Cobalt hydroxide oxide

Endpoint summary

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:

2010-07-28 to 2010-08-02

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Deviations:

no

Qualifier:

according to guideline

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

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2009-03-30

Details on test animals or test system 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 (Lot No.: 10-EKIN-028) was 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 / corrosion parameter:

% tissue viability

Value:

87.6

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

After treatment with the test item cobalt oxyhydroxide the relative absorbance values decreased to 87.6%. This value is well above the threshold for irritancy of ≤ 50%. Therefore, the test item is not considered to possess an irritant potential.

- After treatment with the negative control the absorbance values were well above the required acceptability criterion of mean OD ≥ 0.6 till≤1.5 for the15 minutes treatment interval thus showing the quality of the tissues.

- Treatment with the positive control induced a decrease in the relative absorbance as compared to the negative control to 39.7%. The value is slightly out of the range of the historical control data (3-34%). However, since a markedly reduction in the relative absorbance compared to the negative control cultures could be observed, the positive control is considered valid. The deviation is considered as biological variation with no detrimental effect on the validity.

- The standard deviations between the % variabilities of the test item, the positive and negative controls were below 10% (threshold of the "OECD Guideline for Testing of Chemicals 439: In vitro Skin Irritation: Reconstructed Human Epidermis Test Method": 18%), thus ensuring the validity of the study.

Results after treatment with cobalt oxyhydroxide

 

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	0.683	0.326	0.280	0.331	46.6 39.1 33.5	6.5	39.7*
Test Item	15 min	0.774	0.683	0.732	0.730	92.9 82.0 87.8	5.5	87.6

*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:

GHS criteria not met

Conclusions:

The test item cobalt oxyhydroxide is not irritating to the skin and therefore, 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:

2010-11-04 to 2010-11-15

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

2002-04-24

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

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: 15 - 16 weeks
- Weight at study initiation: 2559 - 2671 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: 6 - 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:

not 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 the 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:

0.67

Max. score:

1

Reversibility:

fully reversible within: 72 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

Irritation parameter:

chemosis score

Basis:

mean

Remarks:

(animal #2)

Time point:

24/48/72 h

Score:

0

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

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 across 3 scoring times (24, 48 and 72 hours after instillation) for each animal for corneal opacity, iris light reflex, redness and chemosis of the conjunctivae, separately. 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 0.67, 0.00 and 0.00 for reddening, respectively and 0.00 for chemosis for all animals.
- Slight reddening of the conjunctivae and slight reddening of the sclera were noted in two animals (#1 and #2) one hour after instillation of the test item. The slight reddening of the conjunctivae persisted in one animal (#1) up to the 48-hour reading. Slight swelling (chemosis) of the conjunctivae was observed in one animal (#1) at the 1-hour reading. At the same observation point, slight ocular discharge was present in two animals (#1 and #2).
- No abnormal findings were observed in the treated eyes of any animals 72 hours after treatment, the end of the observation period for all animals.

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:

GHS criteria not met

Conclusions:

According to Regulation (EC) No. 1272/2008 and subsequent regulations, cobalt hydroxide oxide 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 in an in vitro human skin model test (according to OECD 431) are as follows:

·                   Relative viability 3 min after treatment: 82.4 %

·                   Relative viability 60 min after treatment: 54.0 %

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 87.6%. 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 12.10. 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=0.67, 0.00, 0.00

-         Conjunctival oedema (chemosis)=0.00 for all three animals

The classification criteria acc. to regulation (EC) 1272/2008 as irritating to eyes are not met, since the value of conjunctival redness was below the threshold of ≥ 2 as well as the effect was fully reversible within 72 hours. No classification required.

 

Respiratory irritation

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