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# TOXICOLOGICAL REVIEW

OF

HEXAVALENT CHROMIUM

(CAS No. 18540-29-9)



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

April 2010 (Note: This document is a reassessment of the nonca ncer and cancer health effects associated with the oral route of exposure and incl udes a mode of action analysis

for cancer across all routes of exposure.)

NOTICE

This document is an Interagency Science Consultation draft . This information is distributed solely for the purpose of pre-dissemination peer re view 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 implic ations.

U.S. Environmental Protection Agency

Washington, DC

### ii DRAFT - DO NOT CITE OR QUOTE DISCLAIMER

This document is a preliminary draft for review pur poses only. This information is distributed solely for the purpose of pre-dissemina tion peer review under applicable information quality guidelines. It has not been formally disse minated by EPA. It does not represent and should not be construed to represent any Agency det ermination or policy. Mention of trade names or commercial products does not constitute en dorsement or recommendation for use.

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CONTENTS—TOXICOLOGICAL REVIEW OF HEXAVALENT CHROMIU M (CAS No. 18540-29-9)

DISCLAIMER .........................................

................................................... .................................... ii LIST OF TABLES.....................................

................................................... ...................................v LIST OF FIGURES ....................................

................................................... .............................. viii LIST OF ABBREVIATIONS AND ACRONYMS .................

................................................... .. ix FOREWORD ...........................................

................................................... .................................... x AUTHORS, CONTRIBUTORS, AND REVIEWERS ............... .................................................. x i

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

................................................... ................................1

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

................................................... ...3

3. TOXICOKINETICS .................................

................................................... ...............................8 3.1. ABSORPTION FOLLOWING INGESTION...............

................................................... ... 8 3.2. DISTRIBUTION FOLLOWING INGESTION .............

................................................... 11 3.3. METABOLISM FOLLOWING INGESTION...............

................................................... 15 3.4. ELIMINATION FOLLOWING INGESTION..............

................................................... . 18 3.5. PHYSIOLOGICALLY-BASED TOXICOKINETIC MODELS... ................................... 19

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

................................................... ....................26 4.1. ORAL STUDIES IN HUMANS .......................

................................................... ............. 26 4.1.1. Acute Exposure.............................

................................................... ........................ 26 4.1.2. Environmental Exposure.....................

................................................... .................. 27 4.2. SUBCHRONIC AND CHRONIC STUDIES AND CANCER BIO ASSAYS IN ANIMALS—ORAL.......................................

................................................... ..................... 41 4.2.1. Subchronic Oral Exposure ...................

................................................... ................. 42 4.2.2. Chronic Oral Exposure ......................

................................................... ................... 63 4.3. REPRODUCTIVE AND DEVELOPMENTAL TOXICITY STUDI ES—ORAL........... 84 4.3.1. Effects on Reproductive Tissues and Mating Behavior........................................... 85 4.3.2. Effects on Reproductive Outcomes ...........

................................................... ........... 98 4.3.3. Effects of Pre-gestational Exposure on Repr oductive Outcome and Fetal Development........................................

................................................... .......................... 103 4.3.4. Effects of Gestational and/or Lactational E xposure on Reproductive Outcome and Fetal Development..................................

................................................... ....................... 107 4.4. MECHANISTIC DATA AND OTHER STUDIES IN SUPPORT OF THE MODE OF ACTION.............................................

................................................... ............................... 115 4.4.1. Genotoxicity Studies ......................

................................................... ........................ 115 4.4.1.1. Genotoxicity assays in experimental syste ms................................................. .... 117 4.4.1.2. Genotoxicity studies in humans...........

................................................... ............ 144 4.4.2. Intracellular reduction ....................

................................................... ........................ 150 4.5. SYNTHESIS OF MAJOR NONCANCER EFFECTS - ORAL .. ................................... 158 4.6. EVALUATION OF CARCINOGENICITY ................

................................................... 166 4.6.1. Summary of Overall Weight-of-Evidence ......

................................................... ....... 166 4.6.2. Synthesis of Human, Animal, and Other Suppo rting Evidence................................ 166 4.6.3. Mode of Action Information .................

................................................... ................. 168 4.6.3.1. Hypothesized Mode of Action..............

................................................... ........... 168 4.6.3.2. Experimental Support for the Hypothesized Mode of Action ............................ 170

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4.6.3.3. Other Possible Modes of Action...........

................................................... ........... 179 4.6.3.4. Conclusions About the Hypothesized Mode o f Action ...................................... 179 4.7. SUSCEPTIBLE POPULATIONS AND LIFE STAGES...... .......................................... 181 4.7.1. Possible Childhood Susceptibility..........

................................................... ................ 181 4.7.2. Possible Gender Differences ................

................................................... .................. 181

5. DOSE-RESPONSE ASSESSMENTS......................

................................................... ...........182 5.1. ORAL REFERENCE DOSE (RfD) ....................

................................................... ......... 182 5.1.1. Choice of Principal Study and Critical Effe ct—with Rationale and Justification .... 182 5.1.2. Methods of Analysis—Including Models (PBPK, BMD, etc.)................................. 187 5.1.3. RfD Derivation—Including Application of Unc ertainty Factors (UFs) ................... 189 5.1.4. Previous RfD Assessment ....................

................................................... .................. 190 5.2. UNCERTAINTIES IN THE ORAL REFERENCE DOSE ..... ....................................... 191 5.3. ORAL CANCER ASSESSMENT .......................

................................................... ........ 192 5.3.1. Choice of Study/Data—with Rationale and Jus tification ......................................... 192 5.3.2. Dose-Response Data .........................

................................................... ..................... 193 5.3.3. Dose Adjustments and Extrapolation Method(s ) .................................................. .... 197 5.3.4. Oral Slope Factor ..........................

................................................... ......................... 198 5.3.5. Application of Age-Dependent Adjustment Fac tors............................................... .. 199 5.3.6. Uncertainties in Cancer Risk Values........

................................................... .............. 200 5.3.7. Previous Cancer Assessment.................

................................................... ................. 203

6. MAJOR CONCLUSIONS IN THE CHARACTERIZATION OF HA ZARD AND DOSE RESPONSE...........................................

................................................... ....................................204 6.1. HUMAN HAZARD POTENTIAL .......................

................................................... ....... 204 6.2. DOSE RESPONSE................................

................................................... ....................... 209 6.2.1. Noncancer — Oral ............................

................................................... ................... 209 6.2.2. Cancer — Oral ...............................

................................................... ...................... 209

7. REFERENCES .....................................

................................................... ...............................211

APPENDIX A. SUMMARY OF EXTERNAL PEER REVIEW AND PU BLIC COMMENTS AND DISPOSITION ....................................

................................................... ........................... A-1

APPENDIX B. BENCHMARK DOSE CALCULATIONS ........... ...........................................B-1 APPENDIX B-1. Details of Benchmark Dose Analysis f or the RfD......................................... B-2 APPENDIX B-2. Details of Benchmark Dose Analysis f or the Oral Cancer Slope Factor .....B-16

### v DRAFT - DO NOT CITE OR QUOTE LIST OF TABLES

Table 2-1. Physical properties of selected hexaval ent chromium compounds ............................. . 5 Table 3-1. In vitro kinetic parameters of hexavale nt chromium uptake in erythrocytes of rats and humans .............................................

................................................... ................................... 11

Table 3-2. Terminal tissue chromium levels in rats ingesting K 2 

### Cr 2 

### O 4 

### in drinking water for 1

year...............................................

................................................... ....................................... 12 Table 3-3. Chromium in tissues ( μ g/g wet tissue or μ g/mL blood) of mice and rats after ingesting K 2 

### Cr 2 

### O 7 

### in drinking water (8 mg hexavalent chromium/kg-day) for 4 or 8 weeks 14 Table 3-6. Kinetic parameters of hexavalent chromi um reduction in human liver microsomes from five individuals ..............................

................................................... ............................. 17 Table 3-7. Chemical-specific parameters in the rat and human chromium models..................... 22 Table 4-1. Data pertaining to hexavalent chromium concentrations in drinking water in five villages along path of groundwater contamination fr om alloy plant, western JinZhou, China from 1965 to 1979..................................

................................................... ............................. 30 Table 4-2. Results pertaining to cancer mortality rates in five villages along path of groundwater contamination from alloy plant and other comparison areas, western JinZhou, China from 1970 to 1978, based on analyses by Beaumont et al. (2008) and Kerger et al. (2009).......... 35 Table 4-3. Risk ratios comparing cancer mortality rates in five villages along path of groundwater contamination from alloy plant and othe r comparison areas, western JinZhou, China from 1970 to 1978 ............................

................................................... ........................ 36 Table 4-4. Hematological effects in male and femal e F344/N rats exposed to sodium dichromate dihydrate in drinking water for up to 3 months.....

................................................... .............. 44 Table 4-5. Clinical chemistry effects in male and female F344/N rats exposed to sodium dichromate dihydrate in drinking water for 3 months

................................................... ......... 47 Table 4-6. Selected organ weights in male and fema le F344/N rats exposed to sodium dichromate dihydrate in drinking water for 3 months

................................................... ......... 48 Table 4-7. Incidence of nonneoplastic lesions obse rved in male and female F344/N rats exposed to sodium dichromate dihydrate in drinking water fo r 3 months......................................... .. 50 Table 4-8. Selected organ weights in male and fema le B6C3F 1 

### mice exposed to sodium dichromate dihydrate in drinking water for 3 months

................................................... ......... 53 Table 4-9. Incidence of nonneoplastic lesions obse rved in male and female B6C3F 1 

### mice exposed to sodium dichromate dihydrate in drinking water for 3 months............................. 54 Table 4-10. Hematological effects in male B6C3F 1 

### , BALB/c, and am3 -C57BL/6 mice exposed to sodium dichromate dihydrate in drinking water fo r 3 months......................................... .. 57 Table 4-11. Incidence of nonneoplastic lesions obs erved in male B6C3F 1 

### , BALB/c, and am3

- C57BL/6 mice exposed to sodium dichromate dihydrate in drinking water for 3 months .... 58 Table 4-12. Hematological effects in male F344/N r ats exposed to sodium dichromate dihydrate in drinking water for up to 12 months..............

................................................... ................... 65

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Table 4-13. Serum ALT activity in male F344/N rats exposed to sodium dichromate dihydrate in drinking water for up to 12 months.................

................................................... .................... 67 Table 4-14. Incidence of nonneoplastic lesions obs erved in male and female F344/N rats exposed to sodium dichromate dihydrate in drinking water for 2 years ................................ 70 Table 4-15. Incidence of neoplastic lesions observ ed in the oral cavity of male and female F344/N rats exposed to sodium dichromate dihydrate in drinking water for 2 years ............ 72 Table 4-16. Neoplastic lesions in other tissues (e .g., non-oral cavity) in F344/N rats exposed to sodium dichromate dihydrate in drinking water for 2 years ............................................. ..... 73 Table 4-17. Hematological effects in female B6C3F 1 

### mice exposed to sodium dichromate dihydrate in drinking water for up to 12 months....

................................................... ............. 76 Table 4-18. Incidence of nonneoplastic lesions obs erved in male and female B6C3F 1 

### mice exposed to sodium dichromate dihydrate in drinking water for 2 years ................................ 78 Table 4-19. Incidence of neoplastic lesions observ ed in the small intestine of male and female B6C3F 1 

### mice exposed to sodium dichromate dihydrate in dri nking water for 2 years ......... 79 Table 4-20. Evidence of mutagenicity of hexavalent chromium compounds in experimental systems ............................................

................................................... .................................. 116 Table 4-21. In vitro genotoxicity studies of hexav alent chromium in non-mammalian cells ... 120 Table 4-22. In vitro genotoxicity studies of hexav alent chromium in mammalian cells........... 127 Table 4-23. In vivo genotoxicity studies of hexava lent chromium in D. melanogaster and laboratory mammals.................................

................................................... ......................... 134 Table 4-24. In vivo genotoxicity studies in humans exposed to hexavalent chromium............ 146 Table 4-25. Observed effects and corresponding NOA ELs and LOAELs for subchronic, chronic, and reproductive toxicity studies following oral ex posure to hexavalent chromium .......... 161 Table 5-1. Incidence data for nonneoplastic lesion s from all treatment groups of female F344/N rats and male and female B6C3F 1 

### mice exposed to sodium dichromate dihydrate in dri nking water for 2 years (NTP, 2008)......................

................................................... ..................... 187 Table 5-2. Summary of BMD 10 

### and BMDL 10 

### from the best fitting models for nonneoplastic lesions of the liver, duodenum, mesenteric lymph no des, and pancreas in female rats and male and female mice after exposure to sodium dichr omate dihydrate in drinking water for 2 years (NTP, 2008) ..................................

................................................... ........................... 189 Table 5-3. Incidences of squamous cell papillomas or carcinomas in the oral cavity of male F344/N rats exposed to sodium dichromate dihydrate in drinking water for 2 years (NTP, 2008) ..............................................

................................................... ................................... 194 Table 5-4. Incidences of squamous cell papillomas or carcinomas in the oral cavity of female F344/N rats exposed to sodium dichromate dihydrate in drinking water for 2 years (NTP, 2008) ..............................................

................................................... ................................... 195

Table 5-5. Incidences of adenomas and carcinomas c ombined in the small intestine of male B6C3F 1 

### mice exposed to sodium dichromate dihydrate in dri nking water for 2 years (NTP, 2008) ..............................................

................................................... ................................... 196

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Table 5-6. Incidences of adenomas and carcinomas c ombined in the small intestine of female B6C3F1 mice exposed to sodium dichromate dihydrate in drinking water for 2 years (NTP, 2008) ..............................................

................................................... ................................... 197 Table 5-7. Application of ADAFs for a 70-year expos ure to 0.0001 mg hexavalent chromium/kgday from ages 0 to 70 ..............................

................................................... .......................... 200 Table 5-8. Summary of uncertainties in the cancer risk assessment for hexavalent chromium 201

### viii DRAFT - DO NOT CITE OR QUOTE LIST OF FIGURES

Figure 3-1. A physiologically based model of chrom ium kinetics in the rat and human............ 21 Figure 4-1. Ternary DNA adduct formation by chromi um................................................. ....... 152

### ix DRAFT - DO NOT CITE OR QUOTE LIST OF ABBREVIATIONS AND ACRONYMS

3 β

- ∆ 5-HSH 3 β

‐ ∆ 5

‐ hydroxysteroid dehydrogenase AcP acid phosphatase ALT alanine aminotransferase AP alkaline phosphatase AST asparate aminotransferase CASRN Chemical Abstracts Service Registry Number CI confidence interval ED Enumeration Districts FSH follicle-stimulating hormone GD gestation day GFR glomerular filtration rate GH growth hormone GI gastrointestinal GSH glutathione GST glutathione S-transferase H&E haematoxylin and eosin Hct hematocrit Hgb hemoglobin IOM The Institute of Medicine IRIS Integrated Risk Information System K m 

### Michaelis constant LH luteinizing hormone LOAEL lowest-observed-adverse-effect level MCH mean cell hemoglobin MCHC mean cell hemoglobin concentration MCV mean cell volume NOAEL no-observed-adverse-effect level NRC National Research Council NTP National Toxicology Program PAM Pulmonary alveolar macrophages PBTK Physiologically based toxicokinetic PND postnatal day RfC reference concentration RfD reference dose SDH sorbitol dehydrogenase SMART somatic mutation and recombination test TEM transmission electron microscopy U.S. EPA U.S. Environmental Protection Agency

### x DRAFT - DO NOT CITE OR QUOTE FOREWORD

The purpose of this Toxicological Review is to prov ide the scientific support and rationale for the hazard and dose-response assessme nt in IRIS pertaining to chronic exposure to hexavalent chromium via ingestion. It is not inten ded to be a comprehensive treatise on the chemical or toxicological nature of hexavalent chro mium. This document is a reassessment of

the noncancer and cancer health effects associated with the oral route of exposure and includes a mode of action analysis for cancer across all route s of exposure. A reassessment of the

noncancer and cancer health effects associated with the inhalation route of exposure will be

added at a later date.

Section 5, Dose-Response Assessments , is based largely on the work of four independent groups that have recently evaluated the toxicity of hexavalent chromium via ingestion: 1) U.S. EPA’s Office of Pesticide Programs (OPP), 2) the Ne w Jersey Department of Environmental

Protection (NJDEP), 3) the California Environmental Protection Agency (CalEPA), and 4) the Agency for Toxic Substances and Disease Registry (A TSDR). Section 5.1 was developed based on work conducted by ATSDR and CalEPA, and the refe rence dose (RfD) was derived using ATSDR’s analysis for chronic oral exposure to hexav alent chromium. Section 5.3 was developed based on work conducted by CalEPA and NJD EP, and the oral cancer slope factor (CSF) was derived using NJDEP’s analysis for cancer potency. 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, wher e applicable, and to characterize the overall confidence in the quantitative and qualitative aspe cts 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 ass essor 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 (20 2) 566-1676 (phone), (202) 566-1749 (fax), or hotline.iris@epa.gov (e-mail address).

### xi DRAFT - DO NOT CITE OR QUOTE AUTHORS, CONTRIBUTORS, AND REVIEWERS

CHEMICAL MANAGER/AUTHOR

Ted Berner U.S. EPA, ORD/NCEA 1200 Pennsylvania Ave., NW Washington, D.C. 20460

AUTHORS

Catherine Gibbons U.S. EPA, ORD/NCEA 1200 Pennsylvania Ave., NW Washington, D.C. 20460

Glinda Cooper U.S. EPA, ORD/NCEA 1200 Pennsylvania Ave., NW Washington, D.C. 20460

CONTRACTOR SUPPORT

Julie Klotzbach, Ph.D. Michael H, Lumpkin, Ph.D. Daniel J. Plewak, B.S. SRC Syracuse, NY

REVIEWERS This document has been provided for review to EPA scientists.

### 1 DRAFT - DO NOT CITE OR QUOTE 1. INTRODUCTION 1

2 3 

### This document presents background information and j ustification for the Integrated Risk 4 

### Information System (IRIS) Summary of the hazard and dose-response assessment of ingested 5 

### hexavalent chromium. IRIS Summaries may include or al reference dose (RfD) and inhalation 6 

### reference concentration (RfC) values for chronic an d other exposure durations, and a 7 

### carcinogenicity assessment. This Toxicological Rev iew provides documentation for oral toxicity 8 

### values (i.e., RfD and oral cancer slope factor) onl y. 9 

### The RfD and RfC, if derived, provide quantitative i nformation for use in risk assessments 10 

### for health effects known or assumed to be produced through a nonlinear (presumed threshold) 11 

### mode of action. The RfD (expressed in units of mg/ kg-day) is defined as an estimate (with 12 

### uncertainty spanning perhaps an order of magnitude) of a daily exposure to the human 13 

### population (including sensitive subgroups) that is likely to be without an appreciable risk of 14 

### deleterious effects during a lifetime. The inhalat ion RfC (expressed in units of mg/m 3 

### ) is 15 

### analogous to the oral RfD, but provides a continuou s inhalation exposure estimate. The 16



### inhalation RfC considers toxic effects for both the respiratory system (portal-of-entry) and for 17 

### effects peripheral to the respiratory system (extra respiratory or systemic effects). Reference 18 

### values are generally derived for chronic exposures (up to a lifetime), but may also be derived for 19 

### acute ( ≤ 24 hours), short-term (>24 hours up to 30 days), an d subchronic (>30 days up to 10% of 20 

### lifetime) exposure durations, all of which are deri ved based on an assumption of continuous 21 

### exposure throughout the duration specified. Unless specified otherwise, the RfD and RfC are 22 

### derived for chronic exposure duration. 23 

### The carcinogenicity assessment provides information on the carcinogenic hazard 24 

### potential of the substance in question and quantita tive estimates of risk from oral and inhalation 25 

### exposure may be derived. The information includes a weight-of-evidence judgment of the 26 

### likelihood that the agent is a human carcinogen and the conditions under which the carcinogenic 27 

### effects may be expressed. Quantitative risk estima tes may be derived from the application of a 28 

### low-dose extrapolation procedure. If derived, the oral slope factor is a plausible upper bound on 29 

### the estimate of risk per mg/kg-day of oral exposure . Similarly, an inhalation unit risk is a 30 

### plausible upper bound on the estimate of risk per μ g/m 3 

### air breathed. 31 

### Development of these hazard identification and dose -response assessments for hexavalent 32 

### chromium has followed the general guidelines for ri sk assessment as set forth by the National 33 

### Research Council (NRC, 1983). U.S. Environmental P rotection Agency (U.S. EPA) Guidelines 34

### 2 DRAFT - DO NOT CITE OR QUOTE

and Risk Assessment Forum Technical Panel Reports t hat may have been used in the 1 

### development of this assessment include the followin g: Guidelines for the Health Risk 2 

### Assessment of Chemical Mixtures (U.S. EPA, 1986a), Guidelines for Mutagenicity Risk 3



### Assessment

(U.S. EPA, 1986b), Recommendations for and Documentation of Biological Values 4 

### for Use in Risk Assessment (U.S. EPA, 1988), Guidelines for Developmental Toxicity Risk 5



### Assessment

(U.S. EPA, 1991), Interim Policy for Particle Size and Limit Concentr ation Issues in 6 

### Inhalation Toxicity (U.S. EPA, 1994a), Methods for Derivation of Inhalation Reference 7 

### Concentrations and Application of Inhalation Dosime try (U.S. EPA, 1994b), Use of the 8 

### Benchmark Dose Approach in Health Risk Assessment (U.S. EPA, 1995), Guidelines for 9 

### Reproductive Toxicity Risk Assessment (U.S. EPA, 1996), Guidelines for Neurotoxicity Risk 10



### Assessment

(U.S. EPA, 1998), Science Policy Council Handbook : Risk Characterization (U.S. 11 

### EPA, 2000a), Benchmark Dose Technical Guidance Document (U.S. EPA, 2000b), 12 

### Supplementary Guidance for Conducting Health Risk A ssessment of Chemical Mixtures (U.S. 13 

### EPA, 2000c), A Review of the Reference Dose and Reference Concen tration Processes (U.S. 14 

### EPA, 2002), Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005a) , Supplemental 15 

### Guidance for Assessing Susceptibility from Early-Li fe Exposure to Carcinogens (U.S. EPA, 16 

### 2005b), Science Policy Council Handbook: Peer Review (U.S. EPA, 2006a), and A Framework 17 

### for Assessing Health Risks of Environmental Exposur es to Children (U.S. EPA, 2006b). 18 

### The literature search strategy employed for this co mpound was based on the Chemical 19 

### Abstracts Service Registry Number (CASRN) and at le ast one common name. Any pertinent 20 

### scientific information submitted by the public to t he IRIS Submission Desk was also considered 21 

### in the development of this document. The relevant literature was reviewed through December 22



### 2009. 23

24

### 3 DRAFT - DO NOT CITE OR QUOTE 2. CHEMICAL AND PHYSICAL INFORMATION 1

2 3 

### Chromium is a naturally occurring element present i n the earth’s crust that is typically 4 

### found in several valence states, with trivalent (Cr (III)) and hexavalent (Cr(VI)) chromium being 5 

### the most common. In humans, trivalent chromium is an essential nutrient required for normal 6 

### energy metabolism (ATSDR, 2008). Currently, the bi ological target for the essential effects of 7 

### trivalent chromium is unknown. Chromodulin, also k nown as glucose tolerance factor (GTF), 8 

### has been proposed as one possible candidate (ATSDR, 2008). The function of chromodulin, an 9 

### oligopeptide complex containing four chromic ions, has not been established; however, a 10 

### possible mechanism is that chromodulin facilitates the interaction of insulin with its cellular 11 

### receptor sites and thus improves glucose tolerance, although this has not been proven (ATSDR, 12 

### 2008). In general, hexavalent chromium compounds a re more toxic than trivalent chromium 13 

### compounds. This toxicological review focuses prima rily on hexavalent chromium compounds, 14 

### and the adverse effects associated with exposures t o hexavalent chromium are described in 15 

### Section 4 of this toxicological review. 16 

### Hexavalent chromium compounds are a group of substa nces that contain chromium in the 17 

### hexavalent or +6 oxidation state. The compounds di scussed in this document include 18 

### chromium(VI) oxide, chromic acid, and selected salt s of the chromate (CrO 4 2- 

### ) and dichromate 19 

### (Cr 2 

### O 7 2- 

### ) anions. Sodium chromate, sodium dichromate, and chromium(VI) oxide are obtained 20 

### directly from chromite ore through an oxidative alk aline roasting process (Anger et al., 2005; 21 

### Page and Loar, 2004). Sodium chromate and sodium d ichromate are the starting materials for 22 

### the production of most other chromium compounds (An ger et al., 2005; Page and Loar, 2004). 23 

### Chemical structures and selected physical and chemi cal properties of hexavalent chromium 24 

### compounds are presented in Table 2-1. 25 

### As a class of substances, hexavalent chromium compo unds are oxidizing agents (Anger 26 

### et al., 2005; Cotton et al., 1999). Chromium(VI) o xide and ammonium dichromate can react 27 

### explosively when brought into contact with organic materials (Lewis, 2007; O’Neil, 2006). 28 

### Major (or former) uses of hexavalent chromium compo unds include metal plating, manufacture 29 

### of pigments and dyes, corrosion inhibitors, chemica l synthesis, refractory production, leather 30 

### tanning, and wood preservation (Blade et al., 2007; Shanker et al., 2005; Page and Loar, 2004). 31



### Natural occurrence of hexavalent chromium is rare a s this form of chromium is readily 32 

### reduced by organic matter in the environment (Ashle y et al., 2003; Barceloux, 1999; U.S. EPA, 33 

### 1984). However, hexavalent chromium compounds rele ased to the environment by 34

### 4 DRAFT

-

anthropogenic sources may persist in natural waters and soils that contain low amounts of 1 

### organic matter (Johnson et al., 2006; Loyaux-Lawnic zak et al., 2001; U.S. EPA, 1984). 2 

### Hexavalent chromium compounds are considered to be more soluble in water and have greater 3 

### mobility in soil than other types of chromium compo unds (Loyuax-Lawniczak et al., 2001; 4 

### James et al., 1997). Industrial releases of hexava lent chromium compounds to surface water and 5 

### soil can result in the transport and leaching of th ese substances into groundwater, provided these 6 

### substances remain under oxidizing conditions (Loyua x-Lawniczak et al., 2001; Pellerin and 7 

### Booker, 2000; James et al., 1997). 85 DRAFT - DO NOT CITE OR QUOTETable 2-1. Physical properties of selected hexavalent chromium compoundsName Chromium(VI) oxideaChromic acida,bSodium chromate Sodium dichromate Sodium dichromate, dihydrateCASNumber1333-82-0 7738-94-5 (H2CrO4);13530-68-2 (H2Cr2O7)7775-11-3 10588-01-9 7789-12-0Synonyms(ChemIDPlus,2008)Chromium oxide;hexavalent chromiumoxide; chromictrioxide; chromicanhydride; chromicacid anhydride (Angeret al., 2005)Chromic(VI) acid; chromiumhydroxide oxide; dichromic acid(H2Cr2O7)Sodium chromate(VI);chromium disodiumoxide; disodiumchromate; rachromate;chromic acid, disodiumsalt; chromate of sodaSodium dichromate(VI);sodium bichromate;dichromic acid, disodiumsalt; bichromate of sodaDichromic acid, disodium salt,dihydrateStructure(ChemIDPlus,2008)CrOOOMolecularweight99.994 (Lide, 2008) 118.010 (H2CrO4) (Lide, 2008);218.001 (H2Cr2O7) (ChemID Plus)161.974 (Lide, 2008) 261.968 (Lide, 2008) 297.999 (Lide, 2008)MolecularformulaCrO3(ChemID Plus,2008)H2CrO4;H2Cr2O7(ChemID Plus,2008)Na2CrO4(ChemIDPlus, 2008)Na2Cr2O7(ChemID Plus,2008)Na2Cr2O7•2H2O (ChemID Plus,2008)FormDark red, deliquescentbipyramidal prismaticcrystals, flakes, orgranular powder(O’Neil, 2006)Exists only as an aqueous solution(Lide, 2008); yellow to orange-red(Anger et al., 2005)Yellow, orthorhombiccrystals (Anger et al.,2005)Light brown to orangered plates (Anger et al.,2005)Orange-red, monoclinic,translucent needles (Anger et al.,2005)Stability/reactivityDeliquescent;decomposition beginsabove 198°C (Anger etal., 2005); powerfuloxidizer (O’Neil, 2006)Strong oxidizing agent (Anger et al.,2005)Hygroscopic (Anger etal., 2005)Strongly hygroscopic;decomposes above 400°C(Lide, 2008); strongoxidizing agent (Anger etal., 2005)Very hygroscopic, deliquescesin air; Decomposes above 85°C;strong oxidizing agent in acidsolution (Lide, 2008; Anger etal., 2005)Meltingpoint197°C (Lide, 2008) Not applicable 794°C (Lide, 2008) 357°C (Lide, 2008) Decomposes prior to melting(Lide, 2008)Density2.7 g/cm3(Lide, 2008) Not applicable 2.72 g/cm3(Lide, 2008)2.52 g/cm3(Anger et al.,2005)2.35 g/cm3(Lide, 2008)Watersolubility169 g/100 g H2O at25°C (Lide, 2008)Not applicable 87.6 g/100 g H2O at25°C (Lide, 2008)187 g/100 g H2O at 25°C(Lide, 2008)272.9 g/100 g H2O (73.18 wt%)at 20°C (Anger et al., 2005)OthersolubilitySoluble in alcohol andmineral acids (Lewis,2007)Not applicable Slightly soluble inethanol (Lide, 2008)Not available Soluble in acetic acid (Lide,2008)6 DRAFT - DO NOT CITE OR QUOTETable 2-1. Physical properties of selected hexavalent chromium compoundsName Potassium chromatePotassium dichromate Calcium chromateAmmonium dichromate Zinc chromate Lead chromateCASNumber7789-00-6 7778-50-9 13765-19-0 7789-09-5 13530-65-97758-97-6Synonyms(ChemIDPlus,2008)Potassiumchromate(VI);bipotassium chromate;dipotassium chromate;chromate of potash;tarapacaite;chromic acid,dipotassium saltPotassium dichromate(VI);bichromate of potash;potassium bichromate;dipotassium bichromate;dipotassium dichromate;dipotassium dichromiumheptaoxide; lopezite;dichromic acid,dipotassium saltCalciumchromate(VI);calcium chromeyellow; calciummonochromate;gelbin; yellowultramarine; chromicacid, calcium saltAmmonium bichromate;ammonium dichromate(VI);diammonium dichromate;chromic acid, diammoniumsaltZinc chromate(VI)hydroxide; buttercupyellow; chromicacid, zinc salt; zincchrome yellow; zincteraoxychromateLead chromate(VI);phoenicochroite;plumbouschromate; chromicacid, lead salt;chrome yellow(O’Neil, 2006)Structure(ChemIDPlus,2008)Molecularweight194.191 (Lide, 2008) 294.185 (Lide, 2008) 156.07 (Lide, 2008) 252.065 (Lide, 2008) 181.403 (Lide,2008)323.2 (Lide, 2008)MolecularformulaK2CrO4(ChemID Plus,2008)K2Cr2O7(ChemID Plus,2008)CaCrO4(ChemIDPlus, 2008)(NH4)2Cr2O7(ChemID Plus,2008)ZnCrO4(ChemIDPlus, 2008)PbCrO4(ChemIDPlus, 2008)FormLemon yellow prisms(Anger et al., 2005)Tabular or prismatic, brightorange-red triclinic crystals(Anger et al., 2005)Yellow monoclinicor rhombic crystals(O’Neil, 2006)Large, bright, orange-redcrystals (Anger et al., 2005)Yellow prisms(Lide, 2008)Yellow-orangemonoclinic crystals(Lide, 2008)Stability/reactivityNon-hygroscopic(Anger et al., 2005)Non-hygroscopic;Decomposes at 500°C(Anger et al., 2005; Lide,2008)Decomposes at1,000°C (Lide,2008); oxidizingagent (Lewis, 2007)Flammable; non-hygroscopic;decomposition begins uponheating at 180°C (O’Neil,2006). Strong oxidizing agent,may explode in contact withorganic materials (Lewis,2007)Not available Not availableMeltingpoint974°C (Lide, 2008) 398°C (Lide, 2008) Decomposesprior tomelting (Lide, 2008)Decomposes prior to melting(Lide, 2008)316°C (Lide, 2008) 844°C (Lide, 2008)Density2.73 g/cm3(Lide,2008)2.68 g/cm3(Lide, 2008) 3.12 g/cm3(Anger etal., 2005)2.155 g/cm3(Lide, 2008) 3.40 g/cm3(Lide,2008)6.12 g/cm3(Lide,2008)7 DRAFT - DO NOT CITE OR QUOTETable 2-1. Physical properties of selected hexavalent chromium compoundsName Potassium chromatePotassium dichromate Calcium chromateAmmonium dichromate Zinc chromate Lead chromateWatersolubility65.0 g/100 g H2O at25°C (Lide, 2008)15.1 g/100 g H2O at 25°C(Lide, 2008)4.5 g/100 g H2O(4.3 wt%) at 0°C(Anger et al., 2005)35.6 g/100 g H2O at 20°C(Lide, 2008)3.08 g/100 g H2O(Lide, 2008)0.000017 g/100 gH2O at 20°C (Lide,2008)OthersolubilityInsoluble in alcohol(O’Neil, 2006)Insoluble in alcohol(Lewis, 2007)Soluble in diluteacids; practicallyinsoluble in alcohol(O’Neil, 2006)Soluble in alcohol (Lewis,2007)Dissolves readily inacids (Anger et al.,2005); insoluble inacetone (Lide, 2008)Insoluble in aceticacid; soluble insolutions of fixedalkali hydroxides;soluble in dilutenitric acid (O’Neil,2006)aChromic acid is formed in aqueous solution when chromium(VI) oxide is dissolved in water; it cannot beisolated as a pure compound out of solution (Angeretal., 2005; Page and Loar, 2004). The term, chromicacid, is sometimes used in reference to chromium(VI) oxide; however, it should be noted that there isastructural difference between the anhydrous substance chromium(VI) oxide and the aqueous chromic acidthat forms when the oxide is dissolved in water.bChromic acid exists in solution as both H2CrO4andH2Cr2O7(Anger et al., 2005; Page and Loar, 2004; Cotton etal., 1999). H2CrO4is the main species in basicsolutions (pH > 6) while H2Cr2O7is the main species in strongly acidic solutions (pH<1) (Anger et al., 2005; Page and Loar, 2004; Cotton et al., 1999). Bothspecies are present in equilibrium in solutions that have a pH value between 2 and 6 (Anger et al., 2005; Page and Loar, 2004; Cotton et al., 1999).

### 8 DRAFT - DO NOT CITE OR QUOTE 3. TOXICOKINETICS 1

2

3 

### Experimental evidence has demonstrated that hexaval ent chromium can be absorbed via 4 

### the oral, inhalation, or dermal routes of exposure in both humans and laboratory animals. For 5 

### this toxicological review, however, the focus is on the toxicokinetics of hexavalent chromium 6 

### following ingestion. Once ingested, hexavalent chr omium compounds can interact with 7 

### endogenous fluids and other organic matter in the g astrointestinal (GI) tract, resulting, to some 8 

### extent, in the reduction of hexavalent chromium to trivalent chromium. For the purpose of this 9 

### section, this process, whereby hexavalent chromium is reduced to trivalent chromium in the GI 10 

### tract, is termed “extracellular” reduction. The ex tent of absorption of ingested hexavalent 11 

### chromium into the GI tissues is determined by both the solubility of the hexavalent chromium 12 

### compound ingested and how rapidly hexavalent chromi um is reduced to trivalent chromium in 13 

### the GI tract, as trivalent chromium does not diffus e readily across cell membranes. Reduced 14 

### trivalent chromium, however, can form complexes wit h organic ligands, which allow it to pass 15 

### more easily across cell membranes. Hexavalent chro mium can easily cross cell membranes due 16 

### to its ability to use existing nonspecific sulfate and phosphate anion transport mechanisms. 17 

### Absorbed hexavalent chromium is distributed through out the body. Liver, kidney, and 18 

### bone are the primary sites of chromium accumulation . Once inside the cell, hexavalent 19 

### chromium is reduced to trivalent chromium, either e nzymatically or non-enzymatically. For the 20 

### purpose of this section, this process is called “in tracellular” reduction to distinguish it from the 21 

### extracellular process described above. This intrac ellular reduction yields such reactive 22 

### intermediates as chromium(V) and chromium(IV), alon g with oxygen radicals generated during 23 

### this process. Hexavalent chromium is primarily eli minated in the urine as trivalent chromium. 24 

### Biliary excretion of hexavalent chromium has been m easured in animals following intravenous 25 

### injection. Chromium can also be eliminated in hair , nails, and breast milk. There does not 26 

### appear to be a gender difference in the toxicokinet ics of hexavalent chromium, and inter27 

### individual variability in extracellular reduction a nd subsequent absorption and elimination may 28 

### be primarily driven by differences in gastric conte nts and intervals between meals. 29

30



### 3.1. ABSORPTION FOLLOWING INGESTION 31 

### Most quantitative studies of the GI absorption of c hromium in humans have estimated the 32 

### absorption fraction to be <10% of the ingested dose . In general, these studies suggest that the 33

### 9 DRAFT - DO NOT CITE OR QUOTE

absorbed fraction of soluble hexavalent chromium co mpounds (e.g., K 2 

### Cr 2 

### O 7 

### ) is higher than 1 

### insoluble forms (e.g., Cr 2 

### CO 3 

### ). Furthermore, soluble hexavalent chromium compou nds (e.g., 2



### K 2 

### Cr 2 

### O 7 

### ) are absorbed to a greater extent than soluble tri valent chromium compounds (e.g., 3



### CrCl 3 

### ). 4 

### The absorption of hexavalent chromium in human volu nteers ingesting single or multiple 5



### low doses of K 2 

### CrO 4 

### or K 2 

### Cr 2 

### O 7 

### was reported in a series of studies (Finley et al. , 1997, 1996; 6 

### Kerger et al., 1997, 1996; Paustenbach et al., 1996 ; Gargas et al., 1994). Bolus doses of 5 mg 7 

### trivalent chromium (as CrCl 3 

### ), hexavalent chromium (as K 2 

### Cr 2 

### O 7 

### ), or K 2 

### Cr 2 

### O 7 

### in orange juice 8 

### resulted in 0.13, 6.9, and 0.6% absorption, respect ively, across the GI tract (Kerger et al., 1996). 9



### K 2 

### Cr 2 

### O 7 

### added to orange juice likely resulted in near comp lete reduction to trivalent chromium, 10 

### which was complexed with various organic ligands, s uch as ascorbate (Stearns et al., 1994). 11 

### Although trivalent chromium is relatively nondiffus able across cellular membranes, formation of 12 

### complexes with organic ligands is believed to make trivalent chromium more easily absorbed 13 

### (Kerger et al., 1996). In individuals ingesting 5– 10 mg K 2 

### CrO 4 

### for 4 days, 3–6% of the ingested 14 

### dose was absorbed. Because the erythrocyte and pla sma elimination profiles were so similar 15 

### (i.e., hexavalent chromium was not being sequestere d in erythrocytes), the absorbed chromium 16 

### was probably a trivalent chromium-gastric ligand co mplex (Kerger et al., 1997). An individual 17 

### ingesting 4 mg K 2 

### Cr 2 

### O 7 

### /day for 17 days exhibited 2% absorption of chromiu m (Paustenbach et 18 

### al., 1996). Upon cessation of exposure, levels of chromium in plasma and erythrocytes returned 19 

### to pre-exposure levels within a few days. 20 

### In rats and mice, daily oral doses of 8 mg hexavale nt chromium per day (as K 2 

### CrO 4 

### ) for 21 

### 8 weeks resulted in absorption and accumulation of chromium in the bone, spleen, liver, and 22 

### kidney (Kargacin et al., 1993). Rats given 0.138 μ mol hexavalent chromium per day 23 

### (approximately 7 μg/day as Na 2 51 

### CrO 4 

### ) for 3 days exhibited GI absorption of about 16% ( Febel 24 

### et al., 2001). Absorption of 4–10% of a single dai ly dose of 57 μg hexavalent chromium (as 25



### Na 2 51 

### CrO 4 

### ) was observed in rats, regardless of fasting state (MacKenzie et al., 1959). Sutherland 26 

### et al. (2000) demonstrated that significant tissue accumulation of chromium occurred in rats 27 

### chronically exposed to 3 or 10 ppm of hexavalent ch romium in drinking water for 44 weeks, 28 

### with the effect being most pronounced at a concentr ation of 10 ppm. Chromium was most 29 

### highly concentrated in bone and kidney. These inve stigators concluded that this result confirms 30 

### that a portion of the ingested hexavalent chromium was bioavailable, absorbed, and taken up 31 

### from systemic circulation by a variety of tissues. Because the elevated chromium measured in 32 

### the tissues (i.e., bone, kidney, liver, brain, test is, ovary, and blood) was not speciated, however, 33

### 10 DRAFT - DO NOT CITE OR QUOTE

the investigators posed two nonmutually exclusive e xplanations for these results. A portion of 1 

### the ingested hexavalent chromium may have escaped r eduction, entered systemic circulation, and 2 

### was available for cellular uptake. The other possi bility proposed was that trivalent chromium 3 

### that was formed in the gut and absorbed was not cle ared by the kidneys but rather taken up by 4 

### the cells. In any event, this study suggests that even at relatively low concentrations, hexavalent 5 

### chromium is likely absorbed and retained in the bod y. 6



### Several studies have evaluated the extent to which hexavalent chromium gets reduced to 7



### trivalent chromium in the GI tract of both laborato ry animals and humans. Data from in vitro 8 

### studies show that hexavalent chromium may be reduce d via enzymatic and non-enzymatic 9 

### mechanisms; the extent to which this mechanism is i nvolved is largely determined by the route 10 

### of exposure. For oral exposures, a combination of thermostable reducing compounds in the 11 

### saliva and GI fluids and low pH environment of the stomach dominate the reduction of 12 

### hexavalent to trivalent chromium. This has a signi ficant impact on the extent to which orally 13 

### ingested chromium is absorbed across the GI tract. 14 

### Investigators have reported non-enzymatic reduction of hexavalent chromium in in vitro 15 

### tests utilizing GI fluids, although the exact fluid constituents responsible for reduction have not 16



### been identified. Saliva from five volunteers reduc ed an average of 1.4 μ g hexavalent 17 

### chromium/mL. Intestinal bacteria, cultured from hu man feces from three volunteers, were 18



### observed to reduce 3.8 μ g hexavalent chromium/10 9 

### cells after contact for 4 hours. These fluids 19 

### represent the first line of defense against hexaval ent chromium toxicity from oral exposures. 20 

### In samples of human gastric juices collected from h ospital patients suffering from 21 

### duodenal ulcers, reduction of hexavalent chromium ( in the form of Na 2 

### CrO 4 

### ) occurred at 22 

### maximal rates of 40–60 μg/mL/hour at 3–4 hours foll owing meal consumption (DeFlora et al., 23 

### 1987). Minimal reduction was observed in gastric j uices collected during the night and between 24 

### meals. Artificial acidification did not markedly c hange the reducing capability of the collected 25 

### gastric juices, suggesting that hexavalent chromium reduction is predominantly mediated by 26 

### reducing agents present in the gastric environment and not pH. This is consistent with the 27 

### findings of Donaldson and Barreras (1966), who admi nistered oral solutions of radiolabled 28

51 

### CrCl 3 

### or Na 2 51 

### CrO 4 

### in human volunteers. Almost all of the trivalent chromium was recovered 29 

### in the feces, while 2–11% of hexavalent chromium wa s absorbed. However, when subjects were 30 

### given trivalent chromium or hexavalent chromium via duodenal intubation (bypassing the 31 

### stomach), approximately 50% of the administered hex avalent chromium dose was absorbed, 32 

### while the fractional absorption of trivalent chromi um changed less than 5%. Both pH and gastric 33

### 11 DRAFT - DO NOT CITE OR QUOTE

reducing agents aid in gastric reduction of hexaval ent chromium, as intrajejunal intubation of 1



### Na 2 51 

### CrO 4 

### alone resulted in 25% absorption, while Na 2 51 

### CrO 4 

### pre-incubated with HCl or gastric 2 

### juices resulted in absorption of approximately 25% and 2% of hexavalent chromium, 3 

### respectively. 4

5



### 3.2. DISTRIBUTION FOLLOWING INGESTION 6 

### Absorbed chromium distributes to nearly all tissues , with the highest concentrations 7 

### found in kidney and liver. Bone is also a major de pot and may contribute to the long-term 8



### retention kinetics of chromium. Hexavalent chromiu m is unstable in the body and is reduced 9 

### intracellularly to reactive chromium(V) and chromiu m(IV), and ultimately to trivalent 10 

### chromium, both enzymatically and non-enzymatically. Hexavalent chromium in blood is taken 11 

### up into red blood cells, where it undergoes reducti on and forms complexes with Hgb and other 12 

### intracellular proteins that are sufficiently stable to retain chromium for a substantial fraction of 13 

### the red blood cell lifetime. Over time, the erythr ocyte-associated chromium appears to be 14 

### transferred to the spleen as a result of scavenging of aging erythrocytes from the blood. 15



### Absorbed chromium also can be transferred to fetuse s through the placenta and to infants via 16



### breast milk. 17 

### In humans, hexavalent chromium has been measured in blood and urine following oral 18 

### exposures of ≤ 10 mg/day (Finley et al., 1997, 1996; Kerger et al. , 1997, 1996; Paustenbach et al., 19 

### 1996; Gargas et al., 1994). In the blood, hexavale nt chromium is taken up into the erythrocytes 20 

### via a sulphate anion channel, where it is reduced t o trivalent chromium and bound to Hgb. 21 

### Excess trivalent chromium in the erythrocyte is seq uestered until cell death (Kerger et al., 1997; 22 

### Aaseth et al., 1982). Trivalent chromium in plasma does not readily diffuse into erythrocytes. 23 

### This explains the observation of higher chromium er ythrocyte to plasma ratios following 24 

### exposure to hexavalent chromium. 25 

### Wiegand et al. (1985) described the in vitro uptake kinetics of hexavalent chromium in 26 

### erythrocytes of rats and humans. No species differ ences were observed; both species exhibited 27 

### Michaelis-Menten uptake kinetics, with an initial f ast uptake rate (Table 3-1). 28

29 

### Table 3-1. In vitro kinetic parameters of hexavale nt chromium uptake in

erythrocytes of rats and humans

Hexavalent chromium uptake Human Rat Half-time (whole blood)

### 12 DRAFT - DO NOT CITE OR QUOTE

Initial phase 22.7 s 6.9 s Second phase 10.4 min 10.1 min Initial transport capacity (CrO 4 2- /erythrocyte/min) 3.1 × 10 8 2.5 × 10 8

Whole blood kinetics

V max ( μ mol/mL/min) 2.8 3.0 Michaelis constant (K m ) (mM/l blood) 20.9 14.1

Source: Wiegand et al. (1985).

1 

### The partitioning of hexavalent chromium from plasma into erythrocytes is significant; it 2 

### has been used as a biomonitoring endpoint (Kerger e t al., 1996; Minoia and Cavelleri, 1988) and 3 

### is responsible for the observed residence time of c hromium in whole blood (Paustenbach et al., 4 

### 1996; Langard et al., 1978). K 2 

### Cr 2 

### O 7 

### introduced into plasma and reconstituted whole blo od 5 

### (stabilized with EDTA) from three individuals was r eadily reduced to trivalent chromium in the 6 

### concentration range of 100–1,000 μ g hexavalent chromium/L. Hexavalent chromium was 7 

### detected in spiked plasma at concentrations of 2,00 0 and 10,000 μ g hexavalent chromium/L, but 8 

### not at 1,000 μ g hexavalent chromium/L. Further, the plasma:eryth rocyte ratio of total chromium 9 

### decreased with increasing hexavalent chromium conce ntration. The variability between subjects 10 

### in the ratio of plasma:erythrocyte total chromium d iminished by approximately 1 order of 11 

### magnitude as the hexavalent chromium concentration increased from 200 to 1,000 μ g hexavalent 12 

### chromium/L. These data suggest that the reductive capacity of erythrocytes is much greater than 13 

### plasma, and that the reduction rate of hexavalent c hromium in erythrocytes is greater than the 14 

### rate of uptake from the plasma (Corbett et al., 199 7). 15 

### Three drinking water studies in rats provide data o n the tissue uptake of total chromium 16 

### following ingestion. MacKenzie et al. (1959) measu red tissue chromium levels in rats ingesting 17



### K 2 

### Cr 2 

### O 4 

### in drinking water for 1 year (Table 3-2). Hexavale nt chromium drinking water 18 

### concentrations ranged from 0.45 to 11.2 mg hexavale nt chromium/L, but the stability of 19 

### hexavalent chromium in drinking water was not repor ted. Tissue concentrations (in liver, 20 

### kidney, spleen, and bone) of chromium increased in a dose-related manner. The order of 21 

### chromium concentrations were spleen > bone > kidney > liver. No gender-specific differences in 22



### chromium tissue accumulation were observed. 23

24



### Table 3-2. Terminal tissue chromium levels in rats ingesting K 2 

### Cr 2 

### O 4 

### in

drinking water for 1 year

K 2 Cr 2 O 4 concentration Liver ( μ g/g) Kidney

( μ g/g) Bone

( μ g/g) Spleen

( μ g/g)

### 13 DRAFT - DO NOT CITE OR QUOTE (mg/L) male female male female male female male fem ale

controls 0 0 0 0.25 ± 0.02 0 0.72 ±

0.8 0 0

0.45 0.02 ± 0.002 0.08 ±

0.007 0.14 ±

0.007 0.39 ±

0.04 0.58 ±

0.04 0.76 ± 0.04 0.95 0.91 ±

0.11

2.2 0.08 ± 0.017 0.17 ±

0.03 0.29 ±

0.02 0.48 ±

0.07 1.27 ±

0.06 1.48 ± 0.04 0.68 ±

0.18 1.14 ± 0.1

4.5 0.15 ± 0.04 0.47 ±

0.06 0.45 ±

0.17 1.09 ±

0.13 2.14 ±

0.25 2.44 ± 0.25 3.41 ±

0.44 4.48 ± 0.71

7.7 0.70 ± 0.04 0.55 ±

0.06 3.30 ±

0.03 2.39 ±

0.09 3.43 ±

0.83 5.10 ± 0.35 5.24 ±

0.20 4.73 ± 0.8

11.2 1.22 ± 0.06 1.62 ±

0.14 4.40 ±

0.36 3.98 ±

0.32 3.84 ±

0.49 6.06 ± 0.58 9.91 ±

0.83 11.1 ± 0.86

Source: MacKenzie et al. (1959).

1 

### Kargacin et al. (1993) examined the species differe nces in distribution of chromium in 2 

### male mice and rats exposed to 8 mg hexavalent chrom ium/kg-day as K 2 

### Cr 2 

### O 7 

### in drinking water 3 

### for 4 or 8 weeks. Regardless of duration, chromium accumulated primarily in the spleen, liver, 4 

### and kidney of mice and rats (1–2 orders of magnitud e higher than controls), with mouse liver and 5 

### kidney burdens being about two- to fourfold higher than rats (Table 3-3). Chromium 6 

### accumulation in bone was also significantly higher (four- and sevenfold higher at 4 and 8 weeks) 7 

### in mice than rats. The reason for the higher accum ulation of chromium in mouse liver is 8 

### unknown, but may result from greater reduction of h exavalent chromium in the rat gut prior to 9 

### uptake from the GI tract. Alternatively, the mouse liver may have a higher hexavalent chromium 10 

### reduction capacity than rats, causing more trivalen t chromium to be sequestered in hepatocytes. 11

12

### 14 DRAFT - DO NOT CITE OR QUOTE

Table 3-3. Chromium in tissues ( μ g/g wet tissue or μ g/mL blood) of mice and rats after ingesting K 2 

### Cr 2 

### O 7 

### in drinking water (8 mg hexavalent chromium/kg-day) for 4 or 8 weeks

Controls 4-Week exposure 8-Week exposure

Mice

Liver 0.22 ± 0.14 10.92 ± 5.48 13.83 ± 6.06 Kidney 0.24 ± 0.14 3.77 ± 0.99 4.72 ± 0.68 Spleen 0.53 ± 0.38 5.04 ± 1.45 10.09 ± 2.50

Femur 0.90 ± 0.48 7.43 ± 1.03 12.55 ± 2.99 Lung 0.24 ± 0.12 0.99 ± 0.10 1.08 ± 0.26

Heart 0.32 ± 0.15 0.80 ± 0.23 1.02 ± 0.20

Muscle 0.32 ± 0.23 1.12 ± 0.37 0.60 ± 0.25

Blood 0.14 ± 0.05 0.71 ± 0.07 0.42 ± 0.04

Rats

Liver 0.19 ± 0.14 3.32 ± 0.93 3.59 ± 0.73 Kidney 0.34 ± 0.20 8.62 ± 2.40 9.49 ± 4.38 Spleen 0.43 ± 0.20 3.65 ± 1.87 4.38 ± 0.84

Femur 1.00 ± 0.46 1.85 ± 0.46 1.78 ± 0.99 Lung 0.39 ± 0.43 1.10 ± 0.38 0.67 ± 0.24

Heart 0.38 ± 0.22 0.52 ± 0.12 1.05 ± 0.19

Muscle 0.24 ± 0.14 0.19 ± 0.10 0.17 ± 0.10

Blood 0.19 ± 0.17 0.73 ± 015 0.58 ± 0.13

Source: Kargacin et al. (1993).

1 

### Sutherland et al. (2000) observed significant tissu e accumulation of chromium following 2 

### chronic ingestion of water containing 3 or 10 ppm h exavalent chromium in F344/N rats. In this 3 

### study, chromium was most highly concentrated in kid ney and bone, which is similar to the 4 

### pattern of disposition observed by MacKenzie et al. (1959). Female rats, but not males, had 5 

### significantly higher hepatic chromium concentration s than controls at both 3 and 10 ppm 6 

### hexavalent chromium, which again is in agreement wi th the results of MacKenzie et al. (1959). 7 

### Sutherland et al. (2000) were not able to detect an y chromium in whole blood or brain in any 8 

### rats. The investigators concluded that the absence of detectable chromium in whole blood is 9 

### probably the result of rapid chromium delivery to t issues and clearance of plasma chromium by 10 

### the kidneys. Thus, whole-blood chromium levels are probably not a good indicator of tissue 11 

### chromium levels. Finally, male rats that drank wat er containing 10 ppm hexavalent chromium 12 

### had elevated testicular chromium concentrations, bu t these concentrations were described by the 13 

### investigators as “modest” compared to those found i n bone and kidney. 14

### 15 DRAFT - DO NOT CITE OR QUOTE

Hexavalent chromium is capable of crossing the plac enta, as pregnant mice given a single 1 

### intravenous injection of 10 mg hexavalent chromium/ kg (as Na 2 51 

### CrO 4 

### ) on gestational day (GD) 2 

### 13 exhibited total embryo chromium levels that were 12% of maternal blood levels (Danielsson 3 

### et al., 1982). Intraperitoneal injection of 10 mg trivalent chromi um/kg (as 51 

### CrCl 3 

### ) in pregnant 4 

### mice on GD 8 resulted in approximately equal 51 

### Cr activity in the embryo and maternal blood 5 

### (Iijima et al., 1983). While these studies demonstrate placental transfer of chromium, they are of 6 

### limited use for assessing embryonic exposure to chr omium due to maternal oral exposures to 7



### hexavalent chromium. 8

9



### 3.3. METABOLISM FOLLOWING INGESTION 10 

### Once inside the cell, hexavalent chromium is metabo lized (i.e., reduced) to trivalent 11 

### chromium, either enzymatically (via microsomal enzy mes) or non-enzymatically (via ascorbate 12 

### and GSH). This intracellular reduction yields reac tive intermediates, chromium(V) and 13 

### chromium(IV). These reactive intermediates are for med along with oxygen radicals generated 14 

### via Fenton-like and other possible reactions that o ccur during intracellular reduction. 15



### Hexavalent chromium taken into red blood cells unde rgoes reduction and forms 16 

### complexes with Hgb and other intracellular proteins that are sufficiently stable to retain 17



### chromium for a substantial fraction of the red bloo d cell lifetime. GSH appears to dominate the 18 

### reduction of hexavalent chromium within erythrocyte s (Wiegand et al., 1984). In erythrocyte 19 

### suspensions, the addition of GSH results in intrace llular reduction of hexavalent chromium to 20



### trivalent chromium. The role of GSH was confirmed by the reduction (from 100 to 40%) of 21 

### chromium binding in the erythrocytes following pret reatment with diethylmaleate, a GSH 22 

### depletion agent (Aaseth et al., 1982). Incubation of human erythrocytes with K 2 51 

### Cr 2 

### O 7 

### resulted 23 

### in depletion of the erythrocyte GSH content to abou t 10% of normal. Subsequent analysis of 24 

### erythrocyte lysates suggest that chromium-GSH compl exes are formed and that approximately 25 

### 97% of [ 51 

### Cr] is bound to Hgb (Wiegand et al., 1984). 26 

### Within parenchymal and phagocytic cells, hexavalent chromium may be reduced in the 27 

### cytosolic and microsomal compartments (DeFlora and Wetterhahn, 1989). Isolated liver 28 

### perfusion in rats suggests that the majority of hex avalent chromium reduction is cytosolic, as 60, 29 

### 14, 9, and 2% of [ 51 

### Cr] activity was found in the cytosolic, mitochondr ial, microsomal, and 30 

### nuclear fraction, respectively (Wiegand et al., 198 7). Caution should be used in interpreting cell 31 

### culture data, as the cell culture medium could play a role in hexavalent chromium reduction, 32 

### confounding the extent of intracellular hexavalent chromium reduction. For example, Dulbeco’s 33

### 16 DRAFT - DO NOT CITE OR QUOTE

Modified Eagle’s Medium reduces hexavalent chromium to chromium(V) in the absence of cells 1 

### (Borthiry et al., 2008). In human bronchial epithe lial cells (BEAS-2B), Na 2 

### CrO 4 

### , and to a lesser 2 

### extent, insoluble Zn 2 

### CrO 4 

### , were reduced to two reactive chromium(V) species; one appeared to 3 

### be mediated by a thiol-independent NADP(H) reductas e, and the other possibly via a hexavalent 4 

### chromium-GSH intermediate (Borthiry et al., 2008). Electron paramagnetic resonance studies of 5 

### hexavalent chromium reacting with GSH revealed the generation of two reactive chromium(V) 6 

### intermediates and a glutathione thiyl radical (Aiya r et al., 1991). Pulmonary alveolar 7 

### macrophages (PAM) also reduce hexavalent chromium v ia a NADP(H)-dependent reductase and 8 

### GSH (Petrilli et al., 1986). PAMs in smokers had a pproximately twice the reductive ability than 9 

### cells from nonsmokers, ostensibly due to reductase induction by cigarette smoke (Petrilli et al., 10 

### 1986). 11 

### The predominant mechanism for intracellular hexaval ent chromium reduction via 12 

### microsomal enzymes has been extensively described. Incubation of K 2 

### Cr 2 

### O 7 

### with rat liver 13 

### microsomes or NADP(H) alone resulted in very little hexavalent chromium reduction (Jennette, 14 

### 1982; Gruber and Jennette, 1978). However, incubat ion with microsomes and NADP(H) 15 

### resulted in essentially complete disappearance of h exavalent chromium. Within seconds, 16 

### hexavalent chromium (as K 2 

### C 2 

### O 7 

### ) incubated with rat liver microsomes and NADP(H) w as 17 

### reduced to chromium(V), presumably via 1-electron t ransfer from cytochrome P450 (Jennette, 18 

### 1982). 19 

### In contrast to rat liver microsomes, human lung and liver microsomes do not reduce 20 

### hexavalent chromium via cytochrome P450. Pratt and Myers (1993) showed that human liver 21 

### and lung microsomes reduced hexavalent chromium via an NADP(H) reductase-dependent 22 

### system that was not perturbed by the addition of fi ve different P450 inhibitors. The system was, 23 

### however, inhibited by the addition of TlCl 3 

### , indicating the involvement of flavoproteins, 24 

### specifically cytochrome c reductase. The V max 

### and Michaelis-Menten constant (K m 

### ) for liver 25



### microsomal reduction of hexavalent chromium was 5.0 3 nmol/minute/mg protein and 1.04 mM, 26 

### respectively. The human microsomal K m 

### was 1–3 orders of magnitude lower than those 27 

### measured in rat liver microsomes (16–34 μ M [Mikalsen et al., 1989] to 1.6 mM [Garcia and 28 

### Jenette, 1981]). Another striking difference betwe en rat and human hexavalent chromium 29 

### microsomal reduction is the relative insensitivity to O 2 

### in human microsomes (Pratt and Myers, 30 

### 1993). While rat microsomal hexavalent chromium re duction was markedly inhibited in the 31 

### presence of 0.1% O 2 

### , human microsomal reduction was diminished by only 34–56% in the 32 

### presence of ambient (21%) O 2 

### . These results suggest two things about the spati al distribution of 33

### 17 DRAFT - DO NOT CITE OR QUOTE

microsomal hexavalent chromium reduction in rats an d humans. First, P450-dependent 1 

### hexavalent chromium reduction is likely to be confi ned to the centrilobular region of the rat liver, 2



### since an O 2 

### tension of only 1 mm Hg exists there. Secondly, t he insensitivity to O 2 

### of human 3 

### microsomes makes it possible for enzymatic reductio n to occur in highly aerated tissues, such as 4 

### the lung. 5 

### Myers and Myers (1998) verified and extended the de scription of enzymatic hexavalent 6



### chromium reduction in human liver microsomes. Live r microsomes from five individuals were 7



### incubated with Na 2 

### CrO 4 

### to determine reduction kinetics. Using a series of P450 inhibitors and 8



### TlCl 3 

### , the authors showed that hexavalent chromium reduc tion was mediated by flavoproteins, 9 

### NADP(H)-dependent P450 reductase, and cytochrome b 5 

### . Parameters for reduction kinetics in 10



### these five individuals are shown in Table 3-6. The range of V max 

### and K m 

### values was very similar 11 

### across subjects. Lung microsomes from one individu al exhibited V max 

### and K m 

### values that were 12



### 0.66- and 2.8-fold lower than liver microsome value s. Finally, the addition of iron to the liver 13 

### microsomal system revealed that hexavalent chromium reduction could be stimulated by iron 14



### levels that were 3- to 26-fold lower than the hexav alent chromium levels, suggesting that the iron 15 

### may have a catalytic role in the enzymatic reductio n of hexavalent chromium. 16

17 

### Table 3-6. Kinetic parameters of hexavalent chromi um reduction in human

liver microsomes from five individuals

Parameter Observation

V max 10.4–10.7

K m 1.04–1.68 Inhibition by O 2 26–37% Inhibition by TlCl 3 96–100% Inhibition by P450 inhibitors

Carbon monoxide None Piperonyl butoxide None Aminopyrine None

Source: Myers and Myers (1998).

18 

### Proteoliposomes composed of recombinant human P450 reductase and cytochrome b 5 19 

### were used to verify that electrons from NADP(H) cou ld be transferred to cytochrome b 5 

### during 20 

### the reduction of hexavalent chromium (Jannetto et a l., 2001). Markedly less hexavalent 21 

### chromium reduction occurred in proteoliposomes devo id of cytochrome b 5 

### . Further, hexavalent 22

### 18 DRAFT - DO NOT CITE OR QUOTE

chromium reduction in proteoliposomes was almost id entical to human liver microsomes when 1 

### corrected for the cytochrome b 5 

### concentration. 2



### The available data in human and animal studies did not suggest a significant gender 3



### difference in metabolism of hexavalent chromium. F urther, human liver microsome studies did 4 

### not identify marked variability in enzymatic rates of hexavalent chromium reduction (Myers and 5 

### Myers, 1998), although samples were examined from a small number of individuals. 6

7



### 3.4. ELIMINATION FOLLOWING INGESTION 8 

### Chromium absorbed from the GI tract is excreted pre dominantly in urine. Chromium that 9 

### is secreted in bile is extensively reabsorbed. Uri nary and biliary chromium is excreted 10 

### predominantly as trivalent chromium. Chromium can also be eliminated by transfer to hair, 11 

### nails, and breast milk. The elimination of various species of chromium can be different, as 12 

### elimination of ingested hexavalent chromium (as K 2 

### Cr 2 

### O 7 

### ) appears to have a slower elimination 13 

### rate (half-life approximately 40 hours) than ingest ed trivalent chromium (as CrCl 3 

### -organic 14 

### ligand; half-life of approximately 10 hours). 15 

### The clearance of orally ingested hexavalent chromiu m in humans was addressed in a 16



### series of studies in which human volunteers were do sed with up to 10 mg hexavalent 17 

### chromium/day in bolus or repeated doses. Four volu nteers ingesting a bolus dose of 5 mg 18 

### hexavalent chromium (as K 2 

### Cr 2 

### O 7 

### ) excreted 76–82% of the total as urinary chromium within 19 

### 3 days, resulting in an average urinary elimination half-life of 39.3 hours (range 36–41 hours) 20 

### (Kerger et al., 1997). In a single human volunteer ingesting 4 mg hexavalent chromium/day (as 21



### K 2 

### Cr 2 

### O 7 

### ) for 17 days, 87% of the total chromium was excret ed in the urine in the first 4 days, 22 

### with a urinary elimination half-life of 36 hours (P austenbach et al., 1996). In human volunteers 23 

### ingesting chromium-containing water 3 times/day, to taling 5 or 10 mg hexavalent chromium/day 24 

### for a 3-day period, approximately 1.7 and 3.5% of t he administered doses, respectively, were 25 

### excreted in urine (Finley et al., 1997). This is c onsistent with an observation in a hospital patient 26 

### given Na 2 

### CrO 4 

### , who excreted an average of 2.1% (range 0.2–4.4%) of administered hexavalent 27 

### chromium (Donaldson and Barreras, 1966). Chromium is also excreted into breast milk, 28 

### although no data are available identifying the exte nt to which hexavalent chromium is eliminated 29 

### by this route. Two studies of 45 and 17 lactating women found an average of 0.3 and 0.2 μg/L of 30 

### hexavalent chromium in breast milk, respectively. 31 

### In rats gavaged with a single dose of Na 2 51 

### CrO 4, 

### approximately 99% of the administered 32 

### dose was eliminated in the feces, while 0.8% was el iminated in the urine, both within 4 days 33

### 19 DRAFT - DO NOT CITE OR QUOTE

(Sayato et al., 1980). Approximately 81% and 2.17% of 7 μ g hexavalent chromium/day (as 1



### Na 2 51 

### CrO 4 

### ), administered to rats for 3 days, was eliminated in the feces and urine, respectively 2 

### (Febel et al., 2001). 3

4



### 3.5. PHYSIOLOGICALLY-BASED TOXICOKINETIC MODELS 5 

### Physiologically based toxicokinetic (PBTK) models a re mathematical representations of 6 

### biological systems in animals and humans that are r elevant to the quantitative determination of 7



### internal doses of toxic moieties of xenobiotics res ulting from external doses or exposures 8 

### (Krishnan et al., 1994). By employing chemical- an d species-specific parameter values for tissue 9 

### volumes, process rates, and reaction kinetics, PBTK models are used to extrapolate internal 10 

### dosimetry of chemicals across routes of exposure, d ose ranges, and species. In risk assessment, 11 

### the use of PBTK models quantitatively reduces uncer tainties in these extrapolations, thus 12 

### partially or completely obviating the need to apply uncertainty factors in the derivation of 13 

### exposure limits protective of cancer and noncancer effects (Clewell and Andersen, 1985). 14 

### The development of PBTK models occurs in four seque ntial steps: 1) conceptual 15 

### representation of the body into discrete compartmen ts, 2) parameterization of the model, 16 

### 3) exercise of the model by simulating one or more exposures and comparing model predictions 17 

### against empirical observations, and 4) verification of the ability of the model to adequately 18 

### predict empirical data not used for model exercisin g (Krishnan and Andersen, 1994). PBTK 19 

### models, by nature, are simplified representations o f complex biological systems and often 20 

### contain gaps in the quantitation of a substance’s t oxicokinetics. However, if a model is capable 21 

### of adequately predicting absorption, distribution, metabolism, and clearance of a compound and 22 

### metabolites relevant to the critical toxic endpoint s, it serves to reduce the overall uncertainty in 23 

### the biology acting on that compound. 24 

### For chromium, two PBTK models have been published f or hexavalent and trivalent 25 

### chromium in rats and humans (O’Flaherty et al., 200 1; O’Flaherty, 1996, 1993). The inclusion 26



### of trivalent chromium in the model allows for the u se of trivalent chromium exposure time 27 

### course data to aid in parameterization of chromium elimination and to evaluate the ability of the 28 

### model to predict elimination of hexavalent chromium as trivalent chromium. This section 29 

### describes the structure, development, and performan ce of these models and provides a discussion 30 

### of the utility of these models for use in health ri sk assessment of inhaled or ingested hexavalent 31 

### chromium for subchronic and chronic exposure durati ons. 32

33

### 20 DRAFT - DO NOT CITE OR QUOTE

O’Flaherty Model (O’Flaherty et al., 2001; O’Flaher ty, 1996, 1993) 1 

### The O’Flaherty rat and human model is composed of 1 0 tissue compartments, including 2 

### lung, GI tract, liver, kidney, bone, plasma, erythr ocytes, urine, and well- and poorly-perfused 3 

### tissues (Figure 3-1). The model parameters, values , and definitions are shown in Table 3-7. The 4 

### lung compartment treats an inhaled dose as a dose d eposited to the total lung; it does not 5 

### distinguish particle-dependent deposition into the various levels of the lower respiratory tract. 6 

### As such, it is well suited to simulate intratrachea l instillation exposures. Inhaled doses are first 7 

### deposited into the bioavailable pool A of the lung. From there, doses can be absorbed into the 8 

### plasma, transferred to the GI tract (mucocilliary c learance), or transferred to pool B, which 9 

### represents the non-bioavailable portion of the dose . Chromium in pool B is transferred out of the 10 

### lung to the GI tract. First-order rate constants g overn transfer of chromium from the lung. The 11



### model does not discriminate between free trivalent chromium and trivalent chromium-organic 12 

### ligand complexes in the GI tract. 13

14

KIDNEY Cr(VI) → Cr(III)

URINARY PATH POOL B

POOL A Cr(VI) → Cr(III)

WELL-PERFUSED Cr(VI) → Cr(III)

POORLY-PERFUSED Cr(VI) → Cr(III)

BONE Cr(VI) → Cr(III)

LIVER Cr(VI) → Cr(III)PLASMAPLASMARED CELLSCr(VI)→Cr(III)

GI TRACT Cr(VI) → Cr(III)

URINARY EXCRETION ORAL EXPOSURE

FECAL EXCRETION INHALATION EXPOSURE LUNG

15

### 21 DRAFT - DO NOT CITE OR QUOTE

Source: O’Flaherty (1996). 1

2 

### Figure 3-1. A physiologically based model of chrom ium kinetics in the rat 3 

### and human. 4

### 22 DRAFT - DO NOT CITE OR QUOTE

1 

### Table 3-7. Chemical-specific parameters in the rat and human chromium models

Rat Human Parameter a Cr(III) Cr(VI) Cr(III) Cr(VI) Definition Absorption

KGI 0.01 0.04 0.25 2.5 First-order rate constant for absorption from the GI tract (Da -1 )

KLU 0.2 2.0 NA NA First-order rate constant for absorption from the bioavailable lung pool (pool A) (Da -1 )

KMUCOA 0.8 0.8 NA NA First-order rate constant for mucoci liary clearance from pool A to the GI tract (Da -1 )

KMUCOB 0.025 0.025 NA NA First-order rate constant for mu cociliary clearance from the nonbioavailable lung pool (pool B) to the GI tract (Da -1 )

KLUAB 1.2 1.2 NA NA First-order rate constant fo r transfer from pool A to pool B (Da -1 )

FRLUNG NA NA 0.3 0.3 Fraction of inhaled chromium absorbed to blood

FRTRGI NA NA 0.7 0.7 Fraction of inhaled chromium transferred to GI tract

Distribution

CR 5.0 15.0 NA b NA b Relative clearance of chromium into mineralizing b one (lit ers of blood plasma cleared per liter of new bone forme d) KINRBC 0.0003 1.5 12.0 NA Clearance from plasma t o red cell (L/Da) KDIN 0.007 1.5 3.0 30.0 Clearance from plasma to kidney (L/Da) LDIN 0.0001 1.5 3.0 30.0 Clearance from plasma to liver (L/Da) WDIN 0.0001 1.5 3.0 30.0 Clearance from plasma to other well-perfused tissues (L/Da) PDIN 0.0001 0.01 3.0 30.0 Clearance from plasma t o poorly-perfused tissues (L/Da)

BDIN 0.0001 0.01 NA b NA b Clearance from plasma to bone (L/Da) CR NA NA 5.0 15.0 Fraction deposition from blood t o forming bone

KOUTRBC 0.0003 10.0 12.0 NA Clearance from red cell to pla sma (L/Da) KDOUT 0.001 10.0 3.0 30.0 Clearance from kidney to plasma (L/Da)

LDOUT 0.0003 10.0 3.0 30.0 Clearance from liver to plasma (L/Da)

WDOUT 0.001 10.0 3.0 30.0 Clearance from other wel l-perfused tissues to plasma (L/Da) PDOUT 0.003 10.0 3.0 30.0 Clearance from poorly perfused tissues to plasma (L/Da)

BDOUT 0.003 10.0 NA b NA b Clearance from bone to plasma (L/Da)

Excretion

KFX 1.5 1.5 14.0 14.0 First-order rate constant for loss of chromium from intestinal tract contents to the feces (Da -1 ) QEC 0.065 0.065 NA c NA c Excretion clearance from the plasma (urinary clear ance) (L/kg/Da)

CLEAR c NA NA 12.0 12.0 Parameter in expression for cleara nce from blood plasma to urine (L/day)

MAX c NA NA 0.008 0.008 Parameter in expression for clea rance from blood plasma to urine (mg/day)

KM c NA NA 0.0008 0.0008 Parameter in expression for clearance from blood pl asma to urine (mg/L) FB 0.0 0.0 NA NA Fraction of body burden secreted in the bile FI 0.0 0.0 NA NA Fraction of body burden excreted via the GI tract

### 23 DRAFT - DO NOT CITE OR QUOTE

Table 3-7. Chemical-specific parameters in the rat and human chromium models

Rat Human Parameter a Cr(III) Cr(VI) Cr(III) Cr(VI) Definition

Reduction

KREDRC NA 0.7 NA 7.0 First-order rate constant for reduction of Cr(VI) to Cr(III) in the red cell (Da -1 )

KREDBP NA NA NA 0.2 First-order rate constant for reduction of Cr(VI) to Cr(III) in blood plasma (Da -1 )

KREDKL NA NA NA 500.0 First-order rate constant fo r reduction of Cr(VI) to Cr(III) in kidney (Da -1 )

KREDGI NA 10.0 NA 100.0 First-order rate constant for reduction of Cr(VI) to Cr(III) in GI tract contents (Da -1 )

KRED NA 0.5 NA 5.0 First-order rate constant for r eduction of Cr(VI) to Cr(III) in all other tissues and in lung contents (Da -1 ) Lag time for excretion of urine FRHOLD 0.7 0.7 NA NA Fraction of urinary chromium not excreted immediately; that is, temporarily held in pool

KHOLD 0.05 0.05 NA NA First-order rate constant f or excretion from the retained urine pool (Da -1 )

FR 0.10 0.10 NA NA Fraction of chromium in retain ed urine that is associated with the kidney

a Parameter names are those for human model in cases where the reported rat and human parameter names we re not identical.

b Exchanges between blood plasma and cortical and tra becular bone are simulated as functions of bone for mation and resorption rates.

c

CBP

KM MAX CLEAR QE +

− = , where QE is clearance from blood plasma to urine (L/day) and CBP is plasma concentration of chromium (mg/L).

