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Carcinogenicity

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Description of key information

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

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Endpoint:
carcinogenicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1990-08-23 to 1992-08-27
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Although no guidelines are stated for this study it is reliable without restriction, because it was conducted according to GLP requirements under the National Toxicology Program.
Reason / purpose:
reference to same study
Qualifier:
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:
yes
Species:
mouse
Strain:
B6C3F1
Sex:
male/female
Details on test animals 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.
Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Vehicle:
other: see details on inhalation exposure below
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); 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.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)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:
yes
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
Post exposure period:
No data
Remarks:
Doses / Concentrations:0, 0.3, 1.0 or 3.0 mg/m³ cobalt sulfate heptahydrateBasis:nominal conc.
Remarks:
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 dataDERMAL IRRITATION (if dermal study): 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.
Statistics:
- 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 Neoplasm Incidences: The primary statistical method used was logistic regression analysis. In this approach, neoplasm prevalence was modeled as a logistic function of chemical exposure and time. In addition to logistic regression, other methods of statistical analysis were used. These methods include the life table test (Cox, 1972; Tarone, 1975), appropriate for rapidly lethal neoplasms, and the Fisher exact test and the Cochran-Armitage trend test (Armitage, 1971; Gart et al., 1979), procedures based on the overall proportion of neoplasm-bearing animals.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. The procedures described in the preceding paragraphs were also used to evaluate selected nonneoplastic lesions.- Analysis of Nonneoplastic Lesion Incidences: The 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 severitiy values were analyzed for significance using the Mann-Whitney U test (Hollander and Wolfe,1973).
Clinical signs:
no effects observed
Mortality:
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/m³and 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.
Relevance of carcinogenic effects / potential:
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.
Dose descriptor:
BMCL10
Effect level:
0.414 mg/m³ air
Based on:
test mat.
Basis for effect level:
other: alveolar epithelial hyperplasia and squamous metaplasia
Remarks on result:
other: Effect type: carcinogenicity (migrated information)

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.

Conclusions:
Under the conditions of these 2-year inhalation studies, there was some evidence of carcinogenic activity of cobalt sulfate heptahydrate in male F344/N rats based on increased incidences of alveolar/bronchiolar neoplasms. Marginal increases in incidences of pheochromocytomas of the adrenal medulla may have been related to exposure to cobalt sulfate heptahydrate. There was clear evidence of carcinogenic activity in female F344/N rats based on increased incidences of alveolar/bronchiolar neoplasms and pheochromocytomas of the adrenal medulla in groups exposed to cobalt sulfate heptahydrate. There was clear evidence of carcinogenic activity of cobalt sulfate heptahydrate in male and female B6C3F mice based on increased incidences of alveolar/bronchiolar neoplasms.
Endpoint:
carcinogenicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1990-08-30 to 1992-09-4
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Although no guidelines are stated for this study it is reliable without restriction, because it was conducted according to GLP requirements under the National Toxicology Program.
Reason / purpose:
reference to same study
Qualifier:
no guideline followed
Principles of method if other than guideline:
Groups of 50 male and 50 female rats 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:
yes
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS- Source: Simonsen Laboratories (Gilroy, CA)- Age at study initiation: approx. 6 weeks- Housing: Rats 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 rats 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): 21.3°C–26.6° C- Relative humidity: 31%–89%:- Air changes (per hr): 9-23/hour- Photoperiod (hrs dark / hrs light): 12 hours/dayNo further information on the test animals was stated.
Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Vehicle:
other: see details on inhalation exposure below
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); 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 stream through 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.0.3 mg/m³ concentration: MMAD = 1.5 +/- 0.10 µm (GSD = 2.2 +/- 0.14)1.0 mg/m³ concentration: MMAD = 1.4+/- 0.12 µm (GSD = 2.1 +/- 0.13)3.0 mg/m³ concentration: MMAD = 1.6 +/- 0.12 µm (GSD = 2.2 +/- 0.13)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 9 to 11 minutes and T10 ranged from 8 to 9 minutes for rats. With animals present, T90 values ranged from 11 to 16 minutes and T10 ranged from 12 to 13 minutes for rats. A T90 of 12 minutes was selected for the 2-year studies.Studies of cobalt sulfate heptahydrate degradation and monitoring for impurities were conducted throughout the 2-year studies with ICP/AES. No degradation of cobalt sulfate heptahydrate was observed during the studies. Cageboards were used after the first 8 weeks of the studies to control ammonia in the exposure chambers.No further information on the inhalation exposure was stated.
Analytical verification of doses or concentrations:
yes
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
Post exposure period:
No data
Remarks:
Doses / Concentrations:0, 0.3, 1.0 or 3.0 mg/m³ cobalt sulfate heptahydrateBasis:nominal conc.
Remarks:
Doses / Concentrations:0.31 +/- 0.03 mg/m³, 1.03 +/-0.10 mg/m³ or 2.98 +/- 0.20 mg/m³Basis: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 rats 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 10 and 30 mg/m³ in rats, while these lesions at the 0.3 and 1.0 mg/m³ exposure concentrations were composed of mild orminimal squamous metaplasia and/or chronic inflammation in rats. 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 rats.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 dataDERMAL IRRITATION (if dermal study): 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 AND COMPOUND INTAKE (if feeding study):- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: 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 AND COMPOUND INTAKE (if drinking water study): No dataOPHTHALMOSCOPIC EXAMINATION: No dataHAEMATOLOGY: No dataCLINICAL CHEMISTRY: No dataURINALYSIS: No dataNEUROBEHAVIOURAL EXAMINATION: No dataNo further information on the observations and examinations performed and frequency were stated.
Sacrifice and pathology:
GROSS PATHOLOGY: YesA complete necropsy and microscopic examination were performed on all rats. 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 rats. In addition to gross lesions and tissue masses, the tissues examined included:adrenal gland, bone with marrow, brain, clitoral gland, esophagus, harderian gland, heart, large intestine (cecum,colon, rectum), small intestine (duodenum, jejunum, ileum), kidney, larynx, liver, lungs/bronchi, lymph nodes (mandibular, mesenteric,bronchial, mediastinal), mammary gland, nose, oral cavity, 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.No further information on the sacrifice and pathology was stated.
Statistics:
- 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 Neoplasm Incidences: The primary statistical method used was logistic regression analysis. In this approach, neoplasm prevalence was modeled as a logistic function of chemical exposure and time. In addition to logistic regression, other methods of statistical analysis were used. These methods include the life table test (Cox, 1972; Tarone, 1975), appropriate for rapidly lethal neoplasms, and the Fisher exact test and the Cochran-Armitage trend test (Armitage, 1971; Gart et al., 1979), procedures based on the overall proportion of neoplasm-bearing animals.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. The procedures described in the preceding paragraphs were also used to evaluate selected nonneoplastic lesions.- Analysis of Nonneoplastic Lesion Incidences: The 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 severitiy values were analyzed for significance using the Mann-Whitney U test (Hollander and Wolfe,1973).
Clinical signs:
no effects observed
Mortality:
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 more frequently in female ratsexposed to 3.0 mg/m³ than in the chamber controls or other exposed groups.BODY WEIGHT AND WEIGHT GAINMean body weights of exposed male and female rats were similar to those of the chamber controls throughout the study.GROSS PATHOLOGY AND HISTOPATHOLOGY(NON-NEOPLASTIC AND NEOPLASTIC)Lung: In all exposed groups of male and female rats, the incidences of proteinosis, alveolar epithelial metaplasia, granulomatous alveolar inflammation, and interstitial fibrosis were significantly greater than those in the chamber controls. In general, these lung lesions increased in incidence and severity with increased exposure to cobalt sulfate heptahydrate. The incidence of squamous metaplasia in 1.0 mg/m³ females was significantly greater than in the chamber control group. Multifocally, throughout the lungs, pulmonary architecture was distorted by a combination of inflammatory cells, fibrosis, and epithelial metaplasia. Lesions tended to be subpleural, peripheral, and/or along larger blood vessels and airways. Granulomatous inflammation was characterized by accumulations of alveolar macrophages with foamy cytoplasm, occasional multinucleated giant cells and cholesterol clefts, cell debris and few neutrophils. In these areas, the alveolar interstitium and occasionally the overlying pleura were variably thickened by dense fibrous connective tissue which often effaced alveoli Although a diffuse change, aggregates of homogeneous to granular eosinophilic material within alveolar lumens (alveolar proteinosis) were often pronounced within the areas of chronic inflammation. Metaplasia of the alveolar epithelium in alveoli within and at the periphery of foci of inflammation was characterized by replacement of normal Type I epithelial cells with plump cuboidal or ciliated columnar epithelial cells. The incidences of alveolar epithelial hyperplasia in all groups of exposed males and in females exposed to 3.0 mg/m³ and atypical alveolar epithelial hyperplasia in 3.0 mg/m³ females were significantly greater than those in the chamber control groups.The combined incidence of alveolar/bronchiolar neoplasms (adenoma and/or carcinoma) was significantly greater in 3.0 mg/m³ males than that in the chamber controls and exceeded the historical control range. In females exposed to 1.0 or 3.0 mg/m³, the incidences of alveolar/bronchiolar neoplasms were significantly greater than those in the chamber control group and exceeded the historical control ranges. Although the incidences of alveolar/bronchiolar adenoma in 3.0 mg/m³ males and alveolar/bronchiolar carcinoma in 1.0 mg/m³ males were not significantly increased, they exceeded the historical control ranges for inhalation studies. The spectrum of alveolar/bronchiolar neoplasms and nonneoplastic proliferative lesions observed within the lungs of exposed rats was broad. While many of these lesions were highly cellular and morphologically similar to those observed spontaneously, others were predominantly fibrotic, squamous, or mixtures of alveolar/bronchiolar epithelium and squamous or fibrous components. Hyperplasia generally represented an increase in numbers of epithelial cells along alveolar walls with maintenance of normal alveolar architecture. Multiple hyperplastic lesions were often observed in animals receiving higher concentrations of cobalt sulfate heptahydrate. The benign neoplasms typical of those observed spontaneously were generally distinct masses that often compressed surrounding tissue. Component epithelial cells were often arranged in acinar and/or irregular papillary structures and occasionally in a solid cellular pattern. These epithelial 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 histologic features: heterogeneous growth pattern, cellular pleomorphism and/or atypia, and local invasion or metastasis. In addition to these more typical proliferative lesions, there were “fibroproliferative” lesions ranging from less than 1 mm to greater than 1 cm in diameter. Generally, these lesions had a rounded outline and a central fibrous core containing dispersed glandular (alveolar) structures lined by uniformly cuboidal epithelial cells. Aggregates of mostly necrotic inflammatory cells were also present in adjacent pheoalveoli and often within the glandular structures. Peripherally, the fibroproliferative lesions had one to several layers of epithelium which coursed along and often extended into adjacent alveoli, frequently forming papillary projections. These epithelial cells were often slightly pleomorphic with occasional mitotic figures. The smallest of these lesions were usually observed adjacent to areas of chronic inflammation. Small lesions with modest amounts of peripheral epithelial proliferation were diagnosed as atypical hyperplasia, while larger lesions with florid epithelial proliferation, marked cellular pleomorphism, and/or local invasion were diagnosed as alveolar/bronchiolar carcinomas.While squamous epithelium is not normally observed within the lung, squamous metaplasia of alveolar/ bronchiolar epithelium is a relatively common response to pulmonary injury and occurred in a number of rats in this study. Squamous metaplasia was a minor change consisting of a small cluster of alveoli in which the normal epithelium was replaced by multiple layers of flattened squamous epithelial cells that occasionally formed keratin. One 3.0 mg/m³ male and one 1.0 mg/m³ female had a large cystic squamous lesion rimmed by a variably thick (a few to many cell layers) band of viable squamous epithelium with a large central core of keratin. These were diagnosed as cysts. In one 1.0 mg/m³ and one 3.0 mg/m³ female, proliferative squamous lesions had cystic areas but also more solid areas of pleomorphic cells and invasion into the adjacent lung; these lesions were considered to be squamous cell carcinomas. In general, diagnoses of squamous lesions were made only when the lesion composition was almost entirely squamous epithelium. However, squamous metaplasia/ differentiation was a variable component of other alveolar/bronchiolar proliferative lesions, including the fibroproliferative lesions, and was clearly a part of the spectrum of lesions resulting from exposure to cobalt sulfate heptahydrate.Adrenal medulla: The incidence of benign pheochromocytoma in 3.0 mg/m³ females was significantly greater than that in the chamber controls and exceeded the historical range for inhalation studies. The incidences of benign, complex, or malignant pheochromocytoma (combined) in 1.0 mg/m³ males and in 3.0 mg/m³ females were significantly greater than those in the chamber controls and exceeded the historical control ranges.The incidences of bilateral pheochromocytoma in exposed males slightly exceeded that in the chamber control group. The incidence of hyperplasia was not significantly increased in exposed males or females. Focal hyperplasia and pheochromocytoma are considered to constitute a morphological continuum in the adrenal medulla. Focal hyperplasia consisted of irregular, small foci of small- to normal-sized medullary cells arranged in packets or solid clusters slightly larger than normal; compression of surrounding parenchyma was minimal or absent. Benign pheochromocytomas were well-delineated masses often with altered architecture and variable compression of surrounding parenchyma. Neoplastic cells were arranged in variably sized aggregates, clusters, and/or variably thick trabecular cords. Larger neoplasms usually exhibited greater cellular pleomorphism and atypia than smaller neoplasms. Malignant pheochromocytomas were identified when there was invasion of or beyond the adrenal capsule or when distant metastases were observed. Although a very common spontaneous neoplasm in male F344/N rats, pheochromocytomas have a lower spontaneous occurrence in females. In this study, the incidence of pheochromocytoma in 3.0 mg/m³ females was considered related to the administration of cobalt sulfate heptahydrate. The marginally increased incidence of pheochromocytoma in males was considered an uncertain finding because it occurred only in the 1.0 mg/m³ group and was not supported by increased incidence or severity of hyperplasia.Nose: The incidences of hyperplasia of the lateral wall of the nose and atrophy of the olfactory epithelium in all exposed groups of males and females were significantly greater than those in the chamber controls, and the severities of these lesions increased with increasing exposure concentration. The incidences of squamous metaplasia of the lateral wall of the nose and metaplasia of the olfactory epithelium in 3.0 mg/m³ males and females were significantly greater than those in the chamber controls.Although the incidence and severity of nasal lesions increased with increased exposure to cobalt sulfate heptahydrate, they involved limited portions of nasal epithelium and none were severe. Hyperplasia and squamous metaplasia were minimal to mild, unilateral or bilateral, and involved the transitional epithelium along the walls and turbinates of the anterior nasal passage. Hyperplasia was characterized by an increase in thickness of the epithelium from the normal one to two layers to two or more layers, while squamous metaplasia represented areas where the normal transitional epithelium was replaced by multiple layers of flattened epithelial cells. More posterior in the nose, along the dorsal meatus, atrophy of the olfactory epithelium was characterized by loss of cell layers and disorganization of remaining epithelium, and in some instances, increased prominence of sensory cell nuclei. Metaplasia was characterized by replacement of olfactory epithelium with respiratory-type ciliated columnar epithelium.Larynx: The incidences of squamous metaplasia of the epiglottis in all exposed groups of males and females were significantly greater than those in the chamber controls, and the severity of this lesion increased with increasing exposure concentration. Squamous metaplasia was limited to the base of the epiglottis and was not a severe lesion in exposed rats. 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.
Relevance of carcinogenic effects / potential:
Under the conditions of the 2-year inhalation study, there was some evidence of carcinogenic activity of cobalt sulfate heptahydrate in male F344/N rats based on increased incidences of alveolar/bronchiolar neoplasms. Marginal increases in incidences of pheochromocytomas of the adrenal medulla may have been related to exposure to cobalt sulfate heptahydrate. There was clear evidence of carcinogenic activity in female F344/N rats based on increased incidences of alveolar/bronchiolar neoplasms and pheochromocytomas of the adrenal medulla in groups exposed to cobalt sulfate heptahydrate. Exposure to cobalt sulfate heptahydrate caused a spectrum of inflammatory, fibrotic, and proliferative lesions in the respiratory tract of male and female rats.
Dose descriptor:
BMCL10
Effect level:
0.414 mg/m³ air
Based on:
test mat.
Basis for effect level:
other: alveolar epithelial hyperplasia and squamous metaplasia
Remarks on result:
other: Effect type: carcinogenicity (migrated information)

Under the conditions of the 2-year inhalation study, there was some evidence of carcinogenic activity of cobalt sulfate heptahydrate in male F344/N rats based on increased incidences of alveolar/bronchiolar neoplasms. Marginal increases in incidences of pheochromocytomas of the adrenal medulla may have been related to exposure to cobalt sulfate heptahydrate. There was clear evidence of carcinogenic activity in female F344/N rats based on increased incidences of alveolar/bronchiolar neoplasms and pheochromocytomas of the adrenal medulla in groups exposed to cobalt sulfate heptahydrate. Exposure to cobalt sulfate heptahydrate caused a spectrum of inflammatory, fibrotic, and proliferative lesions in the respiratory tract of male and female rats.

