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NC E RE QU EST ED : PPRT V d oc ume ntatio n f or Cob a lt

ENCL OSED I

NFO RMATI ON: Attachment 1: PRO V I S I ONAL PEER REVI EW ED TOXI CI TY

V A L U E S F O R CO B A L T ( C A SR N 7440-48-4)

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# Provisional Peer Reviewed Toxicity Values for

Cobalt
(CASRN 7440-48-4)

Superfund Health Risk Technical Support Center

National Center for Envi ronmental Assessment Office of Research and Development U.S. Environmental Protection Agency Cincinnati, OH 45268

### Acronyms and Abbreviations

bw body weight

cc cubic centimeters

CD Caesarean Delivered CERCLA Comprehensive Environmen tal Response, Compensation and Liability Act of 1980 CNS central nervous system

cu.m cubic meter DWEL Drinking Water Equivalent Level

FEL frank-effect level FIFRA Federal Insecticide, Fungicide, and Rodenticide Act

g grams GI gastrointestinal HEC human equivalent concentration Hgb hemoglobin

i.m. intramuscular i.p. intraperitoneal IRIS Integrated Risk Information System

IUR inhalation unit risk

i.v. intravenous kg kilogram

L liter

LEL lowest-effect level

LOAEL lowest-observed-adverse-effect level LOAEL(ADJ) LOAEL adjusted to continuous exposure duration LOAEL(HEC) LOAEL adjusted for dosimetric differences across species to a human

m meter
MCL maximum contaminant level MCLG maximum contaminant level goal MF modifying factor mg milligram mg/kg milligrams per kilogram mg/L milligrams per liter

MRL minimal risk level

MTD maximum tolerated dose

MTL median threshold limit NAAQS National Ambient Air Quality Standards

NOAEL no-observed-adverse-effect level NOAEL(ADJ) NOAEL adjusted to continuous exposure duration NOAEL(HEC) NOAEL adjusted for dosimetric differences across species to a human

NOEL no-observed-effect level OSF oral slope factor p-IUR provisional inhalation unit risk p-OSF provisional oral slope factor p-RfC provisional inhala tion reference concentration

iii p-RfD provisional oral reference dose PBPK physiologically based pharmacokinetic ppb parts per billion ppm parts per million

PPRTV Provisional Peer Re viewed Toxicity Value RBC red blood cell(s) RCRA Resource Conservation and Recovery Act RDDR Regional deposited dose rati o (for the indicated lung region) REL relative exposure level

RfC inhalation refe rence concentration

RfD oral reference dose RGDR Regional gas dose ratio (for the indicated lung region)

s.c. subcutaneous SCE sister chromatid exchange SDWA Safe Drinking Water Act sq.cm. square centimeters

TSCA Toxic Substances Control Act UF uncertainty factor

μ g microgram

μ mol micromoles VOC volatile organic compound8-25-2008

PROVISIONAL PEER REVIEWED TOXICITY VALUES FOR COBALT (CASRN 7440-48-4)

Background

On December 5, 2003, the U.S. Environmental Protection Agency's (EPA's) Office of Superfund Remediation and Technology Innovation (OSRTI) revised its hierarchy of human health toxicity values for Superfund risk assessmen ts, establishing the follow ing three tiers as the new hierarchy:

1. EPA's Integrated Risk Information System (IRIS).

2. Provisional Peer-Reviewed Toxicity Va lues (PPRTV) used in EPA's Superfund Program.

3. Other (peer-reviewed) to xicity values, including:

< Minimal Risk Levels produced by the Agency for Toxic Substances and Disease Registry (ATSDR),

< California Environmental Protecti on Agency (CalEPA) values, and

< EPA Health Effects Assessment Summary Table (HEAST) values.

A PPRTV is defined as a toxicity value de rived for use in the Superfund Program when such a value is not available in EPA's Integrated Risk Information System (IRIS). PPRTVs are developed according to a Standard Operating Procedure (SOP) and are derived after a review of

the relevant scientific literatu re using the same methods, sources of data, and Agency guidance for value derivation generally used by the EPA IRIS Program. All provi sional toxicity values receive internal review by two EPA scientists and external peer review by three independently selected scientific experts. PPRTVs differ from IRIS values in that PPRTVs do not receive the multi-program consensus review provided for IRIS values. This is because IRIS values are generally intended to be used in all EPA program s, while PPRTVs are developed specifically for the Superfund Program.

Because new information becomes available and scientific methods improve over time, PPRTVs are reviewed on a five-year basis and upda ted into the active database. Once an IRIS value for a specific chemical becomes availabl e for Agency review, the analogous PPRTV for

that same chemical is retired. It should also be noted that some PPR TV manuscripts conclude

that a PPRTV cannot be derive d based on inadequate data.

Disclaimers

Users of this document should first check to se e if any IRIS values exist for the chemical of concern before proceeding to use a PPRTV. If no IRIS value is available, staff in the regional Superfund and RCRA program offices are advised to carefully re view the information provided

in this document to ensure that the PPRTVs us ed are appropriate for the types of exposures and circumstances at the Superfund site or RCRA f acility in question. PPRTVs are periodically

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updated; therefore, users should en sure that the values contained in the PPRTV are current at the

time of use.

It is important to remember that a provi sional value alone tells very little about the

adverse effects of a chemical or the quality of evidence on which the value is based. Therefore, users are strongly encouraged to read the enti re PPRTV manuscript and understand the strengths and limitations of the derived provisional values . PPRTVs are developed by the EPA Office of Research and Development’s National Center for Environmental Assessment, Superfund Health Risk Technical Support Center for OSRTI. Ot her EPA programs or external parties who may

choose of their own initi ative to use these PPRTVs are advise d that Superfund resources will not generally be used to respond to challenges of PPR TVs used in a context outside of the Superfund Program.

Questions Regarding PPRTVs

Questions regarding the contents of th e PPRTVs and their ap propriate use (e.g., on chemicals not covered, or whether chemicals have pending IRIS toxicity values) may be directed

to the EPA Office of Research and Devel opment’s National Center for Environmental Assessment, Superfund Health Risk Technica l Support Center (513-569-7300), or OSRTI.

INTRODUCTION

The Integrated Risk Information System (IRIS) does not report a Reference Dose (RfD) for cobalt (U.S. EPA, 2007). The Health Eff ects Assessment Summary Tables (HEAST) (U.S. EPA, 1997a) and Drinking Water Standards and Health Advisories lis t (U.S. EPA, 2004)

likewise do not contain an RfD for cobalt. Th e Chemical Assessments and Related Activities (CARA) lists (U.S. EPA, 1991, 1994a) report a Hea lth Effect Assessment (HEA) for cobalt (U.S. EPA, 1987). The 1987 HEA derived a chronic RfD of 0.005 mg cobalt/kg-day based on a noobserved-adverse-effect level ( NOAEL) of 5 mg cobalt/kg-day for testicular effects in a subchronic rat study (Nation et al., 1983). The Agency for Toxic Substances and Disease Registry (ATSDR) Toxicological Profile for co balt and its compounds reports an oral Minimal Risk Level (MRL) for intermediate exposure of 1x10 -2 mg/kg-day (ATSDR, 2004), based on a

lowest-observed-adverse-effect level (LOAEL) of approximate ly 1 mg cobalt/kg-day for polycythemia in humans (Davis and Fields, 1958). ATSDR (2004) did not derive an oral MRL for chronic exposure. This MRL for interm ediate exposure was based on the polycythemic effect of cobalt exposure (1 mg cobalt/kg-day, Da vis and Fields, 1958) by application of an UF

of 10 for a LOAEL and an UF of 10 for human variability. The Worl d Health Organization (WHO, 2005) has not published an Environmental Health Criteria (EHC) document on cobalt. An International Agency for Research on Cancer (IARC) Monograph on cobalt and its compounds (IARC, 2006) and the National Toxico logy Program (NTP) St atus Reports (NTP, 2005) were searched for relevant information.

IRIS (U.S. EPA, 2007) does not report a Refe rence Concentration (R fC) for cobalt. The HEAST (U.S. EPA, 1997a) likewise does not list an RfC for coba lt. The cobalt HEA (U.S. EPA, 1987) derived a subchronic inhalation RfC of 9x10 -5 mg/m 3 based on a LOAEL of 0.1 mg/m 3 for

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respiratory effects in a 3-month study in swine (Kerfoot et al ., 1975). A chronic inhalation RfC

of 9x10 -6 mg/m 3 was derived from the same study. The ATSDR Toxicological Profile for cobalt and its compounds reports an inhalati on MRL for chronic exposure of 1x10 -4 mg/m 3 (ATSDR, 2004), based on a NOAEL of 0.0053 mg cobalt/m 3 for decreased pulmonary function in humans (Nemery et al., 1992). The American Conferen ce of Governmental Industrial Hygienists (ACGIH, 2004) has set a Threshold Limit Va lue-Time-Weighted Average (TLV-TWA) of 0.02 mg/m 3 for cobalt and inorganic cobalt compounds, expr essed as cobalt, based on respiratory and

cardiovascular effects. The National Institut e for Occupational Safety and Health (NIOSH, 2005) Recommended Exposure Limit (REL) TWA for cobalt is 0.05 mg/m 3 , based on effects in the respiratory system. The Occupational Sa fety and Health Admi nistration (OSHA, 2005) Permissible Exposure Limit (PEL) is 0.1 mg/m 3

.

IRIS (U.S. EPA, 2007) does not report a cancer classification, slope fa ctor or unit risk for cobalt. The HEAST (U.S. EPA, 1997a) and Drin king Water Standards and Health Advisories list (U.S. EPA, 2004) likewise do not report carcinogenici ty assessments for cobalt. The CARA lists (U.S. EPA, 1991, 1994a) do not report a can cer classification or an estimate of the carcinogenic potency of stable cobalt compounds due to a lack of pertinent data. An IARC Monograph on cobalt and its compounds (IARC, 2006) cl assified cobalt sulfate and other soluble cobalt (II) salts as “possibly car cinogenic to humans.” ACGIH ( 2004) has classified cobalt in category A3 – confirmed animal carci nogen with unknown relevance to humans.

Literature searches for studies relevant to th e derivation of provisiona l toxicity values for cobalt were conducted initially through 2000 in TOXLINE (supplemented with BIOSIS and NTIS updates), MEDLINE, TSCATS, RTEC S, CCRIS, DART, EMIC/EMICBACK, HSDB,

GENETOX and CANCERLIT and subsequently from 2000 to August 2005 in MEDLINE, TOXLINE (NTIS subfile), TOXCENTER, TSCA TS, CCRIS, DART/ETIC, GENETOX, HSDB, RTECS and Current Contents. An updated litera ture search was performed in MEDLINE from

2005 to June 2008.

REVIEW OF PERTINENT DATA

Human Studies

Overview

Indicators of adverse health effects in hu mans following oral exposure to cobalt include increased erythrocyte number and hemogloblin (Taylor et al., 1977; Duckham and Lee, 1976; Davis and Fields, 1958), cardiomyopathy (Mor in et al., 1971; Alexander, 1969, 1972) and decreased iodine uptake by the thyroid (Roche and Layrisse, 1956). Cardiomyopathy is an endpoint of concern for cobalt in humans; however, it is highly likely that alcohol consumed in “beer-cobalt cardiomyopathy,” as well as other fact ors, such as smoking, played a role in the

effects that were observed. Cobalt is a sensitizer in humans by any route of exposure. Sensitized individuals may react to inhalation of cobalt by developing asthma; ingestion or dermal contact with cobalt may result in development of dermatit is. Several studies have suggested that cross38-25-2008

sensitization may occur between cobalt and nickel (Shirakawa et al., 1990; Lammintausta et al., 1985; Bencko et al., 1983; Rystedt and Fisher, 1983).

Respiratory effects, including respirator y irritation, wheezing, asthma, pneumonia and fibrosis, have been widely reported in humans exposed to cobalt by inhalation (for review, see Barceloux, 1999; Lison, 1996). Epidemiology studi es show decreased pulmonary function in workers exposed to inhaled cobalt (Nemery et al., 1992; Gennart and Lauwerys, 1990). Results of studies investigating cancer incidence in workers exposed to inhaled cobalt are suggestive of a possible association between exposure to cobalt and respiratory tumors (Tuchsen et al., 1996; Mur et al., 1987; Morgan, 1983).

Oral Exposure

In humans, cobalt stimulates production of red blood cells through increased production of the hormone erythropoietin and has been explor ed for use in the treatm ent of anemia (Smith and Fisher, 1973; Duckham and Lee, 1976). Increases in red bl ood cell counts and blood hemoglobin have been reported in non-anemic volunteers (Davis and Fields, 1958) and in anephric anemic patients (Taylor et al., 1977; Duckham and Lee, 1976).

Reversible polycythemia (increase in blood cell number) was reporte d (see Table 1) in six healthy adult males following treatment with 150 mg cobalt chloride per day for 22 days (Davis and Fields, 1958). Five subjects received 150 mg cobalt chloride/day for the entire exposure period and a sixth subject initially re ceived 120 mg cobalt chloride/day, which was later increased (time not specifi ed) to 150 mg/day. Cobalt chlori de was administered as a 2%

solution diluted in either wa ter or milk. Assuming an average body weight of 70 kg, 150 mg cobalt chloride/day corresponds to approximately 1 mg cobalt/kg-day. Outcomes assessed in this study were red blood cell count, hemogl obin percentage, leukocyt e count, reticulocyte percentage and thrombocyte count. Polycythemia was observed in all six patients within 7 to 22 days of treatment as demonstrated by increase s in red blood cell counts ranging from 0.5 to 1.19

Table 1. Hematopoietic, Thyroid and Developmen tal Effects of Cobalt (Co) via Oral Route

Target Organ Species Effect Dosage (mg Co/kg-day)

Hematopoietic Effects Human

Human

Rat Reversible Effect (Polycythemia) ↑ Hemoglobin and ↑ RBC Hematopoietic effect 1.0

0.16 – 0.32*

0.5 – 32.0 Thyroid Human

Mice ↓ Iodine uptake Histopathological ch anges in thyroid 1.0

48.0

Fetus Rat Developmental toxicity 5.2 – 21.0

Heart Rat ↓ Myocardial function 8.0

*Therapeutic doses for anemic patients

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million (approximately 16-20% increase above pre-treatment levels) and increases in hemoglobin levels ranging from 6 to 11% above pret reatment values. In five of the six subjects, reticulocyte levels were elev ated, reaching at least twice the pre-experiment values. Thrombocyte and total leukocyte counts were not significantly different from pretreatment values. Erythrocyte counts retu rned to pre-treatment levels w ithin 9 to 15 days after cobalt

administration was discontinued. The fact that leucocyte counts remained relatively constant throughout the experiment supports th e concept that this is a true polycythemia. As such, based on the results of this study, 1 mg cobalt/kg-day was identified as a LOAEL for cobalt-induced polycythemia in humans.

Duckham and Lee (1976) treated 12 anephric pa tients on dialysis with 25 to 50 mg cobalt chloride daily for approximately 12 weeks. A ssuming an average body weight of 70 kg, doses of 25 and 50 mg cobalt chloride/day are equivalent to 0.16 and 0.32 mg cobalt/kg-day, respectively. During the exposure period, patients also received daily treatment with 100 mg ferrous sulfate and 50 mg ascorbic acid. Within approximately 2 months of initiation of treatment with cobalt, an increase in hemoglobin of 26-70% was observe d in patients treated with 0.32 mg cobalt/kgday. Serum cobalt levels app eared to reach steady state within 2 months of exposure (approximately 40-100 μg cobalt/100 mL). In a subgroup of three patients, continuation of treatment with 0.16 mg cobalt/kg-day for appr oximately 3 months maintained elevated hemoglobin levels. Hemoglobin levels decreased rapidly when cobalt therapy was discontinued. The authors did not report whether therapy w ith ferrous sulfate a nd ascorbic acid was

discontinued at the same time. Results of this study are difficult to interpret because patients were anephric and on dialysis, which may have altered cobalt pharmacokine tics and dose-effect relationships. Furthermore, since it is well established that treatment with ferrous sulfate alone increases hemoglobin concentration (Hillman, 2001), concomitant therapy with iron is a confounding factor. Since this study did not evaluate the resp onse of patients treated with ferrous sulfate alone, it is not possible to dete rmine the relative contribut ions of iron and cobalt to the observed increases in hemoglobin. Thus, adverse effect levels cannot be confidently determined for cobalt. In a separate study, a gr oup of eight anephric patients with refractory anemia were treated with 25 to 50 mg cobalt ch loride daily for 12 to 36 weeks (Taylor et al., 1977). Increased hemoglobin concentration and decreased requirement for blood transfusions were observed (Taylor et al., 1977). Data on he moglobin concentrations (or other indicators of polycythemia) were not reported.