NA = not applicable

Sources: O'Flaherty (1996) (rat parameters); O’Fla herty et al. (2001) (human parameters).

1 

### The GI tract contains two competing transfer proces ses: absorption from the GI lumen to 2 

### the liver, and transfer of the unabsorbed fraction to the feces. All of chromium absorbed from 3



### the GI tract is first transferred to the liver. 4 

### Exchange of hexavalent chromium between compartment s is assumed to be flow limited 5 

### (i.e., exchange between compartments occurs more ra pidly than blood flow), while trivalent 6 

### chromium is diffusion limited. Chromium is exchang ed between the plasma and soft tissues via 7 

### separate first order rate constants for uptake into and passage from the tissue. No partition 8 

### coefficients are used; plasma protein and macromole cule binding is not considered, due to lack 9 

### of data. The rat model represents chromium exchang e between plasma and bone with a single 10

### 24 DRAFT - DO NOT CITE OR QUOTE

constant. The human model represents bone chromium uptake and elimination as a function of 1 

### the bone formation and resorption rates, respective ly. The dynamic model is described in detail 2 

### by O’Flaherty (1995, 1993). 3 

### Systemic elimination of chromium is represented as a one-way exchange from plasma to 4 

### the urine. It proceeds by a first-order constant i n the rat model, but as a variable chromium 5 

### concentration-dependent function of the glomerular filtration rate (GFR) in the human model. 6 

### For example, urinary elimination is 0.7% of GFR at 1 × 10 -4 

### mg/L and 40% of GFR at 7 

### 0.01 mg/L. 8 

### Initial parameter values for the rat and human mode ls were taken from the literature of 9 

### oral, inhalation, and intravenous exposure studies (Cavalleri et al., 1985; Edel and Sabbioni, 10 

### 1985; Bragt and van Dura, 1983; Weber, 1983; Cikrt and Bencko, 1979; Mertz et al., 1969; 11 

### MacKenzie et al., 1959; Thompson and Hollis, 1958). Parameters for reduction of hexavalent 12 

### chromium, exchange of hexavalent chromium and triva lent chromium between the tissues, and 13 

### chromium elimination were optimized against time co urse data provided by intratracheal 14 

### instillation studies (Edel and Sabbioni, 1985; Brag t and van Dura, 1983; Weber, 1983). Once 15 

### optimized, the rat model performance was evaluated by its ability to predict the observations 16



### from studies of ZnCrO 4 

### inhalation (Langård et al., 1978) and chronic Na 2 

### CrO 4 

### or CrCl 3 

### ingestion 17 

### (up to 25 mg/L) in drinking water (MacKenzie et al. , 1958). 18 

### The human model parameters were optimized using dat a for plasma and red blood cell 19 

### chromium burden, and chromium elimination to the ur ine in adults receiving single doses of 20



### CrCl 3 

### or K 2 

### Cr 2 

### O 7 

### (Finley et al., 1997; Kerger et al., 1996). The p erformance of the human 21 

### model was evaluated against data for the time cours e of plasma chromium levels and urinary 22 

### elimination of chromium in a single adult ingesting 4 mg K 2 

### Cr 2 

### O 7 

### /day for 17 days (Paustenbach 23 

### et al., 1996). Since the evaluation data were from a single subject, the GI absorption rate 24 

### constant had to be adjusted to obtain adequate fits

. 25 

### The rat and human models are capable of estimating internal doses of trivalent chromium 26



### and hexavalent chromium in plasma, erythrocytes, kidney, liver, bone, lung, an d GI tract. The 27 

### rat model has not been evaluated against time cours e data from multiple inhalation exposures. 28 

### Although tissue burden predictions were evaluated a gainst experimental observations, the 29 

### observations covered a single timepoint (i.e., term inal sacrifice). Nevertheless, these data should 30 

### be representative of tissue burdens at steady-state , which is advantageous for simulating internal 31 

### dosimetry from chronic exposures. The human model was optimized against data for 32 

### erythrocyte, plasma, and urinary chromium levels re sulting from single oral doses in volunteers. 33

### 25 DRAFT - DO NOT CITE OR QUOTE

It may be useful for predicting internal dosimetry for chronic oral exposures, as it was able to 1 

### replicate plasma and urine chromium burdens from a study of a single individual exposed for 2 

### 17 days, over which steady state chromium levels we re achieved. The model was optimized 3 

### against data from both males and females, although the hexavalent chromium database does not 4 

### imply a significant gender difference in hexavalent chromium toxicokinetics. 5 

### The PBTK model of O’Flaherty (O’Flaherty et al., 20 01; O’Flaherty, 1996, 1993) is not 6 

### designed to predict deposited and absorbed doses fr om the lungs. Furthermore, key human 7 

### model parameters were optimized using short-term or al ingestion data from five or fewer human 8 

### subjects. The human model was also evaluated using ingestion data from a single human 9 

### subject. During the evaluation exercise, the param eter for gastric reduction of hexavalent 10 

### chromium, a key parameter for chromium metabolism, had to be adjusted empirically. This 11 

### suggests that the variability in gastric reduction capacity within a human population may be 12 

### extensive, but is uncertain. The limitations of th e model for replicating inhalation exposures plus 13 

### the performance of the model against limited, oral exposure data sets make it inadequate for use 14 

### in deriving health risks from chronic oral or inhal ation exposures in human populations. 15

16

17

### 26 DRAFT - DO NOT CITE OR QUOTE 4. HAZARD IDENTIFICATION 1

2 3 

### 4.1. ORAL STUDIES IN HUMANS 4 

### The human health effects observed following oral in gestion of hexavalent chromium 5 

### usually come from individuals accidentally or inten tionally ingesting hexavalent chromium 6 

### compounds or from human populations unknowingly con suming food or drinking water 7



### contaminated with hexavalent chromium. 8

9



### 4.1.1. Acute Exposure 10 

### Several case reports have been published on clinica l signs and symptoms in individuals 11 

### following acute accidental or intentional ingestion of high doses (fatal or near fatal) of 12 

### hexavalent chromium compounds, including chromic ac id (Loubieres et al., 1999; Saryan and 13 

### Reedy, 1988; Fristedt et al., 1965), potassium dich romate (Hantson et al., 2005; Clochesy, 1984; 14 

### Iserson et al., 1983; Sharma et al., 1978; Kaufman et al., 1970; Partington, 1950; Goldman and 15 

### Karotkin, 1935), and ammonium dichromate (Hasan, 20 07; Reichelderfer, 1968). Clinical 16 

### presentation of patients following acute, high-dose exposure was similar, regardless of the 17 

### specific hexavalent chromium compound ingested, and included the following: abdominal pain, 18 

### nausea, and vomiting; hematemesis and bloody diarrh ea; caustic burns of mouth, pharynx, 19 

### esophagus, stomach, and duodenum and GI hemorrhage; anemia, decreased blood Hgb, 20 

### abnormal erythrocytes, and intravascular hemolysis; hepatotoxicity (hepatomegaly, jaundice, 21 

### elevated blood bilirubin, and liver enzymes activit ies); renal failure (oliguria and anuria); 22 

### cyanosis; and metabolic acidosis, hypotension, and shock. Findings on tissue biopsies included 23 

### hepatic fatty degeneration and necrosis and renal t ubular degeneration and necrosis (Loubieres et 24 

### al., 1999; Sharma et al., 1978; Kaufman et al., 197 0; Reichelderfer, 1968). Based on estimated 25 

### amounts of hexavalent chromium ingested, the range of lethal doses for hexavalent chromium in 26 

### humans is estimated to range from approximately 4.1 to 357 mg hexavalent chromium/kg body 27 

### weight (Loubieres et al., 1999; Saryan and Reedy, 1 988; Clochesy, 1984; Iserson et al., 1983; 28 

### Kaufman et al., 1970). 29 

### A series of acute and short-term repeated (17-day) ingestion studies were conducted on 30 

### human volunteers to evaluate hexavalent chromium ph armacokinetics (Corbett et al., 1997; 31 

### Finley et al., 1997; Kerger et al., 1997, 1996; Kuy kendall et al., 1996; Paustenbach et al., 1996). 32 

### With the exception of Paustenbach et al. (1996), th ese studies reported that study protocols were 33 

### reviewed and approved by a human use committee comp rised of three board-certified 34

### 27 DRAFT - DO NOT CITE OR QUOTE

occupational physicians and one board-certified tox icologist. In each case, the committee 1 

### determined that participants were properly informed of the reported adverse health effects 2 

### associated with hexavalent chromium exposure. The study by Paustenbach et al. (1996) 3 

### involved a singe male volunteer. The methods secti on of this study noted that “The volunteer 4 

### had a PhD in toxicology, and the test protocol was approved by a human use committee.” As 5 

### part of these studies, standard clinical evaluation s were performed that included blood cell 6 

### counts, blood clinical chemistry (SMA-20), and urin alysis (volume, specific gravity, creatinine). 7 

### In the longest duration exposure, a single subject ingested 2 L/day of a solution of containing 8 

### 2 mg hexavalent chromium/L (as potassium dichromate in water) for 17 consecutive days 9 

### (approximately 0.06 mg hexavalent chromium/kg-day, assuming a 70-kg body weight) 10 

### (Paustenbach et al., 1996). In shorter duration st udies, 3–5 subjects ingested 1 L/day of solutions 11 

### containing 0.1–10 mg hexavalent chromium/L in water (approximately 0.001–0.14 mg 12 

### hexavalent chromium/kg-day, assuming a 70-kg body w eight) for 1–3 days (Finley et al., 1997; 13 

### Kerger et al., 1997, 1996; Kuykendall et al., 1996) . Data from the clinical evaluations were not 14 

### reported; however, results were described in genera l terms that suggested that values for clinical 15 

### chemistry endpoints were “similar” when measured pr ior to, during, or following dosing 16 

### (Paustenbach et al., 2003, 1996). 17

18



### 4.1.2. Environmental Exposure 19 

### Human studies of possible associations between oral exposures to environmental 20



### hexavalent chromium and health outcomes include sev eral epidemiology studies in which health 21 

### outcomes (primarily cancer) were evaluated among po pulations who resided near sources of 22 

### industrial waste containing hexavalent chromium com pounds in Liaoning Province, China 23 

### (Kerger et al., 2009; Beaumont et al., 2008; Zhang and Li, 1997, 1987, 1980), Kings County/San 24 

### Bernadino County, California (Fryzek et al., 2001), Nebraska (Bednar and Kies, 1991), and 25 

### Glasgow, UK (Eizaguirre-Garcia et al., 2000, 1999). In addition to these studies, two cases of 26 

### Hodgkin’s disease in residents of Hinkley, Californ ia, where hexavalent chromium was used as a 27 

### cooling additive at a local gas plant, were describ ed in a case report by Bick et al. (1996). 28

29 

### Liaoning Province, China (Kerger et al., 2009; Beau mont et al., 2008; Zhang and Li, 1997, 30 

### 1987) 31 

### In 1987, Zhang and Li published a paper describing the soil and water contamination by 32 

### chromium in the vicinity of an alloy plant where ch romium was smelted in the Jinzhou area of 33

### 28 DRAFT - DO NOT CITE OR QUOTE

Liaoning Province, China (Zhang and Li, 1987). Thi s paper was based on an earlier unpublished 1 

### report (Zhang and Li, 1980). A more detailed morta lity analysis, which included variation in 2 

### cancer mortality rates among the 5 villages along t he Nuer River, was published in 1997 (Zhang 3 

### and Li, 1997) in the Journal of Occupational and Environmental Medicine. This study has had a 4 

### controversial history that culminated in the retrac tion, in 2006, of the latest report (Zhang and Li, 5 

### 1997) by the editors of the Journal of Occupational and Environmental Medicine because 6



### “ financial and intellectual input to the paper by ou tside parties was not disclosed ” (Brandt-Rauf, 7 

### 2006). The financial and intellectual input in que stion were those from a consulting firm that 8 

### had (or may have had) financial ties with industry clients potentially liable for chromium wastes 9 

### in the United States (Smith, 2008). Two reanalyses of data compiled by Zhang and Li have also 10 

### been reported (Kerger et al., 2009; Beaumont et al. , 2008). The following presentation of the 11 

### studies begins with a description of the geographic area, industrial operations, and resulting 12 

### chromium dispersion in the surrounding communities, with information obtained from the most 13 

### recent reports (Kerger et al. 2009; Beaumont et al. , 2008) and from earlier published and 14 

### unpublished reports (Zhang and Li, 1986; Zhang and Li, 1980; JinZhou Antiepidemic Station, 15 

### 1979). The commonalities and differences in the rea nalyses by Kerger et al. (2009) and 16 

### Beaumont et al. (2008) are then described. 17 

### The study area is west of JinZhou, a city in Liaoni ng province in northeastern China. 18 

### This area was described by Zhang and Li (1987) as b eing primarily agricultural with some 19 

### pockets of industries. One of the industrial plant s is the JinZhou ferrochromium alloy plant, 20 

### located near the Nuer River. The town of TangHeZi d eveloped around the plant (Zhang and Li, 21 

### 1980). A series of small rural villages (Jinchangb ao, Nuer River Village, Yangxing, Shilitai and 22 

### Wenjiatun) are located approximately 1 to 5 km to t he east of the plant along the Nuer River. 23 

### The alloy plant began trial smelting of chromium in 1959, small-scale production in 1961, and 24 

### mass production in 1965 (Zhang and Li, 1987). Liqu id wastes from the production process were 25 

### released to a dry river bed (the “Old Nuer River”) near the plant. The amount of hexavalent 26 

### chromium in the wastewater was considerable (estima ted as 20 mg/L at the end of the discharge 27 

### pipe) (Zhang and Li, 1986). Solid wastes (>300,000 tons by 1986) were stored in outdoor piles 28 

### and were subject to leaching to surface water and g roundwater. These piles of ore residue were 29 

### the main long-term source of underground water cont amination. Hexavalent chromium was also 30 

### released into the air through the various productio n and waste processes, with a northeast 31 

### prevailing wind pattern. An additional source of c hromium exposure was from food grown in 32 

### areas using contaminated well water for irrigation. 33

### 29 DRAFT - DO NOT CITE OR QUOTE

In 1964, residents in the Nuer River Village notic ed a yellowing of the color of their 1 

### drinking water. The local health department (refer red to as the “JinZhou Disease Control and 2



### Prevention Station” or the “JinZhou Health and Anti -epidemic Station” or “JinZhou 3 

### Antiepidemic Station” depending on the translation) initiated testing of well water samples in 4 

### each of the five villages in 1965. Chromium was fo und in 75 (28%) of the first set of samples 5 

### from 266 wells in Jinchangbao and Nuer River Villag e, with levels up to 10 mg/L. By the end of 6 

### 1965, the zone of underground water contamination h ad spread, following a path eastward from 7 

### the plant. In JinChangBao, 41% of the wells contai ned hexavalent chromium, as did 96% of the 8 

### wells in Nuer River Village. The highest concentra tion (5 mg/L) was found in Yangxing and 9 

### Nuer Railway Station, which are east of JinChangBao and Nuer River Village. In 1966, 10



### hexavalent chromium was detected in the Nanshan res ervoir (supplying drinking water to 11 

### JinZhou), 9 km from the alloy plant. Monitoring of well water continued, and the expansion of 12 

### the contamination zone appeared to peak in 1979 (Zh ang and Li, 1986). A variety of efforts to 13



### reduce the chromium run-off were undertaken in 1965 -1967. 14 

### Table 4-1 includes a compilation of the available d ata from the 1965 water sampling 15 

### studies (based on Table 2 from Beaumont et al., 200 8, with the addition of the distance from the 16 

### plant and average chromium levels in the well water samples from Kerger et al., 2009). The 17 

### analytical methods used to quantify chromium were n ot reported, but these values (and all other 18 

### values for chromium concentrations noted below) wer e reported as hexavalent chromium; 19 

### Beaumont et al. (2008) note that other forms would not be expected to be water soluble. 20 

### Beaumont et al. (2008) and Kerger et al. (2009) are in general agreement regarding their 21 

### interpretation of the 1965 water testing data. The re is disagreement, however, as to what can be 22 

### established regarding levels in later years (Table 4-1), and the stability of the relative levels 23 

### among the villages. Beaumont et al. (2008) do not consider the available data to be adequate to 24 

### classify the individual villages with respect to a relative ranking of exposure, given the lack of 25 

### information regarding the selection of wells sample d, lack of information regarding use of 26 

### specific wells by individuals within the villages, paucity of data from later years, and the rapid 27 

### changes in chromium concentrations in various areas due to the groundwater movement as well 28



### as the efforts to curtail the chromium contaminatio n. Kerger et al. (2009), however, use the 1965 29 

### well water sample data to derive two measures of ex posure (average chromium concentration 30 

### and percent of wells >0.05 mg/L) that they applied to each of the 5 villages for an exposure31 

### response analysis of cancer risk. 32

33

### 30 DRAFT - DO NOT CITE OR QUOTE

Table 4-1. Data pertaining to hexavalent chromium concentrations in drinking water in five villages along path of groun dwater contamination from alloy plant, western JinZhou, China from 1965 to 1979

Village (km from alloy plant)

Year Jinchanbao (1.4) Nuer River

Village (1.5) Yangxing

(3.0) Shilitai

(3.5) Wenjiatun

(5.0) Early 1965 a Cr +6 detected in 75 (28%) of 265 wells sampled in Jinchanbao and Nuer River Village; 73 of the 75 wells were in Nuer River Village; 41 (15%) were >2.0 mg/L. Range 0.6 to 10.0 mg/L

Later in 1965 a,b n wells sampled a,b 123 170 50 21 33

Cr +6 (mg/L) a Number of wells (%) <0.001 73 (59) 7 (4) 14 (28) 2 (10) 27 (82) 0.001 – <0.05 35 (28) 1 (1) 16 (32) 19 (90) 6 (18) 0.05 – <0.1 7 (6) 5 (3) 5 (10) 0 (0) 0 (0) 0.01 – <0.5 8 (7) 27 (16) 12 (24) 0 (0) 0 (0) 0.5 – <1.0 0 (0) 17 (10) 2 (4) 0 (0) 0 ( 0) 1.0 – <5.0 0 (0) 76 (45) 1 (2) 0 (0) 0 ( 0)

≥ 5.0 0 (0) 37 (22) 0 (0) 0 (0) 0 (0) maximum (mg/L) a,b 0.4 20.0 <5 <0.05 <0.05 average (mg/L) b 0.031 2.6 0.18 0.02 0.004

1966 c 0.002 – 20.0

1967 b <0.05 <0.05 <0.05

1972 b <0.05

1974 10.5 d 0.01 – 0.05 c

1979 c 0.06 – 4.33 0.001 – 0.03 0.003 – 0.004

Cr +6 : hexavalent chromium.

a As reported by Beaumont et al. (2008)

b As reported by Kerger et al. (2009)

c As reported by Zhang and Li, 1986, number of sample s not stated.

d Zhang and Li (1986) report this concentration as 7 0.5 mg/L, but Zhang and Li (1987), Beumont et al. ( 2008) and Kerger et al. (2009) report a concentration of 10.5 mg/L. The total number of samples and the range i n concentrations were not specified.

1 

### A mortality study was described first by Zhang and Li in 1980 in an unpublished report 2 

### for the JinZhou health department, and later publis hed in a Chinese journal (Zhang and Li, 3 

### 1987). Mortality records for the period 1970–1978 were obtained from local police stations for 4 

### the five villages along the Nuer River, the distric t surrounding the ferrochromium alloy plant 5 

### (Tanghezi), and three other areas to the west (Yaot anghezi) and north (North Thanghezi, North 6 

### Nuer River) of the plant. Tanghezi and the other t hree areas were not affected by the 7

### 31 DRAFT - DO NOT CITE OR QUOTE

groundwater chromium contamination, and these areas serve as one of the comparison groups in 1 

### the analyses. Cause of death was abstracted by tra ined study staff and reviewed by Dr. Zhang 2 

### (Kerger et al., 2009). A study interview was also conducted (with unspecified surrogates), but 3



### the content of the interview was not described in d etail (Zhang and Li, 1980). The mortality 4 

### analysis indicated that the lung cancer rate was re latively high in TangHeZi (the industrial town 5 

### surrounding the ferrochromium alloy plant), but dec reased in areas further to the north (Zhang 6 

### and Li, 1980). In the areas to the east of TangHeZ i (JinChangBao, Neur River Village, ShiLiTai, 7 

### YangZing, and WenJiaTun), total cancer mortality ra tes (71.9 – 92.7 per 100,000 person-years) 8 

### were high relative to the region (65.4 per 100,000 person-years). Similar elevations were seen 9 

### for lung cancer mortality (13.2 – 21.4 compared wit h 11.2 per 100,000 person-years in the 10 

### eastern villages and comparison region, respectivel y, and stomach cancer mortality rates (27.7 – 11 

### 55.2 in the eastern villages; comparison rates not given in the report, but Zhang and Li state these 12 

### rates are “higher than the district as a whole”) (Z hang and Li, 1987). 13 

### A subsequent paper by Zhang and Li (1997) expanded their work to include an analysis 14 

### of variation in cancer rates among the five village s in the contamination zone in relation to 15 

### distance from the plant and other exposure measures . This analysis is also included in the 16 

### Kerger et al. (2009) report, described below. 17 

### The mortality data described in the reports by Zhan g and Li (1987, 1980) are the basis for 18 

### the subsequent analyses by Beaumont et al. (2008) a nd Kerger et al. (2009). The reanalyses by 19 

### Beaumont et al. (2008) and Kerger et al. (2009) pro vide very similar estimates of person-years. 20 

### Beaumont et al. (2008) used 1982 census data for th e study areas and estimated annual growth 21 

### rates from 1970-1982 for Liaoning Province to estim ate yearly population counts for each of the 22 

### nine study areas; the summation of these figures fr om 1970 to 1978 represents the person-years 23 

### for the study period. Kerger et al. (2009) based t he population figures on the estimated 24 

### populations in 1974 and multiplied these numbers by 9 (number of years of follow-up) to 25 

### estimate person-years for each of the study regions . Tanghezi, the industrial area surrounding 26 

### the ferrochromium alloy plant (1975 population appr oximately 17,500) is approximately 3 to 10 27 

### times bigger than the other study areas (Table 4-2)

. 28 

### The number of total cancer deaths, lung cancer deat hs, and stomach cancer deaths was 29 

### used in combination with estimated person-years at risk as the basis of the calculation of area30 

### specific mortality rates in the analyses by Zhang a nd Li (1997, 1987, 1980), Beaumont et al. 31 

### (2008) and Kerger et al. (2009). Because the resul ts of Zhang and Li (1997) are repeated in the 32

### 32 DRAFT - DO NOT CITE OR QUOTE

presentation by Kerger et al. (2009), only the more recent of these analyses is described in more 1



### detail below. 2 

### There are two relatively minor and two relatively m ajor differences between the analyses 3 

### of the cancer mortality data presented by Beaumont et al. (2008) and Kerger et al. (2009). One 4



### of the minor differences is the value used for stom ach cancer mortality for one of the villages in 5 

### the contamination zone, Nuer River Village. Beaumo nt et al. (2008) do not include an estimate 6 

### of stomach cancer mortality for Nuer River Village in their primary analysis because it was 7 

### missing from the original (1980) unpublished report (Zhang and Li, 1980) and Dr. Zhang 8 

### indicated in a faxed communication with the study a uthors that the estimated rate of 28 per 9 

### 100,000 per year (reported in Zhang and Li, 1997) w as of uncertain accuracy. Beaumont et al. 10 

### (2008) did repeat their analysis using the 28 per 1 00,000 rate for stomach cancer mortality in 11 

### Nuer River Village, and found this inclusion had ve ry little effect on their estimates. Kerger et 12 

### al. (2009) used 28 per 100,000 per year as the stom ach cancer rate for Nuer River Village. The 13 

### second relatively minor difference is in the estima tion of age-adjusted mortality rates. The 14 

### original analyses by Zhang and Li (1987) presented age-adjusted rates for all cancer mortality, 15 

### but not for stomach cancer or lung cancer mortality . Kerger et al. (2009) do not attempt to make 16 

### an age-adjustment for lung or stomach cancer becaus e “small numbers of site-specific deaths in 17 

### the villages would have precluded the calculation o f relatable direct standardized site-specific 18 

### rates in the current study.” Beaumont et al. (2008 ) addressed this issue by calculating the ratio of 19 

### unadjusted to adjusted total cancer rates for each study area, which they term the “age20 

### adjustment influence” ratio. This ratio ranged fro m 0.84 to 1.05. The area-specific lung and 21 

### stomach cancer unadjusted rates were multiplied by the respective area-specific age-adjustment 22 

### influence ratio to create estimated age-adjusted lu ng and stomach cancer rates (Table 4-2). 23 

### One of the major differences between the analyses b y Beaumont et al. (2008) and Kerger 24 

### et al. (2009) was described previously: Kerger et a l. (2009) use the 1965 exposure data for 25 

### exposure-response modeling of the variation in canc er rates among the five villages in the 26 

### chromium contamination zone, and Beaumont et al. (2 008) do not believe the available data are 27 

### adequate for this purpose. The other major differe nce between the analyses is the inclusion of 28 

### TangHeZi, the industrial district surrounding the f errochromium alloy plant, in the comparison 29 

### group. Kerger et al. (2009) considered this distri ct to be too different from the smaller villages in 30 

### terms of urban-rural lifestyles and other exposures that could affect cancer risk (specifically 31 

### stomach cancer and lung cancer) and therefore did n ot include it in their comparison group. 32 

### Beaumont et al. (2008) include TangHeZi, presumably because it was part of the original study 33

### 33 DRAFT - DO NOT CITE OR QUOTE

design. They do not explicitly address the compara bility issue with respect to stomach cancer 1 

### risk factors, although they do note the potential f or occupational chromium exposure to 2 

### contribute to a relatively high lung cancer rate in TangHeZi. 3 

### Table 4-3 presents the measures of association betw een chromium exposure and cancer 4 

### mortality, based on the five villages in the contam ination zone and the various comparison 5 

### groups used by Beaumont et al. (2008) and Kerger et al. (2009). These risk ratios are based on 6 

### comparison of the rates shown in Table 4-2, using a Poisson distribution for calculation of 95% 7 

### confidence intervals. With respect to stomach canc er, the primary site of interest from the 8 

### standpoint of drinking water contamination, Beaumon t et al. (2008) report an association using 9 

### the four comparison areas (TangHeZi, North TangHeZi , North Nuer River, and Yao TangHeZi) 10 

### that were the basis for the original analysis (risk ratio = 1.82, 95% CI (1.11, 2.91)) and using 11 

### rates from all of Liaoning province as a comparison (risk ratio = 1.69, 95% CI (1.12, 2.44)). 12 

### Kerger et al. (2009) excluded the most populous are a, TangHeZi from the comparison group, and 13 

### reported a risk ratio = 1.22 (95% CI (0.74, 2.01)), which they interpret as being evidence of no 14 

### association. In the lung cancer analyses, Beaumont et al. (2008) report relatively little difference 15



### between the rates in the contamination zone and the comparison area (risk ratio = 1.15, 95% CI 16 

### (0.62, 2.07)), but a stronger association using Lia oning province as a comparison (risk ratio = 17 

### 1.78, 95% CI (1.03, 2.87)). Kerger et al. (2009) o bserved higher lung cancer rates in the five 18 

### villages in the contamination zone compared with th e three rural areas they included in the 19 

### comparison group (risk ratio = 1.76, 95% CI (0.78, 3.98)), and slightly reduced risk when 20 

### compared to TanHeZi (risk ratio = 0.80, 95% CI (0.4 4, 1.47)). 21 

### Kerger et al. (2009) also presented results of anal yses of variation in cancer rates within 22 

### the five villages in the chromium contamination zon e, using three measures of exposure 23 

### potential: distance from the plant, average hexaval ent chromium concentrations in 1965, and 24 

### percent of wells with >0.05 mg/L hexavalent chromiu m in 1965 (these measures can be found in 25 

### Table 4-2). The analysis was based on Poisson regr ession of the log-transformed cancer rate in 26 

### relation to the exposure measures (separate models run for each measure). For the distance 27 

### measure, a negative value for the coefficient indic ates an increased cancer rate with closer 28 

### proximity to the plant, and for the other exposure measures a positive coefficient indicates an 29 

### increased cancer rate with higher exposure. The res ults for all cancer mortality (given as the 30 

### regression coefficient and p-value) were 0.04 ( p = 0.61), –0.07 (

p = 0.54) and –0.24 (

p = 0.45) 31 

### for the distance, average hexavalent chromium conce ntration in 1965, and percent of wells >0.05 32 

### mg/L hexavalent chromium in 1965 measures, respecti vely. For stomach cancer mortality, the 33

### 34 DRAFT - DO NOT CITE OR QUOTE

coefficients were 0.01 (

p = 0.93), –0.11 (

p = 0.50) and –0.32 (

p = 0.51) for the distance, average 1 

### hexavalent chromium concentration in 1965, and perc ent of wells >0.05 mg/L hexavalent 2 

### chromium in 1965 measures, respectively, and for lu ng cancer, the coefficients were 0.12 ( p = 3 

### 0.50), –0.06 (

p = 0.79) and –0.11 (

p = 0.88) for the distance, average hexavalent chrom ium 4 

### concentration in 1965, and percent of wells >0.05 m g/L hexavalent chromium in 1965 measures, 5 

### respectively. As described previously, Beaumont et al. (2008) did not include this type of 6 

### exposure-response analysis because they believed th e inherent limitations of the exposure data 7 

### precluded a meaningful analysis. 8

935DRAFT - DO NOT CITE OR QUOTETable 4-2. Results pertaining to cancer mortalityrates in five villages along path of groundwatercontamination from alloy plant and other comparisonareas, western JinZhou, China from 1970 to 1978, basedon analyses by Beaumont et al. (2008) and Kerger etal. (2009)Rate per 100,000 person-yearsAll Cancer Stomach Cancer Lung CancerArea (population or person-years)aAge-adjustedrateAge-adjustmentinfluencebCrude rateEstimated ageadjusted ratebCrude rateEstimated age-adjusted ratebAreas in Contamination ZoneJinchanbao (2900) 83.6 0.97 36.7 35.5 13.2 12.8Nuer River Village (2800) 71.9 0.98 28.0 missingb15.0 14.7Shilitai (2600) 93.0 0.94 55.2 51.7 missing missingYangxing (1100) 76.8 0.94 36.5 34.5 21.4 20.2Wenjiatun (1700) 91.1 0.94 27.7 26.0 20.8 19.5Group average (~98,700)c81.3 34.9 35.3 17.1 16.9Comparison areasTangHeZi (17,500) 71.3 0.86 16.9 14.5 21.4 18.3North TangHeZi (3600) 81.8 0.84 26.4d22.1 8.8 7.4North Nuer River (5800) 71.8 1.05 30.5 31.9 7.6 8.0Yao TangHeZi (1500) 61.3 0.90 26.6 23.8 20.0 17.9Group average – all (~252,500)e72.1 19.4 14.7Group average – without TangHeZi(96,826)f73.7 28.6 9.7aArea population figures are based on approximate 1975 data from Beaumont et al. (2008); group values are total person-years for the combined area.bAs calculated by Beaumont et al. (2008). Nuer River Village stomach cancer rate was not included in the primary analysis by Beaumont et al. (2008) becauseit was missing in the original (1980) report; an additional analysis used a rate of 28.0 as reported by Zhang and Li (1987).cBeaumont et al. (2008) estimate was 98,458 and Kerger et al. (2009) estimate was 98,850.dBeaumont et al. (2008) report this value as 26.14 in Table 2, but based on the calculation of the estimated age-adjusted rate it appears that a value close to 26.3was used; Kerger et al. (2009) report this value as26.4.eBeaumont et al. (2008) estimate was 252,277 and Kerger et al. (2009) estimate was 253,282.fAs reported by Kerger et al. (2009).

### 36 DRAFT

1 

### Table 4-3. Risk ratios comparing cancer mortality rates in five villages

along path of groundwater contamination from alloy plant and other comparison areas, western JinZhou, China from 1970 to 1978

All Cancers Stomach Cancer Lung Cancer Comparison Group a Risk ratio (95% CI) Risk ratio (95% CI) Risk ratio (95% CI)

All 4 areas b 1.13 (0.86, 1.46) 1.82 (1.11, 2.91) 1.15 (0.62, 2. 07) Excluding TangHeZi c 1.10 (0.80,1.51) 1.22 (0.74, 2.01) 1.76 (0.78, 3.9 8) Liaoning province b 1.23 (0.97, 1.53) 1.69 (1.12, 2.44) 1.78 (1.03, 2. 87)

a TangHeZi, North TangHeZi, North Nuer River, and Yao TangHeZi.

b Reported by Beaumont et al. (2008).

c Reported by Kerger et al. (2009).

2 

### In addition to the cancer mortality study, the Jin Zhou health department also collected 3 

### data pertaining to symptoms in 1965 in Nuer River V illage, which was one of the highly 4 

### contaminated areas at that time (well water hexaval ent chromium levels 0.1–20.0 mg/L) (Zhang 5 

### and Li, 1987, 1986). Among 156 residents surveyed, 51 (33%) had oral ulcers, 20 (17%) had 6 

### diarrhea, 48 (31%) had abdominal pain, 26 (17%) had dyspepsia, 81 (30%) had stomach pain, 7 

### and 20 (17%) had vomiting (JinZhou Antiepidemic Sta tion, 1979). The authors state that “no 8 

### such symptoms were found among the residents whose water wells were not contaminated.” A 9 

### similar study of 158 people in Shilitai in 1971 fou nd a similar pattern of symptoms, with 92 10 

### (58%) reporting oral ulcers, 48 (30%) diarrhea, and 36 (23%) abdominal pain. In 1974, another 11 

### study of children in Wenjiatun and Sandaohao, at th e eastern edge of the contamination zone, 12 

### also found similar symptoms (data not shown in the 1979 report). The authors speculate that the 13 

### symptoms may have been due to the increased concent rations of sulfates (>300 mg/L) in the 14 

### drinking water in these areas in 1974, rather than the relatively low concentrations of hexavalent 15 

### chromium (0.003–0.05 mg/L) 16 

### Zhang and Li (1987, 1986) also conducted hematolog ical assessments of 12 individuals 17 

### in 1965, and another study of 93 individuals (time not specified). The exact location of the 18 

### participants was not specified, but they were said to be from “highly polluted” or “high density 19



### contamination” areas. White blood cell counts were elevated in the first study, and the number 20 

### of neutrophilic granulocytes and what was termed “j uvenile cells” among these granulocytes was 21 

### elevated in the second study. 22

23

### 37 DRAFT - DO NOT CITE OR QUOTE

Kings County/San Bernadino County, California (Fryz ek et al., 2001) 1 

### A study of areas in Kings County and San Bernardino County, California, compared 2 

### cancer mortality in locations near natural gas comp ressor plants with areas not located near the 3 

### plants (Fryzek et al., 2001). Hexavalent chromium compounds had been used as anti-corrosion 4 

### additives in cooling tower water at the gas plants during the period 1950 to approximately 1980. 5 

### Waste material was released to surface ponds and wa s subject to percolation to groundwater. 6 

### Cooling tower water was also aerosolized and transp orted to the ground surface where it may 7 

### have contacted soil, crops, and surface water. Thu s, exposures to hexavalent chromium may 8 

### have occurred by several routes (i.e., inhalation, ingestion, and dermal contact). Mortality 9 

### records for zip codes for the cities of Kettleman C ity (in Kings County), and Hinkley and 10 

### Topock (in San Bernadino County), in which natural gas compressor plants were located, were 11 

### compared to records from zip codes in Kings County and San Bernadino County, other than 12 

### those encompassing these three cities. The study i ncluded mortality records for the period 1989– 13 

### 1998, during which time 2,226,214 deaths were recor ded. Age-adjusted cancer mortality rate 14 

### ratios (rate in areas near the plant/rate in compar ison areas) were 1.03 (95% CI 0.90–1.17) for 15 

### lung cancer death, 0.93 (95% CI 0.87–1.00) for all cancer deaths, and 0.98 (95% CI 0.95–1.02) 16



### for all deaths. Rate ratios for stomach cancer wer e not reported. This study found no significant 17 

### difference between mortality or cancer mortality am ong residents from zip codes in which gas 18 

### plants that used hexavalent chromium additives in c ooling tower water were located compared to 19 

### residents of other nearby areas without such plants . An important limitation of this study is that 20 

### exposure assignment was based on zip code, rather t han on individual-level data, which is likely 21 

### to result in significant exposure misclassification

. 22

23 

### Nebraska (Bednar and Kies, 1991) 24 

### Bednar and Kies (1991) compared levels of chromium (and other chemicals) in drinking 25



### water in Nebraska counties with death rates in thes e same areas. Data on chromium in drinking 26 

### water were obtained for each of 453 communities (al l incorporated communities of Nebraska) 27 

### for the period 1986–1987, and mortality data for ea ch Nebraska county was obtained for the year 28 

### 1986 (both compiled by the Nebraska Department of H ealth). Mean total chromium 29 

### concentration in drinking water for the 453 communi ties was 0.002 mg chromium/L (range 30 

### <0.001–0.01); the study report did not indicate val ence state of chromium detected in these 31 

### drinking water samples. Possible associations betw een chromium exposure and health outcomes 32 

### were assessed by linear correlation (Pearson) of mo rtality rates (at the county level) and 33

### 38 DRAFT - DO NOT CITE OR QUOTE

chromium concentrations in drinking water (presumab ly aggregated from community data to 1 

### represent counties). Correlations were reportedly explored for mortality from cancer, 2 

### cerebrovascular disease, heart disease, pneumonia, and chronic lung disease; however, only one 3 

### chromium correlation coefficient was reported to be statistically significant, that for death from 4 

### chronic lung disease, and the correlation was negat ive (-0.101, p = 0.03). As with the other 5 

### studies of this design, a major limitation is that exposures to chromium cannot be estimated for 6 

### individual subjects in the study and may not be acc urately represented by the drinking water 7 

### chromium measurements. For example, the 1986–1987 drinking water data do not necessarily 8 

### represent long-term exposure patterns, and an indiv idual represented in a county death record 9 

### does not necessarily mean that the individual resid ed in the county for their lifetime or any 10 

### significant fraction of their lifetime. 11

12 

### Glasgow, UK (Eizaguirre-Garcia et al., 2000, 1999) 13 

### Eizaguirre-Garcia et al. (2000, 1999) examined risk of leukemia and birth defects in 14 

### people residing near the site of a former chromium processing facility in Glasgow, UK. The 15 

### factory was in operation for more than 100 years an d ceased operations in 1967. A survey 16 

### conducted in 1991 found average soil concentrations at the site of operations to be 8,164 mg/kg 17 

### for total chromium and 848 mg/kg for hexavalent chr omium. Soil concentrations of total 18 

### chromium and hexavalent chromium approximately 2–3 km from the factory site were reported 19 

### as “approximately half” of those at the site; no ad ditional information on soil levels off-site were 20 

### reported (Eizaguirre-Garcia et al., 2000, 1999). R eported cases of leukemia for the period 1975– 21 

### 1989 were obtained from the Scottish Cancer Registr ation, during which 1,205 cases of leukemia 22 

### were reported in a population of 873,643 (Eizaguirr e-Garcia et al., 1999). Leukemia cases were 23 

### aggregated at the level of Enumeration Districts (E D) (approximately 350–500 individuals per 24 

### district). When stratified by distance of the EDs from the plant (out to 9–10 km), relative risks of 25 

### leukemia (0–2 km as reference) were unrelated to di stance. When other influential variables 26 

### were included in a Poisson regression model (gender , socioeconomic status, and age) in addition 27 

### to distance of EDs from the plant (0–4, 4–9, 9–10 k m), relative risk was significant (1.29, 95% 28 

### CI: 1.07–1.56) for EDs 4–9 km from the plant (relat ive to 0–4 km), but not for EDs 9–10 km 29 

### from the plant. These results suggest that leukemi a risk increased with distance from the plant 30 

### (i.e., 4–9>0–4 km) and then declined with further d istance (i.e., 9–10 km = 0–4 km). This 31 

### pattern does not strongly implicate the plant as a major contributor to leukemia risk. 32

### 39 DRAFT - DO NOT CITE OR QUOTE

A similar study of risk of birth defects was conduc ted on the same population 1 

### (Eizaguirre-Garcia et al., 2000). In this study, d ata on number of births and congenital 2 

### malformations were collected for the period 1982–19 89. Case definitions (not reported) 3 

### followed those of the European-wide EURCAT network (http://www.eurocat.ulster.ac.uk/). The 4 

### study included 2,778 cases from a population of 81, 057 births; cases were aggregated at the level 5 

### of EDs. When distance from the plant (0–1, 2–4, 4– 10 km) and socioeconomic status were 6 

### included in a Poisson regression model, relative ri sk was significant for the EDs in the 2–4 km 7 

### category (1.47, 95% CI: 1.2–1.7) and the 4–10 km ca tegory (1.25, 95% CI: 1.05–1.49); however, 8 

### both distance categories were associated with highe r risks than the closest distance category, 0– 9 

### 1 km. Similar to the results for leukemia, this pa ttern does not strongly implicate the plant as a 10 

### major contributor to risk of congenital anomalies. Not taken into consideration in this study 11 

### were several other potentially influential variable s on developmental outcomes; for example, 12 

### maternal age and health, smoking, and alcohol consu mption. 13

14 

### Summary 15 

### The Liaoning province studies provide the most deta iled analysis of all of the 16 

### epidemiological studies that have been conducted wi th respect to chromium and cancer mortality 17 

### (specifically stomach cancer or other cancers of th e digestive system). These studies are 18 

### important in that they examined a population expose d to very high levels of chromium in 19 

### drinking water wells (i.e., sufficient to impart a visible yellow color to the water). Sources of 20 

### exposure include the drinking water, food grown in contaminated soil, and possibly air. Levels 21 

### up to 20 mg/L in well water were documented in the first surveys done in 1965 in the two 22 

### villages closest to the source of exposure (a ferro chromium alloy plant). The contamination 23 

### began sometime between 1959 and 1964; the reporting of a yellowing of the water by local 24 

### residents in 1964 is what led to the investigation and identification of this contamination by the 25 

### local health department. 26 

### The interpretation of the mortality data originally collected by Zhang and Li (1980) 27 

### depends in large part on the choice of referent gro up. That choice depends on many factors, 28 

### including the perceived comparability and the size of the populations. Larger populations, such 29 

### as a province or state, have the advantage of provi ding relatively stable estimates, particularly for 30 

### low-incident events such as site-specific cancers. Smaller areas (e.g., a neighboring community) 31 

### offer the advantage of potentially greater similari ties in ethnic background, socioeconomic 32 

### status, and occupational and lifestyle factors that may affect cancer risk. However, small 33

### 40 DRAFT - DO NOT CITE OR QUOTE

comparison groups are likely to produce imprecise e stimates, and the issue of over-controlling 1 

### may arise, for example, if the comparison populatio n shares the specific exposure of interest (for 2 

### example, with the selection of friends or co-worker s in case-control studies). The associations 3 

### presented by Beaumont et al. (2008) using Liaoning province as the comparison group provide 4 

### evidence of an excess risk in the villages in the c ontamination zone of mortality from stomach 5 

### cancer (RR 1.69, 95% CI (1.12, 2.44)) and lung canc er (RR 1.78, 95% CI (1.03, 2.87)), with a 6 

### small increase also suggested in total cancer morta lity (RR 1.23, 95% CI (0.97, 1.53)). The 7 

### association with stomach cancer mortality is also s een when using the four adjacent areas as the 8 

### referent group (RR 1.82, 95% CI (1.11, 2.91)), but is weaker when the industrial area 9 

### surrounding the plant, TangZeHi, is removed from th e comparison group (RR 1.22, 95% CI 10 

### (0.74, 2.01)). Kerger et al. (2009) believe the re latively urban environment of TangHeZi makes 11 

### it an inappropriate comparison group for the villag es in the contamination zone. With respect to 12 

### stomach cancer, historical trends show clear decrea ses in the incidence of this cancer in a variety 13 

### of geographical areas, with improvements that come with economic development and 14 

### urbanization (e.g., sanitation, refrigeration) cont ributing to this decline. An analysis of gastric 15



### cancer rates in China in 1990-1992 showed lower mor tality rates in urban areas (15.3 per 16 

### 100,000) compared with rural areas (24.4 per 100,00 0) (Yang, 2006). However, this same study 17 

### reported little difference between urban and rural rates in 1973-1975 (20.1 and 19.4 per 100,000 18 

### in urban and rural areas, respectively), the releva nt time period with respect to the Liaoning 19 

### province studies. Thus, the EPA does not consider the exclusion of TangZeHi from the 20 

### comparison group to be warranted. 21 

### Another issue regarding the interpretation of the m ortality data is the validity of analyses 22 

### of the variability in cancer rates among the five v illages in the contamination zone in relation to 23 

### the available exposure measures (distance from the plant, average concentration in wells in 1965, 24 

### and percent of wells with hexavalent chromium level s above 0.05 mg/L in 1965). There are 25 

### considerable limitations to these measures, includi ng the lack of individual-level data on use of 26 

### water from specific wells over time and the changes in exposure due to efforts to treat the water 27



### in the most contaminated areas with treatment wells built in 1967. Based on these limitations, 28 

### the EPA concluded that the exposure-response analys es presented by Zhang and Li (1997) and 29 

### Kerger et al. (2009) are not based on the quality o f data that is needed to support a conclusion 30 

### regarding the presence or absence of a dose-respons e among the observed cancer rates in these 31 

### villages. 32

33

### 41 DRAFT - DO NOT CITE OR QUOTE

4.2. SUBCHRONIC AND CHRONIC STUDIES AND CANCER BIO ASSAYS IN 1



### ANIMALS—ORAL 2 

### The effects of subchronic oral exposure to hexavale nt chromium have been evaluated in 3 

### rats (NTP, 2007; Quinteros et al., 2007; Rafael et al., 2007; Acharya et al., 2001; Chopra et al., 4 

### 1996; Vyskocil et al., 1993) and mice (NTP, 2007; A smatullah and Noreen 1999), and the effects 5 

### of chronic oral exposure to hexavalent chromium hav e been evaluated in rats (NTP, 2008, 6 

### MacKenzie et al., 1958), mice (NTP, 2008), and dogs (Anwar et al., 1961). The studies 7 

### conducted by NTP (2008, 2007) provide dose-response data on the effects of oral hexavalent 8 

### chromium exposure based on a comprehensive assessme nt of toxicological endpoints. NTP 9 

### (2008, 2007) identified lowest-observed-adverse-effect levels (LOAELs) and no-observed10 

### adverse-effect levels (NOAELs) in rats and mice for subchronic and chronic exposure durations. 11 

### Results of the NTP (2007) subchronic study identifi ed several hexavalent chromium-induced 12 

### effects, including hematological effects, hepatotox icity, alterations in lipid metabolism, and 13 

### histopathological changes in GI tissues and pancrea tic and mesenteric lymph nodes. The most 14



### sensitive hexavalent chromium-induced effects were microcytic, hypochromic anemia, increased 15 

### serum liver enzyme activities, and histopathologica l changes to the duodenum and pancreatic 16 

### lymph nodes in rats; and histopathological changes in the duodenum in mice. The most sensitive 17 

### noncancer effects in the NTP (2008) two-year toxico logy and carcinogenicity study were 18 

### nonneoplastic histopathological changes to the live r, duodenum, and mesenteric lymph nodes in 19 

### rats and the duodenum, mesenteric lymph nodes, and liver in mice. In addition, based on 20 

### findings of squamous cell neoplasms of the oral cav ity in rats and neoplasms of the small 21 

### intestine in mice, NTP (2008) concluded that result s of this study provide clear evidence of 22 

### carcinogenic activity of sodium dichromate dihydrate. 23 

### Several other oral exposure studies (i.e., Quintero s et al., 2007; Rafael et al., 2007; 24 

### Asmatullah and Noreen, 1999; Vyskocil et al., 1993; Anwar et al., 1961) do not provide suitable 25 

### data for identifying NOAELs or LOAELs because compr ehensive toxicological endpoints were 26



### not evaluated in these studies. LOAELs identified in studies by Acharya et al. (2001) and 27 

### Chopra et al. (1996) were based on evaluation of a limited number of liver endpoints. In 28 

### addition, interpretation of results from these stud ies was limited due to the small number of 29 

### animals evaluated, lack of dose-response data, or i nadequate reporting for estimation of doses in 30 

### mg hexavalent chromium/kg-day. However, results of these studies are useful for identification 31 

### of potential adverse effects of oral hexavalent chr omium exposure. 32

33

### 42 DRAFT - DO NOT CITE OR QUOTE

4.2.1. Subchronic Oral Exposure 1 

### NTP, 2007 2 

### NTP (2007) conducted a 3-month toxicology study of sodium dichromate dihydrate in 3 

### drinking water in rats and mice. This study was di vided into three separate studies evaluating 4 

### effects of treatment in: 1) male and female F344/N rats, 2) male and female B6C3F 1 

### mice, and 3) 5 

### three strains of male mice (B6C3F 1 

### , BALB/c, and am3- C57BL/6). In the 3-month study in 6 

### F344/N rats, groups of 10 males and 10 females (“co re” study animals) were exposed to sodium 7 

### dichromate dihydrate in drinking water at concentra tions of 0, 62.5, 125, 250, 500, or 1,000 mg 8 

### sodium dichromate dihydrate/L (equivalent to 0, 21. 8, 43.6, 87.2, 174.5, or 348 mg hexavalent 9 

### chromium/L, respectively) for 3 months. Based on w ater consumption monitored throughout the 10 

### study, NTP (2007) calculated average daily doses ov er the 3-month treatment duration of 11 

### approximately 0, 5, 10, 17, 32, or 60 mg sodium dic hromate dihydrate/kg-day (equivalent to 0, 12 

### 1.7, 3.5, 5.9, 11.2, or 20.9 mg hexavalent chromium /kg-day, respectively) for both males and 13



### females. An additional 10 male and 10 female rats (“clinical pathology” animals) were exposed 14



### to the same concentrations of sodium dichromate dih ydrate for 4 weeks. “Core” study animals 15 

### were observed twice daily for mortality and clinica l signs of toxicity; water consumption and 16 

### body weights were recorded weekly. Blood was colle cted from “clinical pathology” animals on 17 

### treatment days 5 and 23 and from “core” study anima ls at study termination for comprehensive 18 

### hematology and clinical chemistry endpoints. Urine was collected from “clinical pathology” 19 

### animals on day 16 and analyzed for comprehensive ur inalytic endpoints. At study termination, 20 

### necropsies were performed on all “core” study anima ls, with organ weights recorded for heart, 21 

### right kidney, liver, lung, spleen, right testis, an d thymus. Microscopic examinations of 22 

### comprehensive tissues were conducted in all core st udy animals in the control and 20.9 mg 23 

### hexavalent chromium/kg-day (high-dose) groups and o n six core study animals from each of the 24 

### other treatment groups. In addition, all tissues i dentified as target organs in the 20.9 mg 25 

### hexavalent chromium/kg-day (high-dose) group were e xamined in lower dose groups until a no26



### effect level was identified or all animals were exa mined. 27



### No mortalities were observed in male or female rats exposed to sodium dichromate 28 

### dihydrate in drinking water for 3 months (NTP, 2007 ). Final body weights in male rats were 29 

### significantly decreased by 5% and 11% in the 11.2 a nd 20.9 mg hexavalent chromium/kg-day 30 

### groups, respectively, compared to controls. In fem ales, final body weight was significantly 31 

### decreased by 9% in the 20.9 mg hexavalent chromium/ kg-day group compared to controls. In 32



### males and females in the ≥ 5.9 mg hexavalent chromium/kg-day groups, water con sumption was 33

### 43 DRAFT - DO NOT CITE OR QUOTE

decreased (statistical significance not reported). Data on food consumption were not reported. 1 

### No treatment-related signs of clinical toxicity wer e observed throughout the study. 2 

### Results of hematology analyses show that exposure o f male and female rats to sodium 3 

### dichromate dihydrate in drinking water produced mic rocytic, hypochromic anemia, characterized 4 

### by decreases in mean cell volume (MCV), hematocrit (Hct), hemoglobin (Hgb), and mean cell 5 

### hemoglobin (MCH) (NTP, 2007). The severity of micr ocytic, hypochromic anemia exhibited 6 

### duration- and dose-dependence, with peak effects oc curring at 23 days (Table 4-4). After 5 days 7 

### of exposure, small changes were observed in several hematological parameters; however, 8 

### decreases in all treatment groups were ≤ 5% compared to controls. More severe, dose-related 9 

### effects were observed after 23 days of treatment, w ith changes observed in all treatment groups 10



### in males and females. Similar effects were observe d after 3 months of treatment, although 11 

### severity at 3 months was generally less than that o bserved at 22 days. Blood smears showed 12 

### evidence of erythrocyte injury or increased turnove r, including erythrocyte fragments, 13 

### keratocytes, and blebbing (incidence data not repor ted). Increased reticulocyte counts and 14 

### nucleated erythrocytes, indicative of a compensator y hematopoietic response, were also observed 15 

### in both sexes at 23 days and 3 months; however, the se increases did not exhibit a consistent 16 

### pattern of dose- or duration-dependence. Dose-depe ndent increases in platelet counts occurred at 17 

### 23 days in all treatment groups compared to control s; however, severity was decreased at 18 

### 3 months (Table 4-4). NTP (2007) stated that incre ased platelet counts are consistent with 19 

### compensatory hematopoiesis or an iron deficiency pr ocess. Increased neutrophil and monocyte 20 

### counts were observed at higher doses ( ≥ 5.9 and ≥ 3.5 mg hexavalent chromium/kg-day in males 21 

### and females, respectively) and were considered by N TP (2007) to reflect an inflammatory 22 

### response related to the inflammatory gastric lesion s. Results of hematological analyses show 23 

### that exposure of rats to sodium dichromate dihydrat e in drinking water at daily doses ≥ 1.7 mg 24 

### hexavalent chromium/kg-day produced microcytic, hyp ochromic anemia, but that severity 25 

### decreased slightly as exposure duration increased f rom 23 days to 3 months. 26

### 44 DRAFT - DO NOT CITE OR QUOTE

1 

### Table 4-4. Hematological effects in male and femal e F344/N rats exposed to

sodium dichromate dihydrate in drinking water for u p to 3 months

Treatment group (mg hexavalent chromium/kg-day) Hematological parameter Time on

treatment 0 1.7 3.5 5.9 11.2 20.9

Males 23 Days 48.0 ± 0.5 a 44.7 ± 0.7 b (93.1) 39.8 ± 0.8 b

(82.9) 36.2 ± 1.0 b

(75.4) 34.4 ± 0.5 b

(71.7) 32.3 ± 1.1 b

(67.3) Hct (percent)

3 Months 45.7 ± 0.2 45.2 ± 0.4 (98.9) 45.2 ± 0.3

(98.9) 44.8 ± 0.7

(98.0) 42.9 ± 0.4 b

(93.9) 36.9 ± 0.8 b

(80.7) 23 Days 15.9 ± 0.1 14.2 ± 0.2 b (89.3) 12.0 ± 0.3 b

(75.5) 10.9 ± 0.3 b

(68.6) 10.3 ± 0.3 b

(64.8) 9.2 ± 0.3 b

(57.9) Hgb (g/dL)

3 Months 15.3 ± 0.1 15.2 ± 0.1 (99.3) 15.0 ± 0.1

(98.0) 14.4 ± 0.2 b

(94.1) 13.3 ± 0.2 b

(86.9) 10.9 ± 0.3 b

(71.2) 23 Days 61.1 ± 0.5 53.6 ± 0.6 b (87.7) 48.0 ± 0.4 b

(78.6) 46.4 ± 0.6 b

(75.9) 46.2 ± 0.3 b

(75.6) 46.4 ± 0.5 b

(75.9) MCV (fL)

3 Months 51.8 ± 0.1 50.3 ± 0.2 b (97.1) 49.0 ± 0.1 b

(94.6) 44.4 ± 1.0 b

(85.7) 39.7 ± 0.5 b

(76.6) 36.0 ± 0.4 b

(69.5) 23 Days 20.1 ± 0.2 16.9 ± 0.2 b (84.0) 17.2 ± 0.7 b

(85.6) 18.2 ± 0.4

(90.5) 19.7 ± 0.3

(98.1) 20.7 ± 0.6

(103.0) MCH ( ρ g)

3 Months 17.3 ± 0.1 16.9 ± 0.1 b (97.7) 16.2 ± 0.1 b

(93.6) 14.2 ± 0.4 b

(82.1) 12.3 ± 0.2 b

(71.1) 13.0 ± 0.5 b

(75.1) 23 Days 7.94 ±

0.10 8.38 ± 0.11

(105.5) 7.13 ± 0.35 c

(89.8) 6.0 ± 0.28 b

(75.6) 5.25 ± 0.19 b

(66.1) 4.54 ± 0.33 b

(57.2) Erythrocyte count (10 6 /μL)

3 Months 8.88 ± 0.05 9.04 ± 0.09 c

(101.8) 9.25 ± 0.07 b

(104.2) 10.15 ± 0.22 b

(114.3) 10.87 ± 0.07 b

(122.4) 8.52 ± 0.45 b

(95.9) 23 Days 745.2 ±

22.2 1,065.3 ±

67.9 b (143) 2,768.6 ±

328.5 b (372) 3,504.7 ±

235.0 b (470) 4,226.0 ±

204.5 b (567) 4,688.8 ±

242.7 b (629) Platelet count (10 6 /μL)

3 Months 618.6 ± 20.0 736.1 ± 11.5

(119) 604.3 ± 24.5

(98) 909.8 ± 119.1 b

(147) 1,743.1 ±

178.0 b (282) 5,123.0 ±

638.9 b (828)

Females 23 Days 48.0 ± 0.4 a 46.6 ± 0.9 (97.1) 42.9 ± 0.8 b

(89.4) 39.2 ± 0.7 b

(81.7) 37.2 ± 0.7 b

(79.6) 33.4 ± 0.6 b

(69.6) Hct (percent)

3 Months 44.6 ± 0.4 45.2 ± 0.1 (101.3) 44.1 ± 0.3

(98.9) 42.9 ± 0.2 b

(96.2) 42.6 ± 0.5 b

(95.5) 38.3 ± 0.5 b

(85.9) 23 Days 15.9 ± 0.1 14.7 ± 0.3 b (92.5) 13.0 ± 0.3 b

(81.8) 11.8 ± 0.3 b

(74.2) 10.9 ± 0.2 b

(68.6) 9.7 ± 0.2 b

(61.0) Hgb (g/dL)

3 Months 15.2 ± 0.1 15.4 ± 0.1 (101.3) 14.9 ± 0.1

(98.0) 14.3 ± 0.1 b

(94.1) 14.1 ± 0.2 b

(92.8) 12.0 ± 0.2 b

(78.9) 23 Days 61.1 ± 0.4 53.9 ± 0.5 b (88.2) 48.8 ± 0.5 b

(79.9) 46.6 ± 0.6 b

(76.3) 45.7 ± 0.4 b

(74.8) 46.5 ± 0.5 b

(76.1) MCV (fL)

3 Months 53.3 ± 0.1 53.3 ± 0.1 (100) 52.4 ± 0.2 b

(98.3) 50.5 ± 0.3 b

(94.7) 48.0 ± 0.9 b

(90.1) 40.0 ± 0.7 b

(75.0) 23 Days 20.4 ± 0.1 17.3 ± 0.2 (84.8) 18.0 ± 0.3

(88.2) 18.9 ± 0.7

(92.6) 21.0 ± 0.6

(102.9) 23.1 ± 0.5

(113.2) MCH ( ρ g)

3 Months 18.4 ± 0.1 17.9 ± 0.1 b (97.3) 17.8 ± 0.1 b

(96.7) 16.9 ± 0.1 b

(91.8) 15.9 ± 0.4 b

(86.4) 12.5 ± 0.3 b

(67.9)

### 45 DRAFT - DO NOT CITE OR QUOTE

Table 4-4. Hematological effects in male and femal e F344/N rats exposed to sodium dichromate dihydrate in drinking water for u p to 3 months

Treatment group (mg hexavalent chromium/kg-day) Hematological parameter Time on

treatment 0 1.7 3.5 5.9 11.2 20.9 23 Days 7.82 ±

0.09 8.52 ± 0.14

(109.0) 7.22 ± 0.19

(92.3) 6.32 ± 0.36 b

(80.8) 5.27 ± 0.23 b

(67.4) 4.21 ± 0.16 c

(53.8) Erythrocyte count (10 6 /μL)

3 Months 8.30 ± 0.06 8.60 ± 0.05 b

(103.6) 8.40 ± 0.04 c

(101.2) 8.47 ± 0.04 c

(102.0) 8.93 ± 0.11 b

(107.6) 9.62 ± 0.10 b

(115.9) 23 Days 611.5 ±

43.7 1,156.3 ±

76.4 b (189) 2808.8 ±

198.5 b (459) 3295.0 ±

349.7 b (539) 4,318.4 ±

234.9 b (706) 5,132.8 ±

247.0 b (839) Platelet count (10 6 /μL)

3 Months 588.9 ± 17.1 605.8 ± 17.1

(103) 574.8 ± 21.3

(98) 528.2 ± 14.1

(90) 619.3 ± 55.4

(105) 1,524.9 ±

193.3 b (259)

a Values are means ± SE; values in parenthesis are pe rcent of control; n = 10 rats/group, with the follo wing exceptions: 1.7 mg hexavalent chromium/kg-day group females on days 23 and month 3 (n = 9), 3.5 mg hex avalent chromium/kg-day group females on day 23 (n = 8), 5. 9 mg hexavalent chromium/kg-day group females on da y 23 (n = 9), and 20.9 mg hexavalent chromium/kg-day gro up females on day 23 and month 3 (n = 9).

b Significantly different (

p ≤ 0.01) from the control group by Dunn’s or Shirley’ s test.

c Significantly different (

p ≤ 0.05) from the control group by Dunn’s or Shirley’ s test.

Source: NTP (2007).

1 

### Results of clinical chemistry analyses in male and female rats exposed to sodium 2 

### dichromate dihydrate in drinking water showed treat ment-related increases in serum liver 3 

### enzyme activities, bile acids, and serum creatine k inase activity and alterations in lipid 4 

### metabolism (Table 4-5) (NTP, 2007). Serum alanine aminotransferase (ALT) and sorbitol 5 

### dehydrogenase (SDH) activities were significantly i ncreased compared to controls in all 6 

### treatment groups at 3 months, with less severe effe cts seen at 23 days. A consistent relationship 7 

### between severity and dose was not observed. In mal e rats, elevations of ALT and SDH activities 8 

### increased with increasing dose between 1.7 and 11.2 mg/kg-day; but less severe elevations were 9 

### observed at 20.9 mg/kg-day (Table 4-5). In females , increases in ALT and SDH activities were 10 

### generally indicative of a uniform effect across the dose range (Table 4-5). NTP (2007) suggested 11 

### that increases are consistent with hepatocellular i njury or membrane leakage. At 3 months, bile 12 

### acids were significantly increased compared to cont rols at ≥ 11.2 mg hexavalent chromium/kg13 

### day in males and in all treatment groups (except 5. 9 mg hexavalent chromium/kg-day) in 14 

### females; similar to serum liver enzymes, increases in bile acids were not consistently related to 15 

### dose. NTP (2007) suggested that increased bile aci d was indicative of hepatic toxicity rather 16 

### than colestasis, as markers of colestasis (e.g., al kaline phosphatase [AP] and 5N-nucleotidase) 17 

### were not affected by treatment. At 3 months, decre ased serum cholesterol and triglycerides, 18

### 46 DRAFT - DO NOT CITE OR QUOTE

indicative of altered lipid metabolism, were observ ed; however, a consistent relationship 1 

### between severity and dose was not observed. At 3 m onths, dose-related increases in serum 2 

### creatine kinase activity, indicative of muscle dama ge, were observed in males and females at 3



### ≥ 5.9 mg hexavalent chromium/kg-day. Urinalysis show ed dose-related decreased volume and 4 

### increased specific gravity, consistent with decreas ed water intake. NTP (2007) suggested that 5 

### decreased water intake was due to decreased palatab ility of water. Other changes in clinical 6 

### chemistry and urinalysis parameters were transient, with no apparent relationship to treatment. 7 

### Results of clinical chemistry analyses indicate tha t exposure of rats to sodium dichromate 8 

### dihydrate in drinking water induced hepatocellular membrane damage or cytotoxicity (both 9 

### sexes) and increased bile acids (females) at doses ≥ 1.7 mg hexavalent chromium/kg-day (both 10 

### sexes). 11

12

### 47 DRAFT - DO NOT CITE OR QUOTE

Table 4-5. Clinical chemistry effects in male and female F344/N rats exposed to sodium dichromate dihydrate in drinking water for 3 months

Treatment group (mg hexavalent chromium/kg-day) Clinical chemistry parameter Time on

treatment 0 1.7 3.5 5.9 11.2 20.9

Males ALT (IU/L) 3 Months 98 ± 6 a 274 ± 30 c (280) 461 ± 102 c

(470) 447 ± 121 c

(456) 740 ± 81 c

(755) 191 ± 17 c

(195) SDH (IU/L) 3 Months 31 ± 2 55 ± 5 c (177) 110 ± 24 c

(355) 102 ± 24 c

(329) 173 ± 20 c

(558) 59 ± 6 c

(190) Bile acids (μmol/L) 3 Months 22.0 ± 2.2 24.0 ± 3.4 (109) 34.5 ± 7.0

(157) 32.6 ± 5.3

(148) 45.3 ± 2.8 c

(206) 28.1 ± 2.0 c

(128) Cholesterol (mg/dL) 3 Months 89 ± 2 95 ± 2 (107) 86 ± 4

(97) 65 ± 2 c

(73) 86 ± 3 b

(97) 71 ± 2 c

(80) Triglycerides (mg/dL) 3 Months 170 ± 9 169 ± 8 (99) 172 ± 15

(101) 170 ± 13

(100) 164 ± 12

(96) 98 ± 8 c

(57)

Creatine kinase (IU/L) 3 Months 214 ± 26 286 ± 32 (134) 291 ± 36

(136) 364 ± 23 c

(170) 413 ± 16 c

(193) 374 ± 44 c

(175)

Females ALT (IU/L) 3 Months 64 ± 5 a 437 ± 68 c (683) 218 ± 27 c

(340) 245 ± 30 c

(383) 246 ± 37 c

(384) 248 ± 22 c

(387) SDH (IU/L) 3 Months 22 ± 2 101 ± 17 c (459) 65 ± 10 c

(295) 81 ± 13 c

(368) 96 ± 20 c

(436) 103 ± 12 c

(468) Bile acids (μmol/L) 3 Months 19.7 ± 2.5 50.4 ± 6.0 c (256) 39.9 ± 4.3 c

(203) 35.3 ± 3.5

(179) 45.3 ± 5.6 c

(230) 38.7 ± 3.2 b

(196) Cholesterol (mg/dL) 3 Months 95 ± 2 111 ± 4 (117) 94 ± 2

(99) 87 ± 2

(92) 83 ± 2 b

(87) 79 ± 2 c

(83) Triglycerides (mg/dL) 3 Months 139 ± 18 116 ± 10 (93) 98 ± 9

(70) 81 ± 4 c

(58) 76 ± 7 c

(55) 59 ± 6 c

(42)

Creatine kinase (IU/L) 3 Months 197 ± 23 311 ± 94 (158) 265 ± 23

(135) 296 ± 24 c

(150) 359 ± 23 c

(182) 432 ± 48 c

(219)

a Values are means ± SE; values in parenthesis are pe rcent of control; n = 10 rats/group, with the follo wing exceptions: control group males (n = 9), 1.7 mg hex avalent chromium/kg-day group females (n = 9), and 20.9 mg hexavalent chromium/kg-day group females (n = 9).

b Significantly different (

p ≤ 0.05) from the control group by Dunn’s or Shirley’ s test.

c Significantly different (

p ≤ 0.01) from the control group by Dunn’s or Shirley’ s test.

Source: NTP (2007).

1 

### Changes in organ weights in rats exposed to sodium dichromate dihydrate in drinking 2 

### water for 3 months are summarized in Table 4-6 (NTP , 2007). Treatment-related effects were 3 

### generally observed at doses ≥ 11.2 mg hexavalent chromium/kg-day. In males, decr eases were 4 

### observed in absolute and relative liver weights and in absolute and relative spleen weights; in 5 

### females, relative right kidney weights and relative spleen weights were increased. Changes in 6 

### weights of other organs were considered by NTP (200 7) to be secondary to changes in body 7 

### weight rather than due to adverse effects of treatm ent. 8

### 48 DRAFT - DO NOT CITE OR QUOTE

1 

### Table 4-6. Selected organ weights in male and fema le F344/N rats exposed

to sodium dichromate dihydrate in drinking water fo r 3 months

Treatment group (mg hexavalent chromium/kg-day) Organ 0 1.7 3.5 5.9 11.2 20.9

Males Liver, absolute weight 10.89 ± 0.42 a 10.30 ± 0.28 11.45 ± 0.38 10.51 ± 0.18 9.20 ± 0.17 b 8.88 ± 0.18 b Liver, relative weight d 32.91 ± 0.65 31.91 ± 0.61 33.98 ± 0.75 31.90 ± 0.54 29.15 ± 0.53 c 29.80 ± 0.35 b

Spleen, absolute weight 0.64 ± 0.02 0.60 ± 0.01 0.62 ± 0.02 0.60 ± 0.02 0.53 ± 0.01 b 0.60 ± 0.01 b Spleen, relative weight d 1.94 ± 0.03 1.85 ± 0.03 1.83 ± 0.04 1.81 ± 0.05 c 1.69 ± 0.02 b 2.00 ± 0.03

Females Right kidney, relative weight 3.34 ± 0.09 a 3.32 ± 0.04 3.55 ± 0.05 3.55 ± 0.07 3.58 ± 0.10 c 3.63 ± 0.09 c Spleen, relative weight d 2.12 ± 0.05 2.04 ± 0.03 2.16 ± 0.05 2.22 ± 0.03 2.25 ± 0.05 c 2.39 ± 0.03 c

a Values are means ± SE; n = 10 rats/group.

b Significantly different (

p ≤ 0.01) from the control group by Williams’s or Dunn ett’s test.

c Significantly different (

p ≤ 0.05) from the control group by Williams’s or Dunn ett’s test.

d Relative weight = mg organ weight/g body weight

Source: NTP (2007).

