Toxicology 333 (2015) 195–205

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Comparative toxicity and carcinogenicity of soluble and insoluble cobalt compounds Mamta Behl a, * , Matthew D. Stout a , Ronald A. Herbert a , Jeffrey A. Dill b , Gregory L. Baker b , Barry K. Hayden b , Joseph H. Roycroft a , John R. Bucher a , Michelle J. Hooth a a b

Division of the National Toxicology Program, National Institute of Environmental Health Sciences, Research Triangle Park, NC, USA Battelle Memorial Institute, Columbus, OH, USA

A R T I C L E I N F O

A B S T R A C T

Article history: Received 12 February 2015 Received in revised form 1 April 2015 Accepted 14 April 2015 Available online 17 April 2015

Occupational exposure to cobalt is of widespread concern due to its use in a variety of industrial processes and the occurrence of occupational disease. Due to the lack of toxicity and carcinogenicity data following exposure to cobalt, and questions regarding bioavailability following exposure to different forms of cobalt, the NTP conducted two chronic inhalation exposure studies in rats and mice, one on soluble cobalt sulfate heptahydrate, and a more recent study on insoluble cobalt metal. Herein, we compare and contrast the toxicity profiles following whole-body inhalation exposures to these two forms of cobalt. In general, both forms were genotoxic in the Salmonella T98 strain in the absence of effects on micronuclei. The major sites of toxicity and carcinogenicity in both chronic inhalation studies were the respiratory tract in rats and mice, and the adrenal gland in rats. In addition, there were distinct sites of toxicity and carcinogenicity noted following exposure to cobalt metal. In rats, carcinogenicity was observed in the blood, and pancreas, and toxicity was observed in the testes of rats and mice. Taken together, these findings suggest that both forms of cobalt, soluble and insoluble, appear to be multi-site rodent carcinogens following inhalation exposure. Published by Elsevier Ireland Ltd.

Keywords: Cobalt Cancer Toxicity Rats Mice

1. Introduction Exposure to cobalt is of widespread concern primarily due to its use in a variety of industrial processes. Occupational exposure occurs to a variety of forms ranging from soluble salts to insoluble cobalt metal with the major routes of exposure being dermal and inhalation. Exposure to soluble cobalt salts may occur in electroplating and electrochemical industries, in ceramics as a coloring agent, or when used as a drying agent in inks, paints, varnishes, and linoleum. In contrast, exposure to insoluble cobalt metal occurs in the production of cemented tungsten–cobalt (hard metal) and when used as an alloying element in superalloys, magnetic and hard-facing alloys, cobalt-containing high-strength steels, electrodeposited alloys, and other alloys with special properties (IARC, 1991; Donaldson and Beyersmann, 2005). One of the major

* Corresponding author at: Division of National Toxicology Program, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709, USA. Tel.: +1 9195413340. E-mail address: [email protected] (M. Behl). http://dx.doi.org/10.1016/j.tox.2015.04.008 0300-483X/ Published by Elsevier Ireland Ltd.

concerns associated with cobalt–tungsten carbide exposure is the occurrence of hard-metal disease in workers. The International Agency for Research on Cancer (IARC) has classified cobalt as a Group 2B possible human carcinogen (IARC, 2006). In addition, the report on carcinogens (RoC) listed two cobalt compounds, cobalt sulfate and cobalt–tungsten carbide as “reasonably anticipated to be a human carcinogens” (NTP, 2011). Although there is a suggested correlation between cobalt exposure and lung diseases in humans, including lung cancer, asthma and alveolitis (Mur et al., 1987; Van Goethem et al., 1997), epidemiological studies on the carcinogenicity of cobalt are limited and inconclusive, partially due to co-exposure to established carcinogens, such as nickel and chromium (IARC, 2006). Animal studies previously conducted by the NTP demonstrated an increased incidence of alveolar/bronchiolar neoplasms and pheochromocytomas in rodents following inhalation exposure to soluble cobalt sulfate heptahydrate (NTP, 1991, 1998; Bucher et al., 1999). Additionally, in mice, there were decreases in testis and epididymal weights, spermatid and epididymal spermatozoa counts, and sperm motility coupled with histopathologic findings in the testis and epididymis in the subchronic studies (NTP, 1991)