Pregnant women given 75 to 100 mg cobalt ch loride/day with no ot her treatment for 90 days to 6 months did not experience pregna ncy-induced reductions in hematocrit and hemoglobin levels, compared to untreated controls (Holly, 1955). However, daily treatment with 1 g ferrous sulfate alone or combined daily trea tment with 60 to 90 mg c obalt chloride and 0.8 to 1.2 g ferrous sulfate prevented pr egnancy-related decreases in he matocrit and hemoglobin levels. The response to combined cobalt chloride and iron therapy was more pronounced than the response to iron therapy alone. In patients treat ed with iron only, decreases in hemoglobin and hematocrit were prevented in approximately 80% of patients, compared to 100% of patients

treated with combined cobalt chloride and iron.

Cardiomyopathy has been observed in associ ation with consumpti on of large quantities of beer containing cobalt chlori de (introduced into the beer to stabilize the foam) (Alexander,

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1969, 1972; Morin et al., 1971). Exposure estimates in reported cases range from 0.04 to 0.14 mg cobalt/kg-day (corresponding to approximately 8-30 pints of beer daily) over a period of years (Alexander, 1969, 1972; Morin et al., 1971). The cardiomyopathy in the beer drinkers,

referred to in the literature as “beer-cobalt cardiomyopathy,” wa s fatal to 43% of the subjects within several years, with approximately 18% of these deaths occurring wi thin the first several days following diagnosis. Beer-cobalt cardiomyopa thy appeared to be similar to alcoholic cardiomyopathy and beriberi; however, the onset of the beer-cobalt cardiomyopathy was much more abrupt. The practice of adding cobalt to beer to stabilize the foam has been discontinued. It should be noted, however, that the cardiomyopathy may also have b een due to the fact that the beer drinkers had protein-poor di ets and may have had prior or concurrent cardiac and hepatic damage from alcohol abuse. Due to the potential adverse effects of poor nutrition and/or chronic ethanol exposure on cardiovascular health, it is difficult to delineate the contribution of oral cobalt exposure to the observed cardiomyopathy. As such, no adverse e ffects levels can be determined for cobalt-induced cardiotoxicity.

The thyroid also appears to be a target or gan for cobalt (see Table 1). Treatment of 12 euthyroid (normal thyroid) patients with 150 mg cobalt chloride/day (equivalent to 1 mg cobalt/kg-day, assuming a body weight of 70 kg) for 2 weeks resulted in a greatly reduced uptake of 48-hour radioactive iodi ne by the thyroid when measured after 1 week of exposure to cobalt, with uptake nearly abolished complete ly by the second week of exposure to cobalt (Roche and Layrisse, 1956). It should be not ed that when cobalt treatment was discontinued, iodine uptake returned to pre-tr eatment reported values. No othe r clinical details were provided for the human subjects. Therefore, based on the results of this study, a LOAEL of 1 mg cobalt/kg-day was identified for decreased radioa ctive iodine uptake in human thyroid following oral cobalt exposure. In anothe r small clinical study (Paley et al., 1958), decreased radioactive iodine uptake was reported in two of four (3 males, 1 female) euthyroid patients orally administered 37.5 mg cobalt/day as cobalt ch loride (equivalent to 0.54 mg cobalt/kg-day, assuming a body weight of 70 kg) for 10 to 14 days . One of the two subjects with reported decreased iodine uptake ha d received i.v. cobalt in addition to oral cobalt intake, and had been previously diagnosed with hyperthyroidism (alth ough was clinically euthyroid at the time of study). The i.v. dosing may have raised the intern al cobalt concentration to a level greater than the reported 0.54 mg dosage based upon oral dosing of 37.5 mg/day in other subjects that did not receive i.v. cobalt. Of the remaining three subjects, 24-hour iodine uptak e was not significantly decreased following oral cobalt exposure compared to corresponding pre-treatment values (based on pairwise t-test). The or al cobalt dose of 0.54 mg cobalt/kg-day represents a NOAEL for thyroid effects in humans. It s hould be noted that the Roche and Layrisse (1956) and Paley et al. (1958) studies lack details pertinent to other clinical conditions (e.g. including effects on thyroid stimulating hormone [TSH]) of these patients; thus the mechanism for the effect of cobalt on thyroidal iodine uptake cannot be ascertained. However, cobalt ap pears to increase thiocyanateinduced release of radioiodine from the thyroid, suggesting a possible effect on binding of iodine (e.g., iodination of thyroglobu lin) in the thyroid gland.

Cobalt has been found to be a sensitizer in humans. Individuals are sensitized following dermal or inhalation exposure, but flares of dermatitis may be triggered following cobalt ingestion. In a small clinical study, several patients with eczema of the hands were challenged orally with 1 mg cobalt sulfate (0.005 mg c obalt/kg-day, assuming a body weight of 70 kg) in

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tablet form once per week for 3 weeks; this tran slates to an estimated average daily dose of 0.0007 mg cobalt/kg-day (1 day a week/7 days a week × 0.005 mg cobalt/kg-day). 28/47 patients had a flare of dermatitis following the oral challenge (Veien et al., 1987). All 47 patients had positive dermal patch tests to cobalt ( 13 to cobalt alone and 34 to nickel and cobalt) and 7 of the 13 patients who had patch-tested positive to cobalt alone reacted to the oral challenge. These results suggest th at cobalt allergy can be induced from oral ingestion exposures to cobalt. Although the exposure levels asso ciated with sensitization to cobalt following inhalation or dermal exposure have not been es tablished, interrelationships have been found to

exist between cobalt and nickel sensitization (Bencko et al., 1983; Rystedt and Fisher, 1983; Veien et al., 1987). In guinea pigs , nickel and cobalt sensitization appear to be interrelated and mutually enhancing (Lammintaust a et al., 1985). Ther efore, it is possible that in people sensitized by nickel, exposure to coba lt may result in an allergic reaction.

Inhalation Exposure

Numerous studies have inves tigated health effects in work ers occupationally exposed to cobalt-bearing dust (Linna et al ., 2003; Swennen et al., 1993; Au chincloss et al., 1992; Cugell, 1992; Nemery et al., 1992; Prescott et al., 1992 ; Gennart and Lauwerys, 1990; Meyer-Bisch et al., 1989; Raffn et al., 1988; Shirakawa et al., 1988, 1989; Sprince et al ., 1988; Kusaka et al., 1986a,b; Demedts et al., 1984; Davi son et al., 1983). However, many of these studies are of limited utility for risk assessment due to inadequate characterization of exposure and/or effects. Four studies were considered to be potentially suitable for RfC derivation. Two of these focused exclusively on respiratory effects (Nemery et al., 1992; Gennart and Lauwerys, 1990); one studied only thyroid effects (P rescott et al., 1992) and one considered multiple endpoints (Swennen et al., 1993). The populations studied included diamond-cobalt saw manufacturers, diamond polishers, plate painters and cobalt produc tion workers. All four studies were crosssectional design.

Several studies have examined the effect s of hard metal, a mixture containing approximately 20% cobalt with the remainder be ing primarily tungsten carbide. Exposure of

humans to hard metal has been shown to result in an increase in cancer mortality (Moulin et al., 1998; Lasfargues et al., 1994) as well as a num ber of other diseases, including asthma and pulmonary fibrosis (for reviews, see Bar celoux, 1999; Lison, 1996). There is substantial evidence from animal studies that tungsten, alth ough it acts as an iner t dust by itself, can potentiate the effects of cobalt on the respiratory tract (Lasfar gues et al., 1995; Lison et al., 1995, 1996; Swennen et al., 1993). For this reason, st udies of hard metal were not given further

consideration.

Gennart and Lauwerys (1990) studied vent ilatory function in workers at a plant producing diamond-cobalt circular saws. The fo rm of cobalt used in diamond polishing is primarily metallic cobalt powder; specific coba lt species contained in this powder were not identified. The exposed populat ion consisted of 48 workers (34 males and 14 females) who agreed to participate in the study (an additional 27 workers d eclined). Exposure duration for these workers ranged from 0.1 to 32 years, with an average of approximately 6 years. The work involved weighing and mixing cobalt powder an d microdiamond particles (and possibly small

amounts of other undisclosed s ubstances), cold pressing, heat ing and hot pressing. After

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sintering, the pieces were welded onto steel disks. These operations were performed in two rooms called the mixing room and the oven room, where all the examined workers spent most of

their time. Controls consisted of 23 workers (11 males and 12 females) from other factories in the same area who were not exposed to known pneumotoxic chemicals. Personal air samples were collected at different workplaces during half a workshift. S ubjects filled out a questionnaire regarding occupa tional and medical histories, smoking habits and pulmonary symptoms; gave a urine sample for cobalt determina tion; and participated in lung function tests.

Cobalt concentrations varied from 9.4 to 2875 μ g/m 3 in the mixing room (geometric mean=135.5

μ g/m 3 ) and from 6.2 to 51.2 μ g/m 3 in the oven room (geometric mean=15.2 μ g/m 3 ). The prevalence of respiratory symptoms, such as cough, sputum and dyspnea, were significantly increased in the exposed workers compared to th e control group (numeric data not reported). Mean predicted values of FEV 1 (forced expiratory volume in 1 second adjusted for body size) and FVC (forced vital ca pacity) were significantly lower, an d the prevalence of abnormal values was higher in the cobalt exposed workers (both smokers and non-smokers) compared to the control group. In controls, FEV 1 and FVC were 95.4 and 101.6 per cent of predicted values, respectively. Mean percent predicted FEV 1 and FVC in exposed non-smokers were 87.1 and 92.3, respectively, and in exposed smokers were 83.9 and 93.4, respectively. Among nonsmokers, all measures of pulmonary function were lower in workers exposed for 5 years or more than in those exposed to cobalt for a shorter period of time.

Nemery et al. (1992) conducted a cross-secti onal study of cobalt exposure and respiratory effects in diamond polishers who were primarily exposed to metallic cobalt-containing dust; species of cobalt in the dust samples were not identified. The study group was composed of 194 polishers working in 10 different workshops. In two of these workshops (#1, 2), the workers used cast iron polishing disks almost exclusively, and in the others, they primarily used cobaltcontaining disks. The number of subjects from each workshop varied from 6 to 28 and the participation rate varied from 56 to 100%. The low participation in some workshops reflects the fact that only workers who used cobalt disks were initially asked to be in the study; low participation is not due to a high refusal rate (only eight refusals were documented). More than a year after the polishing workshops were studied, an additional three workshops with workers engaged in sawing diamonds, cleaving diamonds or drawing jewelry were studied as an unexposed control group (n=59 workers). Subjects were asked to fill out a questionnaire regarding employment history, working conditions, medical hist ory, respiratory symptoms and smoking habits; to give a urine sample for coba lt determination; and to undergo a clinical examination and lung function tests. Both ar ea air samples and personal air samples were collected (always on a Thursday). Sampling fo r area air determinations started 2 hours after work began and continued until 1 hour before the end of the work day. Personal air samples were collected from the breathing zone of a few workers per workshop for four successive 1-hour periods. Air samples were analyzed for cobalt and iron. In addition, personal air samplers were used to sample the air 1 cm a bove the polishing disks. These samples were analyzed for the entire spectrum of mineral and metallic compounds. Air samples were not obtained at one of the polishing workshops (#4) ; however, this workshop was reported to be almost identical to an adjoining workshop (#3) for which samples were obtained. Urinary cobalt

levels were similar between workers in these tw o workshops, so exposure was considered to be

similar as well.

88-25-2008

Results of area and personal air sampling were strongly correlated (R =0.92), with area air sampling reporting lower concentrations than pers onal air samples in all workshops except one (#9) (Nemery et al., 1992). In this workshop, pers onal air samples appeared to be artificially low in comparison to area air samples and urinary coba lt levels of the workers. When this workshop was excluded, a strong correlation (R=0.85-0.88) betw een urinary cobalt and cobalt in the air was observed. Based on urinary cobalt levels, the predicted concentration of cobalt expected in personal air samples from workshop #9 was approximately 45 μ g/m 3 (the mean value actually reported was 6 μ g/m 3 ). The polishing workshops were divided into two groups: those with low exposure to cobalt (#1-5, n=102) and those with high exposure to cobalt (#6-10, n=91). Mean cobalt exposure concentra tions were 0.4, 1.6 and 10.2 μ g/m 3 by area air sampling and 0.4, 5.3

and 15.1 μ g/m 3 by personal air sampling in the control, low-exposure and high-exposure groups, respectively. The inclusion of the apparently biased personal air samples from workshop #9 means that the reported mean cobalt exposure in the high-exposure group obtained by personal air sampling (15.1 μ g/m 3 ) may be lower than the true value. Air concentrations of iron were highest in the two polishing workshops that used iron disks and the sawing workshop (highest

value=62 μ g/m 3 ), and were not correlated w ith cobalt levels. Analysis of samples taken near the disks showed the presence of cobalt, with occasi onal traces of copper, zinc, titanium, manganese, chromium, silicates and silicon dioxide. No tungsten was detected. Some workers may have previously been exposed to asbest os since pastes contai ning asbestos had been used in the past to glue the diamonds onto holders. However, sin ce the asbestos was in its non-friable form, exposure was insufficient to pr oduce functional impairment. Sm oking habits were similar in workers from the high-exposure, low-exposure a nd control groups. Duration of exposure was

not discussed.

Workers in the high-exposure group were more likely than those in the other groups to complain about respiratory symptoms; the preval ences of eye, nose and throat irritation and cough, and the fraction of these symptoms related to work, were signifi cantly increased in the high-exposure group (Nemery et al., 1992). Wo rkers in the high-exposure group also had significantly lower lung function compared to controls and low-exposure group workers, as assessed by FVC, FEV 1 , MMEF (forced expiratory flow be tween 25 and 75% of the FVC) and mean PEF (peak expiratory flow rate), although the prevalence of abnormal values did not differ significantly between ex posure categories. In controls, FVC, FEV 1 and MMEF were approximately 110, 107 and 94 percent of pred icted values, respectively, compared to approximately 105, 104 and 87 percent of predicte d values, respectively, in the high-exposure group workers. Results in the low-exposure group did not differ from controls. The effect on spirometric parameters in the high exposure gro up was present in both men and women. Women seemed to be affected more than men; howev er, the interaction betw een exposure and sex was not significant (two-way analys is of variance). Smoking was found to exert a strong effect on lung function; however, lung func tion level remained negatively correlated with exposure to cobalt, independent of smoking.

A cobalt dose-effect relationship is evident from the Nemery et al. (1992) study, based on a multivariate regression analysis of urinary cobalt and lung function measurements. Increasing urinary cobalt concentration (appr oximate range <1-70 μg cobalt/g creatinine) was significantly ( p <0.05) associated with co-variate-adjusted d ecreasing forced expiratory volume (FEV1%) and forced vital capacity (FVC%). Significant co-v ariates retained in the regression analysis

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included gender and smoking. The model predic ted 3% and 4% decreases in FEV1% and FVC%, respectively, in association with a 10-fold increase in ur inary cobalt concentration. The approximate mean urinary cobalt levels of the control and high exposure groups were 2 and 20

: g cobalt/g creatinine, respectively. The magnit ude of the cobalt effect was similar to the predicted effect of smoking, approximately 3-4% decrease in FEV1% and FVC%. Cobalt concentration determined from personal air samp ling may be more representative of airborne cobalt exposure than area sampling. As such, 5.3 μ g/m 3 and 15.1 μ g/m 3 represent a NOAEL and LOAEL, respectively, for decreased pulmonary function and increased symptoms of airway

irritation.

Swennen et al. (1993) conducted a cross-sec tional study of workers exposed to metallic cobalt and various inorganic cobalt salts and oxides (specific species not identified) at a cobalt plant producing these materials from cobalt me tal cathodes and scrap metal. The study group included 82 male workers from the cobalt plant who had no history of lung disease prior to employment and who had never been exposed to other pneumotoxic chem icals. Methods for selection or exclusion of subjec ts in constructing the cohort and participation were not reported. The control group comprised 82 age-matched work ers from the mechanical workshop of a nearby plant owned by the same company. Work ers filled out a questionnaire regarding occupational history, respirator y complaints and smoking habits ; received a routine clinical examination; participated in lung function tests; had a chest radiograph taken; and gave blood and urine samples (before and after worki ng on Monday and Friday of one week) for

determination of cobalt content as well as he matological and serum chemistry analyses. Exposure was monitored by personal air samplers worn by each cobalt worker for 6 hours on both Monday and Friday.

