Cobalt trihydroxide

Endpoint summary

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009-09-23 to 2009-10-09

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study reliable without restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Commission Regulation (EC) No. 440/2008, L 142, Annex Part B, 30 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

2009-04-06

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Winkelmann, D-33178 Borchen
- Age at study initiation: 8 – 9 weeks
- Weight at study initiation: 19 - 23 g
- Housing: Full barrier in an air-conditioned room; the animals were kept in groups of 5 animals in IVC cages, type II L, polysulphone cages on Altromin saw fibre bedding (preliminary study: lot no. 060609; main study: lot no. 040509)
- Diet (ad libitum): Altromin 1324 maintenance diet for rats and mice (lot no. 0654)
- Water (ad libitum): tap water, sulphur acidified to a pH value of approx. 2.8 (drinking water, municipal residue control, microbiol. controlled at regular intervals)
- Acclimation period: at least five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 55 ± 10%
- Air changes (per hr): at least 10 x / hour
- Photoperiod (hrs dark / hrs light): Artificial light, sequence being 12 hours light, 12 hours dark

Vehicle:

other: acetone/olive oil (3+1 (v/v))

Concentration:

6.25%, 12.5% and 25% (w/v) (diluted with acetone/olive oil)

No. of animals per dose:

5 mice per test group
5 mice per negative control group (vehicle)

Details on study design:

VEHICLE:
Due to the solubility properties of the test item the vehicle AOO (3+1 (v/v) Acetone/Olive Oil) was used (Acetone, neoLab, lot no. 1898a4307, expiry date: July 2010; olive oil highly refined, Sigma, lot no. 058K0684, expiry date: January 2010).

RANGE FINDING TESTS:
- Compound solubility: Before the initiation of the preliminary test, a feasibility test was performed to assess the maximum concentration which is technically applicable to the animals. The pasty test-substance was diluted in order to achieve a solution/ suspension suitable for application. The maximum technically applicable concentration of the test item in the vehicle was found to be 25%.
- Conduction of preliminary test: To determine the highest tolerated and non-irritant test concentration a preliminary test was performed. For this purpose, two animals were treated by topical application with the test item on three consecutive days at the following concentrations (diluted in AOO) to the entire dorsal surface of each ear: animal no. 1 was treated with a test item concentration of 25%, animal no. 2 was treated with a test item concentration of 25%, one further animal was treated with 100% AOO and served as negative control.
From day 1 to day 4 the ear thickness of each animal was measured.
During this period also all clinical signs were recorded.
Cageside observations included spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in the skin and fur, eyes and mucous membranes (salivation, discharge).
No signs of systemic toxicity could be detected in the animal.
- Irritation: No signs of irritation at the application site could be detected in the animal.

MAIN STUDY
TREATMENT PREPARATION AND ADMINISTRATION:
- Preparation of the Test Item: Based on the results observed in the preliminary test the following test item concentrations were selected for the main study: 6.25%, 12.5% and 25% (diluted with AOO). The preparations were made immediately prior to each dosing.
- Control: Acetone/olive oil (AOO) was used as vehicle and served as negative control.

TEST REGIME:
- Topical Application: Each mouse was treated by topical application of 25 μL of the selected solution to the entire dorsal surface of each ear. Topical applications were performed once daily over three consecutive days.
- Administration of 3H-methyl thymidine: Five days after the first topical application all mice were dosed with 20 μCi 3H-methyl thymidine by intravenous injection (tail vein) of 250μL of 3H-methyl thymidine, diluted to a working concentration of 80μCi/mL.
- Preparation of cell suspension: Approximately 5 hours after 3H-methyl thymidine-injection all mice were sacrificed by cervical dislocation. The draining “auricular lymph nodes” were excised, individually pooled for each animal (2 lymph nodes per animal) and collected in phosphate buffered saline (PBS). A single cell suspension of pooled lymph node cells was prepared by gentle mechanical disaggregation through polyamide gauze (200 mesh size). After washing the gauze with PBS the cell suspension was pelleted in a centrifuge. The supernatant was discarded and the pellets were resuspended with PBS. This washing procedure was repeated. After the final wash each pellet was resuspended in approx. 1 mL 5% TCA at approx. 4 °C for approximately 18 hours for precipitation of macromolecules. Each precipitate was once washed again, resuspended in 1 mL 5% TCA and 7 mL scintillation fluid was added. Then this solution was transferred into scintillation vials and stored at room temperature overnight.
- Determination of incorporated 3H-methyl thymidine: The 3H-methyl thymidine – incorporation was measured in a ß-counter and expressed as the number of disintegrations per minute (DPM). Similarly, background 3H-methyl thymidine levels were also measured (5% TCA). Determination of radioactivity was performed individually for each animal.

