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Description of key information

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation / corrosion
Remarks:
other: validated "in vitro" test method
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010-07-28 to 2010-08-02
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study reliable without restrictions
Qualifier:
according to
Guideline:
other: OECD 439: In vitro Skin Irritation: Reconstructed Human Epidermis Test Method (Original guideline adopted 22 July 2010)
Deviations:
no
Qualifier:
according to
Guideline:
other: ECVAM international validation study on in vitro tests for acute skin irritation (Altern Lab Anim. 2007 Dec; 35 (6):559-601)
Deviations:
no
Qualifier:
according to
Guideline:
other: Commission Regulation (EC) No. 440/2008 B.46
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
signed 2009-03-30
Details on test animals and environmental conditions:
Not applicable - Since this is an in vitro study there is no information on test animals.
Vehicle:
water
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): Approx. 10 mg of the test item were applied to each of triplicate tissues, spread to match the tissue size, and wetted with 15 µL deionised water.
No further information on the amount/concentration was stated.
Duration of treatment / exposure:
15 minutes
Observation period:
not applicable
Number of animals:
not applicable
Details on study design:
CELL CULTURE:
EpiSkin™ kit was (Lot No.: 10-EKIN-028) purchased from Skinethic Laboratories (06000 Nice, France). The EpiSkin™ tissue consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiSkin™ tissues (surface 0.38 cm²) are cultured on specially prepared cell culture inserts.

The RhE (Reconstructed human Epidermis) model supplier ensured and demonstrated that each batch of the RhE model used met defined production release criteria, e.g. viability, barrier function, no bacterial and mycoplasma contamination and histological scoring.

TREATMENT:
The negative control (deionised water (Lot no. 230710, produced in-house); Volume 10 µL) and positive control (5% SLS (Sodium lauryl sulphate, lot no. 1353471 51508322, Fluka, Sigma-Aldrich, 89555 Steinheim, Germany) solution in deionised water, prepared freshly prior to the performance of the experiment; Volume 10 µL), and the test item were added into the insert atop the concerning EpiSkin™ triplicate tissues each. The 12-well plates were placed into the incubator for 15 min at 37 ± 1.5 °C, 5 ± 0.5% CO2.
After the end of the treatment interval the inserts were removed immediately from the 12-well plate. Using a wash bottle the tissues were gently rinsed with PBS to remove any residual test material. Excess PBS was removed by gently shaking the inserts and blotting the bottom with blotting paper. The inserts were placed in the plates with 2 mL maintenance medium. The tissues were incubated for about 42 hours at 37 ± 1.5 °C, 5 ± 0.5% CO2.

CELL VIABILITY TEST:
Cell viability is measured by dehydrogenase conversion of MTT [(3-4,5-dimethyl thiazole 2-yl) 2,5-diphenyl-tetrazoliumbromide], in cell mitochondria, into a blue formazan salt that is quantitatively measured after extraction from tissues (Faller, C., Bracher, M., Dami, N., Roguet, R., 2002. Predictive ability of reconstructed human epidermis equivalents for assessment of skin irritation of cosmetics. Toxicology in vitro 16 (5), 557-552). The percent reduction of cell viability in comparison of untreated negative controls is used to predict skin irritation potential (see OECD TG 439) and is used for the purpose of classification as irritating or non-irritating according to chemicals law (EU CLP, UN GHS).
After the treatment procedure was completed for all tissues of each time point cell culture inserts were transferred from the holding plates to plates filled with 2 mL assay medium containing 0.3 mg/mL MTT per well. After a 3 hour incubation period (37 ± 1.5 °C, 5 ± 0.5% CO2) MTT solution was aspirated from the wells and the wells were rinsed three times with PBS. Tissue samples were cut out of the inserts with a biopsy punch and transferred into plastic vials. The tissue samples were immersed into extractant solution by gently pipetting 0.5 mL extractant solution (isopropanol) into each vial. The tissue samples were completely covered by isopropanol. The vials were sealed to inhibit isopropanol evaporation. The formazan salt was extracted about 67 hours in the refrigerator.
Per each tissue sample 2 X 200 µL aliquots of the blue formazan solution were transferred into a 96-well flat bottom microtiter plate. OD was read in a microplate reader (Versamax® Molecular Devices, 85737 Ismaning, Germany) at 570 nm without reference filter . Mean values were calculated from the 2 wells per tissue sample.