Conclusions:
Under the conditions of these 2-year inhalation studies, there was some evidence of carcinogenic activity of cobalt sulfate heptahydrate in male F344/N rats based on increased incidences of alveolar/bronchiolar neoplasms. Marginal increases in incidences of pheochromocytomas of the adrenal medulla may have been related to exposure to cobalt sulfate heptahydrate. There was clear evidence of carcinogenic activity in female F344/N rats based on increased incidences of alveolar/bronchiolar neoplasms and pheochromocytomas of the adrenal medulla in groups exposed to cobalt sulfate heptahydrate. There was clear evidence of carcinogenic activity of cobalt sulfate heptahydrate in male and female B6C3F mice based on increased incidences of alveolar/bronchiolar neoplasms.
Endpoint:
carcinogenicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2006-05-15 to 2008-05-15
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study
Reason / purpose:
reference to same study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 451 (Carcinogenicity Studies)
Version / remarks:
adopted 1981-05-12
Principles of method if other than guideline:
With respect to the inhalation route of exposure, the special requirements for inhalation studies with particulate materials as outlined in OECD TG 413 (1981/2009) have been taken into account (aerosol generation, particle size distribution, and determination of nominal and actual exposure concentrations).
GLP compliance:
yes
Species:
mouse
Strain:
other: B6C3F1/N
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS- Source: Taconic Farms, Inc. (Germantown, NY)- Age at study initiation: 5 to 6 weeks old- Weight at study initiation: Males0 mg/m³: 23.6 g1.24 mg/m³: 23.5 g2.49 mg/m³: 23.1 g5.01 mg/m³: 23.2 gFemales0 mg/m³: 19.8g1.24 mg/m³: 19.7 g2.49 mg/m³: 19.6 g5.01 mg/m³: 19.6 g- Housing: housed individually; cages: stainless steel wire bottom (Lab Products, Inc., Seaford, DE), changed weekly, rotated weekly in chambers; cageboard: untreated paper cage pan liner (Techboard, Shepherd Specialty Papers, Kalamazoo, MI), changed daily- Diet (ad libitum, except feed was withheld during exposure periods): irradiated NTP-2000 wafer diet (Zeigler Brothers, Inc., Gardners, PA), changed weekly- Water (ad libitum): tap water (Richland, WA municipal supply)- Acclimation period: 12 daysFive male and five female mice were randomly selected for parasite evaluation and gross observation of disease.
Route of administration:
inhalation: aerosol
Type of inhalation exposure (if applicable):
whole body
Vehicle:
air
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION- Exposure apparatus: the inhalation exposure chambers was designed so that uniform aerosol concentrations could be maintained throughout the chambers with the catch pans in place. The total volume of the chamber was 2.3 m³ with an active mixing volume of 1.7 m³. Tests showed that aerosol concentration could be reliably maintained homogenous within 8% throughout the chambers, provided the aerosol was uniformly mixed before passing through the chamber inlet and provided the test material did not react to a significant extent with animals, animal excrement, or the chamber interior (Griffis et al., 1981)*.Chamber environment:Temperature: 20.6 to 23.9°CRelative humidity: 55% ± 15%Room fluorescent light: 12 hours/dayChamber air changes: 15 ± 2/hour- System of generating particulates/aerosols: the generation system used a linear feed device to meter cobalt metal into the Trost jet mill. Initial particle size reduction was accomplished within the Trost jet mill. From the jet mill, aerosol was directed to the main distribution line where it was diluted with humidified air then conveyed from the exposure control center to the exposure room where it passed through a cyclone separator to further reduce particle size. On exiting the cyclone, the aerosol-laden air was directed to either of two smaller branch lines. From the branch lines, aerosol was delivered to each exposure chamber by a sampling tube. The flow through the sampling tube was induced by a stainless steel ejector pump. The aerosol then entered the chamber inlet duct where it was further diluted with conditioned chamber air to achieve the desired exposure concentration.- Method of particle size determination: particle size distribution was determined once prior to the 2-year study and monthly during the 2-year study. Impactor samples were taken from each exposure chamber using a Mercer-style seven-stage impactor and the stages (glass coverslips lightly coated with silicone to prevent particle bounce) were analyzed using ICP/AES after cobalt was extracted from the slides. The relative mass collected on each stage was analyzed by the CASPACT impactor analysis program developed at Battelle based on probit analysis (Hill et al., 1977)*.1.24 mg/m³: MMAD: 1.5 to 1.8 µm / GSD: 1.7 to 1.92.49 mg/m³: MMAD: 1.6 to 2.1 µm / GSD: 1.6 to 1.85.01 mg/m³: MMAD: 1.7 to 2.0 µm / GSD: 1.6 to 1.8TEST ATMOSPHERE- Brief description of analytical method used: the concentration of cobalt metal in the exposure chambers and room air was monitored using three real-time aerosol monitors (RAMs). Each RAM was calibrated by constructing a response curve using the measured RAM voltages (voltage readings were corrected by subtracting the RAM zero-offset voltage from measured RAM voltages) and cobalt metal concentrations that were determined by analyzing tandem Teflon®-coated, glass-fiber filters collected daily from the exposure chambers. Cobalt was extracted from the filters and analyzed using ICP/AES.Buildup and decay rates for chamber aerosol concentrations were determined with and without animals present in the chambers. At a chamber airflow rate of 15 air changes per hour, the theoretical value for the time to achieve 90% of the target concentration after the beginning of aerosol generation (T90) and the time for the chamber concentration to decay to 10% of the target concentration after aerosol generation was terminated (T10) was approximately 9.4 minutes. A T90 value of 12 minutes was selected.The uniformity of aerosol concentration in the inhalation exposure chambers without animals was evaluated before the 2-year study began; in addition, concentration uniformity with animals current in the chambers was measured every 3 to 4 months during the 2-year study. Chamber concentration uniformity was maintained throughout the study. The persistence of cobalt metal in the exposure chambers after aerosol delivery ended was determined by monitoring the concentration overnight in the 5 mg/m³ mouse chambers in the 2-year study with and without animals current in the chambers. The average cobalt metal concentration decreased to 1% of the target concentration within 19 minutes.Stability studies of the test material in the generation and exposure system were performed before and during the study. In this studiy, XRD analyses consistently indicated two primary phases of cobalt in the samples, cubic and hexagonal, and minimal detectable concentrations of cobalt oxides. Low and acceptable levels of trace element inorganic impurities were detected in these stability samples using PIXE and ICP/AES assays.*References- Griffis, L.C., Wolff, R.K., Beethe, R.L., Hobbs, C.H., and McClellan, R.O. (1981). Evaluation of a multitiered inhalation exposure chamber. Fundam. Appl. Toxicol. 1, 8-12.- Hill, M.A., Watson, C.R., and Moss, O.R. (1977). An Interactive Computer Program for Particle Size Analysis, PNL-2405, UC-32. Pacific Northwest Laboratory, Richland, WA.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Please refer to "Details on inhalation exposure" above.
Duration of treatment / exposure:
Core study: 105 weeksLung burden study: 79 weeks
Frequency of treatment:
6 hours plus T90 (12 minutes) per day, 5 days per week
Remarks:
Doses / Concentrations:1.24 ± 0.06 mg/m³Basis:analytical conc.
Remarks:
Doses / Concentrations:2.49 ± 0.11 mg/m³Basis:analytical conc.
Remarks:
Doses / Concentrations:5.01 ± 0.20 mg/m³Basis:analytical conc.
No. of animals per sex per dose:
Core study: 50 males / 50 femalesLung burden study: 35 females
Control animals:
yes
Details on study design:
- Dose selection rationale: based on reductions in body weights and moderate severity of nose and lung lesions in the 10 mg/m³ groups in the 3-month study (please refer to the endpoint study record k_NTP_2013_mice_14 weeks in section 7.5.2), this exposure concentration was considered too high to be used in the 2-year study. Hence, 5 mg/m³ was selected as the highest exposure concentration for the 2-year inhalation study in mice.
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 and every 4 weeks through week 93, then every 2 weeks, and at terminal kill.-DETAILED CLINICAL OBSERVATIONS: No dataBODY WEIGHT: Yes- Time schedule for examinations: core study animal body weights were recorded on day 1, weekly for the first 13 weeks, every 4 weeks through week 93, every 2 weeks thereafter, and at terminal kill.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 dataOTHER:Lung burden studies: five female lung burden mice per group were randomly selected and sent to necropsy immediately after exposure on days 1, 2, 3, 4, 184, 366, and 548. The lungs were removed, weighed, and analyzed for cobalt metal concentration using an ICP/AES method.
Sacrifice and pathology:
GROSS PATHOLOGY: YesHISTOPATHOLOGY: YesComplete necropsies and microscopic examinations were performed on all core study mice; selected necropsies were performed on lung burden study animals. At necropsy, organs and tissues were examined for grossly visible lesions, and all (core study) major tissues were fixed and preserved, processed and trimmed, embedded, sectioned, and stained for microscopic examination. For all paired organs, samples from each organ were examined. Tissues examined microscopically are the following: adrenal gland, bone with marrow, brain, clitoral gland, esophagus, eyes, gallbladder, Harderian gland, heart, large intestine (cecum, colon, rectum), small intestine (duodenum, jejunum, ileum), kidney, larynx, liver, lung, lymph nodes (bronchial, mandibular, mesenteric, and mediastinal), mammary gland, nose, ovary, pancreas, parathyroid gland, pituitary gland, preputial gland, prostate gland, salivary gland, skin, spleen, stomach (forestomach and glandular), testis with epididymis and seminal vesicle, thymus, thyroid gland, trachea, urinary bladder, and uterus.
Statistics:
Please refer to the field "Any other information on materials and methods incl. tables" below.
Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
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:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
effects observed, treatment-related
Details on results:
CLINICAL SIGNS AND MORTALITY- Survival of males exposed to 2.49 or 5.01 mg/m³ was significantly less than that of the chamber control group.- abnormal breathing and thinness were noted in exposed male and female mice.BODY WEIGHT AND WEIGHT GAIN- mean body weights of 5.01 mg/m³ males and females were at least 10% less than those of the chamber control groups after weeks 85 and 21, respectively. HISTOPATHOLOGY: NON-NEOPLASTIC / HISTOPATHOLOGY: NEOPLASTIC- Lung:The incidences of alveolar/bronchiolar adenoma in males exposed to 2.49 mg/m³ and females exposed to 5.01 mg/m³ and the incidences of alveolar/bronchiolar carcinoma and alveolar adenoma or carcinoma (combined) in all exposed groups of male and female mice were significantly greater than those in the chamber controls. The incidences of alveolar/bronchiolar carcinoma and alveolar/bronchiolar adenoma or carcinoma (combined) occurred with positive trends and exceeded the historical control ranges for inhalation studies and all routes of administration. In addition, significantly increased incidences of multiple alveolar/bronchiolar carcinoma occurred in all exposed groups of males and females.Alveolar/bronchiolar adenomas were small, generally nodular masses that distorted the alveolar architecture and compressed the adjacent parenchyma. Most adenomas consisted of interlacing papillary fronds and folds which projected into alveolar spaces and were lined by well-differentiated cuboidal to low columnar epithelium supported by scant fibrovascular stroma. Other more solid appearing adenomas were composed of closely packed nests or cords of polygonal to cuboidal, lightly eosinophilic cells that completely filled the alveolar spaces. Alveolar/bronchiolar carcinomas were discrete to locally infiltrative, compressive, nodular to irregularly shaped masses that effaced the alveolar parenchyma and ranged in diameter from 1 to a few millimeters to over 1 centimeter. Smaller carcinomas were relatively well-differentiated neoplasms that were morphologically similar to adenomas but were distinguished by their slightly greater cellular pleomorphism and architectural disorganization. Larger carcinomas were clearly malignant neoplasms which were composed of poorly differentiated, pleomorphic anaplastic cells with a variety of growth patterns ranging from complex papillary, tubular, and/or glandular, and less commonly, solid sheets. Many lungs that had carcinomas also had multiple, small nests of neoplastic cells randomly scattered throughout the lung parenchyma and occasionally on the pleura. Among the exposed mice with lung carcinomas, metastases occurred in various other organs including the liver, kidney, heart, nose, trachea, pancreas, cecum, adrenal cortex/medulla, coagulating gland, epididymis, testes, lymph nodes, thymus, skin, and skeletal muscle.A spectrum of nonneoplastic lesions occurred in the lungs of male and female mice. The incidences of alveolar/bronchiolar epithelium hyperplasia and cytoplasmic vacuolization, alveolar epithelium hyperplasia, proteinosis, and alveolus infiltration cellular histiocyte occurred with positive trends in male and female mice and were significantly increased in all exposed groups of males and females. The incidences of bronchiole epithelium hyperplasia occurred with positive trends in males and females and were significantly increased in males exposed to 5.01 mg/m³ and females exposed to 2.49 or 5.01 mg/m³. The incidence of bronchiole epithelium erosion was significantly increased in males exposed to 2.49 mg/m³. The incidences of suppurative inflammation were significantly increased in males exposed to 2.49 or 5.01 mg/m³ and females exposed to 5.01 mg/m³. In general, the severities of these nonneoplastic lesions increased with increasing exposure concentration.Nonneoplastic lesions invariably occurred together and presented as a complex mixture of lesions that at times were difficult to separate as individual lesions. Alveolar/bronchiolar epithelium hyperplasia and alveolar/bronchiolar epithelium cytoplasmic vacuolization were changes that occurred in the epithelium of the periacinar region of the lung which encompassed the terminal bronchioles, associated alveolar ducts, and immediately adjacent alveoli. The primary change throughout the epithelium was cytoplasmic vacuolization of the epithelium of terminal bronchioles and associated alveolar ducts and immediately adjacent alveoli that was also observed to a lesser extent in the larger bronchioles and bronchi. Cells with cytoplasmic vacuolization were swollen, cuboidal to irregularly polygonal, and had finely vacuolated to diffusely clear cytoplasm with small hyperchromic nuclei; generally, there was a decrease or absence of the surface cilia and apical blebbing that are characteristic of the normal ciliated epithelium and mouse Clara cells. Focally to multifocally, the epithelium of the terminal bronchioles appeared thickened by disorganized proliferation and piling up of vacuolated, cuboidal to polygonal epithelial cells that frequently extended into the alveolar ducts and the adjacent alveoli; these changes were diagnosed as alveolar/bronchiolar epithelium hyperplasia. Hyperplasia of the alveolar epithelium occurred as focal, discrete, irregular, noncompressive proliferations of alveolar epithelial (Type II) cells distributed randomly within the parenchyma or adjacent to the terminal bronchioles with preservation of the underlying alveolar architecture. These foci consisted of several contiguous alveolar septa lined by uniformly small cuboidal cells with small, hyperchromatic nuclei; occasional karyomegalic cells were present. Hyperplasia of the bronchiole epithelium was characterized by proliferation of cuboidal to low columnar bronchiole epithelial cells as few to multiple papillary structures that were supported by scant fibrous stroma and which projected into the lumens of the terminal bronchioles. Erosion of the bronchiole epithelium consisted of minimal, focal denudation of bronchiolar epithelial cells with associated minimal necrosis.Alveoli contained a complex mixture of proteinaceous material (diagnosed as proteinosis) and inflammatory cells. Alveolar proteinosis in mice was characterized by accumulation of variable amounts of brightly eosinophilic material within alveolar spaces and ducts with extension into the lumens of the bronchioles in the more extreme cases. The character of this material ranged from pale eosinophilic, flocculent to amorphous aggregates to brightly eosinophilic, dense, round to irregular clumps free within alveolar spaces or alveolar macrophages. In the more severe cases of proteinosis, there were single or aggregated slender, elongate, acicular to rectangular refractile, crystalline or spicule-like structures free within the alveoli or alveolar macrophages. Almost diffusely mixed with this proteinaceous material were increased numbers of histiocytes/macrophages that occurred as small, scattered aggregates to massive accumulations that on occasion completely occluded the alveoli in large regions of the lung. The histiocyte/macrophage infiltrates were mixtures of small to swollen macrophages to multinucleated giant cells. Many were distended with proteinaceous material or, especially in the lower exposure concentration groups, had cytoplasm that was lightly eosinophilic to gray, finely granular or foamy (amphophilic). Large histiocytes/macrophages and multinucleated giant cells contained the refractile, crystalline/specular-like structures. Accumulations of small histiocyte/macrophage infiltrates aggregated adjacent to alveolar/bronchiolar neoplasms (especially larger carcinomas). Also considered a component of this lesion were multifocal accumulations of plump foamy histiocytes/macrophages within the alveolar spaces; such cells were more frequent in the lower exposure concentration groups. Together, these histiocyte/macrophage infiltrates were diagnosed as histiocytic cellular infiltration of the alveolus. Mixed with the alveolar proteinaceous material and histiocyte/macrophage infiltrates were areas of prominent neutrophil accumulation that were diagnosed as suppurative inflammation. This occurred primarily in the male and female mice exposed to 5.01 mg/m³ and consisted of variably sized, localized accumulations of neutrophils and necrotic debris within alveoli. In areas of intense neutrophil accumulation, the alveolar septa were sometimes necrotic or even completely effaced; peribronchiolar edema, intraalveolar hemorrhage, and bacteria were occasionally observed in association with suppurative inflammation.