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# TOXICOLOGICAL REVIEW

OF

VANADIUM PENTOXIDE (V 2 

# O 5 

# )

(CAS No. 1314-62-1)



## In Support of Summary Information on the Integrated Risk Information System (IRIS)

July 2011

NOTICE

This document is an Interagency Science Consultation draft . This information is distributed solely for the purpose of pre-dissemination peer review under applicable information quality guidelines. It has not been formally disseminated by EPA. It does not represent and should not be construed to represent any Agency determination or policy. It is being circulated for review of its technical accuracy and science policy implications.

U.S. Environmental Protection Agency Washington, DC.DRAFT - DO NOT CITE OR QUOTE ii DISCLAIMER

This document is a preliminary draft for review purposes only. This information is distributed solely for the purpose of pre-dissemination peer review under applicable information quality guidelines. It has not been formally disseminated by EPA. It does not represent and should not be construed to represent any Agency determination or policy. Mention of trade names or commercial products does not constitute endorsement or recommendation for use.DRAFT - DO NOT CITE OR QUOTE iii CONTENTS – TOXICOLOGICAL REVIEW OF VANADIUM PENTOXIDE (CAS No. 1314-62-1)

AUTHORS, CONTRIBUTORS, AND REVIEWERS ........................................................... xiii

1. INTRODUCTION .................................................................................................................... 1

2. CHEMICAL AND PHYSICAL INFORMATION ............................................................... 3

3. TOXICOKINETICS ................................................................................................................. 6

3.1 Absorption ............................................................................................................................ 6 3.1.1 Inhalation Exposure ................................................................................................. 6 3.1.2 Oral Exposure ................................................................................................................ 7

3.1.3 Dermal Exposure ..................................................................................................... 8

3.1.4 Other Routes of Exposure ....................................................................................... 8

3.2 Distribution ........................................................................................................................... 8 3.2.1 Inhalation Exposure ................................................................................................. 8

3.2.2 Oral Exposure .......................................................................................................... 9

3.2.3 Dermal Exposure ................................................................................................... 10

3.2.4 Other Routes of Exposure ..................................................................................... 11

3.3 Metabolism ......................................................................................................................... 11 3.4 Elimination and Excretion .................................................................................................. 11 3.4.1 Inhalation Exposure ............................................................................................... 11

3.4.2 Oral Exposure ........................................................................................................ 12

3.4.3 Dermal Exposure ................................................................................................... 12

3.4.4 Other Routes of Exposure ..................................................................................... 12

3.5 Physiologically Based Pharmacokinetic (PBPK)/Pharmacodynamic (PD) Models .......... 12 3.5.1 Animal-to-Human Extrapolations ......................................................................... 12

4. HAZARD IDENTIFICATION ............................................................................................. 14

4.1. STUDIES IN HUMANS – EPIDEMIOLOGY, CASE REPORTS, CLINICAL CONTROLS ..................................................................................................................... 14 4.1.1 Oral Exposure ............................................................................................................... 14 4.1.2 Inhalation Exposure ...................................................................................................... 14 4.2. SUBCHRONIC AND CHRONIC STUDIES AND CANCER BIOASSAYS IN ANIMALS – ORAL AND INHALATION ...................................................................... 31 4.2.1. Oral Exposure ........................................................................................................ 31

4.2.1.1. Subchronic Studies ................................................................................................ 31 4.2.1.2 Chronic Studies ..................................................................................................... 32 4.2.2. Inhalation Exposure ............................................................................................... 33DRAFT - DO NOT CITE OR QUOTE iv 4.2.2.1 Subchronic Studies ................................................................................................ 33 4.2.2.2 Chronic Studies ..................................................................................................... 43 4.3. REPRODUCTIVE/DEVELOPMENTAL STUDIES – ORAL, INHALATION, INTRAPERITONEAL AND INJECTION ....................................................................... 53 4.3.1. Oral Studies ........................................................................................................... 53

4.3.2. Inhalation Studies .................................................................................................. 54

4.3.3. Intraperitoneal and Injection Studies .................................................................... 55

4.4 OTHER DURATION- OR ENDPOINT-SPECIFIC STUDIES ......................................... 57 4.4.1 Acute and Short-Term Studies ...................................................................................... 57

4.4.1.1 Acute Studies .......................................................................................................... 57 4.4.1.2. Short-term Studies ................................................................................................. 59 4.4.2 Immunological Endpoints ............................................................................................. 61

4.4.2.1 Human Studies ....................................................................................................... 61 4.4.2.2 Animal Studies ........................................................................................................ 64 4.4.3 Neurological Endpoints ................................................................................................ 65

4.4.3.1 Human Studies ....................................................................................................... 66 4.4.3.2 Animal Studies ....................................................................................................... 66 4.5. MECHANISTIC DATA AND OTHER STUDIES IN SUPPORT OF THE MODE OF ACTION FOR PULMONARY FIBROSIS AND CANCER ........................................... 68 4.5.1 Genotoxicity .................................................................................................................. 68

4.5.1.1 Human Studies ........................................................................................................ 68 4.5.1.2 Laboratory in vivo and in vitro studies ................................................................... 71 4.5.2 Mechanisms of Inflammation and Fibrosis ................................................................... 78 4.5.3 Mechanisms of Hyperplasia and Carcinogenicity ......................................................... 82

4.6. SYNTHESIS OF MAJOR NONCANCER EFFECTS ....................................................... 86 4.6.1. Oral ........................................................................................................................ 86

4.6.1.1 Acute ....................................................................................................................... 86 4.6.1.2 Subchronic .............................................................................................................. 88 4.6.1.3 Chronic .................................................................................................................... 88 4.6.2. Inhalation ............................................................................................................... 88

4.6.2.1 Acute and Short-term .............................................................................................. 89 4.6.2.2 Subchronic ............................................................................................................ 90 4.6.2.3 Chronic .................................................................................................................... 91 4.6.3 Mode of Action Information ......................................................................................... 98

4.6.3.1 Pulmonary Toxicity .............................................................................................. 98 4.6.3.2 Neurotoxicity ........................................................................................................ 100 4.6.3.3 Reproductive Toxicity .......................................................................................... 100 4.7. EVALUATION OF CARCINOGENICITY ..................................................................... 101 4.7.1 Summary of Overall Weight of Evidence ................................................................ 101

4.7.2. Synthesis of Human, Animal, and Other Supporting Evidence .......................... 101DRAFT - DO NOT CITE OR QUOTE v 4.7.3. Mode of Action Information ............................................................................... 102

4.8. SUSCEPTIBLE POPULATIONS AND LIFE STAGES ................................................. 103 4.8.1. Possible Childhood Susceptibility ....................................................................... 103

4.8.2. Possible Gender Differences ............................................................................... 103

4.8.3. Other Susceptible Populations ............................................................................ 103

5. DOSE-RESPONSE ANALYSIS .......................................................................................... 106

5.1. ORAL REFERENCE DOSE (RfD) .................................................................................. 106 5.1.1 Methods of Analysis ................................................................................................... 108 5.1.2 RfD Derivation – Including Application of Uncertainty Factors (UF) ....................... 109

5.1.2. Previous RfD Assessment ................................................................................... 110 5.2. INHALATION REFERENCE CONCENTRATION (RfC) ............................................. 111 5.2.1 Choice of Principal Study and Critical Effect with Rationale and Justification ......... 111

5.2.2 Methods of Analysis ................................................................................................ 113

5.2.3 RfC Derivation- Including Application of Uncertainty Factors (UFs) .................... 119

5.2.4. Previous RfC Assessment ................................................................................... 120 5.3 UNCERTAINTIES IN THE INHALATION REFERENCE CONCENTRATION (RfC) 120 5.4. CANCER ASSESSMENT ............................................................................................... 123 5.4.1 Choice of Study/Data – with Rationale and Justification ........................................ 123

5.4.2 Dose-Response Data ................................................................................................ 124

5.4.3 Dose Adjustments and Extrapolation Methods ........................................................ 124

5.4.4 Inhalation Unit Risk ................................................................................................. 127 5.4.5 Oral Cancer Slope Factor .......................................................................................... 127

5.4.6 Uncertainties in Cancer Risk Values ....................................................................... 127

6. MAJOR CONCLUSIONS IN THE CH ARACTERIZATION OF HAZARD AND DOSE RESPONSE

.............................................................................................................................. . 132

6.1 Human Hazard Potential .................................................................................................... 132 6.2. Dose Response .................................................................................................................. 136 6.2.1 Noncancer .................................................................................................................... 136

6.2.2 Cancer .......................................................................................................................... 138

7. REFERENCES ...................................................................................................................... 140DRAFT - DO NOT CITE OR QUOTE vi LIST OF TABLES

Table 2-1. Valence states and water solubility of various vanadium compounds. .................. 4

Table 2-2: Chemical and physical properties of Vanadium Pentoxide .................................... 5 Table 4–1. Hematological results of oral van adium pentoxide exposure in rats (Mountain et al. 1953).

.............................................................................................................................. ..... 32

Table 4-2. Body Weight Gain and Lung Weights in Rats (F344/N) Exposed to Vanadium Pentoxide by Inhalation for 3 Months (Values are Means±Standard Error) (NTP, 2002) . 35 Table 4-3. Selected Hematology Parameters in Rats (F344/N) Exposed to Vanadium Pentoxide by Inhalation for 3 Months (NTP, 2002) ................................................................. 35 Table 4-4. Incidences of Selected Nonneoplast ic Lesions of the Lung and Nose in Rats (F344/N) Exposed to Vanadium Pentoxide by Inhalation for 3 Months (NTP, 2002) .......... 37 Table 4-5. Body Weight Gain and Lung Weights in Mice (B6C3F 1 ) Exposed to Vanadium Pentoxide by Inhalation for 3 Months (Values are Means±Standard Error) (NTP, 2002) . 40 Table 4-6. Incidences of Selected Nonneoplastic Lesions of the Lung in Mice (B6C3F 1 ) Exposed to Vanadium Pentoxide by Inhalation for 3 Months (NTP, 2002) .......................... 41

Table 4-7. Selected Nonneoplastic Lesions of the Respiratory System in Rats Exposed to Particulate Aerosols of Vanadium Pentoxide for 2 Years (NTP, 2002) ................................. 44 Table 4-8. Incidences of Respiratory Tumors in Rats Exposed to Vanadium Pentoxide in a 2 Year Inhalation Study (NTP, 2002) a ...................................................................................... 47 Table 4-9. Selected Nonneoplastic Lesions of the Respiratory System in Mice Exposed to Vanadium Pentoxide in a 2 Year Inhalation Study (NTP, 2002) .......................................... 48 Table 4-10. Incidences of Respiratory Tumors in Mice Exposed to Vanadium Pentoxide in the 2 Year Inhalation Study (NTP, 2002) ................................................................................. 51

Table 4 -11. Lung tumor multiplicity in MCA-Treated mice exposed to V 2 O 5 by pharyngeal aspiration (Rondini et al. 2010). a,b ........................................................................ 52 Table 4-12. Genotoxicity Data Following Exposure to Vanadium Pentoxide ....................... 74

Table 4-13: Summary of Noncancer Results of Repeat-Dose Studies for Oral Exposure of Experimental Animals to Vanadium Pentoxide ....................................................................... 86

Table 4-14: Summary of Noncancer Results of Repeat-Dose Studies for Inhalation Exposure of Vanadium Pentoxide ............................................................................................. 92 Table 5–1. Hematological results of oral van adium pentoxide exposure in rats (Mountain et al. 1953).

.............................................................................................................................. ... 107 Table 5-2 Human equivalence dose conversion by BW 3/4 for RfD derivation. ................... 109

Table 5-3. Selected Nonneoplastic Lesions of the Respiratory System in Rats Exposed to Particulate Aerosols of Vanadium Pentoxide for 2 Years (NTP, 2002) ............................... 114DRAFT - DO NOT CITE OR QUOTE vii Table 5-4: Human Equivalent Concentrations of Vanadium Pentoxide in the 2-Year Inhalation Studies in Rats (NTP, 2002) .................................................................................. 116

Table 5-5: Candidate PODs for Vanadium Pentoxide Derived from NTP Studies (2002) through BMDS Modeling. ........................................................................................................ 116 Table 5-6: Benchmark Modeling Results for Inci dence of Larynx Chronic Inflammation in Female Rats, NTP (2002) .......................................................................................................... 118 Table 5-7: Benchmark Modeling Results for In cidence of Larynx Respiratory Epithelium, Epiglottis, Hyperplasia in Female Rats, NTP (2002) ............................................................. 119 Table 5-8. Incidences of Respiratory Tumors in Mice Exposed to Vanadium Pentoxide in the 2 Year Inhalation Study (NTP, 2002) ............................................................................... 123 Table 5-9: Human Equivalent Concentrations of Vanadium Pentoxide in the 2-Year Inhalation Studies (NTP, 2002) ................................................................................................ 125 Table 5- 10. BMDS Modeling Results for Comb ined Incidence of Alveolar/Bronchiolar Adenoma and Carcinoma in Male Mice, NTP (2002). .......................................................... 126 Table 5-11. Summary of uncertainty in the vanadium pentoxide cancer risk assessment.

............................................................................................................................... ...................... 129

Table B-1: Candidate PODs for Vanadium Pentoxide Derived from NTP Studies (2002) through BMDS Modeling. ........................................................................................................ 154

Table B-2: Trend Tests on the Selected Data Sets from the 2-Year Inhalation Studies in Rats (NTP, 2002) ....................................................................................................................... 156 Table B-3: Average Life Time Animal Body Weight of Rats in the 2-Year Inhalation Studies of Vanadium Pentoxide (NTP, 2002) ......................................................................... 157 Table B-4: RDDRs for Different Concentration/Sex Group in the 2-Year Inhalation Studies of Vanadium Pentoxide (NTP, 2002) ....................................................................................... 157 Table B-5: Human Equivalent Concentrations of Vanadium Pentoxide in the 2-Year Inhalation Studies (NTP, 2002) ................................................................................................ 158 Table B-6: Benchmark Modeling Results for Incidence of Lung Alveolar Epithelium Hyperplasia in Male Rats, NTP (2002) ................................................................................... 159 Table B-7: Benchmark Modeling Results for Incidence of Lung Chronic Active Inflammation in Male Rats, NTP (2002) ................................................................................ 162 Table B-8: Benchmark Modeling Results for Inci dence of Larynx Chronic Inflammation in Male Rats, NTP (2002) ............................................................................................................. 164 Table B-9: Benchmark Modeling Results for In cidence of Larynx Respiratory Epithelium, Epiglottis, Hyperplasia in Male Rats, NTP (2002) ................................................................. 167 Table B-10: Benchmark Modeling Results for In cidence of Nose Goblet Cell, Respiratory Epithelium, Hyperplasia in Male Rats, NTP (2002) .............................................................. 169 Table B-11: Benchmark Modeling Results for Incidence of Lung Alveolar Epithelium Hyperplasia in Female Rats, NTP (2002) ............................................................................... 172DRAFT - DO NOT CITE OR QUOTE viii Table B-12: Benchmark Modeling Results for Incidence of Lung Chronic Active Inflammation in Female Rats, NTP (2002) ............................................................................. 174 Table B-13: Benchmark Modeling Results for In cidence of Larynx Chronic Inflammation in Female Rats, NTP (2002) ..................................................................................................... 176 Table B-14: Benchmark Modeling Results for Incidence of Larynx Respiratory Epithelium, Epiglottis, Hyperplasia in Female Rats, NTP (2002) ........................................ 179 Table B-15: Benchmark Modeling Results for Incidence of Larynx Respiratory Epithelium, Epiglottis, Hyperplasia in Female Rats, NTP (2002) ........................................ 181 Table C- 1. Summary of Candidate PODs and Cancer Slope Factors. ............................... 185

Table C-2: Trend Tests on the Inhalation Ca rcinogenesis Data Sets in Mice from the 2- Year Inhalation Studies (NTP, 2002) ...................................................................................... 185 Table C-3: Average Life Time Animal Body We ight of Mice for 2-Year Inhalation Studies of Vanadium Pentoxide (NTP, 2002) ....................................................................................... 186

Table C-4: RDDRs for Different Concentrat ion/Sex Group of Mice in the 2-Year Inhalation Studies of Vanadium Pentoxide (NTP, 2002) ...................................................... 187 Table C-5: Human Equivalent Concentrations of Vanadium Pentoxide in the 2-Year Inhalation Studies (NTP, 2002) ................................................................................................ 187

Table C-6: BMR Estimation for Male and Female Mice Data Sets. .................................... 188 Table C- 7. BMDS Modeling Results for Comb ined Incidence of Alveolar/Bronchiolar Adenoma and Carcinoma in Male Mice, NTP (2002). .......................................................... 189 Table C- 8. BMDS Modeling Results for Comb ined Incidence of Alveolar/Bronchiolar Adenoma and Carcinoma in Male Mice after Dropping the Highest Concentration, NTP (2002).

.............................................................................................................................. ........... 191 Table C- 9. BMDS Modeling Results for Comb ined Incidence of Alveolar/Bronchiolar Adenomas and Carcinomas with All Four Concentrations in Female Mice, NTP (2002). 193 Table C- 10. BMDS Modeling Results for Comb ined Incidence of Alveolar/Bronchiolar Adenoma and Carcinoma in Female Mice after Dropping the Highest Concentration, NTP (2002).

.............................................................................................................................. ........... 194 Table C- 11. Grouped Data for MSW Time-to-tumor Modeling; B6C3F 1 Male Mice Exposed to Vanadium Pentoxide by Inhalation for 2 Years (NTP, 2002). .......................... 197 Table C- 12. Grouped Data for MSW Time-to-tumor Modeling; B6C3F 1 Female Mice Exposed to Vanadium Pentoxide by Inhalation for 2 Years (NTP, 2002). .......................... 201DRAFT - DO NOT CITE OR QUOTE ix LIST OF FIGURES

Figure 2-1. Vanadium pentoxide structure ................................................................................ 3 Figure 4-1: Genomics of V 2 O 5 -Induced Bronchitis (reprinted with permission from Ingram et al., (2007) Respir. Res. Apr 25;8(1):34) ............................................................................... 82DRAFT - DO NOT CITE OR QUOTE x LIST OF ACRONYMS AND ABBREVIATIONS

BMC benchmark concentration BMCL benchmark concentration (lower limit)
BMD benchmark dose BMDL benchmark dose (lower limit)
BMDS benchmark dose software BMR benchmark response bw body weight
cc cubic centimeters
CD caesarean delivered CERCLA Comprehensive Environmental Response, Compensation and Liability Act of 1980 CNS central nervous system
cu.m cubic meter FIFRA Federal Insecticide, Fungicide, and Rodenticide Act

g grams GI gastrointestinal HED human equivalent dose 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
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 MF modifying factor mg milligramDRAFT - DO NOT CITE OR QUOTE xi mg/kg milligrams per kilogram mg/L milligrams per liter
MRL minimal risk level
MTD maximum tolerated dose NAAQS National Ambient Air Quality Standards NMMAPS National Morbidity, Mortality, and Air Pollution Study 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 PBPK physiologically based pharmacokinetic ppb parts per billion ppm parts per million PPRTV Provisional Peer Reviewed Toxicity Value RBC red blood cell(s) RCRA Resource Conservation and Recovery Act RDDR Regional deposited dose ratio (for the indicated lung region)
RfC inhalation reference concentration
RfD oral reference dose RGDR Regional gas dose ratio (for the indicated lung region) 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 compoundDRAFT - DO NOT CITE OR QUOTE xii FOREWORD

The purpose of this Toxicological Review is to provide scientific support and rationale for the hazard and dose-response assessment in IRIS pertaining to chronic exposure to vanadium pentoxide. It is not intended to be a comprehensive treatise on the chemical or toxicological nature of vanadium pentoxide, and does not address other vanadium compounds.
The intent of Section 6, Major Conclusions in the Characterization of Hazard and Dose
Response, is to present the major conclusions reached in the derivation of the reference dose, reference concentration and cancer assessment, where applicable, and to characterize the overall confidence in the quantitative and qualitative aspects of hazard and dose response by addressing the quality of data and related uncertainties. The discussion is intended to convey the limitations of the assessment and to aid and guide the risk assessor in the ensuing steps of the risk

assessment process. For other general information about this assessment or other questions relating to IRIS, the reader is referred to EPA’s IRIS Hotline at (202) 566-1676 (phone), (202) 566-1749 (fax), or hotline.iris@epa.gov (email address).DRAFT - DO NOT CITE OR QUOTE xiii AUTHORS, CONTRIBUTORS, AND REVIEWERS

CHEMICAL MANAGER/AUTHOR Maureen R. Gwinn, Ph.D., DABT
National Center for Environmental Assessment US Environmental Protection Agency Washington, D.C. 20460

IRIS CO-LEAD/AUTHOR Kathleen Newhouse, M.S.
National Center for Environmental Assessment US Environmental Protection Agency Washington, D.C. 20460

CO-AUTHORS Gillian Backus, Ph.D.* Northern Virginia Community College Sterling, VA. 20164

Yuyang Christine Cai, M.S., PMP
National Center for Environmental Assessment US Environmental Protection Agency Washington, D.C. 20460

Sanjivani Diwan, Ph.D.*
National Center for Environmental Assessment US Environmental Protection Agency Washington, D.C. 20460

Barbara Glenn, Ph.D.
National Center for Environmental Assessment US Environmental Protection Agency Washington, D.C. 20460

Kathleen Raffaele, Ph.D.DRAFT - DO NOT CITE OR QUOTE xiv Office of Solid Waste and Emergency Response US Environmental Protection Agency Washington, D.C. 20460

Suryanarayana V. Vulimiri, D.V.M., Ph.D., DABT
National Center for Environmental Assessment US Environmental Protection Agency Washington, D.C. 20460

*Former EPA employeeDRAFT - DO NOT CITE OR QUOTE 1 1. INTRODUCTION

This document presents background information and justification for the Integrated Risk Information System (IRIS) Summary of the hazard and dose-response assessment of vanadium pentoxide. IRIS Summaries may include oral reference dose (RfD) and inhalation reference concentration (RfC) values for chronic and other exposure durations, and a carcinogenicity

assessment. The RfD and RfC, if derived, provide quantitative information for use in risk assessments for health effects known or assumed to be produced through a nonlinear (presumed threshold) mode of action. The RfD (expressed in units of mg/kg-day) is defined as an estimate (with uncertainty spanning perhaps an order of magnitude) of a daily exposure to the human population (including sensitive subgroups) that is lik ely to be without an appreciable risk of deleterious effects during a lifetime. The inhalation RfC (expressed in units of mg/m 3 ) is analogous to the oral RfD, but provides a continuous inhalation exposure estimate. The
inhalation RfC considers toxic effects for both th e respiratory system (portal-of-entry) and for effects peripheral to the respiratory system (ext rarespiratory or systemic effects). Reference values are generally derived for chronic exposures (up to a lifetime), but may also be derived for acute ( ≤ 24 hours), short-term (>24 hours up to 30 days), and subchronic (>30 days up to 10% of lifetime) exposure durations, all of which are derived based on an assumption of continuous exposure throughout the duration specified. Unle ss specified otherwise, the RfD and RfC are derived for chronic exposure duration. The carcinogenicity assessment provides information on the carcinogenic hazard potential of the substance in question, and quantitative estimates of risk from oral and inhalation exposures may be derived. The information includes a weight-of-evidence judgment of the likelihood that the agent is a human carcinogen and the conditions under which the carcinogenic effects may be expressed. Quantitative risk estimates may be derived from the application of a low-dose extrapolation procedure. If derived, the oral slope factor is a plausible upper bound on the estimate of risk per mg/kg-day of oral exposure. Similarly, an inhalation unit risk is a plausible upper bound on the estimate of risk per μ g/m 3 air breathed. Development of these hazard identification and dose-response assessments for vanadium pentoxide has followed the general guidelines for risk assessment as set forth by the National Research Council (1983). EPA Guidelines and Risk Assessment Forum technical panel reports that may have been used in the development of this assessment include the following: Guidelines for the Health Risk Assessment of Chemical Mixtures (U.S. EPA, 1986a), Guidelines for Mutagenicity Risk Assessment (U.S. EPA, 1986b), Recommendations for and Documentation ofDRAFT - DO NOT CITE OR QUOTE 2 Biological Values for Use in Risk Assessment (U.S. EPA, 1988), Guidelines for Developmental Toxicity Risk Assessment (U.S. EPA, 1991), Interim Policy for Particle Size and Limit Concentration Issues in Inhalation Toxicity (U.S. EPA, 1994a), Methods for Derivation of Inhalation Reference Concentrations and Application of Inhalation Dosimetry (U.S. EPA, 1994b), Use of the Benchmark Dose Approach in Health Risk Assessment (U.S. EPA, 1995), Guidelines for Reproductive Toxicity Risk Assessment (U.S. EPA, 1996), Guidelines for Neurotoxicity Risk Assessment (U.S. EPA, 1998), Science Policy Council Handbook : Risk Characterization (U.S. EPA, 2000a), Benchmark Dose Technical Guidance Document (U.S. EPA, 2000b), Supplementary Guidance for Conducting Health Risk Assessment of Chemical

Mixtures (U.S. EPA, 2000c), A Review of the Reference Dose and Reference Concentration

Processes (U.S. EPA, 2002), Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005a)

, Supplemental Guidance for Assessing Susceptibility from Early-Life Exposure to Carcinogens (U.S. EPA, 2005b), Science Policy Council Handbook: Peer Review (U.S. EPA, 2006a), and A Framework for Assessing Health Risks of Environmental Exposures to Children (U.S. EPA, 2006b). The literature search strategy employed for this compound was based on the Chemical Abstracts Service Registry Number (CASRN) and at least one common name. Any pertinent scientific information submitted by the public to the IRIS Submission Desk was also considered in the development of this document. The relevant literature was reviewed through May 2011. Portions of this document were developed under a Memorandum of Understanding with the Agency for Toxic Substances and Disease Registry (ATSDR) as part of a collaborative effort in the development of human health toxicological assessments.DRAFT - DO NOT CITE OR QUOTE 3 2. CHEMICAL AND PHYSICAL INFORMATION

As an element, vanadium (V) exists in several oxidation states from -1 to +5. Vanadium (CASRN 7440-62-2) is a soft silver-grey metal commonly found in ores, tars, coals and oils and is used as an alloy in steel (WHO, 1988). The focus of this toxicological review is on vanadium pentoxide (CASRN 1314-62-1), but a short descrip tion of chemistry of vanadium and related compounds is given below for clarification. The chemistry of vanadium is complex; the valence state of vanadium can shift depending on pH and other factors. In the body, there is an interconversion of two oxidation states of vanadium, the tetravalent form, vanadyl (V +4 ), and the pentavalent form, vanadate (V +5 ). Vanadate is considered more toxic than vanadyl because vanadate is reactive with a number of enzymes and is a potent inhibitor of the Na+K+-ATPase of plasma membranes (Harris et al. 1984; Patterson et al. 1986).

Figure 2-1. Vanadium pentoxide structure

Generally, V 3+ and V 4+ predominate in body tissues while V 5+ predominates in plasma (IPCS, 2001). Vanadium pentoxide (V 2 O 5 ), sodium metavanadate (NaVO 3 ), sodium orthovanadate (Na 3 VO 4 ), and ammonium metavanadate (NH 4 VO 3 ) all contain vanadium in the +5 oxidation state. Of these compounds, V 2 O 5 is the only compound that is covalently bonded. Vanadium compounds differ in their physicochemical properties which influence their solubility under different pH conditions and their accessibility and availability in biological systems [reviewed in ( Assem and Levy 2009 )]. An acidic pH favors tetravalent state (V +4 ) keeping it as vanadyl, while an alkaline pH prefers pentavalent state (V +5 ) as vanadate ( Crans et

al . 2004 ). In the case of oral ingestion, vanadium compounds are exposed to a range of pH solutions in the digestive tract starting from the stomach (pH typically between 1-3.5) followed by the small intestine (pH around 8). Bruyere et al. (1999) state that at pH between 1.3 and 3.3 the predominate form of vanadium is VO 2+ and at higher pH the form is VO(OH) 3 . When the pH is high V( 5+ ) (e.g. VO 4 (OH) 3 ) and polymerized vanadium is predominant (H n V 10 O 29 (n-6) )( Bruyere

et al . 1999 ). At physiological pH vanadium compounds have been shown to exist in monomeric tetravalent [VO(OH) 3 ] - and dimeric [(VO) 2 (OH) 5 ] - forms, as well as pentavalent (H 2 VO 4 - ) formsDRAFT - DO NOT CITE OR QUOTE 4 [reviewed in ( Assem and Levy 2009 )]. Thus, the valence of a vanadium compound will depend on the pH. The solubility of different vanadium compounds in water between 20 and 25 o C differs among different valences as shown in Table 2-1 ( HSDB 2009 ; IPCS 2001 ). The elemental vanadium (V 0 ) is insoluble in water. The tetravalent (V +4 ) compound vanadyl sulfate (VOSO 4 ) is highly soluble 534.64 g/L ( Rahman and Skyllas-Kazacos 1998 ), while the pentavalent vanadium compounds (V +5 ), such as vanadium pentoxide (V 2 O 5 ) is less soluble (8 g/L). Other vanadium compounds such as sodium metavanadate (NaVO 3 ), sodium orthovanadate (Na 3 VO 4 ) and ammonium metavanadate (NH 4 VO 3 ) have solubility of 211 g/L, 100 g/L, 58 g/L, respectively ( IPCS 2001 ). Furthermore, the rate (distinguished from the solubility – an equilibrium or thermodynamic parameter) of dissolution of various vanadium compounds may vary, resulting in different concentrations of specific forms of vanadium. It seems a reasonable hypothesis that these various forms of vanadium will be absorbed differently, which may result in different physiological effects. For example, V(5 + ) compounds can mimic phosphate and can inhibit phosphatases ( Assem and Levy 2009 ).

Table 2-1. Valence states and water so lubility of various vanadium compounds.

Vanadium compound Formula CASRN Valenc
y Solubility (g/L) at 20–25°C ( HSDB 2009 ; IPCS 2001 )

Vanadium V 7440-62-2 0 Insoluble Vanadium pentoxide V 2 O 5 1314-62-1 +5 8 Sodium m -vanadate NaVO 3 13718-26-8 +5 211 Sodium o -vanadate Na 3 VO 4 13721-39-6 +5 100

Ammonium m -vanadate NH 4 VO 3 7803-55-6 +5 58 ( IPCS 2001 ); 5.2 at 15 o C ( HSDB 2009 )

Vanadium oxytrichloride VOCl 3 7727-18-6 +5 Soluble, decomposes in presence of moisture into vanadic acid and HCl.

Vanadyl sulfate VOSO 4 27774-13-6 +4 535 at 20 o C ( Rahman and Skyllas-Kazacos 1998 ) Vanadium tetrachloride VCl 4 7632-51-1 +4 Decomposes Vanadyl oxydichloride VOCl 2 10213-09-9 +3 Decomposes Vanadium trioxide V 2 O 3 1314-34-7 +3 Slightly soluble Adapted from ( Assem and Levy 2009 ).

This Toxicological Review focuses exclusively on vanadium pentoxide (V 2 O 5 , CASRN 1314-62-1) (Figure 2-1), the most common form of vanadium used commercially. VanadiumDRAFT - DO NOT CITE OR QUOTE 5 pentoxide exists in the pentavalent state as a yellow-red powder (Table 2-2) (OSHA 2007).

Table 2-2: Chemical and physical properties of Vanadium Pentoxide

Characteristic Information Reference
Chemical name Vanadium Pentoxide Synonym(s) Vanadium oxide, vanadic anhydride dust, divanadium pentaoxide, divanadium pentoxide, vanadium pentaoxide OSHA 2007

Chemical formula V 2 O 5 CAS

CASRN 1314-62-1 CAS Molecular weight 181.9
Color Yellowish-red powder Yellow to rust-brown orthorhombic powder Yellow-orange powder or dark gray flakes dispersed in air OSHA 2007 O’Neil 2001; NIOSH 2005

Melting point 690 °C OSHA 2007 Boiling point 1750 °C OSHA 2007 Density at 18 °C 3.357 ChemFinder.com, HSDB 2008, Lewis, 1997.
Odor threshold: Odorless OSHA 2007, NIOSH 2005. Solubility: Water

Organic solvents 8 g/L (20 °C) 10 g/L (20 °C)

Soluble in alkalies, concentrated acids, insoluble in alcohol OSHA 2007 ChemFinder.com

O’Neil 2001; HSDB, 2008 Vapor pressure 0 mm Hg HSDB 2008 Specific Gravity 3.4 g/cm 3 NTP 2008 Flash point Not applicable, Non-combustible OSHA 2007
Conversions: ppm to mg/m 3 mg/m 3 to ppm 1 ppm = 7.44 mg/m 3 1 mg/m 3 = 0.134 ppm Calculated CalculatedDRAFT - DO NOT CITE OR QUOTE 6 3

. TOXICOKINETICS

Toxicokinetics of vanadium pentoxide have been investigated in limited studies described below and reviewed by Cooper (2007). Toxicokinetics of several other vanadium compounds have been evaluated in animal models and are reviewed elsewhere (Sabbioni et al. 1996; Barceloux 1999; Mukherjee et al. 2004; ATSD R 2009). Vanadium pentoxide is rapidly absorbed by both inhalation and oral exposures through lungs and the gastrointestinal tract, respectively, although the amount absorbed through the gastrointestinal tract is low. Laboratory animal studies show vanadium pentoxide is ma inly distributed following inhalation and oral exposure to the bone, lungs, liver and kidney. Elimination of vanadium pentoxide has been studied only following inhalation exposure, and is mainly through the urine.

3.1 Absorption

3.1.1 Inhalation Exposure

Several occupational studies indicate that absorption can occur in humans following inhalation exposure. An increase in urinary vanadium levels was found in workers occupationally exposed to <1 ppm (<7.44mg/m 3 ) of vanadium compounds, including vanadium pentoxide (Gylseth et al. 1979; Kiviluoto et al. 1981a; Lewis 1959; NIOSH 1983), with the majority excreted in urine within one day post long-term or moderate exposure to vanadium dust (Kiviluoto et al. 1981a). The vanadium concentration in serum was higher than the nonoccupationally exposed controls following exposure to vanadium pentoxide dust (Kiviluoto et al. 1981b). Indirect evidence of absorption of vanadium in animals is indicated in studies involving inhalation exposure or intratracheal administration. In rats and mice exposed to 0.28–2.2 mg
vanadium/m 3 as vanadium pentoxide 1 for 14 days or 2 years (6 hours/day, 5 days/week), marginal increases in blood vanadium levels were observed, suggesting that vanadium pentoxide was poorly absorbed or rapidly cleared from the blood (NTP 2002; Dill et al. 2004); in the 2-year studies by NTP (2002), the increase in blood vanadium levels were concentration-related, although not statistically significant. Intratracheal studies suggest that vanadium pentoxide is

1 Many studies describe exposures in terms of concentrati on of vanadium, particularly when describing exposure to mixtures. When possible, a concentration is given as amounts of vanadium pentoxide. As listed here (mg vanadium/m 3 as vanadium pentoxide) shows exposure was to vanadium pentoxide, with data shown here in concentration of vanadium.DRAFT - DO NOT CITE OR QUOTE 7 readily absorbed through the lungs. The greatest absorption of a radioactive dose, 48 V, was found to occur 5 minutes after administration in albino rats (gender not specified) (Roshchin et al. 1980). Most of the vanadium, i.e., 80 and 85% of the tetravalent (V 4+ ) and pentavalent (V 5+ ) forms of vanadium, respectively, cleared from the lungs 3 hours after intratracheal exposure in male abino rats (Edel and Sabbioni 1988). After 3 days, 90% of vanadium pentoxide was eliminated from the lungs of female rats following intratracheal instillation (Conklin et al. 1982). In an intratracheal instillation study in female Fischer rats 50% was cleared in 18 minutes, and the rest within a few days (Rhoads and Sanders 1985). Wallenborn et. al. (2007) analyzed the components of a complex particulate matter mixture into its metal components and tracked the absorption of different metals in different tissues following a single intratracheal dose in rats. Healthy male Wistar rats were instilled with a single intratracheal dose of combustion particulate matter (PM) containing a moderate amount of transition metals, including vanadium. The composition of vanadium in the PM was 62.95

μ g/mg, with 7.18 μ g/mg in the water soluble fraction, 26.50 μ g/mg in the acid soluble fraction

and 29.27 μ g/mg in the insoluble fraction. According to calculations, of the 196.63 μ g/rat of vanadium instilled (theoretical), 110.32 μ g/rat was measured in lung 4-hrs post-instillation and

62.76 μ g/rat was measured in lung 24-hrs post-instillation. In the plasma and lung, vanadium was significantly elevated 4-hr post-ins tillation (130,000 ng V/g lung tissue, and 350 ng V/g plasma) compared to 24-hrs (60,000 ng V/g lung tissue, and 110 ng V/g plasma, respectively) suggesting rapid uptake of water-soluble vanadi um. The vanadium component of PM was tracked to lung, plasma, heart and liver and was significantly increased compared to controls at both 4- and 24-hrs post-instillation compared to saline controls. This study permitted detectable changes in component metals of a complex mixture in various organs and provides evidence that metals dissociate from particulate matter and tr anslocate to various target organs, depending on solubility (Wallenborn et al 2007).

3.1.2 Oral Exposure

No studies were available in the published literature regarding the rate and extent of absorption in humans after oral exposure to vanadium pentoxide. The absorption of vanadium through the gastrointestinal tract of animals is low. Lessthan 0.1% of an intragastric dose was detectable in the blood of albino rats at 15 minutes postexposure, and less than 1% at 1 hour (Roshchin et al. 1980). Similarly, only 2.6% of an orally administered radiolabeled dose of vanadium pentoxide was absorbed 3 days after exposure in female Fischer rats (Conklin et al. 1982). Vanadium was reported in tissues and urine of maleDRAFT - DO NOT CITE OR QUOTE 8 albino rats within hours after a single oral dose (Edel and Sabbioni 1988), suggesting that it is rapidly absorbed. Young rats that consumed vanadium in the drinking water and feed were found to have higher tissue vanadium levels 21 days after birth than they did 115 days after birth (Edel et al. 1984). The data suggest that there is a higher absorption of vanadium in these young animals due to a greater nonselective permeability of the undeveloped intestinal barrier. Thus, age of the rodents appears to play an important role in the absorption of vanadium in the gastrointestinal tract.

3.1.3 Dermal Exposure

No specific studies were available in the published literature regarding absorption in humans or animals after dermal exposure to vanadium pentoxide, although absorption by this route is generally considered to be very low (WHO 1988). Vanadium is a metal with low solubility, therefore absorption through the skin is thought to be minimal.

3.1.4 Other Routes of Exposure

No studies were available in the published literature regarding the nature and extent of absorption in humans after other routes of exposure to vanadium pentoxide.

3.2 Distribution

Distribution was measured from autopsy cases with unknown routes of exposure. Vanadium has been detected in the lungs (in 52% of the cases) and intestines (in 16% of the cases) of humans with no known occupational exposure, collected from autopsy data and reviewed in Schroeder et al. (1963). In the gastrointestinal tract, it was primarily found in the ileum (37%), cecum (45.1%), sigmoid colon (15.9%), and rectum (26.2%). Most positive samples had 0.01 μ g or less per g of tissue. The heart, aorta, brain, kidney, muscle, ovary, and

testes were found to have no detectable vanadium concentrations.

3.2.1 Inhalation Exposure

There are limited data on the distribution of vanadium in workers; serum vanadium levels in workers were highest within a day after inhalation exposure followed by a rapid decline in levels upon cessation of exposure (Gylseth et al. 1979; Kiviluoto et al. 1981b). Analytical studies have shown low levels of vanadium in human kidneys and liver, with even less in brain,DRAFT - DO NOT CITE OR QUOTE 9 heart, and milk. Higher levels were detected in hair, bone, and teeth (Byrne and Kosta 1978). Inhalation exposure and intratracheal administrati on studies have examined the distribution of vanadium in rodents. In F344 rats chronically exposed to 0.56 or 1.1 mg vanadium/m 3 as vanadium pentoxide (6 hours/day, 5 days/week), vanadium lung burdens peaked after 173 days of exposure and declined until 542 days; lung levels never reached steady state (NTP 2002). In contrast, lung burdens appeared to reach steady state by exposure day 173 in rats exposed to 0.28 mg vanadium/m 3 (NTP 2002). Similarly, lung burdens di d not reach steady state in B6C3F1 mice exposed to 1.1 or 2.2 mg vanadium/m 3 as vanadium pentoxide, 6 hours/day, 5 days/week for 542 days (NTP 2002). Rather, lung burdens peaked near day 54 and declined through day 535. Steady state was achieved in mice exposed to 0.56 mg vanadium/m 3 during the first 26 days of exposure. Vanadium is found to have a two-phase lung clearance after a single acute exposure in
both male Wistar rats and female Fischer rats (Oberg et al. 1978; Rhoads and Sanders 1985). The initial phase is rapid with a large percentage of the absorbed dose distributed to most organs and blood 24 hours postexposure, followed by a slower clearance phase. Vanadium is transported mainly in the plasma. It is found in appreciable amounts in the blood initially and only at trace levels 2 days after exposure (Roshchin et al. 1980). The pentavalent and tetravalent forms of vanadium compounds were found to have similar distribution patterns in male albino Sprague-Dawley rats (Edel and Sabbioni 1988). Three hours after exposure to the pentavalent or tetravalent form, 15–17% of the absorbed dose was found in the lung, 2.8% in the liver, and 2% in the kidney (Edel and Sabbioni 1988). After intratracheal instillation of pentavalent vanadium, retention of vanadium was observed in lungs, liver, kidneys, bone, testes and spleen with clearance at different timepoints post-exposure with little to no retention observed in stomach, intestines, heart or trachea (Edel and Sabbioni 1988). This is similar to the distribution seen following inhalation and oral exposure.

3.2.2 Oral Exposure

No studies were available in the published literature regarding distribution in humans after oral exposure to vanadium pentoxide. Acute studies with rats showed the highest vanadium concentration in the skeleton. Male rats had approximately 0.05% of the administered 48 V in bones, 0.01% in the liver, and <0.01% in the kidney, blood, testis, or spleen after 24 hours (Edel and Sabbioni 1988). Other authors who found that the bone had the greatest concentration of radiolabeled vanadium, followed by the kidney (Roshchin et al. 1980), noted similar findings. Conklin et al. (1982) reported thatDRAFT - DO NOT CITE OR QUOTE 10 after 3 days, 25% of the absorbed vanadium pe ntoxide was detectable in the skeleton and blood
of female Fischer rats. Oral exposure for an intermediate duration produced the highest accumulation of vanadium in the kidney. In young male rats at 3 weeks of age, the kidneys, heart, and lungs had the highest levels immediately following exposure (Edel et al. 1984). Vanadium in the kidney, liver, and lung decreased significantly at 115 days of age. There was an accumulation in muscle and fat, related to the growing mass of the tissues with age. The higher levels of vanadium in the young rat tissues may be due to the higher retention capacity of the undeveloped tissues, or a greater permeability of the intestinal wall. Adult rats exposed to 5 or 50 ppm vanadium in the drinking water for 3 months had the highest vanadium levels in the kidney, followed by bone, liver, and muscle (Parker and Sharma 1978). The retention in bone may have been due to phosphate displacement. All tissue levels plateaued at the third week of exposure. A possible explanation for the initially higher levels in the kidney during intermediate-duration exposure is the daily excretion of vanadium in the urine. When the treatment is stopped, levels decrease in the kidney. At the cessation of treatment, vanadium mobilized rapidly from the liver and slowly from the bones. Other tissue levels decreased rapidly after oral exposure was discontinued. Thus, retention of vanadium was much longer in the bones (Edel et al. 1984; Parker and Sharma 1978). Radike et al. (2002) assessed the distributi on of various metals, including vanadium, in female B6C3F1 mice. Mice ingested either (1) a metal mixture containing Chromium (Cr), Cadmium (Cd), Arsenic (As), Nickel (Ni) and Va nadium (V) in drinking water or (2) a metal mixture containing Cr, Cd, As, Ni, and V in NI H-31 feed. In water and feed, the calculated vanadium concentration in the mixture was 45 ppm and 1.105 ppm, respectively. Measured vanadium levels in the small intestine were 10 ppm at 5 weeks and 14 ppm at 8 weeks, and were significantly higher compared to controls than any other metal constituent in the small intestine. Mainly, vanadium pentoxide is distributed to bone (10-25% of administered oral dose), liver (~5%), and kidney (~4%). In addition, vanadium levels in the kidneys and the femur were significantly greater than in controls at 4, 8, 12, 16, and 24 week following oral dosing. Vanadium levels in small intestine and kidneys were lower in mice given vanadium as part of a heterogeneous metal mixture in feed vs. water (Radike et al. 2002).

3.2.3 Dermal Exposure

No studies were available in the published literature regarding distribution in humans or animals after dermal exposure to vanadium.DRAFT - DO NOT CITE OR QUOTE 11 3.2.4 Other Routes of Exposure

After intraperitoneal administration to rats, vanadium is distributed to all organs. After 24 hours, the highest concentrations were found in the bones and kidney, although initial levels were highest in the kidney (Roshchin et al. 1980; Sharma et al. 1980).

3.3 Metabolism

Vanadium is an element, and as such, is not metabolized. In the oxygenated blood, it circulates as a polyvanadate (isopolyanions contai ning pentavalent vanadium) but in tissues, it is retained mainly as the vanadyl cation (cationic form of tetravalent vanadium). Depending on the availability of reducing equivalents (such as reduced glutathione GSH, NADPH, NADH) and oxygen, vanadium may be reduced, reoxidized, and/or undergo redox cycling (Byczkowski and Kulkarni 1992).

3.4 Elimination and Excretion

3.4.1 Inhalation Exposure

Occupational studies showed that urinary vanadium levels significantly increased in vanadium pentoxide exposed workers (Gylseth et al. 1979; Kiviluoto et al. 1981a; Lewis 1959; NIOSH 1983; Zenz et al. 1962). Male and female workers exposed to 0.1–0.19 mg/m 3 vanadium in a manufacturing company, had significantly higher urinary levels (20.6 μ g/L) than the nonoccupationally exposed control subjects (2.7 μ g/L) (NIOSH 1983). The correlation between ambient vanadium levels and urinary levels of vanadium is difficult to determine from these epidemiological studies (Kiviluoto et al. 1981b). In most instances, no other excretion routes were monitored. Analytical studies have shown very low levels in human milk (Byrne and Kosta 1978). Evidence from animal studies supports the occupational findings. Vanadium administered intratracheally to rats was reported to be excreted predominantly in the urine (Oberg et al. 1978) at levels twice that found in the feces (Rhoads and Sanders 1985). Three days after intratracheal exposure to radiolabel ed vanadium pentoxide, 40% of the recovered 48 V dose was cleared in the urine while 30% remained in the skeleton, and 2–7% was in the lungs, liver, kidneys, or blood (Conklin et al. 1982). In female rats exposed to 0.56 or 1.1 mg vanadium/m 3 as vanadium pentoxide for 16 daysDRAFT - DO NOT CITE OR QUOTE 12 (6 hours/day, 5 days/week), lung clearance half-times during an 8-day recovery period were 4.42 and 4.96 days, respectively (NTP 2002). In mice similarly exposed to 1.1 or 2.2 mg
vanadium/m 3 as vanadium pentoxide, lung clearance half-times were 2.55 and 2.40 days, respectively (NTP 2002). In contrast to the 16-day exposure data, the lung clearance half-times in female rats exposed to 0.28, 0.56, or 1.1 mg vanadium/m 3 for 2 years (6 hours/day, 5 days/week) were 37.3, 58.6, and 61.4 days, respectively (NTP 2002). In mice, the half-times were 6.26, 10.7, and 13.9 days at 0.56, 1.1, and 2.2 mg vanadium/m 3 exposure levels (NTP 2002). After intratracheal instillation of pentavalent vanadium, clearance from lungs was initially rapid (3h) but with some vanadium (2% original dose) remaining at 12 d post-exposure. All other tissues eliminated 98-99% of original dose by 3h post-exposure (Edel and Sabbioni 1988). Epidemiological studies and animal studies suggest that elimination of vanadium following inhalation exposure is primarily in the urine.

3.4.2 Oral Exposure

No studies were available published literature regarding excretion in humans or laboratory animals after oral exposure to vanadium pentoxide.

3.4.3 Dermal Exposure

No studies were available published literature regarding excretion in humans or laboratory animals after dermal exposure to vanadium pentoxide.

3.4.4 Other Routes of Exposure

No studies were available published literature regarding excretion in humans or laboratory animals after other routes of exposure to vanadium pentoxide.

3.5 Physiologically Based Pharmacokinetic (PBPK)/Pharmacodynamic (PD) Models

No PBPK models for vanadium pentoxide are available.

3.5.1 Animal-to-Human Extrapolations

There are no relevant data available to evaluate potential toxicokinetic differencesDRAFT - DO NOT CITE OR QUOTE 13 between humans and laboratory animals. Similar effects have been reported in humans and animals following inhalation or oral exposure to vanadium pentoxide; however, this conclusion is based on the limited human toxicity data. In absence of data to the contrary, rats or mice appear to be valid models for extrapolation to humans.DRAFT - DO NOT CITE OR QUOTE 14 4. HAZARD IDENTIFICATION

4.1. STUDIES IN HUMANS – EPIDEMIOLOGY, CASE REPORTS, CLINICAL

CONTROLS

4.1.1 Oral Exposure

Few relevant studies investigating the eff ects of acute, subchronic or chronic oral exposure to vanadium pentoxide in humans were identified in the peer-reviewed literature. One study, Kucera et al. (1992), measured vanadium in the hair and blood of children exposed to vanadium by accidental drinking of contaminated water near a vanadium pentoxide plant. Vanadium levels in the hair did not differ signi ficantly for the control and exposed groups. An increase in vanadium concentrations was found in the blood of exposed children (median: 0.078

μ g/L) compared to control children (median: 0.042 μ g/L). No exposure-response relationship could be quantified. These results suggest that the vanadium levels in blood, but not hair is a sensitive or suitable indicator of environmental exposure.

4.1.2 Inhalation Exposure

Health effects of inhalation exposure to vanadium pentoxide and other vanadium compounds reported by case studies and epidemiological investigations include respiratory tract irritation, bronchitis (often called boilermakers’ bronchitis), airway obstruction, chest pain, rhinitis, pharyngitis, laryngitis and conjunctivitis in workers exposed to vanadium-containing dust during vanadium processing (Sjöberg, 1951; Sjöberg, 1956; Zenz et al., 1962; Kiviluoto et al., 1979; Kiviluoto, 1980; Kiviluoto et al., 1981; Musk and Tees, 1982; Irsigler et al., 1999) or to fuel-oil ash containing vanadium during cleaning and maintenence of oil-burning boilers (Williams, 1952; Sjöberg, 1955; Lees, 1980; Ross, 1983; Levy et al., 1984; Hauser et al., 1995a; Hauser et al., 1995b; Woodin et al., 1998; Woodin et al., 1999; Woodin et al., 2000; Hauser et al., 2001; Kim et al., 2004). Most of these st udies did not quantify the inhalation exposure specifically to vanadium pentoxide, although it wa s the primary exposure in vanadium pentoxide production. Exposures to residual oil fly ash (R OFA) involves a mixture of pollutants including elemental vanadium, vanadium oxides, vanadium pentoxide, vanadium sulphates, particulate matter and other metal constituents (Hauser et al., 1995b). Vanadium pentoxide is a majorDRAFT - DO NOT CITE OR QUOTE 15 constituent of ROFA and the studies of health effects among boilermakers evaluated models in relation to vanadium content of respirable particulate matter. Therefore, these papers serve to inform the assessment of the nature and scope of the health response to vanadium pentoxide. More recently, epidemiology studies evaluated the metals content of ambient particulate matter (PM) and found that communities in the United States with higher vanadium content in PM have higher PM-related risk of mortality or hospitalizations for cardiovascular or respiratory disease (Lippmann et al., 2006; Dominici et al., 2007; Bell et al., 2009; Patel et al., 2009; Lagorio et al., 2006). Because exposures to vanadium, not vanadium pentoxide, were evaluated in the studies of ROFA or PM air pollution, information to characterize the exposure-response relationship between inhaled vanadium pentoxide alone and adverse health effects in humans is limited. Moreover, both ROFA and PM are mixtures with several components that also may contribute to
observed health effects.

4.1.2.1 Controlled Human Exposure Study

Zenz and Berg (1967) exposed nine volunteer s to vanadium pentoxide dust to evaluate respiratory effects. Volunteers (gender not reported) were exposed to 0.1 mg/m 3 (n=2), 0.25 mg/m 3 (n=5) or 1 mg/m 3 (n=2) vanadium pentoxide in an environmental chamber for 8 hrs; no control group was included in this study. Particle size analysis revealed that 98% of particles had a diameter <0.5 μm. Post-exposure assessments of chest x-ray, blood, urine, nasal smear samples and pulmonary function were compared with baseline values determined for each subject prior to exposure. All subjects were observed for clinical symptoms for 11-19 months after exposure. Subjects exposed to 1 mg/m 3 vanadium pentoxide developed sporadic cough after 5 hrs of exposure, which progressed to pe rsistent cough during the last 3 hrs of exposure and continued for 8 days. No other signs of respiratory irritation were observed. Results of pulmonary function tests and chest x-ray 1, 2 and 3 weeks after exposure were similar to baseline (data not reported). Hematology and urinalysis parameters were not affected by exposure (data not reported). Nasal smears obtained 24 hrs, 72 hrs and 1 week after exposure were negative for eosinophilia. Three weeks after the initial exposure, two of the subjects were accidentally exposed to a “heavy cloud” of vanadium pentoxide dust (concentration not reported) for 5 minutes. Within 16 hrs of exposure, both subjects developed a “marked” productive cough with rales and expiratory wheeze, which continued for 1 week. However, pulmonary function test results were comparable to baseline (data not reported). Blood and nasal smear samples were negative for eosinophilia. Subjects in the 0.25 mg/m 3 exposure group developed a “loose” productive cough on the day after exposure, which lasted for 7-10 days. No additionalDRAFT - DO NOT CITE OR QUOTE 16 symptoms were observed and post-exposure pulmonary function and laboratory tests were comparable to baseline results (data not reported). Subjects exposed to 0.1 mg/m 3 developed “considerable” mucus formation within 24 hrs after exposure, lasting for 4 days. No other symptoms or positive findings for pulmonary function or laboratory tests were observed. No treatment-related symptoms or clinical findings were reported for any subject during the 11-19 months post-treatment period.