EVALUATION OF RESULTS:
The mean OD of the three negative control tissues was calculated. This value corresponds to 100% tissue viability in the current test. For each individual tissue treated with the test item or the positive control the individual relative tissue viability is calculated according to the following formula:
Relative viability (%) = [OD test item/ OD negative control] X 100
For the test item and the positive control the mean relative viability +/- standard deviation of the three individual tissues are calculated and used for classification according to the following prediction model:
For the current test, an irritation potential of a test item according to EU classification R38 (according to directive 67/548/EEC), H315 (according to regulation (EC) 1272/2008) is recommended if the mean relative tissue viability of three individual tissues is reduced below 50% of the negative control.

ACCEPTABILITY OF THE ASSAY:
The absolute OD 570 nm of the negative control tissues in the MTT test is an indicator of tissue viability obtained after the shipping and storing procedure and under specific conditions of the assay. Tissue viability is meeting the acceptance criterion if the mean OD of the three tissues is ≥ 0.6 till ≤ 1.5.
The standard deviations in between tissues of the same treatment group should be ≤ 18%.
An assay is meeting the acceptance criterion if mean relative tissue viability of the positive control is ≤ 40%.

TEST FOR DIRECT MTT REDUCTION:
It was necessary to assess the ability of the test item to directly reduce MTT. To test for this ability approximately 25 mg of the test item were added to 1 mL of MTT solution and the mixture was incubated in the dark at room temperature for 60 minutes. Untreated MTT medium was used as control. If the MTT solution colour turned blue/purple, the test item was presumed to have reduced the MTT.
No colour change could be observed in the present study.
No further information on the study design was stated.
Irritation parameter:
other: relative viability (%)
Basis:
mean
Time point:
other: after 15 min incubation
Score:
95.3
Max. score:
100.9
Reversibility:
no data
Irritant / corrosive response data:
After treatment with the test item tricobalt tetraoxide the relative absorbance values decreased irrelevantly to 95.3%. This value is well above the threshold for irritancy of ≤ 50%. Therefore, the test item is not considered to possess an irritant potential.

Results after treatment with tricobalt tetraoxide

 

Dose group

Treat-ment Interval

Absor-bance 570 nm
Tissue 1

Absor-bance 570 nm
Tissue 2

Absor-bance 570 nm
Tissue 3

Mean Absor-bance of 3 Tissues

Absorbance [%] Tissue 1, 2 + 3

Standard Deviation [%]

Rel. Absorbance

[% of Negative Control]

Negative Control

15 min

0.924

0.801

0.775

0.834

110.8
96.1
93.0

9.5

100.0

Positive Control

15 min

0.388

0.326

0.280

0.331

46.6
39.1
33.5

6.5

39.7*

Test Item

15 min

0.812

0.731

0.841

0.795

97.4
87.7
100.9

6.8

95.3

*The viability is out of the historical range; nevertheless, since the positive control met the laboratories internal acceptance criteria (viability of 40%) and since it showed a positive effect, it can still be considered as valid.

Optical evaluation of the MTT-reducing capacity of the test item after 1 hour incubation with MTT-reagent did not show blue colour.

Historical data:

