Cobalt(2+) hydrogen citrate

Carcinogenicity

Description of key information

Read-across with cobalt sulfate heptahydrate
No NOAECs were identified neither for rats nor for mice.
LOAEC (local, rat): 0.5 mg/m3 (cobalt hydrogen citrate; recalculated value)
LOAEC (local, mouse): 0.5 mg/m3 (cobalt hydrogen citrate; recalculated value)

Key value for chemical safety assessment

Carcinogenicity: via oral route

Endpoint conclusion

Endpoint conclusion:

no study available

Carcinogenicity: via inhalation route

Link to relevant study records

Reference

Endpoint:

carcinogenicity: inhalation

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study with acceptable restrictions.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

Deviations:

yes

Remarks:

- no haematology, no urinalysis, no clinical chemistry

GLP compliance:

yes

Species:

mouse

Strain:

B6C3F1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Simonsen Laboratories, Gilroy, CA, USA
- Age at study initiation: 6 weeks
- Housing: individually housing; stainless-steel wire bottom cages
- Diet (e.g. ad libitum): NIH 07 open formula pellet diet (Zeigler Bros., Inc., Gardners, PA); ad libitium except during exposure periods; changed weekly
- Water (e.g. ad libitum): tap water (Richland municipal supply) via automatic watering system (Edstrom Industries, Waterford, WI, USA); ad libitium

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.5-27.1
- Humidity (%): 28-93
- Air changes (per hr): 9-23
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

inhalation: aerosol

Type of inhalation exposure (if applicable):

whole body

Vehicle:

unchanged (no vehicle)

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Hazleton 2000 inhalation exposure chambers (Harford Systems Division of Lab Products, Inc. Aberdeen, MD, USA)
- System of generating particulates/aerosols: Cobalt(II)sulfate heptahydrate aerosol was generated and delivered from an aqueous solution by a system composed of three main components: a compressed-air-driven nebulizer (Model PN7002; RETEC Development Laboratory, Portland, OR, USA), an aerosol charge neutralizer, and an aerosol distribution system.
- Method of particle size determination: Aerosol size distribution was determined monthly for each exposure chamber with a Mercer-style sevenstage impactor (In-Tox Products, Albuquerque, MN, USA). The relative mass on each impactor stage was analysed by probit analysis; the mass median aerodynamic diameter for the aerosol was within the specified range of 1 to 3 µm.
- Other information: No degradation of the test item was observed during the study.

TEST ATMOSPHERE
- Brief description of analytical method used: The chamber concentrations of the test item were monitored by computer-controlled real-time aerosol monitors (Model RAM-1; MIE, Inc., Bedford, MA). Chamber aerosol concentrations were sampled at least once per hour during each exposure day.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The chamber concentrations of the test item were monitored by computer-controlled real-time aerosol monitors (Model RAM-1; MIE, Inc., Bedford, MA). Chamber aerosol concentrations were sampled at least once per hour during each exposure day.

Duration of treatment / exposure:

105 weeks

Frequency of treatment:

6 hours per day
5 days per week

Remarks:

Doses / Concentrations:
0.3, 1, 3 mg/m3
Basis:
other: nominal conc.; calculated as anhydrous salt of cobalt(II)sulfate

No. of animals per sex per dose:

50

Control animals:

yes

Details on study design:

Animals were distributed randomly into groups of approx. equal initial mean body weights; cages were distributed randomly into groups from another computer-generated list of random numbers.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Clinical findings were recorded initially, at weeks 5, 9 and 13, monthly through week 92, every 2 weeks thereafter, and at the end of the study.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded initially, weekly for 13 weeks, monthly through week 92, every 2 weeks thereafter, and at the end of the study.

