Cobalt dihydroxide

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:

2010-10-13 to 2010-10-18

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 applied 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], present 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:

110.1

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 dihydroxide the relative absorbance values remained unchanged at 110.1%. 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 within the required acceptability criterion of mean OD ≥ 0.6 till ≤ 1.5 for the 15 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 23.4% thus ensuring the validity of the test system.

- The standard deviations between the % variabilities of the test item, the positive and negative controls were below 9% (threshold of the "OECD Guideline for the 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 dihydroxide:

Dose group	Treatment Interval	Absorbance 570 nm Tissue 1	Absorbance 570 nm Tissue 2	Absorbance 570 nm Tissue 3	Mean Absorbance of 3 Tissues	Absorbance [%] Tissue 1, 2 + 3	Standard Deviation [%]	Rel. Absorbance [% of Negative Control]
Negative Control	15 min	1.057	0.920	0.912	0.963	109.8 95.5 94.7	8.5	100.0
Positive Control	15 min	0.204	0.185	0.285	0.225	21.2 19.2 29.6	5.5	23.4
Test Item	15 min	1.064	1.091	1.025	1.060	110.5 113.4 106.5	3.5	110.1

 

 

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 dihydroxide 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, other

Remarks:

in vivo study

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Well documented with individual Draize Scoring Scheme - The observation time was only until 72 hours after application of the test item. this observation time was not long enough to evaluate the reversibility of the irritating effect, but it could be observed that the irritating effect got better with the time passing. - The eyes were not examined 1 hour after application of the test item. - There was no statement if effect other than ocular were observed. - The purity and stability of the test item was missing.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

1987-02-24

Deviations:

yes

Remarks:

, see "rational for reliability"

GLP compliance:

no

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Sweetwater farms
-No further information on the test animals was stated.

Vehicle:

unchanged (no vehicle)

Controls:

no

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 g of the undiluted sample was applied to the right eye of each of six rabbits. The left eye of all rabbits was untreated and served as a cntrol.
No further information on amount/concentration applied was stated.

Duration of treatment / exposure:

In 3 of 6 rabbits the test material was left in the eye for 30 seconds . For the remaining three rabbits no statement regarding duration of treatment/exposure was made.

Observation period (in vivo):

24, 48 and 72 hours after application of the test material

Number of animals or in vitro replicates:

6 rabbits

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): The treated eyes of three rabbits were irrigated following application with 200 ml of lukewarm tap water. The treated eyes of the three remaining received no further treatment.
- Time after start of exposure: Irrigation after 30 seconds

SCORING SYSTEM: Scoring of irritative effects was according to the method of Draize in which corneal, iris and conjunctival effects are scored separately. In this scoring system, injuries to the cornea and iris may represent as much as 80 percent of the total score. cornea and iris scores are heavily weighted because of their essential role in vision.
No further information on the study design was stated.

Irritation parameter:

cornea opacity score

Basis:

mean

Remarks:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

0

Remarks on result:

other: Eyes were not rinsed after application of the test item.

Irritation parameter:

iris score

Basis:

mean

Remarks:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

0

Remarks on result:

other: Eyes were not rinsed after application of the test item.

Irritation parameter:

conjunctivae score

Basis:

mean

Remarks:

animal #1

Time point:

24/48/72 h

Score:

1.67

Max. score:

2

Reversibility:

not specified

Remarks on result:

other: Eyes were not rinsed after application of the test item.

Irritation parameter:

chemosis score

Basis:

mean

Remarks:

animal #1

Time point:

24/48/72 h

Score:

0.33

Max. score:

1

Reversibility:

fully reversible within: 48 hours

Remarks on result:

other: Eyes were not rinsed after application of the test item.

Irritation parameter:

cornea opacity score

Basis:

mean

Remarks:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

0

Remarks on result:

other: Eyes were not rinsed after application of the test item.

Irritation parameter:

iris score

Basis:

mean

Remarks:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

0

Remarks on result:

other: Eyes were not rinsed after application of the test item.

Irritation parameter:

conjunctivae score

Basis:

mean

Remarks:

animal #2

Time point:

24/48/72 h

Score:

2

Max. score:

3

Reversibility:

not specified

Remarks on result:

other: Eyes were not rinsed after application of the test item.

Irritation parameter:

chemosis score

Basis:

mean

Remarks:

animal #2

Time point:

24/48/72 h

Score:

0.67

Max. score:

2

Reversibility:

fully reversible within: 48 hours

Remarks on result:

other: Eyes were not rinsed after application of the test item.