Positive Control

Negative Control

Number of Studies

85

Number of Studies

85

Period

July 2007 – June 2010

Period

July 2007 – June 2010

Mean Viability

17.8%

Mean OD

1.051

Standard Deviation

10.7%

Standard Deviation

0.185

Range of Viabilities

3% - 34%

Range of ODs

0.7 – 1.5

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test item tricobalt tetraoxide is not irritating to the skin and therefore, the test item should not be classified and labelled as skin irritant according to Directive 67/548/EEC.
Also, the test item should not be classified and labelled as skin irritant according to Regulation (EC) No.: 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:
2010-10-28 to 2010-11-08
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study reliable without restrictions
Qualifier:
according to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Version / remarks:
adopted April 24, 2002
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)
Version / remarks:
May 20, 2008
Deviations:
no
GLP compliance:
yes
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories U.K. Ltd., Hillcrest, Dodgeford Lane, Belton, Loughborough, Leics, LE12 9TE, UK
- Age at study initiation: 12 weeks
- Weight at study initiation: 2301 - 2710 g
- Housing: Individually in stainless steel cages equipped with feed hoppers and drinking water bowls
- Diet (ad libitum): Pelleted standard Teklad Global High Fiber Rabbit Diet 2031C (batch no. 25/10, Provimi Kliba AG, 4303 Kaiseraugst, Switzerland)
- Water (ad libitum): Community tap water from Füllinsdorf
- Acclimation period: 5 - 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17-23
- Humidity (%): 30-70
- Air changes (per hr): Air-conditioned with 10-15 air changes
- Photoperiod (hrs dark / hrs light): automatically controlled light cycle of 12 hours light and 12 hours dark

The animals were provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.
Vehicle:
unchanged (no vehicle)
Controls:
no
Amount / concentration applied:
TEST MATERIAL
- The test item was applied as a weight of 0.1 g/animal, the dose specified in the test guidelines for solid test items.
- On the day of treatment, the test item was applied with an eye glass to the conjunctival sac of the left eye of each animal after gently pulling the lower lid away from the eyeball. The lids were then gently held together for about one second to prevent loss of test item. The right eye
remained untreated and served as the reference control.
- A single animal was treated first. As neither a corrosive effect nor a severe irritant effect was observed after the 1- and 24-hour examinations, the test was completed using the two remaining animals.
Duration of treatment / exposure:
no applicable
Observation period (in vivo):
approximately 1, 24, 48 and 72 hours after test item instillation
Number of animals or in vitro replicates:
3 (male rabbits)
Details on study design:
The eyes of the animals were examined one day prior to test item administration.

Observation and Scoring:
- The eye reactions were assessed according to Draize scale. Scleral reddening and ocular discharge were also assessed according to following scale (See "Any other information on materials and methods incl. tables"). Eye examinations were made with a Varta Cliptrix diagnostic-lamp (Roth AG, 4153 Reinach / Switzerland).
- Viability / Mortality: Daily from acclimatization of the animals to termination of the test.
- Clinical signs (systemic): Daily from acclimatization of the animals to termination of the test.
- Body weights: At start of acclimatization, on the day of application and at termination of observation.
Irritation parameter:
cornea opacity score
Basis:
mean
Remarks:
(animal #1)
Time point:
24/48/72 h
Score:
0
Irritation parameter:
iris score
Basis:
mean
Remarks:
(animal #1)
Time point:
24/48/72 h
Score:
0
Irritation parameter:
conjunctivae score
Basis:
mean
Remarks:
(animal #1)
Time point:
24/48/72 h
Score:
0.33
Max. score:
1
Reversibility:
fully reversible within: 48 hours
Irritation parameter:
chemosis score
Basis:
mean
Remarks:
(animal #1)
Time point:
24/48/72 h
Score:
0
Irritation parameter:
cornea opacity score
Basis:
mean
Remarks:
(animal #2)
Time point:
24/48/72 h
Score:
0
Irritation parameter:
iris score
Basis:
mean
Remarks:
(animal #2)
Time point:
24/48/72 h
Score:
0
Irritation parameter:
conjunctivae score
Basis:
mean
Remarks:
(animal #2)
Time point:
24/48/72 h
Score:
0.33
Max. score:
1
Reversibility:
fully reversible within: 48 hours
Irritation parameter:
chemosis score
Basis:
mean
Remarks:
(animal #2)
Time point:
24/48/72 h
Score:
0.33
Max. score:
1
Reversibility:
fully reversible within: 48 hours
Irritation parameter:
cornea opacity score
Basis:
mean
Remarks:
(animal #3)
Time point:
24/48/72 h
Score:
0
Irritation parameter:
iris score
Basis:
mean
Remarks:
(animal #3)
Time point:
24/48/72 h
Score:
0
Irritation parameter:
conjunctivae score
Basis:
mean
Remarks:
(animal #3)
Time point:
24/48/72 h
Score:
0.33
Max. score:
1
Reversibility:
fully reversible within: 48 hours
Irritation parameter:
chemosis score
Basis:
mean
Remarks:
(animal #3)
Time point:
24/48/72 h
Score:
0
Irritant / corrosive response data:
- The mean score was calculated separately for each animal across three scoring times (24, 48 and 72 hours after instillation) for corneal opacity, iris light reflex, redness and chemosis of the conjunctivae, respectively. The individual mean scores for corneal opacity and iris light reflex were 0.00 for all three animals. The individual mean scores for the conjunctivae were 0.33 for all animals for reddening and 0.00, 0.33 and 0.00 for chemosis, respectively.
- Slight reddening of the conjunctivae was noted in all animals 1 hour and 24 hours after instillation of the test item. At the same time, slight swelling (chemosis) of the conjunctivae was observed in one animal (#2). Slight reddening of the sclerae was recorded in two animals (#2 and #3) at the 1-hour reading and persisted in one animal (#2) up to the 24-hour reading. Slight ocular discharge was present in all animals at the 1-hour reading and persisted in one animal (#2) up to the 24-hour reading.
- No abnormal findings were observed in the treated eyes of any animals 48 hours after treatment.
- No staining produced by the test item was observed in the treated eyes.
- Black test item remnants were observed in the treated eyes of all animals at the 1-hour reading and persisted in one animal (#2) up to the 24-hour reading.
- No corrosion of the cornea was observed at any of the reading times.
Other effects:
- Viability / Mortality: No intercurrent deaths occurred during the course of the study.
- Clinical signs: No clinical signs were recorded throughout the entire observation period.
- Body weights: The body weight of the animals was within the range commonly recorded for this strain and age.
Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
According to 67/548/EEC and subsequent regulations, tricobalt tetraoxide is not classified as an eye irritant.
According to Regulation (EC) No. 1272/2008 and subsequent regulations, tricobalt tetraoxide is not classified as an eye irritant.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Additional information