2 

### Gross and microscopic examinations of male and fema le rats exposed to sodium 3 

### dichromate dihydrate in drinking water for 3 months showed nonneoplastic lesions of the 4 

### duodenum, glandular stomach, pancreatic lymph nodes , liver (females only), and bone marrow 5 

### (females only) (NTP, 2007); incidence data are summ arized in Table 4-7. The incidence of 6 

### minimal-to-mild duodenal histiocytic cellular infil tration was increased in males and females at 7 

### 3.5 and 1.7 mg hexavalent chromium/kg-day, respecti vely, compared to controls; incidence 8 

### increased with dose. Histiocytic cellular inflamma tion appeared as multifocal, randomly 9 

### scattered, small clusters of enlarged macrophages w ith pale foamy cytoplasm. Incidences of 10 

### nonneoplastic lesions of the glandular stomach (ulc er, focal regenerative hyperplasia, and focal 11 

### squamous hyperplasia) were increased in rats in the highest dose group. Microscopically, ulcers 12 

### were characterized by complete loss of the lining o f the mucosal epithelium with necrosis, often 13 

### extending through to the submucosa, and muscle laye rs; mild to marked chronic inflammation 14 

### (infiltrates of neutrophils, macrophages, lymphocyt es, and eosinophils), and proliferation of 15 

### fibrous connective tissue through the submucosa. L esions were not observed in the forestomach. 16 

### Microscopic examinations of the oral mucosa and ton gue were not conducted; NTP (2007) did 17 

### not report lesions of the oral cavity on gross exam ination of rats at necropsy. In males, a dose18 

### dependent increase in the incidence of histiocytic cellular infiltration of pancreatic lymph nodes 19

### 49 DRAFT - DO NOT CITE OR QUOTE

was observed at 1.7 mg hexavalent chromium/kg-day, whereas increased pancreatic lymph node 1 

### sinusoidal ectasia and lymphoid hyperplasia were on ly increased in the highest dose group; in 2 

### females, significant increases in nonneoplastic les ions of pancreatic lymph nodes were only 3 

### observed in the highest dose group. Microscopicall y, lymphoid hyperplasia was characterized by 4 

### minimal-to-mild lymphocyte proliferation, and sinus oid ectasia was characterized by minimal-to5 

### mild dilatation of the subcapsular or medullary sin uses; histiocytic cell infiltration was similar to 6



### that observed in the duodenum. In the liver of fem ales, a dose-dependent increase in the 7 

### incidence of histiocytic cellular infiltration was observed at ≥ 3.5 mg hexavalent chromium/kg8 

### day and chronic inflammation was increased in the h ighest dose group. Although serum liver 9 

### enzymes were increased in treatment groups (discuss ed above), significant histopathological 10 

### changes to the livers of male rats were not observe d. The incidence of bone marrow hyperplasia 11 

### was significantly increased in high-dose females. This observation is consistent with an 12 

### increased hematopoiesis in response to hexavalent c hromium-induced microcytic, hypochromic 13



### anemia. 14

### 50 DRAFT - DO NOT CITE OR QUOTE

1 

### Table 4-7. Incidence of nonneoplastic lesions obse rved in male and female

F344/N rats exposed to sodium dichromate dihydrate in drinking water for 3 months

Treatment Group (mg hexavalent chromium/kg-day) Tissue (lesion type) 0 1.7 3.5 5.9 11.2 20.9

Males Duodenum (histiocytic cellular infiltration) 0/10 a 0/10 7/10 c (1.1) 9/10 c

(1.2) 8/10 c

(1.4) 7/10 c

(1.4) Stomach, glandular (ulcer) 0/10 0/10 0/10 0/10 0/10 8/10 c (3.0) Stomach, glandular (focal regenerative hyperplasia) 0/10 0/10 0/10 0/10 0/10 10/10 c (2.2) Stomach, glandular (focal squamous hyperplasia) 0/10 0/10 0/10 0/10 1/10 (2.0) 7/10 c

(2.6) Pancreatic lymph node (ectasia) 0/10 0/10 0/10 0/10 1/10 (1.0) 10/10 c

(1.7) Pancreatic lymph node (lymphoid hyperplasia) 0/10 0/10 0/10 3/10 (1.0) 3/10

(1.0) 6/10 c

(2.7) Pancreatic lymph node (histiocytic cellular infiltration) 0/10 5/10 b (1.0) 2/10

(1.0) 4/10 b

(1.0) 5/10 b

(1.0) 9/10 c

(1.9)

Females Duodenum (histiocytic cellular infiltration) 0/10 a 1/10 c (1.0) 5/10 c

(1.0) 7/10 c

(1.4) 8/10 c

(1.6) 10/10 c

(1.7) Stomach, glandular (ulcer) 0/10 0/10 0/10 0/10 0/10 10/10 c (3.5) Stomach, glandular (focal regenerative hyperplasia) 0/10 0/10 0/10 0/10 0/10 10/10 c (2.0) Stomach, glandular (focal squamous hyperplasia) 0/10 0/10 0/10 0/10 0/10 10/10 c (2.4) Pancreatic lymph node (ectasia) 0/10 0/10 0/10 0/10 1/10 (1.0) 10/10 c

(1.8) Pancreatic lymph node (lymphoid hyperplasia) 0/10 0/10 2/10 (1.5) 0/10 0/10 10/10 c (2.1) Pancreatic lymph node (histiocytic cellular infiltration) 4/10 (1.0) 8/10

(1.4) 7/10

(1.7) 7/10

(1.3) 7/10

(1.7) 9/10 b

(1.9) Liver (histiocytic cellular infiltration) 0/10 3/10 (1.3) 6/10 c

(1.0) 6/10 c

(1.0) 9/10 c

(1.2) 8/10 c

(1.0) Liver (chronic focal inflammation) 3/10 (1.0) 5/10

(1.0) 2/10

(1.0) 7/10

(1.0) 2/10

(1.0) 10/10 c

(1.0) Bone marrow (hyperplasia) 0/10 0/10 0/10 0/10 0/10 4/10 b (1.0)

a Number of animals with lesion/number of animals exa mined; parenthesis indicate average severity grade, with 1=minimal; 2=mild; 3=moderate; 4=severe.

b Significantly different (

p ≤ 0.05) from the control group by the Fisher exact t est.

c Significantly different (

p ≤ 0.01) from the control group by the Fisher exact t est.

Source: NTP (2007).

### 51 DRAFT - DO NOT CITE OR QUOTE

In conclusion, the NTP (2007) 3-month study in F344 /N rats exposed to sodium 1 

### dichromate dihydrate in drinking water identified s everal effects of subchronic oral hexavalent 2 

### chromium exposure, including adverse hematological effects (microcytic, hypochromic anemia), 3 

### hepatotoxicity (increased serum enzyme activities, increased serum bile acids, and 4 

### histopathological changes), alterations in lipid me tabolism (decreased serum cholesterol and 5 

### triglycerides), possible muscle damage (increased s erum creatine kinase activity), and 6 

### histopathological changes in GI tissues (duodenum a nd glandular stomach) and in pancreatic 7 

### lymph nodes. Results of this study identified a LO AEL in male and female rats of 1.7 mg 8 

### hexavalent chromium/kg-day; a NOAEL was not identif ied. In males, the LOAEL was based on 9 

### observations of microcytic, hypochromic anemia (dec reased Hct, Hgb, MCV, MCH) occurring 10 

### after 23 days to 3 months of exposure, increased se rum liver enzyme activities (ALT and SDH), 11 

### and histopathological changes to pancreatic lymph n odes (histiocytic cellular infiltration), all 12 

### observed at daily doses ≥ 1.7 mg hexavalent chromium/kg-day. In females, the LOAEL was 13 

### based on observations of microcytic, hypochromic an emia (decreased Hgb, MCV, MCH) 14 

### occurring after 23 days to 3 months of exposure, in creased serum liver enzyme activities (ALT 15 

### and SDH) and bile acids, and histopathological chan ges to the duodenum (histiocytic cellular 16 

### infiltration), all observed at daily doses ≥ 1.7 mg hexavalent chromium/kg-day. 17 

### In the 3-month study in B6C3F 1 

### mice, groups of 10 males and 10 females were expos ed 18 

### to sodium dichromate dihydrate in drinking water at concentrations of 0, 62.5, 125, 250, 500, or 19 

### 1,000 mg sodium dichromate dihydrate/L (equivalent to 0, 21.8, 43.6, 87.2, 174,5, or 348 mg 20 

### hexavalent chromium/L, respectively) for 3 months ( NTP, 2007). Based on water consumption 21 

### monitored throughout the study, NTP (2007) calculat ed average daily doses over the 3-month 22 

### treatment duration of approximately 0, 9, 15, 26, 4 5, or 80 mg sodium dichromate dihydrate/kg23 

### day (equivalent to 0, 3.1, 5.3, 9.1, 15.7, or 27.9 mg hexavalent chromium/kg-day, respectively) 24 

### for both males and females. Mice were subjected to the same evaluations and procedures as 25 

### those described above for “core” study rats (NTP, 2 007), except that blood was not analyzed for 26 

### clinical chemistry as the study in mice did not inc lude a group of “clinical pathology” animals 27 

### for evaluation after exposure durations of 5 and 23 days. 28



### No mortalities were observed in male or female mice exposed to sodium dichromate 29 

### dihydrate in drinking water for 3 months (NTP, 2007 ). Dose-related significant decreases were 30 

### observed in final body weights in male mice, with d ecreases reaching 20% (compared with 31 

### control values) in the 27.9 mg hexavalent chromium/ kg-day group; in females, dose-related 32 

### decreases in final body weight were observed at ≥ 5.3 mg hexavalent chromium/kg-day, with 33

### 52 DRAFT - DO NOT CITE OR QUOTE

decreases reaching 13% in the 27.9 mg hexavalent ch romium/kg-day group. Drinking water 1 

### consumption was reduced in males at ≥ 5.3 mg hexavalent chromium/kg-day and in females at 2 

### 27.9 mg hexavalent chromium/kg-day (statistical sig nificance not reported). Data on food 3 

### consumption were not reported. No treatment-relate d signs of clinical toxicity were observed 4 

### throughout the study. 5 

### Results of hematological analyses show that mice ex posed to sodium dichromate 6 

### dihydrate in drinking water for 3 months developed mild erythrocyte microcytosis (NTP, 2007); 7 

### however, compared to hematological effects observed in rats (described above), effects in mice 8 

### were less severe. In male mice, MCV and MCH were s ignificantly decreased in all treatment 9 

### groups, with maximum decreases of approximately 8%, compared to controls, in the highest dose 10 

### group. In females, MCV and MCH were significantly reduced at ≥ 3.1 and ≥ 5.2 mg hexavalent 11 

### chromium/kg-day, respectively, with maximum decreas es of approximately 9% and 10%, 12 

### respectively, compared to controls, in the highest dose group. Although statistically significant 13 

### ( p < 0.05) decreases in MCV were observed in males and females in the 3.1 mg hexavalent 14 

### chromium/kg-day group, decreases were very small (1 –2%, compared to controls); at doses up to 15 

### 9.1 mg hexavalent chromium/kg-day, decreases in MCV were ≤ 5%, compared with controls. 16 

### Thus, mild microcytosis observed at ≥ 9.1 mg hexavalent chromium/kg-day does not appear t o 17 

### represent a clinically significant adverse effect. Erythrocyte counts were slightly increased ( ≤ 6% 18 

### increase, compared with controls) at ≥ 5.2 mg hexavalent chromium/kg-day in females, but n ot in 19



### males. 20 

### Changes in organ weights in mice exposed to sodium dichromate dihydrate in drinking 21 

### water for 3 months are summarized in Table 4-8 (NTP , 2007). In males, absolute liver and right 22 

### kidney weights were decreased at ≥ 9.1 mg hexavalent chromium/kg-day, although the onl y 23 

### significant change in relative organ weight was an increase in relative kidney weight at 27.9 mg 24 

### hexavalent chromium/kg-day. In females, absolute l iver weight was increased at ≥ 15.7 mg 25 

### hexavalent chromium/kg-day, but no changes in relat ive liver weight were observed. Changes in 26 

### weights of other organs were considered by NTP (200 7) to be secondary to changes in body 27 

### weight rather than due to adverse effects of treatm ent. 28

29

### 53 DRAFT - DO NOT CITE OR QUOTE

Table 4-8. Selected organ weights in male and fema le B6C3F 1 

### mice exposed to sodium dichromate dihydrate in drinking water fo r 3 months

Treatment Group (mg hexavalent chromium/kg-day) Organ 0 3.1 5.3 9.1 15.7 27.9

Males Right kidney, absolute weight 0.28 ± 0.01 a 0.28 ± 0.01 0.26 ± 0.01 0.26 ± 0.01 b 0.24 ± 0.01 c 0.26 ± 0.01 c Right kidney, relative weight d 7.25 ± 0.11 7.68 ± 0.29 7.43 ± 0.35 7.75 ± 0.20 7.76 ± 0.30 8.18 ± 0.07 c Liver, absolute weight 1.60 ± 0.08 1.54 ± 0.05 1.50 ± 0.05 1.40 ± 0.05 b 1.33 ± 0.06 c 1.34 ± 0.04 c

Females Liver, relative weight d 1.15 ± 0.03 a 1.14 ± 0.04 1.06 ± 0.02 1.11 ± 0.04 1.04 ± 0.02 b 0.99 ± 0.02 c

a Values are means ± SE; n = 10 mice/group.

b Significantly different (

p ≤ 0.05) from the control group by Williams’s or Dunn ett’s test.

c Significantly different (

p ≤ 0.01) from the control group by Williams’s or Dunn ett’s test.

d Relative weight = mg organ weight/g body weight.

Source: NTP (2007).

1 

### Gross and microscopic examinations of male and fema le mice exposed to sodium 2 

### dichromate dihydrate in drinking water for 3 months showed nonneoplastic lesions of the 3 

### duodenum and mesenteric lymph nodes (NTP, 2007); in cidence data are summarized in 4 

### Table 4-9. In the duodenum, a dose-related increas e in the incidence of minimal-to-mild 5 

### histiocytic cellular infiltration was observed in m ales and females in all treatment groups and in 6 

### the incidence of minimal-to-mild epithelial hyperpl asia at ≥ 5.3 mg hexavalent chromium/kg7 

### day; a slight dose-related increase in severity was observed. The duodenum had short, thick 8 

### duodenal villi, elongated crypts with diffuse hyper plasia, and hyperplastic epithelial cells with 9 

### swollen, vaculolated cytoplasm and increased number s of “mitotic figures” (incidence data not 10 

### reported). NTP (2007) stated that duodenal lesions were indicative of regenerative hyperplasia 11 

### subsequent to epithelial cell injury. Minimal hist iocytic cellular infiltration, morphologically 12 

### similar to that observed in rats (discussed above), was observed in mesenteric lymph nodes in 13



### male and female mice at ≥ 5.3 mg hexavalent chromium/kg-day. 14

### 54 DRAFT - DO NOT CITE OR QUOTE

1 

### Table 4-9. Incidence of nonneoplastic lesions obse rved in male and female

B6C3F 1 

### mice exposed to sodium dichromate dihydrate in dri nking water for 3 months

Treatment group (mg hexavalent chromium/kg-day) Tissue (lesion type) 0 3.1 5.3 9.1 15.7 27.9

Males Duodenum (histiocytic cellular infiltration) 0/10 a 4/10 b (1.0) 5/10 c

(1.0) 10/10 c

(1.3) 10/10 c

(1.7) 10/10 c

(1.9) Duodenum (epithelial hyperplasia) 0/10 0/10 8/10 c (1.3) 10/10 c

(1.8) 10/10 c

(2.1) 10/10 c

(1.8) Mesenteric lymph node (histiocytic cellular infiltration) 0/10 0/9 4/9 b (1.0) 6/8 c

(1.0) 3/8

(2.0) 8/10 c

(1.3)

Females Duodenum (histiocytic cellular infiltration) 0/10 a 7/10 c (1.0) 8/9 c

(1.3) 10/10 c

(1.3) 10/10 c

(1.4) 10/10 c

(1.7) Duodenum (epithelial hyperplasia) 0/10 0/10 9/9 c (1.1) 10/10 c

(1.1) 10/10 c

(1.5) 10/10 c

(1.4) Mesenteric lymph node (histiocytic cellular infiltration) 0/10 0/10 6/10 c (1.0) 6/10 c

(1.0) 4/9 b

(1.3) 9/10 c

(1.1)

a Number of animals with lesion/number of animals exa mined; parenthesis indicate average severity grade, with 1=minimal; 2=mild; 3=moderate; 4=severe.

b Significantly different (

p ≤ 0.05) from the control group by the Fisher exact t est.

c Significantly different (

p ≤ 0.01) from the control group by the Fisher exact t est.

Source: NTP (2007).

2 

### In conclusion, the NTP (2007) 3-month study in B6C3 F 1 

### mice exposed to sodium 3 

### dichromate dihydrate in drinking water identified a dverse treatment-related hematological effects 4 

### (erythrocyte microcytosis) and histopathological ch anges to the small intestine (duodenal 5 

### epithelial hyperplasia and cellular histiocytic inf iltration) and mesenteric lymph nodes (cellular 6 

### histiocytic infiltration). Based on histopathologi cal changes (histiocytic cellular infiltration) in 7 

### the duodenum, a LOAEL of 3.1 mg hexavalent chromium /kg-day was identified for male and 8 

### female mice; in both sexes, a NOAEL was not identif ied because the effects were observed at the 9 

### lowest dose tested. Although a statistically signi ficant decrease in MCV also was observed at 10 

### 3.1 mg hexavalent chromium/kg-day in males and fema les, hematological effects (e.g., 11 

### microcytosis) were not considered as the basis of t he LOAEL, since decreases in MCV were 12 

### small (1–2%) at the lowest dose tested. 13 

### Finally, NTP (2007) conducted a comparative study i n three strains of mice (B6C3F 1 

### , 14 

### BALB/c, and am3 -C57BL/6) on the effects of exposure to sodium dich romate dihydrate in 15

### 55 DRAFT - DO NOT CITE OR QUOTE

drinking water for 3 months. This comparative stud y was conducted to investigate possible 1



### strain differences in mice based on results of an e arlier study reporting hepatotoxicity 2 

### (hepatocellular vacuolization) in BALB/c mice fed 3 2 mg hexavalent chromium/kg-day in the 3 

### diet as potassium dichromate (NTP 1996a); no eviden ce of hepatotoxicity (including 4 

### histopathological changes) was observed in male or female B6C3F 1 

### mice exposed for 3 months 5 

### to sodium dichromate dihydrate in drinking water at doses up to 20.9 mg hexavalent 6 

### chromium/kg-day (NTP, 2007; results summarized abov e). In the “core study”, groups of 10 7



### male B6C3F 1 

### , 10 male BALB/c, and 5 male am3 -C57BL/6 mice were exposed to sodium 8 

### dichromate dihydrate in drinking water at concentra tions of 0, 62.5, 125, or 250 mg/L 9 

### (equivalent to 0, 21.8, 43.6, or 87.2 mg hexavalent chromium/L, respectively) for 3 months. An 10



### additional five male am3 -C57BL/6 mice were exposed to the same concentratio ns of sodium 11 

### dichromate dihydrate for a mutagenicity study. How ever, mutagenicity studies were not 12 

### conducted due to technical problems; blood collecte d from these animals was analyzed for 13 

### hematology and clinical chemistry. Based on water consumption monitored throughout the 14 

### study, NTP (2007) calculated average daily doses ov er the 3-month treatment duration of 15 

### approximately 0, 8, 15 or 25 mg sodium dichromate d ihydrate/kg-day (equivalent to 0, 2.8, 5.2, 16 

### or 8.7 mg hexavalent chromium/kg-day, respectively) for all strains. Animals were observed 17 

### twice daily for mortality and clinical signs of tox icity; body weights were recorded weekly and 18 

### water consumption was recorded at least every 4 day s. Blood was collected at the end of the 3- 19 

### month treatment period and analyzed for hematology and clinical chemistry, as described above 20 

### for “core study” rats (NTP, 2007). At study termin ation, necropsies were performed on all mice, 21 

### with organ weights recorded for heart, right kidney , liver (except B6C3F 1 

### mice), lung, spleen, 22 

### right testis, and thymus. Microscopic examination was conducted on all gross lesions and 23 

### masses and selected tissues (liver, forestomach, gl andular stomach, duodenum, pancreas, kidney, 24 

### and mesenteric and pancreatic lymph nodes). Sperm count and motility were assessed in all 25 

### study animals, including spermatids per testis and per mg testis, spermatids per cauda and per mg 26 

### cauda, and sperm motility, and weights of left caud a, left epididymis, and left testis. 27



### No mortalities were observed in male B6C3F 1 

### , BALB/c, or am3 -C57BL/6 mice exposed 28 

### to sodium dichromate dihydrate in drinking water fo r 3 months (NTP, 2007). In the 5.2 and 29 

### 8.7 mg hexavalent chromium/kg-day groups, final bod y weights were significantly decreased 30 

### (compared to controls) by 9% and 12%, respectively, in B6C3F 1 

### mice and by 7% and 11%, 31 

### respectively, in BALB/c mice. Final body weight wa s reduced in all treatment groups in am3

- 32 

### C57BL/6 mice, with decreases reaching 44% in the 8. 7 mg hexavalent chromium/kg-day group. 33

### 56 DRAFT - DO NOT CITE OR QUOTE

Water consumption was reduced at 8.7 mg hexavalent chromium/kg-day in all three strains. 1 

### Data on food consumption were not reported. No tre atment-related signs of clinical toxicity 2



### were observed in B6C3F 1 

### or am3 -C57BL/6 mice. In BALB/c mice, ruffled fur was obs erved at 3 

### 8.7 mg hexavalent chromium/kg-day. 4 

### Results of hematology analyses show that male B6C3F 1 

### , BALB/c, and am3 -C57BL/6 5 

### mice exposed to sodium dichromate dihydrate in drin king water for 3 months developed mild 6 

### erythrocyte microcytosis (e.g., MCV) and small decr eases in MCH, with changes observed in 7 

### most treatment groups (Table 4-10) (NTP, 2007). In the 2.8 and 5.2 mg hexavalent 8 

### chromium/kg-day groups, decreases in MCV and MCH we re ≤ 7%, compared with controls, with 9 

### slightly greater decreases at 8.7 mg hexavalent chr omium/kg-day. Erythrocyte counts were 10 

### significantly increased in B6C3F 1 

### mice (7% at 8.7 mg hexavalent chromium/kg-day) and in 11 

### BALB/c mice (2% and 5% at 5.2 and 8.7 mg hexavalent chromium/kg-day, respectively), but not 12



### in am3 -C57BL/6 mice. Hemoglobin and Hct were decreased b y approximately 5%, in am3

- 13 

### C57BL/6 mice at 8.7 mg hexavalent chromium/kg-day, compared with controls, but not in 14



### B6C3F 1 

### or BALB/c mice. Compared with hematological effec ts observed in rats (described 15 

### previously), effects in mice were much less severe. Clinical chemistry analysis showed small 16 

### increases (1.2- to 1.3-fold) in ALT at ≥ 5.2 mg hexavalent chromium/kg-day in BALB/c mice an d 17



### a 1.9-fold increase in ALT in am3 -C57BL/6 mice; in B6C3F 1 

### mice, no increases in serum liver 18 

### enzyme activities were observed. Decreases in vari ous absolute and relative organ weights were 19



### observed at ≥ 5.2 mg hexavalent chromium/kg-day. NTP (2007) cons idered all changes to be 20 

### related to decreased body weight, except for a sign ificant decrease (29% compared with controls; 21



### p ≤ 0.05) in absolute thymus weight in B6C3F 1 

### mice in the 8.7 mg hexavalent chromium/kg-day 22 

### group; however, relative thymus weight was not diff erent from controls in any treatment group. 23 

### No treatment-related effects were observed for repr oductive tissue evaluations or other 24 

### reproductive parameters, except for a significant d ecrease (12.4% compared to controls; p ≤ 25 

### 0.01) in absolute left testis weight in am3 -C57BL/6 mice at 8.7 mg hexavalent chromium/kg26 

### day; NTP (2007) stated that this change was related to decreased body weight. 27

### 57 DRAFT - DO NOT CITE OR QUOTE

1 

### Table 4-10. Hematological effects in male B6C3F 1 

### , BALB/c, and

am3 -C57BL/6 mice exposed to sodium dichromate dihydrat e in drinking water for 3 months

Treatment Group (mg hexavalent chromium/kg-day)

0 2.8 5.2 8.7

B6C3F 1 mice MCV (fL) 47.7 ± 0.2 a 46.6 ± 0.2 b (97.7) 46.4 ± 0.2

(97.3) 44.7 ± 0.1

(93.7) MCH ( ρ g) 15.3 ± 0.1 14.9 ± 0.1 b (93.1) 14.7 ± 0.1 b

(96.1) 14.2 ± 0.0 b

(92.8)

BALB/c mice MCV (fL) 44.8 ± 0.2 a 43.8 ± 0.2 b (97.8) 42.9 ± 0.2

(95.8) 42.6 ± 0.2

(95.1) MCH ( ρ g) 15.0 ± 0.1 14.5 ± 0.1 b (96.7) 14.2 ± 0.1 b

(94.7) 14.0 ± 0.1 b

(93.3)

am3 -C57BL/6 mice MCV (fL) 45.8 ± 0.2 a 44.2 ± 0.4 (96.5) 43.7 ± 0.3 b

(95.4) 40.5 ± 0.3

(88.4) MCH ( ρ g) 14.4 ± 0.1 14.1 ± 0.1 b (97.9) 13.8 ± 0.1 b

(95.8) 13.5 ± 0.2 b

(98.8 )

a Values are means ± SE; values in parenthesis are pe rcent of control; n = 10 mice/group, with the follo wing exceptions: in B6C3F 1 mice, controls (n = 7), 2.8 mg hexavalent chromium /kg-day group (n = 9); 5.2 mg hexavalent chromium/kg-day group (n = 9); in am3 -C57BL/6 mice, 8.7 mg hexavalent chromium/kg-day gr oup (n = 9).

b Significantly different (

p ≤ 0.01) from the control group by Dunn’s or Shirley’s test.

Source: NTP (2007).

2 

### Microscopic examinations of gross lesions and masse s and of selected tissues in male 3



### B6C3F 1 

### , BALB/c, and am3 -C57BL/6 mice exposed to sodium dichromate dihydrat e in drinking 4 

### water for 3 months showed changes to the duodenum, liver, pancreas and mesenteric lymph 5 

### nodes (NTP, 2007); incidence data are summarized in Table 4-11. In the duodenum, dose6



### related increases in the incidences of minimal-to-m ild histiocytic cellular infiltration and 7 

### epithelial hyperplasia were observed in all strains , with histopathological changes of the 8 

### duodenum observed in all exposure groups; severity increased with dose. Microscopically, 9



### lesions were similar to those described above for m ale and female B6C3F 1 

### mice. Dose-related 10 

### increases in the incidences of hepatic glycogen dep letion and pancreatic secretory depletion were 11 

### also observed; NTP (2007) stated that these lesions were likely due to depressed food 12 

### consumption, which is frequently observed when wate r consumption is decreased. The 13 

### incidence of minimal-to-mild histiocytic cellular i nfiltration of mesenteric lymph nodes was 14

### 58 DRAFT - DO NOT CITE OR QUOTE

increased at 8.7 mg hexavalent chromium/kg-day in am3 -C57BL/6 mice, but not in B6C3F 1 

### or 1



### BALB/c mice. 2

3 

### Table 4-11. Incidence of nonneoplastic lesions obs erved in male B6C3F 1 

### ,

BALB/c, and am3 -C57BL/6 mice exposed to sodium dichromate dihydrat e in drinking water for 3 months

Treatment Group (mg hexavalent chromium/kg-day) Tissue (lesion type) 0 2.8 5.2 8.7

B6C3F 1 mice Duodenum (histiocytic cellular infiltration) 0/10 a 8/10 c (1.0) 10/10 c

(1.4) 10/10 c

(2.0) Duodenum (epithelial hyperplasia) 0/10 4/10 b (1.0) 10/10 c

(1.1) 10/10 c

(1.6) Liver (glycogen depletion) 1/10 (1.0) 2/10

(1.5) 9/10 c

(1.4) 10/10 c

(2.2) Pancreas (secretory depletion) 0/10 2/10 (1.0) 7/10 c

(1.0) 9/10 c

(1.0)

BALB/c mice Duodenum (histiocytic cellular infiltration) 0/10 a 4/10 b (1.0) 8/10 c

(1.8) 10/10 c

(1.7) Duodenum (epithelial hyperplasia) 0/10 2/10 (1.0) 10/10 c

(1.1) 10/10 c

(1.4) Pancreas (secretory depletion) 0/10 6/10 c (1.0) 9/10 c

(1.3) 10/10 c

(1.5)

am3 -C57BL/6 mice Duodenum (histiocytic cellular infiltration) 0/5 a 2/5 (1.0) 5/5 c

(1.4) 4/5 b

(1.8) Duodenum (epithelial hyperplasia) 0/5 5/5 c (1.0) 5/5 c

(1.2) 5/5 c

(1.8) Liver (glycogen depletion) 0/5 4/5 b (2.0) 5/5 c

(1.6) 5/5 c

(3.8) Pancreas (secretory depletion) 0/5 3/5 (1.0) 4/5 b

(1.0) 5/5 c

(1.6) Mesenteric lymph node (histiocytic cellular infiltration) 0/5 0/5 0/5 4/5 b (1.5)

a Number of animals with lesion/number of animals exa mined; parenthesis indicate average severity grade, with 1=minimal; 2=mild; 3=moderate; 4=severe.

b Significantly different (

p ≤ 0.01) from the control group by Fisher exact test.

c Significantly different (

p ≤ 0.01) from the control group by Fisher exact test.

Source: NTP (2007).

4 

### In conclusion, the comparative 3-month drinking wat er study on sodium dichromate 5 

### dihydrate in male B6C3F 1 

### , BALB/c, and am3 -C57BL/6 mice showed similar effects in the 6 

### 3 strains (NTP, 2007). A LOAEL of 2.8 mg hexavalen t chromium/kg-day was identified based 7

### 59 DRAFT - DO NOT CITE OR QUOTE

on histopathological changes in the duodenum in B6C 3F 1 

### mice (histiocytic cellular infiltration 1 

### and epithelial hyperplasia), BALB/c mice (histiocyt ic cellular infiltration), and am3 -C57BL/6 2 

### mice (epithelial hyperplasia); a NOAEL was not iden tified. Mild erythrocyte microcytosis was 3 

### not considered the basis for the LOAEL, since the m agnitude of decreases in MCV and MCH in 4 

### the 2.8 mg hexavalent chromium/kg-day group was ≤ 7% compared to controls. 5

6 

### Quinteros et al., 2007 7 

### Quinteros et al. (2007) showed that subchronic oral exposure of rats to hexavalent 8 

### chromium in drinking water decreased circulating pr olactin levels. Groups of 15 male Wistar 9 

### rats were exposed to drinking water containing 0 or 500 mg hexavalent chromium/L as 10 

### potassium dichromate for 30 days. Based on water i ntake and body weights measured over the 11 

### course of the study, Quinteros et al. (2007) calcul ated a daily dose of 73.05 mg hexavalent 12 

### chromium/kg-day. At the end of the treatment perio d, blood was collected for analysis of 13 

### prolactin and luteinizing hormone (LH), and the pit uitary gland and hypothalamus were analyzed 14 

### for chromium content (see Section 3.2). At the end of the 30-day treatment period, water 15 

### consumption and body weight in hexavalent chromium- treated rats were decreased by 30.5 and 16 

### 11.5% compared to controls. Serum prolactin levels in treated rats were decreased by 17 

### approximately 59% ( p < 0.001) compared to controls; serum levels of LH w ere comparable in 18 

### control and treatment groups. NOAEL and LOAEL valu es for this study could not be identified 19 

### because only one dose was evaluated and effects on other potential hexavalent chromium target 20



### tissues were not assessed. 21

22 

### Rafael et al., 2007 23 

### Adverse hepatic effects were reported in rats follo wing subchronic oral exposure to 24 

### hexavalent chromium, but details of this study were not available (Rafael et al., 2007). Male 25 

### Wistar rats (9 control and 19 treated) were adminis tered drinking water containing 0 or 20 mg 26 

### hexavalent chromium/L (chromium compound not report ed) for 10 weeks. According to the 27 

### investigators, no clinical signs of toxicity or cha nges in body weight were observed (data not 28 

### reported). Data on drinking water consumption were not reported, and the report did not indicate 29 

### if drinking water consumption was similar between c ontrol and treatment groups; thus, given this 30 

### uncertainty, daily hexavalent chromium doses cannot be estimated from this study. At the end of 31 

### the treatment period, serum glucose was decreased b y 45% ( p = 0.0002) and serum ALT activity 32 

### was increased by 153% ( p = 0.039), compared with controls. Serum levels of total protein, 33

### 60 DRAFT - DO NOT CITE OR QUOTE

gamma glutamyl transferase, alkaline phosphatase, c holesterol, and total bilirubin were not 1 

### affected by treatment. Microscopic examination of livers of treated mice showed increased 2 

### intracellular space, “little” focal necrosis, and d egenerative alteration with vascularization; 3



### fibrosis was not observed. A NOAEL or LOAEL could not be identified from this study. 4

5 

### Acharya et al., 2001 6 

### In a follow-up to a stidu by Chopra et al. (1996), Acharya et al. (2001) explored whether 7 

### Wistar rats demonstrated sex-specific responses to exposures to chromium and chromium plus 8 

### ethanol using a study design similar to Chopra et a l. (1996), but exposing male rather than 9 

### female Wistar rats. Acharya et al. (2001) exposed 1.5-month old male Wistar rats (5 or 6/group) 10 

### to potassium dichromate in drinking water for 22 we eks at concentrations of 0 or 25 ppm 11 

### potassium dichromate. These dose groups were part of a larger study to evaluate the interactive 12 

### effects of ethanol and chromium. The authors repor ted that food and water consumption was 13 

### monitored daily and each animal was weighed once a week, although these results were not 14 

### reported. Using reference values for body weight a nd drinking water consumption (0.217 kg; 15 

### 0.032 L/day) for male Wistar rats (U.S. EPA, 1988), doses of 0 and 1.5 mg hexavalent 16 

### chromium/kg-day were estimated. At study terminati on, animals were sacrificed and blood 17 

### samples were collected for analysis of serum enzyme activities. Liver and kidney tissues were 18 

### examined for histopathological changes, and liver h omogenates were used to measure total 19 

### triglycerides, total cholesterol, glycogen, and tot al glutathione. 20 

### Serum AST and ALT levels were statistically signif icantly elevated (approximately 21 

### twofold) in chromium-treated rats compared to contr ols. Serum succinate dehydrogenase, AP, 22 

### and AcP in chromium-treated rats were not significa ntly different from the control. Liver total 23 

### triglyceride and liver glycogen levels were signifi cantly reduced in chromium-treated rats (by 24 

### approximately 40 and 20%, respectively). There was a significant increase in liver total 25 

### cholesterol levels (approximately 10%) in chromium- treated rats. Liver glutathione levels in 26



### chromium-treated rats were similar to controls. 27 

### Histopathological examination of the livers of chro mium-treated animals showed altered 28 

### hepatic architecture in the periportal area, with i ncreased sinusoidal space, vacuolation, and 29 

### necrosis. Histopathological examination of the kid neys in chromium-treated rats revealed 30 

### vacuolation in glomeruli, degeneration of the basem ent membrane, and renal tubular epithelial 31 

### degeneration. No information regarding the number of animals examined or the number of 32

### 61 DRAFT - DO NOT CITE OR QUOTE

animals displaying histopathology was provided. Th e only dose tested in this study, 1.5 mg 1 

### hexavalent chromium/kg-day, was identified as a LOA EL. A NOAEL was not identified. 2

3 

### Asmatullah and Noreen, 1999 4 

### Asmatullah and Noreen (1999) studied the effects of subchronic exposure to hexavalent 5 

### chromium on growth rate and hepatic histological st ructure in mice. Groups of male albino 6 

### Swiss mice (9 per group) were exposed to drinking w ater containing 0, 500, 750, 1,000, 1,500, or 7 

### 2,000 mg potassium dichromate/L (equivalent to 0, 1 77, 265, 353, 530, or 706 mg hexavalent 8 

### chromium/L, respectively) for 8 weeks. Data on dri nking water consumption were not reported; 9 

### based on findings of other studies (NTP, 2007, 2008 ) showing decreased drinking water 10 

### consumption and body weight in animals treated with drinking water containing ≥ 30 mg 11 

### hexavalent chromium/L, daily doses of hexavalent ch romium cannot be accurately estimated for 12 

### this study. Body weights and feed consumption were recorded weekly. At the end of the 13 

### treatment period, organ weights were determined for liver, heart, and kidney and microscopic 14 

### examination of the liver was conducted. During the last 2 weeks of treatment, body weights 15 

### were decreased in all treatment groups, with decrea ses ranging from 9 to 29%, compared with 16 

### controls; decreases in body weight were accompanied by similar decreases in feed intake in all 17 

### treatment groups. After 8 weeks of treatment, abso lute wet and dry weights of liver and heart 18 

### were increased in all treatment groups, although th e magnitude of these increases did not exhibit 19 

### dose-dependence. No consistent pattern of change w as observed for wet or dry weight of the 20 

### heart. Relative organ weights were not reported. Histopathological changes in the liver were 21 

### observed, with severity increasing with dose (but i ncidence data were not reported). At 265 mg 22 

### hexavalent chromium/L, an increase in the sinusoida l space was observed; at 353 and 530 mg 23 

### hexavalent chromium/L, hepatic cirrhosis and increa sed sinusoidal space were observed, with 24 

### severity increasing with dose; and at 706 mg hexava lent chromium/L, increased sinusoidal space, 25 

### cirrhosis and nuclear pyknosis (a marker for apopto sis) were observed. Results of microscopic 26 

### examination of liver in mice treated with 177 mg he xavalent chromium/L were not reported. A 27



### NOAEL or LOAEL could not be identified from this st udy. 28

29 

### Chopra et al., 1996 30 

### Chopra et al. (1996) exposed 50-day old female Wis tar rats (5 or 6/group) to potassium 31 

### dichromate in drinking water for 22 weeks at concen trations of 0 or 25 ppm potassium 32 

### dichromate. As in the Acharya et al. (2001) study, these dose groups were part of a larger study 33

### 62 DRAFT - DO NOT CITE OR QUOTE

designed to evaluate the interactive effects of eth anol and chromium. The authors reported that 1 

### food and water consumption were monitored daily and each animal was weighed once a week, 2 

### although these results were not reported. Using re ference values for body weight and drinking 3 

### water consumption (0.156 kg; 0.025 L/day) for femal e Wistar rats (U.S. EPA, 1988), doses of 0 4 

### and 1.4 mg hexavalent chromium/kg-day were estimate d. At study termination, animals were 5 

### sacrificed and blood samples were collected for ana lysis of serum enzyme activities and serum 6 

### triglycerides, cholesterol, and glucose. A kidney homogenate was used to measure glutathione, 7 

### and a liver homogenate was used to measure triglyce rides, cholesterol, glycogen, GSH, and lipid 8 

### peroxidation. Liver and kidney tissues were examin ed for histopathological changes. 9 

### Terminal body weights in chromium-treated rats wer e not significantly different from the 10 

### control. The liver to body weight ratio in chromiu m-treated rats was statistically significantly 11 

### increased (approximately twofold) over the control. Serum SDH levels were significantly lower 12 

### (by approximately 20%) in chromium-treated rats com pared to the control, whereas AST, ALT, 13 

### AP, and AcP were statistically significantly increa sed (approximately two- to threefold). Serum 14 

### triglycerides and glucose were statistically signif icantly increased (approximately threefold) in 15 

### chromium-treated rats; serum cholesterol was signif icantly reduced (approximately twofold). 16 

### Analysis of liver homogenates revealed that chromiu m treatment resulted in reduced liver 17 

### glycogen (by approximately twofold); levels of live r cholesterol, GSH, and lipid peroxidation (as 18 

### measured by diene conjugation) did not differ from the control. Kidney GSH in chromium19 

### treated rats was statistically significantly lower than the control (approximately 2.5-fold). 20 

### Histopathological examination of the liver of chro mium-treated animals showed altered 21 

### hepatic architecture in the periportal area, with i ncreased sinusoidal space, vacuolation, and 22 

### necrosis. Histopathological examination of the kid neys in chromium-treated rats revealed 23 

### significant damage to renal tubules and the Bowmans capsule and degeneration of the basement 24 

### membrane. No information regarding the number of a nimals examined or the number of animals 25 

### displaying histopathology was provided. The only d ose tested in this study, 1.4 mg hexavalent 26 

### chromium/kg-day, was identified as a LOAEL. A NOAE L was not identified. 27

28 

### Vyskocil et al., 1993 29 

### Alterations in renal function, as assessed by urina lysis, were observed in rats exposed to 30 

### oral potassium chromate for up to 6 months (Vyskoci l et al., 1993). Groups of Wistar rats 31 

### (20/sex/group) were exposed to drinking water conta ining 0 or 25 mg hexavalent chromium/L. 32 

### Based on water consumption, which was comparable be tween control and treatment groups, 33

### 63 DRAFT - DO NOT CITE OR QUOTE

Vyskocil et al. (1993) calculated average daily hex avalent chromium doses of 2.18 and 2.47 mg 1 

### hexavalent chromium/kg-day in males and females, re spectively, during the first 3 months of 2 

### exposure, and 1.40 and 1.76 mg hexavalent chromium/ kg-day in males and females, 3 

### respectively, during the second 3 months of exposur e. After 3 or 6 months of exposure, urine 4 

### was collected from 10 rats/sex/group and analyzed f or total protein, albumin, β 2 

### -microglobulin, 5 

### β -N-acetyl-D-glucosamine, and lactate dehydrogenase and lysozyme activities, and body and 6 

### kidney weights were determined. Water consumption was monitored throughout the study. No 7 

### effects on body weight gain or kidney weight were o bserved. In male rats, results of urinalysis 8 

### did not show any treatment-related effects. In fem ales, urinary albumin excretion, a marker of 9 

### glomerular function, was significantly increased by approximately twofold ( p < 0.05), compared 10 

### to controls, at both 3 months and 6 months. Urinar y β 2 

### -microglobulin, a marker of renal tubular 11 

### dysfunction, was increased by 2-fold ( p < 0.05) at 3 months and by 1.4-fold at 6 months (no t 12 

### statistically significant) compared to controls. G ross or microscopic examinations of kidneys 13



### were not conducted. NOAEL and LOAEL values from th is study could not be identified because 14 

### only one dose was evaluated and effects on other po tential hexavalent chromium target tissues 15



### were not assessed. 16

17



### 4.2.2. Chronic Oral Exposure 18 

### NTP, 2008 19 

### NTP (2008) conducted a 2-year toxicology and carcin ogenicity study of sodium 20 

### dichromate dihydrate in drinking water in rats and mice. Groups of F344/N rats (“core” study 21 

### animals; 50/sex/group) were exposed to sodium dichr omate dihydrate in drinking water at 22 

### concentrations of 0, 14.3, 57.3, 172, or 516 mg sod ium dichromate dihydrate/L (equivalent to 0, 23 

### 5, 20, 60, or 180 mg hexavalent chromium/L, respect ively). Based on water consumption 24 

### measured throughout the study, NTP (2008) calculate d average daily doses over the 2-year 25 

### treatment duration of approximately 0, 0.6, 2.2, 6, or 17 mg sodium dichromate dihydrate/kg-day 26 

### for males (equivalent to 0, 0.21, 0.77, 2.1, or 5.9 mg hexavalent chromium/kg-day, respectively) 27 

### and 0.7, 2.7, 7, and 20 mg sodium dichromate dihydr ate/kg-day for females (equivalent to 0, 28 

### 0.24, 0.94, 2.4, or 7.0 mg hexavalent chromium/kg-d ay, respectively). Animals were observed 29 

### twice daily for mortality and clinical signs of tox icity; after 5 weeks of treatment, clinical signs 30 

### were recorded at 4-week intervals. Body weights we re recorded weekly for the first 13 weeks, 31



### and then at 4-week intervals for the duration of th e study. Water consumption was recorded 32 

### weekly for the first 13 weeks of treatment and then every 4 weeks. At the end of the 2-year 33

### 64 DRAFT - DO NOT CITE OR QUOTE

treatment period, complete necropsies and microscop ic examinations of comprehensive tissues 1 

### were performed on all core study animals. An addit ional “special study” group of male rats 2 

### (10/group) were exposed to the same drinking water concentrations as core animals for up to 3 

### 53 weeks. For the special study rats only, blood w as collected on days 4 and 22 and at 3, 6, and 4 

### 12 months for hematology (i.e., Hct; Hgb concentrat ion; erythrocyte, reticulocyte, and platelet 5 

### counts; erythrocyte and platelet morphology; MCV; M CH; mean cell hemoglobin concentration 6 

### (MCHC); and leukocyte count and differentials) and clinical chemistry (i.e., urea nitrogen, 7 

### creatinine, total protein, albumin, ALT, AP, creati ne kinase, sorbitol dehydrodrogenase, bile 8 

### acids) analyses. At the end of the 53-week treatme nt period, special study animals were 9 

### evaluated for chromium tissue distribution (see Sec tion 3.2). 10 

### Survival rates of exposed core study rats were simi lar to controls (NTP, 2008). 11 

### Throughout the study, water consumption was decreas ed in the two highest dose groups 12 

### compared to controls. During the second year of th e study, water consumption in the two highest 13 

### dose groups in males was decreased by 15 and 22%, r espectively, and by 15 and 27%, 14 

### respectively, in females (statistical significance not reported). No data on food consumption 15 

### were reported. At the end of the 2-year treatment period, body weight was decreased in males 16 

### and females in the highest dose group by 12 and 11% , respectively, compared with controls 17 

### (statistical significance not reported). NTP (2008 ) suggested that decreased body weights in the 18 

### highest dose group may have been partially due to d ecreased water consumption (due to 19 

### decreased palatability), rather than an adverse eff ect of sodium dichromate dihydrate. No 20 

### treatment-related signs of clinical toxicity were o bserved throughout the study. 21 

### Results of hematologic analyses in special study ma le rats showed that exposure to 22 

### sodium dichromate dihydrate in drinking water produ ced microcytic, hypochromic anemia, 23 

### characterized by decreases in MCV, Hct, Hgb, MCH, a nd MCHC (NTP, 2008). The severity of 24 

### microcytic, hypochromic anemia exhibited duration- and dose-dependence, with peak effects 25 

### occurring at 22 days (Table 4-12). After 4 days of exposure, small changes were observed in 26 

### several hematological parameters; however, decrease s in all treatment groups were ≤ 5%, 27 

### compared to controls. More severe effects were obs erved after 22 days of treatment, with 28 

### significant decreases in MCV, Hct, and Hgb at ≥ 0.77 mg hexavalent chromium/kg-day. At 29 

### 5.9 mg hexavalent chromium/kg-day, MCV, Hct, and Hg b decreased to approximately 76, 73, 30 

### and 65% of control values, respectively; reticulocy te and nucleated erythrocyte counts were 31 

### increased by approximately 66% ( p ≤ 0.01) and 600% (

p ≤ 0.01), respectively, compared to 32 

### controls, indicating compensatory hematopoiesis. B lood smears showed evidence of erythrocyte 33

### 65 DRAFT - DO NOT CITE OR QUOTE

injury or increased turnover, including poikilocyte s, erythrocyte fragments, and keratocytes 1 

### (incidence data not reported). Similar effects wer e observed after 3 months of treatment, 2 

### although severity at 3 months was generally less th an that observed at 22 days. Severity was 3 

### further decreased after 6 and 12 months of exposure ; at 12 months, affected parameters were 4 

### generally only decreased by ≤ 5%, compared to controls. Results of hematological analyses show 5 

### that exposure of rats to sodium dichromate dihydrat e in drinking water produced microcytic, 6 

### hypochromic anemia at subchronic exposure durations (22 days to 3 months), but that severity 7 

### decreased with increasing exposure duration (6–12 m onths). 8

9 

### Table 4-12. Hematological effects in male F344/N r ats exposed to sodium

dichromate dihydrate in drinking water for up to 12 months

Treatment group (mg hexavalent chromium/kg-day) Hematological parameter Time on

treatment 0 0.21 0.77 2.1 5.9 Day 22 59.5 ± 0.4 a 58.6 ± 0.5 (98.5) 54.9 ± 0.5 c

(92.3) 47.4 ± 0.4 c

(80.0) 45.0 ± 0.7 c

(75.6)

Month 3 48.6 ± 0.2 48.3 ± 0.2 (99.4) 47.3 ± 0.2 c

(97.3) 45.7 ± 0.2 c

(94.0) 39.2 ± 0.6 c

(80.7)

Month 6 49.8 ± 0.1 49.5 ± 0.1 (99.4) 48.6 ± 0.1 c

(97.6) 47.8 ± 0.2 c

(96.0) 45.4 ± 0.5 c

(91.2) MCV (fL)

Month 12 52.6 ± 0.2 52.4 ± 0.2 (99.6) 51.9 ± 0.3

(98.7) 51.4 ± 0.3 c

(97.7) 49.9 ± 0.2 c

(94.9) Day 22 46.0 ± 1.1 44.4 ± 0.4 (96.5) 43.2 ± 0.6 b

(93.9) 38.7 ± 0.6 c

(84.1) 33.5 ± 0.8 c

(72.8)

Month 3 45.3 ± 0.4 44.5 ± 0.3 (98.2) 44.5 ± 0.4

(98.2) 44.1 ± 0.5

(97.4) 41.0 ± 0.5 c

(90.5)

Month 6 45.9 ± 0.4 45.7 ± 0.5 (99.6) 45.5 ± 0.4

(99.1) 45.5 ± 0.5

(99.1) 45.0 ± 0.3

(98.0) Hct (percent)

Month 12 47.6 ± 0.5 46.6 ± 0.4 (97.9) 47.4 ± 0.5

(99.6) 47.7 ± 0.4

(100.2) 47.3 ± 0.4

(99.4) Day 22 15.5 ± 0.3 15.1 ± 0.2 (97.4) 14.2 ± 0.2 c

(91.6) 12.0 ± 0.3 c

(77.4) 10.1 ± 0.2 c

(65.2)

Month 3 15.1 ± 0.1 14.9 ± 0.1 (98.7) 14.9 ± 0.2

(98.7) 14.6 ± 0.2 b

(96.7) 12.9 ± 0.2 c

(85.4)

Month 6 15.2 ± 0.1 15.2 ± 0.2 (100) 15.0 ± 0.2

(98.7) 14.9 ± 0.1

(98.0) 14.5 ± 0.1 c

(95.4) Hgb (g/dL)

Month 12 15.8 ± 0.2 15.4 ± 0.2 (97.5) 15.6 ± 0.2

(98.7) 15.6 ± 0.2

(98.7) 15.3 ± 0.1 b

(96.8)

### 66 DRAFT - DO NOT CITE OR QUOTE

Table 4-12. Hematological effects in male F344/N r ats exposed to sodium dichromate dihydrate in drinking water for up to 12 months

Treatment group (mg hexavalent chromium/kg-day) Hematological parameter Time on

treatment 0 0.21 0.77 2.1 5.9 Day 22 19.8 ± 0.1 19.5 ± 0.2 (98.5) 17.7 ± 0.2 c

(89.4) 14.8 ± 0.2 c

(74.7) 16.3 ± 0.5 c

(82.3)

Month 3 16.2 ± 0.1 16.2 ± 0.1 (100) 15.7 ± 0.0 c

(96.9) 15.0 ± 0.1 c

(92.6) 11.9 ± 0.3 c

(73.5)

Month 6 16.3 ± 0.1 16.1 ± 0.1 (98.8) 15.7 ± 0.1 c

(96.3) 15.3 ± 0.1 c

(93.9) 14.3 ± 0.2 c

(87.7) MCH ( ρ g)

Month 12 17.0 ± 0.1 16.8 ± 0.1 (98.8) 16.6 ± 0.1 b

(97.6) 16.2 ± 0.1 c

(95.3) 15.7 ± 0.1 c

(92.4) Day 22 33.3 ± 0.1 33.3 ± 0.1 (100) 32.2 ± 0.2

(96.7) 31.2 ± 0.2 c

(93.7) 36.2 ± 0.8

(108.7)

Month 3 33.4 ± 0.1 33.5 ± 0.2 (100.3) 33.2 ± 0.1

(99.4) 32.7 ± 0.1 c

(97.9) 30.2 ± 0.3 c

(90.4)

Month 6 32.7 ± 0.1 32.5 ± 0.1 (99.4) 32.3 ± 0.1 b

(98.8) 32.1 ± 0.1 c

(98.2) 31.6 ± 0.2 c

(96.6) MCHC (g/dL)

Month 12 32.3 ± 0.2 32.1 ± 0.3 (99.4) 32.0 ± 0.2

(99.1) 31.6 ± 0.2 b

(97.8) 31.5 ± 0.2 b

(97.5) Day 22 7.80 ± 0.13 7.74 ± 0.15 (99.2) 8.06 ± 0.16

(103.3) 8.10 ± 0.14

(103.8) 6.21 ± 0.13 c

(79.6)

Month 3 9.28 ± 0.05 9.24 ± 0.06 (99.6) 9.46 ± 0.11

(101.9) 9.75 ± 0.11 c

(105.1) 10.93 ± 0.16 c

(117.7)

Month 6 9.34 ± 0.06 9.43 ± 0.08 (101.0) 9.54 ± 0.11

(102.1) 9.71± 0.08 c

(104.0) 10.15 ± 0.13 c

(108.7) Erythrocyte count (10 6 /μL)

Month 12 9.27 ± 0.10 9.17 ± 0.07 (98.9) 9.40 ± 0.12

(101.4) 9.61 ± 0.11

(103.7) 9.74 ± 0.08 c

(105.1)

a Values are means ± SE; values in parenthesis are pe rcent of control; n = 10 rats/group, with the follo wing exceptions: control group on day 4 (n = 9), 0.77 mg hexavalent chromium/kg-day group on day 4 (n = 9), and 2.1 mg hexavalent chromium/kg-day group in month 12 (n = 8).

b Significantly different (

p ≤ 0.05) from the control group by Dunn’s or Shirley’ s test.

c Significantly different (

p ≤ 0.01) from the control group by Dunn’s or Shirley’ s test.

Source: NTP (2007).

1 

### Results of clinical chemistry analyses in special s tudy male rats (clinical chemistry was 2 

### not assessed in female rats) show that exposure to sodium dichromate dihydrate in drinking 3 

### water produced dose-dependent increases in serum AL T activity (NTP, 2008). Significant 4 

### increases in serum ALT activity were observed at 4 days and at 6 months in rats treated with 5



### ≥ 2.1 mg hexavalent chromium/kg-day and at 22 days an d 3 and 12 months at ≥ 0.77 mg 6 

### hexavalent chromium/kg-day (Table 4-13). Serum ALT enzyme activity reached maximum 7 

### increases (approximately 170–260% of control values ) in rats treated for 3–12 months at daily 8



### doses of ≥ 2.1 mg hexavalent chromium/kg-day. In rats treated for 12 months with 2.1 and 9

### 67 DRAFT - DO NOT CITE OR QUOTE

5.9 mg hexavalent chromium/kg-day, serum SDH activi ty was 164 and 173% of control values, 1 

### respectively; however, no increases in SDH activity were observed at other doses or time points. 2 

### No increases in serum AP activity were observed in any treatment group throughout the 12- 3 

### month treatment period. Increased serum ALT activi ty is consistent with histopathological 4 

### findings of minimal chronic inflammation of the liv er observed in core study animals (discussed 5 

### below); however, because other clinical chemistry m arkers of hepatic damage were not observed, 6 

### NTP (2008) suggested that increased serum ALT activ ity may reflect enzyme induction rather 7 

### than hepatocellular damage. Changes in other clini cal chemistry outcomes were generally <5% 8 

### compared to controls and did not exhibit dose- or d uration-dependence. 9

10 

### Table 4-13. Serum ALT activity in male F344/N rats exposed to sodium

dichromate dihydrate in drinking water for up to 12 months

Treatment group (mg hexavalent chromium/kg-day) Time on treatment 0 0.21 0.77 2.1 5.9 Day 4 54 ± 2 a 53 ± 2 (98) 60 ± 3

(113) 68 ± 1 c

(126) 70 ± 2 c

(130) Day 22 45 ± 1 46 ± 1 (102) 58 ± 2 c

(129) 75 ± 3 c

(167) 73 ± 4 c

(162)

Month 3 82 ± 4 82 ± 12 (100) 135 ± 18 b

(165) 176 ± 13 c

(215) 216 ± 21 c

(263)

Month 6 122 ± 15 114 ± 9 (93) 150 ± 12

(123) 238 ± 2 c

(195) 210 ± 12 c

(172)

Month 12 102 ± 6 107 ± 8 (105) 135 ± 10 b

(132) 261 ± 23 c

(256) 223 ± 15 c

(219)

a Values are means ± SE; values in parenthesis are pe rcent of control; n = 10 rats/group, with the follo wing exception of 0.77 mg hexavalent chromium/kg-day gro up on day 4 (n = 9). Note: clinical chemistry was not assessed in female rats.

b Significantly different (

p ≤ 0.05) from the control group by Dunn’s or Shirley’ s test.

c Significantly different (

p ≤ 0.01) from the control group by Dunn’s or Shirley’ s test.

Source: NTP (2008).

11 

### Gross and microscopic examinations of core study ra ts exposed to sodium dichromate 12 

### dihydrate in drinking water for 2 years showed nonn eoplastic lesions of the small intestine 13 

### (duodenum), liver, and lymph nodes in both sexes, n onneoplastic lesions of the salivary gland in 14 

### females, and neoplastic lesions of the oral cavity in both sexes (NTP, 2008). Incidence data for 15 

### nonneoplastic lesions are summarized in Table 4-14. The incidence of minimal-to-mild cellular 16 

### histiocytic infiltration of the duodenum was signif icantly increased in males and females at 17



### ≥ 0.77 and ≥ 2.4 mg hexavalent chromium/kg-day, respectively, co mpared with controls; 18

### 68 DRAFT - DO NOT CITE OR QUOTE

increases in both sexes were dose-related. Duodena l histiocytic infiltrate was characterized by 1 

### single or clusters of macrophages in the lamina pro pria of the duodenal villi. Based on incidence 2 

### data, males appeared more sensitive than females to hexavalent chromium-induced 3 

### nonneoplastic changes to the small intestine. 4 

### Significant findings in the liver included histiocy tic cellular infiltration, chronic 5 

### inflammation, fatty change, basophilic foci, and cl ear cell foci. The incidence of histiocytic 6 

### cellular inflammation, which was mild-to-moderate i n severity and characterized by clusters of 7 

### macrophages in parenchymal and portal areas, was si gnificantly increased in males and females 8



### at 5.9 and ≥ 0.94 mg hexavalent chromium/kg-day, respectively (T able 4-14); in females, 9 

### increases in incidence and severity were dose-depen dent. Increased minimal-to-mild hepatic 10 

### inflammation was observed in males at 2.1 mg hexava lent chromium/kg-day and in females in 11 

### all treatment groups, with dose-dependent increases in incidence and severity in females. NTP 12 

### (2008) noted that chronic inflammation is a typical hepatic lesion observed in aged rats; 13 

### however, exposure to sodium dichromate dihydrate ap peared to enhance development of this 14



### lesion. An increase in the incidence of mild-to-mo derate fatty change was observed in females 15 

### only at ≥ 0.94 mg hexavalent chromium/kg-day. Morphologicall y, fatty change was 16 

### characterized by hepatocytes with fat-containing cy toplasmic vacuoles. The incidence of 17 

### basophilic foci was increased in males only at 0.77 and 2.1 mg hexavalent chromium/kg-day, 18



### and the incidence of clear cell foci was increased in females at 2.4 mg hexavalent chromium/kg19 

### day. Based on the dose-response data for histopath ological changes of the liver, female rats 20 

### appear more sensitive to hexavalent chromium than m ale rats to hepatic effects of sodium 21 

### dichromate dihydrate. 22 

### In lymph nodes, lesions were observed in mesenteric lymph nodes (histiocytic cellular 23 

### infiltration and hemorrhage) in both sexes and in p ancreatic lymph nodes (histiocytic cellular 24 

### infiltration) in females only. The incidences of h istiocytic cellular infiltration and hemorrhage of 25 

### mesenteric lymph nodes were significantly increased in males at ≥ 0.77 mg hexavalent 26 

### chromium/kg-day and in females at ≥ 2.4 and 7.0 mg hexavalent chromium/kg-day, respecti vely. 27 

### In males, the severity of histiocytic cellular infi ltration and hemorrhage of mesenteric lymph 28 

### nodes was minimal-to-mild in all groups, but severi ty of histiocytic cellular infiltration was 29 

### slightly increased at ≥ 2.4 mg hexavalent chromium/kg-day. The incidence o f cellular histiocytic 30 

### infiltration of pancreatic lymph nodes was signific antly increased in females in the 2.4 mg 31 

### hexavalent chromium/kg-day group only, with severit y increased at ≥ 0.94 mg hexavalent 32 

### chromium/kg-day group. Morphologically, histiocyti c cellular infiltrate of the lymph nodes was 33

### 69 DRAFT - DO NOT CITE OR QUOTE

similar to that observed in the liver, with random clusters of macrophages located in the cortex 1 

### and medullary sinuses; in mesenteric lymph nodes, s ome clusters merged to form sheets that 2 

### replaced the parenchyma. NTP (2008) suggested that mesenteric lymph node hemorrhage may 3 

### have resulted from histiocytic infiltration. A sig nificant increase in the incidence of salivary 4 

### gland atrophy, appearing as single focal lesions, w as observed in females in the 2.4 mg 5 

### hexavalent chromium/kg-day group only, compared wit h controls. NTP (2008) noted that 6 

### atrophy is an age-related change commonly observed in rats and that the biological significance 7 

### of salivary atrophy in female rats chronically trea ted with 2.4 mg hexavalent chromium/kg-day 8 

### group is unknown. 9

### 70 DRAFT - DO NOT CITE OR QUOTE

1 

### Table 4-14. Incidence of nonneoplastic lesions obs erved in male and female

F344/N rats exposed to sodium dichromate dihydrate in drinking water for 2 years

Treatment group (mg hexavalent chromium/kg-day) Tissue (lesion type) 0 0.21 0.77 2.1 5.9

Males Liver (histiocytic cellular infiltration) 1/50 a (1.0) 0/50 2/49 (1.0) 5/50

(1.4) 34/49 c

(1.4) Liver (chronic inflammation) 19/50 (1.1) 25/50

(1.2) 21/49

(1.3) 28/50 b

(1.1) 26/49

(1.3) Liver (basophilic focus) 22/50 28/50 29/49 b 32/50 b 30/49 Small intestine, duodenum (histiocytic cellular infiltration) 0/48 0/48 6/47 b (1.2) 36/46 c

(1.1) 47/48 c

(1.5) Lymph node, mesenteric (histiocytic cellular infiltration) 13/49 (2.0) 11/50

(1.5) 30/49

(1.9) 39/50 c

(2.1) 41/49 c

(2.1) Lymph node, mesenteric (hemorrhage) 2/49 (1.5) 7/50

(1.1) 9/49 b

(1.3) 8/50 b

(1.1) 17/49 c

(1.3) Treatment group (mg hexavalent chromium/kg-day) Tissue (lesion type) 0 0.24 0.94 2.4 7.0

Females Liver (histiocytic cellular infiltration) 1/50 a (1.0) 5/50

(1.0) 21/50 c

(1.3) 42/50 c

(2.0) 47/50 c

(2.6) Liver (chronic inflammation) 12/50 (1.3) 21/50 b

(1.2) 28/50 c

(1.3) 35/50 c

(1.6) 39/50 c

(2.1) Liver (fatty change) 3/50 (3.3) 7/50

(3.6) 10/50 b

(2.5) 13/50 c

(2.5) 16/50 c

(2.8) Liver (clear cell focus) 7/50 5/50 7/50 20/50 c 7/50 Small intestine, duodenum (histiocytic cellular infiltration) 0/46 0/49 1/48 (1.0) 30/46 c

(1.0) 47/50 c

(1.2) Lymph node, mesenteric (histiocytic cellular infiltration) 21/50 (1.7) 18/50

(1.4) 27/50

(1.5) 36/50 c

(2.0) 42/50 c

(2.4) Lymph node, mesenteric (hemorrhage) 11/50 (1.1) 13/50

(1.3) 16/50

(1.3) 14/50

(1.1) 21/50 b

(1.3) Lymph node, pancreatic (histiocytic cellular infiltration) 17/29 (2.0) 20/36

(1.9) 23/30

(2.6) 32/34 c

(2.8) 27/33

(3.0) Salivary gland (atrophy) 9/50 (1.3) 7/50

(1.4) 10/50

(1.2) 17/50 b

(1.4) 17/50

(2.1)

a Number of animals with lesion/number of animals exa mined; parenthesis indicate average severity grade, with 1=minimal; 2=mild; 3=moderate; 4=severe.

b Significantly different (

p ≤ 0.05) from the control group by the Poly-3 test.

c Significantly different (

p ≤ 0.01) from the control group by the Poly-3 test.

Source: NTP (2008).

2

### 71 DRAFT - DO NOT CITE OR QUOTE

Incidence data for neoplastic lesions of the oral c avity in male and female rats exposed to 1 

### sodium dichromate dihydrate in drinking water for 2 years are summarized in Table 4-15 (NTP, 2 

### 2008). Neoplasms observed in the oral cavity of tr eated rats were squamous cell carcinoma of 3 

### the oral mucosa (both sexes), squamous cell papillo ma of the oral mucosa (males only), 4 

### squamous cell carcinoma of the tongue (both sexes), and squamous cell papilloma and carcinoma 5 

### of the tongue (both sexes). The incidences of squa mous cell carcinoma of the oral mucosa 6 

### (13.6%) and of combined squamous cell papilloma or carcinoma (15.7%) of the oral mucosa 7 

### were significantly increased in male rats treated w ith 5.9 mg hexavalent chromium/kg-day, 8 

### compared with controls. The incidences of squamous cell carcinoma of the oral mucosa (23.9%) 9 

### and of combined squamous cell carcinoma of the oral mucosa or tongue (23.9%) were 10 

### significantly increased in females treated with 7.0 mg hexavalent chromium/kg-day, compared 11 

### with controls. The incidences of other neoplastic lesions of the oral cavity were not significantly 12 

### increased in any treatment group in males or female s compared with controls, although the 13 

### incidence of squamous cell carcinoma of the oral mu cosa in female rats in the 2.4 mg hexavalent 14 

### chromium/kg-day group (4.6%) exceeded that of histo rical controls (0/300 in drinking water 15 

### studies; 5/1,400 by all routes). Other neoplasms o bserved in treated rats included pancreatic 16 

### acinar adenoma and benign pheochromocytomas in male s and mononuclear cell leukemia in 17 

### females (Table 4-16). However, the incidence of th ese neoplasms did not exhibit dose18 

### dependence. Thus, NTP (2008) concluded that the re lationship of neoplastic changes in other 19 

### tissues (e.g., not of the oral cavity) to exposure to sodium dichromate dihydrate was uncertain. 20

### 72 DRAFT - DO NOT CITE OR QUOTE

1 

### Table 4-15. Incidence of neoplastic lesions observ ed in the oral cavity of

male and female F344/N rats exposed to sodium dichr omate dihydrate in drinking water for 2 years

Treatment group (mg hexavalent chromium/kg-day) Neoplasm type 0 0.21 0.77 2.1 5.9

Males Oral mucosa, squamous cell papilloma

Overall rate a ,b 0/50 (0%) 0/50

(0%) 0/49

(0%) 0/50

(0%) 1/49

(2%) Oral mucosa, squamous cell carcinoma

Overall rate a 0/50 (0%) 0/50

(0%) 0/49

(0%) 0/50

(0%) 6/49

(12%) [543] Adjusted rate c 0%

p < 0.001 0% 0% 0% 13.5%

p = 0.015 Tongue, squamous cell papilloma

Overall rate a,b 0/50 (0%) 0/50

(0%) 0/49

(0%) 0/50

(0%) 1/49

(2%) Tongue, squamous cell carcinoma

Overall rate a 0/50 (0%) 1/50

(2%) 0/49

(0%) 0/50

(0%) 0/49

(0%) Oral mucosa or tongue, squamous cell papilloma or c arcinoma

Overall rate a 0/50 (0%) 1/50

(2%) [729T] 0/49

(0%) 0/50

(0%) 7/49

(14.5%) [543] Adjusted rate c 0%

p < 0.001 2.4% 0% 0% 15.7%

p = 0.007 Treatment group (mg hexavalent chromium/kg-day) Neoplasm type 0 0.24 0.94 2.4 7.0

Females Oral mucosa, squamous cell carcinoma

Overall rate a 0/50 (0%) 0/50

(0%) 0/50

(0%) 2/50

(4%) [646] 11/50

(22%) [506] Adjusted rate c 0%

p < 0.001 0% 0% 4.6% 23.9%

p < 0.001 Tongue, squamous cell papilloma

Overall rate a,b 1/50 (2%) 1/50

(2%) 0/50

(0%) 0/50

(0%) 0/50

(0%) Tongue, squamous cell carcinoma

Overall rate a,b 0/50 (0%) 0/50

(0%) 0/50

(0%) 1/50

(2%) 0/50

(0%)

### 73 DRAFT - DO NOT CITE OR QUOTE

Table 4-15. Incidence of neoplastic lesions observ ed in the oral cavity of male and female F344/N rats exposed to sodium dichr omate dihydrate in drinking water for 2 years

Treatment group (mg hexavalent chromium/kg-day) Neoplasm type 0 0.24 0.94 2.4 7.0

Females Oral mucosa or tongue, squamous cell papilloma or c arcinoma

Overall rate a 1/50 (2%) [618] 1/50

(2%) [729T] 0/50

(0%) 2/50

(4%) [646] 11/50

(22%) [506] Adjusted rate c 2.2%

p < 0.001 2.3% 0% 4.6% 23.9%

p = 0.002

a Overall rate: number of animals with lesion/number of animals examined; parenthesis are the percent of animals examined with lesion; brackets are days to first in cidence; T: observed at terminal sacrifice. p -value under treatment group incidence data indicates statistica lly significant Poly-3 test for pairwise comparison between control and exposed group. Statistical analysis us ing overall rates was only conducted if adjusted ra tes were not determined.

b Adjusted rate not reported.

c Adjusted rate: Poly-3 estimated neoplasm incidence (expressed as percent of animals with neoplasm) adj usted for intercurrent mortality. p -Value under control group indicates statistically significant positive Poly-3 trend test.

p -Value under treatment group incidence data indicat es statistically significant Poly-3 test for pairwi se comparison between control and exposed groups, using adjusted rates.

Source: NTP (2008).

1

2 

### Table 4-16. Neoplastic lesions in other tissues (e .g., non-oral cavity) in

F344/N rats exposed to sodium dichromate dihydrate in drinking water for 2 years

Treatment group (mg hexavalent chromium/kg-day) Neoplasm type 0 0.21 0.77 2.1 5.9

Males

Pancreatic acinar adenoma 1/50 a,b 2/50 6/49 c 2/50 2/49 Benign pheochromocytoma (adrenal medulla) 6/49 a,b 13/50 c 14/49 c 5/50 4/49 Treatment group (mg hexavalent chromium/kg-day) Neoplasm type 0 0.24 0.94 2.4 7.0

Females

Mononuclar cell leukemia 8/50 a,b 18/50 c 13/50 7/50 11/50

a Number of animals with lesion/number of animals exa mined.

b Not statistically significant for positive trend ( p > 0.05) by the Poly-3 test.

c Significantly different from controls by the Poly-3 test ( p < 0.05).

Source: NTP (2008).

3

### 74 DRAFT - DO NOT CITE OR QUOTE

In conclusion, the NTP (2008) 2-year drinking water toxicology and carcinogenicity 1 

### study on sodium dichromate dihydrate identified NOA EL and LOAEL values for noncancer 2 

### effects in male rats of 0.21 and 0.77 mg hexavalent chromium/kg-day, respectively, based on 3 

### increased incidences of nonneoplastic histopatholog ical changes to the liver (basophilic foci), 4 

### duodenum (histiocytic cellular infiltrate), and mes enteric lymph nodes (histiocytic cellular 5 

### infiltrate and hemorrhage). Although hematological effects indicative of microcytic, 6 

### hypochromic anemia were observed in male rats expos ed to ≥ 0.77 mg hexavalent chromium/kg7 

### day from 4 days to 6 months, the severity of effect s decreased over time, such that only small 8 

### changes (<5%) were observed at ≥ 2.1 mg hexavalent chromium/kg-day after 12 months o f 9 

### exposure; therefore, hematological effects were not considered as the basis for the chronic 10 

### NOAEL in male rats. In female rats, a LOAEL for no ncancer effects of 0.24 mg hexavalent 11 

### chromium/kg-day was identified based on the increas ed incidence of chronic inflammation of the 12 

### liver (observed in all treatment groups); a NOAEL w as not identified. In addition to noncancer 13 

### effects, exposure of rats to sodium dichromate dihy drate in drinking water for 2 years resulted in 14 

### a significant increase in squamous epithelial neopl asms of the oral mucosa and tongue at the 15 

### highest exposure level (average daily doses of 5.9 and 7.0 mg hexavalent chromium/kg-day in 16 

### males and females, respectively), but not at the th ree lower exposure levels. NTP (2008) 17 

### concluded that results from this study provide clear evidence of carcinogenic activity of sodium 18 

### dichromate dihydrate in male and female F344/N rats based on increased incidences of 19 

### squamous cell neoplasms of the oral cavity. 20



### B6C3F 1 

### mice were exposed to sodium dichromate dihydrate i n drinking water for up to 2 21 

### years (NTP, 2008). Groups of 50 male mice (male “c ore” study animals) were exposed to 22 

### sodium dichromate dihydrate in drinking water at co ncentrations of 0, 14.3, 28.6, 85.7, or 23 

### 257.4 mg sodium dichromate dihydrate/L (equivalent to 0, 5, 10, 30, or 90 mg hexavalent 24 

### chromium/L, respectively). Based on water consumpt ion measured throughout the study, NTP 25 

### (2008) calculated average daily doses for males ove r the 2-year treatment duration of 26 

### approximately 0, 1.1, 2.6, 7, or 17 mg sodium dichr omate dihydrate/kg-day (equivalent to 0, 27 

### 0.38, 0.91, 2.4, or 5.9 mg hexavalent chromium/kg-d ay, respectively). Groups of 50 female mice 28 

### (female “core” study animals) were exposed to sodiu m dichromate dihydrate in drinking water at 29 

### concentrations of 0, 14.3, 57.3, 172, or 516 mg sod ium dichromate dihydrate/L (equivalent to 0, 30 

### 5, 20, 50, or 190 mg hexavalent chromium/L, respect ively). Based on water consumption 31 

### measured throughout the study, NTP (2008) calculate d average daily doses for females over the 32 

### 2-year treatment duration of approximately 0, 1.1, 3.9, 9, or 25 mg sodium dichromate 33

### 75 DRAFT - DO NOT CITE OR QUOTE

dihydrate/kg-day (equivalent to 0, 0.38, 1.4, 3.1, or 8.7 mg hexavalent chromium/kg-day, 1 

### respectively). “Core” study mice were subjected to the same evaluations and procedures as those 2 

### described above for “core” study rats (NTP, 2008). An additional “special study” group of 3 

### female mice (10/group) were exposed to the same dri nking water concentrations of sodium 4 

### dichromate dihydrate as core animals for up to 53 w eeks. For the special study mice only, blood 5 

### was collected on day 22 and at 3, 6, and 12 months for hematologic analyses only (i.e., Hct; Hgb 6 

### concentration; erythrocyte, reticulocyte, and plate let counts; erythrocyte and platelet 7 

### morphology; MCV; MCH; MCHC; and leukocyte count and differentials). At the end of the 8 

### 53-week treatment period, special study animals wer e evaluated for chromium tissue distribution 9 

### (see Section 3.2). 10 

### Survival rates of core study mice exposed to sodium dichromate dihydrate were similar to 11 

### controls (NTP, 2008). Throughout the study, water consumption by males and females was 12 

### decreased in the two highest dose groups compared w ith controls. During the second year of the 13 

### study, water consumption in the two highest dose gr oups was decreased by 15 and 35%, 14 

### respectively, in males and by 25 and 32%, respectiv ely, in females (statistical significance not 15 

### reported). No data on food consumption were report ed. At the end of the 2-year treatment 16 

### period, body weight in males in the highest dose gr oup was decreased by 6% compared with 17 

### controls (statistical significance not reported), a nd body weight in females in the two highest 18 

### dose groups was decreased by 8 and 15%, respectivel y. NTP (2008) suggested that decreased 19 

### body weights in the highest dose group may have bee n partially due to decreased water 20 

### consumption (due to decreased palatability), rather than an adverse effect of sodium dichromate 21 

### dihydrate. No treatment-related signs of clinical toxicity were observed throughout the study. 22 

### Results of hematology analyses in special study fem ale mice (hematology was not 23 

### assessed in male mice) show that exposure to sodium dichromate dihydrate in drinking water 24 

### produced microcytic, hypochromic anemia (NTP, 2008) . Anemia in mice was characterized by 25



### dose-related decreases in MCV and MCH and increases in erythrocyte counts (Table 4-17); the 26 

### magnitude of change in other hematological paramete rs was small ( ≤ 5% compared with 27 

### controls). The pattern of dose- and duration-relat ed severity in female mice was similar to that 28 

### observed in male special study rats (as described a bove); however, severity in mice was less than 29 

### in rats. Thus, exposure of female mice to sodium d ichromate dihydrate in drinking water 30 

### produced microcytic, hypochromic anemia at subchron ic exposure durations (22 days to 31 

### 3 months), with decreased severity at 6–12 months. 32

### 76 DRAFT - DO NOT CITE OR QUOTE

1 

### Table 4-17. Hematological effects in female B6C3F 1 

### mice exposed to sodium

dichromate dihydrate in drinking water for up to 12 months

Treatment group (mg hexavalent chromium/kg-day) Hematological parameter Time on

treatment 0 0.38 1.4 3.1 8.7 Day 22 48.8 ± 0.2 a 48.3 ± 0.1 b (90.0) 47.8 ± 0.2 c

(98.0) 47.0 ± 0.2 c

(96.3) 46.8 ± 0.2 c

(95.9)

Month 3 47.2 ± 0.1 46.9 ± 0.3 (99.4) 46.7 ± 0.1

(98.9) 45.1 ± 0.2 c

(95.6) 43.7 ± 0.3 c

(92.6)

Month 6 45.8 ± 0.2 45.5 ± 0.3 (99.3) 45.1 ± 0.2 b

(98.5) 44.6 ± 0.2 c

(97.4) 42.8 ± 0.3 c

(93.4) MCV (fL)

Month 12 46.9 ± 0.3 46.9 ± 0.3 (100) 46.3 ± 0.3

(98.7) 45.2 ± 0.2 c

(96.4) 43.9 ± 0.5 c

(93.6) Day 22 16.4 ± 0.1 16.2 ± 0.0 b (98.8) 15.9 ± 0.1 c

(97.0) 15.7 ± 0.1 c

(95.7) 15.5 ± 0.1 c

(94.5)

Month 3 15.8 ± 0.0 15.7 ± 0.1 (99.4) 15.6 ± 0.0 c

(98.7) 14.9 ± 0.1 c

(88.6) 14.3 ± 0.1 c

(90.5)

Month 6 15.3 ± 0.1 15.2 ± 0.1 (99.3) 15.1 ± 0.1

(98.7) 14.9 ± 0.1 c

(97.4) 14.1 ± 0.1 c

(92.2) MCH ( ρ g)

Month 12 15.5 ± 0.1 15.7 ± 0.2 (101.3) 15.5 ± 0.1

(100) 15.1 ± 0.1 b

(97.4) 14.4 ± 0.2 c

(92.9) Day 22 10.25 ± 0.15 10.20 ± 0.08 (99.5) 10.47 ± 0.19

(102.1) 10.77 ± 0.13 b

(105.1) 10.61 ± 0.13 b

(103.5)

Month 3 10.10 ± 0.16 10.66 ± 0.13 b (105.5) 10.55 ± 0.17 b

(104.5) 10.95 ± 0.10 c

(108.4) 11.55 ± 0.16 c

(114.4)

Month 6 10.56 ± 0.15 10.81 ± 0.10 (102.4) 10.60 ± 0.13

(100.4) 10.77 ± 0.20

(102.0) 11.50 ± 0.20 c

(108.9) Erythrocyte count (10 6 /μL)

Month 12 9.58 ± 0.10 9.72 ± 0.09 (101.4) 9.77 ± 0.10

(102.0) 9.95 ± 0.13 b

(103.9) 10.30 ± 0.21 c

(107.5)

a Values are means ± SE; values in parenthesis are pe rcent of control; n = 10 mice/group, with the excep tion of 1.4 mg hexavalent chromium/kg-day group on month 12 (n = 9).

b Significantly different (

p ≤ 0.05) from the control group by Dunn’s or Shirley’ s test.

c Significantly different (

p ≤ 0.01) from the control group by Dunn’s or Shirley’ s test.

