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www.epa.gov/iris

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 noncancer and cancer health effects 

associated with the oral route of exposure and includes 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 review under applicable information quality 

guidelines. It has not been formally disseminated by EPA. It does not represent and should not 

be construed to represent any Agency determination or policy. It is being circulated for review 

of its technical accuracy and science policy implications. 

U.S. Environmental Protection Agency 

Washington, DC 
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DISCLAIMER 

This document is a preliminary draft for review purposes only. This information is 

distributed solely for the purpose of pre-dissemination peer review under applicable information 

quality guidelines. It has not been formally disseminated by EPA. It does not represent and 

should not be construed to represent any Agency determination or policy. Mention of trade 

names or commercial products does not constitute endorsement or recommendation for use. 
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CONTENTS—TOXICOLOGICAL REVIEW OF HEXAVALENT CHROMIUM 

(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 ................................................................. xi 

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 BIOASSAYS IN 

ANIMALS—ORAL............................................................................................................... 41 

4.2.1. Subchronic Oral Exposure ....................................................................................... 42 

4.2.2. Chronic Oral Exposure ............................................................................................ 63 

4.3. REPRODUCTIVE AND DEVELOPMENTAL TOXICITY STUDIES—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 Reproductive Outcome and Fetal 

Development..................................................................................................................... 103 

4.3.4. Effects of Gestational and/or Lactational Exposure 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 systems..................................................... 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 Supporting 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 of 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 Effect—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 Uncertainty 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 Justification ......................................... 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 Factors................................................. 199 

5.3.6. Uncertainties in Cancer Risk Values......................................................................... 200 

5.3.7. Previous Cancer Assessment..................................................................................... 203 

6. MAJOR CONCLUSIONS IN THE CHARACTERIZATION OF HAZARD 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 PUBLIC COMMENTS 

AND DISPOSITION .................................................................................................................. A-1 

APPENDIX B. BENCHMARK DOSE CALCULATIONS ......................................................B-1 

APPENDIX B-1. Details of Benchmark Dose Analysis for the RfD.........................................B-2 

APPENDIX B-2. Details of Benchmark Dose Analysis for the Oral Cancer Slope Factor .....B-16 
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LIST OF TABLES 

Table 2-1. Physical properties of selected hexavalent chromium compounds .............................. 5 

Table 3-1. In vitro kinetic parameters of hexavalent chromium uptake in erythrocytes of rats and 

humans ................................................................................................................................... 11 

Table 3-2. Terminal tissue chromium levels in rats ingesting K2Cr2O4 in drinking water for 1 

year......................................................................................................................................... 12 

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

ingesting K2Cr2O7 in drinking water (8 mg hexavalent chromium/kg-day) for 4 or 8 weeks 14 

Table 3-6. Kinetic parameters of hexavalent chromium 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 from 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 other comparison areas, western JinZhou, 

China from 1970 to 1978 ....................................................................................................... 36 

Table 4-4. Hematological effects in male and female 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 female F344/N rats exposed to sodium 

dichromate dihydrate in drinking water for 3 months............................................................ 48 

Table 4-7. Incidence of nonneoplastic lesions observed in male and female F344/N rats exposed 

to sodium dichromate dihydrate in drinking water for 3 months........................................... 50 

Table 4-8. Selected organ weights in male and female B6C3F1 mice exposed to sodium 

dichromate dihydrate in drinking water for 3 months............................................................ 53 

Table 4-9. Incidence of nonneoplastic lesions observed in male and female B6C3F1 mice 

exposed to sodium dichromate dihydrate in drinking water for 3 months............................. 54 

Table 4-10. Hematological effects in male B6C3F1, BALB/c, and am3-C57BL/6 mice exposed 

to sodium dichromate dihydrate in drinking water for 3 months........................................... 57 

Table 4-11. Incidence of nonneoplastic lesions observed in male B6C3F1, 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 rats 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 observed 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 observed 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 B6C3F1 mice exposed to sodium dichromate 

dihydrate in drinking water for up to 12 months.................................................................... 76 

Table 4-18. Incidence of nonneoplastic lesions observed in male and female B6C3F1 mice 

exposed to sodium dichromate dihydrate in drinking water for 2 years ................................ 78

Table 4-19. Incidence of neoplastic lesions observed in the small intestine of male and female 

B6C3F1 mice exposed to sodium dichromate dihydrate in drinking 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 hexavalent chromium in non-mammalian cells ... 120 

Table 4-22. In vitro genotoxicity studies of hexavalent chromium in mammalian cells........... 127 

Table 4-23. In vivo genotoxicity studies of hexavalent 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 NOAELs and LOAELs for subchronic, chronic, 

and reproductive toxicity studies following oral exposure to hexavalent chromium .......... 161 

Table 5-1. Incidence data for nonneoplastic lesions from all treatment groups of female F344/N 

rats and male and female B6C3F1 mice exposed to sodium dichromate dihydrate in drinking 

water for 2 years (NTP, 2008).............................................................................................. 187 

Table 5-2. Summary of BMD10 and BMDL10 from the best fitting models for nonneoplastic 

lesions of the liver, duodenum, mesenteric lymph nodes, and pancreas in female rats and 

male and female mice after exposure to sodium dichromate 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 combined in the small intestine of male 

B6C3F1 mice exposed to sodium dichromate dihydrate in drinking water for 2 years (NTP, 

2008) .................................................................................................................................... 196 
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Table 5-6. Incidences of adenomas and carcinomas combined 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 exposure 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 
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LIST OF FIGURES 

Figure 3-1. A physiologically based model of chromium kinetics in the rat and human............ 21 

Figure 4-1. Ternary DNA adduct formation by chromium........................................................ 152 
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LIST OF ABBREVIATIONS AND ACRONYMS 

3β-∆5-HSH 3β‐∆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 

Km 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 
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FOREWORD 

The purpose of this Toxicological Review is to provide the scientific support and 

rationale for the hazard and dose-response assessment in IRIS pertaining to chronic exposure to

hexavalent chromium via ingestion. It is not intended to be a comprehensive treatise on the 

chemical or toxicological nature of hexavalent chromium. 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 routes 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 New Jersey Department of Environmental 

Protection (NJDEP), 3) the California Environmental Protection Agency (CalEPA), and 4) the 

Agency for Toxic Substances and Disease Registry (ATSDR). Section 5.1 was developed based 

on work conducted by ATSDR and CalEPA, and the reference dose (RfD) was derived using 

ATSDR’s analysis for chronic oral exposure to hexavalent chromium. Section 5.3 was 

developed based on work conducted by CalEPA and NJDEP, 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, where applicable, and to characterize the overall 

confidence in the quantitative and qualitative aspects of hazard and dose response by addressing 

the quality of data and related uncertainties. The discussion is intended to convey the limitations 

of the assessment and to aid and guide the risk assessor in the ensuing steps of the risk 

assessment process. 

For other general information about this assessment or other questions relating to IRIS, 

the reader is referred to EPA’s IRIS Hotline at (202) 566-1676 (phone), (202) 566-1749 (fax), or 

hotline.iris@epa.gov (e-mail address). 
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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. 
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1 1. INTRODUCTION 

2 

3 

4 This document presents background information and justification for the Integrated Risk 

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

6 hexavalent chromium. IRIS Summaries may include oral reference dose (RfD) and inhalation 

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

8 carcinogenicity assessment. This Toxicological Review provides documentation for oral toxicity 

9 values (i.e., RfD and oral cancer slope factor) only. 

10 The RfD and RfC, if derived, provide quantitative information for use in risk assessments 

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

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

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

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

deleterious effects during a lifetime. The inhalation RfC (expressed in units of mg/m315 ) is 

16 analogous to the oral RfD, but provides a continuous inhalation exposure estimate. The 

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

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

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

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

21 lifetime) exposure durations, all of which are derived based on an assumption of continuous 

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

23 derived for chronic exposure duration. 

24 The carcinogenicity assessment provides information on the carcinogenic hazard 

25 potential of the substance in question and quantitative estimates of risk from oral and inhalation 

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

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

28 effects may be expressed. Quantitative risk estimates may be derived from the application of a 

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

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

plausible upper bound on the estimate of risk per μg/m331 air breathed. 

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

33 chromium has followed the general guidelines for risk assessment as set forth by the National 

34 Research Council (NRC, 1983). U.S. Environmental Protection Agency (U.S. EPA) Guidelines 
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1 and Risk Assessment Forum Technical Panel Reports that may have been used in the 

2 development of this assessment include the following: Guidelines for the Health Risk 

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

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

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

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

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

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

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

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

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

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

13 Supplementary Guidance for Conducting Health Risk Assessment of Chemical Mixtures (U.S. 

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

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

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

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

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

19 The literature search strategy employed for this compound was based on the Chemical 

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

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

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

23 2009. 

24 
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1 2. CHEMICAL AND PHYSICAL INFORMATION 

2 

3 

4 Chromium is a naturally occurring element present in the earth’s crust that is typically 

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

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

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

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

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

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

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

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

13 2008). In general, hexavalent chromium compounds are more toxic than trivalent chromium 

14 compounds. This toxicological review focuses primarily on hexavalent chromium compounds, 

15 and the adverse effects associated with exposures to hexavalent chromium are described in 

16 Section 4 of this toxicological review. 

17 Hexavalent chromium compounds are a group of substances that contain chromium in the 

18 hexavalent or +6 oxidation state. The compounds discussed in this document include 

chromium(VI) oxide, chromic acid, and selected salts of the chromate (CrO4

2- 19 ) and dichromate 

(Cr2O7

2- 20 ) anions. Sodium chromate, sodium dichromate, and chromium(VI) oxide are obtained 

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

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

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

24 Chemical structures and selected physical and chemical properties of hexavalent chromium 

25 compounds are presented in Table 2-1. 

26 As a class of substances, hexavalent chromium compounds are oxidizing agents (Anger 

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

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

29 Major (or former) uses of hexavalent chromium compounds include metal plating, manufacture 

30 of pigments and dyes, corrosion inhibitors, chemical synthesis, refractory production, leather 

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

32 Natural occurrence of hexavalent chromium is rare as this form of chromium is readily 

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

34 1984). However, hexavalent chromium compounds released to the environment by 
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1 anthropogenic sources may persist in natural waters and soils that contain low amounts of 

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

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

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

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

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

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

8 Booker, 2000; James et al., 1997). 
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Table 2-1. Physical properties of selected hexavalent chromium compounds 

Name Chromium(VI) oxide

a Chromic acida,b Sodium chromate Sodium dichromate Sodium 

CAS Number 

1333-82-0 7738-94-5 (H2CrO4); 

13530-68-2 (H2Cr2O7) 

7775-11-3 10588-01-9 7789-1

Synonyms (ChemID Plus, 2008) 

Chromium oxide; hexavalent chromium oxide; chromic trioxide; chromic anhydride; chromic acid anhydride (Anger et al., 2005) 

Chromic(VI) acid; chromium hydroxide oxide; dichromic acid (H2Cr2O7) 

Sodium chromate(VI); chromium disodium oxide; disodium chromate; rachromate; chromic acid, disodium salt; chromate of soda 

Sodium dichromate(VI); sodium bichromate; dichromic acid, disodium salt; bichromate of soda 

Dichrodihydr

Structure (ChemID Plus, 2008) 

O Cr

O

O

Molecular weight 

99.994 (Lide, 2008) 118.010 (H2CrO4) (Lide, 2008); 

218.001 (H2Cr2O7) (ChemID Plus) 

161.974 (Lide, 2008) 261.968 (Lide, 2008) 297.99

Molecular formula 

CrO3 (ChemID Plus, 

2008) 

H2CrO4; 

H2Cr2O7 (ChemID Plus, 

2008) 

Na2CrO4 (ChemID 

Plus, 2008) 

Na2Cr2O7 (ChemID Plus, 

2008) 

Na2Cr2

2008) 

Form Dark red, deliquescent 

bipyramidal prismatic crystals, flakes, or granular powder (O’Neil, 2006) 

Exists only as an aqueous solution (Lide, 2008); yellow to orange-red (Anger et al., 2005) 

Yellow, orthorhombic crystals (Anger et al., 2005) 

Light brown to orangered plates (Anger et al., 2005) 

Orange

translu2005) 

Stability/ reactivity 

Deliquescent; decomposition begins above 198°C (Anger et al., 2005); powerful oxidizer (O’Neil, 2006)

Strong oxidizing agent (Anger et al., 2005) 

Hygroscopic (Anger et al., 2005) 

Strongly hygroscopic; decomposes above 400°C (Lide, 2008); strong oxidizing agent (Anger et al., 2005) 

Very hin air; strong solutioal., 200

Melting point 

197°C (Lide, 2008) Not applicable 794°C (Lide, 2008) 357°C (Lide, 2008) Decom

(Lide, 

Density 2.7 g/cm

3 (Lide, 2008) Not applicable 2.72 g/cm

3 (Lide, 2008) 2.52 g/cm

3 (Anger et al., 

2005) 

2.35 g/

Water solubility 

169 g/100 g H2O at 

25°C (Lide, 2008) 

Not applicable 87.6 g/100 g H2O at 

25°C (Lide, 2008) 

187 g/100 g H2O at 25°C 

(Lide, 2008) 

272.9 g

at 20°C 

Other solubility 

Soluble in alcohol and mineral acids (Lewis, 2007) 

Not applicable Slightly soluble in 

ethanol (Lide, 2008) 

Not available Solubl

2008) 
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Table 2-1. Physical properties of selected hexavalent chromium compounds 

Name Potassium chromate Potassium dichromate Calcium chromate Ammonium dichromate Zinc chroma

CAS Number 

7789-00-6 7778-50-9 13765-19-0 7789-09-5 13530-65-9 

Synonyms (ChemID Plus, 2008) 

Potassium chromate(VI); bipotassium chromate; dipotassium chromate; chromate of potash; tarapacaite; chromic acid, dipotassium salt 

Potassium dichromate(VI); bichromate of potash; potassium bichromate; dipotassium bichromate; dipotassium dichromate; dipotassium dichromium heptaoxide; lopezite; dichromic acid, dipotassium salt 

Calcium chromate(VI); calcium chrome yellow; calcium monochromate; gelbin; yellow 

ultramarine; chromic acid, calcium salt 

Ammonium bichromate; ammonium dichromate(VI); diammonium dichromate; chromic acid, diammonium salt 

Zinc chromate(V

hydroxide; buttyellow; chromic acid, zinc salt; zchrome yellow; teraoxychromat

Structure (ChemID Plus, 2008) Molecular weight 

194.191 (Lide, 2008) 294.185 (Lide, 2008) 156.07 (Lide, 2008) 252.065 (Lide, 2008) 181.403 (Lide, 

2008) 

Molecular formula 

K2CrO4 (ChemID Plus, 

2008) 

K2Cr2O7 (ChemID Plus, 

2008) 

CaCrO4 (ChemID 

Plus, 2008)

(NH4)2Cr2O7 (ChemID Plus, 

2008) 

ZnCrO4 (ChemI

Plus, 2008)

Form Lemon yellow prisms 

(Anger et al., 2005) 

Tabular or prismatic, bright orange-red triclinic crystals (Anger et al., 2005) 

Yellow monoclinic or rhombic crystals (O’Neil, 2006) 

Large, bright, orange-red crystals (Anger et al., 2005) 

Yellow prisms (Lide, 2008) 

Stability/ reactivity 

Non-hygroscopic (Anger et al., 2005) 

Non-hygroscopic; Decomposes at 500°C (Anger et al., 2005; Lide, 2008) 

Decomposes at 1,000°C (Lide, 2008); oxidizing agent (Lewis, 2007) 

Flammable; non-hygroscopic; decomposition begins upon heating at 180°C (O’Neil, 

2006). Strong oxidizing agent, may explode in contact with organic materials (Lewis, 2007) 

Not available 

Melting point 

974°C (Lide, 2008) 398°C (Lide, 2008) Decomposes prior to 

melting (Lide, 2008)

Decomposes prior to melting (Lide, 2008) 

316°C (Lide, 20

Density 2.73 g/cm

3 (Lide, 

2008) 

2.68 g/cm

3 (Lide, 2008) 3.12 g/cm

3 (Anger et 

al., 2005) 

2.155 g/cm

3 (Lide, 2008) 3.40 g/cm

3 (Lid

2008) 
7 DRAFT - 

Table 2-1. Physical properties of selected hexavalent chromium compounds 

Name Potassium chromate Potassium dichromate Calcium chromate Ammonium dichromate Zinc chroma

Water solubility 

65.0 g/100 g H2O at 

25°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 H2

(Lide, 2008) 

Other solubility 

Insoluble in alcohol (O’Neil, 2006) 

Insoluble in alcohol (Lewis, 2007) 

Soluble in dilute acids; practically insoluble in alcohol (O’Neil, 2006) 

Soluble in alcohol (Lewis, 2007) 

Dissolves readilacids (Anger et 2005); insoluble acetone (Lide, 2

aChromic acid is formed in aqueous solution when chromium(VI) oxide is dissolved in water; it cannot be isolated as a pure compound 

al., 2005; Page and Loar, 2004). The term, chromic acid, is sometimes used in reference to chromium(VI) oxide; however, it should be 

structural difference between the anhydrous substance chromium(VI) oxide and the aqueous chromic acid that forms when the oxide is 

bChromic acid exists in solution as both H2CrO4 and

H2Cr2O7 (Anger et al., 2005; Page and Loar, 2004; Cotton et al., 1999). H2CrO4 is 

solutions (pH > 6) while H2Cr2O7 is the main species in strongly acidic solutions (pH<1) (Anger et al., 2005; Page and Loar, 2004; Cot

species are present in equilibrium in solutions that have a pH value between 2 and 6 (Anger et al., 2005; Page and Loar, 2004; Cotton e
8 DRAFT - DO NOT CITE OR QUOTE 

1 3. TOXICOKINETICS 

2 

3 

4 Experimental evidence has demonstrated that hexavalent chromium can be absorbed via 

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

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

7 following ingestion. Once ingested, hexavalent chromium compounds can interact with 

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

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

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

11 tract, is termed “extracellular” reduction. The extent of absorption of ingested hexavalent 

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

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

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

15 trivalent chromium, however, can form complexes with organic ligands, which allow it to pass 

16 more easily across cell membranes. Hexavalent chromium can easily cross cell membranes due 

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

18 Absorbed hexavalent chromium is distributed throughout the body. Liver, kidney, and 

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

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

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

22 extracellular process described above. This intracellular reduction yields such reactive 

23 intermediates as chromium(V) and chromium(IV), along with oxygen radicals generated during 

24 this process. Hexavalent chromium is primarily eliminated in the urine as trivalent chromium. 

25 Biliary excretion of hexavalent chromium has been measured in animals following intravenous 

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

27 appear to be a gender difference in the toxicokinetics of hexavalent chromium, and inter28 individual variability in extracellular reduction and subsequent absorption and elimination may 

29 be primarily driven by differences in gastric contents and intervals between meals. 

30 

31 3.1. ABSORPTION FOLLOWING INGESTION 

32 Most quantitative studies of the GI absorption of chromium in humans have estimated the 

33 absorption fraction to be <10% of the ingested dose. In general, these studies suggest that the 
9 DRAFT - DO NOT CITE OR QUOTE 

1 absorbed fraction of soluble hexavalent chromium compounds (e.g., K2Cr2O7) is higher than 

2 insoluble forms (e.g., Cr2CO3). Furthermore, soluble hexavalent chromium compounds (e.g., 

3 K2Cr2O7) are absorbed to a greater extent than soluble trivalent chromium compounds (e.g., 

4 CrCl3). 

5 The absorption of hexavalent chromium in human volunteers ingesting single or multiple 

6 low doses of K2CrO4 or K2Cr2O7 was reported in a series of studies (Finley et al., 1997, 1996; 

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

8 trivalent chromium (as CrCl3), hexavalent chromium (as K2Cr2O7), or K2Cr2O7 in orange juice 

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

10 K2Cr2O7 added to orange juice likely resulted in near complete reduction to trivalent chromium, 

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

12 Although trivalent chromium is relatively nondiffusable across cellular membranes, formation of 

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

14 (Kerger et al., 1996). In individuals ingesting 5–10 mg K2CrO4 for 4 days, 3–6% of the ingested 

15 dose was absorbed. Because the erythrocyte and plasma elimination profiles were so similar 

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

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

18 ingesting 4 mg K2Cr2O7/day for 17 days exhibited 2% absorption of chromium (Paustenbach et 

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

20 to pre-exposure levels within a few days. 

21 In rats and mice, daily oral doses of 8 mg hexavalent chromium per day (as K2CrO4) for 

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

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

(approximately 7 μg/day as Na2

51 24 CrO4) for 3 days exhibited GI absorption of about 16% (Febel 

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

Na2

51 26 CrO4) was observed in rats, regardless of fasting state (MacKenzie et al., 1959). Sutherland 

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

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

29 with the effect being most pronounced at a concentration of 10 ppm. Chromium was most 

30 highly concentrated in bone and kidney. These investigators concluded that this result confirms 

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

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

33 the tissues (i.e., bone, kidney, liver, brain, testis, ovary, and blood) was not speciated, however, 
10 DRAFT - DO NOT CITE OR QUOTE 

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

2 the ingested hexavalent chromium may have escaped reduction, entered systemic circulation, and 

3 was available for cellular uptake. The other possibility proposed was that trivalent chromium 

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

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

6 chromium is likely absorbed and retained in the body. 

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

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

9 studies show that hexavalent chromium may be reduced via enzymatic and non-enzymatic 

10 mechanisms; the extent to which this mechanism is involved is largely determined by the route 

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

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

13 hexavalent to trivalent chromium. This has a significant impact on the extent to which orally 

14 ingested chromium is absorbed across the GI tract. 

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

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

17 been identified. Saliva from five volunteers reduced an average of 1.4 μg hexavalent 

18 chromium/mL. Intestinal bacteria, cultured from human feces from three volunteers, were 

observed to reduce 3.8 μg hexavalent chromium/10919 cells after contact for 4 hours. These fluids 

20 represent the first line of defense against hexavalent chromium toxicity from oral exposures. 

21 In samples of human gastric juices collected from hospital patients suffering from 

22 duodenal ulcers, reduction of hexavalent chromium (in the form of Na2CrO4) occurred at 

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

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

25 meals. Artificial acidification did not markedly change the reducing capability of the collected 

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

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

28 findings of Donaldson and Barreras (1966), who administered oral solutions of radiolabled 

51CrCl3 or Na251 29 CrO4 in human volunteers. Almost all of the trivalent chromium was recovered 

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

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

32 stomach), approximately 50% of the administered hexavalent chromium dose was absorbed, 

33 while the fractional absorption of trivalent chromium changed less than 5%. Both pH and gastric 
11 DRAFT - DO NOT CITE OR QUOTE 

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

Na2

51CrO4 alone resulted in 25% absorption, while Na251 2 CrO4 pre-incubated with HCl or gastric 

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

4 respectively. 

5 

6 3.2. DISTRIBUTION FOLLOWING INGESTION 

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

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

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

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

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

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

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

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

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

16 Absorbed chromium also can be transferred to fetuses through the placenta and to infants via 

17 breast milk. 

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

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

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

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

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

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

24 This explains the observation of higher chromium erythrocyte to plasma ratios following 

25 exposure to hexavalent chromium. 

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

27 erythrocytes of rats and humans. No species differences were observed; both species exhibited 

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

29 

Table 3-1. In vitro kinetic parameters of hexavalent 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 (CrO4

2-/erythrocyte/min) 3.1 × 108

 2.5 × 108

Whole blood kinetics 

Vmax (μmol/mL/min) 2.8 3.0 

Michaelis constant (Km) (mM/l blood) 20.9 14.1 

Source: Wiegand et al. (1985). 

1 

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

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

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

5 1996; Langard et al., 1978). K2Cr2O7 introduced into plasma and reconstituted whole blood 

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

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

8 detected in spiked plasma at concentrations of 2,000 and 10,000 μg hexavalent chromium/L, but 

9 not at 1,000 μg hexavalent chromium/L. Further, the plasma:erythrocyte ratio of total chromium 

10 decreased with increasing hexavalent chromium concentration. The variability between subjects 

11 in the ratio of plasma:erythrocyte total chromium diminished by approximately 1 order of 

12 magnitude as the hexavalent chromium concentration increased from 200 to 1,000 μg hexavalent 

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

14 plasma, and that the reduction rate of hexavalent chromium in erythrocytes is greater than the 

15 rate of uptake from the plasma (Corbett et al., 1997). 

16 Three drinking water studies in rats provide data on the tissue uptake of total chromium 

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

18 K2Cr2O4 in drinking water for 1 year (Table 3-2). Hexavalent chromium drinking water 

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

20 hexavalent chromium in drinking water was not reported. Tissue concentrations (in liver, 

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

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

23 chromium tissue accumulation were observed. 

24 

Table 3-2. Terminal tissue chromium levels in rats ingesting K2Cr2O4 in 

drinking water for 1 year 

K2Cr2O4 

concentration 

Liver 

(μg/g) 

Kidney 

(μg/g) 

Bone 

(μg/g) 

Spleen 

(μg/g) 
13 DRAFT - DO NOT CITE OR QUOTE 

(mg/L) male female male female male female male female 

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 

2 Kargacin et al. (1993) examined the species differences in distribution of chromium in 

3 male mice and rats exposed to 8 mg hexavalent chromium/kg-day as K2Cr2O7 in drinking water 

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

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

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

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

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

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

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

11 reduction capacity than rats, causing more trivalent chromium to be sequestered in hepatocytes. 

12 
14 DRAFT - DO NOT CITE OR QUOTE 

Table 3-3. Chromium in tissues (μg/g wet tissue or μg/mL blood) of mice 

and rats after ingesting K2Cr2O7 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 

2 Sutherland et al. (2000) observed significant tissue accumulation of chromium following 

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

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

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

6 significantly higher hepatic chromium concentrations than controls at both 3 and 10 ppm 

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

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

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

10 probably the result of rapid chromium delivery to tissues and clearance of plasma chromium by 

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

12 chromium levels. Finally, male rats that drank water containing 10 ppm hexavalent chromium 

13 had elevated testicular chromium concentrations, but these concentrations were described by the 

14 investigators as “modest” compared to those found in bone and kidney. 
15 DRAFT - DO NOT CITE OR QUOTE 

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

intravenous injection of 10 mg hexavalent chromium/kg (as Na2

51 2 CrO4) on gestational day (GD) 

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

et al., 1982). Intraperitoneal injection of 10 mg trivalent chromium/kg (as 51 4 CrCl3) in pregnant 

mice on GD 8 resulted in approximately equal 51 5 Cr activity in the embryo and maternal blood 

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

7 limited use for assessing embryonic exposure to chromium due to maternal oral exposures to 

8 hexavalent chromium. 

9 

10 3.3. METABOLISM FOLLOWING INGESTION 

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

12 chromium, either enzymatically (via microsomal enzymes) or non-enzymatically (via ascorbate 

13 and GSH). This intracellular reduction yields reactive intermediates, chromium(V) and 

14 chromium(IV). These reactive intermediates are formed along with oxygen radicals generated 

15 via Fenton-like and other possible reactions that occur during intracellular reduction. 

16 Hexavalent chromium taken into red blood cells undergoes reduction and forms 

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

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

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

20 suspensions, the addition of GSH results in intracellular reduction of hexavalent chromium to 

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

22 chromium binding in the erythrocytes following pretreatment with diethylmaleate, a GSH 

depletion agent (Aaseth et al., 1982). Incubation of human erythrocytes with K2

51Cr2O7 23 resulted 

24 in depletion of the erythrocyte GSH content to about 10% of normal. Subsequent analysis of 

25 erythrocyte lysates suggest that chromium-GSH complexes are formed and that approximately 

97% of [51 26 Cr] is bound to Hgb (Wiegand et al., 1984). 

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

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

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

14, 9, and 2% of [51 30 Cr] activity was found in the cytosolic, mitochondrial, microsomal, and 

31 nuclear fraction, respectively (Wiegand et al., 1987). Caution should be used in interpreting cell 

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

33 confounding the extent of intracellular hexavalent chromium reduction. For example, Dulbeco’s 
16 DRAFT - DO NOT CITE OR QUOTE 

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

2 (Borthiry et al., 2008). In human bronchial epithelial cells (BEAS-2B), Na2CrO4, and to a lesser 

3 extent, insoluble Zn2CrO4, were reduced to two reactive chromium(V) species; one appeared to 

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

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

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

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

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

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

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

11 1986). 

12 The predominant mechanism for intracellular hexavalent chromium reduction via 

13 microsomal enzymes has been extensively described. Incubation of K2Cr2O7 with rat liver 

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

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

16 resulted in essentially complete disappearance of hexavalent chromium. Within seconds, 

17 hexavalent chromium (as K2C2O7) incubated with rat liver microsomes and NADP(H) was 

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

19 1982). 

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

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

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

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

24 however, inhibited by the addition of TlCl3, indicating the involvement of flavoproteins, 

25 specifically cytochrome c reductase. The Vmax and Michaelis-Menten constant (Km) for liver 

26 microsomal reduction of hexavalent chromium was 5.03 nmol/minute/mg protein and 1.04 mM, 

27 respectively. The human microsomal Km was 1–3 orders of magnitude lower than those 

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

29 Jenette, 1981]). Another striking difference between rat and human hexavalent chromium 

30 microsomal reduction is the relative insensitivity to O2 in human microsomes (Pratt and Myers, 

31 1993). While rat microsomal hexavalent chromium reduction was markedly inhibited in the 

32 presence of 0.1% O2, human microsomal reduction was diminished by only 34–56% in the 

33 presence of ambient (21%) O2. These results suggest two things about the spatial distribution of 
17 DRAFT - DO NOT CITE OR QUOTE 

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

2 hexavalent chromium reduction is likely to be confined to the centrilobular region of the rat liver, 

3 since an O2 tension of only 1 mm Hg exists there. Secondly, the insensitivity to O2 of human 

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

5 the lung. 

6 Myers and Myers (1998) verified and extended the description of enzymatic hexavalent 

7 chromium reduction in human liver microsomes. Liver microsomes from five individuals were 

8 incubated with Na2CrO4 to determine reduction kinetics. Using a series of P450 inhibitors and 

9 TlCl3, the authors showed that hexavalent chromium reduction was mediated by flavoproteins, 

10 NADP(H)-dependent P450 reductase, and cytochrome b5. Parameters for reduction kinetics in 

11 these five individuals are shown in Table 3-6. The range of Vmax and Km values was very similar 

12 across subjects. Lung microsomes from one individual exhibited Vmax and Km values that were 

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

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

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

16 may have a catalytic role in the enzymatic reduction of hexavalent chromium. 

17 

Table 3-6. Kinetic parameters of hexavalent chromium reduction in human 

liver microsomes from five individuals 

Parameter Observation 

Vmax 10.4–10.7 

Km 1.04–1.68 

Inhibition by O2 26–37% 

Inhibition by TlCl3 96–100% 

Inhibition by P450 inhibitors 

Carbon monoxide None 

Piperonyl butoxide None 

Aminopyrine None 

Source: Myers and Myers (1998). 

18 

19 Proteoliposomes composed of recombinant human P450 reductase and cytochrome b5

20 were used to verify that electrons from NADP(H) could be transferred to cytochrome b5 during 

21 the reduction of hexavalent chromium (Jannetto et al., 2001). Markedly less hexavalent 

22 chromium reduction occurred in proteoliposomes devoid of cytochrome b5. Further, hexavalent 
18 DRAFT - DO NOT CITE OR QUOTE 

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

2 corrected for the cytochrome b5 concentration. 

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

4 difference in metabolism of hexavalent chromium. Further, human liver microsome studies did 

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

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

7 

8 3.4. ELIMINATION FOLLOWING INGESTION 

9 Chromium absorbed from the GI tract is excreted predominantly in urine. Chromium that 

10 is secreted in bile is extensively reabsorbed. Urinary and biliary chromium is excreted 

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

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

13 elimination of ingested hexavalent chromium (as K2Cr2O7) appears to have a slower elimination 

14 rate (half-life approximately 40 hours) than ingested trivalent chromium (as CrCl3-organic 

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

16 The clearance of orally ingested hexavalent chromium in humans was addressed in a 

17 series of studies in which human volunteers were dosed with up to 10 mg hexavalent 

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

19 hexavalent chromium (as K2Cr2O7) excreted 76–82% of the total as urinary chromium within 

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

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

22 K2Cr2O7) for 17 days, 87% of the total chromium was excreted in the urine in the first 4 days, 

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

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

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

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

27 given Na2CrO4, who excreted an average of 2.1% (range 0.2–4.4%) of administered hexavalent 

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

29 although no data are available identifying the extent to which hexavalent chromium is eliminated 

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

31 hexavalent chromium in breast milk, respectively. 

In rats gavaged with a single dose of Na2

51CrO4, 32 approximately 99% of the administered 

33 dose was eliminated in the feces, while 0.8% was eliminated in the urine, both within 4 days 
19 DRAFT - DO NOT CITE OR QUOTE 

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

Na2

51 2 CrO4), administered to rats for 3 days, was eliminated in the feces and urine, respectively 

3 (Febel et al., 2001). 

4 

5 3.5. PHYSIOLOGICALLY-BASED TOXICOKINETIC MODELS 

6 Physiologically based toxicokinetic (PBTK) models are mathematical representations of 

7 biological systems in animals and humans that are relevant to the quantitative determination of 

8 internal doses of toxic moieties of xenobiotics resulting from external doses or exposures 

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

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

11 dosimetry of chemicals across routes of exposure, dose ranges, and species. In risk assessment, 

12 the use of PBTK models quantitatively reduces uncertainties in these extrapolations, thus 

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

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

15 The development of PBTK models occurs in four sequential steps: 1) conceptual 

16 representation of the body into discrete compartments, 2) parameterization of the model, 

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

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

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

20 models, by nature, are simplified representations of complex biological systems and often 

21 contain gaps in the quantitation of a substance’s toxicokinetics. However, if a model is capable 

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

23 metabolites relevant to the critical toxic endpoints, it serves to reduce the overall uncertainty in 

24 the biology acting on that compound. 

25 For chromium, two PBTK models have been published for hexavalent and trivalent 

26 chromium in rats and humans (O’Flaherty et al., 2001; O’Flaherty, 1996, 1993). The inclusion 

27 of trivalent chromium in the model allows for the use of trivalent chromium exposure time 

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

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

30 describes the structure, development, and performance of these models and provides a discussion 

31 of the utility of these models for use in health risk assessment of inhaled or ingested hexavalent 

32 chromium for subchronic and chronic exposure durations. 

33 
20 DRAFT - DO NOT CITE OR QUOTE 

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

2 The O’Flaherty rat and human model is composed of 10 tissue compartments, including 

3 lung, GI tract, liver, kidney, bone, plasma, erythrocytes, urine, and well- and poorly-perfused 

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

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

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

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

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

9 plasma, transferred to the GI tract (mucocilliary clearance), or transferred to pool B, which 

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

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

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

13 ligand complexes in the GI tract. 

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)

PLASMA

PLASMA

RED CELLS

Cr(VI) 

→Cr(III)

GI TRACT

Cr(VI)→Cr(III)

URINARY

EXCRETION

ORAL

EXPOSURE

FECAL EXCRETION

INHALATION

EXPOSURE LUNG

15 
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1 Source: O’Flaherty (1996). 

2 

3 Figure 3-1. A physiologically based model of chromium kinetics in the rat 

4 and human. 
22 DRAFT - DO NOT CITE OR QUOTE 

1 

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

models 

Rat Human 

Parametera

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 mucociliary clearance from pool 

A to the GI tract (Da-1) 

 KMUCOB 0.025 0.025 NA NA First-order rate constant for mucociliary 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 for 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 NAb NAb Relative clearance of chromium into mineralizing bone (liters 

of blood plasma cleared per liter of new bone formed) 

 KINRBC 0.0003 1.5 12.0 NA Clearance from plasma to 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 to poorly-perfused tissues (L/Da) 

 BDIN 0.0001 0.01 NAb NAb Clearance from plasma to bone (L/Da) 

 CR NA NA 5.0 15.0 Fraction deposition from blood to forming bone 

 KOUTRBC 0.0003 10.0 12.0 NA Clearance from red cell to plasma (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 well-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 NAb NAb 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 NAc NAc Excretion clearance from the plasma (urinary clearance) 

(L/kg/Da) 

 CLEARc NA NA 12.0 12.0 Parameter in expression for clearance from blood plasma to 

urine (L/day) 

 MAXc NA NA 0.008 0.008 Parameter in expression for clearance from blood plasma to 

urine (mg/day) 

 KMc NA NA 0.0008 0.0008 Parameter in expression for clearance from blood plasma 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 

Parametera

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 for 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 reduction 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 for excretion from the retained urine 

pool (Da-1) 

 FR 0.10 0.10 NA NA Fraction of chromium in retained 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 were not 

identical. 

bExchanges between blood plasma and cortical and trabecular bone are simulated as functions of bone formation 

and resorption rates. 

c 

KM CBP

MAX QE CLEAR

+

= −

, 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’Flaherty et al. (2001) (human parameters). 

1 

2 The GI tract contains two competing transfer processes: absorption from the GI lumen to 

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

4 the GI tract is first transferred to the liver. 

5 Exchange of hexavalent chromium between compartments is assumed to be flow limited 

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

7 chromium is diffusion limited. Chromium is exchanged between the plasma and soft tissues via 

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

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

10 of data. The rat model represents chromium exchange between plasma and bone with a single 
24 DRAFT - DO NOT CITE OR QUOTE 

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

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

3 by O’Flaherty (1995, 1993). 

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

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

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

For example, urinary elimination is 0.7% of GFR at 1 × 10-4 7 mg/L and 40% of GFR at 

8 0.01 mg/L. 

9 Initial parameter values for the rat and human models were taken from the literature of 

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

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

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

13 chromium, exchange of hexavalent chromium and trivalent chromium between the tissues, and 

14 chromium elimination were optimized against time course data provided by intratracheal 

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

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

17 from studies of ZnCrO4 inhalation (Langård et al., 1978) and chronic Na2CrO4 or CrCl3 ingestion 

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

19 The human model parameters were optimized using data for plasma and red blood cell 

20 chromium burden, and chromium elimination to the urine in adults receiving single doses of 

21 CrCl3 or K2Cr2O7 (Finley et al., 1997; Kerger et al., 1996). The performance of the human 

22 model was evaluated against data for the time course of plasma chromium levels and urinary 

23 elimination of chromium in a single adult ingesting 4 mg K2Cr2O7/day for 17 days (Paustenbach 

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

25 constant had to be adjusted to obtain adequate fits. 

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

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

28 rat model has not been evaluated against time course data from multiple inhalation exposures. 

29 Although tissue burden predictions were evaluated against experimental observations, the 

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

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

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

33 erythrocyte, plasma, and urinary chromium levels resulting from single oral doses in volunteers. 
25 DRAFT - DO NOT CITE OR QUOTE 

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

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

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

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

5 imply a significant gender difference in hexavalent chromium toxicokinetics. 

6 The PBTK model of O’Flaherty (O’Flaherty et al., 2001; O’Flaherty, 1996, 1993) is not 

7 designed to predict deposited and absorbed doses from the lungs. Furthermore, key human 

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

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

10 subject. During the evaluation exercise, the parameter for gastric reduction of hexavalent 

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

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

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

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

15 in deriving health risks from chronic oral or inhalation exposures in human populations. 

16 

17 
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1 4. HAZARD IDENTIFICATION 

2 

3 

4 4.1. ORAL STUDIES IN HUMANS 

5 The human health effects observed following oral ingestion of hexavalent chromium 

6 usually come from individuals accidentally or intentionally ingesting hexavalent chromium 

7 compounds or from human populations unknowingly consuming food or drinking water 

8 contaminated with hexavalent chromium. 