EVALUATION OF RESULTS:
The proliferative response of lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph node (DPM/NODE) and as the ratio of 3H-methyl thymidine - incorporation into lymph node cells of test group animals relative to that recorded for control group animals (STIMULATION INDEX). Before DPM/NODE values were determined, background values were subtracted.
EC3 values, calculated concentrations which induce stimulation indices of three, are determined by linear interpolation {EC3=c+[(3-d)/(b-d)]x(a-c)}, between two points of the stimulation indices axis, one above (a,b) and one below (c,d) the stimulation index of three. If all measured points are above or below the stimulation index of three, no EC3 value can be stated.
A substance is regarded as a 'sensitiser' in the LLNA if at least one concentration of the test item results in a 3 fold or greater increase in 3H-methyl thymidine - incorporation into lymph node cells of the lymph nodes of the test group animals, relative to that recorded for the lymph nodes of control group animals (Stimulation Index equal to or greater than 3.0).

Positive control substance(s):

other: p-Phenylenediamine (CAS 106-50-3, Sigma GmbH, purity >98%; Lot 128K0093; 1% in acetone/olive oil (Aceton, Merck; olive oil highly refined, Sigma) on three consecutive days)

Statistics:

Please see "Details on study design" above.

Positive control results:

In the positive control group given p-Phenylenediamine at the concentration of 1 % a stimulation index exceeding the threshold value of 3 (SI = 13.8) was noted. The study was therefore considered valid.

Parameter:

SI

Remarks on result:

other: see Remark

Remarks:

None of the three tested concentrations of the test item reached the stimulation index of 3: The stimulation index at a concentration of 6.25% was 2.3 The stimulation index at a concentration of 12.5% was 1.6 The stimulation index at a concentration of 25% was 2.2 The EC3 value (derived by linear interpolation) could not be calculated as the stimulation indices of all concentrations were below 3.

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: 6.25 %: 4051.2 (mean DPM); 2018.0 (mean DPM per node) 12.5 %: 2828.6 (mean DPM); 1406.7 (mean DPM per node) 25 %: 3828.8 (mean DPM); 1906.8 (mean DPM per node) Negative control: 1747.5 (mean DPM); 866.2 (mean DPM per node)

All animals survived throughout the test period without showing any clinical signs.

All animals showed the expected weight development, which includes a weight loss of up to 2 g throughout the study.

Interpretation of results:

not sensitising

Remarks:

Migrated information

Conclusions:

The test item Cobaltic hydroxide (CoRC Study EFF06) is expected to have no sensitising properties and therefore, should not be regarded as a dermal sensitiser.
According to the criteria specified by Directive 67/548/EEC and subsequent regulations, the test item is not classified as skin sensitiser.
According to the EC Regulation No. 1272/2008 and subsequent regulations, the test item is not classified as skin sensitiser.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

Migrated from Short description of key information:
Cobalt trihydroxide is expected to have no sensitising properties and therefore, should not be regarded as a dermal sensitiser. For further information please refer to the document attached to the endpoint summary in IUCLID section 7.4.

Justification for selection of skin sensitisation endpoint:
Key study

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

Occupational exposure studies in cobalt facilities:

Swennen et al (1993): Probability of dyspnoea correlated as function of increasing concentration of airborne Co dust during exercise. Reduction of FEV1/ VC ratio correlated with intensity of current cobalt concentration in air and urine. Exposure: 50% of the workers exposed to TWA cobalt air levels above 50µg/m³. 25% exposed to TWA cobalt air levels above 500µg/m³.

                                                           

Verougstraete et al (2004), follow-up study of Swennen et al.: Strict environmental control implemented between 1988 and 2001 resulted in decreases in airborne and urine cobalt levels. Cobalt exposures (as measured by cobalt in urine) were associated with decreases in FEV1 only in workers who smoked.

 

Roto (1980): Included case-referent and cross-sectional study. Exposure range of 0.06-0.1 mg cobalt/m³ (cobalt metal powder) were given. A correlation in decrease in FEV1 with increasing exposure to cobalt was analysed. Occupational asthma was defined as more than 15% reduction in FEV1. Overall, it was concluded that the risk for asthma is 5-fold higher in cobalt exposed workers as compared to controls.

 

Linna et al.2003, follow-up study of Roto: Process changes to hydrometallurgical workplaces now includes exposures to cobalt sulfate, carbonate, sulfide, oxides, hydroxides, as well as cobalt metal powders. Two new cases of occupational or allergic asthma were reported. Workers who smoked had significantly lower lung function parameters than workers who did not smoke.