Source: NTP (2008).

2 

### Gross and microscopic examinations of core study mi ce exposed to sodium dichromate 3 

### dihydrate in drinking water for 2 years showed nonn eoplastic lesions of the small intestine, liver, 4 

### lymph nodes, and pancreas and neoplastic lesions of the small intestine (NTP, 2008). Incidence 5 

### data for nonneoplastic lesions are summarized in Ta ble 4-18. In the small intestine, significant 6 

### increases in the incidences of diffuse epithelial h yperplasia of the duodenum were observed in 7 

### male and female mice in all treatment groups and of the jejunum in females at 8.7 mg hexavalent 8 

### chromium/kg-day, compared with controls. NTP (2008 ) noted that diffuse epithelial hyperplasia 9 

### is consistent with tissue regeneration following ep ithelial cell damage. Incidences of histiocytic 10

### 77 DRAFT - DO NOT CITE OR QUOTE

cellular infiltration of the duodenum were increase d at ≥ 2.4 and ≥ 3.1 mg hexavalent 1 

### chromium/kg-day in males and females, respectively, and of the jejunum at 8.7 mg hexavalent 2 

### chromium/kg-day in females, compared with controls. Focal epithelial hyperplasia was also 3 

### observed in the duodenum in males and females, alth ough incidences were not significantly 4



### different from controls and did not exhibit dose-de pendence. Due to its morphological similarity 5 

### to adenoma, focal epithelial hyperplasia was classi fied as a preneoplastic lesion by NTP (2008). 6 

### In the liver of female mice, dose-dependent increas es were observed in the incidences of 7 

### histiocytic infiltration at all doses and of chroni c inflammation in the 3.1 mg hexavalent 8 

### chromium/kg-day group. Significant decreases in th e incidences of clear cell and eosinophilic 9 

### foci were observed in the liver of males at 5.9 mg hexavalent chromium/kg-day and of 10 

### eosinophilic foci in the liver of females at ≥ 3.1 mg hexavalent chromium/kg-day; the biological 11 

### significance of these decreases is uncertain. 12 

### Dose-dependent increases in the incidences and seve rity of histiocytic cellular infiltration 13 

### of the mesenteric lymph nodes were observed in male s and females in all treatment groups and 14 

### of the pancreatic lymph nodes in males and females at ≥ 2.4 and ≥ 3.1 mg hexavalent 15 

### chromium/kg-day, respectively, compared with contro ls. 16 

### In the pancreas, the dose-dependent increases in th e incidences and severity of cytoplasm 17 

### alterations, characterized by depletion of cytoplas m zymogen granules, were observed at 18



### ≥ 2.4 mg hexavalent chromium/kg-day in males and in a ll treatment groups in females. NTP 19 

### (2008) stated that the biological significance of t his finding is uncertain. 20

### 78 DRAFT - DO NOT CITE OR QUOTE

1 

### Table 4-18. Incidence of nonneoplastic lesions obs erved in male and female

B6C3F 1 

### mice exposed to sodium dichromate dihydrate in dri nking water for 2 years

Treatment group (mg hexavalent chromium/kg-day) Tissue (lesion type) 0 0.38 0.91 2.4 5.9

Males Small intestine, duodenum (diffuse hyperplasia) 0/50 a 11/50 c (2.0) 18/50 c

(1.6) 42/50 c

(2.1) 32/50 c

(2.1) Small intestine, duodenum (histiocytic cellular infiltration) 0/50 2/50 (1.0) 4/50

(1.0) 37/50 c

(1.2) 35/50 c

(1.7) Lymph node, mesenteric (histiocytic cellular infiltration) 14/47 (1.2) 38/49 c

(1.1) 31/49 c

(1.2) 32/49 c

(1.5) 42/46 c

(2.5) Lymph node, pancreatic (histiocytic cellular infiltration) 0/5 2/13 (1.0) 2/10

(1.0) 5/8 b

(1.4) 12/16 b

(2.3) Pancreas (cytoplasmic alteration) 0/49 1/49 (3.0) 1/50

(3.0) 9/49 c

(2.1) 8/48 c

(2.6) Treatment group (mg hexavalent chromium/kg-day) Tissue (lesion type) 0 0.38 1.4 3.1 8.7

Females Small intestine, duodenum (diffuse hyperplasia) 0/50 a 16/50 c (1.6) 35/50 c

(1.7) 31/50 c

(1.6) 42/50 c

(2.2) Small intestine, duodenum (histiocytic cellular infiltration) 0/50 0/50 4/50 (1.3) 33/50 c

(1.2) 40/50 c

(2.0) Small intestine, jejunum (diffuse hyperplasia) 0/50 2/50 (2.0) 1/50

(1.0) 0/50 8/50 c (1.9) Small intestine, jejunum (histiocytic cellular infiltration) 0/50 0/50 0/50 2/50 (1.0) 8/50 c

(1.6) Liver (histiocytic cellular infiltration) 2/49 (1.0) 15/50 c

(1.1) 23/50 c

(1.0) 32/50 c

(1.0) 45/50 c

(1.9) Liver (chronic inflammation) 16/49 (1.1) 21/50

(1.1) 22/50

(1.10 27/50 c

(1.1) 24/50

(1.0) Lymph node, mesenteric (histiocytic cellular infiltration) 3/46 (1.0) 29/48 c

(1.3) 26/46 c

(1.1) 40/50 c

(1.9) 42/50 c

(2.7) Lymph node, pancreatic (histiocytic cellular infiltration) 0/14 1/12 (1.0) 2/15

(1.5) 7/14 c

(1.9) 8/13 c

(2.5) Pancreas (cytoplasmic alteration) 0/48 6/50 b (2.5) 6/49 b

(2.0) 14/50 c

(2.4) 32/50 c

(2.6)

a Number of animals with lesion/number of animals exa mined; parenthesis indicate average severity grade, with 1=minimal; 2=mild; 3=moderate; 4=severe.

b Significantly different (

p ≤ 0.05) from the control group by the Poly-3 test.

c Significantly different (

p ≤ 0.01) from the control group by the Poly-3 test.

Source: NTP (2008).

2 

### Incidence data for neoplastic lesions of the small intestine in male and female mice 3 

### exposed to sodium dichromate dihydrate in drinking water for 2 years are summarized in 4

### 79 DRAFT - DO NOT CITE OR QUOTE

Table 4-19 (NTP, 2008). In male mice, incidences o f combined small intestine (duodenum, 1 

### jejunum, and ileum) adenoma or carcinoma were signi ficantly increased at ≥ 2.4 mg hexavalent 2 

### chromium/kg-day and incidences of duodenal adenoma, small intestine adenoma, and small 3 

### intestine carcinoma were significantly increased at 5.9 mg hexavalent chromium/kg-day. In 4 

### addition, significant positive dose-related trends were observed for the incidences of duodenal 5 

### adenoma, duodenal carcinoma, jejunal adenoma, small intestine adenoma, small intestine 6



### carcinoma and combined small intestine adenoma or c arcinoma. In female mice, significant 7 

### increases in the incidences of duodenal adenoma, sm all intestine adenoma, and combined small 8



### intestine adenoma or carcinoma were observed at ≥ 3.1 mg hexavalent chromium/kg-day and 9 

### incidences of duodenal carcinoma, jejunal adenoma, and small intestine carcinoma were 10 

### significantly increased at 8.7 mg hexavalent chromi um/kg-day. Significant positive dose-related 11 

### trends were observed for duodenal adenoma, duodenal carcinoma, jejunal adenoma, small 12 

### intestine adenoma, small intestine carcinoma and co mbined small intestine adenoma or 13 

### carcinoma. No other statistically or biologically significant neoplasms were observed in other 14



### tissues. 15

16 

### Table 4-19. Incidence of neoplastic lesions observ ed in the small intestine of

male and female B6C3F 1 

### mice exposed to sodium dichromate dihydrate in drinking water for 2 years

Treatment group (mg hexavalent chromium/kg-day) Tissue and lesion type 0 0.38 0.91 2.4 5.9

Males Duodenum, adenoma

Overall rate a,b 0/50 (0%) 0/50

(0%) 0/50

(0%) 0/50

(0%) 6/50

(12%)

p ≤ 0.05 Duodenum, all adenoma (includes multiple adenomas)

Overall rate a 1/50 (2%) [665] 0/50

(0%) 1/50

(2%) [729T] 5/50

(10%) [729T] 15/50

(30%) [451] Adjusted rate c 2.2%

p < 0.001 0% 2.3% 10.8% 32.9%

p < 0.001 Duodenum, carcinoma

Overall rate a 0/50 (0%) 0/50

(0%) 0/50

(0%) 2/50

(4%) [729T] 3/50

(6%) [729T] Adjusted rate c 0%

p < 0.011 0% 0% 4.3% 6.8%

### 80 DRAFT - DO NOT CITE OR QUOTE

Table 4-19. Incidence of neoplastic lesions observ ed in the small intestine of male and female B6C3F 1 

### mice exposed to sodium dichromate dihydrate in drinking water for 2 years

Jejunum, adenoma

Overall rate a 0/50 (0%) 0/50

(0%) 0/50

(0%) 0/50

(0%) 3/50

(6%) [714] Adjusted rate c 0%

p = 0.002 0% 0% 0% 6.8%

Treatment group (mg hexavalent chromium/kg-day) Tissue and lesion type 0 0.38 0.91 2.4 5.9

Males Jejunum, multiple carcinoma

Overall rate a, b 0/50 1/50 0/50 0/50 0/50 Jejunum, all carcinoma (includes multiple)

Overall rate a,b 0/50 2/50 0/50 1/50 2/50

All small intestine d , adenoma

Overall rate a 1/50 (2%) [665] 1/50

(2%) [729T] 1/50

(2%) [729T] 5/50

(10%) [729T] 17/50

(34%) [451] Adjusted rate c 2.2%

p < 0.001 2.3% 2.3% 10.8% 37.2%

p < 0.001

All small intestine d , carcinoma

Overall rate a 0/50 (0%) 2/50

(4%) [729T] 1/50

(2%) [729T] 3/50

(6%) [729T] 5/50

(10%) [729T] Adjusted rate c 0%

p = 0.014 4.5% 2.3% 6.5% 11.4%

p = 0.028

All small intestine d , adenoma or carcinoma

Overall rate a 1/50 (2%) [665] 3/50

(6%) [729T] 2/50

(4%) [729T] 7/50

(14%) [729T] 20/50

(40%) [451] Adjusted rate c 2.2%

p < 0.001 6.8% 4.6% 15.1%

p = 0.032 43.8%

p < 0.001 Treatment group (mg hexavalent chromium/kg-day) Tissue and lesion type 0 0.38 1.4 3.1 8.7

Females Duodenum, multiple adenoma

Overall rate a,b 0/50 (0%) 0/50

(0%) 0/50

(0%) 1/50

(2%) 6/50

(12%)

p ≤ 0.05 Duodenum, all adenoma (includes multiple)

Overall rate a 0/50 (0%) 0/50

(0%) 2/50

(4%) [729T] 13/50

(25%) [729T] 12/50

(24%) [693] Adjusted rate c 0%

p < 0.001 0% 4.2% 27.8%

p < 0.001 25.2%

p < 0.001 Duodenum, carcinoma

Overall rate a 0/50 (0%) 0/50

(0%) 0/50

(0%) 1/50

(2%) [729T] 6/50

(12%) [625] Adjusted rate c 0%

p < 0.001 0% 0% 2.1% 12.6%

p = 0.019 Jejunum, multiple adenomas

### 81 DRAFT - DO NOT CITE OR QUOTE

Table 4-19. Incidence of neoplastic lesions observ ed in the small intestine of male and female B6C3F 1 

### mice exposed to sodium dichromate dihydrate in drinking water for 2 years

Overall rate a,b 0/50 (0%) 0/50

(0%) 0/50

(0%) 0/50

(0%) 1/50

(2%) Treatment group (mg hexavalent chromium/kg-day) Tissue and lesion type 0 0.38 1.4 3.1 8.7

Females Jejunum, all adenomas (including multiple)

Overall rate a 0/50 (0%) 1/50

(2%) [729T] 0/50

(0%) 2/50

(4%) [729T] 5/50

(10%) [729T] Adjusted rate c 0%

p = 0.002 2.2% 0% 4.3% 10.6%

p = 0.035 Jejunum, carcinoma

Overall rate a,b 1/50 (2%) 0/50

(0%) 2/50

(4%) 2/50

(4%) 1/50

(2%)

All small intestine d , adenoma

Overall rate a 0/50 (0%) 1/50

(2%) [729T] 2/50

(4%) [729T] 15/50

(30%) [729T] 16/50

(32%) [693] Adjusted rate c 0%

p < 0.001 2.2% 4.2% 32.0%

p < 0.001 33.7%

p < 0.001

All small intestine d , carcinoma

Overall rate a 1/50 (2%) [729T] 0/50

(0%) 2/50

(4%) [729T] 3/50

(6%) [729T] 7/50

(14%) [625] Adjusted rate c 2.2%

p < 0.001 0% 4.2% 6.4% 14.7%

p = 0.037

All small intestine d , adenoma or carcinoma

Overall rate a 1/50 (2%) [729T] 1/50

(2%) [729T] 4/50

(8%) [729T] 17/50

(34%) [729T] 22/50

(44%) [625] Adjusted rate c 2.2%

p < 0.001 2.2% 8.3% 36.3%

p < 0.001 45.9%

p < 0.001

a Overall rate: number of animals with lesion/number of animals examined; parenthenesis are the percent of animals examined with lesion; brackets indicate the days to first incidence; T: observed at terminal sacrifice

. p -Value under treatment group incidence data indicates statistica lly significant Poly-3 test for pairwise comparison between control and exposed group. Statistical analysis us ing overall rates were only conducted if adjusted r ates were not determined.

b Adjusted rate not reported.

c Adjusted rate: Poly-3 estimated neoplasm incidence (expressed as % of animals with neoplasm) adjusted for intercurrent mortality. p -Value under control group indicates statistically significant positive Poly-3 trend test. p

Value under treatment group incidence data indicate s statistically significant Poly-3 test for pairwis e comparison between control and exposed groups, using adjusted rates. d Duodenum, jejunum, or ileum.

Source: NTP (2008).

1 

### In conclusion, the NTP (2008) 2-year toxicology and carcinogenicity study on sodium 2 

### dichromate dihydrate identified a LOAEL for noncanc er effects of 0.38 mg hexavalent 3 

### chromium/kg-day in male and female B6C3F 1 

### mice; a NOAEL value was not identified. In 4

### 82 DRAFT - DO NOT CITE OR QUOTE

males, the LOAEL was based on increased incidences of histopathological changes to the 1 

### duodenum (diffuse epithelial hyperplasia) and mesen teric lymph nodes (histiocytic cellular 2 

### infiltration); in females, the LOAEL was based on i ncreased incidences of histopathological 3 

### changes to the duodenum (diffuse epithelial hyperpl asia), mesenteric lymph nodes (histiocytic 4 

### cellular infiltration), liver (histiocytic cellular infiltration), and pancreas (depletion of 5 

### cytoplasmic zymogen granules). Although mild micro cytic, hypochromic anemia was observed 6



### in female mice at ≥ 0.38 mg hexavalent chromium/kg-day after 22 days of exposure, the severity 7 

### of these effects decreased over time, such that onl y small changes (<5%) were observed at 8



### ≥ 3.1 mg hexavalent chromium/kg-day after 12 months o f exposure; therefore, hematological 9



### effects were not considered as the basis for the ch ronic LOAEL value in female mice. In 10 

### addition to noncancer effects, exposure of B6C3F 1 

### mice to sodium dichromate dihydrate in 11 

### drinking water for 2 years resulted in significant increases in the incidences of neoplasms of the 12



### small intestine in males and females at doses ≥ 2.4 and ≥ 3.1 mg hexavalent chromium/kg-day, 13 

### respectively. NTP (2008) concluded that results of this study provide clear evidence of 14 

### carcinogenic activity of sodium dichromate dihydrate in male and female B 6C3F 1 

### mice based on 15 

### increased incidences of neoplasms of the small inte stine. 16

17 

### Borneff et al., 1968 18 

### Borneff et al. (1968) conducted a long-term animal cancer bioassay of hexavalent 19 

### chromium administered in drinking water. Using a t hree-generation study design, Borneff et al. 20 

### (1968) treated 120 female and 10 male NMRI mice wit h 1 mg potassium chromate/day (500 21 

### ppm) in drinking water (containing 3% household det ergent). A control group of animals 22 

### received drinking water (3% detergent) only. An ou tbreak of mousepox (ectromelia) virus 23 

### occurred during the eighth month of the experiment, and within three months, the majority (512) 24 

### of the animals died. All animals received a mousep ox vaccination two months after the 25 

### outbreak, and this effectively ended the epidemic a nd the study continued. Two carcinomas of 26 

### the stomach were observed in female mice exposed to potassium chromate. No malignant 27



### stomach tumors were found in control mice. Nine be nign stomach tumors were observed in 28 

### female mice exposed to potassium chromate. The com bined incidence of malignant and benign 29 

### stomach tumors (11/66) in potassium chromate-expose d-female mice was significantly different 30



### than the combined incidence of tumors in control fe male mice (2/79) [Fisher’s Exact test, 31 

### p<0.05]. 32

33

### 83 DRAFT - DO NOT CITE OR QUOTE

Anwar et al., 1961 1 

### The effects of chronic oral exposure to hexavalent chromium were evaluated in dogs by 2 

### Anwar et al. (1961). Dogs (one control dog and one to two dogs/treatment group) were exposed 3 

### to potassium chromate in drinking water at concentr ations of 0, 0.45, 2.25, 4.5, 6.75, or 11.2 mg 4 

### hexavalent chromium/L for 4 years. Several differe nt breeds of dogs (German shepherds, 5 

### poodles, and beagles) were used and body weights of animals were not reported; thus, daily 6 

### hexavalent chromium doses cannot be accurately esti mated. Throughout the exposure period, 7 

### animals were evaluated for clinical signs of toxici ty, and food consumption and growth rate were 8 

### recorded (frequency of observations not reported). At monthly intervals, blood was obtained for 9 

### evaluation of hematology (i.e., erythrocyte counts, total and differential leukocyte counts, and 10 

### Hgb), and at 6-month intervals, urine was analyzed for albumin, acetone, bile pigments, glucose, 11 

### erythrocytes, and specific gravity. At the end of the 4-year treatment period, weights of the liver, 12 

### kidney, and spleen were recorded, and microscopic e xamination was conducted on selected 13 

### tissues of major organs. No chromium-related effec ts were observed. Interpretation of the study 14 

### results is limited by the small number of animals e valuated and the inability to estimate daily 15 

### doses of hexavalent chromium received by the treate d animals. A NOAEL or LOAEL could not 16 

### be identified from this study. 17

18 

### MacKenzie et al., 1958 19 

### MacKenzie et al. (1958) conducted two experiments i n which Sprague-Dawley rats were 20 

### administered hexavalent chromium in drinking water for 1 year. In the first experiment, groups 21 

### of rats (10/sex in the control group and 8/sex/grou p in the treatment groups) were exposed to 22 

### drinking water containing potassium chromate at con centrations of 0, 0.45, 2.2, 4.5, 7.7, or 23 

### 11 mg hexavalent chromium/L. In the second experim ent, groups of 12 male and 9 female rats 24 

### were exposed to drinking water containing potassium chromate at concentrations of 0 or 25 mg 25 

### hexavalent chromium/L. For experiment 1, MacKenzie et al. (1958) reported that drinking water 26 

### consumption and body weights in the treatment group s were comparable to controls, although 27 

### data were not reported. Using reference values for body weight (males: 0.523 kg; females: 28 

### 0.338 kg) and daily drinking water intake (males: 0.062 L/day; females: 0.045 L/day) for adult 29 

### male and female Sprague-Dawley rats (U.S. EPA, 1988 ), doses of 0.05, 0.26, 0.53, 0.91, or 30 

### 1.3 mg hexavalent chromium/kg-day for males and 0.0 6, 0.29, 0.60, 1.0, or 1.5 mg hexavalent 31 

### chromium/kg-day for females exposed to drinking wat er containing 0.45, 2.2, 4.5, 7.7, or 11 mg 32 

### hexavalent chromium/L, respectively, were estimated . For experiment 2, drinking water 33

### 84 DRAFT - DO NOT CITE OR QUOTE

consumption was decreased by 16 and 27% in male and female rats, respectively. Thus, using 1 

### reference values for body weight and daily drinking water intake for adult male and female 2 

### Sprague-Dawley rats (listed above; U.S. EPA, 1988) and assuming decreases in water 3 

### consumption of 16 and 27% in males and females, res pectively, average daily doses of 2.8 and 4 

### 2.4 mg hexavalent chromium/kg-day in males and fema les, respectively, were estimated. 5 

### Throughout the treatment period in both experiments , animals were examined for clinical signs 6 

### of toxicity, and weight gain and food and water con sumption were recorded (frequency of 7 

### observations not reported). At monthly intervals, blood was analyzed for Hgb, erythrocyte 8 

### counts, and total and differential leukocyte counts . At the end of treatment, microscopic 9 

### examination of selected tissues (kidney, adrenal gl and, liver, spleen, heart, brain, stomach, 10 

### duodenum, ileum, colon, and bone marrow) was conduc ted (as described by Decker et al., 1958). 11 

### No treatment-related clinical signs of toxicity, ef fects on food consumption, body weight gain, or 12 

### histopathological findings were observed. 13

14



### 4.3. REPRODUCTIVE AND DEVELOPMENTAL TOXICITY STUDI ES—ORAL 15 

### Studies evaluating the potential reproductive effec ts of oral exposure to hexavalent 16 

### chromium compounds have been conducted in monkeys ( Aruldhas et al., 2006, 2005, 2004; 17 

### Subramanian et al., 2006), rats (Bataineh et al., 2 007, 1997; Elsaieed and Nada, 2002; Li et al., 18 

### 2001; Kanojia et al., 1998, 1996; NTP, 1996b; Chowd hury and Mitra, 1995;), mice (Al-Hamood 19 

### et al., 1998; Elbetieha and Al-Hamood, 1997; NTP, 1 997; 1996a; Junaid et al., 1996a, b, 1995; 20 

### Murthy et al., 1996; Zahid et al., 1990; Trivedi et al., 1989), and rabbits (Yousef et al., 2006). In 21 

### addition, several studies have specifically evaluat ed the potential effects of pre-gestational, 22 

### gestational, or lactational exposure on fetal devel opment in rats (Banu et al., 2008; Elsaieed and 23 

### Nada, 2002; Kanojia et al., 1998, 1996) and mice (A l-Hamood et al., 1998; Junaid et al., 1996a, 24 

### b, 1995; Trivedi et al., 1989). Studies conducted by NTP (1997, 1996a, b) and Zahid et al. 25 

### (1990) evaluated dietary exposure; all other studie s have evaluated animals exposed to 26 

### hexavalent chromium in drinking water or by gavage. In general, studies that evaluated 27 

### developmental effects of hexavalent chromium were c onducted at higher exposure levels than 28 

### those that evaluated reproductive effects. 29 

### Collectively, the available studies provide evidenc e that oral exposure of laboratory 30 

### animals to hexavalent chromium compounds produces a dverse reproductive effects, including 31 

### histopathological changes to reproductive organs in males (Aruldhas et al., 2006, 2005, 2004; 32 

### Chowdhury and Mitra, 1995; Li et al., 2001; Zahid e t al., 1990) and females (Murthy et al., 33

### 85 DRAFT - DO NOT CITE OR QUOTE

1996); alterations in sperm, including decreased co unt, decreased motility, and abnormal 1 

### morphology (Subramanian et al., 2006; Yousef et al. , 2006; Li et al., 2001; Zahid et al., 1990); 2 

### decreased plasma testosterone levels (Yousef et al. , 2006; Chowdhury and Mitra, 1995); 3 

### increased estrous cycle length (Kanojia et al., 199 8, 1996; Murthy et al., 1996); changes in 4 

### mating behavior and decreased fertility in males (B ataineh et al., 1997); and adverse 5 

### reproductive outcomes, including decreased numbers of live fetuses and implantations, and 6 

### increased numbers of resorptions and pre- and posti mplantation losses (Bataineh et al., 2007; 7 

### Elsaieed and Nada, 2002; Elbetieha and Al-Hamood, 1 997; Junaid et al., 1996a, b, 1995; Kanojia 8 

### et al., 1998, 1996; Trivedi et al., 1989). Develop mental effects observed have included 9 

### decreased fetal weight and length (Elsaieed and Nad a, 2002; Kanojia et al., 1998; Junaid et al., 10 

### 1996a, b, 1995; Trivedi et al., 1989); external (su bdermal hemorrhage and tail malformations) 11 

### and skeletal abnormalities (decreased ossification) (Elsaieed and Nada, 2002; Junaid et al., 12 

### 1996a, b, 1995; Kanojia et al., 1998, 1996; Trivedi et al., 1989); and delayed sexual maturation 13 

### and function in female offspring (Banu et al., 2008 ; Al-Hamood et al., 1998). In contrast to 14 

### results of the above studies, adverse effects were not observed in dietary exposure studies 15 

### conducted by NTP that investigated the potential fo r hexavalent chromium to produce adverse 16 

### effects on male reproductive organs in rats and mic e (NTP, 1996a, b) and on reproductive 17 

### outcomes in a continuous breeding study in mice (NT P, 1997). 18 

### The following review of available reproductive and developmental studies is organized as 19 

### follows: (1) studies evaluating effects on reprodu ctive tissues and mating behavior; (2) studies 20 

### evaluating effects on reproductive outcomes; (3) st udies evaluating pre-gestational exposure on 21 

### reproductive outcomes and fetal development; and (4 ) studies evaluating gestational and/or 22 

### lactational exposure on reproductive outcomes and f etal development. 23

24



### 4.3.1. Effects on Reproductive Tissues and Mating Behavior 25 

### Aruldhas et al., 2006, 2005, 2004; Subramanian et a l., 2006 26 

### In a series of studies conducted by the same resear ch group, adverse effects on male 27 

### reproductive organs were observed in monkeys expose d to hexavalent chromium in drinking 28 

### water (Aruldhas et al., 2006, 2005, 2004; Subramani an et al., 2006). All of these studies 29 

### followed the same exposure protocol; adult male bon net monkeys (6–8 years old) were exposed 30 

### to drinking water containing 0, 100, 200, or 400 mg potassium dichromate/L (Aruldhas et al., 31 

### 2006, 2005, 2004) or 0, 50, 100, 200, or 400 mg pot assium dichromate/L (equivalent to 0, 17.6, 32 

### 35.3, 70.6, and 141.2 mg hexavalent chromium/L, res pectively) (Subramanian et al., 2006) for 33

### 86 DRAFT - DO NOT CITE OR QUOTE

180 days; two studies included a 180-day post-treat ment recovery period (Aruldhas et al., 2006; 1 

### Subramanian et al., 2006). Aruldhas et al. (2004) noted that 400 mg potassium dichromate/L 2



### was selected as the maximum concentration tested si nce exposure to higher concentrations 3 

### resulted in decreased food and drinking water consu mption and death within 3 months. At the 4 

### beginning of the treatment period, body weights of monkeys were reported as 7–8 kg by 5 

### Aruldhas et al. (2005) and as 7–9 kg by Subramanian et al. (2006). Although body weights were 6 

### not reported by Aruldhas et al. (2006, 2004), it is assumed that initial body weights were similar 7 

### in all studies. The study authors did not report b ody weights or drinking water consumption over 8 

### the course of treatment or calculate daily doses of hexavalent chromium. For this review, daily 9 

### doses of 0, 1.0, 2.1, 4.1, and 8.3 mg hexavalent ch romium/kg-day for the 0, 50, 100, 200, or 400 10 

### potassium dichromate/L groups, respectively, were e stimated using the allometric equation for 11 

### drinking water consumption for primates (0.09 × bod y weight 0.7945 

### ; U.S. EPA, 1988) and an 12 

### average reported initial body weight of 8 kg (Subra manian et al., 2006; Aruldhas et al., 2005); 13 

### however, these dose estimates are uncertain due to the absence of data on body weight and 14 

### drinking water consumption over the course of the 6 -month treatment period. In the following 15 

### discussions, the three treatment groups evaluated i n the Aruldhas et al. (2006, 2005, 2004) 16 

### studies (i.e., 100, 200, or 400 mg potassium dichro mate/L, approximately equivalent to 2.1, 4.1, 17 

### or 8.3 mg hexavalent chromium/kg-day, respectively) are referred to as the low-, mid- and high18 

### dose groups, respectively; the four treatment group s evaluated in the Subramanian et al. (2006) 19 

### study (i.e., 50, 100, 200, or 400 mg potassium dich romate/L, approximately equivalent to 1.0, 20 

### 2.1, 4.1, or 8.3 mg hexavalent chromium/kg-day, res pectively) are referred to as the lowest-, 21 

### low-, mid-, and high-dose groups, respectively. 22 

### Aruldhas et al. (2004) conducted histological asses sments of testes and epididymides 23 

### from monkeys (three monkeys/group) following 180 da ys of treatment. Testes and epididymides 24 

### were evaluated by light microscopy (resin-embedded slices) and transmission electron 25 

### microscopy (TEM). In the three treatment groups, e pididymal damage and the development of 26 

### microcanals in the cauda epididymal epithelium were observed; severity of ductal damage 27 

### increased with dose. In the low-dose group, the ca uda epididymal epithelium appeared 28 

### pseudostratified; degeneration of principal cells a nd epithelial rupture, with the lumen occluded 29 

### by principal cells, were observed. In the mid-dose group, the occluded lumen appeared packed 30 

### with immature germ cells and macrophages. In the h igh-dose group, hypertrophy of the caudal 31 

### epithelium and “obliteration” of the ductal lumen w ere observed. The development of two 32 

### morphologically distinct microcanals was observed i n all treatment groups. Arulhhas et al. 33

### 87 DRAFT - DO NOT CITE OR QUOTE

(2004) proposed that microcanal development was an adaptive response to provide passage for 1 

### spermatozoa around the obstructed ducts and to entr ap spermatozoa that had been released into 2 

### the epithelium due to the epithelial rupture. Appe arance of tissues from the control group was 3 

### not reported. Additional TEM evaluations of testes from monkeys (three monkeys/group) in the 4 

### three hexavalent chromium treatment groups showed a dose-related accumulation of basal cells 5 

### along the basal lamina of the epididymis, giving th e epithelium a pseudostratified appearance, 6 

### and intraepithelial macrophages (Aruldhas et al., 2 006). In addition, cells showed an 7 

### accumulation of sperm-derived lipofuscin material, indicative of phagocytosis and processing of 8 

### sperm. In contrast, these findings were not observ ed in testes from control monkeys. 9 

### Aruldhas et al. (2005) evaluated the effects of hex avalent chromium exposure in male 10 

### monkeys at the completion of the 180-day treatment period (three monkeys/group) and following 11 

### an additional 180-day recovery period (three monkey s/group); assessments included plasma 12 

### chromium concentration, absolute and relative testi cular weights, and microscopic (light and 13 

### TEM) evaluations of testes. At the end of the trea tment period, chromium plasma concentration 14 

### was significantly (

p < 0.05) increased in the three treatment groups, w ith increases reaching 15 

### almost ninefold in the high-dose group compared to controls. Relative testicular weight was 16 

### significantly (

p < 0.05) decreased by 23, 35, and 34% in the low-, mid-, and high-dose groups, 17 

### respectively; absolute testicular weight was not af fected by treatment (data not reported). 18 

### Following the recovery period, chromium plasma conc entrations and relative testicular weight in 19 

### treatment groups were comparable to controls. Ligh t microscopic evaluations of testes in control 20 

### monkeys showed seminiferous tubules and Leydig cell s with normal appearance and cellular 21 

### organization. In the three hexavalent chromium tre atment groups, seminiferous tubules appeared 22 

### disorganized, with decreased diameters, epithelial degeneration, and lumens filled with 23 

### prematurely released germ cells and cellular debris ; depletion of germ cells, hyperplasia of 24 

### Leydig cells, and Sertoli cell fibrosis were also o bserved. TEM examination of testes from the 25 

### three treatment groups showed morphological changes in spermatids (granulation of chromatin 26 

### and vacuolization) and spermatocytes (fragmented ch romatin and swollen mitochondria) and the 27 

### presence of macrophages containing phagocytosed spe rm; effects were more severe in the high28 

### dose group. Following the recovery period, no hist opathological findings were observed in 29 

### testes of hexavalent chromium-treated monkeys, with the exception of “a few” prematurely 30 

### released germ cells in the seminiferous tubular lum en (treatment group for this observations was 31 

### not specified). 32

### 88 DRAFT - DO NOT CITE OR QUOTE

Subramanian et al. (2006) evaluated sperm count and sperm straight-line velocity 1 

### at monthly intervals during the 180-day treatment p eriod; the same evaluations were 2 

### conducted monthly in monkeys in the high-dose group during a 180-day recovery period. In the 3 

### lowest-dose group, no effects were observed on sper m count or straight-line velocity. Sperm 4 

### count was significantly decreased in the low-, mid- , and high-dose groups, compared with 5 

### controls; decreases were dose- and duration-depende nt. For example, in the low-dose group, 6 

### significant (

p < 0.05) decreases in sperm count were first observe d after 4 months (11% 7 

### decrease), with a maximum decrease of 25% after 6 m onths; in the high-dose group, sperm 8 

### counts were significantly decreased by 13% after 2 months, with a 30% reduction after 6 months. 9 

### Similar effects were observed for sperm straight-li ne velocity. In the low-dose group, velocity 10 

### was significantly (

p < 0.05) decreased by 10 and 25% after 4 months and 6 months of treatment, 11 

### respectively; in the high-dose group, velocity was significantly decreased by 12% after 2 months 12 

### and by 35% after 6 months. Effects on sperm count and straight-line velocity were reversible 13 

### following withdrawal from treatment. During the fi rst month of the recovery period (high-dose 14 

### monkeys only), sperm count was significantly increa sed compared with that observed at the end 15 

### of the treatment period, with counts returning to p re-treatment levels by month 3 of the recovery 16 

### period; sperm velocity returned to pre-treatment le vels by month 3 of the recovery period. 17 

### Results of these four studies (Aruldhas et al., 200 6, 2005, 2004; Subramanian et al., 18 

### 2006) indicate that exposure of monkeys to hexavale nt chromium as potassium dichromate in 19 

### drinking water produced reversible changes to male reproductive organs, including disruption of 20 

### spermatogenesis. Effects on sperm count and veloci ty and histopathological changes were 21 

### observed in the low-, mid-, and high-dose groups ( ≥ 2.1 mg hexavalent chromium/kg-day), but no 22 

### effects on sperm count and velocity were observed i n monkeys in the lowest treatment group 23 

### (1.0 mg hexavalent chromium/kg-day). However, this dose cannot be considered a NOAEL 24 

### value because microscopic evaluations were not cond ucted in monkeys from this group. 25 

### Although group sizes in these studies were small, r esults provide evidence of adverse male 26 

### reproductive effects in nonhuman primates exposed t o hexavalent chromium in drinking water at 27 

### concentrations as low as 35.3 mg hexavalent chromiu m/L (2.1 mg hexavalent chromium/kg28 

### day). 29

30 

### Chowdhury and Mitra, 1995 31 

### Effects of oral exposure to hexavalent chromium on male reproductive organs was 32 

### evaluated in mature (age not reported) male Charles Foster rats that were administered 0, 20, 40, 33

### 89 DRAFT - DO NOT CITE OR QUOTE

or 60 mg hexavalent chromium/kg-day as sodium dichr omate in saline by gavage for 90 days 1 

### (Chowdhury and Mitra, 1995). Although Chowdhury an d Mitra (1995) stated that the control 2 

### and exposure groups included 10 animals per group, conflicting summaries of the actual group 3 

### sizes are presented in the report. Body weights we re recorded twice weekly. At the end of the 4 

### treatment period, testes were excised, weighed, and prepared for histological or biochemical 5 

### evaluations, and serum testosterone activity was de termined. For biochemical analyses, fresh 6 

### tissue was homogenized and assayed for total choles terol, activities of succinic dehydrogenase 7



### and 3 β

- ∆ 5 

### -hydroxysteroid dehydrogenase (3 β

- ∆ 5 

### -HSH), and total protein, DNA, and RNA. For 8 

### microscopic evaluations, testes were fixed in Bouin ’s fluid, embedded in paraffin, and stained 9 

### with haematoxylin and eosin (H&E). 10 

### Final body weight was significantly reduced by appr oximately 27% compared to controls 11 

### in the mid- and high-dose groups (statistical signi ficance not reported); absolute testis weights 12 

### were significantly reduced by 28% ( p < 0.05) and 35% (

p < 0.001) in the mid- and high-dose 13 

### groups, respectively, compared with controls. Seru m testosterone levels were decreased by 31% 14 

### in the low- (

p < 0.05) and mid-dose (

p < 0.001) groups and by 47% ( p < 0.001) in the high-dose 15 

### group. Biochemical analysis of testes showed signi ficant decreases in total cholesterol by 2% ( p 16 

### < 0.05) and 25% (

p < 0.001) in the mid- and high-dose groups, respecti vely, and significant ( p < 17 

### 0.001) decreases in succinic dehydrogenase activity by 35 and 45% in the mid- and high-dose 18 

### groups, respectively. In all treatment groups, 3 β

- ∆ 5 

### -HSH was significantly decreased by 25% ( p 19 

### < 0.05), 28% (

p < 0.05), and 52% (

p < 0.001) in the low-, mid-, and high-dose groups, 20 

### respectively. Dose-related decreases in total test icular protein were observed, with decreases 21 

### reaching 46% (

p < 0.001) in the high-dose group. Testicular DNA an d RNA levels were 22 

### significantly decreased in the mid- and high-dose g roups, with decreases reaching 45% ( p < 23 

### 0.001) and 37% (

p < 0.001), respectively, in the high-dose group. Mi croscopic evaluation of 24



### testicular tissue showed adverse effects in the mid - and high-dose groups including disintegration 25 

### of peritubular membranes, detachment of seminiferou s cellular components from basement 26 

### membranes, and accumulation of cellular debris in t he mid-dose group, and cellular degeneration 27 

### and complete disruption of the epithelium with fibr ous tissue in the high-dose group; reduction in 28 

### seminiferous tubular diameter, decreased number of Leydig cells, and Leydig cell degeneration 29 

### were observed in the mid- and high-dose groups. No change in the number of spermatogonia 30 

### were observed, although the number of pachytene spe matocytes and stage 7 spermatids were 31 

### decreased in the mid- and high-dose groups and rest ing spermatocytes were decreased in the 32 

### high-dose group. No treatment-related histopatholo gical effects were observed in the testes of 33

### 90 DRAFT - DO NOT CITE OR QUOTE

rats in the low-dose group, although histochemical evaluations of testes showed dose-related loss 1



### of 3 β

- ∆ 5 

### -HSH activity in all treatment groups. 2 

### Results of histological and biochemical analyses sh ow that oral exposure of male rats to 3 

### hexavalent chromium for 90 days produced adverse ef fects on male reproductive tissues, 4 

### including decreased spermatogenic and steroidogenic activities. Based on decreased serum 5



### testosterone levels and loss of 3 β

- ∆ 5 

### -HSH activity in testes observed in all treatment g roups, a 6 

### LOAEL of 20 mg hexavalent chromium/kg-day was ident ified in this gavage study of male 7 

### Charles Foster rats. A LOAEL of 40 mg hexavalent c hromium/kg-day was identified for 8 

### degenerative changes in the testes detected by micr oscopy. 9

10 

### Bataineh et al., 1997 11



### Effects of oral hexavalent chromium administration on mating behavior, aggression, and 12 

### fertility were assessed in male rats by Bataineh et al. (1997). Adult (age not specified) male 13 

### Sprague-Dawley rats (n = 12 or 13) were administere d drinking water containing 0 or 1,000 mg 14 

### potassium dichromate/L (equivalent to 353 mg hexava lent chromium/L) for 12 weeks. No data 15 

### on drinking water consumption were included in the study report. Based on findings of other 16 

### studies (NTP, 2008, 2007) showing decreased drinkin g water consumption and body weight at 17 

### drinking water concentrations ≥ 30 mg hexavalent chromium/L, it is likely that drin king water 18 

### consumption was decreased in the chromium treatment group; thus, daily doses of hexavalent 19 

### chromium cannot be accurately estimated from this s tudy. Following the treatment period, 20



### assessments were conducted for sexual behavior in t he presence of females in estrous (number of 21 

### mounts without penile intromission, time to first m ount, time from presentation of female to first 22 

### intromission, number of penile intromissions, time from first intromission to ejaculation, and 23 

### time from ejaculation to next intromission), aggres sive behavior in the presence of a second 24 

### untreated male (number of lacerations given, boxing bouts, fights, and ventral presenting), 25 

### fertility following a 10-day mating period with unt reated females (numbers of pregnant females, 26 

### viable fetuses, and resorptions), body weight, and weights of reproductive organs (paired testes, 27 

### seminal vesicles, and preputial glands). Histopath ological evaluations of tissues were not 28



### conducted. 29 

### All rats “appeared healthy” throughout the treatmen t period. Assessment of mating 30



### behavior in hexavalent chromium-treated rats showed significant decreases in number of mounts 31 

### (35% decrease; p < 0.001) and percentage of males ejaculating (79% decreases; p < 0.005), and 32 

### increases in the time from first intromission to ej aculation (59% increase; p < 0.001) and time 33

### 91 DRAFT - DO NOT CITE OR QUOTE

from ejaculation to next intromission (37% increase s, p < 0.001), compared with controls. All 1 

### measures of aggressive behavior were decreased in r ats treated with potassium dichromate. All 2 

### measures of fertility were comparable between contr ol and treatment groups. Treatment resulted 3 

### in significant (

p < 0.001) decreases in body weight (19% decrease) a nd absolute weights of testes 4 

### (24% decrease), seminal vesicles (15% decrease), an d preputial gland (23% decrease); however, 5 

### for relative weights of reproductive tissues, only relative testes weight was significantly 6 

### decreased (6% decrease, p < 0.05) compared to controls. 7 

### This study identified a LOAEL of 535 mg hexavalent chromium/L as potassium 8 

### dichromate in drinking water based on adverse effec ts on mating and aggressive behaviors; a 9 

### NOAEL was not identified. Because drinking water c onsumption and body weight data over the 10 

### course of the study was not provided, a LOAEL, expr essed in mg hexavalent chromium/kg-day, 11 

### could not be derived from this study. 12

13 

### Li et al., 2001 14 

### Oral exposure of male rats to chromium(VI) oxide fo r 6 days resulted in adverse 15 

### reproductive effects, including reduced epididymal sperm counts and increased abnormal sperm 16 

### (Li et al., 2001). Groups of 8–11 male Wistar rats (60 days old) were administered 17 

### chromium(VI) oxide by gavage at doses of 0, 10, or 20 mg chromium(VI) oxide/kg-day 18 

### (equivalent to 0, 5.2 or 10.4 mg hexavalent chromiu m/kg-day, respectively) for 6 days. After 19 

### 6 weeks, rats were sacrificed; testes and epididymi s were removed and analyzed for epididymal 20 

### sperm count and abnormal sperm; and testes were pre pared (fixed in formaldehyde, embedded in 21 

### paraffin, sliced, and stained with H&E) for histolo gical evaluations of morphological 22



### abnormalities and diameter of seminiferous tubules. Epididymal sperm counts were significantly 23 

### ( p < 0.05) decreased by 76 and 80%, and the percentage of abnormal sperm was significantly 24 

### ( p < 0.01) increased by 143 and 176% in the 5.2 and 10 .4 mg hexavalent chromium/kg-day 25 

### groups, respectively. Treatment-related histopatho logical findings included decreased diameter 26 

### of seminiferous tubules and disruption of germ cell arrangement within seminiferous tubules in 27 

### both treatment groups. Based on decreased sperm co unts and histopathological changes to the 28 

### testes, 5.2 mg hexavalent chromium/kg-day was ident ified as a LOAEL for male rats exposed to 29 

### gavage doses of chromium(VI) oxide for 6 days; a NO AEL value was not identified. 30

31 

### Zahid et al., 1990 32 

### Zahid et al. (1990) reported adverse effects on the male reproductive system in mice fed 33

### 92 DRAFT - DO NOT CITE OR QUOTE

diets containing potassium dichromate. However, ot her research groups (NTP 1997, 1996a,b; 1 

### Finley et al., 1993) have questioned the validity o f the Zahid et al. (1990) study due to concerns 2 

### regarding study methods and reporting inconsistenci es (as discussed below). Zahid et al. (1990) 3 

### fed male weanling BALB/c albino Swiss mice diets co ntaining 0, 100, 200 or 400 mg potassium 4 

### dichromate/kg diet (equivalent to 0, 35.3, 70.6, or 141.2 mg hexavalent chromium/kg diet, 5 

### respectively) for 35 days. Although Zahid et al. ( 1990) stated that the control and exposed 6 

### groups included seven animals/group, conflicting su mmaries of the actual group sizes are 7 

### presented throughout the report. Body weights were recorded weekly and food consumption was 8 

### recorded every 48 hours. The study report stated t hat body weight gain and food consumption in 9 

### treatment groups were comparable to the control gro up (data not reported); however, Zahid et al. 10 

### (1990) did not calculate daily doses of hexavalent chromium. Since treatment did not affect 11 

### body weight gain or food consumption, doses of 0, 6 .4, 12.7, or 25.5 mg hexavalent 12 

### chromium/kg-day for the 0, 35.3, 70.6, or 141.2 mg hexavalent chromium/kg diet groups, 13 

### respectively, were estimated for this review using reference values for body weight (0.0316 kg) 14 

### and daily food intake (0.0057 kg food/day) for subc hronic exposure of male B6C3F 1 

### mice (U.S. 15 

### EPA, 1988). After 35 days, testes and epididymis w ere weighed, and then minced in buffered 16 

### formalin. Sperm counts were then subsequently dete rmined and sperm were examined for 17 

### morphological abnormalities. Testes were fixed wit h Bouin’s fluid for 1 week, embedded in 18 

### paraffin and were subsequently sectioned to 0.6 mic ron thickness and stained with H&E for 19 

### histological examination. Ten sections were chosen randomly from the anterior, middle, and 20 

### posterior parts of each testis and studied. One se miniferous tubule was chosen and examined to 21 

### determine the cellular stages of spermatogenesis an d the number of degenerated tubules. 22 

### Statistical analyses of the data were conducted usi ng either a t -test or a 2 × 2 contingency chi23 

### square test. Adverse effects observed in the male mouse testes included ambiguous levels of 24 

### degeneration in the outermost cellular layers of th e seminiferous tubules, reduced (or absent) 25 

### spermatogonia per tubule, accumulation of germ cell s in the resting spermatocytes stage, reduced 26 

### sperm count in the epididymis, and increased percen tage of morphologically abnormal sperm. 27 

### Effects were observed in all hexavalent chromium gr oups and severity of effects appeared to 28 

### increase with dose for percentage of degenerated tu bules, percentage of tubules that were not 29 

### degenerated but were without spermatogonia, percent age of abnormal sperm, and number of 30 

### spermatogonia. Based on these findings, the lowest dietary concentration tested (100 mg 31 

### potassium dichromate/kg diet or approximately 6.4 m g hexavalent chromium/kg-day) was 32



### identified as the LOAEL. 33

### 93 DRAFT - DO NOT CITE OR QUOTE

Other research groups (NTP, 1997, 1996a,b; Finley e t al., 1993) have questioned the 1 

### validity of the Zahid et al. (1990) study due to co ncerns regarding study design and methods. 2 

### Finley et al. (1993) noted the following three conc erns: 1) use of immersion fixatives (such as 3 

### Bouin’s fluid and paraffin embedding) that can intr oduce artifacts, such as grains and shrinkage, 4 

### that can mimic tubular or spermatogenic pathology; 2) use of staining methods that were unable 5 

### to detect the acrosome (i.e., the part of the sperm that releases enzymes to penetrate the egg) of 6 

### developing spermatids; and 3) uncertainties regardi ng the actual groupings of animals used, the 7 

### small number of animals assessed per group, and ina ppropriate statistical analysis of the data. 8 

### NTP (1997, 1996a, b) concluded that the methods uti lized by Zahid et al. (1990) were 9 

### insufficient to identify spermatogonia, were likely to have generated nonreproducible counts of 10 

### epididymal sperm, and resulted in the biologically implausible conclusion of reduction in 11 

### spermatogonia numbers concurrent with unchanged spe rmatocyte and spermatid numbers. 12

13 

### Murthy et al., 1996 14 

### Effects on ovarian function were investigated in ad ult Swiss albino mice (age: 90 days; 15 

### mean initial body weight: 30 g) exposed to drinking water containing potassium dichromate for 16 

### 20 or 90 days (Murthy et al., 1996). For the 20-da y study, groups of 30 female mice were 17 

### exposed to drinking water containing 0, 250, 500, o r 750 mg hexavalent chromium/L; the 20-day 18 

### exposure period was selected as it coincides with o ne folliculogenesis cycle. For the 90-day 19 

### study, groups of 10 female mice were administered d rinking water containing 0, 0.05, 0.5, or 20 

### 5 mg hexavalent chromium/L. The study report state s mice in both studies were evaluated daily 21 

### for clinical signs of toxicity, body weight, and wa ter and food consumption; however, no data for 22 

### these outcomes were reported. Based on findings of other studies (NTP, 2008, 2007) showing 23 

### decreased drinking water consumption and body weigh t at drinking water concentrations ≥ 30 mg 24 

### hexavalent chromium/L, it is likely that drinking w ater consumption and body weight were 25 

### decreased in all treatment groups in the 20-day stu dy; thus, daily doses of hexavalent chromium 26 

### cannot be accurately estimated from this study. Fo r the 90-day study, the concentrations of 27 

### hexavalent chromium in drinking water were very low and not likely to affect drinking water 28 

### consumption or body weight. Thus, using reference values for body weight (0.035 kg) and daily 29 

### drinking water (0.0084 L/day) intake for mature fem ale B6C3F 1 

### mice (U.S. EPA, 1988), doses 30 

### of 0, 0.01, 0.12, or 1.2 mg hexavalent chromium/kg- day were estimated for female mice exposed 31 

### to drinking water containing 0, 0.05, 0.5 or 5 mg h exavalent chromium/L, respectively. In the 32 

### 20-day study, three types of assessments were condu cted at the end of the treatment period (each 33

### 94 DRAFT - DO NOT CITE OR QUOTE

in 10 mice/group): 1) ovaries were evaluated by lig ht microscopy and the number of follicles at 1 

### each development stage, based on size (small, mediu m, large) and structural maturity, were 2 

### determined; 2) superovulation was induced (by admin istration of gonadotropin) and the number 3 

### of released ova were counted; and 3) estrous cycle length was assessed (by vaginal smears) for 4 

### 12 consecutive estrous cycles following treatment. In the 90-day study, all mice were sacrificed 5 

### at the end of the treatment period and ovaries were evaluated by electron microscopy for 6 

### ultrastructural changes. 7 

### In mice exposed for 20 days, significant ( p < 0.05) changes in follicular development 8 

### were observed in all treatment groups, with dose-re lated decreases in the number of small 9 

### follicles in the mid- and high-dose groups and medi um and large follicles in all treatment groups. 10 

### In the high-dose group, the numbers of small, mediu m, and large follicles were reduced by 36, 11 

### 53, and 72%, respectively, compared with controls. Ovarian response to gonadotropin was 12 

### affected in the mid- and high-dose groups, with red uctions in the number of ova released of 30 13 

### and 90%, respectively, compared with controls. Est rous cycle length was significantly increased 14 

### ( p < 0.05) by 1.7-fold in the high-dose group, compare d with controls. Histopathological 15 

### evaluation of ovaries after 20 days of treatment sh owed changes in the mid-dose (i.e., 16 

### proliferated, dilated, and congested blood vessels, pyknotic nuclei in follicular cell of mature 17 

### follicles) and high-dose (i.e., undeveloped follicl es with degenerative cumulus cells containing 18 

### dense pyknotic nuclei, neovasculiarization and kary orrhexis of follicular cells, erythrocytes 19 

### located within stromal spaces) groups; histopatholo gical changes were not observed in ovaries 20



### from control and low-dose mice. In mice treated fo r 90 days, ultrastructural changes (i.e., 21 

### disintegrated cell membranes in two-layered follicu lar cells and alterations in mitochondria in 22 

### thecal cells, which are cells of the corpus luteum that secrete estrone, estradiol, and 23 

### progesterone) were observed in the high-dose group; the study report did not provide any 24



### information on ultrastructural evaluations in the l ow- and mid-dose groups. Murthy et al. (1996) 25 

### concluded that hexavalent chromium may induce chang es in ovarian function and ovulation. 26 

### Due to inadequate reporting (i.e., no information o n effects of treatment on body weight or 27 

### drinking water consumption), a LOAEL from this stud y coulc not be identified. 28

29 

### Yousef et al., 2006 30 

### Adverse effects on male reproductive tissues were o bserved in rabbits exposed to 31 

### potassium dichromate for 10 weeks (Yousef et al., 2 006). Groups of six male New Zealand 32 

### white rabbits (age: 7 months) were administered 0 o r 5 mg potassium dichromate/kg-day by 33

### 95 DRAFT - DO NOT CITE OR QUOTE

gavage (vehicle not specified) for 10 weeks. Youse f et al. (2006) reported that the dose of 5 mg 1 

### potassium dichromate/kg-day was equivalent to 3.6 m g hexavalent chromium/kg-day. During 2 

### the treatment period, food intake and body weights were recorded weekly. Semen was collected 3 

### weekly and analyzed for pH and sperm count, motilit y, and morphology. Blood was collected 4 

### every 2 weeks and analyzed for testosterone. At th e end of the treatment period, animals were 5 

### sacrificed and relative testes and epididymis weigh ts were determined. At sacrifice, seminal 6 

### plasma was collected and analyzed for AST, ALT, AP, AcP, and GST activities. 7 

### Histopathological evaluations of tissues were not c onducted. 8 

### No clinical signs of toxicity were observed through out the study. Mean body weight over 9 

### the 10-week treatment period was significantly ( p < 0.05) decreased by 9% compared to controls, 10 

### although average food intake over the 10-week perio d was not affected by treatment; final body 11 

### weight was not reported. After treatment for 10 we eks, relative testes and epididymis weights 12 

### were significantly decreased by 22% ( p < 0.05). The 10-week mean plasma testosterone leve l in 13 

### treated rabbits was decreased by 21% ( p < 0.05) compared with controls. In hexavalent 14 

### chromium-treated rabbits compared with controls, me an values of the following sperm-related 15 

### characteristics were significantly ( p < 0.05) decreased after 10 weeks: 1) packed sperm v olume 16 

### (10% decrease), 2) sperm concentration (18% decreas e), 3) total sperm output (26% decrease), 4) 17 

### sperm motility (5% decrease), 5) total motile sperm per ejaculation (34% decrease), 6) total 18 

### functional sperm fraction (37% decrease), and 7) no rmal sperm (4% decrease). Both percentage 19 

### of dead sperm (24% increase) and seminal fluid pH ( 4% increase) were increased; no effect was 20 

### observed on semen ejaculate volume. Seminal fluid activities of GST, AST and AcP were 21 

### significantly (

p < 0.05) decreased at the end of the treatment perio d, although decreases were 22 

### small ( ≤ 12%) compared with controls. 23 

### The results indicate that exposure of rabbits to or al potassium dichromate gavage doses 24 

### of 3.6 mg hexavalent chromium/kg-day for 10 weeks p roduced adverse effects on male 25 

### reproductive tissues including decreased testes and epididymis weight and decreased sperm 26 

### output. Thus, a LOAEL for hexavalent chromium of 3 .6 mg/kg-day can be identified from this 27 

### study. 28

29

### 96 DRAFT - DO NOT CITE OR QUOTE

NTP, 1996a,b 1 

### The NTP conducted studies to investigate the potent ial effects of dietary hexavalent 2 

### chromium as potassium dichromate on male reproducti ve organs in Sprague-Dawley rats (NTP, 3 

### 1996b) and BALB/c mice (NTP, 1996a). The NTP studi es were designed to replicate the Zahid 4 

### et al. (1990) study (described above) and thereby p rovide data to either refute or confirm findings 5 

### of adverse male reproductive effects. 6 

### Groups of 24 male and 48 female Sprague-Dawley rats were exposed to diets containing 7 

### 0, 15, 50, 100, or 400 mg potassium dichromate/kg d iet (equivalent to 0, 5.3, 17.6, 35.3, or 8 

### 141.2 mg hexavalent chromium/kg diet, respectively) daily for 9 weeks followed by an 8-week 9 

### recovery period (NTP, 1996b). Based on food consum ption measured during the 9-week 10 

### treatment period, NTP (1996a,b) calculated average daily doses of 0, 1, 3, 6, or 24 mg potassium 11 

### dichromate/kg-day (equivalent to 0, 0.35, 1.1, 2.1, or 8.5 mg hexavalent chromium/kg-day, 12 

### respectively) in males and 0, 1, 3, 7, or 28 mg hex avalent chromium/kg-day (equivalent to 0, 13 

### 0.35, 1.1, 2.5, or 9.9 mg hexavalent chromium/kg-da y, respectively) in females for the 0, 15, 50, 14 

### 100, or 400 mg potassium dichromate/kg diet groups, respectively. Animals were examined 15 

### twice daily for mortality and clinical signs of tox icity. Physical examinations and measurement 16 

### of body weight and food and water consumption were conducted weekly. After 3, 6, or 9 weeks 17 

### of treatment or after the full recovery period, 6 m ales and 12 females were sacrificed; necropsies 18 

### were performed; blood was obtained for hematology ( i.e., Hgb, Hct, MCV, MCH, MCHC, mean 19 

### platelet volume, and erythrocyte, leukocyte and pla telet counts); organ weights (not specified, 20 

### but including right and left testes) were recorded; microscopic examinations were conducted on 21 

### liver, kidney, ovary, and testes (testes and epidid ymis were examined for Sertoli nuclei and 22 

### preleptotene spermatocyte counts in Stage X or XI t ubules); and sperm were collected analyzed 23



### for chromatin structure. 24 

### No mortalities or treatment-related clinical signs of toxicity were observed in rats in any 25 

### treatment group (NTP, 1996b). Body weights and foo d and drinking water consumption were 26 

### comparable between controls and treatment groups. Results of hematological analyses showed a 27 

### slight erythrocyte microcytosis in the highest dose group, as indicated by small, but significant, 28 

### decreases in MCV in females exposed for 3 weeks (3% decrease; p < 0.05) and in males exposed 29 

### for 9 weeks (6% decrease; p < 0.05), compared with controls; at 9 weeks, MCV in females was 30 

### decreased by 3%, but the change was not statistical ly significant. No changes in MCV were 31 

### observed in rats exposed for 6 weeks or at the end of the 8-week recovery period. After 9 weeks 32 

### of treatment, MCH was decreased by approximately 6% in males and females (statistical 33

### 97 DRAFT - DO NOT CITE OR QUOTE

significance not reported). No treatment-related f indings were observed on necropsy or on 1 

### microscopic examination of the liver, kidney, ovary , testes, epididymis, or sperm. In conclusion, 2 

### no adverse effects on reproductive organs were obse rved in male or female rats exposed to 3 

### dietary potassium dichromate at doses of 8.5 and 9. 5 mg hexavalent chromium/kg-day, 4 

### respectively, for up to 9 weeks. Based on slight e rythrocyte microcytosis, the results indicate 5 

### respective NOAELs and LOAELs of 2.1 and 8.5 mg hexa valent chromium/kg-day in male 6 

### Sprague-Dawley rats, and 2.5 and 9.5 mg hexavalent chromium/kg-day in females. 7 

### Groups of 24 male and 48 female BALB/c mice were ex posed to diets containing 0, 15, 8 

### 50, 100, or 400 mg potassium dichromate/kg diet (eq uivalent to 0, 5.3, 17.6, 35.3, or 141.2 mg 9 

### hexavalent chromium/kg diet, respectively) daily fo r 9 weeks followed by an 8-week recovery 10 

### period (NTP, 1996a). Based on food consumption mea sured during the 9-week treatment period, 11 

### the study authors calculated average daily doses of 0, 3, 10, 21, or 92 mg potassium 12 

### dichromate/kg-day (equivalent to 0, 1.1, 3.5, 7.4, or 32.5 mg hexavalent chromium/kg-day, 13 

### respectively) in males and 0, 5, 16, 34, or 137 mg hexavalent chromium/kg-day (equivalent to 0, 14 

### 1.8, 5.6, 12.0, or 48.4 mg hexavalent chromium/kg-d ay, respectively) in females for the 0, 15, 15 

### 50, 100, or 400 mg potassium dichromate/kg diet gro ups, respectively. This study followed the 16 

### same protocol and conducted the same evaluations as described in the NTP (1996b) study in rats 17 

### (described above). 18 

### Mortalities occurred in five male mice, but they we re deemed not related to treatment, 19 

### and no treatment-related findings were observed on necropsy. The number of deaths were one, 20 

### one, two, one, and none in the control through high -dose male groups, respectively. All females 21 

### survived to study completion. No treatment-related clinical signs of toxicity were observed. At 22 

### most weekly evaluations, body weight was decreased by 5–9% in males in the highest dose 23 

### group and by 2–4% in females in the two highest dos e groups (statistical significance not 24 

### reported); body weights in these groups remained de pressed during the post-treatment recovery 25 

### period in high-dose males and in females at 12.0 mg hexavalent chromium/kg-day (but not high26 

### dose females). Feed consumption was generally incr eased (5–34%, relative to controls) in all 27 

### treatment groups in males, although changes were no t statistically significant; in females, feed 28 

### consumption was increased in all dose groups (1–37% ), with changes of statistical significance 29 

### in most dose groups during treatment weeks 5 and 6. Water consumption in males and females 30 

### was decreased through the first 3 weeks of treatmen t and comparable to controls for the 31 

### remainder of exposure. Hematological analyses show ed a slight erythrocyte microcytosis. In 32 

### high-dose male and female mice, MCV was decreased b y 2–4% ( p < 0.05) at weeks 3, 6, and 9; 33

### 98 DRAFT - DO NOT CITE OR QUOTE

MCV was also slightly decreased (<2%) at 12.0 mg he xavalent chromium/kg-day in females at 1 

### 6 weeks. Changes in MCV were generally accompanied by small decreases in MCH. At the end 2 

### of the recovery period, a small increase in MCV (2. 8%; p < 0.05) was observed in males; in 3 

### females, MCV in all treatment groups was comparable to controls. No other effects on 4 

### hematological parameters were observed. Microscopi c evaluations revealed a treatment-related 5 

### increase in the incidence of cytoplasmic vacuolizat ion of hepatocytes in male and female mice at 6 

### the end of the 9-week treatment period. Vacuoles w ere demarked and appeared small and clear; 7 

### NTP (1996a) noted that vacuoles were consistent wit h lipid accumulation. Incidences of hepatic 8 

### cytoplasmic vacuolization in the control through hi gh-dose groups were 0/6, 0/6, 1/6, 2/6, and 9 

### 2/5 in males and 1/12, 0/12, 3/12, 2/12, and 4/12 i n females, respectively; lesion severity and 10 

### statistical significance were not reported. No oth er treatment-related histopathological findings 11



### were observed. 12 

### In conclusion, no adverse effects on reproductive o rgans were observed in male or female 13 

### mice exposed to dietary potassium dichromate at dos es up to 32.5 and 48.4 mg hexavalent 14 

### chromium/kg-day, respectively, for 9 weeks. Based on histopathological changes to the liver 15 

### (cytoplasmic vacuolization), the results indicate r espective NOAELs and LOAELs of 3.5 and 16 

### 7.4 mg hexavalent chromium/kg-day in male BALB/c mi ce and 1.8 and 5.6 mg hexavalent 17 

### chromium/kg-day in female mice. 18

19



### 4.3.2. Effects on Reproductive Outcomes 20 

### Elbetieha and Al-Hamood, 1997 21 

### Reproductive effects of drinking water containing 1 ,000–5,000 mg potassium 22 

### dichromate/L (equivalent to 353–1,765 mg hexavalent chromium/L) were evaluated in Swiss 23 

### mice in a series of three experiments (Elbetieha an d Al-Hamood, 1997). No data on drinking 24 

### water consumption were included in the study report . Based on findings of other studies (NTP, 25 

### 2008, 2007) showing decreased drinking water consum ption and body weight at drinking water 26



### concentrations ≥ 30 mg hexavalent chromium/L, it is likely that drin king water consumption was 27 

### decreased in all chromium treatment groups; thus, d aily doses of hexavalent chromium cannot be 28 

### accurately estimated for this study. In the first experiment, sexually mature (age: 50 days) male 29 

### Swiss mice were exposed to drinking water containin g 0 (20 males), 1,000 (19 males), 30 

### 2,000 (11 males), 4,000 (9 males), or 5,000 (13 mal es) mg potassium dichromate/L (equivalent 31 

### to 0, 353, 706, 1,412, or 1,765 mg hexavalent chrom ium/L, respectively) for 12 weeks. After 32 

### 12 weeks, males were mated with untreated sexually mature females for 10 days; 1 week after 33

### 99 DRAFT - DO NOT CITE OR QUOTE

completion of the mating period, females were sacri ficed and evaluated for the number of 1 

### pregnant females, viable fetuses, resorptions, and dead fetuses. Histopathological evaluations of 2 

### tissues were not conducted. No data on body weight s were reported. Exposure of male mice to 3 

### hexavalent chromium did not affect the percentage o f pregnant females. The numbers of 4 

### implantations and viable fetuses were significantly reduced from 33% in controls to 20% ( p < 5 

### 0.01) and 16% (

p < 0.05) in the 706 and 1,412 mg potassium dichromat e/L groups, respectively; 6 

### in the 1,765 mg hexavalent chromium/L group, the nu mbers of implantation and viable fetuses 7 

### were reduced to 19%, although this reduction did no t reach statistical significance. No 8 

### resorptions or dead fetuses were observed in the co ntrol, 706, or 1,412 mg potassium 9 

### dichromate/L groups, but three resorptions were obs erved at 353 mg hexavalent chromium/L and 10 

### six resorptions and six dead fetuses were observed at 1,765 mg hexavalent chromium/L 11 

### (statistical significance not reported). 12 

### In the second experiment, sexually mature (age: 50 days) female Swiss mice were 13 

### exposed to drinking water containing 0 (19 females) , 2,000 (15 females), or 5,000 (11 females) 14 

### mg potassium dichromate/L (equivalent to 0, 706, or 1,765 mg hexavalent chromium/L, 15 

### respectively) for 12 weeks (Elbetieha and Al-Hamood , 1997). After 12 weeks, each female was 16 

### mated with an untreated sexually mature male for 10 days; 1 week after completion of the mating 17 

### period, females were sacrificed and evaluated for t he numbers of pregnant females, viable 18 

### fetuses, and resorptions and dead fetuses. No data on body weights were reported. No 19



### treatment-related effects were observed on the numb er of pregnant mice. The number of 20 

### implantations was significantly reduced from 17% in controls to 14% ( p < 0.01) and 9% (

p < 21 

### 0.05) in the 706 and 1,765 mg hexavalent chromium/L groups, respectively, and the number of 22 

### viable fetuses was significantly reduced from 17% i n controls to 9% in the 706 ( p < 0.05) and 23 

### 1,765 (

p < 0.01) mg hexavalent chromium/L groups, respective ly. The number of mice with 24 

### resorptions was significantly increased from 11% in controls to 53% ( p < 0.01) and 63% (

p < 25 

### 0.005) in the 706 and 1,765 mg hexavalent chromium/ L groups, respectively, and the total 26 

### number of resorptions was increased from 4 in contr ols to 36 and 14 in the 706 and 1,765 mg 27 

### hexavalent chromium/L groups, respectively (statist ical significance not reported). 28 

### In the third experiment, sexually mature (age: 50 d ays) mice were exposed to drinking 29 

### water containing 0 (10 males, 8 females), 2,000 (13 males, no females), or 5,000 (13 males, 30 

### 10 females) mg potassium dichromate/L for 12 weeks (Elbetieha and Al-Hamood, 1997). 31 

### Following treatment, body weights and weights of re productive organs (paired testes, seminal 32 

### vesicles, preputial glands, paired ovaries, and ute ri) were determined. No mortalities or clinical 33

### 100 DRAFT - DO NOT CITE OR QUOTE

signs of toxicity were observed. Final body weight s of males were significantly ( p < 0.01) 1 

### reduced by approximately 10 and 12% in the 706 and 1,765 mg hexavalent chromium/L groups, 2 

### respectively; final mean body weights of treated fe males were similar to controls. Relative testes 3 

### weights were increased by approximately 18% ( p < 0.01) and 22% (

p < 0.05) in the 706 and 4 

### 1,765 mg hexavalent chromium/L groups, respectively , and relative weights of seminal vesicles 5 

### and preputial gland were significantly ( p < 0.001) decreased by approximately 27 and 34%, 6 

### respectively, in the 1,765 mg hexavalent chromium/L group. Relative ovary weight was 7 

### significantly increased by 54% in females in the 1, 765 mg hexavalent chromium/L group, 8 

### although uterine weight was unaffected by treatment . Histopathological assessments of 9 

### reproductive tissues were not conducted. 10 

### In conclusion, results of the three experiments con ducted by Elbetieha and Al-Hamood 11 

### (1997) show that exposure to potassium dichromate i n drinking water affects reproductive 12 

### outcomes in exposed males and females. In female m ice, decreased numbers of implantations 13 

### and viable fetuses and increased resorptions were o bserved at 2,000 mg potassium dichromate/L 14 

### (equivalent to 706 mg hexavalent chromium/L). In m ales, exposure for 12 weeks prior to mating 15 

### reduced the numbers of implantations and viable fet uses at 2,000 and 4,000 mg potassium 16 

### dichromate/L (equivalent to 706 and 1,412 mg hexava lent chromium/L, respectively), but not at 17 

### 1,000 mg potassium dichromate/L (equivalent to 353 mg hexavalent chromium/L). In addition, 18 

### treatment-related changes in weights of male reprod uctive organs were observed at 2,000 and 19 

### 5,000 mg potassium dichromate/L (equivalent to 706 and 1,412 mg hexavalent chromium/L, 20 

### respectively). Although reproductive performance w as not affected at the lowest exposure level, 21 

### weights of male reproductive organs were not evalua ted in male mice treated with 1,000 mg 22 

### potassium dichromate/L. Due to inadequate reportin g (i.e., no information on effects of 23 

### treatment on body weight or drinking water consumpt ion), a NOAEL or LOAEL from this study 24



### could not be identified. 25

26 

### NTP, 1997 27 

### The potential reproductive toxicity of dietary pota ssium dichromate was evaluated in 28 

### BALB/c mice in a continuous breeding study (NTP, 19 97). Groups of 20 male and female pairs 29 

### (F 0 

### ) were exposed to dietary potassium dichromate at 0 , 100, 200, and 400 mg potassium 30 

### dichromate/kg diet (equivalent to 0, 17.6, 35.3, or 141.2 mg hexavalent chromium/kg diet, 31 

### respectively) for 13 weeks (1 week prior to and 12 weeks during cohabitation). During exposure 32



### of the F 0 

### generation, animals were examined daily for mortal ity and clinical signs of toxicity; 33

### 101 DRAFT - DO NOT CITE OR QUOTE

body weights and food consumption were measured per iodically (4–5 times). Litters produced 1 