HAEMATOLOGY: Not examined

CLINICAL CHEMISTRY: Not examined

URINALYSIS: Not examined.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Complete histopathology was performed on all mice: adrenal gland, bone with marrow, brain, clitoral gland, esophagus, gallbladder, heart, large intestine (caecum, colon, rectum), small intestine (duodenum, jejunum, ileum), kidney, larynx, liver, lungs/bronchi, lymph nodes (mandibular, mesenteric, bronchial, mediastinal), mammary gland (only females), nose, ovary, pancreas, pancreatic islets, parathyroid gland, pituitary gland, preputial gland, prostate gland, salivary gland, sciatic nerve, seminal vesicle, skin , spinal cord, spleen, stomach (forestomach and glandular), testes/epididymides, thymus, thyroid gland, trachea, urinary bladder and uterus

Statistics:

Survival analyses: The probability of survival was estimated by the product-limit procedure of Kaplan and Meier (1958). Statistical analyses for possible dose-related effects on survival used Cox´s (1972) method for testing two groups for equality and Tarone´s (1975) life table test to identify dose-related trends.
Analysis of neoplasm incidences: The neoplasm incidences of exposed and control groups were compared on the basis of the likelihood score test for the regression coefficient of dose. Fisher exact test and Cochran-Armitage trend test. Tests of significance included pairwise comparisons of each exposed group with controls and a test for an overall dose-related trend. Continuity-corrected tests were used in the analysis of neoplasm incidence, and reported P values are one sided.
Analysis of nonneoplastic lesion incidences: Primary statistical analysis used was a logistic regression analysis in which nonneoplastic lesion prevalence was modeled as a logistic function of chemical exposure and time.
Analysis of continuous variables: Average severity values were analysed for significance using the Mann-Whitney U test.

Details on results:

CLINICAL SIGNS AND MORTALITY
Survival of exposed male and female mice was similar to that of the control animals (see table 1 and table 2).
Irregular breathing was observed slightly more frequently in female mice exposed to 1 mg/m3 than in the control animals or the other exposed groups.

BODY WEIGHT AND WEIGHT GAIN
Mean body weights of male mice exposed to 3 mg/m3 were less than those of the controls from week 96 until study termination. The mean body weights of all exposed female mice were generally greater than those of the controls from week 20 until study termination.

In the following, only statistically significant or biologically noteworthy changes in the incidences of neoplasms and/or nonneoplastic lesions were described:

Lung:
In all exposed groups of males and females, the incidences of cytoplasmic vacuolisation of the bronchi were significantly greater than those in the control groups. The incidences of diffuse histiocytic cell infiltration in 3 mg/m3 males and of focal histiocytic cell infiltration in 3 mg/m3 females were significantly greater than in the controls.
Cytoplasmic vacuolisation of the bronchial epithelium was minimal change of unknown biological significance confined to the epithelial cells lining the apex of the bronchial bifurcation. The affected cells were somewhat larger than normal with a diffusely clear to finely vacuolated cytoplasm. Histiocyte infiltration was characterised by one or more histiocytes with foamy cytoplasm within variable numbers of alveolar lumens. Focal infiltrate was a localised accumulation of histiocytes, while diffuse infiltrate was more widely scattered. The histiocyte infiltrate was very commonly seen in lungs with alveolar/bronchiolar neoplasms, and the increased incidences of infiltrate in the lungs of exposed animals were considered to reflect the higher incidences of lung neoplasms in these animals rather than a primary effect of the exposure.
The incidences of alveolar/bronchiolar neoplasms (adenoma and/or carcinoma) in 3 mg/m3 males and females and the combined incidence of alveolar/bronchiolar neoplasms in 1 mg/m3 females were significantly greater than those in the control groups. In exposed males and females, the incidences of all lung neoplasms occurred with positive trends.

Nose:
The incidences of atrophy of the olfactory epithelium in 1 and 3 mg/m3 males and females and hyperplasia of the olfactory epithelium in 3 mg/m3 males and females were significantly greater than those in the controls. The incidences of suppurative inflammation in 3 mg/m3 males and in 1 mg/m3 females were significantly greater than those in the control groups. Atrophy of the olfactory epithelium was characterised by loss of cell layers (sensory cells) and a decrease in the number of axons in the lamina propria. Hyperplasia of the olfactory epithelium was observed only in animals exposed to 3 mg/m3 and was characterised by increased numbers of sensory cells that were usually arranged in nests or rosettes. The suppurative inflammation involved only a few animals and was a very mild change. It primarily involved animals that died prior to the end of the study and consisted of focal aggregate of inflammatory cells.