4.1.2.2 Occupational Exposure during Vana dium Pentoxide Mining and Processing

Respiratory and other symptoms have been documented among workers employed at facilities producing and processing vanadium pent oxide (Sjöberg, 1951; Sjöberg, 1956; Zenz et al., 1962; Kiviluoto et al., 1979; Kiviluoto, 1980; Kiviluoto et al., 1981a; Musk and Tees, 1982; Irsigler et al., 1999). Sjöberg (1951) reviewed earlier reports of symptoms among workers exposed to vanadium pentoxide and described symptoms among 36 employees (foremen, workers, builders/repairers) at a V 2 O 5 production factory in Falun, Sweden that began operation in 1946. The employees experienced symptoms on one or multiple occasions while under his medical surveillance between 1947 and 1950. Symptoms generally persisted an average of 13 days. Air samples in different parts of the vanadium pentoxide facility were found to contain dust concentrations of 0.6 – 86.9 mg/m 3 during pulverization of iron ore slag with a vanadium content of 4.8 to 7.5%. A significant proportion of the dust consisted of small, respirable particles (22% < 8μ, 39% < 12μ). Although vanadi um pentoxide concentrations in air were not reported, vanadium was detected in the blood and urine of 23 and 27 individuals, respectively. Sjöberg (1956) reported the main findings of his medical surveillance, including those of a thorough examination of the cohort in October, 1948, comparing them to an external referent population, a group of 703 workers from mines and sawmills in northern Sweden followed during the same time period and examined using the same methods. The authors assumed that the comparison population was exposed only to inert dust, however no sampling data was reported. The main symptoms of the upper respiratory tract were nasal irritation and/or nasal catarrh (inflammation of mucus membranes; 42% versus 20% among unexposed) and throat dryness and pain (86% versus 8% among unexposed). These symptoms were reported to be more prevalent at the final examination in 1948 and included acute and chronic pathological changes in the nose and pharynx. Lower respiratory tract symptoms included cough with no sputum (61%), cough with sputum (39%), wheezing (86%) and shortness of breath (75%). In the comparison group, prevalence of coughing and shortness of breath was 4% and 24%, respectively. Bronchoscopy, measured in five individuals, revealed no severe changes in fourDRAFT - DO NOT CITE OR QUOTE 17 cases and mild chronic bronchitis in one person. Acute changes in the lungs indicative of pneumonitis were noted in five workers. Spirometric measures were reported to be higher than those of the comparison group. A decrease in hemoglobin levels (7.8 ± 2.36% compared to 3.0 ± 2.07%) and red blood cell count was observed, particularly among workers who were permanently or frequently employed at the plant during the observation period, although levels remained within normal limits. The authors did not observe leucopenia or eosnophilia in blood samples. Six cases of dermatitis were observed, and one person had a positive response to a patch test with a sodium vanadate solution. Finally, weakness and fatigue were reported to be common symptoms after heavy exposure to dust, and symptoms of a neurasthenic character were
noted in some cases. Six workers with persistent symptoms were re-examined in December, 1953 to January, 1954 (Sjöberg, 1956). Reported symptoms included cough and wheezing (N=5), and dyspnea and fatigue (N=6). Clinical measurements and blood pressure were normal including sinuses, circulating eosinophils, erythrocyte sedimentati on rate, and radiographic examination of the lungs and heart revealed no evidence of pneumoconiosis. Lung function measures had increased
which the authors attributed to differences in technique. However, bronchoscopic examination and biopsy of bronchial mucosa showed evidence of chronic bronchitis (N=5). Two additional published case summaries described symptoms that appeared among workers after the start of new plant operations producing vanadium pentoxide pellets (N=18) (Zenz et al., 1962) or refining vanadium pe ntoxide (N=4) (Musk and Tees, 1982). Both exposures were to dry vanadium pentoxide powder at high concentrations (> 0.5 mg/m 3 ). Symptoms similar to those reported by Sjöberg (1956) appeared by the end of the first day of exposure including eye and throat irritation and cough with sputum, and persisted 2 – 4 weeks after exposure ended. Lung function tests revealed reversible airflow obstruction in 3 of the 4 case histories reported by Musk and Tees, and bronchial reactivity to histamine was demonstrated in two of the four. One individual with a family history of asthma and positive skin prick tests to common allergens, continued to experience wheezing for 8 weeks. Zenz et al (1962) did not find reductions in airflow among the 18 men examined. Kiviluoto et al. (1979; 1980; 1981a, b) published a series of reports regarding an investigation in 1975 of respiratory symptoms and clinical findings among employees (process workers, repairmen, foremen, and a laboratory wo rker) at a factory making vanadium pentoxide from magnetite ore. A total of 79 men employed at the vanadium factory ≥ 4 months were eligible and 63 men, aged 19-52 years, who were not on holiday or sick leave were enrolled (80% participation rate). A referent group of 63 men living in the same area were selected from workers at the magnetite ore mine (concentrating plant, the mine, the repair shop, and the office)DRAFT - DO NOT CITE OR QUOTE 18 and matched for age (within 2 years) and smoking habit (within 5 cigarettes/day). Vanadium dust concentrations at various sites measured on 8 days over 2 shifts in March-May 1976, were 0.012 mg/m 3 ranging between 0.002 (LOD) and 0.043 mg/m 3 . Vanadium concentrations in the breathing zone were 0.028 mg/m 3 (TWA) with a range of 0.002 – 0.42 mg/m 3 . Higher concentrations were found where grinding and p acking of smelt were conducted (TWA (range): 2.3 mg/m 3 (one sample) and 0.13 mg/m 3 (0.02 – 0.37). Air monitoring results were not reported for the referent population. Urinary vanadium concentrations among the exposed averaged 0.26 ± 0.17 umol/L (18 hour excretion), while concentrations among the referent group did not exceed the < 0.04 umol/L limit of detection (LOD). Clinical assessments were conducted by health personnel with no knowledge of exposure status. The occurrence of wheeze differed by exposure. Workers who reported wheezing were twice as likely to work in the vanadium factory (p<0.05) (Kiviluoto, 1980). Prevalence of nasal catarrh, cough, phlegm, or other respiratory symptoms did not differ between the exposed and referent groups. Spirometric measurements (FVC, FEV 1 , adjusted for height), obtained at the end of workers’ summer holidays, did not differ between the exposed and referent groups (p>0.01) and were not related to duration of exposure to vanadium dust (p>0.1). Inflammation was observed in nasal smears and these results are discussed in the section on immunological endpoints (Section 4.2.1). Nonfasting serum chemistry parameters, analyzed in May – June, 1975, when vanadium concentrations were higher (0.2-0.5 mg/m 3 ) were compared between 16 exposed and 16 referent subjects. Among the several serum chemistry parameters tested, the difference between the exposed and referent gr oups for serum albumin, chloride, urea, bilirubin and conjugated bilirubin were statistically signifi cant, although no values were outside the range of reference values. Hematological results (nonfasting) for 63 exposed and 16 referents, analyzed in March – May, 1976, when vanadium concentrations were lower (0.01-0.04 mg/m 3 ), did not vary by exposure group (p>0.05). In addition, there were no differences noted for serum cholesterol, serum triglyceride, or leukocyte differential. Irsigler et al. (1999) evaluated the clinical histories of 40 men, who were employed at a vanadium pentoxide production plant in South Af rica, and were referred by the plant’s medical staff for more detailed medical assessment because of persistence of respiratory symptoms (cough, breathing difficulty, wheezing) between October 1995 – October 1997. Twelve men, aged 19 – 60 years with bronchial hyperresponsiveness to inhaled histamine or exercise challenge (out of 40 men referred) were selected for analysis along with 12 men, aged 24 – 54, who were referred and did not have bronchial hyperresponsiveness, matched by age and smoking. The authors concluded that the asthma symptoms and bronchial reactivity had occurred as a result of vanadium exposure because all were free of current symptoms or aDRAFT - DO NOT CITE OR QUOTE 19 previous history of asthma when they began employment at the plant. Bronchial reactivity was determined by the repeated inhalation of histamine at successively doubled doses until a decrease of 20% or greater in FEV 1 was measured (Histamine PC20 FEV 1 ). A PC20 > 8 mg/ml was considered to be normal. Alternatively, an exercise challenge consisting of free tread mill running indoors for 6 minutes was used to assess bronchial reactivity. A positive test was defined as a decrease of 200 ml or a 15% or greater fall in FEV 1 . Vanadium pentoxide concentrations in air from area samples were < 0.15 mg/m 3 in the mills, kiln, leaching, and pollution control areas, 1.53 mg/m 3 in the fusion precipitation area, and 0.057 mg/m 3 in the
ferrovanadium area. Concentrations of SO 2 and NH 3 were above their recommended occupational limits in the kiln area. Vanadium pentoxide in spot urine samples was detected in 10 of the 12 workers with bronchial reactivity (5.2 – 180 μg/g creatinine) and was above a level considered to be toxic in 3 individuals (> 50 μg/g creatinine). Levels in the 12 referent men ranged between 12.0 – 55 μg/g creatinine, with one person above 50 μg/g creatinine. Among 9 subjects who returned for a follow-up examination after 5 to 23 months with no vanadium pentoxide exposure, 8 still exhibited bronchial reactivity. Atopy was ruled out as a primary cause because 5 individuals with positive skin prick allergy tests were equally distributed in the reactive and nonreactive groups. In addition, no evidence of viral upper respiratory tract infection was found in the study subjects, and 10 ci garette smokers were also distributed equally in both groups. Work tasks did not appear to be different between the two groups. This small study was not informative regarding associations between vanadium pentoxide exposure and bronchial reactivity. Although the group with br onchial reactivity had two subjects with high urine vanadium levels, differences in biomarker levels or job site between groups were not analyzed statistically, and the sample size and study design were not adequate to determine an association of vanadium pentoxide exposure with bronchial reactivity.

4.1.2.3 Occupational Exposure during Cleaning and Maintenance of Oil-Fired Boilers

Vanadium pentoxide is present in significan t amounts along with other vanadium oxides, vanadium sulphate and metals in ash that accumulate in oil- and coal-fired boilers, as well as other fuel types used in boilers. Several reports of case histories and epidemiology studies of boilermakers involved in the construction, cleaning and maintenance of oil-fired boilers have described upper and lower respiratory symptoms similar to those reported among workers processing vanadium pentoxide (Williams, 1952; Sjöberg, 1955; Lees, 1980; Ross, 1983; Levy et al., 1984; Hauser et al., 1995a; Hauser et al., 1995b; Woodin et al., 1998; Woodin et al., 1999; Woodin et al., 2000; Hauser et al., 2001; Kim et al., 2004). Additional health parameters haveDRAFT - DO NOT CITE OR QUOTE 20 been investigated including pulmonary functi on (Lees, 1980; Levy et al., 1984; Hauser et al., 1995a; Woodin et al., 1999; , biomarkers of infl ammation in nasal fluid (Hauser et al., 1995b; Woodin et al., 1998), and autonomic cardiac func tion (Magari et al., 2002). Studies have investigated acute effects occurring after jobs cleaning or overhauling boilers lasting a few days to several weeks and chronic conditions among boilermakers who had worked in that occupation for several years. Several case summaries described the health response of workers cleaning oil-fired boilers in Great Britain (Williams, 1952; Ross, 1983), Sweden (Sjöberg, 1955), and Canada (Lees, 1980). Two case series reports described exposure to dust during cleaning jobs of a few days to one week during 1946 – 1953. The onset of symptoms, including rhinorrhoea, sneezing, eye irritation, sore throat, and chest pain, began within 1 to 12 hours (Williams, 1952; Sjoberg, 1955). Symptoms with a delayed onset of 6 – 24 hours included a dry cough becoming paroxysmal and productive in some workers, wheezing, dyspnea upon exertion, and fatigue (Williams, 1952; Sjoberg, 1955). Bronchial irritation, bronchitis, and the development of rales in regions of the lung of some of the workers also were reported. Some workers also developed a greenish-black coating on the tongue. Symptoms were reported to persist for 3 days to one week after exposure was ended. Concentrations of vanadium pentoxide particles (10-20 μ in diameter) in the air inside the boilers during cleaning were 17 – 85 mg/m 3

. Lees (1980) reported on a clinical evaluation of 17 men occupationally exposed to bottom ash from cleaning the boiler of an oil-fired electricity generating station. Personal sampling in the breathing zone of four of the men indicated a mean time weighted average of

523 μ g/m 3 (0.52 mg/m 3 ) for dust under 10 μ . Vanadium content in the bottom ash and crusted deposits was 15.3% and 24.2 – 35%, respectively. The men wore cartridge filter type respirators during the cleaning operation, however subsequent testing documented that they leaked up to 9%. The air concentrations of vanadium pentoxide were not specifically reported. Symptoms and lung function after exposure were compared to health status assessed before exposure began. A medical history and clinical examination, pe rformed before work began and the day after, indicated symptoms similar to those described in earlier reports, including cough with sputum, respiratory wheeze, and sore throat reported in 77%, 53%, and 41% of the men, respectively. Serial spirometry measurements at 24 hour intervals showed reductions with the lowest mean
FVC and FEV 1 values at postexposure days 3 and 2, respectively, compared to baseline (percent of baseline: 88.6% and 86.6%, respectively; p<0.05). Reductions in forced mid-expiratory flow of 9-31% also were recorded. These measures had returned to preexposure levels after 4 weeks. No details were provided regarding the years the study was conducted and the characteristics of the men under study.DRAFT - DO NOT CITE OR QUOTE 21 In 1981, the Occupational Safety and Health Administration conducted an investigation of work-related bronchitis among 100 boilermakers exposed to vanadium pentoxide during an oil to coal conversion of a utility company power plant in western Massachusetts (Levy et al., 1984). The conversion occurred over the course of approximately 6 weeks, October 15 – November 30, with most of the men working 10 hour days, six days per week. Air samples obtained in the boiler at approximately 4 weeks during the conversion indicated vanadium pentoxide fume concentrations of 0.05 – 5.3 mg/m 3 . Concentrations of chromium, nickel, and fumes of copper and iron oxide were stated to be within acceptable limits. Nitrogen dioxide and hydrogen sulfide were not detected. Low concen trations of carbon dioxide (<5 ppm) and ozone (< 0.1 ppm) were measured. Sulfur dioxide (< 1 ppm) was measured in the boiler during welding operations and outside the boiler (1 – 35 ppm) when expansion joints were cut with a
torch. In early December, a questionnaire was distributed to all 100 workers through the union president and responses were received from 55 men over the next two months. All of the respondents, aged 23 – 60 years, reported symptoms, with over half describing cough with sputum (85%), sore throat (76%), dyspnea on exertion (71%), chest pain or discomfort (65%), headache (56%), runny nose or sneezing (56%), wheezing (55%) and tiredness (51%). The median time to onset was 7 days with clustering at 0 – 4 days and 6 – 8 days. When the questionnaires were completed, symptoms had resolved or were improving in 41 of the 55 respondents. Although three-fourths of the responde nts stated that they had used a respirator over half the time when in the boiler, more than half stated that the respirator used was a paper mask. Respondents had been boilermakers a median of 10 years. Pulmonary function tests were performed on 35 individuals after they visited a physician. Median FVC was 87% of predicted, but was < 80% of predicted in 5 of 27 individuals. Median FEV 1 was 93% of predicted, but was < 80% of predicted in 8 of 27 men. Median FEV 1 /FVC was 79% of predicted. Median FEF 25-

75% among 24 workers tested was 57% of predicted with only 4 of 24 above 80% of predicted.

FEF 25-75% was not correlated with smoking history among the 69% of workers for whom this
information was obtained. The symptoms and effects on lung function are consistent with previous reports of occupational exposures during the cleaning and maintenance of boilers that had burned oil contaminated with vanadium pentoxide. A marked deficit in pulmonary function, particularly in FEF, was observed in some of the workers. However, pulmonary function was assessed in only 60% of respondents by several different health providers. In addition, this study is limited by a lack of baseline information on health status or comparison to a comparable occupational group, a relatively low response among the exposed workers, and data collection days to weeks afterDRAFT - DO NOT CITE OR QUOTE 22 exposure was discontinued. A subsequent study of lung function among boilermakers before and after four weeks of work overhauling an oil-fired boiler did not observe an association with respirable vanadium dust concentrations (Hauser et al., 1995a). A total of 36 out of 80 eligible workers completed a baseline test, and 26 completed a postexposure test. The men averaged 42.5 years of age (27 – 60) and 16.9 years on the job (6 months – 35 years). Daily exposure estimates were developed for each subject based on work diaries detailing tasks and locations and personal sampling. Between 1 and 10 hour time weighted average sampling was available for 15% of the total number of study days and these data were applied to the task/location information to assign exposure levels for PM 10 and vanadium dust for each worker. Lung function values were analyzed in relation to three exposure indices for the exposure period; average or peak concentrations, and concentrations on the day of the postexposure test. Average, peak and mean day-of vanadium concentrations in particulate matter < 10 μm (range) were estimated to be 12.2

± 9.1 μ g/m 3 (2.2 – 31.3), 20.2 ± 11.4 μ g/m 3 (2.2-32.2) and 12.1 ± 10.9 (1.6-31.1), respectively. For the spirometric measures, the largest value from three acceptable curves was used in the analysis. Reductions in several lung function measures were observed over the average of 27 ±4.1 days between the baseline and postexposure tests that were statistically significant. FEV 1 , FVC, and FEF 25-75% decreased by an average of 140 ± 160 ml (range: -390-420), 140 ± 200 ml (range: -580-320) and 270 ± 450 ml/s (range: -1170-870), respectively. Each lung function index was adjusted by dividing the change by the average of the pre- and post-exposure value. The adjusted value was analyzed for associations with exposure in multiple linear regression models adjusting for age and current smoking status. Peak PM 10 was inversely associated with adjusted Δ FEV 1 (p=0.03), Δ FEV 25% , (p=0.07), Δ FEV 50% , (p=0.01), Δ FVC (p=0.01), but not

Δ FEV 25-75% , (p=0.23) and Δ FEV 75% (p=0.43). However, mean, peak, and day-of respirable
vanadium dust concentrations were not associated with any spirometric indices (the data were not presented). PM 10 and vanadium dust exposure also were not related to bronchial reactivity as measured with methacholine challenge tests before and after the overhaul. The authors noted that the concentrations of vanadium dust were low and the variation in the range of concentrations may not have been wide enough to detect a relation with lung function in this small sample. Alternatively, the deficits in lung function may have been caused by a different
constituent in PM 10
. Woodin et al (1998, 1999, 2000) described in a series of reports a prospective clinical study that evaluated health measures among 18 boilermakers and compared them to 11 utilityworkers involved in the overhaul of a large, oil-fired boiler over a six week period from midMay, 1995 to late-June, 1995. The men had volunteered for the study and did not have allergicDRAFT - DO NOT CITE OR QUOTE 23 symptoms two weeks prior to or during the overhaul. Data from one person who presented with cold or flu symptoms at one of the clinical assessments were excluded. All were white men aged 26 – 61 years and were employed a mean of 20.5 years (range: 3 – 39). Vanadium and PM 10 exposure was calculated for each subject for each work day using information on task duration and location and use of personal protective equipment from job diaries, and PM 10 or vanadium concentrations from personal exposure monitors in the breathing zone. Personal and stationary sampling of PM 10 (< 10 μ m) was conducted over 10 – 12 hour shifts in major work areas in and around the boiler. Environmental concentrations were compared before and during the boiler overhaul, and between the two occupational groups. Utility workers did not enter the boiler during the overhaul. Vanadium levels before the boiler work was comparable for boilermakers and utility workers (geometric mean (SD) μ g/m 3 : 1.2 (1.4) and 1.1 (1.2), respectively). During the boiler work, vanadium levels rose to a geometric mean (SD) of 8.9 (2.3) μ g/m 3 inside the boiler but did not change appreciably outside the boiler (geometric mean (SD) μ g/m 3 : 1.4 (1.6) (p < 0.001)). Exposure estimates were adjusted for the type of protective gear worn and its duration to calculate individual daily dose. The daily dose to the upper and lower airway was estimated using values for minute volume, penetration and deposition rates, and particle size. Quartiles of lung vanadium dose ( μ g) were ≤ 0.90, > 0.90 - ≤ 5.30, > 5.30 - ≤ 22.30 and > 22.30. Quartiles of nasal vanadium dose ( μ g) were ≤ 2.50, > 2.50 - ≤ 23.20, > 23.20 - ≤ 68.30 and >

68.30. The workers recorded symptoms five times per day in a log and scored them for severity with a numerical score from 0 to 3. The highest severity score for each day was used in the analysis. Incidence of upper airway symptoms (nasal congestion/irritation, throat irritation) was 67% (12/18) among boilermakers and 36% (4/11) among utility workers. The incidence of lower airway symptoms (chest tightness, wheeze, cough, and sputum production) was 72% (13/18) among boilers and 27% (3/11) among utility workers. Robust regression models of lower airway maximum severity scores and average symptom frequency in relation to quartiles of lung vanadium dose indicated a dose-related increase. Maximum lower airway severity scores were increased by 0.47 (p=0.01), 0.86 (p<0.01) and 0.24 (0.10) in quartiles of lung vanadium dose 2, 3, and 4 compared to 1. Average lower airway frequency was increased by 0.19 (p=0.02), 0.39 (p<0.01) and 0.14 (0.07) in quartiles of lung vanadium dose 2, 3, and 4 compared to 1. The regression models were adjusted for current smoking. Lung function was assessed on three occasions: before the overhaul, during the overhaul (before the shift on the last day) and 2 weeks after the work ended (Woodin et al., 1999). The highest value from three acceptable curves obtained during each test was used in repeated measures analysis of variance to test for differences over time. No changes in the four airflowDRAFT - DO NOT CITE OR QUOTE 24 measures, FEF 25 , FEF 50 , FEF 75 , and MMEF, were observed over the course of the study. Mean (SD) FEV 1 (l) values before, during and after the overhaul were 3.73 (0.61), 3.76 (0.54) and 3.65 (0.42), respectively. Mean (SD) FVC (l) values before, during and after the overhaul were 5.01 (0.67), 4.94 (0.61) and 4.92 (0.55), respectively. Change in lung function from the beginning to
the end of the overhaul was not associated with upper or lower airway dose levels of either
vanadium or PM 10 when assessed in linear regression models adjusting for smoking and age. In addition, mean dose estimates were not different between individuals who experienced a loss of
either FEV 1 or FVC > 100 ml or those who experienced no change or an increase (two-sample ttest). Boilermakers were exposed to higher PM 10 concentrations compared to utility workers both before (geometric mean (SD): 0.40 (1.60) versus 0.10 (2.70), p < 0.05) and during the overhaul (geometric mean (SD): 0.47 (1.90) versus 0.13 (4.00), p < 0.001). In contrast to the elevations in vanadium concentrations measured during the overhaul, PM 10 concentrations did not increase appreciably (p > 0.05). During the boiler overhaul, ozone concentrations increased somewhat outside the boiler, but did not change inside the boiler. The authors considered the levels of other metals (cadmium, chromium, manganese, lead, arsenic, and nickel) to be low. All samples were 1-3 orders of magnitude below the 1996 TLV, and no significant changes were observed during the overhaul. Analyses of lung function and other pollutants were not reported. Reductions in lung function over two years were investigated by Hauser et al. (2001) in a longitudinal study of boilermaker construction workers exposed to combustion particles from multiple sources including powerplants (oil, coal, natural gas), trash incinerators, paper mill incinerators and other industrial sources with boiler, vessels and tanks requiring maintenance and repair. A total of 118 boilermakers from Local 29 of the International Brotherhood of Boilermakers, Iron Shipbuilders, Blacksmiths, Forgers and Helpers (81% of those contacted) were followed between 1997 and 2000. Participants completed spirometry, a modified American Thoracic Society questionnaire on re spiratory symptoms, and a work history questionnaire at baseline and two annual follow-up visits. The male cohort averaged 42.6 years of age (range: 20.5 – 56.5 years) and 97% were Caucasian. Technicians used standardized techniques and the same equipment to conduct spirometry testing for all workers during the study. Each worker, after some days off work, performed 3 – 7 forced vital capacity maneuvers to obtain at least three acceptable curves between 9:00 am and 2:00 pm on testing days. Baseline

FEV 1 and FVC were 90% and 94% of predicted. The nine participants who were lost to followup had a lower mean baseline FEV 1 compared to those who remained in the study (84% compared to 91.2% predicted). The number of years worked as a boilermaker was a statistically significant predictor of annual FEV 1 (-33.5 ml/years worked (95% CI:-45.9 - -21.1)). InDRAFT - DO NOT CITE OR QUOTE 25 generalized estimating equations adjusting for age, baseline FEV 1 and cigarette smoking status, the number of hours worked at gas-fired powerplants in the previous year was inversely related
to annual FEV 1 (-9.8 ml/100 hours worked (95% CI: -16.0 - -3.5). Adjusted models analyzing the number of hours worked at oil and gas-fired powerplants also showed FEV 1 reductions, however the associations were not statistically significant. Statistically significant reductions in
annual FEV 1 also were observed among boilermakers who had ever worked at a coal, oil or gasfired powerplant in models evaluating each fuel type separately. This study provides evidence of long-term declines in lung function among boilermakers exposed to combustion particles from several fuel types. The study did not estimate exposure to individual substances however, and no conclusions can be drawn regarding a role for vanadium compounds. The effect of occupational PM 2.5 concentrations and metals components on cardiac autonomic function during a work shift was studied among a panel of 39 boilermaker construction workers (Magari et al., 2002). The group of apprentice and journeyman boilermakers was an average of 38 years old (18 – 59 years) and had worked an average of 13 years (0 – 40 years) in that occupation. Metals concentrations over an 8 – 10 hour work shift were determined from particle samples (< 2.5 μ m) collected using personal monitors, and heart rate was monitored using a five-lead Holter monitor during the same period. Heart rate variability was estimated as the mean of 5 minute average SDNN (standard deviation of the normal-to-normal intervals). Vanadium concentrations (corrected for blank filter metal content)
were skewed with a mean of 0.76 ± 1.96 μ g/m 3 and a median of 0.13 ± 1.96 μ g/m 3 (range 0 – 11.62). Fifteen of 48 personal samples were above the limit of detection for vanadium (0.00859

μ g/m 3 ). Average PM 2.5 concentrations were 1.16 ± 1.61 μ g/m 3 with a median of 0.56 ± 1.96

μ g/m 3 (range 0.09 – 7.76). Vanadium concentrations during the shift were associated with a 3.98 msec (95% CI: 1.64 – 6.32) increase in SDNN index per μ g/m 3 in mixed effects regression models with a random effect for each study subject and fixed covariates for smoking status, age, and mean heart rate. Lead also was associated with an increase in SDNN index (11.3
msec/ μ g/m 3 , 95% CI: 2.88 – 19.73). 2 In contrast to earlier studies in this cohort that observed a decrease in SDNN measures, average PM 2.5 concentrations were not associated with SDNN index in this study (-0.77 msec/ μ g/m 3 , 95% CI: -2.36 – 2.81). Vanadium concentrations were
not correlated with either lead or PM 2.5 indicating that these pollutants were not likely to be confounders of the association of heart rate variability with vanadium. No associations with heart rate variability were observed for the other analyzed metals including nickel, chromium,

2 These effect estimates for vanadium and lead were reported as such in the abstract and Table 4, but were transposed in the text of the Results.DRAFT - DO NOT CITE OR QUOTE 26 manganese, or copper. It is possible that the observation of an increase in heart rate variability with unit increases in vanadium and lead concentrations was the result of the temporal framework chosen for the analysis (e.g., averages over the work shift) rather than other time frames for exposure and SDNN averages. Indeed, a subsequent study examining changes in a different index of autonomic cardiac function, rMSSD (square root of the mean squared differences of successive intervals), averaged over the night-time hours (0:00 – 7:00), found an inverse association with average work shift concentrations of the PM 2.5 metal, manganese (Cavallari et al., 2008). The authors did not report on the results of analyses for vanadium. The biological significance of the association of vanadium with increases in heart rate variability is unclear. All-cause mortality has been associated with decreased heart rate variability measured at baseline in longitudinal studies (Dekker et al., 1997). However, the effect on heart rate variability indicates that vanadium exposure may alter autonomic function. Alternatively, the observed association may have been due to chance. In summary, case series, cross-sectional and longitudinal studies of occupational exposure to vanadium pentoxide or vanadium in residual oil fuel ash over a few days to several weeks reported symptoms of upper and lower respiratory tract irritation including headache, runny nose or sneezing, sore throat, cough with sputum, dyspnea on exertion, chest pain or discomfort, wheezing and tiredness (Sjöberg, 1951; Sjöberg, 1956; Zenz et al., 1962; Kiviluoto et al., 1979; Kiviluoto, 1980; Musk and Tees, 1982; Williams, 1952; Sjöberg, 1955; Lees, 1980; Ross, 1983; Levy et al., 1984; Woodin et al., 2000). Some symptoms began after a few hours while the onset of other symptoms occurred after one to several days. Vanadium pentoxide concentrations in production facilities where the studies were conducted varied by work location from 0.06 mg/m 3 in the ferrovanadium area to 1.53 mg/m 3 in the fusion precipitation area at one facility (Irsigler et al., 1999) and > 0.5 mg/m 3 at a pilot-plant operation (Zenz et al., 1962). Concentrations of vanadium pentoxide inside the boiler during a cleaning operation were 85 mg/m 3 (Sjoberg, 1955) and ranged from 0.05 – 5.3 mg/m 3 during conversion of a power plant from coal to oil (Levy et al., 1984). The frequency and severity of symptoms were associated with vanadium among boilermakers exposed to relatively low ambient concentrations. Upper airway symptom severity scores (nasal congestion/irritation, throat irritation) increased across quartiles of estimated vanadium dose in the nose and the elevation was statistically significant in the third quartile compared to the first quartile in regression models adjusted for smoking (Woodin et al., 2000). Increases in lower airway symptom frequency and severity (chest tightness, wheeze, cough, and sputum production) were associated in a dose-related manner with estimates of lung vanadium dose. During the boiler overhaul, geometric mean respirable vanadium dust concentrations (SD)DRAFT - DO NOT CITE OR QUOTE 27 were 8.9 (2.3) μg/m 3 (0.009 mg/m 3 ). Vanadium concentrations in respirable particles (< 10 μm) inside the boiler during an overhaul (geometric mean (SD): 8.9 (2.3) μg/m 3 ) associated with increased upper and lower respiratory symptoms among boilermakers were not related to deficits in pulmonary function. Pulmonary function declines (FEV 1 , FVC, FEF 25%-75% ) over the course of a boiler overhaul were reported among boilermakers (Lees, 1980; Levy et al., 1984; Hauser et al., 1995a; Woodin et al., 1999). However, those studies with more systematic and detailed reporting of methods and
results observed no association of FEV 1 , FVC or flow measures with average vanadium
concentrations in PM 10 (Mean (SD): 12.2 (9.1) μg/m 3 ) or estimates of vanadium or PM 10 dose in nasal passages or the lung (Hauser et al., 1995a; Woodin et al., 1999). Peak PM 10 exposure (the highest concentration (1 – 10 hour TWA) reached on any day during the overhaul) (Mean (SD): 4.25 (1.58) mg/m 3 ) was inversely associated with Δ FEV 1 , Δ FEV 50% , and Δ FVC, however peak vanadium concentrations (Mean (SD): 20.3 (11.4) μg/m 3 ) were not. The authors reported that concentrations of respirable vanadium dust were relatively low and there may not have been enough variation in exposure to allow detection of an association with the small changes in pulmonary function that occurred during the overhaul. Uncertainties in the exposure estimates, particularly inside the boiler, also may have prevented detection of associations between vanadium exposure and pulmonary function. The authors reported that they obtained fewer air samples inside the boiler where wearing the monitor was uncomfortable. Consequently, exposure and dose estimates for these locations may be more uncertain compared to other locations. The cohort had worked in this occupation an average of 20 years and may represent a healthy, less susceptible population. In addition to vanadium, boilermakers are exposed to increases in the levels of other metals during boiler overhauls including nickel, chromium and manganese (Liu et al., 2005). The observed pulmonary function declines amng boilermakers may be explained by exposure to other ROFA constituents.

4.1.2.4 Exposure to PM 2.5 Vanadium in Ambient Air

Vanadium is a constituent of ambient particulate generated by oil combustion. Recent studies of the short-term health effects of particulate matter and its constituents have found higher risks of mortality and hospitalization in locations with a higher fractional content of vanadium, nickel and elemental carbon in PM (Dominici et al., 2007; Bell et al., 2009). Lippmann et al (2006) evaluated the impact of average concentrations of 16 PM 2.5 components across 60 U.S. communities on the association between the daily change in PM 10 concentration on daily all-cause mortality risk in those communities. Community-specificDRAFT - DO NOT CITE OR QUOTE 28 mortality risk estimates per daily change in PM 10 concentrations were obtained from the National Morbidity, Mortality, and Air Pollution Study (NMMAPS) database for the years 1987 – 1994. Annual average concentrations for PM constituents were obtained for 2000-2003 from
the PM 2.5 speciation network. The authors used weighted linear regression to evaluate for each chemical constituent whether annual average concentration altered the association between PM 10 concentration on the previous day and mortality risk. The constituents, nickel and vanadium,
were found to increase PM 10 mortality risk. These results were re-evaluated and extended by Dominici and colleagues (2007) using
NMMAPS data for 90 communities from 1987 – 2000 and data on PM 2.5 composition for 187
U.S. counties for 2000 - 2005. A total of 69 U.S. communities in the NMMAPS database also
had data on PM 2.5 composition and were included in this analysis. Using a Bayesian hierarchical regression model to estimate the association between the l-day lag PM 10 mortality risk and average county-level PM constituent concentrations, counties with high average concentrations of nickel and vanadium had higher PM 10 mortality risk with a one-day lag. When the three counties that comprise the NMMAPS New York community were excluded from the analysis,
the effect of nickel and vanadium was much weaker and lost statistical significance. The authors
stated that the three New York counties had nickel and vanadium concentrations that were 8.9 and 3.4 times higher than the other counties in the analysis. Bell et al (2009) used a Bayesian hierarchical regression model to evaluate the effect of

PM 2.5 chemical constituents as percent of PM 2.5 total mass on PM 2.5 associated cardiovascular and respiratory hospital admissions by county and season for 106 U.S. counties during 1999 - 2005. Counties with a population 200,000 or greater for which data on PM and constituent concentrations were available were selected. Models were adjusted for day of the week, seasonality, long-term trends using a smoothi ng function, daily temperature and dew point temperature, as well as the previous three days temperature and dew point temperature. Countyand season-specific PM 2.5 relative risk for cardiovascular and respiratory hospital admissions were higher in counties and seasons with a nickel, vanadium or elemental carbon fraction of total

PM 2.5 in the 75 th compared to the 25 th percentile. The effect of these PM constituents was statistically significant. The average concentration of vanadium across the counties was 0.003

μ g/m 3 (range: 0.001-0.01) with an interquartile range of 0.001 μ g/m 3 . The interquartile range as percent of PM 2.5 total mass was 0.01%. Each interquartile range increase in the fraction of PM 2.5 total mass for vanadium was associated with a 27.5% (95% posterior interval: 10.6 - 44.4)
increase in PM 2.5 associated cardiovascular hospitalizations and a 392% (95% posterior interval: 46.3 – 738) increase in PM 2.5 associated respiratory hospitalizations. Associations also were observed for elemental carbon and nickel, and effect estimates were not always stable inDRAFT - DO NOT CITE OR QUOTE 29 multipollutant models. The finding that communities with a higher fractional content of vanadium, nickel and elemental carbon in ambient particulate matter have a higher risk of mortality and hospital admissions related to daily change in PM 2.5 concentration is intriguing and indicates the need for further research on the contribution of fuel oil combustion to regional and local air pollution, and the contribution of specific metals, including possi bly vanadium pentoxide to elevated health risks. The time series study design used in these investigations evaluates exposure-disease associations at the county level and therefore, individual-level assessments of exposure and the impact of possible confounders is not possible. However, Bell et al (2009) investigated whether county-level indicators of socioeconomic status , racial composition, and degree of urbanization could be alternative explanations for the observed effect modification by PM 2.5 constituents and
concluded that this was not the case.
Ambient concentrations of PM 2.5 and PM 2.5 fractions of nickel, vanadium, zinc and elemental carbon were evaluated in relation to respiratory symptoms among young Dominican and African American children, aged 3 – 24 months, followed as part of a birth cohort study in Northern Manhattan and the South Bronx in New York City between 1998 and 2007 (Patel et al., 2009). Among 653 24 month old children with questionnaire data (90% of total enrolled) 3- month average ambient vanadium concentrations were associated with an increase in the presence of wheeze during the cold and flu season (September 1 – March 31). After adjusting for elemental carbon, NO 2 , copper, and iron, an interquartile range increase (0.003 μ g/m 3 ) in three-month average vanadium concentrations was associated with a 31% increased probability of wheeze during the cold season (p<0.0003). When not stratified by season, an IQR increase in vanadium concentration was associated with a 14% increased probability of wheeze (p=0.08). However, when the highest 5% of vanadium c oncentrations were excluded, the association of wheeze with vanadium lost significance in the multipollutant model. Vanadium concentrations were not associated with cough. Twenty-four hour average ambient concentrations of PM 2.5 and

PM 2.5 fractions of nickel, vanadium, zinc and el emental carbon, measured every third day at two stationary sites in the Bronx, were obtained from the New York State Department of Environmental Conservation. Exposure levels we re assigned to each subject by calculating 3- month moving average concentrations of each pollutant based on each follow-up questionnaire date and the previous three months. Exposures were assigned to each subject’s address using inverse-distance weighted concentrations from th e two stationary monitors. Associations were evaluated using generalized additive mixed effects models and a first-order autoregressive correlation structure to account for correlation between the up to 8 repeated observations for each individual. The models also adjusted for sex, ethnicity, postnatal ETS exposure, and aDRAFT - DO NOT CITE OR QUOTE 30 smoothed term for calendar time using natural cubic splines. Other pollutants also were associated with increased probability of wheeze (nickel) or cough (elemental carbon, NO 2 ) during the cold/flu season or wheeze in other months (NO 2 ). PM 2.5 mass concentrations were not related to an increase in probability of symptoms. The interpretation of the multi-pollutant models for vanadium is complicated because other PM constituents are correlated with vanadium concentrations resulting in less stable risk
estimates. Nickel was not evaluated in the same model with vanadium for this reason. The association with vanadium was independent of the association with NO 2 , a marker for traffic emissions, and the authors suggested that oil combustion for space heating may contribute to the observed respiratory symptoms in the very young children in this study. Although vanadium cannot be singled out as the responsible agent for the probability of wheeze observed in this study, the association is consistent with the respiratory symptoms observed among boilermakers with exposure to high levels of residual oil fuel ash for periods of days to weeks.
The effect of the metal content of ambient PM 2.5 on lung function also was evaluated in a time-series panel study of 29 patients with chronic obstructive lung disease, asthma, or ischemic heart disease in Rome, Italy in the spring and winter of 1999 (Lagorio et al., 2006). Outpatients of the Pneumology and Cardiology Departments of the Catholic University Hospital in Rome who met eligibility requirements for COPD (N=11), asthma (N=11) or ischemic heart disease (N=7), and who lived in census tracts less than 2 kilometers from one of six air monitoring stations were selected for the study. The subjects volunteered to conduct repeated clinical examinations for two one-month periods. Pulmonary function testing was conducted according American Thoracic Society guidelines, and measures were expressed as the percentage of predicted based on subject-specific age, hei ght and weight. An average of 15, 24 and 9 observations were obtained from each participant in the COPD, ischemic heart disease and asthma panels, respectively. Daily average PM 2.5 concentrations were calculated based on measurements obtained at two fixed site monitors set up for the study. PM content of cadmium, chromium, iron, nickel, lead, platinum, vanadium and zinc was calculated as the ratio of the metal content in each PM sample to the air volume collected during the sampling. The mean 24-
hour PM 2.5 concentrations during the spring and winter of 1999 were 18.2 ± 5.0 μ g/m 3 and 36.7

± 24.1 μ g/m 3 , respectively. The mean 24-hour vanadium concentrations during the spring and winter of 1999 were 2.4 ± 1.6 ng/m 3 and 1.1 ± 0.52 μ g/m 3 , respectively. Vanadium was not associated with daily change in percent predicted pulmonary function among subjects with asthma, ischemic heart disease, or COPD in generalized estimating equations models. Models adjusted for season (all), daily mean temperature (all), relative humidity (all), day of the week (COPD, IHD) and ß-2 agonist use (asthma). Although the repeated measures design was aDRAFT - DO NOT CITE OR QUOTE 31 strength of the study, the number of subjects in each disease panel was small, and may not have been large enough to detect an association with the very low vanadium concentrations analyzed.

4.2. SUBCHRONIC AND CHRONIC STUDIES AND CANCER BIOASSAYS IN

ANIMALS – ORAL AND INHALATION

4.2.1. Oral Exposure

4.2.1.1. Subchronic Studies

No animal studies that have comprehensively examined histopathological, biochemical and clinical endpoints of subchronic oral exposure were identified from the available literature. Mountain et al. (1953) evaluated the effects of subchronic exposure of rats to dietary vanadium pentoxide on body weight gain, erythrocyte count, hemoglobin and cystine content of hair. Groups of 5 male Wistar rats were fed diet s containing 0, 25, 50, 500 or 1000 ppm of vanadium incorporated in the form of pentoxide for 103 days (25 and 50 ppm groups; “low-exposure” groups) or 75 days (500 and 1000 ppm groups; “high-exposure” groups). After 35 days of treatment, dietary vanadium levels of the “low-exposure” groups were increased to 100 and 150 ppm, resulting in average daily doses of 0, 74.5, 116, 500, and 1000 ppm (0, 5.9, 9.2, 39, or 79 mg/kg-day) 3 of vanadium resulting in a doses of 0, 10.5, 16.4, 69.6, or 141.0 mg/kg-day of vanadium pentoxide. 4 At the end of treatment, body weight gain, liver weight, and cystine content of hair were measured in all groups, erythrocyte count and hemoglobin level were measured in control and “low-exposure” groups, and relative liver weight was measured in control and 69.6 mg/kg-day groups. Average body weights of animals at the conclusion of the study were not reported, though average weight gain in grams/rat was reported. Compared to control, average body weight gain was increased in the 10.5 mg/kg-day and 16.4 mg/kg-day groups (54 and 45% increase) and decreased in the 69.6 mg/kg-day group (66%) and 141.0 mg/kg-day group (no gain in body weight over the study period). The increase in body weight gain at the low-exposure levels was not expl ained and statistical significance or standard deviations were not reported for any result. Relative liver weight in the 69.6 mg/kg-day group

3 Calculation: mg/kg-day = ppm(mg of compound per kg food) x mg food consumed/day x 1/kg body weight (using reference food consumption rate of 0.0217 kg/day [U.S. EPA, 1988] and average body weight of 0.275 kg for male rats [Mountain et al., 1953]).

4 Conversion from mg/kg – d of vanadium to amount va nadium pentoxide: [(mg/kg-d vanadium)(MW vanadium pentoxide, 181.9)]/(2xMW vanadium, 101.9).DRAFT - DO NOT CITE OR QUOTE 32 was statistically significantly increased compared to control, reported as a ratio of liver weight/body weight (3.86 compared to 3.51, p < 0.05, F ratio in analysis of variance). Data on relative liver weight were not reported for other dose groups. A dose-dependent decrease in erythrocyte count (12.8 and 21.3%) was observed over the duration of the study, in rats exposed to 10.5 mg/kg-day and 16.4 mg/kg-day vanadium pentoxide respectively, compared to controls (3.8% decrease) (Table 4-1). A 20-30% decrease in erythrocyte count is considered biologically significant, however, no statistical analysis was reported by the study authors, and no measure of variance (SE or SD) was given for the means. Data were not reported for high dose groups. Hemoglobin levels decreased 4.6% and 10.5%, respectively, in the 10.5 and 16.4 mg/kg-day groups over the duration of the study compared to 3.9% in controls. Cystine content of hair significantly decreased in a non-dose dependent manner in all vanadium pentoxide treatment groups compared to controls with the exception of the lowest exposure group. The biological significance of decreased hair cystine content is not established; though the researchers speculated that vanadium may have inhibited enzymes, such as sulfotransferases, that decreased the availability of cystine for hair growth. This study observed potentially dose-related changes in erythrocytes, body weight gain, and liver weight in treated animals. However, no statistical analysis was performed for the decrease in eryt hrocytes and body weight gain and no degree of variance was reported (precluding statistical analysis for this review), therefore, a NOAEL or LOAEL could not be determined from this study. However, compared to hematological data for Wistar rats (Wright et al., 2009; Charles Ri ver 2008), the observed decrease in erythrocyte counts observed in this study is outside of the refe rence range for the historical controls and thus is believed to be a clinically significant finding.

Table 4–1. Hematological results of oral vanadium pentoxide exposure in rats (Mountain et al. 1953). Control 10.5 mg/kg-day 16.4 mg/kg-day Red Cell Count (M/mm 3 ) Start 8.0 7.8 8.0 Finish (103 days) 7.7 6.8 6.3 Percent change between start and finish of expt (%) 3.8 12.8 21.3

Hemoglobin, % Start 15.6 15.2 15.3 Finish (103 days) 15.0 14.5 13.7 Percent change between start and finish of expt (%) 3.9 4.6 10.5

4.2.1.2 Chronic StudiesDRAFT - DO NOT CITE OR QUOTE 33 A 2.5-year dietary study on vanadium in rats (s train not described) was previously used as the basis of the chronic RfD (Stokinger et al., 1953). The results were summarized in Patty's Industrial Hygiene and Toxicology, 3rd ed., 1981. In this chronic study, an unspecified number of rats were exposed to dietary levels of 10 or 100 ppm vanadium (about 17.9 or 179 ppm vanadium pentoxide; 1.41 and 14. 1 mg/kg-day ) 5 for 2.5 years. Endpoints evaluated were limited to growth rate, survival, and hair cystine content. The study did not assess comprehensive toxicity endpoints. Hair cystine content was significantly decreased in exposed animals, compared to controls but no values were given. This study reports the oral NOAEL upon which an RfD can be derived as 17.9 ppm (1.41 mg/kg - day) vanadium pentoxide. However, the biological significance of decreased ha ir cystine is unclear and is not specific to vanadium pentoxide exposure. No additional or al chronic exposure studies in animals were identified in the published literature.

4.2.2. Inhalation Exposure

4.2.2.1 Subchronic Studies

The 3-month exposure studies in F344/N rats were conducted to evaluate the cumulative toxic effects of subchronic inhalation exposure to vanadium pentoxide (NTP, 2002). Chemical identity and purity of vanadium pentoxide was evaluated prior to the beginning of and following the conclusion of all assays. Groups of 10 male and 10 female rats were exposed (whole-body exposure) to aerosols of vanadium pentoxide at concentrations of 0, 1, 2, 4, 8 or 16 mg/m 3 , 6 hrs per day, 5 days/week for 3 months. Additional groups of 10 male and 10 female rats were exposed to 4, 8 or 16 mg/m 3 for 12 (females) or 13 (males) weeks to investigate effects of exposure on cardiovascular function, pulmonary function and pulmonary inflammation. Clinical findings were recorded weekly and animals were weighed weekly and at the end of the study. Blood and urine were collected from core study rats at study termination. Blood was also collected from cardiopulmonary physiology study rats on days 4 and 23 for hematology and clinical chemistry determinations. Necropsy a nd histopathological evaluations (light microscopy of comprehensive tissues 6 ) were performed on all main study rats exposed to 0, 2 (male rats

5 Converted to ppm vanadium pentoxide by [(ppm vana dium)(MW vanadium pentoxide, 181.9)]/(2xMW vanadium, 101.9). No information given on average rat weights so ca nnot be converted to mg/kg-day directly. Conversions based on data from subchronic study from same group (Moutai n et al., 1953). Calculation: mg/kg-day =ppm(mg of compound per kg food) x mg food consumed/day x 1/kg body weight (usingreference food consumption rate of 0.0217 kg/day [U.S. EPA, 1988] and average body weight of 0.275 kg for male rats [Mountain et al., 1953])
6 Complete histopathology was performed on 0, 8 (rats only), and 16 mg/m 3 rats and mice. In addition to grossDRAFT - DO NOT CITE OR QUOTE 34 only), 4, 8 or 16 (female rats only) mg/m 3 at the completion of the study. Sperm motility and vaginal cytology evaluations were analyzed from all core study rats. Seven male rats and three female rats exposed to 16 mg/m 3 vanadium pentoxide died during the study (NTP, 2002). Abnormal breathing, emaciation, lethargy, abnormal posture and ruffled fur were observed in male and female rats exposed to concentrations of 8 mg/m 3 and higher. Diarrhea and nasal/eye discharge were also observed in some rats exposed to 16 mg/m 3

. Weight gain and absolute and relative lung weights are summarized in Table 4-2. Weight gain over the 3-month treatment period was significantly decreased compared to control in males exposed to 4 (6% decrease), 8 (10% decrease) and 16 (60% decrease) mg/m 3 and in females exposed to 16 mg/m 3 (30% decrease). Absolute lung weights were significantly increased in males exposed to concentrations of 2 mg/m 3 and greater and in females exposed to 4 mg/m 3 and greater. Relative lung weights were significantly greater than control in males exposed to 2 (16% increase), 4 (30% increase), 8 (51% increase) or 16 (145% increase) mg/m 3 and in females exposed to 4 (19% increase), 8 (76% increase) or 16 (117% increase) mg/m 3 . Other organ weight differences were considered to be related to body weight decreases. Results of hematology assessments following 3-months of inhalation exposure are presented in Table 4-3. Erythrocyte count wa s significantly increased in the 8 and 16 mg/m 3 groups and hematocrit was significantly increased in the 16 mg/m 3 group in male and female rats. Hemoglobin was increased significantly only in females exposed to 16 mg/m 3

. Microscopic evaluation of the red blood cell mo rphology detected increased polychromasia and hypochromia in rats in the 16 mg/m 3 groups (data not presented). Significantly decreased mean cell hemoglobin concentrations were observed in males exposed to 8 and 16 mg/m 3 and in females exposed to 4, 8, and 16 mg/m 3 . Reticulocyte count was significantly increased in males and females exposed to 16 mg/m 3 . Mean cell volume was significantly decreased, indicative of microcytosis, in male rats at concentrations of 2 mg/m 3 and above and in female rats at concentrations of 4 mg/m 3 and above. The observed hematological changes, including erythrocytosis, are consistent with pulmonary lesions that reduce pulmonary oxygen transfer, resulting in tissue hypoxia and stimulation of erythropoiesis by increased renal production of erythropoietin. Erythrocyte microcytosis is cons istent with ineffective erythropoiesis, suggestive

lesions and tissue masses, the following tissues were exam ined in the 3 month studies: adrenal gland, bone with marrow, brain, clitoral gland, esophagus, gallbladder (mice only), heart and aorta, large intestine (cecum, colon and rectum), small intestine (duodenum, jejunum, and ileum), kidney, larynx, liver, lung and mainstream bronchi, lymph nodes (mandibular, mediastinal, mesenteric, and bronchi al), mammary gland (except mail mice), nose, ovary, pancreas, parathyroid gland, pituitary gland, preputial gla nd, prostate gland, salivary gland, skin, spleen, stomach (forestomach and glandular), testis (with epididymis and seminal vesicle), thymus, thyr oid gland, trachea, urinary bladder, and uterus. The lung of rats and mice and nose of rats in all remaining exposure groups and the thymus in 8 mg/m 3 mice were also examined.DRAFT - DO NOT CITE OR QUOTE 35 of altered iron metabolism and heme/hemoglobin production.

Table 4-2. Body Weight Gain and Lung Weights in Rats (F344/N) Exposed to Vanadium Pentoxide by Inhalation for 3 Months (Values are Means±Standard Error) (NTP, 2002)

Parameter Exposure
Control 1 mg/m 3 2 mg/m 3 4 mg/m 3 8 mg/m 3 16 mg/m 3

Male Rats Weight gain during 3- month
exposure period (g) 197±6 202±5 180±5 173±8 a 161±5 a 1±9 a

Absolute lung weight (g) 2.38±0.17 2.56±0.11 2.65±0.07 2.93±0.09 a 3.26±0.13 a 1.98±0.10 b

Relative lung weight 6.77±0.36 7.40±0.28 7.83±0.19

a 8.88±0.22 a 10.20±0.30 a 16.60±0.33 a

Female Rats Weight gain during 3- month
exposure period (g) 87±3 88±4 96±4 83±3 77±4 25±7 a

Absolute lung weight (g) 1.65±0.11 c 1.58±0.04 1.92±0.12 b 1.95±0.08 b ,c 2.16±0.06 a 2.16±0.12 a

Relative lung weight 8.37±0.58 c 7.84±0.16 9.23±0.53 10.00±0.38 b ,c 11.48±0.33 a 18.15±1.06 a

a Significantly different from control by William’s or Dunnett’s test (p ≤ 0.01)
b Significantly different from control by William’s or Dunnett’s test (p ≤ 0.05)

c n = 9

Table 4-3. Selected Hematology Parameters in Ra ts (F344/N) Exposed to Vanadium Pentoxide by Inhalation for 3 Months (NTP, 2002) a

Parameter Exposure
Control 1 mg/m 3 2 mg/m 3 4 mg/m 3 8 mg/m 3 16 mg/m 3

Male Rats
Number 9 9 10 9 10 3 Erythrocytes (10 6 /μL) 9.2±0.1 9.0±0.1 9.1±0.1 9.3±0.2 9.7±0.2 b 15.1±0.3 c

Reticulocytes 0.2 0.22±0.03 0.19±0.02 0.23±0.03 0.25±0.02 0.8 ±0.08 bDRAFT - DO NOT CITE OR QUOTE 36 Table 4-3. Selected Hematology Parameters in Ra ts (F344/N) Exposed to Vanadium Pentoxide by Inhalation for 3 Months (NTP, 2002) a

Parameter Exposure Control 1 mg/m 3 2 mg/m 3 4 mg/m 3 8 mg/m 3 16 mg/m 3 (10 6 /μL) ±0.02 Hematocrit (%) 48.5±0.6 47.7±0.5 47.6±0.6 48.7±0.9 49.9±0.7 71.2±2.8 b Hemoglobin (g/dL) 15.8±0.1 15.5±0.1 15.5±0.2 15.9±0.2 16.1±0.2 20.4±0.8
Mean cell volume (fL) 52.9±0.2 52.9±0.1 52.3±0.1 b 52.2±0.2 b 51.3±0.2 c 46.8±1.0 c

Mean cell hemoglobin (pg) 17.3±0.2 17.2±0.1 17.1±0.1 17.1±0.02 16.5±0.2 c 13.4±0.4 c

Female Rats
Number 10 10 9 10 10 6 Erythrocytes (10 6 /μL) 8.0±0.1 7.8±0.1 8.2±0.2 8.3±0.1 8.6±0.1 b 12.5±0.34 c

Reticulocytes (10 6 /μL) 0.15 ± 0.02 0.17 ± 0.01 0.17 ± 0.01 0.16 ± 0.02 0.17 ± 0.02 0.45 ± 0.08 c Hematocrit (%) 45.8±0.5 44.3±0.4 46.1±1.2 46.4±0.4 47.2±0.6 60.8±1.4 c Hemoglobin (g/dL) 15.5±0.2 15.0±0.1 15.5±0.2 15.6±0.1 15.8±0.1 18.2±0.3 c

Mean cell volume (fL) 56.9±0.1 56.9±0.1 56.6±0.1 55.8±0.1 c 55.0±0.2 c 48.7±0.6 c

Mean cell hemoglobin (pg) 19.3±0.2 19.3±0.2 19.0±0.2 18.7±0.2 c 18.5±0.2 c 14.6±0.3 c

a Values are means±standard error
b Significantly different from control (p ≤ 0.05)

c Significantly different from control (p ≤ 0.01)

Sporadic alterations in clinical chemistry and urinalysis variables were observed at various time-points in exposed males and females; however, no dose- or duration-related pattern of effect was observed. Occasional changes in serum liver enzyme activities were not consistent with hepatocellular injury. Vanadium pentoxide exposure did not aff ect reproductive endpoints in males (sperm count, spermatid heads, sperm motility), but it did increase estrous cycle length by 10% in females exposed to 8 mg/m 3 , but not to 16 mg/m 3 , and reduced the number of cycling females in surviving rats in the 16 mg/m 3 group (percent reduction not reported) (NTP 2002). Complete histopathological assessments were performed on rats exposed to 0, 8 and 16 mg/m 3 for 3 months; only nonneoplastic lesions of the lung and nose were related to treatment (NTP, 2002). Results of histopathologi cal evaluations of lung and nasal tissue from male and female rats exposed to 1, 2, 4, 8 and 16 mg/m 3 for 3 months are summarized in TableDRAFT - DO NOT CITE OR QUOTE 37 4-4. Significant increases in the incidences of epithelial hyperplasia of the lung were observed in male and female rats exposed to concentrations of 2 mg/m 3 or greater, compared to controls. Epithelial hyperplasia occurred in the distal airways and associated alveolar ducts and alveoli. Inflammation and fibrosis were significantly increased in males (2 mg/m 3 or greater) and females (4 mg/m 3 or greater). In the nasal compartment, incidences of hyperplasia and metaplasia of the respiratory epithelium were significantly increased in males exposed to 8 or 16 mg/m 3 and in females exposed to 4 mg/m 3 or greater. Nasal hyperplasia and metaplasia was localized to respiratory epithelium on the ventral portion of the nasal septum, the vomeronasal organ, and, to a lesser extent, the ventral lateral walls of the anterior portion of the nasal cavity. Nasal inflammation was significantly increased in males and females exposed to 16 mg/m 3

. Cardiopulmonary assessments were conducted in groups of 4-10 male and female rats exposed to 0, 4, 8 and 16 mg/m 3 for 3 months (NTP, 2002). No treatment-related changes in cardiovascular function, as assessed by blood pressure (systolic, diastolic and mean), heart rate and electrocardiogram, were observed in rats exposed to 4 or 8 mg/m 3 . Decreased heart rate and diastolic, systolic and mean blood pressure observed in male and female rats exposed to 16 mg/m 3 were considered to be a reflection of the poor condition of the animals, and complicated by anesthesia. Significant exposure-related decreases in pulmonary function (as assessed by respiratory rate, tidal and minute volume, expiratory resistance, vital and total capacity, diffusing capacity, and dynamic and peak compliance) were observed at all concentrations of vanadium pentoxide-exposed male and female rats. Observed changes in impaired capacity to diffuse carbon monoxide and reduced static and dynamic lung volumes at exposure concentrations of 4 mg/m 3 and greater suggest a restrictive lesion. Changes in forced expiratory maneuvers in rats exposed to 16 mg/m 3 suggest the presence of an obstructive disease. Pulmonary function results may indicate obstructive disease or may reflect the deteriorating condition of the 16 mg/m 3 rats, since histopathological finding in lungs of rats exposed to 8 and 16 mg/m 3 were similar. Taken together, results of pulmonary function tests indicate that a presence of restrictive injury in male and female rats exposed to concentrations of 4 mg/m 3 or greater, while an obstructive lung injury may have been present in rats exposed to 16 mg/m 3

.