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with no evidence of testis toxicity/carcinogenicity in rats or mice in the chronic studies (NTP, 1998). Bioavailability based on the form of exposure (soluble vs insoluble) has been identified as an important factor influencing the toxicity and health effects of cobalt metal, alloys, and salts (Lison and Lauwerys, 1994; Lison, 1996; Lasfargues et al., 1992; Menzel et al., 1989; Rhoads and Sanders, 1985; ATSDR, 2001; Bailey et al., 1989; Elinder and Friberg, 1986). While the NTP study on cobalt sulfate sheds light on the toxicity and carcinogenicity to a soluble cobalt salt, there were still concerns and a lack of data on the potential toxicity of insoluble cobalt metal. There are no animal studies in the literature that have assessed the chronic inhalation effects of insoluble cobalt metal. To address these concerns, the NTP recently conducted prechronic and chronic inhalation studies on cobalt metal in rats and mice (NTP, 2014). It was hypothesized that the toxicity and carcinogenicity of insoluble cobalt would be limited to pulmonary effects potentially due to accumulation and lung overload at the higher exposure concentrations. Furthermore, less systemic toxicity compared to that seen with the previous studies of soluble cobalt sulfate was expected due to the lower solubility and bioavailability of cobalt metal. Herein, we present the major findings from the chronic cobalt metal study, and compare and contrast them with our previously conducted studies on cobalt sulfate. 2. Materials and method Additional details can be found elsewhere (NTP, 1998, 2014); a brief description of methods for both studies is provided below. 2.1. Test agents and inhalation exposures Cobalt sulfate heptahydrate (purity
99%) was obtained from Curtin Matheson Scientific (Kansas City, MO). Cobalt metal (purity > 98%; cubic and hexagonal phases present) was produced by OMG Kokkola Chemicals Oy (Kokkola, Finland) and was provided to the NTP by the Cobalt Development Institute via PEL Technologies. Periodic re-analysis revealed no degradation of either test agent during the course of the studies. Cobalt sulfate was generated and delivered from an aqueous solution by a system consisting of a compressed-air-driven nebulizer, an aerosol charge neutralizer, and an aerosol distribution system. Exposure concentrations of cobalt sulfate heptahydrate in this study are expressed as mg cobalt sulfate/m3; however, it was determined that each mole of aerosol in the exposure chambers contained an approximate 1:1:6 molar ratio of cobalt: sulfate:water, indicating that exposures were primarily to cobalt sulfate hexahydrate. Cobalt metal was generated and delivered as a dry powder by a system consisting of a linear feed device, a Trost jet mill for particle size reduction, and a cyclone separator to further reduce particle size. Chamber aerosol concentrations of cobalt sulfate or cobalt metal were monitored by computer-controlled real time aerosol monitors (RAM) that were calibrated daily by construction of a response curve of background-corrected RAM voltages and cobalt sulfate or cobalt metal concentrations determined by ICP/AES analysis of cobalt on filter samples. 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. Based on experimental data, a T90 of 12 min was selected for the 2-year studies of cobalt sulfate or cobalt metal. Aerosol size distribution was determined monthly using a Mercer-style seven stage cascade impactor. Samples were analyzed by ICP/AES. The relative mass on each impactor stage

was analyzed by probit analysis. The mass median aerodynamic diameter for each aerosol was within the NTP-specified range of 1–3 mm with a geometric standard deviation of less than 3. Studies of cobalt degradation and monitoring for impurities were conducted throughout the 2-year studies with ICP/AES (cobalt sulfate) or XRD, PIXE and ICP/AES (cobalt metal) and demonstrated acceptable purity and stability. Chamber concentration uniformity was maintained throughout the 2-year studies for both test agents. 2.2. Animals and animal maintenance Male and female F344/N rats and B6C3F1/N mice were obtained from Simonsen Laboratories (Gilroy, CA) for the cobalt sulfate studies. Male and female F344/NTac rats and B6C3F1/N mice were obtained from Taconic Farms, Inc. (Germantown, NY) for the cobalt metal studies. Rats and mice were 3–4 weeks upon receipt, were quarantined for 12–14 days and were 5–6 weeks old at the start of the studies. Prior to the start of the studies, rats and mice not selected for study were evaluated for parasites and gross observation of disease and serology samples were collected for viral screening. During the studies, animal health was monitored according to the NTP sentinel animal program. Rats and mice were housed individually in cage units within the exposure chambers. NIH-07 (cobalt sulfate) or NTP-2000 (cobalt metal) wafer feed and tap water were available ad libitum, except feed was withheld during animal exposures. Chambers and racks were rotated weekly. Chamber environmental conditions were set to maintain 72  1 degrees F (temperature) 55  15% (humidity), 12 h (light/ dark cycle) and 15  2 (air changes per hour). Animal care and use were in accordance with the Public Health Service Policy on Humane Care and Use of Animals. All animal studies were conducted in an animal facility accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care International. Studies were approved by the study laboratory Animal Care and Use Committee and conducted in accordance with all relevant NIH and NTP animal care and use policies and applicable federal, state, and local regulations and guidelines. 2.3. Study design Inhalation studies were conducted at Battelle Toxicology Northwest (Richland, WA). Groups of 50 male and 50 female rats and mice were exposed to cobalt sulfate by whole body inhalation of aqueous aerosols containing 0, 0.3, 1.0, or 3.0 mg/m3 cobalt sulfate (corresponding to 0.114, 0.32 or 1.14 mg/m3 cobalt) or to cobalt metal by inhalation of particulate aerosol at concentrations of 0, 1.25, 2.5, or 5 mg/m3, for 6 h plus T90 per day, 5 days per week, for up to 105 weeks. All animals were observed twice daily. Body weights were recorded at the start of the studies, weekly for the first 13 weeks, monthly until week 92 (cobalt sulfate) or 93 (cobalt metal), every two weeks thereafter, and at terminal sacrifice. Clinical findings were recorded at similar durations as body weight except for recording once approximately every four weeks for the first 13weeks of the studies. A complete necropsy and microscopic examination was performed on all rats and 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 5–6 mm, 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. In the cobalt metal study, an extended evaluation was performed on the

