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Description of key information

In a GLP-compliant study according to OECD439, ruthenium (IV) oxide was considered to be non-cytotoxic and predicted to be non-irritant to skin.

In a GLP-compliant study according to OECD405, ruthenium (IV) oxide hydrate (0.1 g) produced very slight eye irritation following instillation into the eyes of rabbits, but did not meet the EU criteria for classification as an eye irritant (Collier, 1982b).

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:
14 May 2018 - 18 May 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study conducted according to GLP
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
Reconstructed Human Epidermis Test method
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
purity: 75.08% Ru content (calculated after loss on reduction = 100% - 24.92%)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: EpiDermTM (EPI-200, Lot no. 28611) MatTek In Vitro Life Science Laboratories, s.r.o, Mlynské Nivy 73, 821 05 Bratislava II, Slovak Republic
Source strain:
other: Reconstructed human epidermis model (see below)
Details on animal used as source of test system:
Not applicable
Justification for test system used:
The reconstructed human epidermis model system is suitable to test solids, liquids, semi-solids and waxes. The liquids may be aqueous or non-aqueous; solids may be soluble or insoluble in water.
Vehicle:
unchanged (no vehicle)
Details on test system:
The test method is based on reconstructed human epidermis models, which in their overall design (the use of human derived epidermal keratinocytes as cell source, representative tissue and cyto-architecture) closely mimic the biochemical and physiological properties of the upper parts of the human skin, i.e. the epidermis. The procedure described under this test method allows the hazard identification of irritant substances in accordance with UN GHS Category 1 or Category 2.

EpiDerm is a three-dimensional reconstructed human epidermis model EpiDermTM, comprised of normal, human-derived epidermal keratinocytes, which had been cultured to form a multilayered, highly differentiated model of the human epidermis. It consisted of organised basal, spinous and granular layers, and a multilayered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo.

The principle of the reconstructed human epidermis model test is based on the premise that irritant substances are able to penetrate the stratum corneum by diffusion and are cytotoxic to the cells in the underlying layers. Cell viability was measured by dehydrogenase conversion of the vital dye MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue), into a blue formazan salt that was quantitatively measured after extraction from tissues. Irritant substances were identified by their ability to decrease cell viability below defined threshold levels (i.e.≤ 50% for UN GHS Category 1 or Category 2).

The EpiDerm™ System was manufactured according to defined quality assurance procedures. All biological components of the epidermis and the culture medium were tested by manufacturer for viral, bacterial, fungal and mycoplasma contamination. MatTek determines the ET50 value following exposure to Triton X-100 (1%) for each EpiDerm lot. The ET50 must fall within a range established based on a historical database of results.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg of test item were applied to the skin model and uniformly covered the skin surface with an area of 0.63 cm2. The test item is a fine powder. For better contact of the test item to the skin, the skin surface was moistened with 25 µL Dulbecco’s phosphate buffered saline (D-PBS). Three tissue replicates were used.

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 μL D-PBS was added to each of the three negative control skin units.

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 μL SDS was added to each of the three positive control skin units.
- Concentration (if solution): 5% aqueous solution
Duration of treatment / exposure:
Exposure for 60 minutes: 35 minutes at 37°C, 5% CO2 and 95% relative humidity followed by 25 minutes at room temperature under sterile hood.
Duration of post-treatment incubation (if applicable):
The post-treatment incubation period of the rinsed tissues in fresh assay medium was 42 hours.
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Remarks:
Score is a percentage of the negative control
Run / experiment:
mean (42 hour time point)
Value:
95.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
The mean optical density (OD) of the negative control of 3 tissues was 1.542 and was well within the acceptable range of ≥ 0.8 to ≤ 2.8.
Positive controls validity:
valid
Remarks:
The viability of cells treated with the positive reference item was 6.6% of the negative control and fulfilled the acceptance criterion of <=20%.
Remarks on result:
no indication of irritation
Remarks:
Mean viability of cells exposed to Ruthenium(IV) oxide was 95.5% of the negative control and was well above the cut-off percentage cell viability value that distinguishes irritant from non-irritant test items of > 50%. Ruthenium(IV) oxide was considered to be non-cytotoxic and predicted to be non-irritant to skin.
Other effects / acceptance of results:
Assay acceptability criteria:
1: The mean optical density (OD) of 3 negative control tissues was 1.542 and was well within the acceptable range of ≥0.8 to ≤2.8.
2: The viability of cells treated with the positive reference item, 5% SDS, was 6.6% of the negative control and fulfilled the acceptance criterion of ≤20%.
3: The assay meets the acceptance criterion if the standard deviation (SD) calculated from individual % tissue viabilities of the 3 identically treated replicates is ≤ 18%.

