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Repeated dose toxicity: inhalation

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repeated dose toxicity: inhalation
combined repeated dose and carcinogenicity
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1990-08-23 to 1992-08-27
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Although no guideline is stated for this study it is reliable without restriction, because it was conducted according to GLP requirements under the National Toxicology Program.
Reason / purpose for cross-reference:
reference to same study

Data source

Referenceopen allclose all

Reference Type:
Reference Type:
Inhalation toxicity and carcinogenicity studies of cobalt sulfate
Bucher, J.R.; et al.
Bibliographic source:
Toxicol. Sci. 49, 56-67

Materials and methods

Test guideline
no guideline followed
Principles of method if other than guideline:
Groups of 50 male and 50 female mice were exposed to aerosols containing 0, 0.3, 1.0, or 3.0 mg/m³ cobalt sulfate heptahydrate 6 hours per day, 5 days per week, for 105 weeks.
GLP compliance:
Limit test:

Test material

Constituent 1
Reference substance name:
cobalt sulfate heptahydrate
cobalt sulfate heptahydrate
Constituent 2
Reference substance name:
Cas Number:
Details on test material:
- Name of test material (as cited in study report): Cobalt sulfate heptahydrate (CoSO4 * 7H2O) (From curtin matheson Scientific (Kansas City, MO)- Physical state: red, crystalline solid- Molecular weight: 281.13 g- Analytical purity: approx. 99 %; Elemental analyses for sulfur and hyfrogen were in agreement with the theoretical values for cobalt sulfate heptahydrate, but results for cobalt wer slightly low. Karl Fischer water analysis indicated 44.6 % +/- 0.5% water- Impurities (identity and concentrations): Spark source mass spectroscopy indicated 140 ppm nickel present as impurity; all other impurities had a combined total of less than 175 ppm.- Lot No.: 412092- Storage condition of test material: To ensure stability, the bulk chemical was stored in its original shipping containers, metal cans, at room temperature.- Other: Cobalt sulfate heptahydrate is stable as a bulk chemical when stored protected from light at normal tempertures. The heptahydrate dehydrates to the hexahydrate at 41.5 °C and to the monohydrate when heated to 71°C, with no further changes expected below the decomposition temperature (708°C). Therefore, an accelerated stability study wa not conducted. Stability was monitored during the study using elemental analysis by inductively coupled plasma/atomic emission spectroscopy (ICP/AES) normalized against a cobalt standard (National Institute of Standards and Technology, Gaithersburg, MD); no degradation of the bulk chemical was detected.No further information on the test material was stated.

Test animals

Details on test animals or test system and environmental conditions:
TEST ANIMALS- Source: Simonsen Laboratories (Gilroy, CA)- Age at study initiation: approx. 6 weeks- Housing: Mice were housed individually. Cages (stainless-steel wire-bottom (Hazleton System, Inc., Aberdeen, MD)) and racks were rotated weekly. Cages were changed weekly. Bedding: cageboard (Bunzl Cincinnati Paper Co., Cincinnati, OH), changed daily (15 October 1990 to study termination)- Chamber Air supply filters: Single HEPA (Flanders Filters, Inc., San Rafael, CA)- Chambers: Stainless-steel with excreta pan suspended below each cage unit (Harford System Division of Lab Products, Inc., Aberdeen, MD), changedweekly- Diet (ad libitum except during exposure period): NIH-07 open formula pellet diet (Zeigler Brothers, Inc., Gardners, PA), changed weekly- Water (ad libitum): Tap water (Richland municipal supply) via automatic watering system (Edstrom Industries, Waterford, WI)- Quarantine period: 14 days before beginning of the study. Five male and five females mice were selected for parasite evaluation and gross observation of disease. Serology samples were collected for viral screening. The health of the animals was monitored during the study according to the protocols of the NTP Sentinel Animal Program.ENVIRONMENTAL CONDITIONS OF CHAMBERS FOR EXPOSURE- Temperature (°C): 19.5°C–27.1° C- Relative humidity: 28%–93%:- Air changes (per hr): 9-23/hour- Photoperiod (hrs dark / hrs light): 12 hours/dayNo further information on the test animals was stated.