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kidney of male rats for renal proliferative lesions. The kidneys were step sectioned at 1 mm intervals, and three to eight additional sections were prepared from each kidney for microscopic evaluation. Pathology specimens and diagnoses from the study laboratory were subjected to NTP pathology quality assessment and Pathology Working Group review; details of these review procedures have been described, in part, by Maronpot and Boorman (1982) and Boorman et al. (1985). For subsequent analyses of the pathology data, the decision of whether to evaluate the diagnosed lesions for each tissue type separately or combined was generally based on the guidelines of McConnell et al. (1986). The studies were conducted in compliance with Food and Drug Administration Good Laboratory Practice Regulations (21CFR, Part 58). In addition, the laboratory reports, pathology tables and technical reports were subjected to a retrospective quality assessment review by an independent organization (NTP, 1998, 2014). 2.4. Statistical analysis and historical control data

197

Table 2 Survival and final mean body weight following 2-years of exposure to cobalt sulfate and cobalt metal in rats and mice. Concentration Survival Mean body weights (mg Co/m3) over second year of study (% control) Cobalt sulfate Rats

Mice

Males 0 0.114 0.38 1.14

17/50 15/50 21/50 15/50

100 102 97

22/50 31/50 24/50 20/50

103 100 98

Females 0 0.114 0.38 1.14

28/50 25/49 26/50 30/50

101 101 101

34/50 37/50 32/50 28/50

107 104 106

Survival Mean body weights over second year of study (% control)

Cobalt metal

The probability of survival was estimated by the product-limit procedure of Kaplan and Meier (1958) and is presented in the form of graphs. Animals found dead of other than natural causes were censored; 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. The primary statistical method used for neoplastic and non-neoplastic lesion incidences for cobalt sulfate was logistic regression analysis, where lesion prevalence was modeled as a logistic function of chemical exposure and time, since most of the lesions were considered to be incidental to the cause of death (Dinse and Haseman, 1986). For cobalt metal, the poly-k test (Bailer and Portier, 1988; Portier and Bailer, 1989; Piegorsch and Bailer, 1997) was used. This test is a survival-adjusted quantal-response procedure that modifies the Cochran–Armitage linear trend test to take survival differences into account. For cobalt sulfate, the historical control data at the time of study reporting was used. The cobalt metal study was the only inhalation study in the F344/NTac database; therefore, the historical data for that study reflected control incidences for all routes and vehicles. Table 1 compares the concentrations of cobalt present in cobalt sulfate to that in the cobalt metal study. The conversions are in mg Co/m3 using the weight percent of cobalt in cobalt sulfate. Note: the exposure concentrations of cobalt in both the studies overlapped at only one concentration, the lowest concentration of cobalt metal (1.25 mg Co/m3) and the highest concentration of cobalt sulfate (3.0 mg/m3; 1.14 mg Co/m3). Hence, direct comparisons of neoplastic and non-neoplastic lesion incidences can only be made at this similar Co concentration. 3. Results 3.1. Survival, body weights and clinical observations There were no exposure-related effects in survival, body weights or clinical observations in rats or mice exposed to cobalt Table 1 Comparison of exposure concentrations of cobalt following exposure to cobalt sulfate or cobalt metal. Cobalt sulfate – mg/m3 (mg Co/m3)

Cobalt metal – mg Co/m3

0.3 (0.114) 1.0 (0.38) 3.0 (1.14)

