladium di(4-oxopent-2-en-2-oate) for skin irritation under EC Regulation 1272/2008.

However, palladium di(4-oxopent-2-en-2-oate) produced effects on the conjunctivae and cornea in rabbit eyes in a reliable in vivo study. As these effects were not fully reversed within a 3-week observation period, the substance should be classified as Category 1 (irreversible effects on the eye) in accordance with EU CLP criteria (EC 1272/2008).