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Skin sensitisation

Link to relevant study records
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2006-05-03 to 2006-05-22
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
according to guideline
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)
Details on test animals and environmental conditions:
- Source: Janvier, Le Genest-Saint-Isle, France
- Age at study initiation: On the first day of the treatment period, the animals were approx. 10 weeks old.
- Weight at study initiation: On the first day of the treatment period, the animals had a mean body weight +/- standardd deviation of 21.2 +/- 1.2 g.
- Housing: The animals were housed individually in disposable crystal polystyrene cages (22.00 cm X 8.50 cm X 8.00 cm). Each cage contained autoclaved sawdust (SICSA, Alfortville, France).
- Diet: Free access to SsniffR/M-H pelleted diet (SSNIFF Spezialdiäten GmbH, Soest, Germany)
- Water: Free access to tap water
- Acclimation period: At least 5 days

- Temperature: 22 +/- 2 °C
- Relative humidity: 30 to 70 %
- Ventilation: approx. 12 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 / 12
No further information on the test material was stated.
Preliminary test: 25, 10, 5, 2.5, 1, 0.5,0.25 and 0.1 %
Main test: 0.05, 0.1, 0.25, 0.5, 1 and 25 %
No. of animals per dose:
2 females per concentration (preliminary test); 4 females per concentration (main test)
Details on study design:
No cutaneous reactions were noted. At the concentration ≥ 2.5 %, a significant increase in ear thickness was recorded, showing the irritant potential of the test item at these concentrations. The highest concentration retained for the main test was therefore 1 %.

Due to the unsatisfactory solubility of the test item in the first recommended vehicle (acetone/olive oil (4/1, v/v)), dimethylformaide was chosen among the other proposed vehicles. A solution was obtained at the maximum concentration of 25 %.

- Name of test method: LLNA
- Criteria used to consider a positive response: The test item was considered as a skin sensitizer when the Stimulation Indices (SI) for a dose group is ≥ 3. Other relevant criteria such as cellularity, radioactivity levels and ear thickness were also taken into account for the interpretation of results.

The test item was prepared in the vehicle at the chosen concentration.
All dosage form preparations were made freshly on the morning of administration and any unused material was discarded that same day.
On days 1, 2 and 3, a dose-volume of 25µL of the control or dosage form preparations was applied to the dorsal surface of both ears, using an adjustable pipette fitted with a plastic tip. In order to avoid licking and to ensure an optimized application of the test materials, the animals were placed under light isoflurane anesthezia during the administration. No massage was performed but the tip was used to spread the preparation over the application sites. No rinsing was performed between each application.
Lymph node cell proliferative responses were measured as described by Kimber and Dearman (1991). On day 6, all animals of all groups received a single intravenous injection of 250 µL of 0.9 % NaCl containing 20 µCi of 3H-TdR (specific activity of 25 Ci/mmol) via the tail vein. Approximately 5 hours later, the animals were killed by cervical dislocation and the auricular lymph nodes were excised. The lymph nodes were pooled for each experimental group.
For each experimental group, a single cell suspension of auricular lymph node cell (ALNC) was prepared by mechanical dissagregation in Petri dishes with the plunger of a syringe. Cell suspensions were washed with 15 mL of 0.9 % NaCl and pellets obtained were re-suspended in exclusion of trypan blue. Each cell suspension was then centrifuged and pellets were precipitated with 3 mL of 5% (w/v) trichloroacetic acid (TCA) in purified water at +5°C overnight. After a last centrifugation, the pellets were precipitated with 1 mL of 5% TCA. Three mL of Ultima Gold scintillation fluid (Packard) were added in order to measure incorporation of 3H-TdR using β-scintillation counting.

The animals were observed at least once a day during the study for clinical signs, sings of morbidity or mortality.

The animals were weighed individually on the first day (day 1) and on the day of sacrifice (day 6).

Ear thickness measurements and recording of local reactions were performed in order to access any possible irritant effect of the test item, as possible irritancy maybe involved in false psoitive lymphoproliferative responses.
On days 1, 2 and 3 (before application) as well as on day 6 (after sacrifice), the thickness of the left ear of each animal of group 1 to 6 was measured using a micrometer.
No measurement of ear thickness was perfomred for the animals of the positive control group.
Any irritation reaction (erythema and oedema) was recorded in parallel. Any other observation (colouration, presence of residual test item, ....) was noted.
The irritation level of the test item was determined according to the table 1 (see "Any other information on materials and methods incl. tables")

No further information on details on study design (LLNA) was stated.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
The results were expressed as disintegration's/mn (dpm) per group and per node. Stimulation Indices (SI) were calculated according to the following fromula: SI = disintegration's/mn of treated group / disintegration's/mn of control group.
Positive control results:
In the positive control group given α-hexyl cinnamic aldehyde at the concentration of 25 %, a moderate increase in cellularity and a stimulation index exceeding the threshold value of 3 (SI = 22.02) were noted. The study was therefore considered valid.
Test group / Remarks:
Test group / Remarks:
Test group / Remarks:
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No mortality and no clincal sings were observed during the study.