9 

10 4.1.1. Acute Exposure

11 Several case reports have been published on clinical signs and symptoms in individuals 

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

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

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

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

16 Karotkin, 1935), and ammonium dichromate (Hasan, 2007; Reichelderfer, 1968). Clinical 

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

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

19 nausea, and vomiting; hematemesis and bloody diarrhea; caustic burns of mouth, pharynx, 

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

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

22 elevated blood bilirubin, and liver enzymes activities); renal failure (oliguria and anuria); 

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

24 hepatic fatty degeneration and necrosis and renal tubular degeneration and necrosis (Loubieres et 

25 al., 1999; Sharma et al., 1978; Kaufman et al., 1970; Reichelderfer, 1968). Based on estimated 

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

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

28 weight (Loubieres et al., 1999; Saryan and Reedy, 1988; Clochesy, 1984; Iserson et al., 1983; 

29 Kaufman et al., 1970). 

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

31 human volunteers to evaluate hexavalent chromium pharmacokinetics (Corbett et al., 1997; 

32 Finley et al., 1997; Kerger et al., 1997, 1996; Kuykendall et al., 1996; Paustenbach et al., 1996). 

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

34 reviewed and approved by a human use committee comprised of three board-certified 
27 DRAFT - DO NOT CITE OR QUOTE 

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

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

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

4 involved a singe male volunteer. The methods section of this study noted that “The volunteer 

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

6 part of these studies, standard clinical evaluations were performed that included blood cell 

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

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

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

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

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

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

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

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

15 reported; however, results were described in general terms that suggested that values for clinical 

16 chemistry endpoints were “similar” when measured prior to, during, or following dosing 

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

18 

19 4.1.2. Environmental Exposure

20 Human studies of possible associations between oral exposures to environmental 

21 hexavalent chromium and health outcomes include several epidemiology studies in which health 

22 outcomes (primarily cancer) were evaluated among populations who resided near sources of 

23 industrial waste containing hexavalent chromium compounds in Liaoning Province, China 

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

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

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

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

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

29 

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

31 1987) 

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

33 chromium in the vicinity of an alloy plant where chromium was smelted in the Jinzhou area of 
28 DRAFT - DO NOT CITE OR QUOTE 

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

29 and were subject to leaching to surface water and groundwater. These piles of ore residue were 

30 the main long-term source of underground water contamination. Hexavalent chromium was also 

31 released into the air through the various production and waste processes, with a northeast 

32 prevailing wind pattern. An additional source of chromium exposure was from food grown in 

33 areas using contaminated well water for irrigation. 
29 DRAFT - DO NOT CITE OR QUOTE 

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

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

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

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

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

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

7 1965, the zone of underground water contamination had spread, following a path eastward from 

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

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

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

11 hexavalent chromium was detected in the Nanshan reservoir (supplying drinking water to 

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

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

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

15 Table 4-1 includes a compilation of the available data from the 1965 water sampling 

16 studies (based on Table 2 from Beaumont et al., 2008, with the addition of the distance from the 

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

18 analytical methods used to quantify chromium were not reported, but these values (and all other 

19 values for chromium concentrations noted below) were reported as hexavalent chromium; 

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

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

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

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

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

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

26 information regarding the selection of wells sampled, lack of information regarding use of 

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

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

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

30 well water sample data to derive two measures of exposure (average chromium concentration 

31 and percent of wells >0.05 mg/L) that they applied to each of the 5 villages for an exposure32 response analysis of cancer risk. 

33 
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Table 4-1. Data pertaining to hexavalent chromium concentrations in 

drinking water in five villages along path of groundwater 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 10.5d 0.01 – 0.05 c 

1979 c 0.06 – 4.33 0.001 – 0.03 0.003 – 0.004 

Cr+6: hexavalent chromium.

aAs reported by Beaumont et al. (2008) 

bAs reported by Kerger et al. (2009) 

cAs reported by Zhang and Li, 1986, number of samples not stated. 

d

 Zhang and Li (1986) report this concentration as 70.5 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 in 

concentrations were not specified. 

1 

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

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

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

5 the five villages along the Nuer River, the district surrounding the ferrochromium alloy plant 

6 (Tanghezi), and three other areas to the west (Yaotanghezi) and north (North Thanghezi, North 

7 Nuer River) of the plant. Tanghezi and the other three areas were not affected by the 
31 DRAFT - DO NOT CITE OR QUOTE 

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

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

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

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

5 analysis indicated that the lung cancer rate was relatively high in TangHeZi (the industrial town 

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

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

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

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

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

11 eastern villages and comparison region, respectively, and stomach cancer mortality rates (27.7 – 

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

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

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

15 of variation in cancer rates among the five villages in the contamination zone in relation to 

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

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

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

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

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

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

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

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

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

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

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

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

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

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

30 used in combination with estimated person-years at risk as the basis of the calculation of area31 specific mortality rates in the analyses by Zhang and Li (1997, 1987, 1980), Beaumont et al. 

32 (2008) and Kerger et al. (2009). Because the results of Zhang and Li (1997) are repeated in the 
32 DRAFT - DO NOT CITE OR QUOTE 

1 presentation by Kerger et al. (2009), only the more recent of these analyses is described in more 

2 detail below. 

3 There are two relatively minor and two relatively major differences between the analyses 

4 of the cancer mortality data presented by Beaumont et al. (2008) and Kerger et al. (2009). One 

5 of the minor differences is the value used for stomach cancer mortality for one of the villages in 

6 the contamination zone, Nuer River Village. Beaumont et al. (2008) do not include an estimate 

7 of stomach cancer mortality for Nuer River Village in their primary analysis because it was 

8 missing from the original (1980) unpublished report (Zhang and Li, 1980) and Dr. Zhang 

9 indicated in a faxed communication with the study authors that the estimated rate of 28 per 

10 100,000 per year (reported in Zhang and Li, 1997) was of uncertain accuracy. Beaumont et al. 

11 (2008) did repeat their analysis using the 28 per 100,000 rate for stomach cancer mortality in 

12 Nuer River Village, and found this inclusion had very little effect on their estimates. Kerger et 

13 al. (2009) used 28 per 100,000 per year as the stomach cancer rate for Nuer River Village. The 

14 second relatively minor difference is in the estimation of age-adjusted mortality rates. The 

15 original analyses by Zhang and Li (1987) presented age-adjusted rates for all cancer mortality, 

16 but not for stomach cancer or lung cancer mortality. Kerger et al. (2009) do not attempt to make 

17 an age-adjustment for lung or stomach cancer because “small numbers of site-specific deaths in 

18 the villages would have precluded the calculation of relatable direct standardized site-specific 

19 rates in the current study.” Beaumont et al. (2008) addressed this issue by calculating the ratio of 

20 unadjusted to adjusted total cancer rates for each study area, which they term the “age21 adjustment influence” ratio. This ratio ranged from 0.84 to 1.05. The area-specific lung and 

22 stomach cancer unadjusted rates were multiplied by the respective area-specific age-adjustment 

23 influence ratio to create estimated age-adjusted lung and stomach cancer rates (Table 4-2). 

24 One of the major differences between the analyses by Beaumont et al. (2008) and Kerger 

25 et al. (2009) was described previously: Kerger et al. (2009) use the 1965 exposure data for 

26 exposure-response modeling of the variation in cancer rates among the five villages in the 

27 chromium contamination zone, and Beaumont et al. (2008) do not believe the available data are 

28 adequate for this purpose. The other major difference between the analyses is the inclusion of 

29 TangHeZi, the industrial district surrounding the ferrochromium alloy plant, in the comparison 

30 group. Kerger et al. (2009) considered this district to be too different from the smaller villages in

31 terms of urban-rural lifestyles and other exposures that could affect cancer risk (specifically 

32 stomach cancer and lung cancer) and therefore did not include it in their comparison group. 

33 Beaumont et al. (2008) include TangHeZi, presumably because it was part of the original study 
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1 design. They do not explicitly address the comparability issue with respect to stomach cancer 

2 risk factors, although they do note the potential for occupational chromium exposure to 

3 contribute to a relatively high lung cancer rate in TangHeZi. 

4 Table 4-3 presents the measures of association between chromium exposure and cancer 

5 mortality, based on the five villages in the contamination zone and the various comparison 

6 groups used by Beaumont et al. (2008) and Kerger et al. (2009). These risk ratios are based on 

7 comparison of the rates shown in Table 4-2, using a Poisson distribution for calculation of 95% 

8 confidence intervals. With respect to stomach cancer, the primary site of interest from the 

9 standpoint of drinking water contamination, Beaumont et al. (2008) report an association using 

10 the four comparison areas (TangHeZi, North TangHeZi, North Nuer River, and Yao TangHeZi) 

11 that were the basis for the original analysis (risk ratio = 1.82, 95% CI (1.11, 2.91)) and using 

12 rates from all of Liaoning province as a comparison (risk ratio = 1.69, 95% CI (1.12, 2.44)). 

13 Kerger et al. (2009) excluded the most populous area, TangHeZi from the comparison group, and 

14 reported a risk ratio = 1.22 (95% CI (0.74, 2.01)), which they interpret as being evidence of no 

15 association. In the lung cancer analyses, Beaumont et al. (2008) report relatively little difference 

16 between the rates in the contamination zone and the comparison area (risk ratio = 1.15, 95% CI 

17 (0.62, 2.07)), but a stronger association using Liaoning province as a comparison (risk ratio = 

18 1.78, 95% CI (1.03, 2.87)). Kerger et al. (2009) observed higher lung cancer rates in the five 

19 villages in the contamination zone compared with the three rural areas they included in the 

20 comparison group (risk ratio = 1.76, 95% CI (0.78, 3.98)), and slightly reduced risk when 

21 compared to TanHeZi (risk ratio = 0.80, 95% CI (0.44, 1.47)). 

22 Kerger et al. (2009) also presented results of analyses of variation in cancer rates within 

23 the five villages in the chromium contamination zone, using three measures of exposure 

24 potential: distance from the plant, average hexavalent chromium concentrations in 1965, and 

25 percent of wells with >0.05 mg/L hexavalent chromium in 1965 (these measures can be found in 

26 Table 4-2). The analysis was based on Poisson regression of the log-transformed cancer rate in 

27 relation to the exposure measures (separate models run for each measure). For the distance 

28 measure, a negative value for the coefficient indicates an increased cancer rate with closer 

29 proximity to the plant, and for the other exposure measures a positive coefficient indicates an 

30 increased cancer rate with higher exposure. The results for all cancer mortality (given as the 

31 regression coefficient and p-value) were 0.04 (p = 0.61), –0.07 (p = 0.54) and –0.24 (p = 0.45) 

32 for the distance, average hexavalent chromium concentration in 1965, and percent of wells >0.05 

33 mg/L hexavalent chromium in 1965 measures, respectively. For stomach cancer mortality, the 
34 DRAFT - DO NOT CITE OR QUOTE 

1 coefficients were 0.01 (p = 0.93), –0.11 (p = 0.50) and –0.32 (p = 0.51) for the distance, average 

2 hexavalent chromium concentration in 1965, and percent of wells >0.05 mg/L hexavalent 

3 chromium in 1965 measures, respectively, and for lung cancer, the coefficients were 0.12 (p = 

4 0.50), –0.06 (p = 0.79) and –0.11 (p = 0.88) for the distance, average hexavalent chromium 

5 concentration in 1965, and percent of wells >0.05 mg/L hexavalent chromium in 1965 measures, 

6 respectively. As described previously, Beaumont et al. (2008) did not include this type of 

7 exposure-response analysis because they believed the inherent limitations of the exposure data 

8 precluded a meaningful analysis. 

9 
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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 197

on analyses by Beaumont et al. (2008) and Kerger et al. (2009) 

Rate per 100,000 person-years 

All Cancer Stomach Cancer Lun

Area (population or person-years)a

Age-adjusted 

rate 

Age-adjustment 

influenceb Crude rate 

Estimated ageadjusted rate b Crude rate 

Areas in Contamination Zone 

Jinchanbao (2900) 83.6 0.97 36.7 35.5 13.2 Nuer River Village (2800) 71.9 0.98 28.0 missing b 15.0 Shilitai (2600) 93.0 0.94 55.2 51.7 missing Yangxing (1100) 76.8 0.94 36.5 34.5 21.4 Wenjiatun (1700) 91.1 0.94 27.7 26.0 20.8 Group average (~98,700)c 81.3 34.9 35.3 17.1 

Comparison areas 

TangHeZi (17,500) 71.3 0.86 16.9 14.5 21.4 North TangHeZi (3600) 81.8 0.84 26.4 d 22.1 8.8 North Nuer River (5800) 71.8 1.05 30.5 31.9 7.6 Yao TangHeZi (1500) 61.3 0.90 26.6 23.8 20.0 Group average – all (~252,500) e 72.1 19.4 Group average – without TangHeZi (96,826) f

73.7 28.6 9.7 

aArea population figures are based on approximate 1975 data from Beaumont et al. (2008); group values are total person-years for the combbAs calculated by Beaumont et al. (2008). Nuer River Village stomach cancer rate was not included in the primary analysis by Beaumont e

it 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 

was used; Kerger et al. (2009) report this value as 26.4. 

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 areasb 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, 3.98) 

Liaoning provinceb 1.23 (0.97, 1.53) 1.69 (1.12, 2.44) 1.78 (1.03, 2.87) 

aTangHeZi, North TangHeZi, North Nuer River, and Yao TangHeZi. 

bReported by Beaumont et al. (2008). 

cReported by Kerger et al. (2009). 

2 

3 In addition to the cancer mortality study, the JinZhou health department also collected 

4 data pertaining to symptoms in 1965 in Nuer River Village, which was one of the highly 

5 contaminated areas at that time (well water hexavalent chromium levels 0.1–20.0 mg/L) (Zhang 

6 and Li, 1987, 1986). Among 156 residents surveyed, 51 (33%) had oral ulcers, 20 (17%) had 

7 diarrhea, 48 (31%) had abdominal pain, 26 (17%) had dyspepsia, 81 (30%) had stomach pain, 

8 and 20 (17%) had vomiting (JinZhou Antiepidemic Station, 1979). The authors state that “no 

9 such symptoms were found among the residents whose water wells were not contaminated.” A 

10 similar study of 158 people in Shilitai in 1971 found a similar pattern of symptoms, with 92 

11 (58%) reporting oral ulcers, 48 (30%) diarrhea, and 36 (23%) abdominal pain. In 1974, another 

12 study of children in Wenjiatun and Sandaohao, at the eastern edge of the contamination zone, 

13 also found similar symptoms (data not shown in the 1979 report). The authors speculate that the 

14 symptoms may have been due to the increased concentrations of sulfates (>300 mg/L) in the 

15 drinking water in these areas in 1974, rather than the relatively low concentrations of hexavalent 

16 chromium (0.003–0.05 mg/L) 

17 Zhang and Li (1987, 1986) also conducted hematological assessments of 12 individuals 

18 in 1965, and another study of 93 individuals (time not specified). The exact location of the 

19 participants was not specified, but they were said to be from “highly polluted” or “high density 

20 contamination” areas. White blood cell counts were elevated in the first study, and the number 

21 of neutrophilic granulocytes and what was termed “juvenile cells” among these granulocytes was 

22 elevated in the second study. 

23 
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1 Kings County/San Bernadino County, California (Fryzek et al., 2001) 

2 A study of areas in Kings County and San Bernardino County, California, compared 

3 cancer mortality in locations near natural gas compressor plants with areas not located near the 

4 plants (Fryzek et al., 2001). Hexavalent chromium compounds had been used as anti-corrosion 

5 additives in cooling tower water at the gas plants during the period 1950 to approximately 1980. 

6 Waste material was released to surface ponds and was subject to percolation to groundwater. 

7 Cooling tower water was also aerosolized and transported to the ground surface where it may 

8 have contacted soil, crops, and surface water. Thus, exposures to hexavalent chromium may 

9 have occurred by several routes (i.e., inhalation, ingestion, and dermal contact). Mortality 

10 records for zip codes for the cities of Kettleman City (in Kings County), and Hinkley and 

11 Topock (in San Bernadino County), in which natural gas compressor plants were located, were 

12 compared to records from zip codes in Kings County and San Bernadino County, other than 

13 those encompassing these three cities. The study included mortality records for the period 1989–

14 1998, during which time 2,226,214 deaths were recorded. Age-adjusted cancer mortality rate 

15 ratios (rate in areas near the plant/rate in comparison areas) were 1.03 (95% CI 0.90–1.17) for 

16 lung cancer death, 0.93 (95% CI 0.87–1.00) for all cancer deaths, and 0.98 (95% CI 0.95–1.02) 

17 for all deaths. Rate ratios for stomach cancer were not reported. This study found no significant 

18 difference between mortality or cancer mortality among residents from zip codes in which gas 

19 plants that used hexavalent chromium additives in cooling tower water were located compared to 

20 residents of other nearby areas without such plants. An important limitation of this study is that 

21 exposure assignment was based on zip code, rather than on individual-level data, which is likely 

22 to result in significant exposure misclassification. 

23 

24 Nebraska (Bednar and Kies, 1991) 

25 Bednar and Kies (1991) compared levels of chromium (and other chemicals) in drinking 

26 water in Nebraska counties with death rates in these same areas. Data on chromium in drinking 

27 water were obtained for each of 453 communities (all incorporated communities of Nebraska) 

28 for the period 1986–1987, and mortality data for each Nebraska county was obtained for the year 

29 1986 (both compiled by the Nebraska Department of Health). Mean total chromium 

30 concentration in drinking water for the 453 communities was 0.002 mg chromium/L (range 

31 <0.001–0.01); the study report did not indicate valence state of chromium detected in these 

32 drinking water samples. Possible associations between chromium exposure and health outcomes 

33 were assessed by linear correlation (Pearson) of mortality rates (at the county level) and 
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1 chromium concentrations in drinking water (presumably aggregated from community data to 

2 represent counties). Correlations were reportedly explored for mortality from cancer, 

3 cerebrovascular disease, heart disease, pneumonia, and chronic lung disease; however, only one 

4 chromium correlation coefficient was reported to be statistically significant, that for death from 

5 chronic lung disease, and the correlation was negative (-0.101, p = 0.03). As with the other 

6 studies of this design, a major limitation is that exposures to chromium cannot be estimated for 

7 individual subjects in the study and may not be accurately represented by the drinking water 

8 chromium measurements. For example, the 1986–1987 drinking water data do not necessarily 

9 represent long-term exposure patterns, and an individual represented in a county death record 

10 does not necessarily mean that the individual resided in the county for their lifetime or any 

11 significant fraction of their lifetime. 

12 

13 Glasgow, UK (Eizaguirre-Garcia et al., 2000, 1999) 

14 Eizaguirre-Garcia et al. (2000, 1999) examined risk of leukemia and birth defects in 

15 people residing near the site of a former chromium processing facility in Glasgow, UK. The 

16 factory was in operation for more than 100 years and ceased operations in 1967. A survey 

17 conducted in 1991 found average soil concentrations at the site of operations to be 8,164 mg/kg 

18 for total chromium and 848 mg/kg for hexavalent chromium. Soil concentrations of total 

19 chromium and hexavalent chromium approximately 2–3 km from the factory site were reported 

20 as “approximately half” of those at the site; no additional information on soil levels off-site were 

21 reported (Eizaguirre-Garcia et al., 2000, 1999). Reported cases of leukemia for the period 1975–

22 1989 were obtained from the Scottish Cancer Registration, during which 1,205 cases of leukemia 

23 were reported in a population of 873,643 (Eizaguirre-Garcia et al., 1999). Leukemia cases were 

24 aggregated at the level of Enumeration Districts (ED) (approximately 350–500 individuals per 

25 district). When stratified by distance of the EDs from the plant (out to 9–10 km), relative risks of 

26 leukemia (0–2 km as reference) were unrelated to distance. When other influential variables 

27 were included in a Poisson regression model (gender, socioeconomic status, and age) in addition 

28 to distance of EDs from the plant (0–4, 4–9, 9–10 km), relative risk was significant (1.29, 95% 

29 CI: 1.07–1.56) for EDs 4–9 km from the plant (relative to 0–4 km), but not for EDs 9–10 km 

30 from the plant. These results suggest that leukemia risk increased with distance from the plant 

31 (i.e., 4–9>0–4 km) and then declined with further distance (i.e., 9–10 km = 0–4 km). This 

32 pattern does not strongly implicate the plant as a major contributor to leukemia risk. 
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1 A similar study of risk of birth defects was conducted on the same population 

2 (Eizaguirre-Garcia et al., 2000). In this study, data on number of births and congenital 

3 malformations were collected for the period 1982–1989. Case definitions (not reported) 

4 followed those of the European-wide EURCAT network (http://www.eurocat.ulster.ac.uk/). The 

5 study included 2,778 cases from a population of 81,057 births; cases were aggregated at the level 

6 of EDs. When distance from the plant (0–1, 2–4, 4–10 km) and socioeconomic status were 

7 included in a Poisson regression model, relative risk was significant for the EDs in the 2–4 km 

8 category (1.47, 95% CI: 1.2–1.7) and the 4–10 km category (1.25, 95% CI: 1.05–1.49); however, 

9 both distance categories were associated with higher risks than the closest distance category, 0–

10 1 km. Similar to the results for leukemia, this pattern does not strongly implicate the plant as a 

11 major contributor to risk of congenital anomalies. Not taken into consideration in this study 

12 were several other potentially influential variables on developmental outcomes; for example, 

13 maternal age and health, smoking, and alcohol consumption. 

14 

15 Summary 

16 The Liaoning province studies provide the most detailed analysis of all of the 

17 epidemiological studies that have been conducted with respect to chromium and cancer mortality 

18 (specifically stomach cancer or other cancers of the digestive system). These studies are 

19 important in that they examined a population exposed to very high levels of chromium in 

20 drinking water wells (i.e., sufficient to impart a visible yellow color to the water). Sources of 

21 exposure include the drinking water, food grown in contaminated soil, and possibly air. Levels 

22 up to 20 mg/L in well water were documented in the first surveys done in 1965 in the two 

23 villages closest to the source of exposure (a ferrochromium alloy plant). The contamination 

24 began sometime between 1959 and 1964; the reporting of a yellowing of the water by local 

25 residents in 1964 is what led to the investigation and identification of this contamination by the 

26 local health department. 

27 The interpretation of the mortality data originally collected by Zhang and Li (1980) 

28 depends in large part on the choice of referent group. That choice depends on many factors, 

29 including the perceived comparability and the size of the populations. Larger populations, such 

30 as a province or state, have the advantage of providing relatively stable estimates, particularly for 

31 low-incident events such as site-specific cancers. Smaller areas (e.g., a neighboring community) 

32 offer the advantage of potentially greater similarities in ethnic background, socioeconomic 

33 status, and occupational and lifestyle factors that may affect cancer risk. However, small 
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1 comparison groups are likely to produce imprecise estimates, and the issue of over-controlling 

2 may arise, for example, if the comparison population shares the specific exposure of interest (for 

3 example, with the selection of friends or co-workers in case-control studies). The associations 

4 presented by Beaumont et al. (2008) using Liaoning province as the comparison group provide 

5 evidence of an excess risk in the villages in the contamination zone of mortality from stomach 

6 cancer (RR 1.69, 95% CI (1.12, 2.44)) and lung cancer (RR 1.78, 95% CI (1.03, 2.87)), with a 

7 small increase also suggested in total cancer mortality (RR 1.23, 95% CI (0.97, 1.53)). The 

8 association with stomach cancer mortality is also seen when using the four adjacent areas as the 

9 referent group (RR 1.82, 95% CI (1.11, 2.91)), but is weaker when the industrial area 

10 surrounding the plant, TangZeHi, is removed from the comparison group (RR 1.22, 95% CI 

11 (0.74, 2.01)). Kerger et al. (2009) believe the relatively urban environment of TangHeZi makes 

12 it an inappropriate comparison group for the villages in the contamination zone. With respect to 

13 stomach cancer, historical trends show clear decreases in the incidence of this cancer in a variety 

14 of geographical areas, with improvements that come with economic development and 

15 urbanization (e.g., sanitation, refrigeration) contributing to this decline. An analysis of gastric 

16 cancer rates in China in 1990-1992 showed lower mortality rates in urban areas (15.3 per 

17 100,000) compared with rural areas (24.4 per 100,000) (Yang, 2006). However, this same study 

18 reported little difference between urban and rural rates in 1973-1975 (20.1 and 19.4 per 100,000 

19 in urban and rural areas, respectively), the relevant time period with respect to the Liaoning 

20 province studies. Thus, the EPA does not consider the exclusion of TangZeHi from the 

21 comparison group to be warranted. 

22 Another issue regarding the interpretation of the mortality data is the validity of analyses 

23 of the variability in cancer rates among the five villages in the contamination zone in relation to 

24 the available exposure measures (distance from the plant, average concentration in wells in 1965, 

25 and percent of wells with hexavalent chromium levels above 0.05 mg/L in 1965). There are 

26 considerable limitations to these measures, including the lack of individual-level data on use of 

27 water from specific wells over time and the changes in exposure due to efforts to treat the water 

28 in the most contaminated areas with treatment wells built in 1967. Based on these limitations, 

29 the EPA concluded that the exposure-response analyses presented by Zhang and Li (1997) and 

30 Kerger et al. (2009) are not based on the quality of data that is needed to support a conclusion 

31 regarding the presence or absence of a dose-response among the observed cancer rates in these 

32 villages. 

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1 4.2. SUBCHRONIC AND CHRONIC STUDIES AND CANCER BIOASSAYS IN 

2 ANIMALS—ORAL 

3 The effects of subchronic oral exposure to hexavalent chromium have been evaluated in 

4 rats (NTP, 2007; Quinteros et al., 2007; Rafael et al., 2007; Acharya et al., 2001; Chopra et al., 

5 1996; Vyskocil et al., 1993) and mice (NTP, 2007; Asmatullah and Noreen 1999), and the effects 

6 of chronic oral exposure to hexavalent chromium have been evaluated in rats (NTP, 2008, 

7 MacKenzie et al., 1958), mice (NTP, 2008), and dogs (Anwar et al., 1961). The studies 

8 conducted by NTP (2008, 2007) provide dose-response data on the effects of oral hexavalent 

9 chromium exposure based on a comprehensive assessment of toxicological endpoints. NTP 

10 (2008, 2007) identified lowest-observed-adverse-effect levels (LOAELs) and no-observed11 adverse-effect levels (NOAELs) in rats and mice for subchronic and chronic exposure durations. 

12 Results of the NTP (2007) subchronic study identified several hexavalent chromium-induced 

13 effects, including hematological effects, hepatotoxicity, alterations in lipid metabolism, and 

14 histopathological changes in GI tissues and pancreatic and mesenteric lymph nodes. The most 

15 sensitive hexavalent chromium-induced effects were microcytic, hypochromic anemia, increased 

16 serum liver enzyme activities, and histopathological changes to the duodenum and pancreatic 

17 lymph nodes in rats; and histopathological changes in the duodenum in mice. The most sensitive 

18 noncancer effects in the NTP (2008) two-year toxicology and carcinogenicity study were 

19 nonneoplastic histopathological changes to the liver, duodenum, and mesenteric lymph nodes in 

20 rats and the duodenum, mesenteric lymph nodes, and liver in mice. In addition, based on 

21 findings of squamous cell neoplasms of the oral cavity in rats and neoplasms of the small 

22 intestine in mice, NTP (2008) concluded that results of this study provide clear evidence of 

23 carcinogenic activity of sodium dichromate dihydrate. 

24 Several other oral exposure studies (i.e., Quinteros et al., 2007; Rafael et al., 2007; 

25 Asmatullah and Noreen, 1999; Vyskocil et al., 1993; Anwar et al., 1961) do not provide suitable 

26 data for identifying NOAELs or LOAELs because comprehensive toxicological endpoints were 

27 not evaluated in these studies. LOAELs identified in studies by Acharya et al. (2001) and 

28 Chopra et al. (1996) were based on evaluation of a limited number of liver endpoints. In 

29 addition, interpretation of results from these studies was limited due to the small number of 

30 animals evaluated, lack of dose-response data, or inadequate reporting for estimation of doses in 

31 mg hexavalent chromium/kg-day. However, results of these studies are useful for identification 

32 of potential adverse effects of oral hexavalent chromium exposure. 

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1 4.2.1. Subchronic Oral Exposure

2 NTP, 2007 

3 NTP (2007) conducted a 3-month toxicology study of sodium dichromate dihydrate in 

4 drinking water in rats and mice. This study was divided into three separate studies evaluating 

5 effects of treatment in: 1) male and female F344/N rats, 2) male and female B6C3F1 mice, and 3) 

6 three strains of male mice (B6C3F1, BALB/c, and am3-C57BL/6). In the 3-month study in 

7 F344/N rats, groups of 10 males and 10 females (“core” study animals) were exposed to sodium 

8 dichromate dihydrate in drinking water at concentrations of 0, 62.5, 125, 250, 500, or 1,000 mg 

9 sodium dichromate dihydrate/L (equivalent to 0, 21.8, 43.6, 87.2, 174.5, or 348 mg hexavalent 

10 chromium/L, respectively) for 3 months. Based on water consumption monitored throughout the 

11 study, NTP (2007) calculated average daily doses over the 3-month treatment duration of 

12 approximately 0, 5, 10, 17, 32, or 60 mg sodium dichromate dihydrate/kg-day (equivalent to 0, 

13 1.7, 3.5, 5.9, 11.2, or 20.9 mg hexavalent chromium/kg-day, respectively) for both males and 

14 females. An additional 10 male and 10 female rats (“clinical pathology” animals) were exposed 

15 to the same concentrations of sodium dichromate dihydrate for 4 weeks. “Core” study animals 

16 were observed twice daily for mortality and clinical signs of toxicity; water consumption and 

17 body weights were recorded weekly. Blood was collected from “clinical pathology” animals on 

18 treatment days 5 and 23 and from “core” study animals at study termination for comprehensive 

19 hematology and clinical chemistry endpoints. Urine was collected from “clinical pathology” 

20 animals on day 16 and analyzed for comprehensive urinalytic endpoints. At study termination, 

21 necropsies were performed on all “core” study animals, with organ weights recorded for heart, 

22 right kidney, liver, lung, spleen, right testis, and thymus. Microscopic examinations of 

23 comprehensive tissues were conducted in all core study animals in the control and 20.9 mg 

24 hexavalent chromium/kg-day (high-dose) groups and on six core study animals from each of the 

25 other treatment groups. In addition, all tissues identified as target organs in the 20.9 mg 

26 hexavalent chromium/kg-day (high-dose) group were examined in lower dose groups until a no27 effect level was identified or all animals were examined. 

28 No mortalities were observed in male or female rats exposed to sodium dichromate 

29 dihydrate in drinking water for 3 months (NTP, 2007). Final body weights in male rats were 

30 significantly decreased by 5% and 11% in the 11.2 and 20.9 mg hexavalent chromium/kg-day 

31 groups, respectively, compared to controls. In females, final body weight was significantly 

32 decreased by 9% in the 20.9 mg hexavalent chromium/kg-day group compared to controls. In 

33 males and females in the ≥5.9 mg hexavalent chromium/kg-day groups, water consumption was 
43 DRAFT - DO NOT CITE OR QUOTE 

1 decreased (statistical significance not reported). Data on food consumption were not reported. 

2 No treatment-related signs of clinical toxicity were observed throughout the study. 

3 Results of hematology analyses show that exposure of male and female rats to sodium 

4 dichromate dihydrate in drinking water produced microcytic, hypochromic anemia, characterized 

5 by decreases in mean cell volume (MCV), hematocrit (Hct), hemoglobin (Hgb), and mean cell 

6 hemoglobin (MCH) (NTP, 2007). The severity of microcytic, hypochromic anemia exhibited 

7 duration- and dose-dependence, with peak effects occurring at 23 days (Table 4-4). After 5 days 

8 of exposure, small changes were observed in several hematological parameters; however, 

9 decreases in all treatment groups were ≤5% compared to controls. More severe, dose-related

10 effects were observed after 23 days of treatment, with changes observed in all treatment groups 

11 in males and females. Similar effects were observed after 3 months of treatment, although 

12 severity at 3 months was generally less than that observed at 22 days. Blood smears showed 

13 evidence of erythrocyte injury or increased turnover, including erythrocyte fragments, 

14 keratocytes, and blebbing (incidence data not reported). Increased reticulocyte counts and 

15 nucleated erythrocytes, indicative of a compensatory hematopoietic response, were also observed 

16 in both sexes at 23 days and 3 months; however, these increases did not exhibit a consistent 

17 pattern of dose- or duration-dependence. Dose-dependent increases in platelet counts occurred at 

18 23 days in all treatment groups compared to controls; however, severity was decreased at 

19 3 months (Table 4-4). NTP (2007) stated that increased platelet counts are consistent with 

20 compensatory hematopoiesis or an iron deficiency process. Increased neutrophil and monocyte 

21 counts were observed at higher doses (≥5.9 and ≥3.5 mg hexavalent chromium/kg-day in males 

22 and females, respectively) and were considered by NTP (2007) to reflect an inflammatory 

23 response related to the inflammatory gastric lesions. Results of hematological analyses show 

24 that exposure of rats to sodium dichromate dihydrate in drinking water at daily doses ≥1.7 mg 

25 hexavalent chromium/kg-day produced microcytic, hypochromic anemia, but that severity 

26 decreased slightly as exposure duration increased from 23 days to 3 months. 
44 DRAFT - DO NOT CITE OR QUOTE 

1 

Table 4-4. Hematological effects in male and female F344/N rats exposed to 

sodium dichromate dihydrate in drinking water for up to 3 months 

Hematological Treatment group (mg hexavalent chromium/kg-day) 

parameter 

Time on 

treatment 0 1.7 3.5 5.9 11.2 20.9 

Males 

23 Days 48.0 ± 0.5a44.7 ± 0.7b

(93.1) 

39.8 ± 0.8b

(82.9)

36.2 ± 1.0b

(75.4)

34.4 ± 0.5b

(71.7)

32.3 ± 1.1b

(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 ± 0.4b

(93.9) 

36.9 ± 0.8b

(80.7) 

23 Days 15.9 ± 0.1 14.2 ± 0.2b

(89.3) 

12.0 ± 0.3b

(75.5) 

10.9 ± 0.3b

(68.6) 

10.3 ± 0.3b

(64.8) 

9.2 ± 0.3b

(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 ± 0.2b

(94.1) 

13.3 ± 0.2b

(86.9) 

10.9 ± 0.3b

(71.2) 

23 Days 61.1 ± 0.5 53.6 ± 0.6b

(87.7) 

48.0 ± 0.4b

(78.6) 

46.4 ± 0.6b

(75.9) 

46.2 ± 0.3b

(75.6) 

46.4 ± 0.5b

(75.9) 

MCV (fL) 

3 Months 51.8 ± 0.1 50.3 ± 0.2b

(97.1) 

49.0 ± 0.1b

(94.6) 

44.4 ± 1.0b

(85.7) 

39.7 ± 0.5b 

(76.6) 

36.0 ± 0.4b

(69.5) 

23 Days 20.1 ± 0.2 16.9 ± 0.2b

(84.0) 

17.2 ± 0.7b

(85.6) 

18.2 ± 0.4 

(90.5) 

19.7 ± 0.3 

(98.1) 

20.7 ± 0.6 

(103.0) 

MCH (ρg) 

3 Months 17.3 ± 0.1 16.9 ± 0.1b

(97.7) 

16.2 ± 0.1b

(93.6) 

14.2 ± 0.4b

(82.1) 

12.3 ± 0.2b

(71.1) 

13.0 ± 0.5b

(75.1) 

23 Days 7.94 ± 

0.10 

8.38 ± 0.11 

(105.5) 

7.13 ± 0.35c

(89.8) 

6.0 ± 0.28b 

(75.6) 

5.25 ± 0.19b 

(66.1) 

4.54 ± 0.33b

(57.2) 

Erythrocyte 

count (106

/μL) 

3 Months 8.88 ± 

0.05 

9.04 ± 0.09c

(101.8) 

9.25 ± 0.07b 

(104.2) 

10.15 ± 0.22b

(114.3) 

10.87 ± 0.07b

(122.4) 

8.52 ± 0.45b

(95.9) 

23 Days 745.2 ± 

22.2 

1,065.3 ± 

67.9b

 

(143) 

2,768.6 ± 

328.5b 

(372) 

3,504.7 ± 

235.0b

(470) 

4,226.0 ± 

204.5b 

(567) 

4,688.8 ± 

242.7b

(629) 

Platelet count 

(106

/μL) 

3 Months 618.6 ± 

20.0 

736.1 ± 11.5 

(119) 

604.3 ± 24.5 

(98) 

909.8 ± 119.1b

(147) 

1,743.1 ± 

178.0b 

(282) 

5,123.0 ± 

638.9b

(828) 

Females 

23 Days 48.0 ± 0.4a46.6 ± 0.9 

(97.1) 

42.9 ± 0.8b 

(89.4) 

39.2 ± 0.7b 

(81.7) 

37.2 ± 0.7b 

(79.6) 

33.4 ± 0.6b

(69.6) 

Hct (percent) 

3 Months 44.6 ± 0.4 45.2 ± 0.1 

(101.3) 

44.1 ± 0.3 

(98.9) 

42.9 ± 0.2b 

(96.2) 

42.6 ± 0.5b 

(95.5) 

38.3 ± 0.5b

(85.9) 

23 Days 15.9 ± 0.1 14.7 ± 0.3b

(92.5) 

13.0 ± 0.3b 

(81.8) 

11.8 ± 0.3b 

(74.2) 

10.9 ± 0.2b 

(68.6) 

9.7 ± 0.2b

(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 ± 0.1b 

(94.1) 

14.1 ± 0.2b 

(92.8) 

12.0 ± 0.2b

(78.9) 

23 Days 61.1 ± 0.4 53.9 ± 0.5b

(88.2) 

48.8 ± 0.5b 

(79.9) 

46.6 ± 0.6b 

(76.3) 

45.7 ± 0.4b 

(74.8) 

46.5 ± 0.5b

(76.1) 

MCV (fL) 

3 Months 53.3 ± 0.1 53.3 ± 0.1 

(100) 

52.4 ± 0.2b 

(98.3) 

50.5 ± 0.3b 

(94.7) 

48.0 ± 0.9b

(90.1) 

40.0 ± 0.7b

(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 (ρg) 

3 Months 18.4 ± 0.1 17.9 ± 0.1b

(97.3) 

17.8 ± 0.1b

(96.7) 

16.9 ± 0.1b 

(91.8) 

15.9 ± 0.4b 

(86.4) 

12.5 ± 0.3b

(67.9) 
45 DRAFT - DO NOT CITE OR QUOTE 

Table 4-4. Hematological effects in male and female F344/N rats exposed to 

sodium dichromate dihydrate in drinking water for up to 3 months 

Hematological Treatment group (mg hexavalent chromium/kg-day) 

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 ± 0.36b 

(80.8) 

5.27 ± 0.23b 

(67.4) 

4.21 ± 0.16c

(53.8) 

Erythrocyte 

count (106

/μL) 

3 Months 8.30 ± 

0.06 

8.60 ± 0.05b

(103.6) 

8.40 ± 0.04c

 

(101.2) 

8.47 ± 0.04c 

(102.0) 

8.93 ± 0.11b 

(107.6) 

9.62 ± 0.10b

(115.9) 

23 Days 611.5 ± 

43.7 

1,156.3 ± 

76.4b

(189) 

2808.8 ± 

198.5b

(459) 

3295.0 ± 

349.7b

(539) 

4,318.4 ± 

234.9b

(706) 

5,132.8 ± 

247.0b

(839) 

Platelet count 

(106

/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 ± 

193.3b

(259) 

aValues are means ± SE; values in parenthesis are percent of control; n = 10 rats/group, with the following 

exceptions: 1.7 mg hexavalent chromium/kg-day group females on days 23 and month 3 (n = 9), 3.5 mg hexavalent 

chromium/kg-day group females on day 23 (n = 8), 5.9 mg hexavalent chromium/kg-day group females on day 23 

(n = 9), and 20.9 mg hexavalent chromium/kg-day group 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 

2 Results of clinical chemistry analyses in male and female rats exposed to sodium 

3 dichromate dihydrate in drinking water showed treatment-related increases in serum liver 

4 enzyme activities, bile acids, and serum creatine kinase activity and alterations in lipid 

5 metabolism (Table 4-5) (NTP, 2007). Serum alanine aminotransferase (ALT) and sorbitol 

6 dehydrogenase (SDH) activities were significantly increased compared to controls in all 

7 treatment groups at 3 months, with less severe effects seen at 23 days. A consistent relationship 

8 between severity and dose was not observed. In male rats, elevations of ALT and SDH activities 

9 increased with increasing dose between 1.7 and 11.2 mg/kg-day; but less severe elevations were 

10 observed at 20.9 mg/kg-day (Table 4-5). In females, increases in ALT and SDH activities were 

11 generally indicative of a uniform effect across the dose range (Table 4-5). NTP (2007) suggested 

12 that increases are consistent with hepatocellular injury or membrane leakage. At 3 months, bile 

13 acids were significantly increased compared to controls at ≥11.2 mg hexavalent chromium/kg14 day in males and in all treatment groups (except 5.9 mg hexavalent chromium/kg-day) in 

15 females; similar to serum liver enzymes, increases in bile acids were not consistently related to 

16 dose. NTP (2007) suggested that increased bile acid was indicative of hepatic toxicity rather 

17 than colestasis, as markers of colestasis (e.g., alkaline phosphatase [AP] and 5N-nucleotidase) 

18 were not affected by treatment. At 3 months, decreased serum cholesterol and triglycerides, 
46 DRAFT - DO NOT CITE OR QUOTE 

1 indicative of altered lipid metabolism, were observed; however, a consistent relationship 

2 between severity and dose was not observed. At 3 months, dose-related increases in serum 

3 creatine kinase activity, indicative of muscle damage, were observed in males and females at 

4 ≥5.9 mg hexavalent chromium/kg-day. Urinalysis showed dose-related decreased volume and 

5 increased specific gravity, consistent with decreased water intake. NTP (2007) suggested that 

6 decreased water intake was due to decreased palatability of water. Other changes in clinical 

7 chemistry and urinalysis parameters were transient, with no apparent relationship to treatment. 

8 Results of clinical chemistry analyses indicate that exposure of rats to sodium dichromate 

9 dihydrate in drinking water induced hepatocellular membrane damage or cytotoxicity (both 

10 sexes) and increased bile acids (females) at doses ≥1.7 mg hexavalent chromium/kg-day (both 

11 sexes). 