Table 4-4. Incidences of Selected Nonneoplastic Lesions of the Lung and Nose in Rats (F344/N) Exposed to Vanadium Pentoxide by Inhalation for 3 Months (NTP, 2002)

Lesion Location and Type Numbers of Animals with Lesions (Avg Severity Score)
Control 1 mg/m 3 2 mg/m 3 4 mg/m 3 8 mg/m 3 16 mg/m 3

Male Rats aDRAFT - DO NOT CITE OR QUOTE 38 Table 4-4. Incidences of Selected Nonneoplastic Lesions of the Lung and Nose in Rats (F344/N) Exposed to Vanadium Pentoxide by Inhalation for 3 Months (NTP, 2002)

Lesion Location and Type Numbers of Animals with Lesions (Avg Severity Score) Control 1 mg/m 3 2 mg/m 3 4 mg/m 3 8 mg/m 3 16 mg/m 3 Lung Epithelium, hyperplasia 0 0 10 b (2.0) 10 b (3.0) 10 b (3.6) 10 b (3.3)

Inflammation 0 0 9 b (1.0) 10 b (1.0) 10 b (1.6) 10 b (2.1)
Fibrosis 0 0 2 (1.0) 10 b (1.9) 10 b (3.2) 10 b (3.1) Bronchiole, exudates 0 0 0 0 7 b (1.0) 8 b (1.4)

Nose Epithelium, hyperplasia 0 0 0 1 (1.0) 10 b (1.2) 10 b (2.0)

Epithelium,
squamous metaplasia 0 0 0 1 (1.0) 10 b (1.2) 10 b (1.8)

Inflammation 0 0 0 0 0 7 b (1.6)

Female Rats a Lung Epithelium, hyperplasia 0 0 10 b (1.3) 10 b (2.9) 10 b (3.5) 10 b (3.2)

Inflammation 0 0 0 10 b (1.0) 10 b (1.9) 10 b (1.2)
Fibrosis 0 0 0 10 b (1.0) 10 b (2.9) 10 b (3.2) Bronchiole, exudates 0 0 0 0 10 b (1.0) 8 b (1.1)

Nose Epithelium, hyperplasia 0 0 0 10 b (1.0) 10 b (1.8) 10 b (2.7)

Epithelium,
squamous metaplasia 0 0 0 8 b (1.0) 10 b (1.8) 10 b (2.8)

Inflammation 0 0 0 0 1 (1.0) 9 b (1.6))

a 10 animals per treatment group; numbers in parentheses i ndicate average severity grade of lesions in affected animals: 1=minimal, 2=mild, 3=moderate, 4=marked
b Significantly different from control by Fisher exact test (p ≤ 0.01)

BAL fluid was analyzed for markers of pulmonary inflammation in rats exposed to 0, 4, 8 and 16 mg/m 3 for 3 months (NTP, 2002). Concentration-related increases were observed in the total numbers of cells, lymphocytes, neutrophils and protein recovered in BAL fluid from ratsDRAFT - DO NOT CITE OR QUOTE 39 exposed to vanadium pentoxide at concentrations of 4 and 8 mg/m 3 , demonstrating a pulmonary inflammatory response in male and female rats. These endpoints also were increased in the 16 mg/m 3 group, but to a lesser extent, vanadium pentoxide was overtly toxic at this dose. Results of this study show that inhalation exposure of male and female rats to vanadium pentoxide aerosol for 3 months produced adverse effects on the hematological system and the lung (NTP, 2002). Microcytic erythrocytosis, which was possibly secondary to impaired pulmonary function, was observed at concentrations of 2 mg/m 3 and greater in males and 4 mg/m 3 and greater in females. Absolute and rela tive lung weights were significantly increased compared to controls at concentrations of 4 mg/m 3 and greater in females and 2 mg/m 3 and greater and 4 mg/m 3 and greater, respectively, in males. The incidence of nonneoplastic lesions of the nose was increased in male and female rats at concentrations of 8 mg/m 3 and greater and 4 mg/m 3 and greater, respectively, and the incidence of nonneoplastic lesions of the lung was increased in male and female rats 2 mg/m 3 and greater. Results of pulmonary function tests consistent with restrictive lung disease were observed at concentrations of 4 mg/m 3 and greater. Based on decreased erythrocyte size in male rats and nonneoplastic lung lesions in male and female rats, the NOAEL and LOAEL values identified for 3-month inhalation exposure to vanadium pentoxide aerosols were 1 and 2 mg/m 3 , respectively. Three-month exposure studies in B6C3F 1 mice were conducted to evaluate the toxicity of subchronic inhalation exposure to vanadium pe ntoxide (NTP, 2002). Groups of 10 male and 10 female mice were exposed (whole-body exposur e) to vanadium pentoxide aerosols at concentrations of 0, 1, 2, 4, 8 or 16 mg/m 3 , 6 hrs per day, 5 days/week for 3 months. Particle size given in mass median aerodynamic diameter (MMAD) ± geometric standard deviation (GSD) for each dose groups was as follows: 1 mg/m 3 =1.2±2.8; 2 mg/m 3 =1.1±2.8; 4 mg/m 3 =1.2±2.8; 8 mg/m 3 =1.0±2.9; 16 mg/m 3 =1.2±2.8. Clinical findings were recorded weekly. Animals were weighed weekly and at the end of the study. All study animals were necropsied. Histopathological examinations of l ungs were performed in all mice in the 0, 1, 2, 4, 8 or 16 mg/m 3 groups and of thymus in all mice in the 0, 8 or 16 mg/m 3 groups. At the end of the 3-month exposure period, samples for sperm motility and vaginal cytology evaluations were collected from mice exposed to 0, 4, 8 or 16 mg/m 3 . Complete histopathological examination was performed in mice in the control and 16 mg/m 3 groups. Assessments of cardiopulmonary function, pulmonary inflammation (analysis of pulm onary lavage), and hematological parameters
were not conducted in mice. One male mouse in the 16 mg/m 3 group died before the end of the study. Other than appearing thin, no other signs of toxicity were reported (NTP, 2002). No other treatment-related clinical findings were observed in any other mice in any treatment group. Weight gain andDRAFT - DO NOT CITE OR QUOTE 40 absolute and relative lung weights are summarized in Table 4-5. Weight gain over the 3-month treatment period was significantly decreased compared to control in males exposed to 8 (6% decrease) and 16 (10% decrease) mg/m 3 and in females exposed to 4 (11% decrease), 8 (10% decrease) and 16 (12% decrease) mg/m 3 . Absolute lung weights were significantly increased compared to control at concentrations of 2 mg/m 3 and higher in males and 4 mg/m 3 and higher in females. Relative lung weights were significantly greater than control in males exposed to 4 (33% increase), 8 (43% increase) or 16 (82% increase) mg/m 3 and in females exposed to 4 (62% increase), 8 (63% increase) or 16 (101% increase) mg/m 3 . Other organ weight differences were considered to be related to decreases in body weight by the researchers. The epididymal spermatozoal motility of males exposed to 8 or 16 mg/m 3 was significantly decreased by 13 and 5%, respectively. No treatment-related effects were observed for assessments of estrous cycle (estrous cycle length and number of cycling females).

Table 4-5. Body Weight Gain and Lung Weights in Mice (B6C3F 1 ) Exposed to Vanadium Pentoxide by Inhalation for 3 Months (Values are Means±Standard Error) (NTP, 2002)

Parameter Exposure
Control 1 mg/m 3 2 mg/m 3 4 mg/m 3 8 mg/m 3 16 mg/m 3

Male Mice Weight gain during 3-month exposure period (g) 8.4±0.9 7.4±0.8 8.2±0.6 7.7±0.5 6.2±0.2 a 5.6±0.7 b

Absolute lung weight (g) 0.2±0.01 0.2±0.01 0.3±0.01 b 0.3±0.01 b 0.3±0.01 b 0.4±0.01 b

Relative lung weight 7.0±0.2 6.9±0.2 7.8±0.3 9.3±0.2 b 10.0±0.3 b 12.7±0.4 b

Female Mice Weight gain during 3-month exposure period (g) 9.7±1.0 10.0±1.0 8.1±0.4 5.8±0.5 b 6.1±0.4 b 5.4±0.3 b

Absolute lung weight (g) 0.2±0.01 0.3±0.01 0.3±0.01 0.3±0.02 b 0.4±0.02 b 0.4±0.02 b

Relative lung weight 8.1±0.5 8.8±0.3 9.7±0.5 13.2±0.9 b 13.2±0.6 b 16.3±0.52 b

a Significantly different from control by William’s test (p ≤ 0.05)
b Significantly different from control by William’s test (p ≤ 0.01)DRAFT - DO NOT CITE OR QUOTE 41 Results of histopathological evaluations of lung tissue from male and female mice exposed to 0, 1, 2, 4, 8 and 16 mg/m 3 for 3 months are summarized in Table 4-6 (NTP, 2002). Epithelial hyperplasia was observed in male and female mice exposed to concentrations of 2 mg/m 3 and above; lesion severity increased with increasing exposure concentration. Hyperplasia involved alveolar and, to a lesser ex tent, bronchiolar epithelium. Inflammation was characterized by multiple foci of a mixed cellular infiltrate oriented around blood vessels and bronchioles and was observed in male mice exposed to 4 mg/m 3 and above and in female mice exposed to 2 mg/m 3 and above. Infiltrate was composed primarily of macrophages with abundant cytoplasm and fewer lymphocytes and ne utrophils. Histopathological evaluations of the thymus of male and female mice exposed to 0, 8 and 16 mg/m 3 for 3 months showed lymphoid depletion in mice exposed to 16 mg/m 3 (males: control, 0/9; 8 mg/m 3 , 0/8; 16 mg/m 3

, 2/7; females: 0/9, 0/9, 1/10). The lung was identified as the most sensitive target organ in the 3-month inhalation study in mice (NTP, 2002). Based on increases in absolute lung weights at concentrations of 2 mg/m 3 and greater (males) and inflammation of the respiratory epithelium at concentrations of 2 mg/m 3 and greater (males and females), NOAEL and LOAEL values were identified as 1 and 2 mg/m 3

, respectively.

Table 4-6. Incidences of Selected Nonneoplastic Lesions of the Lung in Mice (B6C3F 1 ) Exposed to Vanadium Pentoxide by Inhalation for 3 Months (NTP, 2002)

Lesion Type Numbers of Animals with Lesions a

Control 1 mg/m 3 2 mg/m 3 4 mg/m 3 8 mg/m 3 16 mg/m 3

Male
Number 10 10 10 10 10 10
Inflammation 0 1 (1.0) 3 (1.0) 4 b (1.0) 10 c (2.0) 10 c (2.0) Epithelium, hyperplasia 0 1 (1.0) 4 b (1.0) 5 b (1.0) 10 c (1.3) 10 c (3.0)

Female
Number 10 9 10 9 10 10
Inflammation 0 1 (1.0) 7 c (1.0) 9 c (1.9) 10 c (1.9) 10 c (2.5) Epithelium, hyperplasia 0 0 6 c (1.0) 9 c (1.5) 10 c (1.5) 10 c (2.5)

a Numbers in parentheses indicate averag e severity grade of lesions in aff ected animals: 1=minimal, 2=mild, 3=moderate, 4=marked
b Significantly different from control by Fisher exact test, p ≤ 0.05

c Significantly different from control by Fisher exact test, p ≤ 0.01

In a study using cynomolgus monkeys, weekly provocation challenges (single 6-hrDRAFT - DO NOT CITE OR QUOTE 42 exposures to 0.5 or 3.0 mg/m 3 ) with inhaled vanadium pentoxide aerosol for six weeks produced statistically significant pulmonary responses, prior to a subchronic exposure (6 hrs/day, 5 days/week for 26 weeks) (Knecht et al., 1992). The subchronic exposure was divided into three groups; one group (n=8) was exposed to filtered, conditioned air and two exposed groups (n=8 each) received nominally equal weekly vanadium pentoxide exposures (concentration x time) with different exposure profile. The peak e xposure group received an actual concentration of 0.16 ± 0.01) mg/m 3 (0.1 mg/m 3 nominal) vanadium pentoxide on Mondays, Wednesdays and Fridays and 1.38 ± 0.07) mg/m 3 (1.1 mg/m 3 nominal) vanadium pentoxide on Tuesdays and Thursdays, and the constant exposure group received a constant daily actual concentration of 0.57 ± 0.03) mg/m 3 (0.5 mg/m 3 nominal). The constant exposure regimen corresponded to a continuous exposure of 0.10 mg/m 3 after adjusting for exposure protocol (0.57 mg/m 3 x 6/24 x 5/7). The peak exposure regimen averaged to a slightly higher continuous exposure of 0.12 mg/m 3 after adjusting for exposure protocol. Vanadium pentoxide particle size was determined weekly during challenges and biweekly during exposures. Average particle size for the subchronic constant exposure group was 3.15 μm (MMAD), with a GSD of 3.25 μm. Particle sizes (MMAD ± GSD) for the peak exposure group were 3.17 ± 2.48 and 3.10 ± 2.45 for the 0.1 mg/m 3 and 1.1 mg/m 3 exposures, respectively. Pulmonary function tests, cytological and immunological analyses of blood and bronchiolar lavage fluid, and skin sensitivity tests were conducted before the pre- and post-exposure pr ovocation challenges. Immunological analyses are described in Section 4.4.1.2. Pulmonary function tests and bronchiolar lavage fluid analyses were also performed one day after the provoca tion challenges. Cytological endpoints included complete and differential blood cell counts and leukotriene C 4 levels. Pulmonary function endpoints included total pulmonary resistance (RL), forced expiratory flow (FEF), forced vital capacity (FVC), residual volume (RV) and dynamic lung compliance (CL dyn ). Respiratory distress, characterized by audible wheezing and coughing, occurred in 3 out of 8 monkeys from the peak exposure group on peak exposure days during the first few weeks of the 26-week exposure; the responses developed within 3-4 hrs of exposure and occasionally required early removal of the affected monkeys from the exposure chamber. Impaired pulmonary function accompanied pre-exposure provocation challenges with V 2 O 5 at 3.0 mg/m 3 and was characterized by a 14% increase in RL and 13% decrease in FEV 50 /FVC accompanied by a 14% increase in RV and 3% decrease in FVC. Pulmonary function and other study endpoints were not significantly different between the three e xposure groups (control, peak and constant) at either challenge concentration when the monkeys were rechallenged following subchronic exposure. The authors suggested that the absence of increased pulmonary reactivity to vanadium pentoxide following subchronic inhalation may be a ttributed to the development of tolerance.DRAFT - DO NOT CITE OR QUOTE 43 The study establishes a subchronic NOAEL [ADJ] of 0.10 mg/m 3 (continuous exposure) for pulmonary function. No subchronic LOAEL was es tablished. However, an apparent acute, but reversible, LOAEL of 1.38 mg/m 3 is established based on the respiratory distress observed at 1.38 mg/m 3 at an early time point. Hematological effects of vanadium pentoxide were assessed in male CD-1 mice that wereexposed by whole-body inhalation for 1 hr/day, 2 days/week for up to 12 weeks (GonzalezVillalva et al., 2006). A 0.02 M aqueous solution of vanadium pentoxide was aerosolized generating a reported average vanadium of 1436 μg/m 3 (1.44 mg/m 3 V ), as measured by filters following the 12 weeks exposure. This study did not provide reliable exposure information, thus exposure concentrations in mg/m3 could not be determined. Groups of 8 exposed mice and 8 vehicle control mice (inhaling deionized water droplets) were evaluated after 24 hrs and weekly for 12 weeks. Evaluations consisted of a complete blood count and morphological examination of platelets. Platelet count was significantly increased in the exposed mice on weeks 3-12; counts increased from week 3 to a maximum at week 9 and subsequently declined, but still remained above controls (quantitative data inadequately reported). The morphology examinations showed the presence of giant platelets at unspecified longer exposure times. The study establishes an apparent LOAEL for incr eased platelet count and altered platelet morphology from short-term intermittent exposure to vanadium pentoxide at 2.56 mg/m 3 . A continuous exposure equivalent concentration cannot be estimated with any confidence, as the intermittency of the exposure protocol is extreme.

4.2.2.2 Chronic Studies

The toxicity of chronic inhalation exposure to particulate aerosols of vanadium pentoxide was assessed in groups of 50 male and 50 female F344/N rats exposed (whole-body exposure) at concentrations of 0, 0.5, 1 or 2 mg/m 3 6 hrs/day, 5 days/week for 104 weeks (Ress et al., 2003; NTP, 2002). Body weights and clinical findings were recorded throughout the exposure period. Necropsy and comprehensive histopathological evaluation 7 were performed on all animals. No clinical findings related to vanadium pentoxide exposure were observed. Mean body weights of females exposed to 2 mg/m 3 were marginally less (3-6%; statistical significance not reported)

7 In addition to gross lesions and tissue masses, the following tissues were examined in the 2-yr bioassay: adrenal gland, bone with marrow, brain, clitoral gland, esophagus, gallbladder (mice only), heart and aorta, large intestine (cecum, colon and rectum), small intestine (duodenum, jejunum, and ileum), kidney, larynx, liver, lung and mainstream bronchi, lymph nodes (mandibular, mediastinal, mesenteric, and bronchial), mammary gland (except mail mice), nose, ovary, pancreas, parathyroid gland, pituitary gland, preputial gland, pros tate gland, salivary gland, skin, spleen, stomach (forestomach and glandular), tes tis (with epididymis and seminal vesicle), thymus, thyroid gland, trachea, urinary bladder, and uterus.DRAFT - DO NOT CITE OR QUOTE 44 than that of controls throughout the 2-year study; mean body weights of exposed male rats were similar to controls throughout the study. The percent survival of male and female rats for the entire 104-week exposure period ranged from 52-58% in male rats and 30-40% in female rats. The percent survival of controls was 40% for male rats and 28% for female rats (Table 4-7). No infection was reported in this study that may account for these survival rates; however, these survival rates are comparable to historical rates of survival for male and female F344/N rats. The incidences of nonneoplastic lesions of the respiratory tract in male and female rats are summarized in Table 4-7 (Ress et al., 2003; NTP, 2002). In male rats, the incidences of nonneoplastic lesions of the lungs (alveolar and bronchiolar epithelial hyperplasia and alveolar histiocyte infiltration), larynx (inflammation and epiglottis degeneration, hyperplasia and squamous metaplasia) and nose (goblet cell hyperplasia) were significantly increased compared to controls in all vanadium pentoxide exposure groups. In female rats, the incidences of nonneoplastic lesions of the lungs (interstitial fibros is and alveolar histiocyte infiltration) and larynx (inflammation and epiglottis degeneration and hyperplasia) were significantly increased compared to control in all vanadium pentoxide exposure groups. In general, the incidences and severity ratings of respiratory lesions increased with exposure level. No treatment-related histopathological findings were observed in other tissues. A LOAEL of 0.5 mg/m 3 was established for nonneoplastic lesions of the respiratory tract in male and female rats; a NOAEL
was not identified.

Table 4-7. Selected Nonneoplastic Lesions of the Respiratory System in Rats Exposed to Particulate Aerosols of Vanadium Pentoxide for 2 Years (NTP, 2002)

Lesion Type and Location a Exposure Group Control 0.5 mg/m 3 1 mg/m 3 2 mg/m 3
Male Rats Percent survival d 40 58 52 54 Lung Number of animals examined) 50 49 48 50 Alveolar epithelium, hyperplasia 7 (2.3) 24 b (2.0) 34 b (2.0) 49 b (3.3) Bronchiole epithelium, hyperplasia 3 (2.3) 17 b (2.2) 31 b (1.8) 48 b (3.3)

Alveolar epithelium, squamous metaplasia 1 (1.0) 0 0 21 b (3.6)

Bronchiole epithelium, squamous metaplasia 0 0 0 7 b (3/7)

Inflammation, chronic active 5 (1.6) 8 (1.8) 24 b (1.3) 42 b (2.4) Interstitial, fibrosis 7 (1.4) 7 (2.0) 16 c (1.6) 38 b (2.1) Alveolus, histiocyte infiltration 22 (1.3) 40 b (2.0) 45 b (2.3) 50 b (2.1)DRAFT - DO NOT CITE OR QUOTE 45 Table 4-7. Selected Nonneoplastic Lesions of the Respiratory System in Rats Exposed to Particulate Aerosols of Vanadium Pentoxide for 2 Years (NTP, 2002)

Lesion Type and Location a Exposure Group Control 0.5 mg/m 3 1 mg/m 3 2 mg/m 3 Larynx Number of animals examined 49 50 50 50 Inflammation, chronic 3 (1.0) 20 b (1.1) 17 b (1.5) 28 b (1.6) Epiglottis epithelium, degeneration 0 22 b (1.1) 23 b (1.1) 33 b (1.5)

Epiglottis epithelium, hyperplasia 0 18 b (1.5) 34 b (1.5) 32 b (1.9) Epiglottis epithelium, squamous metaplasia 0 9 b (1.7) 16 b (1.8) 19 b (1.9)

Nose Number of animals examined 49 50 49 48 Goblet cell, hyperplasia 4 (1.8) 15 b (1.8) 12 c (2.0) 17 b (2.1) Female Rats Percent survival d 28 40 34 30 Lung Number of animals examined 49 49 50 50 Alveolar epithelium, hyperplasia 4 (1.0) 8 (1.5) 21 b (1.2) 50 b (3.1) Bronchiole epithelium, hyperplasia 6 (1.5) 5 (1.6) 14 c (1.3) 48 b (3.0)

Alveolar epithelium, squamous metaplasia 0 0 0 6 c (3.0)

Bronchiole epithelium, squamous metaplasia 0 0 0 1 (2.0)

Inflammation,chronic active 10 (1.5) 10 (1.1) 14 (1.2) 40 b (1.7) Interstitial, fibrosis 19 (1.4) 7 b (1.3) 12 (1.6) 32 b (1.4) Alveolus, histiocyte infiltration 26 (1.4) 35 c (1.3) 44 b (2.0) 50 b (1.9) Larynx Number of animals examined 50 49 49 50 Inflammation, chronic 8 (1.8) 26 b (1.5) 27 b (1.3) 38 b (1.4) Epiglottis epithelium, degeneration 2 (1.0) 33 b (1.2) 26 b (1.3) 40 b (1.5)

Epiglottis epithelium, hyperplasia 0 25 b (1.4) 26 b (1.3) 33 b (1.5) Epiglottis epithelium, squamous metaplasia 2 (2.0) 7 (1.9) 9 (1.7) 16 b (1.4)

Nose Number of animals examined 50 50 50 50 Goblet cell, hyperplasia 13 (2.0) 19 (2.0) 16 (1.9) 30 b (2.0)

a Number of animals with lesion; numbers in parentheses indicate average severity grade of lesions in affected animals: 1=minimal, 2=mild, 3= moderate, 4=marked
b Significantly different from control by the Poly-3 test (p < 0.01)

c Significantly different from control by the Poly-3 test (p < 0.05)
d Nonneoplastic lesions observed at tim e of sacrifice; percent survival consistent with historical controls for F344 rats in NTPDRAFT - DO NOT CITE OR QUOTE 46 studies.

The NTP (2002) and Ress et. al. (2003) studies also conducted analysis of neoplasms in rats exposed to vanadium pentoxide by inhalation for 2-years, using the protocol described above. Compared to concurrent controls, the incidences of alveolar/bronchiolar adenoma,
alveolar/bronchiolar carcinoma or combined alve olar/bronchiolar adenoma or carcinoma were not significantly different (Poly-3 test) for male or female rats. Compared to historical controls, alveolar/bronchiolar adenoma in 0.5 and 2 mg/m 3 males and 0.5 mg/m 3 females, alveolar/bronchiolar carcinoma in 0.5 and 2 mg/m 3 males, and combined alveolar/bronchiolar carcinoma in 0.5, 1 and 2 mg/m 3 males and in 0.5 mg/m 3 females were statistically significantly different (see Table 4-8 footnotes). NTP (2002) and Ress et al. (2003) conclude d that exposure to vanadium pentoxide
caused alveolar and bronchiolar adenomas and carcinomas in male rats because incidence exceeded historical controls. The marginal increase in lung neoplasms observed in female rats was statistically significant only in the 0.5 mg/m 3 exposure group. This increase was not definitively attributed to vanadium pentoxide exposure since the tumors were observed only at the lowest dose and no dose-response was evident.DRAFT - DO NOT CITE OR QUOTE 47 Table 4-8. Incidences of Respiratory Tumors in Ra ts Exposed to Vanadium Pentoxide in a 2 Year Inhalation Study (NTP, 2002) a

Tumor Type Exposure Group
Historical

Control Control 0.5 mg/m 3 1 mg/m 3 2 mg/m 3

Male Rats
Number of animals examined 1054 50 49 48 50
Alveolar/bronchiolar adenoma b 18 (10%) 4 (8%) 8 (16%) c 5 (10%) 6 (12%) c

Alveolar/bronchiolar carcinoma d 8 (4%) 0 (0%) 3 (6%) c 1 (2%) 3 (6%) c

Alveolar/bronchiolar adenoma or carcinoma e 26 (10%) 4 (8%) 10 (20%) c 6 (12%) c 9 (18%) c

Female Rats
Number of animals examined 1050 49 49 50 50
Alveolar/bronchiolar adenoma f 12 (4%) 0 (0%) 3 (6%) c 1 (2%) 0 (0%)

Alveolar/bronchiolar carcinoma 0 (0%) 0 (0%) 0 (0%) 0 (0%) 1 (2%)

Alveolar/bronchiolar adenoma or carcinoma g 14 (4%) 0 (0%) 3 (6%) c 1 (2%) 1 (2%)

a Numbers in parentheses indicate percent incidence; pa rticle size mass mean aerodynamic diameter + geometric standard deviation (MMAD±GSD): 0.5 mg/m 3 =1.2±2.9; 1 mg/m 3 =1.2±2.9; 2 mg/m 3 =1.3±2.9
b Historical incidence of alveolar/bronchiolar adenoma male F344/N rats fed in inhalation chamber controls given NIH-07 diet.

c Incidence exceeds historical control (statistical compar ison between NTP (2002) data and historical data not conducted)
d Historical incidence of alveolar/bronchiolar carcinoma of male F344/N rats fed in inhalation chamber controls given NIH-07 diet.

e Historical incidence of combined al veolar/bronchiolar adenoma or carcinoma male F344/N rats fed in inhalation chamber controls given NIH-07 diet.
f Historical incidence of alveolar/bronchiolar adenoma female F344/N rats fed in inhalation chamber controls given NIH-07 diet.

g Historical incidence of combined alve olar/bronchiolar adenoma or carcinoma female F344/N rats fed in inhalation chamber controls given NIH-07 diet.

NTP (2002) and Ress et. al. (2003) also repor ted the toxicity of chronic exposure to vanadium pentoxide in mice. Groups of 50 male and 50 female B6C3F 1 mice were exposed (whole-body exposure) to vanadium pentoxide par ticulate aerosol concentrations of 0, 1, 2 or 4 mg/m 3 , 6 hrs per day, 5 days/week, for 104 weeks (Ress et al., 2003; NTP, 2002). Particle MMAD±GSD for each dose group was reported as follows: 1 mg/m 3 =1.3±2.9; 2 mg/m 3 =1.2±2.9; 4 mg/m 3 =1.2±2.9. Body weights and clinical findings were recordedDRAFT - DO NOT CITE OR QUOTE 48 throughout the exposure period. Necropsy and comp rehensive histopathological evaluation were performed on all animals and analysis of both non neoplastic and neoplastic lesions was performed. Many mice exposed to vanadium pentoxide were thin and exhibited abnormal breathing, particularly those exposed to 2 or 4 mg/m 3 vanadium pentoxide (specific incidence data not reported). Mean body weights were generally less than control in males exposed to 4 mg/m 3 (decreases of 5-15%) and in females for all exposure groups (1 mg/m 3 , decreases of 4-10%; 2 mg/m 3 , decreases of 14-20%; and 4 mg/m 3 , decreases of 4-19%) (Statistical significance not reported). The number of mice surviving for the entire 104-week exposure period was similar to control (78% for male mice and 76% for female mice) for all exposure groups for female mice and for males in the 1 and 2 mg/m 3 groups, but survival was significantly decreased in male mice exposed to 4 mg/m 3 (50% survival rate)(Table 4-9). The incidences of nonneoplastic lesions of the respiratory tract in male and female mice are summarized in Table 4-9 (Ress et al., 2003; NTP, 2002). In male mice, the incidences of nonneoplastic lesions of the lungs (hyperplasia of the alveolar and bronchiole epithelium, inflammation, alveolus histiocyte infiltration), larynx (squamous metaplasia of the epiglottis) and nose (olfactory and respiratory epithelium degeneration in males and olfactory epithelial degeneration and atrophy in females) were significantly increased compared to control in all vanadium pentoxide exposure groups. Incidences of interstitial fibrosis were significantly increased in male and female mice exposed to 2 or 4 mg/m 3 . In general, the incidences and severity ratings of lesions increased with exposure level and matched the types of lesions observed in rats. No treatment-related histopat hological findings were observed in other tissues. The LOAEL of 1 mg/m 3 was established for nonneoplastic lesions of the respiratory tract in male and female mice; a NOAEL was not identified.

Table 4-9. Selected Nonneoplastic Lesions of the Respiratory System in Mice Exposed to Vanadium Pentoxide in a 2 Year Inhalation Study (NTP, 2002)

Lesion Type and Location a Exposure Group Control 1 mg/m 3 2 mg/m 3 4 mg/m 3
Male Mice Percent survival 78 66 72 50 b Lung Number of animals examined 50 50 50 50 Alveolar epithelium, hyperplasia 3 (3.0) 41 c (2.2) 49 c (3.3) 50 c (3.9)

Bronchiole epithelium, hyperplasia 0 15 c (1.0) 37 c (1.1) 46 c (1.7)

Inflammation, chronic 6 (1.5) 42 c (1.5) 45 c (1.6) 47 c (2.0) Alveolus, histiocyte 10 (2.4) 36 c (2.4) 45 c (2.6) 49 c (3.0)DRAFT - DO NOT CITE OR QUOTE 49 infiltration Interstitial, fibrosis 1 (1.0) 6 (1.7) 9 c (1.2) 12 c (1.7) Larynx Number of animals examined 49 50 48 50 Epiglottis epithelium, squamous metaplasia 2 (1.0) 45 c (1.0) 41 c (1.0) 41 c (1.0)

Nose Number of animals examined 50 50 50 50 Inflammation, suppurative 16 (1.3) 11 (1.4) 32 c (1.2) 23 b (1.3) Olfactory epithelium, atrophy 6 (1.0) 7 (1.6) 9 (1.3) 12 (1.2) Olfactory epithelium, degeneration 1 (1.0) 7 b (1.0) 23 b (1.1) 30 c (1.2)

Respiratory epithelium, degeneration 8 (1.1) 22 c (1.0) 38 c (1.2) 41 c (1.4)

Bronchial Lymph Node Number of animals examined 40 38 36 40 Hyperplasia 7 (2.1) 7 (2.4) 12 (2.1) 13 (2.2)DRAFT - DO NOT CITE OR QUOTE 50 Female Mice Percent survival 76 64 60 64 Lung Number of animals examined 50 50 50 50 Alveolar epithelium, hyperplasia 31 c (1.6) 38 c (2.0) 50 c (3.3)

Bronchiole epithelium, hyperplasia 0 12 c (1.0) 34 c (1.0) 48 c (1.5)

Inflammation, chronic 0 37 c (1.3) 39 c (1.8) 49 c (2.0) Alveolus, histiocyte infiltration 4 (1.0) 34 c (2.4) 35 c (2.4) 45 c (2.7)

Interstitial, fibrosis 0 1 (2.0) 4 b (2.5) 8 c (1.5) Larynx Number of animals examined 50 50 49 50 Epiglottis epithelium, squamous metaplasia 0 39 c (1.0) 45 c (1.0) 44 c (1.1)

Nose Number of animals examined 50 50 50 50 Inflammation, suppurative 19 (1.1) 14 (1.2) 32 c (1.2) 30 c (1.3) Olfactory epithelium, atrophy 2 (1.5) 8 b (1.3) 5 (1.0) 14 c (1.3) Olfactory epithelium, degeneration 11 (1.2) 23 c (1.0) 34 c (1.2) 48 c (1.3)

Respiratory epithelium, degeneration 35 (1.3) 39 (1.5) 46 c (1.7) 50 c (1.8)

Bronchial Lymph Node (Number of animals examined) 39 40 45 41

Hyperplasia 3 (2.0) 13 c (1.8) 14 c (2.3) 20 c (2.3)

a Number of animals with lesion; numbers in parentheses indicate average severity grad e of lesions in affected animals: 1=minimal, 2=mild, 3=moderate, 4=marked
b Significantly different from control by the Poly-3 test (p < 0.05)

c Significantly different from control by the Poly-3 test (p < 0.01)

The incidences of tumors of the respiratory tract in male and female mice exposed to vanadium pentoxide for 2 years are summarized in Table 4-10 (Ress et al., 2003; NTP, 2002). The incidences of alveolar/bronchiolar adenoma, alveolar/bronchiolar carcinoma and combined
alveolar/bronchiolar adenoma or carcinoma were significantly increased in all groups of exposed female mice. In male mice, the incidences of alveolar/bronchiolar carcinoma and combined alveolar/bronchiolar adenoma or carcinoma were significantly increased compared to control in all vanadium pentoxide treatment groups and alveolar/bronchiolar adenoma was significantly increased in the 2 mg/m 3 group.DRAFT - DO NOT CITE OR QUOTE 51 Table 4-10. Incidences of Respiratory Tumors in Mice Exposed to Vanadium Pentoxide in the 2 Year Inhalation Study (NTP, 2002)

Tumor Type a Exposure Group Historical

Control Control 1 mg/m 3 2 mg/m 3 4 mg/m 3

Male Mice Number of animals examined 1071 50 50 50 50 Alveolar/bronchiolar adenoma b 201 (19%) 13 (26%) 16 (32%) 26 c (53%) 15 (30%)

Alveolar/bronchiolar carcinoma 97(9%) 12 (24%) 29 c (58%) 30 c (60%) 35 c (70%)

Alveolar/bronchiolar adenoma or carcinoma 285 (26.8%) 22 (28%) 42 c (84%) 43 c (86%) 43 c (86)

Female Mice Number of animals examined 1075 50 50 50 50 Alveolar/bronchiolar adenoma 67 (6.3%) 1 (2%) 17 c (34%) 23 c (46%) 19 c (38%)

Alveolar/bronchiolar carcinoma 43 (–3.9%) 0 (0%) 23 c (46%) 18 c (36%) 22 c (44%)

Alveolar/bronchiolar adenoma or carcinoma 109 (– 10.1%) 1 (2%) 32 c (64%) 35 c (70%) 32 c (64%)

a Number of animals with tumor; numbers in parentheses i ndicate percent incidence; particle size mass mean aerodynamic diameter + geometric standard deviation (MMAD±GSD): 1 mg/m 3 = 1.3±2.9; 2 mg/m 3 = 1.2±2.9; 4 mg/m 3 =1.2±2.9 b Historical incidence of alveolar/bronchiolar adenoma male B6C3F 1 mice fed in inhalation chamber controls given NIH-07 diet.

c Significantly different from control by the Poly-3 test (p ≤ 0.01)

A recent study has examined the effect of vanadium pentoxide exposure by aspiration (Rondini et al. 2010). Rondini et al (2010) examined the induction of pulmonary inflammation and tumor promotion in three different mice strains (A/J, BALB/c and C57BL/6J) following oropharyngeal aspiration exposure. This study was designed to test the hypothesis that vanadium pentoxide acts as a tumor promoter in exposed rodents. Three mouse strains were used to further understand potential susceptibility to these effects. These particular mouse strains were selected because of their known differential susceptibility to chronic pulmonary inflammation and carcinogenesis: A/J mice are sensitive, BALB/C are intermediate and C57BL/6J are resistant. The experiment was designed to measure vanadium pentoxide tumor promotion following tumor initiation by 3-methylcholanthrene (MCA). All experimental mice were exposed to MCA (10ug/g bw in corn oil; intraperitoneal injection) in week 1, followed by 5 weekly aspirations of either V2O5 (4 mg/kg) or PBS. Tumor incidence was measured at 20 weeks post-MCA exposure. Statistically significant lung tumor increases were observed in A/J and BALB/C mice as compared to the MCA-treated control (p ≤ 0.05; Table 4 – 11). Differences were also observed between strains, with A/J mice showing increased tumorigenicity in responseDRAFT - DO NOT CITE OR QUOTE 52 to vanadium pentoxide. In the absence of MCA, V2O5 was not sufficient to initiate tumorigenesis in this study. C57BL/6J had no tumors following exposure (data not shown). To evaluate the strain differences in inflammation, mice were aspirated with V2O5 (4 mg/kg bw) 4 times weekly, with BALF collected at 6hr, 1d, 3d, 6d, and 21d following the last aspiration. Cellular infiltrates and protein content was compared, and lungs were snap-frozen for histopathology. Increased pulmonary inflammation and hyperpermeability was increased in all exposed strains in a similar pattern as the tumor induction, with greater increases observed in A/J
mice than in BALB/C. C57BL/6J showed the least amount of increase in all parameters compared to all mouse strains. All results returned to baseline at 21 days post-exposure. The results of PMN increases were confirmed by hist opathology in A/J and C57BL/6J mice. Further analysis was performed to measure inflammatory chemokine production (KC, MIP-2, MCP-1) and transcription factor activity (NFkB, c-Fos) and signaling pathway activation (MAPK). Like the increased inflammatory markers above, increased levels of KC and MCP-1 were observed in A/J and BALB/C mice as compared to the C57BL/6J mice. Similar strain differences were observed for the transcription activity of NFkB and c-Fos and the MAPK signaling activity in A/J mice as compared to C57BL/6J (BALB/C were not analyzed).

Table 4 -11. Lung tumor multiplicity in MCA-Treated mice exposed to V 2 O 5 by pharyngeal aspiration (Rondini et al. 2010). a,b PBS Control V 2 O 5 A/J 3.3 ± 0.75 (n=4) 10 ± 1.4 (n=15) BALB/C 0.78 ± 0.28 (n=8) 2.2 ± 0.36 (n=12)

a No tumors were observed in C57BL/6J mice.
b Number of animals for each treatment in parantheses.

In summary, the identified noncancer health effects following occupational exposure via inhalation to vanadium pentoxide in humans include respiratory irritation, cough and bronchitis; inhalation exposure in animals results in multiple health effects, including pulmonary inflammation, lung and nasal hyplerplasia and pulmonary fibrosis. Vanadium pentoxide exposure for two years was associated with a wide spectrum of nonneoplastic pulmonary lesions in both rats and mice, ranging from hyperplasia to inflammation, fibrosis, and metaplasia. Lesions were detected in the lung, larynx, and nose in both rats and mice exposed to vanadium pentoxide. Bronchial lymph node changes were detected in mice exposed to vanadium pentoxide. There are currently no human epidemiology studies that examined carcinogenesis following exposure to vanadium pentoxide. Howeve r, there is clear evidence of carcinogenicityDRAFT - DO NOT CITE OR QUOTE 53 in male and female mice exposed to vanadium pentoxide, and some evidence of carcinogenicity in male rats, based on observations of alveol ar and bronchiolar neoplasms that exceeded historical controls in groups exposed to vana dium pentoxide (NTP 2002). A more recent study has also shown lung tumor promotion in sensitive mouse strains (Rondini et al. 2010).

4.3. REPRODUCTIVE/DEVELOPMENTAL STUDIES – ORAL, INHALATION,

INTRAPERITONEAL AND INJECTION

4.3.1. Oral Studies

Vanadium pentoxide delivered orally to weanling rats (10 to 200 μ mol/kg) for three days produced a significant increase in alkaline phosphatase activity and DNA content in the diaphysis of femoral bones, and suggests that vanadium pentoxide may be linked to bone formation in the developing rat (Yamaguchi et . al., 1989). Yamaguchi et al. (1989) examined the potential effects of vanadium pentoxide on bone metabolism. Weanling male Wistar rats were exposed to 1.8 to 36.4 mg/kg (10 to 200 μ mol/kg) 8 vanadium pentoxide orally 1 hr following an oral injection of 15.3 μmol Zn/100g) fo r three times at 24-h intervals. The highest dose vanadium pentoxide tested (200 μ mol/kg) led to death in four of nine rats (cause of death not described). Authors state that administration of zinc as well blocked these deaths. All rats were killed 24h following the last vanadium ad ministration, and blood immediately removed by cardiac puncture. Statistically significant increases were observed in serum from high-dose rats for calcium (27.3 – 36.4 mg/kg; p < 0.05) and decreased for phosphorus (36.4 mg/kg; p < 0.01). Administration of zinc completely prevented these serum changes. Femurs were also immediately removed. The diaphysis and epiphysis were used for alkaline phosphatase measures (right femur) and DNA content analyses (left femur). Alkaline phosphatase activity was significantly increased at the lowest dose tested (1.8 mg/kg; p< 0.05) and peaked at 3.6 mg/kg. Further increasing doses led to decreases in alkaline phosphatase activity. A similar pattern was observed for bone DNA content, with statistically significant increases at 1.8 – 18.1 mg/kg (p < 0.05), but decreased at higher doses. Although the authors state the interaction of vanadium and zinc led to an increase in bone calcium, based on the data presented in the figures in this publication, vanadium administration did not lead to alterations of bone calcium. Overall, this study shows an increase in both DNA content and alkaline phosphatase activity in weanling male rats following oral administration of vanadium pentoxide, suggesting a possible role of vanadium in increased bone growth.

8 Conversion factor: μmol = 0.1819 mgDRAFT - DO NOT CITE OR QUOTE 54 Mravcova et. al (1993) assessed the extent of vanadium pentoxide accumulation in the bones of rats following 6 month exposure (full study description in 4.4.4.2). Vanadium accumulated in the epiphyseal cartilage of the tibia in rats with significantly higher concentrations of vanadium in the tibia and incisors of weanling rats compared to adults. However, no dose response data for these endpoints was reported.

4.3.2. Inhalation Studies

Investigations of the reproductive and developmental toxicity of subchronic or chronic inhalation exposure to vanadium pentoxide are limited to two studies. Mussali-Galante et. al (2005), used immunohistochemistry to assess the amount of gamma tubulin accumulating within somatic and testicular germ cells. Mussali-Galante et al. (2005) exposed 60 male CD-1 mice to inhaled vanadium pentoxide (0.02M, apparen tly aqueous solution containing aerosolized vanadium pentoxide) for 1 hr two times a week for 12 weeks. Avila-Costa et. al. (2004), investigators from the same laboratory, used the same protocol and reported that droplets of the vanadium pentoxide mixture had average diameters of 0.5 – 5 μ m (Avila-Costa et. al., 2004). Thirty-six control animals inhaled only vehicle (deionized water). Groups of three exposed animals and three control animals were sacrificed per week for 12 weeks. Results indicated accumulation of vanadium pentoxide in testes (Mussali-Galante et al., 2005). Moreover, gamma tubulin was significantly decreased in testicular samples exposed to vanadium pentoxide compared to control, starting after the first week of exposure. Changes in gamma tubulin may suggest changes in microtubule-involved function, such as cell division, which may impact spermatogenesis. Responses were duration-dependent, with the lowest percentages of immunoreactive cells occurring at the end of the exposure period; values at week 12 ranged from 1.2% for germ cells and 1.5% for Sertoli cells to 10.1% for Leydig cells (compared to 87-88% in controls) (Mussali-Galante et. al., 2005). Fortoul et al. (2007) analyzed testes for ultrastructural changes, testosterone concentration, and vanadium tissue concentration, using the same protocol as that reported above (Mussali-Galante et al 2005). No overt toxicity or changes in body or testicular weight were observed. Histopathological analysis revealed necrotic cell death in spermatocytes (25%) and Sertoli cells (15%) at weeks 5-6 in vanadium-exposed animals. Spermatocytes exhibited cytoplasmic vacuolation, nuclear distortion and intercellular edema in response to vanadium pentoxide exposure. Spermatogonia (40% necrosis during weeks 6-7) were the most susceptible cell type, followed by spermatocytes and Sertoli cells. Moreover, vanadium pentoxide concentrations increased dramatically after one week of exposure and remained consistently elevated (avg concentration 0.05 μ g/g dry tissue in controls, 1.63 μ g/g dry tissue in exposedDRAFT - DO NOT CITE OR QUOTE 55 animals, 33 times higher). Concentrations of testosterone were highly variable and not statistically significant. Vanadium pentoxide exposure did not affect reproductive endpoints in male rats (sperm count, spermatid heads, sperm motility), but it did increase estrous cycle length by 10% in female rats exposed to 8 mg/m 3 , but not to 16 mg/m 3 , and reduced the number of cycling females in surviving rats in the 16 mg/m 3 group (percent reduction not reported). The epididymal spermatozoal motility of male mice exposed to 8 or 16 mg/m 3 was significantly decreased by 13 and 5%, respectively. No treatment-related effects were observed for assessments of estrous cycle in female mice (estrous cycle length and number of cycling females) (NTP 2002).

4.3.3. Intraperitoneal and Injection Studies

Male and female reproductive endpoints were evaluated in young rats following intraperitoneal administration of vanadium pe ntoxide (Altamirano et al., 1991). Newborn male and female rats were injected with 0 or 12.5 mg/kg vanadium pentoxide in saline on every second day from birth to age 21 days; groups sizes were 5 (treated males) or 9 (male and female controls and treated females). Males were sacrificed at 55 days of age and the females were sacrificed on the day of first vaginal estrus. Ot her groups of females were injected with 0 or 12.5 mg/kg-day vanadium pentoxide (n = 10 and 6, respectively) from age 21 days to the day of first vaginal estrus, at which time they were sacrificed. Reported endpoints in the males consisted of absolute weights of testes, prostate, seminal vesicles, adrenals, pituitary, thymus, liver, kidneys and submandibular glands. The only effects in treated males were statistically significant increases in seminal vesicle, thymus and submandibular gland weights (20.1, 29.5 and 19.2% higher than controls, respectively). Endpoints evaluated in the females included body weight, absolute organ weights (ovaries, uterus, adrenals, pituitary, thymus, liver, kidneys and submandibular glands), age at vaginal opening, number of ova in oviducts and ovulation rate. The only effects in treated females occurred in the group treated from 21 days of age; these consisted of statistically significant increases in body weight (14.3% higher than controls) and increased weights of thymus, submandibular gland and liver (31.1, 15.8 and 28.4% above control weights, respectively). Fertility and sperm assessments were also performed in male CD-1 mice following intraperitoneal administration of vanadium pe ntoxide (Altamirano-Lozano et al., 1996). In the fertility assessment, groups of 20 and 15 male mice were injected with 0 and 8.5 mg/kg vanadium pentoxide in saline, respectively, every 3rd day for 60 days and mated 24 hrs after the last injection. Statistically significant effects in the treated group included reduced fertility rate in males (33% compared to 85% in controls), reduced numbers of implantation sites (avg = 5.8DRAFT - DO NOT CITE OR QUOTE 56 compared to 10.88 in controls), reduced numbers of live fetuses (3.4 compared to 10.53 in controls) and increased number of resorptions per dam (2.00 compared to 0.24 average resorptions in controls). In the sperm assessment, 20 males were injected with 8.5 mg/kg vanadium pentoxide every 3 days for up to 60 days with groups of 5 evaluated after 10, 20, 30, 40, 50 or 60 days of treatment. Statistically significant effects included reduced sperm motility
after ≥ 10 days, reduced sperm count and increased percentage of abnormal sperm after ≥ 20 days, decreased absolute testicular weight after ≥ 50 days (relative weight not reported), and decreased body weight after 60 days. Developmental toxicity was evaluated in groups of 13 or 15 female CD-1 mice that were administered 0 or 8.5 mg/kg vanadium pentoxide in distilled water, respectively, by intraperitoneal injection on days 6-15 of gestation (Altamirano-Lozano et al., 1993). No maternal toxicity was reported (endpoints not speci fied). Developmental toxicity endpoints were assessed on gestation day 18. The endpoints included the number of implants, resorptions, and live fetuses. For all fetuses, weight, sex, and external malformations were noted. For two-thirds of the fetuses, skeletal abnormalities were also recorded. Internal soft-tissue examinations do not appear to have been conducted. The treated group had statistically significant increases in the number of litters with abnormal fetuses (9/15 compared to 3/13 in controls), number of abnormal fetuses (15/149 compared to 3/124), and number of fetuses with short limbs (8/149 compared to 0/124 in controls). Additionally, the numbers of ossification centers in forelimbs and hindlimbs were significantly reduced in the treated fetuses. Zhang et al. (1991a) evaluated the developmental toxicity in NIH mice following intraperitoneal injection of 5 mg/kg-day vana dium pentoxide on days 1-5, 6-15, 7, 8, 9, 10, 11 or 14-17 of gestation. There were no adverse effects on preimplantation or implantation, developmental toxicity, or premature births. In creased frequencies of resorption or fetal death were observed for gestation days 6-15, 7 and 14- 17. Delayed ossification (sites not specified) was observed for gestation days 6-15, 8, 10 and 14-17. In a second study, Zhang et al. (1993a) evaluated developmental toxicity in Wistar rats following intraperitoneal injection of 0.33, 1.0 or 3.0 mg/kg-day on days 6-15 of gestation. Decreas ed placental weight and increases in embryofetus mortality and external or skeletal malformations (unspecified) occurred at 1.0 and 3.0 mg/kg-day. Maternal toxic symptoms (unspecifi ed), decreased maternal weight gain during treatment and fetal growth retardation were obs erved at 3.0 mg/kg-day. In the third study, Zhang et al. (1993b) evaluated developmental toxicity in Wistar rats following intraperitoneal injection of vanadium pentoxide in doses of 3 mg/kg-day on days 6-15 of gestation or 5 mg/kg-day on days 9, 10, 11 or 9-12 of gestation. Effects in rats exposed on gestation days 6-15 and 9-12 included decreased maternal weight gain, increased fetal mortality, decreased fetal weight andDRAFT - DO NOT CITE OR QUOTE 57 re test). crown-rump length, delayed ossification of unspecified bones, and increased incidences of subcutaneous hemorrhage, wavy ribs, and dilation of lateral ventricles and renal pelvis. Effects in rats exposed on a single day of gestation included subcutaneous hemorrhage and unspecified visceral anomalies following exposure on days 9, 10 and 11, and increased fetal mortality and delayed ossification of unspecified bones following exposure on day 10. Additional studydetails were not available. Overall, the three studies identified an intraperitoneal developmentaltoxicity LOAEL for vanadium pentoxide of 1 mg/kg-day (Zhang et al., 1991a, 1993a,b). One study examined the effects of vanadium pentoxide (1.1mg/kg in distilled water) following tail vein injection in a total of 20 pregnant NMRI mice (data pooled from three studies with 6-10 mice each) (Wide 1984). Injections were performed before implantation or on gestation day 3 or 8, and animals were euthanized on gestation day 17 (two days before parturition. Pre-implantation exposure had no effect on the fetuses as regards number per litter, weight, or external and internal morphology. Exposure to vanadium pentoxide on gestation day 3 or 8 did not lead to significant changes in resorption frequencies, fetal weights or frequencies of fetal hemorrhages as compared to controls. However, the number of fetuses defined as having
less mature skeletons 9 by the authors was significantly greater in mice given vanadium pentoxide on gestation day 8, but not gestation day 3 (p < 0.001, chi squa Although the intraperitoneal and tail vein injection studies show the potential of vanadium pentoxide to cause reproductive and developmental effects in rodents, the studies are of little use in the quantitation of vanadium pentoxi de toxicity, as equivalent oral or inhalation exposures cannot be established.

4.4 OTHER DURATION- OR ENDPOINT-SPECIFIC STUDIES

4.4.1 Acute and Short-Term Studies

4.4.1.1 Acute Studies

4.4.1.1.1 Oral

According to the Concise International Ch emical Assessment Document 29 (WHO-IPCS, CICAD 29, 2001), rat oral LD 50 values for vanadium pentoxide range from 86-137 mg/kg body weight (Yao et. al., 1986 as cited in WHO-IPCS, 2001). A rat oral LD 50 value of 10 mg/kg body weight was reported in IARC Monographs, volume 86 (IARC, 2006) in a study by Lewis et al.

9 No ossification of three of four elements examined (supraoccipital bone, sternum, metatarsalia, and all caudal vertebrae).DRAFT - DO NOT CITE OR QUOTE 58 (2000). Lewis et al. (2000) also reported a mouse oral LD 50 value at 23 mg/kg body weight. Clinical signs of acute toxicity included leth argy, excessive tearing (lacrimation), and diarrhea but dose-response data was not reported (Yao et. al., 1986 as cited in WHO-IPCS, 2001). Histopathological analysis revealed liver necrosis and swelling of renal tubules. Other studies (WHO-IPCS, 2001) have identified LD 50 s of ~10 mg/kg body weight in rats and 23 mg/kg body weight in mice. An oral LD 50 at 64 mg/kg body weight in rabbits was established (WHO-IPCS, 2001). Signs of toxicity in rabbits mimicked those reported for rats.