Administration / exposure

Route of administration:
other: particulated aerosol from solution; see details on inhalation exposure below
Type of inhalation exposure:
whole body
other: see details on inhalation exposure below
Remarks on MMAD:
MMAD / GSD: 0.3 mg/m³ concentration: MMAD = 1.6 +/- 0.16 µm (GSD = 2.3 +/- 0.19)1.0 mg/m³ concentration: MMAD = 1.5+/- 0.12 µm (GSD = 2.3 +/- 0.13)3.0 mg/m³ concentration: MMAD = 1.6 +/- 0.15 µm (GSD = 2.3 +/- 0.12)
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION- Exposure apparatus: Hazleton 2000 inhalation exposure chambers (Harford Systems Division of Lab Products, Inc., Aberdeen, MD); The inhalation exposure chambers were designed so that uniform aerosol concentrations could be maintained throughout each chamber with the catch pans in place. The total active mixing volume of each chamber was 1.7 m³.- System of generating particulates/aerosols: Cobalt 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), an aerosol charge neutralizer, and an aerosol distribution system. The nebulizer consisted of two orifices of different sizes aligned on opposite sides of a small chamber. Compressed air entered the chamber through the small orifice and, on entering the larger orifice, induced a negative pressure.Cobalt sulfate heptahydrate in deionized water (approx. 400 g/L) was siphoned from the bulk reservoir to the nebulizer reservoir and then aspirated into the nebulizer chamber and expelled as a streamthrough the larger orifice. Shear forces broke the stream into droplets that were evaporated to leave dry particles of cobalt sulfate heptahydrate. The aerosol generation and delivery system included primary and secondary compressed-air-driven nebulizers. The aerosol generated by the compressed-air-driven nebulizer was passed through the aerosol charge neutralizer to remove static charge that formed on the aerosol particles during generation, reducing adhesion of the droplets to the walls of the delivery system. This neutralizer consisted of a length of plastic duct with two 10-mCi 63Ni-plated foils suspended in the center of the tube. The activity of the foils was matched to the diameter of the duct to allow adequate time for the aerosol to approach Boltzmann equilibrium at the system flow rate.A distribution line carried aerosol (20 mg/m³) to exposure chambers on both sides of the exposure room. Aerosol was siphoned from the branches of the distribution line by pneumatic pumps (one pump per exposure chamber). The flow rate in each branch of the distribution line was controlled by an Air-Vac pump (Air-Vac Engineering, Milford, CT) and monitored by a photohelic differential pressure gauge (Dwyer Instruments, Inc., Michigan City, IN) coupled to a Venturi tube. At each chamber, aerosol moving through the chamber inlet was further diluted with HEPA-filtered air to the appropriate concentration for the chamber. - Temperature, humidity, pressure in air chamber: see above under "Details on test animals and environmental conditions"- Method of particle size determination: Aerosol size distribution was determined monthly for each exposure chamber with a Mercer-style sevenstageimpactor (In-Tox Products, Albuquerque, NM). Samples were analyzed for cobalt sulfate heptahydrate with ICP/AES. The relative mass on each impactor stage was analyzed by probit analysis; the mass median aerodynamic diameter for the aerosol was within the specified range of 1 to 3 μm.TEST ATMOSPHERE- Brief description of analytical method used: The chamber aerosol concentrations of cobalt sulfate heptahydrate were monitored by real-time aerosol monitors (Model RAM-1; MIE, Inc., Bedford, MA) controlled by a Hewlett-Packard HP-85B computer (Hewlett-Packard Company, Palo Alto, CA). The RAM-1s detected aerosol particles ranging from 0.1 to 20 μm in diameter. Three RAM-1s were employed in the