Justification for selection of skin irritation / corrosion endpoint:
Key study

Justification for selection of eye irritation endpoint:
Key study

Justification for classification or non-classification

Skin corrosion

Reference Heppenheimer (2010) is considered as the key study for skin corrosion and will be used for classification. The overall irritation results an in vitro human skin model test (according to OECD 431) are as follows:

·                   Relative viability 3 min after treatment: 94.7 %

·                   Relative viability 60 min after treatment: 83.3 %

The classification criteria according to regulation (EC) 1272/2008 as corrosive to skin are not met since the relative viability after 3 and 60 min was above 50 % and 15 % respectively, hence no classification required.

 

Skin irritation

Reference Heppenheimer (2010) is considered as the key study for skin irritation and will be used for classification. The mean relative absorbance (% of the negative control, correlating with mean tissue viability) after 15 minutes incubation in the in vitro human skin model test (EpiSkin, according to OECD TG 439) was 95.3%. The classification criteria according to regulation (EC) 1272/2008 as skin irritant are not met, since the mean tissue viability was above the threshold for skin irritants of 50.0%. No classification required.

 

Eye irritation

The references Heppenheimer (2010) and Sieber (2011) are considered as the key studies for severe eye irritation and will be used for classification.

According to Heppenheimer (2010) the mean in vitro score after 240 minutes incubation results of the in vitro bovine corneal opacity and permeability assay (BCOP, according to OECD TG 437) was 10.39. The value was below the threshold for severe eye irritants of 55.1. The classification criteria according to regulation (EC) 1272/2008 as severe eye irritation are not met, hence no classification required.

According to Sieber (2011) the overall irritation results 24, 48 and 72 hours after application per animal are as follows:

-         Corneal opacity=0.00 for all three animals

-         Iris light reflex=0.00 for all three animals

-         Conjunctival redness=0.33 for all three animals

-         Conjunctival oedema (chemosis)=0.00, 0.33 and 0.00

The classification criteria acc. to regulation (EC) 1272/2008 as irritating to eyes are not met, since the values of the conjunctivae were below the thresholds of ≥ 2 as well as the effects were fully reversible within 48 hours. No classification required.

 

Respiratory irritation

The justification for non classification as respiratory irritant is covered under the endpoint specific target organ toxicity- single exposure: inhalation as given in the acute toxicity section.