### during the cohabitation period were evaluated (i.e. , total pups, live and dead pups, and sex), 2 

### weighed on postnatal day (PND) 1, and euthanized wi th no additional assessments; pregnancy 3 

### index (number of liters/breeding pair) was also det ermined. After the cohabitation period, F 0 4 

### breeding pairs were separated and continued on stud y diets; litters born during the post5 

### separation period (F 1 

### animals) were reared with the F 0 

### dams until weaning (PND 21). Dam and 6 

### pup weights and dam food consumption were monitored during the lactational period. Upon 7 

### weaning, F 0 

### animals were sacrificed and the following terminal evaluations were conducted: 8 

### necropsy; organ weights (liver, kidneys, right caud a epididymis right epididymis, prostate, 9 

### seminal vesicles with coagulating glands, right tes tis, and ovaries); sperm evaluations (testicular 10 

### spermatid head count and epididymal sperm density, motility, and morphology); and 11 

### histopathology (liver and kidneys). Following wean ing of F 1 

### animals, animals were maintained 12 

### on the same study diets as their parents. During p ost-lactational exposure of the F 1 

### generation, 13 

### animals were examined daily for mortality and clini cal signs of toxicity; body weights and food 14 

### consumption were measured periodically (3–4 times). At sexual maturity (approximately 15 

### 74 days), groups of 20 F 1 

### animals of each sex were selected as breeding pair s (avoiding sibling 16 

### matings), cohabitated for 7 days, and then separate d. Reproductive endpoints (numbers of live 17 

### and dead pups, sexes of pups, and total pup weight by sex) were evaluated on PND 1 of the F 2 18 

### offspring; there was no further evaluation of the F 2 

### pups. Estrous cycle (time spent in estrous 19 

### stages, cycle length, number of cycles, number of c ycling females, and number of females with 20 

### regular cycles) was evaluated using 12-day vaginal smears beginning 4 days after the last 21 

### delivery. Terminal evaluations of F 1 

### adults (time from separation to terminal sacrifice not 22 

### reported) were the same as those described above fo r F 0 

### adults, with the addition of hematology 23 

### (i.e., Hgb, Hct, MCV, MCH, MCHC, mean platelet volu me, erythrocyte morphology, and 24 

### erythrocyte, leukocyte, and platelet counts). 25 

### No treatment-related mortalities or clinical signs of toxicity were observed in F 0 26 

### generation BALB/c mice exposed to dietary potassium dichromate (NTP, 1997). Mortalities 27 

### occurred in eight animals (four low-dose males, one mid-dose male, and three mid-dose 28 

### females); however, since no mortalities were observ ed in the high-dose group, NTP (1997) 29



### concluded that these deaths were not related to tre atment. Terminal body weight of males in all 30 

### treatment groups was comparable to controls; mean b ody weight of females in the high-dose 31 

### groups was decreased by 7% ( p < 0.05). In general, food consumption was increase d in 32 

### treatment groups. Based on measured food consumpti on and body weights during the 33

### 102 DRAFT - DO NOT CITE OR QUOTE

cohabitation period, NTP (1997) calculated average daily doses in F 0 

### males and females of 0, 1 

### 19.4, 38.6, or 85.7 mg potassium dichromate/kg-day (equivalent to 0, 6.8, 13.6, or 30.3 mg 2 

### hexavalent chromium/kg-day, respectively). During lactation, sporadic decreases in body 3 

### weights of dams in the mid- and high-dose groups we re observed, but body weights at the end of 4 

### lactation (PND 21) were similar to controls; food c onsumption during lactation was similar 5 

### between control and treatment groups. Based on mea sured food consumption and body weights, 6 

### NTP (1997) calculated average daily doses in lactat ing F 0 

### females of 0, 32.8, 69.0 or 143.1 mg 7 

### potassium dichromate/kg-day (equivalent to 0, 11.6, 24.4, or 50.5 mg hexavalent chromium/kg8 

### day, respectively). At the terminal evaluations of F 0 

### animals, absolute (but not relative) liver 9 

### weights were increased by 17% ( p < 0.05) and 22% (

p < 0.05) in high-dose males and females, 10 

### respectively, compared with controls. No other cha nges in organ weights were observed. No 11 

### treatment-related histopathological findings were o bserved in the F 0 

### generation. Although 12 

### various hepatic lesions were observed, including cy toplasmic vacuolization, study authors 13 

### concluded that these findings were not treatment re lated, since incidence data did not show a 14 

### relationship with dose. Evaluations of male reprod uctive tissues did not reveal any treatment15



### related effects. In the F 0 

### generation, no treatment-related effects on reprod uctive outcomes, 16 

### including pregnancy index, mean cumulative time to litter, litter size, live and dead pups/litter, 17 

### live pup weight, and sex ratio, were observed. 18



### Evaluations conducted on F 1 

### pups during lactational exposure showed no effects on pup 19 

### survival (NTP, 1997). On PND 21, weight of high-do se male pups was decreased by 16% 20 

### compared with controls, but the decrease was not st atistically significant. From weaning to 21 

### sexual maturity, two mortalities occurred (one cont rol male and one high-dose male). No 22 

### treatment-related clinical signs of toxicity were o bserved. At the initiation of the F 1 

### breeding 23 

### phase (approximately PND 74), mean body weights of mid-dose females were decreased by 6% 24 

### compared with controls and by 9% in high-dose F 1 

### male and females (statistical significance not 25 

### reported). Food consumption was generally increase d during the period from weaning to sexual 26 

### maturity. Based on measured food consumption and bo dy weights, NTP (1997) calculated 27 

### average daily doses in F 1 

### animals of 0, 22.4, 45.5 or 104.9 mg potassium dic hromate/kg-day 28 

### (equivalent to 0, 7.9, 16.1, or 37.1 mg hexavalent chromium/kg-day, respectively). Hematology 29 

### analysis at terminal sacrifice of F 1 

### adults revealed slight erythrocyte microcytosis ba sed on the 30 

### following observations (comparisons to controls, st atistical significance not reported): MCV 31 

### decreased by 3% in mid- and high-dose males and by 2, 3, and 4% in low-, mid-, and high-dose 32 

### females, respectively; MCH decreased by 3% in high- dose males; and Hgb decreased by 5% in 33

### 103 DRAFT - DO NOT CITE OR QUOTE

high-dose F1 females. No changes in erythrocyte mo rphology were observed. Relative kidney 1 

### weight was increased by 5% in mid-dose females, but no other organ weight changes were 2 

### observed. No treatment-related histopathological f indings were observed. Although various 3 

### hepatic lesions were observed, including cytoplasmi c vacuolization, NTP (1997) concluded that 4 

### findings were not treatment related, since incidenc e data did not show a relationship with dose. 5 

### Evaluations of male reproductive tissues and female estrous cycle did not reveal any treatment6



### related effects. In the F 1 

### generation, no treatment-related effects on reprod uctive outcomes, 7 

### including pregnancy index, mean cumulative time to litter, gestation length, litter size, live and 8 

### dead pups/litter, and sex ratio, were observed. Li ve pup weight of females in the high-dose 9 

### group was decreased by 11% ( p < 0.05) compared to controls, but no decrease was o bserved for 10 

### live pup weight of males or of combined males and f emales. 11 

### In conclusion, NTP (1997) identified a LOAEL for pa rental toxicity in the F 1 

### generation 12 

### of 7.9 mg hexavalent chromium/kg-day in females exp osed to potassium dichromate in the diet 13 

### based on erythrocyte microcytosis (slight decrease in MCH); a NOAEL for parental toxicity in 14



### the F 1 

### generation was not established. Although NTP (199 7) did not specifically identify a 15 

### NOAEL for reproductive effects, in the absence of r eproductive findings, the highest dose tested 16 

### is identified as a free-standing NOAEL for effects of dietary hexavalent chromium exposure on 17 

### fertility and on male and female reproductive organ histology and weights (30.3 mg hexavalent 18 

### chromium/kg-day in F 0 

### mice and 37.1 mg hexavalent chromium/kg-day in F 1 

### mice). 19

20



### 4.3.3. Effects of Pre-gestational Exposure on Reproductive Outcome and Fetal Development 21 

### Kanojia et al., 1996 22 

### Kanojia et al. (1996) administered adult Swiss albi no female rats (20/group) drinking 23 

### water containing 0, 250, 500, or 750 mg hexavalent chromium/L (as potassium dichromate) for 24 

### 20 days prior to gestation. During the exposure an d gestational periods, body weights and water 25 

### intake were recorded daily. At the end of the expo sure period, rats were mated overnight with 26 

### untreated males. Following mating, the mating inde x (percentage of mated females) and the 27 

### fertility index (percentage of pregnant females) we re determined. On GD 19, 10 rats/group were 28 

### sacrificed and the numbers of copora lutea, fetuses /litter, live and dead fetuses, and resorptions, 29 

### pre- and post-implantation losses, and fetal and pl acental weights were recorded and fetuses were 30 

### examined for internal abnormalities (one third of f etuses) and external and skeletal abnormalities 31 

### (remaining fetuses). In the remaining 10 rats/grou p, estrous cycle length was evaluated for 12 32 

### consecutive cycles. Based on drinking water consum ption during the exposure period, Konijia et 33

### 104 DRAFT - DO NOT CITE OR QUOTE

al. (1996) reported daily hexavalent chromium intak es of 6.4, 12.2, and 15.3 mg hexavalent 1 

### chromium/rat-day. The study report did not include data on body weights over the course of the 2 

### 20-day treatment period, although it is likely that treatment-related effects on body weight 3 

### occurred during the exposure period, as significant decreases in gestational weight gain were 4 

### observed in all treatment groups (decreases of appr oximately 8, 14, and 21% in the low-, mid-, 5 

### and high-dose groups, respectively, compared to con trols). Thus, in the absence of data on the 6 

### effect of treatment on body weights during the expo sure period, daily doses of hexavalent 7 

### chromium in terms of body weight (e.g., mg hexavale nt chromium/kg-day) cannot be accurately 8



### estimated. 9 

### No mortalities or clinical signs of toxicity in dam s were observed. Dose-related 10 

### decreases in mating and fertility indices were obse rved; in the high-dose group, mating and 11 

### fertility indices were decreased by 60 and 68%, res pectively, compared to controls (statistical 12 

### significance not reported). In all treatment group s, the number of live fetuses was decreased, the 13 

### numbers of resorptions and post-implantation loss w ere increased, and placental weight was 14 

### increased. In the mid- and high-dose groups, numbe rs of corpora lutea and implantations were 15 

### decreased and pre-implantation losses were increase d. No treatment-related effects were 16 

### observed for fetal weight or crown-rump length. Ex amination of fetuses showed gross 17 

### abnormalities in the high-dose group, including pat ches of subdermal hemorrhage, kinky tail, 18 

### short tail, and dropping wrist. Skeletal abnormali ties were also observed, including reduced 19 

### caudal ossification in mid- and high-dose groups an d reduced parietal and inter-parietal 20 

### ossification in the high-dose group. No visceral a bnormalities were observed. Postpartum 21 

### estrous cycle length was significantly increased by 37% ( p < 0.05) in the high-dose group. 22 

### Results of this study show that 20-day pre-gestatio nal exposure of Swiss albino rat dams 23 

### to hexavalent chromium adversely affected reproduct ive outcomes (decreased number of live 24 

### fetuses and increased number of resorptions and pos t-implantation loss) at the lowest drinking 25 

### water concentrations of potassium dichromate tested ( ≥ 250 mg hexavalent chromium/L or 26



### ≥ 6.4 mg hexavalent chromium/rat-day) and produced ad verse developmental effects (gross and 27 

### skeletal abnormalities) at the highest drinking wat er concentrations tested (750 mg hexavalent 28 

### chromium/L or 15.3 mg hexavalent chromium/rat-day). Because of the lack of reporting of body 29 

### weight data over the course of the study, NOAELs an d/or LOAELs, expressed in mg hexavalent 30 

### chromium/kg-day, could not be derived from this stu dy. 31

32

### 105 DRAFT - DO NOT CITE OR QUOTE

Kanojia et al., 1998 1 

### Kanojia et al. (1998) administered adult Druckrey f emale rats (20/group; mean initial 2 

### body weight 80 g) drinking water containing 0, 250, 500, or 750 mg hexavalent chromium/L (as 3 

### potassium dichromate) for 3 months prior to gestati on. This study was designed to following the 4 

### same protocol as that used in the Kanojia et al. (1 996) study (described above). However, at the 5 

### end of the 3-month exposure period, rats in all tre atment groups were acyclic (persistent 6 

### diestrous phase). Therefore, since mating could no t take place immediately following 7 

### completion of the exposure period, rats were held f or an additional 15–20 days (treatment-free), 8 

### during which estrous cycle resumed. 9 

### During the exposure period, mortality occurred in 1 5 and 10% of rats in the mid- and 10 

### high-dose groups, respectively; no deaths occurred in the control or low-dose groups. Clinical 11 

### signs of toxicity observed during the exposure peri od in the mid- and high-dose groups included 12 

### hair loss and lethargic and aggressive behavior. A t the end of the exposure period, body weight 13 

### was significantly (

p < 0.05) decreased by approximately 18 and 24% in th e mid- and high-dose 14 

### groups, respectively, compared with controls. Knoj ia et al. (1998) reported average hexavalent 15 

### chromium intakes (based on water consumption) of 5. 57, 10.18, and 13.56 mg hexavalent 16 

### chromium/rat-day in the low-, mid-, and high-dose g roups, respectively. Using these daily 17 

### intake levels and the mean initial body weight of 8 0 g, daily doses of 70, 127, and 170 mg 18 

### hexavalent chromium/kg-day for the low-, mid-, and high-dose groups, respectively, were 19 

### estimated. During the postexposure gestational per iod, maternal weight gain was significantly ( p 20 

### < 0.05) decreased by 17 and 22% in the mid- and hig h-dose groups, respectively, compared with 21 

### controls. The mating index was decreased by 30, 40 and 60% and the fertility index was 22 

### decreased by 32, 41, and 49% in the low-, mid-, and high-dose groups, respectively, compared 23 

### with controls (statistical significance not reporte d). In all treatment groups, pre- and post24 

### implantation losses were significantly ( p < 0.05) increased, with increases in the high-dose group 25 

### reaching 3.1- and 4.2-fold, respectively. In the m id- and high-dose groups, the numbers of 26 

### implantations, live fetuses, and resorptions were s ignificantly ( p < 0.05) increased. Assessments 27 

### of fetuses (on a per litter basis compared with con trols) showed the following (significant 28 

### difference compared with controls; p < 0.05); decreased fetal weight (all treatment grou ps); 29 

### decreased crown-rump length (mid- and high-dose gro ups); gross external abnormalities, 30 

### including subdermal hemorrhagic patches and droopin g wrists in all treatment groups and kinky 31 

### and short tail in mid- and high-dose groups; and sk eletal abnormalities, including decreased 32 

### caudal ossification in all treatment groups and red uced parietal and interparietal ossification in 33

### 106 DRAFT - DO NOT CITE OR QUOTE

mid- and high-dose groups. No internal abnormaliti es in fetuses were observed. Postpartum 1 

### estrous cycle length was significantly ( p < 0.05) increased in all treatment groups, with inc reases 2 

### reaching approximately 1.7-fold in the high-dose gr oup. 3 

### Results of this study show that 3-month pre-gestati onal exposure of Druckrey rat dams to 4 

### hexavalent chromium as potassium dichromate adverse ly affected reproductive outcomes 5 

### (increased pre- and post-implantation losses) and p roduced adverse developmental effects 6 

### (decreased fetal weight and external and skeletal a bnormalities) at all drinking water 7 

### concentrations tested ( ≥ 250 mg hexavalent chromium/L or approximately ≥ 70 mg hexavalent 8 

### chromium/kg-day). Thus, a LOAEL for hexavalent chr omium of 70 mg/kg-day can be identified 9 

### from this study. 10

11 

### Junaid et al., 1996a 12 

### Junaid et al. (1996a) administered Swiss albino fem ale mice drinking water containing 0, 13 

### 250, 500, or 750 mg hexavalent chromium/L (as potas sium dichromate) for 20 days prior to 14 

### gestation. The study followed the same protocol an d conducted the same evaluations as those 15 

### reported in the study by Kanojia et al. (1996) (des cribed above), except that estrous cycle length 16 

### was not evaluated. Evaluations on reproductive out comes and developmental effects were 17 

### conducted in 10 mice/group. 18 

### No clinical signs of toxicity were observed in mice during the exposure period. In the 19 

### high-dose group, mortality occurred in 20% of anima ls; the cause of death was not established. 20 

### Based on drinking water consumption monitored durin g the exposure period, study authors 21 

### reported daily hexavalent chromium intake levels of 1.9, 3.56, and 5.23 mg hexavalent 22 

### chromium/mouse-day in the low-, mid-, and high-dose groups, respectively. No treatment23 

### related effects were observed on body weight (data not reported); thus, using the reported mean 24 

### initial body weight of 30 g, daily doses of 63, 119 , and 174 mg hexavalent chromium/kg-day for 25 

### the low-, mid-, and high-dose groups, respectively, were estimated. During the gestational 26 

### period, maternal weight gain in the low- and mid-do se groups was comparable to controls; no 27 

### weight gain was observed during gestation in high-d ose group dams. In the low-dose group, 28 

### post-implantation loss was significantly ( p < 0.05) increased compared with controls (control: 29 

### 0%; low-dose group: 17.5%); no effects were observe d for the numbers of corpora lutea, 30 

### implantations, live fetuses, or resorptions or for pre-implantation loss. In the mid-dose group, 31 

### the numbers of implantation and live fetuses were s ignificantly ( p < 0.05) decreased and the 32 

### numbers of resorptions and pre- and post-implantati on losses were significantly ( p < 0.05) 33

### 107 DRAFT - DO NOT CITE OR QUOTE

increased; no effect on the number of corpora lutea was observed. In the high-dose group, no 1 

### litters were produced and implantation sites were c ompletely absent; corpora lutea were present, 2 

### but numbers were decreased by 44% compared to contr ols. Assessments of fetuses (on a per 3 

### litter basis compared with controls) showed the fol lowing (significant difference compared to 4 

### controls; p < 0.05); decreased fetal weight and length in the l ow- and mid-dose groups; gross 5 

### (external) abnormalities, including subdermal hemor rhagic patches and short and kinky tail in 6 

### the mid-dose group; and skeletal abnormalities, inc luding reduced caudal ossification in the low7 

### and mid-dose groups and reduced parietal and interp arietal ossification in the mid-dose group. 8



### No internal abnormalities in fetuses were observed. 9 

### Thus, at all drinking water concentrations of potas sium dichromate tested ( ≥ 250 mg 10 

### hexavalent chromium/L or approximately ≥ 63 mg hexavalent chromium/kg-day), pre-gestational 11 

### exposure of Swiss albino female mice for 20 days pr oduced adverse effects on reproductive 12 

### outcome (decreased fertility) and fetal development (decreased fetal body weight and delays in 13 

### skeletal development). Thus, a LOAEL for hexavalen t chromium of 63 mg/kg-day can be 14 

### identified from this study. 15

16



### 4.3.4. Effects of Gestational and/or Lactational Exposure on Reproductive Outcome and 17 

### Fetal Development 18 

### Elsaieed and Nada, 2002 19 

### Effects of gestational exposure to hexavalent chrom ium were investigated in Wistar rats 20 

### (Elsaieed and Nada, 2002). Groups of 10 pregnant r ats (mean initial body weight: 170 g) were 21 

### administered drinking water containing 0 or 50 mg h exavalent chromium/L as potassium 22 

### dichromate on GD 6 through 15. During the exposure period, dams were evaluated for clinical 23 

### signs of toxicity, body weights, and food and drink ing water consumption. One day before 24 

### delivery, rats were sacrificed and the following we re evaluated: numbers of corpora lutea, pre25 

### and post-implantation losses, resorptions, and live and dead fetuses; fetal weight; and visceral 26



### and skeletal anomalies. 27 

### No mortalities or clinical signs of toxicity were o bserved. Elsaieed and Nada (2002) 28 

### stated that food and drinking water consumption was comparable between control and treatment 29 

### groups, although data were not reported. Gestation al weight gain was significantly ( p < 0.05) 30 

### decreased by 40% in hexavalent chromium treated dam s, compared with controls. Based on an 31 

### average gestational body weight of 177 g (average c alculated using body weights at mating and 32 

### at the end of gestation) and the allometric equatio n for drinking water consumption for 33

### 108 DRAFT - DO NOT CITE OR QUOTE

laboratory mammals (0.10 × body weight 0.7377 

### ; U.S. EPA, 1988), a daily dose of 7.9 mg 1 

### hexavalent chromium/kg-day was estimated. In this study, treatment of rats with hexavalent 2 

### chromium resulted in significant ( p < 0.05) increases in pre-implantation loss/litter ( 2.1 vs. 0 in 3 

### control), post-implantation loss/litter (1.5 vs. 0) , resorptions/litter (1.2 vs. 0), and dead 4 

### fetuses/litter (1.2 vs. 0) and decreases in live fe tuses/litter (1.5 vs. 6.8 in control) and fetal wei ght 5 

### (33% decrease). In the exposed group, increased li tters with fetal abnormalities or 6 

### malformations were observed including visceral (ren al pelvis dilation: 2.1/litter) and skeletal 7 

### (incomplete skull ossification: 1.0/litter) changes ; no control fetuses showed these changes. 8 

### The results show that exposure of pregnant Sprague- Dawley rats to drinking water 9 

### containing 50 mg hexavalent chromium/L as potassium dichromate (approximately 7.9 mg 10 

### hexavalent chromium/kg-day) on GDs 6–15 produced ad verse effects on reproductive outcome 11 

### and fetal development. Thus, a LOAEL for hexavalen t chromium of 7.9 mg/kg-day can be 12 

### identified from this study. 13

14 

### Bataineh et al., 2007 15 

### Reproductive outcome was evaluated in adult female rats (age not specified) orally 16 

### exposed to potassium dichromate for 3 days followin g mating (Bataineh et al., 2007). Groups of 17 

### 10 successfully mated female Sprague-Dawley rats we re administered daily doses of 0 or 25 mg 18 

### potassium dichromate/rat (equivalent to 8.8 mg hexa valent chromium/rat-day or approximately 19 

### 35 mg hexavalent chromium/kg-day, based on the aver age reported body weight of 245 g at 20 

### mating) in saline daily by gavage on GDs 1–3 or 4–6 . On GD 20, rats were sacrificed and the 21 

### number of implantation sites, live fetuses, and res orptions along the uterine horns were recorded; 22 

### fetuses were not assessed for external, skeletal, o r visceral abnormalities. 23 

### In rats treated with potassium dichromate on GDs 1– 3, no pregnancies, implantations, 24 

### resorptions, or viable fetuses were observed, compa red with 10/10 pregnancies, 8.2 25 

### implantations/female, 8.2 live fetuses/female, and 0/82 resorptions in controls. In rats treated on 26 

### GDs 4–6, the numbers of pregnant rats and implantat ions/female were comparable to values in 27 

### the control group. However, the number of viable f etuses was decreased by 69% ( p < 0.001) and 28 

### the percentage of resorptions per implantations was increased by 222% ( p < 0.001). The study 29 

### report did not indicate if clinical signs of toxici ty were observed in chromium-treated dams, and 30 

### no additional measures to assess systemic toxicity were reported. 31 

### The results indicate that short-term gavage exposur e of Sprague-Dawley dams to 32 

### potassium dichromate at a dose of 35 mg hexavalent chromium/kg-day on GDs 1–3 completely 33

### 109 DRAFT - DO NOT CITE OR QUOTE

impaired implantation; exposure on GDs 4–6 markedly increased resorptions and decreased the 1 

### number of viable fetuses, compared with controls. Thus, a LOAEL for hexavalent chromium of 2 

### 35 mg/kg-day can be identified from this study. 3

4 

### Trivedi et al., 1989 5 

### Effects on reproductive outcome and fetal developme nt were observed in ITRC-bred 6



### albino mice administered hexavalent chromium in dri nking water (Trivedi et al., 1989). Groups 7 

### of 10–13 pregnant mice (average initial body weight of 30 g) were administered drinking water 8 

### containing 0, 250, 500, or 1,000 mg hexavalent chro mium/L (as potassium dichromate) during 9 

### the entire gestational period. Dams were observed daily for mortality, clinical signs of toxicity, 10 

### body weight, and water consumption. On GD 19, dams were sacrificed and the following were 11 

### recorded: numbers of corpora lutea, total implantat ions, live and dead fetuses, and pre12 

### implantation and postimplantations losses; placenta l weight; fetal weight and crown-rump 13 

### length; number of stunted fetuses; and sex ratio pe r liter. In addition, fetus were examined for 14 

### external (all fetuses), internal (approximately one -third of fetuses), and skeletal (remaining 15 

### fetuses) anomalies. 16 

### No mortalities or clinical signs of toxicity were o bserved. In the low-dose group, body 17 

### weight gain was comparable to controls; however, bo dy weight gain was significantly decreased 18 

### by 21% (

p < 0.05) in the mid-dose group, and dams in the high -dose group lost weight during 19 

### treatment. Daily hexavalent chromium intakes were reported as 1.76, 3.6, and 7.03 mg 20 

### hexavalent chromium/mouse-day, in the low-, mid-, a nd high-dose groups, respectively, based 21 

### on measured drinking water consumption. Using aver age body weights for the gestational period 22 

### (36.8, 36.6, and 29.4 g in the low-, mid-, and high -dose groups, respectively; calculated for this 23 

### review using: [average initial body weight + body weight at the end of gestation]/2) and reported 24 

### daily chromium intakes, daily doses of 48, 98, and 239 mg hexavalent chromium/kg-day were 25 

### estimated. In low-dose mice, the percentages of re sorptions and postimplantation loss were 26 

### significantly increased ( p < 0.001) to 33 and 36%, respectively, compared with 10 and 1.7%, 27 

### respectively, in controls; the number of litters, l itter size, number of copora lutea, and placental 28 

### weight in the low-dose group were comparable to con trols. In the mid-dose group, the 29 

### percentages of resorptions and postimplantation los ses were significantly ( p < 0.001) increased 30 

### to 52 and 88%, respectively. In addition, in the m id-dose group, litter size was significantly 31 

### decreased by 44% ( p < 0.01) compared with controls, and the percentage of preimplantation loss 32 

### was increased to 26.2% ( p < 0.001), compared with 3.6% in controls. No treat ment-related 33

### 110 DRAFT - DO NOT CITE OR QUOTE

effects on placental weight were observed in the lo w- or mid-dose groups. In the high-dose 1 

### group, no litters were produced and implantation si tes were completely absent. In the low- and 2 

### mid-dose groups, mean fetal crown-rump lengths were decreased ( p < 0.001) by 17 and 27%, 3 

### respectively, and mean fetal weights were decreased ( p < 0.001) by 31 and 44%, respectively. 4 

### Sex ratio was unaffected by treatment. Examination of fetuses for external anomalies showed no 5 

### effects in the low-dose group; in the mid-dose grou p, tail kinking and subdermal hemorrhagic 6 

### patches and streaks were observed. An increase in the incidence of minor skeletal anomalies was 7 

### observed in fetuses in the low-dose (reduced ossifi cation of the cranium) and mid-dose (reduced 8 

### ossification of the cranium, forelimb, hindlimb, st ernebrae, and thoracic and caudal vertebrae and 9 

### reduced number of ribs) groups. No internal anomal ies were observed. 10 

### The LOAEL and NOAEL for maternal toxicity, assessed as decreased body weight gain 11 

### in ITRC-bred albino mice exposed to potassium dichr omate in drinking water throughout 12 

### gestation, were 98 and 48 mg hexavalent chromium/kg -day, respectively. Based on increased 13 

### resorptions and postimplantation loss, and decrease d fetal length and weight, the lowest 14 

### concentration tested (250 mg hexavalent chromium/L; 48 mg hexavalent chromium/kg-day) is 15 

### identified as a LOAEL for developmental effects. 16

17 

### Junaid et al., 1996b 18 

### Junaid et al. (1996) evaluated the effects of oral exposure of pregnant mice to hexavalent 19 

### chromium on reproductive outcome and fetal developm ent. Groups of 10 successfully mated 20 

### Swiss albino female mice (average initial body weig ht of 30 g) were administered drinking water 21 

### containing 0, 250, 500, or 750 mg hexavalent chromi um/L (as potassium dichromate) on GDs 6 22 

### though 14. Throughout the exposure period, dams we re evaluated daily for clinical signs of 23 

### toxicity, body weight, and drinking water consumpti on. On GD 19, dams were sacrificed and 24



### evaluations of dams and fetuses were conducted as d escribed by Trivedi et al. (1989) 25 

### (summarized above). 26 

### No mortalities or clinical signs of toxicity were o bserved. Gestational weight gain was 27 

### significantly (

p < 0.05) decreased in the mid- and high-dose groups by 8 and 32%, respectively, 28 

### but was comparable to controls in the low-dose grou p. Daily hexavalent chromium intakes were 29 

### reported as 2.00, 3.75, or 5.47 mg chromium/mouse-d ay in the low-, mid-, and high-dose groups, 30 

### respectively, based on measured drinking water cons umption. Using average body weights for 31 

### the gestational period (37.6, 37.2, and 35.9 g in t he low-, mid-, and high-dose groups, 32 

### respectively; calculated for this report using: [av erage initial body weight + body weight at the 33

### 111 DRAFT - DO NOT CITE OR QUOTE

end of gestation]/2) and reported daily chromium in takes, daily doses of 53, 101, and 152 mg 1 

### hexavalent chromium/kg-day in the low-, mid-, and h igh-dose groups, respectively, were 2 

### estimated. The number of resorptions was significa ntly ( p < 0.05) increased in all treatment 3 

### groups, with increases reaching 7.7-fold in the hig h-dose group. In the mid- and high-dose 4 

### groups, significant (

p < 0.05) decreases in the total number of fetuses an d increases in the 5 

### numbers of dead fetuses and resorption sites were o bserved. Fetal weight was significantly ( p < 6 

### 0.05) decreased by 13 and 19% in the mid- and high- dose groups, respectively; no treatment7 

### related effects were observed on fetal length. Gro ss external examination of fetuses showed 8 

### significant (

p < 0.05) increases in the incidences of minor abnorm alities (subdermal hemorrhagic 9 

### patches, drooping wrist, kinky and short tail) in t he high-dose group. Examination of fetuses for 10 

### skeletal abnormalities showed significant ( p < 0.05) increases in the incidences of reduced caud al 11 

### ossification in the mid- and high-dose groups and o f reduced nasal, frontal, parietal, interparietal, 12 

### carpals, and tarsals ossification. No external or skeletal abnormalities were observed in fetuses 13 

### in the low-dose group. No visceral abnormalities w ere observed in any treatment group. 14 

### Junaid et al. (1996b) concluded that oral exposure of dams during the organogenesis 15 

### phase of gestation produces adverse effects in embr yos and during fetal development. The 16 

### LOAEL and NOAEL for maternal toxicity, assessed as decreased body weight gain in Swiss 17 

### albino mice administered potassium dichromate in dr inking water on GDs 6–14, were 101 and 18 

### 53 mg hexavalent chromium/kg-day, respectively. Ba sed on reduced number of implantation 19 

### sites, the lowest dose tested (approximately 53 mg hexavalent chromium/kg-day) is identified as 20 

### a developmental LOAEL for this study. 21

22 

### Junaid et al., 1995 23 

### The effects of late gestational exposure to hexaval ent chromium on reproductive outcome 24 

### and fetal development were evaluated in mice (Junai d et al., 1995). Groups of 10 successfully 25 

### mated Swiss albino female mice (average initial bod y weight of 30 g) were administered 26 

### drinking water containing 0, 250, 500, or 750 mg he xavalent chromium/L (as potassium 27 

### dichromate) on GD 14 though 19. Throughout the exp osure period, dams were evaluated daily 28 

### for clinical signs of toxicity, body weight and dri nking water consumption. On GD 19, dams 29



### were sacrificed and evaluations of dams and fetuses were conducted as described by Trivedi et 30 

### al. (1989) (summarized above). 31 

### No mortalities or clinical signs of toxicity were o bserved. Gestational weight gain was 32 

### significantly (

p < 0.05) decreased in the mid- and high-dose groups by 11 and 26%, respectively, 33

### 112 DRAFT - DO NOT CITE OR QUOTE

but was comparable to controls and the low-dose gro up. No data on drinking water consumption 1 

### were reported; however, it is likely that daily dos es were similar to those calculated for the study 2 

### by Junaid et al. (1996b) (e.g., approximately 53, 1 01, and 152 mg hexavalent chromium/kg-day 3 

### in the low-, mid-, and high-dose groups, respective ly), which used the same mouse strain and 4 

### drinking water concentrations, and a similar study design. In the mid- and high-dose groups, the 5 

### numbers of dead fetuses and post-implantation losse s were significantly ( p < 0.05) increased; the 6 

### numbers of corpora lutea and total fetuses per litt er were similar to controls in all treatment 7 

### groups. Fetal weight and length were significantly decreased in all treatment groups, with 8 

### decreases reaching approximately 47 and 29%, respec tively, in the high-dose group. Gross 9 

### external examination of fetuses showed significant ( p < 0.05) increases in the incidences of 10 

### minor abnormalities in the mid-dose (drooping wrist s) and high-dose (drooping wrists, 11 

### subdermal hemorrhagic patches, kinky and short tail ) groups. Examination of fetuses for skeletal 12 

### abnormalities showed significant ( p < 0.05) increases in the incidences of reduced caud al 13 

### ossification in all treatment groups, of reduced ta rsals ossification in mid- and high-dose groups, 14 

### and of reduced nasal, parietal, interparietal, carp als, and metatarsals ossifications in the high15 

### dose group. No visceral abnormalities were observe d in any treatment group. 16 

### The NOAEL and LOAEL for maternal toxicity, assessed as decreased body weight gain 17 

### in Swiss albino mice administered potassium dichrom ate in drinking water on GDs 14–19, were 18 

### 53 and 101 mg hexavalent chromium/kg-day, respectiv ely. Based on reduced fetal weight and 19 

### length and increased incidence of reduced caudal os sification in all treatment groups, the lowest 20 

### dose tested (approximately 53 mg hexavalent chromiu m/kg-day) is identified as a developmental 21 

### LOAEL for this study. 22

23 

### Al-Hamood et al., 1998 24 

### The effects of gestational and lactational exposure of mice to hexavalent chromium on 25 

### sexual maturation and fertility in offspring were i nvestigated by Al-Hamood et al. (1998). On 26 

### GD 12 through day 20 of lactation, groups of 25 pre gnant Swiss strain BALB/c mice (mean 27 

### initial body weight: 25 g) were administered drinki ng water containing 0 or 1,000 mg potassium 28 

### dichromate/L (equivalent to 353 mg hexavalent chrom ium/L). Based on drinking water 29 

### consumption by dams, daily hexavalent chromium inta kes of 2.1 and 1.7 mg hexavalent 30 

### chromium/mouse-day were calculated for the gestatio nal and lactational periods, respectively. 31 

### No data on body weights of dams were reported; howe ver, since other studies have shown 32 

### decreased maternal weight gain in pregnant mice exp osed to drinking water containing ≥ 176 33

### 113 DRAFT - DO NOT CITE OR QUOTE

hexavalent chromium/L) (Junaid et al., 1996b, 1995) , it is likely that treatment-related decreases 1 

### in maternal weight gain occurred. Therefore, given this uncertainty, daily hexavalent chromium 2 

### doses expressed in terms of body weight cannot be a ccurately estimated for this study. At birth, 3 

### litters were culled to eight pups per female and of fspring were weaned on PND 21; from 4 

### weaning to day 60 of age, offspring received contro l drinking water. From PND 20 to the onset 5 

### of puberty, female offspring were examined for time to vaginal opening. Fertility in offspring 6 

### was assessed at day 60 of age; male offspring were mated with untreated females and female 7 

### offspring were mated with untreated males for 10 da ys. At completion of the mating period, 8 

### females were examined for numbers of pregnant femal es, implantations, viable fetuses, and 9 

### resorptions. Additional groups (n = 9–12) of offsp ring were sacrificed on day 50 of age, and 10 

### body weights and weights of reproductive organs (pa ired testes, seminal vesicles, and preputial 11 

### glands in males and paired ovaries and uteri in fem ales) were determined. 12 

### In female offspring, time to vaginal opening was si gnificantly ( p < 0.001) increased from 13 

### 24.6 days in controls to 27.1 days in treated rats. Mating studies in female offspring showed 14 

### decreased numbers of pregnant females (35% decrease ; p < 0.025), implantations (12% decrease; 15



### p < 0.05), and viable fetuses (14% decrease; p < 0.05). No treatment-related effects on female 16 

### body weight or relative weights of reproductive org ans were observed. In male offspring, no 17 

### treatment-related effects were observed in mating s tudies or on body weights or weights of 18 

### reproductive organs. 19 

### The results indicate that gestational and lactation al exposure of BALB/c mouse dams to 20 

### drinking water containing 353 mg hexavalent chromiu m/L as potassium dichromate resulted in 21 

### impaired reproductive development and function in f emale offspring. Because of the lack of 22 

### reporting of body weight data over the course of th e study, NOAELs and/or LOAELs, expressed 23 

### in mg hexavalent chromium/kg-day, could not be deri ved from this study. 24

25 

### Banu et al., 2008 26 

### Banu et al. (2008) investigated the effects of lact ational exposure to hexavalent 27 

### chromium on sexual development of female rat offspr ing. Groups of 18 lactating Wistar rats 28 

### were administered drinking water containing 200 mg potassium dichromate (equivalent to 29 

### 70.6 mg hexavalent chromium/L) on postpartum days 1 through 21. No specific assessments of 30 

### dams were conducted. Banu et al. (2008) noted that toxic effects in dams were not “significant,” 31 

### although no additional information regarding matern al toxicity or data on body weights or 32 

### drinking water consumption in dams were reported. As discussed above, exposure of laboratory 33

### 114 DRAFT - DO NOT CITE OR QUOTE

animals to hexavalent chromium in drinking water ma y result in decreased body weight and 1 

### drinking water consumption; thus, in the absence of data on body weight and drinking water 2 

### consumption in dams, daily doses of hexavalent chro mium cannot be accurately estimated for 3 

### this study. At birth, litters were culled to four female pups per dam. Following weaning on PND 4 

### 21, pups were separated from dams. Pups (n = 24) w ere evaluated for the onset of puberty by 5 

### daily examination for vaginal opening. After the o nset of puberty, the time spent in each estrous 6 

### cycle phase (proestrous, estrous, metestrous, and d iestrous) was determined by analysis of 7 

### vaginal smears (n = 24). On PNDs 21, 45, and 65, p ups (n = 24, at each time point) were 8 

### sacrificed; at each time point, blood was analyzed for hormones (estradiol, progesterone, 9 

### testosterone, LH, follicle-stimulating hormone [FSH ], growth hormone [GH], and prolactin) and 10



### ovaries were examined for the number of follicles a nd follicle development stage (primordial, 11 

### primary, secondary, and antral). 12 

### The onset of puberty was significantly ( p < 0.05) increased from 33 days in control rats to 13 

### 55 days in hexavalent chromium-treated rats. Estro us cycle phase was also altered in hexavalent 14 

### chromium-treated rats, with the time spent in diest rous significantly (

p < 0.05) increased by 15 

### approximately 1.4-fold compared with controls (data presented graphically); time spent in other 16 

### estrous phases was unaffected by treatment. Evalua tions of ovaries on PNDs 21 and 45 showed 17 

### significant (

p < 0.05) decreases in the numbers of primordial, pri mary, secondary, and antral 18 

### follicles in hexavalent chromium-treated rats compa red with control rats; on PND 65, the 19 

### numbers of primordial and primary follicles were al so decreased in hexavalent chromium-treated 20 

### rats. At the 21- and 45-day assessments in hexaval ent chromium-treated rats, plasma 21 

### concentrations of estradiol, progesterone, testoste rone, GH, and prolactin were significantly ( p < 22 

### 0.05) decreased (by approximately 40 to 60%) and co ncentrations of FSH were significantly 23 

### increased (by approximately 40%), compared with con trols. Similar effects were observed at the 24 

### 65-day assessment, except that FSH concentrations i n treatment and control groups were 25 

### comparable. Plasma LH concentration was not affect ed by treatment at any time point. 26 

### The results indicate that lactational exposure of W istar rat dams to drinking water 27 

### containing 70.6 mg hexavalent chromium/L as potassi um dichromate resulted in delayed onset of 28 

### puberty and follicular development and impaired ova rian steroidogenesis in female offspring; 29 

### male offspring were not assessed for possible effec ts on sexual maturation. Because of the lack 30 

### of reporting of body weight data over the course of the study, NOAELs and/or LOAELs, 31 

### expressed in mg hexavalent chromium/kg-day, could n ot be derived from this study. 32

33

### 115 DRAFT - DO NOT CITE OR QUOTE

4.4. MECHANISTIC DATA AND OTHER STUDIES IN SUPPORT OF THE MODE OF 1



### ACTION 2 

### 4.4.1. Genotoxicity Studies 3 

### The mutagenic potential of hexavalent chromium has been studied extensively. Although 4 

### study results vary with specific test systems, expe rimental conditions, and hexavalent chromium 5 

### compounds tested, results of in vitro and in vivo s tudies provide substantial evidence for the 6 

### mutagenic activity of hexavalent chromium compounds . A general summary of the evidence 7 

### demonstrating the mutagenic activity of hexavalent chromium compounds in experimental 8 

### systems is provided in Table 4-20. As discussed in detail in Section 4.4.2 (Intracellular 9 

### Reduction), mutagenicity of hexavalent chromium is mediated through the generation of highly 10 

### reactive chromium intermediates (e.g., chromium(IV) and chromium(V)) and reactive oxygen 11 

### species formed during the intracellular reduction o f hexavalent chromium. Reactive chromium 12 

### intermediates and oxygen species react with DNA, le ading to oxidative DNA damage, 13 

### chromium-DNA adducts, DNA strand breaks, and chromo somal aberrations (Wise et al., 2008). 14

15116 DRAFT - DO NOT CITE OR QUOTETable 4-20. Evidence of mutagenicity of hexavalentchromium compounds in experimentalsystemsIn vitro studies(non-mammalian cells)In vitro studies(mammalian cells)In vivo studies(D. melanogasteror mammals)ChemicalDNAdamage MutationsDNAdamage MutationsChromosomaldamageDNAdamage MutationsChromosomaldamageAmmoniumchromateND●ND ND ND ND ND NDCalcium chromate ND●ND ND●ND●(D) NDChromic acid ND●ND ND●ND●(D) NDPotassium chromate●●●●●●(M)●(D)●(M)●(M)Potassiumdichromate●●●●●●(M)●(D)●(M)Sodium chromate ND●●ND●ND ND NDSodium dichromate ND●●ND●●(M)●(D) NDSodium dichromatedihydrateND●ND●ND ND ND●(M)●= positive resultsND = no data identified for this review(D) = study inD. melanogaster(M) = study in laboratory mammal

### 117 DRAFT - DO NOT CITE OR QUOTE

4.4.1.1. Genotoxicity assays in experimental syste ms 1 

### The mutagenic activity of hexavalent chromium has b een demonstrated in numerous studies 2 

### using both in vitro and in vivo experimental system s. In in vitro test systems (see Tables 4-21 and 4 -22 3 

### for studies in non-mammalian and mammalian cells, r espectively), hexavalent chromium compounds 4 

### have mostly tested positive for gene mutations (inc luding reverse mutations, frame shift mutations, an d 5 

### base pair substitutions) and DNA damage (including DNA-protein crosslinks) in bacterial cells 6 

### ( Salmonella typhimurium, Escherichia coli, Bacillus subttilis ). Reverse mutations were observed in 7 

### multiple species and strains, including those that are sensitive to frameshift mutagens ( S. typhimurium 8 

### TA97, TA98, TA1537, and TA1538), G/C base-pair subs titution mutagens ( S. typhimurium TA100 and 9 

### TA1535), and A/T base-pair substitution mutagens ca used by oxidizing and/or cross-linking agents ( S. 10 

### typhimurium TA102; E. coli WP2uvrA and WP2uvrA/pKM101). Positive results were also found for 11 

### forward mutations and mitotic gene conversion in ye ast ( Saccharomyces cerevisiae ); and for DNA 12 

### damage (DNA strand breaks, fragmentation, DNA-prote in crosslinks, DNA-DNA crosslinks), 13 

### chromosomal damage (sister chromatid exchanges and chromosomal aberrations), and DNA synthesis 14 

### inhibition in mammalian cell lines and primary cult ures (including primary cultures of human gastric 15 

### mucosal cells, respiratory tract cells, and lymphoc ytes). 16



### In in vivo test systems (see Table 4-23), hexavalent chromium compounds have tested positive 17



### for mutations in Drosophila melanogaster and for DNA damage (DNA-protein crosslinks, DNA st rand 18 

### breaks), mutations (in mice exposed in utero, in mo use germ cells, and in transgenic mice), 19 

### chromosomal damage (sister chromatid exchanges, chr omosomal aberrations, and micronuclei), and 20 

### DNA synthesis inhibition in rats and mice. The in vivo studies in laboratory mammals have evaluated 21 

### the mutagenic activity of hexavalent chromium follo wing exposure by the oral, parenteral, inhalation, 22



### and intratracheal routes. 23 

### Hexavalent chromium-induced mutagenicity has been d emonstrated following oral exposure. In 24 

### oral exposures studies, DNA damage has been observe d in several tissues in mice and rats following 25 

### gavage administration of hexavalent chromium, inclu ding stomach, colon, liver, lung, brain, and 26 

### peripheral lymphocytes (Wang et al., 2006; Devi et al., 2001; Sekihashi et al., 2001; Coogan et al., 27 

### 1991b). Devi et al. (2001) observed DNA damage via the comet assay in mouse leukocytes following 28 

### an oral dose as low as 0.21 mg/kg, an effect that i ncreased with dose up to 9.5 mg/kg and did not caus e a 29 

### decrease in cell viability. Similarly, Wang et al. (2006) found a dose-dependent increase in DNA 30 

### damage in peripheral lymphocytes using the comet as say that was found to persist for five days post- 31

### 118 DRAFT - DO NOT CITE OR QUOTE

exposure and was accompanied by a significant incre ase in reactive oxygen species and apoptosis in the 1 

### liver. Sekihashi et al. (2001) also found comet da mage in mouse stomach, colon, liver, kidney, bladde r, 2 

### lung, and brain following one gavage dose of 85.7 m g/kg. These effects were not accompanied by 3 

### cytotoxicity, although it is unknown whether a resp onse to dose would have occurred as only one dose 4 

### was administered. Coogan et al. (1991b) observed D NA−protein crosslinks in liver following 3 and 6 5 

### week exposures via drinking water in rats; no cytot oxicity was found in these animals. 6 

### Results of most studies evaluating hexavalent chrom ium-induced chromosomal damage in bone 7 

### marrow or peripheral blood cells following oral exp osure were negative (NTP, 2007; De Flora et al., 8 

### 2006; Mirsalis et al., 1996; Shindo et al., 1989). However, chromosomal damage (as indicated by 9 

### micronuclei formation) was observed in peripheral r ed blood cells of one strain of mice ( am3 -C57BL/6) 10 

### exposed to ≥ 21.8 mg hexavalent chromium/kg-day as sodium dichro mate dihydrate in drinking water for 11 

### 3 months, but not in B6C3F 1 

### or BALB/c mice at daily doses up to 87.2 mg hexava lent chromium/kg-day 12 

### (NTP, 2007). 13 

### Oral exposure studies evaluating the mutagenicity o f hexavalent chromium in tissues from the GI 14 

### tract are of particular relevance in light of the r esults of the NTP (2008) cancer bioassay showing 15 

### neoplasms of the oral cavity in rats (at 5.9–7.0 mg hexavalent chromium/kg-day) and of the small 16 

### intestine in mice (at 2.4–3.1 mg hexavalent chromiu m/kg-day) administered sodium dichromate 17 

### dihydrate in drinking water for 2 years. In ddY mi ce, positive results were reported for DNA damage a s 18 

### measured by the comet assay in the stomach and colo n following gavage administration of a single high 19 

### dose of hexavalent chromium (85.7 mg hexavalent chr omium/kg) (Sekihashi et al., 2001). This dose is 20 

### at least 12-fold greater than chronic dosages assoc iated with oral and GI neoplasms in rats and mice 21 

### (NTP, 2008), although no concurrent cytotoxicity wa s found. Data on the potential for DNA damage in 22 

### cells of the GI tract at lower oral doses (e.g., th ose in the range of the NTP [2008] bioassay) are no t 23 

### available. Negative results were reported for oxid ative DNA damage and DNA-protein crosslinks in 24 

### cells of the forestomach, glandular stomach, and du odenum of female SKH-1 mice administered 25 

### drinking water containing 5 or 20 mg hexavalent chr omium/L (approximately equivalent to 1.20 and 26 

### 4.82 mg hexavalent chromium/kg-day, respectively) a s sodium dichromate dihydrate for 9 months (De 27 

### Flora et al., 2008). The lack of effects seen in D eFlora et al. (2008) are peculiar given that the hi ghest 28 

### dose evaluated in this study is slightly less than chronic dosages associated with neoplasms of the or al 29 

### cavity in rats (5.9–7.0 mg hexavalent chromium/kg-d ay), and slightly greater than those associated wit h 30 

### neoplasms of the small intestine in mice (2.4–3.1 m g hexavalent chromium/kg-day) (NTP, 2008). No 31

### 119 DRAFT - DO NOT CITE OR QUOTE

oral exposure studies on the potential clastogenic activity of hexavalent chromium in oral mucosal or GI 1 