4.4.1.1.2 Inhalation

A 1-hr inhalation exposure to vanadium pentoxide dust in rats led to an LC 67 of 1.44 mg/L (1440 mg/m 3 ) (US EPA, 1992). Clinical signs of t oxicity included respiratory difficulty, irritation of mucosa, and diarrhea (WHO-IPCS, 2001). Knecht et al. (1985) reported air flow restriction, as measured by pulmonary function tests, in sixteen adult male cynomolgus monkeys ( Macaca fasicularis ) exposed to vanadium pentoxide by whole-body inhalation at 5.0 mg/m 3 for 6 hrs but not at the lower dose tested (0.5 mg/m 3 ). From this study, a LOAEL of 5.0 mg/m 3 and a NOAEL of 0.5 mg/m 3 were established. The lung was also identified as a target organ in response to acute inhalation exposure to vanadium pentoxide. Following a baseline measurement of pulmonary function, each of sixteen male cynomolgus monkeys were exposed to aerosols of 0.5 mg/m 3 vanadium pentoxide by whole-body inhalation for 6 hrs (Knecht et al., 1985). One week later, these monkeys were exposed to aerosols of 5 mg/m 3 vanadium pentoxide by whole-body inhalation for 6 hrs. Effects on ai rway function were evaluated in monkeys by comprehensive pulmonary function tests (PFTs) performed 24 hours post-exposure to 0.5 and 5 mg/m 3 and on pulmonary inflammation by analysis of bronchiolar lavage (BAL) fluid in monkeys performed after exposure to 5 mg/m 3 . Significant changes in pulmonary function parameters compared to baseline values were observed only following exposure to 5 mg/m 3 as follows: 16% increase in pulmonary resistance; 11% decrease in peak expiratory flow rate; 5-22% decreases in forced expiratory flow maneuvers; 33% increase in residual volume; and 24% increase in forced residual capacity. Results are consistent with air-flow limitation in both small peripheral and large central airways. An increase (approximately 87%; data presented graphically) in the total number of cells recovered in BAL fluid was observed 1 day after exposure to 5 mg/m 3 vanadium pentoxide. The increase in BAL fluid total cell number was primarily due to a marked increase (approximately 425%; data presented graphically) in the number of polymorphonuclear leukocytes. Results suggest that pulmonary inflammation and release of bronchoconstrictive mediators from inflammatory cells may play a role in vanadiumDRAFT - DO NOT CITE OR QUOTE 59 pentoxide-induced air-flow restriction. An acute (single 6-hr exposure) LOAEL for vanadium pentoxide of 5 mg/m 3 for pulmonary function in monkeys was established in this study, with a NOAEL of 0.5 mg/m 3

. A study in male CD-1 mice (n = 48) by Avila-Costa et al. (2006) noted significantly impaired performance on memory tasks, significantly decreased dendritic spine length, and significant increases in percentages of necrotic cells in the hippocampus compared to controls following a 1 hr inhalation exposure to 0.02 M (2.5 mg/m 3 as Vanadium) vanadium pentoxide (p < 0.05). The dose-response relationship for these effects could not be evaluated, since only one dose was tested, and a NOAEL could not be established.

4.4.1.2. Short-term Studies

4.4.1.2.1 Inhalation and Aspiration

The primary noncancer health effect of short-term inhalation exposure in humans is respiratory irritation where 100 workers were reportedly exposed to 0.05 to5.3 mg/m 3 vanadium for 10 hr/day, 6 days/week, for 4 weeks (Levy et al., 1984). A LOAEL of 0.05 mg/m 3 was established. However, dose-response was not systematically measured, there were no controls, and exposure due to vanadium pentoxide could not be directly correlated to effects. The primary noncancer health effects of short term inhalation exposure in animals include increased pulmonary inflammation, and dose-related decreases in body weight and relative lung weight in rodents (NTP, 2002). Results of the NTP (2002) study in rats and mice provide evidence of toxicity to the upper and lower respiratory tract, including in creased lung weight, inflammation, nonneoplastic lesions, and decreased pulmonary function following 13-day or 16-day inhalation exposure to vanadium pentoxide. A significant increase in pulmonary inflammation and histiocytic infiltrate of minimal to mild severity was observed in female rats (assessments not made in male rats) exposed to vanadium pentoxide for 13 days, with a LOAEL of 1 mg/m 3 ; a NOAEL was not established. Similar results were observed for female mice (assessments not made in male mice) exposed for 13 days, with a LOAEL of 2 mg/m 3 for minimal to mild epithelial hyperplasia and inflammation; a NOAEL was not established. Male rats (22/group) and female mice (50/group) were also assessed for pulmonary inflammation (bronchiolar lavage analysis) and systemic immunotoxicity (pulmonary bacteriocidal activity) following exposure to 0, 4, 8, and 16 mg/m 3 vanadium pentoxide for 16- days (NTP, 2002). Observed effects included significant alterations in the percentage ofDRAFT - DO NOT CITE OR QUOTE 60 recoverable bronchial lavage cells (macrophages and neutrophils) (LOAEL of 8 , mg/m 3 and NOAEL of 4 mg/m 3 .), and increased lung protein and lysozyme in male rats (LOAEL of 4 mg/m 3 ). No NOAEL was established. In female mice exposed to vanadium pentoxide for 16 days, there was a localized inflammatory response in the lungs based on increase in lymphocytes, protein and lysozymes at all concentrations; the NOAEL was not established. A significant decrease in the percentage of macropha ges from bronchiolar lavage fluid led to a LOAEL of 8 mg/m 3 and a NOAEL of 4 mg/m 3 . These responses, NOAEL, and LOAELs are commensurate with those observed in the 3-month study in rats and mice of both genders (NTP, 2002). Thus, the lowest concentrations at whic h adverse effects were observed were 1 and 2 mg/m3 (LOAEL) for lung inflammation in female rats and mice, respectively, exposed to vanadium pentoxide for 13-days. An additional 5 male and 5 female mice were exposed by inhalation to vanadium pentoxide for 6 hrs per day, 5 days a week for 16-days at concentrations of 0, 2, 4, 8, 16, or 32 mg/m 3 (NTP, 2002). All male mice exposed to 32 mg/m 3 died before study completion. Body weight was significantly decreased in male and female mice at 16 and 32 mg/m 3 , respectively. Absolute lung weights were significantly increased in a dose-dependent manner in males at ≥ 4 mg/m 3 , and relative lung weight were significantly increased in males at ≥ 2 mg/m 3 . Among females both absolute and relative lung weights increased in all exposure groups establishing the LOAEL of 2 mg/m3; no NOAEL was established. Additional groups of 40-60 female mice were exposed to 0, 2, 4 or 8 mg/m 3 for 6 hrs per day, 5 days per week for 16 days (NTP, 2002). The nonneoplastic lung lesions noted on day 6 and 13 consisted of hyperplasia of the alvelolar and bronchiolar epithelium at all exposure levels. Increase in severity of lesions was correlated with increasing concentration and time. The LOAEL for nonneoplastic lung lesions was 2 mg/m 3 . A NOAEL was not established.A duration-dependent decrease in the number of immunoreactive TH+ neurons (AvilaCosta et al., 2004) after 4 weeks and increased quantities in metalloproteinase (MMP)-2 and
MMP-9 in CNS after 8 weeks in male mice (Colin-Barenque et. al. 2008) following twice weekly, one hour inhalation exposure to 5.13 mg/m 3 vanadium pentoxide were suggestive of disruption of blood-brain barrier . Turpin et al. (2010) exposed male AKR mice to vanadium pentoxide by intranasal aspiration following exposure to respiratory syncytial virus (RSV) to determine if pre-exposure to the virus exacerbated the vanadium pentoxi de-induced lung inflammation and fibrosis. Animals were exposed intranasally to RSV (6 x 10 5 PFU in 100ul PBS) on day -1 and day 8, then exposed intranasally to V 2 O 5 (4mg/kg in 50ul PBS) on day 0 and day 7. One hour before euthanasia, animals were given BrdU (50 mg/ kg) by i.p. for analyzing cell proliferation inDRAFT - DO NOT CITE OR QUOTE 61 bronchus-associated lymphoid tissue (BALT). Lungs were lavaged with PBS and BALF collected for analyzing differential cell counts (neutrophils, macrophages and lymphocytes). Total RNA from lung was analyzed by real tim e RT-PCR for mRNAs coding for pro-fibrogenic growth factors TGF- β -1, connective tissue growth factor (CTG F), platelet-derived growth factorC (PDGF-C) and collagen Col1A2, anti-fibrogenic type I interferons-alpha (IFN- α) and -beta (IFN- β) and IFN-inducible chemokines CXCL9 and CXCL10. Lung sections stained with Masson’s trichrome staining to show collagen had an inflammation score of two, representing mild fibrosis with vanadium pentoxide exposure alone. However, vanadium pentoxide-induced fibrotic response was less severe in the lungs of micewhich received either pre- or post-RSV exposure, and no difference observed in pre- or postRSV alone and the negative controls. In addition, BALF from mice exposed to vanadium pentoxide alone or with RSV-post exposure had significantly higher total cell count compared to RSV pre-exposure, RSV pre-exposure plus vanadi um pentoxide or controls. In particular, vanadium pentoxide alone caused a significant increase in the levels of neutrophils and lymphoctyes compared to controls. Both pre- and post-exposure to RSV led to a decrease in the severity of V2O5-induced fibrosis, and gene e xpression analysis showed decreases in several pro-fibrinogenic genes associated with innate immunity. The acute and short-term studies described here support those results describe previously in the subchronic and chronic studies, and sugge st progressive lung effects from vanadium pentoxide exposure. These further support that the lung is the most sensitive organ to vanadium pentoxide exposure.

4.4.2 Immunological Endpoints

4.4.2.1 Human Studies

Some of the early case series observed dermatitis among affected workers employed at vanadium pentoxide processing facilities (Sjöber g, 1951; Zenz et al., 1962). Zenz et al. (1962) observed that respiratory symptoms, such as conjunctivitis, nasopharyngitis, hacking cough, fine rales, and wheezing recurred with greater severity when work resumed after three days of no exposure, even with the use of respirators, perhaps indicating immune system involvement. Kiviluoto published a series of reports regarding an investigation in 1975 of respiratory symptoms and clinical findings among employees (process workers, repairmen, foremen, and a laboratory worker) at a factory making vanadium pentoxide from magnetite ore (Kiviluoto, 1980; Kiviluoto et al., 1979; Kiviluoto et al., 1981a). A higher proportion of the exposed group (N = 63) had an elevated number of neutrophils in nasal smears compared to the referent group (N =DRAFT - DO NOT CITE OR QUOTE 62 63) who were employed at the magnetite ore mine and matched to the exposed by age and smoking habit (35% versus 7%, p<0.001, N=55 pairs). In biopsies of the nasal mucosa, a higher proportion in the exposed groups had elevated plasma cells and round cells (26% versus 0%, p<0.05, N=57 pairs and 48% versus 29%, p<0.05, N=56, respectively). The prevalence of elevated numbers of eosinophils did not vary by e xposure, leading the authors to conclude that the cytological and histological response in th e vanadium-exposed group was an irritant, not allergic response. Vanadium concentrations in the breathing zone averaged 0.028 mg/m 3 (TWA) with a range of 0.002 – 0.42 mg/m 3 . Higher concentrations were found where grinding and packing of smelt were conducted (TWA (range): 2.3 mg/m 3 (one sample) and 0.13 mg/m 3 (0.02 – 0.37). Motolese et. al. (1993) assessed whether exposure to vanadium pentoxide in the ceramicsindustry was associated with contact dermatitis or contact sensitization. One hundred and twentysix enamellers and sixty-four decorators from five ceramics factories were tested for contact sensitization using skin patch testing after exposure to a variety of substances, including vanadium pentoxide. Among the 190 workers under study, twenty-two individuals were found to have dermatitis and 17 reported having had skin lesions in the past. One worker responded with a positive skin patch test indicating sensitization to a 10% solution of vanadium pentoxide.

A pilot study using nasal lavage to evaluate an inflammatory response to fuel oil ash exposure, did not find an association of se veral exposure indices of vanadium or PM 10 exposure with counts or percentages of polymorphonuclear cells, eosinophils, and epithelial cells (Hauser et al., 1995b). Thirty-six out of 50 volunteers with no symptoms of cold or flu provided a nasal lavage sample both at baseline after at least 36 hours away from work and after 72 hours of exposure at a local electric company. A total of 19 boilermakers involved in the overhaul of a large oil-fired boiler and 18 utility workers, full-time employees of the power company with lower exposure to fuel oil ash, were studied. Daily exposure estimates for PM 10 (< 10 μ m) and vanadium (adjusted for filter extraction efficiency) were assigned to each individual using data from personal air sampling (1 – 10 hour TWA) and a self-completed work diary completed by each participant listing tasks and job locations during the day. A total of 29 task/location exposure categories were identified, but only 3 or fewer samples were available to estimate
concentrations at 23 of them. Environmental PM 10 concentrations based on personal sampling

were 50 to 4510 μ g/m 3 . Concentrations of respirable vanadium dust were 0.10 to 139.2 μ g/m 3

. Compared to baseline, the number of polymorphonuclear cells/ml recovered nasal fluid (adjusted by dividing the change by the mean of the baseline and postexposure value) increased by 40% (SD 100%, p < 0.05) with a range of -89% - 200%. The adjusted number of epithelial cells/ml recovered nasal fluid increased by 26.7% (SD 81.4%, p > 0.05) with a range of -83% - 200%.DRAFT - DO NOT CITE OR QUOTE 63 Regression models controlled for age and smoking. Models did not control for ozone levels or respirator use. The wide variation in the change in cell counts after exposure, especially among nonsmokers, and the small number of subjects may have precluded the detection of an association with vanadium or particulate exposure. Alternatively, the authors considered the levels of vanadium dust to be low, possibly not enough to cause inflammation in these workers. Woodin et. al (1998) analyzed nasal lavage fluid from 18 boilermakers before, during, and after the overhaul of a large, oil-fired boiler over a six week period from mid-May, 1995 to late-June, 1995. Biomarkers of upper airway inflammation in nasal lavage fluid were more prevalent among boilermakers during the overhaul compared to 11 utility workers (Woodin et al., 1998). Interleukin-8 (pg/ml) levels increased from a mean of 93.7 pg/ml (22.6-235.0) before the overhaul to 140.9 pg/ml (32.4-307.0) during the overhaul (p < 0.05), and decreased to levels comparable to those before the overhaul two weeks later (mean (range): 89.0 pg/ml (20.3-75.0)). Interleukin-8 levels among utility workers did not change substantially during the overhaul (mean (range): 69.2 (24.6-104.5), 58.5 (14.3-108.4) and 47.5 (12.8–74.7), respectively. Myeloperoxidase levels also increased among boilermakers during the overhaul, but not among utility workers. Among boilermakers, myeloperoxidase levels (ng/ml) were 22.7 (2.0-72.8), 33.9 (2.0-103.0) and 24.2 (3.9-58.1) before, during and afte r the overhaul, respectively (before versus during: p < 0.05). Among utility workers, my eloperoxidase levels were 25.6 (10.1-47.6), 27.2 (4.9-66.2) and 25.6 (4.9-51.7), respectively. Mean IL-6 and eosinophilic cationic protein levels did not change during the overhaul work suggesting that the inflammatory response was not due to an allergy or respiratory infection. During the boiler work, vanadium levels rose to a geometric mean (SD) of 8.9 (2.3) μ g/m 3 inside the boiler but did not change appreciably outside the boiler where the utility workers were located (geometric mean (SD) μ g/m 3 : 1.4 (1.6) (p < 0.001)). However, vanadium concentrations in nasal lavage fluid were not associated with levels of either IL-8 or myeloperoxidase using Spearman’s Rank Order Correlation Test. Hauser et. al. (2002) reported on a prospective cohort study of 118 boilermaker construction workers (see Section 4.1.2). The participants provided information on their work at oil, gas and coal-fired powerplants in annual wo rk history questionnaires, but exposure to specific components in the combustion particles were not quantified. Spirometry was used to measure lung function. Among the cohort, 6 workers reported having asthma diagnosed by a physician and 18 workers reported having symptoms of chronic bronchitis. Workers with asthma or chronic bronchitis experienced greater reductions in annualFEV 1 associated with the number of hours worked at gas or coal-fired powerplants during the year compared to the other workers. The generalized estimating equation models adjusted for age and smoking status, and interaction terms were statistically significant. In contrast, models adjusting for age, baseline FEV 1 , andDRAFT - DO NOT CITE OR QUOTE 64 cigarette smoking status did not find a similar e ffect of airway responsiveness, measured by methacholine challenge, on exposure-related reductions in FEV 1 . The annual reduction in FEV 1 associated with the number of hours worked at ga s, oil or coal-fired powerplants was similar between workers with and without reactive airways. In conclusion, case studies of occupational exposure to vanadium pentoxide dust or ROFA have reported individuals with dermatitis, positive skin patch reactions and bronchial reactivity, although this does not appear to be a common occurence. Although increases in the numbers of inflammatory cells in nasal smears or nasal lavage fluid have been observed in exposed workers, no associations were observed in relation to estimates of respirable vanadium dust or vanadium concentrations in nasal fluid. The authors did not report increases in eosinophils suggesting that the inflammation was due to irritation and was not an allergic

response.

4.4.2.2 Animal Studies

Pinon-Zarate et. al. (2008) exposed 112 male CD1 mice to ~1.4 mg/m 3 vanadium pentoxide by inhalation for 1 hr/day, 2x a day, for 12 weeks as measured by filters following exposure. This study did not provide reliable expos ure information, thus exposure concentrations in mg/m 3 could not be more specfically determined. Spleen weight and histology were determined. B-lymphocytes in the spleen were identified by immunohistochemical staining for CD19 (a cell surface marker which acts as a co-receptor for other CD markers). In addition, eight control and eight vanadium pentoxide-treated mice were immunized with Hepatitis B Surface antigen, a well-known T-cell dependent antigen. Avidity to the resulting antibody was measured. Spleen weight of vanadium-pentoxide exposed animals increased significantly and peaked at 9 weeks, and then decreased significantly. Splenic germinal centers were significantly increased in vanadium pentoxide-treated mice and contained a significantly increased number of CD19+ cells compared to controls. The authors suggest that vanadium pentoxide does not act as a direct antigen and does not induce a “host humoral response”. These data suggest that vanadium pentoxide may affect the avidity of antibodies – the ability of antibodies to bind effectively to substrates (affinity) and to engage multiple epitopes of the antigen at once. Mravcova et al (1993) conducted experiments to determine effects of subchronic exposure to low doses of vanadium pentoxide on the immune system. Weanling and adult male and female Wistar rats (n= 10 per group) were given vanadium pentoxide in drinking water (0, 1, 100 mg/L or 0, 0.14, 14 mg/kg-day) for 6 months. Also, male and female ICR mice (groups of 10) were given vanadium pentoxide (0 or 6 mg /kg-day) by gavage 5x a week for 6 weeks.DRAFT - DO NOT CITE OR QUOTE 65 Immunotoxicity endpoints included spleen and t hymus weight, spleen cellularity, number ofperipheral white blood cells, phagocytosis and natural killer cell activity, the extent of plaqueforming cells being converted to T-dependent antigen, and several cell-mediated immunity endpoints (Concanavalin A (ConA), pokeweed mitogen responsiveness (PWM), and phytohemagglutinin responsiveness (PHA) assays). Of these endpoints, spleen weight was significantly elevated over controls at 14 mg/kg-day in rats. The ConA and PHA assays illustrated significant cell-mediated immune activation at 1 mg/L in rats over control (3 and 2.5 times higher than control values, respectively) but statistical significance was not indicated or reported. At the high dose (14 mg/kg-day vanadi um), ConA and PHA assay results were close to control values. Thus, no dose-response pattern was detected. The low dose response may be a transient or compensatory response. Results of other endpoints were not reported. No significant differences in these parameters were reported in mice. The authors suggested that the high Con A response of T suppressor cells i ndicate that vanadium pentoxide may induce hypersensitivity responses at low doses. Immunological endpoints were also analyzed as part of a pulmonary study using cynomolous monkeys weekly provocation challenges (single 6-hr exposures to 0.5 or 3.0 mg/m 3 ). Inhaled vanadium pentoxide aerosol for six weeks produced statistically significant pulmonary responses, prior to a subchronic exposure (6 hrs/day, 5 days/week for 26 weeks) (Knecht et al., 1992; study details in Sec 4.2.2.1). Immunological analyses of blood and bronchiolar lavage fluid, and skin sensitivity tests were conducted before the pre- and postexposure provocation challenges. Bronchiolar lava ge fluid analyses were also performed one day after the provocation challenges. Cytological endpoints included complete and differential
blood cell counts and leukotriene C 4 levels. Immunological endpoints included total IgE, total IgG, albumin and total protein. The skin sensitivity tests assessed immediate and delayed responses to intradermal injections of vanadium pentoxide-monkey serum albumin conjugate. BAL fluid analysis showed a significant infl ux of inflammatory cells (polymorphonuclear leukocytes) into the lung. Other study endpoints were not significantly different between the three exposure groups (control, peak and constant) at either challenge concentration when the monkeys were rechallenged following subchronic exposure. In summary, immunological effects of vana dium pentoxide exposure have not been comprehensively studied. The studies described here have not shown a statistically significant response in the endpoints tested, however, some effects were observed. These results are
therefore inconclusive.

4.4.3 Neurological EndpointsDRAFT - DO NOT CITE OR QUOTE 66 4.4.3.1 Human Studies

No studies on the neurological effects of va nadium pentoxide were reported in humans.

4.4.3.2 Animal Studies

Pazynich (1966) exposed 33 male albino rats (species not specified) to 0.027 mg/m 3 or 0.002 mg/m 3 aerosolized vanadium pentoxide “round the clock” for 70 days. A third group of rats served as control. The animals were evaluated for general condition, body weight, motor chromaxy of antagonistic muscles, whole blood cholinesterase activity, and oxyhemoglobin content. Motor chromaxy of extensor muscles decreased significantly (p<0.01) while that of flexor muscles increased (p<0.001) in animals exposed to 0.027 mg/m 3 . Blood cholinesterase levels were statistically significantly reduced after exposure to 0.027 mg/m 3 and the reduction persisted throughout the 90-day recovery period. There was a statistically significant reduction in venous oxyhemoglobin in rats from the 0.027 mg/m 3 . Recovery was observed after 20 days. No difference in these parameters was reported in the 0.002 mg/m 3 group compared to controls. These results suggest a LOAEL of 0.027 mg/m 3 for hematological and CNS effects in albino rats with a NOAEL of 0.002 mg/m 3

. Three recent studies by Avila-Costa et al. (2004, 2005, 2006) found morphological changes in the central nervous system following inhalation exposure to vanadium pentoxide. Male CD-1 mice (n=48) were exposed to vanadium pentoxide by whole-body inhalation for 1 hr/day, 2 days/week for up to 8 weeks (Avila-Costa et al., 2004, 2005). Particle size was not reported in either study. The exposure concentration was reported as 0.02 M (Avila-Costa et al., 2004, 2005, 2006) or “2.5 mg/m 3 V” (Avila-Costa et al., 2005). The same group of investigators (Gonzalez-Villalva et al., 2006; Mussali-Galante et al., 2005) using the same exposure protocol reported that the 0.02 M solution generated an average chamber concentration of 2.5 mg/m 3 , as vanadium metal (MW = 50.94), corresponding to 2.57 mg/m 3 vanadium pentoxide (MW = 181.9). The number of immunoreactive-TH + neurons in the substantia nigra region of the basal ganglia in the mesencephalon (Avila-Costa et. al., 2004) and the morphology of the blood-brain barrier (Avila-Costa et. al., 2005) were assessed at the end of each week for up to 8 weeks of exposure. No clinical signs of toxicity were reported in either study. A duration-dependent
decrease in the number of immunoreactive-TH + neurons was observed from week 3 (decrease of approximately 30%; data presented graphically) through week 8 (decreased by approximately 63%; data presented graphically) of exposure (Avila-Costa et al., 2004). Morphological changes to the blood-brain barrier (cilia loss, cell sloughing and ependymal cell layer detachment) wereDRAFT - DO NOT CITE OR QUOTE 67 also observed starting at one week and increasing with duration of exposure (Avila-Costa et al., 2005). Using a similar protocol, Avila-Costa et al . (2006) assessed the effects of vanadium pentoxide on memory and morphology of hippocampal neurons in male CD-1 mice that were exposed by whole-body inhalation for 1 hr/day, 2 days/week for up to 4 weeks. Groups of 6 exposed mice and 6 vehicle control mice (inhaling deionized water droplets) were evaluated after 24 hrs and weekly for 4 weeks. No clinical signs or body weight changes were observed.Spatial memory was tested using a modified Morris water maze task that was learned preexposure. Performance on this test, as assessed by latency (swimming time) to locate a hiddenplatform, was significantly impaired in the exposed mice at all time points in an increasing, timerelated manner. Pyramidal neurons from the hippocampus CA1 region were evaluated for cytological and ultrastructural changes, because impairment in spatial memory is frequently seen following damage to this region of the brain. The cytological analysis assessed numbers of dendritic spines in the hippocampal cells; results showed a significant loss of dendritic spines in the exposed mice at all time points; the loss increased with time, in a manner that correlated with the memory impairments. The ultrastructural analysis showed a significantly increased percentage of necrotic hippocampal cells at a ll time points, with a maximum of 33% after 4 weeks of exposure; other findings included hyperdense postsynaptic terminals and edema in mitochrondria, dendrites, dendritic spines and presynaptic terminals. These three studies establish a LOAEL for morphological changes to the central nervous system accompanied by behavioral effects following acute and short-term intermittent exposure to vanadium pentoxide at concentrations of 2.56 mg/m 3 two times per week, for 1-hr duration/exposure. Colin-Barenque et al. (2008) investigated whether the vanadium pentoxide-mediated disruption of the blood-brain barrier was associated with the activation of matrix metalloproteinases (MMPs), protein degrading enzymes that are involved in tissue remodeling. Male CD-1 mice (n= 20 per group) were exposed to 0.02 M (2.56 mg/m 3 ) aerosolized vanadium pentoxide in deionized water or deionized water alone for 1 hr two times a week for up to four weeks. Five mice were sacrificed from each group, per time point (24 h, 1, 2 and 4 weeks). The presence of matrix metalloproteinase (MMP) was determined by gel zymography. In the olfactory bulb, MMP-2 was not different between vanadium pentoxide treated mice and controls, regardless of time point. MMP-9 was significantly elevated (300% and ~420%) in vanadium pentoxide-exposed mice compared to controls at 2 and 4 weeks, respectively. Both MMP-2 and MMP-9 were detected in the prefrontal cortex ; MMP-2 was not different between controls and treated animals at any time point, but , MMP-9 was significantly elevated over control values at 1, 2 and 4 weeks of exposure (150%, ~175%, and 250% of control values, respectively). In theDRAFT - DO NOT CITE OR QUOTE 68 hippocampus, MMP-9 from vanadium pentoxide-t reated mice was significantly elevated over controls after 1, 2 and 4 weeks of exposure (200%, 340%, and ~370%) while MMP-2 in exposed animals was significantly increased at 4 weeks (~150% over controls). In the striatum, MMP-9 from exposed mice was significantly elevated over that of controls after 4 weeks of exposure; MMP-2 levels from exposed mice were significantly elevanted over control values after 2 (160%) and 4 weeks (~250%) of exposure, but were not documented at earlier time points. These findings suggest that vanadium-induced increases in MMP’s in different parts of CNS occur in association with dendritic spine loss as well as neuronal death, and could be related to blood-brain barrier disruption.

4.5. MECHANISTIC DATA AND OTHER STUDIES IN SUPPORT OF THE MODE OF

ACTION FOR PULMONARY FIBROSIS AND CANCER

The preceding paragraphs have highlighted the main noncancer and cancer health effects that result from exposure to vanadium pentoxide. Noncancer effects in the lung range from histiocytic infiltration and alveolar inflammation, to hyperplasia of alveolar epithelium and pulmonary fibrosis. These endpoints exist in a plausible biological response continuum—from inflammation to reparative hyperplasia, to fibrosis. These effects also display a temporal and dose-response continuum, ranging from inflammatory and hyperplastic responses that occur at earlier time points (13 and 16 days) and at lower doses (2 mg/m 3 ) to fibrosis that occurs at later time points (3 months) and at higher doses (4 mg/m 3 ). Inflammation and hyperplasia are biologically relevant as precursor events to pulmonary fibrosis. Several investigators have systematically investigated the molecular mechanisms underlying vanadium pentoxide-induced pulmonary inflammation and fibrosis. These studies are summarized below.

4.5.1 Genotoxicity

The genotoxicity assays of vanadium pentoxide are summarized in Table 4-12.

4.5.1.1 Human Studies

Two studies investigated mutagenic activity in humans exposed to vanadium pentoxide (Ehrlich et al 2008; Ivancsits et al 2002). The in vivo genotoxicity of vanadium pentoxide in lymphocytes and whole blood leukocytes obtained from 49 male workers exposed to vanadium pentoxide at a processing plant was compared to 12 non-exposed controls (Ivancsits et al., 2002). The average exposure duration for workers was 12.4 years. Workers reported using protectiveDRAFT - DO NOT CITE OR QUOTE 69 masks at least occasionally. Measurements or estimates of worker exposure to vanadium pentoxide were not reported, although expos ure to vanadium was confirmed throughmeasurement of serum and urine vanadium. No significant differences between vanadiumexposed and control workers were observed for DNA strand breaks (as assessed by alkaline comet assay), 8-hydroxy-2'deoxyguanosine (8 -OHdG),an oxidized DNA base common indicative of oxidative stress or the frequency of sister chromatid exchange (SCE) in leukocytes. When normal human leukocytes or human fibroblasts were cultured in vitro and exposed to vanadate (25-500 μ g/L), both whole blood cells and isolated non-proliferating lymphocytesexhibited a significant increase in DNA migration in the alkaline comet assay compared to nonexposed cells only at the highest doses tested (250-500 μ g/L). Cultured human fibroblasts, however, exhibited a dramatic dose-dependent increase in DNA migration after vanadate treatment at lower concentrations as well (as low as 0.5 μ g/L) and suggest that fibroblasts are more sensitive to DNA damage in the presence of vanadate than are blood cells when exposed in vitro (Ivancsitis et al. 2002). Ehrlich et al (2008) investigated the im pact of inhaled vanadium pentoxide on DNA stability in vanadium production workers (n= 52) compared to non-exposed jail wardens (n=52) during October 2004 – May 2005. All subjects studied were male, and were exposed for their entire 8-hr shift while wearing protective masks. However, the duration of exposure and concentration of the inhaled vanadium was not determined. The median concentration (25 th

– 75 th percentile) of vanadium in plasma was 7-fold higher in exposed workers compared to the unexposed reference group. Leukocytes were then assayed (Comet assay) for DNA damage, and endogenous levels of oxidized purines and pyrimidines were determined. No differences in DNA migration by exposure were noted in leukocyt es under standard conditions, demonstrating that exposure is not associated with increases in single- and double-strand breaks. However, increases were observed in both oxidized purine (7% increase, p = 0.02) and pyrimidine (33% increase, p = 0.002) formation in exposed individuals. Moreover, DNA damage induced by bleomycin was 25% greater in leukocytes from the exposed workers (p < 0.0001) and DNA repair after bleomycin administration was less evident (p < 0.0001). The extent of micronuclei formation, necrosis and apoptosis was determined in isolated lymphocytes using the CBMN Cyt assay. The number of micronuclei was 2.5 fold higher in 24 workers than 23 non-exposed referents (p = 0.01). The frequency of nucleoplas mic bridges and nuclear buds (which indicate evidence of misrepaired DNA breaks and gene amplification, respectively) were significantly increased (7-fold and 3-fold) over controls. Numbers of necrotic and apoptotic cells were increased by 55% and 50% respectively in exposed workers. Together, these results suggest that occupational exposure to inhaled vanadium pe ntoxide may affect DNA stability by increasingDRAFT - DO NOT CITE OR QUOTE 70 levels of oxidized bases and affecting DNA re pair. Age, body mass index and smoking habit (cigarettes/day) were similar between the two groups. Folate levels also were similar but
vitamin B 6 and B 12 levels were lower among the unexposed indicating that the effects on DNA in the exposed were not due to vitamin deficits.Kim and colleagues (2004) assessed the cross-shift change in urine levels of 8-hydroxy2’-deoxyguanosine (8-OHdG), a marker of DNA repair of oxidative DNA damage, over a 5-day period in 1999 among a group of 20 boilermakers involved in the overhaul of oil-fired boilers at a power plant (74% of eligible). Median total PM 2.5 8-hour TWA concentration, measured using personal exposure monitoring, was 0.44 mg/m 3 (Q 25% - Q 75% : 0.29 – 0.76 mg/m 3 ). Total vanadium 8-hour TWA concentration, including vanadium oxides, was 1.23 μ g/m 3 (Q 25% - Q 75% : 0.47 – 3.53 μ g/m 3 ). The workers were 18 – 59 years old (mean ± SD: 45.5 ± 12.0) and had been employed as boilermakers for 0.04 to 40 years ((mean ± SD: 21.7 ± 12.9). The mean cross-shift change in creatinine adjusted 8-OHdG levels in urine was 1.88 μ g/g creatinine (SD 0.74). Preshift levels, measured an average of two days aw ay from work, were significantly different from post-shift levels (p = 0.02). In linear mixed regression models, a 1 mg/m 3 increase in total PM 2.5 8-hour TWA concentration was associated with an increase in urinary 8-OHdG concentrations of

1.67 μ g/g creatinine (95% CI: 0.21 – 3.14), adjusting for urinary cotinine, chronic bronchitis status, and age. A 1 μ g/m 3 increase in PM 2.5 vanadium concentration was associated with an increase in urinary 8-OHdG concentrations of 0.23 μ g/g creatinine (95% CI: 0.04 – 0.42) in a
model with the same covariates. PM 2.5 manganese, nickel and lead concentrations also were associated with 8-OHdG levels in urine when analyzed separately in similar models. The concentrations of the metals were correlated (0.52 < r < 0.92) and so the association with vanadium may not have been independent of the associations with the other metals. However, the finding of oxidative DNA injury and repair among healthy boilermakers is consistent with similar reports among vanadium pentoxide workers. Another marker of oxidative DNA damage, 7-hydro-8-oxo-2’-deoxyguanosine (8- oxodG), was assessed in relation to water soluble transition metal content in ambient PM 2.5 among male and female nonsmoking students, 20 – 33 years of age, living in central Copenhagen (Sorensen et al., 2005). Personal samples of PM 2.5 were collected over two days twice during one year, once during summer and once during autumn. Median (interquartile range)
concentrations of PM 2.5 were 20.1 μ g/m 3 (13.1 – 27.7) in November and 12.6 μ g/m 3 (9.4 – 24.3) in August. The median (interquartile range) concentration of vanadium in PM 2.5 was 3.0 (0.3 – 4.7) in November and 3.2 (1.4 – 5.7) in August. Of 66 participating students, 32 provided measurements for both seasons. Median (interquartile range) levels of 8-OxodG in lymphocytes (per 105 dG) were 0.55 (0.34 – 0.78) and 0.58 (0.47 – 0.70) in November and August,DRAFT - DO NOT CITE OR QUOTE 71 respectively. Vanadium and chromium concentrations in aqueous suspensions of PM 2.5 were associated with the 8-oxodG concentration in lymphocytes in mixed regression models with subject as a random factor and adjusting for PM 2.5 mass and season. A 1 μ g/L increase in either
vanadium or chromium was associated with a 1.9% (95% CI: 0.6 – 3.3) or 2.2% (95% CI: 0.8 – 3.5) increase in 8-oxodG concentrations in lymphocytes, respectively. Platinum, nickel, copper, and iron were not associated with 8-oxodG concentration in lymphocytes and no metals were associated with 8-oxodG concentration in urine. This study suggests that metal content of ambient fine particulate matter increases oxidative DNA damage in lymphocytes and that vanadium may be one of the responsible agents along with other metal constituents in particulate air pollution at levels common in urban settings. Three other studies demonstrated a genotoxic effect of vanadium pentoxide on primary human lymphocytes in vitro (Rojas et al. 1996; Ramirez et al. 1997; Roldan and Altamirano 1990). These studies examined chromosomal aberrations and aneuploidy by fluorescence in situ hybridization (FISH) and SCE assays, as well as DNA damage by the comet assay. Cells exposed to vanadium pentoxide had significan tly increased DNA migration indicative of DNA damage at all doses tested in primary human leukocytes (p<0.05) and in the high doses in three of four donor lymphocyte cell strains (p<0.05). Vanadium pentoxide (0 – 0.1uM) lead to an increase in aneuploidy with some interindividual variation observed in four primary human lymphocyte cell strains (Ramirez et al. 1997). An increase in aneuploidy was observed in one primary human cell strain exposed to vanadium pentoxide (0 – 6ug/ml) but no chromosomal aberrations were observed (Roldan and Altamirano 1990).

4.5.1.2 Laboratory in vivo and in vitro studies

Vanadium pentoxide produced gene mutations in two bacterial test systems ( Bacillus

subtilus and Escherichia coli ) (Kada et. al., 1980, Kanematsu et. al., 1980); although negative results were reported by NTP (2002) in a reverse mutation assay in Salmonella typhimurium (TA97,TA98,TA100, TA102,TA1535 with or without metabolic activation). Negative results were also reported in a gene mutation assay in Chinese hamster V79 fibroblast cells (Zhong et al., 1994). However, DNA damage and/or aneupl oidy was observed in all in vitro studies performed in primary human cells (Ivancsits et al. 2002; Kleinsasser et al. 2003; Ramirez et al. 1997; Rojas et al. 1996; Roldan and Altamirano 1990). Positive results were observed for DNA strand breaks in cultured human lymphocytes (Rojas et. al, 1996) at high doses of vanadate (Ivancsits et. al, 2002) and in cultured human fibroblasts at lower, more environmentally relevant doses of vanadate (0.5 μ g/L) (Ivancsits et. al., 2002). Positive results have been noted forDRAFT - DO NOT CITE OR QUOTE 72 aneuploidy (Ramirez et. al., 1997), and polyploi dy (Roldan and Altamirano et. al., 1990). Negative results were reported for chromosomal aberrations (Roldan and Altamirano et. al., 1990). In Chinese hamster V79 lung fibroblast cells, positive results were observed for micronuclei formation (Zhong et. al., 1994), altered mitosis (Zhong et. al., 1994), and cell transformation in Syrian hamster embryo cells (Kerckaert et al., 1996) at concentrations that were not cytotoxic. Negative results were reported for sister chromatid exchanges and gene mutations in vanadium pentoxide-treated Chinese hamster V79 firbroblast cells (Zhong et. al., 1994). One study evaluated the genotoxicity of vanadium pentoxide in primary human cell cultures. Kleinsasser et. al. (2003) took mucosal bi opsies from inferior nasal turbinates and blood samples from seventeen healthy volunteers. Isolated lymphocytes and mucosal cells were cultured and exposed to 0, 0.06, 0.12, 0.24, and 0.47 mM vanadium pentoxide in vitro for 1hr. Mucosal cells and lymphocytes were assessed for DNA migration by the Comet assay. Extent of migration was measured qualitatively (image analysis) and quantitatively (“Olive Tail Moment” method). DNA migration was not significantly diffe rent in exposed human nasal mucosal cells compared to controls. However, DNA migration appeared to increase dose-dependently in exposed human lymphocytes compared to controls (p = 0.001). Cytotoxicity was limited in both cell types at all doses as measured by trypan blue exclusion assay. These results suggest that human lymphocytes, but not nasal mucosal cells demonstrate genotoxic damage (single strand breaks and/or alkali-labile damage) in response to vanadium pentoxide. Experimental data in animals provide evidence of some types of genotoxicity following

in vivo exposure to vanadium pentoxide. Vanadium pentoxide administered for 3 months by inhalation to male and female mice (1, 2, 4, 8 or 16 mg/m 3 ) did not increase the frequency of micronucleated normochromatic erythrocytes in peripheral blood (NTP, 2002). Additional details of exposure are provided in the NTP (2002) study summary (see Section 4.2.1.2). Genotoxicity was evaluated in male CD-1 mice following single intraperitoneal injections of 5.75, 11.5 or 23 mg/kg vanadium pentoxide (A ltamirano-Lozano et al., 1993, 1996). Exposure caused no treatment-related effects on mitotic index, average generational time or sister chromatid exchanges in bone marrow cells (Altamirano-Lozano et al., 1993), although all doses induced DNA damage in testicular germ cells (Altamirano-Lozano et al., 1996). Altamirano-Lozano et al. (1999) assessed DNA damage in male CD-1 mice 24 hrs following single intraperitoneal injections of 0, 23.0, 11.5 or 5.75 mg/kg vanadium pentoxide (corresponding approximately to the LD 50 , 1 / 2 LD 50 and 1 / 4 LD 50 , respectively). Comet test results show the number of cells with DNA damage (primarily single strand breaks and alkaliDRAFT - DO NOT CITE OR QUOTE 73 labile damage) was increased in liver, kidney, lung, spleen and heart, although increases did not exhibit dose-dependence. No evidence of DNA damage was observed in bone marrow. In summary, the evidence for mutagenicity in humans is limited. There are few studies examining genotoxicity in humans in vivo, with equivocal results. Ivancsits et. al. (2002) reported no differences in DNA strand breaks, oxidative damage, or sister chromatid exchange frequency in leukocytes between control and vanadium pentoxide-exposed workers. Ehrlich et.al., (2008) noted changes in DNA stability and DNA repair in leukocytes of occupationallyexposed workers as compared to controls. Studies have demonstrated a genotoxic effect of vanadium pentoxide on human cells in vitro. Iv ancsits et al. (2002) demonstrated significant increases in DNA damage as measured by the Comet assay in both leukocytes and fibroblasts but with different dose sensitivity, while Kleinsasse r et. al (2003) noted DNA migration differences occurred dose-dependently in peripheral blood lymphocytes but not in nasal mucosa. Earlier studies in human lymphocyte cultures also demonstrated increased aneuploidy (Ramirez et al. 1997; Rojas et al. 1996) and DNA damage (Rolda n and Altamirano 1990) following exposure to vanadium pentoxide. Thus, vanadium pentoxide-induced mutagenicity may occur at doses higher than those measured in these occupational exposures, may be tissue-specific and may be associated with oxidative stress rather than direct DNA damage. In vitro tests in bacterial and yeast systems provide mixed evidence of vanadium pentoxide-induced mutagenicity. In general, classic gene mutation assays were negative, as were tests that assessed sister chromatid exchange and other chromosomal aberrations. DNA strand breaks (Rojas et al., 1996; Ivancsits et . al., 2002) and micronuclei formation (Zhong et. al., 1994) were indicated in some studies in cultured cells but were dependent on cell type. Fibroblasts appear to be more sensitive to vanadium exposure in vitro than are blood cells. Similarly, experimental data from animal studi es is equivocal. NTP (2002) reported that the frequency of micronucleated normochromatic eryt hrocytes in peripheral blood was not increased in exposed compared to control mice. However, a number of studies by Altamirano-Lozano et al. (1993, 1996, 1999) have noted DNA damage in speci fic target tissues in vanadium pentoxidetreated mice. It should be noted that Altamirano-Lozano et al (1993) consistently used intraperitoneal injection as the route of exposure for these studies.Table 4-12. Genotoxicity Data Following Exposure to Vanadium PentoxideTest System/SpeciesResultsExposureDoseEffectsEndpointReferenceIn vivoHuman49 exposed maleworkers at vanadiumpentoxide processingplant; 12 nonexposure controls_avg 12.4ynot reported -exposureconfirmedthrough bloodand urinemeasurementsof vanadiumGenotoxicity measured in isolated lymphocytes andwhole blood leukocytes. Study also examined invitro exposure (below).Comet AssayIvancsits etal. 2002_DNA damage(8OHdG)_Sisterchromatidexchange52 exposed workers;52 non-exposedworkers (jailwardens)-inhalationnot reported -8hr shift,protectivemasks;exposureconfirmedthrough bloodlevels ofvanadiumGenotoxicity was measured in isolated leukocytesby Comet assay, with no increases in single- anddouble-DNA strand breaks observed. However,increases were observed in oxidized purines andpyrimidine formation in exposed workers. CBMNCyt assay demonstrated increased MN induction,nucleoplasmic bridges and nuclear bud formation.Necrosis and apoptosis levels were also increased inexposed individuals.Comet AssayEhrlich etal. 2008+MNinduction,oxidativenucleotidesLaboratory AnimalsMale CD-1 mice (n= 4)+intraperitonealinjection,sacrificed 24hpost injection0, 5.75, 11.5,23 μg/g bwDNA damage was observed in all tissues examinedexcept for bone marrow. This included liver,kidney, lung, spleen, and heart.Comet assayAltamiranoLozano etal. 1999Male CD-1 mice (n= 2)+intraperitonealinjection,sacrificed 24hpost injection0, 5.75, 11.5,23 μg/g bwAs part of a larger study on reprotoxicity, DNAdamage in sperm cells was analyzed. Significantincreases were observed in a dose-dependentmanner (p <0.05).Comet assayAltamiranoLozano etal. 1996DRAFT - DO NOT CITE OR QUOTE74Male CD-1 mice (n= 4)-intraperitonealinjection,sacrificed 24hpost injection0, 5.75, 11.5,23 μg/g bwAnalysis of sister chromatid exchange demonstratedno effect of vanadium pentoxide exposure in thisstudy.cytogeneticassayAltamiranoLozano etal. 1993B6C3F1 mice (M, F)+inhalation,2yr0, 1,2, or 4mg/m3DNA was isolated from lung tumros and normaltissue from exposed animals. Of the 20 tumorsanalyzed, 13 had either K-ras mutations or LOH atchromosome 6 or both.cytogeneticassayDevereux etal. 2002B6C3F1 mice (M, F)+inhalation,2yr0, 1,2, or 4mg/m3Analysis of frequency of micronuclei in peripheralblood normochromatic erythrocytes demonstratedon effect of vanadium pentoxide exposure in thisstudy.MicronucleiassayNTP 2002In vitroprimary humanlymphocytes+25 – 500μg/LV2O5 exposure led to significant increase in DNAmigration as measured by Comet assay at thehighest doses tested (250 - 500ug/L) for wholeblood lymphocytes and leukocytes, and at all dosestested in cultured fibroblasts (p values not given).Comet AssayIvancsits etal. 2002primary humanwhole bloodleukocytes+cultured humanfibroblasts+primary humanlymphocytes+0 – 47 mMExposure to vanadium pentoxide led to a dosedependent increase in DNA migration inlymphocytes but not in mucosal cells.Comet assayKleinsasseret al. 2003primary human nasalmucosal cells-primary humanlymphocytes+0 - 0.1 μMVanadium pentoxide lead to an increase inaneuploidy with some interindividual variationobserved between the four primary cell strains.Disruption of spindle formation may be due tointeraction with microtubules.FISHRamirez etal. 1997DRAFT - DO NOT CITE OR QUOTE75primary humanlymphocytes (n=4)+24h0.3, 30, 3000μMCells exposed to vanadium pentoxide hadsignificantly increased DNA migration at all dosestested in the leukocytes (p < 0.05) and in the highdoses in lymphocytes for three of the four donors (p< 0.05). DNA repair occurred generally within in45 min post-exposure.Comet AssayRojas et al.1996primary humanlymphocytes (n = 1)-72h0, 2, 4, 6μg/mlVanadium pentoxide exposed cells had an increasein aneuploidy and a decrease in mitotic index, withno changes in SCE or chromosomal aberrations.SCE assayRoldan andAltamirano1990+aneuploidySyrian HamsterEmbryo cells+0, 24h, 7d0 to 0.875μg/mlVanadium pentoxide exposed cells were positive at7d exposure but not at 24h, similar to other tumorpromotion chemicals studied by this group.SHEtransformationassayKerckaertet al. 1996Chinese hamsterV79 cells+24h0, 1, 3, 6, 9,12 μg/mlVanadium pentoxide exposure led to increased MNinduction (p < 0.005), apparently due to damage tothe spindle apparatus but no significant increases inmutations or SCE.MN inductionZhong et al.1994-SCE assay-HGPRTmutationBacterial systemsBacillus subtilispositivewith andwithoutactivationStudy details not available.recombinationrepairKada et al.1980DRAFT - DO NOT CITE OR QUOTE76DRAFT - DO NOT CITE OR QUOTE77Escherichia colipositivewithoutactivation(not testedwithactivation)Study details not available.genemutationKanematsuet al. 1980SalmonellatyphimuriumSalmonellatyphimurium(TA97,TA98,TA100, TA102,TA1535with or withoutmetabolic activation)All strainsnegativewith andwithoutactivation(bothhamsterand rat S9fractions)48 h0 - 333μg/plateNo increase in revertant colonies was observedfollowing exposure.gene mutationNTP 2002DRAFT - DO NOT CITE OR QUOTE 78 4.5.2 Mechanisms of Inflammation and Fibrosis

Increases in markers of pulmonary inflammation have been observed in the BAL fluid of rats and susceptible mice following intratracheal instillation exposure to vanadium pentoxide (Pierce et al. 1996; Bonner et al. 2002). Thes e include macrophage inflammatory protein-2 (MIP-2), keratinocyte-derived chemokine (KC), interleukin-6 (IL-6), and IL-8. Further, the increased expression of prostaglandin-ge nerating enzymes cyclooxygenases (COX) and prostaglandin E synthases have been associated with exposure to vanadium pentoxide (Bonner et al 2002; Pierce et al 1996), further suggesting increased inflammation. Pierce et. al. (1996) investigated the ability of several vanadium compounds to increase mRNA levels of cytokines in bronchoalveolar lava ge (BAL) fluid. Female CD rats received 42

or 420 μ g of vanadium pentoxide or phosphate-buffered saline (PBS) by intratracheal

instillation. BAL fluid was recovered one hr to ten days after exposure. Significant neutrophil influx was observed after 24 hrs exposure to va nadium pentoxide and peaked at 48-hr postexposure with 20% neutrophils. MIP-2 mRNA expr ession levels were significantly elevated in vanadium pentoxide-treated rats compared to c ontrols at early time points (1 hr to 48 hrs) suggesting pulmonary inflammation. Pro-inflammatory prostaglandins such as PGE 2 produced by the enzymes COX-1 and COX-2, and PGE synthase mediate tissue homeost asis and/or known to be associated with various inflammatory diseases. Bonner et. al . (2002) assessed the role of COX-1 and COX-2 enzymes in vanadium pentoxide-induced pulmonary inflammatory and fibrotic responses using 6-8 month-old male and female mice (of a hybr id C57BL/6J and 129/Ola genotype) that were deficient in either COX-1 (COX1 -/- ) or COX-2 (COX2 -/- ) enzyme. These COX-deficient mice and genotype-matched controls (wildtype) were instilled with 50 μ L of saline (n = 3 to 4) or 1mg/kg vanadium pentoxide in saline (n = 5 to 6). Lungs were lavaged at 1, 3, 6, or 15 days post-instillation and BAL fluid was collected and analyzed for tumor necrosis factor-alpha (TNF- α ) and prostanoids (e.g., PGE2) by ELISA. Lungs were removed and preserved for histopathology, hydroxyproline assay, or COX immunoblotting. Histopathology showed marked inflammation and increased injury in COX2 -/- mice compared to wild-type and COX1 -/- mice 3 days following vanadium pentoxide instillation. Hydroxyproline content was not different in wildtype or COX1 -/- mice in response to vanadium pentoxide compared to saline-instilled controls. Hydroxyproline content in vanadium pentoxide-exposed COX2 -/- mice was increased twofold compared to saline-instilled COX2 -/- mice suggesting enhancement of lung fibrosisfollowing vanadium pentoxide exposure. PGE2 levels increased from ~500 pg/mL in salineinstilled wildtype mice to ~1000 pg/mL in vanadium pentoxide-treated wildtype mice at 24 hrsDRAFT - DO NOT CITE OR QUOTE 79 but not at other time points. The PGE2 level in saline-treated COX1 -/- mice was ~10 pg/mL and about ~225 pg/mL after 24 hrs of vanadium pentoxide. PGE2 levels were ~200 pg/mL at 24 hrs
in saline-treated COX2 -/- mice and did not differ significantly from vanadium pentoxide-treated

COX2 -/- mice regardless of time point. This study suggests that vanadium pentoxide-induced inflammation may also be at least partially mediated by prostaglandins such as PGE2 generated by COX-2. Myofibroblasts, the principal proliferating cells that produce collagen, are involved in fibrogenic response of lungs following exposure to pulmonary irritants. Bonner et. al. (2000) observed that proliferating myofibroblasts were the principle cell type that contributed to the observed fibrosis. Male Sprague-Dawley ra ts weighing ~200 g received intratreacheal instillation of sterile saline or 1 mg/kg vanadium pentoxide. Rats were additionally injected with BrdU (50 mg/kg, i.p.) one hr prior to sacrifice. Sacrifice occurred at 3, 6, and 15 days after vanadium pentoxide instillation. Excised lung tissue was assessed morphometrically and by immunohistochemistry for vimentin and desmin, two biomarkers for myofibroblasts and smooth muscle cells, respectively. Trichrome staining was used to assess collagen levels, an indicator of the extent of fibrosis. Vanadium pentoxide exposure induced thickening of the desmin-positive bronchiolar smooth muscle cell layer by day 6 post exposure and were identified as myofibroblasts. A 2.3-fold increase in airway smooth muscle cell nuclear profile, suggesting increase in smooth muscle cell proliferation was due to hyperplasia. Serial sections of the peribronchiolar region stained positive for vimentin and desmin, and were mainly myofibroblasts. The thickness of the subepithelial trichrome-positive layer was 3.1-3.9-fold higher at day 15 in vanadium pentoxide vs. control samples. The peak appearance of peribronchiloar myofibroblasts occurred at day 6 and declined by day 15. A thickened collagen ring was apparent by day 15 in vanadium pentoxide-exposed samples compared to controls. Rice et. al. (1999) using both in vitro and in vivo models showed that myofibroblasts do proliferate in response to vanadium pentoxide, and are dependent on platelet-derived growth factor (PDGF) and epidermal growth factor (EGF). Rat lung myofibroblasts were isolated from exposed male Sprague-Dawley rats, as stated in Bonner (1998) above, and were grown to confluency. Cultures were incubated for 24 hrs with increasing concentrations of one of two inhibitors of the PDGF-R (AG1296) and EGF-R (AG1478), respectively, at a concentration of

100 μ mol/L. Autophosphorlyation of PDGF-R and EG F-R in vitro was specifically blocked by AG1296 and AG1478, respectively. Tritiated [ 3 H] thymidine uptake, a measure of mitogenesis, was blocked by selective inhibition of PDGF- and EGF- receptors. An in vivo study was carried out at the same time. Male Sprague-Dawley rats were treated with AG1296 or AG1478 (50 mg/kg) by intraperitoneal injection 1 hr prior to intratracheal instillation of vanadium pentoxideDRAFT - DO NOT CITE OR QUOTE 80 (1 mg/kg) and again two days after vanadium pentoxide was administered. Rats were sacrificed 3, 6, and 15 days after instillation and lungs were preserved for bromodeoxyuridine (BrdU) immunohistochemistry and hydroxyproline assays to measure DNA replication and cell division, respectively. Quantitation of BrdU-labeled cells in the nuclei of rat lung tissue was significantly reduced in vanadium pentoxide-treated animal s that had received injections of AG1296 and AG1478, compared to vanadium pentoxide treated an imals that were injected with vehicle alone. Vanadium pentoxide treatment induced a five-fold increase in lung hydroxyproline content, anindicator of lung collagen and potentially fibrosis, 15 days after instillation. Prior and posttreatment with AG1296 reduced hydroxyproline content in vanadium pentoxide-treated animals to quantities similar to saline-instilled animals. Pre- and post-treatments with AG1478 reduced hydroxyproline content by ~50%, but were still significantly higher than in saline-instilled
controls. Two recent studies examined the mechanism of inflammation in mice (Rondini et al. 2010; Turpin et al. 2010). Rondini et al (2010) examined the effect of exposure to vanadium pentoxide in three mouse strains of varying susceptibility to lung cancer (A/J, BALB/C and C57BL/6J) in an initiation/promotion model (full study description in Section 4.2.2.2). Significantly higher transcriptional activity was observed for NFkB (A/J mice; peaked at 1 daypost-treatment) and AP-1 (A/J and B6 mice; peaked at 6 hrs post-treatment) compared to PBScontrols, the activities in the order of A/J mice > B6 mice; in vanadium pentoxide-treated mice. Overall, the differential inflammatory responses observed in the three strains of mice appear to positively correlate with increased levels of chemokines, such as keratinocyte-derived chemokine (KC) and monocyte chemotactic protein-1 (MCP-1), and increased binding of transcriptional factors NF κ B and AP-1 (c-Fos), and sustained activation of MAP kinases (MAPKs) and extracellular signal-regulated kinase s 1 and 2 (ERK 1/2) suggesting inflammation as a major response in mice. Turpin et al. (2010) examined pulmonary inflammation and fibrosis following intranasal aspiration exposure to vana dium pentoxide with and without respiratory syncytial virus (RSV) exposure (full study description in Section 4.4.1.2). In this study, vanadium pentoxide exposure also caused a significant increase in cell proliferation in the airways and lung parenchyma, lung mRNAs for TGF- β -1, CTGF, PDGF-C, Col1A2, and

mRNAs for IFN- α and - β and IFN-inducible chemokines CXCL9 and CXCL10 compared to controls. Pre- or post-treatment with RSV caused a significant reduction in the all mRNAs. Together, results from this study showed that vanadium pentoxide induces inflammatory and fibrogenic response in mouse lung and these effects were suppressed by RSV infection.To elucidate the potential cell signaling cascades associated with these endpoints, AntaoMenezes et. al. (2008) investigated the role of the signaling molecule, signal transducer andDRAFT - DO NOT CITE OR QUOTE 81 activator of transcription (STAT)-1 in vanadium pentoxide-induced pulmonary fibrosis. Their work identified another inflammatory molecule, interferon-beta (IFN- β ), as a mediator of vanadium pentoxide-induced STAT-1 activation in normal human lung fibroblasts. Briefly, confluent, quiescent cultures of human lung fibroblasts were either placed in serum-free defined medium (SFDM) or SFDM supplemented with 10 μ g/cm 2 vanadium pentoxide. Neutralizing

anti-IFN- α and anti-IFN- β antibodies were used to quench activity of IFN- α and IFN- β and the ratio of phosphor-STAT-1 to STAT-1 was then measured by quantitative RT-PCR or Western blot. Vanadium pentoxide-induced STAT-1 activ ation could be inhibited by a broad spectrum

NADPH inhibitor at 24 hr. Vanadium pentoxide induced significant IFN- β expression after 18 and 24 hrs. This effect was nearly completely abolished by addition of the NAPDH inhibitor. Activation of STAT-1 (as measured by a ratio of phosphor-STAT1 to total STAT-1) was significantly decreased by addition of neutralizing IFN- β antibodies and also by addition of a Janus Associated Kinase (JAK) inhibitor. In summary, STAT-1 was activated in response to vanadium pentoxide exposure, and was linked to IFN- β as its primary mediator. This study identifies a putative signaling pathway leading to expression of genes that control proliferation of myofibroblasts. Ingram et. al. (2007) performed gene array analysis to determine a list of candidate genes altered by exposure to vanadium pentoxide. Norm al human lung fibroblasts were exposed to 10

μ g/cm 2 vanadium pentoxide or saline in vitro. RNA from cells was harvested 1, 4 8, 12, and 24 hrs post-treatment. Labeled cRNA hybridized to the Affymetrix Human Genome Array U133A 2.0 gene chip was used to assess gene expression at various time points up to 24 hrs of exposure. About 300 genes were found to be upregulated in response to vanadium pentoxide including inflammatory and immunomodulatory genes. Ov er 1,000 genes were downregulated in response to vanadium pentoxide including genes from the ubiquitin cycle and cell cycle genes. A dozen genes were confirmed by RT-PCR and included growth factors (heparin-binding EGF-like growth factor [HB-EGF], vascular endothelial cell growth factor [VEGF], and connective tissue growth factor [CTGF]), chemokines (IL-8, CXCL9, CXCL10), oxidative response genes (superoxide dismutase [SOD]2, pipecolic acid oxidase [PIPOX], oxidative stress response [OXR]1) and DNA-binding proteins (growth arrest specific [GAS]1, STAT1). The gene array analysis thus confirms that a number of mit ogens, growth factors, chemokines, cytokines, oxidative response genes, and DNA-binding proteins are all critical to the formation of fibroproliferative lesions in response to vanadium pentoxide exposure in vitro (Figure 4-1).DRAFT - DO NOT CITE OR QUOTE 82 Figure 4-1: Genomics of V 2 O 5 -Induced Bronchitis (reprinted with permission from Ingram et al., (2007) Respir. Res. Apr 25;8(1):34)

Together, these results indicate that proliferating myofibroblasts are the primary cell type associated with vanadium pentoxide-induced pulm onary fibrosis, and that cellular proliferation depends on activated mitogens such as PDGF and EGF, in addition to HB-EGF. The STAT-1 and MAPKinase pathways may play key roles in this process. Moreover, fibroproliferative lesions contain collagen.