The mean viability of cells exposed to Ruthenium(IV) oxide was 95.5% of the negative control and, hence, was well above the cut-off percentage cell viability value that distinguishes irritant from non-irritant test items of >50%. Ruthenium(IV) oxide was considered to be non-cytotoxic and predicted to be non-irritant to skin.

The mean optical density (OD) of 3 negative control tissues was 1.542 and was well within the acceptable range of ≥0.8 to ≤2.8. The viability of cells treated with the positive reference item, 5% SDS, was 6.6% of the negative control and fulfilled the acceptance criterion of ≤20%.

The summary of the results is given below:

   Optical density (n = 3 tissues) CV (%)

 

 viability (%)

 

 SD

 Negative control (D-PBS)

 1.542

 10.5

 

 100

 

 10.6

 Ruthenium(IV) oxide

 1.473

 16.4

 95.5

 

 15.7

 Positive control (5% SDS)

 0.102

 16.9

 6.6

 

 1.1

Interpretation of results:
GHS criteria not met
Conclusions:
In an in vitro skin irritation test, using a reconstructed human epidermis model (EpiDermTM), Ruthenium(IV) oxide tested at an exposure time of 60 minutes and a 42-hour post-treatment incubation period, did not show irritant properties and is therefore not classified as irritant (UN GHS no category).
Executive summary:

The cytotoxic properties of Ruthenium( IV) oxide to skin cells, which might lead to irritation of human skin, were determined by using an artificial three dimensional model of human skin (EpiDerm TM model). Three replicates were used for each treatment and concurrent control group. The

optical density (OD) was determined by using the MTT reduction assay and expressed as relative percentage of viability of the negative control-treated tissues.

Ruthenium( IV) oxide was applied as solid test item to the model skin surface, which was moistened with Dulbecco's phosphate buffered saline (D-PBS). D-PBS was used as the negative control. 5% aqueous sodium dodecyl sulphate (SOS) was used as the positive reference item. An exposure time of 60 minutes was employed followed by a 42-hour post-treatment incubation period in fresh medium.

The mean viability of cells exposed to Ruthenium(IV) oxide was 95.5% of the negative control and, hence, was well above the cut-off percentage cell viability value that distinguishes irritant from non-irritant test items of > 50%. Ruthenium(IV) oxide was considered to be non-cytotoxic and predicted to be non-irritant to skin.

All acceptance criteria required were fulfilled and the study can be considered as valid.

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:
17 March 1982 – 24 March 1982
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted according to OECD guidelines, and to GLP
Qualifier:
according to guideline
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Deviations:
yes
Remarks:
see below:
Principles of method if other than guideline:
In one group of animals the eye remained unwashed and in the other group the eye was washed 30 seconds after instillation of the test material. The guideline states that “the eyes of the test animals should not be washed for at least 24 hours following instillation of the test substance, except for solids, and in case of immediate corrosive or irritating effects. At 24 hours a washout may be used if considered appropriate.” It goes on to say, in the case of solids, that “if the solid test substance has not been removed from the eye of the test animal by physiological mechanisms at the first observation time point of 1 hour after treatment, the eye may be rinsed with saline or distilled water.”
GLP compliance:
yes
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: Nottingham University, School of Agriculture, Sutton Bonington, Leicestershire
- Weight at study initiation: 2.02-2.51 kg
- Housing: individually in suspended metal cages
- Diet: ad libitum rabbit diet supplied by J Waring Ltd, Shardlow, Derbyshire
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 ± 3
- Air changes (per hr): minimum of 20 air changes/hr
- Photoperiod (hrs dark / hrs light): lighting cycle - 12 hours on / 12 hours off with no daylight
Vehicle:
unchanged (no vehicle)
Controls:
other: Yes, untreated eye
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 100 mg
Duration of treatment / exposure:
“Unwashed group” - Eye not rinsed, therefore possibly up to 7 days
“Washed group” - 30 seconds
Observation period (in vivo):
1, 24, 48 and 72 hrs after application and, if irritation persisted, an additional reading was made at day 7 to assess reversibility or irreversibility.
Number of animals or in vitro replicates:
3/group; sex not specified
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing (if done): yes, in one of the groups
- Time after start of exposure: 30 seconds