monitoring system; these monitorswere exchanged with different RAM-1s when the on-line monitor performance deteriorated. Chamber aerosol concentrations were sampled at least once per hour during each exposure day. Sample lines connecting the exposure chambers to the RAM-1s were designed to minimize aerosol particle losses due to settling or impaction. Throughout the 2-year studies, the background concentrations of total suspended particles in the control chambers were less than the limit of detection.The RAM-1 voltage output was calibrated against cobalt sulfate heptahydrate concentrations of chamber filter samples. Samples were collected on Teflon®-coated, glass-fiber filters with a calibrated flow sampler. Equations for the calibration curves contained in the HP-85B computer converted the RAM-1 voltages into exposure concentrations. Solutions of filter samples in 2% nitric acid were analyzed quantitatively for cobalt sulfate heptahydrate by inductively coupled plasma/atomic emission spectroscopy (ICP/AES). Calibration samples were collected every 2 weeks. Additional samples for monitoring the accuracy of calibration were collected daily from at least one chamber monitored by each RAM-1 and were analyzed two to three times per week. The ICP/AES was calibrated with a solution of standard cobalt diluted with nitric acid.The stability of aerosol concentrations in the 0.3 and 3.0 mg/m³ chambers was monitored by analyzing samples collected on Gelman A/E glass fibers using a calibrated flow sampler. X-ray diffraction analyses were performed by a Philips 3600 diffraction unit with Cu Ka radiation. Results indicated that cobalt sulfate hexahydrate was the primary species delivered to the chambers.The time required for the chamber concentration to reach 90% of the target value following the beginning of exposure (T90) and the time required for the chamber concentration to reach 10% of the target value following termination of the exposure (T10) were determined for each exposure chamber. Without animals present, T90 values ranged from 7 to 12 minutes and T10 ranged from 8 to 9 minutes for mice. With animals present, T90 values ranged from 8 to 12 minutes and T10 ranged from 11 to 12 minutes for mice. A T90 of 12 minutes was selected for the 2-year studies.No further information on the inhalation exposure was stated.
Analytical verification of doses or concentrations:
Details on analytical verification of doses or concentrations:
see "Details on inhalation exposure" above
Duration of treatment / exposure:
6 hours plus T90 (12 minutes) per day
Frequency of treatment:
5 days per week, for 105 weeks
Doses / concentrationsopen allclose all
Doses / Concentrations:0, 0.3, 1.0 or 3.0 mg/m³ cobalt sulfate heptahydrateBasis:nominal conc.
Doses / Concentrations:0.30 +/- 0.04 mg/m³, 1.02 +/- 0.08 mg/m³ or 3.01 +/- 0.19 mg/m³ cobalt sulfate heptahydrateBasis:analytical conc.
No. of animals per sex per dose:
50 males / 50 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The exposure concentrations for the 2-year cobalt sulfate heptahydrate studies were based on the findings of 16-day and 13-week studies. The most sensitive tissue was the larynx, with squamous metaplasia observed in mice at the lowest exposure concentration of 0.3 mg/m³. A NOAEL was not reached for this tissue. Inflammatory polyps, some nearly obstructing the esophagus, were observed at the 0.3 and 1.0 mg/m³ exposure concentrations. They were composed of mild or minimal squamous metaplasia and/or chronic inflammation in mice. The severity of the laryngeal changes and other lesions in the respiratory tract at 3.0 mg/m³ was not considered life threatening, and, therefore, exposure concentrations of 0.3, 1.0, and 3.0 mg/m³ were chosen for the 2-year study with mice.No further information on the study design was stated.
Positive control:
No data


Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes- Time schedule: All animals were observed twice daily. 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 studies.DETAILED CLINICAL OBSERVATIONS: No dataBODY 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 studies.FOOD CONSUMPTION:- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No dataFOOD EFFICIENCY:- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No dataWATER CONSUMPTION: No dataOPHTHALMOSCOPIC EXAMINATION: No dataHAEMATOLOGY: No dataCLINICAL CHEMISTRY: No dataURINALYSIS:No dataNEUROBEHAVIOURAL EXAMINATION: No dataNo further information on the observations and examinations perofrmed and frequency were stated.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes A complete necropsy and microscopic examination were performed on all mice. At necropsy, all organs and tissues were examined for grossly visible lesions, and all major tissues were fixed and preserved in 10% neutral buffered formalin, processed and trimmed, embedded in paraffin, sectioned to a thickness of 5 to 6 μm, and stained with hematoxylin and eosin for microscopic examination. For all paired organs (i.e., adrenal gland, kidney, ovary), samples from each organ were examined. HISTOPATHOLOGY: YesComplete histopathology was performed on all mice. In addition to gross lesions and tissue masses, the tissues examined included:adrenal gland, bone with marrow, brain, clitoral gland, esophagus, gallbladder, heart, large intestine (cecum,colon, rectum), small intestine (duodenum, jejunum, ileum), kidney, larynx, liver, lungs/bronchi, lymph nodes (mandibular, mesenteric,bronchial, mediastinal), mammary gland (except male mice), nose, ovary, pancreas, pancreatic islets, parathyroid gland,pituitary gland, preputial gland, prostate gland, salivary gland, sciatic nerve, seminal vesicle, skin, spinal cord, spleen, stomach (forestomachand glandular), testes/epididymides, thymus, thyroid gland, trachea, urinary bladder, and uterus.
- Survival analyses: The probability of survival was estimated by the product-limit procedure of Kaplan and Meier (1958). Animals found dead of other than natural causes or pregnant were censored from the survival analyses; animals dying from natural causes were not censored. 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. All reported P values for the survival analyses are two sided.- Analysis of continuous variables: Average severity values were analyzed for significance using the Mann-Whitney U test (Hollander and Wolfe, 1973).