4.5.3 Mechanisms of Hyperplasia and Carcinogenicity

Molecular events underlying the mechanism of reparative hyperplasia and carcinogenesis have been documented. Bonner et. al. (1998) exposed male Sprague-Dawley rats to vanadium pentoxide or sterile saline by intratracheal instilla tion at 1 mg/kg. Animals were sacrificed at 3, 6 and 15 days after instillation. Lungs were preserved and analyzed for PDGFR- α by immunohistochemical analysis and morphometry fo r fibroproliferative lesions. Smooth muscle thickening was observed beneath ciliated epithelial cells, as indicated by increased desmin localization, on day 6 in exposed samples. Trichrome staining revealed increased collagen deposition around bronchioles in vanadium-pentoxide exposed samples. The thickness of the subepithelial layer increased by 3.1 to 3.9 fold at day 15 after instillation of vanadium pentoxide, determined by morphometric techniques. Platelet-derived growth factor (PDGF) is a mitogen and chemoattractant for fibroblasts.DRAFT - DO NOT CITE OR QUOTE 83 Male Sprague-Dawley rats were intratracheally instilled with sterile saline or 2 mg/kg of vanadium pentoxide (Bonner et al 1998). Tissues were harvested at 24, 48, and 72 hr post exposure as well as at 6 and 15 days. Pulmonary myofibroblasts and alveolar macrophages were isolated. Isolated total lung RNA was assesse d for quantitation of PDGF by Northern blot analysis. PDGF-receptor alpha mRNA and protein expression were significantly elevated in vanadium pentoxide exposed animals compared to controls at 24 and 48 hrs. PDGF-receptor beta was not significantly elevated over controls at any time points. Confluent cultures of lung myofibroblasts were stimulated with vanadium pentoxide. Similarly, cultures of lung macrophages were stimulated with vanadium pent oxide. Levels of PDGF in myofibroblasts were not affected by direct stimulation by vana dium pentoxide, but were affected by a factor released by vanadium pentoxide stimulated macr ophages, and were associated with interleukin (IL)-1B, an inflammatory cytokine. Together, these results suggest that hyperplasia occurs under the control of inflammatory mediatiors such as IL-1B that can recruit mitogens such as PDGF that can stimulate growth of myofibroblasts, the main cell type involved in the development of fibrotic lesions.Zhang et al (2001a) investigated the ability of vanadium pentoxide to induce heparinbinding epidermal growth factor-like growth factor (HB-EGF) (another mitogen) in vitro, using normal human bronchial epithelial cells (NHBECs). Mature cultures of NHBECs were incubated with vanadium pentoxide at 0, 1, 10 and 50 μ g/cm 3 for 3 hrs. Total RNA was then isolated and RT-PCR was used to quantitate HB-EGF. HB-EGF mRNA was significantly and dose-dependently increased in response to vanadium pentoxide compared to controls. In a second time course study, (using only the high dose 50 μ g/cm 3 of vanadium pentoxide) the peak

of HB-EGF induction occurred after 3hrs of e xposure and persisted until 8 hrs of exposure. In a follow-up study, Ingram et al. (2003) sim ilarly showed a peak induction of HB-EGF in quiescent cultured human lung fibroblasts exposed to 10 μ g/cm 2 vanadium pentoxide at 3 hrs. Quiescent cultured human lung fibroblasts were exposed for 3 hrs to 0, 10, 30 or 100 μ g/cm 2 vanadium pentoxide. HB-EGF RNA was isolated and detected by Northern blot. HB-EGF was significantly elevated in a dose-dependent manne r in vanadium pentoxide-exposed fibroblasts compared to controls. Stimulating human lung fibroblasts with H 2 O 2 (10 μ M) similarly induced HB-EGF, with a peak mRNA expression at 1hr post exposure. HB-EGF protein expression peaked at 6 hr post--exposure, as measured by Western blot. Quiescent human lung fibroblasts

stimulated with 10 μ g/cm 2 vanadium pentoxide were found to initially quench spontaneous H 2 O 2 production (at early time points) and then boost H 2 O 2 production at a peak of 12 hrs post exposure. To assess the role of extracellular signal-regulated protein kinase (ERK), and the p38 subunit of mitogen-activated protein (MAP) kina se in vanadium pentoxide induced HB-EGFDRAFT - DO NOT CITE OR QUOTE 84 production, Ingram et al (2003) exposed quiescent confluent human lung fibroblast cells in

culture to 500 μ M H 2 O 2 or 10 μ g/cm 2 vanadium pentoxide for 15 min, 30 min, 1 hr, 3 hr, 6 hr or 24 hr. Cell lysates were collected and Western blot was performed for the phosphorylated form of ERK or total ERK protein or for the phosphorylated p38 subunit of MAPkinase and total p38. Peak phosphorylated ERK occurred at 30 min post exposure to H 2 O 2 . Vanadium-induced increases to p-ERK were biphasic, with one peak at 30 min post-exposure and the next at 24 hr post-exposure. Phosphorylated p38 was similarly maximally elevated at 30 min post-treatment
with H 2 O 2 and 24 hrs post treatment with vanadium pentoxide. Thus, this study suggests that HB-EGF expression occurs as a result of ac tivation of the MAPKinase and ERK pathways. NTP (2002) and Ress et al. (2003) conclude d that exposure to vanadium pentoxide
caused alveolar and bronchiolar adenomas and carcinomas in male and female mice and there is some evidence of carcinogenicity in male rats, ba sed on observations of alveolar and bronchiolar neoplasms in groups exposed to vanadium pentoxi de that exceeded historical controls. The body of evidence that has investigated the mode of action (MOA) underlying cancer effects due toexposure of vanadium pentoxide is not as well characterized as mechanisms underlying noncancer fibrotic effects. However, loss of heterozygosity (LOH) and DNA damage has been
documented. Using mouse lung tumor tissues from the NTP (2002) chronic inhalation study from mice exposed to 0, 1, 2 or 4 mg/m 3 vanadium pentoxide, Zhang et. al. (2001b), observed LOH on chromosome 6 (in the region of the K- ras gene) in 17 of 19 vanadium pentoxide-induced mouse tumor samples. Moreover, 29 of 40 (73%) vanadium pentoxide-induced murine adenocarcinomas from the 2002 NTP study had mutations in Kras2 . The Kras2 mutations typically were the result of either a GA Æ AT transition or a GA Æ TA transversion in the second base of codon 12 (Zhang et. al., 2001b). To determine the effect of the K-ras mutations and LOH on activated MAP kinase, Devereux et al (2002), used tissues from the NTP (2002) 2-year carcinogenicity study in female and male B6C3F1 mice to isolate protein from 17 vanadium pentoxide-induced alveolar and bronchiolar car cinomas, one spontaneous carcinoma, and two normal (untreated) lung tissue samples. Levels of total MAP kinase and activated MAP kinase were assessed by probing isolated lung protein for total and phosphorylated MAP kinase with anti-phospho-MAP kinase antibody in all samples. Only qualitative analysis was reported. Total MAP kinase was not different between normal tissue and lung tumor tissue. Activated
MAP kinsase was elevated in five of six tumors that had both LOH and K- ras mutations, and was barely detectable in all seven tumors examined where no K- ras mutations were detected.
Four of five tumors with K- ras mutations but were not positive for LOH had elevated phosphorylated MAP kinase levels. However, these results should be interpreted cautiously asDRAFT - DO NOT CITE OR QUOTE 85 LOH was difficult to detect due to interference from infiltrating lymphocytes. In summary, mouse tumor tissue excised from mice used in the NTP 2002 study showed LOH in the region of the k-ras oncogene location in 17 or 19 samples tested. However, the signaling events, and specifically the role of the MAPkinase pathway, associated with this oncogenic mutation remain
unclear. Pierce et al. (1996) identified proinflammatory cytokines associated with vanadium pentoxide exposure. Pierce et al. (1996) repor ted increased mRNA expression of macrophage inflammatory protein (MIP-2) and keratinocyte-de rived cytokine (KC) in bronchiolar lavage (BAL) fluid in vanadium pentoxide-treated female CD rats compared to controls at early time points (1 hr to 48 hrs). In summary, hyperplastic responses following exposure to vanadium pentoxide are associated with increased expression of various mitogens such as PDGF and HB-EGF.Moreover, PDGF activation may be dependent on inflammatory mediators such as IL-1B. HBEGF expression is dependent on activation of the MAPKinase and ERK signaling pathways. No information is available concerning the rat neoplasm data. Indeed, the marginal increase in lung neoplasms observed in female rats was not st atistically significant. It is not known whether lung neoplasms from male rats exhibit elevated activated MAP kinase or whether the rat tumors
have increased K- ras mutation and LOH on chromosome 6.DRAFT - DO NOT CITE OR QUOTE 86 4.6. SYNTHESIS OF MAJOR NONCANCER EFFECTS

4.6.1. Oral

Limited studies have been published examining the effects following oral exposure to vanadium pentoxide. Table 4-13 presents a summary of the noncancer results for the subchronic and chronic oral studies of vanadium pentoxi de toxicity in experimental animals.

4.6.1.1 Acute

The only acute studies available report oral LD 50 values in rats that range from ~10-137 mg/kg body weight and 64 mg/kg-body weight in mice depending on the source (Yao et. al., 1986b and CICAD 29, 2001). Clinical signs of t oxicity included lethargy, excessive tearing (lacrimation), and diarrhea. Histopathological anal ysis revealed liver necrosis and swelling of renal tubules. No acute dose-response studies are available for any animal species.

Table 4-13: Summary of Noncancer Results of Repeat-Dose Studies for Oral Exposure of Experimental Animals to Vanadium Pentoxide Species (sex) Avg Daily Dose (mg/kg-day) Exposure Duration and Route Response at LOAEL NOAEL(mg/kgday) LOAEL (mg/kg/day) Comments Reference

Wistar Rat (male) 0, 10.5, 16.4, 69.6, 141.0 mg/kg-day (correspond s to 0, 74.5 a

, 116.1 b , 500 and 1000 ppm) 103days in food Decreased erythrocyt
e count 10.5mg/kgday 16.4 mg/kgday Dietary
exposure

was increased at day 35 of study (from 25 to 100 and from 50 to 150 ppm) (See notes below) Mountain et al., 1953

0, 10.5, 16.4, 69.6, 141.0 mg/kg-day (correspond s to 0, 74.5 a

, 116.1 b , 500 and 1000 ppm) 103 days in food Decreased relative liver weight -- 69.6 mg/kgdayDRAFT - DO NOT CITE OR QUOTE 87 Table 4-13: Summary of Noncancer Results of Repeat-Dose Studies for Oral Exposure of Experimental Animals to Vanadium Pentoxide 0, 10.5, 16.4, 69.6, 141.0 mg/kg-day (correspond s to 0, 74.5 a

, 116.1 b , 500 and 1000 ppm) 103 days in food Decreased Hair Cystine 10.5mg/kgday 16.4 mg/kgday

Rat (male) 1.41 and14.1 mg/kgday (correspond s to 17.9 and 179 ppm) 2.5 yrs in food Decreased Hair Cystine 1.41mg/kgday 14.1 mg/kgday Study published in Patty’s Industrial Hygiene and Toxicology 3 rd ed. 1981 Original data not available. Stokinger et. al., 1953

a Represents an average dose based on 25 ppm for 35 days, and 100 ppm for 68 days
b Represents an average dose based on 50 ppm for 35 days and 150 ppm for 68 days.

.DRAFT - DO NOT CITE OR QUOTE 88 4.6.1.2 Subchronic

Data on toxicity following subchronic oral exposure to vanadium pentoxide are extremely limited. No comprehensive studies outlining toxic effects in response to vanadium pentoxide in humans have been reported. The primary noncancer health effects of subchronic oral exposure in animals include changes in relative liver weight, decreased erythrocyte count and hemoglobin counts, and decreased hair cystine content (Mountain et. al., 1953) in rats. Erythrocyte count and hemoglobin decreases are co rrelated and cannot be considered as separate effects. The hematological parameters were measured at low dose exposure levels. Relative liver weights were only measured for control and in the highest dose (69.6 mg/kg-day) group. Changes in hair cystine were measured in all dose groups but the biological significance of this effect is unknown; changes in hair cystine content may serve as a biomarker of exposure, rather
than as an indication of an adverse effect. Fr om these data, and using standard conversions for body weight and food consumption for Wistar rats, the NOAEL of 74.5 ppm was converted to 10.5 mg/kg-day for decreased erythrocyte count.

4.6.1.3 Chronic

No studies reporting chronic exposure to vanadium pentoxide in humans are available in the published literature. Chronic animal studies evaluating effects of oral exposure to vanadium pentoxide are limited. Previously, a study base d on a 2.5 yr dietary exposure to vanadium (Stokinger et. al., 1953 reported in Patty’s Industrial Hygiene and Toxicology, 3 rd Ed., 1981.) utilized hair cystine as the critical effect, since hair cystine was significantly decreased (no details provided regarding the percent decrease or dose-response) in exposed rats and this change may reflect changes to enzymatic pathways. However, comprehensive toxicity endpoints were not evaluated, and the number and strain of rats used was not reported in this 1953 study. Decreased hair cystine may or may not be a biologically relevant effect. Decreased hair cystine may not be related to vanadium exposure, but rather an effect of poor nutritional status, as a result of an aversion to vanadium pentoxide in the feed. EPA has been unable to gain access to the raw data. No additional oral chronic exposure studies in animals were identified in the
literature.

4.6.2. Inhalation

Table 4-20 presents a summary of the noncancer results for the acute subchronic and chronic studies of vanadium pentoxide toxicity in humans and experimental animals. TheDRAFT - DO NOT CITE OR QUOTE 89 identified noncancer health effects following occupational exposure to vanadium pentoxide in humans include respiratory irritation, airway obstruction, chest pain, bronchitis and similar effects; inhalation exposure in animals results in multiple health effects, including pulmonary inflammation, lung and nasal hyplerplasia, pulmonary fibrosis, changes in nervous system structure and function, and reproductive/devel opmental effects (Table 4-14). The most comprehensive animal studies evaluated toxicity following inhalation of vanadium pentoxide in

F344/N rats and B6C3F 1 mice (NTP, 2002); these studies included acute, short-term, subchronic and chronic duration exposures and multiple toxicological endpoints. In those studies, the lung was identified as the most sensitive organ. The reported neurotoxic effects appear after as little as a single one hour exposure to inhaled vanadium pentoxide; although multiple effects were identified that increased in severity with in creased duration of exposure, the studies were conducted using only one exposure level. Thus, a no-effect level for neurotoxicity could not be determined. Major data gaps also exist for studies on developmental toxicity, reproductive toxicity, and immunotoxicity following inhala tion exposure to vanadium pentoxide, although effects have been seen in available studies (some of which used an exposure paradigm similar to that used for neurotoxicity studies).

4.6.2.1 Acute and Short-term

The primary identified noncancer health effect following acute inhalation exposure in humans is respiratory irritation, cough, and mucus formation. A human controlled exposure study (n = 100) performed by Zenz and Berg ( 1967) reported respiratory irritation, cough, and mucus formation in humans exposed to vanadium pentoxide at 0, 0.1, 0.5, and 1.0 mg/m 3 , for 8 hrs. A NOAEL of 0.5 mg/m 3 was established. These effects are confirmed in male monkeys exposed to vanadium pentoxide at 0.5 or 5.0 mg/m 3 for 6 hrs (Knecht et. al., 1985). Air flow restriction, as measured by pulmonary function tests, was reported at 5.0 mg/m 3 (LOAEL); the NOAEL was 0.5 mg/m. 3 Moreover, a WHO-IPCS (CICAD) document (2001) reports a 1-hr inhalation exposure to vanadium pentoxide dust in rats led to an LC 67 of 1.44 mg/L (1440 mg/m 3 ). Clinical signs of toxicity included resp iratory difficulty, increased respiratory tract mucus production, and irritation of the eyes, nose, and throat (WHO-IPCS, 2001, section 11.1.1). Neurotoxicity has also been observed in mi ce following a single inhalation exposure; AvilaCosta et al. (2006) noted significant changes in brain morphology and impairment in memory inDRAFT - DO NOT CITE OR QUOTE 90 male CD-1 mice compared to controls following a single 1 hr exposure to 0.02 M (2.5 mg/m 3 V ) inhaled vanadium pentoxide. Respiratory irritation was observed following short-term inhalation exposure in humans. 100 workers were reportedly exposed to 0.05 to5.3 mg/m 3 vanadium for 10 hr/day, 6 days/week, for 4 weeks (Levy et al., 1984). A LOAEL of 0.05 mg/m 3 was established. However, doseresponse was not systematically measured, there were no controls, and exposure to vanadium pentoxide could not be directly correlated to e ffects. The primary noncancer health effects ofshort term inhalation exposure in animals include increased pulmonary inflammation, and doserelated decreases in body weight and relative lung weight in rodents (NTP, 2002).

4.6.2.2 Subchronic

No comprehensive subchronic studies have evaluated inhalation effects in humans. The primary noncancer health effects of subchronic inhalation exposure in animals include nonneoplastic lung and nasal lesions (NTP, 2002), hematological parameter changes (NTP, 2002), decreased pulmonary function in male monkeys (Knecht et al., 1992), morphological changes to the CNS (Avila-Costa et al., 2004; 2005; 2006), increased platelet counts (GonzalezVillalva et al., 2006), and testicular malformati on (Mussali-Galante et al., 2005; Fortoul et al., 2007). The NTP (2002) study evaluated pulmonary and nasal endpoints in both male and female rats and mice after 3 months exposure to vanadium pentoxide at 0, 1, 2, 4, 8 and 16 mg/m 3

. Lung inflammation, lung hyperplasia and increased relative lung weight were observed at similar low doses in rats and mice. A LOAEL of 2.0 and NOAEL of 1.0 mg/m 3 were established. In addition, rats exhibited bronchiolar exudates, micr ocytic erythrocytosis, lung fibrosis, and nasal lesions. Associated LOAELs and NOAELs were established and are listed in Table 4-20. Body weight loss and increased absolute lung weight were reported in mice and resulted in a LOAEL of 8.0 mg/m 3 and NOAEL of 4.0 mg/m 3 . An increased absolute and relative lung weight LOAEL was set at 4.0 mg/m 3 and 2.0 mg/m 3 for these lesions observed in mice. In summary, rats appear to be more sensitive to inhalation exposure to vanadium pentoxide than mice, based on the occurrence of a wider variety of nonneoplastic lesions throughout the respiratory tract. Knecht et. al. (1992) observed that inhaled vanadium pentoxide leads to adverse lung effects by measuring decreased pulmonary function in male monkeys exposed to vanadium pentoxide at 0.1, 0.5, or 1.1 mg/m 3 for 26 weeks (6 hrs/day, 5 days/week). A LOAEL of 0.5

mgm 3 and a NOAEL of 0.1 mg/m 3 was established. Pulmonary function parameters were reversible and did not repeat following subsequent challenge. Selected hematology parameters (number of erythrocytes and reticulocytes and percentDRAFT - DO NOT CITE OR QUOTE 91 hematocrit) were significantly altered in male and female rats at the highest dose level (NTP, 2002). A LOAEL of 16.0 mg/m 3 and a NOAEL of 8.0 mg/m 3 were established. Other effects such as increased platelet count following 12 weeks and altered testicular morphology following 20 weeks were tested at only one dose (5.13 mg/m 3 ).

4.6.2.3 Chronic

Limited information is available specific to the vanadium pentoxide exposure levels in human studies. Respiratory and other symptoms have been documented among workers employed at facilities producing and processing vanadium pentoxide (Sjoberg, 1951; Sjoberg, 1956; Zenz et al., 1962; Kiviluoto et al., 1979; Kiviluoto, 1980; Kiviluoto et al., 1981a; Musk and Tees, 1982; Irsigler et al., 1999). Similar effects were observed in occupational studies of boilermakers involved in the construction, cleaning and maintenance of oil-fired boilers (Williams, 1952; Sjoberg, 1955; Lees, 1980; Ross, 1983; Levy et al., 1984; Hauser et al., 1995a; 1995b; 2001; Woodin et al., 1998; 1999; 2000; Kim et al., 2004). Noncancer health effects of chronic inhalation exposure in animals included nonneoplastic pulmonary lesions in male and female rats and mice exposed to vanadium pentoxide for 2 yrs at 0, 0.5, 1.0 and 2.0 mg/m 3 and 0, 1.0, 2.0, and 4.0 mg/m 3 , respectively (NTP, 2002). Increased incidence of alveolar and bronchiolar epithelial hyperplasia in male rats, alveolar histiocyte infiltration, laryngeal inflammation, and epiglottis epithelial degeneration, hyperplasia, and squamous metaplasia in male and female rats, and goblet cell hyperplasia in nasal compartments in male rats were reported. Based on these findings, the lowest dose for which a LOAEL could be established in rats was 0.5 mg/m 3 . Additional findings observed in females included increased interstitial fibrosis. No NOAEL was established. In both male and female mice, increased incidences of pulmonary inflammation, and hyperplasia were reporte d. Increased incidences of nasal olfactory and/or respiratory epithelium degeneration, and epiglottis metaplasia were reported 1.0 mg/m 3 (LOAEL) in both male and female mice. The lung fibrosis and nasal inflammation were noted at

≥ 2 mg/m 3 in both sexes. In general, the incidence and severity of pulmonary lesions increased with increasing dose. No treatment-related histopathological lesions were observed in other evaluated tissues. Interstitial fibrosis was significantly elevated in exposed male and female mice compared to controls at 2 and 4 mg/m 3 . No treatment-related findings were observed in
other tissues.Table 4-14: Summary of Noncancer Results of Repeat-DoseStudies for Inhalation Exposure of Vanadium PentoxideSpecies(sex)AvgDailyDose(mg/m3)ExposureDurationResponse at LOAELNOAEL(mg/m3)LOAEL(mg/m3)CommentsReferenceAcute ExposureMonkey(male)0.5 or 5.0mg/m36 hrs (singleexposure)Air-flow restriction (measuredby pulmonary function tests)0.5a5.0Knecht et.al., 1985Human(notreported)0, 0.1,0.5, 1.0mg/m38 hrs (singleexposure)Respiratory irritation, cough,mucus formation0.51.0Small sample size (n = 9)Zenz andBerg, 1967Mouse(male)2.56mg/m31 hr (singleexposure)Impaired performance inspatial memory, decreaseddendritic spines increasedpercentage of necrotic cells inhippocampus---2.56Only one dose testedAvila-Costaet. al., 2006Short-term ExposureRatand mice(female)0, 1, 2, 4mg/m313 days, 6hrs/day, 5days/weekHistiocytic infiltrate and lunginflammation (rats); alveolarand bronchial hyperplasia(mice)--1.0(rats)2.0(mice)No study performed inmale rats or miceNTP, 2002Rat(male)0, 4, 8,16, 32mg/m316 days, 6hr/day, 5day/weekBALFluid analysis: increasedneutrophilia and decreasedmacrophage infiltrationSignificant increase in total cellcount8.0416.08Sample size = 22 rats.Rats exposed to 32 mg/m3were emaciated, 3 malerats died in the 32 mg/m3group,Mouse(male)Mouse(female)0, 2, 4, 8, 16,32 mg/m316 days, , 6hr/day, 5day/weekAbsolute lung weightRelative lung weightDecreased percentage ofmacrophages in BAL Fluid2.0--4.04.02.08.0Sample size = 5 of eachsex. All male mice died at32 mg/m3Sample size 40-60.DRAFT - DO NOT CITE OR QUOTE92Table 4-14: Summary of Noncancer Results of Repeat-DoseStudies for Inhalation Exposure of Vanadium PentoxideIncreased protein leakage intoBAL Fluid and increasedlymphocytes- -4.0Females only.Mouse(male)2.56mg/m330 days (1 hr,2 times aweek for up to4 weeks)Changes in matrixmetalloproteinase levels inmultiple regions of the brain--2.56Signficant increasesoccurred after 1 month ofexposure. Only one doselevel was used.ColinBarenque et.al., 2008Human(notreported)Range of0.05 to5.3mg/m34 weeks, 10hr/day, 6days/weekRespiratory irritation--0.05bDose and response werenot systematicallymeasured, vanadiumpentoxide exposure notaccurateLevy et. al.,1984Subchronic ExposureMouse(male)2.56mg/m38 weeks, 1 hr,2 times/weekMorphological changes to CNS(cilia loss, increased cellsloughing, ependymal celllayer detachment, decreaseddendritic spines inhippocampus and substantianigra, increased cell loss,decreased performance inMorris water maze)---2.56No clinical signs oftoxicity or othertoxicologic endpointswere reportedAvila-Costaet. al., 2004,2005, 2006Mouse(male)2.56mg/m312 week,(1hr/day, 2days/weeks)Increased platelet counts andaltered platelet morphology--2.56GonzalezVillalva et.al., 2006Mouse(male)2.56mg/m312 weeks (1hr 2 times perweek)Decreased gamma globulin intestes, Increased cell death inspermatogonia--2.56Only one dose usedMussaliGalante et.al., 2005,DRAFT - DO NOT CITE OR QUOTE93Table 4-14: Summary of Noncancer Results of Repeat-DoseStudies for Inhalation Exposure of Vanadium PentoxideFortoul et.al., 2007Monkey(male)0.1, 0.5,or 1.1mg/m326 weeks (6hrs per day, 5days perweek)Impaired pulmonary function0.10.5Exposures occurred onalternate days for two setsof animals (some received0.1 or 1.1 mg/m3 onalternate days, while asecond group wasexposed to a constantconcentration (0.5mg/m3) for all 5 days.Pulmonary functionparameters werereversible and did notreappear followingsubsequent challenge. N=26 animals total, n-= 8-9per group.Knecht et.al., 1992Rat (both)andmouse(both)0, 1, 2, 4,8, 16mg/m33 months (6hr/day, 5days/week)Lung hyperplasia1.02.0NTP, 2002Rat (both)0, 1, 2, 4,8, 16mg/m33 months (6hr/day, 5days/week)Lung fibrosis2.04.0Rat(male)Microcytic erythrocytosis, lunginflammation, and , lungepithelial hyperplasia,1.02.0Mouse(male)0, 1, 2, 4,8, 16mg/m33 months (6hr/day, 5days/week)Lung inflammation2.04.0Mouse(female)1.02.0Rat0, 1, 2, 4,3 months (6Nasal hyperplasia and4.08.0NTP, 2002DRAFT - DO NOT CITE OR QUOTE94Table 4-14: Summary of Noncancer Results of Repeat-DoseStudies for Inhalation Exposure of Vanadium Pentoxide(male)8, 16mg/m3hr/day, 5days/week)squamous metaplasia, bodyweight gainRat(female)2.04.0Ratandmouse(male)Rat andMouse(female)0, 1, 2, 4,8, 16mg/m33 months (6hr/day, 5days/week)Body weight loss4.02.08.04.0NTP, 2002Rat andMouse(male)Rat andMouse(female)0, 1, 2, 4,8, 16mg/m30, 1, 2, 4,8, 16mg/m33 months (6hr/day, 5days/week)3 months (6hr/day, 5days/week)Increased Relative lung weightIncreased absolute lung weight2.01.02.04.02.04.0NTP, 2002Rat (both)0, 1, 2, 4,8, 16mg/m33 months (6hr/day, 5days/week)Increased levels ofhematological parameters8.016.0NTP, 2002Chronic ExposureRat(male)0, 0.5, 1,2 mg/m32 yrs (6hr/day, 5days/week)Lung hyperplasia, histiocyticinfiltration, epiglottisdegeneration, , hyperplasia andsquamous metaplasia, chronicinflammation of the larynx, ,goblet cell hyperplasia--0.5No other clinical findingsor altered survivalNTP, 2002Rat(female)Interstititial fibrosis, andhistiocytic infiltration,epiglottis degeneration,hyperplasia, and squamous--0.5DRAFT - DO NOT CITE OR QUOTE95Table 4-14: Summary of Noncancer Results of Repeat-DoseStudies for Inhalation Exposure of Vanadium Pentoxidemetaplasia, chronicinflammation of the larynxRat(female)0, 0.5, 1,2 mg/m32 yrs (6hr/day, 5days/week)Lung inflammation1.02.0No other clinical findingsor altered survivalNTP, 2002Rat(female)0, 0.5, 1,2 mg/m32 yrs (6hr/day, 5days/week)Lung hyperplasia0.51.0No other clinical findingsor altered survivalNTP, 2002Mouse(both)0, 1, 2, 4mg/m32 yrs (6hr/day, 5days/week)Lung inflammation, andhyperplasia--1.0Mice reported as thin,survival significantlydecreased in male mice at4 mg/m3NTP, 2002Rat(female)0, 0.5, 1,2 mg/m32 yrs (6hr/day, 5days/week)Lung squamous metaplasia ofalveolar and bronchiolarepithelium1.02.0No other clinical findingsor altered survivalRat(male)0, 0.5, 1,2 mg/m32 yrs (6hr/day, 5days/week)Lung fibrosis0.51.0No other clinical findingsor altered survivalNTP, 2002Mouse(both)0, 1, 2, 4mg/m32 yrs (6hr/day, 5days/week)Lung fibrosis1.02.0Mice reported as thin,survival significantlydecreased in male mice at4 mg/m3NTP, 2002Rat(male)0, 0.5, 1,2 mg/m32 yrs (6hr/day, 5days/week)Nasal olfactory degenerationand respiratory degeneration0.5No other clinical findingsor altered survivalNTP, 2002Mouse(both)0, 1, 2, 4mg/m32 yrs (6hr/day, 5days/week)Nasal inflammation1.02.0DRAFT - DO NOT CITE OR QUOTE96DRAFT - DO NOT CITE OR QUOTE97Table 4-14: Summary of Noncancer Results of Repeat-DoseStudies for Inhalation Exposure of Vanadium PentoxideMouse(both)0, 1, 2, 4mg/m32 yrs (6hr/day, 5days/week)Nasal olfactory degenerationand respiratory degeneration- -1.0NTP, 2002Mouse(both)0, 1, 2, 4mg/m32 yrs (6hr/day, 5days/week)Epiglottis metaplasia--1.0NTP, 2002aSingle exposures not adjusted for continuous exposurebNot adjusted for continuous exposure because of the highly intermittent exposure protocol (1 hr/day, 2 days/week)DRAFT - DO NOT CITE OR QUOTE 98 4.6.3 Mode of Action Information

There is currently insufficient evidence to establish the mode of action for vanadium pentoxide toxicity. However, the limited data to inform the mechanisms of various non-cancer health effects (pulmonary toxicity, neurotoxic ity and reproductive toxicity) following inhalation exposure to vanadium pentoxide are summarized below.

4.6.3.1 Pulmonary Toxicity

The mechanism of action underlying the formation of pulmonary fibroproliferative lesions has been linked to inflammation, leading to regenerative hyperplastic responses, as evidenced by the presence of mitogens and observed smooth muscle thickening. Oxidative stress has also been implicated. Oxidative stress induced directly or indirectly by vanadium pentoxide may work in combination with vanadium pentoxide-induced inflammatory responses to activate signaling molecules such as ERK 1 or 2 and p38 kinase that lead to induction to growth factors and generation of fibrotic lesions. These potential modes of action are supported by a limited number of mechanistic analyses of inflammation and fibrosis following exposure to vanadium pentoxide. Inflammation. Pierce et. al. (1996) identified proinflammatory cytokines associated with vanadium pentoxide exposure. Oxidative stress has been implicated in the mechanism underlying vanadium pentoxide induced pulmonary injury. All species of vanadium may participate in redox cycling and can generate reactive oxygen species (Carter et. al.1997).
Intracellular and extracellular H 2 O 2 production increases significantly after 18 hr exposure to vanadium pentoxide compared to controls (A ntao-Menezes et. al. 2008). Vanadium pentoxide induced significant IFN- β expression after 18 and 24 hrs but could be inhibited by catalase, an

inhibitor of H 2 O 2 . Oxidative stress was also a suggested mechanism underlying STAT-1 activation, since addition of either an NAPDH inhi bitor or a xanthine oxidase inhibitor ablated STAT-1 activation in cells exposed to vanadium pentoxide (Antao-Menezes et. al. 2008). Moreover, spontaneous hydrogen peroxide generation by fibroblasts was depleted within minutes by addition of vanadium pentoxide (Ingr am et. al. 2003). In addition, vanadium can contribute to inhibition of protein tyrosine phosphatases through the generation of reactive oxygen species (Zhang et. al. 2001). It is possible that hydrogen peroxide and vanadium pentoxide reacted to form peroxovanadium interm ediates and/or ROS that led to the production of HB-EGF in fibroblasts. Oxidative stress could also be inducing ERK and p38 kinase that leadDRAFT - DO NOT CITE OR QUOTE 99 to the production of HB-EGF (Ingram et. al. 2003) or by ROS-mediated competitive inhibition of protein tyrosine phosphatases (Zhang et. al., 2001, Samet et. al., 1999). Gene array analysis and subsequent confirmation by PCR revealed that various oxidative stress genes such as superoxide dismutase (SOD2), pipecolic acid oxidase (PIPOX), and oxidative stress response (OXR1) were altered by vanadium pentoxide e xposure (Ingram et. al., 2007). Thus vanadium pentoxide-mediated production of ROS may lead to oxidative stress and induce downstream signaling events that result in activation of mitogens and proinflammatory cytokines that contribute to the formation of fibroproliferative lesions in the lung.

Fibrosis. Bonner et al. has published numerous studies describing the mechanism underlying the formation of fibroproliferative lesions in response to vanadium pentoxide exposure. Specifically, smooth muscle thickening and increased collagen deposition was observed beneath ciliated epithelial cells in vanadium pentoxide-exposed male Sprague-Dawley rats (Bonner et. al. 1998). Further, proliferating m yofibroblasts were the principle cell type that contributed to the observed fibrosis (Bonner et. al. 2000). Using both in vitro and in vivo models, Rice et. al. (1999) showed that inhibition of autophosphorylation of tyrosine kinases reduced vanadium pentoxide-induced pulmonary fibr osis, thus implicating the tyrosine kinases as key signaling mediators underlying the mechanism of PDGF release and ultimately, fibrinogenesis.Zhang et. al. (2001) and Ingram et. al. (2003) identified a second mitogen, heparinbinding epidermal growth factor-like growth fact or (HB-EGF) as an important mediator of vanadium pentoxide-induced injury in vitro in normal human bronchial epithelial cells (NHBECS). Further, two signaling molecules (ERK and the p38 subunit of MAPkinase) were activated in response to vanadium pentoxide (Ingram et. al. 2003). Gene array analysis confirmed the importance of HB-EGF and IL-8 in vanadium pentoxide-induced lung injury and identified several new candidate genes incl uding growth factors (VEGF, and CTGF),chemokines (CXCL9, CXCL10), oxidative response genes (SOD2, PIPOX, OXRI) and DNAbinding proteins (GAS1, STAT 1) (Ingram et. al., 2007). Bonner et. al. (2003) has also reported that fibroproliferative lesions are resolved and repair initiated in response to vanadium pentoxide. Their work illustrates that mice deficient in prostaglandins (PG) such as the enzyme cycl ooxygenase (COX)-2, are protected from vanadium pentoxide-induced fibroproliferative lesions and indicate the potential important role of cyclooxygenases in mitigating vanadium pentoxide induced injury. Antao-Menezes et. al. (2008) characterized the role of STAT-1 in vanadium pentoxide pulmonary fibrosis. Their work identified Interferon-beta (IFN- β ) as a mediator of vanadium pentoxide-induced STAT-1 activation in normal human lung fibroblasts, and linked STAT-1 activation with STAT-1DRAFT - DO NOT CITE OR QUOTE 100 dependent production of a chemokine, CXCL10, that was identified in the Ingram et. al. (2007) gene array analysis (Figure 4-1). Thus fibroblasts appear to synthesize IFN- β that activates STAT-1. STAT-1 activation simultaneously causes growth arrest and increases levels of CXCL10 which then diminish fibrinogenesis, as a negative feedback loop. In summary, vanadium pentoxide stimulated production of IFN- β activates signaling pathways that lead to the resolution of fibrosis after vanadium pentoxide induced injury.

4.6.3.2 Neurotoxicity

The mechanism(s) underlying nervous system toxicity in response to vanadium pentoxide is not well-characterized. A duration-dependent decrease in the number of immunoreactive TH+ neurons and morphological changes to the blood-brain barrier were observed in response to vanadium pentoxide. It has been suggested that blood-brain barrier disruption may be related to brain region-specific changes in metalloproteinases (MMP-2 and MMP-9) that have been seen following vanadium pentoxide exposure (Colin-B arenque et. al. 2008), however more work is needed to fully characterize these findings.

4.6.3.3 Reproductive Toxicity

Limited studies of vanadium pentoxide have examined reproductive and developmental toxicity. Short-term oral exposures in wean ling rats demonstrated a potential effect on bone growth as measured by serum calcium concentrations and bone alkaline phosphatase activity (Yamaguchi et al. 1989). Reproductive effects due to vanadium pentoxide inhalation exposure included morphological changes to spermatogonia, spermatocytes and Sertoli cells (Fortoul et. al., 2007). Estrous cycle length was increased in female rats but not mice following inhalation exposure at high doses, while decreased spermatozoal motility was observed in male mice but not male rats at high doses (NTP 2002). Excess vanadium pentoxide was found to accumulate in the testes following inhalation exposure (Mussali-Galante et al., 2005). Moreover, significant decreases in gamma globulin were observed in testicular samples exposed to vanadium pentoxide (Mussali-Galante et al., 2005). Decreased gamma-globulin levels may lead to changes in microtubule formation that would impact spermatogenesis. Injection studies described by Wide (1984) demonstrated decreased ossification in fetuses exposed to vanadium pentoxide in utero. Determination of a specific mode of acti on for reproductive toxicity is not possible due to the limited studies examining these effects.DRAFT - DO NOT CITE OR QUOTE 101 4.7. EVALUATION OF CARCINOGENICITY

4.7.1 Summary of Overall Weight of Evidence

Under the U.S. EPA Guidelines for Carcinogenic Risk Assessment (U.S. EPA, 2005a) vanadium pentoxide is “likely to be carcinogeni c to humans” by the inhalation route of exposure based on inhalation studies in male rats and male and female mice (NTP 2002). No studies evaluating the carcinogenic potential in humans exposed to inhaled vanadium pentoxide were identified. No studies suitable for evaluation of the oral carcinogenic potential for vanadium pentoxide were located in the published literature. There was clear evidence of carcinogenesis in both male and female mice based on the statistically significant and dose-related increased
incidence of alveolar/bronchiolar tumors (NTP, 2002; Ress et al., 2003). There was some evidence of carcinogenic activity in male rats and equivocal evidence in female rats (NTP, 2002). Although the incidence of bronchiolar tumors in vanadium-pentoxide-treated rats was not significantly increased compared to control, tumor incidence was elevated relative to historical control in most treatment groups in male rats and some treatment groups in female rats (Table 4- 8). No other tumor type was significantly increased in either rats or mice in this study. These results are supported by a recent study by Rondini et al (2010) also showed increased lung tumors in male mice (A/J, BALB/C) following exposure to vanadium pentoxide along with an initiator (MCA).

U.S. EPA’s Guidelines for Carcinogenic Risk Assessment (U.S. EPA, 2005a) indicate for that for tumors occurring at a site other than the initial point of contact, the weight of evidence for carcinogenic potential may apply to all routes of exposure that have not been adequately tested at sufficient doses. An exception occurs when there is convincing information (e.g., toxicokinetic data) that absorption does not occur by other routes. Information available on the carcinogenic effects of V 2 O 5 via the inhalation route is limited to examination of the respiratory tumors. Information on the carcinogenic effects of V 2 O 5 via the oral and dermal routes in humans or animals is absent. Based on the observance of only portal-of-entry tumors (respiratory tumors) following inhalation exposure, and in the absence of information to establish a mode of action, this cancer descriptor applies only to th e inhalation route of exposure. Therefore, V 2 O 5 is “likely to be carcinogenic to humans” by the inhalation route of exposure, and the database has “inadequate information to assess carcinogenic potential” via the oral or dermal route.

4.7.2. Synthesis of Human, Animal, and Other Supporting Evidence

Few studies are available that assess the carcinogenic potential of vanadium pentoxide.DRAFT - DO NOT CITE OR QUOTE 102 Although both epidemiology and laboratory animal studies show similar respiratory tract toxicity, there are currently no epidemiology studies available in the published literature examining carcinogenicity. Only two published laboratory animal studies provide evidence for the carcinogenic potential of vanadium pentoxide (NTP 2002; Rondini et al., 2010). Vanadium pentoxide has been shown to induce pulmonary tumors following inhalation exposure in a study performed by NTP (NTP 2002). F344 rats (50/sex/group) and B6C3F1 mice (50/sex/group) were exposed to vanadium pentoxide particles for six hours a day, fi ve days per week for two years. Rats were exposed to 0.5, 1.0, or 2.0 mg/m 3 of vanadium pentoxide, with mice exposed to 1.0, 2.0 or 4.0 mg/m 3 of vanadium pentoxide. Lung tumors were observed in male and female rats, but incidence exceeded historical controls in male rats only (Table 4-15). Both male and female mice showed statistically significant increases in lung tumors as compared to controls (p ≤ 0.01). These increases were observed in both sexes at all doses, with 50% of the male mice in the highest exposure group dying before the end of the study. Survival rates in all other exposed groups for both rats and mice was not significantly different from controls. Both rats and mice showed other lesions of the respiratory tract , including inflammation, fibrosis and hyperplasia (Tables 4-7, 4-9). Decreased body weight gain wa s observed as early as 3 months post-exposure in the high dose groups of all exposed animals. Along with the NTP study (2002), a recent study by Rondini et al (2010) examined tumor promotion of vanadium pentoxide in three different mice strains. Lung tumors were
observed in two of three mouse strains 20 weeks after MCA tumor initiation, followed by exposure (only males exposed) to V 2 O 5 (4 mg/kg, 5 times weekly) (Table 4-11). A/J and BALB/C male mice showed increases in lung tumors following exposure to both MCA (initiator)
and V 2 O 5 , but not V 2 O 5 alone, suggesting V 2 O 5 works as a tumor promoter.

4.7.3. Mode of Action Information

The U.S. EPA (2005a) Guidelines for Carcinogen Risk Assessment defines mode of action (MOA) 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 action include mutagenic, mitogenic, anti-apoptotic (inhibition of programmed cell death), and cytotoxic mechanisms with reparative cell proliferation and immunologic suppression. There is insufficient information to establish a carcinogenic mode of action for bronchiolar tumors observed in animals following inhalation exposure to vanadium pentoxide.DRAFT - DO NOT CITE OR QUOTE 103 4.8. SUSCEPTIBLE POPULATIONS AND LIFE STAGES

4.8.1. Possible Childhood Susceptibility

There are no reports of childhood susceptibility due to exposure to vanadium pentoxide. There are also no reports indicating increased susceptibility in developings, however the data on developmental effects of vanadium pentoxide are very limited (see discussion of data gaps below). Young rats that consumed vanadium in the drinking water and feed were found to have higher tissue vanadium levels 21 days after birth than they did 115 days after birth (Edel et al. 1984). The data suggest that there is a higher absorption of vanadium in these young animals due to a greater nonselective permeability of the undeveloped intestinal barrier. Thus, age of the rodents appears to play an important role in the absorption of vanadium in the gastrointestinal tract. Mravcova et. al (1993) assessed the extent of vanadium pentoxide accumulation in the bones of rats following 6 month exposure. Vanadium accumulated in the epiphyseal cartilage of the tibia in rats with significantly higher concentra tions of vanadium in the tibia and incisors of weanling rats compared to adults. However, no dose response data for these endpoints was reported.

4.8.2. Possible Gender Differences

Reports of gender differences are limited to the carcinogenicity data from NTP, 2002 where clear evidence of carcinogenicity was reported in male and female mice exposed to vanadium pentoxide, and some evidence of carcinogenicity was reported in male rats, based on observations of alveolar and bronchiolar neoplasms that exceeded historical controls in groups exposed to vanadium pentoxide. The number of neoplasms in female rats was not higher than
that observed in historical controls and thus , a relationship between neoplasms and vanadium pentoxide could not be established in female rats (NTP, 2002 and Ress, 2002). Thus, increased tumor incidence in rats is equivocal overall, but the lack of any increase in females may suggest a gender-related increase in susceptibility in males. It is unknown whether this observed difference is applicable to humans. There are no reported differences for response to vanadium pentoxide between genders for either animals or humans for any non-cancer endpoint.

4.8.3. Other Susceptible Populations

No data exists on the role of genetic polymorphisms in differentially susceptible human populations in response to vanadium pentoxide exposure. In mice, research suggests that all strains have an inflammatory response to vanadium pentoxide exposure, but the severity ofDRAFT - DO NOT CITE OR QUOTE 104 inflammation varies greatly from strain to strain. Such variability in response suggests that a genetic component may contribute to the sever ity of vanadium pentoxide-induced pulmonary inflammation and tumorigenicity in mice (Rondi ni et al., 2010). The NTP study (2002) used B6C3F1 mice in all of their exposure protocols for 16-day, 3 month, and 2 yr exposure studies. Pulmonary fibrosis was observed in this hybrid strain. Kyono et. al. (1999) used a rat model of acute bronchiolitis (Br) to investigate whether animals with pre-existing lung conditions would be differentially susceptible to inhaled vanadium pentoxide. Compared to exposed normal rats, Br rats exhibited delayed recovery from pre-existing lesions and exacerbated lung inflammation. Sensitive rats also showed reductions in the deposition and clearance rates of inhaled particles. Rondini et al (2010) examined the effect of exposure to vanadium pentoxide in three mouse strains of varying susceptibility to l ung cancer (A/J, BALB/C and C57BL/6J) in an initiation/promotion model (full study description in Section 4.2.2.2). Three mouse strains were used to further understand potential susceptibility to these effects. These particular mouse strains were selected because of their known differential susceptibility to chronic pulmonary inflammation and carcinogenesis: A/J mice are sensitive, BALB/C are intermediate and C57BL/6J are resistant. Statistically significan t lung tumor increases were observed in A/J and BALB/C mice as compared to the MCA-treated control (p ≤ 0.05; Table 4 – 11). Differences were also observed between strains, with A/J mice showing increased tumorigenicity in response to vanadium pentoxide. In the absence of MCA, V 2 O 5 was not sufficient to initiate tumorigenesis in this study. C57BL/6J had no tumors following exposure (data not shown). Overall, the differential inflammatory responses observed in the three strains of mice appear to positively correlate with increased levels of chemokines, such as keratinocyte-derived chemokine (KC) and monocyte chemotactic protein-1 (MCP-1), and increased binding of
transcriptional factors NF κ B and AP-1 (c-Fos), and sustained activation of MAP kinases (MAPKs) and extracellular signal-regulated kinase s 1 and 2 (ERK 1/2) suggesting inflammation as a major response in mice. Turpin et al. (2010) examined pulmonary inflammation and fibrosis following intranasal aspiration exposure to vana dium pentoxide with and without respiratory syncytial virus (RSV) exposure (full study description in Section 4.4.1.2). In this study, vanadium pentoxide exposure also caused a significant increase in cell proliferation in the airways and lung parenchyma, lung mRNAs for TGF- β -1, CTGF, PDGF-C, Col1A2, and

mRNAs for IFN- α and - β and IFN-inducible chemokines CXCL9 and CXCL10 compared toDRAFT - DO NOT CITE OR QUOTE 105 controls. Pre- or post-treatment with RSV caused a significant reduction in the all mRNAs. Together, results from this study showed that vanadium pentoxide induces inflammatory and fibrogenic response in mouse lung and these effects were suppressed by RSV infection.DRAFT - DO NOT CITE OR QUOTE 106 5. DOSE-RESPONSE ANALYSIS

5.1. ORAL REFERENCE DOSE (RfD)

Only two studies exist on human oral exposure to vanadium pentoxide, and neither examined health effects related to this exposure. Two studies measured cystine levels in hair and fingernails and vanadium levels in urine (K ucera et al., 1994) or blood (Kucera et al., 1992) following oral exposure to vanadium pentoxide. Ku cera et al. (1994) detected vanadium in urine
of workers from a Czechoslovakian vanadium pentoxide production plant, however, it is expected that these urinary levels of vanadium resulted from multiple exposure routes. Kucera et al. (1992) measured vanadium in the hair and blood of children and the blood of adults potentially exposed through ingestion of vanadi um-contaminated drinking water (concentration range: 0.001-0.1 mg/L). Vanadium concentrations in water supply wells exceeded the maximum permissible limit in drinking water (0.01 mg/L) with the contamination continuing over 2 years. Significantly increased vanadium concentrations were found in blood of exposed children compared to unexposed children and adults, whereas vanadium levels in hair of exposed children (adults not measured) were no different from the control group. No exposure-response relationship could be determined for either endpoint, and changes in hair cystine levels have not been correlated with adverse health effects. Additional studies of workers occupationally exposed to vanadium pentoxide exist, although it is presumed that these workers were exposed by multiple routes, inhalation was likely the prim ary route of exposure (Sjöberg, 1951; Sjöberg, 1956; Zenz et al., 1962; Kiviluoto et al., 1979; Kiviluoto, 1980; Kiviluoto et al., 1981b; Musk and Tees, 1982; Irsigler et al., 1999). Mountain et al. (1953) is the only published, peer-reviewed study that evaluated the effects of subchronic oral vanadium pentoxide expos ure in laboratory animals. Male Wistar rats (5/group; 200 – 350 gm bw) were exposed to vanadium pentoxide for 103 days using average daily doses of 0, 10.5, 16.4, 69.6, 141.0 mg/kg-day vanadi um pentoxide in feed. Changes were observed in body weight gain, erythrocyte count, hemoglobin, and cystine content of hair. Other endpoints of toxicity were not reported. The study authors reported increased body weight gain in the low exposure groups (0 –16.4 mg/kg-day) and decreased body weight gain in the highest exposure group (141.0 mg/kgday). However, these data were not accompanied by any explanation, statistical analysis, or measures of variability (SE or SD). Cystine content of hair was significantly decreased compared to control at doses ≥ 16.4 mg/kg-day vanadium pentoxide. Alterations in hair cystine levels can be associated with dietary changes or altered health status (Kleinfeld et al. 1961). TheDRAFT - DO NOT CITE OR QUOTE 107 authors speculated that vanadium may have inhibited enzymes, such as sulfur transferases, that decreased the availability of cystine for hair growth. However, data to support a relationship between decreased hair cystine levels and adverse health outcomes are not available in the published literature. Thus, the biological significance of decreased hair cystine content is
unclear.

Table 5–1. Hematological results of oral vanadium pentoxide exposure in rats (Mountain et al. 1953). Control 10.5 mg/kg-day 16.4 mg/kg-day Red Cell Count (M/mm 3 ) Start 8.0 7.8 8.0 Finish (103 days) 7.7 6.8 6.3 Percent change between start and finish of expt (%) 3.8 12.8 21.3

Hemoglobin, % Start 15.6 15.2 15.3 Finish (103 days) 15.0 14.5 13.7 Percent change between start and finish of expt (%) 3.9 4.6 10.5

Relative liver weight increases and decreases in erythrocytes and hemoglobin levels were also reported by Mountain et al. (1953). Mean rela tive liver weights, reported as a ratio of liver weight to body weight, were statistically signifi cantly elevated above controls at 69.6 mg/kg-day (i.e., 3.51 ± 0.06 versus 3.86 ± 0.07). Liver weight data were not reported for other doses. Apparent dose-related decreases in RBC count (21.3%) and hemoglobin concentration (10.5%) were observed in the 10.5 and 16.4 mg/kg-day vanadium pentoxide dose groups compared to controls (Table 5-1). No statistical analysis was performed by the study authors on these data, and no measure of variance was reported, precluding independent statistical analysis. The effects on RBC count and hemoglobin concentration observed in this oral study are consistent with the hematological effects observed in a 3-month inhalation study of vanadium pentoxide in rats (NTP, 2002). Decreases in mean cell volume (MCV) and mean cell hemoglobin (MCH) accompanied by erythrocyte microcytosis were suggestive of altered iron metabolism and heme/hemoglobin production following vanadium pe ntoxide inhalation exposure. Based on the dose-related decreases in RBC count in the oral study supported by the hematological effects observed in the inhalation study, decreased RBC c ount was selected as the critical effect. Although a dose-related decrease in RBC count was seen at 10.5 and 16.4 mg/kg-day vanadium pentoxide (12.8% and 21.3%, respectivel y) in the Mountain et al. (1953) study, the magnitude of this change is considered biologically significant only at 16.4 mg/kg-day, whichDRAFT - DO NOT CITE OR QUOTE 108 EPA thus identified as the LOAEL. The lowest dose of 10.5 mg/kg-day was then identified by

EPA as a NOAEL.

