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Description of key information

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010-04-26 to 2010-05-12
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study relaible without restrictions
according to guideline
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
adopted 2002-04-24
GLP compliance:
yes (incl. QA statement)
signed 2009-04-06
Type of study:
mouse local lymph node assay (LLNA)
Details on test animals and environmental conditions:
- Source: Harlan Winkelmann, 33178 Borchen, Germany
- Age at study initiation: 8 - 9 weeks
- Weight at study initiation: 18 - 22 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 (lot no. 291109, preliminary test; 101109, main test)
- Diet (ad libitum): Altromin 1324 maintenance diet for rats and mice (lot no. 1130)
- Water (ad libitum): Tap water, sulphur acidified to a pH value of approx. 2.8 (drinking water, municipal resdue control, microbiological controls at regular intervals).
- Acclimation period: At least five days

- Temperature: 22 +/- 3°C
- Relative humidity: 55 +/- 10 %
- Air changes: At least 10 X / hour
- Photoperiod (hrs dark / hrs light): 12/12

No further information on the test animals was stated.
other: acetone/olive oil (3+1 (v/v))
50 %, 25 % and 12.5 % of cobalt stearate (diluted in acetone/olive oil)
No. of animals per dose:
5 mice per test group
5 mice per negative control group (vehicle)
Details on study design:
Due to the solubility properties of the test item the vehicle acetone/olive oil was used. (Acetone, Prolabo, lot no. 09J140506, expiry date: 10/2013; olive oil highly refined, Sigma, lot no. 0001423657-22709119, expiry date: 05/2010)

- Compound solubility: Before the initiation of the preliminary test, a solubility test was performed to define the maximum concentration which is technically applicable to the animals. The maximum technically applicable concentration of the test item in the vehicle was found to be 50%.
To determined 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 a concentration of 50 % (diluted in acetone/olive oil) to the entire dorsal surface of each ear.
One further animal was treated with 100 % acetone/olive oil 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, aspyxia, vocalisation, diarrhoea, changes in the skin and fur, eyes and mucous membranes (salivation, discharge).
No signs of systemic toxicity could be detected in any animal.
- Irritation: No signs of irritation at the application site could be detected in any animal.

Based on the results observed in the preliminary test the following test item concentrations were selected for the main study:
50 %, 25 % and 12.5 % (diluted in acetone/olive oil). The preparations were made immediately prior to each dosing.
Acetone/olive oil 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 ((TRK 300, 20 Ci/mmol; Perkin Elmer, lot no. 201003E), diluted to a working concentration of 80 µCi/mL):
Five days after the first topical application all mice were dosed with 20 µCi 3H-methyl thymidine by intravenous injection (tail vein) of 250 µL of 3H-methyl thymidine, diluted to a working concentration of 80 µCi/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; BSL BIOSERVICE, lot no. PBS-22042010, expiry date:10/2010). 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 (Trichloroacetic acid; Sigma, lot no. 105K0708, expiry date:11/2010) 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 β-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.

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).
No further information on the study design was stated.
Positive control substance(s):
other: P-Phenylenediamine (CAS 106-50-3, Sigma GmbH, purity > 98 %; Lot 128K0093; Vehicle: acetone/olive oil (3+1 (v/v acetone olive oil; 1 % concentration on 3 consecutive days)
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 = 5.1) was noted. The study was therefore considered valid.
other: disintegrations per minute (DPM)
Remarks on result:
other: 12.5 %: 7546.0 (mean DPM); 3763.8 (mean DPM per node) 25 %: 13042.0 (mean DPM); 6511.8 (mean DPM per node) 50 %: 10832.0 (mean DPM); 5406.8 (mean DPM per node) Negative control: 1355.6 (mean DPM); 668.6 (mean DPM per node)
Test group / Remarks:
Test group / Remarks:
Test group / Remarks:

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:
Migrated information
The test item cobalt stearate (CoRC Study EFF18) is expected to have sensitising properties and therefore, should be regarded as a dermal sensitiser.
According to the criteria specified by Directive 67/548/EEC and subsequent regulations, the test item is classified as skin sensitiser.
According to the EC Regulation No. 1272/2008 and subsequent regulations, the test item is classified as Category 1.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:
Migrated from Short description of key information:
Stearic acid, cobalt salt is expected to have sensitising properties and therefore, should be regarded as a dermal sensitiser. For further information please refer to the document attached to the endpoint summary in IUCLID section 7.4 (Sensitisation) or Appendix 2 of the CSR.

Justification for selection of skin sensitisation endpoint:
Key study.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:
Migrated from Short description of key information:
There is no known direct experience or information implicating this substance as occupational respiratory sensitiser.

Justification for classification or non-classification


The reference Ahuja (2010) 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 12.5%: SI 5.6

·        concentration 25%: SI 9.7

·        concentration 50%: SI 8.1


The EC3 value could not be calculated as the stimulation indices of all three concentrations were above 3. The classification criteria acc. to regulation (EC) 1272/2008 as skin sensitiser are met. Stearic acid, cobalt salt is classified as skin sensitiser category 1 (H317).


Respiratory sensitisation

There is no known direct experience or information implicating this substance as occupational respiratory sensitiser, thus classification criteria according to regulation (EC) 1272/2008 as respiratory sensitiser are not met.