- Nose:A spectrum of nonneoplastic lesions occurred in exposed groups of males and females and the incidences of these lesions were generally significantly greater than those in the chamber control groups. For some lesions, the severities increased with increasing exposure concentration.Suppurative inflammation in the nasal cavity of mice occurred primarily in the Level II nasal section and consisted of accumulations of neutrophils, proteinaceous fluid, and cellular debris in the nasal passages at all levels of the nose and was occasionally associated with fragments of plant material. Neutrophils sometimes infiltrated the nasal epithelium and lamina propria, and occasionally, the inflammatory process extended into the nasolacrimal duct, maxillary sinuses, and vomeronasal organ.Atrophy of the olfactory epithelium was of minimal to mild severity and occurred in the epithelium of the dorsal meatus in Levels II and III and the ethmoturbinates of Level III. Olfactory epithelial atrophy in mice was characterized by focal to diffuse hypocellularity and disorganization of the epithelium, often with increased extent of clear intercellular spaces with or without an overall decrease in height of the epithelium. There were variable decreases in the size and number of the nerve bundles and submucosal glands in the adjacent lamina propria. Hyperplasia of the olfactory epithelium was of minimal to mild severity and consisted of scattered focal proliferations of basal olfactory epithelial cells that extended through the basal lamina into the adjacent lamina propria often associated with Bowman’s gland ducts. The cells were sometimes clustered in small intraepithelial nests or extended into the lamina propria around the Bowman’s gland ducts.Respiratory metaplasia of the olfactory epithelium was of minimal to mild severity and observed more frequently in the dorsal meatus of Level II and on the nasal septum and ethmoturbinates in Level III. These lesions consisted of replacement of olfactory epithelium by ciliated, cuboidal to tall columnar, respiratory-type epithelial cells. The metaplastic epithelium occurred as crypt-like folds and invaginations and extended into the ducts of the submucosal Bowman’s glands. In 1.24 and 2.49 mg/m³ animals of both sexes, there were focal, highly site-specific, often bilaterally symmetrical, exophytic lesions located on the dorsal surface of the dorsal scroll. These focal lesions seemingly arose in areas that resembled respiratory metaplasia. The lesions were slightly to prominently elevated above the surface of the ethmoturbinates and in extreme cases, formed synechia with opposing dorsal turbinates and the roof of the dorsal meatus. Morphology ranged from small, single, or few rosette- or gland-like structures, to larger more complex formations of glands lined by flattened to ciliated respiratory type epithelium. These unusual lesions were diagnosed as olfactory epithelium atypical respiratory metaplasia.Hyaline droplet accumulation in the respiratory epithelium occurred with minimal severity and involved the respiratory epithelium adjacent to the junction with squamous epithelium lining the ventral aspects of the nasal passages in Levels I and II, adjacent to the incisive duct in Level II, and overlying the vomeronasal organ. Hyaline droplet accumulation consisted of intracytoplasmic, homogenous, eosinophilic, globular material in the cytoplasm of the respiratory epithelial cells.Cytoplasmic vacuolization of the respiratory epithelium primarily occurred with minimal to mild severity and affected the respiratory epithelium of the dorsal to mid-septum and/or the dorsal meatus of Level I and occasionally Level II (lateral walls and metaplastic region of the dorsal meatus). The morphology of cytoplasmic vacuolization was similar to that observed in the bronchiolar epithelium. In affected sites, the normally tall, pseudostratified,ciliated, columnar epithelium was replaced by plump, variably ciliated, cuboidal to polygonal epithelial cells that had finely vacuolated to diffusely clear cytoplasm.Squamous metaplasia of the respiratory epithelium of minimal to mild severity was generally similar to this lesion in the rat study. It occurred in the Level I and to a lesser extent, Level II sections along the dorsal to mid-septum and on the tips of the nasoturbinates and maxilloturbinates. Squamous metaplasia was characterized by replacement of the normally current single layer of ciliated columnar respiratory epithelium by nonkeratinized, flat, squamous epithelial cells.Turbinate atrophy was a minimal to moderate change. It was characterized by often prominent attenuation of the bone and structures of the lamina propria including the glands, vessels, nerve bundles, interstitial stroma on the naso-, maxilla-, and ethmoturbinates and the medial septum at Levels I, II, III. This resulted in narrowing, shortening, distortion , and sometimes loss of the turbinates, as well as occasional adhesions of turbinate remnants to each other, the nasal septum, or the lateral walls. The nasal septum was often buckled, bent, and sometimes perforated.- Larynx:The incidences of respiratory epithelium squamous metaplasia and cytoplasmic vacuolization in all exposed groups of males and females were significantly greater than those in the chamber controls. The incidences of squamous epithelium hyperplasia were significantly increased in all exposed groups of females and in males exposed to 5.01 mg/m³. The incidence of squamous epithelium erosion was significantly increased in females exposed to 2.49 mg/m³. All of these laryngeal lesions were of minimal severity.Respiratory epithelium squamous metaplasia involved the epithelium at the base of the epiglottis overlying the medial submucosal glands and consisted of one to a few layers of flattened, non-ciliated, low-cuboidal to squamous epithelial cells replacing the ciliated, tall, columnar epithelium that is normally current in this location. Respiratory epithelium cytoplasmic vacuolization was a subtle focal to diffuse change that occurred in the epithelium lining the dorsal aspects and lateral walls of Levels II and III laryngeal sections and was morphologically similar to cytoplasmic vacuolization observed in the bronchiolar epithelium. The ciliated columnar epithelial cells normally current in the sites were shorter (cuboidal), in general had lost their cilia, and had slightly vacuolated to clear cytoplasm. Squamous epithelium hyperplasia was a focal change most common in the epithelium along the medial aspects and tips of the vocal processes of the arytenoid cartilages and consisted of increased layers of lining epithelial cells from the normal two to three cell layers to four to six cell layers. Erosion was characterized by small focal areas of epithelial necrosis and loss of the superficial epithelium in areas of hyperplastic squamous epithelium.- Trachea: The incidences of minimal to mild epithelium cytoplasmic vacuolization were significantly increased in all exposed groups of males and females. Cytoplasmic vacuolization in the epithelium lining the trachea and the submucosal tracheal glands was morphologically similar to that observed in the bronchiolar and laryngeal epithelia.- Testes: The incidence of minimal to mild germinal epithelium degeneration in male mice exposed to 5.01 mg/m³ was significantly greater than that in the chamber controls (chamber control, 9/50; 1.24 mg/m³, 14/49; 2.49 mg/m3, 8/50; 5.01 mg/m³, 21/50). Germinal epithelium hyperplasia was generally a minimal to mild lesion usually affecting one to a few scattered seminiferous tubules. Affected tubules were characterized by partial to complete absence of spermatogenic cells often with concurrent swelling of the Sertoli cells with resultant hypocellularity and decreased height of the germinal epithelium. The lumens were generally empty but sometimes contained few spermatozoa, sloughed germinal epithelial cells, or cellular debris.OTHER FINDINGS - Tissue burden studiesLung weights and lung cobalt burdens (female mice): - Lung weights of female mice were significantly increased starting on day 4 in groups exposed to 2.49 or 5.01 mg/m³ and continuing until day 548. At 1.24 mg/m³, lung weights were increased on days 366 and 548; because of these increases in lung weights, lung cobalt burdens rather than lung cobalt concentrations were evaluated for toxicokinetic parameters.- Cobalt concentrations and burdens in the lung increased with increasing exposure concentration and were significantly increased in all exposed groups of female mice at all time points compared to those in the chamber control group. Cobalt concentrations in the chamber control group were at or below the LOD at all time points. By day 184, lung cobalt concentrations for all exposed groups appeared to reach steady state and did not change significantly through day 548. Lung cobalt burdens increased rapidly by day 4, but by day 184, the rate of increase slowed as lung burdens asymptotically approached steady state. Analysis of lung cobalt burdens normalized to exposure concentration indicated that there were proportional increases between the 1.24 and 2.49 mg/m³ groups, but nonproportional increases were observed between the 2.49 and 5.01 mg/m³ groups. At the earlier time points, normalized lung cobalt burdens were lower in animals exposed to 5.01 mg/m³ than in those exposed to 2.49 mg/m³; however the opposite was true at the longer exposure durations, where normalized cobalt burdens were greater than proportional relative to the 2.49 mg/m³ group.- The lung cobalt burden data from the exposure phases of the 3-month (please refer to endpoint study record k_NTP_2013_rats_14 weeks) and 2-year studies were modeled using a two-compartment model; these data showed the following:- rapid clearance phase half-lives were 1.2, 1.1, and 5.2 days, respectively, for the 1.24, 2.49, and 5.01 mg/m³ groups, indicating a slightly longer half-life in animals exposed to 5.01 mg/m³. - cobalt deposition rates for the rapid clearance phase were 0.87, 1.84, and 1.18 μg cobalt/day at 1.24, 2.49, and 5.01 mg/m³, respectively. - slow clearance phase half-lives revealed the opposite trend, with half-lives of 409, 172, and 118 days with increasing exposure concentration. - cobalt deposition rates for the slow clearance phase were 0.027, 0.075, and 0.25 μg cobalt/day. - overall theoretical steady-state lung cobalt burdens, including both the rapid and slow clearance phases (LSSa + LSSb), were approximately 17.8, 21.4, and 51.8 μg cobalt/lung in the 1.24, 2.49, and 5.01 mg/m³ groups, respectively; these data support the achievement of steady state in the 2.49 and 5.01 mg/m³ groups but not in the 1.24 mg/m³ group. The fractions of deposition in the slow clearance phase (FB) for the exposed groups were quite low, increasing from 0.031 to 0.176 as exposure concentration increased, corresponding to total slow phase lung cobalt clearances of 3.1% to 17.6%; clearances of total deposited cobalt during the rapid clearance phase ranged from 96.9% to 82.4% [(1–FB) × 100] with increasing exposure concentration.
Dose descriptor:
LOAEC
Effect level:
1.24 mg/m³ air (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Remarks on result:
other: Effect type: carcinogenicity (migrated information)
Conclusions:
The brief conclusions given below for the chronic inhalation study in rats and mice with cobalt metal powder were made based on a draft report, which might be subject to revisions. Hence, the conclusions will be revised and extended, once the final version of the report is made available by the NTP.Respiratory tract as major target organ for cancerogenicity was confirmed and was previously observed with Co sulfate hepathydrate at comparable exposure levels. Under the conditions of these 2-year inhalation studies, there was clear evidence of carcinogenic activity of cobalt metal in male and female F344/NTac rats based on increased incidences of alveolar/bronchiolar adenoma and carcinoma in the lung. There was clear evidence of carcinogenic activity of cobalt metal in male and female B6C3F1/N mice based on increased incidences of alveolar/bronchiolar neoplasms of the lung (predominantly carcinoma), including multiple carcinoma.The systemic effects are rat specific, and were not observed in the 2nd species (mouse). The lack of a finding in the 2nd species indicates that these findings are not relevant for humans (e.g. findings in the pancreas). This indication is supported by further arguments:- Typical Fisher rat specific illness of ageing (leukemia)- Sensitivity of male rats to develop kidney abnormalities- Weak statistics, no dose response - Some findings occurred not only exclusively in rats, but also only in one sex (Leukemia: female rats only; kidney findings: male rats only) - The weight of evidence strongly supports that only the local findings need to be taken forward to a human hazard assessment.
Endpoint:
carcinogenicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2006-05-08 to 2008-05-08
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study
Reason / purpose:
reference to same study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 451 (Carcinogenicity Studies)
Version / remarks:
adopted 1981-05-12
Principles of method if other than guideline:
With respect to the inhalation route of exposure, the special requirements for inhalation studies with particulate materials as outlined in OECD TG 413 (1981/2009) have been taken into account (aerosol generation, particle size distribution, and determination of nominal and actual exposure concentrations).
GLP compliance:
yes
Species:
rat
Strain:
other: F344/NTac rats
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS- Source: Taconic Farms, Inc. (Germantown, NY)- Age at study initiation: 5 to 6 weeks old- Weight at study initiation: Males0 mg/m³: 86 g1.24 mg/m³: 86 g2.50 mg/m³: 85 g5.01 mg/m³: 86 gFemales0 mg/m³: 77g1.24 mg/m³: 78 g2.50 mg/m³: 77 g5.01 mg/m³: 78 g- Housing: housed individually; cages: stainless steel wire bottom (Lab Products, Inc., Seaford, DE), changed weekly, rotated weekly in chambers; cageboard: untreated paper cage pan liner (Techboard, Shepherd Specialty Papers, Kalamazoo, MI), changed daily- Diet (ad libitum, except feed was withheld during exposure periods): irradiated NTP-2000 wafer diet (Zeigler Brothers, Inc., Gardners, PA), changed weekly- Water (ad libitum): tap water (Richland, WA municipal supply)- Acclimation period: 12 daysFive male and five female rats were randomly selected for parasite evaluation and gross observation of disease.
Route of administration:
inhalation: aerosol
Type of inhalation exposure (if applicable):
whole body
Vehicle:
air
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION- Exposure apparatus: the inhalation exposure chambers was designed so that uniform aerosol concentrations could be maintained throughout the chambers with the catch pans in place. The total volume of the chamber was 2.3 m³ with an active mixing volume of 1.7 m³. Tests showed that aerosol concentration could be reliably maintained homogenous within 8% throughout the chambers, provided the aerosol was uniformly mixed before passing through the chamber inlet and provided the test material did not react to a significant extent with animals, animal excrement, or the chamber interior (Griffis et al., 1981)*.Chamber environment:Temperature: 20.6 to 23.9°CRelative humidity: 55% ± 15%Room fluorescent light: 12 hours/dayChamber air changes: 15 ± 2/hour- System of generating particulates/aerosols: the generation system used a linear feed device to meter cobalt metal into the Trost jet mill. Initial particle size reduction was accomplished within the Trost jet mill. From the jet mill, aerosol was directed to the main distribution line where it was diluted with humidified air then conveyed from the exposure control center to the exposure room where it passed through a cyclone separator to further reduce particle size. On exiting the cyclone, the aerosol-laden air was directed to either of two smaller branch lines. From the branch lines, aerosol was delivered to each exposure chamber by a sampling tube. The flow through the sampling tube was induced by a stainless steel ejector pump. The aerosol then entered the chamber inlet duct where it was further diluted with conditioned chamber air to achieve the desired exposure concentration.- Method of particle size determination: particle size distribution was determined once prior to the 2-year study and monthly during the 2-year study. Impactor samples were taken from each exposure chamber using a Mercer-style seven-stage impactor and the stages (glass coverslips lightly coated with silicone to prevent particle bounce) were analyzed using ICP/AES after cobalt was extracted from the slides. The relative mass collected on each stage was analyzed by the CASPACT impactor analysis program developed at Battelle based on probit analysis (Hill et al., 1977)*.1.24 mg/m³: MMAD: 1.4 to 1.7 µm / GSD: 1.7 to 1.92.50 mg/m³: MMAD: 1.6 to 2.0 µm / GSD: 1.6 to 1.85.01 mg/m³: MMAD: 1.6 to 2.0 µm / GSD: 1.7 to 1.8TEST ATMOSPHERE- Brief description of analytical method used: the concentration of cobalt metal in the exposure chambers and room air was monitored using three real-time aerosol monitors (RAMs). Each RAM was calibrated by constructing a response curve using the measured RAM voltages (voltage readings were corrected by subtracting the RAM zero-offset voltage from measured RAM voltages) and cobalt metal concentrations that were determined by analyzing tandem Teflon®-coated, glass-fiber filters collected daily from the exposure chambers. Cobalt was extracted from the filters and analyzed using ICP/AES.Buildup and decay rates for chamber aerosol concentrations were determined with and without animals present in the chambers. At a chamber airflow rate of 15 air changes per hour, the theoretical value for the time to achieve 90% of the target concentration after the beginning of aerosol generation (T90) and the time for the chamber concentration to decay to 10% of the target concentration after aerosol generation was terminated (T10) was approximately 9.4 minutes. A T90 value of 12 minutes was selected.The uniformity of aerosol concentration in the inhalation exposure chambers without animals was evaluated before the 2-year study began; in addition, concentration uniformity with animals current in the chambers was measured every 3 to 4 months during the 2-year study. Chamber concentration uniformity was maintained throughout the study. The persistence of cobalt metal in the exposure chambers after aerosol delivery ended was determined by monitoring the concentration overnight in the 5 mg/m³ rat chambers in the 2-year study with and without animals current in the chambers. The average cobalt metal concentration decreased to 1% of the target concentration within 19 minutes.Stability studies of the test material in the generation and exposure system were performed before and during the study. In this studiy, XRD analyses consistently indicated two primary phases of cobalt in the samples, cubic and hexagonal, and minimal detectable concentrations of cobalt oxides. Low and acceptable levels of trace element inorganic impurities were detected in these stability samples using PIXE and ICP/AES assays.*References- Griffis, L.C., Wolff, R.K., Beethe, R.L., Hobbs, C.H., and McClellan, R.O. (1981). Evaluation of a multitiered inhalation exposure chamber. Fundam. Appl. Toxicol. 1, 8-12.- Hill, M.A., Watson, C.R., and Moss, O.R. (1977). An Interactive Computer Program for Particle Size Analysis, PNL-2405, UC-32. Pacific Northwest Laboratory, Richland, WA.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Please refer to "Details on inhalation exposure" above.
Duration of treatment / exposure:
Core study: 105 weeksLung burden study: 79 weeks
Frequency of treatment:
6 hours plus T90 (12 minutes) per day, 5 days per week
Remarks:
Doses / Concentrations:1.24 ± 0.07 mg/m³Basis:analytical conc.
Remarks:
Doses / Concentrations:2.50 ± 0.10 mg/m³Basis:analytical conc.
Remarks:
Doses / Concentrations:5.01 ± 0.18 mg/m³Basis:analytical conc.
No. of animals per sex per dose:
1.24 mg/m³: MMAD: 1.4 to 1.7 µm / GSD: 1.7 to 1.92.50 mg/m³: MMAD: 1.6 to 2.0 µm / GSD: 1.6 to 1.85.01 mg/m³: MMAD: 1.6 to 2.0 µm / GSD: 1.7 to 1.8
Control animals:
yes
Details on study design:
- Dose selection rationale: there were no significant effects on survival or body weight in the 5 mg/m³ groups in the 3-month study (please refer to the endpoint study record k_NTP_2013_rats_14 weeks in section 7.5.2). In addition, increases in lung weights, and incidences of nonneoplastic lesions in the nose and lung, and alterations of erythroid parameters were not considered sufficiently severe to preclude exposure at this concentration. Hence, 5 mg/m³ was selected as the highest exposure concentration for the 2-year inhalation study in rats.
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 and every 4 weeks through week 93, then every 2 weeks, and at terminal kill.-DETAILED CLINICAL OBSERVATIONS: No dataBODY WEIGHT: Yes- Time schedule for examinations: core study animal body weights were recorded on day 1, weekly for the first 13 weeks, every 4 weeks through week 93, every 2 weeks thereafter, and at terminal kill.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 dataOTHER:Lung burden studies: five female lung burden rats per group were randomly selected and sent to necropsy immediately after exposure on days 1, 2, 3, 4, 184, 366, and 548. The lungs were removed, weighed, and analyzed for cobalt metal concentration using an ICP/AES method.
Sacrifice and pathology:
GROSS PATHOLOGY: YesHISTOPATHOLOGY: YesComplete necropsies and microscopic examinations were performed on all core study rats; selected necropsies were performed on lung burden study animals. At necropsy, organs and tissues were examined for grossly visible lesions, and all (core study) major tissues were fixed and preserved, processed and trimmed, embedded in paraffin, sectioned, and stained for microscopic examination. For all paired organs, samples from each organ were examined. Tissues examined microscopically are the following: adrenal gland, bone with marrow, brain, clitoral gland, oesophagus, eyes, Harderian gland, heart, large intestine (cecum, colon, rectum), small intestine (duodenum, jejunum, ileum), kidney, larynx, liver, lung, lymph nodes (bronchial, mandibular, mesenteric, and mediastinal), mammary gland, nose, ovary, pancreas, parathyroid gland, pituitary gland, preputial gland, prostate gland, salivary gland, skin, spleen, stomach (forestomach and glandular), testis with epididymis and seminal vesicle, thymus, thyroid gland, trachea, urinary bladder, and uterus. For extended evaluation of renal proliferative lesions in male rats, kidneys were step sectioned and three to eight additional sections were obtained from each kidney.
Statistics:
Please refer to the field "Any other information on materials and methods incl. tables" below.
Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
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
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
effects observed, treatment-related
Details on results:
CLINICAL SIGNS AND MORTALITY- survival of female rats exposed to 2.5 mg/m³ was significantly less than that of the chamber control group.- exposure-related clinical findings included abnormal breathing and thinness in male and female rats.BODY WEIGHT AND WEIGHT GAIN- mean body weights of 2.5 and 5.01 mg/m³ males were at least 10% less than those of the chamber control group after weeks 99 and 12, respectively, and those of 2.5 and 5.01 mg/m³ females were at least 10% less after weeks 57 and 21, respectively.HISTOPATHOLOGY: NON-NEOPLASTIC / HISTOPATHOLOGY: NEOPLASTIC- Lung: The incidences of alveolar/bronchiolar adenoma, alveolar/bronchiolar carcinoma, and alveolar/bronchiolar adenoma or carcinoma (combined) occurred with positive trends in male and female rats and with the exception of the incidence of alveolar/bronchiolar adenoma in 1.24 mg/m³ females, the incidences were significantly greater than those in the chamber controls. The incidences of these neoplasms in all exposed groups exceeded the historical control ranges for all routes of administration. Incidences of multiple alveolar/bronchiolar adenoma generally increased with increasing exposure concentrations in males and females. Significantly increased incidences of multiple alveolar/bronchiolar carcinoma occurred in all exposed groups of males and in females exposed to 5.01 mg/m³. Increased incidences of cystic keratinizing epithelioma occurred in exposed groups of female rats; however, the increases were not statistically significant. In male rats, single incidences of cystic keratinizing epithelioma occurred the 1.24 and 5.01 mg/m³ exposure groups. One female rat exposed to 5.01 mg/m³ had a squamous cell carcinoma. Cystic keratinizing epithelioma and squamous cell carcinoma have not been observed in the lung of 100 historical controls for all routes of administration.Alveolar/bronchiolar adenomas were discrete, expansile, densely cellular masses that compressed the surrounding lung parenchyma. They were composed of relatively well-differentiated, uniform, cuboidal to columnar cells supported by a fine fibrovascular stroma and arranged in solid nests or papillary fronds that projected into alveolar spaces. Alveolar/bronchiolar carcinomas were larger, irregular, poorly circumscribed, unencapsulated, expansile, locally invasive masses that effaced the lung parenchyma. They were composed of poorly differentiated, moderately to markedly pleomorphic (anaplastic) cuboidal, columnar, or polygonal cells with pleomorphic nuclei; occasionally, cells had mitotic figures. The cells were arranged in single to multiple layers, formed irregular papillary or acinar structures and/or solid sheets and were supported by fibrovascular stroma. Some carcinomas had areas of squamous differentiation, and many contained extensive areas of necrosis, desmoplastic tissue, and inflammation. Other carcinomas had a core of dense fibrous tissue with embedded islands of malignant cells arranged in irregular cords, clusters, and acini. In several animals, metastases were observed in other tissues. Cystic keratinizing epitheliomas were well-circumscribed, unencapsulated, irregularly expansive masses that effaced the lung parenchyma. The epitheliomas consisted of an irregular wall of well-differentiated squamous epithelium surrounding a core of concentrically arranged keratin. Invariably the walls of these neoplasms had areas that lacked orderly maturation with foci of basal cell disorganization. The outer portion of the lesion grew by expansion into the adjacent lung, but evidence of invasion was not observed. The squamous cell carcinoma was an infiltrative mass that obliterated the normal lung architecture. The neoplastic cells formed swirling clusters, often around laminated keratin, separated by small to moderate amounts of fibrous stroma. The cells were polygonal, variable in size and shape, and contained small to moderate amounts of eosinophilic cytoplasm.The incidences of alveolar epithelium hyperplasia, alveolar proteinosis, chronic active inflammation, and bronchiole epithelium hyperplasia in all exposed groups of male and female rats were significantly greater than those in the chamber control groups. The severities of these lesions generally increased with increasing exposure concentration. This spectrum of nonneoplastic lesions invariably occurred together and presented as a complex mix of changes, and at times it was difficult to separate the individual components. Alveolar epithelium hyperplasia was a multifocal and sometimes focally extensive, discrete, randomly distributed but frequently subpleural lesion characterized by proliferation of flat to cuboidal to low columnar epithelial cells (presumed to be Type II pneumocytes) lining the alveolar septa; however, the underlying alveolar architecture was generally maintained. The interstitium of the alveolar septa was also variably expanded by increased amounts of collagen. Alveolar proteinosis was characterized by accumulations of brightly eosinophilic, wispy to globular, homogeneous, proteinaceous material filling the alveolar spaces; this proteinaceous material frequently contained acicular cholesterol crystals or cleft-like spaces. These lesions were invariably accompanied by chronic active inflammation which consisted of complex mixtures of predominantly macrophages and lymphocytes mixed with lesser numbers of neutrophils within the alveolar spaces and septa and low numbers of multinucleated giant cells; clear cleft-like spaces (cholesterol clefts) were frequently present among the inflammatory cells. Also associated with areas of chronic active inflammation, there was frequently variable proliferation of the alveolar epithelial (Type II) cells and variable alveolar septal interstitial fibrosis. Frequently, there were large numbers of inflammatory cell infiltrates, mostly macrophages, accumulated around the alveolar/bronchiolar neoplasms. The alveolar macrophages were frequently engorged with an intensely eosinophilic material similar to that in the alveolar spaces, and many macrophages also contained acicular cholesterol crystals. Multifocal accumulations of plump foamy macrophages within the alveolar spaces were considered a component of the inflammation changes. Bronchiole epithelium hyperplasia was characterized by proliferation and disorganized crowding of ciliated, cuboidal columnar to pleomorphic epithelial cells lining terminal bronchioles with extension onto adjacent alveolar septa. There was often minimal to mildly increased amounts of collagen in the interstitium of the bronchiolar wall.- Nose:A spectrum of nonneoplastic lesions occurred with positive trends in male and female rats, and the incidences were often significantly greater than those in the chamber controls. For some lesions, the severities increased with increasing exposure concentration. Chronic active inflammation was most prominent in Levels I and II and less often in Level III nasal section. Chronic active inflammation consisted of infiltrates of mostly lymphocytes, plasma cells, neutrophils, and fewer macrophages within the lamina propria and overlying epithelium accompanied by cellular debris in the nasal passages. Suppurative inflammation occurred primarily in Level II and consisted of accumulations of nondegenerate and degenerate neutrophils mixed with eosinophilic proteinaceous material, occasional macrophages, cellular debris, and sometimes colonies of coccobacilli and foreign material within the nasal passages and adjacent epithelium and lamina propria of the nasal turbinates. More pronounced suppurative inflammation was often accompanied by florid hyperplasia of the adjacent epithelium.Respiratory metaplasia and/or atrophy of the olfactory epithelium occurred in the dorsal meatuses of Level II and sometimes Level III. When the predominant change in the affected segment was replacement of the olfactory epithelium by respiratory type columnar epithelial cells, olfactory epithelium respiratory metaplasia was diagnosed. When the olfactory epithelium was attenuated due to loss of olfactory epithelial cells, olfactory epithelium atrophy was diagnosed. Olfactory epithelium hyperplasia mostly occurred in rats exposed to 5.01 mg/m³ and consisted of small, focal, intraepithelial proliferations of epithelial cells that formed clusters or rosettes that sometimes extended into the lamina propria. Olfactory epithelium basal cell hyperplasia was invariably associated with olfactory epithelium hyperplasia and consisted of disorganized proliferation and crowding of the basal olfactory epithelial cells. Necrosis of the olfactory epithelium was a minimal to mild lesion mostly affecting male rats and a few females and was associated with inflammatory lesions. In sites of necrosis, the epithelium was effaced and replaced by cellular and karyorrhectic debris.Respiratory epithelium hyperplasia occurred in the epithelium lining the tips of the nasoturbinates, maxilloturbinates, and the septa in Levels I and II. In affected sites, the epithelium was thickened by increased numbers of cuboidal to ciliated columnar epithelial cells crowded in multiple layers sometimes forming undulations with invaginations into the underlying lamina propria. This lesion was most prominent in areas of suppurative inflammation. Respiratory epithelium squamous metaplasia was most common at the tips of the nasoturbinates and along the lateral walls of Level I and less often Level II. In affected sites, flattened squamous epithelium of variable thickness replaced the ciliated columnar epithelium normally present in this location. Necrosis of the respiratory epithelium was associated with the inflammatory lesions and primarily affected rats in the 5.01 mg/m³ groups. In areas of necrosis, the epithelium was effaced and replaced by cellular and karyorrhectic debris; necrosis would sometimes extend into the submucosa and sinuses.Turbinate atrophy was a minimal to mild change that primarily affected the naso- and maxilloturbinates in Levels I and II and occasionally Level III. Affected turbinates were short, thin, and blunted due to attenuation of the turbinate bone and loss of structures in the lamina propria, including the glands, vessels, nerve bundles, and connective tissue. As a result, the nasal passages appeared wider than normal. The nasal septum was sometimes similarly affected and had a noticeable decrease in width.- Adrenal medulla: The incidences of benign pheochromocytoma, malignant pheochromocytoma, and benign or malignant pheochromocytoma (combined) occurred with positive trends in male and female rats and with the exception of the incidence of malignant pheochromocytoma in 2.5 mg/m³ females, the incidences in rats exposed to 2.5 or 5.01 mg/m³ were significantly greater than those in the chamber controls and exceeded the historical control ranges for all routes of administration. The incidences of bilateral benign pheochromocytoma were significantly increased in all exposed groups of males and in 2.5 and 5.01 mg/m³ females, and the incidences of bilateral malignant pheochromocytoma were significantly increased in male and female rats exposed to 5.01 mg/m³.Benign pheochromocytoma occurred as variably sized, well-demarcated, expansile proliferations of medullary cells that formed large trabeculae or solid clusters separated by delicate fibrous stroma and/or sinusoids. The cells were polygonal to spindyloid and moderately uniform in size and shape. Malignant pheochromocytomas were generally larger, irregular, poorly demarcated invasive masses that effaced the adrenal gland extending through the capsule into the periadrenal tissue; the neoplastic cells were poorly differentiated and pleomorphic. In some animals, malignant pheochromocytomas metastasized to other organs.The incidences of medullary hyperplasia in the adrenal gland were significantly increased in female rats exposed to 1.24 or 2.5 mg/m³; incidences of this lesion were significantly decreased in male rats exposed to 2.5 or 5.01 mg/m³. Hyperplasia occurred as focally discrete proliferations of medullary epithelial cells that blended with, but did not compress, the surrounding medullary parenchyma. The cells were generally smaller and more basophilic than the surrounding normal medullary epithelial cells.- Pancreatic islets: The incidences of carcinoma and adenoma or carcinoma (combined) occurred with positive trends in male rats and the incidences of adenoma, carcinoma, and adenoma or carcinoma (combined) generally exceeded the historical control ranges for all routes of administration. The incidences of adenoma in 2.5 mg/m³ males and of adenoma or carcinoma (combined) in males exposed to 2.5 or 5.01 mg/m³ were significantly greater than those in the chamber controls. Incidences of adenoma, carcinoma, and adenoma or carcinoma (combined) in 5.01 mg/m³ females were slightly increased; the increases were not statistically significant but did exceed the historical control ranges for all routes of administration. Adenomas were well-circumscribed, expansile masses that compressed the acini. The neoplastic cells were well-differentiated with minimal to mild cellular atypia and slightly altered growth patterns. Carcinomas were poorly circumscribed, unencapsulated, irregular, expansile, and invasive masses that effaced the parenchyma. Carcinomas had a heterogeneous growth pattern with cells that were moderately to markedly pleomorphic.The increases were not statistically significant.These tumours are rare and they were seen for the first time in NTP studies. They were also not observed in other studies with cobalt substances as described in this report. In this context it needs to be considered that the historical control data for this strain and exposure route in this institution are weak. The exact mechanism for induction of these tumours is not well understood and they may result from chronic inflammation of the endocrine hormone producing tissue of the pancreas. However, no evidence of pancreatic inflammation was seen in the other studies with cobalt. Therefore, these findings should be interpreted with caution. - Mononuclear cell leukemia:The incidences of mononuclear cell leukemia were significantly increased in all exposed groups of female rats and exceeded the historical control range for all routes of administration.The relevance of this result for an assessment of human cancer risk is questionable because it was obtained in a rat strain highly predisposed to developing MNCL.- Kidney:In the standard evaluation of the kidney, the incidences of renal tubule adenoma, carcinoma, and adenoma or carcinoma (combined) were slightly increased in male rats exposed to 5.01 mg/m³. Although not statistically significant, the incidences in this group exceeded the historical control ranges for all routes of administration. In the standard evaluation, a single section of each kidney is routinely examined microscopically. Because the incidences of renal tubule neoplasms in the standard evaluation suggested the possibility of a treatment-related carcinogenic effect, an extended evaluation of the kidney was performed in male rats to explore this possibility. In the extended evaluation, additional renal tubule adenomas and renal tubule hyperplasias were identified but no additional renal tubule carcinomas; a renal tubule oncocytoma was identified in one male exposed to 2.5 mg/m³. In the combined standard and extended evaluation, the incidences of renal tubule hyperplasia in the exposed groups were similar to that in the chamber controls. The incidence of renal tubule adenoma in the 5.01 mg/m³ group was greater than that in the chamber control group, but the increase was not statistically significant. The incidences of renal tubule carcinomas were unchanged.Renal tubule adenomas were small, well-circumscribed proliferations of renal tubule epithelial cells with a cross-sectional area greater than five times that of a single, normal renal tubule. The cells were well-differentiated and uniform in size and shape and formed poorly defined papillary structures or solid clusters of cells. Renal tubule carcinomas were larger, expansive, and invasive masses that effaced and replaced much of the renal parenchyma.- Liver:The incidences of basophilic focus occurred with positive trends in male and female rats and in all exposed groups of males (chamber control, 5/50; 1.24 mg/m³, 17/50; 2.5 mg/m³, 17/50; 5.01 mg/m³, 19/50) and females exposed to 5.01 mg/m³ (16/50, 20/50, 22/50, 33/50), and the incidences were significantly greater than those in the chamber control groups. Basophilic foci occur spontaneously in rats, and the incidences are sometimes increased with exposure to chemicals. They are considered putative preneoplastic lesions; however, the incidences of hepatocellular neoplasms were not increased in male or female rats exposed to cobalt metal.- Testes: The incidence of infarct was significantly increased in male rats exposed to 5.01 mg/m³ (1/50, 0/50, 2/50, 12/50). Infarcts were mostly unilateral, and in affected testes, there was complete effacement of the parenchyma due to necrosis with loss of differential staining (tissue was diffusely hypereosinophilic) and cellular detail. Multifocal intratubular mineralization was current in a few of the affected testes.OTHER FINDINGS - Tissue burden studiesLung weights and lung cobalt burdens (female rats): - lung weights increased in all exposed groups; increases in lung weights occurred earlier in the study (day 184) in the 2.5 and 5.01 mg/m³ groups than in the 1.24 mg/m³ group (day 366).- cobalt concentrations and burdens in the lung increased with increasing exposure concentration and were significantly increased in all exposed groups of female rats at all time points compared to those in the chamber control group. - cobalt concentrations in the chamber control group were at or below the LOD at all time points except day 548 (one animal had a lung cobalt concentration exceeding the LOD but less than the experimental limit of quantitation (ELOQ). - by day 184, lung cobalt concentrations for all exposed groups appeared to reach steady state and did not change significantly through day 548- lung cobalt burdens increased rapidly by day 4, but by day 184 the rate of increase slowed as lung burdens asymptotically approached steady state.- analysis of normalized lung cobalt burdens revealed no tendency toward disproportionate changes and no biologically significant differences in normalized burdens with increasing exposure concentration.- the lung cobalt burden data from the exposure phases of the 3-month (please refer to endpoint study record k_NTP_2013_rats_14 weeks) and 2-year studies were modeled using a two-compartment model; these data showed the following:- steady state was clearly reached at 2.5 and 5 mg/m³ but not at 1.25 mg/m³ (all target concentrations). - rapid clearance phase half-lives were between 1.53 days and 2.37 days, while slow clearance phase half-lives were 789 days, 167 days, and 83 days for 1.25 mg/m³, 2.5 mg/m³, and 5 mg/m³, respectively. The apparent lack of achievement of steady state and long half-life at 1.25 mg/m³ are likely spurious findings due to uncertainty in the model. - cobalt deposition rates were 1.4, 2.1, and 5.6 μg cobalt/day during the rapid clearance phase and 0.018, 0.078, and 0.29 μg cobalt/day during the slow clearance phase at 1.25, 2.5, and 5 mg/m³, respectively. - steady-state lung cobalt burdens including both the rapid and slow clearance phases (LSSa + LSSb) were approximately 25.4, 27.8, and 46.8 μg cobalt/lung in animals exposed to 1.25, 2.5, and 5 mg/m³, respectively. - the fractions of deposition in the slow clearance phase (FB) for the exposed groups were quite low, increasing from 0.012 to 0.049 as exposure concentrations increased, corresponding to total slow phase lung cobalt clearances of 1.2% to 4.9%; clearances of total deposited cobalt during the rapid clearance phase ranged from 98.8% to 95.1% [(1-FB) × 100] with increasing exposure concentration.
Dose descriptor:
LOAEC
Effect level:
1.24 mg/m³ air (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Remarks on result:
other: Effect type: carcinogenicity (migrated information)
Conclusions:
The brief conclusions given below for the chronic inhalation study in rats and mice with cobalt metal powder were made based on a draft report, which might be subject to revisions. Hence, the conclusions will be revised and extended, once the final version of the report is made available by the NTP.Respiratory tract as major target organ for cancerogenicity was confirmed and was previously observed with Co sulfate hepathydrate at comparable exposure levels. Under the conditions of these 2-year inhalation studies, there was clear evidence of carcinogenic activity of cobalt metal in male and female F344/NTac rats based on increased incidences of alveolar/bronchiolar adenoma and carcinoma in the lung. There was clear evidence of carcinogenic activity of cobalt metal in male and female B6C3F1/N mice based on increased incidences of alveolar/bronchiolar neoplasms of the lung (predominantly carcinoma), including multiple carcinoma.The systemic effects are rat specific, and were not observed in the 2nd species (mouse). The lack of a finding in the 2nd species indicates that these findings are not relevant for humans (e.g. findings in the pancreas). This indication is supported by further arguments:- Typical Fisher rat specific illness of ageing (leukemia)- Sensitivity of male rats to develop kidney abnormalities- Weak statistics, no dose response - Some findings occurred not only exclusively in rats, but also only in one sex (Leukemia: female rats only; kidney findings: male rats only) - The weight of evidence strongly supports that only the local findings need to be taken forward to a human hazard assessment.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Study duration:
chronic
Species:
other: rat and mouse
System:
respiratory system: lower respiratory tract
Organ:
lungs