1.25 2.5 5.0

Rats

Mice

Males 0 1.25 2.5 5

17/50 20/50 16/50 16/50

98 93 77

39/50 31/50 29/50* 25/50**

99 98 89

Females 0 1.25 2.5 5

35/50 26/50 24/50* 25/50

101 88 79

36/50 36/50 27/50 26/50

97 94 75*

Survival analysis by Kaplan-Meier determinations. * p < 0.05. ** p < 0.01.

sulfate (Table 2). Exposure to cobalt metal resulted in decreased survival in 2.5 mg/m3 female rats and 2.5 and 5.0 mg/m3 male mice, decreased body weights of at least 10% compared to controls in the 2.5 and 5 mg/m3 female rats and at 5 mg/m3 in male rats, and male and female mice (Table 2). Clinical findings included abnormal breathing and thinness in some of the animals exposed to cobalt metal (NTP, 2014). 3.2. Common sites of toxicity and carcinogenicity seen in soluble and insoluble cobalt studies 3.2.1. Respiratory system In both chronic studies, the lung was a common site of toxicity and carcinogenicity (Tables 3 and 4). Alveolar/bronchiolar adenomas and carcinomas occurred in male and female rats and mice in both studies with generally higher incidences following cobalt metal exposure (Table 5, Figs. 1A–H and 2A–H ). Whereas the response in the cobalt sulfate study was equally driven by adenomas or carcinomas at the highest concentration (1.14 mg/m3 Co), the effects in the cobalt metal study were predominantly driven by carcinomas at a comparable cobalt concentration (1.25 mg/m3 Co). In addition, exposure to cobalt metal induced cystic keratinizing epitheliomas (CKE) in the lungs of rats with a higher incidence in males than females (Fig. 3). CKE are rare chemically induced lung neoplasms. In general, the alveolar/bronchiolar neoplasms induced in rats and mice were morphologically similar. Alveolar/bronchiolar adenomas were composed of a relatively uniform population of neoplastic epithelial cells that formed glandular and/or irregular papillary

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Table 3 Comparison of neoplastic sites between cobalt sulfate and cobalt metal (overall findings). Cobalt sulfate

Cobalt metal

Lung Male rats Female rats Male mice Female mice

+ + + +

+ + + +

Adrenal gland Male rats Female rats Male mice Female mice

 + – –

+ + – –

Pancreas Male rats Female rats Male mice Female mice

– – – –

+  – –

Kidney Male rats Female rats Male mice Female mice

– – – –

 – – –

MCL Male rats Female rats Male mice Female mice

– – – –

– + – –

N = 49–50 animals/sex/group; +, evidence of exposure-related carcinogenicity; –, no evidence of carcinogenicity, , carcinogenicity may have been related to chemical exposure.

structures, and occasionally as solid sheets (Figs. 1A–D and 2A–D). Alveolar/bronchiolar carcinomas had similar cellular patterns but were generally larger masses that effaced the alveolar architecture and had one or more of the following Table 4 Comparison of non-neoplastic sites between cobalt sulfate and cobalt metal dust (overall findings). Cobalt sulfate

Cobalt metal

Nose Male rats Female rats Male mice Female mice

+ + + +

+ + + +

Trachea Male rats Female rats Male mice Female mice

– – – –

– – + +

Larynx Male rats Female rats Male mice Female mice

+ + + +

– – + +

Lung Male rats Female rats Male mice Female mice

+ + + +

+ + + +

Testis Male rats Male mice

– –

+ +

N = 49–50 animals/sex/group; +, evidence of exposure-related non-neoplastic lesions; –, no evidence of non-neoplastic lesions.