5.1.1 Methods of Analysis

The most sensitive endpoint following oral exposure to vanadium pentoxide is decreased
RBC count with a NOAEL of 10.5 mg/kg-day vanadium pentoxide (Mountain et. al., 1953). Because the study authors reported the decrease in RBC count as a mean with no measure of variability (SE or SD), this continuous endpoint could not be subjected to benchmark dose (BMD) modeling. Therefore, the NOAEL of 10.5 mg/kg-day is identified as the point of departure (POD) for use in deriving the RfD for vanadium pentoxide. The Agency endorses a hierarchy of a pproaches to derive human equivalent oral exposures from data from laboratory animal species, with the preferred approach being physiologically based toxicokinetic modeling (U.S . EPA, 2011). Other approaches may include using some chemical-specific information, without a complete physiologically based toxicokinetic model. In lieu of data to support either of these types of approaches, body weight scaling to the 3⁄4 power (i.e., BW 3/4 ) is endorsed as a general default to extrapolate toxicologically equivalent doses of orally administered agents from all laboratory animals to humans for the purpose of deriving an oral Reference Dose (RfD). In general, the use of BW 3/4 scaling is considered appropriate for deriving an oral RfD when the observed effects are associated with the parent compound or a stable metabolite, not related to a portal-of-entry effect, and not related to developmental endpoints. Use of BW 3/4 scaling in combination with consideration of a reduced interspecies uncertainty factor, UF A , is recommended as the Agency default approach. No physiologically based toxicokinetic modeling information exists for vanadium pentoxide. The selected critical effect (decreased red blood cell counts) is associated with the parent compound, is not considered a portal-of-entry effect and was observed in mature rats. Therefore, consistent with U.S. EPA guidance (U.S. EPA, 2011), the POD identified in rats (i.e., 10.5 mg/kg-day) is converted to a human equivalent dose (HED) through application of a dosimetric adjustment factor (DAF) derived as follows:

DAF = (BW a 1/4 / BW h 1/4 ),

where DAF = dosimetric adjustment factor

BW a = animal body weight

BW h = human body weightDRAFT - DO NOT CITE OR QUOTE 109 Using a BW a of 0.25 kg for rats and a BW h of 70 kg for humans, the resulting DAF is 0.244. Applying this DAF to the POD identified in rats yields a HED of 2.56 mg/kg-day as
follows:

HED = Laboratory animal dose (mg/kg-day) x DAF = 10.5 mg/kg-day x 0.244 = 2.56 mg/kg-day

Table 5-2 Human equivalence dose conversion by BW 3/4 for RfD derivation.
Species BW a 1/4 / BW h 1/4 = DAF HED UFs RfD (mg/kg-day)

Rat (0.25kg) 0.25kg 1/4 ÷ 70kg 1/4 = 0.244 10.5 mg/kgd x 0.244 =2.56 mg/kgd Total = 3000 UF A = 3 UF H = 10 UF S = 10 UF D = 10 2.56 ÷ 3000 = 8.5 x 10 -4

a. Using the BW a 0.25 kg for rats and BW h 70 kg for humans, and multiplying it by the NOAEL of 10.5 mg/kg-d from Mountain et al. (1953).

5.1.2 RfD Derivation – Including Applic ation of Uncertainty Factors (UF)

The NOAEL of 10.5 mg/kg-day for decreased RBC count in male rats (Mountain et. al., 1953) was used as the POD to derive an RfD. The application of uncertainty factors to the POD, converted to a HED, results in a composite uncertainty factor of 3,000 covering four areas of uncertainty: 3 for interspecies extrapolation from animals to humans (UF A ); 10 for human intraspecies variability (UF H ), 10 for extrapolation from a subchronic to a chronic study (UF S ), and 10 for database insufficiencies (UF D ). No UF is needed for extrapolation from a LOAEL to NOAEL because a NOAEL was used to identify the POD (US EPA, 2002). An interspecies uncertainty factor, UF A , of 3 (10 0.5 = 3.16, rounded to 3) was applied to account for uncertainty in extrapolating from laboratory animals to humans (i.e., interspecies variability). This interspecies uncertainty factor is comprised of two separate but equal sources of uncertainty, toxicokinetic and toxicodynamic differences between animals and humans. For vanadium pentoxide, toxicokinetic uncertainty was accounted for by calculation of a human equivalent dose (HED) through application of a dos imetric adjustment factor (DAF) as outlined in the U.S. EPA guidance on the use of BW 3/4 scaling in the derivation of the oral RfD (U.S.DRAFT - DO NOT CITE OR QUOTE 110 EPA, 2011). As the toxicokinetic differences are thus accounted for, only the toxicodynamic uncertainty remains, and an UF A of 3 is retained to account for this residual uncertainty. An intraspecies uncertainty factor, UF H , of 10 for intraspecies differences (human variability) was used to account for potentially susceptible individuals in the absence of quantitative information on the variability of response in humans.

An UF S of 10 for extrapolation from a subchronic to chronic study is applied because a subchronic study was used for the POD.

An UF L for LOAEL to NOAEL extrapolation was not used because a NOAEL was identified from the principal study and used as the POD.

An UF D of 10 was used for database insufficiencies due to the lack of a developmental toxicity study and a multi-generation reproductive study for V 2 O 5 by the oral route. Studies using alternate routes of exposure (intraper itoneal) have indicated adverse reproductive and developmental effects in response to vanadium pentoxide, including statistically significant increases in seminal vesicle, thymus and submandibular gland weights in male mice and body weight, thymus, submandibular gland, and liver weights in female mice (Altamirano et al., 1991) as well as reduced fertility (Altamirano-Lozano et al., 1996). No physiologically based toxicokinetic models are available for conducting a route-to-route extrapolation.

Therefore, the RfD for vanadium pentoxide is calculated as follows:

RfD = NOAEL HED ÷ UF = 2.56 mg/kg-day ÷ 3000 = 0.000085 mg/kg-day or 9E-04 mg/kg-day

Note: Because vanadium exists in several different valence states, all of which are not equivalent toxicologically (WHO-IPCS, 2001), the values generated here apply to vanadium pentoxide and should not be applied to other vanadium compounds.

5.1.2. Previous RfD Assessment

U.S. EPA previously derived a chronic reference dose (RfD) of 9 x 10 -3 mg/kg-day for vanadium pentoxide based on a 2.5-year dietar y NOAEL of 0.89 mg/kg-day vanadium pentoxide for decreased hair cystine content that was entered on the IRIS database in 1987 (U.S. EPA, 1987; Stokinger et. al., 1953 reported in Patty’s Industrial Hygiene and Toxicology, 3 rd Ed., 1981). Limited details were provided, and this study is not available for analysis. The rats (number and species unspecified) were exposed to dietary levels of vanadium pentoxide (0.89 or 8.9 mg/kg-day for 2.5 yrs) and assessed for growth rate, survival, and hair cystine content. OfDRAFT - DO NOT CITE OR QUOTE 111 the endpoints reported in the book chapter, decreased hair cystine was selected as the critical effect with a NOAEL of 0.89 mg/kg-day. Upon furthe r analysis of this study as described in Section 5.1.1, it was determined to be inadequate for use in deriving an RfD.

5.2. INHALATION REFERENCE CONCENTRATION (RfC)

5.2.1 Choice of Principal Study and Critical Effect with Rationale and Justification

The available human and animal data identify the respiratory tract as the primary target for chronic exposure to vanadium pentoxide. Irr itation of the upper and lower respiratory tract has been reported in several acute, subchronic and chronic occupational and case studies of workers exposed to vanadium pentoxide in fuel-oil ash and vanadium dust (Woodin et al., 2000; Irsigler et al., 1999; Woodin et al., 1999; Hauser et al., 1995; Levy et al., 1984; Musk and Tees, 1982; Kiviluoto, 1980; Lees, 1980; Kiviluoto et al., 1979; Zenz et al., 1962; Sjöberg, 1955; Vintinner et al., 1955; Williams, 1952, Lewis, 1959). These results are supported by effects observed in rodents, including inflammation, hyperplasia, and fibrosis. Although subchronic occupational exposure studies provide supportive evidence for the respiratory tract as a target for inhaled vanadium pentoxide, studies often failed to quantify vanadium pentoxide concentration as a constituent in an inhaled mixture. Thus, the available occupational exposure studies are not
suitable as the basis for the RfC. The toxicity database for inhalation exposure in laboratory animals includes two chronic studies (Knecht et al., 1992; NTP, 2002). A 6 month cynomologus monkey study (Knecht et al., 1992) exposed monkeys (n=8) to vanadium pentoxide aerosol (0.5 or 3.0 mg/m 3 ) for 6 hrs/day, 5 days/week for 26 weeks. This study was not selected as the principal study due to limitations in the number of test animals, study duration and number of doses used. Results of the NTP (2002) study in rats and mice provide evidence of toxicity to the upper and lower respiratory tract, including incr eased lung weight, inflammation, histological lesions, and decreased pulmonary function followi ng a 3-month inhalation exposure to vanadium pentoxide. Pulmonary lesions were observed in F344/N rats (10 male and 10 female) and B6C3F1 mice (10 male and 10 female) exposed for 3 months, with NOAEL and LOAEL values of 1 and 2 mg/m 3 , respectively, for minimal to mild epithelial hyperplasia (Tables 4-4 and 4-6). Other observed endpoints include increased lung br onchiolar exudate, lung fibrosis, altered nasal morphology, and increased nasal inflammation observed in rats. Significant exposure-related decreases in pulmonary function were also observed in male and female rats, with a LOAEL of 4 mg/m 3 (pulmonary function not assessed in mice) (NTP, 2002). Other effects identified fromDRAFT - DO NOT CITE OR QUOTE 112 the NTP (2002) 3 month inhalation study are the erythrocytosis , body weight and lung weight changes. Erythrocytosis was observed in male and female rats exposed to inhaled vanadium pentoxide for 3 months; hematological endpoints were not examined in mice (NTP, 2002). The NOAELs for mild erythrocytosis were 1 mg/m 3 in male rats and 2 mg/m 3 in female rats. The erythrocytosis is considered to be a secondary effect arising from the primary lung lesions and
was not considered as a critical effect. Abnormal breathing, emaciation, and lethargy were observed in male and female rats exposed to concentrations of 8 mg/m 3 or higher (NOAEL 4 mg/m 3 , LOAEL 8 mg/m 3 ) in response to vanadium pentoxide after 3 months of exposure. Final body weight was statistically significantly decreased as compared to the respective controls at 16 mg/m 3 in male rats (60%) and male mice (10%); 8 mg/m 3 in male rats (10%) and mice (6%); 16 mg/m 3 in female rats (30%) and female mice (12%); 8 mg/m 3 in female mice (10%); and 4 mg/m 3 in female mice (11%). Thus, a NOAEL of 4 mg/m 3 and a LOAEL of 8 mg/m 3 suggested for body weight loss and associated behavioral changes in rats and male mice. Relative lung weights were significantly increased in male and female rats (4 mg/m 3 ) following 3-months inhalation exposure to vanadium pentoxide. Similarly, relativ e lung weights were significantly increased in male and female mice starting at 4 mg/m 3

. Other studies identified morphological changes to the central nervous system (CNS) in male mice exposed to vanadium pentoxide for up to 8 weeks (Avila-Costa et al., 2004, 2005). Avila-Costa et al. (2004, 2005) also reported morphological changes in the substantia nigra region of the basal ganglia and the blood-brain barrier in male mice exposed to 1.4 mgV/m 3 for up to 8 weeks; effects on central nervous syst em function or other comprehensive endpoints were not reported. Using the same dose regimen (1.4 mgV/m 3 two times a week for up to onemonth) Avila Costa et. al. (2006) reported a time-dependent loss of dendritic spines, necroticlike cell death, and morphological changes to the hippocampal region and that these changes may be related to the associated spatial memory loss. Morphological changes in the CNS reported by Avila-Costa et al. (2004; 2005; 2006) were not considered as the critical effect due to lack of information on the exposure-response relationship for morphological changes to the central nervous system, as only one exposure level was tested. Moreover, the lung appears to be the most sensitive target to inhalation exposure to vanadium pentoxide. Results of the NTP (2002) study in rats and mice provide evidence of toxicity to the upper and lower respiratory tract, including incr eased lung weight, inflammation, histological lesions, and decreased pulmonary function following 3-month inhalation exposure to vanadium pentoxide. Though body weights and a complete necropsy and histological analysis were performed, adverse effects in other target organs were not identified in the chronic exposureDRAFT - DO NOT CITE OR QUOTE 113 study. The series of effects described in th e NTP (2002) bioassay and supporting studies from the available inhalation database reflect a dose-response with increase in severity with vanadium pentoxide concentration, and a progression of respiratory effects (infiltration of macrophages, inflammation, hyperplastic response, fibrosis). Based on the dose-response and temporal relationship of effects throughout the course of the 2-year study, and the concordance with effects seen in humans, the NTP (2002) study is selected as the critical study. The 2-year exposure study in F344/N rats and B6C3F1 mice (50/sex/group) (NTP, 2002) examined the effects of chronic inhalation (0, 0.5, 1 or 2mg/m 3 in rats and 0, 1, 2, 4 mg/m 3 in mice for 6hrs/d, 5d/wk, 104 weeks) of vanadium pentoxide. Th e NTP (2002) observed nonneoplastic lesions of the respiratory tract in male and female rats and mice. Numerous lesions of the upper and lower respiratory tract were observed in male and female rats and mice at the lowest exposure concentrations tested. Specifically, epiglottis epithelial hyperplasia in male and female rats (LOAELs of 0.5 mg/m 3 ), and alveolar and bronchiolar hyperplasia was observed in male rats (LOAELs of 0.5 mg/m 3 ) and in male and female mice (1 mg/m 3 ); NOAELs were not identified (Tables 4-7 and 4-9). Other histological lesions observed at a LOAEL of 0.5 mg/m 3 include multiple lesions in the nose and larynx of male and female rats including epiglottis epithelial degeneration, hyperplasia, and squamous metaplasia in both sexes and nasal goblet cell hyperplasia in males. In male and female mice, histological lesions also occurred in the larynx and nasal tissues at 1 mg/m 3 ; it should be noted that 0.5 mg/m 3 was not used as a dose in mice. In mice exposed to 1 mg/m 3 , lesions were observed in the nose, bronchioles and lung of male mice and the nose, larynx, bronchioles and lung of female mice. Chronic, active inflammation and interstitial fibrosis was observed in male rats at a LOAEL of 1 mg/m 3 (NOAEL 0.5 mg/m 3 ). The NTP (2002) chronic study is well-designed, well-controlled, and well-reported. Numerous toxicological endpoints were assessed. As with the subchronic exposure studies, results of this two-year inhalation study identify the upper and lower respiratory tract as the target for chronic inhalation exposure to vanadium pentoxide. The nasal and laryngeal lesions observed by NTP (2002) are among the most sensitive effects observed and were observed in both sexes of rats and mice (Table 5-3). Irritation of the upper and lower respiratory tract has been reported in several occupational and case studies of workers exposed for days or weeks to vanadium pentoxide in fuel-oil ash and vanadium dust (Sjöberg, 1951; Sjöberg, 1956; Zenz et al., 1962; Kiviluoto et al., 1979; Kiviluoto, 1980; Kiviluoto et al., 1981a, b; Musk and Tees, 1982; Irsigler et al., 1999). Therefore, NTP (2002) was selected as the principal study, with effects on the upper and lower respiratory tract selected as the critical effects.

5.2.2 Methods of AnalysisDRAFT - DO NOT CITE OR QUOTE 114 To analyze the concentration response effect of vanadium pentoxide, the reported concentrations of vanadium pentoxide were conve rted to human equivalent concentrations prior to any modeling (Table 5.4, Appendix B). The Me thods for Derivation of Inhalation Reference Concentrations and Application of Inhalation Dosimetry (hereafter referred to as the RfC Methodology) recommends converting the POD [ADJ] to a human equivalent concentration (HEC) (U.S. EPA, 1994b). The RfC Methodology separates gases into three categories based on their water solubility and reactivity with tissues in the respiratory tract, and recommends the use of regional deposited dose ratios (RDDRs) for converting to HECs for particles (e.g., vanadium pentoxide). RDDRs were calculated for rats w ith the RDDR computer program (U.S. EPA, 1994) using mean body weights for male and female rats and the average particle size MMAD±GSD of 1.24±1.89 for rats as reported by NT P (2002). Human equivalent concentration (HEC) conversions (in mg vanadium pentoxide/m 3 ) were calculated by multiplying Conc [ADJ] by the pulmonary RDDR for lesions in the lung, or the extrathoracic RDDR for lesions in the larynx and nose and are summarized in Table 5-4. Duration-adjusted exposure concentrations (Conc [ADJ] ) of 0.09, 0.18 and 0.36 mg/m 3 , corresponding to nominal exposure concentrations of 0.5, 1 and 2 mg/m 3 , were calculated to account for continuous ambient exposure:

Conc [ADJ] = Conc × 6/24 × 5/7

HECs were calculated by multiplying Conc [ADJ] by the extrathoracic RDDR for lesions of the larynx in female rats (Appendix B, Table B-5).

Table 5-3. Selected Nonneoplastic Lesions of the R espiratory System in Rats Exposed to Particulate Aerosols of Vanadium Pentoxide for 2 Years (NTP, 2002)

Lesion Type and Location a Exposure Group Control (% incidence) 0.5 mg/m 3 (% incidence) 1 mg/m 3 (% incidence) 2 mg/m 3 (% incidence) Male Rats Percent survival (%) 40 58 52 54 Lung Number of animals examined 50 49 48 50 Alveolar epithelium, hyperplasia 7 (14) 24 b (49) 34 b (71) 49 b (98) Bronchiole epithelium, hyperplasia 3 (6) 17 b (35) 31 b (65) 48 b (96)

Inflammation, chronic active 5 (10) 8 (16) 24 b (50) 42 b (84)DRAFT - DO NOT CITE OR QUOTE 115 Table 5-3. Selected Nonneoplastic Lesions of the R espiratory System in Rats Exposed to Particulate Aerosols of Vanadium Pentoxide for 2 Years (NTP, 2002)

Lesion Type and Location a Exposure Group Control (% incidence) 0.5 mg/m 3 (% incidence) 1 mg/m 3 (% incidence) 2 mg/m 3 (% incidence) Interstitial, fibrosis 7 (14) 7 (14) 16 c (33) 38 b (76) Alveolus, histiocyte infiltration 22 (44) 40 b (82) 45 b (94) 50 b (100) Larynx Number of animals examined 49 50 50 50 Inflammation, chronic 3 (6) 20 b (40) 17 b (34) 28 b (56) Epiglottis epithelium, degeneration 0 (0) 22 b (44) 23 b (46) 33 b (66)

Epiglottis epithelium, hyperplasia 0 (0) 18 b (36) 34 b (68) 32 b (64) Epiglottis epithelium, squamous metaplasia 0 (0) 9 b (18) 16 b (32) 19 b (38)

Nose Number of animals examined 49 50 49 48 Goblet cell, hyperplasia 4 (8) 15 b (30) 12 c (24) 17 b (35) Female Rats Percent survival (%) 28 40 34 30 Lung Number of animals examined 49 49 50 50 Alveolar epithelium, hyperplasia 4 (8) 8 (16) 21 b (42) 50 b (100) Bronchiole epithelium, hyperplasia 6 (12) 5 (10) 14 c (28) 48 b (96)

Inflammation,chronic active 10 (20) 10 (20) 14 (28) 40 b (80) Interstitial, fibrosis 19 (39) 7 b (14) 12 (24) 32 b (64) Alveolus, histiocyte infiltration 26 (53) 35 c (71) 44 b (88) 50 b (100) Larynx
Number of animals examined 50 49 49 50 Inflammation, chronic 8 (16) 26 b (53) 27 b (55) 38 b (76) Epiglottis epithelium, degeneration 2 (4) 33 b (67) 26 b (53) 40 b (80)

Epiglottis epithelium, hyperplasia 0 (0) 25 b (51) 26 b (53) 33 b (66) Epiglottis epithelium, squamous metaplasia 2 (4) 7 (14) 9 (18) 16 b (32)

Nose
Number of animals examined 50 50 50 50 Goblet cell, hyperplasia 13 (26) 19 (38) 16 (32) 30 b (60)

a Number of animals with lesion; numbers in paranthe ses indicate percent incidence compared to control.DRAFT - DO NOT CITE OR QUOTE 116 b Significantly different from control by the Poly-3 test, p ≤ 0.01

c Significantly different from control by the Poly-3 test, p ≤ 0.05

Table 5-4: Human Equivalent Concentrations of Vanadium Pentoxide in the 2-Year Inhalation Studies in Rats (NTP, 2002)

Concentration as Reported a (mg/m 3 ) Continuous Exposure Adjustment

Factor b RDDR c Human Equivalent Concentration d (mg/m 3 )
Extrathoraci

c Pulmonary Extrathoraci

c Pulmonary
Male Rats (F344/N) 0 0.179 0.516 0.496 0.00 0.00 0.5 0.179 0.530 0.494 0.05 0.04

1 0.179 0.520 0.495 0.09 0.09 2 0.179 0.503 0.498 0.18 0.18 Female Rats (F344/N) 0 0.179 0.263 0.524 0.00 0.00 0.5 0.179 0.259 0.524 0.02 0.05

1 0.179 0.263 0.524 0.05 0.09 2 0.179 0.245 0.524 0.09 0.19

a “Toxicology and carcinogenesis studies of vanadium pentoxide in F344/N rats and B6C3F 1 Mice”, NTP, 2002.
b “Continuous Exposure Adjustment Factor” = (6/24) * (5/7 ); animals were exposed to vanadium pentoxide 6 hours per day and 5 days per week.

c Please refer to Appendix Table B-4.
d “Human Equivalent Concentration” = “Concentration as Reported” * “Continuous Exposure Adjustment Factor ” * “RDDR”

To determine the POD for derivation of the RfC, benchmark dose modeling was conducted on lesions observed in both male and female rats in the NTP study (2002; chronic lung inflammation, alveolar epithelium hyperplasia, chronic inflammation of the larynx, respiratory epithelial hyperplasia of the larynx and nose) with the best-fitting model selected for each endpoint (Appendix B). As shown in Table 5-5, the lowest BMCL [HEC] of 0.003 mg/m 3 was observed for chronic inflammation of the larynx of female rats, indicating that the larynx was the most sensitive target for chronic inhalation exposure to vanadium pentoxide. Thus, it was chosen as the POD for the
basis of the RfC calculation.

Table 5-5: Candidate PODs for Vanadium Pent oxide Derived from NTP Studies (2002) through BMDS Modeling. Endpoint Selected BM HEC bDRAFT - DO NOT CITE OR QUOTE 117 Model a R (Extr

a Risk) BMC (mg/m 3 ) BMCL c (mg/m 3 ) (Candidate POD) Male F344/N Rats Lung Alveolar Epithelium Hyperplasia Probit 0.1 0.016 0.013 Chronic Active Inflammation Logistic 0.1 0.035 0.029 Larynx Chronic Inflammation LogLogistic 0.1 0.017 0.012 Respiratory Epithelium, Epiglottis, Hyperplasia LogLogistic 0.1 0.008 0.006

Nose Goblet Cell, Respiratory Epithelium, Hyperplasia LogLogistic 0.1 0.044 0.026

Female F344/N Rats Lung Alveolar Epithelium Hyperplasia Gamma 0.1 0.076 0.063

Chronic Active Inflammation Multistage (Stage3) 0.1 0.080 0.048
Larynx Chronic Inflammation LogLogistic 0.1 0.005 0.003 Respiratory Epithelium, Epiglottis, Hyperplasia LogLogistic 0.1 0.004 0.003

Nose Goblet Cell, Respiratory Epithelium, Hyperplasia Multistage (Stage2) 0.1 0.038 0.014

a Selected model is the best-fitting model for the data set based on the draft Benchmark Dose Technical Guidance (2000).
b HEC=Human Equivalent Concentration.

c BMCL= the lower bound of BMC at 95% confidence level.

Degeneration of the epiglottis epithelium was not selected for BMD modeling because the incidence of this lesion did not exhibit dose-dependence, with the same incidence observed in the low and high dose groups. Epithelial squamous metaplasia was not selected for BMD modeling because the incidence of this lesion was not significantly different from control at the low- and mid-dose groups; thus, other lesions of the larynx were more sensitive endpoints. In all vanadium pentoxide groups, lesion severity was classified as minimal to mild. Modeling was performed using the Benchm ark Dose Modeling Software (BMDS; Version 2.1.2) (U.S. EPA, 2000b). Biological and st atistical considerations were taken into account in the selection of a benchmark response (BMR) level for all data sets. In the absence of information indicating what magnitude of inflammatory changes in the larynx and epiglottis areDRAFT - DO NOT CITE OR QUOTE 118 considered biologically significant, the benchmark response (BMR) of 10% increase in extra risk was used as the basis for the BMC (BMC 10 ), with the BMCL 10 representing by the 95% lower
confidence limit on the BMC 10 (US EPA 2000b). Following the model selection steps outlined in the draft Benchmark Dose Technical Guidance (US EPA, 2000b), the best-fitting model was selected. Biological and statistical considerations were taken into account in the selection of a benchmark response (BMR) level for this data set. Statistically, a 10% level of response is intended to represent a response level near the lower range of detectable observations in typical studies conducted with 50 animals per dose group (U.S. EPA, 2000b). Results of the BMDS modeling for chronic inflammation of the larynx in female rats are summarized in Table 5-6, and for epiglottis hyperplasia in Table 5-7. As assessed by the chi-square goodness-of-fit test, several models demonstrated adequate goodness of fit p -value ≥ 0.1 and good visual fit (Appendix B). In accordance with the draft Benchmark Dose Technical Guidance (US EPA, 2000b), the LogLogistic model was selected as the best fitting model. The

BMC 10 and BMCL 10 were estimated as 0.005 and 0.003 mg/m 3

.

Table 5-6: Benchmark Modeling Results for Inciden ce of Larynx Chronic Inflammation in Female Rats, NTP (2002)

Model a Goodness of Fit

BMR HEC c

P

Value AIC b Largest Scaled Residual Scaled Residual of

Interest BMC (mg/

m 3 ) BMC L d (mg/m
3 ) LogLogistic 0.46 242.0 -0.88 -0.13

Extra Risk 10% 0.005 0.003
LogProbit 0.27 243.6 -0.89 -0.02 0.003 0.000

Gamma

0.19 243.7 1.58 -0.57 0.007 0.006 0.013 0.011 Multistage e

(Stage1)

0.014 0.011 Weibull

Probit 0.03 247.5 1.99 1.99

Logistic 0.03 247.6 1.97 1.97

a Selected (best-fitting) model shown in first row, in boldface type.
b AIC=Akaike Information Criterion.

c HEC=Human Equivalent Concentration.
d BMCL= the lower bound of BMC at 95% confidence level.

e For Multistage model, up to 3 stages were tested. Only the model with the lowest AIC and lowest stage was reported here.DRAFT - DO NOT CITE OR QUOTE 119 Table 5-7: Benchmark Modeling Results for In cidence of Larynx Respiratory Epithelium, Epiglottis, Hyperplasia in Female Rats, NTP (2002)

Model a Goodness of Fit

BMR HEC c

P

Value AIC
b Largest Scaled Residual Scaled Residual of

Interest BMC (mg/m
3 ) BMCL d (mg/m 3 )
LogLogist ic 0.30 205.3 1.53 0.000

Extra Risk 10% 0.004 0.003 LogProbit 0.78 204.2 -0.57 0.000 0.000 failed

Gamma

0.01 212.3 2.85 0.000 0.006 0.005 Multistage

e (Stage1) Weibull

Probit 0.00 234.6 3.258 3.258 0.016 0.013 Logistic 0.00 235.3 -3.295 3.174 0.016 0.014

a Selected (best-fitting) model shown in first row, in boldface type.
b AIC=Akaike Information Criterion.

c HEC=Human Equivalent Concentration.
d BMCL= the lower bound of BMC at 95% confidence level.

e For Multistage model, up to 3 stages were tested. Only the model with the lowest AIC and lowest stage was reported here.

5.2.3 RfC Derivation- Including Applic ation of Uncertainty Factors (UFs)

Benchmark dose modeling of incidence data for chronic inflammation of the larynx in female rats yielded the BMCL 10 of 0.003 mg/m 3 . A total UF of 300 was applied to the BMCL 10 of 0.003 mg/m 3 : 3 for interspecies extrapolation from animals to humans (UF A ); 10 for human interspecies variability (UF H ), and 10 for database insufficiencies (UF D ). An uncertainty factor (UF S ) was not applied for extrapolation of subchronic to chronic study as data was used from a chronic 2-yr study. An UF of 3 (10 1/2 = 3.16, rounded to 3) was applied for interspecies extrapolation (UF A ) to account for uncertainty in extrapolating from laboratory animals to humans (i.e., interspecies variability). This uncertainty factor is comprised of two separate and equal areas of uncertainty to account for differences in the toxicokinetics and toxicodynamics of animals and humans. In this assessment, toxicokinetic uncertainty was accounted for by the calculation of a human equivalent concentration by the application of a dosimetric adjustment factor as outlined in the RfC methodology ( U.S. EPA, 1994 ). As the toxicokinetic differences are thus accounted for, only the toxicodynamic uncertainties remain, and a UF of 3 is retained to account for this residual uncertainty.DRAFT - DO NOT CITE OR QUOTE 120 An UF H of 10 for intraspecies differences (human variability) was used to account for potentially susceptible individuals in the absence of quantitative information or information on the variability of response in humans. Considering the pulmonary effects of V 2 O 5 , individuals with pre-existing respiratory disorders may be more susceptible to inhaled vanadium pentoxide.

An UF L for LOAEL to NOAEL extrapolation was not used because the current approach
is to address this factor as one of the consid erations in selecting a BMR for BMD modeling (US EPA 2000b; 1994b). In this case, a BMR of a 10% increase in the incidence of chronic inflammation of the larynx and epithelial hyperplasia of the epiglottis was selected under the assumption that it represents a minimal, biologically significant change.

An UF D of 10 was used for database insufficiencies due to the lack of a developmental toxicity study and a multi-generation reproductive study for V 2 O 5 by the inhalation route. Studies using alternate routes of exposure (int raperitoneal) have indicated adverse reproductive and developmental effects in response to vanadi um pentoxide, including statistically significant increases in seminal vesicle, thymus and submandibular gland weights in male mice and body weight, thymus, submandibular gland, and liver weights in female mice (Altamirano et al., 1991) as well as reduced fertility (Altamirano-Lozano et al., 1996). No pharmacokinetic models are available for conducting a route-to-route extrapolation.

The chronic RfC for vanadium pentoxide is calculated as follows:

RfC = BMCL 10 ÷ UF

= 0.003 mg/m 3 ÷ 300 = 0.00001 mg/m 3 or 1E-05 mg/m 3

Note: Because vanadium exists in several different valence states, all of which are not equivalent toxicologically (WHO-IPCS, 2001), the values generated here apply to vanadium pentoxide and should not be applied to other vanadium compounds.

5.2.4. Previous RfC Assessment

An inhalation assessment for V 2 O 5 was not previously available on IRIS.

5.3 UNCERTAINTIES IN THE INHALATION REFERENCE CONCENTRATION (RfC)

The following discussion identifies uncertainties associated with the subchronic and chronic RfCs. As presented earlier in this s ection, EPA standard practices and RfC guidance (U.S. EPA 1994a,b, 1995, 2002b) were followed in applying an uncertainty factor approach to aDRAFT - DO NOT CITE OR QUOTE 121 point of departure (POD). A BMDL 10 approach was used for derivation of the chronic RfC. Factors accounting for uncertainties associated with a number of steps in the analyses were adopted to account for extrapolating from an animal bioassay to human exposure, a diverse human population of varying susceptibilities, and to account for database insufficiencies. These extrapolations are carried out with standard a pproaches given the paucity of experimental and human data on vanadium pentoxide to inform individual steps. An adequate range of animal toxicology da ta is available for the inhalation hazard assessment of vanadium pentoxide, as described in Section 4. Included in these studies are shortterm, subchronic, and chronic bioassays in rats, as well as a range of supporting genotoxity studies. Toxicity associated with inhalation exposure to vanadium pentoxide is observed in reproductive organs, the CNS, and particularly in the resp iratory system, including a range of nasal and pulmonary nonneoplastic lesions su ch as pulmonary inflammation, tissue morphology changes, and development of pulmonary fibrosis. Recent mechanistic studies have investigated vanadium pentoxide-induced pulmonary fibrosis and have contributed to some understanding of a putative mode of action for pulmonary fibrosis in response to vanadium pentoxide. In addition to respiratory effects, immunological and neurological effects have been seen following vanadium pentoxide exposure in expe rimental animals. An inhalation study using vanadium pentoxide (12 weeks, 1.4 mg/m 3 ) in male mice noted altered immune status, including a temporary increase in spleen weight, and alterations in antibody avidity following exposure to vanadium pentoxide (Pinon-Zarate et. al. 2008). This in part supports the results of an earlier oral study (6 months, 0 – 14 mg/kg-day in drinking water) in rats that also observed increased spleen weight (Mravcova et al., 1993). Several recent studies document neurotoxic morphological and behavioral effects (decreased de ndritic spine length and increased necrosis in neural cells) in animals following inhalation exposur e to vanadium pentoxide (Avila-Costa et al., 2004; 2005; 2006; Colin-Barenque et al., 2008). T hough these immune and neurological effects occurred at a low dose (1.4 mg/m 3 ), in most cases only one dose was tested and precludes formation of an adequate dose-response.
For derivation of the chronic RfC for vanadium pentoxide, a two-year inhalation study in rats and mice (NTP, 2002) was selected as the prin cipal study and inflammation of the larynx and epithelial hyperplasia of the epiglottis were selected as the critical effects. Lung hyperplasia, nasal inflammation, and lung fibrosis were also sens itive effects. Inflammation of the larynx and epithelial hyperplasia were selected as the critical effects because they are sensitive indicators of vanadium pentoxide-induced respiratory t oxicity and yielded the lowest POD. The selection of the benchmark dose model for the quantitation of the RfC does not lead to significant uncertainty in estimating the POD since benchmark effect levels were within the range ofDRAFT - DO NOT CITE OR QUOTE 122 experimental data for chronic inflammation of th e larynx. However, the selected model, the loglogistic model, while the best fitting model, is not the only model that adequately describes the data. Other models could be selected to yield more extreme results, both higher and lower than those
included in this assessment. Extrapolating from animals to humans entails further issues and uncertainties as the magnitude of the effect and the effect itself asso ciated with the concentration at the point of departure in rodents are extrapolated to human response. Pharmacokinetic models are useful in examining species differences in pharmacokinetics; however, a PBPK model for vanadium pentoxide was not available. Therefore, toxicokinetic species differences were addressed by the determination of a HEC through inhalation dosimetry adjustments (as described in the RfC methodology, U.S. EPA, 1994b). A UF of 3 was applied to account for the remaining toxicokinetic uncertainties in the extrapolation from rats and humans. Information was unavailable for quantitative a ssessment of toxicokinetic or toxicodynamic differences between animals and humans, so the th ree-fold uncertainty factor (UF) was used to account for uncertainty in extrapolating from labor atory animals to humans in the derivation of the
subchronic and chronic RfC values. Heterogeneity among humans is another uncertainty associated with the Rf C. In the absence of vanadium pentoxide-specific data on human varia tion, an uncertainty factor of 10 was applied to account for uncertainty associated with variation in the human population in the derivation of the subchronic and chronic RfCs. The range of human re sponse to vanadium pentoxi de may be larger or smaller. Vanadium pentoxide-specific data to examine the potential magnitude of over- or
underestimation are not available. No NOAEL was identified in the NTP, 2002 study for either the subchronic or chronic RfCs. Effects in this study were found at the lowest exposure concentrations measured, therefore, the LOAEL determined for this study for both subchronic and chronic effects does not indicate where a threshold of effects would lie and the data provided in the study is not sufficient for a dose response analysis. The data were amenable to BMD analysis and a BMCL was used so the UF for LOAEL to NOAEL extrapolation is not applicable. The selected critical effect was observed in longer term studies (3-months and 2-yrs) at similar doses and appeared to be a persistent and sensitive effect. Data gaps have been identified that are associated with uncertainties specific to the developmental and reproductive toxicities of vana dium pentoxide following inhalation exposure. Studies that used intraperitoneal injection to st udy pregnant rats exposed to vanadium pentoxide suggest that exposure to the developing fetus may have adverse health effects. However, no inhalation studies have assessed the risk to the developing fetus. The database also does notDRAFT - DO NOT CITE OR QUOTE 123 include a comprehensive multigenerational reproductive study to help establish the full range of developmental endpoints and thus represents a gap in the database. To account for the lack of inhalation developmental studies and for the lack of multigenerational reproductive toxicity studies to establish the range of toxicities across development, a full uncertainty factor of 10 was applied to the chronic RfC derivations. Overall confidence in the chronic RfC is medium. Confidence in the principal study (NTP, 2002) is high. It is a well-conducted study that used rats and mice, an adequate number of animals, and exposure at a range of doses. Multiple toxicity endpoints were assessed. However, confidence in the overall database is medium. The NTP study is the only chronic animal study available. Recent mechanistic studies have investigated vanadium pentoxide-induced pulmonary fibrosis and have contributed to a better understanding of a putative mode of action for pulmonary fibrosis in response to vanadium pentoxide. Health effects reported among workers exposed to vanadium pentoxide and other vanadium compounds in dust are consistent with effects observed in experimental studies. Refl ecting high confidence in the principal study and medium confidence in the database, confidence in the RfC is medium.

5.4. CANCER ASSESSMENT

5.4.1 Choice of Study/Data – with Rationale and Justification

The 2-year NTP (2002) inhalation cancer bioassay reported an increased incidence of
alveolar/bronchiolar adenomas or carcinomas in male F344/N rats and equivocal evidence of carcinogenic activity of vanadium pentoxide in female F344/N rats at the high dose. Male and female B6C3F1 mice had even greater incidences of these lesions, with a statistically significantly increased incidence of alveolar/br onchiolar adenomas or carcinomas in both male and female B6C3F1 mice following inhalation exposure to ≥ 1 mg/m 3 of vanadium pentoxide (NTP, 2002) (Table 5-8). These tumor types are considered relevant to humans. Human data on the carcinogenic potential of inhalation exposure to vanadium pentoxide are not available. There are no human or laboratory animal data to dete rmine the carcinogenicity of vanadium pentoxide by the oral or dermal route.

Table 5-8. Incidences of Respiratory Tumors in Mice Exposed to Vanadium Pentoxide in the 2 Year Inhalation Study (NTP, 2002) Exposure GroupDRAFT - DO NOT CITE OR QUOTE 124 Tumor Type a Historical Control (% Historical Control) Control 1 mg/m 3 2 mg/m 3 4 mg/m 3

Male Mice Number of animals examined 1071 50 50 50 50 Alveolar/bronchiolar adenoma b 201 (19%) 13 (26%) 16 (32%) 26 c (53%) 15 (30%)

Alveolar/bronchiolar carcinoma 97(9%) 12 (24%) 29 c (58%) 30 c (60%) 35 c (70%)

Alveolar/bronchiolar adenoma or carcinoma 285 (26.8%) 22 (28%) 42 c (84%) 43 c (86%) 43 c (86)

Female Mice Number of animals examined 1075 50 50 50 50 Alveolar/bronchiolar adenoma 67 (6.3%) 1 (2%) 17 c (34%) 23 c (46%) 19 c (38%)

Alveolar/bronchiolar carcinoma 43 (3.9%) 0 (0%) 23 c (46%) 18 c (36%) 22 c (44%)

Alveolar/bronchiolar adenoma or carcinoma 109 (10.1%) 1 (2%) 32 c (64%) 35 c (70%) 32 c (64%)

a Number of animals with tumor; numbers in parentheses i ndicate percent incidence; particle size mass mean aerodynamic diameter + geometric standard deviation (MMAD±GSD): 1 mg/m 3 = 1.3±2.9; 2 mg/m 3 = 1.2±2.9; 4 mg/m 3 =1.2±2.9 b Historical incidence of alveolar/bronchiolar adenoma male B6C3F 1 mice fed in inhalation chamber controls given NIH-07 diet.

c Significantly different from control by the Poly-3 test (p ≤ 0.01)

5.4.2 Dose-Response Data

Data on the incidences of alveolar/bronchiolar adenomas or carcinomas in male and female mice from the NTP (2002) study were used for cancer dose-response assessment. These
data are shown in Table 5-9.

5.4.3 Dose Adjustments and Extrapolation Methods

The NTP (2002) 2-year carcinogenicity study in mice was used for the derivation of an inhalation unit risk, based on the dose-response relationship for alveolar/bronchiolar neoplasms (adenoma and carcinoma). Using the RDDR computer program, as specified in the RfC guidelines (U.S. EPA, 1994b), HECs (in mg/m 3 ) were calculated at each exposure level for male and female mice using mean body weights for males and females reported by NTP (2002) and the average particle size MMAD±GSD of 1.26±1.87 as reported by NTP (2002). HECs were calculated by multiplying

Conc [ADJ] by the RDDR for male and female mice (Table 5-9).DRAFT - DO NOT CITE OR QUOTE 125 Table 5-9: Human Equivalent Concentrations of Vanadium Pentoxide in the 2-Year Inhalation Studies (NTP, 2002)
Concentration as Reported a (mg/m 3 ) Continuous Exposure Adjustment Factor b RDDR c HEC d (mg/m 3 ) Pulmonary Pulmonary Male Mice (B6C3F 1 ) 0 0.179 1.168 0.00 1 0.179 1.168 0.21 2 0.179 1.168 0.42 4 0.179 1.134 0.81 Female Mice (B6C3F 1 ) 0 0.179 1.168 0.00 1 0.179 1.143 0.20 2 0.179 1.077 0.38 4 0.179 1.023 0.73

a “Toxicology and carcinogenesis studies of vanadium pentoxide in F344/N rats and B6C3F 1 Mice”, NTP, 2002.
b “Continuous Exposure Adjustment Factor” = (6/24) * (5/7 ); animals were exposed to vanadium pentoxide 6 hours per day and 5 days per week.

c Please refer to Appendix Table C-4.
d HEC=Human Equivalent Concentration = “Concentrati on as Reported” * “Continuous Exposure Adjustment Factor ” * “RDDR”.

According to the U.S. EPA Guidelines for Carcinogen Risk Assessment (US EPA, 2005a), for each tumor response, a POD from the observed data should be estimated to mark the beginning of extrapolation to lower doses. The POD is an estimated dose near the lower end of the observed range without significant extrapolation to lower doses. 10 Since all non-control concentrations from the NTP carcinogenesis studies (2002) showed a plateau response, the data set provided limited information about the concentration-response relationship because the complete range of response from background to maximum must occur somewhere below the lowest dose. Therefore, a BMR based on the response at the control concentration and the first non-control concentration was calculated (Table C-6), and then used for estimation of POD (BMCL, extra risk of 0.71 for male mice, 0.67 for female mice). Modeling was performed using the Benchm ark Dose Modeling Software (BMDS; Version 2.1.2) developed by the National Center for Environmental Assessment (U.S. EPA, 2000b). The incidence of alveolar/bronchiolar adenomas and carcinomas in mice were combined; males and females were modeled separately (Table 5-10).Models were run using the

10 If the POD is above some data points, it can fail to refl ect the shape of the concentration-response curve at the lowest doses and can introduce bias into subsequent extra polations. However, if the POD is far below all observed data points, it can introduce model uncertainty and paramete r uncertainty that increase with the distance between the data and the POD. Use of a POD at the lowest level s upported by the data seeks to balance these considerations.DRAFT - DO NOT CITE OR QUOTE 126 default restrictions on parameters built into the BMD software. Goodness-of-fit was evaluated using the Chi-square statistic calculated by the BMDS program. Acceptable global goodness of fit was a p-value greater than or equal to 0.1. Each data set was first fitted with the dichotomous multi-stage cancer model; if the goodness-of-fit p-value was < 0.05, other dichotomous models were fitted; if still no model showed adequate goodness of fit p-value ≥ 0.05, the highest dose was dropped for further modeling.

Table 5- 10. BMDS Modeling Results for Combined Incidence of Alveolar/Bronchiolar Adenoma and Carcinoma in Male Mice, NTP (2002).

Model a Goodness of Fit BM R HEC P

Valu

e AIC
b Largest Scaled Residual Scaled Residual of

Interest BMC (mg/m 3 ) BMCL c (mg/m 3 ) Primary Cancer ModelsMultistageCancer Stage 1,2 and 3 0.01 207. 0 2.05 0.72 Extr

a Risk 71% 0.532 0.379

Other Dichotomous Models
LogLogistic 0.19 200. 6 -1.42 0.04

Extr

a Risk 71% 0.360 0.208

LogProbit 0.88 199. 6 0.13 -0.06 0.146 failed

Gamma 0.01 207. 0 2.05 0.72 0.532 0.379 Weibull Logistic 0.00 209. 4 2.15 0.96 0.609 0.447

Probit 0.00 210. 4 2.19 -1.54 0.654 0.495

a Selected model is the best-fitting model for the data set based on the draft Benchmark Dose Technical Guidance (2000).
b HEC=Human Equivalent Concentration.

c BMCL= the lower bound of BMC at 95% confidence level.

The BMDS modeling results for models meeting goodness-of-fit criteria are summarized in Appendix C. For incidence data in male mice, the log-logistic model was the only model that met goodness-of-fit criteria when all three dose groups were included, predicting BMC 71 and

BMCL 71 values of 0.360 and 0.208 mg/m 3 , respectively (Appendix C). The multi-stage model fit the incidence data for male mice when the high dose was dropped, predicting BMC 71 and

BMCL 71 values of 0.306 and 0.220 mg/m 3 , respectively.DRAFT - DO NOT CITE OR QUOTE 127 None of the dichotomous models fit tumor incidence data for female mice when all three dose groups were included. One model (log-logistic) fit the incidence data for female mice when the high dose was dropped, predicting BMC 67 and BMCL 67 values of 0.237 and 0.161 mg/m 3

, respectively.
The BMCL 71 of 0.208 mg/m 3 for male mice was selected as the point of departure (POD) for derivation of the inhalation unit risk as as this was the only model fit when including all doses for analysis of lung tumor formation following inhalation exposure to vanadium pentoxide.

5.4.4 Inhalation Unit Risk

Data to support a mode of action for the carcinogenicity of vanadium pentoxide are insufficient, although some data suggest that either a mutagenic or a cytotoxic and reparative proliferation mode of action is operative. In the absence of such data, extrapolation from the point of departure to lower doses was conducted by using a linear approach. The inhalation unit risk represents an upper bound, continuous lifetime exposure risk estimate and is calculated as BMR/BMCL [0.71/(0.208 mg/m 3 )]. The HEC BMCL 71 for extra
risk of alveolar/bronchiolar adenomas or carci nomas in male B6C3F1 mice exposed to vanadium pentoxide results in an inhalation unit risk of 3.4 (mg/m 3 ) –1 . This value was derived by linear extrapolation to the origin from the point of departure of 0.208 mg/m 3 and represents an upperbound estimate.

5.4.5 Oral Cancer Slope Factor

No human data or animal studies relevant to the carcinogenicity of vanadium pentoxide following oral exposure were located in the published literature. Therefore, an oral cancer slope
factor is not derived.

5.4.6 Uncertainties in Cancer Risk Values

Extrapolation of study data to estimate potential risks to human populations from exposure to vanadium pentoxide has engendered some uncertainty in the results. Several types of uncertainty may be considered quantitatively, but other important uncertainties cannot be considered quantitatively. Section 5.4.5.1 and Table 5-11 summarize principal uncertainties. Carcinogenicity due to chronic exposure of vanadium pentoxide was observed in two species (NTP, 2002), with the carcinogenicity more definitive in mice than in rats, particularly female rats, where the increased incidence of neoplasms did not exceed what would be expected due to spontaneous tumor formation. Similarly, spontaneous tumors were observed in male ratsDRAFT - DO NOT CITE OR QUOTE 128 at control levels, though tumor incidence increased in male rats in response to exposure to vanadium pentoxide. The confidence in the database is low. Respiratory tract carcinogenicity was reported in two rodent species (i.e., mice and rats) in awell-conducted NTP study (2002) and supported by a study in mice (Rondini et al., 2010). Human variability in response to vanadium pentoxide is unknown and, humans occupationally exposed to vanadium pentoxide are often simultaneously exposed to other valence states of vanadium and to other inhaled environmental toxicants simultaneously. Genotoxicity is equi vocal and no further mode of action information
is available. Overall confidence in the inhalation unit risk is low. Choice of low-dose extrapolation approach . The Mode of Action (MOA) is a keyconsideration in clarifying how risks should be estimated for low-dose exposure. A linear lowdose extrapolation approach was used to estimate human carcinogenic risk associated with vanadium pentoxide exposure due to the unavailability of data that supports any specific mode of carcinogenic action of vanadium pentoxide.

Dose metric. Vanadium exists in the +5 valence state in vanadium pentoxide. Other valence states ranging from -1 to +5 exist. Frequently, vanadium exposures involve a mixture of vanadium compounds ranging mostly from +3 to +5 valence state. The carcinogenic potential of
other valence states of vanadium has not been established. It is not known whether vanadium pentoxide dissociates to other valence states with known carcinogenic potential or whether some other valence state or some combination of +5 and other valence states is responsible for the observed toxicity. If the actual carcinogenic moie ty is proportional to administered exposure, then use of administered exposure as the dose metric is the preferred choice. Statistical uncertainty at the point of departure. Parameter uncertainty can be assessed through confidence intervals. Each description of parameter uncertainty assumes that the underlying model and associated assumptions are valid. For the log-logistic cancer model applied to the male mice data, there is a reasonably small degree of uncertainty at a 71% increase in tumor incidence (the point of departure for linear low-dose extrapolation). Bioassay selection . The study by NTP (2002) was selected for development of an inhalation unit risk. This was a well-designed study, conducted in both sexes in two species with an adequate number of animals per dose group. The number of test animals allocated among the two dose levels and an untreated control group was adequate, with examination of appropriate toxicological endpoints in both sexes of rats a nd mice. Both genders of mice exhibited a statistically significant increased incidence of lung tumors. Choice of species/gender : The inhalation unit risk for vanadium pentoxide was quantified using the tumor incidence data for male mice which was more amenable to BMD modeling than tumor incidence in female mice. In addition, lung neoplasm incidence was reported in male andDRAFT - DO NOT CITE OR QUOTE 129 female rats, though neither gender of rat was as sensitive as mice. A 71% tumor incidence level was observed in female mice at the lowest exposure level (1 mg/m 3 ), suggesting that lower doses may have revealed more information about low dose region of the dose response curve. Male mice demonstrated a high background rate of lung tumors, with spontaneous lung neoplasms observed in male mice at control levels (up to 28% of male mice). Tumor incidence increased significantly (84%) at the lowest dose level tested (1 mg/m 3 ). While these incidence response rates were higher in male mice than those of the females at the comparable exposure level,suggesting greater sensitivity of the male mice, there is no information concerning the doseresponse relationships at lower exposure levels.In other words, the behavior of vanadium pentoxide at 1.0 mg/m 3 in male mice may not inform the tumor response to vanadium pentoxide at lower exposures.

Relevance to humans . In the absence of direct human data, the most appropriate animal bioassays to use in the derivation of cancer risk values are chronic (i.e., lifetime) studies in two species of rodents. The inhalation unit risk was derived from the combined tumor incidence of lung adenomas and carcinomas in male mice. The information investigating the mode of action of the lung tumors observed in the chronic animal bioassay, however, is limited. The genotoxicity studies provide inadequate eviden ce of a genotoxic mode of action, and there are inadequate data to support alternative mode-of-action hypotheses. Human population variability

. The extent of inter-individual variability in animals for vanadium pentoxide metabolism has not been characterized. Strain differences in the response to vanadium pentoxide-induced pulmonary fibrosis in rodents suggests a genetic component to susceptibility. Moreover, humans occupationally exposed to vanadium pentoxide are often simultaneously exposed to other valence states of vanadium and to other inhaled environmental toxicants simultaneously. This lack of understanding about potential differences in metabolism and susceptibility across exposed human populations thus represents a source of uncertainty.

Table 5-11. Summary of uncertainty in th e vanadium pentoxide cancer risk assessment

.

Consideration/ Approach Impact on inhalation unit risk Decision JustificationDRAFT - DO NOT CITE OR QUOTE 130 Consideration/ Approach Impact on inhalation unit risk Decision Justification Low-dose extrapolation procedure Departure from EPA’s Guidelines for Carcinogen Risk Assessment POD paradigm, if justified, could ↓

or ↑ slope factor an unknown
extent Log-logistic cancer model to determine POD, linear low-dose extrapolation from POD Available MOA data do not inform selection of dose-response model.

Dose metric Alternatives could ↑ or ↓ slope factor by an unknown extent Used administered
exposure Experimental evidence supports a role of other valence states of vanadium in the body on health effects, but no data exists to support carcinogenicity due to other forms of vanadium. Cross-species scaling Alternative methods could ↓

or ↑ inhalation unit risk [e.g., 3.5-fold ↓ (scaling by BW)

or ↑ 2-fold (scaling by BW 2/3 )] RDDR RDDR software was used to adjust for toxicokinetic differences in inhalation dosimetry.

Statistical uncertainty at POD ↓ slope factor if MLE used rather than lower bound on POD LEC (method for calculating reasonable upper bound slope factor) Lower bound is 95% confidence interval on administered exposure.

Bioassay Alternatives could ↑ or ↓ slope factor by an unknown extent NTP study Alternative bioassays were unavailable.

Species /gender combination Human risk could ↓ or ↑ , depending on relative sensitivity Male mice lung cancer There are no MOA data to inform extrapolation approach for any choice. It was assumed that humans are as sensitive as the most sensitive rodent gender/species tested; true correspondence is unknown. The carcinogenic response occurs across genders and species.DRAFT - DO NOT CITE OR QUOTE 131 Consideration/ Approach Impact on inhalation unit risk Decision Justification Human relevance of
mouse tumor data Human relevance of mouse tumor data could ↓ slope factor Lung tumors in mice are relevant to human
exposure Vanadium pentoxide may be carcinogenic through an unknown mode of action.

