Cobalt trihydroxide

Skin sensitisation

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Deviations:

no

Test guideline 3

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)

Test material

Test material information

Constituent 1

Reference substance name:

Cobalt trihydroxide

EC Number:

215-153-0

EC Name:

Cobalt trihydroxide

Cas Number:

1307-86-4

Molecular formula:

CoH3O3

IUPAC Name:

cobalt(3+) trihydroxide

Details on test material:

- Name of test material (as cited in study report): Cobaltic Hydroxide (CoRC Study EFF06)
- Chemical name: Cobalt trihydroxide
- Physical state: black paste
- Storage condition of test material: room temperature

No further information on the test material was stated.

In vivo test system

Test animals

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

Study design: in vivo (LLNA)

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.

Results and discussion

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.

In vivo (LLNA)

Results

Results 1

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.

Results 2

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)

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.