Carcinogenicity: via dermal route

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

1. Database

 

There are 54 references of publicly available scientific literature and 10 unpublished statements and comments, which address the potential carcinogenicity of cobalt compounds. Upon screening, a total of 29 references were evaluated further (please refer to the section on disregarded studies on the carcinogenicity endpoint at the end of this discussion (IUCLID) or in Appendix 3 of the CSR. These references were evaluated for their relevance and reliability for the assessment of carcinogenicity in the context of the chemical risk assessment of cobalt compounds, specifically under the REACH Regulation (ECHA Guidance on information requirements and chemical safety assessment Chapter R.4: Evaluation of available information, Version 1.1 December 2011).

 

Only three animal studies investigated the induction of tumours following chronic inhalation exposure; one in hamsters with cobalt(II)oxide (Wehner et al., 1977), and two studies with rats and mice to cobalt sulfate hexahydrate and cobalt metal powder. Out of these, only the 2-year inhalation carcinogenicity studies on rats and mice (Bucher et al., 1999; NTP 1998; NTP 2014) were evaluated in detail. Both studies were performed under GLP and represent highly reliable studies without restrictions (RL 1). These studies provide reliable data, based on sufficient size of the groups, three dose graduations and an exposure time of 2 years.

 

There is a complete lack of studies regarding cancer in experimental animals after oral or dermal exposure to any cobalt compound.

 

 

2. Chronic studies in animals

 

2.1 NTP – Cobalt sulfate

In chronic inhalation toxicity/carcinogenicity studies by Bucher et al. (1999; NTP 1998), groups of 50 F344/N rats and 50 B6C3F1 mice of each sex were exposed by inhalation to aerosols of cobalt sulfate hexahydrate at chamber concentrations of 0, 0.3, 1.0, and 3.0 mg/m³ (equivalent to 0, 0.067, 0.224, and 0.673 mg Co/m³) for 6 hours/day, 5 days/week, over a period of 105 weeks. The MMAD (µm) ± GSD was in the range from 1.4 ± 2.1 to 1.6 ± 2.2. The survival time and the body weight development were not affected in both species. The treatment resulted in changes in the incidences of neoplasms and/or non-neoplastic lesions of the lung, adrenal medulla, nose, and larynx. The respiratory tract was the primary site of non-neoplastic lesions and neoplasms in both species.

In rats, proteinosis, alveolar epithelial metaplasia, granulatomous alveolar inflammation, and interstitial fibrosis were observed in the lung of all exposed groups. The incidence of hyperplasia of the respiratory epithelium of the lateral wall of the nose and atrophy of the olfactory epithelium in all exposed groups was significantly greater than those in controls, and the severity of these lesions increased with increasing exposure concentration. Nasal lesions in mice were less severe than in rats, but olfactory epithelial atrophy was observed at ≥ 1.0 mg/m³. The incidence of squamous metaplasia of the epiglottis in all exposed groups of rats and mice was significantly increased, and the severity of this lesion increased in rats with higher concentrations as well. Hence, 2-year exposure of rats and mice to cobalt sulfate resulted in non-neoplastic lesions of the nose, larynx and lung at all concentrations studied.

 

Rats revealed an exposure-related increase in the incidence of benign and malignant alveolar/bronchiolar neoplasms (adenoma or carcinoma) in both sexes. The incidence of benign and malignant alveolar/bronchiolar neoplasms were 1/50, 4/50, 4/48, and 7/50 for 0, 0.3, 1.0, and 3.0 mg/m³, respectively, in males and 0/50, 3/49, 16/50, and 16/50 in females (including squamous cell carcinoma). Significantly increased rates of tumours (alveolar/ bronchiolar adenoma or carcinoma) were seen in the lung of female rats in the medium group and above and in male rats at 3.0 mg/m³ as compared with those in the controls and exceeded the historical control ranges of NTP inhalation studies. The frequency of tumours in the low dose groups of both sexes was non-significantly greater than that in the chamber controls, but was within the range of historical controls. In addition, increased incidences of adrenal pheochromocytoma were seen in mid-dose male rats, but not in high-dose males, and in high dose females.

In mice, the incidence of alveolar/bronchiolar adenoma or carcinoma were 11/50, 14/50, 19/50, and 28/50 for 0, 0.3, 1.0, and 3.0 mg/m³, respectively, in males and 4/50, 7/50, 13/50, and 18/50 in females. Significantly increased incidences of alveolar/bronchiolar adenoma or carcinoma were found in 3.0 mg/m³ male and female mice, too. The combined incidence of alveolar/bronchiolar neoplasms in 1.0 mg/m³ females was significantly greater than that in the control. The frequency of tumours in the low dose groups of both sexes was non-significantly increased compared to that in the chamber controls, but was within the range of historical controls. The frequent occurrence of haemangiosarcoma of the liver in male animals of this study is based on an infection and not classified by the authors as being related to the treatment.

 

Taken together, 2-year exposure of rats and mice to cobalt sulfate by inhalation resulted in increased incidences of alveolar/bronchiolar neoplasms and a spectrum of inflammatory, fibrotic and proliferative lesions in the respiratory tracts of male and female rats and mice. Tumours developed in rats and mice of both sexes at concentrations ≥ 0.3 mg/m³ cobalt sulfate hexahydrate (equivalent to ≥ 0.067 mg Co/m³), thus this concentration represents a LOAEC for inhalation carcinogenicity. A NOAEC for tumour formation as well as non-neoplastic local effects was not reached in these studies.