### cells were identified. Although the NTP (2007) 3-m onth drinking water study evaluated micronuclei 2 

### formation in peripheral red blood cells of mice (wi th positive results in the am3 -C57BL/6 strain, as 3 

### discussed above), mutagenic effects of hexavalent c hromium exposure in GI tissues were not evaluated 4 

### in this study. 5 

### Results of parenteral exposure studies are uniforml y positive for hexavalent chromium-induced 6 

### mutagenicity. Following parenteral exposure, DNA d amage has been observed in numerous tissues, 7 

### including peripheral lymphocytes, stomach, colon, l iver, kidney, bladder, lung, and brain (Patlolla an d 8 

### Tchounwou, 2006; Sekihashi et al., 2001; Ueno et al ., 2001); mutations have been observed in liver 9 

### (Knudsen, 1980); and chromosomal damage (micronucle i) has been observed in peripheral erythrocytes 10 

### and bone marrow (De Flora et al., 2006; Itoh and Sh imada, 1997; Shindo et al., 1989; Hayashi et al., 11 

### 1982; Wild, 1978). 12 

### Mutagenic activity of hexavalent chromium has also been demonstrated in lung cells of animals 13 

### following intratracheal exposure. DNA damage (DNA fragmentation, DNA-protein crosslinks and 14 

### DNA adducts) was reported in lung cells of Sprague- Dawley rats administered 0.09 mg hexavalent 15 

### chromium/kg by intratracheal instillation for 3 day s (Izzotti et al., 1998) and mutations were reporte d in 16 

### lung cells of C57BL/6 mice administered a single in tratracheal dose of 7.65 mg hexavalent 17 

### chromium/kg. Results of these studies are relevant to occupational exposure studies showing increased 18 

### respiratory tract cancers in hexavalent chromium wo rkers (see Section 4.4.1.2). No inhalation or 19 

### intratracheal exposure studies on the potential cla stogenic activity of hexavalent chromium in respira tory 20 

### tract cells were identified. Chromosomal damage (c hromosome aberrations and sister chromatid 21 

### exchange) was observed in peripheral lymphocytes, b ut not bone marrow, of Sprague-Dawley rats 22 

### exposed to chromium fumes for 1 week (1.84 mg/m 3 

### ) or 2 months (0.55 mg/m 3 

### ) (Koshi et al., 1987). 23

24120 DRAFT - DO NOT CITE OR QUOTE1Table 4-21. In vitro genotoxicity studies of hexavalent chromium in non-mammalian cellsResultsaEndpoint Chemical form Test systemWithoutactivationWithactivationReferenceReverse mutations Ammonium chromateS. typhimuriumTA97,TA1538, TA98, TA100+ NSDeFlora et al., 1984Reverse mutations Ammonium chromateS. typhimuriumTA1537 ± NS DeFlora et al., 1984Reverse mutations Ammonium chromateS. typhimuriumTA1535 – – DeFlora et al., 1984Reverse mutations Calcium chromateS. typhimuriumTA97,TA1538, TA98, TA100+ NSDeFlora et al., 1984Reverse mutations Calcium chromateS. typhimuriumTA1537 ± NS DeFlora et al., 1984Reverse mutations Calcium chromateS. typhimuriumTA1535 – – DeFlora et al., 1984Reverse mutations Calcium chromateS. typhimuriumTA98 – ± Dunkel et al., 1984Reverse mutations Calcium chromateS. typhimuriumTA100,TA1535, TA1537, TA1538– –Dunkel et al., 1984Reverse mutations Calcium chromateE. coliWP2 uvrA – ± Dunkel et al., 1984Reverse mutations Chromic acidS. typhimuriumTA102,TA2638+ ND Watanabe et al., 1998Reverse mutations Chromic acidE. coli, WP2/pKM101, WP2uvrA/pKM101+ NDWatanabe et al., 1998Reverse mutations Chromium trioxideS. typhimuriumTA97,TA1538, TA98, TA100+ NSDeFlora et al., 1984Reverse mutations Chromium trioxideS. typhimuriumTA1537± NS DeFlora et al., 1984Reverse mutations Chromium trioxideS. typhimuriumTA1535– – DeFlora et al., 1984Reverse mutations Potassium chromateS. typhimuriumTA102 + ND Marzin and Phi, 1985Reverse mutations Potassium chromateS. typhimuriumTA97,TA1538, TA98, TA100+ NSDeFlora et al., 1984Reverse mutations Potassium chromateS. typhimuriumTA1537± NS DeFlora et al., 1984Reverse mutations Potassium chromateS. typhimuriumTA1535– – DeFlora et al., 1984Reverse mutations Potassium chromateE. coliHs30R + ND Nakamuro et al., 1978121 DRAFT - DO NOT CITE OR QUOTETable 4-21. In vitro genotoxicity studies of hexavalent chromium in non-mammalian cellsResultsaEndpoint Chemical form Test systemWithoutactivationWithactivationReferenceReverse mutations Potassium chromateE. coliWp2 hcr- try-,B/rWP2+ (Wp2 hcr) NDKanematsu et al., 1980Reverse mutations Potassium chromateE. coliWp2(try-) + ND Venitt and Levy, 1974Reverse mutations Potassium chromateE. coliWP2uvrA, CM571 + ND Seo and Lee, 1993Reverse mutations Potassium dichromateS. typhimuriumTA97, TA98,TA100, TA1535, TA1537+ +Zeiger et al., 1992Reverse mutations Potassium dichromateS. typhimuriumTA102 + ND Marzin and Phi, 1985Reverse mutations Potassium dichromateS. typhimuriumTA100 + + Venier et al., 1982Reverse mutations Potassium dichromateE. coliWp2 hcr- try-,B/rWP2+ (Wp2 hcr) NDKanematsu et al., 1980Reverse mutations Potassium dichromateE. coliHs30R + ND Nakamuro et al., 1978Reverse mutations Potassium dichromateE. coliWP2, WP2uvrA,CM571+ ND Nishioka, 1975Reverse mutations Potassium dichromateE. coliWP2uvrA, CM571 + ND Seo and Lee, 1993Reverse mutations Potassium dichromateS. cerevisiaeD7 + ND Sing, 1983Reverse mutations Potassium dichromateS. typhimuriumTA98 ± – Venier et al., 1982Reverse mutations Potassium dichromateS. typhimuriumTA1538 – – Venier et al., 1982Reverse mutations Sodium chromateE. coliWp2(try-) + ND Venitt and Levy, 1974Reverse mutations Sodium dichromateS. typhimuriumTA102,TA2638+ ND Watanabe et al., 1998Reverse mutations Sodium dichromateS. typhimuriumTA102+ +Bennicelli et al., 1983Reverse mutations Sodium dichromateS. typhimuriumTA100 + – DeFlora, 1978Reverse mutations Sodium dichromateS. typhimuriumTA97+ NS DeFlora et al., 1984Reverse mutations Sodium dichromateS. typhimuriumTA1537,TA1538, TA98, TA100± NSDeFlora et al., 1984Reverse mutations Sodium dichromateS. typhimuriumTA1535– – DeFlora et al., 1984122 DRAFT - DO NOT CITE OR QUOTETable 4-21. In vitro genotoxicity studies of hexavalent chromium in non-mammalian cellsResultsaEndpoint Chemical form Test systemWithoutactivationWithactivationReferenceReverse mutations Sodium dichromateE. coli, WP2/pKM101, WP2uvrA/pKM101+ NDWatanabe et al., 1998Reverse mutations Sodium dichromatedihydrateS. typhimuriumTA102,TA2638a+ –Ryden et al., 2000Reverse mutations Sodium dichromatedihydrateS. typhimuriumTA100, TA98+ +NTP, 2007Reverse mutations Sodium dichromatedihydrateE. coli, WP2 uvrA/pKM101+ +NTP, 2007Induction of SOSresponseChromic acidE. coliAB1157, GC2375,UA4202, PQ30+ NDLlagostera et al., 1986Induction of SOSresponsePotassium chromateE. coliPQ37, PQ35+ –Olivier and Marzin, 1987Induction of SOSresponsePotassium chromateE. coliAB1157, GC2375,UA4202, PQ30+ NDLlagostera et al., 1986Induction of SOSresponsePotassium dichromateE. coliAB1157, GC2375,UA4202, PQ30+ NDLlagostera et al., 1986Induction of SOSresponsepotassium dichromateE. coliPQ37, PQ35+ –Olivier and Marzin, 1987Mutations Ammonium chromateS. typhimuriumTA1978(rec+), TA1538 (rec - )+ ND Gentile et al., 1981Mutations Ammonium chromateB. subtilis+ ND Gentile et al., 1981Mutations Calcium chromateS. typhimuriumTA97, TA98,TA100– –Brams et al., 1987Mutations Chromic acidS. typhimuriumTA1978(rec+), TA1538 (rec - )+ ND Gentile et al., 1981Mutations Chromic acid B. subtilis + ND Gentile etal., 1981Mutations Potassium chromateS. typhimuriumTA98,TA100, TA1537+ NDArlauskas et al., 1985Mutations Potassium chromateS. typhimuriumTA100 + ND Arlauskas et al., 1985123 DRAFT - DO NOT CITE OR QUOTETable 4-21. In vitro genotoxicity studies of hexavalent chromium in non-mammalian cellsResultsaEndpoint Chemical form Test systemWithoutactivationWithactivationReferenceMutations Potassium chromateE. coliWP2 uvrA pKm 101 + ND Arlauskas et al., 1985Mutations Potassium chromateB. subtilisH17 + ND Nishioka, 1975Mutations Potassium chromateS. typhimuriumTA1535,TA1538– NDArlauskas et al., 1985Mutations Potassium dichromateS. typhimuriumTA 1535pSK1002+ + Yamamoto et al., 2002Mutations Potassium dichromateS. typhimuriumTA100,TA1025, TA98+ NDLe Curieux et al., 1993Mutations Potassium dichromateS. typhimuriumTA1978(rec+), TA1538 (rec - )+ ND Gentile et al., 1981Mutations Potassium dichromateE. coliWP2uvrA + ND Venier et al., 1987Mutations Potassium dichromateB. subtilis+ ND Gentile et al., 1981Mutations Sodium dichromateB. subtilis+ ND Gentile et al., 1981Mutations Potassium dichromateB. subtilisNIG45, NIG17 + ND Matsui, 1980Mutations Potassium dichromateB. subtilisH17 + ND Nishioka, 1975Mutations Sodium dichromateS. typhimuriumTA1978(rec+),TA1538 (rec - )+ ND Gentile et al., 1981Frame shift mutations Calcium chromateS. typhimuriumTA98,TA1537+ ND Haworth et al., 1983Frame shift mutation Potassium chromateS. typhimuriumTA 1537 + ND La Velle, 1986Frame shift mutation Potassium chromateE. coli343/358, /415, /435,/477+ NDLa Velle, 1986Frame shift mutations Potassium dichromateS. typhimuriumTA97a, TA98+ + Tagliari et al., 2004Frame shift mutations Potassium dichromateS. typhimuriumTA100,TA1537, TA1538– ND Kanematsu et al., 1980Frame shift mutations Sodium dichromateS. typhimuriumTA97,TA1978+ NDBennicelli et al., 1983124 DRAFT - DO NOT CITE OR QUOTETable 4-21. In vitro genotoxicity studies of hexavalent chromium in non-mammalian cellsResultsaEndpoint Chemical form Test systemWithoutactivationWithactivationReferenceFrame shift mutations Sodium dichromateS. typhimuriumTA1537,TA1538– NDBennicelli et al., 1983Frame shift mutations,base pair substitutionsCalcium chromateS. typhimuriumTA1537,TA98, TA100+ + Petrilli and De Flora, 1977Frame shift mutations,base pair substitutionsCalcium chromateS. typhimuriumTA1535 – – Petrilli and De Flora, 1977Frame shift mutations,base pair substitutionsChromic acidS. typhimuriumTA1537,TA98, TA100+ + Petrilli and De Flora, 1977Frame shift mutations,base pair substitutionsChromic acidS. typhimuriumTA1535 – – Petrilli and De Flora, 1977Frame shift mutations,base pair substitutionsPotassium chromateS. typhimuriumTA1537,TA98, TA100+ + Petrilli and De Flora, 1977Frame shift mutations,base pair substitutionsPotassium chromateS. typhimuriumTA1535 – – Petrilli and De Flora, 1977Frame shift mutations,base substitutionsPotassium dichromateS. typhimuriumTA98 TA100,TA1535, TA1538+ + Bianchi et al., 1983Frame shift mutations,base pair substitutionsSodium dichromateS. typhimuriumTA1537,TA98, TA100+ + Petrilli and De Flora, 1977Frame shift mutations,base pair substitutionsSodium dichromateS. typhimuriumTA1535 – – Petrilli and De Flora, 1977Base pair substitutions Ammonium chromateS. typhimuriumTA1537,TA1538, TA98, TA100± NSDeFlora et al., 1984;DeFlora, 1981Base pair substitutions Ammonium chromateS. typhimuriumTA1535 – – DeFlora, 1981Base pair substitutions Calcium chromateS. typhimuriumTA100,TA1535+ ND Haworth et al., 1983Base pair substitutions Calcium chromateS. typhimuriumTA1537,TA1538, TA98, TA100± NSDeFlora et al., 1984;DeFlora, 1981Base pair substitutions Calcium chromateS. typhimuriumTA1535 – – DeFlora, 1981Base pair substitutions Chromic acidS. typhimuriumTA1537,TA1538, TA98, TA100± NS DeFlora et al., 1984;DeFlora, 1981125 DRAFT - DO NOT CITE OR QUOTETable 4-21. In vitro genotoxicity studies of hexavalent chromium in non-mammalian cellsResultsaEndpoint Chemical form Test systemWithoutactivationWithactivationReferenceBase pair substitutions Chromic acidS. typhimuriumTA1537,TA1538, TA98, TA100± NS DeFlora et al., 1984;DeFlora, 1981Base pair substitutions Potassium chromateS. typhimuriumTA1537,TA1538, TA98, TA100± NS DeFlora et al., 1984;DeFlora, 1981Base pair substitutions Potassium chromateS. typhimuriumTA1535 – – DeFlora, 1981Base pair substitutions Potassium dichromateS. typhimuriumTA100,TA102+ + Tagliari et al., 2004Base pair substitutions Potassium dichromateS. typhimuriumTA100,TA1535– ND Kanematsu et al., 1980Base pair substitutions Sodium dichromateS. typhimuriumTA100,TA102, TA92+ ND Bennicelli et al., 1983Base pair substitutions Sodium dichromateS. typhimuriumTA1537,TA1538, TA98, TA100± NS DeFlora, 1981, DeFlora etal., 1984Base pair substitutions Sodium dichromateS. typhimuriumTA1535 – – DeFlora, 1981Base pair substitutions Sodium dichromateS. typhimuriumTA1535 – ND Bennicelli et al., 1983Reverse mutation,induction of geneconversionPotassium dichromate S.cerevisiaeD7 + ND Kharab and Singh, 1985Forward mutation Potassium dichromateSchizosaccharomyces pombe972, h-± ND Bonatti et al., 1976Mitotic cross-over Chromic acidS. cerevisiaeD7 + ND Fukunaga et al., 1982Mitotic geneconversionsChromic acidS. cerevisiaeD7 + ND Singh, 1983; Fukunaga etal., 1982Mitotic gene conversion,point reverse mutationSodium chromateS. cerevisiaeD7 + ND Bronzetti and Galli, 1989Mitotic gene conversionat trp5 locus, reversemutation of ilvl-92 alleleChromic acidS. cerevisiaeD7 + ND Vashishat and Vasudeva,1987126 DRAFT - DO NOT CITE OR QUOTETable 4-21. In vitro genotoxicity studies of hexavalent chromium in non-mammalian cellsResultsaEndpoint Chemical form Test systemWithoutactivationWithactivationReferenceMitotic gene conversionat trp5 locus, reversemutation of ilvl-92 allelePotassium dichromateS.cerevisiaeD7 + ND Vashishat and Vasudeva,1987Induction of disomic anddiploid sporesPotassium dichromateS. cerevisiaeD1S13 + ND Sora et al., 1986umugene expression Potassium dichromateS. typhimuriumTA1535 ± – Nakamura et al., 1987DNA damage Potassium dichromateE. coliPQ37 + ND Le Curieux et al., 1993DNA-protein crosslinks Potassium chromateE. coliDNA – ND Fornace et al., 1981DNA polymerase arrest Sodium dichromate PSV2neo-based plasmidDNA– + Bridgewater et al., 1998,1994a+ = positive, ± = equivocal or weakly positive, – =negative, ND = no data; NS = not specified.1127 DRAFT - DO NOT CITE OR QUOTETable 4-22. In vitro genotoxicity studies of hexavalent chromium in mammalian cellsResultsaEndpointChemicalform Test systemWithoutactivationWithactivationReferenceDNA damage PotassiumdichromateHuman lymphocytes + ND Blasiak and Kowalik, 2000DNA damage PotassiumdichromateHuman gastric mucosa + ND Trzeciak et al., 2000DNA damage PotassiumdichromateHuman peripheral blood lymphocytes + ND Trzeciak etal., 2000DNA damage PotassiumdichromateHuman lymphocytes, humanlymphoblastoid TK-6 cells+ ND Cemeli et al., 2003DNA damage SodiumdichromateHuman gastric mucosa cells, Rat gastricmucosa cells+ ND Pool-Zobel et al., 1994DNA adducts, [32P] postlabelingPotassiumchromateCalf thymus DNA ––(+1 mMH2O2)Adams et al., 1996DNA fragmentation PotassiumchromateHuman bronchial epithelial cells + ND Fornace et al., 1981DNA fragmentation PotassiumchromateHuman embryonic lung fibroblasts (IMR90)+ ND Fornace et al., 1981DNA fragmentation PotassiumchromateMouse L1210 leukemia cells + ND Fornace et al., 1981DNA fragmentation sodiumchromateChinese hamster ovary cells + ND Blankenship et al., 1997DNA strand breaks PotassiumdichromateVero kidney fibroblsts, Pam 212keratinocytes+ ND Flores and Perez, 1999DNA strand breaks SodiumdichromateRat primary lymphocytes + ND Gealy et al., 2007DNA strand breaks SodiumdichromateRat hepatocytes + ND Gao et al., 1993DNA strand breaks PotassiumchromateHuman lymphocytes + ND Depault et al., 2006128 DRAFT - DO NOT CITE OR QUOTETable 4-22. In vitro genotoxicity studies of hexavalent chromium in mammalian cellsResultsaEndpointChemicalform Test systemWithoutactivationWithactivationReferenceDNA strand breaks PotassiumchromateHuman fibroblast + ND Fornace 1982DNA strand breaks PotassiumchromateBacteriophageλDNA + +(+1mMH2O2)Adams et al., 1996DNA strand breaks SodiumdichromateRat primary lymphocytes + ND Elia et al., 1994DNA strand breaks PotassiumdichromateHuman lymphocytes, human gastricmucosa cells+ ND Blasiak et al., 1999DNA-DNA crosslinks SodiumchromateHuman lung fibroblasts + ND Xu et al., 1996DNA-protein crosslinks PotassiumchromateHuman embryonic lung fibroblasts (IMR90)+ ND Fornace et al., 1981DNA-protein crosslinks PotassiumchromateHuman fibroblast + ND Fornace, 1982DNA-protein crosslinks PotassiumchromateChinese hamster cells (V79-UL) + ND Merk et al., 2000DNA-protein crosslinks PotassiumchromateMouse L1210 leukemia cells + ND Fornace et al., 1981DNA-protein crosslinks SodiumchromateHuman HL-60 cells + ND Capellmann et al., 1995Induced DNA methylationPotassiumchromateChinese hamster V79 cells (hpr-1gpt+transgenic cell line G12)+ (T) ND Klein et al., 2002Unscheduled DNAsynthesisSodiumdichromateRat hepatocytes + (T) ND Gao et al., 1993DNA synthesis inhibition PotassiumchromateHeLa S3 cells + ND Heil and Reifferscheid, 1992DNA synthesis inhibition PotassiumdichromateMouse L cells + ND Nishio and Uyeki, 1985129 DRAFT - DO NOT CITE OR QUOTETable 4-22. In vitro genotoxicity studies of hexavalent chromium in mammalian cellsResultsaEndpointChemicalform Test systemWithoutactivationWithactivationReferenceDNA polymerase arrest SodiumchromateHuman lung fibroblasts + ND Xu et al., 1996Mutations at the HGPRTlocusPotassiumdichromateChinese hamster ovary cells (AT3-2) + ND Paschin etal., 1983Mutations at the HGPRTlocusPotassiumdichromateChinese hamster cells (V79) + ND Paschin et al., 1983Forward mutation CalciumchromateMouse lymphoma cells (L5178Y tk+/tk-) + + McGregor et al., 1987Forward mutation CalciumchromateMouse lymphoma cells (L5178Y tk+) + + Mitchell et al., 1988Forward mutation CalciumchromateMouse lymphoma cells (L5178Y tk+) + + Myhr and Caspary, 1988Forward mutation CalciumchromateMouse lymphoma cells (L5178Y tk+) + + Oberly et al., 1982MorphologicaltransformationCalciumchromateSyrian hamster embryo cells + ND Elias et al., 1991MorphologicaltransformationSodiumchromatedihydrateSyrian hamster cells + ND DiPaolo and Casto, 1979Cell transformation CalciumchromateBalb/3T3, Syrian hamster embryo,R-MuLV-RE cells+ ND Dunkel et al., 1981Transformations PotassiumchromateRat liver epithelial cells + ND Briggs and Briggs,1988Chromosomal damage CalciumchromateChinese hamster ovary cells + ND Levis and Majone,1979Chromosomal damage Chromic acid Chinese hamster ovary cells + ND Levis and Majone, 1979Chromosomal damage PotassiumchromateChinese hamster ovary cells + ND Levis and Majone,1979Chromosomal damage PotassiumdichromateChinese hamster ovary cells + ND Seoane and Dulout,1999130 DRAFT - DO NOT CITE OR QUOTETable 4-22. In vitro genotoxicity studies of hexavalent chromium in mammalian cellsResultsaEndpointChemicalform Test systemWithoutactivationWithactivationReferenceChromosomal damage PotassiumdichromateChinese hamster ovary cells + ND Levis and Majone,1979Chromosomal damage PotassiumdichromateChinese hamster ovary cells + ND Majone and Levis,1979Chromosomal damage SodiumchromateChinese hamster ovary cells + ND Levis and Majone,1979Chromosomal damage SodiumdichromateChinese hamster ovary cells + ND Levis and Majone,1979Chromosomal damage SodiumdichromateChinese hamster ovary cells + ND Majone and Levis,1979Chromosome aberrations CalciumchromateChinese hamster lung DON cells + ND Koshi and Iwaski 1983;Koshi 1979Chromosome aberrations CalciumchromateChinese hamster ovary cells (C3H10T1/2) + ND Sen etal., 1987Chromosome aberrations Chromic acid BHK and Chinesehamster ovary cells + ND Bianchi et al., 1980Chromosome aberrations Chromic acid Mouse mammary FM3A carcinoma cells + ND Umeda and Nishmura, 1979Chromosome aberrations Chromic acid Chinese hamsterlung DON cells + ND Koshi and Iwaski 1983;Koshi 1979Chromosome aberrations PotassiumchromateHuman fibroblasts + ND MacRae et al., 1979Chromosome aberrations PotassiumchromateChinese hamster lung DON cells + ND Koshi and Iwaski 1983;Koshi 1979Chromosome aberrations PotassiumchromateChinese hamster ovary cells + ND MacRae et al., 1979Chromosome aberrations PotassiumchromateBHK and Chinese hamster ovary cells + ND Bianchi etal., 1980Chromosome aberrations PotassiumchromateMouse mammary FM3A carcinoma cells + ND Umeda and Nishmura, 1979Chromosome aberrations PotassiumdichromateHuman fibroblasts + ND MacRae et al., 1979131 DRAFT - DO NOT CITE OR QUOTETable 4-22. In vitro genotoxicity studies of hexavalent chromium in mammalian cellsResultsaEndpointChemicalform Test systemWithoutactivationWithactivationReferenceChromosome aberrations PotassiumdichromateChinese hamster ovary cells + ND MacRae et al., 1979Chromosome aberrations PotassiumdichromateBHK and Chinese hamster ovary cells + ND Bianchi etal., 1980Chromosome aberrations PotassiumdichromateMouse mammary FM3A carcinoma cells + ND Umeda and Nishmura, 1979Chromosome aberrations SodiumchromateHuman primary bronchial fibroblasts + ND Wise et al., 2004, 2002Chromosome aberrations SodiumchromateHuman bronchial fibroblasts (WTHBF6 cells)+ ND Holmes et al., 2006Chromosome aberrations SodiumchromateHuman bronchial epithelial cells (BEP2Dcells)+ ND Wise et al., 2006aChromosome aberrations SodiumchromateChinese hamster ovary cells + ND Blankenship et al., 1997Chromosome aberrations SodiumchromateChinese hamster ovary cells (AA8(parental), EM9 (XRCC1 mutant), andH9T3+ ND Grlickova-Duzevik, 2006Chromosome aberrations SodiumdichromateBHK and Chinese hamster ovary cells + ND Bianchi etal., 1980Chromosome andchromatid aberrationsPotassiumdichromateHuman lymphocytes + ND Imreh and Radulescu, 1982Sister chromatidexchangesCalciumchromateHuman lymphocytes + ND Gomez-Arroyo et al., 1981Sister chromatidexchangseCalciumchromateChinese hamster lung DON cells + ND Koshi and Iwaski 1983;Koshi 1979Sister chromatidexchangesCalciumchromateChinese hamster ovary cells + ND Levis and Majone,1979Sister chromatidexchangesChromic acid Chinese hamster ovary cells + ND Levisand Majone, 1979132 DRAFT - DO NOT CITE OR QUOTETable 4-22. In vitro genotoxicity studies of hexavalent chromium in mammalian cellsResultsaEndpointChemicalform Test systemWithoutactivationWithactivationReferenceSister chromatidexchangesChromic acid Chinese hamster cells DON + ND Ohno etal., 1982Sister chromatidexchangesChromic acid Chinese hamster lung DON cells + ND Koshi and Iwaski 1983;Koshi 1979Sister chromatidexchangesChromic acid BHK and Chinese hamster ovary cells +ND Bianchi et al., 1980Sister chromatidexchangesPotassiumchromateHuman fibroblasts + ND MacRae et al., 1979Sister chromatidexchangesPotassiumchromateChinese hamster lung DON cells + ND Koshi and Iwaski 1983;Koshi 1979Sister chromatidexchangesPotassiumchromateChinese hamster ovary cells + ND Levis and Majone,1979Sister chromatidexchangesPotassiumchromateChinese hamster ovary cells + ND MacRae et al., 1979Sister chromatidexchangesPotassiumchromateChinese hamster cells DON + ND Ohno et al., 1982Sister chromatidexchangesPotassiumchromateBHK and CHO cells + ND Bianchi et al., 1980Sister chromatidexchangesPotassiumdichromateHuman lymphocytes + ND Gomez-Arroyo et al., 1981Sister chromatidexchangesPotassiumdichromateHuman lymphocytes + ND Imreh and Radulescu, 1982Sister chromatidexchangesPotassiumdichromateHuman fibroblasts + ND MacRae et al., 1979Sister chromatidexchangesPotassiumdichromateChinese hamster ovary cells + ND Levis and Majone,1981Sister chromatidexchangesPotassiumdichromateChinese hamster ovary cells + ND Levis and Majone,1979Sister chromatidexchangesPotassiumdichromateChinese hamster ovary cells + ND Majone and Levis,1979133 DRAFT - DO NOT CITE OR QUOTETable 4-22. In vitro genotoxicity studies of hexavalent chromium in mammalian cellsResultsaEndpointChemicalform Test systemWithoutactivationWithactivationReferenceSister chromatidexchangesPotassiumdichromateChinese hamster ovary cells + ND MacRae et al., 1979Sister chromatidexchangesPotassiumdichromateChinese hamster cells DON + ND Ohno et al., 1982Sister chromatidexchangesPotassiumdichromateBHK and Chinese hamster ovary cells + ND Bianchi etal., 1980Sister chromatidexchangesPotassiumdichromateMouse blastocysts + ND Iijima et al., 1983Sister chromatidexchangesSodiumchromateChinese hamster ovary cells + ND Levis and Majone,1979Sister chromatidexchangesSodiumchromateBHK and Chinese hamster ovary cells + ND Bianchi etal., 1980Sister chromatidexchangesSodiumdichromateChinese hamster ovary cells + ND Levis and Majone,1979Sister chromatidexchangesSodiumdichromateChinese hamster ovary cells + ND Majone and Levis,1979Sister chromatidexchangesSodiumdichromateBHK and Chinese hamster ovary cells + ND Bianchi etal., 1980Disruption of mitosis SodiumchromateHuman bronchial fibroblasts (WTHBF-6cells)+ ND Wise et al., 2006ba+ = positive, ± = equivocal or weakly positive, –= negative, (T) = toxicity, ND = no data.1134 DRAFT - DO NOT CITE OR QUOTE1Table 4-23. In vivo genotoxicity studies of hexavalent chromium inD. melanogasterand laboratorymammalsEndpointChemicalTest systemTest conditionsResultsaDosebReferenceDNA damage,Comet assayPotassiumchromateDNA damage instomach, colon,bladder, lung,brain; but not inliver, kidney orbone marrowParenteral exposure: ddY mice were administeredsingle intraperitoneal doses of potassium chromateof 0 or 120 mg/kg (0 or 32.1 mg hexavalentchromium/kg).The cells from the stomach, colon,liver, kidney, bladder, lung, brain and bonemarrow were collected 3, 8, and 24 hours aftertreatment and analyzed for DNA damage using thecomet assay+ 32.1 mg hexavalentchromium/kg(intraperitoneal)Sekihashi etal., 2001DNA damage,Comet assayPotassiumchromateDNA damage instomach, colon,liver, kidney,bladder, lung, andbrain; but not inbone marrowOral exposure: ddY mice were administeredsingle gavage doses of potassium chromate of 0 or320 mg/kg (0 or 85.7 mg hexavalentchromium/kg). The cells from the stomach, colon,liver, kidney, bladder, lung, brain, and bonemarrow were collected 3, 8, and 24 hours aftertreatment and analyzedfor DNA damage using thecomet assay.+ 85.7 mg hexavalentchromium/kg(gavage)Sekihashi etal., 2001DNA damage,Comet assayPotassiumdichromateDNA damage inperipherallymphocytesOral exposure: Swiss albino mice wereadministered potassium dichromate by gavage atdoses of 0, 25, 50, and 100 mg/kg for 1 day ordaily for 5 consecutive days (0, 8.8, 17.7, and35.4 mg hexavalent chromium/kg).+ 8.8 mg hexavalentchromium/kg(gavage)Wang et al.,2006DNA damage,Comet assayPotassiumdichromateMouse leukocytes Oral exposure: Swiss albino mice wereadministered potassium dichromate by singlegavage doses of 0, 0.59, 1.19, 2.38, 4.75, 9.5, 19,38, or 76 mg/kg (0, 0.21, 0.42, 0.84, 1.68, 3.37,6.7, 13.5, or 26.9 mg hexavalent chromium/kg).Samples of whole blood were collected at 24, 48,72, and 96 hour, and 1 and 2 week post-treatmentfor alkaline SCGE comet assay analysis ofleukocytes.+ 0.21 mg hexavalentchromium/kg-bodyweight(gavage)Devi et al.,2001DNA damage,Comet assayPotassiumdichromateDNA damage inliver and kidney,but not in spleen,lung, or brainParenteral exposure: Male albino mice weretreated administered potassium dichromate as asingle intraperitoneal dose of 0 or 20 mghexavalent chromium/kg. Organs were removedand cells were collected for DNA strand breakanalysis by single-cell gel electrophoresis.+ 20 mg hexavalentchromium/kg(intraperitonealinjection)Ueno et al.,2001135 DRAFT - DO NOT CITE OR QUOTETable 4-23. In vivo genotoxicity studies of hexavalent chromium inD. melanogasterand laboratorymammalsEndpointChemicalTest systemTest conditionsResultsaDosebReferenceDNA damage,Comet assayPotassiumdichromateRat leukocytes Parenteral exposure: Sprague-Dawley rats wereadministered potassium dichromateintraperitoneally at doses of 2.5, 5, 7.5, and10 mg/kg-day for 5 days (0, 0.88, 1.77, 2.65, or3.54 mg hexavalent chromium/kg-day). Wholeblood was sampled at 24, 48, 72, and 96 hoursafter treatment for alkaline single-cell gelelectrophoresis analysis of leukocytes, Cometassay.+ 0.88 mg hexavalentchromium/kg-day(intraperitonealinjection)Patlolla andTchounwou,2006DNAalterationsSodiumdichromatedihydrateDNA-proteincrosslinks, DNAfragmentation, andDNA adducts inlung, but not liverIntratracheal exposure: Sprague-Dawley rats wereadministered intratracheal instillations of sodiumdichromate at a dose of 0 or 0.25 mg/kg for3 consecutive days (0 or 0.09 mg hexavalentchromium/kg). After last treatment, lung andlivers were removed to analyze for DNAfragmentation, DNA-protein crosslinks, andadducts by [32P] post-labeling.+ 0.09 mg hexavalentchromium/kg(intratrachealinstillation)Izzotti et al.,1998DNA-proteincrosslinksPotassiumchromateDNA-proteincrosslinks detectedin liver but notspleniclymphocytesOral exposure: Male Fischer 344 rats wereadministered potassium chromate in drinkingwater for 3 and 6 weeks at 100 and 200 ppmhexavalent chromium. Liver and spleniclymphocytes were examined for DNA-proteincrosslinks.+ 100 hexavalentchromium/L(drinking water)Coogan et al.,1991bDNA-proteincrosslinksSodiumdichromatedihydrateDNA-proteincrosslinks detectedin lung, liver, andkidney nucleiParenteral exposure: Male Sprague-Dawley ratswere given a single intraperitoneal injection of 20or 40 mg/kg sodium dichromate (7 or 14 mghexavalent chromium/kg). There was no controlgroup used in this study. Nuclei from the rightrenal cortex, the front hepatic lobe, and the wholelung were used for analysis.+ 7 mg hexavalentchromium/kg(intraperitonealinjection)Tsapakos etal., 1983136 DRAFT - DO NOT CITE OR QUOTETable 4-23. In vivo genotoxicity studies of hexavalent chromium inD. melanogasterand laboratorymammalsEndpointChemicalTest systemTest conditionsResultsaDosebReferenceOxidativeDNA damage,DNA proteincrosslinksSodiumdichromatedihydrateMouseforestomach,glandular stomach,and duodenumcellsOral exposure: Female SKH-1 hairless mice wereadministered sodium dichromate dihydrate indrinking water at concentrations of 0, 5, and 20mg hexavalent chromium/L for 9 months. Usingreference values for body weight (0.0353 kg) anddaily drinking water intake (0.0085 L/day) forfemale B6C3F1mice (U.S. EPA, 1988), doses of1.20 and 4.82 mg hexavalent chromium/kg-dayforthe 5 and 20 mg hexavalent chromium/L groups,respectively, were estimated. DNA-proteincrosslinks, and oxidative DNA damage (8-oxo2’deoxyguanosine) were measured in forestomach,glandular stomach, and duodenum cells.– 20 mg hexavalentchromium/L(drinking water)De Flora etal., 2008Suppressednuclear DNAsynthesisPotassiumdichromateMouse tubularrenal cellsParenteral exposure: Mice given singleintraperitoneal injection at a concentration of 15–30% of the LD50(unspecified) in a thymidineincorporation inhibiting screening system; anintraperitoneal injection of3H thymidine wasadministered 15 hours later.+ NSAmlacher andRudolph,1981UnscheduledDNA synthesisPotassiumdichromateRat hepatocytes Oral exposure: Fischer 344 rats wereadministered potassium dichromate atconcentrations of 0, 1, 5, or 20 mg hexavalentchromium/L in drinking water ad libitum, for 48hours, while a second group was administeredsingle gavage doses (20 mL/kg) at the sameconcentrations. Hepatocytes were collected fromthe rat livers and analyzed in the in vivo- in vitrohepatocyte DNA repair assay.– 20 mg hexavalentchromium/L(drinking water)Mirsalis etal., 1996Mutation PotassiumchromateMutations in liver,but not bonemarrow cellsParenteral exposure: Male lacZ transgenicMutaTMmice were administered potassiumchromate by an intraperitoneal dose of 0 or40 mg/kg once a day for 2 consecutive days (0 or14.1 mg hexavalent chromium/kg).+ 14.1 mg hexavalentchromium/kg(intraperitonealinjection)Itoh andShimada,1998, 1997137 DRAFT - DO NOT CITE OR QUOTETable 4-23. In vivo genotoxicity studies of hexavalent chromium inD. melanogasterand laboratorymammalsEndpointChemicalTest systemTest conditionsResultsaDosebReferenceMutation PotassiumchromateMouse offspring Parenteral exposure: Female C57BL/6J mice wereadministered potassium chromate byintraperitoneal injection at a dose of 0, 10, or20 mg/kg on days 8, 9, and 10 of pregnancy in amammalian spot test (0, 2.7, or 5.4 mg hexavalentchromium/kg). The offspring’s fur was checkedfor colored spots from week 2 through week 5after birth.+ 2.7 mg hexavalentchromium/kg(intraperitonealinjection)Knudsen,1980Mutations PotassiumdichromateMouse (C57BL/6 Big Blue) lungand kidney tissue,but not in livertissueIntratracheal exposure: Mice were given singledoses of potassium dichromate via intratrachealinstillation of 0 or 6.75 mg hexavalentchromium/kg and allowed 4 weeks for geneexpression. Isolated DNA samples from lung,liver, and kidney tissues were used for LacI genemutagenesis assay. Depletion of tissueglutathione by pretreatment with buthioninesulfoximine decreased the mutagenic response,suggesting that reduced glutathione plays a role inproducing reactive intermediates duringintracellular reduction of chromium (VI).+ 6.75 mg hexavalentchromium/kg(intratrachealinstillation)Cheng et al.,2000Gene mutation CalciumchromateD. melanogaster24-Hour old males were fed calcium chromate for72 hours at doses of 0, 500, or 750 ppm. Themales were removed and mated.+ 500 ppm(in diet)Zimmering etal., 1985Gene mutation Chromic acidD. melanogaster24–48-Hour old males were treated byintraperitoneal injection of 0, 100, 200, 300, and400 ppm potassium dichromate or 0, 100, 200, and300 ppm chromium trioxide. The F2 generationof flies was scored for sex-linked recessive lethal.+ 100 ppm(intraperitonealinjection)RodriguezArnaiz andMartinez,1986Gene mutation(wing somaticmutation)Chromium oxideD. melanogaster2–3-Day-old larvae were fed potassium chromateor chromium(VI) oxide for 3 days atconcentrations of 0, 1, or 5 mM.+ 1 mM(in diet)Graf andWurgler,1996Gene mutation(white-ivoryeye spot test)Chromium oxideD. melanogaster2–3-Day-old larvae were fed potassium chromateor chromium(VI) oxide for 2 days atconcentrations of 0, 1, or 5 mM.– 5 mM(in diet)Graf andWurgler,1996138 DRAFT - DO NOT CITE OR QUOTETable 4-23. In vivo genotoxicity studies of hexavalent chromium inD. melanogasterand laboratorymammalsEndpointChemicalTest systemTest conditionsResultsaDosebReferenceGene mutation PotassiumchromateD. melanogasterLarvae fed test substance in wing spot test atconcentrations of 0, 0.1, 0.5, 1.0, and 2.5 mM forthe duration of their development. Survivingtransheterozygous (mwh/flr3) and inversionheterozygous (mwh/TM3) flies were used.+ 0.1 mM(in diet)Amrani et al.,1999Gene mutation(wing somaticmutation)PotassiumchromateD. melanogaster2–3-Day-old larvae were fed potassium chromateor chromium(VI) oxide for 3 days atconcentrations of 0, 1 or 5 mM.+ 1 mM(in diet)Graf andWurgler,1996Gene mutation PotassiumchromateD. melanogaster3-Day-old larvae were fed potassium chromate for6 hours at concentrations ranging from 0 to 100mM or 48 hours at concentrations ranging from 0to 5.0 mM. Marker-heterozygous and balancerheterozygous wings from adult flies were thenexamined in the wing somatic mutation andrecombination test (SMART).+ 0.5 mM (48 hours)(in diet)5 mM (6 hours)(in diet)Spano et al.,2001Gene mutation(white-ivoryeye spot test)PotassiumchromateD. melanogaster2–3-Day-old larvae were fed potassium chromateor chromium(VI) oxide for 2 days atconcentrations of 0, 1 or 5 mM.– 5 mM(in diet)Graf andWurgler,1996Gene mutation PotassiumdichromateD. melanogaster3-Day-old transheterozygous larvae were fedpotassium dichromate at 0 or 0.5 mM andanalyzed for multiple wing hair and flare genemutations in the Drosophila wing SMART.+ 0.5 mM(in diet)Kaya et al.,2002Gene mutation PotassiumdichromateD. melanogasterLarvae fed test substance in wing spot test atconcentrations of 0, 0.1, 0.5, 1.0, and 2.5 mM forthe duration of their development. Survivingtransheterozygous (mwh/flr3) and inversionheterozygous (mwh/TM3) flies were used.+ 0.1 mM(in diet)Amrani et al.,1999Gene mutation PotassiumdichromateD. melanogaster24–48-Hour old males were treated byintraperitoneal injection of 0, 100, 200, 300, and400 ppm potassium dichromate or 0, 100, 200, and300 ppm chromium trioxide. The F2 generationof flies was scored for sex-linked recessive lethal.+ 100 ppm(intraperitonealinjection)RodriguezArnaiz andMartinez,1986139 DRAFT - DO NOT CITE OR QUOTETable 4-23. In vivo genotoxicity studies of hexavalent chromium inD. melanogasterand laboratorymammalsEndpointChemicalTest systemTest conditionsResultsaDosebReferenceGene mutation SodiumdichromateD. melanogasterLarvae were treated on filter papers soaked withsodium dichromate at doses of 1.17 and 2.34 mMfor 6 hours and then transferred to vials withsubstrate. Adult males were checked for wild-typepigmented spots in the eyes.+ 2.34 mM Rasmuson,1985DominantlethalityPotassiumdichromateMouse Parenteral exposure: CBA x C57Bl/6J hybridmale mice were treated with a singleintraperitoneal injection of 0, 0.5, 1.0, 2.0, 10,or20 mg/kg potassium dichromate (0, 0.18, 0.35,0.70, 3.5, or 7.1 mg hexavalent chromium/kg) orwith intraperitoneal injections of 0, 1.0, or2.0 mg/kg potassium dichromate daily for 21 days(0, 0.35, 0.70 mg hexavalent chromium/kg). Eachmale was mated with two untreated females for 7days, and then replaced by two more femalesevery 7 days for 4 consecutive weeks. Pregnantdams were sacrificed 12–14 days after conception.The frequency of dominant lethal mutations inmale mice was determined based on the postimplantation loss.++7.1 mg hexavalentchromium/kg(acuteintraperitonealinjection)0.7 mg hexavalentchromium/kg(repeatedintraperitonealinjection)Paschin et al.,1982Chromosomeaberrations,sisterchromatidexchangeChromiumfumes; no furtherinformation wasgiven andchromiumvalence state wasnot specifiedChromosomeaberrations andsister chromatidexchange in ratperipherallymphocytes, butnot in bonemarrow cellsInhalation exposure: Sprague-Dawley rats wereexposed to chromium fumes that were generatedfrom a plasma flame sprayer and chromium metalpowders at a concentration of 1.84 mg/m3for1 week (5 hour/day, 5 day/week) or 0.55 mg/m3for 2 months (5 hour/day, 5 day/week).Cytogenetic analysis was performed 20 hour,3 days, 7 days, and 1 month after the lastexposure.+ 1.84 mg/m3(1-week inhalationexposure)0.55 mg/m3(2-month inhalationexposure)Koshi et al.,1987Micronuclei PotassiumchromateMouse bonemarrow cellsOral exposure: Male MS/Ae and CD-1 mice wereadministered potassium chromate by singlegavage doses 0, 10, 20, 40, 80, 160, or 320 mg/kg(0, 3.5, 7.1, 14.1, 28.3, 56.6, or 113.1 mghexavalent chromium/kg).– 113.1 mghexavalentchromium/kg(gavage)Shindo et al.,1989140 DRAFT - DO NOT CITE OR QUOTETable 4-23. In vivo genotoxicity studies of hexavalent chromium inD. melanogasterand laboratorymammalsEndpointChemicalTest systemTest conditionsResultsaDosebReferenceMicronuclei PotassiumchromateMouse bonemarrow cellsParenteral exposure: Male MS/Ae and CD-1 micewere administered potassium chromate by singleintraperitoneal doses 0, 10, 20, 40, or 80 mg/kg (0,3.5, 7.1, 14.1, or 28.3 mg hexavalentchromium/kg).+ 14.1 mg hexavalentchromium/kg(intraperitonealinjection)Shindo et al.,1989Micronuclei PotassiumchromateMouse peripheralred blood cellsParenteral exposure: Male lacZ transgenicMutaTMmice were administered potassiumchromate by an intraperitoneal dose of 0 or40 mg/kg once a day for 2 consecutive days (0 or14.1 mg hexavalent chromium/kg).+ 14.1 mg hexavalentchromium/kg(intraperitonealinjection)Itoh andShimada,1997Micronuclei PotassiumchromateMouse bonemarrow cellsParenteral exposure: Slc:ddY mice wereadministered potassium chromate intraperitonealinjection once a day for 2 consecutive days atdoses of 0, 30, 40, and 50 mg/kg (0, 10.6, 14.1, or17.7 mg hexavalent chromium/kg).+ 10.6 mg hexavalentchromium/kg(intraperitonealinjection)Itoh andShimada,1996Micronuclei PotassiumchromateMS and ddYmouse bonemarrow cellsParenteral exposure: MS and ddY mice wereadministered potassium chromate by singleintraperitoneal doses of 0, 12.5, 25, or 50 mg/kg(0, 4.4, 8.8, or 17.7 mg hexavalent chromium/kg).+ 17.7 mg hexavalentchromium/kg(intraperitonealinjection)Hayashi etal., 1982Micronuclei PotassiumchromateMouse bonemarrowParenteral exposure: NMRI mice wereadministered potassium chromate by2 intraperitoneal injections with 24 hours betweeneach injection at doses of 0, 12.12, 24.25, or48.5 mg/kg (0, 3.2, 6.49, or 13.0 mg hexavalentchromium/kg).+ 13 mg hexavalentchromium/kg(intraperitonealinjection)Wild, 1978Micronuclei PotassiumdichromateMouse (BDF1)bone marrow cellsand peripheralblood cellsOral exposure: BDF1 male mice wereadministered potassium dichromate in drinkingwater at 0, 10, or 20 mg hexavalent chromium/Lfor 20 days.– 20 mg hexavalentchromium/L(drinking water)De Flora etal., 2006Micronuclei PotassiumdichromateMouse (BDF1)bone marrow cellsOral exposure: BDF1 male mice were given asingle doses of potassium dichromate by gavageof 0 or 50 mg hexavalent chromium/kg.– 50 mg hexavalentchromium/kg(gavage)De Flora etal., 2006141 DRAFT - DO NOT CITE OR QUOTETable 4-23. In vivo genotoxicity studies of hexavalent chromium inD. melanogasterand laboratorymammalsEndpointChemicalTest systemTest conditionsResultsaDosebReferenceMicronuclei PotassiumdichromateMouse bonemarrow cellsOral exposure: Swiss-Webster mice wereadministered potassium dichromate atconcentrations of 0, 1, 5, or 20 mg hexavalentchromium/L in drinking water. One set of micewas allowed access to drinking water ad libitum,for 48 hours, while a second group wasadministered two bolus doses (20 mL/kg) of thesame concentrations at 24 and 48 hours beforesacrifice. Bone marrow cells were collected fromthe femur of the mice.– 20 mg hexavalentchromium/L(drinking water)Mirsalis etal., 1996Micronuclei PotassiumdichromateMouse (Swiss)bone marrow—dams; fetal livercells andperipheral bloodcellsOral exposure: Pregnant Swiss albino mice wereadministered potassium dichromate in drinkingwater at concentrations of 0, 5, or 10 mghexavalent chromium/L throughout the duration ofpregnancy. Mice were sacrificed on day 18 ofpregnancy and bone marrow cells were collectedfrom dams and liver cells were collected fromfetuses.– 10 mg hexavalentchromium/L(drinking water)De Flora etal., 2006Micronuclei PotassiumdichromateMouse (BDF1)bone marrow cellsParenteral exposure: BDF1 male mice were givensingle intraperitoneal doses of potassiumdichromate of 0 or 50 mg hexavalentchromium/kg.+ 50 mg hexavalentchromium/kg(intraperitonealinjection)De Flora etal., 2006Micronuclei PotassiumdichromateMouse bonemarrowParenteral exposure: Balb C mice wereadministered potassium dichromate as a singleintraperitoneal injection at a dose of 0 or 400μmol(20.8 mg hexavalent chromium/kg).+ 20.8 mg hexavalentchromium/kg(intraperitonealinjection)WronskaNofer et al.,1999Micronuclei PotassiumdichromateMouse bonemarrowParenteral exposure: CBA x C57Bl/6J hybridmice were treated with potassium dichromate witha single intraperitoneal injection of 0, 1, 5, or10 mg/kg (0.35, 1.77, or 3.54 mg hexavalentchromium/kg). Bone marrow was sampled 24, 48,and 72 hours after treatment for the micronucleustest.+ 0.35 mg hexavalentchromium/kg(intraperitonealinjection)Paschin andToropzev,1982142 DRAFT - DO NOT CITE OR QUOTETable 4-23. In vivo genotoxicity studies of hexavalent chromium inD. melanogasterand laboratorymammalsEndpointChemicalTest systemTest conditionsResultsaDosebReferenceMicronuclei PotassiumdichromateMouse (Swiss):bone marrowdams; fetal liverand peripheralblood cellsParenteral exposure: Pregnant Swiss albino micewere administered potassium dichromate as asingle intraperitoneal injection at 0 or 50 mghexavalent chromium/kg on day 17 of pregnancy.Mice were sacrificed on day 18 of pregnancy.Theliver and peripheral blood were collected from thefetuses and bone marrows from the dams.+ 50 mg hexavalentchromium/kg(intraperitonealinjection)De Flora etal., 2006Micronuclei SodiumdichromatedihydrateMouse (B6C3F1)peripheral redblood cellsOral exposure: B6C3F1(10/sex/group)mice wereadministered sodium dichromate dihydrate indrinking water for 3 months at concentrations 0,62.5, 125, 250, 500, or 1,000 mg/L (0, 21.8, 43.6,87.2, 174.5, or 349 mg hexavalent chromium/L).NTP estimated daily doses at 0, 3.1, 5.2, 9.1, 15.7,or 27.9 mg hexavalent chromium/kg.– 349 mg hexavalentchromium/L(drinking water)NTP, 2007Micronuclei SodiumdichromatedihydrateMouse (B6C3F1,BALB/coram3-C57BL/6)peripheral redblood cellsOral exposure: B6C3F1(5/group), BALB/c(5/group) andam3-C57BL/6 (10/group) malemice were administered sodium dichromatedihydrate in drinking water for 3 months atconcentrations of 0, 62.5, 125, or 250 mg/L (0,21.8, 43.6, or 87.2 mg hexavalent chromium/L).NTP estimated average daily doses at 0, 2.8, 5.2,or 8.7 mg hexavalent chromium/kg.±–+(drinking water)87.2 mg hexavalentchromium/L(B6C3F1)87.2 mg hexavalentchromium/L(BALB/c)21.8 mg hexavalentchromium/L(am3-C57BL/6)NTP, 2007Micronuclei SodiumdichromatedihydrateMouse (BDF1)bone marrow orperipheral bloodcellsOral exposure: Male and female BDF1 mice wereadministered sodium dichromate dihydrate indrinking water at 0, 5, 50, and 500 mg hexavalentchromium/L for 210 days. Peripheral blood cellswere collected on days 0, 14, 28, 56, and 147;bone marrow cells were collected on day 210.– 500 mg hexavalentchromium/L(drinking water)De Flora etal., 2006143 DRAFT - DO NOT CITE OR QUOTETable 4-23. In vivo genotoxicity studies of hexavalent chromium inD. melanogasterand laboratorymammalsEndpointChemicalTest systemTest conditionsResultsaDosebReferenceMicronuclei SodiumdichromatedihydrateMouse (Swiss)bone marrowdams; fetal liverand peripheralblood cellsOral exposure: Pregnant Swiss albino mice wereadministered sodium dichromate dihydrate indrinking water at concentrations of 0, 5, or 10 mghexavalent chromium/L throughout the duration ofpregnancy. Mice were sacrificed on day 18 ofpregnancy and bone marrow cells were collected.Liver and peripheral blood samples were collectedfrom the fetuses.– 10 mg hexavalentchromium/L(drinking water)De Flora etal., 2006Micronuclei SodiumdichromatedihydrateMouse (Swiss):bone marrowdams; fetal liverand peripheralblood cellsParenteral exposure: Pregnant Swiss albino micewere administered sodium dichromate dihydrateas a single intraperitoneal injection at 0 or50 mg/kg on day 17 of pregnancy. Mice weresacrificed on day 18 of pregnancy and bonemarrow cells were collected. Liver and peripheralblood samples were collected from the fetuses.+ 50 mg hexavalentchromium/kg(intraperitonealinjection)De Flora etal., 2006a+ = positive, ± = equivocal or weakly positive, – =negative, (T) = toxicity.bLowest effective dose for positive results, highestdose tested for negative results, NS = not specified.

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4.4.1.2. Genotoxicity studies in humans 1 

### In addition to mutagenicity evaluations in experime ntal systems, several studies have 2 

### evaluated mutagenicity in humans occupationally exp osed to hexavalent chromium; 3 

### experimental details and citations are summarized i n Table 4-24. Data from available 4 

### mutagenicity studies in exposed workers are limited to assessments of tissues with easy 5 

### accessibility (e.g., circulating lymphocytes and bu ccal and nasal mucosal cells). Data on 6 

### mutagenicity in cancer target tissues (e.g., lung a nd GI tract) are not available. Available data 7 

### provide some evidence of hexavalent chromium-induce d mutagenicity in occupationally exposed 8 

### humans, although results of studies in workers have yielded mixed results. In general, 9 

### associations between hexavalent chromium exposure a nd mutagenicity in workers are uncertain 10 

### because exposure levels were often not quantified o r estimated, past exposure history was not 11 

### well characterized in all studies, small numbers of workers were evaluated, and/or workers were 12 

### potentially co-exposed to other compounds with muta genic activity. 13 

### In a comet assay in Italian chrome platers, positiv e results were reported for DNA strand 14 

### breaks in peripheral lymphocytes; although urine ch romium concentrations were determined, 15 

### hexavalent chromium exposure levels were not report ed (Gambelunghe et al., 2003). However, 16 

### no DNA damage was observed in peripheral lymphocyte s in dichromate production workers 17 

### exposed to 0.001–0.055 mg hexavalent chromium/m 3 

### (Gao et al., 1994) or in volunteers 18 

### ingesting single oral doses of 71 μg hexavalent chr omium/kg (Kuykendall et al., 1996). In 19 

### chrome electroplaters, chromosome aberrations and s ister chromatid exchanges were observed in 20 

### whole blood of workers exposed to relatively high c oncentrations estimated at 5.99 mg 21



### hexavalent chromium/m 3 

### (Wu et al., 2001). However, chromosome aberration s and sister 22 

### chromatid exchanges in peripheral lymphocytes from chrome platers were not observed at lower 23 

### exposure levels (0.0075 and 0.0249 mg chromium[tota l]/m 3 

### ) (Benova et al., 2002). Other studies 24 

### reporting positive (Sarto et al., 1982; Stella et a l., 1982; Wu et al., 2000) or negative (Nagaya et 25 

### al., 1986, 1991) results for chromosome aberrations or sister chromatid exchanges in peripheral 26 

### lymphocytes of workers did not report hexavalent ch romium exposure levels. Micronuclei 27 

### formation in peripheral lymphoctes was also observe d in chrome platers at exposure levels of 28 

### 0.043–0.083 mg chromium(total)/m 3 

### (Vaglenov et al., 1999) and 0.0075–0.0249 mg 29 

### chromium(total)/m 3 

### (Benova et al., 2002). In buccal mucosal cells co llected from chrome 30 

### platers, micronuclei formation was increased at exp osure levels of 0.0075–0.0249 mg 31 

### chromium(total)/m 3 

### , although chromosome aberrations and sister chroma tid exchanges were not 32

### 145 DRAFT - DO NOT CITE OR QUOTE

observed (Benova et al., 2002). Sarto et al. (1990 ) reported negative results for micronuclei in 1 

### buccal and nasal cells of chrome platers, but expos ure levels were not reported. 2 

### In summary, results of available studies in hexaval ent chromium-exposed workers 3 

### provide some evidence of the mutagenic activity of hexavalent chromium in occupationally 4 

### exposed humans, but results have not been consisten t across studies and endpoints. For example, 5 

### associations with increased micronuclei in peripher al lymphocytes or buccal mucosal cells have 6 

### been reported in chrome platers at estimated exposu re levels as low as 0.0075–0.0249 mg 7 

### chromium(total)/m 3 

### (Benova et al., 2002; Vaglenov et al., 1999), alth ough chromosome 8 

### aberrations and sister chromatid exchanges were not observed (Benova et al., 2002). In contrast, 9 

### increased frequencies of chromosome aberrations and sister chromatid exchanges were observed 10 

### in another group of chrome platers exposed to highe r concentrations estimated at 5.99 hexavalent 11



### chromium/m 3 

### (Wu et al., 2001). 12146 DRAFT - DO NOT CITE OR QUOTE1Table 4-24. In vivo genotoxicity studies in humansexposed to hexavalent chromiumEndpointExposure type(chemical form)Cell type Test conditionsResultsaExposure levelbReferenceDNA strandbreaksOccupational -Chromium plating(chromic acid)HumanperipherallymphocytesNineteen chromium plating workers in Italy (meanemployment of 6.3 years), and two groups of controlsubjects(18 hospital workers and 20 university personnel) gave preand post-shift urine samples and blood samples foranalysis inthe comet assay. Duration of employment ranged from 4months to 14 years with a mean duration of 6.3 years. Meanchromium concentrations in urine were determined tobe5.29 μg/g creatinine (pre-shift) and 7.31 μg/g creatinine (postshift). Mean erythrocyte and lymphocyte concentrations in theexposed workers were 4.94 μg/L and 50.3 μg/1012cells,respectively. Air concentrations of chromium werenotreported.+ NS Gambelungheet al., 2003DNA strandbreaks,hydroxylation ofdeoxyquanosineOccupational -Production ofdichromate(includedexposure tochromic acid,potassiumdichromate andsodiumdichromate)HumanperipherallymphocytesUrine and blood samples were taken from 10 exposedworkersand 10 non-exposed workers at the end of a workweekat abichromate production plant in England.The mean duration ofexposure was 15 years. Chromium concentrations inthefactory ranged from 0.001 to 0.055 mg hexavalentchromium/m3(obtained from personal and area samplers).Mean chromium concentrations in urine (5.97 μg/g creatinine),whole blood (5.5 μg/l), plasma (2.8 μg/L), and lymphocytes(1.01 μg/1010cells) of exposed workers were significantlyhigher than in non-exposed workers.– 0.001–0.055 mghexavalentchromium/m3(measuredexposure range)Gao et al.,1994DNA-proteincrosslinksExperimental oralexposure -(potassiumdichromate)HumanperipherallymphocytesFour adult volunteers ingested a single bolus doseof 5,000 μghexavalent chromium as potassium dichromate(approximatelyequivalent to 71 μg hexavalent chromium/kg, assuming a bodyweight of 70 kg). Blood samples were collected at0, 60, 120,180, and 240 minutes after ingestion. Pre-ingestionbackground DNA-protein crosslink levels for each individualserved as the controls.– 71 μg hexavalentchromium/kgKuykendall etal., 1996147 DRAFT - DO NOT CITE OR QUOTETable 4-24. In vivo genotoxicity studies in humansexposed to hexavalent chromiumEndpointExposure type(chemical form)Cell type Test conditionsResultsaExposure levelbReferenceChromosomeaberrations,sister chromatidexchangesOccupational -Chromiumelectroplating(chemical notspecified)HumanperipherallymphocytesBlood from seven chromium electroplating workers ataChinese electroplating facility (mean employment period of12.8 years) and 10 control subjects were analyzed.Airsamples from the electroplating room were collected, alongwith stool and hair samples to determine exposure.The meanchromium (total) air concentration (by random air collection)was 8.1 μg/mm3, the mean chromium concentration in stoolsamples was 8.5 μg/g stool, and the mean chromiumconcentration in hair was 35.68 μg/g. The valenceofchromium that workers were exposed to was unspecified.+ 8.1 μgchromium/mm3cDeng et al.,1988Sister chromatidexchangesOccupational -Chromiumelectroplating(chemical notspecified)Humanwhole bloodcellsThirty-five chromium electroplating factory workersemployedat three electroplating plants in Tawain and 35 control subjectsgave blood samples to analyze the frequency of sisterchromatid exchange. Exposure duration ranged from2 to14 years with a mean of 6.5 years. Mean chromium exposure(determined by personal monitoring samplers) was 5.99 mghexavalent chromium/m3. The mean urinary chromiumconcentration of the exposed workers was 3.67 μg/gcreatinine.+ 5.99 mghexavalentchromium/m3Wu et al.,2001Chromosomalaberrations,sister chromatidexchangesOccupational -Chromium plating(chromic acid)HumanperipherallymphocytesThirty-eight male chromium plating factory workersin Italywere examined for urinary concentrations of chromium andchromosomal aberrations and sister chromatid exchanges.Chromium exposure levels were not reported. Therewere 35unexposed control individuals.+ NS Sarto et al.,1982Sister chromatidexchangesOccupational -Chromium plating(chromic acidfumes)HumanperipherallymphocytesThe frequency of sister chromatid exchanges was determinedin lymphocytes from 12 chromium plating workers inItaly and10 control subjects. Exposure durations ranged from 0.5 to 18years (mean exposure duration was not reported). Hexavalentchromium exposure levels or hexavalent chromium, bloodconcentrations were not reported.+ NS Stella et al.,1982Sister chromatidexchangesOccupational -Chromiumelectroplating(chemical notspecified)HumanperipherallymphocytesThirty-five chromium electroplating factory workersin Taiwanand 35 control subjects (matched for age and gender) gaveblood samples to determine sister chromatid exchangefrequency. The mean duration of employment was 6.5years.Exposure concentrations were not reported.+ NS Wu et al.,2000148 DRAFT - DO NOT CITE OR QUOTETable 4-24. In vivo genotoxicity studies in humansexposed to hexavalent chromiumEndpointExposure type(chemical form)Cell type Test conditionsResultsaExposure levelbReferenceChromosomeaberrations,sister chromatidexchangesOccupational -Chromium plating(chemical notspecified)Humanperipherallymphocytesand buccalmucosalcellsBlood samples and buccal mucosal cells from 15 Bulgarianchromium platers occupationally exposed were taken;exposure was estimated with personal air samplers and in urinesamples. Control subjects were matched with exposedindividuals. Duration of exposure ranged from 2 to>20 years;mean duration of exposure was not reported. Mean airconcentration of total chromium was 0.0075 mg chromium/m3in the low-exposure group and 0.0249 mg chromium/m3in thehigh-exposure group (number of workers in each exposuregroup was not reported). Mean concentrations of chromium inurine were 18.63 μg/L (low) and 104.22 μg/L (high)– Results reportedfor combinedgroups (0.0075and 0.0249 mgchromium/m3)Benova et al.,2002Sister chromatidexchangesOccupational -Chromium plating(chemical notspecified)HumanperipherallymphocytesVenous blood and urine sample were collected from 12 malechromium platers in Japan over a 5-year period. Nocontrolsubjects were used in this study. Employment duration rangedfrom 6.6 to 25.1 years, with mean employment duration of15.5 years. Exposure concentrations were not reported.Urinary chromium concentrations ranged from 1.2 to57.0 μg/gwith a mean urinary chromium concentration of 17.9μg/gcreatinine. Sister chromatid exchange frequency inlymphocytes was determined in blood-urine paired samples.– NS Nagaya et al.,1991Sister chromatidexchangesOccupational -Chromium plating(chemical notspecified)HumanperipherallymphocytesVenous blood and urine sample were collected from 24 malechromium platers in Japan and 24 control subjects.Durationof employment ranged from 0.5 to 30.5 years with ameanemployment of 11.6 years. Exposure concentrationswere notreported. The mean concentration of chromium in the urinewas 13.1 μg/L.– NS Nagaya, 1986149 DRAFT - DO NOT CITE OR QUOTETable 4-24. In vivo genotoxicity studies in humansexposed to hexavalent chromiumEndpointExposure type(chemical form)Cell type Test conditionsResultsaExposure levelbReferenceMicronuclei Occupational -Chromiumelectroplating(chemical notspecified)HumanperipherallymphocytesForty electroplating workers in Bulgaria and 18 controlsubjects gave blood samples to analyze for the frequency ofmicronuclei. The workers were split into two groups based onlevels of exposure. Mean air chromium (total) concentrationswere 43 and 83 μg/m3in the low- and high-exposure groups,respectively. Duration of employment ranged from 4to25 years with mean durations of 10.44 and 11.63 years in thelow- and high-exposure groups, respectively. Meanchromiumconcentrations in erythrocytes and urine of the lowexposuregroup were 4.31 and 3.97 μg/L, respectively. The meanchromium concentrations in erythrocytes and urine of the highexposure group were 8.4 and 5.0 μg/L, respectively.+ 0.043 and0.083 mgchromium/m3Vaglenov etal., 1999Micronuclei Occupational -Chromium plating(chemical notspecified)Humanperipherallymphocytesand buccalmucosalcellsBlood samples and buccal mucosal cells from 15 Bulgarianchromium platers occupationally exposed were taken.Exposure was estimated with personal air samplers and inurine samples. Control subjects were matched withexposedindividuals. Duration of exposure ranged from 2 to>20 years;mean duration of exposure was not reported. Mean airconcentration of total chromium was 0.0075 mg chromium/m3in the low-exposure group and 0.0249 mg chromium/m3in thehigh-exposure group. Mean concentrations of chromium inurine were 18.63 (low) and 104.22 μg/L (high).+ Positive resultsreported forcombined groups(0.0075 and0.0249 mgchromium/m3)Benova et al.,2002Micronuclei Occupational -Chromium plating(chromic acid)Humanbuccal andnasal cellsSixteen exposed Italian electroplating factory workers and27 unexposed control subjects gave samples of exfoliatedbuccal and nasal swabs. Duration of exposure ranged from 0.5to 23 years with a mean duration of 8 years. Urinesampleswere collected at the end of work days to determinechromiumexposure. Urinary chromium concentrations ranged from 2.5to 88 μg/g creatinine; the mean urinary chromiumconcentration was not reported. Chromium levels inair werenot determined.– NS Sarto et al.,1990a+ = positive; – = negativebAll exposure levels associated with positive results, highest exposure level for negative results; NS= not specified.cThe exposure level of 8.1 μg chromium/mm3is as reported by Deng et al., (1988); however, this appears to be a reporting error, as this concentration isequivalent to 8,100,000 mg chromium/m3.1

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4.4.2. Intracellular reduction 1 

### The mutagenic effects of hexavalent chromium are co ntingent upon its reduction within 2 

### the cell. Extracellularly, soluble hexavalent chro mium exists as a chromate oxyanion. The 3 

### tetrahedral arrangement of the oxygen groups makes it structurally similar to phosphate and 4 

### sulfate, allowing it to easily be taken up by the n on-specific phosphate/sulfate anionic 5 

### transporters and cross the cell membrane (Zhitkovic h, 2005). This method of cellular uptake 6



### also allows an accumulation of chromium in the cell at concentrations much higher than that 7 

### found extracellularly (Zhang et al., 2002). Chromi um in its hexavalent state is 8 

### thermodynamically stable in pure water, and is not reactive with DNA at physiological 9 

### concentrations. However, hexavalent chromium is a strong oxidizer, and once inside the cell it 10 

### can undergo rapid reduction. This is most often me diated by the non-enzymatic reductants 11 

### ascorbate (vitamin C) and low molecular weight thio ls including glutathione and cysteine. Other 12 

### potential reductants include cytochrome P450 reduct ase, NAD(P)H-dependent flavoenzymes, 13 

### and mitochondrial electron transport complexes (O’B rien et al., 2003; Sugden and Stearns, 2000; 14 

### Standeven and Wetterhahn, 1989). 15 

### The hexavalent chromium-reductant substrate comple xes that are formed upon 16



### intracellular interaction of hexavalent chromium wi th these reductants are considered the first 17 

### step in the reduction process, although the actual mechanisms of how these reactions proceed are 18 

### unknown (Levina and Lay, 2005). There are two theo rized pathways for the intracellular 19



### reduction of hexavalent chromium. When reductants are present in abundance, the process can 20



### occur with a two electron reduction to tetravalent chromium, immediately followed by a one 21



### electron reduction to trivalent chromium. If lower levels of reductants are available, the first step 22 

### of this process will occur as two distinct one elec tron transfers, producing the intermediates 23 

### pentavalent and tetravalent chromium, and ultimatel y trivalent chromium (O’Brien et al., 2003). 24 

### Either process can produce oxidative states of chro mium localized within the cell that are able to 25 

### damage DNA directly, forming DNA adducts and subseq uent DNA breakage. These chromium 26 

### species can also indirectly cause genetic damage vi a associated radical species derived from the 27 

### reductants that can be involved in secondary DNA da mage (Sugden and Stearns, 2000) and 28 

### disruption of DNA replication. 29

30 

### Final reduction product: Trivalent chromium. 31 

### Trivalent chromium is the ultimate product of hexav alent chromium reduction within the 32 

### cell. It contains six coordination sites, allowing it to form stable complexes with amino acids, 33

### 151 DRAFT - DO NOT CITE OR QUOTE

proteins, RNA, and DNA. In vitro studies of the ki netics of chromium

− 

### DNA binding have 1 

### shown that most of the DNA binding occurs within 1 hour of incubation (Quievryn et al., 2003). 2 

### When hexavalent chromium is reduced by ascorbate or cysteine in the presence of the trivalent 3 

### chromium chelator EDTA, the mutagenic response is a ll but eliminated and very little 4



### chromium

− 

### DNA binding is detected, indicating that the trival ent state is the most DNA reactive 5 

### of all the valence states of chromium (O’Brien et a l., 2003; Quievryn et al., 2003; Zhitkovich et 6 

### al., 2001). Several types of chromium

− 

### DNA adducts have been detected following the 7



### intracellular reduction of hexavalent to trivalent chromium. 8 

### DNA−peptide/amino acid ligand−trivalent chromium cr osslinks. Trivalent chromium 9 

### can form ternary DNA crosslinks with glutathione, a scorbate, cysteine, and histidine. Although 10



### the ascorbate

− trivalent chromium

− DNA adducts are recovered less frequently in vitro due to the 11 

### low concentrations of vitamin C present in commonly used tissue culture media (Zhitkovich, 12 

### 2005), these adducts have been shown to be the most mutagenic of all the ternary adducts 13 

### (Quievryn et al., 2003). These ternary adducts for m by the attachment of trivalent chromium (in 14 

### a binary complex with the ligand) to phosphate grou ps in DNA (Zhitkovich et al., 1995), 15 

### primarily through coordinate covalent binding or el ectrostatic/ionic interactions (O’Brien et al., 16 

### 2003) (Figure 4-1). They have been detected in vit ro in CHO cells following exposure to 17 

### hexavalent chromium, and account for up to 50% of a ll chromium

− DNA adducts. The ternary 18 

### adducts have been found to cause mutagenic and repl ication-blocking lesions in human 19 

### fibroblasts in vitro (Quievryn et al., 2003; Voitku n et al., 1998). 20

21

22

23

24

25

26

27

28

29

30

31

32

33

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1

2

3

4

5

6

7

8

9

10

11

12

13

14 

### Source: Zhitkovich (2005). 15

16 

### Figure 4-1. Ternary DNA adduct formation by chromi um. Hexavalent chromium, 17 

### when reduced intracellularly to trivalent chromium, can form ternary DNA crosslinks 18 

### with the peptide or amino acid ligand (L) involved in the reduction. Here, chromium(III) 19 

### directly coordinates to the 5’-phosphate in the DNA backbone and forms a hydrogen 20 

### bond with the N-7 of deoxyguanosine. 21

22



### DNA−trivalent chromium crosslinks. Reduction of hexavalent chromium in vitro 23 

### produces a large proportion of binary trivalent chr omium

− 

### DNA adducts, but these have not been 24



### detected in vivo. It has been theorized that the f ormation of the ternary adducts described above 25 

### occurs far more frequently due to the high concentr ation of ligands capable of complexing with 26 

### trivalent chromium before it can bind to DNA (Zhitk ovich, 2005). In addition, these adducts 27 

### have been found to be less mutagenic than the terna ry adducts in vitro (Quievryn et al., 2003; 28 

### Zhitkovich et al., 2001). 29 

### DNA−protein crosslinks. These bulky lesions have been detected in hexavalen t 30



### chromium-treated cells in vitro in Chinese hamster ovary cells (Costa, 1991) and in vivo in chick 31 

### embryos (Hamilton and Wetterhahn, 1986). They are not detected in the presence of the 32 

### trivalent chromium chelator EDTA, indicating that t rivalent chromium is the species involved in 33 

### their formation (Miller and Costa, 1989). It has b een recently shown that the mechanism 34 

### forming DNA

− protein crosslinks induced by hexavalent chromium r equires intracellular 35 OH O

O 

### Cr(III) O

N N

NH 2 N NH

O O

O O P O O

O O P L L L L

O H

H

DNA DNA

7

OH OH O

O 

### Cr(III) O

N N

NH 2 N NH

O

N N

NH 2 N NH

O O

O O P O O

O O P L L L L

O H

H

DNA DNA

7

OH

### 153 DRAFT - DO NOT CITE OR QUOTE

reduction to trivalent chromium, formation of DNA

− trivalent chromium adducts, and subsequent 1 

### capture of proteins by the DNA bound to trivalent c hromium (Macfie et al., 2010). Tests for the 2 

### mutagenicity of these crosslinks have proved inconc lusive (reviewed in Macfie et al., 2010), but 3 

### the bulkiness of these lesions indicates potential for genotoxicity resulting from replication fork 4 

### stalling (Costa, 1991). 5



### DNA−DNA crosslinks. These inter- or intra-strand DNA crosslinks are li kely formed by 6 

### oligomers of trivalent chromium. They have been de tected following hexavalent chromium 7 

### exposure, although only when the reductants are asc orbate or cysteine, and not glutathione 8 

### (Zhitkovich, 2005). However, these adducts have on ly been detected in vitro and are not 9 

### expected to form in significant amounts in vivo; th e high intracellular concentrations of ligands 10 

### available to form complexes with trivalent chromium make it unlikely that these oligomers 11 

### would have a chance to form (Salnikow and Zhitkovic h, 2008). 12 

### Repair of chromium−DNA adducts. Repair processes have been shown to be effectively 13 

### carried out by nucleotide excision repair (NER), a DNA repair mechanism responsible for 14 

### removal of bulky DNA lesions. Exposing NER-deficie nt human cells to hexavalent chromium 15 

### was shown to induce apoptosis and clonogenic cell d eath. The most efficient substrates for this 16 

### repair process are lesions that create major distor tions in the DNA structure. Chromium

− DNA 17 

### adducts do not create major helix distortions, but their bulkiness makes them adequate substrates 18 

### for NER, although they are less efficiently removed than optimal NER substrates such as UV 19 

### light-induced lesions (Reynolds et al., 2004). 20 

### Another closely related repair mechanism, mismatch repair (MMR), is responsible for the 21 

### correction of errors in DNA replication. MMR enzym es recognize misincorporated bases during 22 

### DNA replication and homologous recombination, and r epair single base mispairings and small 23 

### insertions or deletions. However, MMR has also bee n shown to be a causative factor in many of 24 

### the toxic and genotoxic effects of hexavalent chrom ium, when processing the repair of the bulky 25 

### lesions formed by chromium lead to the formation of DNA double-strand breaks (Peterson-Roth 26 

### et al., 2005). In this study, mouse and human cell lines deficient in MMR exposed to hexavalent 27 

### chromium had greatly increased clonogenic survival due to a diminished apoptotic response as 28 

### compared to MMR-proficient cells. The apoptotic re sponse in the MMR-proficient cells was 29 

### preceded by a significant induction of DNA double-s trand breaks, indicated by an increased 30 

### formation of gamma-H2AX foci. These discrete foci form when phosphorylation of this histone 31 

### H2A variant occurs in response to DNA double-strand breaks, and can be visualized and 32 

### quantified by immunofluorescence. This increase in gamma-H2AX foci was not detected at 33

### 154 DRAFT - DO NOT CITE OR QUOTE

significant levels until 6 hours post-exposure to h exavalent chromium, suggesting that the DNA 1 

### double-strand breaks were not induced directly by h exavalent chromium, but rather from 2 

### processing of the damaged DNA. These foci also co- localized with cyclin B1 staining, 3 

### indicating the breaks occurred in the G2 phase of t he cell cycle and providing evidence that 4 

### passage through S phase, where MMR would be taking place, was necessary for the induction of 5 

### this damage (Salnikow and Zhitkovich, 2008). The m echanism of this toxic response mediated 6 

### by MMR proteins is unknown, but has been theorized to involve the futile repair of damaged 7 

### bases or the initiation of a stress response (Peter son-Roth et al., 2005). 8 

### As mentioned above, apoptosis, or programmed cell d eath, has been observed in cells 9 

### exposed to hexavalent chromium as a response to ext ensive DNA damage that cannot be 10 

### adequately repaired by the cell. Ye et al. (1999) found hexavalent chromium induced apoptosis 11 

### in human lung epithelial cells exposed to doses ran ging from 75 to 300 μM in vitro; the authors 12 

### theorized that this response involved reactive oxyg en species formed both directly during the 13 

### process of hexavalent chromium reduction and indire ctly through the induction of p53. Flores 14 

### and Perez (1999), using doses close to the IC50 val ues, observed apoptosis concurrent with DNA 15 

### interstrand crosslinks and DNA single-strand breaks in murine keratinocytes transformed with 16 

### the H-ras oncogene. These studies indicate that mu ltiple mechanisms induced by hexavalent 17 

### chromium exposure, including oxidative stress and D NA binding, can lead to cell death. 18 

### However, no in vivo studies have been able to confi rm the extent of apoptosis following 19 

### hexavalent chromium exposure. In addition, several studies specifically measuring genotoxicity 20 

### in vitro and in vivo have observed positive results at doses that did not elicit cytotoxicity (see 21 

### previous section). 22 

### An interesting addendum to the effects involving DN A repair mechanisms is the finding 23 

### that hexavalent chromium, after intracellular reduc tion to the +3 oxidation state, can interfere 24 

### with normal DNA replication and transcription proce sses. Intracellular trivalent chromium has 25 

### been shown to inhibit the enzymatic activity of DNA polymerases, simultaneously increasing the 26 

### rate of replication and the processivity of the DNA polymerase, thereby decreasing its fidelity 27 

### and causing more frequent errors, with a dose-depen dent increase in mutation frequency in vitro 28 

### (Snow, 1991). There can also be replication arrest as a result of the bulky chromium−DNA 29 

### lesions, creating a physical obstruction to the pro gression of DNA polymerases (Bridgewater et 30 

### al., 1998). These effects were recently confirmed in a study utilizing the DNA synthesome, an in 31 

### vitro DNA replication model system that is fully co mpetent to carry out all phases of the DNA 32 

### replication process mediated by human cells (Dai et al., 2009). This study found a reduction of 33

### 155 DRAFT - DO NOT CITE OR QUOTE

the fidelity and an inhibition of DNA synthesis tha t led to a dose-dependent increase in mutation 1 

### frequency following intracellular exposure to triva lent chromium. Gene transcription has also 2 

### been recently shown to be affected by exposure to h exavalent chromium in vitro via epigenetic 3 

### mechanisms. Sun et al. (2009) found alterations in the levels of histone methylation in human 4 

### lung A549 cells exposed to hexavalent chromium, ind icating the capability of these exposures to 5 

### lead directly to changes in gene expression. Thus, hexavalent chromium can lead to the 6 

### disruption of DNA synthesis and gene transcription at multiple levels, corresponding to an 7 

### observable, dose-dependent increase in mutation fre quency in human cells. 8

9



### Reduction intermediates: Pentavalent and tetravalen t chromium. 10 

### Depending on the reductant involved and the concent ration of hexavalent chromium 11 

### present, various amounts of the unstable intermedia tes pentavalent and tetravalent chromium can 12 

### be generated prior to reduction to the final stable oxidative +3 state. At lower levels of 13 

### hexavalent chromium exposure, intracellular concent rations of these reductants are sufficient to 14 

### complete the reduction of hexavalent chromium to it s trivalent state. However, at higher 15 

### hexavalent chromium exposures, these levels are dep leted, resulting in a higher yield of 16 

### pentavalent chromium from the one-electron reducing thiols, glutathione and cysteine, as well as 17 

### tetravalent chromium from the two-electron donating ascorbate. While pentavalent and 18



### tetravalent chromium can be short-lived states of c hromium within the cell, they are DNA 19 

### reactive and can participate in redox reactions, fo rming free radical species that can also damage 20 

### DNA (Stearns and Wetterhahn, 1994). 21 

### Redox cycling of the chromium ions can occur intrac ellularly when they are formed 22 

### during reduction of hexavalent chromium. The proce ss of hexavalent chromium reduction by 23 

### glutathione is accompanied by the reduction of mole cular oxygen, yielding superoxide radicals. 24 

### Reduction by glutathione has been shown to involve the formation of glutathione-derived thiyl 25 

### radicals that can directly damage DNA or react with other thiols to also generate superoxide 26 

### radicals. These radical species will react with hy drogen peroxide to produce hydroxyl radicals 27 

### via Haber-Weiss reactions (Shi et al., 1999). Both hydrogen peroxide and superoxide radicals 28 

### can participate in redox reactions involving both t he pentavalent and tetravalent transition states 29 

### of chromium that can generate hydroxyl radicals via Fenton and Haber-Weiss reactions (Shi et 30 

### al., 1999). Hydroxyl radicals can directly react w ith genetic material, forming DNA−protein 31 

### crosslinks and DNA adducts with proteins and amino acids, damaging DNA bases, and 32 

### producing DNA single- and double-strand breaks (rev iewed in Kasprzak, 1996). 33

### 156 DRAFT - DO NOT CITE OR QUOTE

Although less frequent than the low molecular weigh t non-enzymatic reductants, 1 

### reduction of hexavalent chromium can also occur by NAD(P)H-dependent flavoenzymes, 2 

### including glutathione reductase, lipoyl dehydrogena se, and ferredoxin-NADP+ oxidoreductase 3 

### (Shi and Dalal, 1990). These enzymes catalyze a on e-electron reduction that can result in the 4 

### formation of stable pentavalent chromium−NADPH comp lexes that can react with hydrogen 5 

### peroxide to generate hydroxyl radicals (Shi et al., 1999). The ability to form complexes with 6 

### biological ligands allows stabilization of pentaval ent, but not tetravalent, chromium 7 

### intermediates (Levina and Lay, 2005). These pentav alent chromium−NADPH complexes have 8 

### been shown to form in vitro in E. coli (Shi et al., 1991) and in vivo in mice (Liu et al., 1995). 9 

### Two other important non-enzymatic reducers of hexav alent chromium are ascorbate and 10 

### cysteine. Ascorbate and cysteine are present at lo wer concentrations intracellularly than 11 

### glutathione, but they have kinetically faster rates of hexavalent chromium reduction. Ascorbate 12 

### has been shown to yield pentavalent and tetravalent chromium and radical species when the 13



### intracellular ratio of ascorbate to chromium is les s than 3:1 (Stearns and Wetterhahn, 1994). The 14 

### precise nature of the radical species relevant to D NA damage is not known, however, and the 15 

### degree of damage attributable to oxidative mechanis ms is the subject of much debate. One study 16 

### found an increase in mutations and replication-bloc king DNA lesions in human fibroblasts 17 

### resulting from the ascorbate-driven reduction of he xavalent chromium, but concluded that the 18 

### mechanism responsible did not involve oxidative rad icals, in part because the DNA damage 19 

### anticipated by species including hydroxyl radicals and pentavalent chromium-peroxo complexes, 20 

### namely abasic sites and strand breaks, was not obse rved (Quievryn et al., 2003). This study also 21 

### found that no mutagenesis occurred in the presence of a trivalent chromium chelator, indicating 22 

### the involvement of trivalent chromium−DNA adducts ( see previous section). Similarly, studies 23 

### of the DNA damage resulting from the intracellular reduction of hexavalent chromium by 24 

### cysteine have shown that, while the intermediate sp ecies pentavalent and tetravalent chromium 25 

### and thiyl radicals were formed, they were not respo nsible for DNA damage; rather, the trivalent 26 

### chromium−DNA adducts were found to be the mutagenic species (Zhitkovich et al., 2001). The 27 

### same group also found an elimination of mutagenicit y when glutathione reduction of hexavalent 28 

### chromium occurred in the presence of phosphate ions that led to the sequestration of trivalent 29 

### chromium, preventing its binding to DNA (Guttmann e t al., 2008). 30 

### The ability of these intermediate chromium species to generate damaging free radicals is 31 

### not in doubt, however, and there is evidence of rea ctive oxygen species generated by pentavalent 32 

### chromium causing DNA damage. A decrease in DNA str and breaks was observed when 33

### 157 DRAFT - DO NOT CITE OR QUOTE

hexavalent chromium reduction with glutathione occu rred in the presence of free radical 1 

### scavengers (Kortenkamp et al., 1990). In addition, DNA double-strand breaks in subcellular 2 

### systems were observed when ascorbate-mediated reduc tion of hexavalent chromium generated 3 

### hydroxyl radicals via a Fenton-like reaction (Shi e t al., 1994). 4 

### In an attempt to explain these conflicting results, it has been theorized that the 5 

### responsible free radicals may be chromium-based and not oxygen-based radicals. This is due to 6 

### the observation that the mutational spectra observe d by chromium-induced radicals differs from 7 

### that expected by damage due to reactive oxygen spec ies that are generated following exposure to 8 

### hydrogen peroxide, X-rays, or ionizing radiation (S ugden and Stearns, 2000). Hexavalent 9



### chromium has been shown to induce the formation of 8-oxo-deoxyguanosine adducts (8-oxo-dG) 10 

### that are known to be induced by oxidative damage (S ander et al., 2005), but these lesions have 11 

### also been shown to be induced directly by pentavale nt chromium, with the subsequent addition 12 

### of molecular oxygen (Sugden and Martin, 2002). In addition, the oxididant-sensitive dyes used 13 

### to detect reactive oxygen species intracellularly c an also be oxidized directly by pentavalent 14 

### chromium and chromium-based radicals (O’Brien et al ., 2003). Therefore, the induction of 15 

### mutagenic lesions by the intracellular reduction of hexavalent chromium could be attributed to 16 

### non-oxygen-dependent mechanisms. 17 

### Pentavalent chromium has been detected using electr on paramagnetic resonance (EPR) 18 

### spectroscopy following intraperitoneal administrati on of hexavalent chromium in vivo, both in 19 

### the liver and red blood cells of chick embryos (Lie bross and Wetterhahn, 1992), and in mouse 20 

### liver and blood (Liu et al., 1994). In in vitro, l evels of DNA strand breaks were found to 21 

### correlate with increasing levels of pentavalent chr omium in Chinese hamster V79 cells 22 

### (Sugiyama et al., 1989). Another in vitro study in human leukemic T-lymphocyte MOLT4 cells 23 

### detected pentavalent chromium species and hydroxyl radicals with EPR following exposure to 24 

### hexavalent chromium (Mattagajasingh et al., 2008). The same study also observed a dose25 

### dependent increase in protein carbonyls and malondi aldehyde (MDA) generated via protein 26 

### oxidation and lipid peroxidation, respectively, alt hough the lipid peroxidation only occurred 27 

### significantly at much higher exposures of chromate ( ≥ 100 μM) compared with the protein 28 

### oxidation, which was significant as low as 10 μM. Tetravalent chromium has been more 29



### difficult to observe due to its unstable nature com pared to pentavalent chromium, but this species 30



### was shown to induce mitotic recombination in the so matic wing spot assay in Drosophila (Katz 31 

### et al., 2001). Both species caused an induction of NF-kB, a nuclear transcription factor involved 32 

### in the cellular response to oxidative damage, in cu ltured Jurkat cells. This activation was 33

### 158 DRAFT - DO NOT CITE OR QUOTE

enhanced by hydrogen peroxide and eliminated when c atalase was added to decompose 1 

### hydrogen peroxide, indicating that hydroxyl radical s may have had a role (Shi et al., 1999). 2 

### In summary, there are many potential mechanisms inv olved in the genotoxicity of 3 

### hexavalent chromium when reduced intracellularly. Intermediate valence states can react 4 

### directly and indirectly through coordinate complexe s with DNA as well as form radical species, 5 

### and the final reduction product, trivalent chromium , can form various damaging DNA adducts. 6 

### Additionally, significant evidence points to the ab errant processing of DNA mismatches induced 7 

### by chromium−DNA adducts, leading to apoptosis of th e damaged cells, or further promotion of 8 

### these mutagenic lesions as the DNA double-strand br eaks generated are substrates for error9 

### prone repair processes such as non-homologous end j oining. 10

11



### 4.5. SYNTHESIS OF MAJOR NONCANCER EFFECTS - ORAL 12 

### In humans, several case reports have been published on clinical signs and symptoms in 13 

### individuals following acute accidental or intention al ingestion of high doses (fatal or near fatal) 14 

### of hexavalent chromium compounds, including chromic acid, potassium dichromate, and 15 

### ammonium dichromate. Clinical presentation of pati ents following acute, high-dose exposure 16 

### was similar, regardless of the specific hexavalent chromium compound ingested, and included 17 

### the following: abdominal pain, nausea, and vomiting ; hematemesis and bloody diarrhea; caustic 18 

### burns of mouth, pharynx, esophagus, stomach, and du odenum and GI hemorrhage; anemia, 19 

### decreased blood Hgb, abnormal erythrocytes, and int ravascular hemolysis; hepatotoxicity 20 

### (hepatomegaly, jaundice, elevated blood bilirubin, and liver enzyme activities); renal failure 21 

### (oliguria and anuria); cyanosis; and metabolic acid osis, hypotension, and shock. Findings on 22 

### tissue biopsies included hepatic fatty degeneration and necrosis and renal tubular degeneration 23



### and necrosis. 24



### Information on chronic human health effects resulti ng from exposure to hexavalent 25 

### chromium comes from several studies of human popula tions unknowingly consuming food or 26 

### drinking water contaminated with hexavalent chromiu m over some extended time period. These 27 

### studies have been primarily focused on cancer. How ever, the noncancer effects that have been 28



### recorded are consistent with the GI effects observe d following acute exposures to hexavalent 29 

### chromium and have included oral ulcers, diarrhea, a bdominal pain, dyspepsia, stomach pain, and 30 

### vomiting (JinZhou Antiepidemic Station, 1979). 31 

### Table 4-25 presents a summary of studies of the non cancer effects of hexavalent 32 

### chromium exposure from repeated-dose oral toxicity studies in experimental animals. The most 33

### 159 DRAFT - DO NOT CITE OR QUOTE

sensitive targets of toxicity identified in these s tudies included the blood, liver, and GI tract. Th e 1 

### effects seen in these target organs are more specif ically discussed below. 2 

### In regard to hematological effects, NTP (2007) obse rved microcytic, hypochromic 3 

### anemia (i.e., decreased Hct, Hgb, MCV, and MCH) at a dose of 1.7 mg/kg-day of hexavalent 4



### chromium in both male and female F344/N rats in a t hree-month (subchronic) study. In this 5 

### same study, NTP (2007) also saw histopathological c hanges (i.e., histiocytic cellular infiltration) 6 

### in the pancreatic lymph nodes in male F344/N rats a t 1.7 mg/kg-day of hexavalent chromium. 7 

### Finally, in a chronic (two-year) study, NTP (2008) observed histopathological changes (i.e., 8 

### histiocytic cellular infiltration) in the mesenteri c lymph nodes in male F344/N rats at 0.77 9 

### mg/kg-day of hexavalent chromium and male and femal e B6C3F 1 

### mice at 0.38 mg/kg-day of 10



### hexavalent chromium. 11 

### In the NTP (2007) subchronic study referenced above , liver effects were also observed at 12 

### 1.7 mg/kg-day of hexavalent chromium and included i ncreased serum liver enzyme activities 13 

### (i.e., ALT and SDH) in both males and females and i ncreased bile acids in females. In their two14 

### year bioassay, NTP (2008) found an increased incide nce of chronic inflammation of the liver at 15 

### 0.24 mg/kg-day of hexavalent chromium in female F34 4/N rats and increased incidences of 16 

### histopathological changes to the liver (i.e., basop hilic foci) at 0.77 mg/kg-day of hexavalent 17



### chromium in male F344/N rats. In this same bioassa y, increased incidences of histopathological 18 

### changes to the liver (i.e., histiocytic cellular in filtration) were seen at 0.38 mg/kg-day of 19



### hexavalent chromium in female B6C3F 1 

### mice. 20 

### Effects of hexavalent chromium ingestion on the GI tract have been primarily observed in 21 

### the small intestine (duodenum). In a three-month s tudy, NTP (2007) saw histopathological 22 

### changes to the duodenum in male F344/N rats at 1.7 mg/kg-day of hexavalent chromium, in male 23



### and female B6C3F 1 

### mice at 5.3 mg/kg-day of hexavalent chromium, and in male BALB/c and 24



### am3 -C57BL/6 mice at 2.8 mg/kg-day of hexavalent chromi um. These changes included 25 

### epithelial hyperplasia and histiocytic cellular inf iltration. In their two-year study, NTP (2008) 26 

### also found increased incidences of histopathologica l changes to the duodenum in male F344/N 27 

### rats at 0.77 mg/kg-day of hexavalent chromium and i n male and female B6C3F 1 

### mice at 0.38 28 

### mg/kg-day of hexavalent chromium. Similar to that observed in the subchronic study, these 29 

### changes in the duodenum included epithelial hyperpl asia and histiocytic cellular infiltration. 30 

### Animal studies also provide evidence that oral expo sure to hexavalent chromium 31 

### compounds produces reproductive effects, including histopathological changes to reproductive 32 

### organs in males (Aruldhas et al., 2006, 2005, 2004; Chowdhury and Mitra, 1995; Li et al., 2001; 33

### 160 DRAFT - DO NOT CITE OR QUOTE

Zahid et al., 1990) and females (Murthy et al., 199 6); alterations in sperm, including decreased 1 