 

Sauni et al 2010: Characterised all asthma cases from 1980 until 2003. The incidence of occupational asthma (>15% decrease in FEV1) correlated with an increase on cobalt exposure. Median cobalt air levels ranged from 0.1 mg/m³ in sulfatising and roasting workplaces to 0.03mg/m³ in leaching and solution preparation workplaces. Some work areas had concomitant exposures to sulfur dioxide and hydrochloride gases. The authors concluded that the evidence indicated that irritant gases may enhance the risk of respiratory sensitisation to cobalt. Cobalt air exposure levels below 0.120 mg cobalt/m³ (in the absence of irritant gases) were not associated with occupational asthma.

 

Pilliere et al (1990), Single-case study on occupational exposure to cobalt resinate: Cobalt resinate and cobalt stearate administration precipitated a positive finding in a bronchio-constriction test. Whereas the administration of cobalt tallate resulted in a negative test result. Cobalt resinate or cobalt stearate exposure decreased FEV1 by 30%. The chemical identity of occupational substance was not verified in the report. Inhalation administration of cobalt stearate, -resinate and -tallate was supervised under clinical conditions.

 

Cobalt industry-wide questionnaire

A cobalt industry-wide questionnaire exercise for cases of occupational asthma following cobalt exposure was conducted in 2010. A total of 13 facilities producing inorganic cobalt substances or inorganic cobalt substances with an organic anion (“cobalt carboxylates”) responded. The facilities reported in the occupational exposure studies cited above were not included in the questionnaire results. Three facilities reported some experience with cobalt asthma in either cobalt carboxylate production or inorganic cobalt substance production. The questionnaire indicates that there is some cobalt industry experience with occupational asthma in addition to the occupational exposure studies reported in the literature.

 

Immune vs. non-immune responses

Cobalt exposure has been reported to induce immune responses in some hardmetal workers diagnosed with occupational asthma or reduced lung function (as indicated by measured IgE titres). It is currently accepted that lung function is reduced by inflammatory processes occurring in the lungs. It is not clear (clinically) whether the inflammatory process leads to an immune response or whether the process itself, causes lung function changes in the absence of immune-related mechanisms. The current thinking is that inflammatory mechanisms are associated with reduced lung function by both immune-related and non-immune-related mechanisms. The studies used for the basis if this proposed hazard classification did not evaluate the presence of an immune response. The single-case-study on cobalt resinate and cobalt stearate indicated a late response in the bronchio-provocation test. This finding would favour a non-immune-related response as responses mediated by IgE (immunoglobulin E) are likely to be more-immediate.

 

Conclusion

Five well-characterised exposure studies in two cobalt facilities producing cobalt substances support observations that occupational exposures to inorganic cobalt substances (in the absence of other metal exposures) is associated with occupational asthma. In these cases occupational asthma was defined by clinically-compliant lung function testing. Neither study was able to discriminate between specific cobalt substances and their individual potential to reduce lung function. Neither study indicated a high frequency of occurrence of occupational asthma among the worker population.

 

A case report of occupational exposure to cobalt resinate verified respiratory sensitivity of a worker to cobalt resinate and cobalt stearate by bronchio-provocation-testing with each substance. The worker did not respond to bronchio-provocation after the inhalation administration of cobalt tallate. The cobalt industry-wide questionnaire showed that there is industry experience with cobalt resinates and cases of occupational asthma. Based on available information, there is no indication the frequency of occupational asthma in workers is high.

 

Based on the above argumentation, the following substances will be classified as respiratory sensitiser, category 1B:

Cobalt Powders, Cobalt Sulfate, Cobalt di-Chloride, Cobalt di-Nitrate, Cobalt Carbonate, Cobalt Acetate, Cobalt Monoxide, Tricobalt Tetraoxide, Cobalt Sulfide, Cobalt di-Hydroxide, Cobalt tri-Hydroxide, Cobalt Oxy-Hydroxide, Cobalt Resinate and Cobalt Stearate.

Justification for selection of respiratory sensitisation endpoint:
Weight of evidence information

Justification for classification or non-classification

Sensitisation

The reference Ahuja (2009) is considered as the key study on skin sensitisation (Local lymph node assay (LLNA) in mice) and will be used for classification. The overall sensitisation results are as follows:

·        concentration 6.25%: SI 2.3

·        concentration 12.5%: SI 1.6

·        concentration 25%: SI 2.2

None of the three tested concentrations of the test item reached the stimulation index of 3. The EC3 value (derived by linear interpolation) could not be calculated as the stimulation indices of all concentrations were below 3. The classification criteria acc. to regulation (EC) 1272/2008 as skin sensitiser are not met, hence no classification required.

 

Respiratory sensitisation

Cobalt trihydroxide will be classified as respiratory sensitiser according to section 3.4, annex I of regulation (EC) 1272/2008 based on human epidemiological data showing cases of occupational asthma following prolonged exposure towards cobalt salts, -oxides and hydroxides. Cobalt trihydroxide will be classified as respiratory sensitiser category 1B (H334). Thus further testing is not required, according to section 1.1.3 and 1.2, annex XI of regulation (EC) 1907/2006.