SCORING SYSTEM: Assessment of damage/irritation in both the washed and unwashed groups was made 1, 24, 48 and 72 hr following instillation of the test material, according to Draize (1965) scores. The test material was then assigned a Group Total Score for Eye Irritation.

TOOL USED TO ASSESS SCORE: standard ophthalmoscope (Keeler). The degree of irritation was further investigated at 24 hr under ultra-violet illumination (Mineralight UVSL-58) preceded by the instillation into the eye of fluorescein B.P.

Irritation parameter:
overall irritation score
Basis:
other: Group Total Score, unwashed group
Time point:
other: 1, 24, 48 and 72 hrs
Score:
ca. 21
Max. score:
195
Reversibility:
fully reversible within: 72 hrs
Irritation parameter:
cornea opacity score
Basis:
other: Total score, unwashed group
Time point:
other: 1, 24, 48 and 72 hrs
Score:
0
Max. score:
60
Reversibility:
other: Not applicable
Irritation parameter:
iris score
Basis:
other: Total score, unwashed group
Time point:
other: 1, 24, 48 and 72 hrs
Score:
ca. 1
Max. score:
30
Reversibility:
fully reversible within: 24 hrs
Remarks on result:
other: One rabbit showed slight iridial congestion at the 1 hr observation period, which had disappeared within 24 hrs.
Irritation parameter:
conjunctivae score
Basis:
other: Total score, unwashed group
Time point:
other: 1, 24, 48 and 72 hrs
Score:
ca. 9
Max. score:
45
Reversibility:
fully reversible within: 72 hrs
Remarks on result:
other: Minimal to slight conjunctival redness was seen in all three animals at the 1 hr observation time and had completely disappeared by the 72 hr observation time.
Irritation parameter:
chemosis score
Basis:
other: Total score, unwashed group
Time point:
other: 1, 24, 48 and 72 hrs
Score:
ca. 11
Max. score:
60
Reversibility:
fully reversible within: 72 hrs
Remarks on result:
other: Conjunctival chemosis was evident in all of the treated animals after 1 hr, but had completely disappeared by 72 hrs.
Irritation parameter:
overall irritation score
Basis:
other: Group Total Score, washed group
Time point:
other: 1, 24, 48 and 72 hrs
Score:
ca. 15
Max. score:
195
Reversibility:
fully reversible within: 72 hrs
Irritation parameter:
cornea opacity score
Basis:
other: Total score, washed group
Time point:
other: 1, 24, 48 and 72 hrs
Score:
ca. 0
Max. score:
60
Reversibility:
other: Not applicable
Irritation parameter:
iris score
Basis:
other: Total score, washed group
Time point:
other: 1, 24, 48 and 72 hrs
Score:
ca. 1
Max. score:
30
Reversibility:
fully reversible within: 24 hrs
Remarks on result:
other: Slight iridial congestion was evident in one rabbit after 1 hr but had completely disappeared by the 24 hr observation time.
Irritation parameter:
conjunctivae score
Basis:
other: Total score, washed group
Time point:
other: 1, 24, 48 and 72 hrs
Score:
ca. 6
Max. score:
45
Reversibility:
fully reversible within: 72 hrs
Remarks on result:
other: At the 1 hr observation period, minimal to slight conjunctival redness occurred in all of the treated animals. Complete recovery was seen after 72 hrs.
Irritation parameter:
chemosis score
Basis:
other: Total score, washed group
Time point:
other: 1, 24, 48 and 72 hrs
Score:
ca. 8
Max. score:
60
Reversibility:
fully reversible within: 72 hrs
Remarks on result:
other: Conjunctival chemosis was seen in all of the rabbits at the 1 hr observation time and had completely disappeared after 72 hrs.
Irritant / corrosive response data:
In the unwashed group, minimal to slight ocular irritation was seen in 2 of the 3 animals. Minimal (grade 1) to slight (grade 2) conjunctival redness and chemosis was seen in all of the animals at the 1 hr reading and one rabbit also showed slight iridial congestion. All evidence of iridial congestion had disappeared at 24 hrs but minimal chemosis and slight redness persisted in the conjunctiva of one rabbit and minimal redness and minimal chemosis in the remaining two animals. Irritation continued to subside and all lesions had disappeared within 72 hrs.