Workers in the cobalt plant were exposed to cobalt concentrations ranging from 1 to 7772

μ g/m 3 (Swennen et al., 1993). The geometri c mean exposure concentration was 125 μ g/m 3

. Exposure duration ranged from 0.3 to 39.4 years, with an average exposure of 8.0 years. A significantly higher number of exposed worker s reported dyspnea than did controls. The increase occurred primarily among smokers although no significant interaction was found between smoking and exposure to cobalt. Based on a logistic regression model, the probability of dyspnea during exercise was significantly associ ated with increasing cobalt concentration in the air or urine. The parameters of the mode l were not reported. The clinical examinations detected significantly increased prevalence of skin disorders (eczema, erythema) (51 vs. 25%) and wheezing (16 vs. 6%) in the exposed group compared to controls. Lung function tests did not differ between the two groups; however, a few significant trends were noted: the FEV 1 /VC (forced expiratory volume in one second/vital capacity) ra tio decreased with increasing concentration of cobalt in the air and urine, a nd the RV (residual volum e) and TLC (total lung capacity) increased with increasing duration of exposure. No lung abnormalities were found by chest radiographs in either group. Blood analyses did not show polycythemia, and in fact, there were slight, but significant, decreases in red blood cell count, hemoglobin and hematocrit in the exposed workers. White blood cell counts were significantly increased. Serum levels of the thyroid hormone T3 (triiodothyron ine) were slightly (7%), but significantly, decreased in the exposed group, while T4 (thyroxin e) and TSH (thyrotropin) were not affected. Serum markers for cardiomyopathy (i.e., myocardial creatine kinase) were unchanged.

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Prescott et al. (1992) conducte d a cross-sectional study to inve stigate the effects of cobalt exposure on thyroid volume in female plate pain ters. The test group included 61 female plate painters exposed to cobalt blue dyes in two porce lain factories. The control group consisted of 48 unexposed women working at the same factories. The dyes used in the two factories differed; factory I (36 workers) used cobalt aluminate, whic h is insoluble, and factory II (25 workers) used cobalt-zinc silicate, which was re ported to be "semi-soluble." Wo rkers were exposed to cobalt during the painting procedure when the plates were spray-pain ted (under a fume hood) two or

three times with the water-based cobalt blue und erglaze and when the excess color was removed with a brush after drying. C obalt concentrations were reporte d to be approximately 0.05 mg/m 3
in the workplaces (no further details on air leve ls were reported). The average duration of exposure was 14.6 years in group I workers and 16.2 years in group II workers. Subjects filled out a questionnaire regarding heal th, use of medicines, day of menstrual cycle, employment information and smoking habits and agreed to gi ve blood and urine samples for determination of thyroid hormone levels (e.g. t hyroxine (T4), triiodothyronine (T 3), and thyroid stimulating hormone) and cobalt concentration, respectively, and to undergo ultrasonography to determine volume of the thyroid gland.

Urinary cobalt levels were similar in group I exposed workers and c ontrols (Prescott et al., 1992). Group II workers exposed to semi-sol uble cobalt-zinc silicat e had urinary cobalt levels that were approximately 10-fold higher th an controls. Group I workers did not differ from controls for any of the thyroid paramete rs measured; however, Group II workers had a significant 22% increase in serum T4 (thyroxine) le vels. Mean thyroid volume was lower in this group as well, although the difference from controls (16.1 mL in group II vs. 19.2 mL in controls and 18.7 mL in group I) was not stat istically significant. The occurr ence of respiratory effects in these workers was not reported.

Results of three stud ies investigating cancer incidence in workers exposed to cobalt by the inhalation route (Tuchsen et al., 1996; Mu r et al., 1987; Morgan, 1983) are suggestive of a possible association between exposure to cobalt and respiratory tumors. Morgan (1983) investigated the health and cause s of death of 49 men occupationall y exposed to cobalt salts and oxides (specific species not iden tified) in a manufacturing plant in South Wales. During the study period, 33 men died (five w ith lung cancer and three with cancer at other sites). The expected number of deaths was 3.0 for lung cance r and 4.1 for cancers at other sites, based on national statistics, resulting in mortality ratios of 1.7 and 0.73, respectively (s tatistical analysis of data not reported).

Mur et al. (1987) analyzed the mortality of a cohort of 1143 workers in a plant that refined and processed cobalt and sodium. The pl ant workers may have been involved in multiple processing applications utilizing different forms of cobalt including cobalt chloride, oxides and other salts (specific species not identified). An increase in de aths [Standard Mortality Ratio (SMR) = 4.66; 95% confidence interval (CI) = 1.46-10.64] resulting from lung cancer was

observed in workers based on four cases observe d in the exposed group and one case expected

based on French national statistics . In a study within the cohort that controlled for age and smoking habits, 44% (four workers) in the expose d group and 17% (three wo rkers) in the control group died of lung cancer. The authors indicated that the differences were not statistically

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significant and that the workers were exposed to arsenic and nickel in addition to cobalt. The exposure levels of cobalt were not reported.

Tuchsen et al. (1996) analyzed the can cer incidence of a c ohort of 874 women who worked in one of two factories (382 from one factory, 492 from a second factory) applying a cobalt-based (cobalt-aluminate sp inel) plate underglaze. From une xposed areas of factory I , 520 referents were selected. Both groups were compar ed to statistics for all Danish women in the same calendar year. During the 5-year follow-up period, the overall canc er incidence was only slightly elevated in exposed workers, while the incidence of lung cancers was significantly increased [Standard Incidence Ratio (SIR) = 2.35; 95% CI = 1.01-4.6]. The incidence of lung cancers in the referents (not exposed to cobalt) was greater than that of all Danish women, but

the difference was not statistica lly significant. Exposure charact erization prior to 1980 was not described, while exposures after 1980 were variable and reported as a mean concentration for a given year. Exposures we re generally in the range of 0-1 mg cobalt/m 3 except for 2 years, during which they were greater.

Animal Studies

Overview

Studies in animals show that oral exposure to cobalt produces effects similar to those observed in humans, including increases in re d blood cells and hemoglobin (Domingo et al., 1984; Krasovskii and Fridlyand, 1971; Murdoc k, 1959; Holly, 1955; Stanley et al., 1947), thyroid effects (Shrivastava et al., 1996) and cardiac effects (H aga et al., 1996; Pehrsson et al., 1991; Mohiuddin et al., 1970). Ot her findings in animals not reported in humans include neurobehavioral changes (Singh and Junnarkar , 1991; Bourg et al ., 1985; Krasovskii and Fridlyand, 1971) and testicular toxicity (Anderson et al., 1992, 1993; Pedigo et al., 1988; Corrier et al., 1985; Mollenhauer et al ., 1985; Domingo et al., 1984; Nation et al., 1983). Developmental toxicity studies in rats and mice provide evidence that high oral doses of cobalt may produce developmental effects in animals, in some cases in the absence of overt maternal toxicity (Szakmary et al., 2001; Paternain et al., 1988; Domingo et al., 1985).

Animal data support the conclusion that the re spiratory tract is the critical target for inhaled cobalt (NTP, 1991; Bucher et al., 1990; Wehner et al., 1977). Subchronic inhalation exposure to cobalt resulted in cy totoxicity and reparative prolif eration in all regions of the respiratory tract in rats and mice (NTP, 1991; Bu cher et al., 1990). Available chronic animal studies have demonstrated the carcinogenic potentia l of inhaled cobalt in male and female rats and mice, with alveolar and br onchiolar tumors being the most prevalent (Bucher et al., 1999; NTP, 1998).

Oral Exposure

Studies in rats show that s ubchronic oral exposure to cobalt chloride increases red blood cell counts and hemoglobin levels with NOA ELs ranging from 0.05 to 0.62 mg cobalt/kg-day (Krasovskii and Fridlyand, 1971; Stanley et al., 1947) and LOA ELs ranging from 0.5 to 32 mg cobalt/kg-day (Domingo et al., 1984; Krasovs kii and Fridlyand, 1971; Murdock, 1959; Holly,

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1955; Stanley et al., 1947). In general, effect s in animal studies were observed at higher exposure levels than those reported in humans.

Effects of cobalt on red blood cells and hemogl obin were investigated in Sprague-Dawley rats treated with 2.5, 10, and 40 mg cobalt chlo ride hexahydrate/kg-day (equivalent to 0.62, 2.5, and 9.9 mg cobalt/kg-day, respectively) for 8 week s (Stanley et al., 1947). After 8 weeks of exposure, increases in hemogl obin and red blood cell number were observed in the 2.5 and 9.9 mg cobalt/kg-day treatment groups. Sta tistical significance was not reported.

Hemoglobin and hematocrit were significantl y increased in male Sprague-Dawley rats exposed to 500 ppm cobalt chloride in drinki ng water, equivalent to approximately 32 mg cobalt/kg-day (assuming a water intake of 0.139 L/ kg-day for male Sprague-Dawley rats; U.S. EPA, 1988), for 3 months (Domingo et al., 1984). Compared to controls, hematocrit and hemoglobin were both increased by approximately 30% at the end of the 3-month exposure period, with increases observed within the fi rst 2 weeks of exposure (numeric data not presented). Following the 3-month exposure pe riod, histopathological examination showed no treatment-related morphological or ultrastructural changes to any organ. Increased tissue weights were observed for spleen, heart and l ungs, and testicular weight was decreased compared to controls. Based on the results of this study, 32 mg cobalt/kg- day was identified as a

subchronic LOAEL for increased he matocrit and hemoglobin and decr eased testicular weight in

rats.

In rats exposed to 40 mg cobalt chloride/kg- day (equivalent to 18 mg cobalt/kg-day) for 4 months, hemoglobin and red blood cell count were increased by 37 and 21%, respectively, compared to controls (Holly, 1955). Simila r effects were observed following concomitant administration of 40 mg cobalt ch loride/kg-day and 200 mg ferrous sulfate, with increases of 30% for hemoglobin and 32% for red blood cell count, compared to controls. Statistical significance was not reported.

Oral exposure of rats to 10 mg cobalt/kg-day (a s cobalt chloride) for 5 months resulted in increases in hemoglobin, hematocrit and red bloo d cell count compared to untreated controls, with effects reaching a plateau after approxi mately 60 days of exposure (Murdock, 1959). Statistical significance was not reported. No changes were observed for mean corpuscular hemoglobin concentration and mean cell volume compared to untre ated controls, indicating that stimulation of erythropoiesis by cobalt did not result in the production of abnormal red blood

cells.

The effects of exposure to 0.05, 0.5, and 2.5 mg cobalt/kg-day (as cobalt chloride) for 7 months were examined in rats (Krasovskii a nd Fridlyand, 1971). Trea tment with 0.5 and 2.5 mg cobalt/kg-day, but not 0.05 mg cobalt/kg-day, for 7 months increased red blood cells and hemoglobin. Stimulation of hematopoiesis wa s more pronounced in the 2.5 mg cobalt/kg-day group than in the 0.5 mg cobalt/kg-day group, wi th polycythemia in the 0.5 mg cobalt/kg-day group described as mild and transient. Results of this study are difficult to evaluate since

numeric data and statistical analyses were not reported.

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Studies in animals have noted cardiac effect s following cobalt (c obalt sulfate) exposure (Haga et al., 1996; Pehrsson et al., 1991; Mohi uddin et al., 1970), alt hough at higher exposure

levels than observed in human studies. The effect of cobalt on myocardial function was examined in rats exposed to 8.4 mg cobalt/kg-da y for 16 or 24 weeks (Haga et al. 1996). After 24 weeks of exposure, decreased left ventricula r systolic and diastolic function was observed. An increase in the ventricular weight to body we ight ratio indicates th at left ventricular hypertrophy is a contributory factor in coba lt-induced myocardial dysfunction although a mechanism was not identified. Significant effects on cardiac function were not observed following 16 weeks of exposure. In guinea pi gs, exposure to 20 mg cobalt/kg-day as cobalt

sulfate in the diet for 5 weeks resulted in decr eased absolute and relative heart weights and a greater incidence of abnormal electrocardiograms compared to animals fed on diets not supplemented with cobalt (Mohiuddin et al., 1970). Cardiac arrhythmias, including bradycardia, and repolarization abnormalities, we re observed in 65% of cobalt-treated animals compared to 5% of control animals. Cellular alterations, observed at the light and electron microscopic levels, in cardiac tissues included pericardi al thickening and inflammation, myocardial degeneration and vacuolization, endocardial thicke ning and myofibrillar damage. In contrast, no

effects on cardiac function were observed in male rats (12/group) exposed to protein-restricted diets containing 8.4 mg cobalt/kg-da y for 8 weeks (Pehrsson et al., 1991). Treated rats showed a significant decrease in body weight but no differences in left ve ntricular function relative to animals treated with protein-restricted diets with out added cobalt. Althou gh the results from the Pehrsson et al. (1991) and Haga et al. (1996) rat st udies conflict, it appe ars that oral cobaltinduced myocardial injury/dysf unction may have a significant tim e-dependence. Oral cobalt (as cobalt sulfate) at the same dose level (8.4 mg co balt/kg-day) did not a ppear to alter cardiac structure or function following exposure for up to 16 weeks (Pehr sson et al., 1991; Haga et al., 1996). However, ventricular hypertrophy with a concom itant decrease in left ventricular systolic

and diastolic function was observed in rats after 24 weeks of oral cobalt (Haga et al., 1996). Thus, based on the results of th is study, 8.4 mg cobalt/kg-day represents a subchronic LOAEL for myocardial toxicity in ra ts; Based on the results of th e Mohiuddin et al. (1970) study, a LOAEL of 20 mg cobalt/kg-day was identified for myocardial toxicity in guinea pigs.

Histopathological changes in the thyroid gla nd have been observed following exposure of female mice to 400 ppm cobalt chloride (~48 mg cobalt/kg-day, assuming an average water intake of 0.265 L/kg-day for female mice; U.S. EPA, 1988) in drinking water for 15 to 45 days (Shrivastava et al., 1996). The severity of e ffect increased with exposure duration. After 15 days of exposure, a reduction in thyroid epithelial cell height with dege nerated nuclei and reduced amount of colloid with peripheral resorption vacuoles was observed, with more pronounced effects after 30 days of exposure. More significant degene rative changes were observed after 45 days of exposure, including necr otic epithelial cells, re duced connective tissue between follicles, lymphocytic in filtrate and larger amounts of co lloid within the lumen. Based upon significant thyroid toxicity observed in th is study, a LOAEL of 48 mg cobalt/kg-day was

identified in mice.

Developmental effects of orally administered cobalt have been studied in rats, rabbits and mice (Szakmary et al., 2001; Pedigo and Vernon, 1993; Paternain et al., 1988; Seidenberg et al., 1986; Domingo et al., 1985; Elbeti eha et al., 2008). Szakmary et al. (2001) evaluated the developmental effects of oral cobalt sulfate expo sure in rats, mice and rabbits. Exposure of

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pregnant rats to 5.2-21.0 mg cobalt/kg-day (o ral gavage) decreased perinatal growth and survival, retarded skeletal development and pr oduced skeletal and ur ogenital malformations, with a LOAEL of 5.2 mg cobalt/ kg-day. Maternal toxicity (inc reased relative liver, adrenal, spleen weights; increased BUN, serum creatinine) was only observed at the highest dose (21.0 mg cobalt/kg-day). Thus, embryotoxicity in rats was observed at exposure levels below the LOAEL for maternal toxicity. In pregnant mi ce exposed to 10.5 mg cobalt/kg-day, retarded skeletal development and malformations of the eye, kidney and skeleton were observed in the absence of maternal toxicity. In pregnant rabbits exposed to 4.2 mg cobalt/kg-day, 20% mortality was observed in dams. Fetal resorpti ons were observed in 30% of surviving dams. Results of the studies in rats and mice provide evidence that adverse developmental effects can occur in the absence of maternal toxicity, and that rabbits are more sensitive to oral cobalt.

Domingo et al. (1985) treated pregnant female rats (15 animals/group) with 5.4 to 21.8 mg cobalt/kg-day as cobalt chloride from gest ation day 14 through lactation day 21. Offspring were examined for mortality, body weight, body a nd tail length and general signs of toxicity after 1, 4 and 21 days of nursing. In contrast to the study by Szakmary et al. (2001), results of the Domingo et al. (1985) study re ported maternal toxicity at all doses that produced adverse developmental effects (specific maternal effects observed were not reported). Fetal effects at 5.4 mg cobalt/kg-day included stunted growth of the pups of both sexes, decreased body length and tail length in male offspring a nd decreased spleen and liver weight in female offspring. Effects at the 10.9 mg cobalt/kg-day dose included decreas ed body weight in female pups, while at 21.8 mg cobalt/kg-day, decreased number of living yo ung and decreased survival were seen. Blood parameters (liver enzymes, bilirubin, total protein, uric acid, urea, creatinine, hemoglobin and hematocrit) in pups did not show any treatment-re lated changes. No signs of toxicity were observed in surviving pups in a ny of the cobalt exposure groups.