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 

Clinical chemistry Treatment group (mg hexavalent chromium/kg-day) 

parameter 

Time on 

treatment 0 1.7 3.5 5.9 11.2 20.9 

Males 

ALT (IU/L) 3 Months 98 ± 6a 274 ± 30c 

(280) 

461 ± 102c 

(470) 

447 ± 121c 

(456) 

740 ± 81c 

(755) 

191 ± 17c

(195) 

SDH (IU/L) 3 Months 31 ± 2 55 ± 5c 

(177) 

110 ± 24c 

(355) 

102 ± 24c 

(329) 

173 ± 20c 

(558) 

59 ± 6c

(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 ± 2.8c 

(206) 

28.1 ± 2.0c

(128) 

Cholesterol (mg/dL) 3 Months 89 ± 2 95 ± 2 

(107) 

86 ± 4 

(97) 

65 ± 2c

(73) 

86 ± 3b

(97) 

71 ± 2c

(80) 

Triglycerides 

(mg/dL) 

3 Months 170 ± 9 169 ± 8 

(99) 

172 ± 15 

(101) 

170 ± 13 

(100) 

164 ± 12 

(96) 

98 ± 8c

(57) 

Creatine kinase 

(IU/L) 

3 Months 214 ± 26 286 ± 32 

(134) 

291 ± 36 

(136) 

364 ± 23c 

(170) 

413 ± 16c 

(193) 

374 ± 44c

(175) 

Females 

ALT (IU/L) 3 Months 64 ± 5a 437 ± 68c 

(683) 

218 ± 27c 

(340) 

245 ± 30c 

(383) 

246 ± 37c 

(384) 

248 ± 22c

(387) 

SDH (IU/L) 3 Months 22 ± 2 101 ± 17c 

(459) 

65 ± 10c 

(295) 

81 ± 13c

(368) 

96 ± 20c 

(436) 

103 ± 12c

(468) 

Bile acids (μmol/L) 3 Months 19.7 ± 2.5 50.4 ± 6.0c

(256) 

39.9 ± 4.3c 

(203) 

35.3 ± 3.5 

(179) 

45.3 ± 5.6c 

(230) 

38.7 ± 3.2b

(196) 

Cholesterol (mg/dL) 3 Months 95 ± 2 111 ± 4 

(117) 

94 ± 2 

(99) 

87 ± 2 

(92) 

83 ± 2b

(87) 

79 ± 2c

(83) 

Triglycerides 

(mg/dL) 

3 Months 139 ± 18 116 ± 10 

(93) 

98 ± 9 

(70) 

81 ± 4c

(58) 

76 ± 7c

(55) 

59 ± 6c

(42) 

Creatine kinase 

(IU/L) 

3 Months 197 ± 23 311 ± 94 

(158) 

265 ± 23 

(135) 

296 ± 24c

(150) 

359 ± 23c 

(182) 

432 ± 48c

(219) 

aValues are means ± SE; values in parenthesis are percent of control; n = 10 rats/group, with the following 

exceptions: control group males (n = 9), 1.7 mg hexavalent 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 

2 Changes in organ weights in rats exposed to sodium dichromate dihydrate in drinking 

3 water for 3 months are summarized in Table 4-6 (NTP, 2007). Treatment-related effects were 

4 generally observed at doses ≥11.2 mg hexavalent chromium/kg-day. In males, decreases were 

5 observed in absolute and relative liver weights and in absolute and relative spleen weights; in 

6 females, relative right kidney weights and relative spleen weights were increased. Changes in 

7 weights of other organs were considered by NTP (2007) to be secondary to changes in body 

8 weight rather than due to adverse effects of treatment. 
48 DRAFT - DO NOT CITE OR QUOTE 

1 

Table 4-6. Selected organ weights 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) 

Organ 0 1.7 3.5 5.9 11.2 20.9 

Males 

Liver, absolute weight 10.89 ± 0.42a 10.30 ± 0.28 11.45 ± 0.38 10.51 ± 0.18 9.20 ± 0.17b8.88 ± 0.18b

Liver, relative weightd 32.91 ± 0.65 31.91 ± 0.61 33.98 ± 0.75 31.90 ± 0.54 29.15 ± 

0.53c

29.80 ± 0.35b

Spleen, absolute weight 0.64 ± 0.02 0.60 ± 0.01 0.62 ± 0.02 0.60 ± 0.02 0.53 ± 0.01b0.60 ± 0.01b

Spleen, relative weightd 1.94 ± 0.03 1.85 ± 0.03 1.83 ± 0.04 1.81 ± 0.05c1.69 ± 0.02b2.00 ± 0.03 

Females 

Right kidney, relative weight 3.34 ± 0.09a 3.32 ± 0.04 3.55 ± 0.05 3.55 ± 0.07 3.58 ± 0.10c3.63 ± 0.09c

Spleen, relative weightd 2.12 ± 0.05 2.04 ± 0.03 2.16 ± 0.05 2.22 ± 0.03 2.25 ± 0.05c2.39 ± 0.03c

aValues are means ± SE; n = 10 rats/group. 

b

Significantly different (p ≤ 0.01) from the control group by Williams’s or Dunnett’s test. 

c

Significantly different (p ≤ 0.05) from the control group by Williams’s or Dunnett’s test. 

dRelative weight = mg organ weight/g body weight 

Source: NTP (2007). 

2 

3 Gross and microscopic examinations of male and female rats exposed to sodium 

4 dichromate dihydrate in drinking water for 3 months showed nonneoplastic lesions of the 

5 duodenum, glandular stomach, pancreatic lymph nodes, liver (females only), and bone marrow 

6 (females only) (NTP, 2007); incidence data are summarized in Table 4-7. The incidence of 

7 minimal-to-mild duodenal histiocytic cellular infiltration was increased in males and females at 

8 3.5 and 1.7 mg hexavalent chromium/kg-day, respectively, compared to controls; incidence 

9 increased with dose. Histiocytic cellular inflammation appeared as multifocal, randomly 

10 scattered, small clusters of enlarged macrophages with pale foamy cytoplasm. Incidences of 

11 nonneoplastic lesions of the glandular stomach (ulcer, focal regenerative hyperplasia, and focal 

12 squamous hyperplasia) were increased in rats in the highest dose group. Microscopically, ulcers 

13 were characterized by complete loss of the lining of the mucosal epithelium with necrosis, often 

14 extending through to the submucosa, and muscle layers; mild to marked chronic inflammation 

15 (infiltrates of neutrophils, macrophages, lymphocytes, and eosinophils), and proliferation of 

16 fibrous connective tissue through the submucosa. Lesions were not observed in the forestomach. 

17 Microscopic examinations of the oral mucosa and tongue were not conducted; NTP (2007) did 

18 not report lesions of the oral cavity on gross examination of rats at necropsy. In males, a dose19 dependent increase in the incidence of histiocytic cellular infiltration of pancreatic lymph nodes 
49 DRAFT - DO NOT CITE OR QUOTE 

1 was observed at 1.7 mg hexavalent chromium/kg-day, whereas increased pancreatic lymph node 

2 sinusoidal ectasia and lymphoid hyperplasia were only increased in the highest dose group; in 

3 females, significant increases in nonneoplastic lesions of pancreatic lymph nodes were only 

4 observed in the highest dose group. Microscopically, lymphoid hyperplasia was characterized by 

5 minimal-to-mild lymphocyte proliferation, and sinusoid ectasia was characterized by minimal-to6 mild dilatation of the subcapsular or medullary sinuses; histiocytic cell infiltration was similar to 

7 that observed in the duodenum. In the liver of females, a dose-dependent increase in the 

8 incidence of histiocytic cellular infiltration was observed at ≥3.5 mg hexavalent chromium/kg9 day and chronic inflammation was increased in the highest dose group. Although serum liver 

10 enzymes were increased in treatment groups (discussed above), significant histopathological 

11 changes to the livers of male rats were not observed. The incidence of bone marrow hyperplasia 

12 was significantly increased in high-dose females. This observation is consistent with an 

13 increased hematopoiesis in response to hexavalent chromium-induced microcytic, hypochromic 

14 anemia. 
50 DRAFT - DO NOT CITE OR QUOTE 

1 

Table 4-7. Incidence of nonneoplastic lesions observed 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) 

0/10a 0/10 7/10c

(1.1) 

9/10c

(1.2) 

8/10c

(1.4) 

7/10c

(1.4) 

Stomach, glandular (ulcer) 0/10 0/10 0/10 0/10 0/10 8/10c

(3.0) 

Stomach, glandular (focal 

regenerative hyperplasia) 

0/10 0/10 0/10 0/10 0/10 10/10c

(2.2) 

Stomach, glandular (focal 

squamous hyperplasia) 

0/10 0/10 0/10 0/10 1/10 

(2.0) 

7/10c

(2.6) 

Pancreatic lymph node (ectasia) 0/10 0/10 0/10 0/10 1/10 

(1.0) 

10/10c

(1.7) 

Pancreatic lymph node (lymphoid 

hyperplasia) 

0/10 0/10 0/10 3/10 

(1.0) 

3/10 

(1.0) 

6/10c

(2.7) 

Pancreatic lymph node (histiocytic 

cellular infiltration) 

0/10 5/10b

(1.0) 

2/10 

(1.0) 

4/10b

(1.0) 

5/10b

(1.0) 

9/10c

(1.9) 

Females 

Duodenum (histiocytic cellular 

infiltration) 

0/10a 1/10c

(1.0) 

5/10c

(1.0) 

7/10c

(1.4) 

8/10c

(1.6) 

10/10c

(1.7) 

Stomach, glandular (ulcer) 0/10 0/10 0/10 0/10 0/10 10/10c

(3.5) 

Stomach, glandular (focal 

regenerative hyperplasia) 

0/10 0/10 0/10 0/10 0/10 10/10c

(2.0) 

Stomach, glandular (focal 

squamous hyperplasia) 

0/10 0/10 0/10 0/10 0/10 10/10c

(2.4) 

Pancreatic lymph node (ectasia) 0/10 0/10 0/10 0/10 1/10 

(1.0) 

10/10c

(1.8) 

Pancreatic lymph node (lymphoid 

hyperplasia) 

0/10 0/10 2/10 

(1.5) 

0/10 0/10 10/10c

(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) 

9/10b

(1.9) 

Liver (histiocytic cellular 

infiltration) 

0/10 3/10 

(1.3) 

6/10c

(1.0) 

6/10c

(1.0) 

9/10c

(1.2) 

8/10c

(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) 

10/10c

(1.0) 

Bone marrow (hyperplasia) 0/10 0/10 0/10 0/10 0/10 4/10b

(1.0) 

aNumber of animals with lesion/number of animals examined; 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 test. 

c

Significantly different (p ≤ 0.01) from the control group by the Fisher exact test. 

Source: NTP (2007). 
51 DRAFT - DO NOT CITE OR QUOTE 

1 In conclusion, the NTP (2007) 3-month study in F344/N rats exposed to sodium 

2 dichromate dihydrate in drinking water identified several effects of subchronic oral hexavalent 

3 chromium exposure, including adverse hematological effects (microcytic, hypochromic anemia), 

4 hepatotoxicity (increased serum enzyme activities, increased serum bile acids, and 

5 histopathological changes), alterations in lipid metabolism (decreased serum cholesterol and 

6 triglycerides), possible muscle damage (increased serum creatine kinase activity), and 

7 histopathological changes in GI tissues (duodenum and glandular stomach) and in pancreatic 

8 lymph nodes. Results of this study identified a LOAEL in male and female rats of 1.7 mg 

9 hexavalent chromium/kg-day; a NOAEL was not identified. In males, the LOAEL was based on 

10 observations of microcytic, hypochromic anemia (decreased Hct, Hgb, MCV, MCH) occurring 

11 after 23 days to 3 months of exposure, increased serum liver enzyme activities (ALT and SDH), 

12 and histopathological changes to pancreatic lymph nodes (histiocytic cellular infiltration), all 

13 observed at daily doses ≥1.7 mg hexavalent chromium/kg-day. In females, the LOAEL was 

14 based on observations of microcytic, hypochromic anemia (decreased Hgb, MCV, MCH) 

15 occurring after 23 days to 3 months of exposure, increased serum liver enzyme activities (ALT 

16 and SDH) and bile acids, and histopathological changes to the duodenum (histiocytic cellular 

17 infiltration), all observed at daily doses ≥1.7 mg hexavalent chromium/kg-day. 

18 In the 3-month study in B6C3F1 mice, groups of 10 males and 10 females were exposed 

19 to sodium dichromate dihydrate in drinking water at concentrations of 0, 62.5, 125, 250, 500, or 

20 1,000 mg sodium dichromate dihydrate/L (equivalent to 0, 21.8, 43.6, 87.2, 174,5, or 348 mg 

21 hexavalent chromium/L, respectively) for 3 months (NTP, 2007). Based on water consumption 

22 monitored throughout the study, NTP (2007) calculated average daily doses over the 3-month 

23 treatment duration of approximately 0, 9, 15, 26, 45, or 80 mg sodium dichromate dihydrate/kg24 day (equivalent to 0, 3.1, 5.3, 9.1, 15.7, or 27.9 mg hexavalent chromium/kg-day, respectively) 

25 for both males and females. Mice were subjected to the same evaluations and procedures as 

26 those described above for “core” study rats (NTP, 2007), except that blood was not analyzed for 

27 clinical chemistry as the study in mice did not include a group of “clinical pathology” animals 

28 for evaluation after exposure durations of 5 and 23 days. 

29 No mortalities were observed in male or female mice exposed to sodium dichromate 

30 dihydrate in drinking water for 3 months (NTP, 2007). Dose-related significant decreases were 

31 observed in final body weights in male mice, with decreases reaching 20% (compared with 

32 control values) in the 27.9 mg hexavalent chromium/kg-day group; in females, dose-related 

33 decreases in final body weight were observed at ≥5.3 mg hexavalent chromium/kg-day, with 
52 DRAFT - DO NOT CITE OR QUOTE 

1 decreases reaching 13% in the 27.9 mg hexavalent chromium/kg-day group. Drinking water 

2 consumption was reduced in males at ≥5.3 mg hexavalent chromium/kg-day and in females at

3 27.9 mg hexavalent chromium/kg-day (statistical significance not reported). Data on food 

4 consumption were not reported. No treatment-related signs of clinical toxicity were observed 

5 throughout the study. 

6 Results of hematological analyses show that mice exposed to sodium dichromate 

7 dihydrate in drinking water for 3 months developed mild erythrocyte microcytosis (NTP, 2007); 

8 however, compared to hematological effects observed in rats (described above), effects in mice 

9 were less severe. In male mice, MCV and MCH were significantly decreased in all treatment 

10 groups, with maximum decreases of approximately 8%, compared to controls, in the highest dose 

11 group. In females, MCV and MCH were significantly reduced at ≥3.1 and ≥5.2 mg hexavalent 

12 chromium/kg-day, respectively, with maximum decreases of approximately 9% and 10%, 

13 respectively, compared to controls, in the highest dose group. Although statistically significant 

14 (p < 0.05) decreases in MCV were observed in males and females in the 3.1 mg hexavalent 

15 chromium/kg-day group, decreases were very small (1–2%, compared to controls); at doses up to 

16 9.1 mg hexavalent chromium/kg-day, decreases in MCV were ≤5%, compared with controls. 

17 Thus, mild microcytosis observed at ≥9.1 mg hexavalent chromium/kg-day does not appear to 

18 represent a clinically significant adverse effect. Erythrocyte counts were slightly increased (≤6% 

19 increase, compared with controls) at ≥5.2 mg hexavalent chromium/kg-day in females, but not in 

20 males. 

21 Changes in organ weights in mice exposed to sodium dichromate dihydrate in drinking 

22 water for 3 months are summarized in Table 4-8 (NTP, 2007). In males, absolute liver and right 

23 kidney weights were decreased at ≥9.1 mg hexavalent chromium/kg-day, although the only 

24 significant change in relative organ weight was an increase in relative kidney weight at 27.9 mg 

25 hexavalent chromium/kg-day. In females, absolute liver weight was increased at ≥15.7 mg 

26 hexavalent chromium/kg-day, but no changes in relative liver weight were observed. Changes in 

27 weights of other organs were considered by NTP (2007) to be secondary to changes in body 

28 weight rather than due to adverse effects of treatment. 

29 
53 DRAFT - DO NOT CITE OR QUOTE 

Table 4-8. Selected organ weights in male and female B6C3F1 mice exposed 

to sodium dichromate dihydrate in drinking water for 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 ± 0.01a0.28 ± 0.01 0.26 ± 0.01 0.26 ± 0.01b0.24 ± 0.01c0.26 ± 0.01c

Right kidney, relative weightd 7.25 ± 0.11 7.68 ± 0.29 7.43 ± 0.35 7.75 ± 0.20 7.76 ± 0.30 8.18 ± 0.07c

Liver, absolute weight 1.60 ± 0.08 1.54 ± 0.05 1.50 ± 0.05 1.40 ± 0.05b1.33 ± 0.06c1.34 ± 0.04c

Females 

Liver, relative weightd 1.15 ± 0.03a1.14 ± 0.04 1.06 ± 0.02 1.11 ± 0.04 1.04 ± 0.02b0.99 ± 0.02c

aValues are means ± SE; n = 10 mice/group. 

b

Significantly different (p ≤ 0.05) from the control group by Williams’s or Dunnett’s test. 

c

Significantly different (p ≤ 0.01) from the control group by Williams’s or Dunnett’s test. 

dRelative weight = mg organ weight/g body weight. 

Source: NTP (2007). 

1 

2 Gross and microscopic examinations of male and female mice exposed to sodium 

3 dichromate dihydrate in drinking water for 3 months showed nonneoplastic lesions of the 

4 duodenum and mesenteric lymph nodes (NTP, 2007); incidence data are summarized in 

5 Table 4-9. In the duodenum, a dose-related increase in the incidence of minimal-to-mild 

6 histiocytic cellular infiltration was observed in males and females in all treatment groups and in 

7 the incidence of minimal-to-mild epithelial hyperplasia at ≥ 5.3 mg hexavalent chromium/kg8 day; a slight dose-related increase in severity was observed. The duodenum had short, thick 

9 duodenal villi, elongated crypts with diffuse hyperplasia, and hyperplastic epithelial cells with 

10 swollen, vaculolated cytoplasm and increased numbers of “mitotic figures” (incidence data not 

11 reported). NTP (2007) stated that duodenal lesions were indicative of regenerative hyperplasia 

12 subsequent to epithelial cell injury. Minimal histiocytic cellular infiltration, morphologically 

13 similar to that observed in rats (discussed above), was observed in mesenteric lymph nodes in 

14 male and female mice at ≥5.3 mg hexavalent chromium/kg-day. 
54 DRAFT - DO NOT CITE OR QUOTE 

1 

Table 4-9. Incidence of nonneoplastic lesions observed in male and female 

B6C3F1 mice exposed to sodium dichromate dihydrate in drinking 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) 

0/10a 4/10b

(1.0) 

5/10c

(1.0) 

10/10c

(1.3) 

10/10c

(1.7) 

10/10c

(1.9) 

Duodenum (epithelial hyperplasia) 0/10 0/10 8/10c

(1.3) 

10/10c

(1.8) 

10/10c

(2.1) 

10/10c

(1.8) 

Mesenteric lymph node (histiocytic 

cellular infiltration) 

0/10 0/9 4/9b

(1.0) 

6/8c

(1.0) 

3/8 

(2.0) 

8/10c

(1.3) 

Females 

Duodenum (histiocytic cellular 

infiltration) 

0/10a 7/10c

(1.0) 

8/9c

(1.3) 

10/10c

(1.3) 

10/10c

(1.4) 

10/10c

(1.7) 

Duodenum (epithelial hyperplasia) 0/10 0/10 9/9c

(1.1) 

10/10c

(1.1) 

10/10c

(1.5) 

10/10c

(1.4) 

Mesenteric lymph node (histiocytic 

cellular infiltration) 

0/10 0/10 6/10c

(1.0) 

6/10c

(1.0) 

4/9b

(1.3) 

9/10c

(1.1) 

aNumber of animals with lesion/number of animals examined; 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 test. 

c

Significantly different (p ≤ 0.01) from the control group by the Fisher exact test. 

Source: NTP (2007). 

2 

3 In conclusion, the NTP (2007) 3-month study in B6C3F1 mice exposed to sodium 

4 dichromate dihydrate in drinking water identified adverse treatment-related hematological effects 

5 (erythrocyte microcytosis) and histopathological changes to the small intestine (duodenal 

6 epithelial hyperplasia and cellular histiocytic infiltration) and mesenteric lymph nodes (cellular 

7 histiocytic infiltration). Based on histopathological changes (histiocytic cellular infiltration) in 

8 the duodenum, a LOAEL of 3.1 mg hexavalent chromium/kg-day was identified for male and 

9 female mice; in both sexes, a NOAEL was not identified because the effects were observed at the 

10 lowest dose tested. Although a statistically significant decrease in MCV also was observed at 

11 3.1 mg hexavalent chromium/kg-day in males and females, hematological effects (e.g., 

12 microcytosis) were not considered as the basis of the LOAEL, since decreases in MCV were 

13 small (1–2%) at the lowest dose tested. 

14 Finally, NTP (2007) conducted a comparative study in three strains of mice (B6C3F1, 

15 BALB/c, and am3-C57BL/6) on the effects of exposure to sodium dichromate dihydrate in 
55 DRAFT - DO NOT CITE OR QUOTE 

1 drinking water for 3 months. This comparative study was conducted to investigate possible 

2 strain differences in mice based on results of an earlier study reporting hepatotoxicity 

3 (hepatocellular vacuolization) in BALB/c mice fed 32 mg hexavalent chromium/kg-day in the 

4 diet as potassium dichromate (NTP 1996a); no evidence of hepatotoxicity (including 

5 histopathological changes) was observed in male or female B6C3F1 mice exposed for 3 months 

6 to sodium dichromate dihydrate in drinking water at doses up to 20.9 mg hexavalent 

7 chromium/kg-day (NTP, 2007; results summarized above). In the “core study”, groups of 10 

8 male B6C3F1, 10 male BALB/c, and 5 male am3-C57BL/6 mice were exposed to sodium 

9 dichromate dihydrate in drinking water at concentrations of 0, 62.5, 125, or 250 mg/L 

10 (equivalent to 0, 21.8, 43.6, or 87.2 mg hexavalent chromium/L, respectively) for 3 months. An 

11 additional five male am3-C57BL/6 mice were exposed to the same concentrations of sodium 

12 dichromate dihydrate for a mutagenicity study. However, mutagenicity studies were not 

13 conducted due to technical problems; blood collected from these animals was analyzed for 

14 hematology and clinical chemistry. Based on water consumption monitored throughout the 

15 study, NTP (2007) calculated average daily doses over the 3-month treatment duration of 

16 approximately 0, 8, 15 or 25 mg sodium dichromate dihydrate/kg-day (equivalent to 0, 2.8, 5.2, 

17 or 8.7 mg hexavalent chromium/kg-day, respectively) for all strains. Animals were observed 

18 twice daily for mortality and clinical signs of toxicity; body weights were recorded weekly and 

19 water consumption was recorded at least every 4 days. Blood was collected at the end of the 3-

20 month treatment period and analyzed for hematology and clinical chemistry, as described above 

21 for “core study” rats (NTP, 2007). At study termination, necropsies were performed on all mice, 

22 with organ weights recorded for heart, right kidney, liver (except B6C3F1 mice), lung, spleen, 

23 right testis, and thymus. Microscopic examination was conducted on all gross lesions and 

24 masses and selected tissues (liver, forestomach, glandular stomach, duodenum, pancreas, kidney, 

25 and mesenteric and pancreatic lymph nodes). Sperm count and motility were assessed in all 

26 study animals, including spermatids per testis and per mg testis, spermatids per cauda and per mg 

27 cauda, and sperm motility, and weights of left cauda, left epididymis, and left testis. 

28 No mortalities were observed in male B6C3F1, BALB/c, or am3-C57BL/6 mice exposed 

29 to sodium dichromate dihydrate in drinking water for 3 months (NTP, 2007). In the 5.2 and 

30 8.7 mg hexavalent chromium/kg-day groups, final body weights were significantly decreased 

31 (compared to controls) by 9% and 12%, respectively, in B6C3F1 mice and by 7% and 11%, 

32 respectively, in BALB/c mice. Final body weight was reduced in all treatment groups in am3-

33 C57BL/6 mice, with decreases reaching 44% in the 8.7 mg hexavalent chromium/kg-day group. 
56 DRAFT - DO NOT CITE OR QUOTE 

1 Water consumption was reduced at 8.7 mg hexavalent chromium/kg-day in all three strains. 

2 Data on food consumption were not reported. No treatment-related signs of clinical toxicity 

3 were observed in B6C3F1 or am3-C57BL/6 mice. In BALB/c mice, ruffled fur was observed at 

4 8.7 mg hexavalent chromium/kg-day. 

5 Results of hematology analyses show that male B6C3F1, BALB/c, and am3-C57BL/6 

6 mice exposed to sodium dichromate dihydrate in drinking water for 3 months developed mild 

7 erythrocyte microcytosis (e.g., MCV) and small decreases in MCH, with changes observed in 

8 most treatment groups (Table 4-10) (NTP, 2007). In the 2.8 and 5.2 mg hexavalent 

9 chromium/kg-day groups, decreases in MCV and MCH were ≤7%, compared with controls, with 

10 slightly greater decreases at 8.7 mg hexavalent chromium/kg-day. Erythrocyte counts were 

11 significantly increased in B6C3F1 mice (7% at 8.7 mg hexavalent chromium/kg-day) and in 

12 BALB/c mice (2% and 5% at 5.2 and 8.7 mg hexavalent chromium/kg-day, respectively), but not 

13 in am3-C57BL/6 mice. Hemoglobin and Hct were decreased by approximately 5%, in am3-

14 C57BL/6 mice at 8.7 mg hexavalent chromium/kg-day, compared with controls, but not in 

15 B6C3F1 or BALB/c mice. Compared with hematological effects observed in rats (described 

16 previously), effects in mice were much less severe. Clinical chemistry analysis showed small 

17 increases (1.2- to 1.3-fold) in ALT at ≥5.2 mg hexavalent chromium/kg-day in BALB/c mice and 

18 a 1.9-fold increase in ALT in am3-C57BL/6 mice; in B6C3F1 mice, no increases in serum liver 

19 enzyme activities were observed. Decreases in various absolute and relative organ weights were 

20 observed at ≥5.2 mg hexavalent chromium/kg-day. NTP (2007) considered all changes to be 

21 related to decreased body weight, except for a significant decrease (29% compared with controls; 

22 p ≤ 0.05) in absolute thymus weight in B6C3F1 mice in the 8.7 mg hexavalent chromium/kg-day 

23 group; however, relative thymus weight was not different from controls in any treatment group. 

24 No treatment-related effects were observed for reproductive tissue evaluations or other 

25 reproductive parameters, except for a significant decrease (12.4% compared to controls; p ≤

26 0.01) in absolute left testis weight in am3-C57BL/6 mice at 8.7 mg hexavalent chromium/kg27 day; NTP (2007) stated that this change was related to decreased body weight. 
57 DRAFT - DO NOT CITE OR QUOTE 

1 

Table 4-10. Hematological effects in male B6C3F1, BALB/c, and 

am3-C57BL/6 mice exposed to sodium dichromate dihydrate in drinking 

water for 3 months 

Treatment Group (mg hexavalent chromium/kg-day) 

0 2.8 5.2 8.7 

B6C3F1 mice 

MCV (fL) 47.7 ± 0.2a 46.6 ± 0.2b

(97.7) 

46.4 ± 0.2 

(97.3) 

44.7 ± 0.1 

(93.7) 

MCH (ρg) 15.3 ± 0.1 14.9 ± 0.1b

(93.1) 

14.7 ± 0.1b

(96.1) 

14.2 ± 0.0b

(92.8) 

BALB/c mice 

MCV (fL) 44.8 ± 0.2a 43.8 ± 0.2b

(97.8) 

42.9 ± 0.2 

(95.8) 

42.6 ± 0.2 

(95.1) 

MCH (ρg) 15.0 ± 0.1 14.5 ± 0.1b

(96.7) 

14.2 ± 0.1b

(94.7) 

14.0 ± 0.1b

(93.3) 

am3-C57BL/6 mice 

MCV (fL) 45.8 ± 0.2 a 44.2 ± 0.4 

(96.5) 

43.7 ± 0.3b

(95.4) 

40.5 ± 0.3 

(88.4) 

MCH (ρg) 14.4 ± 0.1 14.1 ± 0.1b

(97.9) 

13.8 ± 0.1b 

(95.8) 

13.5 ± 0.2b

(98.8 ) 

aValues are means ± SE; values in parenthesis are percent of control; n = 10 mice/group, with the following 

exceptions: in B6C3F1 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 group (n 

= 9). 

b

Significantly different (p ≤0.01) from the control group by Dunn’s or Shirley’s test. 

Source: NTP (2007). 

2 

3 Microscopic examinations of gross lesions and masses and of selected tissues in male 

4 B6C3F1, BALB/c, and am3-C57BL/6 mice exposed to sodium dichromate dihydrate in drinking 

5 water for 3 months showed changes to the duodenum, liver, pancreas and mesenteric lymph 

6 nodes (NTP, 2007); incidence data are summarized in Table 4-11. In the duodenum, dose7 related increases in the incidences of minimal-to-mild histiocytic cellular infiltration and 

8 epithelial hyperplasia were observed in all strains, with histopathological changes of the 

9 duodenum observed in all exposure groups; severity increased with dose. Microscopically, 

10 lesions were similar to those described above for male and female B6C3F1 mice. Dose-related 

11 increases in the incidences of hepatic glycogen depletion and pancreatic secretory depletion were 

12 also observed; NTP (2007) stated that these lesions were likely due to depressed food 

13 consumption, which is frequently observed when water consumption is decreased. The 

14 incidence of minimal-to-mild histiocytic cellular infiltration of mesenteric lymph nodes was 
58 DRAFT - DO NOT CITE OR QUOTE 

1 increased at 8.7 mg hexavalent chromium/kg-day in am3-C57BL/6 mice, but not in B6C3F1 or 

2 BALB/c mice. 

3 

Table 4-11. Incidence of nonneoplastic lesions observed in male B6C3F1, 

BALB/c, and am3-C57BL/6 mice exposed to sodium dichromate dihydrate in 

drinking water for 3 months 

Treatment Group (mg hexavalent chromium/kg-day) 

Tissue (lesion type) 0 2.8 5.2 8.7 

B6C3F1 mice 

Duodenum (histiocytic cellular 

infiltration) 

0/10a 8/10c

(1.0) 

10/10c

(1.4) 

10/10c

(2.0) 

Duodenum (epithelial hyperplasia) 0/10 4/10b

(1.0) 

10/10c

(1.1) 

10/10c

(1.6) 

Liver (glycogen depletion) 1/10 

(1.0) 

2/10 

(1.5) 

9/10c

(1.4) 

10/10c

(2.2) 

Pancreas (secretory depletion) 0/10 2/10 

(1.0) 

7/10c

(1.0) 

9/10c

(1.0) 

BALB/c mice 

Duodenum (histiocytic cellular 

infiltration) 

0/10a 4/10b

(1.0) 

8/10c

(1.8) 

10/10c

(1.7) 

Duodenum (epithelial hyperplasia) 0/10 2/10 

(1.0) 

10/10c

(1.1) 

10/10c

(1.4) 

Pancreas (secretory depletion) 0/10 6/10c

(1.0) 

9/10c

(1.3) 

10/10c

(1.5) 

am3-C57BL/6 mice 

Duodenum (histiocytic cellular 

infiltration) 

0/5a 2/5 

(1.0) 

5/5c

(1.4) 

4/5b

(1.8) 

Duodenum (epithelial hyperplasia) 0/5 5/5c

(1.0) 

5/5c

(1.2) 

5/5c

(1.8) 

Liver (glycogen depletion) 0/5 4/5b

(2.0) 

5/5c

(1.6) 

5/5c

(3.8) 

Pancreas (secretory depletion) 0/5 3/5 

(1.0) 

4/5b

(1.0) 

5/5c

(1.6) 

Mesenteric lymph node (histiocytic 

cellular infiltration) 

0/5 0/5 0/5 4/5b

(1.5) 

aNumber of animals with lesion/number of animals examined; 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 

5 In conclusion, the comparative 3-month drinking water study on sodium dichromate 

6 dihydrate in male B6C3F1, BALB/c, and am3-C57BL/6 mice showed similar effects in the 

7 3 strains (NTP, 2007). A LOAEL of 2.8 mg hexavalent chromium/kg-day was identified based 
59 DRAFT - DO NOT CITE OR QUOTE 

1 on histopathological changes in the duodenum in B6C3F1 mice (histiocytic cellular infiltration 

2 and epithelial hyperplasia), BALB/c mice (histiocytic cellular infiltration), and am3-C57BL/6 

3 mice (epithelial hyperplasia); a NOAEL was not identified. Mild erythrocyte microcytosis was 

4 not considered the basis for the LOAEL, since the magnitude of decreases in MCV and MCH in 

5 the 2.8 mg hexavalent chromium/kg-day group was ≤7% compared to controls. 

6 

7 Quinteros et al., 2007 

8 Quinteros et al. (2007) showed that subchronic oral exposure of rats to hexavalent 

9 chromium in drinking water decreased circulating prolactin levels. Groups of 15 male Wistar 

10 rats were exposed to drinking water containing 0 or 500 mg hexavalent chromium/L as 

11 potassium dichromate for 30 days. Based on water intake and body weights measured over the 

12 course of the study, Quinteros et al. (2007) calculated a daily dose of 73.05 mg hexavalent 

13 chromium/kg-day. At the end of the treatment period, blood was collected for analysis of 

14 prolactin and luteinizing hormone (LH), and the pituitary gland and hypothalamus were analyzed 

15 for chromium content (see Section 3.2). At the end of the 30-day treatment period, water 

16 consumption and body weight in hexavalent chromium-treated rats were decreased by 30.5 and 

17 11.5% compared to controls. Serum prolactin levels in treated rats were decreased by 

18 approximately 59% (p < 0.001) compared to controls; serum levels of LH were comparable in 

19 control and treatment groups. NOAEL and LOAEL values for this study could not be identified 

20 because only one dose was evaluated and effects on other potential hexavalent chromium target 

21 tissues were not assessed. 

22 

23 Rafael et al., 2007 

24 Adverse hepatic effects were reported in rats following subchronic oral exposure to 

25 hexavalent chromium, but details of this study were not available (Rafael et al., 2007). Male 

26 Wistar rats (9 control and 19 treated) were administered drinking water containing 0 or 20 mg 

27 hexavalent chromium/L (chromium compound not reported) for 10 weeks. According to the 

28 investigators, no clinical signs of toxicity or changes in body weight were observed (data not 

29 reported). Data on drinking water consumption were not reported, and the report did not indicate 

30 if drinking water consumption was similar between control and treatment groups; thus, given this 

31 uncertainty, daily hexavalent chromium doses cannot be estimated from this study. At the end of 

32 the treatment period, serum glucose was decreased by 45% (p = 0.0002) and serum ALT activity 

33 was increased by 153% (p = 0.039), compared with controls. Serum levels of total protein, 
60 DRAFT - DO NOT CITE OR QUOTE 

1 gamma glutamyl transferase, alkaline phosphatase, cholesterol, and total bilirubin were not 

2 affected by treatment. Microscopic examination of livers of treated mice showed increased 

3 intracellular space, “little” focal necrosis, and degenerative alteration with vascularization; 

4 fibrosis was not observed. A NOAEL or LOAEL could not be identified from this study. 

5 

6 Acharya et al., 2001 

7 In a follow-up to a stidu by Chopra et al. (1996), Acharya et al. (2001) explored whether 

8 Wistar rats demonstrated sex-specific responses to exposures to chromium and chromium plus 

9 ethanol using a study design similar to Chopra et al. (1996), but exposing male rather than 

10 female Wistar rats. Acharya et al. (2001) exposed 1.5-month old male Wistar rats (5 or 6/group) 

11 to potassium dichromate in drinking water for 22 weeks at concentrations of 0 or 25 ppm 

12 potassium dichromate. These dose groups were part of a larger study to evaluate the interactive 

13 effects of ethanol and chromium. The authors reported that food and water consumption was 

14 monitored daily and each animal was weighed once a week, although these results were not 

15 reported. Using reference values for body weight and drinking water consumption (0.217 kg; 

16 0.032 L/day) for male Wistar rats (U.S. EPA, 1988), doses of 0 and 1.5 mg hexavalent 

17 chromium/kg-day were estimated. At study termination, animals were sacrificed and blood 

18 samples were collected for analysis of serum enzyme activities. Liver and kidney tissues were 

19 examined for histopathological changes, and liver homogenates were used to measure total 

20 triglycerides, total cholesterol, glycogen, and total glutathione. 

21 Serum AST and ALT levels were statistically significantly elevated (approximately 

22 twofold) in chromium-treated rats compared to controls. Serum succinate dehydrogenase, AP, 

23 and AcP in chromium-treated rats were not significantly different from the control. Liver total 

24 triglyceride and liver glycogen levels were significantly reduced in chromium-treated rats (by 

25 approximately 40 and 20%, respectively). There was a significant increase in liver total 

26 cholesterol levels (approximately 10%) in chromium-treated rats. Liver glutathione levels in 

27 chromium-treated rats were similar to controls. 

28 Histopathological examination of the livers of chromium-treated animals showed altered 

29 hepatic architecture in the periportal area, with increased sinusoidal space, vacuolation, and 

30 necrosis. Histopathological examination of the kidneys in chromium-treated rats revealed 

31 vacuolation in glomeruli, degeneration of the basement membrane, and renal tubular epithelial 

32 degeneration. No information regarding the number of animals examined or the number of 
61 DRAFT - DO NOT CITE OR QUOTE 

1 animals displaying histopathology was provided. The only dose tested in this study, 1.5 mg 

2 hexavalent chromium/kg-day, was identified as a LOAEL. A NOAEL was not identified. 

3 

4 Asmatullah and Noreen, 1999 

5 Asmatullah and Noreen (1999) studied the effects of subchronic exposure to hexavalent 

6 chromium on growth rate and hepatic histological structure in mice. Groups of male albino 

7 Swiss mice (9 per group) were exposed to drinking water containing 0, 500, 750, 1,000, 1,500, or 

8 2,000 mg potassium dichromate/L (equivalent to 0, 177, 265, 353, 530, or 706 mg hexavalent 

9 chromium/L, respectively) for 8 weeks. Data on drinking water consumption were not reported; 

10 based on findings of other studies (NTP, 2007, 2008) showing decreased drinking water 

11 consumption and body weight in animals treated with drinking water containing ≥30 mg 

12 hexavalent chromium/L, daily doses of hexavalent chromium cannot be accurately estimated for 

13 this study. Body weights and feed consumption were recorded weekly. At the end of the 

14 treatment period, organ weights were determined for liver, heart, and kidney and microscopic 

15 examination of the liver was conducted. During the last 2 weeks of treatment, body weights 

16 were decreased in all treatment groups, with decreases ranging from 9 to 29%, compared with 

17 controls; decreases in body weight were accompanied by similar decreases in feed intake in all 

18 treatment groups. After 8 weeks of treatment, absolute wet and dry weights of liver and heart 

19 were increased in all treatment groups, although the magnitude of these increases did not exhibit 

20 dose-dependence. No consistent pattern of change was observed for wet or dry weight of the 

21 heart. Relative organ weights were not reported. Histopathological changes in the liver were 

22 observed, with severity increasing with dose (but incidence data were not reported). At 265 mg 

23 hexavalent chromium/L, an increase in the sinusoidal space was observed; at 353 and 530 mg 

24 hexavalent chromium/L, hepatic cirrhosis and increased sinusoidal space were observed, with 

25 severity increasing with dose; and at 706 mg hexavalent chromium/L, increased sinusoidal space, 

26 cirrhosis and nuclear pyknosis (a marker for apoptosis) were observed. Results of microscopic 

27 examination of liver in mice treated with 177 mg hexavalent chromium/L were not reported. A 

28 NOAEL or LOAEL could not be identified from this study. 

29 

30 Chopra et al., 1996

31 Chopra et al. (1996) exposed 50-day old female Wistar rats (5 or 6/group) to potassium 

32 dichromate in drinking water for 22 weeks at concentrations of 0 or 25 ppm potassium 

33 dichromate. As in the Acharya et al. (2001) study, these dose groups were part of a larger study 
62 DRAFT - DO NOT CITE OR QUOTE 

1 designed to evaluate the interactive effects of ethanol and chromium. The authors reported that 

2 food and water consumption were monitored daily and each animal was weighed once a week, 

3 although these results were not reported. Using reference values for body weight and drinking 

4 water consumption (0.156 kg; 0.025 L/day) for female Wistar rats (U.S. EPA, 1988), doses of 0 

5 and 1.4 mg hexavalent chromium/kg-day were estimated. At study termination, animals were 

6 sacrificed and blood samples were collected for analysis of serum enzyme activities and serum 

7 triglycerides, cholesterol, and glucose. A kidney homogenate was used to measure glutathione, 

8 and a liver homogenate was used to measure triglycerides, cholesterol, glycogen, GSH, and lipid 

9 peroxidation. Liver and kidney tissues were examined for histopathological changes. 

10 Terminal body weights in chromium-treated rats were not significantly different from the 

11 control. The liver to body weight ratio in chromium-treated rats was statistically significantly 

12 increased (approximately twofold) over the control. Serum SDH levels were significantly lower 

13 (by approximately 20%) in chromium-treated rats compared to the control, whereas AST, ALT, 

14 AP, and AcP were statistically significantly increased (approximately two- to threefold). Serum 

15 triglycerides and glucose were statistically significantly increased (approximately threefold) in 

16 chromium-treated rats; serum cholesterol was significantly reduced (approximately twofold). 