Larynx:
The incidences of squamous metaplasia in all exposed animals were significantly greater than those in the control groups. Squamous metaplasia was limited tot the base of the epiglottis and was not a severe lesion in exposed mice. It was characterised by replacement of the ciliated respiratory epithelium by one or more layers of flattened epithelial cells overlying a basal layer of cuboidal cells. Keratinisation was sometimes observed.

Thyroid gland:
The incidences of follicular cell hyperplasia in all exposed groups of males were significantly greater than the incidences in the controls. Minimal hyperplasias are commonly observed in untreated male and female mice, suggesting that the rate in the concurrent control group is low. The severity of most hyperplasias in theses animals was minimal to mild and did not differ between control and exposed groups. The incidence of hyperplasia did not increase with the exposure, nor was the incidence of neoplasms of the follicular cells increased.

Liver:
High incidences of chronic inflammation, karyomegaly, oval cell hyperplasia, and regeneration occurred in all groups of male mice and were usually observed together in the same liver. These changes were generally mild to moderate in severity and observed throughout the liver (usually not within proliferative lesions), but they appeared most pronounced in the portal regions. Similar lesions were observed in only a few females, and the severity was also much less than that observed in most males. This spectrum of lesions is consistent with those observed with Heliobacter hepaticus infection. Liver sections from 4/5 males with liver lesions were positive for bacterial organisms consistent with Heliobacter hepaticus when examined using Steiner´s modification of the Warthin Starry silver stain.
The incidence of hemangiosarcoma in 1 mg/m3 males was significantly greater than that in the controls. Hemangiosarcomas were morphologically similar to those observed spontaneously and consisted of multiple variably sized blood-filled spaces that were separated by cords of hepatocytes and lined by plump endothelial cells.

Dose descriptor:

LOAEC

Effect level:

0.3 mg/m³ air

Sex:

male/female

Basis for effect level:

other: calculated as the anhydrous salt of cobalt(II)sulfate; increased incidence of benign and malignant alveolar/bronchiolar neoplasms

Remarks on result:

other: Effect type: carcinogenicity (migrated information)

Dose descriptor:

LOAEC

Effect level:

0.5 mg/m³ air

Sex:

male/female

Basis for effect level:

other: CoC6H6O7; recalculated value; incidences of benign and malignant alveolar/bronchiolar neoplasms

Remarks on result:

other: Effect type: carcinogenicity (migrated information)

Table 1: Survival of male animals

	control animals	0.3 mg/m3	1 mg/m3	3 mg/m3
moribund	19	16	17	23
natural deaths	8	3	9	6
animals surviving to study termination	22	31	24	20

Table 2: Survival of female animals

	control animals	0.3 mg/m3	1 mg/m3	3 mg/m3
moribund	11	10	13	16
natural deaths	5	3	5	6
animals surviving to study termination	34	37	32	28

Table 3: Tumor incidence in males

	control animals	0.3 mg/m3	1 mg/m3	3 mg/m3
Alveolar/bronchiolar adenoma or carcinoma	11/50	14/50	19/50	28/50

Table 4: Tumor incidence in females

	control animals	0.3 mg/m3	1 mg/m3	3 mg/m3
Alveolar/bronchiolar adenoma or carcinoma	4/50	7/50	13/50	18/50

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEC

0.5 mg/m³

Study duration:

chronic

Species:

mouse

Quality of whole database:

The available information comprises adequate, reliable (Klimisch score 2) and consistent studies from a reference substance with similar structure and intrinsic properties. Read-across is justified based on the basic assumption that the cobalt ion is the determining factor for biological activity. Please refer to the endpoint discussion for further details.

Carcinogenicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on an analogue approach, the available data on carcinogenicity meet the criteria for classification as Category 1B (H350) according to Regulation (EC) 1272/2008 and as R49, Category 2 according to Directive 67/548/EEC.

Additional information

There are no data available on carcinogenicity for cobalt hydrogen citrate. However, there are reliable data for soluble cobalt compounds considered suitable for read-across using the analogue approach.