No significant effects on fetal growth or su rvival were found in rats exposed to 6.2 to 24.8 mg cobalt/kg-day as cobalt ch loride (oral gavage) during gestation days 6-15 (Paternain et al., 1988). The incidence of stunted fetuses was higher in the animals treated with 12.4 or 24.8 mg cobalt/kg-day (0.3 stunted fetuses per litter in the 12.4 mg cobalt/kg-day group; 1.0 stunted fetuses per litter in the 24.8 mg cobalt/kg-day group) compar ed to the control group (0 stunted fetuses per litter); however, the di fferences were not statistically significant. No treatmentrelated effects were observed for the number of corpora lutea, total implants, resorptions, the

number of dead and live fetuse s or fetal size parameters. No gross external abnormalities,

skeletal malformations or other si gns of fetal toxicity were observ ed. Maternal effects, including reduced body weight gain and food consump tion and altered hematological parameters (increased hematocrit, hemoglobin and reticulocyte s), were reported at al l exposure levels. No fetal effects were reported in mice exposed to 81.7 mg cobalt/kg-day (oral gavage) during gestation days 8-12 (Seidenberg et al., 1986), but a significant ( p <0.05) decrease in maternal weight was found. Additional details were not reported.

Pedigo and Vernon (1993) expos ed male B6C3F1 mice to 40 0 ppm cobalt chloride (~45 mg cobalt/kg-day, assuming a water intake of 0.247 L/kg-day for male B6C3F1 mice; U.S. EPA, 1988) in the drinking water for 10 weeks, after which the males were mated with control females

to examine for dominant lethal effects. Relati ve to the control group, the cobalt treatment group had a lower percentage of pregnant females (c ontrol, 29/32; cobalt, 18 /31), lower number of

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implantations per female (control, 8.3; cobalt, 6.5) and higher preimpla ntation losses (control, 0.43; cobalt, 2.4). At the end of the 10-week treatment period, sperm concentration was decreased to 15.3% and motility decreased to 18.3% of controls. Several measures of sperm velocity were also depressed relative to c ontrols. All sperm parameters, except sperm concentration, returned to contro l levels 8 weeks after the cobalt exposure was terminated. The increase in preimplantation losses in the dominant lethal assay appears rela ted to adverse effects on spermatogenesis rather than to effects on preimplantation development of embryos.

Several studies reported te sticular degeneration and atr ophy in rats exposed to 11.7 to 46.9 mg cobalt/kg-day as cobalt chloride for 2-3 months in the diet or in the drinking water (Anderson et al., 1992, 1993; Pedigo et al., 1988; Corrier et al., 1985; Mollenhauer et al., 1985; Domingo et al., 1984; Nation et al ., 1983). Pedigo et al. (1988) exposed male CD-1 mice to 100, 200 or 400 ppm of cobalt chloride (~11.7, 23.4 or 46.9 mg cobalt/kg-day, respectively, assuming an average water intake of 0.258 L/kg-day for male mice; U.S. EPA, 1988) in the drinking water for 13 weeks. High-dose animals showed a signifi cantly decreased testicular weight beginning at week 9 of treatment and a decreased epididymal sperm concentration by week 11 of treatment. All dose groups showed signifi cantly decreased testicular we ight and epididymal sperm

concentration and increased se rum testosterone levels by we ek 12 of exposure, with the magnitude increasing with dose. Effects on seru m testosterone levels may be secondary to effects on spermatogenesis and related to inhibiti on of local inhibitory feed-back mechanisms. Based on the results of this study, 11.7 mg cobalt /kg-day was identified as a subchronic LOAEL for decreased testicular weight and epidi dymal sperm concentration in male rats.

Anderson et al. (1992, 1993) e xposed groups of male CD-1 mice to 400 ppm of cobalt chloride (~46.9 mg cobalt/kg-day, assuming an av erage water intake of 0.258 L/kg-day for male mice; U.S. EPA, 1988) in the drinking water for up to 13 weeks. A decrease in testicular weight and a progressive degeneration of the seminifer ous tubules were seen beginning at 9 weeks of exposure. Initial changes were vacuolization of Sertoli cells and abnormal spermatid nuclei, followed by sloughing of cells, shrinkage of tubules and thickened endothelium. No recovery was reported after a 20-week non-e xposure recovery period. Co-a dministration of 800 ppm of zinc chloride provided a partial protection against the effects of cobalt. Based on the results of this study, 46.9 mg cobalt/kg-day was identified as a subchronic LOAEL for decreased testicular weight and degeneration of semini ferous tubules in male mice. Similar histology (degeneration of the testes, particularly the seminiferous tubul es) was noted in Sprague-Dawley rats exposed to 20 mg cobalt/kg-day in the diet for up to 98 days (Corrier et al., 1985; Mollenhauer et al., 1985). Decreased testicular weight was seen in Sprague-Dawley ra ts exposed to 500 ppm cobalt chloride (~32 mg cobalt/kg-day, assuming a wa ter intake of 0.139 L/kg-day for male SpragueDawley rats; U.S. EPA, 1988) fo r 3 months (Domingo et al., 1984).

Elbetieha et al. (2008) examined the potential effects of coba lt on male fertility in forty adult (60 day-old) male Swiss mice exposed to cobalt chloride hexahydrate via drinking water at concentrations of 200, 400, or 800 ppm for 12 weeks. Based on daily water intake reported in the study, daily average doses of cobalt chloride were estimated at 26, 47, or 93 mg/kg-day (equivalent to 6.5, 11.7, or 23 mg cobalt/kg-day); c ontrol animals received untreated tap water. Mice were observed daily for signs of clinical to xicity during the exposure period. At the end of the 12-week cobalt exposure peri od, male mice were separated in to individual cages containing

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two virgin Swiss female mice and given ad libitum access to food and untreated tap water. Mice were cohabitated for 10 days during which it wa s estimated that the females completed two estrus cycles. Male control and cobalt-treated mice were necropsied after day 10 of cohabitation and testes, seminal vesicles, epididymides and preputial glands were harvested, weighed, and prepared for analysis. The left testis and ep ididymis from each male mouse was processed for determination of sperm count, wh ile the right testis was proces sed for histopathology. Ten days later, female mice were necropsied and examin ed for number of pregnancies, number of implantation sites, number of viable fetuses, tota l number of resorptions, and incidence rate of resorptions.

Ingestion of cobalt chloride wa s associated with 1/10 and 2/ 10 deaths in the mid- and high-dose treatment groups, respectively, during week 10 of exposure. Average body weight gain was significantly reduced in all cobalt treatment groups ( p < 0.01). No other signs of clinical toxicity were observed in surviving male mice. Relative to the control group, the number of pregnant females mated with male mice from the mid- and high-dose groups was significantly ( p < 0.05) reduced (control, 19/20; mid-dos e, 12/18; high-dose, 7/16). The number of implantation sites was significantly ( p < 0.01) reduced in females mated with low- and middose males (control, 7.89; low-dose, 5.67; mid-dose, 5.42), and the number of viable fetuses was significantly ( p < 0.05) reduced in females mated with males from all cobalt treatment groups (control, 7.74; low-dose, 5.0; mid-dose, 4.67; high- dose, 5.83). In additio n, the total number of resorptions (control, 3/150; low- dose, 9/81; mid-dose, 9/65; high- dose, 10/45) and the number of animals with resorptions (control, 3/19; low-dose, 10/15; mid-dose, 10/16; high-dose, 5/7) were significantly ( p < 0.05) increased in females mated with males from all three cobalt-treatment groups. Analysis of male reproductiv e organs revealed a significant ( p < 0.005) decrease in absolute epididymal weight in mice of the high-dose treatment group. Testes weights were significantly ( p < 0.01) reduced in males at all do ses of cobalt, and a significant ( p < 0.005) increase in the absolute weight of seminal vesicles of the mid- and high-dose males only. Compared to controls, testicul ar sperm counts and daily sperm production were decreased in the mid- and high-dose males, but not in the low- dose animals. Epididymal sperm counts were

decreased in male mice from all three cobalt trea tment groups. Histopathological examination of testis tissue from males of the mid- and high- dose revealed a number of abnormalities including

necrosis of the seminiferous tubules and inters titium, congested blood vessels, hypertrophy of the interstitial Leydig cells, and degeneration of the spermatogonial ce lls; incidence rate of these observations was not reported. These testicular histopathologies were not observed in the testes

of control and low-dose treated males. Base d on the results of this study, a LOAEL of 6.5 mg/kg-day was identified for decreased testicular weight, epididymal sperm counts, and associated reproductive abnormalities in pregnant females. A NOAEL was not identified.

Nation et al. (1983) expose d groups (n=6) of male Sprague-Dawley rats (weighing 200-210 g) to diets containing 0, 5 or 20 mg cobalt/kg-day as coba lt chloride for a total of 69 days. Following 14 days of exposure, animal s were trained for scheduled (operant) or conditioned suppression neurobehavior al tests. Other than two se izures in the same high-dose animal, no overt signs of neurotoxicity were repor ted at any exposure level. A trend toward a decreased response rate in the schedule trai ning behavior was observe d in both the exposed groups but only attained statistical significance in the high-dose animals near the end of the operant testing period (sessions 28-35, on exposure days 44-51). A trend toward decreased

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conditioned suppression beha vior did not attain statistical si gnificance in either group. Animals exposed to 20 mg cobalt/kg-day, but not 5 mg cobalt/kg-day, showed a significantly decreased weight of the testes following 69 days of expos ure. Based on the results of this study, a NOAEL of 5 mg cobalt/kg-day and a LOAEL of 20 mg cobalt/kg-day was identified for decreased testicular weight and changes in operant behavior in male Sprague-Dawley rats.

Several other studies have examined the e ffects of cobalt on neurobehavioral parameters (Singh and Junnarkar, 1991; Krasovskii and Fridly and, 1971; Bourg et al., 1985). In groups of male Sprague-Dawley rats (n=8) exposed to 20 mg cobalt/kg-day as cobalt chloride for 57 days in the drinking water, cobalt enhanced behavioral r eactivity to stress (the animals were less likely to descend from a safe platform to an electrif ied grid) (Bourg et al., 1985 ). Singh and Junnarkar (1991) reported a moderate reduction in spontaneous activity and mild hypothermia in rats exposed orally to cobalt chloride (approxima tely 8 mg cobalt/kg-da y) or cobalt sulfate (approximately 35 mg cobalt/kg-day). Krasovskii and Fridlyand (1971) exposed groups of rats (number and sex not specified) to 0.05, 0.5 or 2.5 mg cobalt/kg-day as cobalt chloride for up to 7

months. Neurobehavioral tests showed that tr eatment with cobalt resulted in a significant ( p <0.05) increase in the latent reflex period at 0.5 mg cobalt/kg and above, and a pronounced neurotropic effect (disturbed condi tioned reflexes) at 2.5 mg cobalt/kg.

Inhalation Exposure

In a subchronic inhalation study, groups of 10 F344/N rats and 10 B6C3F1 mice of each sex were exposed to cobalt sulf ate hexahydrate aerosol (MMAD=0.83-1.10 μ m; σ g not reported) at concentrations of 0, 0.3, 1, 3, 10 or 30 mg/m 3 (equivalent to 0, 0.067, 0.22, 0.67, 2.2 or 6.7 mg

cobalt/m 3 ) 6 hours/day, 5 days/week for 13 weeks (B ucher et al., 1990; NTP, 1991). Although this report indicates that exposur e was to cobalt sulfate heptahydrat e aerosol, detailed analysis of the cobalt aerosol in the 2-year continuation study (Bucher et al., 1999; NTP, 1998) reports that the aerosol was actually compos ed of cobalt sulfate hexahyd rate; thus, exposure to the hexahydrate form is assumed for the 13-week st udy. Animals were monitored for body weight and observed for clinical signs during the exposure period. Urine samples for urinalysis and cobalt determination were collected from rats prior to sacrifice. Following termination of exposure, all animals were sacrificed and necropsied. Blood samples were collected and analyzed for hematological parameters (rats a nd mice) and serum chemistry and thyroid function parameters (rats only). The major organs we re weighed. Animals from the control and highdose groups received comprehensive histopathologi cal examinations, while those from the lower dose groups received more limited examina tions focused on the respiratory tissues.

All rats survived until sc heduled necropsy (NTP, 1991; Bu cher et al., 1990). Gross evidence of toxicity was noted only in rats exposed to 6.7 mg cobalt/m 3 , and they displayed clinical signs of toxicity (ruffl ed fur, hunched posture) and redu ced body weights. Polycythemia, indicated by significant increases in red blood cell count, hemogl obin and hematocrit, was noted in males exposed to > 0.67 mg cobalt/m 3 and females exposed to > 2.2 mg cobalt/m 3 . In addition, platelets were significantly reduc ed in rats of both sexes at > 2.2 mg cobalt/m 3 and reticulocytes were increased in females at 6.7 mg cobalt/m 3 . Leukocyte counts and differentials were unaffected. Serum cholesterol was significantly reduced in males at > 2.2 mg cobalt/m 3 and females at 6.7 mg cobalt/m 3 . No other serum chemistry parameters were affected, including

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creatine kinase isozymes indicative of damage to cardiac muscle cells. Among the thyroid hormones, T3 (triiodothyronine) was significan tly reduced in females at 2.2 mg cobalt/m 3 (83% of control) and males at 6.7 mg cobalt/m 3 (62% of control) an d TSH (thyrotropin) was significantly reduced in males at 6.7 mg cobalt/m 3 (30% of control), but T4 (thyroxine) was not affected in either sex at any dose and the res earchers concluded that thyroid function was not consistently affected in this study. Urinalysis revealed a dose-related in crease in the number of epithelial cells and granular cast s in the urine of many exposed ma le rats (3-7 per group exposed

to > 0.67 mg cobalt/m 3 ) but not in the urine of control male rats. The researchers interpreted this finding as indicating minimal nephropathy in ex posed male rats although histopathological lesions were not detected in the kidney. No effects on sperm counts, sperm motility or the incidence of abnormal sperm were noted. Averag e estrus cycle of females exposed to 6.7 mg

cobalt/m 3 was slightly longer than controls, but the di fference was not significant. Absolute and relative lung weights were si gnificantly increased in both male and female rats at > 0.22 mg

cobalt/m 3 . Other organ weights were not affected by treatment. Compound-related lesions were found only in the respiratory tissues of expos ed rats. Degenerative, inflammatory and regenerative lesions were found throughout the re spiratory tract (see Tabl e 2). Incidence and severity of lesions were similar in males and fe males. The most sensitive tissue was the larynx, with squamous metaplasia pr esent at all exposure levels.

Among mice, 2/10 males exposed to 6.7 mg cobalt/m 3 died during the study (NTP, 1991; Bucher et al., 1990). The only clinical signs of toxicity observed were rapid breathing and skin

discoloration in one of the mice that died. Body weights were reduced throughout the study in both males and females exposed to 6.7 mg cobalt/m 3 . No dose-related hematological effects

were found. Absolute and relativ e lung weights were significantly increased in male and female mice exposed to > 2.2 mg cobalt/m 3 . Respiratory lesions were sim ilar to those observed in rats. As with rats, the most sensitive tissue was the larynx, with squamous metaplasia present at all exposure levels. Reproductive system effects were more prominent in mice than rats. Males had significantly decreased testicular weight (48% compared to c ontrol), decreased epididymal weight (81% compared to control), testicular at rophy consisting of loss of germinal epithelium in

the seminiferous tubules and foci of mineraliz ation and an increased percentage of abnormal sperm at 6.7 mg cobalt/m 3 (295% compared to control). Si gnificant reductions in sperm motility of 90, 87 and 54% were observed in the 0.67, 2.2 and 6.7 mg cobalt/m 3 exposure groups, respectively (lower doses were not tested). Females had a significantly increased length of the estrus cycle at 6.7 mg cobalt/m 3 (119% longer compared to control).