Irritation parameter:

cornea opacity score

Basis:

mean

Remarks:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

0

Remarks on result:

other: Eyes were not rinsed after application of the test item.

Irritation parameter:

iris score

Basis:

mean

Remarks:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

0

Remarks on result:

other: Eyes were not rinsed after application of the test item.

Irritation parameter:

conjunctivae score

Basis:

mean

Remarks:

animal #3

Time point:

24/48/72 h

Score:

2.67

Max. score:

3

Reversibility:

not specified

Remarks on result:

other: Eyes were not rinsed after application of the test item.

Irritation parameter:

chemosis score

Basis:

mean

Remarks:

animal #3

Time point:

24/48/72 h

Score:

1

Max. score:

2

Reversibility:

fully reversible within: 72 hours

Remarks on result:

other: Eyes were not rinsed after application of the test item.

Irritant / corrosive response data:

Following application of cobalt hydrate powder without a rinse, no corneal opacity or iritis was noted at any time diuring the study. moderate to severe conjunctivitis was noted in all three rabbits and blistering of the lower lids was noted in two rabbits at the 24 to 48-hour reading. Mild to moderate conjunctivitis was generally noted in all of the rabbits at the 72-hour reading (for results see "Overall irritation/ corrosion results" above).
Following a 200 ml rinse 30 seconds after application of cobalt hydrate powder, no corneal opacity or iritis was noted during the study. At the 24-hour reading irritative effects noted included mild to moderate conjunctival erythema in all three rabbits, with bistering of the lower lids in one rabbit. At the 48-hour reading, mild conjunctival erythema was noted in one rabbit. No irritative effects were noted at the 72-hour reading (see "Remarks on results including tables and figures" for results; table 1).

Table 1. Eye irritation scores in albino rabbits following application of 0.1 gram of cobalt hydrate powder followed in 30 seconds by irrigation with 200 ml of lukewarm tap water

Irritation parameter	Basis	Time point		Max. Score	Reversibility	Remarks
Cornea score	Mean animal #4	24, 48, and 72 hours	0	0		Eyes rinsed after 30 seconds
Iris score	Mean animal #4	24, 48, and 72 hours	0	0		Eyes rinsed after 30 seconds
Conjunctivae score	Mean animal #4	24, 48, and 72 hours	1	2		Eyes rinsed after 30 seconds
Chemosis score	Mean animal #4	24, 48, and 72 hours	0	0		Eyes rinsed after 30 seconds
Cornea score	Mean animal #5	24, 48, and 72 hours	0	0		Eyes rinsed after 30 seconds
Iris score	Mean animal #5	24, 48, and 72 hours	0	0		Eyes rinsed after 30 seconds
Conjunctivae score	Mean animal #5	24, 48, and 72 hours	0.67	2		Eyes rinsed after 30 seconds
Chemosis score	Mean animal #5	24, 48, and 72 hours	0	0		Eyes rinsed after 30 seconds
Cornea score	Mean animal #6	24, 48, and 72 hours	0	0		Eyes rinsed after 30 seconds
Iris score	Mean animal #6	24, 48, and 72 hours	0	0		Eyes rinsed after 30 seconds
Conjunctivae score	Mean animal #6	24, 48, and 72 hours	0.33	1		Eyes rinsed after 30 seconds
Chemosis score	Mean animal #6	24, 48, and 72 hours	0	0		Eyes rinsed after 30 seconds

Interpretation of results:

Category 2 (irritating to eyes) based on GHS criteria

Conclusions:

The following classification is based on the results obtained without rinsing the eyes after application.
According to the EC Regulation No. 1272/2008 and subsequent regulations, the test item is classified as Category 2.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Additional information

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

Justification for selection of eye irritation endpoint:
Key study

Effects on eye irritation: irritating

Justification for classification or non-classification

Skin irritation

Reference Heppenheimer (2011) 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 110.1%. 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

Reference Vinegar (1977) is considered as the key study for eye irritation and will be used for classification. The overall irritation results 24, 48 and 72h after application per animal are as follows:

·                   Corneal opacity: mean score=0.0 for all 3 animals

·                   Iritis: mean score=0.0 for all 3 animals

·                   Conjunctival redness: 1.67, 2.0, 2.67

·                   Conjunctival oedema (chemosis): 0.33, 0.67, 1.0

The classification criteria acc. to regulation (EC) 1272/2008 as irritating to eyes are met, since 2 of 3 animals showed a mean conjunctival score after 24, 48 and 72hours of 2.0 and above. Cobalt dihydroxide will be classified as irritating to eyes category 2 (H319).

 

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.