Results and discussion

Results of examinations

Clinical signs:
no effects observed
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
effects observed, treatment-related
Details on results:
CLINICAL SIGNS AND MORTALITYSurvival of exposed males and females was similar to that of the chamber controls. Irregular breathing was observed slightly more frequently in female mice exposed to 1.0 mg/m³ than in the chamber controls or other exposed groups.BODY WEIGHT AND WEIGHT GAINMean body weights of 3.0 mg/m³ male mice were less than those of the chamber controls from week 96 until the end of the study. The mean body weights of all exposed female mice were generally greater than those of the chamber controls from week 20 until the end of the study. GROSS PATHOLOGY AND HISTOPATHOLOGY(NON-NEOPLASTIC AND NEOPLASTIC):- Lung: In all exposed groups of males and females, the incidences of cytoplasmic vacuolization of the bronchi were significantly greater than those in the chamber control groups. The incidences of diffuse histiocytic cell infiltration in 3.0 mg/m³ males and of focal histiocytic cell infiltration in 3.0 mg/m³ females were significantly greater than those in the chamber controls.Cytoplasmic vacuolization of the bronchial epithelium was a 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 characterized by one or more histiocytes with foamy cytoplasm within variable numbers of alveolar lumens. Focal infiltrate was a localized 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 cobalt sulfate heptahydrate exposure.The incidences of alveolar/bronchiolar neoplasms (adenoma and/or carcinoma) in 3.0 mg/m³ males and females and the combined incidence of alveolar/ bronchiolar neoplasms in 1.0 mg/m³ females were significantly greater than those in the chamber control groups and generally exceeded the historical control ranges for inhalation studies. In exposed males and females, the incidences of all lung neoplasms occurred with positive trends.All the alveolar/bronchiolar proliferative lesions observed within the lungs of exposed mice were typical of those observed spontaneously. Hyperplasia generally represented an increase in numbers of epithelial cells along alveolar walls which retained normal alveolar structure. Adenomas generally were distinct masses that often compressed surrounding tissue. Component cells were arranged in acinar and/or irregular papillary structures and occasionally in a solid cellular pattern. These cells were typically uniform and similar to hyperplastic counterparts. Malignant alveolar/bronchiolar neoplasms had similar cellular patterns but were generally larger and had one or more of the following: heterogeneous growth pattern, cellular pleomorphism, and/or atypia and local invasion or metastasis. Although similar in appearance to “spontaneous” lung neoplasms in chamber controls, alveolar/ bronchiolar neoplasms in mice exposed to cobalt sulfate heptahydrate had different molecular lesions in the Kras gene. Of the K-ras mutations detected at the second base of codon 12, a higher frequency (5/9, 55%) of G to T transversions was detected compared to concurrent (0/1) and historical control lung neoplasms (1/24, 4%). K-ras codon 61 CTA or CGA mutations were not present in cobalt sulfate heptahydrate-induced lung neoplasms.- Nose: The incidences of atrophy of the olfactory epithelium in 1.0 and 3.0 mg/m³ males and females and hyperplasia of the olfactory epithelium in 3.0 mg/m³ males and females were significantly greater than those in the chamber controls. The incidences of suppurative inflammation in 3.0 mg/m³ males and in 1.0 mg/m³ females were significantly greater than those in the chamber controls. The nasal lesions in mice involved limited segments of the olfactory epithelium located further back in the nasal passage. Atrophy of the olfactory epithelium was characterized 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 3.0 mg/m³ and was characterized 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 a focal aggregate of inflammatory cells.-Larynx: The incidences of squamous metaplasia in all exposed groups of males and females were significantly greater than those in the chamber controls. Squamous metaplasia was limited to the base of the epiglottis and was not a severe lesion in exposed mice. It was characterized by replacement of the ciliated respiratory epithelium by one or more layers of flattened epithelial cells overlying a basal layer of cuboidal cells. Keratinization was sometimes observed.- Thyroid Gland: The incidences of follicular cell hyperplasia in all exposed groups of males were significantly greater than the incidence in the chamber controls (chamber control, 3/49; 0.3 mg/m³, 17/50; 1.0 mg/m³, 11/50; 3.0 mg/m³, 10/50). Minimal hyperplasias are commonly observed in untreated male and female mice, suggesting that the rate in the concurrent chamber control group is low. The severity of most hyperplasias in these mice was minimal to mild and did not differ between chamber control and exposed groups. The incidence of hyperplasia did not increase with exposure to cobalt sulfate heptahydrate, 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 Helicobacter hepaticus infection. Liver sections from four of five male mice with liver lesions were positive for bacterial organisms consistent with H. hepaticus when examined using Steiner’s modification of the Warthin Starry silver stain.The incidences of hemangiosarcoma in all exposed groups of male mice and in 1.0 mg/m³ in female mice exceeded the range observed in historical controls for inhalation studies. In addition, the incidence of hemangiosarcoma in 1.0 mg/m³ males was significantly greater than that in the chamber 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.

Effect levels

open allclose all
Dose descriptor:
Effect level:
0.414 mg/L air
Based on:
test mat.
Basis for effect level:
other: squamous metaplasia lesions in the larynx
Dose descriptor:
Effect level:
0.3 mg/L air (analytical)
Based on:
test mat.
Basis for effect level:
other: squamous metaplasia lesions in the larynx

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

There was clear evidence of carcinogenic activity of cobalt sulfate heptahydrate in male and female B6C3F1 mice based on increased incidences of alveolar/bronchiolar neoplasms. Exposure to cobalt sulfate heptahydrate caused a spectrum of inflammatory, fibrotic, and proliferative lesions in the respiratory tract of male and female mice.

Applicant's summary and conclusion

There was clear evidence of carcinogenic activity of cobalt sulfate heptahydrate in male and female B6C3F1 mice based on increased incidences of alveolar/bronchiolar neoplasms.Exposure to cobalt sulfate heptahydrate caused a spectrum of inflammatory, fibrotic, and proliferative lesions in the respiratory tract of male and female mice.