Other studies in animals have also reported respiratory le sions and altered respiratory function following inhalation exposure to cobalt. Kyono et al. (1992) observed mild pulmonary lesions in rats exposed to 2.12 mg/m 3 of cobalt aerosols (generated from an aqueous suspension

of ultrafine metallic coba lt particles) 5 hours/day for 4 days. Lesions were characterized by focal hypertrophy of the epithelium, a bnormal macrophages, vacuolizati on of type I epithelial cells and proliferation of type II epit helial cells, which are indicative of an initial inflammatory response. Kerfoot et al. (1975) exposed groups of five miniature swine to 0, 0.1 or 1.0 mg/m 3 of pure cobalt metal powder for 6 hours/day, 5 days/w eek for 3 months. Wheezing was observed in animals from both cobalt groups after 4 weeks of exposure (numeric data not reported). Tidal

volume was decreased to 73% and 64% of controls in the low and high dose groups, respectively, and total respiratory compliance was decreased relative to cont rols (low dose, 66%

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Table 2. Rats with Selected Lesions in th e 13-Week Cobalt Sulfa te Inhalation Study a

Exposure Group (mg Cobalt (Co) per m 3 )

Site Lesion Control 0.067 mg Co/m 3 0.22 mg Co/m 3 0.67 mg Co/m 3 2.2 mg Co/m 3 6.7 mg Co/m 3

Inflammation M: 0

F: 1 M: 2

F: 2 M: 8 c

F: 7 c M: 9 c

F: 10 c M: 9 c

F: 10 c M: 9 c

F: 10 c Larynx

Squamous metaplasia M: 0

F: 1 M: 9 c

F: 7 c M: 10 c

F: 10 c M: 10 c

F: 10 c M: 10 c

F: 10 c M: 10 c

F: 10 c

Inflammation M: 0

F: 0 M: 0

F: 0 M: 6 b

F: 2 M: 10 c

F: 9 c M: 10 c

F: 10 c M: 10 c

F: 10 c

Fibrosis M: 0

F: 0 M: 0

F: 0 M: 0

F: 0 M: 0

F: 1 M: 1

F: 4 b M: 10 c

F: 5 b Lung

Bronchiolar epithelium regeneration M: 0

F: 0 M: 0

F: 0 M: 0

F: 0 M: 0

F: 0 M: 0

F: 0 M: 7 c

F: 5 b

M: number of males with lesions out of 50 animals.

F: number of females with lesions out of 50 animals.

a NTP, 1991; Bucher et al., 1990

b p <0.05 vs controls by Fisher exact test

c p <0.01 vs controls by fisher exact test

of control; high dose, 56% of control). Statistica l significance was not reported. Examination of lung tissue by electron microscopy revealed se pta thickened by colla gen, elastic tissue and fibroblasts in both exposure groups, with mo re pronounced effects in the high dose group. Johansson et al. (1987) exposed rabb its (8/group) to 0.4 or 2 mg cobalt/m 3 as cobalt chloride, 6 hours/day, 5 days/week for 14-16 weeks. Nodular accumulation of alveolar type II cells (8/8 rabbits in both cobalt groups), abnormal accu mulation of enlarged, vaculolated alveolar macrophages (5/8 in the low dose group and 8/8 in the high dose group) and interstitial inflammation (4/8 rabbits in the low dose group and 8/8 rabbits in the high dose group) were observed, with more pronounced effects in the high dose group.

The carcinogenicity of inhaled cobalt was i nvestigated in groups of 50 F344/N rats and 50 B6C3F1 mice of each sex exposed to coba lt sulfate hexahydrate aerosol (MMAD=1.4-1.6

μ m; σ g =2.1-2.2) at concentrations of 0, 0.3, 1 or 3 mg/m 3 (equivalent to 0, 0.067, 0.22 or 0.67 mg

cobalt/m 3 ) 6 hours/day, 5 days/week for 105 weeks (B ucher et al., 1999; NTP, 1998). Animals were monitored for body weight and observed fo r clinical signs duri ng the exposure period. Following termination of exposure, all animals we re sacrificed and necropsied. At necropsy, all organs and tissues were examined for gross le sions, trimmed and examined histologically.

In F344 rats, there were no changes in surviv al or mean body weights in males or females of any exposure group (Bucher et al., 1999; NTP, 1998). Irregular breathing was noticed more frequently in female rats exposed to 0.67 mg cobalt/m 3 than in controls or other treatment groups; no changes in clinical signs were noted in any of the trea ted male rats. Incidence of selected neoplasms and nonneoplastic lesions of the lung in rats is summarized in Table 3. Both male and female rats in all exposure groups showed a high incidence (94% or greater) of squamous metaplasia of the alveolar epitheliu m, fibrosis of the pulmonary interstitium and granulomatous inflammation, with all lesions increasing in seve rity with increasing exposure level. Significant increases in alveolar/bronchi olar adenomas or carcinomas were seen in highdose male rats, while significant increases in alve olar/bronchiolar adenomas or carcinomas were

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Table 3. Incidence of Selected Neoplasms a nd Nonneoplastic Lesions in the Respiratory Tract of Rats in the 2-Year Inhalation Study of Cobalt Sulfate a

Exposure Group (mg Cobalt (Co) per m 3 )

Site Lesion Type Control 0.067 mg Co/m 3 0.22 mg Co/m 3 0.67 mg Co/m 3
Alveolar epithelium hyperplasia M: 9

F: 15 M: 20 b

F: 7 M: 20 b

F: 20 M: 23 c

F: 33 c Alveolar epithelium metaplasia M: 0

F: 2 M: 50 c

F: 47 c M: 48 c

F: 50 c M: 49 c

F: 49 c Inflammation granulomatous M: 2

F: 9 M: 50 c

F: 47 c M: 48 c

F: 50 c M: 50 c

F: 49 c

Alveolar/bronchiolar adenoma M: 1

F: 0 M: 4

F: 1 M: 1

F: 10 c M: 6

F: 9 c

Alveolar/bronchiolar carcinoma M: 0

F: 0 M: 0

F: 2 M: 3

F: 6 b M: 1

F: 6 b

A/B adenoma or carcinoma M: 1

F: 0 M: 4

F: 3 M: 4

F: 15 c M: 7 b

F: 15 c Lung

Squamous cell carcinoma M: 0

F: 0 M: 0

F: 0 M: 0

F: 1 M: 0

F: 1 Lateral wall hyperplasia M: 2

F: 1 M: 14 c

F: 8 b M: 21 c

F: 26 c M: 21 c

F: 38 c Nose

Olfactory epithelium atrophy M: 8

F: 5 M: 24 c

F: 29 c M: 42 c

F: 46 c M: 48 c

F: 47 c
Larynx Squamous metaplasia M: 0

F: 1 M: 10 c

F: 22 c M: 37 c

F: 39 c M: 50 c

F: 48 c

M: Incidence of lesions in male rats out of 50 animals.

F: Incidence of lesions in female rats out of 50 animals.

a Bucher et al., 1999; NTP, 1998

b p <0.05 compared to control by logistic regression test

c p <0.01 compared to control by logistic regression test

seen in the mid- and high-dose female rats. Th e combined incidence of alveolar/bronchiolar neoplasms (adenoma and carcinoma) in male rats a nd female rats was significantly greater than that in control animals, and a si gnificant linear trend occurred in both sexes. Rats of both sexes showed treatment-related increases in hyperpla sia of the lateral nasal wall, atrophy of the olfactory epithelium and squamous metaplasia of the larynx. A signi ficant increase in the incidence of pheochromocytoma in 0.67 mg cobalt/m 3 dosed females was also noted (2/48, 1/49, 4/50 and 10/50 in control, 0.067, 0.22 and 0.67 mg cobalt/m 3 groups, respectively). A marginally increased incidence of pheochromo cytoma in males exposed to 0.22 mg cobalt/m 3

, but not in those exposed to 0.67 mg cobalt/m 3 , was considered by the study authors not to be

related to treatment.

In B6C3F1 mice, no changes in survival we re observed in any exposure group (Bucher et al., 1999; NTP, 1998). Male mice exposed to 0.67 mg cobalt/m 3 showed a decreased mean body weight relative to controls from week 96 thr ough the end of the study (105 weeks). Mean body weights of exposed female mice were generally greater than those of controls throughout the study. Irregular breathing was noted slightly more frequently in female mice exposed to 0.22 mg

cobalt/m 3 than in controls or other exposed group s. Incidence of selected neoplasms and nonneoplastic lesions of the lung in mice is summa rized in Table 4. A dose-related increase in

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Table 4. Incidence of Selected Neoplasms a nd Nonneoplastic Lesions in the Respiratory Tract of Mice in the 2-Year Inhalation Study of Cobalt Sulfate a

Exposure Group (mg cobalt (Co) per cubic meter) Lesion Type Control 0.067 mg Co/m 3 0.22 mg Co/m 3 0.67 mg Co/m 3
Bronchus cytoplasmic vacuolization M: 0

F: 0 M: 18 c

F: 6 b M: 34 c

F: 31 c M: 38 c

F: 43 c

Alveolar/bronchiolar adenoma M: 9

F: 3 M: 12

F: 6 M: 13

F: 9 M: 18 b

F: 10 b

Alveolar/bronchiolar carcinoma M: 4

F: 1 M: 12

F: 1 M: 13

F: 4 M: 18 b

F: 9 c Site

A/B adenoma or carcinoma M: 11

F: 4 M: 14

F: 7 M: 19

F: 13 c M: 28 c

F: 18 c Olfactory epithelium atrophy M: 0

F: 0 M: 0

F: 2 M: 28 c

F: 12 c M: 48 c

F: 46 c Nose

Hyperplasia M: 0

F: 0 M: 0

F: 0 M: 0

F: 0 M: 10 c

F: 30 c
Larynx Squamous metaplasia M: 0

F: 0 M: 37 c

F: 45 c M: 48 c

F: 40 c M: 44 c

F: 50 c

M: Incidence of lesions in male mice out of 50 animals.

F: Incidence of lesions in female mice out of 50 animals.

a Bucher et al., 1999; NTP, 1998

b p <0.05 compared to control by logistic regression test

c p <0.01 compared to control by logistic regression test

the occurrence of cytoplasmic vacuolization of th e bronchus was seen in both sexes of mice, with incidences at all exposure levels being significantly different from controls. As in rats, both sexes of mice showed a significant linear trend toward increased alveolar/bronchiolar tumors, with the 0.67 mg cobalt/m 3 male and the 0.22- and 0.67 mg cobalt/m 3 female groups attaining statistical significance. Mice of both sexes showed significan tly increased incidences of squamous metaplasia of the larynx ( p <0.05) at all exposure levels examined. In male mice, but not in females, the incidence of hemangiosarcoma was significantly elevated in animals exposed to 0.22 mg cobalt/m 3 , but not in other exposure groups (2/ 50, 4/50, 8/50 and 7/50 in the control, 0.067, 0.22 and 0.67 mg cobalt/m 3 groups, respectively).

Wehner et al. (1977, 1979) exposed 2-month-old male Syrian golden hamsters to inhaled cobalt oxide at 0 or 10 mg/m 3 (51 animals/group), 7 hours/day, 5 days/week for approximately

15 months. The incidence of tumors in treated hamsters was not statis tically different from

controls. There was “limited” histopathologic and ultrastructura l examination in the study. No developmental toxicity studies were locat ed following inhalation exposure to cobalt.

Other Studies

Parenteral Administration

Heath (1956) injected groups of 10 male and 20 female rats with a single intramuscular 28 mg dose of powdered cobalt in th e thigh. Injection-site sarcomas appeared in 18 (60%) of the

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treated rats within 5-12 months. Similar result s were observed in Wistar rats by Gilman (1962) and Gilman and Ruckerbauer (1962), with single intramuscular dos es of 20 mg of cobalt oxide

and cobalt sulfide. Cobalt oxid e and cobalt sulfide given intram uscularly at doses twice those used in rats did not induce sarcomas in mice (G ilman and Ruckerbauer, 1962). Shabaan et al. (1977) observed a high incidence of fibrosarcomas in rats given subcutaneous injections of cobalt chloride at 40 mg/kg-day for 10 days. Tu mors developed in 8-12 months. Stoner et al. (1976) tested cobalt acetate in the strain A mous e pulmonary tumor test. Groups of 20 mice/sex received three times per week in traperitoneal injections for a total of 19 cumulative doses of 0, 95, 237 or 475 mg/kg. Survival was high over the 30-week observation period, and the incidence of lung tumors in treated mice was not statistically diffe rent from controls.

Genotoxicity Studies

The genetic toxicity of coba lt was reviewed by Beyersman and Hartwig (1992) and more recently by De Boeck et al. (2003b), Hartwig and Schwerdtle (2002) and Lison et al. (2001). Cobalt compounds have generally tested negative in bacterial mutagenicity assays, with occasional positive results occurring with the ad dition of an exogenous metabolic system. In contrast, cobalt compounds have generally tested positive in yeast and plant cells. In mammalian cell systems, cobalt has been shown to induce DNA strand breaks, sister-chromatid exchanges and morphological cell transformation.

Results of in vitro studies using human peripheral bl ood mononucleated cells show that

cobalt metal and cobalt chloride induced DNA st rand breaks at non-cytotoxi c concentrations (De Boeck et al., 1998, 2003a). Evidence dem onstrating mutagenic activity of cobalt in vivo in humans is lacking. No significant change in DNA strand breaks were ob served in lymphocytes from nonsmoking workers who had been occupationally exposed to cobalt or hard metal dust although a positive association was observed between DNA strand breaks and smoking (De Boeck et al., 2000).

Experimental data in animals provide evidence of genotoxicity following in vivo exposure to cobalt. Single oral exposur e of male Swiss mice to 0, 4.96, 9.92 or 19.8 mg cobalt/kg-day, as cobalt chlori de, resulted in significantly increased percentages of both

chromosomal breaks and chromosomal aberrations in bone marrow cells, with significant linear trends toward increasing aberra tions with increased exposure (P alit et al., 1991a,b,c,d). Thirty hours following single intraperitoneal injection of cobalt chloride at doses of 6.19, 12.4, or 22.3 mg cobalt/kg in BALB/c mice, an increase in micronucleus formation was seen in the mid- and high-dose mice but not in low-dose mice (Suzuki et al., 1993). Single injection of 12.4 mg cobalt/kg resulted in significan tly increased micronucleus form ation at 24 hours post-injection but not at 12, 48, 72 or 96 hours. Pedigo and Vernon (1993) reported that treatment with 400 ppm cobalt chloride (~45 mg cobalt/kg-day, assu ming a water intake of 0.247 L/kg-day for male B6C3F1 mice; U.S. EPA, 1988) in the drinki ng water of male B6C3F1 mice for 10 weeks

resulted in an increase in dominant lethal e ffects as indicated by changes in the number of pregnant females, percentage of live embryos an d number of pre-implantation losses per female.

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DERIVATION OF PROVISIONAL SUBCHRONIC AND CHRONIC

ORAL RfD VALUES FOR COBALT

Indicators of human health effects follo wing oral exposure to cobalt (Co) include increased erythrocyte production and hemogloblin le vels, decreased iodine uptake by the thyroid gland, elicitation of dermatitis in sensitized in dividuals and cardiomyopathy. Observations in humans for effects on the heart, blood and the th yroid gland are supported by results of studies in animals. Other effects, including neurobehavioral , developmental and testicular toxicity were observed in animals and at relatively high doses; these endpoints were not considered further for the development of the subchronic or chronic provional RfD (p-RfD).

Cardiomyopathy was considered as an endpoi nt of concern for cobalt exposure in humans; however, it is probable th at alcohol consumed in “beer -cobalt cardiomyopathy,” as well

as other associated factors such as nutritional defi ciency, played a role in the cardiotoxic effects observed. Therefore, a dose-response relationship could not be determined for cobalt exposure

from these studies. Studies in animals have not ed cardiac effects following cobalt exposure at higher exposure levels than observed in human st udies of “beer-cobalt cardiomyopathy.” On this basis, cardiomyopathy was not selected as the critical endpoint for p-RfD derivation.

Allergic response in cobalt-s ensitized workers was consider ed as a potential critical endpoint for the derivation of an oral p-RfD. However, the available data provide no information on the dose-response relationship of cobalt sensitization, nor is a no observable adverse effect level (NOAEL) for the elicitation of an allergic response in humans defined. Interrelationships also exist betw een cobalt and nickel (Ni) sensitizat ion so that people sensitized by (Ni) may have an allergic reaction followi ng cobalt exposure. A llergic response was, therefore, not chosen as the cri tical effect for p-RfD derivation.