histologic features: locally invasive, heterogeneous growth pattern, and marked cellular pleomorphism and/or atypia (Figs. 1E–H and 2E–H). Cystic keratinizing epitheliomas were irregular nodular masses composed of abundant amounts of concentrically arranged keratin surrounded by an irregular wall of well-differentiated squamous epithelium (Fig. 3A and B). Exposure to cobalt metal resulted in significantly increased incidences of hyperplasia in the alveolar and bronchiolar epithelia of male and female rats and mice, whereas exposure to cobalt sulfate resulted in hyperplasia in the alveolar but not bronchiolar epithelium of rats only. In both the alveolar and bronchiolar epithelia, hyperplasia is considered a preneoplastic lesion that can potentially progress to neoplasia. At all exposure concentrations, cobalt sulfate but not cobalt metal caused significantly increased incidences of squamous metaplasia in rats. Both compounds induced non-neoplastic lesions in the nose of male and female rats and mice. The larynx, however, showed a slightly different pattern following exposure whereby cobalt sulfate induced lesions in male and female rats and mice while cobalt metal affected only mice but not rats. 3.2.2. Adrenal gland The adrenal medulla was another common site of toxicity and carcinogenicity in rats following exposure to both compounds (Table 6). At comparable concentrations (highest concentration group in the cobalt sulfate study and lowest concentration group in cobalt metal study) there were significant increases in the incidences of benign, malignant and benign, complex or malignant pheochromocytomas (combined) to approximately a similar extent. Whereas exposure to cobalt sulfate was not accompanied by increased incidences of adrenal medullary hyperplasia in rats, increased incidences of adrenal medullary hyperplasia occurred in female rats exposed to 1.25 or 2.5 mg/m3 cobalt metal. 3.3. Distinct sites of toxicity and carcinogenicity between cobalt sulfate and cobalt metal In addition to the above sites, which were common target organs in both the studies, there were distinct sites of carcinogenicity that were specific to cobalt metal exposure. 3.3.1. Pancreas The incidences of pancreatic islet adenoma or carcinoma (combined) in male rats (2/50, 2/50, 10/48, 9/49) were significantly increased in the 2.5 and 5 mg/m3 groups, compared to concurrent and historical controls. In female rats, there was an increase in the incidence of pancreatic islet carcinoma in the 5 mg/m3 group compared to concurrent chamber controls and historical controls (Table 3). Due to the absence of statistically significant trends or pairwise comparisons, we are uncertain about the linkage between pancreatic tumors and cobalt exposure in female rats (NTP, 2014). 3.3.2. Mononuclear cell leukemia (MCL) In female rats, the incidences of mononuclear cell leukemia were significantly increased at all exposure concentrations (16/50, 29/50, 28/50, 27/50); however, no clear exposure–concentration relationship was seen (Table 3). Although mononuclear cell leukemia is a common spontaneous neoplasm in F344 rats, the increased incidences in females in the cobalt metal study were considered related to cobalt exposure (NTP, 2014). 3.3.3. Testes The testis was a prominent site of toxicity in the cobalt metal study in both rats and mice. In rats, the incidence of infarct in the testes was significantly increased in male rats exposed to 5 mg/m3

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199

Table 5 Comparison of incidences of neoplastic lesions of the lung between cobalt sulfate and cobalt metal in rats and mice in the 2-year inhalation studies. Cobalt sulfate (mg Co/m3)

Cobalt metal (mg Co/m3)

Lungs

0

0.114

Male rats A/B adenoma A/B carcinoma A/B adenoma or carcinoma CKE

1 0 1# –

4 0 4 –

Female rats A/B adenoma A/B carcinoma A/B adenoma or carcinoma CKE

0## 0# 0## –

Male mice A/B adenoma A/B carcinoma A/B adenoma or carcinoma Female mice A/B adenoma A/B carcinoma A/B adenoma or carcinoma

0.38

1.14

0

1.25

2.5

5

6 1 7#

–

–

2* 0** 2** 0

10* 16** 25** 1

10* 34** 39** 0

14** 36** 44** 1

1 2 3 –

10## 6# 15## –

9## 6# 15## –

2** 0** 2** 0

7 9** ** 15 4

9* 17** 20** 1

13** 30** 38** 2

9# 4## 11#

12 5 14

13 7 19

18# 11# 28##

7 11** ** 16

11 38** 41**

15* 42** 43**

3 46** ** 47

3# 1## 4##

6 1 7

9 4 13#

10# 9## 18##

3* 5** 8**

9 25** 30**

8 38** 41**

10* 43** 45**

1 3 4

Number of animals per exposure–concentration = 49–50/sex/group. A/B; alveolar/bronchiolar; CKE; cystic keratinizing epithelioma. * Significantly different (P  0.05) by the poly-3 test from the chamber control or a significant trend if assigned to a control group. ** Significantly different (P  0.01) from the chamber control group by the poly-3 test. # Significantly different (P  0.05) from the chamber control group by the logistic regression test. ## Significantly different (P  0.01) from the chamber control by the logistic regression test.