The body weight change of treated animals was similar to that of control animals.

No cutaneous reactions and no noteworthy increase in ear thickness were observed at any of the tested concentrations.

The quantity of cells obtained in each group was satisfactory and the celluarity correlated with incorporation of 3H-TdR. The cell viability was higher than 80 % in each group.

The stimulation indices were calculated by dividing the dpm of the treated group by the dpm of the control group.A

dose related increased in the SI was noted at all the tested concentrations.

The irritation level was I.

In the absence of local irritation, the positive lymphoproliferative responses observed were attributed to delayed contact hypersensitivity.

Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Under the experimental conditions, the test item cobalt acetyl acetonate induced delayed contact hypersensitivity in the murine Local Lymph Node Assay.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

Cobalt(2+) and C2/C8/C20 carboxylates will be classified as skin sensitiser according to section 3.4, annex I of regulation (EC) 1272/2008 based on animal and human data from the read-across substances cobalt(II) 4-oxopent-2-en-2-olate and  cobalt chloride hexahydrate showing similar bioaccessibility in artificial perspiration than cobalt diacetate (Group 1: High release of cobalt ion in artificial sweat – marked skin sensitisation - Source substances: Co acetyl acetonate, Co dichloride with similar bioavailability - worst case approach with reference to Cobalt diacetate).

Read-across entails the use of relevant information from analogous substances (the ‘source’ information) to predict properties for the ‘target’ substance(s) under consideration. Substances whose physicochemical or toxicological properties are likely to be similar or follow a regular pattern as a result of structural similarity may be considered as a category of substances. Structural similarity is a pre-requisite for any read-across approach under REACH (ECHA Read-Across Assessment Framework, 2015).

In accordance with Annex XI, 1.5 of the REACH regulation and the ECHA Guidance Read-Across Assessment Framework (ECHA, 2017), the similarities may be based on:

1) A common functional group (i.e. chemical similarity within the group);

2) Common precursors and/or likelihood of same breakdown products through physical and/or biological processes which result in structurally-similar degradation products (i.e. similarity through (bio) transformation); or

3) A constant pattern in the changing of the potency of the properties across the group (i.e. of physical-chemical and/or biological properties).

Due to the absence of substance specific information for the majority of substances within the cobalt category, the approach will read-across data from representative source substances to all other members of the read-across group.

Due to the route-specific toxicological properties of the cobalt category substances, several read-across groups are formed. For the endpoint skin sensitisation, the following read-across groups are formed:

Group 1: High release of cobalt ion in artificial sweat – marked skin sensitisation

Source substances: Co acetyl acetonate, Co dichloride

Group 2: Moderate release of cobalt ion in artificial sweat – moderate skin sensitisation

Source substances: Co metal powder

Group 3: Moderate release of cobalt ion in artificial sweat – mild skin sensitisation

Source substances: Co monoxide

Group 4: Low release of cobalt ion in artificial sweat – no skin sensitisation

Source substances: Tricobalt tetraoxide, Co lithium oxide

Group 5: Variable release of cobalt ion in artificial sweat, moderate skin sensitisation (Cobalt substances with long-chain (>C10) ligands)

Source substances: Co stearate, Co naphthenate

Group 6: Variable release of cobalt ion in artificial sweat, marked skin sensitisation (Cobalt substances with long-chain (>C10) ligands)

Source substances: Co resinate

Further details on the read-across approach for skin sensitisation and discussion of the underlying hazard data are given in the read-across report attached in IUCLID section 13.2.

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.



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.

As a worst case approach and by read-across with cobalt di(acetate) and cobalt resinate, Salt reaction of cobalt(2 +) and C2/C8/C20 carboxylates will be classified as respiratory sensitiser, category 1B.

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

Justification for classification or non-classification

Skin sensitisation

Cobalt(2+) and C2/C8/C20 carboxylates will be classified as skin sensitiser, category 1A (H317) according to section 3.4, annex I of regulation (EC) 1272/2008 based on animal and human data from the read-across substance cobalt chloride hexahydrate showing similar bioaccessibility in artificial perspiration than cobalt diacetate (Group 1: High release of cobalt ion in artificial sweat – marked skin sensitisation - Source substances: Co acetyl acetonate, Co dichloride - worst case approach with reference to Cobalt diacetate).


Respiratory sensitisation

Based on occupational exposure studies in cobalt facilities and by read-across with cobalt di(acetate) and cobalt resinate taken as worst case approach, Salt reaction of cobalt(2 +) and C2/C8/C20 carboxylates will be classified as respiratory sensitiser, category 1B.