Cobalt has been shown to induce polycythemia wh ich is characterized by an increase in erythrocyte number and hemoglobin levels thro ugh stimulation of erythropoietin, a hormone produced primarily in the kidney. The hematol ogical effects of cobalt treatment have been reported in healthy, non-anemic adu lts (Davis and Fields, 1958) and in anephric anemic dialysis patients (Taylor et al., 1977; Duckham and Lee, 1976). However, the effects observed in healthy adults were reversible and erythr ocyte counts returned to pre-treatm ent levels within 9 to 15 days

after cobalt administration was disc ontinued. In anephric dialysis patients, treatment with cobalt resulted in an increase in hemoglobin from levels clinically described as “a nemic” to levels at or near “normal.” Thus, the effect of cobalt ad ministration in these patients was clinically beneficial. Furthermore, the re sults of this study are difficult to interpret due to confounding factors, including the anephric status of patients and the conc omitant administration of iron. Hematologic effects of cobalt were also found in several studies in rats (Domingo et al., 1984; Krasovskii and Fridlyand, 1971; Murdock, 1959; Holly, 1955, Stanley et al., 1947), supporting the plausibility for the effects observed in hum ans. However, the effects in animals were generally observed at higher doses than that us ed in the Davis and Fiel ds (1958) human study. It is not known whether cobalt exposure in human s at higher dose levels would increase erythrocytes sufficiently above nor mal physiological levels to signi ficantly increase the risk of cardiovascular effects. Therefore, polycythemia was not chosen as the critical effect for p-RfD

derivation.

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Effects of cobalt on thyroidal iodine uptake were identified as an endpoint of concern in humans, based on a preliminary report by Roche a nd Layrisse (1956). This report showed that oral exposure to cobalt (1 mg cobalt/kg-day) for 2 weeks markedly inhibited radioactive iodine uptake in the human thyroid. In a smaller human clinical study, reduced iodine uptake was reported in 2 of 4 euthyroid patients exposed to 0.54 mg cobalt/kg-day by the oral route for up to 14 days (Paley et al., 1958). A confounding factor in this study is that on e of the two subjects reported to have reduced iodine uptake had received intravenous (i.v.) cobalt in addition to oral cobalt intake. The i.v. loading dose regimen may ha ve raised the internal concentration of cobalt to a level greater than the estimated 0.54 mg c obalt/kg-day based on oral intake alone, rendering the Paley et al. (1958) study in appropriate for consideration. Importantly, long-term cobalt exposure (up to 7 months) at 2-4 mg/kg-day in an emic children has been reported to cause goiter (Gross et al., 1954; Kriss et al., 1955; Little and Sunico, 1958). Th erefore, while reduced iodine uptake is reported in humans following short-term exposures at low doses (Roche and Layrisse, 1956; Paley et al., 1958), potential ly more severe thyroid lesion s may occur as a function of

increased duration or dose. Based on observation s from rodent models of cobalt exposure, the severity of thyroid toxicity appe ars to be related to duration of exposure. Indeed, necrosis and inflammation of the thyroid has been reporte d in mice exposed to approximately 48 mg cobalt/kg-day with an in crease in severity over a period of 15-45 days (Shrivastava et al., 1996).

Subchronic provisional RfD

Although cobalt exposure induces decreased radioactive iodi ne uptake in the thyroid (Roche and Layrisse, 1956), and polycythemia (D avis and Fields, 1958) in humans at similar daily exposure levels (1 mg/kg- day and 0.97 mg/kg-day, respectively) , thyroid toxicity is chosen

as the critical effect for de rivation of provisional oral reference values. Cobalt-induced polycythemia and decreased iodine uptake by the thyroid were reversible following relatively short-term exposure in humans, however suppor ting studies indicate the potential for more severe thyroid effects (e.g., Kri ss et al., 1955). The point of depa rture (POD) of 1 mg cobalt/kgday for decreased iodine uptake in human t hyroid is the LOAEL; dividing this POD by a composite uncertainty (UF) of 300 yields a subchronic p-RfD of 3E-3 mg/kg-day as follows:

Subchronic p-RfD = LOAEL ÷ UF = 1 mg/kg-d ÷ 300

= 0.003 or 3E-3 mg/kg-day

The composite UF of 300 is composed of three un certainty factors: An UF of 10 for LOAEL to NOAEL extrapolation was applied because the P OD is based on a LOAEL. An UF of 10 was applied due to the lack of data regarding inter-individual human variability or information on sensitive subpopulations. Specifically, because th e critical study (Roche and Layrisse, 1956) for oral cobalt was based on healthy (euthyroid) adults, an UF of 10 was applied to protect sensitive human populations. The available database includes several short-term human studies, multiple developmental studies in animals and animal studies investigating he matological, cardiac, neurological, neurobehavioral, and thyroid endpoints. The lack of a multi-generation reproductive toxicity study is of particular concern because the database includes several animal studies indicating effects on sper m function and testicul ar degeneration which raises concerns

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that cobalt exposure may affect reproductive capability. Th erefore, an UF of 3 was applied to account for lack of a multi-generation toxicity study.

Chronic provisional RfD

Using the same LOAEL of 1 mg/kg-day for d ecreased iodine uptake in humans, and an additional UF of 10 for extrapolating from s ubchronic to chronic dura tion (composite UF of 3000), a chronic p-RfD of 3E-4 mg/kg-day is derived as follows:

p-RfD = LOAEL ÷ UF = 1 mg/kg-d ÷ 1000

= 0.0003 or 3E-4 mg/kg-day

An UF of 10 for extrapolation from subchroni c to chronic duration wa s applied because the

critical effect was chosen from a principal study of a relatively s hort duration (2 weeks) of oral exposure in humans. The temporal relationshi p between cobalt-induced decreased radioactive iodine uptake and more severe thyroid toxicity should be consid ered carefully. One postulated temporal relationship is that chronic exposure may have no greater effect than that resulting from short-term exposure, because if the precursor even t of inhibition of iodine uptake does not occur, then there may be no change in thyroid functi on in the short- or long-term. Prolonged cobaltexposure could have less of an effect because of the compensatory response of the pituitarythyroid axis to iodine deficien cy, via increasing iodine uptake. However, although plausible, there are no data to suggest that this postulate d temporal relationship exist for cobalt-induced thyroid toxicity. Indeed, a limited number of clinical observations primarily in children exposed to oral cobalt at doses of 2-4 mg/kg-day for up to 7 months suggest the potential for more severe thyroid toxicity (e.g., Kriss et al ., 1955). In addition, cobalt may not be readily eliminated from the body; for example, the biologic al half-life of cobalt chloride in rats is 25 hours (Rosenberg, 1993). Therefore, an UF of 10 for extrapolat ion from subchronic to chronic duration was applied.

Confidence in the principal study is low- to-medium. Roche and Layrisse (1956) examined twelve subjects over a two-week expo sure period. Since only a single dose level was evaluated, a NOAEL for decreased iodine uptake was not identified. Other human and animal studies support the plausibility of cobalt producing thyroid toxicity (Paley et al., 1958; Prescott et al., 1992; Shirivistava et al., 1996) . Confidence in the database is low-to-medium. Although some studies (Gross et al., 1954; Kriss et al., 195 5; Little and Sunico, 1957) of longer duration reported increased severity of thyroid effects (e .g., goiter) in children exposed to cobalt at higher doses (2-4 mg cobalt/kg-day), critical details of these studies are unavailable for assessment. Therefore, a temporal relationship between pr olonged oral cobalt e xposure and increased severity of thyroid effects in humans (or experime ntal animals) is not cl ear, based upon available data. As such, a low confidence in the provi sional subchronic and chronic RfDs results.

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DERIVATION OF PROVISIONAL SUBCHRONIC AND CHRONIC

INHALATION RfC VALUES FOR COBALT

The human and animal database indicates th at respiratory effects are sensitive endpoints of inhaled cobalt. Symptoms of respiratory tr act irritation and altered pulmonary function have been widely reported in workers exposed to coba lt-containing airborne media. Of the four human epidemiology studies discussed above, th e study by Nemery et al. (1992) provides the strongest basis for derivation of a provisional RfC (p-RfC). Work ers in this study were exposed

to lower air concentrations of metallic cobalt dust than in the studies by Gennart and Lauwerys (1990), Prescott et al. (1992) and Swennen et al. (1993). The valu es obtained from personal air samples from the Nemery et al. (1992) study, indicate a NOAEL of 5.3 μ g/m 3 and a LOAEL of

15.1 μ g/m 3 . Furthermore, the Nemery et al. (1992) study demonstrated a dos e-effect relationship on lung function which correlated with urinary co balt-levels, after adju sting for effects of smoking and gender.

Animal data support the conclusion that the re spiratory tract is the critical target for inhaled cobalt (NTP, 1991; Bucher et al., 1990; Wehner et al., 1977). Subchronic and chronic inhalation exposure to cobalt resulted in inflam mation, fibrosis, and bronchiolar regeneration in all regions of the respirator y tract in both rats and mice (NTP, 1991, 1998; Bucher et al., 1990, 1999) at doses higher than those identified in the Nemery et al (1992) study. The NTP (1991) study further demonstrated that cobalt can produ ce testicular effects in male mice following inhalation exposure, but the eff ects were produced only at high dos e levels. Oral studies have also identified the testes as a target for coba lt toxicity. Multi-gene ration reproduction studies following inhalation or oral expos ure to cobalt are not available. Although developmental toxicity studies following inhala tion exposure to cobalt are not av ailable, oral studies provide evidence that high oral doses of cobalt may produ ce developmental effects in animals (Szakmary et al., 2001; Paternain et al., 1988; Domingo et al., 1985).

Decreased pulmonary function and respiratory tract irritation were identified as the cocritical effects for derivation of the subchroni c and chronic p-RfCs. Assuming the personal air samples to be more representative of worker e xposure than the area air samples, the study by Nemery et al. (1992) identified a NOAEL of 5.3 μ g/m 3 and a LOAEL of 15.1 μ g/m 3 for metallic cobalt for effects on pulmonary function (e.g. forced expiratory volume (FEV), forced vital capacity (FVC) and forced expiratory flow [refe rred to as MMEF]) and an increased prevalence of symptoms of respiratory tr act irritation (e.g. nose/throat ir ritation, cough, phlegm, dyspnea). Although the LOAEL may be biased low due to inclusion of data from workshop #9, this does not affect the p-RfC derivation. A NOAEL/LOAEL approach is taken for the derivation of

inhalation RfC values because the critical eff ect data are not amenable to benchmark dose modeling. For example, workers in the low c obalt exposure group experienced a slight but nonstatistically significant increase in ventilator y function compared to controls, whereas a significant decrease in ventil atory function was observed in the high cobalt exposure group compared to both the control and the low c obalt exposure groups. The NOAEL for occupational exposure was adjusted to c ontinuous exposure as follows:

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5.3 μ g/m 3 (10 m 3 /day / 20 m 3 /day) (5 days / 7 days) = 1.9 μ g/m 3

Using the NOAEL ADJ of 1.9 μ g/m 3 as the POD, the subchronic p-RfC and chronic p-RfC for

cobalt was derived as shown below.

Subchronic p-RfC

Dividing the NOAEL ADJ of 1.9 μ g/m 3 by a composite UF of 100 yields a subchronic pRfC of 2E-5 mg/m 3 for metallic cobalt as follows:

Subchronic p-RfC = NOAEL ADJ ÷ UF

= 1.9 μ g/m 3 ÷ 100

= 0.00002 or 2E-5 mg/m 3

The composite UF of 100 is composed of two uncer tainty factors: 10 for da tabase insufficiencies and 10 for inter-individual variability. Nemery et al. (1992) did not repo rt exposure duration for any worker in this study; an assumption is made that worker exposure was at least of subchronic duration. A factor of 10 was applied to account fo r database insufficiencies due to the lack of inhalation developmental toxicity studies and a multi-generation reproduction study. A factor of 10 was applied to account for human variability, including sensitive subgroups. Individuals with underlying respiratory diseases (a sthma, chronic obstructive pulmonary disease) may be more sensitive to the respiratory effects of inhale d cobalt. This subchronic p-RfC may not be protective for people with hypersensitivity to cobalt.

Chronic p-RfC

Dividing the NOAEL ADJ of 1.9 μ g/m 3 by a composite UF of 300 yields a chronic p-RfC of 6E-6 mg/m 3 for metallic cobalt as follows:

Chronic p-RfC = NOAEL ADJ ÷ UF

= 1.9 μ g/m 3 ÷ 300

= 0.000006 or 6E-6 mg/m 3

The composite UF of 300 is composed of three uncer tainty factors: 3 to account for extrapolating from an assumed subchronic exposure duration to a chronic exposure duration, 10 for database

insufficiencies and 10 for human in ter-individual variability. A fact or of 3 is applied to account for extrapolating from an assumed subchronic to chronic exposure duration. Since Nemery et al. (1992) did not report duration for any worker in this study, it is possible that exposure duration may have been subchronic or l onger for some workers. A factor of 10 is applied to account for

database insufficiencies due to the lack of i nhalation developmental toxicity studies and a multigeneration reproduction study. A factor of 10 is applied to account for human variability, including sensitive subgroups. Individuals with underlying respiratory diseases (asthma, chronic obstructive pulmonary disease) may be more sensitive to the respiratory effects of inhaled cobalt. This chronic p-RfC may not be protective for people with hypersensitivity to cobalt.

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Confidence in the key study (Nemer y et al., 1992) is low because this cross-sectional study:

• looked at only respiratory endpoints;

• included a control group that was studie d more than 1 year after the exposed population;

• included a study group exposed to iron a nd diamond dust in addition to cobalt (and possibly to asbestos in the past);

• did not report durati on of exposure; and

• encountered a number of procedural difficulties during its course (e.g., construction of control group).

Confidence in the database is medium. The choi ce of the critical endpoint is well supported by

other studies in humans and animals. Subchroni c exposure studies in rats and mice (NTP, 1991) found histopathological changes in the upper respiratory tract. Other studies in animals support these findings. Reproductive and developmental effects have not been adequately studied. Furthermore, oral studies reported large doses were required to produce reproductive or developmental effects. It woul d be difficult to get a large eno ugh internal dose via inhalation to produce these effects. For these reasons, there is medium-to-low confidence in the subchronic and chronic p-RfCs.

PROVISIONAL CARCINOGENICITY ASSESSMENT FOR COBALT

Weight-of-Evidence Descriptor

Under the 2005 Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005a), cobalt sulfate (soluble) is described as “likely to be carcinogenic to humans by the inhalation route,” based on both the limited evidence of carcinogeni city in humans and sufficient evidence of carcinogenicity in animals as shown by a statis tically significant in creased incidence of

alveolar/bronchiolar tumors in both sexes of rats and mice, pheoc hromocytomas in female rats, and hemangiosarcomas in male mice (Bucher et al., 1999). While availabl e studies in humans have suggested a possible association between exposure to cobalt and respiratory tumors in cobalt workers (Tuchsen et al., 1996; Mur et al ., 1987; Morgan et al., 1983), limitations within these studies, including small numbers of subject s, inadequate exposure assessment and potential exposure to other chemicals make them inadequa te for assessing the carcinogenic potential of

cobalt. Studies for evaluation of the oral carc inogenic potential for cobalt were not located.

Mode-of-Action Discussion

The U.S. EPA (2005a) Guidelines for Carci nogen Risk Assessment defines mode of action as “a sequence of key events and processes, starting with the interaction of an agent with a cell, proceeding through operational and anatomical changes, and resulting in cancer formation.” Examples of possible modes of carcinogenic actio n, in general, include mutagenic, mitogenic, anti-apoptotic (inhibition of programmed cell deat h), cytotoxic with repa rative cell proliferation, and immunologic suppression.

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While the mode of action of cobalt-induced carcinogenicity has not been determined, data suggests a number of potential biological events that might be involved including nonmutagenic genotoxicity (e.g. clas togenicity). A recent review by Lison et al. (2001) of in vitro

and in vivo experiments in animal models indica tes that two different mechanisms of genotoxicity may contribute to the carci nogenic potential of cobalt compounds: DNA strand breakage and inhibition of DNA re pair. DNA strand breaks have b een reported at non-cytotoxic concentrations in human peripheral blood monocytes (De Boeck et al., 1998, 2003a). Furthermore, oral exposure of mice to cobalt ch loride resulted in si gnificantly increased percentages of both chromosomal breaks and ch romosomal aberrations in bone marrow cells (Palit et al., 1991a,b,c,d). Mechan istic studies suggest cobalt-induced oxida tive stress may be involved. Exposure to cobalt compounds increase s indices of oxidative stress, including diminished levels of reduced glutathione, increased levels of oxidized glutathione, increased levels of oxygen radicals and increased free-ra dical-induced DNA damage (Kawanishi et al., 1994; Lewis et al., 1991; Kadiiska et al., 1989; Zhang et al., 1998; Moorehouse et al., 1985). To compound the potential DNA strand br eaking effects of cobalt, it a ppears that cobalt may also inhibit the repair of such genetic damage. A review by Hartwig and Schwerdtle (2002) concluded that cobalt may specifi cally target zinc finger struct ures in DNA repair proteins, interfering with base and nucleot ide excision repair. Collectively, while data indicate that cobalt induces DNA damage and repair inhibition, there is weak evidence to suggest direct or indirect mutagenicity in bacterial or mammalian systems.