(1/50,0/50,2/50,12/50) while in mice, there was an exposurerelated increase in the incidence of germinal epithelium degeneration (9/50, 14/49, 8/50, 21/50). 3.3.4. Kidney In the kidney, we noted increased incidences of renal tubule adenoma or carcinoma in male rats compared to the chamber control group; however, the increases were not statistically significant. There was also no supporting evidence of preneoplastic renal lesions. Since these lesions are relatively rare, it is possible that they may have been related to cobalt exposure (NTP, 2014). 4. Discussion The NTP conducted two 2-year inhalation studies in rats and mice, one on soluble cobalt sulfate heptahydrate (NTP, 1998) and a more recent one on insoluble cobalt metal (NTP, 2014). Despite differences in methodology including different animal strains (F344/N vs F344/NTac rats), generation systems, diets (NIH-07 vs NTP-2000), and slightly different statistical methods for analyzing lesion incidence (logistic regression vs poly-k), both forms of cobalt were toxic to the upper and lower respiratory tracts, and carcinogenic in the lung and adrenal medulla. There was one concentration with comparable cobalt exposure between the studies; the highest exposure concentration of cobalt sulfate heptahydrate (3.0 mg/m3) resulted in a cobalt concentration (1.14 mg/m3) that is similar to the lowest exposure concentration of cobalt metal (1.25 mg/m3). Contrary to the expectation that cobalt metal would show less systemic toxicity compared with the soluble salt, cobalt metal was more toxic and carcinogenic at a similar cobalt concentration as evident by the incidence and spectrum of lung neoplasms and the extent of systemic lesions. While exposure to cobalt sulfate resulted in a combination of adenomas and carcinomas in the lung, carcinomas predominated following exposure to cobalt metal (Fig. 4). Morphologically, the alveolar/bronchiolar adenomas and carcinomas were generally typical of those that occur

spontaneously. The development of alveolar/bronchiolar neoplasms is considered to progress through a continuum of proliferative lung lesions that includes preneoplastic epithelial hyperplasia, benign alveolar/bronchiolar adenoma and malignant alveolar/bronchiolar carcinomas. In general, the range of proliferative lung lesions observed in both studies are similar to those that that are observed in NTP inhalation studies with particulates (NTP, 1998, 2000, 2001). However, in both rats and mice, at equivalent exposure concentrations given the greater incidence of lung neoplasms particularly carcinomas, cobalt metal appears to be more effective in potentiating the progression of lung proliferative lesions than cobalt sulfate heptahydrate. Exposure to cobalt metal also resulted in the occurrence of cystic keratinizing epitheliomas in rats but not mice. In experimental rodent species, CKE are rare chemically-induced lung neoplasms, but have been induced in the lungs of rats following intratracheal instillation or chronic inhalation exposure to various particulate compounds (Martin et al., 1977; Lee et al., 1985; Muhle et al., 1989; Hobbs et al., 1993; Pott et al., 1993; Mauderly et al., 1994; Boorman et al., 1996; Rittinghausen and Kaspareit, 1998; Dixon et al., 2008). CKE are benign neoplasms that grow by expansion rather than invasion. They are considered part of a continuum of lesions that can progress from squamous metaplasia to keratin cysts to CKE to malignant squamous cell carcinoma, and have been previously noted in several NTP studies (NTP, 2006a,b,c, d,e, 2010a,b). Additionally, both forms of cobalt showed a spectrum of nonneoplastic inflammatory, fibrotic and proliferative lesions in the upper respiratory tract following subchronic and chronic exposure (NTP, 1998, 2014). Amongst other lesions, the larynx appeared to be the most sensitive to cobalt sulfate in the subchronic studies in both rats and mice while it appeared to be a target organ for toxicity only in mice following exposure to cobalt metal. In the adrenal gland, the incidence of pheochromocytomas was similar between the two studies at comparable cobalt concentrations. Although a common spontaneous neoplasm in male

200

[(Fig._1)TD$IG]

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Fig. 1. Photomicrographs of alveolar/bronchiolar neoplasms in the lungs of rats exposed to 1.14 mg Co/m3 cobalt sulfate heptahydrate or 1.25 mg Co/m3 cobalt metal for 2 years. The morphology of the alveolar/bronchiolar adenomas (Fig. 1A–D) and carcinomas (Fig. 1E–H) induced by cobalt sulfate heptahydrate and cobalt metal were generally similar. (A) Alveolar/bronchiolar adenoma from a male rat exposed to 1.14 mg Co/m3 cobalt sulfate heptahydrate, scale bar = 500 mm. (B) Higher magnification of 1A. The neoplasm is composed of a closely packed, morphologically uniform population of neoplastic cells, scale bar = 100 mm. (C) Alveolar/bronchiolar adenoma from a male rat exposed to 1.25 mg Co/m3 cobalt metal, scale bar = 1 mm. (D) Higher magnification of 1C. The neoplasm is composed of a morphologically uniform population of neoplastic cells that form papillary structures, scale bar = 80 mm. (E) Alveolar/bronchiolar carcinoma from a male rat exposed to 1.14 mg Co/m3 cobalt sulfate heptahydrate, scale bar = 4 mm. (F) Higher magnification of 1E. The neoplasm is composed of a pleomorphic population of neoplastic cells arranged in irregular cords, papillary and poorly defined glandular structures separated by collagen, scale bar = 100 mm. (G) Alveolar/bronchiolar carcinoma from a male rat exposed to 1.25 mg Co/m3 cobalt metal, scale bar = 2 mm. (H) Higher magnification of 1G. The neoplasm is composed of a pleomorphic population of neoplastic cells arranged in cords, papillary structure and well-defined glandular structures irregularly separated by vascularized collagen, scale bar = 100 mm.