17 Analysis of liver homogenates revealed that chromium treatment resulted in reduced liver 

18 glycogen (by approximately twofold); levels of liver cholesterol, GSH, and lipid peroxidation (as 

19 measured by diene conjugation) did not differ from the control. Kidney GSH in chromium20 treated rats was statistically significantly lower than the control (approximately 2.5-fold). 

21 Histopathological examination of the liver of chromium-treated animals showed altered 

22 hepatic architecture in the periportal area, with increased sinusoidal space, vacuolation, and 

23 necrosis. Histopathological examination of the kidneys in chromium-treated rats revealed 

24 significant damage to renal tubules and the Bowmans capsule and degeneration of the basement 

25 membrane. No information regarding the number of animals examined or the number of animals 

26 displaying histopathology was provided. The only dose tested in this study, 1.4 mg hexavalent 

27 chromium/kg-day, was identified as a LOAEL. A NOAEL was not identified. 

28 

29 Vyskocil et al., 1993 

30 Alterations in renal function, as assessed by urinalysis, were observed in rats exposed to 

31 oral potassium chromate for up to 6 months (Vyskocil et al., 1993). Groups of Wistar rats 

32 (20/sex/group) were exposed to drinking water containing 0 or 25 mg hexavalent chromium/L. 

33 Based on water consumption, which was comparable between control and treatment groups, 
63 DRAFT - DO NOT CITE OR QUOTE 

1 Vyskocil et al. (1993) calculated average daily hexavalent chromium doses of 2.18 and 2.47 mg 

2 hexavalent chromium/kg-day in males and females, respectively, during the first 3 months of 

3 exposure, and 1.40 and 1.76 mg hexavalent chromium/kg-day in males and females, 

4 respectively, during the second 3 months of exposure. After 3 or 6 months of exposure, urine 

5 was collected from 10 rats/sex/group and analyzed for total protein, albumin, β2-microglobulin, 

6 β-N-acetyl-D-glucosamine, and lactate dehydrogenase and lysozyme activities, and body and 

7 kidney weights were determined. Water consumption was monitored throughout the study. No 

8 effects on body weight gain or kidney weight were observed. In male rats, results of urinalysis 

9 did not show any treatment-related effects. In females, urinary albumin excretion, a marker of 

10 glomerular function, was significantly increased by approximately twofold (p < 0.05), compared 

11 to controls, at both 3 months and 6 months. Urinary β2-microglobulin, a marker of renal tubular 

12 dysfunction, was increased by 2-fold (p < 0.05) at 3 months and by 1.4-fold at 6 months (not 

13 statistically significant) compared to controls. Gross or microscopic examinations of kidneys 

14 were not conducted. NOAEL and LOAEL values from this study could not be identified because 

15 only one dose was evaluated and effects on other potential hexavalent chromium target tissues 

16 were not assessed. 

17 

18 4.2.2. Chronic Oral Exposure

19 NTP, 2008 

20 NTP (2008) conducted a 2-year toxicology and carcinogenicity study of sodium 

21 dichromate dihydrate in drinking water in rats and mice. Groups of F344/N rats (“core” study 

22 animals; 50/sex/group) were exposed to sodium dichromate dihydrate in drinking water at 

23 concentrations of 0, 14.3, 57.3, 172, or 516 mg sodium dichromate dihydrate/L (equivalent to 0, 

24 5, 20, 60, or 180 mg hexavalent chromium/L, respectively). Based on water consumption 

25 measured throughout the study, NTP (2008) calculated average daily doses over the 2-year 

26 treatment duration of approximately 0, 0.6, 2.2, 6, or 17 mg sodium dichromate dihydrate/kg-day 

27 for males (equivalent to 0, 0.21, 0.77, 2.1, or 5.9 mg hexavalent chromium/kg-day, respectively) 

28 and 0.7, 2.7, 7, and 20 mg sodium dichromate dihydrate/kg-day for females (equivalent to 0, 

29 0.24, 0.94, 2.4, or 7.0 mg hexavalent chromium/kg-day, respectively). Animals were observed 

30 twice daily for mortality and clinical signs of toxicity; after 5 weeks of treatment, clinical signs 

31 were recorded at 4-week intervals. Body weights were recorded weekly for the first 13 weeks, 

32 and then at 4-week intervals for the duration of the study. Water consumption was recorded 

33 weekly for the first 13 weeks of treatment and then every 4 weeks. At the end of the 2-year 
64 DRAFT - DO NOT CITE OR QUOTE 

1 treatment period, complete necropsies and microscopic examinations of comprehensive tissues 

2 were performed on all core study animals. An additional “special study” group of male rats 

3 (10/group) were exposed to the same drinking water concentrations as core animals for up to 

4 53 weeks. For the special study rats only, blood was collected on days 4 and 22 and at 3, 6, and 

5 12 months for hematology (i.e., Hct; Hgb concentration; erythrocyte, reticulocyte, and platelet 

6 counts; erythrocyte and platelet morphology; MCV; MCH; mean cell hemoglobin concentration 

7 (MCHC); and leukocyte count and differentials) and clinical chemistry (i.e., urea nitrogen, 

8 creatinine, total protein, albumin, ALT, AP, creatine kinase, sorbitol dehydrodrogenase, bile 

9 acids) analyses. At the end of the 53-week treatment period, special study animals were 

10 evaluated for chromium tissue distribution (see Section 3.2). 

11 Survival rates of exposed core study rats were similar to controls (NTP, 2008). 

12 Throughout the study, water consumption was decreased in the two highest dose groups 

13 compared to controls. During the second year of the study, water consumption in the two highest 

14 dose groups in males was decreased by 15 and 22%, respectively, and by 15 and 27%, 

15 respectively, in females (statistical significance not reported). No data on food consumption 

16 were reported. At the end of the 2-year treatment period, body weight was decreased in males 

17 and females in the highest dose group by 12 and 11%, respectively, compared with controls 

18 (statistical significance not reported). NTP (2008) suggested that decreased body weights in the 

19 highest dose group may have been partially due to decreased water consumption (due to 

20 decreased palatability), rather than an adverse effect of sodium dichromate dihydrate. No 

21 treatment-related signs of clinical toxicity were observed throughout the study. 

22 Results of hematologic analyses in special study male rats showed that exposure to 

23 sodium dichromate dihydrate in drinking water produced microcytic, hypochromic anemia, 

24 characterized by decreases in MCV, Hct, Hgb, MCH, and MCHC (NTP, 2008). The severity of 

25 microcytic, hypochromic anemia exhibited duration- and dose-dependence, with peak effects 

26 occurring at 22 days (Table 4-12). After 4 days of exposure, small changes were observed in 

27 several hematological parameters; however, decreases in all treatment groups were ≤5%, 

28 compared to controls. More severe effects were observed after 22 days of treatment, with 

29 significant decreases in MCV, Hct, and Hgb at ≥0.77 mg hexavalent chromium/kg-day. At 

30 5.9 mg hexavalent chromium/kg-day, MCV, Hct, and Hgb decreased to approximately 76, 73, 

31 and 65% of control values, respectively; reticulocyte and nucleated erythrocyte counts were 

32 increased by approximately 66% (p ≤ 0.01) and 600% (p ≤ 0.01), respectively, compared to 

33 controls, indicating compensatory hematopoiesis. Blood smears showed evidence of erythrocyte 
65 DRAFT - DO NOT CITE OR QUOTE 

1 injury or increased turnover, including poikilocytes, erythrocyte fragments, and keratocytes 

2 (incidence data not reported). Similar effects were observed after 3 months of treatment, 

3 although severity at 3 months was generally less than that observed at 22 days. Severity was 

4 further decreased after 6 and 12 months of exposure; at 12 months, affected parameters were 

5 generally only decreased by ≤5%, compared to controls. Results of hematological analyses show 

6 that exposure of rats to sodium dichromate dihydrate in drinking water produced microcytic, 

7 hypochromic anemia at subchronic exposure durations (22 days to 3 months), but that severity 

8 decreased with increasing exposure duration (6–12 months). 

9 

Table 4-12. Hematological effects in male F344/N rats exposed to sodium 

dichromate dihydrate in drinking water for up to 12 months 

Hematological Treatment group (mg hexavalent chromium/kg-day) 

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 ± 0.5c 

 (92.3) 

47.4 ± 0.4c 

(80.0) 

45.0 ± 0.7c 

(75.6) 

Month 3 48.6 ± 0.2 48.3 ± 0.2 

(99.4) 

47.3 ± 0.2c 

 (97.3) 

45.7 ± 0.2c 

(94.0) 

39.2 ± 0.6c 

(80.7) 

Month 6 49.8 ± 0.1 49.5 ± 0.1 

(99.4) 

48.6 ± 0.1c 

(97.6) 

47.8 ± 0.2c 

(96.0) 

45.4 ± 0.5c 

(91.2) 

MCV (fL) 

Month 12 52.6 ± 0.2 52.4 ± 0.2 

(99.6) 

51.9 ± 0.3 

(98.7) 

51.4 ± 0.3c 

(97.7) 

49.9 ± 0.2c 

(94.9) 

Day 22 46.0 ± 1.1 44.4 ± 0.4 

(96.5) 

43.2 ± 0.6b 

(93.9) 

38.7 ± 0.6c

(84.1) 

33.5 ± 0.8c

(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 ± 0.5c

(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 ± 0.2c 

(91.6) 

12.0 ± 0.3c 

(77.4) 

10.1 ± 0.2c

(65.2) 

Month 3 15.1 ± 0.1 14.9 ± 0.1 

(98.7) 

14.9 ± 0.2 

(98.7) 

14.6 ± 0.2b

(96.7) 

12.9 ± 0.2c

(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 ± 0.1c

(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 ± 0.1b

(96.8) 
66 DRAFT - DO NOT CITE OR QUOTE 

Table 4-12. Hematological effects in male F344/N rats exposed to sodium 

dichromate dihydrate in drinking water for up to 12 months 

Hematological Treatment group (mg hexavalent chromium/kg-day) 

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 ± 0.2c 

(89.4) 

14.8 ± 0.2c 

(74.7) 

16.3 ± 0.5c

(82.3) 

Month 3 16.2 ± 0.1 16.2 ± 0.1 

(100) 

15.7 ± 0.0c 

(96.9) 

15.0 ± 0.1c 

(92.6) 

11.9 ± 0.3c

(73.5) 

Month 6 16.3 ± 0.1 16.1 ± 0.1 

(98.8) 

15.7 ± 0.1c 

(96.3) 

15.3 ± 0.1c 

(93.9) 

14.3 ± 0.2c

(87.7) 

MCH (ρg) 

Month 12 17.0 ± 0.1 16.8 ± 0.1 

(98.8) 

16.6 ± 0.1b 

(97.6) 

16.2 ± 0.1c 

(95.3) 

15.7 ± 0.1c

(92.4) 

Day 22 33.3 ± 0.1 33.3 ± 0.1 

(100) 

32.2 ± 0.2 

(96.7) 

31.2 ± 0.2c 

(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 ± 0.1c 

(97.9) 

30.2 ± 0.3c

(90.4) 

Month 6 32.7 ± 0.1 32.5 ± 0.1 

(99.4) 

32.3 ± 0.1b 

(98.8) 

32.1 ± 0.1c 

(98.2) 

31.6 ± 0.2c

(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 ± 0.2b

 

(97.8) 

31.5 ± 0.2b

(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 ± 0.11c

(105.1) 

10.93 ± 0.16c

(117.7) 

Month 6 9.34 ± 0.06 9.43 ± 0.08 

(101.0) 

9.54 ± 0.11 

(102.1) 

9.71± 0.08c 

(104.0) 

10.15 ± 0.13c 

(108.7) 

Erythrocyte count 

(106

/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 ± 0.08c 

(105.1) 

aValues are means ± SE; values in parenthesis are percent of control; n = 10 rats/group, with the following 

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 

2 Results of clinical chemistry analyses in special study male rats (clinical chemistry was 

3 not assessed in female rats) show that exposure to sodium dichromate dihydrate in drinking 

4 water produced dose-dependent increases in serum ALT activity (NTP, 2008). Significant 

5 increases in serum ALT activity were observed at 4 days and at 6 months in rats treated with 

6 ≥2.1 mg hexavalent chromium/kg-day and at 22 days and 3 and 12 months at ≥0.77 mg 

7 hexavalent chromium/kg-day (Table 4-13). Serum ALT enzyme activity reached maximum 

8 increases (approximately 170–260% of control values) in rats treated for 3–12 months at daily 

9 doses of ≥2.1 mg hexavalent chromium/kg-day. In rats treated for 12 months with 2.1 and 
67 DRAFT - DO NOT CITE OR QUOTE 

1 5.9 mg hexavalent chromium/kg-day, serum SDH activity was 164 and 173% of control values, 

2 respectively; however, no increases in SDH activity were observed at other doses or time points. 

3 No increases in serum AP activity were observed in any treatment group throughout the 12-

4 month treatment period. Increased serum ALT activity is consistent with histopathological 

5 findings of minimal chronic inflammation of the liver observed in core study animals (discussed 

6 below); however, because other clinical chemistry markers of hepatic damage were not observed, 

7 NTP (2008) suggested that increased serum ALT activity may reflect enzyme induction rather 

8 than hepatocellular damage. Changes in other clinical chemistry outcomes were generally <5% 

9 compared to controls and did not exhibit dose- or duration-dependence. 

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 ± 2a53 ± 2 

(98) 

60 ± 3 

(113) 

68 ± 1c

(126) 

70 ± 2c

(130) 

Day 22 45 ± 1 46 ± 1 

(102) 

58 ± 2c

(129) 

75 ± 3c

(167) 

73 ± 4c

(162) 

Month 3 82 ± 4 82 ± 12 

(100) 

135 ± 18b

(165) 

176 ± 13c

(215) 

216 ± 21c

(263) 

Month 6 122 ± 15 114 ± 9 

(93) 

150 ± 12 

(123) 

238 ± 2c 

(195) 

210 ± 12c

(172) 

Month 12 102 ± 6 107 ± 8 

(105) 

135 ± 10b

(132) 

261 ± 23c

(256) 

223 ± 15c

(219) 

aValues are means ± SE; values in parenthesis are percent of control; n = 10 rats/group, with the following 

exception of 0.77 mg hexavalent chromium/kg-day group 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 

12 Gross and microscopic examinations of core study rats exposed to sodium dichromate 

13 dihydrate in drinking water for 2 years showed nonneoplastic lesions of the small intestine 

14 (duodenum), liver, and lymph nodes in both sexes, nonneoplastic lesions of the salivary gland in 

15 females, and neoplastic lesions of the oral cavity in both sexes (NTP, 2008). Incidence data for 

16 nonneoplastic lesions are summarized in Table 4-14. The incidence of minimal-to-mild cellular 

17 histiocytic infiltration of the duodenum was significantly increased in males and females at 

18 ≥0.77 and ≥2.4 mg hexavalent chromium/kg-day, respectively, compared with controls; 
68 DRAFT - DO NOT CITE OR QUOTE 

1 increases in both sexes were dose-related. Duodenal histiocytic infiltrate was characterized by 

2 single or clusters of macrophages in the lamina propria of the duodenal villi. Based on incidence 

3 data, males appeared more sensitive than females to hexavalent chromium-induced 

4 nonneoplastic changes to the small intestine. 

5 Significant findings in the liver included histiocytic cellular infiltration, chronic 

6 inflammation, fatty change, basophilic foci, and clear cell foci. The incidence of histiocytic 

7 cellular inflammation, which was mild-to-moderate in severity and characterized by clusters of 

8 macrophages in parenchymal and portal areas, was significantly increased in males and females 

9 at 5.9 and ≥0.94 mg hexavalent chromium/kg-day, respectively (Table 4-14); in females, 

10 increases in incidence and severity were dose-dependent. Increased minimal-to-mild hepatic 

11 inflammation was observed in males at 2.1 mg hexavalent chromium/kg-day and in females in 

12 all treatment groups, with dose-dependent increases in incidence and severity in females. NTP 

13 (2008) noted that chronic inflammation is a typical hepatic lesion observed in aged rats; 

14 however, exposure to sodium dichromate dihydrate appeared to enhance development of this 

15 lesion. An increase in the incidence of mild-to-moderate fatty change was observed in females 

16 only at ≥0.94 mg hexavalent chromium/kg-day. Morphologically, fatty change was 

17 characterized by hepatocytes with fat-containing cytoplasmic vacuoles. The incidence of 

18 basophilic foci was increased in males only at 0.77 and 2.1 mg hexavalent chromium/kg-day, 

19 and the incidence of clear cell foci was increased in females at 2.4 mg hexavalent chromium/kg20 day. Based on the dose-response data for histopathological changes of the liver, female rats 

21 appear more sensitive to hexavalent chromium than male rats to hepatic effects of sodium 

22 dichromate dihydrate. 

23 In lymph nodes, lesions were observed in mesenteric lymph nodes (histiocytic cellular 

24 infiltration and hemorrhage) in both sexes and in pancreatic lymph nodes (histiocytic cellular 

25 infiltration) in females only. The incidences of histiocytic cellular infiltration and hemorrhage of 

26 mesenteric lymph nodes were significantly increased in males at ≥0.77 mg hexavalent 

27 chromium/kg-day and in females at ≥2.4 and 7.0 mg hexavalent chromium/kg-day, respectively. 

28 In males, the severity of histiocytic cellular infiltration and hemorrhage of mesenteric lymph 

29 nodes was minimal-to-mild in all groups, but severity of histiocytic cellular infiltration was 

30 slightly increased at ≥2.4 mg hexavalent chromium/kg-day. The incidence of cellular histiocytic 

31 infiltration of pancreatic lymph nodes was significantly increased in females in the 2.4 mg 

32 hexavalent chromium/kg-day group only, with severity increased at ≥0.94 mg hexavalent 

33 chromium/kg-day group. Morphologically, histiocytic cellular infiltrate of the lymph nodes was 
69 DRAFT - DO NOT CITE OR QUOTE 

1 similar to that observed in the liver, with random clusters of macrophages located in the cortex 

2 and medullary sinuses; in mesenteric lymph nodes, some clusters merged to form sheets that 

3 replaced the parenchyma. NTP (2008) suggested that mesenteric lymph node hemorrhage may 

4 have resulted from histiocytic infiltration. A significant increase in the incidence of salivary 

5 gland atrophy, appearing as single focal lesions, was observed in females in the 2.4 mg 

6 hexavalent chromium/kg-day group only, compared with controls. NTP (2008) noted that 

7 atrophy is an age-related change commonly observed in rats and that the biological significance 

8 of salivary atrophy in female rats chronically treated with 2.4 mg hexavalent chromium/kg-day 

9 group is unknown. 
70 DRAFT - DO NOT CITE OR QUOTE 

1 

Table 4-14. Incidence of nonneoplastic lesions observed 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) 1/50a

(1.0) 

0/50 2/49 

(1.0) 

5/50 

(1.4) 

34/49c

(1.4) 

Liver (chronic inflammation) 19/50 

(1.1) 

25/50 

(1.2) 

21/49 

(1.3) 

28/50b

(1.1) 

26/49 

(1.3) 

Liver (basophilic focus) 22/50 28/50 29/49b 32/50b 30/49 

Small intestine, duodenum (histiocytic 

cellular infiltration) 

0/48 0/48 6/47b

(1.2) 

36/46c

(1.1) 

47/48c

(1.5) 

Lymph node, mesenteric (histiocytic 

cellular infiltration) 

13/49 

(2.0) 

11/50 

(1.5) 

30/49 

(1.9) 

39/50c 

(2.1) 

41/49 c

(2.1) 

Lymph node, mesenteric (hemorrhage) 2/49 

(1.5) 

7/50 

(1.1) 

9/49b

(1.3) 

8/50b

(1.1) 

17/49c

(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) 1/50a

(1.0) 

5/50 

(1.0) 

21/50c

(1.3) 

42/50c

(2.0) 

47/50c

(2.6) 

Liver (chronic inflammation) 12/50 

(1.3) 

21/50b

(1.2) 

28/50c

(1.3) 

35/50c

(1.6) 

39/50c

(2.1) 

Liver (fatty change) 3/50 

(3.3) 

7/50 

(3.6) 

10/50b

(2.5) 

13/50c

(2.5) 

16/50c

(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) 

30/46c

(1.0) 

47/50c

(1.2) 

Lymph node, mesenteric (histiocytic 

cellular infiltration) 

21/50 

(1.7) 

18/50 

(1.4) 

27/50 

(1.5) 

36/50c

(2.0) 

42/50c

(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) 

32/34c

(2.8) 

27/33 

(3.0) 

Salivary gland (atrophy) 9/50 

(1.3) 

7/50 

(1.4) 

10/50 

(1.2) 

17/50b

(1.4) 

17/50 

(2.1) 

aNumber of animals with lesion/number of animals examined; 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 

1 Incidence data for neoplastic lesions of the oral cavity in male and female rats exposed to 

2 sodium dichromate dihydrate in drinking water for 2 years are summarized in Table 4-15 (NTP, 

3 2008). Neoplasms observed in the oral cavity of treated rats were squamous cell carcinoma of 

4 the oral mucosa (both sexes), squamous cell papilloma of the oral mucosa (males only), 

5 squamous cell carcinoma of the tongue (both sexes), and squamous cell papilloma and carcinoma 

6 of the tongue (both sexes). The incidences of squamous cell carcinoma of the oral mucosa 

7 (13.6%) and of combined squamous cell papilloma or carcinoma (15.7%) of the oral mucosa 

8 were significantly increased in male rats treated with 5.9 mg hexavalent chromium/kg-day, 

9 compared with controls. The incidences of squamous cell carcinoma of the oral mucosa (23.9%) 

10 and of combined squamous cell carcinoma of the oral mucosa or tongue (23.9%) were 

11 significantly increased in females treated with 7.0 mg hexavalent chromium/kg-day, compared 

12 with controls. The incidences of other neoplastic lesions of the oral cavity were not significantly 

13 increased in any treatment group in males or females compared with controls, although the 

14 incidence of squamous cell carcinoma of the oral mucosa in female rats in the 2.4 mg hexavalent 

15 chromium/kg-day group (4.6%) exceeded that of historical controls (0/300 in drinking water 

16 studies; 5/1,400 by all routes). Other neoplasms observed in treated rats included pancreatic 

17 acinar adenoma and benign pheochromocytomas in males and mononuclear cell leukemia in 

18 females (Table 4-16). However, the incidence of these neoplasms did not exhibit dose19 dependence. Thus, NTP (2008) concluded that the relationship of neoplastic changes in other 

20 tissues (e.g., not of the oral cavity) to exposure to sodium dichromate dihydrate was uncertain. 
72 DRAFT - DO NOT CITE OR QUOTE 

1 

Table 4-15. Incidence of neoplastic lesions observed in the oral cavity of 

male and female 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 

Oral mucosa, squamous cell papilloma 

Overall ratea ,b 0/50 

(0%) 

0/50 

(0%) 

0/49 

(0%) 

0/50 

(0%) 

1/49 

(2%) 

Oral mucosa, squamous cell carcinoma 

Overall ratea 0/50 

(0%) 

0/50 

(0%) 

0/49 

(0%) 

0/50 

(0%) 

6/49 

(12%) [543] 

Adjusted ratec 0% 

p < 0.001 

0% 0% 0% 13.5% 

p = 0.015 

Tongue, squamous cell papilloma 

Overall ratea,b 0/50 

(0%) 

0/50 

(0%) 

0/49 

(0%) 

0/50 

(0%) 

1/49 

(2%) 

Tongue, squamous cell carcinoma 

Overall ratea 0/50 

(0%) 

1/50 

(2%) 

0/49 

(0%) 

0/50 

(0%) 

0/49 

(0%) 

Oral mucosa or tongue, squamous cell papilloma or carcinoma 

Overall ratea 0/50 

(0%) 

1/50 

(2%) [729T] 

0/49 

(0%) 

0/50 

(0%) 

7/49 

(14.5%) [543] 

Adjusted ratec 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 ratea 0/50 

(0%) 

0/50 

(0%) 

0/50 

(0%) 

2/50 

(4%) [646] 

11/50 

(22%) [506] 

Adjusted ratec 0% 

p < 0.001 

0% 0% 4.6% 23.9%

p < 0.001 

Tongue, squamous cell papilloma 

Overall ratea,b 1/50 

(2%) 

1/50 

(2%) 

0/50 

(0%) 

0/50 

(0%) 

0/50 

(0%) 

Tongue, squamous cell carcinoma 

Overall ratea,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 observed in the oral cavity of 

male and female F344/N rats exposed to sodium dichromate dihydrate in 

drinking water for 2 years 

Treatment group (mg hexavalent chromium/kg-day) 

Neoplasm type 0 0.24 0.94 2.4 7.0 

Females 

Oral mucosa or tongue, squamous cell papilloma or carcinoma 

Overall ratea 1/50 

(2%) [618] 

1/50 

(2%) [729T] 

0/50 

(0%) 

2/50 

(4%) [646] 

11/50 

(22%) [506] 

Adjusted ratec 2.2% 

p < 0.001 

2.3% 0% 4.6% 23.9% 

p = 0.002 

aOverall rate: number of animals with lesion/number of animals examined; parenthesis are the percent of animals 

examined with lesion; brackets are days to first incidence; T: observed at terminal sacrifice. p-value under 

treatment group incidence data indicates statistically significant Poly-3 test for pairwise comparison between 

control and exposed group. Statistical analysis using overall rates was only conducted if adjusted rates were not 

determined. 

bAdjusted rate not reported. 

cAdjusted rate: Poly-3 estimated neoplasm incidence (expressed as percent 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 indicates statistically significant Poly-3 test for pairwise 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 1/50a,b 2/50 6/49c 2/50 2/49 

Benign pheochromocytoma (adrenal medulla) 6/49a,b 13/50c 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 8/50a,b 18/50c 13/50 7/50 11/50 

aNumber of animals with lesion/number of animals examined. 

bNot 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 

1 In conclusion, the NTP (2008) 2-year drinking water toxicology and carcinogenicity 

2 study on sodium dichromate dihydrate identified NOAEL and LOAEL values for noncancer 

3 effects in male rats of 0.21 and 0.77 mg hexavalent chromium/kg-day, respectively, based on 

4 increased incidences of nonneoplastic histopathological changes to the liver (basophilic foci), 

5 duodenum (histiocytic cellular infiltrate), and mesenteric lymph nodes (histiocytic cellular 

6 infiltrate and hemorrhage). Although hematological effects indicative of microcytic, 

7 hypochromic anemia were observed in male rats exposed to ≥0.77 mg hexavalent chromium/kg8 day from 4 days to 6 months, the severity of effects decreased over time, such that only small 

9 changes (<5%) were observed at ≥2.1 mg hexavalent chromium/kg-day after 12 months of 

10 exposure; therefore, hematological effects were not considered as the basis for the chronic 

11 NOAEL in male rats. In female rats, a LOAEL for noncancer effects of 0.24 mg hexavalent 

12 chromium/kg-day was identified based on the increased incidence of chronic inflammation of the 

13 liver (observed in all treatment groups); a NOAEL was not identified. In addition to noncancer 

14 effects, exposure of rats to sodium dichromate dihydrate in drinking water for 2 years resulted in 

15 a significant increase in squamous epithelial neoplasms of the oral mucosa and tongue at the 

16 highest exposure level (average daily doses of 5.9 and 7.0 mg hexavalent chromium/kg-day in 

17 males and females, respectively), but not at the three lower exposure levels. NTP (2008) 

18 concluded that results from this study provide clear evidence of carcinogenic activity of sodium 

19 dichromate dihydrate in male and female F344/N rats based on increased incidences of 

20 squamous cell neoplasms of the oral cavity. 

21 B6C3F1 mice were exposed to sodium dichromate dihydrate in drinking water for up to 2 

22 years (NTP, 2008). Groups of 50 male mice (male “core” study animals) were exposed to 

23 sodium dichromate dihydrate in drinking water at concentrations of 0, 14.3, 28.6, 85.7, or 

24 257.4 mg sodium dichromate dihydrate/L (equivalent to 0, 5, 10, 30, or 90 mg hexavalent 

25 chromium/L, respectively). Based on water consumption measured throughout the study, NTP 

26 (2008) calculated average daily doses for males over the 2-year treatment duration of 

27 approximately 0, 1.1, 2.6, 7, or 17 mg sodium dichromate dihydrate/kg-day (equivalent to 0, 

28 0.38, 0.91, 2.4, or 5.9 mg hexavalent chromium/kg-day, respectively). Groups of 50 female mice 

29 (female “core” study animals) were exposed to sodium dichromate dihydrate in drinking water at 

30 concentrations of 0, 14.3, 57.3, 172, or 516 mg sodium dichromate dihydrate/L (equivalent to 0, 

31 5, 20, 50, or 190 mg hexavalent chromium/L, respectively). Based on water consumption 

32 measured throughout the study, NTP (2008) calculated average daily doses for females over the 

33 2-year treatment duration of approximately 0, 1.1, 3.9, 9, or 25 mg sodium dichromate 
75 DRAFT - DO NOT CITE OR QUOTE 

1 dihydrate/kg-day (equivalent to 0, 0.38, 1.4, 3.1, or 8.7 mg hexavalent chromium/kg-day, 

2 respectively). “Core” study mice were subjected to the same evaluations and procedures as those 

3 described above for “core” study rats (NTP, 2008). An additional “special study” group of 

4 female mice (10/group) were exposed to the same drinking water concentrations of sodium 

5 dichromate dihydrate as core animals for up to 53 weeks. For the special study mice only, blood 

6 was collected on day 22 and at 3, 6, and 12 months for hematologic analyses only (i.e., Hct; Hgb 

7 concentration; erythrocyte, reticulocyte, and platelet counts; erythrocyte and platelet 

8 morphology; MCV; MCH; MCHC; and leukocyte count and differentials). At the end of the 

9 53-week treatment period, special study animals were evaluated for chromium tissue distribution 

10 (see Section 3.2). 

11 Survival rates of core study mice exposed to sodium dichromate dihydrate were similar to 

12 controls (NTP, 2008). Throughout the study, water consumption by males and females was 

13 decreased in the two highest dose groups compared with controls. During the second year of the 

14 study, water consumption in the two highest dose groups was decreased by 15 and 35%, 

15 respectively, in males and by 25 and 32%, respectively, in females (statistical significance not 

16 reported). No data on food consumption were reported. At the end of the 2-year treatment 

17 period, body weight in males in the highest dose group was decreased by 6% compared with 

18 controls (statistical significance not reported), and body weight in females in the two highest 

19 dose groups was decreased by 8 and 15%, respectively. NTP (2008) suggested that decreased 

20 body weights in the highest dose group may have been partially due to decreased water 

21 consumption (due to decreased palatability), rather than an adverse effect of sodium dichromate 

22 dihydrate. No treatment-related signs of clinical toxicity were observed throughout the study. 

23 Results of hematology analyses in special study female mice (hematology was not 

24 assessed in male mice) show that exposure to sodium dichromate dihydrate in drinking water 

25 produced microcytic, hypochromic anemia (NTP, 2008). Anemia in mice was characterized by 

26 dose-related decreases in MCV and MCH and increases in erythrocyte counts (Table 4-17); the 

27 magnitude of change in other hematological parameters was small (≤5% compared with 

28 controls). The pattern of dose- and duration-related severity in female mice was similar to that 

29 observed in male special study rats (as described above); however, severity in mice was less than 

30 in rats. Thus, exposure of female mice to sodium dichromate dihydrate in drinking water 

31 produced microcytic, hypochromic anemia at subchronic exposure durations (22 days to 

32 3 months), with decreased severity at 6–12 months. 
76 DRAFT - DO NOT CITE OR QUOTE 

1 

Table 4-17. Hematological effects in female B6C3F1 mice exposed to sodium 

dichromate dihydrate in drinking water for up to 12 months 

Hematological Treatment group (mg hexavalent chromium/kg-day) 

parameter 

Time on 

treatment 0 0.38 1.4 3.1 8.7 

Day 22 48.8 ± 0.2a 48.3 ± 0.1b 

(90.0) 

47.8 ± 0.2c

(98.0) 

47.0 ± 0.2c

(96.3) 

46.8 ± 0.2c

(95.9) 

Month 3 47.2 ± 0.1 46.9 ± 0.3 

(99.4) 

46.7 ± 0.1 

(98.9) 

45.1 ± 0.2c

(95.6) 

43.7 ± 0.3c

(92.6) 

Month 6 45.8 ± 0.2 45.5 ± 0.3 

(99.3) 

45.1 ± 0.2b 

(98.5) 

44.6 ± 0.2c

(97.4) 

42.8 ± 0.3c

(93.4) 

MCV (fL) 

Month 12 46.9 ± 0.3 46.9 ± 0.3 

(100) 

46.3 ± 0.3 

(98.7) 

45.2 ± 0.2c

(96.4) 

43.9 ± 0.5c

(93.6) 

Day 22 16.4 ± 0.1 16.2 ± 0.0b 

(98.8) 

15.9 ± 0.1c 

(97.0) 

15.7 ± 0.1c 

(95.7) 

15.5 ± 0.1c

(94.5) 

Month 3 15.8 ± 0.0 15.7 ± 0.1 

(99.4) 

15.6 ± 0.0c 

(98.7) 

14.9 ± 0.1c 

(88.6) 

14.3 ± 0.1c

(90.5) 

Month 6 15.3 ± 0.1 15.2 ± 0.1 

(99.3) 

15.1 ± 0.1 

(98.7) 

14.9 ± 0.1c 

(97.4) 

14.1 ± 0.1c

(92.2) 

MCH (ρg) 

Month 12 15.5 ± 0.1 15.7 ± 0.2 

(101.3) 

15.5 ± 0.1 

(100) 

15.1 ± 0.1b 

(97.4) 

14.4 ± 0.2c

(92.9) 

Day 22 10.25 ± 0.15 10.20 ± 0.08 

(99.5) 

10.47 ± 0.19 

(102.1) 

10.77 ± 0.13b

(105.1) 

10.61 ± 0.13b

(103.5) 

Month 3 10.10 ± 0.16 10.66 ± 0.13b 

(105.5) 

10.55 ± 0.17b 

(104.5) 

10.95 ± 0.10c 

(108.4) 

11.55 ± 0.16c 

(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 ± 0.20c

(108.9) 

Erythrocyte 

count (106/μL) 

Month 12 9.58 ± 0.10 9.72 ± 0.09 

(101.4) 

9.77 ± 0.10 

(102.0) 

9.95 ± 0.13b

 

(103.9) 

10.30 ± 0.21c

(107.5) 

aValues are means ± SE; values in parenthesis are percent of control; n = 10 mice/group, with the exception 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 

3 Gross and microscopic examinations of core study mice exposed to sodium dichromate 

4 dihydrate in drinking water for 2 years showed nonneoplastic lesions of the small intestine, liver, 

5 lymph nodes, and pancreas and neoplastic lesions of the small intestine (NTP, 2008). Incidence 

6 data for nonneoplastic lesions are summarized in Table 4-18. In the small intestine, significant 

7 increases in the incidences of diffuse epithelial hyperplasia of the duodenum were observed in 

8 male and female mice in all treatment groups and of the jejunum in females at 8.7 mg hexavalent 

9 chromium/kg-day, compared with controls. NTP (2008) noted that diffuse epithelial hyperplasia 

10 is consistent with tissue regeneration following epithelial cell damage. Incidences of histiocytic 
77 DRAFT - DO NOT CITE OR QUOTE 

1 cellular infiltration of the duodenum were increased at ≥2.4 and ≥3.1 mg hexavalent 

2 chromium/kg-day in males and females, respectively, and of the jejunum at 8.7 mg hexavalent 

3 chromium/kg-day in females, compared with controls. Focal epithelial hyperplasia was also 

4 observed in the duodenum in males and females, although incidences were not significantly 

5 different from controls and did not exhibit dose-dependence. Due to its morphological similarity 

6 to adenoma, focal epithelial hyperplasia was classified as a preneoplastic lesion by NTP (2008). 

7 In the liver of female mice, dose-dependent increases were observed in the incidences of 

8 histiocytic infiltration at all doses and of chronic inflammation in the 3.1 mg hexavalent 

9 chromium/kg-day group. Significant decreases in the incidences of clear cell and eosinophilic 

10 foci were observed in the liver of males at 5.9 mg hexavalent chromium/kg-day and of 

11 eosinophilic foci in the liver of females at ≥3.1 mg hexavalent chromium/kg-day; the biological 

12 significance of these decreases is uncertain. 

13 Dose-dependent increases in the incidences and severity of histiocytic cellular infiltration 

14 of the mesenteric lymph nodes were observed in males and females in all treatment groups and 

15 of the pancreatic lymph nodes in males and females at ≥2.4 and ≥3.1 mg hexavalent 

16 chromium/kg-day, respectively, compared with controls. 

17 In the pancreas, the dose-dependent increases in the incidences and severity of cytoplasm 

18 alterations, characterized by depletion of cytoplasm zymogen granules, were observed at 

19 ≥2.4 mg hexavalent chromium/kg-day in males and in all treatment groups in females. NTP 

20 (2008) stated that the biological significance of this finding is uncertain. 
78 DRAFT - DO NOT CITE OR QUOTE 

1 

Table 4-18. Incidence of nonneoplastic lesions observed in male and female 

B6C3F1 mice exposed to sodium dichromate dihydrate in drinking 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) 

0/50a 11/50c

(2.0) 

18/50c

(1.6) 

42/50c

(2.1) 

32/50c

(2.1) 

Small intestine, duodenum (histiocytic 

cellular infiltration) 

0/50 2/50 

(1.0) 

4/50 

(1.0) 

37/50c

(1.2) 

35/50c

(1.7) 

Lymph node, mesenteric (histiocytic 

cellular infiltration) 

14/47 

(1.2) 

38/49c

(1.1) 

31/49c

(1.2) 

32/49c

(1.5) 

42/46c

(2.5) 

Lymph node, pancreatic (histiocytic 

cellular infiltration) 

0/5 2/13 

(1.0) 

2/10 

(1.0) 

5/8b

(1.4) 

12/16b

(2.3) 

Pancreas (cytoplasmic alteration) 0/49 1/49 

(3.0) 

1/50 

(3.0) 

9/49c

(2.1) 

8/48c

(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) 

0/50a 16/50c

(1.6) 

35/50c

(1.7) 

31/50c

(1.6) 

42/50c

(2.2) 

Small intestine, duodenum (histiocytic 

cellular infiltration) 

0/50 0/50 4/50 

(1.3) 

33/50c

(1.2) 

40/50c

(2.0) 

Small intestine, jejunum (diffuse 

hyperplasia) 

0/50 2/50 

(2.0) 

1/50 

(1.0) 

0/50 8/50c

(1.9) 

Small intestine, jejunum (histiocytic 

cellular infiltration) 

0/50 0/50 0/50 2/50 

(1.0) 

8/50c

(1.6) 

Liver (histiocytic cellular infiltration) 2/49 

(1.0) 

15/50c

(1.1) 

23/50c

(1.0) 

32/50c

(1.0) 

45/50c

(1.9) 

Liver (chronic inflammation) 16/49 

(1.1) 

21/50 

(1.1) 

22/50 

(1.10 

27/50c

(1.1) 

24/50 

(1.0) 

Lymph node, mesenteric (histiocytic 

cellular infiltration) 

3/46 

(1.0) 

29/48c

(1.3) 

26/46c

(1.1) 

40/50c

(1.9) 

42/50c

(2.7) 

Lymph node, pancreatic (histiocytic 

cellular infiltration) 

0/14 1/12 

(1.0) 

2/15 

(1.5) 

7/14c

(1.9) 

8/13c

(2.5) 

Pancreas (cytoplasmic alteration) 0/48 6/50b

(2.5) 

6/49b

(2.0) 

14/50c

(2.4) 

32/50c

(2.6) 

aNumber of animals with lesion/number of animals examined; 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 

3 Incidence data for neoplastic lesions of the small intestine in male and female mice 

4 exposed to sodium dichromate dihydrate in drinking water for 2 years are summarized in 
79 DRAFT - DO NOT CITE OR QUOTE 

1 Table 4-19 (NTP, 2008). In male mice, incidences of combined small intestine (duodenum, 

2 jejunum, and ileum) adenoma or carcinoma were significantly increased at ≥2.4 mg hexavalent 

3 chromium/kg-day and incidences of duodenal adenoma, small intestine adenoma, and small 

4 intestine carcinoma were significantly increased at 5.9 mg hexavalent chromium/kg-day. In 

5 addition, significant positive dose-related trends were observed for the incidences of duodenal 

6 adenoma, duodenal carcinoma, jejunal adenoma, small intestine adenoma, small intestine 

7 carcinoma and combined small intestine adenoma or carcinoma. In female mice, significant 

8 increases in the incidences of duodenal adenoma, small intestine adenoma, and combined small 

9 intestine adenoma or carcinoma were observed at ≥3.1 mg hexavalent chromium/kg-day and 

10 incidences of duodenal carcinoma, jejunal adenoma, and small intestine carcinoma were 

11 significantly increased at 8.7 mg hexavalent chromium/kg-day. Significant positive dose-related 

12 trends were observed for duodenal adenoma, duodenal carcinoma, jejunal adenoma, small 

13 intestine adenoma, small intestine carcinoma and combined small intestine adenoma or 

14 carcinoma. No other statistically or biologically significant neoplasms were observed in other 

15 tissues. 