Potential for a Mutagenic Mode of Action

Key events

The precise mechanism of cobalt- induced carcinogenicity has not been fully determined. There is evidence that cobalt is capable of elic iting genotoxic effects. While evaluations for mutagenic effects in bacteria have generally yielded negative result s, results in several mammalian cell systems have suggested that coba lt is genotoxic in mammalian cells. Limited

data from in vivo animal studies show that cobalt induces genotoxic effects, including chromosomal breaks, chromosomal aberrations and micronucleus formation. The most likely mechanisms for the genotoxic effects of coba lt are DNA strand breakage and the inhibition of DNA repair.

Strength, consistency, speci ficity of association

Although the carcinogenic potential of inhaled cobalt has been demonstrated in rats and mice by increased incidence of alveolar/bronchi olar tumors (Bucher et al., 1999; NTP, 1998), direct evidence demonstrating that cobalt ca n induce mutagenic cha nges in cells of the respiratory tract is lacking. In vivo exposure to hard metal dust containing 6.3% cobalt, 84% tungsten and 5.4% carbon induced DNA strand break s in rat type II epith elial lung cells (De Boeck et al., 2003c). Chromosome/genome mu tations were observed within 12 hours of exposure to a single intratracheal instillation of 16.6 mg hard metal dust/kg body weight. Since the mutagenic potential of cobalt alone was not evaluated in this study, a causal relationship between type II epithelial cell mutations and cobalt exposure could not be es tablished. Potential mutagenic changes in respiratory tract cells co uld also be mediated through activated oxygen

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species released by inflammatory cells (e.g., macrophages, polymorpohnuclear neutrophils), rather than directly by cobalt (Lison et al., 2001).

Dose-response concordance

A dose-response concordance has not been established between the development of bronchoalveolar tumors and mutagenesis followi ng inhalation exposure to cobalt. Doseresponse information on mutagenicity is availabl e for acute oral and parenteral exposure to cobalt in mice (Suzuki et al., 1993; Palit et al ., 1991a,b,c,d). No carc inogenicity data are available for the oral or parent eral routes upon which to base a dose-response concordance. Furthermore, no data are available on the mutage nic potential of cobalt in respiratory tract cells following in vitro or in vivo exposure.

Temporal relationships

In vivo studies in animals show that acute oral and parenteral expos ure to cobalt produces genotoxicity to bone marrow cells (Suzuki et al., 1993; Palit et al ., 1991a,b,c,d). Due to the lack of data on the mutagenic potential of cobalt in respiratory tract cells, the temporal relationship between potential mutagenic mechanisms and the development of bronchoalveolar tumors cannot be assessed. Development of lung tumo rs in animals exposed to cobalt occurred following chronic exposure (NTP, 1998).

Biological plausibility and coherence

In vivo mutagenicity studies in mice show that or al and intraperitoneal exposure to single doses of cobalt chloride induced mutagenic ch anges in bone marrow cells (Suzuki et al., 1993; Palit et al., 1991a,b,c,d). Although it has been hypot hesized that the bronchoalveolar tumors are the result of genotoxicity (De Boeck et al., 2003b; Hartwig an d Schwerdtle, 2002; Lison et al., 2001), no direct evidence is available linking coba lt-induced mutagenesis to the development of cancer. Carcinogenicity through an indirect mu tagenic mode of action may be mediated by activated inflammatory cells (macrophages, pol ymorpohnuclear neutrophils ) (Lison et al., 2001).

Other Potential Mode(s) of Action: Cy totoxicity and Cellular Regeneration

Subchronic and chronic inhalation studies (Bucher et al., 1990, 1999; NTP, 1991, 1998) in rodents provide some evidence that cobalt cau ses cell injury with subsequent reparative cell proliferation, which may be involved in the development of bronchoalveo lar tumors. Following inhalation exposure to cobalt su lfate hexahydrate aerosol at c oncentrations of 0.3 to 30 mg/m 3
(equivalent to 0.067 to 6.7 mg cobalt/m 3 ) for 3 months, rats and mi ce developed several lesions indicative of cell damage and prol iferation throughout the entire respiratory tr act, including nasal epithelial degeneration and metaplasia, laryngeal inflamma tion and metaplasia, bronchiolar epithelial regeneration a nd ectasia, alveolar hyperplasia and l ung fibrosis (NTP, 1991; Bucher et al., 1990). Squamous hyperplasia of the larynx was the most sensitive effect (LOAEL=0.067 mg

cobalt/m 3 ). The results of the 2-year carcinogene sis study (Bucher et al., 1999; NTP, 1998) in

rats and mice revealed a stat istically significant increase in combined alveolar/bronchiolar

adenomas and carcinomas in the 0.67 mg cobalt/m 3 group, but not in the 0.067 and 0.22 mg

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cobalt/m 3 groups for male rats and mice. In female rats and mice, a st atistically significant

increase in combined alveolar /bronchiolar adenomas and car cinomas was observed in the 0.22 and 0.67 mg cobalt/m 3 groups, but not in the 0.067 mg cobalt/m 3 group. In this same study, granulomatous inflammation of the lung was obs erved at all exposure levels (0.067, 0.22 and 0.67 mg cobalt/m 3 ) in rats. Other markers of cell da mage and proliferation, including hyperplasia, metaplasia and fibrosis, we re observed in the 0.22 and 0.67 mg cobalt/m 3 exposure groups. Compared to rats, mice appeared to be less sensitive to cobalt-induced cytotoxic changes. Results of this study show that broncho alveolar tumors develop at exposure levels that also produce cell damage and reparative proliferat ion, although cell damage and repair are also observed at lower exposure levels than tumorigene sis. These observations suggest the possibility that cell injury in the respiratory tract may have preceded the development of cancers although direct evidence for this assertion is lacking.

Although limited evidence of carcinogenicity in humans is available, results of several epidemioloic studies suggest a possible associa tion between exposure to cobalt and respiratory tumors (Tuchsen et al., 1996; Mur et al., 1987; Morgan, 1983). Subchronic exposure studies in

cobalt workers show an associa tion between cobalt exposure and diminished pulmonary function (Nemery et al., 1992; Gennart and La uwerys, 1990). Taken together, results of studies in rodents and humans suggest that inhaled cobalt may produce a cytotoxic response in the respiratory tract that may contribute to decreases in pulmonary function and the development of bronchoalveolar

tumors.

Sustained cell proliferati on, in response to cytotoxicity, can be a significant risk factor for cancer (Correa, 1996). Sustained cytotoxicity and regenerative cell proliferation may result in the perpetuation of mutations (spontaneous or dir ectly or indirectly i nduced by the chemical), resulting in uncontrolled growth. It is also possible that conti nuous proliferation may increase the probability that damaged DNA will not be repa ired. No data on cobalt are available to directly evaluate the relations hip between cell damage and repa rative proliferation and the development of bronchoalveolar tumors.

Conclusions Regarding Cancer Mode of Action

Limited evidence supports genotoxicity a nd cytotoxicity followed by cellular regeneration as potentia l modes of action for cobalt tumorigenicity. In vitro and in vivo studies provide evidence that cobalt is capable of eliciting genotoxic effects in mammalian cells; however, two key uncertainties remain:

(1) No direct evidence linking cobalt-induced mu tagenesis to the development of cancer is available and (2) the mutagenic potential of cobalt in respiratory cells has not been evaluated.

Results of the 3-month and 2-year inhalation studies in rats and mice (Bucher et al., 1990, 1999; NTP, 1991, 1998) are also consistent with th e hypothesis that cobalt acts through a mode of action involving cytotoxicity and cellular regeneration, based on the observations that these effects occur following subchroni c exposure and bronchoalveolar tumors develop at exposure levels that produce cytotoxicity and reparative proliferation. These obs ervations suggest the possibility that cell injury in the respiratory tract may have preceded the development of cancers

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although direct evidence for this assertion is lacking. No mode of action data are available to explain the statistically significant increases in the incidences of pheochromocytomas and hemangiosarcomas that were observed in female rats and male mice, respectively.

Because a mutagenic mode of action is plausi ble, but cannot be clearly established for carcinogenicity of inhaled cobalt, it is recommended that an age-de pendent adjustment factor not be applied to the unit risk to account for possible age-dependence of carcinogenic potency as described in U.S. EPA (2005b).

Quantitative Estimates of Carcinogenic Risk

Oral Exposure

Human or animal studies examining the carc inogenicity of cobalt following oral exposure were not located. Therefore, derivation of an oral slope factor is precluded.

Inhalation Exposure

As available human inhalation studies were not sufficiently detailed, particularly with regards to analysis of exposure, the NTP (1998; Bucher et al., 1999) 2-year carcinogenicity study in rats and mice was chosen as the principal st udy for the derivation of an inhalation unit risk, based on the dose-response relationship for sta tistically significant increased incidences of alveolar/bronchiolar (A/B) neoplasms (ade noma and carcinoma). Although statistically significant increases in the incidences of phe ochromocytomas and hemangiosarcomas were observed in female rats and male mice, respectiv ely, these tumors were not considered for the derivation of the inhalation unit risk because a higher and more consistent response across species was observed for alveolar/b ronchiolar tumors. The exposur e concentrations in this study were adjusted to continuous exposure as follows:

[ ] 

### Conc Conc days week

days week hours day

hours day ADJ 

### =× × 5

7 6

24 /

/ /

/

This adjustment resulted in duration-adju sted concentrations of 0, 0.012, 0.040 and 0.120 mg

cobalt/m 3 , respectively, for exposure to cobalt su lfate hexahydrate at 0.0, 0.3, 1.0 and 3.0 mg/m 3
exposure levels. Using the RDDR computer prog ram, as specified in the RfC guidelines (U.S. EPA, 1994b), human equivalent concen trations (HECs, in mg cobalt/m 3 ) were calculated at each exposure level for each species and sex using bod y weight default values (U.S. EPA, 1994b), assuming exposure to particulates (MMAD=1.5 μ m, σ g =2.2) with effects occurring in the thoracic region of the respiratory trac t. Table 5 shows the resulting HECs.

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Table 5. Human Equivalent Co ncentrations (mg Cobalt/m 3 ) Corresponding to Exposure Concentrations in the NTP (1998; Bucher et al., 1999) Chronic Cancer Bioassay

Study Male Rat Female Rat Male Mouse Female Mouse

RDDR Multiplier 0.83 0.79 1.48 1.44

Control 0 0 0 0

Low 0.010 0.0095 0.018 0.017

Medium 0.033 0.032 0.059 0.058

High 0.10 0.095 0.18 0.17

All models for quantal data in the U.S. EPA Benchmark Dose (BMD) software (version 1.3.2) were fit to incidence for tumors (combine d A/B adenomas and carcinomas), in rats and mice; males and females were modeled separately. All data sets modeled showed a statistical

trend for increased tumor incidence with increas ing exposure concentration. In accordance with the U.S. EPA (2000) BMD methodology, the de fault benchmark response (BMR) of 10%

increase in extra risk was used as the basis fo r the BMD, with the BMDL represented by the 95%

lower confidence limit on the BMD. Models were run using the defa ult restrictions on parameters built into the BMD software. Ta ble 6 shows the exposur e concentration and

incidence data that were modeled.

Table 7 summarizes the BMD modeling resu lts. BMDLs shown were derived from acceptable model fits ( p >0.5). As is shown in Table 7, BM DLs were similar across study groups (range: 0.011-0.035 mg/m 3 ). Lung tumors in female rats were chosen as the endpoint for use as a point of departure for derivati on of the inhalation unit risk. The BMDL for this endpoint was the lowest for all study groups (i.e., male and fe male rats and mice) and was based on a model that showed a good fit to the data ( p =0.84), as reflected in the pr oximity of the BMDL to the BMD, after dropping the high exposure gr oup. Dropping the high exposure group is recommended according to U.S. EPA (2000) procedure when no models achieve adequate fit using all exposure levels. Although this left only two exposure levels (in addition to the control), these exposure levels are in the low-dose portionof the curve within the region of the doseresponse relationship in which response is incr easing with exposure leve l (i.e., the region of interest for deriving the point of departure) a nd bracket the derived BMD. Appendix A presents

the results from all model runs used to support this toxicity assessment.

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Table 6. Neoplasm Incidence Observed in the NTP (1998; Bucher et al., 1999) Chronic Cancer Bioassay

Animal/Strain/Site Incidence of Neoplasms

Human Equivalent Concentration of Cobalt (mg/m 3 )

F-344 Rats (male) 0 0.010 0.033 0.10

Lung: A/B adenoma or carcinoma 1/50 4/50 4/48 7/50

Human Equivalent Concentration of Cobalt (mg/m 3 )

F-344 Rats (female) 0 0.0095 0.032 0.095

Lung: A/B adenoma or carcinoma 0/50 3/49 15/50 15/50

Human Equivalent Concentration of Cobalt (mg/m 3 )

B6C3F1 Mice (male) 0 0.018 0.059 0.18

Lung: A/B adenoma or carcinoma 11/50 14/50 19/50 28/50

Human Equivalent Concentration of Cobalt (mg/m 3 )

B6C3F1 Mice (female) 0 0.017 0.058 0.17

Lung: A/B adenoma or carcinoma 4/50 7/50 13/50 18/50

Table 7. Summary of BMD Modeling Results for Cobalt Cancer Data

Tumor Species Sex BMD (mg/m 3 ) BMDL (mg/m 3 )

Lung: A/B adenoma or carcinoma rat male 0.085 0.035

Lung: A/B adenoma or carcinoma rat female 0.014 a 0.011 a

Lung: A/B adenoma or carcinoma mouse male 0.026 0.015

Lung: A/B adenoma or carcinoma mouse female 0.038 0.023

a Based on control, low and middle exposure levels; high exposure level was dropped due to failure of models to achieve adequate fit using all exposure levels.

In the absence of mode of action data to in form the low dose extrapolation for cobalt, an

inhalation cancer unit risk was calculated by linear extrapolation of the BMDL to zero exposure level (U.S. EPA, 2005a). The provisional inhalation unit risk of 9 (mg/m 3 ) -1 for cobalt sulfate (soluble) was calculated as follows:

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Provisional Unit Risk = BMR / BMDL

= 0.1 / 0.011

= 9 (mg/m 3 ) -1

Table 8 shows continuous life-tim e exposure concentrations that correspond with specified risk levels (i.e., 1x10 -4 , 1x10 -5 , 1x10 -6 ).

Table 8. Continuous Life-time Exposure Concentrations Corresponding to Specified Cancer Risk

Exposure Concentration at 1x10 -4 Risk 1.1x10 -5 mg/m 3

Exposure Concentration at 1x10 -5 risk 1.1x10 -6 mg/m 3

Exposure Concentration at 1x10 -6 Risk 1.1x10 -7 mg/m 3

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APPENDIX A: SUMMARY OF BMD MODELING OF TUMOR INCIDENCE DATA IN

MALE AND FEMALE RATS AND MICE (NTP, 1998; BUCHER ET AL., 1999)

Male rat – A/B adenoma or carcinoma : All models show acceptable fit (p > 0.1) Log-logistic model yielded best fit (highest p-value and lowest AIC) Best estimate of BMDL = 0.035 mg/m 3

Model p AIC BMD BMDL mg/m 3 mg/m 3
gamma (power > 1) 0.502 111.12 0.087 0.043 logistic 0.446 111.52 0.099 0.066 log logistic (slope > 1) 0.510 111.07 0.085 0.035 2 degree polynomial (pos betas) 0.502 111.12 0.087 0.043 1 degree polynomial (pos betas) 0.502 111.12 0.087 0.043 probit 0.453 111.47 0.098 0.063 log probit (slope > 1) 0.357 112.11 0.104 0.064 quantal linear 0.502 111.12 0.087 0.043 quantal quadratic 0.373 111.99 0.010 0.069 weibull (power > 1) 0.502 111.12 0.087 1.043

Output from BMD v1.3.2 is shown below:

======================== ==================================== ======== Logistic Model $Revisi on: 2.1 $ $Date: 2000/02/26 03:38:20 $ Input Data File: C:\PROJE CTS\COBALT\BMDS\RAMALULOG.(D) Gnuplot Plotting File: C :\PROJECTS\COBALT\BMDS\RAMALULOG.plt Fri Sep 09 11:46:38 2005

======================== ==================================== ========

BMDS MODEL RUN

~~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~~~~~~~

The form of the pr obability function is

P[response] = background+(1-backgr ound)/[1+EXP(-intercept -slope*Log(dose))]

Dependent variable = INRM Independent variable = ECRM Slope parameter is restricted as slope > 1

Total number of observations = 4 Total number of records with missing values = 0

Maximum number of iterations = 250 Relative Function Converge nce has been set to 1e-008 Parameter Convergence has been set to 1e-008

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User has chosen the log transformed model

Default Initial Parameter Values background = 0.02 intercept = 0.683504 slope = 1

Asymptotic Correlation Matrix of Parameter Estimates

( *** The model parameter(s) -slope

have been estimated at a boundary point or have been specified by the user and do not appear in the correlation matrix )

background intercept

background 1 -0.63

intercept -0.63 1

Parameter Estimates

Variable Estimate Std. Err. background 0.0398603 0.0231667 intercept 0.272287 0.592931 slope 1 NA

NA - Indicates that this parameter has hit a bound implied by some inequality constraint and thus

has no standard error.