F344/N rats, pheochromocytomas have a lower spontaneous occurrence in females. In the cobalt sulfate study, the incidence of pheochromocytoma in the highest exposure–concentration group of females (1.14 mg Co/m3) was considered related to exposure while the marginally increased incidence in male rats was considered an uncertain finding due to the occurrence in the mid exposure concentration group only and the absence of any supporting non-neoplastic findings. In contrast, there was a significant increase in the incidence of pheochromocytomas in both male and female rats following exposure to cobalt metal; however, these were seen at higher exposure concentrations of

cobalt metal compared to the cobalt sulfate study. The mechanism (s) responsible for the development of pheochromocytomas in rat inhalation studies is not understood. An analysis of the results of several NTP inhalation exposure studies in rats, found an apparent association between the occurrence of variably extensive chronic pulmonary non-neoplastic lesions similar to those that occurred in the cobalt studies, and the development of pheochromocytomas (Ozaki et al., 2002). Additional studies are needed to investigate whether the adrenal response is related to the presence of these extensive space occupying pulmonary lesions rather than due to a chemical specific response.

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[(Fig._2)TD$IG]

201

Fig. 2. Photomicrographs of alveolar/bronchiolar neoplasms in the lungs of mice exposed to 1.14 mg Co/m3 cobalt sulfate heptahydrate or 1.25 mg Co/m3 cobalt metal for 2 years. The morphology of the alveolar/bronchiolar adenomas (Fig. 2A–D) and carcinomas (Fig. 2E–H) induced by cobalt sulfate heptahydrate and cobalt metal were generally similar and were morphologically similar to those induced in the 2-year study in rats. (A) Alveolar/bronchiolar adenoma from a female mouse exposed to 1.14 mg Co/ m3 cobalt sulfate heptahydrate, scale bar = 100 mm. (B) Higher magnification of 2A. Scale bar = 100 mm. (C) Alveolar/bronchiolar adenoma from a female mouse exposed to 1.25 mg, Co/m3 cobalt metal, scale bar = 400 mm. (D) Higher magnification of 2C. Scale bar = 100 mm. (E) Alveolar/bronchiolar carcinoma from a female mouse exposed to 1.14 mg Co/m3 cobalt sulfate heptahydrate, scale bar = 2 mm. (F) Higher magnification of 2E. Scale bar = 100 mm. (G) Alveolar/bronchiolar carcinoma from a female mouse exposed to 1.25 mg Co/m3 cobalt metal, scale bar = 2 mm. (H) Higher magnification of 2G. Scale bar = 100 mm.

[(Fig._3)TD$IG]

Fig. 3. Photomicrograph of a cystic keratinizing epithelioma in the lung of a male rat exposed to cobalt metal (1.25 mg Co/m3) for 2 years. (A) The neoplasm is composed of abundant amounts of concentrically arranged keratin surrounded by an irregular wall of well-differentiated squamous epithelium, scale = 1 mm. (B) Higher magnification of 3A, scale bar = 200 mm.

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Table 6 Comparison of incidences of Neoplastic lesions of the adrenal medulla between cobalt sulfate and cobalt metal in rats in the 2-year inhalation studies. Cobalt sulfate (mg Co/m3) Adrenal gland Male rats Benign, complex or malignant Female rats Benign, complex or malignant

Cobalt metal (mg Co/m3) 0

0.114

0.38

1.14

0

1.25

2.5

5

15

19

25#

20

17**

23

38**

41**

2##

1

4

10#

6**

13

23**

40**

Number of animals per exposure–concentration = 49–50/sex/group. ** Significantly different (P  0.01) from the chamber control group by the poly-3 test or a significant trend if assigned to a control group. # Significantly different (P  0.05) from the chamber control by the logistic regression test or a significant trend if assigned to a control group. ## Significantly different from the chamber control by the logistic regression test or a significant trend if assigned to a control group.

[(Fig._4)TD$IG]

Fig. 4. Comparison of incidence of lung tumors between cobalt sulfate and cobalt metal.

The respiratory tract and adrenal gland lesions were common to both the cobalt sulfate and cobalt metal studies. In addition, there were increased incidences of neoplasms in additional extrapulmonary tissues in the cobalt metal study, including increases in pancreatic neoplasms in male rats and leukemias in female rats; these lesions were observed at cobalt concentrations higher than those resulting from exposure to cobalt sulfate. Non-neoplastic testicular lesions were noted in the chronic study of cobalt metal but not in the chronic study of cobalt sulfate. However, in both the cobalt sulfate and cobalt metal studies, testicular lesions were noted in male mice in the prechronic studies thereby suggesting that cobalt is perhaps a reproductive toxicant in male mice irrespective of the exposure form.