16 

Table 4-19. Incidence of neoplastic lesions observed in the small intestine of 

male and female B6C3F1 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 ratea,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 ratea 1/50 

(2%) [665] 

0/50 

(0%) 

1/50 

(2%) [729T] 

5/50 

(10%) [729T] 

15/50 

(30%) [451] 

Adjusted ratec 2.2% 

p < 0.001 

0% 2.3% 10.8% 32.9% 

p < 0.001 

Duodenum, carcinoma 

Overall ratea 0/50 

(0%) 

0/50 

(0%) 

0/50 

(0%) 

2/50 

(4%) [729T] 

3/50 

(6%) [729T] 

Adjusted ratec 0% 

p < 0.011 

0% 0% 4.3% 6.8% 
80 DRAFT - DO NOT CITE OR QUOTE 

Table 4-19. Incidence of neoplastic lesions observed in the small intestine of 

male and female B6C3F1 mice exposed to sodium dichromate dihydrate in 

drinking water for 2 years 

Jejunum, adenoma 

Overall ratea 0/50 

(0%) 

0/50 

(0%) 

0/50 

(0%) 

0/50 

(0%) 

3/50 

(6%) [714] 

Adjusted ratec 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 ratea, b 0/50 1/50 0/50 0/50 0/50 

Jejunum, all carcinoma (includes multiple) 

Overall ratea,b 0/50 2/50 0/50 1/50 2/50 

All small intestined, adenoma 

Overall ratea 1/50 

(2%) [665] 

1/50 

(2%) [729T] 

1/50 

(2%) [729T] 

5/50 

(10%) [729T] 

17/50 

(34%) [451] 

Adjusted ratec 2.2% 

p < 0.001 

2.3% 2.3% 10.8% 37.2% 

p < 0.001 

All small intestined, carcinoma 

Overall ratea 0/50 

(0%) 

2/50 

(4%) [729T] 

1/50 

(2%) [729T] 

3/50 

(6%) [729T] 

5/50 

(10%) [729T] 

Adjusted ratec 0% 

p = 0.014 

4.5% 2.3% 6.5% 11.4% 

p = 0.028 

All small intestined, adenoma or carcinoma 

Overall ratea 1/50 

(2%) [665] 

3/50 

(6%) [729T] 

2/50 

(4%) [729T] 

7/50 

(14%) [729T] 

20/50 

(40%) [451] 

Adjusted ratec 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 ratea,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 ratea 0/50 

(0%) 

0/50 

(0%) 

2/50 

(4%) [729T] 

13/50 

(25%) [729T] 

12/50 

(24%) [693] 

Adjusted ratec 0% 

p < 0.001 

0% 4.2% 27.8% 

p < 0.001 

25.2% 

p < 0.001 

Duodenum, carcinoma 

Overall ratea 0/50 

(0%) 

0/50 

(0%) 

0/50 

(0%) 

1/50 

(2%) [729T] 

6/50 

(12%) [625] 

Adjusted ratec 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 observed in the small intestine of 

male and female B6C3F1 mice exposed to sodium dichromate dihydrate in 

drinking water for 2 years 

Overall ratea,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 ratea 0/50 

(0%) 

1/50 

(2%) [729T] 

0/50 

(0%) 

2/50 

(4%) [729T] 

5/50 

(10%) [729T] 

Adjusted ratec 0% 

p = 0.002 

2.2% 0% 4.3% 10.6% 

p = 0.035 

Jejunum, carcinoma 

Overall ratea,b 1/50 

(2%) 

0/50 

(0%) 

2/50 

(4%) 

2/50 

(4%) 

1/50 

(2%) 

All small intestined, adenoma 

Overall ratea 0/50 

(0%) 

1/50 

(2%) [729T] 

2/50 

(4%) [729T] 

15/50 

(30%) [729T] 

16/50 

(32%) [693] 

Adjusted ratec 0% 

p < 0.001 

2.2% 4.2% 32.0% 

p < 0.001 

33.7% 

p < 0.001 

All small intestined, carcinoma 

Overall ratea 1/50 

(2%) [729T] 

0/50 

(0%) 

2/50 

(4%) [729T] 

3/50 

(6%) [729T] 

7/50 

(14%) [625] 

Adjusted ratec 2.2% 

p < 0.001 

0% 4.2% 6.4% 14.7% 

p = 0.037 

All small intestined, adenoma or carcinoma 

Overall ratea 1/50 

(2%) [729T] 

1/50 

(2%) [729T] 

4/50 

(8%) [729T] 

17/50 

(34%) [729T] 

22/50 

(44%) [625] 

Adjusted ratec 2.2% 

p < 0.001 

2.2% 8.3% 36.3% 

p < 0.001 

45.9% 

p < 0.001 

aOverall 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 statistically significant Poly-3 test for pairwise comparison between 

control and exposed group. Statistical analysis using overall rates were only conducted if adjusted rates were not 

determined. 

bAdjusted rate not reported. 

cAdjusted 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. pValue under treatment group incidence data indicates statistically significant Poly-3 test for pairwise comparison 

between control and exposed groups, using adjusted rates. 

dDuodenum, jejunum, or ileum. 

Source: NTP (2008). 

1 

2 In conclusion, the NTP (2008) 2-year toxicology and carcinogenicity study on sodium 

3 dichromate dihydrate identified a LOAEL for noncancer effects of 0.38 mg hexavalent 

4 chromium/kg-day in male and female B6C3F1 mice; a NOAEL value was not identified. In 
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1 males, the LOAEL was based on increased incidences of histopathological changes to the 

2 duodenum (diffuse epithelial hyperplasia) and mesenteric lymph nodes (histiocytic cellular 

3 infiltration); in females, the LOAEL was based on increased incidences of histopathological 

4 changes to the duodenum (diffuse epithelial hyperplasia), mesenteric lymph nodes (histiocytic 

5 cellular infiltration), liver (histiocytic cellular infiltration), and pancreas (depletion of 

6 cytoplasmic zymogen granules). Although mild microcytic, hypochromic anemia was observed 

7 in female mice at ≥0.38 mg hexavalent chromium/kg-day after 22 days of exposure, the severity 

8 of these effects decreased over time, such that only small changes (<5%) were observed at 

9 ≥3.1 mg hexavalent chromium/kg-day after 12 months of exposure; therefore, hematological 

10 effects were not considered as the basis for the chronic LOAEL value in female mice. In 

11 addition to noncancer effects, exposure of B6C3F1 mice to sodium dichromate dihydrate in 

12 drinking water for 2 years resulted in significant increases in the incidences of neoplasms of the 

13 small intestine in males and females at doses ≥2.4 and ≥3.1 mg hexavalent chromium/kg-day, 

14 respectively. NTP (2008) concluded that results of this study provide clear evidence of 

15 carcinogenic activity of sodium dichromate dihydrate in male and female B6C3F1 mice based on 

16 increased incidences of neoplasms of the small intestine. 

17 

18 Borneff et al., 1968 

19 Borneff et al. (1968) conducted a long-term animal cancer bioassay of hexavalent 

20 chromium administered in drinking water. Using a three-generation study design, Borneff et al. 

21 (1968) treated 120 female and 10 male NMRI mice with 1 mg potassium chromate/day (500 

22 ppm) in drinking water (containing 3% household detergent). A control group of animals 

23 received drinking water (3% detergent) only. An outbreak of mousepox (ectromelia) virus 

24 occurred during the eighth month of the experiment, and within three months, the majority (512) 

25 of the animals died. All animals received a mousepox vaccination two months after the 

26 outbreak, and this effectively ended the epidemic and the study continued. Two carcinomas of 

27 the stomach were observed in female mice exposed to potassium chromate. No malignant 

28 stomach tumors were found in control mice. Nine benign stomach tumors were observed in 

29 female mice exposed to potassium chromate. The combined incidence of malignant and benign 

30 stomach tumors (11/66) in potassium chromate-exposed-female mice was significantly different 

31 than the combined incidence of tumors in control female mice (2/79) [Fisher’s Exact test, 

32 p<0.05]. 

33 
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1 Anwar et al., 1961 

2 The effects of chronic oral exposure to hexavalent chromium were evaluated in dogs by 

3 Anwar et al. (1961). Dogs (one control dog and one to two dogs/treatment group) were exposed 

4 to potassium chromate in drinking water at concentrations of 0, 0.45, 2.25, 4.5, 6.75, or 11.2 mg 

5 hexavalent chromium/L for 4 years. Several different breeds of dogs (German shepherds, 

6 poodles, and beagles) were used and body weights of animals were not reported; thus, daily 

7 hexavalent chromium doses cannot be accurately estimated. Throughout the exposure period, 

8 animals were evaluated for clinical signs of toxicity, and food consumption and growth rate were 

9 recorded (frequency of observations not reported). At monthly intervals, blood was obtained for 

10 evaluation of hematology (i.e., erythrocyte counts, total and differential leukocyte counts, and 

11 Hgb), and at 6-month intervals, urine was analyzed for albumin, acetone, bile pigments, glucose, 

12 erythrocytes, and specific gravity. At the end of the 4-year treatment period, weights of the liver, 

13 kidney, and spleen were recorded, and microscopic examination was conducted on selected 

14 tissues of major organs. No chromium-related effects were observed. Interpretation of the study 

15 results is limited by the small number of animals evaluated and the inability to estimate daily 

16 doses of hexavalent chromium received by the treated animals. A NOAEL or LOAEL could not 

17 be identified from this study. 

18 

19 MacKenzie et al., 1958 

20 MacKenzie et al. (1958) conducted two experiments in which Sprague-Dawley rats were 

21 administered hexavalent chromium in drinking water for 1 year. In the first experiment, groups 

22 of rats (10/sex in the control group and 8/sex/group in the treatment groups) were exposed to 

23 drinking water containing potassium chromate at concentrations of 0, 0.45, 2.2, 4.5, 7.7, or 

24 11 mg hexavalent chromium/L. In the second experiment, groups of 12 male and 9 female rats 

25 were exposed to drinking water containing potassium chromate at concentrations of 0 or 25 mg 

26 hexavalent chromium/L. For experiment 1, MacKenzie et al. (1958) reported that drinking water 

27 consumption and body weights in the treatment groups were comparable to controls, although 

28 data were not reported. Using reference values for body weight (males: 0.523 kg; females: 

29 0.338 kg) and daily drinking water intake (males: 0.062 L/day; females: 0.045 L/day) for adult 

30 male and female Sprague-Dawley rats (U.S. EPA, 1988), doses of 0.05, 0.26, 0.53, 0.91, or 

31 1.3 mg hexavalent chromium/kg-day for males and 0.06, 0.29, 0.60, 1.0, or 1.5 mg hexavalent 

32 chromium/kg-day for females exposed to drinking water containing 0.45, 2.2, 4.5, 7.7, or 11 mg 

33 hexavalent chromium/L, respectively, were estimated. For experiment 2, drinking water 
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1 consumption was decreased by 16 and 27% in male and female rats, respectively. Thus, using 

2 reference values for body weight and daily drinking water intake for adult male and female 

3 Sprague-Dawley rats (listed above; U.S. EPA, 1988) and assuming decreases in water 

4 consumption of 16 and 27% in males and females, respectively, average daily doses of 2.8 and 

5 2.4 mg hexavalent chromium/kg-day in males and females, respectively, were estimated. 

6 Throughout the treatment period in both experiments, animals were examined for clinical signs 

7 of toxicity, and weight gain and food and water consumption were recorded (frequency of 

8 observations not reported). At monthly intervals, blood was analyzed for Hgb, erythrocyte 

9 counts, and total and differential leukocyte counts. At the end of treatment, microscopic 

10 examination of selected tissues (kidney, adrenal gland, liver, spleen, heart, brain, stomach, 

11 duodenum, ileum, colon, and bone marrow) was conducted (as described by Decker et al., 1958). 

12 No treatment-related clinical signs of toxicity, effects on food consumption, body weight gain, or 

13 histopathological findings were observed. 

14 

15 4.3. REPRODUCTIVE AND DEVELOPMENTAL TOXICITY STUDIES—ORAL 

16 Studies evaluating the potential reproductive effects of oral exposure to hexavalent 

17 chromium compounds have been conducted in monkeys (Aruldhas et al., 2006, 2005, 2004; 

18 Subramanian et al., 2006), rats (Bataineh et al., 2007, 1997; Elsaieed and Nada, 2002; Li et al., 

19 2001; Kanojia et al., 1998, 1996; NTP, 1996b; Chowdhury and Mitra, 1995;), mice (Al-Hamood 

20 et al., 1998; Elbetieha and Al-Hamood, 1997; NTP, 1997; 1996a; Junaid et al., 1996a, b, 1995; 

21 Murthy et al., 1996; Zahid et al., 1990; Trivedi et al., 1989), and rabbits (Yousef et al., 2006). In

22 addition, several studies have specifically evaluated the potential effects of pre-gestational, 

23 gestational, or lactational exposure on fetal development in rats (Banu et al., 2008; Elsaieed and 

24 Nada, 2002; Kanojia et al., 1998, 1996) and mice (Al-Hamood et al., 1998; Junaid et al., 1996a, 

25 b, 1995; Trivedi et al., 1989). Studies conducted by NTP (1997, 1996a, b) and Zahid et al. 

26 (1990) evaluated dietary exposure; all other studies have evaluated animals exposed to 

27 hexavalent chromium in drinking water or by gavage. In general, studies that evaluated 

28 developmental effects of hexavalent chromium were conducted at higher exposure levels than 

29 those that evaluated reproductive effects. 

30 Collectively, the available studies provide evidence that oral exposure of laboratory 

31 animals to hexavalent chromium compounds produces adverse reproductive effects, including 

32 histopathological changes to reproductive organs in males (Aruldhas et al., 2006, 2005, 2004; 

33 Chowdhury and Mitra, 1995; Li et al., 2001; Zahid et al., 1990) and females (Murthy et al., 
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1 1996); alterations in sperm, including decreased count, decreased motility, and abnormal 

2 morphology (Subramanian et al., 2006; Yousef et al., 2006; Li et al., 2001; Zahid et al., 1990); 

3 decreased plasma testosterone levels (Yousef et al., 2006; Chowdhury and Mitra, 1995); 

4 increased estrous cycle length (Kanojia et al., 1998, 1996; Murthy et al., 1996); changes in 

5 mating behavior and decreased fertility in males (Bataineh et al., 1997); and adverse 

6 reproductive outcomes, including decreased numbers of live fetuses and implantations, and 

7 increased numbers of resorptions and pre- and postimplantation losses (Bataineh et al., 2007; 

8 Elsaieed and Nada, 2002; Elbetieha and Al-Hamood, 1997; Junaid et al., 1996a, b, 1995; Kanojia 

9 et al., 1998, 1996; Trivedi et al., 1989). Developmental effects observed have included 

10 decreased fetal weight and length (Elsaieed and Nada, 2002; Kanojia et al., 1998; Junaid et al., 

11 1996a, b, 1995; Trivedi et al., 1989); external (subdermal hemorrhage and tail malformations) 

12 and skeletal abnormalities (decreased ossification) (Elsaieed and Nada, 2002; Junaid et al., 

13 1996a, b, 1995; Kanojia et al., 1998, 1996; Trivedi et al., 1989); and delayed sexual maturation 

14 and function in female offspring (Banu et al., 2008; Al-Hamood et al., 1998). In contrast to 

15 results of the above studies, adverse effects were not observed in dietary exposure studies 

16 conducted by NTP that investigated the potential for hexavalent chromium to produce adverse 

17 effects on male reproductive organs in rats and mice (NTP, 1996a, b) and on reproductive 

18 outcomes in a continuous breeding study in mice (NTP, 1997).

19 The following review of available reproductive and developmental studies is organized as 

20 follows: (1) studies evaluating effects on reproductive tissues and mating behavior; (2) studies 

21 evaluating effects on reproductive outcomes; (3) studies evaluating pre-gestational exposure on 

22 reproductive outcomes and fetal development; and (4) studies evaluating gestational and/or 

23 lactational exposure on reproductive outcomes and fetal development. 

24 

25 4.3.1. Effects on Reproductive Tissues and Mating Behavior

26 Aruldhas et al., 2006, 2005, 2004; Subramanian et al., 2006 

27 In a series of studies conducted by the same research group, adverse effects on male 

28 reproductive organs were observed in monkeys exposed to hexavalent chromium in drinking 

29 water (Aruldhas et al., 2006, 2005, 2004; Subramanian et al., 2006). All of these studies 

30 followed the same exposure protocol; adult male bonnet monkeys (6–8 years old) were exposed 

31 to drinking water containing 0, 100, 200, or 400 mg potassium dichromate/L (Aruldhas et al., 

32 2006, 2005, 2004) or 0, 50, 100, 200, or 400 mg potassium dichromate/L (equivalent to 0, 17.6, 

33 35.3, 70.6, and 141.2 mg hexavalent chromium/L, respectively) (Subramanian et al., 2006) for 
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1 180 days; two studies included a 180-day post-treatment recovery period (Aruldhas et al., 2006; 

2 Subramanian et al., 2006). Aruldhas et al. (2004) noted that 400 mg potassium dichromate/L 

3 was selected as the maximum concentration tested since exposure to higher concentrations 

4 resulted in decreased food and drinking water consumption and death within 3 months. At the 

5 beginning of the treatment period, body weights of monkeys were reported as 7–8 kg by 

6 Aruldhas et al. (2005) and as 7–9 kg by Subramanian et al. (2006). Although body weights were 

7 not reported by Aruldhas et al. (2006, 2004), it is assumed that initial body weights were similar 

8 in all studies. The study authors did not report body weights or drinking water consumption over 

9 the course of treatment or calculate daily doses of hexavalent chromium. For this review, daily 

10 doses of 0, 1.0, 2.1, 4.1, and 8.3 mg hexavalent chromium/kg-day for the 0, 50, 100, 200, or 400 

11 potassium dichromate/L groups, respectively, were estimated using the allometric equation for 

drinking water consumption for primates (0.09 × body weight0.7945 12 ; U.S. EPA, 1988) and an 

13 average reported initial body weight of 8 kg (Subramanian et al., 2006; Aruldhas et al., 2005); 

14 however, these dose estimates are uncertain due to the absence of data on body weight and 

15 drinking water consumption over the course of the 6-month treatment period. In the following 

16 discussions, the three treatment groups evaluated in the Aruldhas et al. (2006, 2005, 2004) 

17 studies (i.e., 100, 200, or 400 mg potassium dichromate/L, approximately equivalent to 2.1, 4.1, 

18 or 8.3 mg hexavalent chromium/kg-day, respectively) are referred to as the low-, mid- and high19 dose groups, respectively; the four treatment groups evaluated in the Subramanian et al. (2006) 

20 study (i.e., 50, 100, 200, or 400 mg potassium dichromate/L, approximately equivalent to 1.0, 

21 2.1, 4.1, or 8.3 mg hexavalent chromium/kg-day, respectively) are referred to as the lowest-, 

22 low-, mid-, and high-dose groups, respectively. 

23 Aruldhas et al. (2004) conducted histological assessments of testes and epididymides 

24 from monkeys (three monkeys/group) following 180 days of treatment. Testes and epididymides 

25 were evaluated by light microscopy (resin-embedded slices) and transmission electron 

26 microscopy (TEM). In the three treatment groups, epididymal damage and the development of 

27 microcanals in the cauda epididymal epithelium were observed; severity of ductal damage 

28 increased with dose. In the low-dose group, the cauda epididymal epithelium appeared 

29 pseudostratified; degeneration of principal cells and epithelial rupture, with the lumen occluded 

30 by principal cells, were observed. In the mid-dose group, the occluded lumen appeared packed 

31 with immature germ cells and macrophages. In the high-dose group, hypertrophy of the caudal 

32 epithelium and “obliteration” of the ductal lumen were observed. The development of two 

33 morphologically distinct microcanals was observed in all treatment groups. Arulhhas et al. 
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1 (2004) proposed that microcanal development was an adaptive response to provide passage for 

2 spermatozoa around the obstructed ducts and to entrap spermatozoa that had been released into 

3 the epithelium due to the epithelial rupture. Appearance of tissues from the control group was 

4 not reported. Additional TEM evaluations of testes from monkeys (three monkeys/group) in the 

5 three hexavalent chromium treatment groups showed a dose-related accumulation of basal cells 

6 along the basal lamina of the epididymis, giving the epithelium a pseudostratified appearance, 

7 and intraepithelial macrophages (Aruldhas et al., 2006). In addition, cells showed an 

8 accumulation of sperm-derived lipofuscin material, indicative of phagocytosis and processing of 

9 sperm. In contrast, these findings were not observed in testes from control monkeys. 

10 Aruldhas et al. (2005) evaluated the effects of hexavalent chromium exposure in male 

11 monkeys at the completion of the 180-day treatment period (three monkeys/group) and following 

12 an additional 180-day recovery period (three monkeys/group); assessments included plasma 

13 chromium concentration, absolute and relative testicular weights, and microscopic (light and 

14 TEM) evaluations of testes. At the end of the treatment period, chromium plasma concentration 

15 was significantly (p < 0.05) increased in the three treatment groups, with increases reaching 

16 almost ninefold in the high-dose group compared to controls. Relative testicular weight was 

17 significantly (p < 0.05) decreased by 23, 35, and 34% in the low-, mid-, and high-dose groups, 

18 respectively; absolute testicular weight was not affected by treatment (data not reported). 

19 Following the recovery period, chromium plasma concentrations and relative testicular weight in 

20 treatment groups were comparable to controls. Light microscopic evaluations of testes in control 

21 monkeys showed seminiferous tubules and Leydig cells with normal appearance and cellular 

22 organization. In the three hexavalent chromium treatment groups, seminiferous tubules appeared 

23 disorganized, with decreased diameters, epithelial degeneration, and lumens filled with 

24 prematurely released germ cells and cellular debris; depletion of germ cells, hyperplasia of 

25 Leydig cells, and Sertoli cell fibrosis were also observed. TEM examination of testes from the 

26 three treatment groups showed morphological changes in spermatids (granulation of chromatin 

27 and vacuolization) and spermatocytes (fragmented chromatin and swollen mitochondria) and the 

28 presence of macrophages containing phagocytosed sperm; effects were more severe in the high29 dose group. Following the recovery period, no histopathological findings were observed in 

30 testes of hexavalent chromium-treated monkeys, with the exception of “a few” prematurely 

31 released germ cells in the seminiferous tubular lumen (treatment group for this observations was 

32 not specified). 
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1 Subramanian et al. (2006) evaluated sperm count and sperm straight-line velocity 

2 at monthly intervals during the 180-day treatment period; the same evaluations were 

3 conducted monthly in monkeys in the high-dose group during a 180-day recovery period. In the 

4 lowest-dose group, no effects were observed on sperm count or straight-line velocity. Sperm 

5 count was significantly decreased in the low-, mid-, and high-dose groups, compared with 

6 controls; decreases were dose- and duration-dependent. For example, in the low-dose group, 

7 significant (p < 0.05) decreases in sperm count were first observed after 4 months (11% 

8 decrease), with a maximum decrease of 25% after 6 months; in the high-dose group, sperm 

9 counts were significantly decreased by 13% after 2 months, with a 30% reduction after 6 months. 

10 Similar effects were observed for sperm straight-line velocity. In the low-dose group, velocity 

11 was significantly (p < 0.05) decreased by 10 and 25% after 4 months and 6 months of treatment, 

12 respectively; in the high-dose group, velocity was significantly decreased by 12% after 2 months 

13 and by 35% after 6 months. Effects on sperm count and straight-line velocity were reversible 

14 following withdrawal from treatment. During the first month of the recovery period (high-dose 

15 monkeys only), sperm count was significantly increased compared with that observed at the end 

16 of the treatment period, with counts returning to pre-treatment levels by month 3 of the recovery 

17 period; sperm velocity returned to pre-treatment levels by month 3 of the recovery period. 

18 Results of these four studies (Aruldhas et al., 2006, 2005, 2004; Subramanian et al., 

19 2006) indicate that exposure of monkeys to hexavalent chromium as potassium dichromate in 

20 drinking water produced reversible changes to male reproductive organs, including disruption of 

21 spermatogenesis. Effects on sperm count and velocity and histopathological changes were 

22 observed in the low-, mid-, and high-dose groups (≥2.1 mg hexavalent chromium/kg-day), but no 

23 effects on sperm count and velocity were observed in monkeys in the lowest treatment group 

24 (1.0 mg hexavalent chromium/kg-day). However, this dose cannot be considered a NOAEL 

25 value because microscopic evaluations were not conducted in monkeys from this group. 

26 Although group sizes in these studies were small, results provide evidence of adverse male 

27 reproductive effects in nonhuman primates exposed to hexavalent chromium in drinking water at 

28 concentrations as low as 35.3 mg hexavalent chromium/L (2.1 mg hexavalent chromium/kg29 day). 

30 

31 Chowdhury and Mitra, 1995 

32 Effects of oral exposure to hexavalent chromium on male reproductive organs was 

33 evaluated in mature (age not reported) male Charles Foster rats that were administered 0, 20, 40, 
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1 or 60 mg hexavalent chromium/kg-day as sodium dichromate in saline by gavage for 90 days 

2 (Chowdhury and Mitra, 1995). Although Chowdhury and Mitra (1995) stated that the control 

3 and exposure groups included 10 animals per group, conflicting summaries of the actual group 

4 sizes are presented in the report. Body weights were recorded twice weekly. At the end of the 

5 treatment period, testes were excised, weighed, and prepared for histological or biochemical 

6 evaluations, and serum testosterone activity was determined. For biochemical analyses, fresh 

7 tissue was homogenized and assayed for total cholesterol, activities of succinic dehydrogenase 

and 3β-∆

5

-hydroxysteroid dehydrogenase (3β-∆

5

8 -HSH), and total protein, DNA, and RNA. For 

9 microscopic evaluations, testes were fixed in Bouin’s fluid, embedded in paraffin, and stained 

10 with haematoxylin and eosin (H&E). 

11 Final body weight was significantly reduced by approximately 27% compared to controls 

12 in the mid- and high-dose groups (statistical significance not reported); absolute testis weights 

13 were significantly reduced by 28% (p < 0.05) and 35% (p < 0.001) in the mid- and high-dose 

14 groups, respectively, compared with controls. Serum testosterone levels were decreased by 31% 

15 in the low- (p < 0.05) and mid-dose (p < 0.001) groups and by 47% (p < 0.001) in the high-dose 

16 group. Biochemical analysis of testes showed significant decreases in total cholesterol by 2% (p 

17 < 0.05) and 25% (p < 0.001) in the mid- and high-dose groups, respectively, and significant (p < 

18 0.001) decreases in succinic dehydrogenase activity by 35 and 45% in the mid- and high-dose 

groups, respectively. In all treatment groups, 3β-∆

5

19 -HSH was significantly decreased by 25% (p 

20 < 0.05), 28% (p < 0.05), and 52% (p < 0.001) in the low-, mid-, and high-dose groups, 

21 respectively. Dose-related decreases in total testicular protein were observed, with decreases 

22 reaching 46% (p < 0.001) in the high-dose group. Testicular DNA and RNA levels were 

23 significantly decreased in the mid- and high-dose groups, with decreases reaching 45% (p < 

24 0.001) and 37% (p < 0.001), respectively, in the high-dose group. Microscopic evaluation of 

25 testicular tissue showed adverse effects in the mid- and high-dose groups including disintegration 

26 of peritubular membranes, detachment of seminiferous cellular components from basement 

27 membranes, and accumulation of cellular debris in the mid-dose group, and cellular degeneration 

28 and complete disruption of the epithelium with fibrous tissue in the high-dose group; reduction in 

29 seminiferous tubular diameter, decreased number of Leydig cells, and Leydig cell degeneration 

30 were observed in the mid- and high-dose groups. No change in the number of spermatogonia 

31 were observed, although the number of pachytene spematocytes and stage 7 spermatids were 

32 decreased in the mid- and high-dose groups and resting spermatocytes were decreased in the 

33 high-dose group. No treatment-related histopathological effects were observed in the testes of 
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1 rats in the low-dose group, although histochemical evaluations of testes showed dose-related loss 

of 3β-∆

5

2 -HSH activity in all treatment groups. 

3 Results of histological and biochemical analyses show that oral exposure of male rats to 

4 hexavalent chromium for 90 days produced adverse effects on male reproductive tissues, 

5 including decreased spermatogenic and steroidogenic activities. Based on decreased serum 

testosterone levels and loss of 3β-∆

5

6 -HSH activity in testes observed in all treatment groups, a 

7 LOAEL of 20 mg hexavalent chromium/kg-day was identified in this gavage study of male 

8 Charles Foster rats. A LOAEL of 40 mg hexavalent chromium/kg-day was identified for 

9 degenerative changes in the testes detected by microscopy. 

10 

11 Bataineh et al., 1997 

12 Effects of oral hexavalent chromium administration on mating behavior, aggression, and 

13 fertility were assessed in male rats by Bataineh et al. (1997). Adult (age not specified) male 

14 Sprague-Dawley rats (n = 12 or 13) were administered drinking water containing 0 or 1,000 mg 

15 potassium dichromate/L (equivalent to 353 mg hexavalent chromium/L) for 12 weeks. No data 

16 on drinking water consumption were included in the study report. Based on findings of other 

17 studies (NTP, 2008, 2007) showing decreased drinking water consumption and body weight at 

18 drinking water concentrations ≥30 mg hexavalent chromium/L, it is likely that drinking water 

19 consumption was decreased in the chromium treatment group; thus, daily doses of hexavalent 

20 chromium cannot be accurately estimated from this study. Following the treatment period, 

21 assessments were conducted for sexual behavior in the presence of females in estrous (number of 

22 mounts without penile intromission, time to first mount, time from presentation of female to first 

23 intromission, number of penile intromissions, time from first intromission to ejaculation, and 

24 time from ejaculation to next intromission), aggressive behavior in the presence of a second 

25 untreated male (number of lacerations given, boxing bouts, fights, and ventral presenting), 

26 fertility following a 10-day mating period with untreated females (numbers of pregnant females, 

27 viable fetuses, and resorptions), body weight, and weights of reproductive organs (paired testes, 

28 seminal vesicles, and preputial glands). Histopathological evaluations of tissues were not 

29 conducted. 

30 All rats “appeared healthy” throughout the treatment period. Assessment of mating 

31 behavior in hexavalent chromium-treated rats showed significant decreases in number of mounts 

32 (35% decrease; p < 0.001) and percentage of males ejaculating (79% decreases; p < 0.005), and 

33 increases in the time from first intromission to ejaculation (59% increase; p < 0.001) and time 
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1 from ejaculation to next intromission (37% increases, p < 0.001), compared with controls. All 

2 measures of aggressive behavior were decreased in rats treated with potassium dichromate. All 

3 measures of fertility were comparable between control and treatment groups. Treatment resulted 

4 in significant (p < 0.001) decreases in body weight (19% decrease) and absolute weights of testes 

5 (24% decrease), seminal vesicles (15% decrease), and preputial gland (23% decrease); however, 

6 for relative weights of reproductive tissues, only relative testes weight was significantly 

7 decreased (6% decrease, p < 0.05) compared to controls. 

8 This study identified a LOAEL of 535 mg hexavalent chromium/L as potassium 

9 dichromate in drinking water based on adverse effects on mating and aggressive behaviors; a 

10 NOAEL was not identified. Because drinking water consumption and body weight data over the 

11 course of the study was not provided, a LOAEL, expressed in mg hexavalent chromium/kg-day, 

12 could not be derived from this study. 

13 

14 Li et al., 2001 

15 Oral exposure of male rats to chromium(VI) oxide for 6 days resulted in adverse 

16 reproductive effects, including reduced epididymal sperm counts and increased abnormal sperm 

17 (Li et al., 2001). Groups of 8–11 male Wistar rats (60 days old) were administered 

18 chromium(VI) oxide by gavage at doses of 0, 10, or 20 mg chromium(VI) oxide/kg-day 

19 (equivalent to 0, 5.2 or 10.4 mg hexavalent chromium/kg-day, respectively) for 6 days. After 

20 6 weeks, rats were sacrificed; testes and epididymis were removed and analyzed for epididymal 

21 sperm count and abnormal sperm; and testes were prepared (fixed in formaldehyde, embedded in 

22 paraffin, sliced, and stained with H&E) for histological evaluations of morphological 

23 abnormalities and diameter of seminiferous tubules. Epididymal sperm counts were significantly 

24 (p < 0.05) decreased by 76 and 80%, and the percentage of abnormal sperm was significantly 

25 (p < 0.01) increased by 143 and 176% in the 5.2 and 10.4 mg hexavalent chromium/kg-day 

26 groups, respectively. Treatment-related histopathological findings included decreased diameter 

27 of seminiferous tubules and disruption of germ cell arrangement within seminiferous tubules in 

28 both treatment groups. Based on decreased sperm counts and histopathological changes to the 

29 testes, 5.2 mg hexavalent chromium/kg-day was identified as a LOAEL for male rats exposed to 

30 gavage doses of chromium(VI) oxide for 6 days; a NOAEL value was not identified. 

31 

32 Zahid et al., 1990 

33 Zahid et al. (1990) reported adverse effects on the male reproductive system in mice fed 
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1 diets containing potassium dichromate. However, other research groups (NTP 1997, 1996a,b; 

2 Finley et al., 1993) have questioned the validity of the Zahid et al. (1990) study due to concerns 

3 regarding study methods and reporting inconsistencies (as discussed below). Zahid et al. (1990) 

4 fed male weanling BALB/c albino Swiss mice diets containing 0, 100, 200 or 400 mg potassium 

5 dichromate/kg diet (equivalent to 0, 35.3, 70.6, or 141.2 mg hexavalent chromium/kg diet, 

6 respectively) for 35 days. Although Zahid et al. (1990) stated that the control and exposed 

7 groups included seven animals/group, conflicting summaries of the actual group sizes are 

8 presented throughout the report. Body weights were recorded weekly and food consumption was 

9 recorded every 48 hours. The study report stated that body weight gain and food consumption in 

10 treatment groups were comparable to the control group (data not reported); however, Zahid et al. 

11 (1990) did not calculate daily doses of hexavalent chromium. Since treatment did not affect 

12 body weight gain or food consumption, doses of 0, 6.4, 12.7, or 25.5 mg hexavalent 

13 chromium/kg-day for the 0, 35.3, 70.6, or 141.2 mg hexavalent chromium/kg diet groups, 

14 respectively, were estimated for this review using reference values for body weight (0.0316 kg) 

15 and daily food intake (0.0057 kg food/day) for subchronic exposure of male B6C3F1 mice (U.S. 

16 EPA, 1988). After 35 days, testes and epididymis were weighed, and then minced in buffered 

17 formalin. Sperm counts were then subsequently determined and sperm were examined for 

18 morphological abnormalities. Testes were fixed with Bouin’s fluid for 1 week, embedded in 

19 paraffin and were subsequently sectioned to 0.6 micron thickness and stained with H&E for 

20 histological examination. Ten sections were chosen randomly from the anterior, middle, and 

21 posterior parts of each testis and studied. One seminiferous tubule was chosen and examined to 

22 determine the cellular stages of spermatogenesis and the number of degenerated tubules. 

23 Statistical analyses of the data were conducted using either a t-test or a 2 × 2 contingency chi24 square test. Adverse effects observed in the male mouse testes included ambiguous levels of 

25 degeneration in the outermost cellular layers of the seminiferous tubules, reduced (or absent) 

26 spermatogonia per tubule, accumulation of germ cells in the resting spermatocytes stage, reduced 

27 sperm count in the epididymis, and increased percentage of morphologically abnormal sperm. 

28 Effects were observed in all hexavalent chromium groups and severity of effects appeared to 

29 increase with dose for percentage of degenerated tubules, percentage of tubules that were not 

30 degenerated but were without spermatogonia, percentage of abnormal sperm, and number of 

31 spermatogonia. Based on these findings, the lowest dietary concentration tested (100 mg 

32 potassium dichromate/kg diet or approximately 6.4 mg hexavalent chromium/kg-day) was 

33 identified as the LOAEL. 
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1 Other research groups (NTP, 1997, 1996a,b; Finley et al., 1993) have questioned the 

2 validity of the Zahid et al. (1990) study due to concerns regarding study design and methods. 

3 Finley et al. (1993) noted the following three concerns: 1) use of immersion fixatives (such as 

4 Bouin’s fluid and paraffin embedding) that can introduce artifacts, such as grains and shrinkage, 

5 that can mimic tubular or spermatogenic pathology; 2) use of staining methods that were unable 

6 to detect the acrosome (i.e., the part of the sperm that releases enzymes to penetrate the egg) of 

7 developing spermatids; and 3) uncertainties regarding the actual groupings of animals used, the 

8 small number of animals assessed per group, and inappropriate statistical analysis of the data. 

9 NTP (1997, 1996a, b) concluded that the methods utilized by Zahid et al. (1990) were 

10 insufficient to identify spermatogonia, were likely to have generated nonreproducible counts of 

11 epididymal sperm, and resulted in the biologically implausible conclusion of reduction in 

12 spermatogonia numbers concurrent with unchanged spermatocyte and spermatid numbers. 

13 

14 Murthy et al., 1996 

15 Effects on ovarian function were investigated in adult Swiss albino mice (age: 90 days; 

16 mean initial body weight: 30 g) exposed to drinking water containing potassium dichromate for 

17 20 or 90 days (Murthy et al., 1996). For the 20-day study, groups of 30 female mice were 

18 exposed to drinking water containing 0, 250, 500, or 750 mg hexavalent chromium/L; the 20-day 

19 exposure period was selected as it coincides with one folliculogenesis cycle. For the 90-day 

20 study, groups of 10 female mice were administered drinking water containing 0, 0.05, 0.5, or 

21 5 mg hexavalent chromium/L. The study report states mice in both studies were evaluated daily 

22 for clinical signs of toxicity, body weight, and water and food consumption; however, no data for 

23 these outcomes were reported. Based on findings of other studies (NTP, 2008, 2007) showing 

24 decreased drinking water consumption and body weight at drinking water concentrations ≥30 mg 

25 hexavalent chromium/L, it is likely that drinking water consumption and body weight were 

26 decreased in all treatment groups in the 20-day study; thus, daily doses of hexavalent chromium 

27 cannot be accurately estimated from this study. For the 90-day study, the concentrations of 

28 hexavalent chromium in drinking water were very low and not likely to affect drinking water 

29 consumption or body weight. Thus, using reference values for body weight (0.035 kg) and daily 

30 drinking water (0.0084 L/day) intake for mature female B6C3F1 mice (U.S. EPA, 1988), doses 

31 of 0, 0.01, 0.12, or 1.2 mg hexavalent chromium/kg-day were estimated for female mice exposed 

32 to drinking water containing 0, 0.05, 0.5 or 5 mg hexavalent chromium/L, respectively. In the 

33 20-day study, three types of assessments were conducted at the end of the treatment period (each 
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1 in 10 mice/group): 1) ovaries were evaluated by light microscopy and the number of follicles at 

2 each development stage, based on size (small, medium, large) and structural maturity, were 

3 determined; 2) superovulation was induced (by administration of gonadotropin) and the number 

4 of released ova were counted; and 3) estrous cycle length was assessed (by vaginal smears) for 

5 12 consecutive estrous cycles following treatment. In the 90-day study, all mice were sacrificed 

6 at the end of the treatment period and ovaries were evaluated by electron microscopy for 

7 ultrastructural changes. 

8 In mice exposed for 20 days, significant (p < 0.05) changes in follicular development 

9 were observed in all treatment groups, with dose-related decreases in the number of small 

10 follicles in the mid- and high-dose groups and medium and large follicles in all treatment groups. 

11 In the high-dose group, the numbers of small, medium, and large follicles were reduced by 36, 

12 53, and 72%, respectively, compared with controls. Ovarian response to gonadotropin was 

13 affected in the mid- and high-dose groups, with reductions in the number of ova released of 30 

14 and 90%, respectively, compared with controls. Estrous cycle length was significantly increased 

15 (p < 0.05) by 1.7-fold in the high-dose group, compared with controls. Histopathological 

16 evaluation of ovaries after 20 days of treatment showed changes in the mid-dose (i.e., 

17 proliferated, dilated, and congested blood vessels, pyknotic nuclei in follicular cell of mature 

18 follicles) and high-dose (i.e., undeveloped follicles with degenerative cumulus cells containing 

19 dense pyknotic nuclei, neovasculiarization and karyorrhexis of follicular cells, erythrocytes 

20 located within stromal spaces) groups; histopathological changes were not observed in ovaries 

21 from control and low-dose mice. In mice treated for 90 days, ultrastructural changes (i.e., 

22 disintegrated cell membranes in two-layered follicular cells and alterations in mitochondria in 

23 thecal cells, which are cells of the corpus luteum that secrete estrone, estradiol, and 

24 progesterone) were observed in the high-dose group; the study report did not provide any 

25 information on ultrastructural evaluations in the low- and mid-dose groups. Murthy et al. (1996) 

26 concluded that hexavalent chromium may induce changes in ovarian function and ovulation. 

27 Due to inadequate reporting (i.e., no information on effects of treatment on body weight or 

28 drinking water consumption), a LOAEL from this study coulc not be identified. 