Taking into account the lack of a NOAEC in the concentration-response assessment of cobalt sulfate a benchmark dose (BMD) was calculated using the US EPA BMD software (Version 2.0) with the Gamma Model (Version 2.13) assuming a threshold mode of action. The numbers of alveolar/bronchiolar adenoma or carcinoma in the lung of rats and mice were selected as benchmark response. The 95% lower confidence limit of the BMD for a treatment-related increase in response of 10% was calculated (BMDL10). The lowest BMDL10 value was that for female rat tumours with 0.414 mg/m³ cobalt sulfate hexahydrate.

 

2.2 NTP-Cobalt metal powder

 In the carcinogenicity studies by NTP (NTP, 2015) groups of 50 male and 50 female F344/NTac rats and B6C3F1/N mice were exposed to cobalt metal particulate aerosol by inhalation at chamber concentrations of 0, 1.25, 2.5, or 5.0 mg/m³ for 6 hours/day, 5 days/week for up to 105 weeks. In an accompanying toxicokinetic study, additional groups of 35 female rats and mice were exposed to the same concentrations of cobalt metal for up to 79 weeks to provide data on pulmonary retention, clearance, and systemic distribution. The exposure concentrations for the 2-year cobalt metal studies were based on the findings of the 16-day and 13-week studies that revealed cytotoxic effects on the epithelia of the upper and lower respiratory tract of F344/N rats and B6C3F1/N mice accompanied by nasal inflammation in mice and pulmonary inflammation in rats. The MMAD of the aerosols was in the range from 1.4 to 2.1 µm, GSDs were in the range from 1.6 to 1.9.

In rats, the survival time of females exposed to 2.5 mg/m³ was significantly decreased in comparison to that of the chamber control group. Mean body weights of 2.5 and 5 mg/m³ males were at least 10% less than those of the control group after weeks 99 and 12, respectively, and those of 2.5 and 5 mg/m³ females were at least 10% less after weeks 57 and 21, respectively.

In mice, survival of males exposed to 2.5 or 5 mg/m³ was significantly less than that of the chamber control group. Mean body weights of 5 mg/m³ males and females were at least 10% less than those of the chamber control groups after weeks 85 and 21, respectively. Exposure-related clinical findings included abnormal breathing and thinness in male and female animals of both species.

 

In rats, the incidences of alveolar/bronchiolar adenoma (including multiple) were in males 2/50, 10/50, 10/50, and 14/50 for the exposure concentrations of 0, 1.25, 2.5, and 5.0 mg/m³ cobalt metal, respectively. Incidences of alveolar/bronchiolar carcinoma (including multiple) were 0/50, 16/50, 34/50, and 36/50, and of alveolar/bronchiolar adenoma and carcinoma (combined) 2/50, 25/50, 39/50, and 44/50 for the respective concentrations. A single cystic keratinizing epithelioma (CKE) was found in each group of male rats exposed to 1.25 and 5.0 mg/m³. In females, the incidences of alveolar/bronchiolar adenoma (including multiple) were 2/50, 7/50, 9/50, and 13/50, and those of alveolar/bronchiolar carcinoma (including multiple) were 0/50, 9/50, 17/50, and 30/50. Alveolar/bronchiolar adenoma and carcinoma (combined) occurred with incidences of 2/50, 15/50, 20/50, and 38/50. Statistically non-significant increases in the incidence of CKE were observed in all exposed groups of female rats. One squamous cell carcinoma was found in a female rat exposed to 5 mg/m³.

The incidences of all neoplasms except that of alveolar/bronchiolar adenoma in 1.25 mg/m³ females were above the concurrent chamber controls. The incidences of multiple alveolar/bronchiolar adenoma and carcinoma generally increased with increasing exposure concentration in males and females. Incidences of multiple carcinomas were significantly increased in all exposed males and in 5 mg/m³ females.

 

In male mice, the incidences of alveolar/bronchiolar adenoma (including multiple) were 7/50, 11/49, 15/50, and 3/50 for the exposure concentrations of 0, 1.25, 2.5, and 5.0 mg/m³. The incidences of alveolar/bronchiolar carcinoma (including multiple) were 11/50, 38/49, 42/50, and 46/50. Alveolar/bronchiolar adenoma and carcinoma (combined) occurred with statistically significantly increased incidences in all exposed males (16/50, 41/49, 43/50, 47/50. In female mice, the incidences of alveolar/bronchiolar adenoma (including multiple) were 3/49, 9/50, 8/50, and 10/50, and those of alveolar/bronchiolar carcinoma (including multiple) were 5/49, 25/50, 38/50, and 43/50. Alveolar/bronchiolar adenoma and carcinoma (combined) occurred with incidences of 8/49, 30/50, 41/50, and 45/50. Significantly increased incidences of alveolar/bronchiolar carcinoma were found in all exposed male and female mice as compared with those in the chamber controls.

 

2.3 Conclusion

There is strong evidence that inhalation of cobalt metal and cobalt sulfate causes lung tumours in both rats and mice (NTP 2014, 1998). In summary, long-term inhalation exposure of rats and mice to cobalt metal and cobalt sulfate heptahydrate clearly shows local carcinogenicity of both cobalt compounds due to the formation of alveolar/bronchiolar carcinomas in the lungs of both rats and mice.

 

A comparison of the results of the long-term studies with cobalt sulfate heptahydrate and cobalt metal reveals that both cobalt substances cause the formation of alveolar/bronchiolar carcinoma in the lungs of both rats and mice. Taking into account the molecular stoichiometry of cobalt sulfate to cobalt metal, the highest exposure concentration of 3.0 mg/m³ of cobalt sulfate which leads to tumour formation (equivalent to 0.67 mg Co/m³) being half the lowest exposure concentration (1.25 mg/m³) delivered to the animals in the cobalt metal studies and shown to be tumorigenic. Thus, this tumour data would almost quantitatively be in line with the concentration-response relationship for alveolar/bronchiolar carcinomas caused by cobalt metal.

 

3. Genotoxic effects in vitro and in vivo

The genotoxicity of cobalt metal and cobalt compounds has been widely studied. Several publications show induction of chromosomal aberrations, micronuclei or DNA damage in mammalian cells in vitro in the absence of S9. Mixed results were seen in gene mutation studies in bacteria and mammalian cells in vitro, and in chromosomal aberration or micronucleus assays in vivo. To resolve these inconsistencies, new studies were performed with soluble and poorly soluble cobalt compounds according to OECD-recommended protocols. Induction of chromosomal damage was confirmed in vitro, but data suggest this may be due to oxidative stress. No biologically significant mutagenic responses were obtained in bacteria, Tk+/- or Hprt mutation tests. Negative results were also obtained for chromosomal aberrations (in bone marrow and spermatogonia) and micronuclei at maximum tolerated doses in vivo. Poorly soluble cobalt compounds do not appear to be genotoxic. Soluble compounds do induce some DNA and chromosomal damage in vitro, probably due to reactive oxygen. The absence of chromosome damage in robust GLP studies in vivo suggests that effective protective processes are sufficient to prevent oxidative DNA damage in whole mammals. Overall, there is no evidence of genetic toxicity with relevance for humans of cobalt substances and cobalt metal.

 

This conclusion has been confirmed by the OECD CoCAM in the Cobalt SIAP, stating that soluble cobalt salts do not elicit any mutagenic activity either in bacterial or mammalian test systems. However they induce some genotoxic effects in vitro, mainly manifest as DNA strand or chromosome breaks, which are consistent with a reactive oxygen mechanism, as has been proposed by various authors. A weight-of-evidence approach was applied, considering positive as well as negative in vivo clastogenicity studies and the absence of such chromosome damage in humans that are occupationally exposed to inorganic cobalt substances. It was concluded that effective protective processes exist in vivo to prevent genetic toxicity with relevance for humans from the soluble cobalt salts category. (http://webnet.oecd.org/hpv/ui/handler.axd?id=e5e60085-1f3f-4df5-92f6-8f32c26c3082).

 

Following a thorough investigation of the genotoxic properties of a variety of cobalt substances it is concluded that (i) cobalt substances are not directly mutagenic, and (ii) genotoxicity is not the predominant key event in the mode of action (MoA) of cancer. A non-threshold mode of action for the carcinogenicity of cobalt sulfate and cobalt metal could therefore be excluded.

 

4. Mode of action analysis

A postulated mode of action (MoA) for carcinogenesis is a biologically plausible sequence of key events leading to an observed effect, supported by robust experimental observations and mechanistic data (Sonich-Mullin et al., 2001; Boobis et al., 2006). It describes key cytological and biochemical events – i.e., those that are both measurable and necessary to the observed effect – in a logical framework. MoA contrasts with mechanism of action, which generally involves a much greater understanding of the molecular basis to establish causality for an effect.

Originally, the IPCS developed a framework for assessment of the MoA for carcinogenesis in experimental animals (Sonich-Mullin et al., 2001). More recently, this IPCS framework has been updated and extended to consider the human relevance (Boobis et al., 2006; Meek, 2008).

In the following, the MoA HRF is applied to analyse the formation of lung tumours (alveolar/bronchiolar carcinomas) in rats and mice caused by inhalation exposure to cobalt with the objective to evaluate their relevance to humans and to contribute to a quantitative assessment of human cancer risk.

 

4.1 Postulated mode of action

Chronic inhalation exposure of rats and mice to cobalt metal and cobalt sulfate resulted in inflammation, hyperplasia, and formation of tumours in the lung. The available toxicological data – long-term inhalation studies in animals, generation of reactive oxygen species (ROS) and subsequent DNA damage – give support to the hypothesis that the cancer mode of action for cobalt and cobalt sulfate-induced lung tumours involves inflammation, alveolar proteinosis, hyperplasia of the alveolar and bronchiolar epithelia, alveolar/bronchiolar adenoma, and alveolar/bronchiolar carcinoma. It has been hypothesised that cobalt metal and cobalt sulfate induce alveolar/bronchiolar adenoma and carcinoma through cobalt-mediated generation of ROS, which subsequently might cause oxidative damage to cells. The postulated MoA is mainly based on observations of consistent concentration-response relationships for the key events inflammation, hyperplasia, and formation of carcinoma.

 

4.2 Key events, and associated critical parameters

The inflammation and non-neoplastic alterations in the lower regions of the lung observed in the toxicity and carcinogenicity studies after repeated/prolonged inhalation exposure give an indication that cobalt metal and cobalt sulfate-induced cytotoxicity in target cells contributes to the development of lung tumours in rats and mice. Non-neoplastic lesions (alveolar proteinosis, chronic active inflammation) and pre-neoplastic lesions (hyperplasia in the alveolar and bronchiolar epithelia) were found in the lungs of cobalt-exposed male and female rats. The spectrum of these non- and pre-neoplastic lesions invariably occurred together and presented as a complex mixture of changes, with the difficulty to separate the individual components at times. Subsequently, the most decisive alterations in lung tissue of rats regarded as key events will be detailed based on results of histopathological examinations.

 

Alveolar epithelial hyperplasia is a multifocal and sometimes focally extensive, discrete, randomly distributed but frequently sub-pleural lesion characterised by proliferation of flat to cuboidal to low columnar epithelial cells lining the alveolar septa; however, the underlying alveolar architecture was generally maintained in cobalt studies. Alveolar proteinosis included accumulations of brightly eosinophilic, homogeneous, proteinaceous material, frequently containing acicular cholesterol crystals or cleft-like spaces. These lesions were invariably accompanied by chronic active inflammation consisting predominantly of macrophages and lymphocytes mixed with a lesser number of neutrophils within the alveolar spaces and septa. Bronchiolar epithelial hyperplasia was characterised by proliferation and disorganised crowding of ciliated, cuboidal columnar to pleomorphic epithelia cells lining terminal bronchioles with an extension into adjacent alveolar septa.

 

Alveolar/bronchiolar adenoma were discrete, expansive, densely cellular masses composed of relatively well-differentiated, uniform, cuboidal to columnar cells supported by a fine fibrovascular stroma and arranged in solid nests or papillary fronds that projected into alveolar space. Alveolar/bronchiolar carcinoma were larger, irregular, poorly circumscribed, un-encapsulated, expansive, locally invasive masses that effaced the lung parenchyma. They were composed of poorly differentiated, moderately to markedly pleomorphic cuboidal, columnar, or polygonal cells with pleomorphic nuclei. The cells were arranged in single to multiple layers, forming irregular papillary or acinar structures and/or solid sheets supported by fibrovascular stroma. Large numbers of inflammatory cell infiltrates, mostly macrophages, accumulated frequently around the alveolar/bronchiolar neoplasms.

 

Considering that benign and malignant tumours finally occurred in the lung, alveolar proteinosis, chronic inflammation, hyperplasia of the alveolar epithelium and hyperplasia of the bronchiolar epithelium could be interpreted to represent site-specific, sequential steps in the cascade of tumorigenic events induced by cobalt metal and cobalt sulfate. This sequential occurrence of key events is in line with the assumed MoA in which cobalt metal induced alveolar/bronchiolar adenoma and carcinoma through generation of ROS, which in turn causes DNA damage (NTP, 2013; NTP, 1998).

 

A number of in vitro studies have shown that cobalt catalyses the generation of different reactive oxygen species. Out of them, hydroxyl radicals and singlet oxygen cause mainly oxidative DNA damage. Hydroxyl radicals were able to generate DNA single-strand breaks, whereas DNA single-base damage was caused by the specific reaction of singlet oxygen with guanine producing 8-hydroxy-7,8-dihydroguanine (8-oxo-guanine) thus leading to G→T transversion mutations (Klaunig et al., 2010).

 

Uncertainties remain as to the exact mechanisms of the alterations in the alveolar and bronchiolar epithelia and the disturbances of the control of regenerating cell proliferation leading to carcinogenesis. Enhanced cell proliferation could trigger the accumulation of cells with DNA damage and thereby may ultimately lead to tumour formation.

 

4.3 Concordance of dose-response relationships

Cobalt: Considerably increased incidences of alveolar/bronchiolar carcinoma were noted in male and female rats at the lowest exposure concentrations of 1.25 mg/m³ in the cobalt metal powder inhalation study (Behl, 2013), at 38.2% and 21.3%, respectively. A NOAEC for pulmonary tumour formation was not established in the 2-year bioassays in rats and mice. Incidences for alveolar/bronchiolar carcinomas were 76.8% and 80.6% for male rats and 42.0% and 69.2% for female rats at higher exposures of 2.5 mg/m³ and 5 mg/m³, respectively.

With respect to the dose-response of hyperplastic effects assumed to be precursors of tumour development, no NOAEC was noted. In lieu thereof, nearly maximal incidences in the range between 88% and 100% for bronchiolar epithelial hyperplasia and alveolar epithelial hyperplasia were found in rats of each sex at the lowest exposure concentrations of 1.25 mg/m³. At both higher exposure levels, the nearly maximal incidences remained elevated for both types of hyperplasia. With respect to inflammation it is to be noted that hyperplastic lesions were invariably accompanied by chronic inflammation of moderate severity in all cobalt-exposed rats of each sex, while its incidence in controls amounted to about 40%.