With cobalt sulfate, the high urinary concentrations following 13-week inhalation exposure indicated systemic distribution, and excretion at levels comparable to human exposure (NTP, 1991; Bucher et al., 1999). In the more recent studies on cobalt metal, tissues burden evaluations were included as part of the subchronic and chronic studies (NTP, 2014). Following prechronic or chronic exposure to cobalt metal, cobalt concentrations and burdens were generally increased in all tissues examined, indicating systemic exposure of rats and mice to cobalt. In particular, liver cobalt concentrations and burdens, which were evaluated in the 2-week and 3-month studies, approached or even exceeded lung cobalt concentrations, particularly at higher exposure concentrations. In addition, cobalt metal was rapidly cleared from lung and blood. In

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the lung, the majority (>95% in rats and >82% in mice) of the deposited cobalt was cleared rapidly, with half lives of
1–5 days across prechronic and chronic studies; these short half lives are characteristic of a soluble particle. One possible explanation for the apparent solubility of cobalt is that while cobalt metal has been reported to be insoluble in aqueous environments, it can be solubilized by strong mineral acids (Takahashi and Koshi, 1981; Kyono et al., 1992). Studies in the literature suggest that cobalt taken up by macrophages is solubilized by lysosomes, despite toxicity to the cell (Rae, 1975; Stopford et al., 2003). Collectively, the toxicity/carcinogenicity and internal dosimetry data indicate that cobalt produces systemic exposure and toxicity and/or carcinogenicity, following exposure to cobalt sulfate or cobalt metal. There were some differences in the genotoxic effects between cobalt sulfate and cobalt metal in the bacterial mutagenicity assay. Cobalt sulfate was positive in Salmonella typhimurium strain TA100 in the presence and absence of S9. In contrast, cobalt metal showed borderline effects in the TA100 strain in the absence of S9 metabolizing enzymes; however, the mutagenic activity was eliminated in the presence of S9. A plausible mechanism for cobalt genotoxicity in human and rodent cells may occur by the induction of chromosome aberrations, single and double strand breaks, sister chromatid exchanges and micronuclei (ATSDR, 2004; Beyersmann and Hartwig, 2008; Lison et al., 2001). There were no increases noted in the frequencies of micronucleated normochromatic erythrocytes or reticulocytes in peripheral blood of male or female mice exposed to cobalt metal (0.625–10 mg/m3) for 3 months by inhalation. To identify an underlying basis for cobalt-induced carcinogenesis, mutation analysis was conducted from the lung tumors following cobalt exposure in both studies. Kras was evaluated in both sets of studies due to its known role in human lung cancer (Boch et al., 2013; Ellis and Clark, 2000; Roberts and Der, 2007). In both sets of studies, there was a distinct pattern in G ! T transversion of Kras mutations in lung tumors from cobalt-exposed animals (Hong et al., 2015). No such mutations were noted in spontaneous lung tumors or normal lung tissues examined. The pattern in the Kras mutation suggests that oxidative damage to DNA may be a contributing factor in the carcinogenesis since G to T transversions are commonly detected DNA base changes associated with active oxygen species during oxidative damage to DNA (Janssen et al., 1993; Shigenaga and Ames, 1991; Tchou et al., 1991). Taken together, our findings point toward the possibility of a common underlying mechanism of cobalt toxicity irrespective of the form of cobalt exposure based on: (i) common sites of carcinogenicity (lung and adrenal gland), (ii) similar mutation spectrum in the Kras gene in lung tumors from cobalt-exposed animals, (iii) toxicity in common extra-pulmonary sites (e.g., testes), and (iv) similar clinical findings (erythrocytosis) (NTP, 1998, 2014). Based on this, one potential mechanism supported by the literature is that solubilization of cobalt ions generate reactive oxygen species, and also stabilization of hypoxia-inducible factor1a (HIF-1a), which in turn results in erythrocytosis (Epstein et al., 2001; Yuan et al., 2003). Continued oxidative stress may further result in DNA damage and inhibition of DNA repair as evidenced by the bacterial mutagenicity assay and by the distinct molecular signature of the lung tumors (Hong et al., 2015) thereby resulting in local and systemic toxicity and carcinogenicity as indicated by the spectrum of neoplastic and non-neoplastic lesions. Recent in vitro studies of low-solubility cobalt oxide particles in human lung cells indicate that intracellular release of Co(II) ions results in cytotoxicity and genotoxicity (Ortega et al., 2014; Smith et al., 2014)

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