Cobalt hydrogen citrate is a metal-organic compound, which is water soluble and nearly completely dissociates in aqueous solutions. For identifying hazardous properties of cobalt hydrogen citrate concerning human health effects, the existing forms of the target chemical at physiological and very acidic pH conditions (e. g. in the stomach) are relevant for risk assessment. For cobalt hydrogen citrate, it can be assumed that cobalt cations are released under biological conditions that are considered to be toxicologically relevant. Furthermore it is anticipated that the cobalt cation released by the parent compound is the determining factor for toxicological effects, same as for other soluble cobalt compounds. Therefore, data originating from soluble cobalt compounds can be used in the derivation of toxicological endpoints for cobalt hydrogen citrate. For further details, please refer to the analogue justification attached in section 13 of the technical dossier.

Groups of mice and rats were exposed to cobalt(II)sulfate heptahydrate aerosols by inhalation at concentrations of 0, 0.3, 1 and 3 mg/m³ (calculated as anhydrous salt; equivalent to 0.5, 1.6 and 4.8 mg/m³ cobalt hydrogen citrate) 6 hours/day, 5 days/week, for 105 weeks (NTP, 1998).

Mean body weights were increased in all treated female mice and decreased only in the high-dose male mice. Survival was not adversely affected by treatment. The incidences of benign and malignant alveolar/bronchiolar neoplasms were increased in a concentration-dependent manner in male and female mice.

Males: 11/50, 14/50, 19/50, and 28/50 for 0, 0.5, 1.6, and 4.8 mg/m³, respectively.

Females: 4/50, 7/50, 13/50, and 18/50 for 0, 0.5, 1.6, and 4.8 mg/m³, respectively.

In rats, mean body weights and survival were unaffected by treatment. Female animals exhibited a concentration-related increase in the incidence of benign and malignant alveolar/bronchiolar neoplasms and of benign and malignant pheochromocytomas of the adrenal medulla. The incidences of benign and malignant alveolar/bronchiolar neoplasms were 0/50, 3/49, 15/50 and 15/50 for 0, 0.5, 1.6 and 4.8 mg/m³, respectively and of benign and malignant pheochromocytomas were 2/48, 1/49, 4/50, and 10/48 for 0, 0.5, 1.6, and 4.8 mg/m³, respectively.

In males, increased incidence of benign and malignant alveolar/bronchiolar neoplasms was observed, but only a marginally increased incidence of pheochromocytomas of the adrenal medulla. The incidences of benign and malignant alveolar/bronchiolar neoplasms were 1/50, 4/50, 4/48, and 7/50 for 0, 0.5, 1.6, and 4.8 mg/m³, respectively and of begnin and malignant pheochromocytomas were 15/50, 19/50, 25/49, and 20/50 for 0, 0.5, 1.6, 4.8 mg/m³, respectivley.

Although many of the alveolar/bronchiolar lesions were morphologically similar to those that arise spontaneously, the lesions in rats, unlike those in mice, were predominantly fibrotic, squamous or mixtures of alveolar/bronchiolar epithelium and squamous or fibrous components. Squamous metaplasia of alveolar/bronchiolar epithelium, which is a common response to pulmonary injury, was observed in number of rats.

The marginally increased incidence of pheochromocytomas in males was considered an uncertain finding because it occurred only in the 1 mg/m³ group and was not supported by increased incidence or severity of hyperplasia. Marginal increases in adrenal medullary tumors may have been exposure related.

In summary, cobalt(II)sulfate heptahydrate was found to be carcinogenic in mice and rats when administered by inhalation. There was clear evidence of carcinogenicity in male mice, female mice and female rats, based on increased incidences of lung tumors. In addition, female rats had an increase incidence of pheochromocytoma of the adrenal medulla. Some evidence of carcinogenicity in male rats was observed, based on increased incidences of lung tumors at the highest exposure level. No NOAECs were identified, neither for rats nor for mice. The local LOAECs were determined to be 0.5 mg/m³ for mice and rats.

Justification for selection of carcinogenicity via inhalation route endpoint:
Hazard assessment is conducted by means of read-across from a structural analogue. The selected study is most adequate and reliable study based on the identified similarities in structure and intrinsic properties between source and target substance and overall assessment of quality, duration and dose descriptor level.

Carcinogenicity: via inhalation route (target organ): respiratory: lung