Human population variability in metabolism and response/ sensitive subpopulations Low-dose risk ↑ to an unknown
extent Considered qualitatively No data to support range of human variability/sensitivity.DRAFT - DO NOT CITE OR QUOTE 132 6. MAJOR CONCLUSIONS IN THE CH ARACTERIZATION OF HAZARD AND DOSE RESPONSE

6.1 Human Hazard Potential

Vanadium is a metal commonly found in ores, tars, coals and oils and is used as an alloy in steel. The toxicity of vanadium depends on its valence state, which can range from -1 to +5, depending on pH and other factors. Vanadium pentoxide (V 2 O 5 ), sodium metavanadate (NaVO 3 ), and ammonium metavanadate (NH 4 VO 3 ) all contain vanadium in the +5 oxidation state. This Toxicological Review focu ses exclusively on vanadium pentoxide (V 2 O 5 ), the most common form of vanadium used commercially. In addition, V 2 O 5 is the only compound that is covalently bonded. Occupational exposure to vanadium pentoxide occurs primarily via inhalation of dust generated during vanadium processing and fuel-oil ash generation during cleaning of oil-burning boilers and furnaces. Toxicokinetics of orally administered vanadium pentoxide is not available in humans. Kucera et. al. (1992) attempted to measure vanadium in the hair of children who had been exposed to vanadium in drinking water, but exposure could not be tied to vanadium pentoxide specifically. Oral toxicokinetic studies in animals are limited. One study investigated the toxicokinetics of components of various metals , including vanadium, in female B6C3F1 mice (Radike et al., 2002). In this study, vanadium was detected in small intestine, kidney and the femur. Similarly, there are a few studies that evaluated the toxicokinetics of inhaled vanadium, but there are no data in humans. Occupational st udies of inhaled vanadium pentoxide indicate that vanadium is absorbed by humans, and levels in blood and urine rapidly declined following exposure (Kiviluoto et al., 1981b). Results of toxic okinetics studies of inhaled or intratracheally administered vanadium pentoxide in rats show that vanadium pentoxide is absorbed from the lung, undergoes a wide distribution to liver, kidney, bone, blood, gastrointestinal tract, and ovary, and is eliminated primarily in the urine (NTP 2002; Dill et al., 2004; Roshchin et al., 1980). The animal oral toxicity database reported decreased hair cystine (Mountain et. al., 1953, Stokinger et. al., 1953), hematological effects, including a decrease in RBC and hemoglobin (Mountain et. al., 1953), and a variety of reproductive and developmental effects following exposure to vanadium pentoxide (Alta mirano et. al., 1993; Zhang et. al., 1993). Studies of human exposure to inhaled vanadium pentoxide include primarily occupational reports and one controlled human exposure study. Several case study summaries reported upper and lower respiratory tract irritation and inflammation among workers withDRAFT - DO NOT CITE OR QUOTE 133 inhalation exposure to vanadium pentoxide and other vanadium compounds in dust during vanadium processing or to fuel oil ash during cleaning and maintenence of oil burning boilers (section 4.1.2). However, the chemical composition of the fuel-oil ash and vanadium dust or exposure measurements for vanadium pentoxide generally were not reported. Thus, in many cases, specific relationships between vanadium pentoxide and adverse respiratory effects cannot be definitively determined. Sjöberg (1955) published seven case reports of vanadium-induced bronchitis in workers cleaning boilers where the concentrations of vanadium pentoxide particles (10-20 μ in diameter) in the air were 2 – 85 mg/m 3 . Self-reported respiratory symptoms included cough, rhinitis, wheeze, sore throat, and conjunctivitis. All symptoms resolved by two weeks post-exposure, but would reappear upon re-exposure. Of 100 workers occupationally exposed to vanadium pentoxide fume (0.05 – 5.3 mg/m 3 ) via an oil-to-coal power plant conversion, 74 reported severe respiratory tract irritation (Levy et al., 1984). Estimated daily nasal and lung dose of vanadium was associated with incidence and severity of upper airway symptoms (nasal congestion/irritation, throat irritation) and lower airway symptoms (chest tightness, wheeze, cough, and sputum production) in a dose-related manner in a prospective clinical study of boilermakers and utility workers involved in the overhaul of a large, oil-fired boiler over a six week period (Woodin et al., 1998, 1999, 2000). Geometric mean concentrations of vanadium (9-10 hour shifts) measured in the breathing zone ranged from 1.1 – 8.9 μg/m 3

. Reductions in pulmonary function were measured among boilermakers with exposure to fly ash for weeks or years, however an association with vanadium has not been established (Woodin et al., 1999; Hauser et al., 1995a; 2001).There are no reported cases of cancer in workers examined over sufficient latency period as a result of exposure to vanadium pentoxide. Respiratory effects in experimental animals are well documented and include lesions in the nasal compartment, larynx, and lung. In addition to respiratory effects observed in animals (NTP, 2002; Knecht et. al., 1985, 1992), there are some animal studies documenting changes to spermatogenesis and testicular ultrastructural changes (Mussali-Galante et. al., 2005) and CNS effects (Colin-Barenque et. al., 2007; Avila-Costa et. al., 2004, 2005, 2006) in response to inhaled vanadium pentoxide. Following repeated inhalation to vanadium pentoxide, the respiratory system is the most sensitive target for noncancer toxicity in rats and mice. Short term inhalation exposure (13-days) in F344 rats and B6C3F1 mice was associated with histiocytic infiltration at 1.0 mg/m 3 , and short-term and subchronic inhalation exposure (3 month) in F344 rats and B6C3F1 mice was associated with increased lung epithelial hyperplasia and inflammation at 2.0 mg/m 3 (NTP, 2002). Long term exposure (2yr) in F344 rats and B6C3F1 mice similarly resulted in increased incidence of respiratory toxicity and carcinogenicity (NTP, 2002). Lung inflammation was observed at 1.0 mg/m 3 and lung hyperplasia was observed at 2.0DRAFT - DO NOT CITE OR QUOTE 134 mg/m 3 in female rats. Lung inflammation and hyperplasia were observed at 1.0 mg/m 3 in both male and female mice. In male and female mice, nonneoplastic lesions also occurred in the larynx (squamous metaplasia of epiglottis epithelium) and nasal tissues (degeneration and atrophy of olfactory epithelium and degeneration of respiratory epithelium) at 1 mg/m 3 ; it should be noted that 1 mg/m 3 was the lowest dose in mice. In mice exposed to 1 mg/m 3 , lesions were observed in the nose, bronchioles and lung of male mice and the nose, larynx, bronchioles and lung of female mice. Chronic, active inflammation and interstitial fibrosis was observed in male rats at a LOAEL of 1 mg/m 3 (NOAEL 0.5 mg/m 3 ). The nasal and laryngeal lesions appear to be among the most sensitive effects observed at the lowest dose tested. Moreover, multiple lesion types were observed in both sexes of rats and thus, the LOAEL of 0.5 mg/m 3 for nasal and laryngeal lesions was selected as the critical effect. Reproductive and developmental studies reveal various altered endpoints in response to vanadium pentoxide via oral and inhalation rout es. Vanadium pentoxide delivered orally to weanling rats for three days caused a significant increase in alkaline phosphatase activity and DNA content in the diaphysis of femoral bones suggesting that vanadium pentoxide may be linked to bone formation in the developing rat (Yamaguchi et. al., 1989). Mussali-Galante et. al., (2005) identified accumulation of vanadium pentoxide in the testes after 1 week of inhalation exposure in male CD-1 mice (0.02M or 1.4 mg V/m 3 ). Gamma tubulin was significantly decreased in testes exposed to vanadium pentoxide and may suggest changes in microtubule function that may impact spermatogenesis. In a follow-up study, Fortoul et. al. (2007) reported necrotic cell death in spermatogonia and increased nuclear distortion in spermatocytes in male CD-1 mice exposed by inhalation to vanadium pentoxide (0.02M or 1.4 mg V/m 3 ). A number of intraperitoneal studies have evaluated reproductive and developmental endpoints. Common reproductive effects include increased reproductive organ weights in male rats (Altamirano et. al., 1991), increased incidence of apoptotic spermatogonia in male mice (Aragon et. al., 2005) and reduced sperm motility, reduced sperm count, and increased numbers of abnormal sperm in male CD1 mice (Altamirano-Lozano et. al., 1996). In developmental studies, reduced ossification in the developing fore and hindlimbs of fetuses have been reported in response to intraperitoneal exposure to vana dium pentoxide during gestation days 6-15 in pregnant CD-1 mice (Altamirano-Lozano et. al., 1993) and Wistar rats (Zhang et. al., 1993a, b). Other reported effects range from reduced fe tal weight and increased placenta weight. The genotoxicity database for vanadium pentoxide is limited. The evidence for genotoxicity in humans is limited. There are few studies examining genotoxicity in humans in vivo, with equivocal results. Ivancsits et. al. (2002) reported no differences in DNA strand breaks, oxidative damage, or sister chromatid exchange frequency in leukocytes between controlDRAFT - DO NOT CITE OR QUOTE 135 and vanadium pentoxide-exposed workers. Ehrlich et. al., (2008) noted changes in DNA stability and DNA repair in leukocytes of occupationally-exposed workers as compared to controls. Studies have demonstrated a genotoxic effect of vanadium pentoxide on human cells in vitro. Ivancsits et al. (2002) demonstrated significant increases in DNA damage as measured by the Comet assay in both leukocytes and fibroblasts but with different dose sensitivity, while Kleinsasser et. al (2003) noted DNA migration differences occurred dose-dependently in peripheral blood lymphocytes but not in nasal mucosa cells. Earlier studies in human lymphocyte cultures also demonstrated increased aneuploidy (Ramirez et al. 1997; Rojas et al. 1996) and DNA damage (Roldan and Altamira no 1990) following exposure to vanadium pentoxide. Thus, vanadium pentoxide-induced mutagenicity may occur at doses higher than those measured in these occupational exposures, may be tissue-specific and may be associated with oxidative stress rather than direct DNA damage. Experimental data in animals provide evidence of some types of genotoxicity following in vivo exposure to vanadium pentoxide. Vanadium pentoxide administered by inhalation to mice or rats did not increase the frequency of micronucleated normochromatic erythrocytes in peripheral blood (NTP, 2002). Genotoxicity assessed in male CD-1 mice following intraperitoneal injection caused no treatment relatedeffects in mitotic index, average generational time, or sister chromatid exchange (AltamiranoLozano et. al., 1993). However, DNA damage was detected in six organs from vanadium pentoxide-treated mice via intraperitoneal inj ection (Altamirano-Lorano et. al., 1999). Vanadium pentoxide produced gene mutations in two bacterial test systems (Kada et. al., 1980; Kanematsu et. al., 1980) but negative results in the NTP (2002) study. Vanadium pentoxide produced DNA strand breaks, aneuploidy, and micronuclei induction but did not produce chromosomal aberrations or sister chromatid exchange in various cell lines (Ivancsits et al. 2002; Kleinsasser et al. 2003; Ramirez et al. 1997; Rojas et al . 1996; Roldan and Altamirano 1990; Zhong et al., 1994). Most of the effects of vanadium pentoxi de are thought to be produced by the parent compound, primarily by inducing cell damage and pulmonary fibrosis (NTP, 2002, Bonner et. al., 1998, Bonner et. al., 2000). Pulmonary fibrosis is mediated either directly by vanadium pentoxide-induced changes to cell signaling molecules or via vanadium pentoxide-induced oxidative stress that induces cellular changes leading to fibrosis (Bonner et. al., 1998, Rice et. al., 1999, Ingram et. al., 2003). It is possible that pulmonary fibrosis could be a key event leading to eventual tumorigenesis. Vanadium pentoxide is “likely carcinogenic to humans” by the inhalation route of exposure under the Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005a). Existing cancer data comes from two animal species (mice, rats) reported in one well-characterized studyDRAFT - DO NOT CITE OR QUOTE 136 (NTP, 2002). Increased incidence of pulmonary adenomas and carcinomas were observed in male and female mice exposed to vanadium pentoxide for 2 years. There is evidence of carcinogenicity in male rats exposed to vanadium pentoxide for 2 years, as respiratory tumor incidence exceeded that in historical controls. In female rats, the incidence of respiratory tumorigenesis in response to vanadium pentoxide did not exceed historical controls. Tumor
incidence has not been documented in humans. Information available on the carcinogenic effects of V 2 O 5 via the inhalation route is limited to examination of the respiratory tumors. Information on the carcinogenic effects of

V 2 O 5 via the oral and dermal routes in humans or animals is absent. Based on the observance of only respiratory tumors following inhalation exposure, and in the absence of information to establish a mode of action, this cancer descriptor applies only to the inhalation route of exposure. Therefore, the database has “inadequate information to assess carcinogenic potential” of V 2 O 5 via the oral or dermal route. The mode of action underlying tumorigenicity in rats and mice has not been established. The genotoxicity database for vanadium pentoxide is equivocal, including both positive and negative studies for mutation, DNA damage and chromosomal aberrations. A nongenotoxic mode of action hypothesis involving hyperplasia and development of fibrotic pulmonary lesions is supported by the presence of hyperplastic lesions and pulmonary fibrosis at earlier time points and at lower doses. However, the dose-response relationship is not robust and a clear relationship linking these effects to the tumor response has not been established. It is unknown whether cytotoxicity may be a required precursor event for vanadium pentoxide-induced cell proliferation. Sufficient data regarding a plausible dose response and temporal progression from cytotoxicity to hyperplasia to fibrosis to tumorigenesis are not available.

6.2. Dose Response

6.2.1 Noncancer

Limited studies are available examining the toxicity of vanadium pentoxide following oral exposure in humans or laboratory anim als. Decreased RBC count and hemoglobin was observed following subchronic oral exposure to va nadium pentoxide in rats (Mountain et. al. 1953). The authors reported the decrease of RBC as a mean with no measure of variability between animals, therefore the continuous data was not amenable to BMD modeling. Derivation of an RfD was based on a NOAEL of 10.5 mg/kg-d for the critical effect decreased RBC counts and divided by a total UF of 3000, 3 to represent interspecies toxicodynamic uncertainties, 10 for interhuman variability in the absence of quantita tive information on the variability of response inDRAFT - DO NOT CITE OR QUOTE 137 humans, 10 for extrapolation from subchronic to chronic study and 10 for database deficiencies
to arrive at the chronic RfD of 9 x 10 -4 mg/kg-day. Confidence in the principal study for derivation of the RfD (Mountain et al., 1953) is low. Mountain et al. (1953) is a well-conducted study with numerous doses, but is a subchronic study with a small sample size (n = 5) for one species of rodents (Wistar rat), using one gender (male), limited endpoints and time points. Confidence in the critical effect is medium. Hematological effects have been documented in other rodent species in response to inhaled vanadium pentoxide (NTP, 2002). However, confidence in the overall database is low. Mountain et al. (1953) is the single relevant p eer-reviewed study for derivation of the chronic RfD. Thus, overall confidence in the chronic RfD is low. Pulmonary effects have been documented in numerous species, including humans and primates, in response to inhaled vanadium pentoxide. Inflammation and histiocytic infiltrate are common hallmarks of initial vanadium-induced pulmonary injury. The database also includes occupational (inhalation) and laboratory animal (inhalation and oral) studies demonstrating possible immunotoxicity following exposure to vanadium pentoxide. In addition, the database includes studies of neurotoxicity and reproductive and developmental toxicity studies following inhalation exposure to vanadium pentoxide in rodents. Overall, the lung is the most sensitive target for noncancer toxicity in rats and mice following chronic inhalation exposure to vanadium pentoxide. Nonneoplastic lung lesions (specifically laryngeal lesions) were selected as the most sensitive endpoint from a well-conducted chronic study (NTP, 2002; 2-year rodent bioassay). The chronic inflammation of the larynx and epithelial hyperplasia of the epiglottis in rats were chosen as critical effects because they are the most sensitive effects and the most proximal to route of exposure (inhalation). These effects were observed in both male and female rats and
mice. The dose-response pattern for laryngeal lesions (NTP, 2002) was amenable to BMD modeling and was used for derivation of the chronic RfC. Two laryngeal lesions were selected for modeling because they had the lowest Regional Deposited Dose Ratio (RDDR) value and corresponding BMCL [HEC]. In accordance with U.S. EPA Benchmark Dose methodology (2000b), a benchmark response (BMR) of 10% increase in extra risk was selected to represent a minimally adverse level. All available dichotomous models were fit to the incidence data for chronic inflammation and for epithelial hyperplasia of the epiglottis. The model with the lowest Akaike’s Information Criteria (AIC) value was considered to provide a superior fit. Benchmark dose modeling of incidence data for chronic inflammation of the larynx and epithelial hyperplasia of the epiglottis in female rats yielded the same BMCL 10 of 0.003 mg/m 3 . The
shared BMCL 10 of 0.003 mg/m 3 for either chronic inflammation of the larynx or epithelialDRAFT - DO NOT CITE OR QUOTE 138 hyperplasia of the epiglottis was divided by a total UF of 300, 3 to represent interspecies toxicodynamic uncertainties, 10 for interhuman variability in the absence of quantitative information on the variability of response in humans, and 10 for database deficiencies to arrive at
the chronic RfC of 1 x 10 -5 mg/m 3

. Confidence in the principal study for derivation of the RfC (NTP, 2002) is high. NTP (2002) is a well-conducted study with numerous doses, a large sample size of two species of rodents, using both genders, numerous endpoints and time points. Confidence in the critical effect is high. Pulmonary effects have been documented in numerous species, including humans and primates, in response to inhaled vanadium pentoxide. Laryngeal lesions are a relevant proximal portal of entry target tissue for inhalation exposure and serves as an indicator of vanadium pentoxide-induced pulmonary injury. However, confidence in the overall database is medium. NTP (2002) remains the single relevant study for use in the derivation of the chronic RfC. Thus, overall confidence in the chronic RfC is medium.

6.2.2 Cancer

There are no studies identifying cancer effects in following oral exposure to vanadium pentoxide in either humans or animals, or following inhalation exposure in humans.
Under the Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005a), the database for vanadium pentoxide indicates that it is “likely to be carcinogenic to humans” via the inhalation route of exposure. This determination is based predominantly on the NTP (2002) study, which found positive evidence of lung tumors in both sexes of mice and male rats after chronic vanadium pentoxide inhalation exposure. This weight of evidence conclusion takes into consideration the NTP (2002) cancer bioassay, the available human studies, and other laboratory animal studies. Information available on the carcinogenic effects of V 2 O 5 via the inhalation route is limited to examination of the respiratory tumors. Information on the carcinogenic effects of

V 2 O 5 via the oral and dermal routes in humans or animals is absent. Based on the observance of only respiratory tumors following inhalation exposure, and in the absence of information to establish a mode of action, this cancer descriptor applies only to the inhalation route of exposure. Therefore, the database has “inadequate information to assess carcinogenic potential” of V 2 O 5 via the oral or dermal route. The increased incidence of lung tumors in male mice observed in the NTP (2002) 104 week inhalation study was used to calculate the inhalation unit risk for vanadium pentoxide. The calculated cancer inhalation unit risk for vanadium pentoxide is 3.4 ug/m 3 for the development of
alveolar/bronchiolar adenomas or carcinomas in male B6C3F1 mice. This value was derived from BMCL10, the 95% lower bound on the dose associated with 71% extra cancer risk ofDRAFT - DO NOT CITE OR QUOTE 139 respiratory carcinoma in male B6C3F1 mice, by dividing the BMR (0.71) by the BMCL10, and represents the upper bound, continuous lifetime exposure estimate of cancer potency. The BMCL10, lower 95% bound on exposure at 71% risk, is 2.08 x 10 -1 mg/m 3 and the slope of the linear extrapolation from the BMCL to the origin = 0.71/2.08 x 10 -1 mg/m 3 = 3.4 (mg/m 3 ) -1 . A linear low-dose extrapolation approach was used to estimate human carcinogenic risk associated with vanadium pentoxide exposure due to a lack of data that supports any specific mode of carcinogenic action of vanadium pentoxide. Ther efore, the derived inhalation unit risk to vanadium pentoxide is 3.4 per mg/m 3

. Areas of uncertainty exist for this cancer assessment. The log-logistic model was selected to model lung tumor incidence in male mice; however, it is unknown how well this model or the linear low-dose extrapolation predicts low-dose risks for vanadium pentoxide. The selected model, while the best fitting model, is not the only model that adequately describes the data. Other models could conceivably be selected to yield different results consistent with the observed data, both higher and lower than those included in this assessment. The human equivalent inhalation unit risks estimated from the statistically significant increase in lung tumors ranged from 1.4 mg/m 3 in male mice to 4.2 mg/m 3 in female mice. These tumors are considered to be relevant to humans. As there is no information to inform which species or gender of animals would be most applicable to humans, the most sensitive group was selected
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Zhong, BZ; Gu, ZW; Wallace, WE; et al. (1994) Genotoxicity of vanadium pentoxide in Chinese hamster V79 cells. Mutation Research 321:35-42.DRAFT - DO NOT CITE OR QUOTE 152 APPENDIX A - SUMMARY OF EXTERNAL PEER REVIEW AND PUBLIC COMMENTS AND DISPOSITION

This page intentionally left blank.DRAFT - DO NOT CITE OR QUOTE 153 APPENDIX B - BENCHMARK CONCENTRAT ION MODELING OF INHALATION STUDIES IN RATS FROM NTP, 2002

To derive the RfC for vanadium pentoxide, i nhalation toxicity effects observed in rats (NTP, 2002) were modeled to estimate the candidate PODs. Five endpoints in both male and female rats (Table B-2) were selected because of two reasons:

• The study in rats was designed with lower concentrations of vanadium pentoxide;

• The results showed both biological significance and statistically significant
trends. Each data set was fitted with the dichotomous models available in EPA BMDS (version 2.1.2). Following the model selection steps outlined in the draft Benchmark Dose Technical Guidance (US EPA, 2000b), the best-fitting model for each data set was used to estimate the candidate POD, which was the BMCL at the selected BMR as 10% extra risk. The highlights of the benchmark dose modeling results are:

• The POD value of 0.003 mg/m 3 , from two endpoints in female rats, was the lowest POD derived and used for the calculation of RfC. The two endpoints were larynx chronic inflammation and larynx respiratory epithelium epiglottis hyperplasia.

• All candidate PODs and the selected models are summarized in Table B-1.DRAFT - DO NOT CITE OR QUOTE 154 Table B-1: Candidate PODs for Vanadium Pent oxide Derived from NTP Studies (2002) through BMDS Modeling.

Endpoint Selected Model a BM R (Extr

a Risk) HEC b

BMC (mg/m 3 ) BMCL c (mg/m 3 ) (Candidate POD) Male F344/N Rats Lung Alveolar Epithelium Hyperplasia Probit 0.1 0.016 0.013 Chronic Active Inflammation Logistic 0.1 0.035 0.029 Larynx Chronic Inflammation LogLogistic 0.1 0.017 0.012 Respiratory Epithelium, Epiglottis, Hyperplasia LogLogistic 0.1 0.008 0.006

Nose Goblet Cell, Respiratory Epithelium, Hyperplasia LogLogistic 0.1 0.044 0.026

Female F344/N Rats Lung Alveolar Epithelium Hyperplasia Gamma 0.1 0.076 0.063

Chronic Active Inflammation Multistage(Stag e3) 0.1 0.080 0.048
Larynx Chronic Inflammation LogLogistic 0.1 0.005 0.003 Respiratory Epithelium, Epiglottis, Hyperplasia LogLogistic 0.1 0.004 0.003

Nose Goblet Cell, Respiratory Epithelium, Hyperplasia Multistage(Stag e2) 0.1 0.038 0.014

a Selected model is the best-fitting model for the data set based on the draft Benchmark Dose Technical Guidance (2000).

b HEC=Human Equivalent Concentration.

c BMCL= the lower bound of BMC at 95% confidence level.

B.1. TREND TESTS ON THE INHALATION DATA SETS FOR BMDS MODELING Ten inhalation toxicology data sets in rats were selected because of the lower concentrations of vanadium pentoxide administrated (as compared to mice), the significantly biologically adverse effects observed and the statistical significance reported by NTP (2002). A Cochran-Armitage test was conducted to confirm the significance of the statistical trend for the selected data sets (Table B-2) before modeling.DRAFT - DO NOT CITE OR QUOTE 155DRAFT - DO NOT CITE OR QUOTE 156 Table B-2: Trend Tests on the Selected Data Sets from the 2-Year Inhalation Studies in Rats (NTP, 2002)

Endpoint Concentration as Reported (mg/m 3 ) a Trend Test b

0.0 0.5 1.0 2.0 Z -Score p -value Incidence c in Male F344/N Rats
L un g Alveolar Epithelium Hyperplasia d 7/50 24/49 35/48 50/50 8.83 <0.0001
Chronic Active Inflammation 5/50 8/49 24/48 42/50 8.36 <0.0001 Lar y nx Chronic Inflammation 3/49 20/50 17/50 28/49 4.79 <0.0001 Respiratory Epithelium, Epiglottis Hyperplasia 0/49 18/50 34/50 32/49 -6.56 <0.0001

N

ose Goblet Cell, Respiratory Epithelium, Hyperplasia 4/49 15/50 12/49 17/48 2.68 0.0037

Incidence c in Female F344/N Rats
L un g Alveolar Epithelium Hyperplasia d 7/49 8/49 21/50 50/50 9.53 <0.0001 Chronic Active Inflammation 10/49 10/49 14/50 40/50 6.71 <0.0001 Lar y nx Chronic Inflammation 8/50 26/49 27/49 37/50 5.38 <0.0001 Respiratory Epithelium, Epiglottis Hyperplasia 0/50 25/49 26/49 33/50 -5.98 <0.0001

N

ose Goblet Cell, Respiratory Epithelium, Hyperplasia 13/50 19/50 16/50 30/50 3.46 0.0003

a Concentrations are as reported by NTP (2002).

b One-sided Cochran-Armitage trend test.

c Incidence=(number of animals affected)/(number of animals examined).

d This data set was calculated by combining the incidences of alveolar epithelium hyperplasia and bronchiole epithelium hyperplasia.

B.2. DOSE CONVERSION In the toxicology and carcinogenesis studies reported by NTP (2002), rats (F344/N) were exposed to vanadium pentoxide through inhalation. To analyze the concentration response effect of vanadium pentoxide, the reported concentrati ons of vanadium pentoxide were converted to human equivalent concentrations before any modeling and extrapolation. First, the average life time animal body weights of rats were estimated based on the mean body weight at different weeks (Table B-3). S econdly, following the Methods for Derivation ofDRAFT - DO NOT CITE OR QUOTE 157 Inhalation Reference Concentrations and Appli cation of Inhalation Dosimetry (US EPA, 1994b), RDDRs (regional deposited dose ratios) were calculated with the RDDR program designed by US EPA (1994). Although six regional RDDRs we re reported by the RDDR program for each concentration/sex group, only two regional RDDRs (i.e. Extrathoracic and Pulmonary) are relevant to the selected endpoints, which are summarized in Table B-4. Then, the human equivalent concentration for each concentration/sex group was calculated from the reported concentration in rats by multiplying the continuous exposure adjustment factor and RDDR (Table B-5). For endpoints in the lung, the pulmonary RDDR was applied; for endpoints in the larynx and nose, the extrathoracic RDDR was applied.

Table B-3: Average Life Time Animal Body Weight of Rats in the 2-Year Inhalation Studies of Vanadium Pentoxide (NTP, 2002)

Vanadium Pentoxide Concentration as Reported (mg/m 3 ) Mean Animal Body Weight a (g) Average Life Time Animal Body Weight b (g) 1-13 weeks 14-52 weeks 53-104 weeks Male Rats (F344/N) 0 238 411 504 440 0.5 241 422 513 449

1 242 414 508 443 2 233 404 494 432 Female Rats (F344/N) 0 151 227 326 269 0.5 150 229 319 266

1 150 227 326 269 2 147 217 308 256

a As reported in Table 11-12 of the NTP report (2002).

b “ Average Life Time Animal Body Weight” = ( “Mean Body Weight 1-13 Weeks” * 13 +“ Mean Body Weight 14 -52 Weeks” * 39 + “Mean Body Weight 53-104 Weeks” * 52) /104

Table B-4: RDDRs for Different Concentration/Sex Group in the 2-Year Inhalation Studies of Vanadium Pentoxide (NTP, 2002) Vanadium Pentoxide Concentration as Reported (mg/m 3 ) Average Life Time Animal Body Weight a (g) Average MMAD
b Average GSD c RDDR d

Extrathoraci

c Pulmonary
Male Rats (F344/N) 0 440 1.24 1.89 0.516 0.496 0.5 449 0.530 0.494

1 443 0.520 0.495DRAFT - DO NOT CITE OR QUOTE 158 2 432 0.503 0.498 Female Rats (F344/N) 0 269

1.24 1.89 0.263 0.524 0.5 266 0.259 0.524

1 269 0.263 0.524 2 256 0.245 0.524

a All average life time animal body weights were calculated in Table B-3.

b MMAD =mass median aerodynamic diameter; calculated ba sed on the MMADs reported for 2-year studies (NTP, 2002) .

c GSD = geometric standard deviation; calculated base d on the GDSs reported for 2-year studies (NTP, 2002) .

d RDDR = regional deposited dose ratio; calculated with the RDDR program (V.2.3. US EPA);

Table B-5: Human Equivalent Concentrations of Vanadium Pentoxide in the 2-Year Inhalation Studies (NTP, 2002)

Concentration as Reported a (mg/m 3 ) Continuous Exposure Adjustment

Factor b RDDR c Human Equivalent Concentration d (mg/m 3 )
Extrathoraci

c Pulmonary Extrathoraci

c Pulmonary
Male Rats (F344/N) 0 0.179 0.516 0.496 0.00 0.00 0.5 0.179 0.530 0.494 0.05 0.04

1 0.179 0.520 0.495 0.09 0.09 2 0.179 0.503 0.498 0.18 0.18 Female Rats (F344/N) 0 0.179 0.263 0.524 0.00 0.00 0.5 0.179 0.259 0.524 0.02 0.05

1 0.179 0.263 0.524 0.05 0.09 2 0.179 0.245 0.524 0.09 0.19

a “Toxicology and carcinogenesis studies of vanadium pentoxide in F344/N rats and B6C3F 1 Mice”, NTP, 2002.

b “Continuous Exposure Adjustment Factor” = (6/24) * (5/7 ); animals were exposed to vanadium pentoxide 6 hours per day and 5 days per week.

c Please refer to Appendix Table B-4.

d “Human Equivalent Concentration” = “Concentration as Reported” * “Continuous Exposure Adjustment Factor ” * “RDDR”

B.3. BMDS MODELING FOR INHALATION DATA SETS
Each selected inhalation data set was fitted with all the standard dichotomous models available in EPA BMDS (version 2.1.2.). Following the model selection steps outlined in the Benchmark Dose Technical Guidance (US EPA, 2000b), the best-fitting model was used to estimate the candidate POD for each endpoint, which was the BMCL at the selected BMR as
10% extra risk.DRAFT - DO NOT CITE OR QUOTE 159 The selected models and candidate PODs for all endpoints are summarized in Table B-1.

Data Set 1: Incidence of Lung Alveolar Epithelium Hyperplasia in Male Rats, NTP (2002) Summary All three concentration groups (0.5, 1 and 2 mg/m 3 ) showed statistically significant differences from the control as reported. The severity of these nonneoplastic lesions increased from mild to moderate as reported when the concentration of vanadium pentoxide increased. The Cochran-Armitage test confirmed the statistically significant trend with Z score as 8.83 and
one-sided p -value as <0.0001. Seven dichotomous models available in EPA BMDS (version 2.1.2.) were fitted to this
data set. A 10% extra risk of the combined incidence was used as the BMR to determine the POD. The goodness of fit, BMC and BMCL for each model are summarized in Table B-6. Five models demonstrated adequate goodness of fit p -value ≥ 0.1 and good visual fit. Based on the draft Benchmark Dose Technical Guidance (U.S. EPA, 2000b), the Probit model was selected as the model for POD computation. The BMCL 10 of this model was 0.013 mg/m 3 and regarded as one of the candidate PODs.

Table B-6: Benchmark Modeling Results for Inciden ce of Lung Alveolar Epithelium Hyperplasia in Male Rats, NTP (2002)

Model a Goodness of Fit

BMR HEC c

P

Value AIC b Largest Scaled Residual Scaled Residual of

Interest BMC (mg/m 3 ) BMCL d (mg/m 3 ) Probit 0.53 170.0 -0.70 -0.23

Extra Risk 10% 0.016 0.013 Logistic 0.38 171.0 -0.87 -0.20 0.016 0.013 Multistage e

(Stage3) 0.43 171.3 -0.50 -0.08 0.011 0.007

Weibull 0.18 172.7 -0.90 -0.15 0.019 0.010 Gamma 0.13 173.6 -1.05 -0.10 0.021 0.009 LogProbit 0.06 175.1 -1.31 0.67 0.026 0.016 LogLogisti

c 0.04 176.4 -1.38 0.67 0.025 0.015

a Selected (best-fitting) model shown in first row, in boldface type.

b AIC=Akaike Information Criterion.

c HEC=Human Equivalent Concentration.

d BMCL= the lower bound of BMC at 95% confidence level.

e For Multistage model, up to 3 stages were tested. Only the model with the lowest AIC and lowest stage was reported here.

BMDS output fileDRAFT - DO NOT CITE OR QUOTE 160 ==================================================================== Probit Model. (Version: 3.2; Date: 10/28/2009) Input Data File: C:\USEPA\BMDS212\Data\V2O5\NTP_2002NonCancerMaleRats\MaleRatsLungAlBrEpHyperplasia\NTP_2002_Lung Alveolar Bronchiole Combined Epithelium Hyperplasia_Probit_0.1.(d) Gnuplot Plotting File: C:\USEPA\BMDS212\Data\V2O5\NTP_2002NonCancerMaleRats\MaleRatsLungAlBrEpHyperplasia\NTP_2002_Lung Alveolar Bronchiole Combined Epithelium Hyperplasia_Probit_0.1.plt

==================================================================== The form of the probability function is: P[response] = CumNorm(Intercept+Slope*Dose), where CumNorm(.) is the cumulative normal distribution function

Dependent variable = Response Independent variable = Dose Slope parameter is not restricted

Total number of observations = 4 Total number of records with missing values = 0 Maximum number of iterations = 250 Relative Function Convergence has been set to: 1e-008 Parameter Convergence has been set to: 1e-008

Default Initial (and Specified) Parameter Values background = 0 Specified intercept = -1.04337 slope = 20.1821 Asymptotic Correlation Matrix of Parameter Estimates ( *** The model parameter(s) -background have been estimated at a boundary point, or have been specified by the user, and do not appear in the correlation matrix )

intercept slope intercept 1 -0.78 slope -0.78 1

Parameter Estimates 95.0% Wald Confidence Interval Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit intercept -1.02981 0.180284 -1.38316 -0.676464 slope 20.0578 2.82623 14.5184 25.5971

Analysis of Deviance Table Model Log(likelihood) # Param's Deviance Test d.f. P-value Full model -82.2383 4 Fitted model -82.9994 2 1.52225 2 0.4671 Reduced model -133.424 1 102.372 3 <.0001

AIC: 169.999

Goodness of Fit Scaled Dose Est._Prob. Expected Observed Size Residual

------------------------------------------------------------------------ 0.0000 0.1515 7.577 7.000 50 -0.228 0.0441 0.4423 21.673 24.000 49 0.669 0.0884 0.7713 37.023 35.000 48 -0.695 0.1779 0.9944 49.721 50.000 50 0.530

Chi^2 = 1.26 d.f. = 2 P-value = 0.5316

Benchmark Dose Computation Specified effect = 0.1 Risk Type = Extra risk Confidence level = 0.95 BMD = 0.0155483 BMDL = 0.0129496DRAFT - DO NOT CITE OR QUOTE 161 0 0.2 0.4 0.6 0.8

1

0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16 0.18Fraction Affected

dose Probit Model with 0.95 Confidence Level

11:00 09/10 2010 BMDL BMD ProbitDRAFT - DO NOT CITE OR QUOTE 162 Data Set 2: Incidence of Lung Chronic Active Inflammation in Male Rats, NTP (2002) Summary Two concentration groups (1 and 2 mg/m 3 ) showed statistically significant difference from the control group as reported. The severity increased from minimal to mild as reported when the concentration of vanadium pentoxide increased. The Cochran-Armitage test confirmed the statistically significant trend with Z score as 8.36 and one-sided p -value as <0.0001. Seven dichotomous models available in EPA BMDS (version 2.1.2.) were fitted to this
data set. A 10% extra risk of the incidence was used as the BMR to determine the POD. The goodness of fit, BMC and BMCL for each model are summarized in Table B-7. All models demonstrated adequate goodness of fit p -value ≥ 0.1 and good visual fit. Based on the draft Benchmark Dose Technical Guidance (US EPA, 2000b), the Logistic model was selected as the model for POD computation. The BMCL 10 of this model was 0.029 mg/m 3 and regarded as one of the candidate PODs.

Table B-7: Benchmark Modeling Results for Incide nce of Lung Chronic Active Inflammation in Male Rats, NTP (2002)

Model a Goodness of Fit BM R HEC c

P

Value AIC
b Largest Scaled Residual Scaled Residual of

Interest BMC (mg/m 3 ) BMCL d (mg/m 3 )

Logistic 0.40 192. 5 -0.92 -0.92

Extr

a Risk 10% 0.035 0.029

Probit 0.37 192. 7 -1.00 -1.00 0.032 0.027

LogProbit 0.67 192. 8 0.29 -0.26 0.046 0.032 LogLogist ic 0.60 192. 9 -0.34 -0.34 0.045 0.031

Gamma 0.40 193. 3 0.58 -0.52 0.042 0.027

Weibull 0.27 193. 9 -0.75 -0.75 0.038 0.024 Multistage

e (Stage2) 0.24 194. 0 0.88 -0.66 0.040 0.019

a Selected (best-fitting) model shown in first row, in boldface type.
b AIC=Akaike Information Criterion.

c HEC=Human Equivalent Concentration.
d BMCL= the lower bound of BMC at 95% confidence level.

e For Multistage model, up to 3 stages were tested. Only the model with the lowest AIC and lowest stage was reported here.

BMDS output file

====================================================================DRAFT - DO NOT CITE OR QUOTE 163 Logistic Model. (Version: 2.13; Date: 10/28/2009) Input Data File: C:\USEPA\BMDS212\Data\V2O5\NTP_2002NonCancerMaleRats\MaleRatsLungChronicActiveInflammation\NTP_20 02_Lung Chronic Active Inflammation_Logistic_0.1.(d) Gnuplot Plotting File: C:\USEPA\BMDS212\Data\V2O5\NTP_2002NonCancerMaleRats\MaleRatsLungChronicActiveInflammation\NTP_20 02_Lung Chronic Active Inflammation_Logistic_0.1.plt

==================================================================== The form of the probability function is: P[response] = 1/[1+EXP(-intercept-slope*dose)]

Dependent variable = Response Independent variable = Dose Slope parameter is not restricted

Total number of observations = 4 Total number of records with missing values = 0 Maximum number of iterations = 250 Relative Function Convergence has been set to: 1e-008 Parameter Convergence has been set to: 1e-008

Default Initial Parameter Values background = 0 Specified intercept = -2.2183 slope = 21.8588 Asymptotic Correlation Matrix of Parameter Estimates ( *** The model parameter(s) -background have been estimated at a boundary point, or have been specified by the user, and do not appear in the correlation matrix )

intercept slope intercept 1 -0.82 slope -0.82 1

Parameter Estimates 95.0% Wald Confidence Interval Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit intercept -2.29582 0.320666 -2.92431 -1.66732 slope 23.0148 3.22048 16.7027 29.3268

Analysis of Deviance Table Model Log(likelihood) # Param's Deviance Test d.f. P-value Full model -93.3159 4 Fitted model -94.2613 2 1.8908 2 0.3885 Reduced model -132.664 1 78.6961 3 <.0001 AIC: 192.523 Goodness of Fit Scaled Dose Est._Prob. Expected Observed Size Residual

------------------------------------------------------------------------ 0.0000 0.0915 4.574 5.000 50 0.209 0.0441 0.2174 10.654 8.000 49 -0.919 0.0884 0.4350 20.880 24.000 48 0.908 0.1779 0.8578 42.892 42.000 50 -0.361 Chi^2 = 1.84 d.f. = 2 P-value = 0.3976

Benchmark Dose Computation Specified effect = 0.1 Risk Type = Extra risk Confidence level = 0.95 BMD = 0.0345486 BMDL = 0.0285337DRAFT - DO NOT CITE OR QUOTE 164 0 0.2 0.4 0.6 0.8

1

0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16 0.18Fraction Affected

dose Logistic Model with 0.95 Confidence Level

11:24 09/10 2010 BMDL BMD Logistic

Data Set 3: Incidence of Larynx Chronic Inflammation in Male Rats, NTP (2002) Summary Three concentration groups (0.5, 1 and 2 mg/m 3 ) showed statistically significant difference from the control group as reported. The severity increased from minimal to about mild as reported when the concentration of vanadium pentoxide increased. The Cochran-Armitage test confirmed the statistically significant trend with Z score as 4.79 and one-sided p -value as

<0.0001. Seven dichotomous models available in EPA BMDS (version 2.1.2.) were fitted to this
data set. A 10% extra risk of the incidence was used as the BMR to determine the POD. The goodness of fit, BMC and BMCL for each model are summarized in Table B-8. Only one model demonstrated adequate goodness of fit p -value ≥ 0.1 and good visual fit. Based on the draft Benchmark Dose Technical Guidance (US EPA, 2000b), the LogLogistic model was selected as the model for POD computation. The BMCL 10 of this model was 0.012 mg/m 3 and regarded as one of the candidate PODs.

Table B-8: Benchmark Modeling Results for Incide nce of Larynx Chronic Inflammation in Male Rats, NTP (2002)

Model a Goodness of Fit

BMR HEC c

P

Value AIC
b Largest Scaled Residual Scaled Residual of

Interest BMC (mg/m 3 ) BMCL d (mg/m 3 ) LogLogistic 0.12 229.1 1.63 -0.27 Extra Risk 10% 0.017 0.012 LogProbit 0.09 229.8 -1.37 -0.01 0.005 0.000 Multistage e

(Stage1) 0.05 230.7 2.14 -0.65 0.023 0.017DRAFT - DO NOT CITE OR QUOTE 165 Gamma Weibull

Probit 0.01 234.8 2.45 2.45 0.043 0.036 Logistic 0.01 235.2 2.43 2.43 0.046 0.038

a Selected (best-fitting) model shown in first row, in boldface type.
b AIC=Akaike Information Criterion.

c HEC=Human Equivalent Concentration.
d BMCL= the lower bound of BMC at 95% confidence level.

e For Multistage model, up to 3 stages were tested. Only the model with the lowest AIC and lowest stage was reported here.

BMDS output file

==================================================================== Logistic Model. (Version: 2.13; Date: 10/28/2009) Input Data File: C:\USEPA\BMDS212\Data\V2O5\NTP_2002NonCancerMaleRats\MaleRatsLarynxChronicInflammation\NTP_2002_L arynx Chronic Inflammation_LogLogistic_0.1.(d) Gnuplot Plotting File: C:\USEPA\BMDS212\Data\V2O5\NTP_2002NonCancerMaleRats\MaleRatsLarynxChronicInflammation\NTP_2002_L arynx Chronic Inflammation_LogLogistic_0.1.plt

==================================================================== The form of the probability function is: P[response] = background+(1-background)/[1+EXP(-intercept-slope*Log(dose))]

Dependent variable = Response Independent variable = Dose 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 Convergence 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 = 0.0612245 intercept = 1.9744 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 -0.53 intercept -0.53 1

Parameter Estimates 95.0% Wald Confidence Interval Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit background 0.0712314 * * * intercept 1.9037 * * * slope 1 * * * * - Indicates that this value is not calculated.

Analysis of Deviance Table Model Log(likelihood) # Param's Deviance Test d.f. P-value Full model -110.451 4 Fitted model -112.55 2 4.19924 2 0.1225 Reduced model -127.371 1 33.8403 3 <.0001

AIC: 229.101

Goodness of Fit Scaled Dose Est._Prob. Expected Observed Size Residual

------------------------------------------------------------------------ 0.0000 0.0712 3.490 3.000 49 -0.272 0.0473 0.2951 14.754 20.000 50 1.627 0.0929 0.4278 21.390 17.000 50 -1.255DRAFT - DO NOT CITE OR QUOTE 166 0.1796 0.5789 28.366 28.000 49 -0.106

Chi^2 = 4.31 d.f. = 2 P-value = 0.1162

Benchmark Dose Computation Specified effect = 0.1 Risk Type = Extra risk Confidence level = 0.95 BMD = 0.0165573 BMDL = 0.0117279

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7

0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16 0.18Fraction Affected

dose Log-Logistic Model with 0.95 Confidence Level

13:22 09/10 2010 BMDL BMD Log-Logistic

Data Set 4: Incidence of Larynx Respiratory Epithelium, Epiglottis, Hyperplasia in Male Rats, NTP (2002) Summary Three concentration groups (0.5, 1 and 2 mg/m 3 ) showed statistically significant difference from the control group as reported. The severity increased slightly when the concentration of vanadium pentoxide increased. The Cochran-Armitage test confirmed the statistically significant trend with Z score as -6.56 and one-sided p -value as <0.0001. Seven dichotomous models available in EPA BMDS (version 2.1.2.) were fitted to this
data set. A 10% extra risk of the incidence was used as the BMR to determine the POD. The goodness of fit, BMC and BMCL for each model are summarized in Table B-9. Both LogLogistic and LogProbit models demonstrated adequate goodness of fit p -value ≥ 0.1 and good visual fit, but the BMC/BMCL ratio in LogProbit was infinite, which was not acceptable. Based on the draft Benchmark Dose Technical Guidance (US EPA, 2000b), the LogLogistic model was selected as the model for POD computation. The BMCL 10 of this model was 0.006 mg/m 3 and regarded as one of the candidate PODs.DRAFT - DO NOT CITE OR QUOTE 167 Table B-9: Benchmark Modeling Results for In cidence of Larynx Respiratory Epithelium, Epiglottis, Hyperplasia in Male Rats, NTP (2002)

Model a Goodness of Fit BM R HEC c

P

Value AIC
b Largest Scaled Residual Scaled Residual of

Interest BMC (mg/m 3 ) BMCL d (mg/m 3 )
LogLogist ic 0.27 197. 3 1.56 0.000

Extr

a Risk 10% 0.008 0.006

LogProbit 0.14 199. 3 1.60 0.000 0.007 0.000

Gamma

0.04 201. 5 -2.12 0.000 0.013 0.010 Multistage

e (Stage1) Weibull

Probit 0.00 226. 4 3.16 1.180 0.031 0.027

Logistic 0.00 227. 1 3.05 1.078 0.032 0.027

a Selected (best-fitting) model shown in first row, in boldface type.
b AIC=Akaike Information Criterion.

c HEC=Human Equivalent Concentration.
d BMCL= the lower bound of BMC at 95% confidence level.

e For Multistage model, up to 3 stages were tested. Only the model with the lowest AIC and lowest stage was reported here.

BMDS output file

==================================================================== Logistic Model. (Version: 2.13; Date: 10/28/2009) Input Data File: C:\USEPA\BMDS212\Data\V2O5\NTP_2002NonCancerMaleRats\MaleRatsLarynxEpiglottisHyperplasia\NTP_2002 _Larynx Epiglottis Hyperplasia_LogLogistic_0.1.(d) Gnuplot Plotting File: C:\USEPA\BMDS212\Data\V2O5\NTP_2002NonCancerMaleRats\MaleRatsLarynxEpiglottisHyperplasia\NTP_2002 _Larynx Epiglottis Hyperplasia_LogLogistic_0.1.plt

==================================================================== The form of the probability function is: P[response] = background+(1-background)/[1+EXP(-intercept-slope*Log(dose))]

Dependent variable = Response Independent variable = Dose 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 Convergence 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 = 2.65432 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 )DRAFT - DO NOT CITE OR QUOTE 168 intercept

intercept 1

Parameter Estimates 95.0% Wald Confidence Interval Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit background 0 * * * intercept 2.66112 * * * slope 1 * * *

* - Indicates that this value is not calculated.

Analysis of Deviance Table Model Log(likelihood) # Param's Deviance Test d.f. P-value Full model -95.6454 4 Fitted model -97.6263 1 3.96181 3 0.2656 Reduced model -134.962 1 78.6325 3 <.0001

AIC: 197.253

Goodness of Fit Scaled Dose Est._Prob. Expected Observed Size Residual

------------------------------------------------------------------------ 0.0000 0.0000 0.000 0.000 49 0.000 0.0473 0.4038 20.190 18.000 50 -0.631 0.0929 0.5706 28.532 34.000 50 1.562 0.1796 0.7200 35.279 32.000 49 -1.043

Chi^2 = 3.93 d.f. = 3 P-value = 0.2694

Benchmark Dose Computation Specified effect = 0.1 Risk Type = Extra risk Confidence level = 0.95 BMD = 0.00776335 BMDL = 0.00584847

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8

0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16 0.18Fraction Affected

dose Log-Logistic Model with 0.95 Confidence Level

13:40 09/10 2010 BMDL BMD Log-Logistic

Data Set 5: Incidence of Nose Goblet Cell, Respiratory Epithelium, Hyperplasia in Male Rats, NTP (2002) Summary Three concentration groups (0.5, 1 and 2 mg/m 3 ) showed statistically significant difference from the control group as reported. The severity increase slightly as reported when theDRAFT - DO NOT CITE OR QUOTE 169 concentration of vanadium pentoxide increased. The Cochran-Armitage test confirmed the statistically significant trend with Z score as 2.68 and one-sided p -value as 0.0037. Seven dichotomous models available in EPA BMDS (version 2.1.2.) were fitted to this
data set. A 10% extra risk of the incidence was used as the BMR to determine the POD. The goodness of fit, BMC and BMCL for each model are summarized in Table B-10. Five models demonstrated adequate goodness of fit p -value ≥ 0.1 and good visual fit, but LogProbit model failed to compute a reasonable BMCL. Based on the draft Benchmark Dose Technical Guidance (U.S. EPA, 2000), the LogLogistic model was selected as the model for POD computation. The BMCL 10 of this model was 0.026 mg/m 3 and regarded as the one of the
candidate PODs.

Table B-10: Benchmark Modeling Results for Incidence of Nose Goblet Cell, Respiratory Epithelium, Hyperplasia in Male Rats, NTP (2002)

Model a Goodness of Fit

BMR HEC c

P

Value AIC
b Largest Scaled Residual Scaled Residual of

Interest BMC (mg/m 3 ) BMCL d (mg/m 3 ) LogLogistic 0.16 213.2 1.66 1.656

Extra Risk 10% 0.044 0.026 LogProbit 0.31 212.8 -0.83 -0.003 0.002 failed

Gamma

0.12 213.7 1.75 1.75 0.052 0.033 Multistage e

(Stage1) Weibull

Probit 0.08 214.9 1.787 -0.155 0.077 0.056 Logistic 0.07 215.0 1.78 -0.128 0.081 0.060

a Selected (best-fitting) model shown in first row, in boldface type.
b AIC=Akaike Information Criterion.

c HEC=Human Equivalent Concentration.
d BMCL= the lower bound of BMC at 95% confidence level.

e For Multistage model, up to 3 stages were tested. Only the model with the lowest AIC and lowest stage was reported here.

BMDS output file

==================================================================== Logistic Model. (Version: 2.13; Date: 10/28/2009) Input Data File: C:\USEPA\BMDS212\Data\V2O5\NTP_2002NonCancerMaleRats\MaleRatsNoseGobletEpiHyperplasia\NTP_2002_La rynx Goblet Hyperplasia _LogLogistic_0.1.(d) Gnuplot Plotting File: C:\USEPA\BMDS212\Data\V2O5\NTP_2002NonCancerMaleRats\MaleRatsNoseGobletEpiHyperplasia\NTP_2002_La rynx Goblet Hyperplasia _LogLogistic_0.1.plt

==================================================================== The form of the probability function is: P[response] = background+(1-background)/[1+EXP(-intercept-slope*Log(dose))]

Dependent variable = Response Independent variable = Dose Slope parameter is restricted as slope >= 1

Total number of observations = 4DRAFT - DO NOT CITE OR QUOTE 170 Total number of records with missing values = 0 Maximum number of iterations = 250 Relative Function Convergence 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 = 0.0816327 intercept = 1.21717 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 -0.72 intercept -0.72 1

Parameter Estimates 95.0% Wald Confidence Interval Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit background 0.110444 * * * intercept 0.926382 * * * slope 1 * * *

* - Indicates that this value is not calculated.

Analysis of Deviance Table Model Log(likelihood) # Param's Deviance Test d.f. P-value Full model -102.873 4 Fitted model -104.611 2 3.47486 2 0.176 Reduced model -109.105 1 12.4644 3 0.00595

AIC: 213.221

Goodness of Fit Scaled Dose Est._Prob. Expected Observed Size Residual

------------------------------------------------------------------------ 0.0000 0.1104 5.412 4.000 49 -0.643 0.0473 0.2054 10.270 15.000 50 1.656 0.0929 0.2794 13.691 12.000 49 -0.539 0.1796 0.3881 18.627 17.000 48 -0.482

Chi^2 = 3.68 d.f. = 2 P-value = 0.1590

Benchmark Dose Computation Specified effect = 0.1 Risk Type = Extra risk Confidence level = 0.95 BMD = 0.0439982 BMDL = 0.026051DRAFT - DO NOT CITE OR QUOTE 171 0 0.1 0.2 0.3 0.4 0.5

0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16 0.18Fraction Affected

dose Log-Logistic Model with 0.95 Confidence Level

14:08 09/10 2010 BMDL BMD Log-Logistic

Data Set 6: Incidence of Lung Alveolar Epithelium Hyperplasia in Female Rats, NTP (2002) Summary Two concentration groups (1 and 2 mg/m 3 ) showed statistically significant difference from the control as reported. The severity of these nonneoplastic lesions increased from minimalto moderate as reported when the concentration of vanadium pentoxide increased. The CochranArmitage test confirmed the statistically significant trend with Z score as 9.53 and one-sided p

value as <0.0001. Seven dichotomous models available in EPA BMDS (version 2.1.2.) were fitted to this
data set. A 10% extra risk of the combined incidence was used as the BMR to determine the POD. The goodness of fit, BMC and BMCL for each model are summarized in Table B-11. Five models demonstrated adequate goodness of fit p -value ≥ 0.1, but the extreme curvature of LogLogistic model did not reelect in the observed data. Based on the draft Benchmark Dose Technical Guidance (US EPA, 2000b), the Gamma model was selected as the model for POD computation. The BMCL 10 of this model was 0.063 mg/m 3 and regarded as one
of the candidate PODs.DRAFT - DO NOT CITE OR QUOTE 172 Table B-11: Benchmark Modeling Results for Incide nce of Lung Alveolar Epithelium Hyperplasia in Female Rats, NTP (2002)

Model a Goodness of Fit

BMR HEC c

P

Value AIC
b Largest Scaled Residual Scaled Residual of

Interest BMC (mg/m 3 ) BMCL d (mg/m 3 ) Gamma 0.89 156.2 0.37 -0.12

Extra Risk 10% 0.076 0.063 LogLogisti

c 0.96 155.9 0.20 0.00 0.086 0.070

Weibull 0.91 157.9 0.08 -0.01 0.071 0.053 LogProbit 0.78 157.9 0.20 0.00 0.085 0.068 Multistage

(Stage 3) 0.46 158.1 0.87 -0.24 0.055 0.041 Logistic 0.00 168.1 2.34 -0.78 0.035 0.028

Probit 0.00 168.4 2.32 -1.11 0.031 0.025

a Selected (best-fitting) model shown in first row, in boldface type.
b AIC=Akaike Information Criterion.

c HEC=Human Equivalent Concentration.
d BMCL= the lower bound of BMC at 95% confidence level.

e For Multistage model, up to 3 stages were tested. Only the model with the lowest AIC and lowest stage was reported here.