### count, decreased motility, and abnormal morphology (Subramanian et al., 2006; Yousef et al., 2 

### 2006; Li et al., 2001; Zahid et al., 1990); decreas ed plasma testosterone levels (Yousef et al., 3 

### 2006; Chowdhury and Mitra, 1995); increased estrous cycle length (Kanojia et al., 1998, 1996; 4 

### Murthy et al., 1996); changes in mating behavior an d decreased fertility in males (Bataineh et al., 5 

### 1997); and adverse reproductive outcomes, including decreased numbers of live fetuses and 6 

### implantations, and increased numbers of resorptions and pre- and postimplantation losses 7 

### (Bataineh et al., 2007; Elsaieed and Nada, 2002; El betieha and Al-Hamood, 1997; Junaid et al., 8 

### 1996a, b, 1995; Kanojia et al., 1998, 1996; Trivedi et al., 1989). These studies are summarized 9



### in Table 4-25. 10 

### Developmental effects observed in animal studies ha ve included decreased fetal weight 11 

### and length (Elsaieed and Nada, 2002; Kanojia et al. , 1998; Junaid et al., 1996a, b, 1995; Trivedi 12 

### et al., 1989); external (subdermal hemorrhage and t ail malformations) and skeletal abnormalities 13 

### (decreased ossification) (Elsaieed and Nada, 2002; Junaid et al., 1996a, b, 1995; Kanojia et al., 14 

### 1998, 1996; Trivedi et al., 1989); and delayed sexu al maturation and function in female offspring 15 

### (Banu et al., 2008; Al-Hamood et al., 1998). These effects were seen at hexavalent chromium 16 

### doses ranging from about 2 to 100 mg/kg-day. 17 

### In contrast to results of the above studies on repr oductive toxicity, reproductive effects 18 

### were not observed in dietary exposure studies condu cted by NTP that investigated the potential 19 

### effects of hexavalent chromium on male reproductive organs in rats and mice (NTP, 1996a,b) 20 

### and on reproductive outcomes in a continuous breedi ng study in mice (NTP, 1997). The reason 21



### for the inconsistent results between the NTP studie s and the other reproductive toxicity studies of 22 

### hexavalent chromium are not readily apparent, as da ily dose ranges evaluated in the NTP studies 23 

### overlapped with those used in the other studies sho wing hexavalent chromium-induced 24 

### reproductive effects. 25

26161 DRAFT - DO NOT CITE OR QUOTETable 4-25. Observed effects and corresponding NOAELs and LOAELs for subchronic, chronic, andreproductive toxicity studies following oral exposure to hexavalent chromiumSpeciesSexExposure level1ExposuredurationNOAEL(mg/kg-day)LOAEL(mg/kg-day)Effects at the NOAEL/LOAELReferenceSubchronic StudiesF344/NratF, M 0, 1.7, 3.5, 5.9,11.2, or 20.9mg/kg-day viadrinking water3 monthsF: NDM: ND1.71.7F: Microcytic, hypochromic anemia (decreased Hgb,MCV, MCH), increased serum liver enzyme activities(ALT and SDH) and bile acids, and histopathologicalchanges to the duodenum (histiocytic cellularinfiltration)M: Microcytic, hypochromic anemia (decreased Hct,Hgb, MCV, MCH), increased serum liver enzymeactivities (ALT and SDH), and histopathologicalchanges to pancreatic lymph nodes (histiocytic cellularinfiltration)NTP (2007)B6C3F1mouseF, M 0, 3.1, 5.3, 9.1,15.7,or 27.9mg/kg-day viadrinking water3 monthsF: NDM: ND3.13.1Histopathological changes (histiocytic cellularinfiltration) in the duodenumNTP (2007)B6C3F1,BALB/c,andam3-C57BL/6mouseM 0, 2.8, 5.2, or 8.7mg/kg-day viadrinking water3 months ND 2.8 Histopathological changes in the duodenum in B6C3F1mice (histiocytic cellular infiltration and epithelialhyperplasia), BALB/c mice (histiocytic cellularinfiltration), andam3-C57BL/6 mice (epithelialhyperplasia)NTP (2007)Wistar ratM 0, 73.05 mg/kgday via drinkingwater30 days ND ND Decreased serum prolactin levels. Data not adequatefor estimation of a NOAEL or LOAEL.Quinteros et al.(2007)Wistar ratM 0, 20 mg/L indrinking water10 weeks ND NDLiver histopathologic changes. Doses in mg hexavalentchromium/kg-day could not be estimated.Rafael et al.(2007)Wistar ratM 0, 1.5 mg/kg-dayvia drinkingwater22 weeks ND 1.5 Changes in serum enzymes; liver triglycerides,glycogen and cholesterol; liver histopathologicchanges.Acharya et al.(2001)SwissmouseM 0, 177, 265, 353,530, or 706 mg/Lin drinking water8 weeks ND ND Liver histopathologic changes. Dosesin mg hexavalentchromium/kg-day could not be estimated.Asmatullah andNoreen (1999)162 DRAFT - DO NOT CITE OR QUOTETable 4-25. Observed effects and corresponding NOAELs and LOAELs for subchronic, chronic, andreproductive toxicity studies following oral exposure to hexavalent chromiumSpeciesSexExposure level1ExposuredurationNOAEL(mg/kg-day)LOAEL(mg/kg-day)Effects at the NOAEL/LOAELReferenceWistar ratF 0, 1.4 mg/kg-dayvia drinkingwater22 weeks ND 1.4 Changes in liver weight; serum enzyme levels,triglycerides, glucose; liver glycogen; liverhistopathology.Chopra et al.(1996)Wistar ratF, M F: 0, 1.76-2.47mg/kg-day viadrinking waterM: 0, 1.4-2.18mg/kg-day viadrinking water6 months ND ND Changes in urinary markers of renalfunction. Nohistopathologic examination of the kidney.Vyskocil et al.(1993)Chronic StudiesF344/NratF, M F: 0.24, 0.94, 2.4or 7.0 mg/kg-dayvia drinkingwaterM: 0.21, 0.77,2.1, or 5.9mg/kg-day viadrinking water2 years F: NDM: 0.210.240.77F: Increased incidence of chronic inflammation of theliverM: Increased incidences of nonneoplastichistopathological changes to the liver (basophilicfoci),duodenum (histiocytic cellular infiltrate), andmesenteric lymph nodes (histiocytic cellular infiltrateand hemorrhage)NTP (2008)B6C3F1mouseF, M F: 0.38, 1.4, 3.1or 8.7 mg/kg-dayvia drinkingwaterM: 0.38, 0.91,2.4, or 5.9mg/kg-day viadrinking water2 years F: NDM: ND0.380.38F: Increased incidences of histopathological changes tothe duodenum (diffuse epithelial hyperplasia),mesenteric lymph nodes (histiocytic cellularinfiltration), liver (histiocytic cellular infiltration), andpancreas (depletion of cytoplasmic zymogen granules)M:Increased incidences of histopathological changes tothe duodenum (diffuse epithelial hyperplasia) andmesenteric lymph nodes (histiocytic cellularinfiltration)NTP (2008)163 DRAFT - DO NOT CITE OR QUOTETable 4-25. Observed effects and corresponding NOAELs and LOAELs for subchronic, chronic, andreproductive toxicity studies following oral exposure to hexavalent chromiumSpeciesSexExposure level1ExposuredurationNOAEL(mg/kg-day)LOAEL(mg/kg-day)Effects at the NOAEL/LOAELReferenceDog Notspecified0, 0.45, 2.25, 4.5,6.75, 11.2 mg/Lin drinking water4 years ND ND No effects were observed. Doses in mg hexavalentchromium/kg-day could not be estimated.Anwar et al.(1961)SpragueDawleyratF, M 0.05 to 2.8mg/kg-day viadrinking water1 year 2.4-2.8 ND No adverse effects observed at the highest dose tested MacKenzie etal. (1958)Reproductive/Developmental StudiesBonnetmonkeyM0, 1.0, 2.1, 4.1,and 8.3 mg/kgday via drinkingwater180 days ND 2.1 Reversible changes to male reproductive organs,including disruption of spermatogenesis, effects onsperm count and velocity, and histopathologicalchangesAruldhas et al.(2006, 2005,2004);Subramanian etal. (2006)CharlesFoster ratM 0, 20, 40, or60 mg/kg-dayviagavage90 days ND 20Decreased serum testosterone levels and loss of 3β-∆5-HSH activity in testesChowdhuryand Mitra(1995)Wistar ratM 0, 5.2 or10.4 mg/kg-dayvia gavage6 days ND 5.2 Decreased sperm counts and histopathological changesto the testesLi et al. (2001)BALB/cmouseM 0, 6.4, 12.7, or25.5 mg/kg-dayvia gavage35 days ND 6.4 Increased percentage of degeneratedtubules,undergenerated tubules without spermatogonia,abnormal sperm, and reduced number of spermatogoniaZahid et al.(1990)NewZealandWhiterabbitM 0 or 3.6 mg/kgday via gavage10 weeks ND 3.6 Decreased testes and epididymis weight and decreasedsperm outputYousef et al.(2006)164 DRAFT - DO NOT CITE OR QUOTETable 4-25. Observed effects and corresponding NOAELs and LOAELs for subchronic, chronic, andreproductive toxicity studies following oral exposure to hexavalent chromiumSpeciesSexExposure level1ExposuredurationNOAEL(mg/kg-day)LOAEL(mg/kg-day)Effects at the NOAEL/LOAELReferenceSpragueDawleyratF, M F: 0, 0.25, 1.1,2.5, or 9.5mg/kg-day viathe dietM: 0, 0.35, 1.1,2.1, or8.5 mg/kg-dayvia the diet9 weeks F: 2.5M: 2.1F: 9.5M: 8.5F: Slight erythrocyte microcytosisM: Slight erythrocyte microcytosisNTP (1996b)BALB/cmouseF, M F: 0, 1.8, 5.6,12.0, 48.4mg/kg-day viathe dietM: 0, 1.1, 3.5,7.4, or32.5 mg/kg-dayvia the diet9 weeks F: 1.8M: 3.5F: 5.6M: 7.4F: Histopathological changes to the liver (cytoplasmicvacuolization)M: Histopathological changes to the liver (cytoplasmicvacuolization)NTP (1996a)BALB/cmouseF 0, 7.9, 16.1, or37.1 mg/kg-dayvia the diet (F1generation)Continuousbreeding studyND 7.9 Erythrocyte microcytosis (slight decrease inMCH) inthe F1generationNTP (1997)DruckreyratF0, 70, 127, or170 mg/kg-dayvia drinkingwater3 months ND 70 Dam: Increased pre- and post-implantation lossesOffspring: Decreased fetal weight and external andskeletal abnormalitiesKanojia et al.(1998)SwissmouseF 0, 63, 119, or174 mg/kg-dayvia drinkingwaterGDs 6 though 14ND 63 Dam: Decreased fertilityOffspring: Decreased fetal body weight and delays inskeletal developmentJunaid et al.(1996a)165 DRAFT - DO NOT CITE OR QUOTETable 4-25. Observed effects and corresponding NOAELs and LOAELs for subchronic, chronic, andreproductive toxicity studies following oral exposure to hexavalent chromiumSpeciesSexExposure level1ExposuredurationNOAEL(mg/kg-day)LOAEL(mg/kg-day)Effects at the NOAEL/LOAELReferenceWistar ratF 0 or 7.9 mg/kgday via drinkingwaterGD 6 through 15ND 7.9 Dam: Increased pre-implantation loss/litter,postimplantation loss/litter, resorptions/litter, and deadfetuses/litter and decreased live fetuses/litterOffspring: Decreased fetal weight and increased litterswith fetal abnormalities or malformations includingvisceral and skeletal changes.Elsaieed andNada (2002)SpragueDawleyratF 0 or 35 mg/kgday via drinkingwaterGDs 1–3 or 4–6 ND 35 Dam: Impaired implantation, increased resorptions, anddecreased number of viable fetusesBataineh et al.(2007)ITRCBredmouseF 0, 48, 98, or 239mg/kg-day viadrinking waterEntire gestationalperiodDam: 48Offspring: NDDam: 98Offspring: 48Dam: Decreased body weight gain and increasedresorptions and postimplantation lossOffspring: Decreased fetal length and weightTrivedi et al.(1989)SwissmouseF0, 53, 101, or152 mg/kg-dayvia drinkingwaterGD 14 though 19Dam: 53Offspring: NDDam: 101Offspring: 53Dam: Decreased body weight gain and reduced numberof implantation sitesJunaid et al.(1996b)SwissmouseF 0, 53, 101, or152 mg/kg-dayvia drinkingwaterGD 14 though 19Dam: 53Offspring: NDDam: 101Offspring: 53Dam: Decreased body weight gainOffspring: Reduced fetal weight and length andincreased incidence of reduced caudal ossificationJunaid et al.(1995)1Unless otherwise noted, dose or concentration expressed as hexavalent chromium.F = female; M = male; ND = not determined

### 166 DRAFT - DO NOT CITE OR QUOTE

1 

### Based on a review of the NOAELs and LOAELs in Table 4-25, the most sensitive 2



### hexavalent chromium-induced effects in rats were in creased incidence of chronic inflammation 3 

### of the liver in females; and increased incidences o f nonneoplastic histopathological changes to 4 

### the liver (basophilic foci), duodenum (histiocytic cellular infiltrate), and mesenteric lymph nodes 5 

### (histiocytic cellular infiltrate and hemorrhage) in males. In mice, the most sensitive hexavalent 6



### chromium-induced effects were increased incidences of histopathological changes to the 7 

### duodenum (diffuse epithelial hyperplasia), mesenter ic lymph nodes (histiocytic cellular 8 

### infiltration), liver (histiocytic cellular infiltra tion), and pancreas (depletion of cytoplasmic 9 

### zymogen granules) in females; and increased inciden ces of histopathological changes to the 10 

### duodenum (diffuse epithelial hyperplasia) and mesen teric lymph nodes (histiocytic cellular 11 

### infiltration) in males. All of these effects were observed in the two-year chronic study by NTP 12 

### (2008), and in general, occurred at lower doses tha n the reproductive or developmental effects. 13

14



### 4.6. EVALUATION OF CARCINOGENICITY 15 

### 4.6.1. Summary of Overall Weight-of-Evidence 16



### Under the U.S. EPA Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005a), 17 

### hexavalent chromium is “likely to be carcinogenic t o humans” via the oral route of exposure 18 

### based on a statistically significant increase in th e incidence of tumors of the oral mucosa and 19 

### tongue of rats and of the small intestine of mice; and evidence of an association between oral 20 

### exposure to hexavalent chromium and stomach cancer in humans. Additionally, available 21



### evidence indicates that chromium interacts with DNA , resulting in DNA damage and 22 

### mutagenesis. Thus, hexavalent chromium is proposed to induce carcinogenicity via a mutagenic 23



### mode of action. 24

25 

### 4.6.2. Synthesis of Human, Animal, and Other Suppo rting Evidence 26 

### Human studies in which health outcomes (primarily cancer) were evaluated among 27 

### populations who resided near sources of industrial waste containing hexavalent chromium 28 

### compounds provide some evidence of possible associa tions between oral exposure to hexavalent 29 

### chromium and cancer. These epidemiological studies evaluated populations in Liaoning 30 

### Province, China (Kerger et al., 2009; Beaumont et a l., 2008; Zhang and Li, 1997, 1987), Kings 31 

### County/San Bernadino County, California (Fryzek et al., 2001), Nebraska (Bednar and Kies, 32 

### 1991), and Glasgow, UK (Eizaguirre-Garcia et al., 2 000, 1999) that unknowingly were exposed 33

### 167 DRAFT - DO NOT CITE OR QUOTE

to hexavalent chromium over some time period. Of t hese studies, the most detailed analyses 1



### were of data collected from the Jinzou area of Liao ning Province, China, where groundwater, 2 

### surface water, and agricultural soils were contamin ated with chromium derived from hexavalent 3 

### chromium production (e.g., 0.001–20 mg chromium/L i n residential well water). This study 4 

### found evidence of an excess risk of mortality from stomach cancer from 1970-1978 in residents 5 

### of the area, relative to the reference populations (four other areas in Liaoning Province, and the 6 

### total population of the province) (Beaumont et al., 2008). The association with stomach cancer 7 

### mortality was weaker when an urban area was exclude d from the reference population (Kerger et 8 

### al., 2009). However, there was little difference b etween stomach cancer rates in urban compared 9 

### to rural areas during this period; indicating no so und rationale for excluding this urban area from 10 

### the reference group. Studies of chromium-exposed p opulations in California and Nebraska 11 

### (Fryzek et al., 2001; Bednar and Kies, 1991) found no significant correlation between cancer 12 

### mortality and drinking water concentration, and the study of the population in Glasgow 13 

### (Eizaguirre-Garcia et al., 2000, 1999) found no cor relation between leukemia risk and distance 14 

### from a former chromium processing facility (where e levated soil concentrations for hexavalent 15 

### chromium were measured). Interpretation of the fin dings from these three studies is limited by 16 

### the analysis of all cancer mortality (rather than i ndividual cancer types) in the case of the 17 

### California and Nebraska studies and leukemia only i n the case of the Glasgow study. 18 

### Evidence of carcinogenicity in animals was provide d by the NTP (2008) bioassay 19 

### conducted in rats and mice. In this study, exposur e of F344/N rats to sodium dichromate 20 

### dihydrate in drinking water for 2 years resulted in a statistically significant increase in the 21 

### incidence of squamous epithelial papillomas and car cinomas of the oral mucosa and tongue 22 

### (noted by NTP as rare when compared with historical controls) at the highest exposure level 23 

### (average daily doses of 5.9 and 7.0 mg hexavalent c hromium/kg-day in males and females, 24 

### respectively), but not at the three lower exposure levels. NTP (2008) also exposed B6C3F1 mice 25 

### to sodium dichromate dihydrate in drinking water fo r 2 years and reported statistically significant 26



### increases in the incidence of adenomas and carcinom as of the small intestine in males and 27



### females at doses ≥ 2.4 and ≥ 3.1 mg hexavalent chromium/kg-day, respectively. 28 

### As discussed in detail in Section 4.6.3, hexavalen t chromium is proposed to induce 29 

### carcinogenicity via a mutagenic mode of action. Th e key precursor events leading to 30 

### mutagenicity have been identified in animals and th ese events are anticipated to occur in humans 31 

### and progress to tumors. 32

### 168 DRAFT - DO NOT CITE OR QUOTE

The “likely to be carcinogenic to humans” descripto r is appropriate when the weight of 1 

### the evidence is adequate to demonstrate carcinogeni c potential to humans but does not reach the 2 

### weight of evidence for the descriptor “carcinogenic to humans”. The database supports this 3 

### descriptor for hexavalent chromium exposure via the oral route. On the other hand, available 4 

### evidence to support the descriptor of “carcinogenic to humans” was also considered. 5 

### The “carcinogenic to humans” descriptor indicates s trong evidence of human 6 

### carcinogenicity, and can be characterized by differ ent combinations of evidence. One line of 7 

### evidence indicates this descriptor is appropriate w hen there is convincing epidemiologic 8 

### evidence of a causal association between human expo sure and cancer (U.S. EPA, 2005a). This is 9 

### not the case for exposure to hexavalent chromium vi a ingestion. A moderately elevated risk of 10 

### stomach cancer mortality was seen in Jinzou (Liaoni ng Province, China), but this risk has not 11 

### been established (or examined) in other populations exposed to drinking water contaminated 12 

### with hexavalent chromium. The epidemiologic data a re not sufficient to establish a causal 13 

### association between exposure to hexavalent chromium by ingestion and cancer. 14 

### A second line of evidence under which this descrip tor may be appropriate involves a 15 

### lesser weight of epidemiologic evidence that is str engthened by other information, including 16 

### strong evidence of an association between human exp osure and either cancer or the key events of 17 

### the MOA and extensive evidence of carcinogenicity i n animals (U.S. EPA, 2005a). As discussed 18 

### above, the epidemiologic evidence for the oral rout e of hexavalent chromium exposure is not 19 

### considered strong. In addition, extensive eviden ce of the carcinogenicity of hexavalent 20 

### chromium in animals via ingestion does not exist. Only one multiple dose chronic oral 21 

### carcinogenicity study of hexavalent chromium in ani mals is available (i.e., the two-year bioassay 22 

### in rodents conducted by NTP [2008]). Taken togethe r, these considerations do not provide a 23



### basis for the characterization of hexavalent chromi um as “carcinogenic to humans” via oral 24 

### exposure. Therefore, EPA concluded that, based on the available information, the descriptor 25 

### “likely to be carcinogenic to humans” is the most a ppropriate descriptor for the carcinogenic 26 

### potential of hexavalent chromium via ingestion. 27

28



### 4.6.3. Mode of Action Information 29 

### 4.6.3.1. Hypothesized Mode of Action 30 

### The hypothesized mode of action for carcinogenicity induced by hexavalent chromium is 31 

### via mutagenesis. The hypothesis is that carcinogen icity can be induced directly by reduced 32 

### forms of chromium interacting with DNA to form addu cts and crosslinks that can lead to DNA 33

### 169 DRAFT - DO NOT CITE OR QUOTE

breaks and mutations, and indirectly by free radica l species generated during the reduction 1 

### process that can also lead to DNA breakage and muta genesis. 2

3 

### Key events 4 

### Hexavalent chromium is readily taken up by cells th rough sulfate transporters, due to the 5 

### structural similarity of hexavalent chromium to the tetrahedral sulfate and phosphate anions 6 

### (Bridges and Zalups, 2005). Once inside the cell, hexavalent chromium quickly undergoes a 7 

### series of reduction reactions to yield pentavalent, tetravalent, and ultimately the 8 

### thermodynamically stable trivalent chromium. Many potential enzymes as well as non9 

### enzymatic cellular reductants capable of reducing h exavalent chromium exist within the cell. 10 

### These reductants include glutathione, ascorbate, cy steine, lipoic acid, NAD(P)H, fructose, and 11 

### ribose (reviewed in McCarroll et al., 2009). Follo wing this intracellular reduction, several 12 

### possible mechanisms leading to mutagenicity can occ ur. 13



### Hexavalent chromium itself does not interact direct ly with DNA. However, the products 14 

### of its reduction within the cell (pentavalent, tetr avalent, and trivalent chromium) have all been 15 

### shown to be DNA reactive (O’Brien et al., 2003). H exavalent chromium is reduced by 16 

### glutathione to yield pentavalent chromium and thiyl radicals, which can react with other thiol 17 

### molecules to produce superoxide radicals. Both pen tavalent and tetravalent chromium can 18 

### participate in Fenton reactions, generating hydroxy l radicals (Salnikow and Zhitkovich, 2008; 19 

### Volko et al., 2006). All of these species can caus e DNA single- and double-strand breaks, base 20 

### modifications, and lipid peroxidation, which may le ad to mutations if not adequately repaired. 21 

### Trivalent chromium is the ultimate product of the i ntracellular reduction of hexavalent 22 

### chromium. Trivalent chromium is capable of interac ting directly with DNA, forming stable 23 

### coordination complexes with nucleic acids and pepti des (Salnikow and Zhitkovich, 2008). In 24 

### particular, trivalent chromium is capable of formin g ternary complexes with DNA and an 25 

### intracellular reducer, such as ascorbate, glutathio ne, or cysteine (Salnikow et al., 1992; 26 

### Zhitkovich et al., 1996), as well as crosslinking D NA and proteins, and forming intrastrand 27 

### DNA-DNA crosslinks (Voitkun et al., 1998; Zhitkovic h, 2005). These chromium-DNA 28 

### complexes, as well as DNA-protein and DNA-DNA cross links, all have the capability of causing 29 

### DNA single- and double-strand breaks, which, if not adequately repaired, could lead to cell 30 

### death, or if misrepaired, could result in mutation. 31 

### Thus, once inside the cell, hexavalent chromium, th rough reduction to its pentavalent, 32 

### tetravalent, and trivalent forms, is capable of ind ucing a wide range of mutagenic and genotoxic 33

### 170 DRAFT - DO NOT CITE OR QUOTE

damage, including the formation of DNA adducts, DNA -protein and DNA-DNA crosslinks, 1 

### mutations, DNA single and double-strand breaks, aba sic sites, oxidized DNA bases, 2 

### chromosomal aberrations, sister chromatid exchanges , and micronuclei. 3

4 

### 4.6.3.2. Experimental Support for the Hypothesized Mode of Action 5 

### Strength, consistency, and specificity of associati on 6 

### A large database of experimental data exists on the mutagenic activity of hexavalent 7 

### chromium compounds (these results are summarized in Section 4.5.1 and in the corresponding 8 

### tables). In vitro, positive results were found in the majority of tests performed on hexavalent 9 

### chromium compounds in bacterial test systems (see T able 4-21). Similarly, in yeast ( S. 10



### cerevisiae and S. pombe ), all available studies described positive results for the detection of gene 11 

### mutations, mitotic gene conversion, and mitotic cro ssing over. 12 

### In mammalian cell lines and primary cells, all stud ies using whole cells in vitro yielded 13 

### positive results (Table 4-22). Evidence of mutatio n induction was shown at the tk locus in the 14 

### mouse lymphoma assay, as well as at the hgprt locus in Chinese hamster ovary cells (V79 and 15 

### AT3-2). In human cells, chromosome aberrations, DN A damage, and DNA-DNA and DNA16 

### protein crosslinks were detected in primary culture s and established cell lines originating from 17 

### target organs, including the gastric mucosa, bronch ial epithelium, and fibroblasts from the 18 

### bronchial tubes and lung. Chromosome aberrations, sister chromatid exchanges, and DNA 19 

### damage were observed in primary human dermal fibrob lasts and lymphocytes as well as 20 

### bronchial fibroblasts and epithelial cells. Chromo some aberrations and DNA damage were 21 

### found in mouse carcinogenic cell lines, and sister chromatid exchanges were detected in mouse 22 

### blastocysts. In rats, DNA damage and unscheduled D NA synthesis were observed in rat gastric 23 

### mucosal cells and hepatocytes as well as in primary lymphocytes, and transformation was 24 

### observed in rat liver epithelial cells upon exposur e to hexavalent chromium. A number of 25 

### studies have been performed using cultured Chinese hamster ovary cells, showing chromosomal 26 

### aberrations and sister chromatid exchanges as well as DNA damage, DNA-protein crosslinks, 27 

### and induced DNA methylation, and three studies show ed induced transformation in cultured 28 

### Syrian hamster embryo cells. 29 

### In vivo, most studies of the mutagenicity of hexava lent chromium compounds have 30 

### yielded positive results (Table 4-23). Somatic and germ cell mutations were detected in 3-day31



### old Drosophila melanogaster larvae fed potassium chromate, potassium dichromate , or calcium 32 

### chromate (Kaya et al., 2002; Spano et al., 2001; Am rani et al., 1999; Graf and Wurgler, 1996; 33

### 171 DRAFT - DO NOT CITE OR QUOTE

Zimmering et al., 1985). A number of in vivo oral exposure studies of the mutagenicity of 1 

### hexavalent chromium in mice and rats are available, with slightly differing results depending on 2 

### the method used. In the two studies in rats, Cooga n et al. (1991b) found DNA-protein crosslinks 3 

### in liver and not in splenic lymphocytes following 3 or 6 week exposures of 100 or 200 mg/L in 4 

### drinking water, but Mirsalis et al. (1996) did not find any evidence of DNA repair via 5 

### unscheduled DNA synthesis in rat hepatocytes follow ing 48-hour exposures of up to 20 mg/L in 6 

### drinking water or a single gavage dose of 20 mL/kg at the same concentration. In other studies 7 

### of mice exposed via gavage, DNA damage as measured by the comet assay was found in 8 

### peripheral leukocytes (including isolated lymphocyt es), stomach, colon, liver, kidney, bladder, 9 

### lung, and brain (Wang et al., 2006; Devi et al., 20 01; Sekihashi et al., 2001), but neither DNA 10 

### damage nor micronuclei were found in bone marrow (D e Flora et al., 2006; Sekihashi et al., 11 

### 2001; Shindo et al., 1989). Similarly, in studies of mice exposed via drinking water, De Flora et 12 

### al. (2008, 2006) reported negative results for the detection of micronuclei in the bone marrow of 13 

### pregnant Swiss albino mice and in the fetal polychr omatic erythrocytes after exposures up to 20 14 

### mg/L and also in adult BDF 1 

### mice following 500 mg/L exposure for 210 days. 15 

### Interestingly, NTP (2007) investigated micronuclei induction in male mouse bone 16 

### marrow following a three-month drinking water expos ure and found differing results depending 17 

### on the strain of mouse used. In one phase of the s tudy, results were negative in B6C3F 1 

### mice 18 

### exposed to doses as high as 349 mg/L, while in anot her phase, following exposures of 0, 21.8, 19 

### 43.6, or 87.2 mg/L hexavalent chromium, results wer e negative in BALB/c mice, equivocal in 20



### B6C3F 1 

### mice, and significantly positive at ≥ 43.6 mg/L exposures in am 3-C57BL/6 mice, with a 21 

### statistically significant positive trend starting a t 21.8 mg/L. 22 

### Somatic and germ cell mutations were detected in Drosophila melanogaster treated 23 

### intraperitoneally with chromic acid or potassium di chromate (Rodriguez-Arnaiz and Martinez, 24 

### 1986) or with sodium dichromate via filter paper (R asmuson, 1985). Following parenteral 25 

### exposure in mice, DNA damage was detected in the st omach, colon, bladder, lung, brain, liver, 26 

### and kidney (Sekihashi et al., 2001; Ueno et al., 20 01; Amlacher and Rudolph, 1981); mutations 27 

### were found in the liver of transgenic mice (Itoh an d Shimada, 1998; 1997), in the germ cells of 28 

### hybrid male mice (Paschin et al., 1982), and in the offspring of exposed female mice (Knudsen, 29 

### 1980); and micronuclei were increased in bone marro w and polychromatic erythrocytes (De 30 

### Flora et al., 2006; Wronska-Nofer et al., 1999; Ito h and Shimada, 1996; Hayashi et al., 1982; 31 

### Paschin and Toropzev, 1982; Wild, 1978), as well as in the liver and peripheral blood of mice 32 

### exposed prenatally (De Flora et al., 2006). In rat s exposed parenterally, DNA damage was 33

### 172 DRAFT - DO NOT CITE OR QUOTE

detected in leukocytes (Patlolla and Tchounwou, 200 6), and DNA-protein crosslinks were found 1 

### in lung, liver, and kidney (Tsapakos et al., 1983). Mutations were observed in the lung and 2 

### kidney from transgenic mice exposed intratracheally to hexavalent chromium (Cheng et al., 3 

### 2000); DNA-protein crosslinks and DNA fragmentation and adducts were found in the lung of 4 

### rats similarly exposed (Izzotti et al., 1998), whil e in rats exposed via inhalation, chromosomal 5 

### aberrations and sister chromatid exchanges were obs erved in peripheral lymphocytes (Koshi et 6 

### al., 1987). 7



### In addition to the in vivo evidence in animals for the genotoxicity of hexavalent 8 

### chromium, several studies are available in humans ( Table 4-24). In the only mutagenicity study 9 

### following oral doses, DNA-protein crosslinks were n ot detected in peripheral lymphocytes up to 10 

### 4 hours after the 4 volunteers were given 71 μg hex avalent chromium/kg (Kuykendall et al., 11 

### 1996). Another study (Gao et al., 1994) failed to detect DNA damage in peripheral lymphocytes 12 

### of workers inhalationally exposed to 0.001-0.055 mg /m 3 

### . However, several studies of 13 

### occupational exposures via inhalation provide evide nce of significant levels of chromium14 

### induced DNA damage (Gambelunghe et al., 2003), and the formation of micronuclei (Benova et 15 

### al., 2002; Vaglenov et al., 1999), chromosomal aber rations (Deng et al., 1988; Sarto et al., 1982), 16 

### and sister chromatid exchanges (Wu et al., 2001, 20 00; Deng et al., 1988; Sarto et al., 1982; 17 

### Stella et al., 1982) in peripheral lymphocytes and/ or buccal mucosal cells. These studies 18 

### detected genotoxicity in workers exposed to mean ai r concentrations as low as 0.0075 and 19 

### 0.0249 mg/m 3 

### (Benova et al., 2002). In addition, three studies found negative results for 20 

### micronuclei and sister chromatid exchange, but the exposure concentrations were not reported 21 

### (Nagaya et al., 1991, 1986; Sarto et al., 1990). 22

23 

### Dose-response concordance and temporal relationship 24 

### As noted above, hexavalent chromium is hypothesized to induce carcinogenicity via a 25 

### mutagenic mode of action. The initial key events in the hypothesized mutagenic mode of action 26 

### are the capability of the hexavalent form of chromi um to pass through the cell membrane and, 27 

### once inside, to be reduced to pentavalent, tetraval ent, and trivalent chromium. 28



### The available studies show that hexavalent chromium induces tumors in the stomach of 29 

### humans (Beaumont et al., 2008) and in the tongue, o ral mucosa, and intestines of rodents (NTP, 30 

### 2008). Studies of a cohort in Liaoning Province, C hina, exposed to 0.001–20 mg chromium/L in 31 

### residential well water (Beaumont et al., 2008; Zhan g and Li, 1997, 1987) reported an excess risk 32 

### of mortality from stomach cancer in residents of th e area. NTP (2008) reported a statistically 33

### 173 DRAFT - DO NOT CITE OR QUOTE

significant increase in the incidence of tumors of the oral mucosa and tongue in rats exposed to 1 

### hexavalent chromium for two years in drinking water at average daily doses of 5.9 and 7.0 2 

### mg/kg-day for males and females, respectively, and tumors of the small intestine in mice 3 

### exposed to average daily doses of ≥ 2.4 and 3.1 mg/kg-day in males and females, respect ively. 4 

### Correlating these data with mutagenicity testing by establishing temporal and dose and/or site 5 

### concordance can be difficult, as in vivo assays des igned to detect mutagenicity are conducted 6 

### within a relatively short time after the exposure p eriod has ended, and tend to rely mainly on 7



### cells from tissues such as bone marrow and/or blood that are actively replicating and therefore 8 

### sensitive to mutagenic agents. There is evidence, however, that hexavalent chromium can 9 

### accumulate and induce mutagenicity in tissues at th e site of entry and systemically, at doses 10 

### relevant to human exposures. 11 

### Following drinking water exposures, only one animal study has directly investigated 12 

### target tissue genotoxicity (De Flora et al., 2008). With regard to dose, the De Flora et al. (2008) 13 

### study tested levels (5 and 20 mg/L, or 1.2 and 4.82 mg/kg-day of hexavalent chromium) that 14 

### were just below those leading to murine intestinal (duodenum, jejunum, and ileum) tumors in the 15 

### two-year NTP study (30 and 50 mg/L for males and fe males, respectively). Negative results 16 

### were reported for DNA-protein crosslinks and DNA ad ducts when measuring the forestomach, 17 

### glandular stomach, and duodenum of mice exposed to hexavalent chromium for 9 months via 18 

### drinking water. However, the shorter study duratio n makes a direct comparison of these results 19 

### to the duodenal tumors reported in the chronic NTP bioassay infeasible. 20 

### Other studies have shown evidence of in vivo genoto xicity in non-target tissues at early 21 

### time points following exposure. In three studies t hat used the comet assay to detect DNA 22 

### damage following oral gavage exposures in mice, Dev i et al. (2001) found evidence of DNA 23 

### damage in leukocytes that peaked at 48 hours post-e xposure, Wang et al. (2006) detected DNA 24 

### damage in lymphocytes after 1-day or 5-day consecut ive exposures, and Seikihashi et al. (2001) 25 

### detected DNA damage in stomach, colon, liver, kidne y, bladder, lung, and brain within 8 hours 26 

### of dosing that subsided by 24 hours post-treatment. 27 

### Devi et al. (2001) found positive dose-dependent re sults at >10-fold lower doses (0.21, 28 

### 0.42, 0.84, 1.68, and 3.37 mg hexavalent chromium/k g). In fact, many of the positive in vivo 29 

### mutagenicity studies found a positive trend with do se, including oral exposures (Devi et al., 30 

### 2001; Wang et al., 2006) and parenteral exposures ( Paschin and Toropzev, 1982; Knudsen, 1980; 31 

### Itoh and Shimada, 1996; Wild, 1978; Shindo et al., 1989; Hayashi et al., 1982) and rats (Patlolla 32 

### et al., 2008) in mice. 33

### 174 DRAFT - DO NOT CITE OR QUOTE

Therefore, the detection of DNA damage, a key event for the mutagenic mode of action, 1 

### following oral exposure to hexavalent chromium, tha t exhibits dose dependence and that is 2 

### observed at time points prior to tumor development, strengthens the causal nature of this 3 

### association. Although DNA-protein crosslinks and D NA adducts were not detected in target 4 

### tissues following drinking water exposure in mice ( De Flora et al., 2008), the lack of these 5 

### findings did not preclude the observation of mutati ons in other tissues and organs, considered to 6 

### be early events following hexavalent chromium expos ure leading to carcinogenesis. 7

8 

### Biological plausibility and coherence 9 

### Mutagenicity as a mode of action for carcinogenicit y in humans is a biologically 10 

### plausible mechanism for tumor induction. Hexavalen t chromium has been shown to be 11 

### mutagenic in vitro and in vivo, across species and tissue types. Human studies have shown 12 

### induction of DNA damage, chromosomal aberrations, a nd micronucleus induction following 13 

### exposure to hexavalent chromium, and in vivo animal studies show that hexavalent chromium 14 

### induces DNA damage in rat blood, bone marrow, lung, liver, and kidney, and in mouse blood, 15 

### lung, liver, kidney, bladder, colon, and brain. Ex posures that induced a mutagenic response in 16 

### these studies included doses within the range causi ng tumors in rats and mice in a chronic 17 

### exposure bioassay (NTP, 2008). 18 

### Only one study examined tumor target tissue for evi dence of mutagenicity (De Flora et 19 

### al., 2008). De Flora et al. (2008) found negative results for DNA-protein crosslinks and DNA 20 

### adducts in the duodenum in mice following drinking water exposures. Other available drinking 21 

### water exposure studies of hexavalent chromium that measured mutagenicity in mice failed to 22



### show evidence of micronucleus induction in the bloo d or bone marrow (De Flora et al., 2008, 23 

### 2006; NTP, 2007; Mirsalis et al., 1996). 24 

### It has been proposed that the positive results for DNA damage found in mice following 25 

### oral gavage exposures (Wang et al., 2006; Devi et a l., 2001; Sekihashi et al., 2001) were the 26 

### result of overwhelming the reductive capacity of th e gastrointestinal tract in mice, allowing the 27 

### accumulation and subsequent absorption of hexavalen t chromium. This proposal would indicate 28 

### that the comparatively lower concentrations of hexa valent chromium administered in the 29 

### drinking water studies (De Flora et al., 2008; 2006 ) are effectively reduced to trivalent chromium 30 

### when ingested, thereby inhibiting cellular uptake a nd subsequent DNA damage. 31 

### While this is a plausible explanation for these res ults, which are unusual in that they 32 

### represent the only component of the hexavalent chro mium mutagenicity database that does not 33

### 175 DRAFT - DO NOT CITE OR QUOTE

show overwhelmingly positive results, there are inc onsistencies with this explanation. For 1 

### example, although the doses administered in De Flor a et al. (2008) were lower than those in 2 

### Wang et al. (2006) and Sekihashi et al. (2001), Dev i et al. (2001) found positive results at doses 3 

### approximately 6-fold lower than the lowest dose use d by De Flora et al. (2008). 4 

### In addition, genetic differences have been implicat ed in predicting the severity of 5 

### genotoxic responses to hexavalent chromium exposure . In the three-month NTP bioassay 6 

### (2007), three different strains of mice (B6C3F 1 

### , BALB/c, and am3 -C57BL/6) were exposed to 7 

### hexavalent chromium in drinking water at concentrat ions of 21.8, 43.6, or 87.2 mg/L, and found 8 

### different results for micronucleus induction in pol ychromatic erythrocytes among strains. The 9 

### BALB/c mice showed no micronucleus induction, and t he B6C3F 1 

### mice were positive only at the 10 

### highest dose of 87.2 mg/L. However, the am3 -C57BL/6 mice responded with an overall positive 11 

### trend, with the two highest doses statistically sig nificant, and the lowest dose nearly so. Based 12 

### on the expected reduction capacity of an average 50 g mouse, it does not appear that the 13 

### reductive capacities were overwhelmed in the NTP bi oassay. The average rate of hexavalent 14 

### chromium exposure for all three strains of mice was estimated to have been 2.9 x 10 -2 

### mg/hour at 15 

### the highest dose (NTP, 2007). This rate is within the estimated reductive capacity of the mouse 16 

### gastrointestinal tract of 4.4 x 10 -2 

### mg/hour that is based on an estimated 0.33 mL/hour rate of 17 

### drinking water consumption. However, the micronucl eus results could reflect minor differences 18 

### in the capacities of these three strains of mice to reduce hexavalent chromium extracellularly, 19 

### since the exact reductive capacity of each mouse st rain used is unknown. 20 

### The transgenic am3 -C57BL/6 mouse contains multiple copies of the Φ X174 am3 allele, 21 

### which is sensitive to A/T base-pair substitution mu tagens. Finding positive results in this strain 22 

### is consistent with DNA damage due to oxidative and/ or crosslinking mechanisms. This suggests 23 

### that interindividual differences in the capacity an d fidelity of DNA repair processes could 24 

### determine susceptibility to ingested hexavalent chr omium. In keeping with this, one DNA repair 25 

### pathway important in resolving mismatched bases dur ing DNA replication, mismatch repair 26 

### (MMR), has recently been implicated in the genotoxi c responses to hexavalent chromium 27



### exposure. 28 

### It has been shown that the processing of chromium-D NA adducts by the mismatch repair 29 

### (MMR) pathway is responsible for turning these lesi ons into frank DNA double-strand breaks 30 

### (Peterson-Roth et al., 2005). This study found tha t cells deficient in MMR were not subject to 31 

### the same toxic responses to hexavalent chromium as were cells with these repair processes intact. 32



### This loss of MMR function leads to an unstable muta tor phenotype, in which replication errors, 33

### 176 DRAFT - DO NOT CITE OR QUOTE

particularly those occurring in simple nucleotide r epeat sequences known as microsatellites, are 1 

### not corrected, leading to an increase in mutation f requency (Loeb et al., 2008). Further, these 2 

### effects would be exacerbated by the physical and ch emical interference with DNA replication 3 

### that occurs when trivalent chromium is present intr acellularly (Eastmond et al., 2008). 4



### There are several forms of cancer that exhibit micr osatellite instability. For example, 5 

### microsatellite instability has been implicated as t he cause of the majority of cases of hereditary 6 

### nonpolyposis colorectal cancer due to the inactivat ion of genes involved in the MMR pathway. 7 

### In an epidemiological study of chromate-exposed wor kers, microsatellite instability was reported 8 

### to occur in 79% of hexavalent chromium-induced lung tumors compared to only 15% in the non9 

### chromate lung cancer group (Hirose et al., 2002). The same group also reported finding 10 

### increased DNA methylation in the promoter region of the tumor suppressor gene p16 and the 11 

### MMR gene hMLH1 in human lung cancers in these chrom ate-exposed workers, indicating that 12 

### chromium can induce epigenetic effects (Kondo et al ., 2006; Takahashi et al., 2005). These 13 

### findings reflect a loss of functional MMR capabilit y that could be mechanistically involved in 14 

### chromate-induced lung cancer. 15 

### It was found that all four proteins responsible for MMR function were required for the 16 

### processing of chromium-DNA adducts into DNA double- strand breaks (Peterson-Roth et al., 17 

### 2005). The genes involved in MMR are known to be highly polymorphic in humans (Goode et 18 

### al., 2002), and given spontaneous background rates of mutation in human cells, it would not be 19 

### unexpected to find small populations of cells that have acquired mutations in one of these four 20 

### MMR genes. An inactivating mutation in any one of these would result in a growth advantage to 21 

### cells exposed to hexavalent chromium, allowing them to evade apoptotic responses to these 22 

### genotoxic lesions, as well as incurring further mic rosatellite instability, leading to a mutator 23 

### phenotype. Thus, a selective advantage upon chroni c exposure to even low levels of hexavalent 24 

### chromium could translate into a clonal expansion of these MMR-deficient cells, leading to 25 

### further evasion of cell death and increasing mutati on frequencies, resulting in a state of genomic 26 

### instability. 27 

### In addition, it is of note that among the available oral exposure studies in mice, all studies 28 

### that investigated DNA damage or micronucleus induct ion in bone marrow cells found negative 29 

### results, including the study by Sekihashi et al. (2 001), which found DNA damage in every tissue 30 

### examined (liver, kidney, lung, brain, stomach, colo n, and bladder) except for the bone marrow. 31 

### The reason for the negative findings in these assay s is unknown. 32

33

### 177 DRAFT - DO NOT CITE OR QUOTE

Bioavailability 1 

### As noted above, there is uncertainty surrounding th e ability of hexavalent chromium to 2 

### induce mutagenicity and carcinogenicity in humans c onsidering the potential for reduced 3 

### bioavailablity. Intrinsic to the mutagenic and car cinogenic processes of hexavalent chromium is 4 

### its ability to reach relevant tissues prior to bein g reduced to pentavalent, tetravalent, and trivalen t 5



### chromium. When hexavalent chromium is reduced to t he trivalent form extracellularly, this 6 

### reduction process effectively detoxifies hexavalent chromium, since trivalent chromium is nearly 7 

### impermeable to the cell. 8 

### Quantitative studies of GI absorption of hexavalent chromium in humans have estimated 9 

### that as much as 10% of an ingested dose of 5 mg is absorbed (Kuykendall et al., 1996), 10 

### indicating that not all hexavalent chromium is redu ced by the gastric juices of the stomach. In 11 

### rats and mice, daily oral doses of 8 mg hexavalent chromium/day for 8 weeks resulted in 12 

### absorption and accumulation of chromium in the bone , spleen, liver, and kidney (Kargacin et al., 13 

### 1993); rats given 0.138 μmol hexavalent chromium/da y for 3 days exhibited GI absorption of 14 

### about 16% (Febel et al., 2001); and the absorption of 4–10% of a single daily dose of 57 μg 15 

### hexavalent chromium (as Na 51 

### CrO 4 

### ) was observed in rats, regardless of fasting state (MacKenzie 16 

### et al., 1959). Distribution studies have shown tha t hexavalent chromium, once absorbed, 17 

### distributes to nearly all tissues, particularly con centrating in the kidney, liver, bone, and red 18 

### blood cells. Thus, at oral doses within human expo sure ranges, hexavalent chromium was not 19 

### completely reduced by the GI tract, making availabl e some portion of ingested hexavalent 20 

### chromium to be absorbed directly by the mucosal cel ls of the GI tract, or to be distributed to 21 

### other tissues throughout the body. 22 

### However, based on an understanding of chromium che mistry, as well as in vitro and in 23 

### vivo studies conducted by De Flora et al. (1997, 20 08), the reduction of at least some portion of 24 

### ingested hexavalent chromium to trivalent chromium likely occurs in the GI tract (see Section 3). 25 

### No data are currently available on the capacity of the rodent stomach to reduce hexavalent 26 

### chromium. However, based on in vitro measurements De Flora et al. (1997) estimated that the 27 

### reductive capacity of the human GI tract is suffici ently large to effectively reduce even high 28 

### doses of ingested hexavalent chromium to the less t oxic trivalent form. Given this assertion, it is 29 

### appropriate to ask whether the observed effects at the doses employed in the NTP (2008) study 30 

### resulted from an exceedance of the reductive capaci ty of the rodent GI tract. This is important 31 

### because if the effects observed only occurred due t o the reductive capacity of the rodent GI tract 32

### 178 DRAFT - DO NOT CITE OR QUOTE

being exceeded, these results may be less relevant to human risk at the lower doses that humans 1 

### are more likely to be exposed. 2 

### In discussing the results of the NTP (2008) study, the original NTP investigators, Stout et 3 

### al. (2009), specifically addressed this extracellul ar reduction issue. Qualitatively, Stout et al. 4 

### (2009) noted that, in the two-year NTP study, the o bserved increases in neoplasms of the small 5 

### intestine of mice and the toxicity to the erythron, histiocytic infiltration, and uptake of hexavalent 6 

### chromium into the tissues of rats and mice suggeste d that, under the conditions of this study, at 7 

### least a portion of the administered hexvalent chrom ium was not reduced in the stomach. 8 

### Moreover, Stout et al. (2009) also pointed out the significant disparity in the oral toxicity and 9 

### carcinogenicity of hexavalent chromium versus triva lent chromium in rodents, including the 10 

### absence of increases in neoplasms or nonneoplastic lesions of the small intestine in rats or mice 11 

### exposed to chromium picolinate monohydrate, a triva lent chromium compound tested in an 12 

### earlier NTP bioassay. Stout et al. (2009) believe that these data provide additional evidence that 13 

### hexavalent chromium is not completely reduced in th e stomach and is responsible for the 14



### observed effects. 15 

### In addressing the De Flora et al. (2008) suggestion that increases in neoplasms of the 16



### small intestine observed in mice are the result of a saturation of the gastric reduction capacity, 17 

### Stout et al. (2009) took a more quantitative approa ch. Stout et al. (2009) postulated that if the 18 

### threshold mechanism proposed by De Flora et al. (20 08) actually existed, the dose that saturated 19 

### the reduction capacity would likely represent an in flection point on a sublinear dose-response 20 

### curve, with doses above the inflection point demons trating an increasing rate of response per unit 21 

### dose. To test this hypothesis, Stout et al. (2009) evaluated tissue concentration and mouse small 22 

### intestine neoplasm data for linearity and found tha t data that were statistically nonlinear were 23 

### supralinear (i.e., exhibited a decreasing rate of r esponse per unit dose), which does not support 24 

### the presence of a reduction threshold. 25 

### Finally, De Flora et al. (1997) estimated the redu ctive capacity of human gastric juice to 26 

### be about 84 to 88 mg of hexavalent chromium per day . Similar data are not available for the 27 

### reductive capacity of mouse gastric juice. However , Stout et al. (2009) assumed that hexavalent 28 

### chromium reduction is equally effective in mice and humans and that gastric secretion scales 29 

### across species by body weight 3/4 

### . Then, they estimated the reductive capacity of t he gastric juice 30 

### from a 50-g mouse to be approximately 0.4 mg/day (8 mg/kg-day). Stout et al. (2009) then 31 

### pointed out that this value is greater than all of the male mouse doses and is nearly equivalent to 32 

### the average daily dose of hexavalent chromium in th e high-dose group of female mice in the 33

### 179 DRAFT - DO NOT CITE OR QUOTE

NTP (2008) study. Therefore, Stout et al. (2009) c oncluded from their analysis that the 1 

### neoplasms in the small intestine of mice occurred a t dose levels that did not exceed the estimated 2 

### hexavalent chromium reduction capacity of the gastr ic juices in mice. 3

4



### 4.6.3.3. Other Possible Modes of Action 5 

### It has been proposed that cellular proliferation su bsequent to cytotoxicity may be 6 

### involved in the carcinogenicity of hexavalent chrom ium. Evidence of diffuse duodenal 7 

### hyperplasia in mice in all exposure groups was obse rved in the 3-month NTP (2007) study. The 8 

### sites where hyperplasia was observed correlated wit h the site of tumors observed in the two-year 9 

### bioassay (NTP, 2008). However, there is no evidenc e that this hyperplasia occurred prior to 10 

### mutagenicity. Temporal evidence exists of mutageni city occurring within 24 hours of exposure 11 

### (see above), making it unlikely that this mutagenes is was the result of regenerative proliferation. 12 

### In addition, several mutagenicity studies specifica lly measuring cytotoxicity reported positive 13 

### findings at doses below those inducing toxicity, in cluding in vivo rodent studies by Itoh and 14 

### Shimada (1996), Sekihashi et al. (2001), Devi et al . (2001), NTP (2007), and Coogan et al. 15 

### (1991b). Therefore, although hyperplasia is involv ed in the carcinogenic process, a mode of 16 

### action involving cytotoxicity and hyperplasia is no t regarded as an initial causative event. 17

18 

### 4.6.3.4. Conclusions About the Hypothesized Mode o f Action 19 

### As noted above, hexavalent chromium is hypothesized to be carcinogenic by a mutagenic 20



### mode of action. The key events in the hypothesized mutagenic mode of action are t he uptake of 21 

### hexavalent chromium into the cell followed by intra cellular reduction to pentavalent, tetravalent, 22



### and trivalent chromium. These reduced forms of hex avalent chromium and the free radicals that 23 

### are formed during the reduction process are capable of directly interacting with cellular 24 

### components, giving rise to mutagenicity (including DNA adduct formation, DNA damage, gene 25 

### mutations, chromosomal aberrations, and micronuclei formation). Considering the database, 26



### there is evidence that hexavalent chromium can accu mulate and induce mutagenicity in various 27 

### tissues throughout the body at doses relevant to hu man exposures and, for oral exposures, within 28 

### the reductive capacity of the gastrointestinal trac t. 29

30



### 1. Is the hypothesized mode of action sufficiently sup ported in the test animals? 31 

### The experimental evidence that hexavalent chromium is mutagenic, as presented in 32 

### Section 4.5.1, includes multiple adverse genetic ef fects including DNA adduct formation, DNA 33

### 180 DRAFT - DO NOT CITE OR QUOTE

damage, gene mutations, chromosomal aberrations, an d the formation of micronuclei. In 1 

### addition to the evidence supporting a mutagenic mod e of action in test animals, alternative or 2 

### additional hypothesized modes of action for hexaval ent chromium carcinogenicity have not been 3



### demonstrated. 4

5



### 2. Is the hypothesized mode of action relevant to huma ns? 6 

### Mutagenicity is a well-established cause of carcin ogenicity. The evidence discussed 7



### above demonstrates that hexavalent chromium is a mu tagen in bacteria, yeast, cultured rodent 8 

### and human cells, fruit flies, mice, and rats, suppo rting the presumption that it could also be a 9 

### mutagen in humans. Moreover, several studies of ex posed workers provide direct evidence of 10 

### DNA damage by hexavalent chromium. In conclusion, the weight of evidence supports a 11 

### mutagenic mode of action for hexavalent chromium ca rcinogenicity. 12

13



### 3. Which populations or lifestages can be particularly susceptible to the hypothesized mode of 14



### action? 15 

### The mutagenic mode of action is considered relevan t to all populations and lifestages. 16 

### According to EPA’s Supplemental Guidance (U.S. EPA, 2005b), there may be increased 17 

### susceptibility to early-life exposures for carcinog ens with a mutagenic mode of action. 18 

### Therefore, because the weight of evidence supports a mutagenic mode of action for hexavalent 19 

### chromium carcinogenicity and in the absence of chem ical-specific data to evaluate differences in 20 

### susceptibility, early-life susceptibility should be assumed and the age-dependent adjustment 21 

### factors (ADAFs) should be applied, in accordance wi th the Supplemental Guidance . In addition, 22 

### individuals with genetic polymorphisms conveying de ficiencies in DNA repair capacity may 23 

### have increased susceptibility to hexavalent chromiu m carcinogenicity. 24

25 

### 4.6.3.5 Mutagenic Across All Routes of Exposure 26 

### There is evidence that ingested hexavalent chromium can reach the systemic circulation 27 

### and affect tissues beyond those at or near the site of entry. 28 

### Following inhalation exposures, hexavalent chromium has been shown to induce lung 29



### tumors in a number of human studies. In addition t o hexavalent chromium activity in the lungs, 30



### evidence exists that hexavalent chromium is absorbe d by the lung when inhaled and can then 31 

### enter systemic circulation. Consistent with this, DNA damage, micronucleus induction, and 32 

### sister chromatid exchanges have been observed in ci rculating peripheral lymphocytes from 33

### 181 DRAFT - DO NOT CITE OR QUOTE

workers exposed to inhalation concentrations as low as 7.5 and 24.9 μg/m 3 

### (Benova et al., 2002), 1 

### and for durations of 4 months to 14 years (Gambelun ghe et al., 2003), 0.5 to 18 years (Stella et 2 

### al., 1982), 2 to >20 years (Benova et al., 2002), o r 4 to 25 years (Vaglenov et al., 1999). These 3 

### mutagenicity studies indicate that, while tumor inc idence following inhalation exposure to 4 

### hexavalent chromium occurs primarily in the lungs, hexavalent chromium also has the capacity 5 

### to damage DNA in other tissues at timepoints and co ncentrations relevant to human exposures. 6



### EPA has concluded that hexavalent chromium is carci nogenic by a mutagenic mode of 7 

### action. Considering the available oral and inhalat ion evidence for mutagenicity and subsequent 8 

### carcinogenicity and that t 

### hese events are capable of occurring in all cells, this mode of action is 9 

### applicable to all routes of exposure and tumor type s. 10

11



### 4.7. SUSCEPTIBLE POPULATIONS AND LIFE STAGES 12 

### 4.7.1. Possible Childhood Susceptibility 13 

### No studies are available that address the possible adverse effects of hexavalent chromium 14 

### in children. However, there is evidence that hexav alent chromium may act through a mutagenic 15



### mode of action. In accordance with the Supplemental Guidance (U.S. EPA, 2005b), the 16 

### mutagenic mode of carcinogenic action for hexavalen t chromium would indicate an increased 17 

### carcinogenic susceptibility for early-life exposure s. In addition, developmental toxicity also is of 18 

### concern due to the mutagenicity of hexavalent chrom ium and the possibility for genetic damage 19 

### to the germ cells of the F 1 

### generation that could be transmitted to the F 2 

### generation. The 20 

### reproductive and developmental toxicity studies tha t have been conducted employing hexavalent 21 

### chromium suggest that the developing fetus may be a target of toxicity, as well as male and 22 

### female reproductive organs, which may result in a r eduction in fertility. 23

24



### 4.7.2. Possible Gender Differences 25 

### The extent to which men and women differ in suscept ibility to hexavalent chromium is 26 

### unknown. However, animal data exist that imply a d ifference between males and females in 27 

### their response to ingestion of hexavalent chromium. For example, in the NTP (2008) study, at 28 

### the highest concentration administered (516 mg/L), female rats exhibited a higher incidence of 29 

### tumors of the oral cavity than male rats (i.e., 11/ 48 (23%) versus 7/50 (14%), respectively). The 30 

### biological significance of this finding at lower do ses and for other species, including humans, is 31



### unknown. 32

33

### 182 DRAFT - DO NOT CITE OR QUOTE 5. DOSE-RESPONSE ASSESSMENTS 1

2 3 

### 5.1. ORAL REFERENCE DOSE (RfD) 4 

### 5.1.1. Choice of Principal Study and Critical Effe ct—with Rationale and Justification 5 

### Two types of studies are available that provide inf ormation on the toxicological effects of 6 

### ingested chromium in humans. The first type of stu dy provides evidence of acute human health 7 

### effects in individuals who accidentally or intentio nally ingested high (fatal or near-fatal) doses of 8 

### hexavalent chromium. The second type of study prov ides evidence of chronic human health 9 

### effects (primarily cancer) in populations exposed u nintentionally to food or drinking water 10 

### containing high levels of hexavalent chromium over an extended time period. Because both 11 

### types of studies provide little information on dose -response relationships and because the second 12 

### type of study is primarily concerned with cancer as an outcome, these available human data are 13 

### not useful for quantifying the risk of noncancer ef fects resulting from chronic exposure to 14



### hexavalent chromium. 15 

### In animals, the effects of subchronic oral exposure to hexavalent chromium have been 16 

### evaluated in rats (NTP, 2007; Quinteros et al., 200 7; Rafael et al., 2007; Acharya et al., 2001; 17 

### Chopra et al., 1996; Vyskocil et al., 1993) and mic e (NTP, 2007; Asmatullah and Noreen, 1999), 18 

### and the effects of chronic oral exposure to hexaval ent chromium have been evaluated in rats 19 

### (NTP, 2008; MacKenzie et al., 1958), mice (NTP, 200 8), and dogs (Anwar et al., 1961). In 20 

### particular, the subchronic and chronic studies cond ucted by NTP (2008, 2007) provide the most 21 

### useful dose-response data on the noncancer effects of oral hexavalent chromium exposure 22 

### because of their comprehensive assessments of numer ous toxicological endpoints at multiple 23 

### dose levels. A number of other studies of reproduc tive and developmental toxicity of hexavalent 24 

### chromium have been conducted in rats, mice and rabb its. These studies are summarized in Table 25



### 4-25. 26 

### Results from the NTP (2007) subchronic (i.e., 90-da y) study identified several hexavalent 27 

### chromium-induced noncancer effects, including hemat ological effects, hepatotoxicity, alterations 28 

### in lipid metabolism, and histopathological changes in GI tissues, and pancreatic and mesenteric 29 

### lymph nodes. The most sensitive hexavalent chromiu m-induced noncancer effects were 30 

### microcytic, hypochromic anemia, increased serum liv er enzyme activities, and histopathological 31 

### changes to the duodenum and pancreatic lymph nodes in rats; and histopathological changes in 32

### 183 DRAFT - DO NOT CITE OR QUOTE

the duodenum in mice. In the two-year toxicology a nd carcinogenicity study by NTP (2008), the 1



### most sensitive noncancer effects identified were hi stopathological changes to the liver, 2 

### duodenum, and mesenteric lymph nodes in rats; and i n the duodenum, mesenteric lymph nodes, 3 

### and liver in mice. LOAELs of 1.7—3.1 mg hexavalent chromium/kg-day were identified in the 4 

### subchronic NTP (2007) study, and LOAELs of 0.24—0.7 7 mg hexavalent chromium/kg-day 5 

### were identified in the chronic NTP (2008) study. 6 

### Other subchronic and chronic oral exposure studies of hexavalent chromium compounds 7 

### do not provide suitable data for identifying points of departure (PODs) for RfD derivation 8 

### because comprehensive toxicological evaluations wer e not conducted in these studies. In 9 

### addition, interpretation of results from these stud ies was compromised because of the small 10 

### number of animals evaluated, the lack of a dose-res ponse relationship, or inadequate reporting of 11 

### results (see Table 4-25). Where LOAELs were identif ied based on examination of a limited set 12 

### of endpoints (e.g., Acharya et al, 2001; Chopra et al, 1996), the LOAELs were higher than those 13 

### identified in the chronic NTP (2008) bioassay. 14 

### Studies of reproductive and developmental toxicity indicate that hexavalent chromium 15 

### exposure can affect reproductive organs, increase p re- and postnatal implantation loss, and cause 16 

### reduced fetal weight and fetal abnormalities. In g eneral, the NOAELs or LOAELs associated 17 

### with reproductive and developmental effects are hig her than those identified in the subchronic 18 

### and chronic toxicity studies summarized in Table 4- 25. 19 

### Thus, based on the comprehensive examination of end points and measurement of 20 

### sensitive endpoints of toxicity, the bioassays by N TP (2008, 2007) were deemed the best 21 

### candidates for use in deriving an oral RfD for hexa valent chromium. Specifically, five studies, 22 

### three subchronic (i.e., one in rats and two in mice ) (NTP, 2007) and two chronic (i.e., one in rats 23 

### and one in mice) (NTP, 2008), were identified as ca ndidate principal studies. The key results 24



### from these five studies are summarized below. 25

26



### Subchronic Studies 27 

### NTP (2007) 90-day studies in rats and mice 28 

### In F344/N rats, sodium dichromate dihydrate was adm inistered in drinking water to 29 

### groups of males and females at five different conce ntrations for 90 days. Based on average 30 

### water consumption rates, the mean effective doses o f hexavalent chromium were estimated by 31

### 184 DRAFT - DO NOT CITE OR QUOTE

NTP to be 0, 1.7, 3.5, 5.9, 11.2 and 20.9 mg/kg-day for both males and females. Results of this 1 

### study identified a LOAEL in male and female rats of 1.7 mg hexavalent chromium/kg-day; a 2



### NOAEL was not identified because effects were obser ved at the lowest dose tested. This 3 

### LOAEL was based on observations of microcytic, hypo chromic anemia, increased serum liver 4 

### enzyme activities, and histopathological changes to pancreatic lymph nodes (in males) and 5 

### histopathological changes to the duodenum (in femal es) at daily doses ≥ 1.7 mg hexavalent 6 

### chromium/kg-day. 7



### In B6C3F 1 

### mice, groups of males and females were exposed to sodium dichromate 8 

### dihydrate in drinking water for 90 days. Based on water consumption monitored throughout the 9 

### study, NTP calculated average daily doses over the 90-day treatment duration of approximately 10 

### 0, 3.1, 5.3, 9.1, 15.7, and 27.9 mg hexavalent chro mium/kg-day for both males and females. 11 

### Based on histopathological changes (histiocytic cel lular infiltration) in the duodenum in both 12 

### sexes, a LOAEL of 3.1 mg hexavalent chromium/kg-day was identified for male and female 13 

### mice; a NOAEL was not identified because the effect s observed were at the lowest dose tested. 14 

### In a comparative 90-day drinking water study in mal e B6C3F 1 

### , BALB/c, and am3

- 15 

### C57BL/6 mice, groups of each strain were exposed to three different concentrations of sodium 16 

### dichromate dihydrate. Based on water consumption a nd body weights monitored throughout the 17 

### study, NTP calculated average daily doses over the 90-day treatment duration of approximately 18 

### 0, 2.8, 5.2, or 8.7 mg hexavalent chromium/kg-day f or all strains. At the end of the study, 19



### similar effects were observed in all 3 strains. A LOAEL of 2.8 mg hexavalent chromium/kg-day 20 

### was identified based on histopathological changes i n the duodenum in B6C3F 1 

### mice (histiocytic 21 

### cellular infiltration and epithelial hyperplasia), BALB/c mice (histiocytic cellular infiltration), 22



### and am3 -C57BL/6 mice (epithelial hyperplasia); a NOAEL was not identified because effects 23



### seen were at the lowest dose tested. 24

25



### Chronic Studies 26 

### NTP (2008) two-year studies in rats and mice 27 

### In F344/N rats, groups of 50 males and females wer e administered sodium dichromate 28 

### dihydrate in drinking water at four different conce ntrations for two years. Based on measured 29 

### water consumption rates and body weights in rats, N TP estimated that male rats received time30 

### weighted average doses of hexavalent chromium of 0. 21, 0.77, 2.1, or 5.9 mg/kg-day, while 31

### 185 DRAFT - DO NOT CITE OR QUOTE

female rats received 0.24, 0.94, 2.4 or 7.0 mg/kg-d ay. This study identified NOAEL and 1



### LOAEL values for noncancer effects in male rats of 0.21 and 0.77 mg hexavalent chromium/kg2 

### day, respectively, based on increased incidences of nonneoplastic histopathological changes to 3 

### the liver (basophilic foci), duodenum (histiocytic cellular infiltrate), and mesenteric lymph nodes 4 

### (histiocytic cellular infiltrate and hemorrhage). In female rats, a LOAEL for noncancer effects of 5 

### 0.24 mg hexavalent chromium/kg-day was identified b ased on the increased incidence of chronic 6 

### inflammation of the liver (observed in all treatmen t groups); a NOAEL was not identified 7



### because effects observed were at the lowest dose te sted. 8



### In B6C3F 1 

### mice, groups of 50 males and females were administ ered sodium dichromate 9 

### dihydrate in drinking water at four different conce ntrations for two years. Based on measured 10 

### amounts of water consumption and body weights in mi ce, NTP estimated that male mice 11 

### received average doses of hexavalent chromium of 0. 38, 0.91, 2.4, or 5.9 mg/kg-day, while 12 

### female mice received 0.38, 1.4, 3.1 or 8.7 mg/kg-da y. This study identified a LOAEL for 13 

### noncancer effects of 0.38 mg hexavalent chromium/kg -day in both male and female B6C3F 1 14 

### mice; a NOAEL value was not identified because effe cts seen were at the lowest dose 15 

### administered. In males, the LOAEL was based on inc reased incidences of histopathological 16 

### changes to the duodenum (diffuse epithelial hyperpl asia) and mesenteric lymph nodes 17 

### (histiocytic cellular infiltration); in females, th e LOAEL was based on increased incidences of 18 

### histopathological changes to the duodenum (diffuse epithelial hyperplasia), mesenteric lymph 19 

### nodes (histiocytic cellular infiltration), liver (h istiocytic cellular infiltration), and pancreas 20 

### (depletion of cytoplasmic zymogen granules). 21 

### The NTP (2008) study was of chronic duration (i.e., 2 years), involved the use of multiple 22 

### dose groups, and included a comprehensive evaluatio n of multiple endpoints. Also, this bioassay 23 

### used lower doses than the subchronic (90-day) studi es also conducted by NTP (2007), and thus 24 

### provided dose-response information at lower exposur e levels than the 90-day studies. 25 

### Additionally, the chronic NTP (2008) study was more sensitive, yielding lower LOAELs than 26 

### the subchronic studies. Thus, the chronic NTP (200 8) study was selected as the principal study. 27 

### As indicated, NTP (2008) observed several hexavale nt chromium-induced noncancer 28



### effects in their chronic studies in rats and mice. Based on a comparison of LOAELs in rats and 29 

### mice (Table 4-25), the lowest LOAELs were observed for the following seven effects: 30

31

### 186 DRAFT - DO NOT CITE OR QUOTE

1. Chronic liver inflammation in female rats, 1 

### 2. Histiocytic cellular infiltration in the liver o f female mice, 2 

### 3. Diffuse epithelial hyperplasia in the duodenum o f male mice, 3 

### 4. Diffuse epithelial hyperplasia in the duodenum o f female mice, 4 

### 5. Histiocytic cellular infiltration in the mesente ric lymph nodes of male mice 5 

### 6. Histiocytic cellular infiltration in the mesente ric lymph nodes of female mice, 6 

### 7. Cytoplasmic cellular alteration of acinar epithe lial cells in the pancreas of female mice. 7

8



### All of these effects occurred at the lowest doses tested (i.e., 0.24 mg/kg-day in female 9 

### rats and 0.38 mg/kg-day in male and female mice), a nd were considered as possible critical 10



### effects for derivation of the RfD for hexavalent ch romium. The incidences of these seven effects 11 

### across all treatment groups in NTP (2008) are shown below in Table 5-1. 12

13

### 187 DRAFT - DO NOT CITE OR QUOTE

Table 5-1. Incidence Data for Lesions From All Tre atment Groups of Female F344/N Rats and Male and Female B6C3F 1 

### Mice Exposed to Sodium Dichromate Dihydrate in Drinking Water for 2 Years (NTP, 2008)

Dose (mg hexavalent chromium/kg-day)

0 0.24 0.94 2.4 7.0

Female Rats Liver, chronic inflammation 12/50 21/50 a 28/50 b 35/50 b 39/50 b

Dose (mg hexavalent chromium/kg-day)

0 0.38 0.91 2.4 5.9

Male Mice Duodenum: Diffuse epithelial hyperplasia 0/50 11/50 b 18/50 b 42/50 b 32/50 a

Mesenteric lymph node: Histiocytic cellular infiltration 14/47 38/47 b 31/49 b 32/49 b 42/46 a

Dose (mghexavalent chromium/kg-day)

0 0.38 1.4 3.1 8.7

Female Mice Duodenum: Diffuse epithelial hyperplasia 0/50 16/50 b 35/50 b 31/50 b 42/50 b

Mesenteric lymph node: Histiocytic cellular infiltration 3/46 29/48 b 26/46 b 40/50 b 42/50 b

Liver: Histiocytic cellular infiltration 2/49 15/50 b 23/50 b 32/50 b 45/50 b Pancreas: Acinus, cytoplasmic alteration 0/48 6/50 a 6/49 a 14/50 b 32/50 b

a Significantly different (p ≤ 0.05) from the control group by Dunn’s or Shirley’s test. b Significantly different (p ≤ 0.01) from the control group by Dunn’s or Shirley’s test.

Source: NTP (2008)

1 

### 5.1.2. Methods of Analysis—Including Models (PBPK, BMD, etc.) 2 

### To determine the specific endpoint for use in deriv ation of the RfD, all available 3



### dichotomous models in the EPA’s Benchmark Dose Soft ware (BMDS) were fit to the incidence 4 

### data for the seven selected endpoints in female rat s and male and female mice administered 5 

### sodium dichromate dihydrate in drinking water for t wo years (NTP, 2008). The incidence data 6 

### employed in the BMD modeling of these seven endpoin ts are shown in Table 5-1. To provide 7 

### candidate PODs based on these endpoints, 10% extra risk was selected as the benchmark 8 

### response (BMR) in accordance with U.S. EPA’s Benchmark Dose Technical Guidance (U.S. 9

### 188 DRAFT - DO NOT CITE OR QUOTE

EPA, 2000), which recommends selecting a response l evel near the lower range of detectable 1 

### observations that also facilitates comparisons acro ss endpoints. 2 

### For chronic liver inflammation in female rats, the log-logistic model provided the best fit, 3 

### yielding BMD 10 

### and BMDL 10 

### values of 0.22 and 0.14 mg hexavalent chromium/kg- day, 4 

### respectively. For diffuse epithelial hyperplasia i n the duodenum of male mice, the multistage 5 

### and quantal linear models provided the best fit, yi elding BMD 10 

### and BMDL 10 

### values of 0.16 and 6 

### 0.13 mg hexavalent chromium/kg-day, respectively. For diffuse epithelial hyperplasia in the 7 

### duodenum of female mice, the best fit was provided by several models (i.e., gamma, multistage, 8 

### quantal linear, and Weibull), yielding BMD 10 

### and BMDL 10 

### values of 0.12 and 0.09 mg 9 

### hexavalent chromium/kg-day, respectively. For both histiocytic cellular infiltration in the liver 10 

### and acinar cytoplasm alteration in the pancreas of female mice, the log-logistic model provided 11 

### the best fit, yielding BMD 10 

### and BMDL 10 

### values of 0.17 and 0.12 mg hexavalent chromium/kg12 

### day, respectively, for the liver lesions and 0.68 a nd 0.52 mg hexavalent chromium/kg-day, 13 

### respectively, for the pancreatic lesions. Finally, for the lesions of the mesenteric lymph nodes 14 

### (i.e., histiocytic cellular infiltration) in male a nd female mice, none of the available dichotomous 15 

### models in BMDS provided adequate fit to the data, e ven with the two highest doses dropped 16 

### from the analysis; thus, data sets for these lesion s were considered to be not suitable for BMD 17 

### analysis. A summary of this BMD modeling informati on is presented in Table 5-2, and further 18 

### details of this modeling are contained in Appendix B-1. 19

20

### 189 DRAFT - DO NOT CITE OR QUOTE

Table 5-2. Summary of BMD 10 

### and BMDL 10 

### from the best fitting models for lesions of the liver, duodenum, mesenteric lymp h nodes, and pancreas in female rats and male and female mice after expos ure to sodium dichromate dihydrate in drinking water for 2 years (NTP, 2008)

End point Species/sex Model Number of doses BMD a

(mg/kg-day) BMDL a

(mg/kg-day) Liver, chronic inflammation Rat/female Log-logistic 5 0.22 0.14

Duodenum: diffuse epithelial hyperplasia Mouse/male 1-Degree polynomial multistage/quantal linear 4 0.16 0.13

Mesenteric lymph node: histiocytic cellular infiltration b Mouse/male –— –— –— –—

Duodenum: diffuse epithelial hyperplasia Mouse/female Gamma/Multistage/quantal linear/Weibull 3 0.12 0.09

Mesenteric lymph node: histiocytic cellular infiltration b Mouse/female –— –— –— –—

Liver: histiocytic cellular infiltration Mouse/female Log-logistic 5 0.17 0.12

Pancreas: acinus, cytoplasmic alteration Mouse/female Log-logistic 5 0.68 0.52

a BMDs and BMDLs from dichotomous data are associated with a 10% extra risk; doses are in terms of mg hexavalent chromium/kg-day.

b None of the models provided an adequate fit to the data.