 

Cobalt sulfate: In rats, concentration-related changes of the incidences of neoplasms of the lung, adrenal medulla, nose, and larynx were observed. The incidences of alveolar/bronchiolar neoplasms in males were 2.3%, 17.7%, 13.4%, and 33.9% for 0, 0.3, 1.0, and 3.0 mg/m³, respectively, and in females 0%, 11.2%, 50.6%, and 46.1% (including two squamous cell carcinoma). Significantly increased rates of tumours (alveolar/bronchiolar adenoma or carcinoma) were seen in the lung of females in the medium and high group and in male rats at 3.0 mg/m³ as compared with those in the chamber controls. Markedly increased non-neoplastic lesions in the lungs of all cobalt sulfate-exposed rats comprised alveolar epithelial metaplasia, granulomatous inflammation, interstitial fibrosis, and proteinosis. Alveolar epithelial hyperplasia was observed in all males and in females exposed to 3.0 mg/m³. The incidences of squamous metaplasia and of atypical alveolar epithelial hyperplasia were increased in 1.0 mg/m³ and 3.0 mg/m³ females, respectively. In mice, significantly higher incidences of alveolar/bronchiolar neoplasms were found as well in all high dose groups. The non-neoplastic lesions in the lung were less severe than in rats. The incidences of cytoplasmic vacuolisation of the bronchi (all exposed groups), diffuse and focal histiocytic cell infiltration (3.0 mg/m³ males and 3.0 mg/m³ females, respectively) were significantly increased. Also, the pattern of non-neoplastic lesions observed in the cobalt sulfate studies (observed even at lower concentrations of elementary cobalt) is very consistent with that of the cobalt metal studies.

 

In summary, the neoplastic changes co-occurred with the non-neoplastic lesions. A plateau-like concentration-response relationship with cobalt metal for hyperplastic changes considered as precursor lesion and a dose-response relationship for the development of alveolar/bronchiolar carcinomas were identified. A maximum incidence of hyperplastic lesions of nearly 100% became evident at the LOAEC for tumour formation of 1.25 mg/m³ with an incidence of 38.2% and 21.3% in male and female rats, respectively.

Thus, the hypothesis that inflammation and hyperplasia of the alveolar and bronchiolar epithelia are key events in the induction of alveolar/bronchiolar carcinomas is supported by the observation that the incidence of the preceding changes is greater than for the latter (carcinomas) at a similar dose. The dose dependency of the increase in the incidence of hyperplasia in the alveolar and bronchiolar epithelia is in concordance with the ultimate incidence of alveolar/bronchiolar carcinomas.

 

4.4 Temporal association

Temporal concordance refers to the observation of key events in sequential order as described in the hypothesised MoA.

A temporal relationship of cytotoxicity-related tumour growth can be assumed for the tumours of the lung (alveolar/bronchiolar carcinomas), because early non-neoplastic lesions (alveolar proteinosis), chronic active inflammation, and hyperplastic findings were seen in rat studies with 90-day duration at the same concentrations of cobalt metal (NTP 2013) and cobalt sulfate (NTP 1998). Minimal to mild bronchiolar epithelial hyperplasia occurred in all male and female rats exposed to 1.25 mg/m³ cobalt metal or greater. The severity of the latter hyperplasia generally increased with increasing exposure concentrations. Alveolar epithelium hyperplasia occurred in 6/20, 7/20, 7/20 rats exposed to cobalt sulfate at 3, 10 and 30mg/m³ respectively.

Thus, the finding of occurrence of bronchiolar epithelial hyperplasia in a single 3-month study before appearance of tumours (adenomas and carcinomas) after two-year exposure can be regarded as strong support of the postulated MoA. Two-year carcinogenicity assays with satellite groups for interim evaluation and recovery studies are however not available.

 

4.5 Strength, consistency, and specificity of association of key events and tumour response

Stop/recovery studies are considered important tests to show the association of tumour response with key events. In case of cobalt metal or cobalt sulfate, however, stop/recovery studies showing absence or reduction of subsequent events or tumours when a key event is blocked or diminished are not available.

There is a good correlation between key events and regional tumour incidences and tumour sites. Chronic inflammation, alveolar proteinosis, and hyperplasia of the alveolar and bronchiolar epithelia were seen in that region of the lower respiratory tract where adenoma and carcinoma have been observed.

Consistency was redefined to reflect support of the pattern of effects across species, strains, organs, and test systems for the hypothesised MoA (Meek et al., 2014).

The observation of statistically significant increases in alveolar/bronchiolar carcinomas in both sexes of rats and mice in two-year carcinogenicity studies on analogous cobalt substances (cobalt metal and cobalt(II) sulfate (NTP, 1998)) and in different strains of rats (cobalt metal: F344/NTac rats; cobalt sulfate: F344/N rats) is consistent with the hypothesised MoA.

 

 

4.6 Biological plausibility and coherence

The sequence of events (inflammation, cytotoxicity, hyperplasia, benign and malignant tumours) at the alveolar and bronchiolar epithelia is consistent with general knowledge on the tumour pathogenesis of substances for which genotoxicity could be excluded as mode of action.

The same key events that are considered key events in the pulmonary carcinogenicity of cobalt metal have been demonstrated in rats and mice. Histopathological effects in the lower respiratory tract (chronic inflammation, alveolar proteinosis, and hyperplasia of the bronchiolar epithelium) were consistent in sub-chronic and chronic animal studies.

The observation of statistically significant increases in alveolar/bronchiolar carcinomas in both sexes of rats and mice in two-year carcinogenicity studies on analogous cobalt substances (cobalt metal and cobalt(II) sulfate (NTP, 1998); cf. below) and in different strains of rats (cobalt metal: F344/NTac rats; cobalt sulfate: F344/N rats) is also consistent with the hypothesised MoA.

 

F344/N rats and B6C3F1 mice were exposed by inhalation to a dried aqueous aerosol of cobalt sulfate hexahydrate at chamber concentrations of 0, 0.3, 1.0, and 3.0 mg/m³ (equivalent to cobalt concentrations of 0, 0.067, 0.224, and 0.673 mg Co/m³) for 6 hours/day, 5 days/week, over a period of 105 weeks.

In rats, concentration-related changes of the incidences of neoplasms of the lung, adrenal medulla, nose, and larynx were observed. The incidences of alveolar/bronchiolar neoplasms in males were 2.3%, 17.7%, 13.4%, and 33.9% for 0, 0.3, 1.0, and 3.0 mg/m³, respectively, and in females 0%, 11.2%, 50.6%, and 46.1% (including two squamous cell carcinoma). Significantly increased rates of tumours (alveolar/bronchiolar adenoma or carcinoma) were seen in the lung of females in the medium and high group and in male rats at 3.0 mg/m³ as compared with those in the chamber controls. Markedly increased non-neoplastic lesions in the lungs of all cobalt sulfate-exposed rats comprised alveolar epithelial metaplasia, granulomatous inflammation, interstitial fibrosis, and proteinosis. Alveolar epithelial hyperplasia was observed in all males and in females exposed to 3.0 mg/m³. The incidences of squamous metaplasia and of atypical alveolar epithelial hyperplasia were increased in 1.0 mg/m³ and 3.0 mg/m³ females, respectively.

In mice, significantly higher incidences of alveolar/bronchiolar neoplasms were found as well in all high dose groups. The non-neoplastic lesions in the lung were less severe than in rats. The incidences of cytoplasmic vacuolisation of the bronchi (all exposed groups), diffuse and focal histiocytic cell infiltration (3.0 mg/m³ males and 3.0 mg/m³ females, respectively) were significantly increased. Also, the pattern of non-neoplastic lesions observed in the cobalt sulfate studies (observed even at lower concentrations of elementary cobalt) is very consistent with that of the cobalt metal studies.

 

A comparison of the results of the long-term studies with cobalt sulfate heptahydrate and cobalt metal reveals that both cobalt substances cause the formation of alveolar/bronchiolar carcinoma in the lungs of both rats and mice. Taking into account the molecular stoichiometry of cobalt sulfate hexahydrate to cobalt metal the highest exposure concentration of 3.0 mg/m³ of cobalt sulfate hexahydrate which leads to tumour formation is equivalent to a concentration of elementary cobalt of 0.67 mg/m³ being half the lowest exposure concentration (1.25 mg/m³) delivered to the animals in the cobalt metal studies and shown to be tumorigenic. Thus, this tumour data would almost quantitatively be in line with the concentration-response relationship for alveolar/bronchiolar carcinomas caused by cobalt metal.

 

5. Read-across approach for inhalation carcinogenicity

With the possible exception of the endpoint “oxidative stress”, it is assumed that each pre-carcinogenic event (e.g., hypoxia, inflammation, modulation of cytokines or oncogenes) is related to the amount of bioavailable Co ion in a relevant compartment of the lung. This indicates that Co compounds which release Co ion in vivo may cause pre-carcinogenic effects and ultimately cancer.

It is probable that the release of Co ion is not the only determinant of inhalation toxicity. Particle- and local effects are likely to also play a role. Further, exposure of the relevant lung compartments to Co compounds is determined by the particle size distribution and aerodynamic behaviour of each compound.

 

For this reason, the Co compounds are investigated in a tiered approach. Several tiers of testing have been defined, which range from the lower (less weighted in terms of relevance) tiers of in vitro testing up to mid-level tiers of in vivo acute and repeated-dose inhalation testing and conclude with the highest tier, a two-year rodent carcinogenicity study. The lower tiers are aimed at making distinctions between the various cobalt compounds based on common toxicological drivers (e.g. hypoxia, cytotoxicity), whereas the higher tier testing is aimed at “proving the concept” of the existence of different groups of compounds regarding chronic inhalation toxicity endpoints (e.g. chronic pulmonary inflammation).

 

Tier 1 – Bioaccessibility in artificial lung fluids (i.e. interstitial, alveolar and lysosomal)

Tier 2 – In vitro biomarkers (hypoxia and cytotoxicity)

Tier 3 – In vivo ‘persistent’ inflammation and/or upper respiratory tract meta- and hyperplasia

Tier 4 – 28-day RDT inhalation testing

Tier 5 – 90-day RDT inhalation testing

Tier 6 – Carcinogenicity study

 

At the current status of testing, it is concluded that:

A – By weight of evidence, the in vivo finding of “persistent inflammation” is more relevant than the in vitro markers. Data for this endpoint (tier 3) will be used to group for RDT inhalation, irrespective of the lower tier findings (bioaccessibility and in vitro markers).

B – Where possible, substances will be tested in tier 2 and 3. This will allow the formation of a database, where a correlation between tier 1, 2 and 3 findings can be investigated. 

C – Not all Co substances can be tested in tier 3, due to the physical form of some of the substances, where an inhalation atmosphere cannot be generated. In cases where tier 3 testing cannot be performed, a draft testing strategy outlines subsequent steps meant to be taken (see inhalation read-across document appendix 3.4). The physical form of the substance during its complete life cycle will be examined, and if an inhalation atmosphere can be generated at any point during manufacture, handling or use, a chronic classification can be derived by grouping as follows:

– According to lower tier data, if a correlation can be observed in “B”

– A weight of evidence judgement will be made according to all tiers of data if no correlation can be observed in “B”.

 

Grouping is based on the knowledge that there is a group of Co substances which are toxic by acute and RDT inhalation, and on the assumption that there is a (group of) Co substances(s) that is non-toxic by acute and RDT inhalation.

 

6. Outlook

Further information will be included in all cobalt substance dossiers of the Cobalt REACH Consortium upon availability. Following the chronic inhalation exposure of cobalt sulfate in rats and mice, the 95% lower confidence limit of the BMD for a treatment-related increase in response of 10% was calculated (BMDL10), in which the numbers of alveolar/bronchiolar adenoma or carcinoma in the lung of rats and mice were selected as benchmark response. The lowest BMDL10 of 0.414mg/m³ will be used for the hazard assessment for carcinogenicity via inhalation, local effects.

A read-across is applied in which the DNEL derived from the existing chronic inhalation study with cobalt sulfate will be recalculated as DNEL for all other cobalt substances being grouped in the reactive cobalt substances group, taking into account the molecular weight.

Details on the substance specific derivation of DNEL for carcinogenicity, local are given in the report which can be found as attachment to the endpoint summary in section 7 of the IUCLID.

 

References

Boobis AR, Cohen SM, Dellarco V, McGregor D, Meek ME, Vickers C, Willcocks D, and Farland W (2006) IPCS framework for analyzing the relevance of a cancer mode of action for humans. Crit Rev Toxicol 36, 781-792

 

Bucher JR, Hailey JR, Roycroft JR, Haseman JK, Sills RC, Grumbein SL, Mellick PW, Chou BJ (1999) Inhalation toxicity and carcinogenicity studies of cobalt sulfate. Toxicol Sci 49, 56-67

 

Cappellini, D (2009). Report to Duke University Medical Center on the testing of cobalt compounds for solubility in serum and artificial body fluids. Testing laboratory: Kirby Memorial Health Center, 71 N Franklin Street, Wilkes-Barre, PA 18701-1391, USA. Owner company: Cobalt Development Institute, 167 High Street Guildford, Surrey GU1 3AJ, United Kingdom. Study number: CDI Study 48.

 

Meek ME (2008) Recent developments in frameworks to consider human relevance of hypothesized modes of action for tumours in animals. Environ Mol Mutagen 49, 110-116

 

NTP (1998) Toxicology and carcinogenesis studies of cobalt sulfate heptahydrate (CAS No. 10026-24-1) in F344/N rats and B6C3F1 mice (Inhalation studies). Technical Report Series No. 471, NIH Publication No. 98-3961. U.S. Department of Health and Human Services, Public Health Service, National Institutes of Health, Research Triangle Park, NC.

 

NTP (2014) Toxicology studies of cobalt metal (CAS No. 7440-48-4) in F344/N rats and B6C3F1/N mice and toxicology and carcinogenesis studies of cobalt metal in F344/NTac rats and B6C3F1/N mice (Inhalation studies). Technical Report Series No. 581. NIH Publication No. 14-5923. U.S. Department of Health and Human Services, Public Health Service, National Institutes of Health, Research Triangle Park, NC.

 

Sonich-Mullin C, Fielder R, Wiltse J, Baetke K, Dempsey J, Fenner-Crisp P, Grant D, Hartley M, Knaap A, Kroese D, Mangelsdorf I, Meek E, Rice JM, and Younes M (2001) IPCS Conceptual framework for evaluating a mode of action for chemical carcinogenesis. Regul Toxicol Pharmacol 34, 146-152

 

Stopford, W.; Turner, J.; Cappellini, D.; Brock, T. (2003). Bioaccessibility testing of cobalt compounds. J. Environ. Monit. 5, 675-680.

 

 


Disregarded carcinogenicity references

Section

Brief description / test system / guideline / test substance

GLP study

Key findings / Results

Comment and reliability rating where applicable

Reference

8.09

Carcinogenicity

Test system:

Test substance:

Guideline:

 

GLP: N

 

RL = not rated

follow up specific investigation of the 2y NTP inhalation study with CoSO4

Ozaki, K.; et al. (2002): Association of adrenal pheochromocytoma and lung pathology in inhalation studies with particulate compounds in the male F344 rat- The national toxicology program experience.Toxicology Pathology 30, 263-270

8.09

Carcinogenicity

Test system: Review on effects of carcinogenic metals on gene expression (including Co)

Test substance:

Guideline:

 

GLP: N

Brief overview on putative mechanisms of induction of growth factor and other genes by cobalt.

RL = 4

Review of literature on effects of metals on gene expression

Beyersmann, D. (2002): Effects of carcinogenic metals on gene expression.Toxicol. Letters 127, 63-68

8.09

Carcinogenicity

Test system:

Test substance: cobalt metal powder

Guideline: not applicable

 

30 male rats (aged 2-3 months) were injected in the thigh muscle of the right leg with 28 mg Co powder suspended in 0.4 ml fowl serum; 15 animals served as controls. Animals were killed at intervals of 1 to 28 days after injection. The portion of the thigh muscle surrounding the injection site was excised and stained for histological examination.

GLP: N

Not evaluated further because of irrelevant route of administration.

RL = 3

Reasonably well-documented publication, but not relevant because of non-physiological application route via intramuscular injection of cobalt powder.

Heath, J.C. (1960): The histogenesis of malignant tumours induced by cobalt in the rat.Br. J. Cancer 14, 478-482

8.09

Carcinogenicity

Test system: Long-term effects of cobalt in the rat as a model for experimental hyperlipaemia

Test substance: CoCl2

Guideline: not applicable

 

Male Wistar albino rats aged about 4 weeks were used.