BMDS output file

==================================================================== Gamma Model. (Version: 2.15; Date: 10/28/2009) Input Data File: C:\USEPA\BMDS212\Data\V2O5\NTP_2002NonCancerFemaleRats\FemaleRatsLungAlBrEpHyperplasia\NTP_2002_L ung Alveolar Bronchiole Combined Epithelium Hyperplasia_Gamma_0.1.(d) Gnuplot Plotting File: C:\USEPA\BMDS212\Data\V2O5\NTP_2002NonCancerFemaleRats\FemaleRatsLungAlBrEpHyperplasia\NTP_2002_L ung Alveolar Bronchiole Combined Epithelium Hyperplasia_Gamma_0.1.plt

==================================================================== The form of the probability function is:

P[response]= background+(1-background)*CumGamma[slope*dose,power], where CumGamma(.) is the cummulative Gamma distribution function

Dependent variable = Response Independent variable = Dose Power parameter is restricted as power >=1

Total number of observations = 4 Total number of records with missing values = 0 Maximum number of iterations = 250 Relative Function Convergence has been set to: 1e-008 Parameter Convergence has been set to: 1e-008

Default Initial (and Specified) Parameter Values Background = 0.156863 Slope = 139.011 Power = 16.8546

Asymptotic Correlation Matrix of Parameter Estimates

( *** The model parameter(s) -Power have been estimated at a boundary point, or have been specified by the user, and do not appear in the correlation matrix )

Background Slope Background 1 -0.32 Slope -0.32 1

Parameter EstimatesDRAFT - DO NOT CITE OR QUOTE 173 95.0% Wald Confidence Interval Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit Background 0.151066 0.03605 0.080409 0.221723 Slope 169.414 9.58875 150.621 188.208 Power 18 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) # Param's Deviance Test d.f. P-value Full model -75.9175 4 Fitted model -76.0968 2 0.358619 2 0.8358 Reduced model -135.531 1 119.227 3 <.0001

AIC: 156.194

Goodness of Fit Scaled Dose Est._Prob. Expected Observed Size Residual

------------------------------------------------------------------------ 0.0000 0.1511 7.402 7.000 49 -0.160 0.0468 0.1523 7.462 8.000 49 0.214 0.0936 0.4286 21.431 21.000 50 -0.123 0.1871 0.9973 49.864 50.000 50 0.369

Chi^2 = 0.22 d.f. = 2 P-value = 0.8945

Benchmark Dose Computation Specified effect = 0.1 Risk Type = Extra risk Confidence level = 0.95 BMD = 0.0756821 BMDL = 0.0626004

0 0.2 0.4 0.6 0.8

1

0 0.05 0.1 0.15Fraction Affected

dose Gamma Multi-Hit Model with 0.95 Confidence Level

14:40 09/10 2010 BMDL BMD Gamma Multi-Hit

Data Set 7: Incidence of Lung Chronic Active Inflammation in Female Rats, NTP (2002) Summary One concentration group (2 mg/m 3 ) showed statistically significant difference from the control group as reported. The severity did not change much when the concentration of vanadium pentoxide increased. The Cochran-Armita ge test confirmed the statistically significant
trend with Z score as 6.71 and one-sided p -value as <0.0001.DRAFT - DO NOT CITE OR QUOTE 174 Seven dichotomous models available in EPA BMDS (version 2.1.2.) were fitted to this
data set. A 10% extra risk of the incidence was used as the BMR to determine the POD. The goodness of fit, BMC and BMCL for each model are summarized in Table B-12. Five models demonstrated adequate goodness of fit p -value ≥ 0.1 and good visual fit. Based on the draft Benchmark Dose Technical Guidance (US EPA, 2000b), the Multistage (Stage3) model was selected as the model for POD computation. The BMCL 10 of this model was 0.048 mg/m 3 and regarded as one of the candidate PODs.

Table B-12: Benchmark Modeling Results for Incidence of Lung Chronic Active Inflammation in Female Rats, NTP (2002)

Model a Goodness of Fit

BMR HEC c

P

Value AIC
b Largest Scaled Residual Scaled Residual of

Interest BMC (mg/m 3 ) BMCL d (mg/m 3 ) Multistage

e (Stage3) 0.82 212.9 -0.51 -0.51

Extra Risk 10% 0.080 0.048

LogProbit 1.00 214.5 0.00 0.00 0.094 0.068

Gamma 0.99 214.5 -0.01 0.00 0.094 0.063 LogLogisti

c 0.97 214.5 -0.03 0.00 0.094 0.065

Weibull 0.94 214.5 -0.06 0.01 0.094 0.059 Logistic 0.04 218.8 1.64 -0.48 0.040 0.033

Probit 0.03 219.4 -1.71 -0.62 0.037 0.031

a Selected (best-fitting) model shown in first row, in boldface type.
b AIC=Akaike Information Criterion.

c HEC=Human Equivalent Concentration.
d BMCL= the lower bound of BMC at 95% confidence level.

e For Multistage model, up to 3 stages were tested. Only the model with the lowest AIC and lowest stage was reported here.

BMDS output file

==================================================================== Multistage Model. (Version: 3.2; Date: 05/26/2010) Input Data File: C:\USEPA\BMDS212\Data\V2O5\NTP_2002NonCancerFemaleRats\FemaleRatsLungChronicActiveInflammation\NT P_2002_Lung Chronic Active Inflammation_Multi3_0.1.(d) Gnuplot Plotting File: C:\USEPA\BMDS212\Data\V2O5\NTP_2002NonCancerFemaleRats\FemaleRatsLungChronicActiveInflammation\NT P_2002_Lung Chronic Active Inflammation_Multi3_0.1.plt

==================================================================== The form of the probability function is: P[response] = background + (1-background)*[1-EXP( -beta1*dose^1-beta2*dose^2-beta3*dose^3)]

The parameter betas are restricted to be positive Dependent variable = Response Independent variable = Dose

Total number of observations = 4 Total number of records with missing values = 0 Total number of parameters in model = 4 Total number of specified parameters = 0DRAFT - DO NOT CITE OR QUOTE 175 Degree of polynomial = 3

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

Default Initial Parameter Values Background = 0.17928 Beta(1) = 0 Beta(2) = 0 Beta(3) = 214.579

Asymptotic Correlation Matrix of Parameter Estimates

( *** The model parameter(s) -Beta(1) -Beta(2) have been estimated at a boundary point, or have been specified by the user, and do not appear in the correlation matrix )

Background Beta(3) Background 1 -0.41 Beta(3) -0.41 1

Parameter Estimates 95.0% Wald Confidence Interval Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit Background 0.186734 * * * Beta(1) 0 * * * Beta(2) 0 * * * Beta(3) 206.262 * * *

* - Indicates that this value is not calculated.

Analysis of Deviance Table Model Log(likelihood) # Param's Deviance Test d.f. P-value Full model -104.257 4 Fitted model -104.451 2 0.389305 2 0.8231 Reduced model -130.861 1 53.209 3 <.0001

AIC: 212.903

Goodness of Fit Scaled Dose Est._Prob. Expected Observed Size Residual

------------------------------------------------------------------------ 0.0000 0.1867 9.150 10.000 49 0.312 0.0468 0.2037 9.983 10.000 49 0.006 0.0936 0.3132 15.659 14.000 50 -0.506 0.1871 0.7896 39.478 40.000 50 0.181

Chi^2 = 0.39 d.f. = 2 P-value = 0.8246

Benchmark Dose Computation Specified effect = 0.1 Risk Type = Extra risk Confidence level = 0.95 BMD = 0.079938 BMDL = 0.0483842 BMDU = 0.0900697 Taken together, (0.0483842, 0.0900697) is a 90% two-sided confidence interval for the BMDDRAFT - DO NOT CITE OR QUOTE 176 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9

0 0.05 0.1 0.15Fraction Affected

dose Multistage Model with 0.95 Confidence Level

15:28 09/10 2010 BMD BMDL Multistage

Data Set 8: Incidence of Larynx Chronic Inflammation in Female Rats, NTP (2002) Summary Three concentration groups (0.5, 1 and 2 mg/m 3 ) showed statistically significant difference from the control group as reported. The severity did not change much when the concentration of vanadium pentoxide increased. The Cochran-Armitage test confirmed the statistically significant trend with Z score as 5.38 and one-sided p -value as <0.0001. Seven dichotomous models available in EPA BMDS (version 2.1.2.) were fitted to this
data set. A 10% extra risk of the incidence was used as the BMR to determine the POD. The goodness of fit, BMC and BMCL for each model are summarized in Table B-13. Five models demonstrated adequate goodness of fit p -value ≥ 0.1 and good visual fit. Based on the draft Benchmark Dose Technical Guidance (US EPA, 2000b), the LogLogistic model was selected as the model for POD computation. The BMCL 10 of this model was 0.003 mg/m 3 and regarded as one of the candidate PODs.

Table B-13: Benchmark Modeling Results for Inci dence of Larynx Chronic Inflammation in Female Rats, NTP (2002)

Model a Goodness of Fit

BMR HEC c

P

Value AIC
b Largest Scaled Residual Scaled Residual of

Interest BMC (mg/m 3 ) BMCL d (mg/m 3 ) LogLogistic 0.46 242.0 -0.88 -0.13 Extra Risk 10% 0.005 0.003 LogProbit 0.27 243.6 -0.89 -0.02 0.003 0.000

Gamma 0.19 243.7 1.58 -0.57 0.007 0.006 Multistage e

(Stage1)DRAFT - DO NOT CITE OR QUOTE 177 Weibull

Probit 0.03 247.5 1.99 1.99 0.013 0.011 Logistic 0.03 247.6 1.97 1.97 0.014 0.011

a Selected (best-fitting) model shown in first row, in boldface type.
b AIC=Akaike Information Criterion.

c HEC=Human Equivalent Concentration.
d BMCL= the lower bound of BMC at 95% confidence level.

e For Multistage model, up to 3 stages were tested. Only the model with the lowest AIC and lowest stage was reported here.

BMDS output file

==================================================================== Logistic Model. (Version: 2.13; Date: 10/28/2009) Input Data File: C:\USEPA\BMDS212\Data\V2O5\NTP_2002NonCancerFemaleRats\FemaleRatsLarynxChronicInflammation\NTP_20 02_Larynx Chronice Inflammation_LogLogistic_0.1.(d) Gnuplot Plotting File: C:\USEPA\BMDS212\Data\V2O5\NTP_2002NonCancerFemaleRats\FemaleRatsLarynxChronicInflammation\NTP_20 02_Larynx Chronice Inflammation_LogLogistic_0.1.plt

==================================================================== The form of the probability function is: P[response] = background+(1-background)/[1+EXP(-intercept-slope*Log(dose))]

Dependent variable = Response Independent variable = Dose 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 Convergence 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 = 0.16 intercept = 3.2396 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 -0.54 intercept -0.54 1

Parameter Estimates 95.0% Wald Confidence Interval Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit background 0.166778 * * * intercept 3.19725 * * * slope 1 * * *

* - Indicates that this value is not calculated.

Analysis of Deviance Table

Model Log(likelihood) # Param's Deviance Test d.f. P-value Full model -118.217 4 Fitted model -118.997 2 1.55893 2 0.4587 Reduced model -137.233 1 38.0314 3 <.0001

AIC: 241.994

Goodness of Fit Scaled Dose Est._Prob. Expected Observed Size Residual

------------------------------------------------------------------------ 0.0000 0.1668 8.339 8.000 50 -0.129 0.0231 0.4678 22.925 26.000 49 0.881 0.0470 0.6123 30.001 27.000 49 -0.880DRAFT - DO NOT CITE OR QUOTE 178 0.0875 0.7347 36.735 37.000 50 0.085

Chi^2 = 1.57 d.f. = 2 P-value = 0.4553

Benchmark Dose Computation Specified effect = 0.1 Risk Type = Extra risk Confidence level = 0.95 BMD = 0.00454158 BMDL = 0.00312534

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9

0 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09Fraction Affected

dose Log-Logistic Model with 0.95 Confidence Level

16:29 09/10 2010 BMDL BMD Log-Logistic

Data Set 9: Incidence of Larynx Respiratory Epithelium, Epiglottis, Hyperplasia in Female Rats, NTP (2002) Summary Three concentration groups (0.5, 1 and 2 mg/m 3 ) showed statistically significant difference from the control group as reported. The severity did not change much when the concentration of vanadium pentoxide increased. The Cochran-Armitage test confirmed the statistically significant trend with Z score as -5.98 and one-sided p -value as <0.0001. Seven dichotomous models available in EPA BMDS (version 2.1.2.) were fitted to this
data set. A 10% extra risk of the incidence was used as the BMR to determine the POD. The goodness of fit, BMC and BMCL for each model are summarized in Table B-14. Both LogLogistic and LogProbit models demonstrated adequate goodness of fit p -value ≥ 0.1 and good visual fit, but LogProbit model failed to compute a reasonable BMCL. Based on the draft Benchmark Dose Technical Guidance (U.S. EPA, 2000), the LogLogistic model was selected as the model for POD computation. The BMCL 10 of this model was 0.003 mg/m 3 and regarded as one of the candidate PODs.DRAFT - DO NOT CITE OR QUOTE 179 Table B-14: Benchmark Modeling Results for In cidence of Larynx Respiratory Epithelium, Epiglottis, Hyperplasia in Female Rats, NTP (2002)

Model a Goodness of Fit BM R HEC c

P

Value AIC
b Largest Scaled Residual Scaled Residual of

Interest BMC (mg/m 3 ) BMCL d (mg/m 3 )
LogLogist ic 0.30 205.3 1.53 0.000

Extr

a Risk 10% 0.004 0.003 LogProbit 0.78 204.2 -0.57 0.000 0.000 failed

Gamma

0.01 212.3 2.85 0.000 0.006 0.005 Multistage

e (Stage1) Weibull

Probit 0.00 234.6 3.258 3.258 0.016 0.013 Logistic 0.00 235.3 -3.295 3.174 0.016 0.014

a Selected (best-fitting) model shown in first row, in boldface type.
b AIC=Akaike Information Criterion.

c HEC=Human Equivalent Concentration.
d BMCL= the lower bound of BMC at 95% confidence level.

e For Multistage model, up to 3 stages were tested. Only the model with the lowest AIC and lowest stage was reported here.

BMDS output file

==================================================================== Logistic Model. (Version: 2.13; Date: 10/28/2009) Input Data File: C:\USEPA\BMDS212\Data\V2O5\NTP_2002NonCancerFemaleRats\FemaleRatsLarynxEpiglottisHyperplasia\NTP_ 2002_Larynx Epiglottis Hyperplasia_LogLogistic_0.1.(d) Gnuplot Plotting File: C:\USEPA\BMDS212\Data\V2O5\NTP_2002NonCancerFemaleRats\FemaleRatsLarynxEpiglottisHyperplasia\NTP_ 2002_Larynx Epiglottis Hyperplasia_LogLogistic_0.1.plt

==================================================================== The form of the probability function is: P[response] = background+(1-background)/[1+EXP(-intercept-slope*Log(dose))]

Dependent variable = Response Independent variable = Dose 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 Convergence 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 = 3.38546 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 95.0% Wald Confidence Interval Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit background 0 * * * intercept 3.3735 * * *DRAFT - DO NOT CITE OR QUOTE 180 slope 1 * * * * - Indicates that this value is not calculated.

Analysis of Deviance Table Model Log(likelihood) # Param's Deviance Test d.f. P-value Full model -99.8781 4 Fitted model -101.66 1 3.56345 3 0.3126 Reduced model -134.962 1 70.1671 3 <.0001

AIC: 205.32

Goodness of Fit Scaled Dose Est._Prob. Expected Observed Size Residual

------------------------------------------------------------------------ 0.0000 0.0000 0.000 0.000 50 0.000 0.0231 0.4029 19.743 25.000 49 1.531 0.0470 0.5781 28.329 26.000 49 -0.674 0.0875 0.7186 35.929 33.000 50 -0.921

Chi^2 = 3.65 d.f. = 3 P-value = 0.3022

Benchmark Dose Computation Specified effect = 0.1 Risk Type = Extra risk Confidence level = 0.95 BMD = 0.00380772 BMDL = 0.0028644

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8

0 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09Fraction Affected

dose Log-Logistic Model with 0.95 Confidence Level

11:10 09/16 2010 BMDL BMD Log-Logistic

Data Set 10: Incidence of Nose Goblet Cell, Respiratory Epithelium, Hyperplasia in Female Rats, NTP (2002) Summary One concentration groups (2 mg/m 3 ) showed statistically significant difference from the control group as reported. The severity did not change much when the concentration of vanadium pentoxide increased. The Cochran-Armita ge test confirmed the statistically significant
trend with Z score as 3.46 and one-sided p -value as 0.0003. Seven dichotomous models available in EPA BMDS (version 2.1.2.) were fitted to this
data set. A 10% extra risk of the incidence was used as the BMR to determine the POD. The goodness of fit, BMC and BMCL for each model are summarized in Table B-15. All models demonstrated adequate goodness of fit p -value ≥ 0.1 and good visual fit.DRAFT - DO NOT CITE OR QUOTE 181 Based on the draft Benchmark Dose Technical Guidance (US EPA, 2000b), the Multistage (Stage2) model was selected as the model for POD computation. The BMCL 10 of this model was 0.014 mg/m 3 and regarded as one of the candidate PODs.

Table B-15: Benchmark Modeling Results for In cidence of Larynx Respiratory Epithelium, Epiglottis, Hyperplasia in Female Rats, NTP (2002)

Model a Goodness of Fit

BMR HEC c

P

Value AIC
b Largest Scaled Residual Scaled Residual of

Interest BMC (mg/m 3 ) BMCL d (mg/m 3 ) Multistage

(Stage2) 0.43 258.3 -0.90 -0.896

Extra Risk 10% 0.038 0.014 Logistic 0.33 258.9 -1.252 0.586 0.024 0.018

Probit 0.32 258.9 -1.273 0.564 0.023 0.018 Weibull ( Quantal

Linear) 0.22 259.7 -1.45 0.337 0.019 0.012

LogLogisti

c 0.28 259.8 0.78 -0.027 0.064 0.014

Gamma 0.28 259.8 0.77 -0.005 0.063 0.014 LogProbit 0.28 259.8 0.765 -0.001 0.062 0.015

a Selected (best-fitting) model shown in first row, in boldface type.
b AIC=Akaike Information Criterion.

c HEC=Human Equivalent Concentration.
d BMCL= the lower bound of BMC at 95% confidence level.

e For Multistage model, up to 3 stages were tested. Only the model with the lowest AIC and lowest stage was reported here.

BMDS output file

==================================================================== Multistage Model. (Version: 3.2; Date: 05/26/2010) Input Data File: C:\USEPA\BMDS212\Data\V2O5\NTP_2002NonCancerFemaleRats\FemaleRatsNoseGobletEpiHyperplasia\NTP_200 2_Nose Goblet Hyperplasia_Multi2_0.1.(d) Gnuplot Plotting File: C:\USEPA\BMDS212\Data\V2O5\NTP_2002NonCancerFemaleRats\FemaleRatsNoseGobletEpiHyperplasia\NTP_200 2_Nose Goblet Hyperplasia_Multi2_0.1.plt

==================================================================== The form of the probability function is: P[response] = background + (1-background)*[1-EXP( -beta1*dose^1-beta2*dose^2)]

The parameter betas are restricted to be positive

Dependent variable = Response Independent variable = Dose

Total number of observations = 4 Total number of records with missing values = 0 Total number of parameters in model = 3 Total number of specified parameters = 0 Degree of polynomial = 2

Maximum number of iterations = 250 Relative Function Convergence has been set to: 1e-008 Parameter Convergence has been set to: 1e-008DRAFT - DO NOT CITE OR QUOTE 182 Default Initial Parameter Values Background = 0.270822 Beta(1) = 0 Beta(2) = 75.6009

Asymptotic Correlation Matrix of Parameter Estimates

( *** The model parameter(s) -Beta(1) have been estimated at a boundary point, or have been specified by the user, and do not appear in the correlation matrix )

Background Beta(2) Background 1 -0.59 Beta(2) -0.59 1

Parameter Estimates 95.0% Wald Confidence Interval Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit Background 0.276279 * * * Beta(1) 0 * * * Beta(2) 71.2588 * * *

* - Indicates that this value is not calculated.

Analysis of Deviance Table Model Log(likelihood) # Param's Deviance Test d.f. P-value Full model -126.318 4 Fitted model -127.169 2 1.70254 2 0.4269 Reduced model -133.292 1 13.9479 3 0.002977

AIC: 258.338

Goodness of Fit Scaled Dose Est._Prob. Expected Observed Size Residual

------------------------------------------------------------------------ 0.0000 0.2763 13.814 13.000 50 -0.257 0.0231 0.3033 15.167 18.000 50 0.872 0.0470 0.3815 19.077 16.000 50 -0.896 0.0875 0.5806 29.030 30.000 50 0.278

Chi^2 = 1.71 d.f. = 2 P-value = 0.4262

Benchmark Dose Computation Specified effect = 0.1 Risk Type = Extra risk Confidence level = 0.95 BMD = 0.0384521 BMDL = 0.0135524 BMDU = 0.0544996

Taken together, (0.0135524, 0.0544996) is a 90% two-sided confidence interval for the BMDDRAFT - DO NOT CITE OR QUOTE 183 0.1 0.2 0.3 0.4 0.5 0.6 0.7

0 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09Fraction Affected

dose Multistage Model with 0.95 Confidence Level

16:49 09/10 2010 BMD BMDL MultistageDRAFT - DO NOT CITE OR QUOTE 184 APPENDIX C - BENCHMARK CONCENTRAT ION MODELING OF COMBINED LUNG ALVEOLAR AND BRONCHIOLAR TUMOR DATA SETS FROM NTP STUDIES (2002)

To derive the cancer slope factor of vanadi um pentoxide, combined alveolar/bronchiolar adenoma and carcinoma data sets in B6C3F1 mice (Table C-2) by NTP (2002) were selected because of their biological and statistical significance. The highlights of the benchmark concentration modeling results are:

• Cancer slope factor: The cancer slope factor was estimated as 3.4 per mg/m 3 after linear extrapolation. The selected model for each data set, the candidate PODs and the calculation of cancer slope factor
are summarized in Table C-1.

• BMDS modeling:Each data set was firstly fitted with the recommended dichotomous model, multistagecancer model, through EPA BMDS (version 2.1.2); if the goodness of fit p -value < 0.05, other dichotomous models available in EPA BMDS (version 2.1.2) were used; if still no model showed adequate goodness of fit p -value ≥ 0.05, the highest dose was dropped for further modeling. Following the general model selection steps outlined in the draft Benchmark Dose Technical Guidance (U.S. EPA, 2000), the best-fitting model was selected for each data set to estimate the candidate POD, which was the BMCL at the pre-determined BMR.

• BMR: Since all the non-control concentrations showed essentially a plateau response, the tumor data sets provided limited information about the concentration-response relationship. So, BMR for each data set was calculated based on the response at the control and the first non-control concentration groups.

• Multistage Weibull (MSW) time-to-tumor modeling: Since the survival curves reported by NTP (2002) showed a high percentage of deaths(22-46%) at the end of 2-year studies across the different concentrations groups, MSW time-totumor model was also used with these data sets. However, no adequate fit was noticed for either
male or female mice data set.DRAFT - DO NOT CITE OR QUOTE 185 Table C- 1. Summary of Candidate PODs and Cancer Slope Factors.

Animal BMR (Extra Risk) a HEC b Cancer Slope Factor d (per mg/m 3 ) BMC (mg/m 3 ) BMCL c (Candidate POD, mg/m 3 )

Male B6C3F 1 Mice 0.71 0.360 0.208 3.4

Female B6C3F 1 Mice 0.67 0.237 0.161 4.2

a BMR was calculated based on the response at the contro l concentration and the first non-control concentration (Table C-6).
b HEC=Human Equivalent Concentration.

c BMCL= the lower bound of BMC at 95% confidence level.
d Cancer Slope Factor = BMR/BMCL, as linear extrapolation was used.

C.1. TREND TESTS BEFORE MODELING Two inhalation carcinogenesis data sets were selected because of the biological and the statistical significance reported by NTP (2002). Cochran-Armitage test was conducted to confirm the significance of the statistical trend for the selected data sets (Table C-2) before modeling.

Table C-2: Trend Tests on the Inhalation Carcinogenesis Data Sets in Mice from the 2-Year Inhalation Studies (NTP, 2002)
Endpoint Concentration as Reported (mg/m 3 ) a Trend Test b
0.0 1.0 2.0 4.0 Z -Score p -value Incidence c in Male B6C3F 1 Mice Lung Alveolar/Bronchiolar Adenoma and Carcinoma 22/50 42/50 43/50 43/50 4.14 <0.0001

Incidence c in Female B6C3F 1 Mice Lung Alveolar/Bronchiolar Adenoma and Carcinoma 1/48 32/47 35/48 32/49 5.19 <0.0001

a Concentrations are as reported by NTP (2002).
b One-sided Cochran-Armitage trend test.

c Incidence = (number of animals with alveolar/bronchiol ar adenoma and/or carcinoma)/( animal sample size). Only the animals alive at 52 weeks or when the first tumor appeared, whichever was earlier, was counted toward the sample size.

C.2. DOSE CONVERSION In the carcinogenesis studies reported by NTP (2002), mice (B6C3F 1 ) were exposed toDRAFT - DO NOT CITE OR QUOTE 186 vanadium pentoxide through inhalation. To analyze the concentration response effect, the reported concentrations of vanadium pentoxide were converted to human equivalent concentrations before any modeling and extrapolation. Firstly, the average life time animal body weights of rats were estimated based on the mean body weight at different weeks (Table C-3). Secondly, following the US EPA inhalation methods (1994), RDDRs (regional deposited dose ratio) were calculated with the RDDR program designed by US EPA (1994) and summarized in Table C-4. Then, the human equivalent concentration for each concentration/sex group was calculated from the reported concentration inmice by multiply the continuous exposure adjustment factor and the pulmonary RDDR (Table C5).

Table C-3: Average Life Time Animal Body Weight of Mice for 2-Year Inhalation Studies of Vanadium Pentoxide (NTP, 2002)

Vanadium Pentoxide Concentration as Reported (mg/m 3 ) Mean Animal Body Weight a (g) Average Life Time Animal Body Weight b (g) 1-13 weeks 14-52 weeks 53-104 weeks Male Mice (B6C3F 1 ) 0 30.8 46.7 54.1 48.9 1 30.5 47.0 52.9 48.3 2 30.3 45.4 51.4 46.9 4 29.8 43.7 46.1 43.6 Female Mice (B6C3F 1 ) 0 25.5 42.7 56.1 47.7 1 24.9 40.9 50.7 44.2 2 24.8 36.8 44.8 39.7 4 24.5 34.2 40.1 36.3

a As reported in Table 20-21of the NTP report (2002).
b “ Average Life Time Animal Body Weight” = ( “Mean Body Weight 1-13 Weeks” * 13 +“ Mean Body Weight 14 - 52 Weeks” * 39 + “Mean Body Weight 53-104 Weeks” * 52) /104DRAFT - DO NOT CITE OR QUOTE 187 Table C-4: RDDRs for Different Concentration/Sex Gr oup of Mice in the 2-Year Inhalation Studies of Vanadium Pentoxide (NTP, 2002)

Vanadium Pentoxide Concentration as Reported (mg/m 3 ) Average Life Time Animal Body Weight a (g) Average MMAD
b Average GSD c RDDR d

Pulmonary

Male Mice (B6C3F 1 )
0 48.9 e

1.26 1.87 1.168 e

1 48.3 e 1.168 e

2 46.9 e 1.168 e

4 43.6 1.134 Female Mice (B6C3F 1 )
0 47.7 e

1.26 1.87 1.168 e

1 44.2 1.143
2 39.7 1.077
4 36.3 1.023

a Average life time animal body weight of each concen tration/sex group was calculated in Table C-3.
b MMAD =mass median aerodynamic diameter; calculated ba sed on the MMADs reported for 2-year studies (NTP, 2002) .

c GSD = geometric standard deviation; calculated base d on the GDSs reported for 2-year studies (NTP, 2002) .
d RDDR = regional deposited dose ratio; calculated with the RDDR program from USA EPA (1994);

e Since the average life time animal body weight was out of the mice weight range (17-46g) accepted by RDDR program (V.2.3, US EPA), 46g was used to calculate the corresponding RDDRs.

Table C-5: Human Equivalent Concentrations of Vanadium Pentoxide in the 2-Year Inhalation Studies (NTP, 2002)

Concentration as Reported a

(mg/m 3 ) Continuous Exposure Adjustment

Factor b RDDR c HEC d (mg/m 3 )
Pulmonary Pulmonary
Male Mice (B6C3F 1 ) 0 0.179 1.168 0.00 1 0.179 1.168 0.21 2 0.179 1.168 0.42 4 0.179 1.134 0.81 Female Mice (B6C3F 1 ) 0 0.179 1.168 0.00 1 0.179 1.143 0.20 2 0.179 1.077 0.38 4 0.179 1.023 0.73

a “Toxicology and carcinogenesis studies of vanadium pentoxide in F344/N rats and B6C3F 1 Mice”, NTP, 2002.
b “Continuous Exposure Adjustment Factor” = (6/24) * (5/7 ); animals were exposed to vanadium pentoxide 6 hours per day and 5 days per week.

c Please refer to Appendix Table C-4.DRAFT - DO NOT CITE OR QUOTE 188 d HEC=Human Equivalent Concentration = “Concentrati on as Reported” * “Continuous Exposure Adjustment Factor ” * “RDDR”

C.3. BMR CALCULATION
Since all non-control concentrations from the NTP carcinogenesis studies (2002) showedessentially a plateau response, the data set provided limited information about the concentrationresponse relationship because the complete range of response from background to maximum must occur somewhere below the lowest dose, thus the BMD may be just below the first dose or orders of magnitudes lower. So, a BMR estimated based on the response at the control concentration and the first non-control concentration was calculated (Table C-6), and then used
for estimation of BMCL.

Table C-6: BMR Estimation for Male and Female Mice Data Sets.

Animal P(Control)

a P(1st Noncontrol Concentration)
b Calculated BMR (Extra Risk) c Male B6C3F 1 Mice 0.44 0.84 0.71 Female B6C3F 1 Mice 0.02 0.68 0.67

a The combined alveolar/bronchiolar adenoma and carcinoma incidence in the control group.
b The combined alveolar/bronchiolar ad enoma and carcinoma incidence in the 1 st non-control concentration group.

c Calculated BMR = [P(1st Non-control Con centration) - P(Control)]/[1- P(Control)].

C.4. BMDS MODELING FOR INHALATION CARCINOGENESIS DATA SETS Each data set was firstly fitted with dichotomous multistage-cancer model provided in EPA BMDS (version 2.1.2); if the goodness of fit p -value < 0.05, other dichotomous models available in EPA BMDS (version 2.1.2) were fitted; if still no model showed adequate goodness
of fit p -value ≥ 0.05, the highest dose was dropped for further modeling. Following the general model selection steps outlined in the draft Benchmark Dose Technical Guidance (U.S. EPA, 2000), the best-fitting model was selected for each data set to estimate the candidate POD, which was the BMCL at the calculated BMR for each data set. The selected models and candidate PODs for all endpoints are summarized in Table C-1.

Lung Tumor Data Set 1: Combined Incidence of Alveolar/Bronchiolar Adenoma and Carcinoma in Male Mice, NTP (2002). Summary The Cochran-Armitage test confirmed the statistically significant trend with Z score as 4.14 and one-sided p -value as <0.0001.DRAFT - DO NOT CITE OR QUOTE 189 Because all the non-control concentrations showed a similar effect, a BMR as 71% extra risk was calculated and used to estimate the POD. The goodness of fit, BMC and BMCL for each
model are summarized in Table C- 7 and C-8. Since the primary cancer model (Multistage-cancer) did not show an adequate fit, six other dichotomous models available in EPA BMDS (version 2.1.2.) were fitted to this data set. Two models demonstrated adequate goodness of fit p -value ≥ 0.05 and good visual fit, but LogProbit model failed to compute a reasonable BMCL. Based on the draft Benchmark Dose Technical Guidance (U.S. EPA, 2000), the LogLogistic model was selected and BMCL 71 of this model was 0.208 mg/m 3

. Further BMD modeling was performed for comparison purposes. Since the primary cancer model (Multistage-cancer) did not show adequate fit with all doses, the highest concentration was dropped for further modeling. After dropping the high dose, the primary cancer model (Multistage-cancer) demonstrated adequate goodness of fit p -value ≥ 0.05 and good visual fit.

Table C- 7. BMDS Modeling Results for Combined Incidence of Alveolar/Bronchiolar Adenoma and Carcinoma in Male Mice, NTP (2002).

Model a Goodness of Fit BM R HEC P

Valu

e AIC
b Largest Scaled Residual Scaled Residual of

Interest BMC (mg/m 3 ) BMCL c (mg/m 3 ) Primary Cancer ModelsMultistageCancer Stage 1,2 and 3 0.01 207. 0 2.05 0.72 Extr

a Risk 71% 0.532 0.379

Other Dichotomous Models
LogLogistic 0.19 200. 632 -1.42 0.04

Extr

a Risk 71% 0.360 0.208

LogProbit 0.88 199. 6 0.13 -0.06 0.146 failed

Gamma 0.01 207. 0 2.05 0.72 0.532 0.379 Weibull Logistic 0.00 209. 4 2.15 0.96 0.609 0.447

Probit 0.00 210. 4 2.19 -1.54 0.654 0.495

a Selected model is the best-fitting model for the data set based on the draft Benchmark Dose Technical Guidance (2000).
b HEC=Human Equivalent Concentration.

c BMCL= the lower bound of BMC at 95% confidence level.DRAFT - DO NOT CITE OR QUOTE 190 BMDS output file

==================================================================== Logistic Model. (Version: 2.13; Date: 10/28/2009) Input Data File: C:\USEPA\BMDS212\Data\V2O5\NTP_2002Cancer\MaleMiceTumor4DosesAllModel\NTP_2002_MaleMiceTum or4DosesAllModels_LogLogistic_0.71.(d) Gnuplot Plotting File: C:\USEPA\BMDS212\Data\V2O5\NTP_2002Cancer\MaleMiceTumor4DosesAllModel\NTP_2002_MaleMiceTum or4DosesAllModels_LogLogistic_0.71.plt

==================================================================== The form of the probability function is: P[response] = background+(1-background)/[1+EXP(-intercept-slope*Log(dose))]

Dependent variable = Effect Independent variable = Dose 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 Convergence 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 = 0.44 intercept = 1.9384 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 -0.58 intercept -0.58 1

Parameter Estimates 95.0% Wald Confidence Interval Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit background 0.451808 * * * intercept 1.91813 * * * slope 1 * * * * - Indicates that this value is not calculated.

Analysis of Deviance Table Model Log(likelihood) # Param's Deviance Test d.f. P-value
Full model -96.7763 4 Fitted model -98.3159 2 3.07913 2 0.2145
Reduced model -112.467 1 31.3814 3 <.0001

AIC: 200.632

Goodness of Fit
Scaled Dose Est._Prob. Expected Observed Size Residual

------------------------------------------------------------------------ 0.0000 0.4518 22.590 22.000 50 -0.168

0.2100 0.7744 38.719 42.000 50 1.110

0.4200 0.8580 42.898 43.000 50 0.041DRAFT - DO NOT CITE OR QUOTE 191 0.8100 0.9159 45.793 43.000 50 -1.423 Chi^2 = 3.29 d.f. = 2 P-value = 0.1934

Benchmark Dose Computation Specified effect = 0.71 Risk Type = Extra risk Confidence level = 0.95

BMD = 0.359607

BMDL = 0.208416

0.3 0.4 0.5 0.6 0.7 0.8 0.9

1

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8Fraction Affected

dose Log-Logistic Model with 0.95 Confidence Level

15:37 09/20 2010 BMDL BMD Log-Logistic

Table C- 8. BMDS Modeling Results for Combined Incidence of Alveolar/Bronchiolar Adenoma and Carcinoma in Male Mice after Dropping the Highest Concentration, NTP (2002).

Model a Goodness of Fit

BMR HEC

P

Value AIC b Largest Scaled Residual Scaled Residual of Interest BMC (mg/m 3 ) BMCL c (mg/m 3 )
Primary Cancer ModelsMultistageCancer Stage 1,2

and 3 0.12 159.5 1.19 Extra Risk 71% 0.310 0..220

Other Dichotomous Models LogLogistic 0.43 157.7 0.52

Extra Risk 71% 0.260 0.140 Gamma 0.12 159.5 1.19 0.310 0.220 Weibull

Probit 0.04 161.5 -1.03 0.340 0.270 Logistic 0.05 160.1 -1.09 0.330 0.250 LogProbit NA 159.1 0.00 0.190 failed

a Selected model is the best-fitting model for the data set based on the draft Benchmark Dose Technical Guidance (2000).
b HEC=Human Equivalent Concentration.

c BMCL= the lower bound of BMC at 95% confidence level.DRAFT - DO NOT CITE OR QUOTE 192 ==================================================================== Multistage Cancer Model. (Version: 1.9; Date: 05/26/2010) Input Data File: C:\USEPA\BMDS212\Data\V2O5\NTP_2002Cancer\MaleMiceTumor3DosesAllModel\NTP_2002_MaleMiceTumor3Dose sAllModels_MultiCanc1_0.71.(d) Gnuplot Plotting File: C:\USEPA\BMDS212\Data\V2O5\NTP_2002Cancer\MaleMiceTumor3DosesAllModel\NTP_2002_MaleMiceTumor3Dose sAllModels_MultiCanc1_0.71.plt Thu Feb 17 17:24:36 2011

==================================================================== The form of the probability function is: P[response] = background + (1-background)*[1-EXP( -beta1*dose^1)] The parameter betas are restricted to be positive Dependent variable = Effect Independent variable = Dose Total number of observations = 3 Total number of records with missing values = 0 Total number of parameters in model = 2 Total number of specified parameters = 0 Degree of polynomial = 1

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

Default Initial Parameter Values Background = 0.535297 Beta(1) = 3.3007 Asymptotic Correlation Matrix of Parameter Estimates Background Beta(1) Background 1 -0.6 Beta(1) -0.6 1

Parameter Estimates 95.0% Wald Confidence Interval Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit Background 0.461149 * * * Beta(1) 4.04221 * * * * - Indicates that this value is not calculated.

Analysis of Deviance Table Model Log(likelihood) # Param's Deviance Test d.f. P-value Full model -76.5282 3 Fitted model -77.7667 2 2.47711 1 0.1155 Reduced model -89.871 1 26.6857 2 <.0001 AIC: 159.533

Goodness of Fit Scaled Dose Est._Prob. Expected Observed Size Residual

------------------------------------------------------------------------ 0.0000 0.4611 23.057 22.000 50 -0.300 0.2100 0.7694 38.471 42.000 50 1.185 0.4200 0.9013 45.067 43.000 50 -0.980 Chi^2 = 2.45 d.f. = 1 P-value = 0.1172

Benchmark Dose Computation Specified effect = 0.71 Risk Type = Extra risk Confidence level = 0.95 BMD = 0.306237 BMDL = 0.220508 BMDU = 0.471328 Taken together, (0.220508, 0.471328) is a 90 % two-sided confidence interval for the BMD Multistage Cancer Slope Factor = 3.21985DRAFT - DO NOT CITE OR QUOTE 193 0.3 0.4 0.5 0.6 0.7 0.8 0.9

1

0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4Fraction Affected

dose Multistage Cancer Model with 0.95 Confidence Level

17:34 02/17 2011 BMD BMDL Multistage Cancer Linear extrapolation

Lung Tumor Data Set 2: Combined Incidence of Alveolar/Bronchiolar Adenoma and Carcinoma in Female Mice, NTP (2002). Summary The Cochran-Armitage test confirmed the statistically significant trend with Z score as 5.19 and one-sided p -value as <0.0001. Because all the non-control concentrations showed a similar effect, a BMR as 67% extra risk was calculated and used to determine the POD. The goodness of fit, BMC and BMCL for
each model are summarized in Table C-9 and C-10. Since the primary cancer model (Multistage-cancer) did not show an adequate fit, six other dichotomous models available in EPA BMDS (version 2.1.2.) were fitted to this data set. Only one model demonstrated adequate goodness of fit p -value ≥ 0.05, however, it failed to compute BMC and BMCL. So, the highest concentration was dropped for further modeling. After dropping the highest dose, the primary cancer model (Multistage-cancer) still did not show an adequate fit. Then six other dic hotomous models available in EPA BMDS (version 2.1.2.) were tried. Only the LogLogistc model demonstrated adequate goodness of fit p -value ≥ 0.05 and good visual fit. Based on the draft Benchmark Dose Technical Guidance (U.S. EPA, 2000), the LogLogistic model was selected and BMCL 67 of this model was 0.161 mg/m 3

.

Table C- 9. BMDS Modeling Results for Combined Incidence of Alveolar/Bronchiolar Adenomas and Carcinomas with All Four Concentrations in Female Mice, NTP (2002). Model a Goodness of Fit BMR HECDRAFT - DO NOT CITE OR QUOTE 194 P

Value AIC b Largest Scaled Residual Scaled Residual of Interest BMC (mg/m 3 ) BMCL c (mg/m 3 )
Primary Cancer ModelsMultistageCancer Stage 1,2 and 3 0.00 218.8 -3.90 1.33 Extra Risk 67% 0.435 0.359

Other Dichotomous Models LogProbit 0.72 192.6 0.62 -999.00

Extra Risk 67% Failed d Failed d LogLogistic 0.00 202.7 -2.87 0.52 0.351 0.255 Gamma 0.00 218.8 -3.90 1.33 0.435 0.359 Weibull Logistic 0.00 239.4 -4.00 -2.62 0.657 0.532

Probit 0.00 239.6 -3.97 -2.48 0.670 0.553

a Selected model is the best-fitting model for the data set based on the draft Benchmark Dose Technical Guidance (2000).
b HEC=Human Equivalent Concentration.

c BMCL= the lower bound of BMC at 95% confidence level.

Table C- 10. BMDS Modeling Results for Combined Incidence of Alveolar/Bronchiolar Adenoma and Carcinoma in Female Mice after Dropping the Highest Concentration, NTP (2002).

Model a Goodness of Fit

BMR HEC

P

Value AIC b Largest Scaled Residual Scaled Residual of Interest BMC (mg/m 3 ) BMCL c (mg/m 3 )
Primary Cancer ModelsMultistageCancer Stage 1,2 and 3 0.06 132.2 1.42 1.42 Extra Risk 67% 0.264 0.213

Other Dichotomous Models LogLogistic 0.37 129.453 -0.67 0.60

Extra Risk 67% 0.237 0.161 Gamma 0.06 132.2 1.42 1.42 0.264 0.213 Weibull

Probit 0.00 145.9 3.00 -1.85 0.309 0.272 Logistic 0.00 146.5 2.84 -1.99 0.303 0.263 LogProbit NA 130.7 0.00 0.00 0.190 failed

a Selected model is the best-fitting model for the data set based on the draft Benchmark Dose Technical Guidance (2000).
b HEC=Human Equivalent Concentration.

c BMCL= the lower bound of BMC at 95% confidence level.

BMDS output fileDRAFT - DO NOT CITE OR QUOTE 195 ==================================================================== Logistic Model. (Version: 2.13; Date: 10/28/2009) Input Data File: C:\USEPA\BMDS212\Data\V2O5\NTP_2002Cancer\FemaleMiceTumor3DosesAllModels\NTP_2002_FemaleMiceTumor 3DosesAllModels_LogLogistic_0.67.(d) Gnuplot Plotting File: C:\USEPA\BMDS212\Data\V2O5\NTP_2002Cancer\FemaleMiceTumor3DosesAllModels\NTP_2002_FemaleMiceTumor 3DosesAllModels_LogLogistic_0.67.plt

==================================================================== The form of the probability function is:

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

Dependent variable = Effect Independent variable = Dose Slope parameter is restricted as slope >= 1

Total number of observations = 3 Total number of records with missing values = 0 Maximum number of iterations = 250 Relative Function Convergence 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 = 0.0208333 intercept = 2.36858 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 -0.14 intercept -0.14 1

Parameter Estimates 95.0% Wald Confidence Interval Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit background 0.0210689 * * * intercept 2.14725 * * * slope 1 * * * * - Indicates that this value is not calculated.

Analysis of Deviance Table Model Log(likelihood) # Param's Deviance Test d.f. P-value Full model -62.3295 3 Fitted model -62.7264 2 0.79377 1 0.373 Reduced model -98.9486 1 73.2384 2 <.0001 AIC: 129.453

Goodness of Fit Scaled Dose Est._Prob. Expected Observed Size Residual

------------------------------------------------------------------------ 0.0000 0.0211 1.011 1.000 48 -0.011 0.2000 0.6391 30.036 32.000 47 0.596 0.3800 0.7698 36.952 35.000 48 -0.669 Chi^2 = 0.80 d.f. = 1 P-value = 0.3699

Benchmark Dose Computation Specified effect = 0.67 Risk Type = Extra risk Confidence level = 0.95 BMD = 0.23715 BMDL = 0.160575DRAFT - DO NOT CITE OR QUOTE 196 0 0.2 0.4 0.6 0.8

0 0.05 0.1 0.15 0.2 0.25 0.3 0.35Fraction Affected

dose Log-Logistic Model with 0.95 Confidence Level

16:29 09/21 2010 BMDL BMD Log-Logistic

C.5. EXTRAPOLATION METHOD AND INHALATION CANCER SLOPE FACTOR As explained in Chapter 5, linear extrapolation was applied in this assessment and BMCLat the calculated BMR was regarded as POD. The inhalation cancer slope factor was the upperbound estimation of risk and calculated as BMR/BMCL. It was used to estimate the lifetime lung tumor risk in human for vanadium pentoxide exposure through inhalation. Data are summarized
in Table C-1.

C.6. MSW TIME-TO-TUMOR MODELING OF THE INHALATION

CARCINOGENESIS DATA SETS Multistage Weibull Time-to-tumor modeling was also used to model these data sets because the survival curves reported by NTP (2002) showed a high percentage (22-46%) of deaths at the end of 2-year studies across th e different concentrations groups. The individual animal data was obtained through the NTP website and summarized according to concentration and week of death (Table C-11). Based on the results, MSW time-to-tumor model was not recommended to derive cancer slope factor for these data sets because of two reasons:

• The visual fit near the 1 st non-control concentration was not adequate.

• The parameters, including c, beta_0 and beta_1, were very close to the initial values, which suggested the model was non-convergence.

.

Lung Tumor Data Set 1: Combined Incidence of Alveolar/Bronchiolar Adenomas andDRAFT - DO NOT CITE OR QUOTE 197 Carcinomas in Male Mice, NTP (2002).

Table C- 11. Grouped Data for MSW Time-to-tumor Modeling; B6C3F 1 Male Mice Exposed to Vanadium Pentoxide by Inhalation for 2 Years (NTP, 2002). Human Equivalent Concentration (mg/m 3 ) Week of Death Response Category for Alveolar/Bronchiolar Adenoma and/or Carcinoma
b Number of Animals

0.0 2 C 1 26 C 1 72 C 1 89 C 1 92 C 1 95 C 1 97 C 1 98 C 2 99 C 1 101 C 1 102 C 1 104 C 6 104 I 1 105 C 31

0.21 3 C 3 64 C 1 75 I 1 76 I 1 81 I 1 83 I 1 86 C 1 87 I 1 91 I 1 93 I 1 94 I 2 95 C 1 101 I 1 103 I 1 104 I 8 104 C 1 105 C 11 105 I 13DRAFT - DO NOT CITE OR QUOTE 198 Table C- 11. Grouped Data for MSW Time-to-tumor Modeling; B6C3F 1 Male Mice Exposed to Vanadium Pentoxide by Inhala tion for 2 Years (NTP, 2002). (Continued) Human Equivalent Concentration (mg/m 3 ) Week of Death Response Category for Alveolar/Bronchiolar Adenoma and/or Carcinoma
b Number of Animals

0.42 36 C 1 39 C 1 40 I 1 45 I 1 50 C 1 59 C 1 70 C 1 72 I 1 76 I 1 82 C 1 83 I 1 91 I 1 92 I 1 95 I 1 95 C 1 99 I 1 101 I 2 101 C 1 104 I 7 105 I 17 105 C 7

0.81 36 C 1 68 I 1 77 I 1 79 I 1 79 C 1 81 C 1 81 I 1 87 C 2 91 I 1 93 I 1 98 I 1 99 C 1 101 I 2 104 I 7 104 C 3 105 I 16 105 C 9DRAFT - DO NOT CITE OR QUOTE 199 a Concentration was the original doses reported in th e publication from NTP(2002). No adjustment was applied.
b Categories of response: “C”=Neither carcinoma nor adenom a was detected when the subject was removed from the study due to scheduled sacrifice or uns cheduled death; “I”= carcinoma and/or adenoma were detected when the subjected was removed from the study due to scheduled sacrifice or unscheduled death.

MSW Time-to-tumor output

======================================================================= Multistage Weibull Model. (Version: 1.6.1; Date: 11/24/2009) Solutions are obtained using donlp2-intv, (c) by P. Spellucci Input Data File: V2O5_NTP_MaleMiceHEC_Poly1.(d)

======================================================================= V2O5_NTP_MaleMiceHEC

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ The form of the probability function is: P[response] = 1-EXP{-(t - t_0)^c * (beta_0+beta_1*dose^1)}

The parameter betas are restricted to be positive

Dependent variable = CONTEXT Independent variables = DOSE, TIME

Total number of observations = 200 Total number of records with missing values = 0 Total number of parameters in model = 4 Total number of specified parameters = 1 Degree of polynomial = 1

User specifies the following parameters: t_0 = 0

Maximum number of iterations = 64 Relative Function Convergence has been set to: 2.22045e-016 Parameter Convergence has been set to: 1.49012e-008

Default Initial Parameter Values c = 1.05882 t_0 = 0 Specified beta_0 = 0.0054829 beta_1 = 0.0193401

Asymptotic Correlation Matrix of Parameter Estimates ( *** The model parameter(s) -c -t_0 have been estimated at a boundary point, or have been specified by the user, and do not appear in the correlation matrix )

beta_0 beta_1 beta_0 1 -0.61 beta_1 -0.61 1

Parameter Estimates 95.0% Wald Confidence Interval Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit c 1 NA beta_0 0.00720175 0.00201449 0.00325342 0.0111501 beta_1 0.0252605 0.00794841 0.00968191 0.0408391

NA - Indicates that this parameter has hit a bound implied by some inequality constraint and thus has no standard error.

Log(likelihood) # Param AIC Fitted Model -101.116 3 208.233

Data Summary CONTEXT C F I U Total Expected Response DOSE 0 28 0 22 0 50 25.86 0.21 8 0 42 0 50 35.33 0.42 7 0 43 0 50 41.53 0.81 7 0 43 0 50 46.15

Benchmark Dose ComputationDRAFT - DO NOT CITE OR QUOTE 200 Risk Response = Incidental Risk Type = Extra Specified effect = 0.71 Confidence level = 0.9 Time = 104 BMD = 0.471196 BMDL = 0.390136 BMDU = 0.716564

0 20 40 60 80 1000.0 0.4 0.8 Dose = 0.00

TimeProbability

0 20 40 60 80 1000.0 0.4 0.8 Dose = 0.21

TimeProbability

0 20 40 60 80 1000.0 0.4 0.8 Dose = 0.42

TimeProbability

0 20 40 60 80 1000.0 0.4 0.8 Dose = 0.81

TimeProbability Incidental Risk: V2O5_NTP_MaleMiceHEC_Poly1DRAFT - DO NOT CITE OR QUOTE 201 Lung Tumor Data Set 2: Combined Incidence of Alveolar/Bronchiolar Adenoma and Carcinoma in Female Mice, NTP (2002).

Table C- 12. Grouped Data for MSW Time-to-tumor Modeling; B6C3F 1 Female Mice Exposed to Vanadium Pentoxide by Inhalation for 2 Years (NTP, 2002). Human Equivalent Concentration (mg/m 3 ) Week of Death Response Category for Alveolar/Bronchiolar Adenoma and/or Carcinoma
b Number of Animals

0.00 2 C 1 26 C 1 72 C 1 89 C 1 92 C 1 95 C 1 97 C 1 98 C 2 99 C 1 101 C 1 102 C 1 104 C 6 104 I 1 105 C 31

0.20 3 C 3 64 C 1 75 I 1 76 I 1 81 I 1 83 I 1 86 C 1 87 I 1 91 I 1 93 I 1 94 I 2 95 C 1 101 I 1 103 I 1 104 I 8 104 C 1 105 C 11 105 I 13DRAFT - DO NOT CITE OR QUOTE 202 Table C-12 . Grouped Data for MSW Time-to-tumor Modeling; B6C3F 1 Female Mice Exposed to Vanadium Pentoxide by Inhala tion for 2 Years (NTP, 2002). (Continued) Human Equivalent Concentration (mg/m 3 ) Week of Death Response Category for Alveolar/Bronchiolar Adenoma and/or Carcinoma
b Number of Animals

0.38 36 C 1 39 C 1 40 I 1 45 I 1 50 C 1 59 C 1 70 C 1 72 I 1 76 I 1 82 C 1 83 I 1 91 I 1 92 I 1 95 I 1 95 C 1 99 I 1 101 I 2 101 C 1 104 I 7 105 I 17 105 C 7

0.73 36 C 1 68 I 1 77 I 1 79 I 1 79 C 1 81 C 1 81 I 1 87 C 2 91 I 1 93 I 1 98 I 1 99 C 1 101 I 2 104 I 7 104 C 3 105 I 16DRAFT - DO NOT CITE OR QUOTE 203 105 C 9

a Concentration was the original one reported in the publication from NTP(2002). No adjustment was applied.
b Categories of response: “C”=Neither carcinoma nor adenom a was detected when the subject was removed from the study due to scheduled sacrifice or uns cheduled death; “I”= carcinoma and/or adenoma were detected when the subjected was removed from the study due to scheduled sacrifice or unscheduled death.

MSW Time-to-tumor output

======================================================================= Multistage Weibull Model. (Version: 1.6.1; Date: 11/24/2009) Solutions are obtained using donlp2-intv, (c) by P. Spellucci Input Data File: V2O5_NTP_FemaleMiceHEC_Poly1.(d)

======================================================================= V2O5_NTP_FemaleMiceHEC

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ The form of the probability function is: P[response] = 1-EXP{-(t - t_0)^c * (beta_0+beta_1*dose^1)}

The parameter betas are restricted to be positive

Dependent variable = CONTEXT Independent variables = DOSE, TIME

Total number of observations = 200 Total number of records with missing values = 0 Total number of parameters in model = 4 Total number of specified parameters = 1 Degree of polynomial = 1

User specifies the following parameters: t_0 = 0

Maximum number of iterations = 64 Relative Function Convergence has been set to: 2.22045e-016 Parameter Convergence has been set to: 1.49012e-008

Default Initial Parameter Values c = 1.125 t_0 = 0 Specified beta_0 = 0.000224032 beta_1 = 0.0144984

Asymptotic Correlation Matrix of Parameter Estimates ( *** The model parameter(s) -t_0 have been estimated at a boundary point, or have been specified by the user, and do not appear in the correlation matrix )

c beta_0 beta_1 c 1 -0.93 -1 beta_0 -0.93 1 0.93 beta_1 -1 0.93 1

Parameter Estimates 95.0% Wald Confidence Interval Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit c 1.12496 0.770137 -0.384483 2.6344 beta_0 0.000224043 0.000862599 -0.00146662 0.00191471 beta_1 0.0144991 0.0511302 -0.0857143 0.114712

Log(likelihood) # Param AIC Fitted Model -109.298 3 224.597

Data Summary CONTEXT C F I U Total Expected Response DOSE 0 49 0 1 0 50 1.94 0.2 18 0 32 0 50 19.95 0.38 15 0 35 0 50 30.09 0.73 18 0 32 0 50 42.08

Benchmark Dose Computation Risk Response = Incidental Risk Type = Extra Specified effect = 0.67DRAFT - DO NOT CITE OR QUOTE 204 Confidence level = 0.9 Time = 104 BMD = 0.411511 BMDL = 0.339589 BMDU = 0.516352

0 20 40 60 80 1000.0 0.4 0.8 Dose = 0.00

TimeProbability

0 20 40 60 80 1000.0 0.4 0.8 Dose = 0.20

TimeProbability

0 20 40 60 80 1000.0 0.4 0.8 Dose = 0.38

TimeProbability

0 20 40 60 80 1000.0 0.4 0.8 Dose = 0.73

TimeProbability Incidental Risk: V2O5_NTP_FemaleMiceHEC_Poly1