29 

30 Yousef et al., 2006 

31 Adverse effects on male reproductive tissues were observed in rabbits exposed to 

32 potassium dichromate for 10 weeks (Yousef et al., 2006). Groups of six male New Zealand 

33 white rabbits (age: 7 months) were administered 0 or 5 mg potassium dichromate/kg-day by 
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1 gavage (vehicle not specified) for 10 weeks. Yousef et al. (2006) reported that the dose of 5 mg 

2 potassium dichromate/kg-day was equivalent to 3.6 mg hexavalent chromium/kg-day. During 

3 the treatment period, food intake and body weights were recorded weekly. Semen was collected 

4 weekly and analyzed for pH and sperm count, motility, and morphology. Blood was collected 

5 every 2 weeks and analyzed for testosterone. At the end of the treatment period, animals were 

6 sacrificed and relative testes and epididymis weights were determined. At sacrifice, seminal 

7 plasma was collected and analyzed for AST, ALT, AP, AcP, and GST activities. 

8 Histopathological evaluations of tissues were not conducted. 

9 No clinical signs of toxicity were observed throughout the study. Mean body weight over 

10 the 10-week treatment period was significantly (p < 0.05) decreased by 9% compared to controls, 

11 although average food intake over the 10-week period was not affected by treatment; final body 

12 weight was not reported. After treatment for 10 weeks, relative testes and epididymis weights 

13 were significantly decreased by 22% (p < 0.05). The 10-week mean plasma testosterone level in 

14 treated rabbits was decreased by 21% (p < 0.05) compared with controls. In hexavalent 

15 chromium-treated rabbits compared with controls, mean values of the following sperm-related 

16 characteristics were significantly (p < 0.05) decreased after 10 weeks: 1) packed sperm volume 

17 (10% decrease), 2) sperm concentration (18% decrease), 3) total sperm output (26% decrease), 4) 

18 sperm motility (5% decrease), 5) total motile sperm per ejaculation (34% decrease), 6) total 

19 functional sperm fraction (37% decrease), and 7) normal sperm (4% decrease). Both percentage 

20 of dead sperm (24% increase) and seminal fluid pH (4% increase) were increased; no effect was 

21 observed on semen ejaculate volume. Seminal fluid activities of GST, AST and AcP were 

22 significantly (p < 0.05) decreased at the end of the treatment period, although decreases were 

23 small (≤12%) compared with controls. 

24 The results indicate that exposure of rabbits to oral potassium dichromate gavage doses 

25 of 3.6 mg hexavalent chromium/kg-day for 10 weeks produced adverse effects on male 

26 reproductive tissues including decreased testes and epididymis weight and decreased sperm 

27 output. Thus, a LOAEL for hexavalent chromium of 3.6 mg/kg-day can be identified from this 

28 study. 

29 
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1 NTP, 1996a,b 

2 The NTP conducted studies to investigate the potential effects of dietary hexavalent 

3 chromium as potassium dichromate on male reproductive organs in Sprague-Dawley rats (NTP, 

4 1996b) and BALB/c mice (NTP, 1996a). The NTP studies were designed to replicate the Zahid 

5 et al. (1990) study (described above) and thereby provide data to either refute or confirm findings 

6 of adverse male reproductive effects. 

7 Groups of 24 male and 48 female Sprague-Dawley rats were exposed to diets containing 

8 0, 15, 50, 100, or 400 mg potassium dichromate/kg diet (equivalent to 0, 5.3, 17.6, 35.3, or 

9 141.2 mg hexavalent chromium/kg diet, respectively) daily for 9 weeks followed by an 8-week 

10 recovery period (NTP, 1996b). Based on food consumption measured during the 9-week 

11 treatment period, NTP (1996a,b) calculated average daily doses of 0, 1, 3, 6, or 24 mg potassium 

12 dichromate/kg-day (equivalent to 0, 0.35, 1.1, 2.1, or 8.5 mg hexavalent chromium/kg-day, 

13 respectively) in males and 0, 1, 3, 7, or 28 mg hexavalent chromium/kg-day (equivalent to 0, 

14 0.35, 1.1, 2.5, or 9.9 mg hexavalent chromium/kg-day, respectively) in females for the 0, 15, 50, 

15 100, or 400 mg potassium dichromate/kg diet groups, respectively. Animals were examined 

16 twice daily for mortality and clinical signs of toxicity. Physical examinations and measurement 

17 of body weight and food and water consumption were conducted weekly. After 3, 6, or 9 weeks 

18 of treatment or after the full recovery period, 6 males and 12 females were sacrificed; necropsies 

19 were performed; blood was obtained for hematology (i.e., Hgb, Hct, MCV, MCH, MCHC, mean 

20 platelet volume, and erythrocyte, leukocyte and platelet counts); organ weights (not specified, 

21 but including right and left testes) were recorded; microscopic examinations were conducted on 

22 liver, kidney, ovary, and testes (testes and epididymis were examined for Sertoli nuclei and 

23 preleptotene spermatocyte counts in Stage X or XI tubules); and sperm were collected analyzed 

24 for chromatin structure. 

25 No mortalities or treatment-related clinical signs of toxicity were observed in rats in any 

26 treatment group (NTP, 1996b). Body weights and food and drinking water consumption were 

27 comparable between controls and treatment groups. Results of hematological analyses showed a 

28 slight erythrocyte microcytosis in the highest dose group, as indicated by small, but significant, 

29 decreases in MCV in females exposed for 3 weeks (3% decrease; p < 0.05) and in males exposed 

30 for 9 weeks (6% decrease; p < 0.05), compared with controls; at 9 weeks, MCV in females was 

31 decreased by 3%, but the change was not statistically significant. No changes in MCV were 

32 observed in rats exposed for 6 weeks or at the end of the 8-week recovery period. After 9 weeks 

33 of treatment, MCH was decreased by approximately 6% in males and females (statistical 
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1 significance not reported). No treatment-related findings were observed on necropsy or on 

2 microscopic examination of the liver, kidney, ovary, testes, epididymis, or sperm. In conclusion, 

3 no adverse effects on reproductive organs were observed in male or female rats exposed to 

4 dietary potassium dichromate at doses of 8.5 and 9.5 mg hexavalent chromium/kg-day, 

5 respectively, for up to 9 weeks. Based on slight erythrocyte microcytosis, the results indicate 

6 respective NOAELs and LOAELs of 2.1 and 8.5 mg hexavalent chromium/kg-day in male 

7 Sprague-Dawley rats, and 2.5 and 9.5 mg hexavalent chromium/kg-day in females. 

8 Groups of 24 male and 48 female BALB/c mice were exposed to diets containing 0, 15, 

9 50, 100, or 400 mg potassium dichromate/kg diet (equivalent to 0, 5.3, 17.6, 35.3, or 141.2 mg 

10 hexavalent chromium/kg diet, respectively) daily for 9 weeks followed by an 8-week recovery 

11 period (NTP, 1996a). Based on food consumption measured during the 9-week treatment period, 

12 the study authors calculated average daily doses of 0, 3, 10, 21, or 92 mg potassium 

13 dichromate/kg-day (equivalent to 0, 1.1, 3.5, 7.4, or 32.5 mg hexavalent chromium/kg-day, 

14 respectively) in males and 0, 5, 16, 34, or 137 mg hexavalent chromium/kg-day (equivalent to 0, 

15 1.8, 5.6, 12.0, or 48.4 mg hexavalent chromium/kg-day, respectively) in females for the 0, 15, 

16 50, 100, or 400 mg potassium dichromate/kg diet groups, respectively. This study followed the 

17 same protocol and conducted the same evaluations as described in the NTP (1996b) study in rats 

18 (described above). 

19 Mortalities occurred in five male mice, but they were deemed not related to treatment, 

20 and no treatment-related findings were observed on necropsy. The number of deaths were one, 

21 one, two, one, and none in the control through high-dose male groups, respectively. All females 

22 survived to study completion. No treatment-related clinical signs of toxicity were observed. At 

23 most weekly evaluations, body weight was decreased by 5–9% in males in the highest dose 

24 group and by 2–4% in females in the two highest dose groups (statistical significance not 

25 reported); body weights in these groups remained depressed during the post-treatment recovery 

26 period in high-dose males and in females at 12.0 mg hexavalent chromium/kg-day (but not high27 dose females). Feed consumption was generally increased (5–34%, relative to controls) in all 

28 treatment groups in males, although changes were not statistically significant; in females, feed 

29 consumption was increased in all dose groups (1–37%), with changes of statistical significance 

30 in most dose groups during treatment weeks 5 and 6. Water consumption in males and females 

31 was decreased through the first 3 weeks of treatment and comparable to controls for the 

32 remainder of exposure. Hematological analyses showed a slight erythrocyte microcytosis. In 

33 high-dose male and female mice, MCV was decreased by 2–4% (p < 0.05) at weeks 3, 6, and 9; 
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1 MCV was also slightly decreased (<2%) at 12.0 mg hexavalent chromium/kg-day in females at 

2 6 weeks. Changes in MCV were generally accompanied by small decreases in MCH. At the end 

3 of the recovery period, a small increase in MCV (2.8%; p < 0.05) was observed in males; in 

4 females, MCV in all treatment groups was comparable to controls. No other effects on 

5 hematological parameters were observed. Microscopic evaluations revealed a treatment-related 

6 increase in the incidence of cytoplasmic vacuolization of hepatocytes in male and female mice at 

7 the end of the 9-week treatment period. Vacuoles were demarked and appeared small and clear; 

8 NTP (1996a) noted that vacuoles were consistent with lipid accumulation. Incidences of hepatic 

9 cytoplasmic vacuolization in the control through high-dose groups were 0/6, 0/6, 1/6, 2/6, and 

10 2/5 in males and 1/12, 0/12, 3/12, 2/12, and 4/12 in females, respectively; lesion severity and 

11 statistical significance were not reported. No other treatment-related histopathological findings 

12 were observed. 

13 In conclusion, no adverse effects on reproductive organs were observed in male or female 

14 mice exposed to dietary potassium dichromate at doses up to 32.5 and 48.4 mg hexavalent 

15 chromium/kg-day, respectively, for 9 weeks. Based on histopathological changes to the liver 

16 (cytoplasmic vacuolization), the results indicate respective NOAELs and LOAELs of 3.5 and 

17 7.4 mg hexavalent chromium/kg-day in male BALB/c mice and 1.8 and 5.6 mg hexavalent 

18 chromium/kg-day in female mice. 

19 

20 4.3.2. Effects on Reproductive Outcomes

21 Elbetieha and Al-Hamood, 1997 

22 Reproductive effects of drinking water containing 1,000–5,000 mg potassium 

23 dichromate/L (equivalent to 353–1,765 mg hexavalent chromium/L) were evaluated in Swiss 

24 mice in a series of three experiments (Elbetieha and Al-Hamood, 1997). No data on drinking 

25 water consumption were included in the study report. Based on findings of other studies (NTP, 

26 2008, 2007) showing decreased drinking water consumption and body weight at drinking water 

27 concentrations ≥30 mg hexavalent chromium/L, it is likely that drinking water consumption was 

28 decreased in all chromium treatment groups; thus, daily doses of hexavalent chromium cannot be 

29 accurately estimated for this study. In the first experiment, sexually mature (age: 50 days) male 

30 Swiss mice were exposed to drinking water containing 0 (20 males), 1,000 (19 males), 

31 2,000 (11 males), 4,000 (9 males), or 5,000 (13 males) mg potassium dichromate/L (equivalent 

32 to 0, 353, 706, 1,412, or 1,765 mg hexavalent chromium/L, respectively) for 12 weeks. After 

33 12 weeks, males were mated with untreated sexually mature females for 10 days; 1 week after 
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1 completion of the mating period, females were sacrificed and evaluated for the number of 

2 pregnant females, viable fetuses, resorptions, and dead fetuses. Histopathological evaluations of 

3 tissues were not conducted. No data on body weights were reported. Exposure of male mice to 

4 hexavalent chromium did not affect the percentage of pregnant females. The numbers of 

5 implantations and viable fetuses were significantly reduced from 33% in controls to 20% (p < 

6 0.01) and 16% (p < 0.05) in the 706 and 1,412 mg potassium dichromate/L groups, respectively; 

7 in the 1,765 mg hexavalent chromium/L group, the numbers of implantation and viable fetuses 

8 were reduced to 19%, although this reduction did not reach statistical significance. No 

9 resorptions or dead fetuses were observed in the control, 706, or 1,412 mg potassium 

10 dichromate/L groups, but three resorptions were observed at 353 mg hexavalent chromium/L and 

11 six resorptions and six dead fetuses were observed at 1,765 mg hexavalent chromium/L 

12 (statistical significance not reported). 

13 In the second experiment, sexually mature (age: 50 days) female Swiss mice were 

14 exposed to drinking water containing 0 (19 females), 2,000 (15 females), or 5,000 (11 females) 

15 mg potassium dichromate/L (equivalent to 0, 706, or 1,765 mg hexavalent chromium/L, 

16 respectively) for 12 weeks (Elbetieha and Al-Hamood, 1997). After 12 weeks, each female was 

17 mated with an untreated sexually mature male for 10 days; 1 week after completion of the mating 

18 period, females were sacrificed and evaluated for the numbers of pregnant females, viable 

19 fetuses, and resorptions and dead fetuses. No data on body weights were reported. No 

20 treatment-related effects were observed on the number of pregnant mice. The number of 

21 implantations was significantly reduced from 17% in controls to 14% (p < 0.01) and 9% (p < 

22 0.05) in the 706 and 1,765 mg hexavalent chromium/L groups, respectively, and the number of 

23 viable fetuses was significantly reduced from 17% in controls to 9% in the 706 (p < 0.05) and 

24 1,765 (p < 0.01) mg hexavalent chromium/L groups, respectively. The number of mice with 

25 resorptions was significantly increased from 11% in controls to 53% (p < 0.01) and 63% (p < 

26 0.005) in the 706 and 1,765 mg hexavalent chromium/L groups, respectively, and the total 

27 number of resorptions was increased from 4 in controls to 36 and 14 in the 706 and 1,765 mg 

28 hexavalent chromium/L groups, respectively (statistical significance not reported). 

29 In the third experiment, sexually mature (age: 50 days) mice were exposed to drinking 

30 water containing 0 (10 males, 8 females), 2,000 (13 males, no females), or 5,000 (13 males, 

31 10 females) mg potassium dichromate/L for 12 weeks (Elbetieha and Al-Hamood, 1997). 

32 Following treatment, body weights and weights of reproductive organs (paired testes, seminal 

33 vesicles, preputial glands, paired ovaries, and uteri) were determined. No mortalities or clinical 
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1 signs of toxicity were observed. Final body weights of males were significantly (p < 0.01) 

2 reduced by approximately 10 and 12% in the 706 and 1,765 mg hexavalent chromium/L groups, 

3 respectively; final mean body weights of treated females were similar to controls. Relative testes 

4 weights were increased by approximately 18% (p < 0.01) and 22% (p < 0.05) in the 706 and 

5 1,765 mg hexavalent chromium/L groups, respectively, and relative weights of seminal vesicles 

6 and preputial gland were significantly (p < 0.001) decreased by approximately 27 and 34%, 

7 respectively, in the 1,765 mg hexavalent chromium/L group. Relative ovary weight was 

8 significantly increased by 54% in females in the 1,765 mg hexavalent chromium/L group, 

9 although uterine weight was unaffected by treatment. Histopathological assessments of 

10 reproductive tissues were not conducted. 

11 In conclusion, results of the three experiments conducted by Elbetieha and Al-Hamood 

12 (1997) show that exposure to potassium dichromate in drinking water affects reproductive 

13 outcomes in exposed males and females. In female mice, decreased numbers of implantations 

14 and viable fetuses and increased resorptions were observed at 2,000 mg potassium dichromate/L 

15 (equivalent to 706 mg hexavalent chromium/L). In males, exposure for 12 weeks prior to mating 

16 reduced the numbers of implantations and viable fetuses at 2,000 and 4,000 mg potassium 

17 dichromate/L (equivalent to 706 and 1,412 mg hexavalent chromium/L, respectively), but not at 

18 1,000 mg potassium dichromate/L (equivalent to 353 mg hexavalent chromium/L). In addition, 

19 treatment-related changes in weights of male reproductive organs were observed at 2,000 and 

20 5,000 mg potassium dichromate/L (equivalent to 706 and 1,412 mg hexavalent chromium/L, 

21 respectively). Although reproductive performance was not affected at the lowest exposure level, 

22 weights of male reproductive organs were not evaluated in male mice treated with 1,000 mg 

23 potassium dichromate/L. Due to inadequate reporting (i.e., no information on effects of 

24 treatment on body weight or drinking water consumption), a NOAEL or LOAEL from this study 

25 could not be identified. 

26 

27 NTP, 1997 

28 The potential reproductive toxicity of dietary potassium dichromate was evaluated in 

29 BALB/c mice in a continuous breeding study (NTP, 1997). Groups of 20 male and female pairs 

30 (F0) were exposed to dietary potassium dichromate at 0, 100, 200, and 400 mg potassium 

31 dichromate/kg diet (equivalent to 0, 17.6, 35.3, or 141.2 mg hexavalent chromium/kg diet, 

32 respectively) for 13 weeks (1 week prior to and 12 weeks during cohabitation). During exposure 

33 of the F0 generation, animals were examined daily for mortality and clinical signs of toxicity; 
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1 body weights and food consumption were measured periodically (4–5 times). Litters produced 

2 during the cohabitation period were evaluated (i.e., total pups, live and dead pups, and sex), 

3 weighed on postnatal day (PND) 1, and euthanized with no additional assessments; pregnancy 

4 index (number of liters/breeding pair) was also determined. After the cohabitation period, F0

5 breeding pairs were separated and continued on study diets; litters born during the post6 separation period (F1 animals) were reared with the F0 dams until weaning (PND 21). Dam and 

7 pup weights and dam food consumption were monitored during the lactational period. Upon 

8 weaning, F0 animals were sacrificed and the following terminal evaluations were conducted: 

9 necropsy; organ weights (liver, kidneys, right cauda epididymis right epididymis, prostate, 

10 seminal vesicles with coagulating glands, right testis, and ovaries); sperm evaluations (testicular 

11 spermatid head count and epididymal sperm density, motility, and morphology); and 

12 histopathology (liver and kidneys). Following weaning of F1 animals, animals were maintained 

13 on the same study diets as their parents. During post-lactational exposure of the F1 generation, 

14 animals were examined daily for mortality and clinical signs of toxicity; body weights and food 

15 consumption were measured periodically (3–4 times). At sexual maturity (approximately 

16 74 days), groups of 20 F1 animals of each sex were selected as breeding pairs (avoiding sibling 

17 matings), cohabitated for 7 days, and then separated. Reproductive endpoints (numbers of live 

18 and dead pups, sexes of pups, and total pup weight by sex) were evaluated on PND 1 of the F2

19 offspring; there was no further evaluation of the F2 pups. Estrous cycle (time spent in estrous 

20 stages, cycle length, number of cycles, number of cycling females, and number of females with 

21 regular cycles) was evaluated using 12-day vaginal smears beginning 4 days after the last 

22 delivery. Terminal evaluations of F1 adults (time from separation to terminal sacrifice not 

23 reported) were the same as those described above for F0 adults, with the addition of hematology 

24 (i.e., Hgb, Hct, MCV, MCH, MCHC, mean platelet volume, erythrocyte morphology, and 

25 erythrocyte, leukocyte, and platelet counts). 

26 No treatment-related mortalities or clinical signs of toxicity were observed in F0

27 generation BALB/c mice exposed to dietary potassium dichromate (NTP, 1997). Mortalities 

28 occurred in eight animals (four low-dose males, one mid-dose male, and three mid-dose 

29 females); however, since no mortalities were observed in the high-dose group, NTP (1997) 

30 concluded that these deaths were not related to treatment. Terminal body weight of males in all 

31 treatment groups was comparable to controls; mean body weight of females in the high-dose 

32 groups was decreased by 7% (p < 0.05). In general, food consumption was increased in 

33 treatment groups. Based on measured food consumption and body weights during the 
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1 cohabitation period, NTP (1997) calculated average daily doses in F0 males and females of 0, 

2 19.4, 38.6, or 85.7 mg potassium dichromate/kg-day (equivalent to 0, 6.8, 13.6, or 30.3 mg 

3 hexavalent chromium/kg-day, respectively). During lactation, sporadic decreases in body 

4 weights of dams in the mid- and high-dose groups were observed, but body weights at the end of 

5 lactation (PND 21) were similar to controls; food consumption during lactation was similar 

6 between control and treatment groups. Based on measured food consumption and body weights, 

7 NTP (1997) calculated average daily doses in lactating F0 females of 0, 32.8, 69.0 or 143.1 mg 

8 potassium dichromate/kg-day (equivalent to 0, 11.6, 24.4, or 50.5 mg hexavalent chromium/kg9 day, respectively). At the terminal evaluations of F0 animals, absolute (but not relative) liver 

10 weights were increased by 17% (p < 0.05) and 22% (p < 0.05) in high-dose males and females, 

11 respectively, compared with controls. No other changes in organ weights were observed. No 

12 treatment-related histopathological findings were observed in the F0 generation. Although 

13 various hepatic lesions were observed, including cytoplasmic vacuolization, study authors 

14 concluded that these findings were not treatment related, since incidence data did not show a 

15 relationship with dose. Evaluations of male reproductive tissues did not reveal any treatment16 related effects. In the F0 generation, no treatment-related effects on reproductive outcomes, 

17 including pregnancy index, mean cumulative time to litter, litter size, live and dead pups/litter, 

18 live pup weight, and sex ratio, were observed. 

19 Evaluations conducted on F1 pups during lactational exposure showed no effects on pup 

20 survival (NTP, 1997). On PND 21, weight of high-dose male pups was decreased by 16% 

21 compared with controls, but the decrease was not statistically significant. From weaning to 

22 sexual maturity, two mortalities occurred (one control male and one high-dose male). No 

23 treatment-related clinical signs of toxicity were observed. At the initiation of the F1 breeding 

24 phase (approximately PND 74), mean body weights of mid-dose females were decreased by 6% 

25 compared with controls and by 9% in high-dose F1 male and females (statistical significance not 

26 reported). Food consumption was generally increased during the period from weaning to sexual 

27 maturity. Based on measured food consumption and body weights, NTP (1997) calculated 

28 average daily doses in F1 animals of 0, 22.4, 45.5 or 104.9 mg potassium dichromate/kg-day 

29 (equivalent to 0, 7.9, 16.1, or 37.1 mg hexavalent chromium/kg-day, respectively). Hematology 

30 analysis at terminal sacrifice of F1 adults revealed slight erythrocyte microcytosis based on the 

31 following observations (comparisons to controls, statistical significance not reported): MCV 

32 decreased by 3% in mid- and high-dose males and by 2, 3, and 4% in low-, mid-, and high-dose 

33 females, respectively; MCH decreased by 3% in high-dose males; and Hgb decreased by 5% in 
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1 high-dose F1 females. No changes in erythrocyte morphology were observed. Relative kidney 

2 weight was increased by 5% in mid-dose females, but no other organ weight changes were 

3 observed. No treatment-related histopathological findings were observed. Although various 

4 hepatic lesions were observed, including cytoplasmic vacuolization, NTP (1997) concluded that 

5 findings were not treatment related, since incidence data did not show a relationship with dose. 

6 Evaluations of male reproductive tissues and female estrous cycle did not reveal any treatment7 related effects. In the F1 generation, no treatment-related effects on reproductive outcomes, 

8 including pregnancy index, mean cumulative time to litter, gestation length, litter size, live and 

9 dead pups/litter, and sex ratio, were observed. Live pup weight of females in the high-dose 

10 group was decreased by 11% (p < 0.05) compared to controls, but no decrease was observed for 

11 live pup weight of males or of combined males and females. 

12 In conclusion, NTP (1997) identified a LOAEL for parental toxicity in the F1 generation 

13 of 7.9 mg hexavalent chromium/kg-day in females exposed to potassium dichromate in the diet 

14 based on erythrocyte microcytosis (slight decrease in MCH); a NOAEL for parental toxicity in 

15 the F1 generation was not established. Although NTP (1997) did not specifically identify a 

16 NOAEL for reproductive effects, in the absence of reproductive findings, the highest dose tested 

17 is identified as a free-standing NOAEL for effects of dietary hexavalent chromium exposure on 

18 fertility and on male and female reproductive organ histology and weights (30.3 mg hexavalent 

19 chromium/kg-day in F0 mice and 37.1 mg hexavalent chromium/kg-day in F1 mice). 

20 

21 4.3.3. Effects of Pre-gestational Exposure on Reproductive Outcome and Fetal Development 

22 Kanojia et al., 1996 

23 Kanojia et al. (1996) administered adult Swiss albino female rats (20/group) drinking 

24 water containing 0, 250, 500, or 750 mg hexavalent chromium/L (as potassium dichromate) for 

25 20 days prior to gestation. During the exposure and gestational periods, body weights and water 

26 intake were recorded daily. At the end of the exposure period, rats were mated overnight with 

27 untreated males. Following mating, the mating index (percentage of mated females) and the 

28 fertility index (percentage of pregnant females) were determined. On GD 19, 10 rats/group were 

29 sacrificed and the numbers of copora lutea, fetuses/litter, live and dead fetuses, and resorptions, 

30 pre- and post-implantation losses, and fetal and placental weights were recorded and fetuses were 

31 examined for internal abnormalities (one third of fetuses) and external and skeletal abnormalities 

32 (remaining fetuses). In the remaining 10 rats/group, estrous cycle length was evaluated for 12 

33 consecutive cycles. Based on drinking water consumption during the exposure period, Konijia et 
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1 al. (1996) reported daily hexavalent chromium intakes of 6.4, 12.2, and 15.3 mg hexavalent 

2 chromium/rat-day. The study report did not include data on body weights over the course of the 

3 20-day treatment period, although it is likely that treatment-related effects on body weight 

4 occurred during the exposure period, as significant decreases in gestational weight gain were 

5 observed in all treatment groups (decreases of approximately 8, 14, and 21% in the low-, mid-, 

6 and high-dose groups, respectively, compared to controls). Thus, in the absence of data on the 

7 effect of treatment on body weights during the exposure period, daily doses of hexavalent 

8 chromium in terms of body weight (e.g., mg hexavalent chromium/kg-day) cannot be accurately 

9 estimated. 

10 No mortalities or clinical signs of toxicity in dams were observed. Dose-related 

11 decreases in mating and fertility indices were observed; in the high-dose group, mating and 

12 fertility indices were decreased by 60 and 68%, respectively, compared to controls (statistical 

13 significance not reported). In all treatment groups, the number of live fetuses was decreased, the 

14 numbers of resorptions and post-implantation loss were increased, and placental weight was 

15 increased. In the mid- and high-dose groups, numbers of corpora lutea and implantations were 

16 decreased and pre-implantation losses were increased. No treatment-related effects were 

17 observed for fetal weight or crown-rump length. Examination of fetuses showed gross 

18 abnormalities in the high-dose group, including patches of subdermal hemorrhage, kinky tail, 

19 short tail, and dropping wrist. Skeletal abnormalities were also observed, including reduced 

20 caudal ossification in mid- and high-dose groups and reduced parietal and inter-parietal 

21 ossification in the high-dose group. No visceral abnormalities were observed. Postpartum 

22 estrous cycle length was significantly increased by 37% (p < 0.05) in the high-dose group. 

23 Results of this study show that 20-day pre-gestational exposure of Swiss albino rat dams 

24 to hexavalent chromium adversely affected reproductive outcomes (decreased number of live 

25 fetuses and increased number of resorptions and post-implantation loss) at the lowest drinking 

26 water concentrations of potassium dichromate tested (≥250 mg hexavalent chromium/L or 

27 ≥6.4 mg hexavalent chromium/rat-day) and produced adverse developmental effects (gross and 

28 skeletal abnormalities) at the highest drinking water concentrations tested (750 mg hexavalent 

29 chromium/L or 15.3 mg hexavalent chromium/rat-day). Because of the lack of reporting of body 

30 weight data over the course of the study, NOAELs and/or LOAELs, expressed in mg hexavalent 

31 chromium/kg-day, could not be derived from this study. 

32 
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1 Kanojia et al., 1998 

2 Kanojia et al. (1998) administered adult Druckrey female rats (20/group; mean initial 

3 body weight 80 g) drinking water containing 0, 250, 500, or 750 mg hexavalent chromium/L (as 

4 potassium dichromate) for 3 months prior to gestation. This study was designed to following the 

5 same protocol as that used in the Kanojia et al. (1996) study (described above). However, at the 

6 end of the 3-month exposure period, rats in all treatment groups were acyclic (persistent 

7 diestrous phase). Therefore, since mating could not take place immediately following 

8 completion of the exposure period, rats were held for an additional 15–20 days (treatment-free), 

9 during which estrous cycle resumed. 

10 During the exposure period, mortality occurred in 15 and 10% of rats in the mid- and 

11 high-dose groups, respectively; no deaths occurred in the control or low-dose groups. Clinical 

12 signs of toxicity observed during the exposure period in the mid- and high-dose groups included 

13 hair loss and lethargic and aggressive behavior. At the end of the exposure period, body weight 

14 was significantly (p < 0.05) decreased by approximately 18 and 24% in the mid- and high-dose 

15 groups, respectively, compared with controls. Knojia et al. (1998) reported average hexavalent 

16 chromium intakes (based on water consumption) of 5.57, 10.18, and 13.56 mg hexavalent 

17 chromium/rat-day in the low-, mid-, and high-dose groups, respectively. Using these daily 

18 intake levels and the mean initial body weight of 80 g, daily doses of 70, 127, and 170 mg 

19 hexavalent chromium/kg-day for the low-, mid-, and high-dose groups, respectively, were 

20 estimated. During the postexposure gestational period, maternal weight gain was significantly (p 

21 < 0.05) decreased by 17 and 22% in the mid- and high-dose groups, respectively, compared with 

22 controls. The mating index was decreased by 30, 40 and 60% and the fertility index was 

23 decreased by 32, 41, and 49% in the low-, mid-, and high-dose groups, respectively, compared 

24 with controls (statistical significance not reported). In all treatment groups, pre- and post25 implantation losses were significantly (p < 0.05) increased, with increases in the high-dose group 

26 reaching 3.1- and 4.2-fold, respectively. In the mid- and high-dose groups, the numbers of 

27 implantations, live fetuses, and resorptions were significantly (p < 0.05) increased. Assessments 

28 of fetuses (on a per litter basis compared with controls) showed the following (significant 

29 difference compared with controls; p < 0.05); decreased fetal weight (all treatment groups); 

30 decreased crown-rump length (mid- and high-dose groups); gross external abnormalities, 

31 including subdermal hemorrhagic patches and drooping wrists in all treatment groups and kinky 

32 and short tail in mid- and high-dose groups; and skeletal abnormalities, including decreased 

33 caudal ossification in all treatment groups and reduced parietal and interparietal ossification in 
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1 mid- and high-dose groups. No internal abnormalities in fetuses were observed. Postpartum 

2 estrous cycle length was significantly (p < 0.05) increased in all treatment groups, with increases 

3 reaching approximately 1.7-fold in the high-dose group. 

4 Results of this study show that 3-month pre-gestational exposure of Druckrey rat dams to 

5 hexavalent chromium as potassium dichromate adversely affected reproductive outcomes 

6 (increased pre- and post-implantation losses) and produced adverse developmental effects 

7 (decreased fetal weight and external and skeletal abnormalities) at all drinking water 

8 concentrations tested (≥250 mg hexavalent chromium/L or approximately ≥70 mg hexavalent 

9 chromium/kg-day). Thus, a LOAEL for hexavalent chromium of 70 mg/kg-day can be identified 

10 from this study. 

11 

12 Junaid et al., 1996a 

13 Junaid et al. (1996a) administered Swiss albino female mice drinking water containing 0, 

14 250, 500, or 750 mg hexavalent chromium/L (as potassium dichromate) for 20 days prior to 

15 gestation. The study followed the same protocol and conducted the same evaluations as those 

16 reported in the study by Kanojia et al. (1996) (described above), except that estrous cycle length 

17 was not evaluated. Evaluations on reproductive outcomes and developmental effects were 

18 conducted in 10 mice/group. 

19 No clinical signs of toxicity were observed in mice during the exposure period. In the 

20 high-dose group, mortality occurred in 20% of animals; the cause of death was not established. 

21 Based on drinking water consumption monitored during the exposure period, study authors 

22 reported daily hexavalent chromium intake levels of 1.9, 3.56, and 5.23 mg hexavalent 

23 chromium/mouse-day in the low-, mid-, and high-dose groups, respectively. No treatment24 related effects were observed on body weight (data not reported); thus, using the reported mean 

25 initial body weight of 30 g, daily doses of 63, 119, and 174 mg hexavalent chromium/kg-day for 

26 the low-, mid-, and high-dose groups, respectively, were estimated. During the gestational 

27 period, maternal weight gain in the low- and mid-dose groups was comparable to controls; no 

28 weight gain was observed during gestation in high-dose group dams. In the low-dose group, 

29 post-implantation loss was significantly (p < 0.05) increased compared with controls (control: 

30 0%; low-dose group: 17.5%); no effects were observed for the numbers of corpora lutea, 

31 implantations, live fetuses, or resorptions or for pre-implantation loss. In the mid-dose group, 

32 the numbers of implantation and live fetuses were significantly (p < 0.05) decreased and the 

33 numbers of resorptions and pre- and post-implantation losses were significantly (p < 0.05) 
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1 increased; no effect on the number of corpora lutea was observed. In the high-dose group, no 

2 litters were produced and implantation sites were completely absent; corpora lutea were present, 

3 but numbers were decreased by 44% compared to controls. Assessments of fetuses (on a per 

4 litter basis compared with controls) showed the following (significant difference compared to 

5 controls; p < 0.05); decreased fetal weight and length in the low- and mid-dose groups; gross 

6 (external) abnormalities, including subdermal hemorrhagic patches and short and kinky tail in 

7 the mid-dose group; and skeletal abnormalities, including reduced caudal ossification in the low8 and mid-dose groups and reduced parietal and interparietal ossification in the mid-dose group. 

9 No internal abnormalities in fetuses were observed. 

10 Thus, at all drinking water concentrations of potassium dichromate tested (≥250 mg 

11 hexavalent chromium/L or approximately ≥63 mg hexavalent chromium/kg-day), pre-gestational 

12 exposure of Swiss albino female mice for 20 days produced adverse effects on reproductive 

13 outcome (decreased fertility) and fetal development (decreased fetal body weight and delays in 

14 skeletal development). Thus, a LOAEL for hexavalent chromium of 63 mg/kg-day can be 

15 identified from this study. 

16 

17 4.3.4. Effects of Gestational and/or Lactational Exposure on Reproductive Outcome and 

18 Fetal Development

19 Elsaieed and Nada, 2002 

20 Effects of gestational exposure to hexavalent chromium were investigated in Wistar rats 

21 (Elsaieed and Nada, 2002). Groups of 10 pregnant rats (mean initial body weight: 170 g) were 

22 administered drinking water containing 0 or 50 mg hexavalent chromium/L as potassium 

23 dichromate on GD 6 through 15. During the exposure period, dams were evaluated for clinical 

24 signs of toxicity, body weights, and food and drinking water consumption. One day before 

25 delivery, rats were sacrificed and the following were evaluated: numbers of corpora lutea, pre26 and post-implantation losses, resorptions, and live and dead fetuses; fetal weight; and visceral 

27 and skeletal anomalies. 

28 No mortalities or clinical signs of toxicity were observed. Elsaieed and Nada (2002) 

29 stated that food and drinking water consumption was comparable between control and treatment 

30 groups, although data were not reported. Gestational weight gain was significantly (p < 0.05) 

31 decreased by 40% in hexavalent chromium treated dams, compared with controls. Based on an 

32 average gestational body weight of 177 g (average calculated using body weights at mating and 

33 at the end of gestation) and the allometric equation for drinking water consumption for 
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laboratory mammals (0.10 × body weight0.7377 1 ; U.S. EPA, 1988), a daily dose of 7.9 mg 

2 hexavalent chromium/kg-day was estimated. In this study, treatment of rats with hexavalent 

3 chromium resulted in significant (p < 0.05) increases in pre-implantation loss/litter (2.1 vs. 0 in 

4 control), post-implantation loss/litter (1.5 vs. 0), resorptions/litter (1.2 vs. 0), and dead 

5 fetuses/litter (1.2 vs. 0) and decreases in live fetuses/litter (1.5 vs. 6.8 in control) and fetal weight 

6 (33% decrease). In the exposed group, increased litters with fetal abnormalities or 

7 malformations were observed including visceral (renal pelvis dilation: 2.1/litter) and skeletal 

8 (incomplete skull ossification: 1.0/litter) changes; no control fetuses showed these changes. 

9 The results show that exposure of pregnant Sprague-Dawley rats to drinking water 

10 containing 50 mg hexavalent chromium/L as potassium dichromate (approximately 7.9 mg 

11 hexavalent chromium/kg-day) on GDs 6–15 produced adverse effects on reproductive outcome 

12 and fetal development. Thus, a LOAEL for hexavalent chromium of 7.9 mg/kg-day can be 

13 identified from this study. 

14 

15 Bataineh et al., 2007 

16 Reproductive outcome was evaluated in adult female rats (age not specified) orally 

17 exposed to potassium dichromate for 3 days following mating (Bataineh et al., 2007). Groups of 

18 10 successfully mated female Sprague-Dawley rats were administered daily doses of 0 or 25 mg 

19 potassium dichromate/rat (equivalent to 8.8 mg hexavalent chromium/rat-day or approximately 

20 35 mg hexavalent chromium/kg-day, based on the average reported body weight of 245 g at 

21 mating) in saline daily by gavage on GDs 1–3 or 4–6. On GD 20, rats were sacrificed and the 

22 number of implantation sites, live fetuses, and resorptions along the uterine horns were recorded; 

23 fetuses were not assessed for external, skeletal, or visceral abnormalities. 

24 In rats treated with potassium dichromate on GDs 1–3, no pregnancies, implantations, 

25 resorptions, or viable fetuses were observed, compared with 10/10 pregnancies, 8.2 

26 implantations/female, 8.2 live fetuses/female, and 0/82 resorptions in controls. In rats treated on 

27 GDs 4–6, the numbers of pregnant rats and implantations/female were comparable to values in 

28 the control group. However, the number of viable fetuses was decreased by 69% (p < 0.001) and 

29 the percentage of resorptions per implantations was increased by 222% (p < 0.001). The study 

30 report did not indicate if clinical signs of toxicity were observed in chromium-treated dams, and 

31 no additional measures to assess systemic toxicity were reported. 

32 The results indicate that short-term gavage exposure of Sprague-Dawley dams to 

33 potassium dichromate at a dose of 35 mg hexavalent chromium/kg-day on GDs 1–3 completely 
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1 impaired implantation; exposure on GDs 4–6 markedly increased resorptions and decreased the 

2 number of viable fetuses, compared with controls. Thus, a LOAEL for hexavalent chromium of 

3 35 mg/kg-day can be identified from this study. 

4 

5 Trivedi et al., 1989 

6 Effects on reproductive outcome and fetal development were observed in ITRC-bred 

7 albino mice administered hexavalent chromium in drinking water (Trivedi et al., 1989). Groups 

8 of 10–13 pregnant mice (average initial body weight of 30 g) were administered drinking water 

9 containing 0, 250, 500, or 1,000 mg hexavalent chromium/L (as potassium dichromate) during 

10 the entire gestational period. Dams were observed daily for mortality, clinical signs of toxicity, 

11 body weight, and water consumption. On GD 19, dams were sacrificed and the following were 

12 recorded: numbers of corpora lutea, total implantations, live and dead fetuses, and pre13 implantation and postimplantations losses; placental weight; fetal weight and crown-rump 

14 length; number of stunted fetuses; and sex ratio per liter. In addition, fetus were examined for 

15 external (all fetuses), internal (approximately one-third of fetuses), and skeletal (remaining 

16 fetuses) anomalies. 

17 No mortalities or clinical signs of toxicity were observed. In the low-dose group, body 

18 weight gain was comparable to controls; however, body weight gain was significantly decreased 

19 by 21% (p < 0.05) in the mid-dose group, and dams in the high-dose group lost weight during 

20 treatment. Daily hexavalent chromium intakes were reported as 1.76, 3.6, and 7.03 mg 

21 hexavalent chromium/mouse-day, in the low-, mid-, and high-dose groups, respectively, based 

22 on measured drinking water consumption. Using average body weights for the gestational period 

23 (36.8, 36.6, and 29.4 g in the low-, mid-, and high-dose groups, respectively; calculated for this 

24 review using: [average initial body weight + body weight at the end of gestation]/2) and reported 

25 daily chromium intakes, daily doses of 48, 98, and 239 mg hexavalent chromium/kg-day were 

26 estimated. In low-dose mice, the percentages of resorptions and postimplantation loss were 

27 significantly increased (p < 0.001) to 33 and 36%, respectively, compared with 10 and 1.7%, 

28 respectively, in controls; the number of litters, litter size, number of copora lutea, and placental 

29 weight in the low-dose group were comparable to controls. In the mid-dose group, the 

30 percentages of resorptions and postimplantation losses were significantly (p < 0.001) increased 

31 to 52 and 88%, respectively. In addition, in the mid-dose group, litter size was significantly 

32 decreased by 44% (p < 0.01) compared with controls, and the percentage of preimplantation loss 

33 was increased to 26.2% (p < 0.001), compared with 3.6% in controls. No treatment-related 
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1 effects on placental weight were observed in the low- or mid-dose groups. In the high-dose 

2 group, no litters were produced and implantation sites were completely absent. In the low- and 

3 mid-dose groups, mean fetal crown-rump lengths were decreased (p < 0.001) by 17 and 27%, 

4 respectively, and mean fetal weights were decreased (p < 0.001) by 31 and 44%, respectively. 