Analysis of Deviance Table

Model Log(likelihood) Deviance Test DF p-value

Full model -52.8567

Fitted model -53.5353 1.35715 2 0.5073

Reduced model -55.5862 5.45902 3 0.1411

AIC: 111.071

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Goodness of Fit

Scaled Dose Est._Prob. Expected Observed Size Residual

---------------- -------------------------------------------------------- 0.0000 0.0399 1.993 1 50 -0.7178

0.0100 0.0523 2.615 4 50 0.8797

0.0330 0.0797 3.827 4 48 0.09207

0.1000 0.1513 7.565 7 50 -0.2228

Chi-square = 1.35 DF = 2 p-value = 0.5099

Benchmark Dose Computation

Specified effect = 0.1

Risk Type = Extra risk

Confidence level = 0.95

BMD = 0.0846262

BMDL = 0.0394914

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0 0.05

0.1 0.15

0.2 0.25

0.3

0 0.02 0.04 0.06 0.08 0.1Fraction Affected

dose Log-Logistic Model with 0.95 Confidence Level

09:11 09/10 2005 BMDL BMD Log-Logistic

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Female rat – A/B adenoma or carcinoma : Most models showed poor fit (p < 0.05) with hi ghest exposure level included (no increase in incidence at the highe st exposure level. The log-logistic model showed the best fit ( p =0.11, lowest AIC)

Model p AIC BMD BMDL mg/m 3 mg/m 3
gamma (power > 1) 0.025 155.21 0.018 0.043 logistic 0.000 167.02 0.045 0.036 log logistic (slope > 1) 0.090 152.86 0.015 0.011 2 degree polynomial (pos betas) 0.025 155.21 0.018 0.014 1 degree polynomial (pos betas) 0.025 155.21 0.018 0.014 probit 0.000 166.25 0.042 0.033 log probit (slope > 1) 0.000 166.51 0.032 0.023 quantal linear 0.025 155.21 0.018 0.014 quantal quadratic 0.000 170.16 0.052 0.040 weibull (power > 1) 0.025 155.21 0.018 0.014

Omitting the data from the highest exposure level improved fit of all models (p > 0.1) Log-probit model yielded best fit (h ighest p-value and lowest AIC) Best estimate of BMDL=0.011 mg/m 3

Model p AIC BMD BMDL mg/m 3 mg/m 3
gamma (power > 1) 1.000 87.66 0.013 0.0077 logistic 0.242 89.69 0.020 0.0164 log logistic (slope > 1) 1.000 87.66 0.013 0.0071 2 degree polynomial (pos betas) 0.710 87.66 0.014 0.0077 1 degree polynomial (pos betas) 1.000 86.40 0.011 0.0073 probit 0.289 89.31 0.019 0.0152 log probit (slope > 1) 0.843 85.98 0.014 0.0110 quantal linear 0.710 86.40 0.011 0.0073 quantal quadratic 0.535 86.70 0.017 0.0139 weibull (power > 1) 1.000 87.66 0.014 0.0077

Output from BMD v1.3.2 (all data included) is shown below:

======================== ==================================== ======== Logistic Model $Revisi on: 2.1 $ $Date: 2000/02/26 03:38:20 $ Input Data File: C:\PROJECTS\ COBALT\BMDS\RAFELU\R AFELULOGLOG.(D) Gnuplot Plotting File:

C:\\PROJECTS\COBALT\BMDS\ RAFELU\RAFELULOGLOG.plt

508-25-2008

Fri Sep 09 16:34:38 2005

======================== ==================================== ========

BMDS MODEL RUN

~~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~~~~~~~

The form of the pr obability function is

P[response] = background + (1-bac kground)/[1+EXP(-intercept-slope*Log(dose))]

Dependent variable = INRF Independent variable = ECRF Slope parameter is restricted as slope > 1

Total number of observations = 4 Total number of records with missing values = 0

Maximum number of iterations = 250 Relative Function Converge nce has been set to 1e-008 Parameter Convergence has been set to 1e-008

User has chosen the log transformed model

Default Initial Parameter Values background = 0 intercept = 1.93572 slope = 1

Asymptotic Correlation Matrix of Parameter Estimates

( *** The model parameter(s) -background -slope

have been estimated at a boundary point, or have been specified by the user, and do not appear in the correlation matrix )

intercept

intercept 1

Parameter Estimates

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Variable Estimate Std. Err. background 0 NA intercept 1.98253 0.20995 slope 1 NA

NA - Indicates that this parameter has hit a bound implied by some inequality constraint and thus

has no standard error.

Analysis of Deviance Table

Model Log(likelihood) Deviance Test DF P-value

Full model -72.3723

Fitted model -75.4299 6.1152 3 0.1061

Reduced model -89.3929 34.0413 3 <.0001

AIC: 152.86

Goodness of Fit

Scaled Dose Est._Prob. Expected Observed Size Residual

---------------- -------------------------------------------------------- 0.0000 0.0000 0.000 0 50 0

0.0095 0.0645 3.162 3 49 -0.09415

0.0320 0.1885 9.427 15 50 2.015

0.0950 0.4082 20.411 15 50 -1.557

Chi-square = 6.49 DF = 3 p-value = 0.0900

Benchmark Dose Computation

Specified effect = 0.1

Risk Type = Extra risk

Confidence level = 0.95

BMD = 0.0153022

BMDL = 0.0109172

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0 0.1 0.2 0.3 0.4

0 0.02 0.04 0.06 0.08 0.1Fraction Affected

dose Log-Logistic Model with 0.95 Confidence Level

09:21 09/10 2005 BMDL BMD Log-Logistic

Output from BMD v1.3.2 (highest exposu re level excluded) is shown below:

======================== ==================================== ======== Probit Model $Revision: 2.1 $ $Date: 2000/02/26 03:38:53 $ Input Data File: C:\PROJECTS\COBALT\BMDS\RAFELUSE\RAFELUSEPROLOG.(D) Gnuplot Plotting File:

C:\PROJECTS\COBALT\BMDS\RA FELUSE\RAFELUSEPROLOG.plt Fri Sep 09 16:41:28 2005

======================== ==================================== ========

BMDS MODEL RUN

~~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~~~~~~~

The form of the pr obability function is:

P[response] = Background + (1-Background) * CumNorm(Intercept+Slope*Log(Dose)),

where CumNorm(.) is the cumulative normal distribution function

538-25-2008

Dependent variable = INRF Independent variable = ECRF Slope parameter is restricted as slope > 1

Total number of observations = 4 Total number of records with missing values = 1

Maximum number of iterations = 250 Relative Function Converge nce has been set to 1e-008 Parameter Convergence has been set to 1e-008

User has chosen the log transformed model

Default Initial (and Specified) Parameter Values background = 0 intercept = 3.0285 slope = 1

Asymptotic Correlation Matrix of Parameter Estimates

( *** The model parameter(s) -background -slope

have been estimated at a boundary point, or have been specified by the user, and do not appear in the correlation matrix )

intercept

intercept 1

Parameter Estimates

Variable Estimate Std. Err. background 0 NA intercept 2.97347 0.157916 slope 1 NA

NA - Indicates that this parameter has hit a bound implied by some inequality constraint and thus

has no standard error.

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Analysis of Deviance Table

Model Log(likelihood) Deviance Test DF P-value

Full model -41.8291

Fitted model -41.9887 0.319256 2 0.8525

Reduced model -54.9105 26.1628 2 <.0001

AIC: 85.9774

Goodness of Fit

Scaled Dose Est._Prob. Expected Observed Size Residual

---------------- -------------------------------------------------------- 0.0000 0.0000 0.000 0 50 0

0.0095 0.0462 2.263 3 49 0.5015

0.0320 0.3197 15.985 15 50 -0.2987

Chi-square = 0.34 DF = 2 p-value = 0.8434

Benchmark Dose Computation

Specified effect = 0.1

Risk Type = Extra risk

Confidence level = 0.95

BMD = 0.0141927

BMDL = 0.0109984

558-25-2008

0 0.1 0.2 0.3 0.4

0 0.005 0.01 0.015 0.02 0.025 0.03 0.035Fraction Affected

dose Probit Model with 0.95 Confidence Level

09:26 09/10 2005 BMDL BMD Probit

568-25-2008

Male mouse – A/B adenoma or carcinoma : All models show acceptable fit (p > 0.1) Log-logistic model yielded best fit (highest p-value and lowest AIC) Best estimate of BMDL = 0.015 mg/m 3

Model p AIC BMD BMDL mg/m 3 mg/m 3
gamma (power > 1) 0.944 251.10 0.033 0.0215 logistic 0.759 251.54 0.048 0.0359 log logistic (slope > 1) 0.999 250.99 0.026 0.0150 2 degree polynomial (pos betas) 0.944 251.10 0.033 0.0215 1 degree polynomial (pos betas) 0.944 251.10 0.033 0.0215 probit 0.775 251.50 0.046 0.0349 log probit (slope > 1) 0.594 252.03 0.059 0.0397 quantal linear 0.944 251.10 0.033 0.0215 quantal quadratic 0.412 252.76 0.080 0.0633 weibull (power > 1) 0.944 251.10 0.033 0.0215

Output from BMD v1.3.2 is shown below:

======================== ==================================== ======== Logistic Model $Revisi on: 2.1 $ $Date: 2000/02/26 03:38:20 $ Input Data File: C:\ROJECTS\C OBALT\BMDS\MOMALU\MO MALULOGLOG.(D) Gnuplot Plotting File: C:\PROJECTS\COBALT\BMDS\MOMALU\MOMALULOGLOG.plt Fri Sep 09 16:57:38 2005

======================== ==================================== ========

BMDS MODEL RUN

~~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~~~~~~~

The form of the pr obability function is

P[response] = background+(1-backgr ound)/[1+EXP(-intercept -slope*Log(dose))]

Dependent variable = INMM Independent variable = ECMM Slope parameter is restricted as slope > 1

Total number of observations = 4 Total number of records with missing values = 0

Maximum number of iterations = 250 Relative Function Converge nce has been set to 1e-008 Parameter Convergence has been set to 1e-008

578-25-2008

User has chosen the log transformed model

Default Initial Parameter Values background = 0.22 intercept = 1.47367 slope = 1

Asymptotic Correlation Matrix of Parameter Estimates

( *** The model parameter(s) -slope

have been estimated at a boundary point, or have been specified by the user, and do not appear in the correlation matrix )

background intercept

background 1 -0.62

intercept -0.62 1

Parameter Estimates

Variable Estimate Std. Err. background 0.22179 0.0478621 intercept 1.45848 0.375385 slope 1 NA

NA - Indicates that this parameter has hit a bound implied by some inequality constraint and thus

has no standard error.

Analysis of Deviance Table

Model Log(likelihood) Deviance Test DF p-value

Full model -123.493

Fitted model -123.494 0.00271986 2 0.9986

Reduced model -130.684 14.3818 3 0.002429

588-25-2008

AIC: 250.988

Goodness of Fit

Scaled Dose Est._Prob. Expected Observed Size Residual

---------------- -------------------------------------------------------- 0.0000 0.2218 11.090 11 50 -0.03047

0.0180 0.2777 13.884 14 50 0.03649

0.0590 0.3793 18.963 19 50 0.0109

0.1800 0.5613 28.065 28 50 -0.0185

Chi-square = 0.00 DF = 2 p-value = 0.9986

Benchmark Dose Computation

Specified effect = 0.1

Risk Type = Extra risk

Confidence level = 0.95

BMD = 0.0258434

BMDL = 0.0149697

598-25-2008

0.1 0.2 0.3 0.4 0.5 0.6 0.7

0 0.05 0.1 0.15Fraction Affected

dose Log-Logistic Model with 0.95 Confidence Level

09:32 09/10 2005 BMDL BMD Log-Logistic

608-25-2008

Male mouse – A/B adenoma or carcinoma : All models show acceptable fit (p > 0.1) Log-logistic model yielded best fit (highest p-value and lowest AIC) Best estimate of BMDL = 0.023 mg/m 3

Model p AIC BMD BMDL mg/m 3 mg/m 3
gamma (power > 1) 0.571 196.12 0.0455 0.0296 logistic 0.273 197.61 0.0735 0.0562 log logistic (slope > 1) 0.700 195.72 0.0384 0.0231 2 degree polynomial (pos betas) 0.571 196.12 0.0455 0.0296 1 degree polynomial (pos betas) 0.571 196.12 0.0455 0.0296 probit 0.300 197.42 0.0697 0.0528 log probit (slope > 1) 0.167 198.57 0.0768 0.0524 quantal linear 0.571 196.12 0.0455 0.0296 quantal quadratic 0.117 199.26 0.0959 0.0739 weibull (power > 1) 0.571 196.12 0.0455 0.0296

Output from BMD v1.3.2 is shown below:

======================== ==================================== ======== Logistic Model $Revisi on: 2.1 $ $Date: 2000/02/26 03:38:20 $ Input Data File: C:\PROJECTS\ COBALT\BMDS\MOFELU\M OFELULOGLOG.(D) Gnuplot Plotting File:

C:\PROJECTS\COBALT\BMDS\ MOFELU\MOFELULOGLOG.plt Fri Sep 09 17:05:08 2005

======================== ==================================== ========

BMDS MODEL RUN

~~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~~~~~~~

The form of the pr obability function is

P[response] = background+(1-backgr ound)/[1+EXP(-intercept -slope*Log(dose))]

Dependent variable = INMF Independent variable = ECMF Slope parameter is restricted as slope > 1

Total number of observations = 4 Total number of records with missing values = 0

Maximum number of iterations = 250 Relative Function Converge nce has been set to 1e-008 Parameter Convergence has been set to 1e-008

618-25-2008

User has chosen the log transformed model

Default Initial Parameter Values background = 0.08 intercept = 1.17812 slope = 1

Asymptotic Correlation Matrix of Parameter Estimates

( *** The model parameter(s) -slope

have been estimated at a boundary point, or have been specified by the user, and do not appear in the correlation matrix )

background intercept

background 1 -0.6

intercept -0.6 1

Parameter Estimates

Variable Estimate Std. Err. background 0.0920048 0.035283 intercept 1.06119 0.354864 slope 1 NA

NA - Indicates that this parameter has hit a bound implied by some inequality constraint and thus

has no standard error.

Analysis of Deviance Table

Model Log(likelihood) Deviance Test DF p-value

Full model -95.5104

Fitted model -95.8619 0.702985 2 0.7036

Reduced model -102.791 14.5619 3 0.002232

AIC: 195.724

628-25-2008

Goodness of Fit

Scaled Dose Est._Prob. Expected Observed Size Residual

---------------- -------------------------------------------------------- 0.0000 0.0920 4.600 4 50 -0.2937

0.0170 0.1345 6.726 7 50 0.1135

0.0580 0.2223 11.117 13 50 0.6403

0.1700 0.3911 19.556 18 50 -0.451

Chi-square = 0.71 DF = 2 p-value = 0.7003

Benchmark Dose Computation

Specified effect = 0.1

Risk Type = Extra risk

Confidence level = 0.95

BMD = 0.0384492

BMDL = 0.0231

638-25-2008

64 0 0.1 0.2 0.3 0.4 0.5

0 0.05 0.1 0.15Fraction Affected

dose Log-Logistic Model with 0.95 Confidence Level

09:36 09/10 2005 BMDL BMD Log-Logistic