BMD = benchmark dose; BMDL = lower confidence limit (95%) on the benchmark dose

Source: ATSDR (2008)

1



### The lowest BMDL 10 

### value of 0.09 mg hexavalent chromium/kg-day, based on the 2 

### selection of the incidence of diffuse epithelial hy perplasia of the duodenum in female mice as the 3 

### critical effect, was identified as the POD from whi ch to derive the RfD for hexavalent chromium. 4

5 

### 5.1.3. RfD Derivation—Including Application of Unc ertainty Factors (UFs) 6 

### The following uncertainty factors (UFs) were appl ied to the POD of 0.09 mg/kg-day, 7 

### based on the incidence of diffuse epithelial hyperp lasia of the duodenum in female mice from 8 

### NTP (2008), to derive the RfD for hexavalent chromi um. 9

10

### 190 DRAFT - DO NOT CITE OR QUOTE • 

### An UF of 10 was used to account for uncertainty in extrapolating from laboratory 1 

### animals to humans (i.e., interspecies variability) because information was 2 

### unavailable to quantitatively assess toxicokinetic or toxicodynamic differences 3 

### between animals and humans. 4 5

• 

### An UF of 10 was used to account for variation in su sceptibility among members of 6 

### the human population (i.e., interindividual variabi lity) because information is 7 

### unavailable to predict potential variability in hum an susceptibility. 8

9

• 

### An UF was not needed to account for extrapolation f rom subchronic-to-chronic 10 

### exposure because a chronic study was used to derive the chronic RfD. 11

12

• 

### An UF for LOAEL to NOAEL extrapolation was not used because the current 13 

### approach is to address this extrapolation as one of the considerations in selecting a 14 

### BMR for BMD modeling. In this case, a BMR represen ted by a 10% extra risk of 15 

### diffuse epithelial hyperplasia was selected under a n assumption that it represents a 16 

### minimal biologically significant change. 17 18

• 

### An UF of 1 was used to account for database deficie ncies. The toxicity of ingested 19 

### hexavalent chromium has been extensively examined i n a range of animal 20 

### toxicology studies. The database for oral toxicity includes a chronic drinking water 21 

### study in rats and mice, a chronic drinking water st udy in rats, a subchronic drinking 22 

### water study in rats and mice, and a number of repro ductive/developmental toxicity 23 

### studies in monkeys, rabbits, rats, and mice. The r eproductive toxicity database 24 

### includes a continuous breeding study (NTP, 1997), i n which F 0 

### and F 1 

### generation 25 

### animals were exposed to hexavalent chromium in the diet, and the offspring of F 1 26 

### animals were evaluated on PND 21. 27 28 

### For this assessment, the RfD of 0.0009 or 9 x 10 -4 

### mg/kg-day for hexavalent chromium 29 

### was derived by dividing the BMDL 10 

### (or POD) of 0.09 mg/kg-day by a composite uncertai nty 30 

### factor of 100 (10 for extrapolation from animals to humans and 10 for human variability). 31

32



### 5.1.4. Previous RfD Assessment 33 

### The previous RfD assessment for hexavalent chromi um was completed in September 34 

### 1998. The previous RfD was based on a NOAEL identi fied from a one-year drinking water 35 

### study in rats in which animals were exposed to hexa valent chromium (as potassium chromate) at 36 

### a dose of 2.5 mg/kg-day (MacKenzie et al., 1958). No toxicity was reported in these animals at 37 

### this dose, resulting in identification of a NOAEL o f 2.5 mg/kg-day, the only dose administered in 38 

### the study, as the POD. A composite uncertainty fac tor of 300 (10 for interspecies extrapolation, 39 

### 10 for intraspecies extrapolation, and 3 for subchr onic to chronic extrapolation) and a modifying 40

### 191 DRAFT - DO NOT CITE OR QUOTE

factor of 3 (to account for concerns raised by the epidemiology study of Zhang and Li, 1987) 1 

### were applied to this POD to yield an oral RfD of 3 x 10 -3 

### mg/kg-day. 2

3



### 5.2. UNCERTAINTIES IN THE ORAL REFERENCE DOSE 4 

### The following discussion identifies uncertainties associated with the RfD for 5 

### hexavalent chromium. As presented above, an RfD of 9 x 10 -4 

### mg/kg-day was derived based on 6 

### the incidence of diffuse epithelial hyperplasia of the duodenum in female mice from a two-year 7 

### drinking water study (NTP, 2008). UFs were applied to the POD, a BMDL 10 

### generated through 8 

### BMD modeling. Factors accounting for uncertainties associated with a number of steps in the 9 

### analyses were adopted to account for extrapolating from an animal bioassay to humans with 10 

### varying susceptibilities. 11 

### An adequate range of animal toxicology data is av ailable for the hazard assessment of 12 

### hexavalent chromium via ingestion, as described pre viously in Section 4. The database of oral 13 

### toxicity studies includes a chronic drinking water study in rats and mice, a chronic drinking 14 

### water study in rats, a subchronic drinking water st udy in rats and mice, and several 15 

### reproductive/developmental toxicity studies in monk eys, rabbits, rats, and mice. Toxicity 16 

### associated with oral exposure to hexavalent chromiu m is observed in the liver, GI tract, and 17 

### reproductive organs, with the liver and GI tract be ing the most sensitive target organs. In 18 

### addition to oral toxicity data, there are absorptio n, distribution, metabolism, and excretion 19 

### studies, although information on internal or target organ dose of hexavalent chromium is not 20



### available. 21 

### Consideration of the available dose-response data to determine an estimate of oral 22 

### exposure that is likely to be without an appreciabl e risk of adverse health effects over a lifetime 23 

### led to the selection of the two-year drinking water study in F344/N rats and B6C3F 1 

### mice (NTP, 24 

### 2008) and increased incidence of diffuse epithelial hyperplasia in the duodenum of female mice 25 

### as the principal study and critical effect, respect ively, for deriving the RfD for hexavalent 26



### chromium. 27 

### The selection of the BMD model for identifying th e POD does not lead to significant 28



### uncertainties since benchmark effect levels were wi thin the range of the experimental data. 29 

### However, the selected models do not represent all p ossible models one might fit, and other 30

### 192 DRAFT - DO NOT CITE OR QUOTE

models could be selected to yield more extreme resu lts, both higher and lower than those 1



### included in this assessment. 2 

### Animal to human extrapolation yields further unce rtainties. The effect and the 3 

### magnitude of this effect associated with the dose a t the POD in mice are extrapolated to humans. 4 

### Pharmacokinetic models are useful to examine specie s differences in pharmacokinetic 5 

### processing; however, dosimetric adjustment using ph armacokinetic modeling was not possible 6 

### for the toxicity observed following oral exposure t o hexavalent chromium. Information was 7 

### unavailable to quantitatively assess toxicokinetic or toxicodynamic differences between animals 8 

### and humans. Accordingly, a 10-fold UF was used to account for uncertainty in extrapolating 9 

### from laboratory animals to humans in the derivation of the RfD. 10 

### Heterogeneity among humans is another area of unc ertainty. In the absence of 11 

### hexavalent chromium-specific data on variation in h uman response, a factor of 10 was used in 12 

### the derivation of the RfD. Human variation may be larger or smaller than this 10-fold factor; 13 

### however, hexavalent chromium-specific data to exami ne the potential magnitude of over- or 14



### underestimation are unavailable. 15

16



### 5.3. ORAL CANCER ASSESSMENT 17 

### 5.3.1. Choice of Study/Data—with Rationale and Jus tification 18 

### Several epidemiology studies have examined the ass ociation between oral exposure to 19 

### environmental hexavalent chromium and cancer in pop ulations that resided near sources of 20 

### industrial waste containing hexavalent chromium com pounds, including studies of populations in 21 

### Liaoning Province, China (Kerger et al., 2009; Beau mont et al., 2008; Zhang and Li, 1997, 1987, 22 

### 1980), Kings County/San Bernadino County, Californi a (Fryzek et al., 2001), Nebraska (Bednar 23 

### and Kies, 1991), and Glasgow, UK (Eizaguirre-Garcia et al., 2000, 1999). The Liaoning 24 

### Province studies provide some evidence of an excess risk of mortality from stomach cancer; 25 

### however, because of various limitations, including limited characterization of exposure, the 26 

### Liaoning Province studies are not considered adequa te for dose-response analysis. 27 

### The NTP rodent bioassay, in which F344/N rats and B6C3F 1 

### mice were administered 28 

### sodium dichromate dihydrate, a hexavalent chromium compound, in drinking water for two years 29 

### (NTP, 2008), was selected as the basis for deriving the oral cancer slope factor for hexavalent 30 

### chromium. This bioassay was selected for dose-resp onse assessment because it is a well- 31

### 193 DRAFT - DO NOT CITE OR QUOTE

conducted lifetime animal study of hexavalent chrom ium carcinogenicity via ingestion (see 1 

### detailed summary of the study in Section 4.2.1.2). No other adequate studies of hexavalent 2 

### chromium carcinogenicity by ingestion are available

. 3

4 

### 5.3.2. Dose-Response Data 5 

### The dose-response data considered in the derivati on of the cancer slope factor (CSF) for 6 

### hexavalent chromium were the incidence of benign an d malignant tumors in rat oral mucosa and 7 

### mouse small intestine observed in the NTP (2008) bi oassay. 8 

### Incidence data for neoplastic lesions of the oral cavity in male and female rats exposed to 9 

### sodium dichromate dihydrate in drinking water for 2 years are summarized in Table 4-15. 10 

### Neoplasms observed in the oral cavity of treated ra ts were squamous cell carcinoma of the oral 11 

### mucosa (both sexes), squamous cell papilloma of the oral mucosa (males only), squamous cell 12 

### carcinoma of the tongue (both sexes), and squamous cell papilloma of the tongue (both sexes). 13 

### The incidences of squamous cell carcinoma of the or al mucosa (13.6%) and of combined 14 

### squamous cell papilloma or carcinoma (15.7%) of the oral mucosa were statistically significantly 15 

### increased (at p < 0.05) in male rats treated with 5.9 mg/kg-day he xavalent chromium (the highest 16 

### dose tested) compared with controls. The incidence s of squamous cell carcinoma of the oral 17 

### mucosa (23.9%) and of combined squamous cell carcin oma of the oral mucosa or tongue 18 

### (23.9%) were statistically significantly increased (at p < 0.05) in female rats treated with 7.0 mg 19 

### hexavalent chromium/kg-day (the highest dose tested ) compared with controls. The incidences 20 

### of other neoplastic lesions of the oral cavity were not statistically significantly increased in any 21 

### treatment group in male or female rats compared wit h controls, although the incidence of 22 

### squamous cell carcinoma of the oral mucosa in femal e rats in the penultimate (2.4 mg/kg-day) 23 

### dose group (4.6%) exceeded that of historical contr ols (i.e., 0/300 in drinking water studies; 24 

### 5/1,400 (0.4%) by all routes of exposure). Other n eoplasms observed in treated rats included 25 

### pancreatic acinar adenomas and benign pheochromocyt omas in males and mononuclear cell 26 

### leukemias in females (see Table 4-16); however, the incidence of these neoplasms did not exhibit 27 

### dose-dependence. Thus, NTP (2008) concluded that e vidence of a relationship between 28 

### neoplastic changes in tissues other than the oral c avity and exposure to sodium dichromate 29 

### dihydrate was equivocal. In summary, exposure of r ats to sodium dichromate dihydrate in 30 

### drinking water for 2 years resulted in a significan t increase in squamous epithelial neoplasms of 31

### 194 DRAFT - DO NOT CITE OR QUOTE

the oral mucosa and tongue at the highest exposure levels (average daily doses of 5.9 and 7.0 mg 1 

### hexavalent chromium/kg-day in males and females, re spectively), but not at the three lower 2 

### exposure levels. The incidences of squamous cell p apillomas or carcinomas in the oral cavity of 3 

### male and female F344/N rats exposed to sodium dichr omate dihydrate in drinking water for 2 4 

### years in the NTP (2008) study are presented in Tabl e 5-3 (for male rats) and Table 5-4 (for 5 

### female rats). 6

7 

### Table 5-3. Incidences of squamous cell papillomas or carcinomas in the

oral cavity of male F344/N rats exposed to sodium d ichromate dihydrate in drinking water for 2 years (NTP, 2008)

Sodium dichromate dihydrate concentration (mg/L) Estimated

daily intake of hexavalent chromium a

(mg/kg-day) Incidence of squamous cell papillomas or carcinomas b 0 0 0/50 (0%) 14.3 0.21 1/50 (2%) 57.3 0.77 0/49 (0%) 172 2.1 0/50 (0%) 516 5.9 7/49 (14.5%) c

a Intakes were reported by NTP (2008) based on drinki ng water intakes and mean body weights observed dur ing the study.

b Number of animals with lesion/number of animals exa mined. Incidence estimates include all animals that were examined for oral tumors unadjusted for survival.

c Statistically significantly elevated above control at p < 0.05 using Fisher’s Exact Test.

8

### 195 DRAFT - DO NOT CITE OR QUOTE

Table 5-4. Incidences of squamous cell papillomas or carcinomas in the oral cavity of female F344/N rats exposed to sodium dichromate dihydrate in drinking water for 2 years (NTP, 2008)

Sodium dichromate dihydrate concentration (mg/L) Estimated

daily intake of hexavalent chromium a

(mg/kg-day) Incidence of squamous cell papillomas or carcinomas b 0 0 1/50 (2%) 14.3 0.24 1/50 (2%) 57.3 0.94 0/50 (0%) 172 2.4 2/50 (4%) 516 7.0 11/50 (22%) c

a Intakes were reported by NTP (2008) based on drinki ng water intakes and mean body weights observed dur ing the study.

b Number of animals with lesion/number of animals exa mined. Incidence estimates include all animals that were examined for oral tumors unadjusted for survival.

c Statistically significantly elevated above control at p < 0.05 using Fisher’s Exact Test.

1 

### Also from the NTP (2008) study, incidence data fo r neoplastic lesions of the small 2



### intestine in male and female B6C3F 1 

### mice exposed to sodium dichromate dihydrate in dri nking 3 

### water for 2 years are summarized in Table 4-19. In male mice, statistically significant increases 4 

### (at p < 0.05) were observed in the incidences of adenoma s or carcinomas combined in the small 5 

### intestine (duodenum, jejunum, and ileum) at hexaval ent chromium doses ≥ 2.4 mg/kg-day (i.e., at 6 

### the two highest doses tested). Furthermore, signif icant positive trends were observed in the 7 

### incidences of duodenal adenomas, duodenal carcinoma s, jejunal adenomas, small intestine 8 

### adenomas, small intestine carcinomas and small inte stine adenomas or carcinomas combined in 9 

### male mice. In female mice, statistically significa nt increases (at p < 0.05) were observed in the 10 

### incidences of duodenal adenomas, small intestine ad enomas, and small intestine adenomas or 11



### carcinomas combined at hexavalent chromium doses ≥ 3.1 mg/kg-day (i.e., at the two highest 12 

### doses tested). Furthermore, significant positive t rends were observed in the incidences of 13 

### duodenal adenomas, duodenal carcinomas, jejunal ade nomas, small intestine adenomas, and 14



### small intestine adenomas or carcinomas combined in female mice. No other statistically or 15 

### biologically significant increases in neoplasms wer e observed in other tissues. 16 

### In summary, exposure of B6C3F 1 

### mice to sodium dichromate dihydrate in drinking wa ter 17 

### for 2 years resulted in statistically significant i ncreases in the incidences of neoplasms of the 18



### small intestine in males and females at hexavalent chromium doses ≥ 2.4 and ≥ 3.1 mg/kg-day, 19

### 196 DRAFT - DO NOT CITE OR QUOTE

respectively. The incidences of adenomas and carci nomas combined in the small intestine of 1



### male and female B6C3F 1 

### mice exposed to sodium dichromate dihydrate in dri nking water for 2 2 

### years are summarized in Tables 5-2 and 5-3, respect ively. In evaluating the tumor incidences in 3 

### rats and mice, the mouse was determined to be the m ost sensitive species because tumor 4 

### incidences were statistically significantly elevate d at lower doses and a greater response was 5 

### exhibited by the mice at the two highest doses. Th erefore, the mouse tumor incidence data were 6 

### used as the basis for the oral CSF derived employin g BMD modeling. 7

8 

### Table 5-5. Incidences of adenomas and carcinomas c ombined in the small

intestine of male B6C3F 1 

### mice exposed to sodium dichromate dihydrate in drinking water for 2 years (NTP, 2008)

Sodium dichromate dihydrate concentration (mg/L) Estimated daily intake of hexavalent chromium a (mg/kg-day) Incidence of adenomas or carcinomas b

0 0 1/49 (2%) 14.3 0.38 3/49 (6.1%) 28.6 0.91 2/49 (4.1%) 85.7 2.4 7/50 (14%) c 257.4 5.9 20/48 (41.7%) c

a Intakes were reported by NTP (2008) based on drinking water intakes and mean body weigh ts observed during the study.

b Calculated from reported percentages of mice with a denomas or carcinomas. Incidence estimates included all animals that were examined for intestinal tumors an d survived for at least 451 days. In each of the c ontrol and first two dose groups, one animal died prior to day 451. In the high-dose group, two animals died pri or to day 451. None of these animals were found to have inte stinal adenomas or carcinomas at the time of death.

c Statistically significantly elevated above control at p < 0.05 using Fisher’s Exact Test. 9 10

11

### 197 DRAFT - DO NOT CITE OR QUOTE

Table 5-6. Incidences of adenomas and carcinomas c ombined in the small intestine of female B6C3F 1 

### mice exposed to sodium dichromate dihydrate in drinking water for 2 years (NTP, 2008)

Sodium dichromate dihydrate concentration (mg/L) Estimated daily intake of hexavalent chromium a (mg/kg-day) Incidence of adenomas or carcinomas b

0 0 1/49 (2%) 14.3 0.38 1/50 (2%) 57.3 1.4 4/49 (8.2%) 172 3.1 17/49 (34.7%) c 516 8.7 22/49 (44.9%) c

a Intakes were reported by NTP (2008) based on drinki ng water intakes and mean body weights observed dur ing the study.

b Calculated from reported percentages of mice with a denomas or carcinomas. Incidence estimates included all animals that were examined for intestinal tumors an d survived for at least 451 days. In all of the do se groups except the low-dose group, one animal died prior to day 451. None of these animals were observed to h ave intestinal adenomas or carcinomas at the time of de ath.

c Statistically significantly elevated above control at p < 0.05 using Fisher’s Exact Test.

1

2 

### 5.3.3. Dose Adjustments and Extrapolation Method(s ) 3



### The EPA Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005a) recommend that 4 

### the method used to characterize and quantify cancer risk from a chemical is determined by what 5 

### is known about the mode of action of the carcinogen and the shape of the cancer dose-response 6 

### curve. The dose response is assumed to be linear i n the low-dose range when evidence supports 7 

### a mutagenic mode of action because of DNA reactivit y, or if another mode of action that is 8 

### anticipated to be linear is applicable. A linear l ow-dose extrapolation approach was used to 9 

### estimate human carcinogenic risk associated with he xavalent chromium exposure due to the 10 

### mutagenic mode of carcinogenic action of this chemi cal. 11 

### In order to derive an oral CSF, BMD modeling was carried out using the EPA’s BMDS 12 

### (U.S. EPA, 2007). The EPA’s BMDS offers several po ssible mathematical dose-response 13 

### functions for use with dichotomous data including l ogistic, gamma, Weibull, quantal linear, 14 

### probit, and multistage models. For this assessment , EPA relied on the results obtained from the 15 

### multistage model only, as this is the model preferr ed by the Agency for conducting cancer dose16 

### response assessments. In applying the benchmark do se approach to the derivation of a CSF, the 17 

### standard procedure is to calculate a lower 95% conf idence bound on the dose corresponding to 18

### 198 DRAFT - DO NOT CITE OR QUOTE

the benchmark response (BMR), where the BMR is typi cally set at 10% extra risk. This lower 1



### confidence bound is referred to as the BMDL 10 

### . The CSF is then calculated by dividing the 2 

### BMR by the BMDL 10 

### and then converting this slope value to human equi valents. 3 

### In estimating the CSF, the incidence of neoplasms in the small intestine of mice was 4 

### employed, as this species was deemed to be more sen sitive than the rat. Only animals that 5 

### survived for at least 451 days, the time until appe arance of the first tumor, were considered at 6 

### risk for tumor development. Consequently, the incid ence estimates included all animals that were 7



### examined for intestinal tumors and survived for at least 451 days (see Tables 5-5 and 5-6). The 8 

### BMD modeling results for the incidence of neoplasms in the small intestine of male and female 9 

### mice are shown in Appendix B-2. For male mice, the two-stage multistage model exhibited the 10 

### best fit to the data yielding a slope of 0.09 (mg/k g-day) -1 

### . For female mice, the two-stage 11 

### multistage model also exhibited the best fit to the data yielding a slope of 0.10 (mg/kg-day) -1



### . 12 

### In order to estimate an oral CSF, these slopes wer e converted to human equivalents. For 13 

### this conversion, body weight to the 3⁄4 power scaling was used, where the time-weighted average 14 

### male and female mouse body weights of controls (i.e ., 50 and 53 grams, respectively) were 15 

### employed, along with an assumed human body weight o f 70 kg. The mouse body weights were 16 

### taken from the NTP (2008) study report. The follow ing equation was then used to convert the 17 

### slopes derived from the BMD modeling to oral CSFs e xpressed in human equivalents: 18

19 

### Slope x (W H 

### /W A 

### ) 0.25 

### = CSF, 20



### where 21



### W H 

### = animal body weight (kg) 22



### W A



### = human body weight (kg) 23

24 

### Using the above equation, the CSFs resulting from the fitting of the two-stage multistage 25 

### model in BMDS to the incidence of neoplasms in the small intestine of male or female mice 26 

### were 0.5 and 0.6 (mg/kg-day) -1 

### , respectively, expressed in human equivalents. 27

28 

### 5.3.4. Oral Slope Factor 29



### The CSF values based on the incidence of small in testine tumors in male and female mice 30 

### are similar (i.e., 0.5 (mg/kg-day) -1 

### for males and 0.6 (mg/kg-day) -1 

### for females). Given the 31

### 199 DRAFT - DO NOT CITE OR QUOTE

poorer fit of the multistage model to the female mo use data, a CSF estimate based on the male 1



### mouse data was considered to be associated with les s uncertainty. Therefore, the CSF of 0.5 2 

### (mg/kg-day) -1 

### , based on the incidence of neoplasms in the small intestine of male mice, was 3 

### selected as the most appropriate CSF for hexavalent chromium. 4

5 

### 5.3.5. Application of Age-Dependent Adjustment Fac tors 6 

### Because a mutagenic mode of action for hexavalent chromium carcinogenicity is 7 

### sufficiently supported in laboratory animals and is relevant to humans (see Section 4.6.3.4), and 8 

### in the absence of chemical-specific data to evaluat e differences in age-specific susceptibility, 9 

### increased early-life susceptibility to hexavalent c hromium is assumed and age-dependent 10 

### adjustment factors (ADAFs) should be applied, as ap propriate, in accordance with the 11 

### Supplemental Guidance for Assessing Susceptibility from Early-Life Exposure to Carcinogens 12 

### (U.S. EPA, 2005b). The oral slope factor of 0.5 (m g/kg-day) -1 

### , calculated from data applicable 13 

### to adult exposures, does not reflect presumed early -life susceptibility to this chemical. Example 14 

### calculations for estimating cancer risks based on a ge at exposure are provided in Section 6 of the 15 

### Supplemental Guidance for Assessing Susceptibility from Early-Life Exposure to Carcinogens 16 

### (U.S. EPA, 2005b). 17



### The Supplemental Guidance for Assessing Susceptibility from Early-Life Exposure to 18 

### Carcinogens establishes ADAFs for three specific age groups. T he current ADAFs and their 19 

### corresponding age groups are 10 for exposed individ uals <2 years old, 3 for exposed individuals 20 

### 2 to <16 years old, and 1 for exposed individuals 1 6 years old and older (U.S. EPA, 2005b). The 21 

### 10-fold and 3-fold adjustments to the slope factor are to be combined with age-specific exposure 22 

### estimates when estimating cancer risks from early l ife (<16 years of age) exposures to hexavalent 23



### chromium. 24



### To illustrate the use of the ADAFs established in the Supplemental Guidance for 25 

### Assessing Susceptibility from Early-Life Exposure t o Carcinogens (U.S. EPA, 2005b), sample 26 

### calculations are presented for three exposure durat ion scenarios, including full lifetime, assuming 27 

### the exposure rate to hexavalent chromium remains co nstant at an average daily dose of 0.0001 28 

### mg hexavalent chromium/kg-day (Table 5-7). 29

30

### 200 DRAFT - DO NOT CITE OR QUOTE

Table 5-7. Application of ADAFs for a 70-year expos ure to 0.0001 mg hexavalent chromium/kg-day from ages 0 to 70

Age group ADAF Slope Factor (per mg/kg-d) Average Daily Dose (mg/kg-d) Duration

adjustment Partial risk 0–<2 yrs 10 0.5 0.0001 2 yrs/70 yrs 1 × 10 -5

2–<16 yrs 3 0.5 0.0001 14 yrs/70 yrs 3 × 10 -5

≥ 16 yrs 1 0.5 0.0001 54 yrs/70 yrs 4 × 10 -5

Total risk 8 × 10 -5

1 

### Note that the partial risk for each age group is t he product of the values in columns 2–5 2 

### (e.g., 10 × 0.5 × 0.0001 × 2/70 = 0.00001 for expos ures from age 0 to <2 years), and the total 3 

### risk is the sum of the partial risks. Thus, a 70-y ear risk estimate for a constant average daily 4 

### dose of 0.0001 mg/kg-day starting at birth is 0.000 08 or 8 × 10 -5



### . 5 

### If calculating the cancer risk for a 30-year expos ure to a constant average daily dose of 6 

### 0.0001 mg hexavalent chromium/kg-day from ages 0 to 30 years, the duration adjustments would 7 

### be 2/70, 14/70, and 14/70, and the partial risks wo uld be 0.00001, 0.00003, and 0.00001, 8 

### resulting in a total risk estimate of 0.00005 or 5 × 10 -5



### . 9 

### If calculating the cancer risk for a 30-year expos ure to a constant average daily dose of 10 

### 0.0001 mg hexavalent chromium/kg-day from ages 20 t o 50 years, the duration adjustments 11 

### would be 0/70, 0/70, and 30/70, and the partial ris ks would be 0, 0, and 0.00002, resulting in a 12



### total risk estimate of 0.00002 or 2 × 10 -5



### . 13

14



### 5.3.6. Uncertainties in Cancer Risk Values 15 

### As in most risk assessments, extrapolation of data from experimental animals to estimate 16 

### potential lifetime cancer risks to human population s from exposure to hexavalent chromium 17 

### yields uncertainties. Some of these uncertainties can be evaluated for th eir quantitative impact 18 

### on the final result, while for others, only their q ualitative impact can be assessed. The principal 19



### uncertainties in the assessment of the cancer risk from exposure to hexavalent chromium are 20 

### summarized below in Table 5-8, and discussed in mor e detail in the following text. 21

### 201 DRAFT - DO NOT CITE OR QUOTE

1 

### Table 5-8. Summary of uncertainties in the cancer risk assessment for

hexavalent chromium

Consideration/ approach Impact on oral slope

factor Decision Justification

Low-dose extrapolation procedure Alternatives could ↓ or

↑ CSF by an unknown

extent Multistage model

used to determinePOD, linear lowdose extrapolation from POD A linear-low-dose extrapolation approach was used

to estimate human carcinogenic risk associate d with hexavalent chromium exposure consistent with a mutagenic mode of carcinogenic action.

Cross-species scaling Alternatives could ↓ or

↑ CSF [e.g., sixfold ↓ (scaling by BW) or ↑ twofold (scaling by BW 2/3 )] BW 3/4 (default

approach) In the absence of hexavalent chromium-specific

information on interspecies differences in toxicokinetics, the default scaling factor of BW 3/4 was used to calculate equivalent cumulative exposures for estimating equivalent human risks (U.S. EPA, 1992).

Statistical uncertainty at POD ↓ CSF 25% if MLE

(i.e., BMD 10 ) used rather than lower bound (BMDL 10 ) for POD BMDL (default

approach for calculating reasonable upper bound CSF) Size of bioassay results in sampling variability;

lower bound is 95% confidence interval on administered dose.

Species/gender combination Human risk could ↓ or

↑ , depending on relative sensitivity Male mouse tumors

(adenomas or carcinomas of the small intestine) 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 species. Generally, direct site concordance is not assumed; consistent with this view, some human tumor types are not found in rodents and rat and mouse tumor types also differ.

Human relevance of rodent tumor data Lack of human

relevance of tumor data would ↓ CSF Tumors with

significant doseresponse considered for estimating potential human

cancer response Hexavalent chromium is judged to be carcinogenic

through a mutagenic mode of action and is a multisite carcinogen in rodents; therefore, the carcinogenicity observed in rodent studies is assumed to be relevant to human exposure.

Human population variability in metabolism and response/ sensitive subpopulations Low-dose risk ↑ or ↓ to

an unknown extent Considered

qualitatively No data are available to support the range of human

variability/sensitivity to hexavalent chromium.

2 

### Choice of low-dose extrapolation approach . The mode of action is a key consideration in 3 

### clarifying how risks should be estimated for low-do se exposure. A linear, low-dose 4 

### extrapolation approach was used to estimate human c arcinogenic risk associated with hexavalent 5

### 202 DRAFT - DO NOT CITE OR QUOTE

chromium exposure consistent with a hypothesized mu tagenic mode of carcinogenic action of 1 

### hexavalent chromium (U.S. EPA, 2005a). 2 

### The multistage model was used to model the tumor i ncidence data because this is the 3 

### model preferred by the Agency for conducting cancer dose-response assessments; however, it is 4



### unknown how well this model or the linear low-dose extrapolation predicts low-dose risks for 5



### hexavalent chromium. The selected model does not r epresent all possible models one might fit, 6 

### and other models could conceivably be selected to y ield more extreme results consistent with the 7 

### observed data, both higher and lower than those inc luded in this assessment. 8 

### Cross-species scaling . The default cross-species scaling factor (BW 3/4 

### ) was applied to 9 

### address toxicological equivalence of internal doses between rodent species and humans, 10



### consistent with the 2005 Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005a). 11



### Because it is unknown whether there are differences in the pharmacokinetic pathways in animals 12 

### and humans following hexavalent chromium exposure, it is not possible to estimate the 13 

### magnitude of the uncertainty in the use of this def ault beyond that associated with other choices 14 

### for default cross-species scaling factors (e.g., BW 2/3 

### or BW 1 

### ). 15 

### Statistical uncertainty at the POD

. Measures of statistical uncertainty require assumin g 16 

### that the underlying model and associated assumption s are valid for the data under consideration. 17 

### For the multistage model applied to the incidence o f male mice GI tract tumors, there is a 18 

### reasonably typical degree of uncertainty at the 10% extra risk level (the POD for linear low-dose 19 

### extrapolation). That is, the BMDL 10 

### for male mice is approximately 25% lower than the BMD 10



### . 20 

### Choice of species/gender . The oral CSF for hexavalent chromium was quantif ied using 21 

### the tumor incidence data for mice, which were thoug ht to be more sensitive than rats to the 22 

### carcinogenicity of hexavalent chromium. While tumo r responses in the mouse were higher than 23 

### those of rats at a comparable dose level, suggestin g greater sensitivity of the mouse, it is 24 

### unknown whether this higher sensitivity would be ma intained at lower exposures. 25



### Relevance to humans . The Guidelines for Carcinogen Risk Assessment (EPA, 2005a) 26 

### state that site concordance is not a prerequisite f or evaluating the implications of animal study 27 

### results for humans. Chemicals that are mutagenic a nd cause tumors at multiple sites in animals 28 

### are likely relevant to human carcinogenesis. Hexav alent chromium is thought to be carcinogenic 29 

### through a mutagenic mode of action and is a multisi te carcinogen in rodents. Considering all of 30 

### the available information, the carcinogenicity obse rved in rodent studies is considered relevant to 31

### 203 DRAFT - DO NOT CITE OR QUOTE

human exposure. In addition, the concordance of th e alimentary system tumors across rats and 1 

### mice lends strength to the concern for human carcin ogenic potential. 2 

### Human population variability

. The extent of inter-individual variability in respo nse to 3 

### hexavalent chromium is unknown. Although a mutagen ic mode of action would indicate 4 

### increased early-life susceptibility, the data explo ring whether there is differential sensitivity to 5 

### hexavalent chromium carcinogenicity across life sta ges is unavailable. This lack of 6 

### understanding about potential differences in metabo lism and susceptibility across exposed 7 

### human populations thus represents a source of uncer tainty. The uncertainties associated with this 8 

### lack of data and knowledge about human variability can, at present, only be considered in 9 

### qualitative terms; however, EPA has developed ADAFs to quantitatively account for some of the 10 

### potential differences in age-dependent response to carcinogens with a mutagenic mode of action. 11 

### ADAFs are to be applied to the CSF for hexavalent c hromium when assessing cancer risks in 12 

### exposed populations composed of individuals less th an 16 years old (U.S. EPA, 2005b). More 13 

### specific guidance in applying these ADAFs was provi ded in Section 5.3.5. 14

15



### 5.3.7. Previous Cancer Assessment 16 

### The previous IRIS assessment for hexavalent chromi um was posted to the IRIS database 17 

### in 1998. In that assessment, EPA concluded that th e oral carcinogenicity of hexavalent 18 

### chromium could not be determined (and was thus clas sified as Group D ) because no data were 19 

### located in the available literature that suggested that hexavalent chromium is carcinogenic by the 20 

### oral route of exposure. Therefore, no oral CSF was derived. 21

22

### 204 DRAFT - DO NOT CITE OR QUOTE

6. MAJOR CONCLUSIONS IN THE CHARACTERIZATION OF HA ZARD AND DOSE 1



### RESPONSE 2

3

4



### 6.1. HUMAN HAZARD POTENTIAL 5 

### Hexavalent chromium compounds are a group of substa nces that contain chromium in the 6 

### hexavalent or +6 oxidation state. As a class, hexa valent chromium compounds are strong 7 

### oxidizing agents, and thus it is rare to find hexav alent chromium naturally occurring in the 8 

### environment because it is readily reduced to trival ent chromium (i.e., chromium in the +3 9 

### oxidation state) by organic matter. However, hexav alent chromium compounds released to the 10 

### environment by anthropogenic sources may persist in natural waters and soils that contain low 11 

### amounts of reducing materials. Major uses or forme r uses of hexavalent chromium compounds 12 

### include metal plating, manufacture of pigments and dyes, corrosion inhibitors, chemical 13 

### synthesis, refractory production, leather tanning, and wood preservation. Individuals may be 14 

### exposed to hexavalent chromium compounds through in gestion of drinking water or contact with 15



### soils or other media contaminated with these substa nces. 16



### Toxicokinetic studies in mice and rats have examine d the absorption, distribution, 17 

### metabolism, and elimination of hexavalent chromium compounds. Hexavalent chromium can be 18 

### absorbed via oral, inhalation, or dermal routes of exposure in humans and laboratory animals. 19 

### For this toxicological review, however, the focus i s on the toxicokinetics of hexavalent 20 

### chromium following ingestion. Once ingested, hexav alent chromium compounds can interact 21 

### with endogenous fluids and other organic matter in the GI tract, resulting, to some extent, in the 22



### reduction of hexavalent chromium to trivalent chrom ium. This process, whereby hexavalent 23



### chromium is reduced to trivalent chromium in the GI tract, is termed “extracellular” reduction. 24 

### The extent of absorption of ingested hexavalent chr omium into the GI tissues appears to be 25 

### determined by both the solubility of the hexavalent chromium compound ingested and how 26 

### rapidly hexavalent chromium is reduced to trivalent chromium in the GI tract, as trivalent 27 

### chromium does not diffuse readily across cell membr anes. Hexavalent chromium can easily 28 

### cross cell membranes due to its ability to use exis ting nonspecific sulfate and phosphate anion 29 

### transport mechanisms. Reduced trivalent chromium, however, can form complexes with organic 30 

### ligands, which allow it to pass more easily across cell membranes. 31 

### Ingested hexavalent chromium is distributed through out the body. Liver, kidney, and 32 

### bone are the primary sites of chromium accumulation . Once inside the cell, hexavalent 33

### 205 DRAFT - DO NOT CITE OR QUOTE

chromium is reduced to trivalent chromium, either e nzymatically or non-enzymatically. This 1 

### process is called “intracellular” reduction to dist inguish it from the extracellular process 2 

### described above. This intracellular reduction yiel ds such reactive intermediates as chromium(V) 3 

### and chromium(IV). These reactive intermediates, al ong with oxygen radicals generated during 4 

### this intracellular reduction, can indirectly damage DNA. In addition, trivalent chromium, the 5 

### final product of the intracellular reduction of hex avalent chromium, can form adducts with a 6 

### number of macromolecules, including DNA

. 7 

### Hexavalent chromium is eliminated primarily in the urine as trivalent chromium. Biliary 8



### excretion of hexavalent chromium has been measured in animals following intravenous injection. 9 

### However, this elimination route is relatively minor , with excreted hexavalent chromium or 10 

### trivalent chromium-ligand complexes being readily r eabsorbed from the gut. Chromium can also 11 

### be eliminated in hair, nails, and breast milk. The re does not appear to be a gender difference in 12 

### the toxicokinetics of hexavalent chromium, and inte r-individual variability in the pre-systemic 13 

### reduction and subsequent absorption and elimination may be primarily driven by differences in 14 

### gastric contents and intervals between meals. 15 

### Two PBTK models have been developed for hexavalent and trivalent chromium in rats 16 

### and humans (O’Flaherty et al., 2001; O’Flaherty, 19 96, 1993). The inclusion of trivalent 17



### chromium in the model allows for the use of trivale nt chromium exposure time course data to aid 18 

### in parameterization of chromium elimination and to evaluate the ability of the model to predict 19



### elimination of hexavalent chromium as trivalent chr omium. However, the limitations of the 20 

### performance of the model against limited oral expos ure data sets make it inadequate for use in 21 

### deriving reference values. 22 

### Two types of studies provide information on the tox icological effects in humans resulting 23 

### from exposure to ingested hexavalent chromium. In the first type of study, acute human health 24 

### effects have been observed following oral ingestion of hexavalent chromium in individuals 25 

### accidentally or intentionally ingesting high (fatal or near-fatal) doses of hexavalent chromium. 26 

### In the second type of study, chronic human health e ffects have been reported in human 27 

### populations exposed unintentionally to elevated lev els of hexavalent chromium in food or 28 

### drinking water over an extended time period. 29 

### In animals, the effects of subchronic oral exposure to hexavalent chromium have been 30 

### evaluated in rats (NTP, 2007; Quinteros et al., 200 7; Rafael et al., 2007; Acharya et al., 2001; 31 

### Chopra et al., 1996; Vyskocil et al., 1993) and mic e (NTP, 2007; Asmatullah and Noreen 1999;), 32 

### and the effects of chronic oral exposure to hexaval ent chromium have been evaluated in rats 33

### 206 DRAFT - DO NOT CITE OR QUOTE

(NTP, 2008, MacKenzie et al., 1958), mice (NTP, 200 8), and dogs (Anwar et al., 1961). Results 1 

### from the NTP (2007) subchronic study identified sev eral hexavalent chromium-induced effects, 2 

### including hematological effects, hepatotoxicity, al terations in lipid metabolism, and 3 

### histopathological changes in GI tissues and pancrea tic and mesenteric lymph nodes. The most 4



### sensitive hexavalent chromium-induced effects in ra ts were microcytic, hypochromic anemia, 5 

### increased serum liver enzyme activities, and histop athological changes to the duodenum and 6 

### pancreatic lymph nodes; in mice, the most sensitive effect was histopathological changes in the 7



### duodenum. The most sensitive noncancer effects in the NTP (2008) two-year toxicology and 8 

### carcinogenicity study were histopathological change s to the liver, duodenum, and mesenteric 9 

### lymph nodes in rats; and in the duodenum, mesenteri c lymph nodes, and liver in mice. 10 

### A number of animal studies have evaluated the repro ductive/developmental toxicity of 11 

### hexavalent chromium via the oral route of exposure. Collectively, these studies provide 12 

### evidence that oral exposure to hexavalent chromium compounds produces reproductive effects, 13 

### including histopathological changes to reproductive organs in males (Aruldhas et al., 2006, 2005, 14 

### 2004; Chowdhury and Mitra, 1995; Li et al., 2001; Z ahid et al., 1990) and females (Murthy et al., 15 

### 1996); alterations in sperm, including decreased co unt, decreased motility, and abnormal 16 

### morphology (Subramanian et al., 2006; Yousef et al. , 2006; Li et al., 2001; Zahid et al., 1990); 17 

### decreased plasma testosterone levels (Yousef et al. , 2006; Chowdhury and Mitra, 1995); 18 

### increased estrous cycle length (Kanojia et al., 199 8, 1996; Murthy et al., 1996); changes in 19 

### mating behavior and decreased fertility in males (B ataineh et al., 1997); and adverse 20 

### reproductive outcomes, including decreased numbers of live fetuses and implantations, and 21 

### increased numbers of resorptions and pre- and posti mplantation losses (Bataineh et al., 2007; 22 

### Elsaieed and Nada, 2002; Elbetieha and Al-Hamood, 1 997; Junaid et al., 1996a, b, 1995; Kanojia 23 

### et al., 1998, 1996; Trivedi et al., 1989). Develop mental effects observed have included 24 

### decreased fetal weight and length (Elsaieed and Nad a, 2002; Kanojia et al., 1998; Junaid et al., 25 

### 1996a, b, 1995; Trivedi et al., 1989); external (su bdermal hemorrhage and tail malformations) 26 

### and skeletal abnormalities (decreased ossification) (Elsaieed and Nada, 2002; Junaid et al., 27 

### 1996a, b, 1995; Kanojia et al., 1998, 1996; Trivedi et al., 1989); and delayed sexual maturation 28 

### and function in female offspring (Banu et al., 2008 ; Al-Hamood et al., 1998). In contrast to 29 

### results of the above studies, effects were not obse rved in dietary exposure studies conducted by 30 

### NTP that investigated the potential for hexavalent chromium to produce effects on male 31 

### reproductive organs in rats and mice (NTP, 1996a,b) and on reproductive outcomes in a 32 

### continuous breeding study in mice (NTP, 1997). The reasons for these inconsistent results are 33

### 207 DRAFT - DO NOT CITE OR QUOTE

not readily apparent, as daily dose ranges evaluate d in the NTP studies overlapped with those 1 

### used in other studies showing hexavalent chromium-i nduced adverse reproductive effects. 2 

### Human studies of possible associations between oral exposure to hexavalent chromium 3 

### and cancer are limited to a few epidemiology studie s in which health outcomes (primarily 4 

### cancer) were evaluated among populations who were e xposed to drinking water contaminated 5 

### with hexavalent chromium in Liaoning Province, Chin a (Kerger et al., 2009, Beaumont et al., 6 

### 2008; Zhang and Li, 1997, 1987), Kings County/San B ernadino County, California (Fryzek et 7 

### al., 2001; Bick et al., 1996), Nebraska (Bednar and Kies, 1991), and Glasgow, UK (Eizaguirre8 

### Garcia et al., 2000, 1999). Analyses of data colle cted from the Jinzou area of Liaoning Province, 9 

### China, where groundwater, surface water, and agricu ltural soils were heavily contaminated with 10 

### chromium derived from hexavalent chromium productio n (e.g., 0.001–20 mg chromium/L in 11 

### residential well water), provide evidence of an exc ess risk of mortality from stomach cancer 12 

### from 1970-1978 in residents of the area, relative t o the reference populations in the province 13 

### (four other areas in Lianoning Province, and the to tal population of the province) (Beaumont et 14 

### al., 2008). The other epidemiologic studies did no t find a significant correlation between 15 

### hexavalent chromium concentrations in drinking wate r (or proximity to the source of hexavalent 16 

### chromium soil contamination) and cancer. 17 

### Exposure of rats to sodium dichromate dihydrate in drinking water for 2 years resulted in 18 

### a significant increase in squamous epithelial neopl asms of the oral mucosa and tongue at the 19 

### highest exposure level (average daily doses of 5.9 and 7.0 mg hexavalent chromium/kg-day in 20 

### males and females, respectively), but not at the th ree lower exposure levels (NTP, 2008). 21 

### Exposure of B6C3F 1 

### mice to sodium dichromate dihydrate in drinking wa ter for 2 years resulted 22 

### in significant increases in the incidences of neopl asms of the small intestine in males and females 23



### at doses ≥ 2.4 and ≥ 3.1 mg hexavalent chromium/kg-day, respectively. NTP (2008) concluded 24 

### that results from these studies provide clear evidence of carcinogenic activity of sodium 25 

### dichromate dihydrate in male and female F344/N rats based on increased incidences of 26 

### squamous cell neoplasms of the oral cavity and clear evidence of carcinogenic activity of sodium 27 

### dichromate dihydrate in male and female B6C3F 1 

### mice based on increased incidences of 28 

### neoplasms of the small intestine. 29 

### The potential mutagenicity of hexavalent chromium h as been studied extensively. 30 

### Although study results vary with specific test syst ems, experimental conditions, and hexavalent 31 

### chromium compounds tested, results of in vitro and in vivo studies provide substantial evidence 32 

### for mutagenic activity of hexavalent chromium compo unds. The mutagenicity of hexavalent 33

### 208 DRAFT - DO NOT CITE OR QUOTE

chromium is mediated through the generation of high ly reactive chromium intermediates (e.g., 1 

### chromium(IV) and chromium(V)) and reactive oxygen s pecies formed during the intracellular 2



### reduction of hexavalent chromium. Reactive chromiu m intermediates and oxygen species react 3 

### with DNA, leading to oxidative DNA damage, chromium -DNA adducts, DNA strand breaks, and 4 

### chromosomal aberrations (Wise et al., 2008). 5 

### In in vitro test systems, hexavalent chromium compo unds have mostly tested positive for 6 

### gene mutations (including reverse mutations, frame shift mutations, and base pair substitutions) 7 

### and DNA damage (including DNA-protein crosslinks) i n bacterial cells (Salmonella 8 

### typhimurium, Escherichia coli, Bacillus subtilis); for forward mutations and mitotic gene 9 

### conversion in yeast (Saccharomyces cerevisiae); and for DNA damage (DNA strand breaks, 10 

### fragmentation, DNA-protein crosslinks, DNA-DNA cros slinks), chromosomal damage (sister 11 

### chromatid exchanges and chromosomal aberrations), a nd DNA synthesis inhibition in 12 

### mammalian cell lines and primary cultures (includin g primary cultures of human gastric mucosal 13 

### cells, respiratory tract cells, and lymphocytes). In in vivo test systems, hexavalent chromium 14 

### compounds have tested positive for mutations in Dro sophila melanogaster and for DNA damage 15 

### (DNA-protein crosslinks, DNA strand breaks), mutati ons, chromosomal damage (sister 16 

### chromatid exchanges, chromosomal aberrations, and m icronuclei), and DNA synthesis inhibition 17 

### in rats and mice. Thus, the mutagenic activity of hexavalent chromium has been demonstrated in 18 

### numerous studies using both in vitro and in vivo ex perimental systems. Given the weight of the 19 

### available evidence, hexavalent chromium is proposed to act through a mutagenic mode of 20 

### carcinogenic action, and thus age-dependent adjustm ent factors (ADAFs) should be applied. 21



### Under the Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005a), hexavalent 22 

### chromium is “likely to be carcinogenic to humans” v ia the oral route of exposure based on a 23 

### statistically significant increase in the incidence of tumors of the oral mucosa and tongue of rats 24 

### and of the small intestine of mice; and evidence of an association between oral exposure to 25



### hexavalent chromium and stomach cancer in humans. Additionally, available evidence indicates 26 

### that chromium interacts with DNA, resulting in DNA damage and mutagenesis. Thus, 27 

### hexavalent chromium is proposed to induce carcinoge nicity via a mutagenic mode of action. 28

29

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6.2. DOSE RESPONSE 1



### 6.2.1. Noncancer — Oral 2 

### NTP (2008), a 2-year animal bioassay that used mult iple dose groups and included a 3 

### comprehensive assessment of endpoints, was selected as the principal study for derivation of the 4 

### RfD. Dose-response analysis using BMD methods was conducted for the following endpoints 5 

### from this study: histopathological changes of the l iver (chronic inflammation in female rats and 6 

### histiocytic cellular infiltration in female mice), duodenum (diffuse epithelial hyperplasia in male 7 

### and female mice), mesenteric lymph node (histiocyti c cellular infiltration in male and female 8 

### mice), and pancreas (cytoplasm cellular alteration of acinar epithelial cells in female mice). 9



### All available dichotomous models in the EPA’s BMD S were fit to the incidence data for 10 

### the selected endpoints, using 10% extra risk as the BMR in accordance with U.S. EPA’s 11



### Benchmark Dose Technical Guidance (U.S. EPA, 2000). 12



### Based on the lowest BMDL 10 

### value of 0.09 mg hexavalent chromium/kg-day, diffu se 13 

### epithelial hyperplasia of the duodenum in female mi ce was selected as the POD for derivation of 14



### the RfD. The RfD of 0.0009 or 9 x 10 -4 

### mg/kg-day for hexavalent chromium was derived by 15 

### dividing the BMDL 10 

### (or POD) of 0.09 mg/kg-day by a composite uncertai nty factor of 100 (10 16 

### for extrapolation from animals to humans and 10 for human variability). 17

18



### 6.2.2. Cancer — Oral 19 

### The mode of action is a key consideration in clari fying how risks should be estimated for 20 

### low-dose exposure. A linear low-dose extrapolation approach was used to estimate human 21 

### carcinogenic risk associated with hexavalent chromi um exposures. This approach is supported 22 

### by the evidence for genotoxicity and a mutagenic mo de of action. 23



### The CSF for hexavalent chromium is based on tumor incidence data from the NTP (2008) 24 

### animal bioassay. The incidence of neoplasms in the small intestine of mice was used to derive 25 

### the CSF. Only animals that survived for at least 4 51 days, the time until appearance of the first 26 

### tumor, were considered at risk for tumor developmen t. 27 

### BMD modeling was carried out using the multistage model in EPA’s BMDS (U.S. EPA, 28 

### 2007) to identify a POD. In applying the BMD appro ach to the derivation of a CSF, the lower 29 

### 95% confidence bound on the dose corresponding to t he BMR (defined as 10% extra risk of 30 

### small intestine tumors) was calculated. This lower confidence bound is referred to as the 31 

### BMDL. The CSF was calculated by dividing the BMR b y the BMDL, and then converting this 32 

### CSF to human equivalents using body weight to the 3⁄4 power scaling. 33

### 210 DRAFT - DO NOT CITE OR QUOTE

The CSF resulting from the fitting of the multist age model in BMDS to the incidence of 1 

### neoplasms in the small intestine of male and female mice was 0.5 (mg/kg-day) -1 

### and 0.6 (mg/kg2 

### day) -1 

### , respectively, expressed in human equivalents. Be cause of the poorer fit of the multistage 3 

### model to the female mouse data, the cancer potency estimate of 0.5 (mg/kg-day) -1 

### based on the 4



### male mouse data was selected as the CSF for hexaval ent chromium. 5

6

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APPENDIX A. SUMMARY OF EXTERNAL PEER REVIEW AND PU BLIC 1



### COMMENTS AND DISPOSITION 2

3

4

5 

### [page intentionally left blank] 6

### B-1 DO NOT CITE OR QUOTE APPENDIX B. BENCHMARK DOSE CALCULATIONS

### B-2 DO NOT CITE OR QUOTE APPENDIX B-1. Details of Benchmark Dose Analysis f or the RfD

Table B-1.1 Incidence data for nonneoplastic lesio ns from all treatment groups of female F344/N rats and male and female B6C3F 1 

### mice exposed to sodium dichromate dihydrate in drinking water for 2 years (NTP, 2008)

Dose (mg hexavalent chromium/kg-day)

0 0.24 0.94 2.4 7.0

Female Rats Liver, chronic inflammation 12/50 21/50 a 28/50 b 35/50 b 39/50 b

Dose (mg hexavalent chromium/kg-day)

0 0.38 0.91 2.4 5.9

Male Mice Duodenum: Diffuse epithelial hyperplasia 0/50 11/50 b 18/50 b 42/50 b 32/50 a

Mesenteric lymph node: Histiocytic cellular infiltration 14/47 38/47 b 31/49 b 32/49 b 42/46 a

Dose (mg hexavalent chromium/kg-day)

0 0.38 1.4 3.1 8.7

Female Mice Duodenum: Diffuse epithelial hyperplasia 0/50 16/50 b 35/50 b 31/50 b 42/50 b

Mesenteric lymph node: Histiocytic cellular infiltration 3/46 29/48 b 26/46 b 40/50 b 42/50 b

Liver: Histiocytic cellular infiltration 2/49 15/50 b 23/50 b 32/50 b 45/50 b

Pancreas: Acinus, cytoplasmic alteration 0/48 6/50 a 6/49 a 14/50 b 32/50 b

a Significantly different (p ≤ 0.05) from the control group by Dunn’s or Shirley’s test. b Significantly different (p ≤ 0.01) from the control group by Dunn’s or Shirley’s test.

Source: ATSDR



### Chronic Inflammation of the Liver in Female Rats. As assessed by the chi-square goodnessof-fit statistic, only the log-logistic model provi ded an adequate fit ( χ 2 

### p -value ≥ 0.1) to the data (Table B-1.2). Based on the log-logistic model, th e BMD associated with a 10% extra risk was 0.22 mg hexavalent chromium/kg-day and its lower 95 % confidence limit (BMDL) was 0.14 mg hexavalent chromium/kg-day (Figure B-1.1).

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Table B-1.2. BMD 10 

### and BMDL 10 

### values and goodness-of-fit statistics from models fit to incidence data for chronic inflammation of t he liver in female rats exposed to sodium dichromium dihydrate in drinking water for 2 years

Model BMD 10

(mg/kg-day) BMDL 10

(mg/kg-day) x 2 p-value AIC

Gamma a 0.51 0.37 0.04 317.97 Logistic 0.84 0.65 0.01 321.45 Log-logistic b 0.22 0.14 0.37 312.57 Multi-stage c 0.51 0.37 0.04 317.97

Probit 0.88 0.70 0.01 321.80 Log-probit b 0.89 0.61 0.01 320.86 Quantal linear 0.51 0.37 0.04 317.97

Weibull a 0.51 0.37 0.04 317.97

a Restrict power ≥ 1

b Slope restricted to >1

c Restrict betas ≥ 0; lowest degree polynomial (up to n-2) with an ade quate fit is reported; no degree of polynomial provided a fit, a 3-degree polynomial is reported.

AIC = Akaike information criterion; BMD = benchmark dose; BMDL = lower confidence limit (95%) on the benchmark dose

Source: ATSDR

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Figure B-1.1. Predicted and observed incidence of chronic inflammation of the liver in female rats exposed to sodium dichromium dihydra te in drinking water for 2 years*

0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9

0 1 2 3 4 5 6 7Fraction Affected

dose Log-Logistic Model with 0.95 Confidence Level

13:34 04/08 2008 BMDL BMD Log-Logistic



### *BMDs and BMDLs indicated are associated with a 10% extra risk, and are in units of mg hexavalent chromium/kg-day.

Source: ATSDR

Diffuse Epithelial Hyperplasia of the Duodenum in M ale Mice. As assessed by the chi-square goodness-of-fit statistic, none of the models provi ded an adequate fit ( χ 2 

### p -value ≥ 0.1) to the full dataset (Table B-1.3). In order to achieve a stati stically fit model, the highest dose was dropped. This is determined to be appropriate, as the area o f concern is with the low-dose region of the response curve. After dropping the highest dose, t he gamma, log-logistic, multistage, log-probit, quantal linear, and Weibull models provided adequat e fits to the data ( χ 2 

### p -value > 0.1). Comparing across models, a better fit is generally indicated by a lower AIC (EPA, 2000b). As assessed by AIC, the 1-degree polynomial multistage model provided the best fit to the data (Figure B-1.2). Based on the multistage model, the BMD associated with a 10% extra risk was 0.16 mg hexavalent chromium/kg-day and its lower 95 % confidence limit (BMDL) was 0.13 mg hexavalent chromium/kg-day.

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Table B-1.3. BMD 10 

### and BMDL 10 

### values and goodness-of-fit statistics from models fit to incidence data for diffuse epithelial hyperp lasia in the duodenum in male mice exposed to sodium dichromium dihydrate in drinking water for 2 years

Model BMD 10

(mg/kg-day) BMDL 10

(mg/kg-day) x 2 p-value AIC

All doses

Gamma a 0.31 0.25 0.00 270.99 Logistic 0.90 0.74 0.00 296.25 Log-logistic b 0.15 0.12 0.00 247.93 Multi-stage c 0.31 0.25 0.00 270.99

Probit 0.90 0.76 0.00 296.18 Log-probit b 0.48 0.36 0.00 274.38 Quantal linear 0.31 0.25 0.00 270.99

Weibull a 0.31 0.25 0.00 270.99 Highest dose dropped (four doses modeled)

Gamma a 0.22 0.14 0.43 167.67 Logistic 0.47 0.39 0.03 177.09 Log-logistic b 0.26 0.15 0.20 169.23 Multi-stage d 0.16 0.13 0.52 166.34

Probit 0.45 0.37 0.04 176.19 Log-probit b 0.28 0.23 0.33 167.41 Quantal linear 0.16 0.13 0.52 166.34

Weibull a 0.22 0.14 0.47 167.50

a Restrict power ≥ 1

b Slope restricted to >1

c Restrict betas ≥ 0; lowest degree polynomial (up to n-2) with an ade quate fit is reported; no degree of polynomial provided a fit, a 3-degree polynomial is reported.

d Restrict betas ≥ 0; lowest degree polynomial (up to n-2) with an ade quate fit is reported; degree polynomial =1.

AIC = Akaike information criterion; BMD = benchmark dose; BMDL = lower confidence limit (95%) on the benchmark dose

Source: ATSDR

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Figure B-1.2. Predicted and observed incidence of diffuse epithelial hyperplasia in the duodenum of male mice exposed to sodium dichrom ium dihydrate in drinking water for 2 years*

0 0.2 0.4 0.6 0.8 1

0 0.5 1 1.5 2 2.5Fraction Affected

dose Multistage Model with 0.95 Confidence Level

15:45 04/08 2008 BMD

BMDL Multistage



### *BMDs and BMDLs indicated are associated with a 10% extra risk, and are in units of mg hexavalent chromium/kg-day.

Source: ATSDR

Histiocytic Cellular Infiltration of the Mesenteric Lymph Nodes in Male Mice. As assessed by the chi-square goodness-of-fit statistic, none of t he models provided an adequate fit ( χ 2 

### p -value ≥ 0.1) to the full dataset (Table B-1.4). In order t o achieve a statistically fit model, the highest dose was dropped. This is determined to be appropr iate, as the area of concern is with the lowdose region of the response curve. Dropping the hi ghest dose did not result in adequately fitting models, nor did dropping the two highest doses. Th is dataset is considered not suitable for benchmark dose modeling.

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Table B-1.4. BMD 10 

### and BMDL 10 

### values and goodness-of-fit statistics from models fit to incidence data for histiocytic cellular infi ltration in mesenteric lymph nodes of male mice exposed to sodium dichromium dihydrate in drinking water for 2 years

Model BMD 10

(mg/kg-day) BMDL 10

(mg/kg-day) x 2 p-value AIC

All doses Gamma a 0.38 0.26 0.00 285.94 Logistic 0.53 0.39 0.00 286.38 Log-logistic b 0.16 0.08 0.00 284.48 Multi-stage c 0.43 0.26 0.00 287.88 Probit 0.56 0.43 0.00 286.35 Log-probit b 0.83 0.52 0.00 289.36 Quantal linear 0.38 0.26 0.00 285.94 Weibull a 0.38 0.26 0.00 285.94 Highest dose dropped (four doses modeled) Gamma a 0.47 0.24 0.00 258.50 Logistic 0.61 0.35 0.00 259.04 Log-logistic b 0.21 0.08 0.00 256.81 Multi-stage d 0.47 0.24 0.00 258.50 Probit 0.63 0.37 0.00 259.08 Log-probit b 1.24 0.56 0.00 261.28 Quantal linear 0.47 0.24 0.00 258.50 Weibull a 0.47 0.24 0.00 258.50 Two highest doses dropped (three doses modeled) Gamma a 0.11 0.07 0.00 187.77 Logistic 0.17 0.12 0.00 189.97 Log-logistic b 0.05 0.03 0.00 183.77 Multi-stage e 0.11 0.07 0.00 187.77 Probit 0.17 0.12 0.00 190.12 Log-probit b 0.17 0.11 0.00 190.37 Quantal linear 0.11 0.07 0.00 187.77 Weibull a 0.11 0.07 0.00 187.77

a Restrict power ≥ 1

b Slope restricted to >1

c Restrict betas ≥ 0; lowest degree polynomial (up to n-2) with an ade quate fit is reported; no degree of polynomial provided a fit, a 3-degree polynomial is reported.

d Restrict betas ≥ 0; lowest degree polynomial (up to n-2) with an ade quate fit is reported; no degree of polynomial provided a fit, a 2-degree polynomial is reported.

e Restrict betas ≥ 0; lowest degree polynomial (up to n-2) with an ade quate fit is reported; no degree of polynomial provided a fit, a 1-degree polynomial is reported.

AIC = Akaike information criterion; BMD = benchmark dose; BMDL = lower confidence limit (95%) on the benchmark dose

Source: ATSDR

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Diffuse Epithelial Hyperplasia of the Duodenum in F emale Mice. As assessed by the chisquare goodness-of-fit statistic, none of the model s provided an adequate fit ( χ 2 

### p -value ≥ 0.1) to the data (Table B-1.5). In order to achieve a stat istically fit model, the highest dose was dropped. This is determined to be appropriate, as the area of concern is with the low-dose region of the response curve. After dropping the highest dose, an adequate fit was still not achieved. After dropping the two highest doses, all of the mo dels except for the logistic and probit models provided an adequate fit ( χ 2 

### p -value ≥ 0.1) to the data. Comparing across models, a bett er fit is generally indicated by a lower AIC (EPA, 2000b). A s assessed by AIC, the gamma, multistage, quantal linear, and Weibull models generated identi cal goodness of fit statistics and benchmark doses, as these models all took the form of a 1-deg ree polynomial multistage model which provides the best fit (Figure B-1.3). Based on the se models, the BMD associated with a 10% extra risk was 0.12 mg hexavalent chromium/kg-day a nd its lower 95% confidence limit (BMDL) was 0.09 mg hexavalent chromium/kg-day.

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Table B-1.5. BMD 10 

### and BMDL 10 

### values and goodness-of-fit statistics from models fit to incidence data for diffuse epithelial hyperp lasia in the duodenum of female mice exposed to sodium dichromium dihydrate in drin king water for 2 years

Model BMD 10

(mg/kg-day) BMDL 10

(mg/kg-day) x 2 p-value AIC

All doses Gamma a 0.34 0.27 0.00 275.34 Logistic 0.88 0.72 0.00 293.17 Log-logistic b 0.12 0.09 0.04 245.54 Multi-stage c 0.34 0.27 0.00 275.34 Probit 0.93 0.78 0.00 294.03 Log-probit b 0.52 0.38 0.00 279.54 Quantal linear 0.34 0.27 0.00 275.34 Weibull a 0.34 0.27 0.00 275.34 Highest dose dropped (four doses modeled) Gamma a 0.20 0.16 0.00 213.41 Logistic 0.55 0.46 0.00 236.10 Log-logistic b 0.11 0.08 0.04 200.07 Multi-stage d 0.20 0.16 0.00 213.41 Probit 0.54 0.45 0.00 235.61 Log-probit b 0.29 0.24 0.00 220.04 Quantal linear 0.20 0.16 0.00 213.41 Weibull a 0.20 0.16 0.00 213.41 Two highest doses dropped (three doses modeled) Gamma a 0.12 0.09 0.87 126.06 Logistic 0.34 0.27 0.00 141.77 Log-logistic b 0.12 0.06 1.00 127.77 Multi-stage e 0.12 0.09 0.87 126.06 Probit 0.32 0.26 0.00 140.65 Log-probit b 0.20 0.16 0.48 127.17 Quantal linear 0.12 0.09 0.87 126.06 Weibull a 0.12 0.09 0.87 126.06

a Restrict power ≥ 1

b Slope restricted to >1

c Restrict betas ≥ 0; lowest degree polynomial (up to n-2) with an ade quate fit is reported; no degree of polynomial provided a fit, a 3-degree polynomial is reported.

d Restrict betas ≥ 0; lowest degree polynomial (up to n-2) with an ade quate fit is reported; no degree of polynomial provided a fit, a 2-degree polynomial is reported.

e Restrict betas ≥ 0; lowest degree polynomial (up to n-2) with an ade quate fit is reported; no degree of polynomial provided a fit, a 1-degree polynomial is reported.

AIC = Akaike information criterion; BMD = benchmark dose; BMDL = lower confidence limit (95%) on the benchmark dose

Source: ATSDR

### B-10 DO NOT CITE OR QUOTE

Figure B-1.3. Predicted and observed incidence of diffuse epithelial hyperplasia in the duodenum of female mice exposed to sodium dichr omium dihydrate in drinking water for 2 years*

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8

0 0.2 0.4 0.6 0.8 1 1.2 1.4Fraction Affected

dose Multistage Model with 0.95 Confidence Level

09:36 04/09 2008 BMD

BMDL Multistage



### *BMDs and BMDLs indicated are associated with a 10% extra risk, and are in units of mg hexavalent chromium/kg-day.

Source: ATSDR

Histiocytic Cellular Infiltration of the Mesenteric Lymph Nodes in Female Mice. As assessed by the chi-square goodness-of-fit statistic, none o f the models provided an adequate fit ( χ 2



### p -value ≥ 0.1) to the full dataset (Table B-1.6). In order to achieve a statistically fit model, the highest dose was dropped. This is determined to be appropriate, as the area of concern is with the low-dose region of the response curve. Droppin g the highest dose did not result in adequately fitting models, nor did dropping the two highest doses. This dataset is not suitable for benchmark dose modeling.

### B-11 DO NOT CITE OR QUOTE

Table B-1.6. BMD 10 

### and BMDL 10 

### values and goodness-of-fit statistics from models fit to incidence data for histiocytic cellular infi ltration in mesenteric lymph nodes of female mice exposed to sodium dichromium dihydra te in drinking water for 2

years

Model BMD 10

(mg/kg-day) BMDL 10

(mg/kg-day) x 2 p-value AIC

All doses Gamma a 0.41 0.30 0.00 282.46 Logistic 0.77 0.61 0.00 290.18 Log-logistic b 0.09 0.06 0.00 263.55 Multi-stage c 0.41 0.30 0.00 282.46 Probit 0.85 0.69 0.00 291.41 Log-probit b 0.68 0.47 0.00 285.85 Quantal linear 0.41 0.30 0.00 282.46 Weibull a 0.41 0.30 0.00 282.46 Highest dose dropped (four doses modeled) Gamma a 0.20 0.15 0.00 224.84 Logistic 0.40 0.33 0.00 230.81 Log-logistic b 0.07 0.05 0.00 215.19 Multi-stage d 0.20 0.15 0.00 224.84 Probit 0.40 0.34 0.00 230.85 Log-probit b 0.37 0.24 0.00 231.76 Quantal linear 0.20 0.15 0.00 224.84 Weibull a 0.20 0.15 0.00 224.84 Two highest doses dropped (three doses modeled) Gamma a 0.14 0.10 0.00 172.32 Logistic 0.31 0.24 0.00 178.99 Log-logistic b 0.07 0.04 0.00 164.47 Multi-stage e 0.14 0.10 0.00 172.32 Probit 0.30 0.23 0.00 178.74 Log-probit b 0.21 0.15 0.00 178.11 Quantal linear 0.14 0.10 0.00 172.32 Weibull a 0.14 0.10 0.00 172.32

a Restrict power ≥ 1

b Slope restricted to >1

c Restrict betas ≥ 0; lowest degree polynomial (up to n-2) with an ade quate fit is reported; no degree of polynomial provided a fit, a 3-degree polynomial is reported.

d Restrict betas ≥ 0; lowest degree polynomial (up to n-2) with an ade quate fit is reported; no degree of polynomial provided a fit, a 2-degree polynomial is reported.

e Restrict betas ≥ 0; lowest degree polynomial (up to n-2) with an ade quate fit is reported; no degree of polynomial provided a fit, a 1-degree polynomial is reported.

AIC = Akaike information criterion; BMD = benchmark dose; BMDL = lower confidence limit (95%) on the benchmark dose

Source: ATSDR

### B-12 DO NOT CITE OR QUOTE

Histiocytic Cellular Infiltration of the Liver in F emale Mice. As assessed by the chi-square goodness-of-fit statistic, only the log-logistic mo del provided an adequate fit ( χ 2 

### p -value ≥ 0.1) to the data (Table B-1.7). Based on the log-logistic model, the BMD associated with a 10% extra risk was 0.17 mg hexavalent chromium/kg-day and its lower 95% confidence limit (BMDL) was 0.12 mg hexavalent chromium/kg-day (Figure B-1.4).

Table B-1.7. BMD 10 

### and BMDL 10 

### values and goodness-of-fit statistics from models fit to incidence data for histiocytic cellular infi ltration in the liver of female rats exposed to sodium dichromium dihydrate in drinking water for 2 years

Model BMD 10 (mg/kg-day) BMDL 10

(mg/kg-day) x 2 p-value AIC

Gamma a 0.35 0.28 0.08 255.40 Logistic 0.85 0.70 0.00 267.56 Log-logistic b 0.17 0.12 0.44 251.36 Multi-stage c 0.35 0.28 0.08 255.40

Probit 0.88 0.75 0.00 268.64 Log-probit b 0.62 0.48 0.01 260.00 Quantal linear 0.35 0.28 0.08 255.40

Weibull a 0.35 0.28 0.08 255.40

a Restrict power ≥ 1

b Slope restricted to >1

c Restrict betas ≥ 0; lowest degree polynomial (up to n-2) with an ade quate fit is reported; no degree of polynomial provided a fit, a 3-degree polynomial is reported.

AIC = Akaike information criterion; BMD = benchmark dose; BMDL = lower confidence limit (95%) on the benchmark dose

Source: ATSDR

### B-13 DO NOT CITE OR QUOTE

Figure B-1.4. Predicted and observed incidence of histiocytic cellular infiltration in the livers of female mice exposed to sodium dichrom ium dihydrate in drinking water for 2 years*

0 0.2 0.4 0.6 0.8 1

0 1 2 3 4 5 6 7 8 9Fraction Affected

dose Log-Logistic Model with 0.95 Confidence Level

11:17 04/09 2008 BMDL BMD Log-Logistic



### *BMDs and BMDLs indicated are associated with a 10% extra risk, and are in units of mg hexavalent chromium/kg-day.

Source: ATSDR

Cytoplasmic Alteration of Acinar Epithelial Cells o f the Pancreas in Female Mice. As assessed by the chi-square goodness-of-fit statisti c, all of the models provide adequate fits ( χ 2 

### p

value ≥ 0.1) to the data (Table B-1.8). Comparing across m odels, a better fit is generally indicated by a lower Akaike’s Information Criteria (AIC) (EPA, 2000b). As assessed by AIC, the log-logistic model provides the best fit (Figur e B-1.5). Based on the log-logistic model, the BMD associated with a 10% extra risk was 0.68 mg he xavalent chromium/kg-day and its lower 95% confidence limit (BMDL) was 0.52 mg hexavalent chromium/kg-day.

### B-14 DO NOT CITE OR QUOTE

Table B-1.8. BMD 10 

### and BMDL 10 

### values and goodness-of-fit statistics from models fit to incidence data for pancreas: acinus, cytopl asmic alteration in female mice exposed to sodium dichromium dihydrate in drinking water for 2 years

Model BMD 10

(mg/kg-day) BMDL 10

(mg/kg-day) x 2 p-value AIC

Gamma a 0.92 0.72 0.13 206.82 Logistic 2.43 2.03 0.09 211.78 Log-logistic b 0.68 0.52 0.19 205.22 Multi-stage c 0.92 0.72 0.13 206.82

Probit 2.24 1.89 0.11 210.99 Log-probit b 1.77 1.40 0.11 209.99 Quantal linear 0.92 0.72 0.13 206.82

Weibull a 0.92 0.72 0.13 206.82

a Restrict power ≥ 1

b Slope restricted to >1

c Restrict betas ≥ 0; lowest degree polynomial (up to n-2) with an ade quate fit is reported; a 1-degree polynomial is reported.

AIC = Akaike information criterion; BMD = benchmark dose; BMDL = lower confidence limit (95%) on the benchmark dose

Source: ATSDR

### B-15 DO NOT CITE OR QUOTE

Figure B-1.5. Predicted and observed incidence of pancreas: acinus, cytoplasmic alteration in female mice exposed to sodium dichrom ium dihydrate in drinking water for 2 years*

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8

0 1 2 3 4 5 6 7 8 9Fraction Affected

dose Log-Logistic Model with 0.95 Confidence Level

11:41 04/09 2008

BMDL BMD Log-Logistic



### *BMDs and BMDLs indicated are associated with a 10% extra risk, and are in units of mg hexavalent chromium/kg-day.

Source: ATSDR

### B-16 DO NOT CITE OR QUOTE

APPENDIX B-2. Details of Benchmark Dose Analysis f or the Oral Slope Factor

The fit of the multistage model to the incidence o f neoplasms in the small intestine of male mice administered sodium dichromate dihydrate in drinking water for 2 years (NTP, 2008):

Source: NJDEP

================================================== ================== Multistage Cancer Model. (Version: 1.7; Date : 05/16/2008) Input Data File: M:\ChromiumVI\msc_MALE_MICE_INTESTINAL_TUMORS_NTP_2 008_Setting.(d) Gnuplot Plotting File: M:\ChromiumVI\msc_MALE_MICE_INTESTINAL_TUMORS_NTP_2 008_Setting.plt Fri Feb 05 09:42:31 2010

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

BMDS Model Run

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

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 positiv e

### B-17 DO NOT CITE OR QUOTE

Dependent variable = Response Independent variable = Dose

Total number of observations = 5 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-008

Default Initial Parameter Values Background = 0.0291151 Beta(1) = 0.0232273 Beta(2) = 0.0107072

Asymptotic Correlation Matrix of Paramet er Estimates

Background Beta(1) Beta(2)

Background 1 -0.73 0.62

Beta(1) -0.73 1 -0.96

Beta(2) 0.62 -0.96 1

Parameter Estimate s

95.0% Wald Confidence Interval Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit Background 0.0287353 * * * Beta(1) 0.024191 * * * Beta(2) 0.0105146 * * *

* - Indicates that this value is not calculated.

Analysis of Deviance Table

Model Log(likelihood) # Param's Devia nce Test d.f. P-value Full model -77.3728 5 Fitted model -77.8649 3 0.98 4149 2 0.6114 Reduced model -96.8272 1 38. 9088 4 <.0001

AIC: 161.73

### B-18 DO NOT CITE OR QUOTE

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

------------------------------------------------- ----------------------- 0.0000 0.0287 1.408 1.000 49 -0.349 0.3800 0.0391 1.915 3.000 49 0.800 0.9100 0.0581 2.848 2.000 49 -0.518 2.4000 0.1374 6.869 7.000 50 0.054 5.9000 0.4160 19.969 20.000 48 0.009

Chi^2 = 1.03 d.f. = 2 P-value = 0.5968

Benchmark Dose Computation

Specified effect = 0.1

Risk Type = Extra risk

Confidence level = 0.95

BMD = 2.21769

BMDL = 1.16524

BMDU = 3.23024

Taken together, (1.16524, 3.23024) is a 90 % tw o-sided confidence interval for the BMD

Multistage Cancer Slope Factor = 0.085819

### B-19 DO NOT CITE OR QUOTE

The fit of the multistage model to the incidence o f neoplasms in the small intestine of female mice administered sodium dichromate dihydrat e in drinking water for 2 years (NTP, 2008):

Source: NJDEP

================================================== ================== Multistage Cancer Model. (Version: 1.7; Date : 05/16/2008) Input Data File: M:\ChromiumVI\msc_FEMALE_MICE_INTESTINAL_TUMORS_NTP _2008_Setting.(d) Gnuplot Plotting File: M:\ChromiumVI\msc_FEMALE_MICE_INTESTINAL_TUMORS_NTP _2008_Setting.plt Fri Feb 05 09:54:51 2010

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

BMDS Model Run

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

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 positiv e

Dependent variable = Response Independent variable = Dose

### B-20 DO NOT CITE OR QUOTE

Total number of observations = 5 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-008

Default Initial Parameter Values Background = 0.0398439 Beta(1) = 0.0695693 Beta(2) = 0

Asymptotic Correlation Matrix of Paramet er Estimates

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

Background Beta(1)

Background 1 -0.62

Beta(1) -0.62 1

Parameter Estimates

95.0% Wald Confidence Interval Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit Background 0.0140838 * * * Beta(1) 0.0792034 * * * Beta(2) 0 * * *

* - Indicates that this value is not calculated.

Analysis of Deviance Table

Model Log(likelihood) # Param's Devia nce Test d.f. P-value Full model -88.9774 5 Fitted model -91.8504 2 5.7 4595 3 0.1246 Reduced model -117.047 1 56. 1401 4 <.0001

AIC: 187.701

### B-21 DO NOT CITE OR QUOTE

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

------------------------------------------------- ----------------------- 0.0000 0.0141 0.690 1.000 49 0.376 0.3800 0.0433 2.166 1.000 50 -0.810 1.4000 0.1176 5.761 4.000 49 -0.781 3.1000 0.2287 11.208 17.000 49 1.970 8.7000 0.5050 24.746 22.000 49 -0.785

Chi^2 = 5.90 d.f. = 3 P-value = 0.1164

Benchmark Dose Computation

Specified effect = 0.1

Risk Type = Extra risk

Confidence level = 0.95

BMD = 1.33025

BMDL = 1.02757

BMDU = 1.93668

Taken together, (1.02757, 1.93668) is a 90 % tw o-sided confidence interval for the BMD

Multistage Cancer Slope Factor = 0.0973173