5 Sex ratio was unaffected by treatment. Examination of fetuses for external anomalies showed no 

6 effects in the low-dose group; in the mid-dose group, tail kinking and subdermal hemorrhagic 

7 patches and streaks were observed. An increase in the incidence of minor skeletal anomalies was 

8 observed in fetuses in the low-dose (reduced ossification of the cranium) and mid-dose (reduced 

9 ossification of the cranium, forelimb, hindlimb, sternebrae, and thoracic and caudal vertebrae and 

10 reduced number of ribs) groups. No internal anomalies were observed. 

11 The LOAEL and NOAEL for maternal toxicity, assessed as decreased body weight gain 

12 in ITRC-bred albino mice exposed to potassium dichromate in drinking water throughout 

13 gestation, were 98 and 48 mg hexavalent chromium/kg-day, respectively. Based on increased 

14 resorptions and postimplantation loss, and decreased fetal length and weight, the lowest 

15 concentration tested (250 mg hexavalent chromium/L; 48 mg hexavalent chromium/kg-day) is 

16 identified as a LOAEL for developmental effects. 

17 

18 Junaid et al., 1996b 

19 Junaid et al. (1996) evaluated the effects of oral exposure of pregnant mice to hexavalent 

20 chromium on reproductive outcome and fetal development. Groups of 10 successfully mated 

21 Swiss albino female mice (average initial body weight of 30 g) were administered drinking water 

22 containing 0, 250, 500, or 750 mg hexavalent chromium/L (as potassium dichromate) on GDs 6 

23 though 14. Throughout the exposure period, dams were evaluated daily for clinical signs of 

24 toxicity, body weight, and drinking water consumption. On GD 19, dams were sacrificed and 

25 evaluations of dams and fetuses were conducted as described by Trivedi et al. (1989) 

26 (summarized above). 

27 No mortalities or clinical signs of toxicity were observed. Gestational weight gain was 

28 significantly (p < 0.05) decreased in the mid- and high-dose groups by 8 and 32%, respectively, 

29 but was comparable to controls in the low-dose group. Daily hexavalent chromium intakes were 

30 reported as 2.00, 3.75, or 5.47 mg chromium/mouse-day in the low-, mid-, and high-dose groups, 

31 respectively, based on measured drinking water consumption. Using average body weights for 

32 the gestational period (37.6, 37.2, and 35.9 g in the low-, mid-, and high-dose groups, 

33 respectively; calculated for this report using: [average initial body weight + body weight at the 
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1 end of gestation]/2) and reported daily chromium intakes, daily doses of 53, 101, and 152 mg 

2 hexavalent chromium/kg-day in the low-, mid-, and high-dose groups, respectively, were 

3 estimated. The number of resorptions was significantly (p < 0.05) increased in all treatment 

4 groups, with increases reaching 7.7-fold in the high-dose group. In the mid- and high-dose 

5 groups, significant (p < 0.05) decreases in the total number of fetuses and increases in the 

6 numbers of dead fetuses and resorption sites were observed. Fetal weight was significantly (p < 

7 0.05) decreased by 13 and 19% in the mid- and high-dose groups, respectively; no treatment8 related effects were observed on fetal length. Gross external examination of fetuses showed 

9 significant (p < 0.05) increases in the incidences of minor abnormalities (subdermal hemorrhagic 

10 patches, drooping wrist, kinky and short tail) in the high-dose group. Examination of fetuses for 

11 skeletal abnormalities showed significant (p < 0.05) increases in the incidences of reduced caudal 

12 ossification in the mid- and high-dose groups and of reduced nasal, frontal, parietal, interparietal, 

13 carpals, and tarsals ossification. No external or skeletal abnormalities were observed in fetuses 

14 in the low-dose group. No visceral abnormalities were observed in any treatment group. 

15 Junaid et al. (1996b) concluded that oral exposure of dams during the organogenesis 

16 phase of gestation produces adverse effects in embryos and during fetal development. The 

17 LOAEL and NOAEL for maternal toxicity, assessed as decreased body weight gain in Swiss 

18 albino mice administered potassium dichromate in drinking water on GDs 6–14, were 101 and 

19 53 mg hexavalent chromium/kg-day, respectively. Based on reduced number of implantation 

20 sites, the lowest dose tested (approximately 53 mg hexavalent chromium/kg-day) is identified as 

21 a developmental LOAEL for this study. 

22 

23 Junaid et al., 1995 

24 The effects of late gestational exposure to hexavalent chromium on reproductive outcome 

25 and fetal development were evaluated in mice (Junaid et al., 1995). Groups of 10 successfully 

26 mated Swiss albino female mice (average initial body weight of 30 g) were administered 

27 drinking water containing 0, 250, 500, or 750 mg hexavalent chromium/L (as potassium 

28 dichromate) on GD 14 though 19. Throughout the exposure period, dams were evaluated daily 

29 for clinical signs of toxicity, body weight and drinking water consumption. On GD 19, dams 

30 were sacrificed and evaluations of dams and fetuses were conducted as described by Trivedi et 

31 al. (1989) (summarized above). 

32 No mortalities or clinical signs of toxicity were observed. Gestational weight gain was 

33 significantly (p < 0.05) decreased in the mid- and high-dose groups by 11 and 26%, respectively, 
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1 but was comparable to controls and the low-dose group. No data on drinking water consumption 

2 were reported; however, it is likely that daily doses were similar to those calculated for the study 

3 by Junaid et al. (1996b) (e.g., approximately 53, 101, and 152 mg hexavalent chromium/kg-day 

4 in the low-, mid-, and high-dose groups, respectively), which used the same mouse strain and 

5 drinking water concentrations, and a similar study design. In the mid- and high-dose groups, the 

6 numbers of dead fetuses and post-implantation losses were significantly (p < 0.05) increased; the 

7 numbers of corpora lutea and total fetuses per litter were similar to controls in all treatment 

8 groups. Fetal weight and length were significantly decreased in all treatment groups, with 

9 decreases reaching approximately 47 and 29%, respectively, in the high-dose group. Gross 

10 external examination of fetuses showed significant (p < 0.05) increases in the incidences of 

11 minor abnormalities in the mid-dose (drooping wrists) and high-dose (drooping wrists, 

12 subdermal hemorrhagic patches, kinky and short tail) groups. Examination of fetuses for skeletal 

13 abnormalities showed significant (p < 0.05) increases in the incidences of reduced caudal 

14 ossification in all treatment groups, of reduced tarsals ossification in mid- and high-dose groups, 

15 and of reduced nasal, parietal, interparietal, carpals, and metatarsals ossifications in the high16 dose group. No visceral abnormalities were observed in any treatment group. 

17 The NOAEL and LOAEL for maternal toxicity, assessed as decreased body weight gain 

18 in Swiss albino mice administered potassium dichromate in drinking water on GDs 14–19, were 

19 53 and 101 mg hexavalent chromium/kg-day, respectively. Based on reduced fetal weight and 

20 length and increased incidence of reduced caudal ossification in all treatment groups, the lowest 

21 dose tested (approximately 53 mg hexavalent chromium/kg-day) is identified as a developmental 

22 LOAEL for this study. 

23 

24 Al-Hamood et al., 1998 

25 The effects of gestational and lactational exposure of mice to hexavalent chromium on 

26 sexual maturation and fertility in offspring were investigated by Al-Hamood et al. (1998). On 

27 GD 12 through day 20 of lactation, groups of 25 pregnant Swiss strain BALB/c mice (mean 

28 initial body weight: 25 g) were administered drinking water containing 0 or 1,000 mg potassium 

29 dichromate/L (equivalent to 353 mg hexavalent chromium/L). Based on drinking water 

30 consumption by dams, daily hexavalent chromium intakes of 2.1 and 1.7 mg hexavalent 

31 chromium/mouse-day were calculated for the gestational and lactational periods, respectively. 

32 No data on body weights of dams were reported; however, since other studies have shown 

33 decreased maternal weight gain in pregnant mice exposed to drinking water containing ≥176 
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1 hexavalent chromium/L) (Junaid et al., 1996b, 1995), it is likely that treatment-related decreases 

2 in maternal weight gain occurred. Therefore, given this uncertainty, daily hexavalent chromium 

3 doses expressed in terms of body weight cannot be accurately estimated for this study. At birth, 

4 litters were culled to eight pups per female and offspring were weaned on PND 21; from 

5 weaning to day 60 of age, offspring received control drinking water. From PND 20 to the onset 

6 of puberty, female offspring were examined for time to vaginal opening. Fertility in offspring 

7 was assessed at day 60 of age; male offspring were mated with untreated females and female 

8 offspring were mated with untreated males for 10 days. At completion of the mating period, 

9 females were examined for numbers of pregnant females, implantations, viable fetuses, and 

10 resorptions. Additional groups (n = 9–12) of offspring were sacrificed on day 50 of age, and 

11 body weights and weights of reproductive organs (paired testes, seminal vesicles, and preputial 

12 glands in males and paired ovaries and uteri in females) were determined. 

13 In female offspring, time to vaginal opening was significantly (p < 0.001) increased from 

14 24.6 days in controls to 27.1 days in treated rats. Mating studies in female offspring showed 

15 decreased numbers of pregnant females (35% decrease; p < 0.025), implantations (12% decrease; 

16 p < 0.05), and viable fetuses (14% decrease; p < 0.05). No treatment-related effects on female 

17 body weight or relative weights of reproductive organs were observed. In male offspring, no 

18 treatment-related effects were observed in mating studies or on body weights or weights of 

19 reproductive organs. 

20 The results indicate that gestational and lactational exposure of BALB/c mouse dams to 

21 drinking water containing 353 mg hexavalent chromium/L as potassium dichromate resulted in 

22 impaired reproductive development and function in female offspring. Because of the lack of 

23 reporting of body weight data over the course of the study, NOAELs and/or LOAELs, expressed 

24 in mg hexavalent chromium/kg-day, could not be derived from this study. 

25 

26 Banu et al., 2008 

27 Banu et al. (2008) investigated the effects of lactational exposure to hexavalent 

28 chromium on sexual development of female rat offspring. Groups of 18 lactating Wistar rats 

29 were administered drinking water containing 200 mg potassium dichromate (equivalent to 

30 70.6 mg hexavalent chromium/L) on postpartum days 1 through 21. No specific assessments of 

31 dams were conducted. Banu et al. (2008) noted that toxic effects in dams were not “significant,” 

32 although no additional information regarding maternal toxicity or data on body weights or 

33 drinking water consumption in dams were reported. As discussed above, exposure of laboratory 
114 DRAFT - DO NOT CITE OR QUOTE 

1 animals to hexavalent chromium in drinking water may result in decreased body weight and 

2 drinking water consumption; thus, in the absence of data on body weight and drinking water 

3 consumption in dams, daily doses of hexavalent chromium cannot be accurately estimated for 

4 this study. At birth, litters were culled to four female pups per dam. Following weaning on PND 

5 21, pups were separated from dams. Pups (n = 24) were evaluated for the onset of puberty by 

6 daily examination for vaginal opening. After the onset of puberty, the time spent in each estrous 

7 cycle phase (proestrous, estrous, metestrous, and diestrous) was determined by analysis of 

8 vaginal smears (n = 24). On PNDs 21, 45, and 65, pups (n = 24, at each time point) were 

9 sacrificed; at each time point, blood was analyzed for hormones (estradiol, progesterone, 

10 testosterone, LH, follicle-stimulating hormone [FSH], growth hormone [GH], and prolactin) and 

11 ovaries were examined for the number of follicles and follicle development stage (primordial, 

12 primary, secondary, and antral). 

13 The onset of puberty was significantly (p < 0.05) increased from 33 days in control rats to 

14 55 days in hexavalent chromium-treated rats. Estrous cycle phase was also altered in hexavalent 

15 chromium-treated rats, with the time spent in diestrous significantly (p < 0.05) increased by 

16 approximately 1.4-fold compared with controls (data presented graphically); time spent in other 

17 estrous phases was unaffected by treatment. Evaluations of ovaries on PNDs 21 and 45 showed 

18 significant (p < 0.05) decreases in the numbers of primordial, primary, secondary, and antral 

19 follicles in hexavalent chromium-treated rats compared with control rats; on PND 65, the 

20 numbers of primordial and primary follicles were also decreased in hexavalent chromium-treated 

21 rats. At the 21- and 45-day assessments in hexavalent chromium-treated rats, plasma 

22 concentrations of estradiol, progesterone, testosterone, GH, and prolactin were significantly (p < 

23 0.05) decreased (by approximately 40 to 60%) and concentrations of FSH were significantly 

24 increased (by approximately 40%), compared with controls. Similar effects were observed at the 

25 65-day assessment, except that FSH concentrations in treatment and control groups were 

26 comparable. Plasma LH concentration was not affected by treatment at any time point. 

27 The results indicate that lactational exposure of Wistar rat dams to drinking water 

28 containing 70.6 mg hexavalent chromium/L as potassium dichromate resulted in delayed onset of 

29 puberty and follicular development and impaired ovarian steroidogenesis in female offspring; 

30 male offspring were not assessed for possible effects on sexual maturation. Because of the lack 

31 of reporting of body weight data over the course of the study, NOAELs and/or LOAELs, 

32 expressed in mg hexavalent chromium/kg-day, could not be derived from this study. 

33 
115 DRAFT - DO NOT CITE OR QUOTE 

1 4.4. MECHANISTIC DATA AND OTHER STUDIES IN SUPPORT OF THE MODE OF 

2 ACTION 

3 4.4.1. Genotoxicity Studies 

4 The mutagenic potential of hexavalent chromium has been studied extensively. Although 

5 study results vary with specific test systems, experimental conditions, and hexavalent chromium 

6 compounds tested, results of in vitro and in vivo studies provide substantial evidence for the 

7 mutagenic activity of hexavalent chromium compounds. A general summary of the evidence 

8 demonstrating the mutagenic activity of hexavalent chromium compounds in experimental 

9 systems is provided in Table 4-20. As discussed in detail in Section 4.4.2 (Intracellular 

10 Reduction), mutagenicity of hexavalent chromium is mediated through the generation of highly 

11 reactive chromium intermediates (e.g., chromium(IV) and chromium(V)) and reactive oxygen 

12 species formed during the intracellular reduction of hexavalent chromium. Reactive chromium 

13 intermediates and oxygen species react with DNA, leading to oxidative DNA damage, 

14 chromium-DNA adducts, DNA strand breaks, and chromosomal aberrations (Wise et al., 2008). 

15 
116 DRAFT - DO NO

Table 4-20. Evidence of mutagenicity of hexavalent chromium compounds in experimental 

systems 

In vitro studies 

(non-mammalian cells) 

In vitro studies 

(mammalian cells) 

In vivo stud

(D. melanogaster or 

Chemical 

DNA 

damage Mutations 

DNA 

damage Mutations 

Chromosomal 

damage 

DNA 

damage Mutations 

Ammonium chromate 

ND ● ND ND ND ND ND N

Calcium chromate ND ● ND ND ● ND ● (D) NChromic acid ND ● ND ND ● ND ● (D) NPotassium chromate ● ● ● ● ● ● (M) ● (D) 

● (M) 

● 

Potassium dichromate 

● ● ● ● ● ● (M) ● (D) ● 

Sodium chromate ND ● ● ND ● ND ND NSodium dichromate ND ● ● ND ● ● (M) ● (D) NSodium dichromate dihydrate 

ND ● ND ● ND ND ND ● 

● = positive results ND = no data identified for this review (D) = study in D. melanogaster (M) = study in laboratory mammal 
117 DRAFT - DO NOT 

1 4.4.1.1. Genotoxicity assays in experimental systems 

2 The mutagenic activity of hexavalent chromium has been demonstrated in numerous studies 

3 using both in vitro and in vivo experimental systems. In in vitro test systems (see Tables 4-21 and 4-22 

4 for studies in non-mammalian and mammalian cells, respectively), hexavalent chromium compounds 

5 have mostly tested positive for gene mutations (including reverse mutations, frame shift mutations, and 

6 base pair substitutions) and DNA damage (including DNA-protein crosslinks) in bacterial cells 

7 (Salmonella typhimurium, Escherichia coli, Bacillus subttilis). Reverse mutations were observed in 

8 multiple species and strains, including those that are sensitive to frameshift mutagens (S. typhimurium 

9 TA97, TA98, TA1537, and TA1538), G/C base-pair substitution mutagens (S. typhimurium TA100 and 

10 TA1535), and A/T base-pair substitution mutagens caused by oxidizing and/or cross-linking agents (S. 

11 typhimurium TA102; E. coli WP2uvrA and WP2uvrA/pKM101). Positive results were also found for 

12 forward mutations and mitotic gene conversion in yeast (Saccharomyces cerevisiae); and for DNA 

13 damage (DNA strand breaks, fragmentation, DNA-protein crosslinks, DNA-DNA crosslinks), 

14 chromosomal damage (sister chromatid exchanges and chromosomal aberrations), and DNA synthesis 

15 inhibition in mammalian cell lines and primary cultures (including primary cultures of human gastric 

16 mucosal cells, respiratory tract cells, and lymphocytes). 

17 In in vivo test systems (see Table 4-23), hexavalent chromium compounds have tested positive 

18 for mutations in Drosophila melanogaster and for DNA damage (DNA-protein crosslinks, DNA strand 

19 breaks), mutations (in mice exposed in utero, in mouse germ cells, and in transgenic mice), 

20 chromosomal damage (sister chromatid exchanges, chromosomal aberrations, and micronuclei), and 

21 DNA synthesis inhibition in rats and mice. The in vivo studies in laboratory mammals have evaluated 

22 the mutagenic activity of hexavalent chromium following exposure by the oral, parenteral, inhalation, 

23 and intratracheal routes. 

24 Hexavalent chromium-induced mutagenicity has been demonstrated following oral exposure. In 

25 oral exposures studies, DNA damage has been observed in several tissues in mice and rats following 

26 gavage administration of hexavalent chromium, including stomach, colon, liver, lung, brain, and 

27 peripheral lymphocytes (Wang et al., 2006; Devi et al., 2001; Sekihashi et al., 2001; Coogan et al., 

28 1991b). Devi et al. (2001) observed DNA damage via the comet assay in mouse leukocytes following 

29 an oral dose as low as 0.21 mg/kg, an effect that increased with dose up to 9.5 mg/kg and did not cause a 

30 decrease in cell viability. Similarly, Wang et al. (2006) found a dose-dependent increase in DNA 

31 damage in peripheral lymphocytes using the comet assay that was found to persist for five days post-
118 DRAFT - DO NOT CITE OR QUOTE 

1 exposure and was accompanied by a significant increase in reactive oxygen species and apoptosis in the

2 liver. Sekihashi et al. (2001) also found comet damage in mouse stomach, colon, liver, kidney, bladder, 

3 lung, and brain following one gavage dose of 85.7 mg/kg. These effects were not accompanied by 

4 cytotoxicity, although it is unknown whether a response to dose would have occurred as only one dose 

5 was administered. Coogan et al. (1991b) observed DNA−protein crosslinks in liver following 3 and 6 

6 week exposures via drinking water in rats; no cytotoxicity was found in these animals. 

7 Results of most studies evaluating hexavalent chromium-induced chromosomal damage in bone 

8 marrow or peripheral blood cells following oral exposure were negative (NTP, 2007; De Flora et al., 

9 2006; Mirsalis et al., 1996; Shindo et al., 1989). However, chromosomal damage (as indicated by 

10 micronuclei formation) was observed in peripheral red blood cells of one strain of mice (am3-C57BL/6) 

11 exposed to ≥21.8 mg hexavalent chromium/kg-day as sodium dichromate dihydrate in drinking water for 

12 3 months, but not in B6C3F1 or BALB/c mice at daily doses up to 87.2 mg hexavalent chromium/kg-day 

13 (NTP, 2007). 

14 Oral exposure studies evaluating the mutagenicity of hexavalent chromium in tissues from the GI 

15 tract are of particular relevance in light of the results of the NTP (2008) cancer bioassay showing 

16 neoplasms of the oral cavity in rats (at 5.9–7.0 mg hexavalent chromium/kg-day) and of the small 

17 intestine in mice (at 2.4–3.1 mg hexavalent chromium/kg-day) administered sodium dichromate 

18 dihydrate in drinking water for 2 years. In ddY mice, positive results were reported for DNA damage as 

19 measured by the comet assay in the stomach and colon following gavage administration of a single high 

20 dose of hexavalent chromium (85.7 mg hexavalent chromium/kg) (Sekihashi et al., 2001). This dose is 

21 at least 12-fold greater than chronic dosages associated with oral and GI neoplasms in rats and mice 

22 (NTP, 2008), although no concurrent cytotoxicity was found. Data on the potential for DNA damage in 

23 cells of the GI tract at lower oral doses (e.g., those in the range of the NTP [2008] bioassay) are not 

24 available. Negative results were reported for oxidative DNA damage and DNA-protein crosslinks in 

25 cells of the forestomach, glandular stomach, and duodenum of female SKH-1 mice administered 

26 drinking water containing 5 or 20 mg hexavalent chromium/L (approximately equivalent to 1.20 and 

27 4.82 mg hexavalent chromium/kg-day, respectively) as sodium dichromate dihydrate for 9 months (De 

28 Flora et al., 2008). The lack of effects seen in DeFlora et al. (2008) are peculiar given that the highest 

29 dose evaluated in this study is slightly less than chronic dosages associated with neoplasms of the oral 

30 cavity in rats (5.9–7.0 mg hexavalent chromium/kg-day), and slightly greater than those associated with 

31 neoplasms of the small intestine in mice (2.4–3.1 mg hexavalent chromium/kg-day) (NTP, 2008). No 
119 DRAFT - DO NOT CITE OR QUOTE 

1 oral exposure studies on the potential clastogenic activity of hexavalent chromium in oral mucosal or GI 

2 cells were identified. Although the NTP (2007) 3-month drinking water study evaluated micronuclei 

3 formation in peripheral red blood cells of mice (with positive results in the am3-C57BL/6 strain, as 

4 discussed above), mutagenic effects of hexavalent chromium exposure in GI tissues were not evaluated 

5 in this study. 

6 Results of parenteral exposure studies are uniformly positive for hexavalent chromium-induced 

7 mutagenicity. Following parenteral exposure, DNA damage has been observed in numerous tissues, 

8 including peripheral lymphocytes, stomach, colon, liver, kidney, bladder, lung, and brain (Patlolla and 

9 Tchounwou, 2006; Sekihashi et al., 2001; Ueno et al., 2001); mutations have been observed in liver 

10 (Knudsen, 1980); and chromosomal damage (micronuclei) has been observed in peripheral erythrocytes 

11 and bone marrow (De Flora et al., 2006; Itoh and Shimada, 1997; Shindo et al., 1989; Hayashi et al., 

12 1982; Wild, 1978). 

13 Mutagenic activity of hexavalent chromium has also been demonstrated in lung cells of animals 

14 following intratracheal exposure. DNA damage (DNA fragmentation, DNA-protein crosslinks and 

15 DNA adducts) was reported in lung cells of Sprague-Dawley rats administered 0.09 mg hexavalent 

16 chromium/kg by intratracheal instillation for 3 days (Izzotti et al., 1998) and mutations were reported in 

17 lung cells of C57BL/6 mice administered a single intratracheal dose of 7.65 mg hexavalent 

18 chromium/kg. Results of these studies are relevant to occupational exposure studies showing increased

19 respiratory tract cancers in hexavalent chromium workers (see Section 4.4.1.2). No inhalation or 

20 intratracheal exposure studies on the potential clastogenic activity of hexavalent chromium in respiratory 

21 tract cells were identified. Chromosomal damage (chromosome aberrations and sister chromatid 

22 exchange) was observed in peripheral lymphocytes, but not bone marrow, of Sprague-Dawley rats 

exposed to chromium fumes for 1 week (1.84 mg/m3) or 2 months (0.55 mg/m323 ) (Koshi et al., 1987). 

24 
120 DRAFT - DO NO

1 

Table 4-21. In vitro genotoxicity studies of hexavalent chromium in non-mammalian cells 

Results

a

Endpoint Chemical form Test system 

Without 

activation 

With 

activation 

Reverse mutations Ammonium chromate S. typhimurium TA97, 

TA1538, TA98, TA100 + NS DeFlora 

Reverse mutations Ammonium chromate S. typhimurium TA1537 ± NS DeFlora Reverse mutations Ammonium chromate S. typhimurium TA1535 – – DeFlora Reverse mutations Calcium chromate S. typhimurium TA97, 

TA1538, TA98, TA100 + NS DeFlora 

Reverse mutations Calcium chromate S. typhimurium TA1537 ± NS DeFlora Reverse mutations Calcium chromate S. typhimurium TA1535 – – DeFlora Reverse mutations Calcium chromate S. typhimurium TA98 – ± Dunkel Reverse mutations Calcium chromate S. typhimurium TA100, 

TA1535, TA1537, TA1538 – – 

Dunkel 

Reverse mutations Calcium chromate E. coli WP2 uvrA – ± Dunkel Reverse mutations Chromic acid S. typhimurium TA102, 

TA2638 

+ ND Watana

Reverse mutations Chromic acid E. coli, WP2/pKM101, WP2 

uvrA/pKM101 + ND Watana

Reverse mutations Chromium trioxide S. typhimurium TA97, 

TA1538, TA98, TA100 + NS DeFlora 

Reverse mutations Chromium trioxide S. typhimurium TA1537 ± NS DeFlora Reverse mutations Chromium trioxide S. typhimurium TA1535 – – DeFlora Reverse mutations Potassium chromate S. typhimurium TA102 + ND Marzin Reverse mutations Potassium chromate S. typhimurium TA97, 

TA1538, TA98, TA100 + NS DeFlora 

Reverse mutations Potassium chromate S. typhimurium TA1537 ± NS DeFlora Reverse mutations Potassium chromate S. typhimurium TA1535 – – DeFlora Reverse mutations Potassium chromate E. coli Hs30R + ND Nakamu
121 DRAFT - DO NO

Table 4-21. In vitro genotoxicity studies of hexavalent chromium in non-mammalian cells 

Results

a

Endpoint Chemical form Test system 

Without 

activation 

With 

activation 

Reverse mutations Potassium chromate E. coli Wp2 hcr- try-, 

B/rWP2 + (Wp2 hcr) ND Kanema

Reverse mutations Potassium chromate E. coli Wp2(try-) + ND Venitt aReverse mutations Potassium chromate E. coli WP2uvrA, CM571 + ND Seo and Reverse mutations Potassium dichromate S. typhimurium TA97, TA98, 

TA100, TA1535, TA1537 + + Zeiger e

Reverse mutations Potassium dichromate S. typhimurium TA102 + ND Marzin Reverse mutations Potassium dichromate S. typhimurium TA100 + + Venier eReverse mutations Potassium dichromate E. coli Wp2 hcr- try-, 

B/rWP2 + (Wp2 hcr) ND Kanema

Reverse mutations Potassium dichromate E. coli Hs30R + ND NakamuReverse mutations Potassium dichromate E. coli WP2, WP2uvrA, 

CM571 

+ ND Nishiok

Reverse mutations Potassium dichromate E. coli WP2uvrA, CM571 + ND Seo and Reverse mutations Potassium dichromate S. cerevisiae D7 + ND Sing, 19Reverse mutations Potassium dichromate S. typhimurium TA98 ± – Venier eReverse mutations Potassium dichromate S. typhimurium TA1538 – – Venier eReverse mutations Sodium chromate E. coli Wp2(try-) + ND Venitt aReverse mutations Sodium dichromate S. typhimurium TA102, 

TA2638 

+ ND Watana

Reverse mutations Sodium dichromate S. typhimurium TA102 + + BenniceReverse mutations Sodium dichromate S. typhimurium TA100 + – DeFloraReverse mutations Sodium dichromate S. typhimurium TA97 + NS DeFlora Reverse mutations Sodium dichromate S. typhimurium TA1537, 

TA1538, TA98, TA100 ± NS DeFlora 

Reverse mutations Sodium dichromate S. typhimurium TA1535 – – DeFlora 
122 DRAFT - DO NO

Table 4-21. In vitro genotoxicity studies of hexavalent chromium in non-mammalian cells 

Results

a

Endpoint Chemical form Test system 

Without 

activation 

With 

activation 

Reverse mutations Sodium dichromate E. coli, WP2/pKM101, WP2 

uvrA/pKM101 + ND Watana

Reverse mutations Sodium dichromate 

dihydrate 

S. typhimurium TA102, 

TA2638a + – Ryden e

Reverse mutations Sodium dichromate 

dihydrate 

S. typhimurium TA100, TA98 + + NTP, 20

Reverse mutations Sodium dichromate 

dihydrate 

E. coli, WP2 uvrA/pKM101 + + NTP, 20

Induction of SOS response 

Chromic acid E. coli AB1157, GC2375, 

UA4202, PQ30 + ND Llagost

Induction of SOS response 

Potassium chromate E. coli PQ37, PQ35 + – Olivier 

Induction of SOS response 

Potassium chromate E. coli AB1157, GC2375, 

UA4202, PQ30 + ND Llagost

Induction of SOS response 

Potassium dichromate E. coli AB1157, GC2375, 

UA4202, PQ30 + ND Llagost

Induction of SOS response 

potassium dichromate E. coli PQ37, PQ35 + – Olivier 

Mutations Ammonium chromate S. typhimurium TA1978 

(rec+), TA1538 (rec - ) 

+ ND Gentile 

Mutations Ammonium chromate B. subtilis + ND Gentile Mutations Calcium chromate S. typhimurium TA97, TA98, 

TA100 – – 

Brams e

Mutations Chromic acid S. typhimurium TA1978 

(rec+), TA1538 (rec - ) 

+ ND Gentile 

Mutations Chromic acid B. subtilis + ND Gentile Mutations Potassium chromate S. typhimurium TA98, 

TA100, TA1537 + ND Arlausk

Mutations Potassium chromate S. typhimurium TA100 + ND Arlausk
123 DRAFT - DO NO

Table 4-21. In vitro genotoxicity studies of hexavalent chromium in non-mammalian cells 

Results

a

Endpoint Chemical form Test system 

Without 

activation 

With 

activation 

Mutations Potassium chromate E. coli WP2 uvrA pKm 101 + ND ArlauskMutations Potassium chromate B. subtilis H17 + ND NishiokMutations Potassium chromate S. typhimurium TA1535, 

TA1538 – ND Arlausk

Mutations Potassium dichromate S. typhimurium TA 1535 

pSK1002 

+ + Yamam

Mutations Potassium dichromate S. typhimurium TA100, 

TA1025, TA98 + ND Le Curi

Mutations Potassium dichromate S. typhimurium TA1978 

(rec+), TA1538 (rec - ) 

+ ND Gentile 

Mutations Potassium dichromate E. coli WP2uvrA + ND Venier eMutations Potassium dichromate B. subtilis + ND Gentile Mutations Sodium dichromate B. subtilis + ND Gentile Mutations Potassium dichromate B. subtilis NIG45, NIG17 + ND Matsui, Mutations Potassium dichromate B. subtilis H17 + ND NishiokMutations Sodium dichromate S. typhimurium TA1978 

(rec+), TA1538 (rec - ) 

+ ND Gentile 

Frame shift mutations Calcium chromate S. typhimurium TA98, 

TA1537 

+ ND Hawort

Frame shift mutation Potassium chromate S. typhimurium TA 1537 + ND La VellFrame shift mutation Potassium chromate E. coli 343/358, /415, /435, 

/477 + ND La Vell

Frame shift mutations Potassium dichromate S. typhimurium TA97a, TA98 + + Tagliari Frame shift mutations Potassium dichromate S. typhimurium TA100, 

TA1537, TA1538 

– ND Kanema

Frame shift mutations Sodium dichromate S. typhimurium TA97, 

TA1978 + ND Bennice
124 DRAFT - DO NO

Table 4-21. In vitro genotoxicity studies of hexavalent chromium in non-mammalian cells 

Results

a

Endpoint Chemical form Test system 

Without 

activation 

With 

activation 

Frame shift mutations Sodium dichromate S. typhimurium TA1537, 

TA1538 – ND Bennice

Frame shift mutations, base pair substitutions 

Calcium chromate S. typhimurium TA1537, 

TA98, TA100 

+ + Petrilli 

Frame shift mutations, base pair substitutions 

Calcium chromate S. typhimurium TA1535 – – Petrilli 

Frame shift mutations, base pair substitutions 

Chromic acid S. typhimurium TA1537, 

TA98, TA100 

+ + Petrilli 

Frame shift mutations, base pair substitutions 

Chromic acid S. typhimurium TA1535 – – Petrilli 

Frame shift mutations, base pair substitutions 

Potassium chromate S. typhimurium TA1537, 

TA98, TA100 

+ + Petrilli 

Frame shift mutations, base pair substitutions 

Potassium chromate S. typhimurium TA1535 – – Petrilli 

Frame shift mutations, base substitutions 

Potassium dichromate S. typhimurium TA98 TA100, 

TA1535, TA1538 

+ + Bianchi 

Frame shift mutations, base pair substitutions 

Sodium dichromate S. typhimurium TA1537, 

TA98, TA100 

+ + Petrilli 

Frame shift mutations, base pair substitutions 

Sodium dichromate S. typhimurium TA1535 – – Petrilli 

Base pair substitutions Ammonium chromate S. typhimurium TA1537, 

TA1538, TA98, TA100 ± NS DeFlora 

DeFlora

Base pair substitutions Ammonium chromate S. typhimurium TA1535 – – DeFloraBase pair substitutions Calcium chromate S. typhimurium TA100, 

TA1535 

+ ND Hawort

Base pair substitutions Calcium chromate S. typhimurium TA1537, 

TA1538, TA98, TA100 ± NS DeFlora 

DeFlora

Base pair substitutions Calcium chromate S. typhimurium TA1535 – – DeFloraBase pair substitutions Chromic acid S. typhimurium TA1537, 

TA1538, TA98, TA100 

± NS DeFlora 

DeFlora
125 DRAFT - DO NO

Table 4-21. In vitro genotoxicity studies of hexavalent chromium in non-mammalian cells 

Results

a

Endpoint Chemical form Test system 

Without 

activation 

With 

activation 

Base pair substitutions Chromic acid S. typhimurium TA1537, 

TA1538, TA98, TA100 

± NS DeFlora 

DeFlora

Base pair substitutions Potassium chromate S. typhimurium TA1537, 

TA1538, TA98, TA100 

± NS DeFlora 

DeFlora

Base pair substitutions Potassium chromate S. typhimurium TA1535 – – DeFloraBase pair substitutions Potassium dichromate S. typhimurium TA100, 

TA102 

+ + Tagliari 

Base pair substitutions Potassium dichromate S. typhimurium TA100, 

TA1535 

– ND Kanema

Base pair substitutions Sodium dichromate S. typhimurium TA100, 

TA102, TA92 

+ ND Bennice

Base pair substitutions Sodium dichromate S. typhimurium TA1537, 

TA1538, TA98, TA100 

± NS DeFlora

al., 1984 

Base pair substitutions Sodium dichromate S. typhimurium TA1535 – – DeFloraBase pair substitutions Sodium dichromate S. typhimurium TA1535 – ND BenniceReverse mutation, induction of gene conversion 

Potassium dichromate S. cerevisiae D7 + ND Kharab 

Forward mutation Potassium dichromate Schizosaccharomyces pombe

972, h- 

± ND Bonatti 

Mitotic cross-over Chromic acid S. cerevisiae D7 + ND FukunaMitotic gene conversions 

Chromic acid S. cerevisiae D7 + ND Singh, 1

al., 198

Mitotic gene conversion, point reverse mutation 

Sodium chromate S. cerevisiae D7 + ND Bronzet

Mitotic gene conversion at trp5 locus, reverse mutation of ilvl-92 allele

Chromic acid S. cerevisiae D7 + ND Vashish

1987 
126 DRAFT - DO NO

Table 4-21. In vitro genotoxicity studies of hexavalent chromium in non-mammalian cells 

Results

a

Endpoint Chemical form Test system 

Without 

activation 

With 

activation 

Mitotic gene conversion at trp5 locus, reverse mutation of ilvl-92 allele

Potassium dichromate S. cerevisiae D7 + ND Vashish

1987 

Induction of disomic and diploid spores 

Potassium dichromate S. cerevisiae D1S13 + ND Sora et 

umu gene expression Potassium dichromate S. typhimurium TA1535 ± – NakamuDNA damage Potassium dichromate E. coli PQ37 + ND Le CuriDNA-protein crosslinks Potassium chromate E. coli DNA – ND Fornace DNA polymerase arrest Sodium dichromate PSV2neo-based plasmid 

DNA 

– + Bridgew

1994 

a+ = positive, ± = equivocal or weakly positive, – = negative, ND = no data; NS = not specified. 

1 
127 DRAFT - DO NO

Table 4-22. In vitro genotoxicity studies of hexavalent chromium in mammalian cells 

Results

a

Endpoint 

Chemical 

form Test system 

Without 

activation 

With 

activation 

DNA damage Potassium 

dichromate 

Human lymphocytes + ND Blasiak a

DNA damage Potassium 

dichromate 

Human gastric mucosa + ND Trzeciak 

DNA damage Potassium 

dichromate 

Human peripheral blood lymphocytes + ND Trzeciak 

DNA damage Potassium 

dichromate 

Human lymphocytes, human lymphoblastoid TK-6 cells 

+ ND Cemeli e

DNA damage Sodium 

dichromate 

Human gastric mucosa cells, Rat gastric mucosa cells 

+ ND Pool-Zob

DNA adducts, [32P] postlabeling Potassium chromate Calf thymus DNA – – (+1 mM 

H2O2) 

Adams e

DNA fragmentation Potassium 

chromate 

Human bronchial epithelial cells + ND Fornace 

DNA fragmentation Potassium 

chromate 

Human embryonic lung fibroblasts (IMR90) + ND Fornace 

DNA fragmentation Potassium 

chromate 

Mouse L1210 leukemia cells + ND Fornace 

DNA fragmentation sodium 

chromate 

Chinese hamster ovary cells + ND Blanken

DNA strand breaks Potassium 

dichromate 

Vero kidney fibroblsts, Pam 212 keratinocytes 

+ ND Flores an

DNA strand breaks Sodium 

dichromate 

Rat primary lymphocytes + ND Gealy et 

DNA strand breaks Sodium 

dichromate 

Rat hepatocytes + ND Gao et a

DNA strand breaks Potassium 

chromate 

Human lymphocytes + ND Depault 
128 DRAFT - DO NO

Table 4-22. In vitro genotoxicity studies of hexavalent chromium in mammalian cells 

Results

a

Endpoint 

Chemical 

form Test system 

Without 

activation 

With 

activation 

DNA strand breaks Potassium 

chromate 

Human fibroblast + ND Fornace 

DNA strand breaks Potassium 

chromate 

Bacteriophage 

λ DNA + + 

(+1mM H2O2)

Adams e

DNA strand breaks Sodium 

dichromate 

Rat primary lymphocytes + ND Elia et al

DNA strand breaks Potassium 

dichromate 

Human lymphocytes, human gastric mucosa cells 

+ ND Blasiak e

DNA-DNA crosslinks Sodium 

chromate 

Human lung fibroblasts + ND Xu et al.

DNA-protein crosslinks Potassium 

chromate 

Human embryonic lung fibroblasts (IMR90) + ND Fornace 

DNA-protein crosslinks Potassium 

chromate 

Human fibroblast + ND Fornace, 

DNA-protein crosslinks Potassium 

chromate 

Chinese hamster cells (V79-UL) + ND Merk et 

DNA-protein crosslinks Potassium 

chromate 

Mouse L1210 leukemia cells + ND Fornace 

DNA-protein crosslinks Sodium 

chromate 

Human HL-60 cells + ND Capellm

Induced DNA methylation Potassium 

chromate 

Chinese hamster V79 cells (hpr

-

1gpt

+

transgenic cell line G12) 

+ (T) ND Klein et 

Unscheduled DNA synthesis 

Sodium dichromate 

Rat hepatocytes + (T) ND Gao et a

DNA synthesis inhibition Potassium 

chromate 

HeLa S3 cells + ND Heil and 

DNA synthesis inhibition Potassium 

dichromate 

Mouse L cells + ND Nishio a
129 DRAFT - DO NO

Table 4-22. In vitro genotoxicity studies of hexavalent chromium in mammalian cells 

Results

a

Endpoint 

Chemical 

form Test system 

Without 

activation 

With 

activation 

DNA polymerase arrest Sodium 

chromate 

Human lung fibroblasts + ND Xu et al.