In the washed group, minimal to slight ocular irritation was noted in all of the treated rabbits. All animals showed minimal to slight conjunctival redness and chemosis at the 1 hr reading, with slight iridial congestion also being seen in one. All iridial congestion had disappeared at the 24 hr reading, but minimal redness and chemosis was still evident in 2 animals. All lesions had disappeared within 72 hrs.
Other effects:
Not reported.
Interpretation of results:
GHS criteria not met
Conclusions:
In a guideline eye irritation study, to GLP, ruthenium (IV) oxide hydrate (0.1 g, powdered) produced a group total score of 21 (out of 195) following instillation onto the conjunctival sac of three rabbits and, in a further three rabbits, a group total score of 15 (out of 195) when the test material was washed from the eye 30 seconds following instillation. Although slight irritation was seen, under the EU CLP regulations the test material would not be classified as an eye irritant.
Executive summary:

In an OECD Test Guideline 405 study, ruthenium (IV) oxide hydrate (0.1 g, powdered) was instilled into the conjunctival sac of the right eye of groups of three New Zealand white rabbits and both lids were briefly closed for about 1 second. The left eye remained untreated and acted as the control. In one of the groups, the treated eye was irrigated with 100 ml sterile distilled water, 30 seconds following instillation. Assessment of damage / irritation to the cornea, iris and conjunctiva of the treated eye, using the Draize scale, was made 1, 24, 48 and 72 hr following treatment. If irritation was persistent at 72 hr, an additional reading was made at day 7 in order to assess the reversibility of the effects.

 

These scores were combined to give a Group Total Score for Eye Irritation of 21 (out of 195) for the unwashed eye and 15 (out of 195) for the washed eye. In the unwashed group, observed at 24 hours after instillation, no cornea or iris irritation was reported, although slight (grade 1) to minimal (grade 2) conjunctival redness and chemosis was seen in all three animals. No irritation was seen at 72 hours in any animal (washed or unwashed groups).

 

Based on the results of this study, ruthenium (IV) oxide hydrate should not be classified for eye irritation according to EU CLP criteria (EC 1272/2008).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

In a GLP-compliant study according to OECD439, ruthenium (IV) oxide was considered to be non-cytotoxic and predicted to be non-irritant to skin.

 

In an OECD Test Guideline 405 study, ruthenium (IV) oxide hydrate (0.1 g, powdered) was instilled into the eyes of groups of three New Zealand white rabbits. One eye remained untreated and acted as control in each animal. In one group of three rabbits, the treated eye was rinsed 30 secs following instillation, whilst in another group of three rabbits the eyes remained unwashed. Only slight evidence of irritation (conjunctival redness and chemosis) was evident at 24 hr in the unwashed group, although this had fully reversed within 72 hr (Collier, 1982b).

No data were identified relating to irritation of the respiratory tract.

Justification for classification or non-classification

Based on the results of the available and reliable skin and eye irritation studies in rabbits, ruthenium (IV) oxide does not require classification for skin irritation/corrosion and eye irritation according to EU CLP criteria (EC 1272/2008).

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