Daily subcutaneous injections of cobalt chloride solution (8 mg/ml in saline) were given into the central abdominal wall of 20 rats at a dose of 40 mg/kg bw for 5 days. After 9 days without application, the injections were continued for 5 furthers days. Controls were injected with 0.5 ml saline in a similar manner. The animals were kept for 12 months (or 8 months in the 2nd experiment) and killed by i.p. injection of phenobarbitone sodium. A post-mortem examination was made on the surviving animals and the number, size, position and appearance of any tumours present were recorded.

GLP: N

In the 1st experiment, 9 of 20 treated rats and 1 of the control rat died within the first year. 8 of the surviving animals developed fibrosarcomas. In 4 of these the tumour was remote from the injection sites. There was no evidence of involvement of the mammary gland or other tissues. No metastases were found in any of the animals. 

In the 2nd experiment, 6 of the 16 surviving animals developed fibrosarcomas.

RL = 3

The paper is considered of limited relevance for an assessment of carcinogenic effects of cobalt due to the use of the physiologically non-relevant route of subcutaneous administration.

Shabaan, A.A. (1977): Fibrosarcomas induced by cobalt chloride in rats.Lab. Animals 11, 43-46

8.09

Carcinogenicity

Test system: Repeated intraperitoneal injections in mice

Test substance: cobalt acetate

Guideline: not stated

 

cobalt acetate 97-99% [ J.T. Backer, USA]

 

Repeated intraperitoneal injections to Strain A mice for evaluation of the development pulmonary tumours.

GLP: N

No significant increase in the average no of lung tumours.

RL = 3

Route (intraperitoneal) of exposure not relevant for risk assessment.

tumour incidence of control: 7/19

Stoner, G.D.; et al. (1976): Test for carcinogenicit of metallic compounds by the pulmonary tumor response in strain A mice.Cancer Res. 36, 1744-1747

8.09

Carcinogenicity

Test system: Evaluation of the carcinogenic effects after s.c., i.p. injections and intratracheal instillation in Sprague Dawley rats

Test substance: cobalt (II) oxide

Guideline: not stated

 

Intratracheal instillation: 50 males and 50 females per group; control and 1 x 10 mg/kg bw and 1x 2 mg/kg bw every 14 days total dose 78 or 390 mg/kg); 2 years treatment.

Additional orientating studies:

s.c. injection: 10 males per group; control and 5 x 2 and 1 x 10 mg/kg/week (total dose 1000 mg/kg); 2 years treatment.

Intraperitoneal injection: 10 male and 10 females per group; control and 3 x 200 mg/kg at intervals of 2 month (total dose 600 mg/kg).

Intratracheal instillation: 20 males and 20 females per group; control and 10 mg/kg once a week (20 mg/kg every 14 days from week 8, 27 treatments in total 470 mg/kg) plus benzo[a]pyrene 20 mg/kg once a week (every 14 days from week 8, 10 treatments in total 200 mg/kg).

Animals were observed over their lifetime.

GLP: N

Intratracheal instillation of CoO resulted in 2 benign pulmonary tumours in rats of the low dose group and 2 benign and 4 malignant pulmonary tumours in the high dose group.

Subcutaneous injections resulted in local malignant tumours in 5 or 4 of 10 rats.

Following i.p. injections 14 of 20 rats developed malignant intraperitoneal tumours.

Intratracheal instillation of CoO and benzo[a]pyrene caused 9 malignant tumours in 20 rats. Benzo[a] pyrene alone caused only 1 malignant tumour.

The latter findings are considered by the German MAK Commission as relevant for Cat3 carc. classification.

RL = 3

The paper is considered of limited relevance for an assessment of carcinogenic effects of cobalt due to the use of the physiologically non-relevant route of intratracheal instillation. The results of the orientating studies are presented only very briefly in a table.

Steinhoff, D; Mohr, U. (1991): On the question of a carcinogenic action of cobalt-containing compounds.Exp. Pathol. 41, 169-174

8.09

Carcinogenicity

Test system: Single intramuscular injection, rats

Test substance: cobalt metal powder

Guideline: not stated

 

Single intramuscular injection into the right dome of the diaphragm or through the intracostal space of rats for evaluation of the development of intrathoracic tumours.

GLP: N

Not evaluated further because of irrelevant route of administration.

RL = 3

Route (intramuscular) of exposure not relevant for risk assessment.

Heath, J.C.; Daniel, M.R. (1962): The production of malignant tumours by cobalt in the rat: intrathoracic tumours.Br. J. Cancer 16, 473-478

8.09

Carcinogenicity

Test system: Single intramuscular injection, rats

Test substance: cobalt metal powder

Guideline: not stated

 

Single intramuscular injection into the right or left leg of rats for evaluation of the development of local tumours.

GLP: N

Not evaluated further because of irrelevant route of administration.

RL = 3

Route (intramuscular) of exposure not relevant for risk assessment.

Heath, J.C. (1956): The production of malignant tumours by cobalt in the rat.Br. J. Cancer 10, 668-673

8.09

Carcinogenicity

Test system: Single intrarenal injection, rat

Test substance: cobalt metal (and cobalt sulphide)

Guideline: not stated

 

Single intrarenal injection into the renal pole of rats for evaluation of the development of renal carcinomas and erythrocytosis.

GLP: N

Not evaluated further because of irrelevant route of administration

RL = 3

Route (intrarenal) of exposure not relevant for risk assessment

Jasmin, G.; Riopelle, J.L. (1976): Renal carcinomas and erythrocytosis in rats following intrarenal injection of nickel subsulfide.Lab. Invest. 35, 71-78

8.09

Carcinogenicity

Test system:

Test substance: cobalt naphthenate activator containing 1% metallic cobalt

Guideline: not stated

 

Chronic intramuscular injection into the right hind limb of rats for evaluation of the development of local tumours.

GLP: N

Not evaluated further because of irrelevant route of administration.

RL = 3

Route (intramuscular) of exposure not relevant for risk assessment.

Nowak, H.F. (1966): Neoplasia in mouse skeletal muscles under the influence of polyester resin activator.Arch. Immunol. Ther. Exp. 14, 774-778

8.09

Carcinogenicity

Test system: single intratracheal application

Test substance: cobalt dust (not specified)

Guideline: not stated

 

Evaluation of the effects of single intratracheal applied dust suspended in saline.

Hamsters (no details given)

GLP: N

Intratracheal instillation resulted in a remarkable hyperplasia of the bronchial epithelium which was rapidly induced (no further details).

RL = 4

No details presented.

Saffiotto, U.; et al. (1963): Intratracheal injection of particulate carcinogens into hamster lungs. Proc. Am.Assoc. Cancer Res. 4, 59

8.09

Carcinogenicity

Test system: occ. Exposure, mortality study

Test substance:

Guideline:

 

A mortality study in 267 male workers of a treatment plant who had been exposed for at least 12 years between 1933 and 1960.

 (production of nickel, cobalt and selenium compounds and salts)

GLP: N

Only 13 death from lung cancer were observed. Therefore, it is very unlikely that a significantly high mortality rate form lung cancer could have been observed in any subgroup.

Conclusion: it is possible that a hazard existed, but there is insufficient evidence to indicate whether it was related to exposure.

RL = human data

Results are presented appropriately in tables and text.

The size of the study was limited because of the too small exposed population.

Relevance questionable, because workers were exposed to a wide range of Co, Ni and Se compounds and salts (including black cobalt oxide and cobalt metal).

Cuckle, H.; et al. (1980): Mortality study of men working with soluble nickel coumpounds. In: Brown, S.S. & Sunderman, F.W. (Eds.): Nickel Toxicology, London, Academic Press, 11-14

8.09

Carcinogenicity

Test system: genesis of neoplasm after injections in rabbits.

Test substance: cobalt naphthene

Guideline: not stated

 

Evaluation of the properties in the genesis of neoplasm following intramuscular, intravascular, intrapleural and intrahepatic injections in rabbits.

GLP: N

Not evaluated further because of irrelevant route of administration.

RL = 4

The routes of exposure and the study design are not relevant for risk assessment.

Nowak, H.F. (1961): The pathogenesis of neoplasia in the rabbit under the influence of polyester resin additions.Akad. Medycz. Jul. Marchl. Bialymstoku 7, 323-348

8.09

Carcinogenicity

Test system: Single human case report, oral

Test substance: cobalt phthalocyanine

Guideline:

 

Case report on a worker (mineral oil refinery) who was exposed accidentally with cobalt phthalocyanine by the oral route.

GLP: N

A fast growing giant cell tumour was observed in the oral cavity about 5 month after exposure. The presence of cobalt salts was determined in the tumour.

RL = human data

Case described appropriately in the text.

Substance not relevant for hazard assessment of inorganic cobalt compounds.

Schulz, G. (1978): Giant-cell tumors after exposure to a dust containing cobaltous phthalocyanine (Merx-catalyst).Staub-Reinh. Luft 38, 480-481

8.09

Carcinogenicity

Test system: Determination of the effects of intratracheal instillation of 5 particulates, including cobalt oxide, on diethylnitrosamine (DEN) carcinogenesis in hamsters.

Guideline: not stated

 

25 male and 25 female hamsters per group; s.c. pre-treatment for 12 weeks with DEN (0.5 mg) or saline (o.25 mg); followed by 30 weeks of intratracheal treatment with cobalt oxide; 0.2 ml of a formulation containing 2g particulate in 100 ml (mixture of gelatine in saline combined with an anaesthetic); control groups were run concurrently

GLP: N

Pre-treatment with DEN (without particulates) resulted in respiratory tract tumours in about 62-73% of control animals, while saline pre-treatment resulted in 5/44 tumour bearing animals (11%). In the groups treated with cobalt oxide, the number of tumour bearing animals (77%) in the DEN pre-treatment group was increased in comparison to the group pre-treated with saline (4%).

The distribution, frequency and morphological appearance of the respiratory tract tumours was modified by treatment with particulates.

RL = 3

The paper is considered of limited relevance for an assessment of carcinogenic effects of cobalt due to the use of the physiologically non-relevant route of intratracheal instillation. Substance not relevant for hazard assessment of inorganic cobalt compounds.

Farrell, R.L.; Davis, G.W. (1974): The effects of particulates on respiratory cacinogenesis by diethylnitrosamine. In: Karbe, E. & Park, J.F.: Experimental Lung Cancer, New York, Springer, 219-233

8.09

Carcinogenicity

Test system:

Test substance: cobalt-chromium alloy

Guideline: not applicable

 

GLP: N

Upon review, not considered to provide information of relevance for this endpoint.

RL = NOT RATED

Not assessed for lack of relevance.

Heath, J.C: et al. (1971): Carcinogenic properties of weak particles from protheses made in cobalt chromium alloy.Lancet 297, 564-566

8.09

Carcinogenicity

Test system:

Test substance: cobalt-chromium alloy

Guideline: not applicable

 

GLP: N

Upon review, not considered to provide information of relevance for this endpoint.

RL = NOT RATED

Not assessed for lack of relevance.

Swansson, S.A.V.; et al. (1973): Laboratory tests on total joint replacement protheses. J. Bone Joint Surg. 55B, 759-773

8.09

Carcinogenicity

Test system:

Test substance: dust, cobalt oxide

Guideline: not applicable

 

GLP: N

Upon review, not considered to provide information of relevance for this endpoint.

RL = NOT RATED

Not assessed for lack of relevance

(intramuscular administration).

Gilman, J.P.W.; Ruckerbauer, G.M. (1961): Metal carcinogenesis.I. Observation on the carcinogenicity of a refinery dust, cobalt oxide, and colloidal thorium oxide.Cancer Res. 22, 152-157

8.09

Carcinogenicity

Test system:

Test substance: cobalt sulphide, cobalt oxide

Guideline: not applicable

 

GLP: N

Upon review, not considered to provide information of relevance for this endpoint.

RL = NOT RATED

Not assessed for lack of relevance.

Gilman, J.P.W. (1961): Metal carcinogenesis. II. A study on the carcinogenic activity of cobalt, copper, iron, and nickel compounds.Cancer Res. 22, 158-165

8.09

Carcinogenicity

Test system: Evaluation of an exposure technique

Test substance:

Guideline:

 

Evaluation of an exposure technique

GLP: N

Upon review, no specific info on cobalt or cobalt compounds given

RL = NOT RATED

Not assessed for lack of relevance.

Driscoll, K.E.; et al. (2000): Intratracheal instillation as an exposure technique for the evaluation of respiratory tract toxicity: uses and limitations.Toxicol. Sci. 55, 24-35

8.09

Carcinogenicity

Test system:

Test substance: cobalt-chromium alloy

Guideline: not applicable

 

GLP: N

Upon review, not considered to provide information of relevance for this endpoint.

RL = NOT RATED

Not assessed for lack of relevance.

Gaechter, A.; et al. (1977): Metal carcinogenesis. A study of the carcinogenesis activity of solid metal alloys in rats.J. Bone Joint Surg. 59A, 622-624

8.09

Carcinogenicity

Test system:

Test substance:

Guideline:

 

Review genotoxicity

GLP: N

Upon review, not considered to provide information of relevance for this endpoint.

RL = NOT RATED

Not assessed for lack of relevance.

Hartwig, A. (1998): Carcinogenicity of metal cmpounds: possible role of DNA repair inhibition.Toxicol. Letters 102-103, 235-239

8.09

Carcinogenicity

Test system:

Test substance: Co(II)diethylnitrosamine, Co(II)3-Metylcholanthrene

Guideline: not applicable

 

GLP: N

Upon review, not considered to provide information of relevance for this endpoint.

RL = NOT RATED

Not assessed for lack of relevance.

Beyersmann, D. (1994): Interactions in metal carcinogenicity.Toxicol. Letters 72, 333-338

8.09

Carcinogenicity

Test system:

Test substance:

Guideline:

 

Preliminary letter report on a combination of cytotoxicity and animal experimental data on cobalt carcinogenicity

GLP: N

Not evaluated further because of irrelevant route of administration.

RL = 3

Route (intramuscular) of exposure not relevant for risk assessment.

Heath, J.C. (1954): Cobalt as a carcinogen.Nature 173, 822-823

8.09

Carcinogenicity

Test system: occ. Exposure, mortality study

Test substance:

Guideline:

 

A mortality study in 267 male workers of a treatment plant who had been exposed for at least 12 years between 1933 and 1960.

 (production of nickel, cobalt and selenium compounds and salts)

GLP: N

Only 13 death from lung cancer were observed. Therefore, it is very unlikely that a significantly high mortality rate form lung cancer could have been observed in any subgroup.

Conclusion: it is possible that a hazard existed, but there is insufficient evidence to indicate whether it was related to exposure.

RL = human data

The size of the study was limited because of the too small exposed population.

Relevance questionable, because workers were exposed to a wide range of Co, Ni and Se compounds and salts (including black cobalt oxide and cobalt metal).

Cuckle, H.; et al. (1980): Mortality study of men working with soluble nickel coumpounds. In: Brown, S.S. & Sunderman, F.W. (Eds.): Nickel Toxicology, London, Academic Press, 11-14

8.09

Carcinogenicity

Test system: Single human case report, oral

Test substance: cobalt phthalocyanine

Guideline:

 

Case report on a worker (mineral oil refinery) who was exposed accidentally with cobalt phthalocyanine by the oral route.

GLP: N

A fast growing giant cell tumour was observed in the oral cavity about 5 month after exposure. The presence of cobalt salts was determined in the tumour.

RL = human data

Case described appropriately in the text.

Substance not relevant for hazard assessment of inorganic cobalt compounds.

Schulz, G. (1978): Giant-cell tumors after exposure to a dust containing cobaltous phthalocyanine (Merx-catalyst).Staub-Reinh. Luft 38, 480-481

 

Justification for classification or non-classification