Mutations at the HGPRT locus 

Potassium dichromate 

Chinese hamster ovary cells (AT3-2) + ND Paschin 

Mutations at the HGPRT locus 

Potassium dichromate 

Chinese hamster cells (V79) + ND Paschin 

Forward mutation Calcium 

chromate 

Mouse lymphoma cells (L5178Y tk

+/tk

-) + + McGreg

Forward mutation Calcium 

chromate 

Mouse lymphoma cells (L5178Y tk

+) + + Mitchell 

Forward mutation Calcium 

chromate 

Mouse lymphoma cells (L5178Y tk

+) + + Myhr an

Forward mutation Calcium 

chromate 

Mouse lymphoma cells (L5178Y tk

+) + + Oberly e

Morphological transformation 

Calcium chromate 

Syrian hamster embryo cells + ND Elias et a

Morphological transformation 

Sodium chromate dihydrate 

Syrian hamster cells + ND DiPaolo 

Cell transformation Calcium 

chromate 

Balb/3T3, Syrian hamster embryo, R-MuLV-RE cells 

+ ND Dunkel e

Transformations Potassium 

chromate 

Rat liver epithelial cells + ND Briggs a

Chromosomal damage Calcium 

chromate 

Chinese hamster ovary cells + ND Levis an

Chromosomal damage Chromic acid Chinese hamster ovary cells + ND Levis anChromosomal damage Potassium 

chromate 

Chinese hamster ovary cells + ND Levis an

Chromosomal damage Potassium 

dichromate 

Chinese hamster ovary cells + ND Seoane a
130 DRAFT - DO NO

Table 4-22. In vitro genotoxicity studies of hexavalent chromium in mammalian cells 

Results

a

Endpoint 

Chemical 

form Test system 

Without 

activation 

With 

activation 

Chromosomal damage Potassium 

dichromate 

Chinese hamster ovary cells + ND Levis an

Chromosomal damage Potassium 

dichromate 

Chinese hamster ovary cells + ND Majone 

Chromosomal damage Sodium 

chromate 

Chinese hamster ovary cells + ND Levis an

Chromosomal damage Sodium 

dichromate 

Chinese hamster ovary cells + ND Levis an

Chromosomal damage Sodium 

dichromate 

Chinese hamster ovary cells + ND Majone 

Chromosome aberrations Calcium 

chromate 

Chinese hamster lung DON cells + ND Koshi an

Koshi 19

Chromosome aberrations Calcium 

chromate 

Chinese hamster ovary cells (C3H10T1/2) + ND Sen et al

Chromosome aberrations Chromic acid BHK and Chinese hamster ovary cells + ND Bianchi Chromosome aberrations Chromic acid Mouse mammary FM3A carcinoma cells + ND Umeda aChromosome aberrations Chromic acid Chinese hamster lung DON cells + ND Koshi an

Koshi 19

Chromosome aberrations Potassium 

chromate 

Human fibroblasts + ND MacRae 

Chromosome aberrations Potassium 

chromate 

Chinese hamster lung DON cells + ND Koshi an

Koshi 19

Chromosome aberrations Potassium 

chromate 

Chinese hamster ovary cells + ND MacRae 

Chromosome aberrations Potassium 

chromate 

BHK and Chinese hamster ovary cells + ND Bianchi 

Chromosome aberrations Potassium 

chromate 

Mouse mammary FM3A carcinoma cells + ND Umeda a

Chromosome aberrations Potassium 

dichromate 

Human fibroblasts + ND MacRae 
131 DRAFT - DO NO

Table 4-22. In vitro genotoxicity studies of hexavalent chromium in mammalian cells 

Results

a

Endpoint 

Chemical 

form Test system 

Without 

activation 

With 

activation 

Chromosome aberrations Potassium 

dichromate 

Chinese hamster ovary cells + ND MacRae 

Chromosome aberrations Potassium 

dichromate 

BHK and Chinese hamster ovary cells + ND Bianchi 

Chromosome aberrations Potassium 

dichromate 

Mouse mammary FM3A carcinoma cells + ND Umeda a

Chromosome aberrations Sodium 

chromate 

Human primary bronchial fibroblasts + ND Wise et a

Chromosome aberrations Sodium 

chromate 

Human bronchial fibroblasts (WTHBF6 cells) + ND Holmes 

Chromosome aberrations Sodium 

chromate 

Human bronchial epithelial cells (BEP2D cells) 

+ ND Wise et a

Chromosome aberrations Sodium 

chromate 

Chinese hamster ovary cells + ND Blanken

Chromosome aberrations Sodium 

chromate 

Chinese hamster ovary cells (AA8 (parental), EM9 (XRCC1 mutant), and H9T3 

+ ND Grlickov

Chromosome aberrations Sodium 

dichromate 

BHK and Chinese hamster ovary cells + ND Bianchi 

Chromosome and chromatid aberrations 

Potassium dichromate 

Human lymphocytes + ND Imreh an

Sister chromatid exchanges 

Calcium chromate 

Human lymphocytes + ND Gomez-A

Sister chromatid exchangse 

Calcium chromate 

Chinese hamster lung DON cells + ND Koshi an

Koshi 19

Sister chromatid exchanges 

Calcium chromate 

Chinese hamster ovary cells + ND Levis an

Sister chromatid exchanges 

Chromic acid Chinese hamster ovary cells + ND Levis an
132 DRAFT - DO NO

Table 4-22. In vitro genotoxicity studies of hexavalent chromium in mammalian cells 

Results

a

Endpoint 

Chemical 

form Test system 

Without 

activation 

With 

activation 

Sister chromatid exchanges 

Chromic acid Chinese hamster cells DON + ND Ohno et 

Sister chromatid exchanges 

Chromic acid Chinese hamster lung DON cells + ND Koshi an

Koshi 19

Sister chromatid exchanges 

Chromic acid BHK and Chinese hamster ovary cells + ND Bianchi 

Sister chromatid exchanges 

Potassium chromate 

Human fibroblasts + ND MacRae 

Sister chromatid exchanges 

Potassium chromate 

Chinese hamster lung DON cells + ND Koshi an

Koshi 19

Sister chromatid exchanges 

Potassium chromate 

Chinese hamster ovary cells + ND Levis an

Sister chromatid exchanges 

Potassium chromate 

Chinese hamster ovary cells + ND MacRae 

Sister chromatid exchanges 

Potassium chromate 

Chinese hamster cells DON + ND Ohno et 

Sister chromatid exchanges 

Potassium chromate 

BHK and CHO cells + ND Bianchi 

Sister chromatid exchanges 

Potassium dichromate 

Human lymphocytes + ND Gomez-A

Sister chromatid exchanges 

Potassium dichromate 

Human lymphocytes + ND Imreh an

Sister chromatid exchanges 

Potassium dichromate 

Human fibroblasts + ND MacRae 

Sister chromatid exchanges 

Potassium dichromate 

Chinese hamster ovary cells + ND Levis an

Sister chromatid exchanges 

Potassium dichromate 

Chinese hamster ovary cells + ND Levis an

Sister chromatid exchanges 

Potassium dichromate 

Chinese hamster ovary cells + ND Majone 
133 DRAFT - DO NO

Table 4-22. In vitro genotoxicity studies of hexavalent chromium in mammalian cells 

Results

a

Endpoint 

Chemical 

form Test system 

Without 

activation 

With 

activation 

Sister chromatid exchanges 

Potassium dichromate 

Chinese hamster ovary cells + ND MacRae 

Sister chromatid exchanges 

Potassium dichromate 

Chinese hamster cells DON + ND Ohno et 

Sister chromatid exchanges 

Potassium dichromate 

BHK and Chinese hamster ovary cells + ND Bianchi 

Sister chromatid exchanges 

Potassium dichromate 

Mouse blastocysts + ND Iijima et 

Sister chromatid exchanges 

Sodium chromate 

Chinese hamster ovary cells + ND Levis an

Sister chromatid exchanges 

Sodium chromate 

BHK and Chinese hamster ovary cells + ND Bianchi 

Sister chromatid exchanges 

Sodium dichromate 

Chinese hamster ovary cells + ND Levis an

Sister chromatid exchanges 

Sodium dichromate 

Chinese hamster ovary cells + ND Majone 

Sister chromatid exchanges 

Sodium dichromate 

BHK and Chinese hamster ovary cells + ND Bianchi 

Disruption of mitosis Sodium 

chromate 

Human bronchial fibroblasts (WTHBF-6 cells) 

+ ND Wise et a

a + = positive, ± = equivocal or weakly positive, – = negative, (T) = toxicity, ND = no data. 

1 
134 DRAFT - DO NO

1 

Table 4-23. In vivo genotoxicity studies of hexavalent chromium in D. melanogaster and labora

mammals 

Endpoint Chemical Test system Test conditions Results

a Dose

b 

DNA damage, Comet assay 

Potassium chromate 

DNA damage in stomach, colon, bladder, lung, brain; but not in liver, kidney or bone marrow 

Parenteral exposure: ddY mice were administered 

single intraperitoneal doses of potassium chromate of 0 or 120 mg/kg (0 or 32.1 mg hexavalent chromium/kg). The cells from the stomach, colon, liver, kidney, bladder, lung, brain and bone marrow were collected 3, 8, and 24 hours after treatment and analyzed for DNA damage using the comet assay 

+ 32.1 mg hexa

chromium/kg (intraperitone

DNA damage, Comet assay 

Potassium chromate 

DNA damage in stomach, colon, liver, kidney, bladder, lung, and brain; but not in bone marrow 

Oral exposure: ddY mice were administered 

single gavage doses of potassium chromate of 0 or 320 mg/kg (0 or 85.7 mg hexavalent chromium/kg). The cells from the stomach, colon, liver, kidney, bladder, lung, brain, and bone marrow were collected 3, 8, and 24 hours after treatment and analyzed for DNA damage using the comet assay. 

+ 85.7 mg hexa

chromium/kg (gavage) 

DNA damage, Comet assay 

Potassium dichromate 

DNA damage in peripheral lymphocytes 

Oral exposure: Swiss albino mice were 

administered potassium dichromate by gavage at doses of 0, 25, 50, and 100 mg/kg for 1 day or daily for 5 consecutive days (0, 8.8, 17.7, and 35.4 mg hexavalent chromium/kg). 

+ 8.8 mg hexav

chromium/kg (gavage) 

DNA damage, Comet assay 

Potassium dichromate 

Mouse leukocytes Oral exposure: Swiss albino mice were 

administered potassium dichromate by single gavage 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-treatment for alkaline SCGE comet assay analysis of leukocytes. 

+ 0.21 mg hexa

chromium/kgweight (gavage) 

DNA damage, Comet assay 

Potassium dichromate 

DNA damage in liver and kidney, but not in spleen, lung, or brain 

Parenteral exposure: Male albino mice were 

treated administered potassium dichromate as a single intraperitoneal dose of 0 or 20 mg hexavalent chromium/kg. Organs were removed and cells were collected for DNA strand break analysis by single-cell gel electrophoresis. 

+ 20 mg hexava

chromium/kg (intraperitoneinjection) 
135 DRAFT - DO NO

Table 4-23. In vivo genotoxicity studies of hexavalent chromium in D. melanogaster and labora

mammals 

Endpoint Chemical Test system Test conditions Results

a Dose

b 

DNA damage, Comet assay 

Potassium dichromate 

Rat leukocytes Parenteral exposure: Sprague-Dawley rats were 

administered potassium dichromate intraperitoneally at doses of 2.5, 5, 7.5, and 10 mg/kg-day for 5 days (0, 0.88, 1.77, 2.65, or 3.54 mg hexavalent chromium/kg-day). Whole blood was sampled at 24, 48, 72, and 96 hours after treatment for alkaline single-cell gel electrophoresis analysis of leukocytes, Comet assay. 

+ 0.88 mg hexa

chromium/kg(intraperitoneinjection) 

DNA alterations 

Sodium dichromate dihydrate 

DNA-protein crosslinks, DNA fragmentation, and DNA adducts in lung, but not liver 

Intratracheal exposure: Sprague-Dawley rats were 

administered intratracheal instillations of sodium dichromate at a dose of 0 or 0.25 mg/kg for 3 consecutive days (0 or 0.09 mg hexavalent chromium/kg). After last treatment, lung and livers were removed to analyze for DNA fragmentation, DNA-protein crosslinks, and adducts by [32P] post-labeling. 

+ 0.09 mg hexa

chromium/kg (intratracheal instillation) 

DNA-protein crosslinks 

Potassium chromate 

DNA-protein crosslinks detected in liver but not splenic lymphocytes 

Oral exposure: Male Fischer 344 rats were 

administered potassium chromate in drinking water for 3 and 6 weeks at 100 and 200 ppm hexavalent chromium. Liver and splenic lymphocytes were examined for DNA-protein crosslinks. 

+ 100 hexavale

chromium/L (drinking wat

DNA-protein crosslinks 

Sodium dichromate dihydrate 

DNA-protein crosslinks detected in lung, liver, and kidney nuclei 

Parenteral exposure: Male Sprague-Dawley rats 

were given a single intraperitoneal injection of 20or 40 mg/kg sodium dichromate (7 or 14 mg hexavalent chromium/kg). There was no control group used in this study. Nuclei from the right renal cortex, the front hepatic lobe, and the wholelung were used for analysis. 

+ 7 mg hexaval

chromium/kg (intraperitoneinjection) 
136 DRAFT - DO NO

Table 4-23. In vivo genotoxicity studies of hexavalent chromium in D. melanogaster and labora

mammals 

Endpoint Chemical Test system Test conditions Results

a Dose

b 

Oxidative DNA damage, DNA protein crosslinks 

Sodium dichromate dihydrate 

Mouse forestomach, glandular stomach, and duodenum cells 

Oral exposure: Female SKH-1 hairless mice were 

administered sodium dichromate dihydrate in drinking water at concentrations of 0, 5, and 20 mg hexavalent chromium/L for 9 months. Using reference values for body weight (0.0353 kg) and daily drinking water intake (0.0085 L/day) for female B6C3F1 mice (U.S. EPA, 1988), doses of 

1.20 and 4.82 mg hexavalent chromium/kg-day for 

the 5 and 20 mg hexavalent chromium/L groups, respectively, were estimated. DNA-protein crosslinks, and oxidative DNA damage (8-oxo2’deoxyguanosine) were measured in forestomach, glandular stomach, and duodenum cells. 

– 20 mg hexava

chromium/L (drinking wat

Suppressed nuclear DNA synthesis 

Potassium dichromate 

Mouse tubular renal cells 

Parenteral exposure: Mice given single 

intraperitoneal injection at a concentration of 15–30% of the LD50 (unspecified) in a thymidine 

incorporation inhibiting screening system; an intraperitoneal injection of 

3H thymidine was 

administered 15 hours later. 

+ NS 

Unscheduled DNA synthesis

Potassium dichromate 

Rat hepatocytes Oral exposure: Fischer 344 rats were 

administered potassium dichromate at concentrations of 0, 1, 5, or 20 mg hexavalent chromium/L in drinking water ad libitum, for 48 hours, while a second group was administered single gavage doses (20 mL/kg) at the same concentrations. Hepatocytes were collected from the rat livers and analyzed in the in vivo- in vitro 

hepatocyte DNA repair assay. 

– 20 mg hexava

chromium/L (drinking wat

Mutation Potassium 

chromate 

Mutations in liver, but not bone marrow cells 

Parenteral exposure: Male lacZ transgenic 

MutaTM mice were administered potassium 

chromate by an intraperitoneal dose of 0 or 40 mg/kg once a day for 2 consecutive days (0 or 14.1 mg hexavalent chromium/kg). 

+ 14.1 mg hexa

chromium/kg (intraperitoneinjection) 
137 DRAFT - DO NO

Table 4-23. In vivo genotoxicity studies of hexavalent chromium in D. melanogaster and labora

mammals 

Endpoint Chemical Test system Test conditions Results

a Dose

b 

Mutation Potassium 

chromate 

Mouse offspring Parenteral exposure: Female C57BL/6J mice were 

administered potassium chromate by intraperitoneal injection at a dose of 0, 10, or 20 mg/kg on days 8, 9, and 10 of pregnancy in a mammalian spot test (0, 2.7, or 5.4 mg hexavalent chromium/kg). The offspring’s fur was checked for colored spots from week 2 through week 5 after birth. 

+ 2.7 mg hexav

chromium/kg (intraperitoneinjection) 

Mutations Potassium 

dichromate 

Mouse (C57BL/6 Big Blue) lung and kidney tissue, but not in liver tissue 

Intratracheal exposure: Mice were given single 

doses of potassium dichromate via intratracheal instillation of 0 or 6.75 mg hexavalent chromium/kg and allowed 4 weeks for gene expression. Isolated DNA samples from lung, liver, and kidney tissues were used for LacI gene mutagenesis assay. Depletion of tissue glutathione by pretreatment with buthionine sulfoximine decreased the mutagenic response, suggesting that reduced glutathione plays a role inproducing reactive intermediates during intracellular reduction of chromium (VI). 

+ 6.75 mg hexa

chromium/kg (intratracheal instillation) 

Gene mutation Calcium 

chromate 

D. melanogaster 24-Hour old males were fed calcium chromate for 

72 hours at doses of 0, 500, or 750 ppm. The males were removed and mated. 

+ 500 ppm 

(in diet) 

Gene mutation Chromic acid D. melanogaster 24–48-Hour old males were treated by 

intraperitoneal injection of 0, 100, 200, 300, and 

400 ppm potassium dichromate or 0, 100, 200, and 300 ppm chromium trioxide. The F2 generation of flies was scored for sex-linked recessive lethal. 

+ 100 ppm 

(intraperitoneinjection) 

Gene mutation (wing somatic mutation) 

Chromium oxide D. melanogaster 2–3-Day-old larvae were fed potassium chromate 

or chromium(VI) oxide for 3 days at concentrations of 0, 1, or 5 mM. 

+ 1 mM 

(in diet) 

Gene mutation (white-ivory eye spot test) 

Chromium oxide D. melanogaster 2–3-Day-old larvae were fed potassium chromate 

or chromium(VI) oxide for 2 days at concentrations of 0, 1, or 5 mM. 

– 5 mM 

(in diet) 
138 DRAFT - DO NO

Table 4-23. In vivo genotoxicity studies of hexavalent chromium in D. melanogaster and labora

mammals 

Endpoint Chemical Test system Test conditions Results

a Dose

b 

Gene mutation Potassium 

chromate 

D. melanogaster Larvae fed test substance in wing spot test at 

concentrations of 0, 0.1, 0.5, 1.0, and 2.5 mM for the duration of their development. Surviving transheterozygous (mwh/flr

3) and inversion 

heterozygous (mwh/TM3) flies were used. 

+ 0.1 mM 

(in diet) 

Gene mutation (wing somatic mutation) 

Potassium chromate 

D. melanogaster 2–3-Day-old larvae were fed potassium chromate 

or chromium(VI) oxide for 3 days at concentrations of 0, 1 or 5 mM. 

+ 1 mM 

(in diet) 

Gene mutation Potassium 

chromate 

D. melanogaster 3-Day-old larvae were fed potassium chromate for 

6 hours at concentrations ranging from 0 to 100 mM or 48 hours at concentrations ranging from 0 to 5.0 mM. Marker-heterozygous and balancerheterozygous wings from adult flies were then examined in the wing somatic mutation and recombination test (SMART). 

+ 0.5 mM (48 h

(in diet) 5 mM (6 hour(in diet) 

Gene mutation (white-ivory eye spot test) 

Potassium chromate 

D. melanogaster 2–3-Day-old larvae were fed potassium chromate 

or chromium(VI) oxide for 2 days at concentrations of 0, 1 or 5 mM. 

– 5 mM 

(in diet) 

Gene mutation Potassium 

dichromate 

D. melanogaster 3-Day-old transheterozygous larvae were fed 

potassium dichromate at 0 or 0.5 mM and analyzed for multiple wing hair and flare gene mutations in the Drosophila wing SMART. 

+ 0.5 mM 

(in diet) 

Gene mutation Potassium 

dichromate 

D. melanogaster Larvae fed test substance in wing spot test at 

concentrations of 0, 0.1, 0.5, 1.0, and 2.5 mM for the duration of their development. Surviving transheterozygous (mwh/flr

3) and inversion 

heterozygous (mwh/TM3) flies were used. 

+ 0.1 mM 

(in diet) 

Gene mutation Potassium 

dichromate 

D. melanogaster 24–48-Hour old males were treated by 

intraperitoneal injection of 0, 100, 200, 300, and 

400 ppm potassium dichromate or 0, 100, 200, and 300 ppm chromium trioxide. The F2 generation of flies was scored for sex-linked recessive lethal. 

+ 100 ppm 

(intraperitoneinjection) 
139 DRAFT - DO NO

Table 4-23. In vivo genotoxicity studies of hexavalent chromium in D. melanogaster and labora

mammals 

Endpoint Chemical Test system Test conditions Results

a Dose

b 

Gene mutation Sodium 

dichromate 

D. melanogaster Larvae were treated on filter papers soaked with 

sodium dichromate at doses of 1.17 and 2.34 mM for 6 hours and then transferred to vials with substrate. Adult males were checked for wild-type 

pigmented spots in the eyes. 

+ 2.34 mM 

Dominant lethality 

Potassium dichromate 

Mouse Parenteral exposure: CBA x C57Bl/6J hybrid 

male mice were treated with a single intraperitoneal injection of 0, 0.5, 1.0, 2.0, 10, or 20 mg/kg potassium dichromate (0, 0.18, 0.35, 0.70, 3.5, or 7.1 mg hexavalent chromium/kg) or with intraperitoneal injections of 0, 1.0, or 2.0 mg/kg potassium dichromate daily for 21 days (0, 0.35, 0.70 mg hexavalent chromium/kg). Each male was mated with two untreated females for 7 days, and then replaced by two more females every 7 days for 4 consecutive weeks. Pregnant dams were sacrificed 12–14 days after conception. The frequency of dominant lethal mutations in male mice was determined based on the postimplantation loss. 

+ + 

7.1 mg hexavchromium/kg (acute intraperitoneainjection) 0.7 mg hexavchromium/kg (repeated intraperitoneainjection) 

Chromosome aberrations, sister chromatid exchange 

Chromium fumes; no further information was given and chromium 

valence state was not specified 

Chromosome aberrations and sister chromatid exchange in rat peripheral lymphocytes, but not in bone marrow cells 

Inhalation exposure: Sprague-Dawley rats were 

exposed to chromium fumes that were generated from a plasma flame sprayer and chromium metal powders at a concentration of 1.84 mg/m

3 for 

1 week (5 hour/day, 5 day/week) or 0.55 mg/m

3

for 2 months (5 hour/day, 5 day/week). Cytogenetic analysis was performed 20 hour, 3 days, 7 days, and 1 month after the last exposure. 

+ 1.84 mg/m

3 (

week inhalatiexposure) 0.55 mg/m

3 (

month inhalaexposure) 

Micronuclei Potassium 

chromate 

Mouse bone marrow cells 

Oral exposure: Male MS/Ae and CD-1 mice were 

administered potassium chromate by single gavage 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 mg hexavalent chromium/kg). 

– 113.1 mg 

hexavalent chromium/kg (gavage) 
140 DRAFT - DO NO

Table 4-23. In vivo genotoxicity studies of hexavalent chromium in D. melanogaster and labora

mammals 

Endpoint Chemical Test system Test conditions Results

a Dose

b 

Micronuclei Potassium 

chromate 

Mouse bone marrow cells 

Parenteral exposure: Male MS/Ae and CD-1 mice 

were administered potassium chromate by single intraperitoneal doses 0, 10, 20, 40, or 80 mg/kg (0, 3.5, 7.1, 14.1, or 28.3 mg hexavalent chromium/kg). 

+ 14.1 mg hexa

chromium/kg (intraperitoneinjection) 

Micronuclei Potassium 

chromate 

Mouse peripheral red blood cells 

Parenteral exposure: Male lacZ transgenic 

MutaTM mice were administered potassium 

chromate by an intraperitoneal dose of 0 or 40 mg/kg once a day for 2 consecutive days (0 or 14.1 mg hexavalent chromium/kg). 

+ 14.1 mg hexa

chromium/kg (intraperitoneinjection) 

Micronuclei Potassium 

chromate 

Mouse bone marrow cells 

Parenteral exposure: Slc:ddY mice were 

administered potassium chromate intraperitoneal injection once a day for 2 consecutive days at doses of 0, 30, 40, and 50 mg/kg (0, 10.6, 14.1, or17.7 mg hexavalent chromium/kg). 

+ 10.6 mg hexa

chromium/kg (intraperitoneinjection) 

Micronuclei Potassium 

chromate 

MS and ddY mouse bone marrow cells 

Parenteral exposure: MS and ddY mice were 

administered potassium chromate by single intraperitoneal 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 hexa

chromium/kg (intraperitoneinjection) 

Micronuclei Potassium 

chromate 

Mouse bone marrow 

Parenteral exposure: NMRI mice were 

administered potassium chromate by 2 intraperitoneal injections with 24 hours between each injection at doses of 0, 12.12, 24.25, or 48.5 mg/kg (0, 3.2, 6.49, or 13.0 mg hexavalent chromium/kg). 

+ 13 mg hexava

chromium/kg (intraperitoneinjection) 

Micronuclei Potassium 

dichromate 

Mouse (BDF1) 

bone marrow cells and peripheral blood cells 

Oral exposure: BDF1 male mice were 

administered potassium dichromate in drinking water at 0, 10, or 20 mg hexavalent chromium/L for 20 days. 

– 20 mg hexava

chromium/L (drinking wat

Micronuclei Potassium 

dichromate 

Mouse (BDF1) 

bone marrow cells 

Oral exposure: BDF1 male mice were given a single doses of potassium dichromate by gavage of 0 or 50 mg hexavalent chromium/kg. 

– 50 mg hexava

chromium/kg (gavage) 
141 DRAFT - DO NO

Table 4-23. In vivo genotoxicity studies of hexavalent chromium in D. melanogaster and labora

mammals 

Endpoint Chemical Test system Test conditions Results

a Dose

b 

Micronuclei Potassium 

dichromate 

Mouse bone marrow cells 

Oral exposure: Swiss-Webster mice were 

administered potassium dichromate at concentrations of 0, 1, 5, or 20 mg hexavalent chromium/L in drinking water. One set of mice was allowed access to drinking water ad libitum, for 48 hours, while a second group was administered two bolus doses (20 mL/kg) of the same concentrations at 24 and 48 hours before sacrifice. Bone marrow cells were collected from the femur of the mice. 

– 20 mg hexava

chromium/L (drinking wat

Micronuclei Potassium 

dichromate 

Mouse (Swiss) bone marrow—dams; fetal liver cells and peripheral blood cells 

Oral exposure: Pregnant Swiss albino mice were 

administered potassium dichromate in drinking water at concentrations of 0, 5, or 10 mg hexavalent chromium/L throughout the duration of pregnancy. Mice were sacrificed on day 18 of pregnancy and bone marrow cells were collected from dams and liver cells were collected from fetuses. 

– 10 mg hexava

chromium/L (drinking wat

Micronuclei Potassium 

dichromate 

Mouse (BDF1) 

bone marrow cells 

Parenteral exposure: BDF1 male mice were given single intraperitoneal doses of potassium dichromate of 0 or 50 mg hexavalent chromium/kg. 

+ 50 mg hexava

chromium/kg (intraperitoneinjection) 

Micronuclei Potassium 

dichromate 

Mouse bone marrow 

Parenteral exposure: Balb C mice were 

administered potassium dichromate as a single intraperitoneal injection at a dose of 0 or 400 

μmol 

(20.8 mg hexavalent chromium/kg). 

+ 20.8 mg hexa

chromium/kg (intraperitoneinjection) 

Micronuclei Potassium 

dichromate 

Mouse bone marrow 

Parenteral exposure: CBA x C57Bl/6J hybrid 

mice were treated with potassium dichromate with a single intraperitoneal injection of 0, 1, 5, or 10 mg/kg (0.35, 1.77, or 3.54 mg hexavalent chromium/kg). Bone marrow was sampled 24, 48, and 72 hours after treatment for the micronucleus test. 

+ 0.35 mg hexa

chromium/kg (intraperitoneinjection) 
142 DRAFT - DO NO

Table 4-23. In vivo genotoxicity studies of hexavalent chromium in D. melanogaster and labora

mammals 

Endpoint Chemical Test system Test conditions Results

a Dose

b 

Micronuclei Potassium 

dichromate 

Mouse (Swiss): bone marrowdams; fetal liver and peripheral blood cells 

Parenteral exposure: Pregnant Swiss albino mice 

were administered potassium dichromate as a single intraperitoneal injection at 0 or 50 mg hexavalent chromium/kg on day 17 of pregnancy. Mice were sacrificed on day 18 of pregnancy. The liver and peripheral blood were collected from the fetuses and bone marrows from the dams. 

+ 50 mg hexava

chromium/kg (intraperitoneinjection) 

Micronuclei Sodium 

dichromate dihydrate 

Mouse (B6C3F1)

peripheral red blood cells 

Oral exposure: B6C3F1 (10/sex/group) mice were 

administered sodium dichromate dihydrate in drinking 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 hexav

chromium/L (drinking wat

Micronuclei Sodium 

dichromate dihydrate 

Mouse (B6C3F1, 

BALB/c or am3-C57BL/6) peripheral red blood cells 

Oral exposure: B6C3F1 (5/group), BALB/c (5/group) and am3-C57BL/6 (10/group) male mice were administered sodium dichromate dihydrate in drinking water for 3 months at concentrations 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 wat87.2 mg hexachromium/L (B6C3F1) 

87.2 mg hexachromium/L (BALB/c) 21.8 mg hexachromium/L (am3-C57BL

Micronuclei Sodium 

dichromate dihydrate 

Mouse (BDF1) 

bone marrow or peripheral blood cells 

Oral exposure: Male and female BDF1 mice were 

administered sodium dichromate dihydrate in drinking water at 0, 5, 50, and 500 mg hexavalent chromium/L for 210 days. Peripheral blood cells were collected on days 0, 14, 28, 56, and 147; bone marrow cells were collected on day 210. 

– 500 mg hexav

chromium/L (drinking wat
143 DRAFT - DO NO

Table 4-23. In vivo genotoxicity studies of hexavalent chromium in D. melanogaster and labora

mammals 

Endpoint Chemical Test system Test conditions Results

a Dose

b 

Micronuclei Sodium 

dichromate dihydrate 

Mouse (Swiss) bone marrowdams; fetal liver and peripheral blood cells 

Oral exposure: Pregnant Swiss albino mice were 

administered sodium dichromate dihydrate in drinking water at concentrations of 0, 5, or 10 mg hexavalent chromium/L throughout the duration of pregnancy. Mice were sacrificed on day 18 of pregnancy and bone marrow cells were collected. Liver and peripheral blood samples were collected from the fetuses. 

– 10 mg hexava

chromium/L (drinking wat

Micronuclei Sodium 

dichromate dihydrate 

Mouse (Swiss): bone marrowdams; fetal liver and peripheral blood cells 

Parenteral exposure: Pregnant Swiss albino mice 

were administered sodium dichromate dihydrate as a single intraperitoneal injection at 0 or 50 mg/kg on day 17 of pregnancy. Mice were sacrificed on day 18 of pregnancy and bone marrow cells were collected. Liver and peripheral blood samples were collected from the fetuses. 

+ 50 mg hexava

chromium/kg (intraperitoneinjection) 

a+ = positive, ± = equivocal or weakly positive, – = negative, (T) = toxicity. bLowest effective dose for positive results, highest dose tested for negative results, NS = not specified. 
144 DRAFT - DO NOT CITE OR QUOTE 

1 4.4.1.2. Genotoxicity studies in humans 

2 In addition to mutagenicity evaluations in experimental systems, several studies have 

3 evaluated mutagenicity in humans occupationally exposed to hexavalent chromium; 

4 experimental details and citations are summarized in Table 4-24. Data from available 

5 mutagenicity studies in exposed workers are limited to assessments of tissues with easy 

6 accessibility (e.g., circulating lymphocytes and buccal and nasal mucosal cells). Data on 

7 mutagenicity in cancer target tissues (e.g., lung and GI tract) are not available. Available data 

8 provide some evidence of hexavalent chromium-induced mutagenicity in occupationally exposed 

9 humans, although results of studies in workers have yielded mixed results. In general, 

10 associations between hexavalent chromium exposure and mutagenicity in workers are uncertain 

11 because exposure levels were often not quantified or estimated, past exposure history was not 

12 well characterized in all studies, small numbers of workers were evaluated, and/or workers were 

13 potentially co-exposed to other compounds with mutagenic activity. 

14 In a comet assay in Italian chrome platers, positive results were reported for DNA strand 

15 breaks in peripheral lymphocytes; although urine chromium concentrations were determined, 

16 hexavalent chromium exposure levels were not reported (Gambelunghe et al., 2003). However, 

17 no DNA damage was observed in peripheral lymphocytes in dichromate production workers 

exposed to 0.001–0.055 mg hexavalent chromium/m318 (Gao et al., 1994) or in volunteers 

19 ingesting single oral doses of 71 μg hexavalent chromium/kg (Kuykendall et al., 1996). In 

20 chrome electroplaters, chromosome aberrations and sister chromatid exchanges were observed in 

21 whole blood of workers exposed to relatively high concentrations estimated at 5.99 mg 

hexavalent chromium/m322 (Wu et al., 2001). However, chromosome aberrations and sister 

23 chromatid exchanges in peripheral lymphocytes from chrome platers were not observed at lower 

exposure levels (0.0075 and 0.0249 mg chromium[total]/m324 ) (Benova et al., 2002). Other studies 

25 reporting positive (Sarto et al., 1982; Stella et al., 1982; Wu et al., 2000) or negative (Nagaya et 

26 al., 1986, 1991) results for chromosome aberrations or sister chromatid exchanges in peripheral 

27 lymphocytes of workers did not report hexavalent chromium exposure levels. Micronuclei 

28 formation in peripheral lymphoctes was also observed in chrome platers at exposure levels of 

0.043–0.083 mg chromium(total)/m329 (Vaglenov et al., 1999) and 0.0075–0.0249 mg 

chromium(total)/m330 (Benova et al., 2002). In buccal mucosal cells collected from chrome 

31 platers, micronuclei formation was increased at exposure levels of 0.0075–0.0249 mg 

chromium(total)/m332 , although chromosome aberrations and sister chromatid exchanges were not 
145 DRAFT - DO NOT CITE OR QUOTE 

1 observed (Benova et al., 2002). Sarto et al. (1990) reported negative results for micronuclei in 

2 buccal and nasal cells of chrome platers, but exposure levels were not reported. 

3 In summary, results of available studies in hexavalent chromium-exposed workers 

4 provide some evidence of the mutagenic activity of hexavalent chromium in occupationally 

5 exposed humans, but results have not been consistent across studies and endpoints. For example, 

6 associations with increased micronuclei in peripheral lymphocytes or buccal mucosal cells have 

7 been reported in chrome platers at estimated exposure levels as low as 0.0075–0.0249 mg 

chromium(total)/m38 (Benova et al., 2002; Vaglenov et al., 1999), although chromosome 

9 aberrations and sister chromatid exchanges were not observed (Benova et al., 2002). In contrast, 

10 increased frequencies of chromosome aberrations and sister chromatid exchanges were observed 

11 in another group of chrome platers exposed to higher concentrations estimated at 5.99 hexavalent 

chromium/m312 (Wu et al., 2001). 
146 DRAFT - DO NO

1 

Table 4-24. In vivo genotoxicity studies in humans exposed to hexavalent chromium 

Endpoint 

Exposure type 

(chemical form) Cell type Test conditions Results

a Exposu

DNA strand breaks 

Occupational - Chromium plating

(chromic acid) 

Human peripheral lymphocytes

Nineteen chromium plating workers in Italy (mean employment of 6.3 years), and two groups of control subjects (18 hospital workers and 20 university personnel) gave preand post-shift urine samples and blood samples for analysis in the comet assay. Duration of employment ranged from 4 months to 14 years with a mean duration of 6.3 years. Mean chromium concentrations in urine were determined to be 5.29 μg/g creatinine (pre-shift) and 7.31 μg/g creatinine (postshift). Mean erythrocyte and lymphocyte concentrations in the exposed workers were 4.94 μg/L and 50.3 μg/1012cells, 

respectively. Air concentrations of chromium were not reported. 

+ NS 

DNA strand breaks, hydroxylation of deoxyquanosine 

Occupational - Production of dichromate (included exposure to chromic acid, potassium dichromate and sodium dichromate) 

Human peripheral lymphocytes

Urine and blood samples were taken from 10 exposed workers and 10 non-exposed workers at the end of a workweek at a bichromate production plant in England. The mean duration of exposure was 15 years. Chromium concentrations in the factory ranged from 0.001 to 0.055 mg hexavalent chromium/m

3 (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/1010 cells) of exposed workers were significantly higher than in non-exposed workers. 

– 0.001–0

hexavalchromiu

(measurexposur

DNA-protein crosslinks 

Experimental oral exposure - (potassium dichromate) 

Human peripheral lymphocytes

Four adult volunteers ingested a single bolus dose of 5,000 μg hexavalent chromium as potassium dichromate (approximately equivalent to 71 μg hexavalent chromium/kg, assuming a body weight of 70 kg). Blood samples were collected at 0, 60, 120, 180, and 240 minutes after ingestion. Pre-ingestion background DNA-protein crosslink levels for each individual served as the controls. 

– 71 μg h

chromiu
147 DRAFT - DO NO

Table 4-24. In vivo genotoxicity studies in humans exposed to hexavalent chromium 

Endpoint 

Exposure type 

(chemical form) Cell type Test conditions Results

a Exposu

Chromosome aberrations, sister chromatid exchanges 

Occupational - Chromium electroplating (chemical not specified) 

Human peripheral lymphocytes

Blood from seven chromium electroplating workers at a Chinese electroplating facility (mean employment period of 12.8 years) and 10 control subjects were analyzed. Air samples from the electroplating room were collected, along with stool and hair samples to determine exposure. The mean chromium (total) air concentration (by random air collection) was 8.1 μg/mm

3, the mean chromium concentration in stool 

samples was 8.5 μg/g stool, and the mean chromium concentration in hair was 35.68 μg/g. The valence of chromium that workers were exposed to was unspecified. 

+ 8.1 μg 

chromiu

Sister chromatid exchanges 

Occupational - Chromium electroplating (chemical not specified) 

Human whole blood cells 

Thirty-five chromium electroplating factory workers employed at three electroplating plants in Tawain and 35 control subjects gave blood samples to analyze the frequency of sister chromatid exchange. Exposure duration ranged from 2 to 14 years with a mean of 6.5 years. Mean chromium exposure (determined by personal monitoring samplers) was 5.99 mg hexavalent chromium/m

3. The mean urinary chromium 

concentration of the exposed workers was 3.67 μg/g creatinine.

+ 5.99 mg 

hexavalchromiu

Chromosomal aberrations, sister chromatid exchanges 

Occupational - Chromium plating

(chromic acid) 

Human peripheral lymphocytes

Thirty-eight male chromium plating factory workers in Italy were examined for urinary concentrations of chromium and chromosomal aberrations and sister chromatid exchanges. Chromium exposure levels were not reported. There were 35 unexposed control individuals. 

+ NS 

Sister chromatid exchanges 

Occupational - Chromium plating

(chromic acid fumes) 

Human peripheral lymphocytes

The frequency of sister chromatid exchanges was determined in lymphocytes from 12 chromium plating workers in Italy and 10 control subjects. Exposure durations ranged from 0.5 to 18 years (mean exposure duration was not reported). Hexavalent chromium exposure levels or hexavalent chromium, blood concentrations were not reported. 

+ NS 

Sister chromatid exchanges 

Occupational - Chromium electroplating (chemical not specified) 

Human peripheral lymphocytes

Thirty-five chromium electroplating factory workers in Taiwanand 35 control subjects (matched for age and gender) gave blood samples to determine sister chromatid exchange frequency. The mean duration of employment was 6.5 years. Exposure concentrations were not reported. 

+ NS 
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Table 4-24. In vivo genotoxicity studies in humans exposed to hexavalent chromium 

Endpoint 

Exposure type 

(chemical form) Cell type Test conditions Results

a Exposu

Chromosome aberrations, sister chromatid exchanges 

Occupational - Chromium plating

(chemical not specified) 

Human peripheral lymphocytes and buccal mucosal cells 

Blood samples and buccal mucosal cells from 15 Bulgarian chromium platers occupationally exposed were taken;exposure was estimated with personal air samplers and in urine samples. Control subjects were matched with exposed individuals. Duration of exposure ranged from 2 to >20 years; mean duration of exposure was not reported. Mean air concentration of total chromium was 0.0075 mg chromium/m

3

in the low-exposure group and 0.0249 mg chromium/m

3 in the 

high-exposure group (number of workers in each exposure group was not reported). Mean concentrations of chromium in urine were 18.63 μg/L (low) and 104.22 μg/L (high) 

– Results 

for comgroups and 0.02chromiu

Sister chromatid exchanges 

Occupational - Chromium plating (chemical not specified) 

Human peripheral lymphocytes

Venous blood and urine sample were collected from 12 male chromium platers in Japan over a 5-year period. No control subjects were used in this study. Employment duration ranged from 6.6 to 25.1 years, with mean employment duration of 15.5 years. Exposure concentrations were not reported. Urinary chromium concentrations ranged from 1.2 to 57.0 μg/g with a mean urinary chromium concentration of 17.9 μg/g creatinine. Sister chromatid exchange frequency inlymphocytes was determined in blood-urine paired samples. 

– NS 

Sister chromatid exchanges 

Occupational - Chromium plating (chemical not specified) 

Human peripheral lymphocytes

Venous blood and urine sample were collected from 24 male chromium platers in Japan and 24 control subjects. Duration of employment ranged from 0.5 to 30.5 years with a mean employment of 11.6 years. Exposure concentrations were not reported. The mean concentration of chromium in the urine was 13.1 μg/L. 

– NS 
149 DRAFT - DO NO

Table 4-24. In vivo genotoxicity studies in humans exposed to hexavalent chromium 

Endpoint 

Exposure type 

(chemical form) Cell type Test conditions Results

a Exposu

Micronuclei Occupational - 

Chromium electroplating (chemical not specified) 

Human peripheral lymphocytes

Forty electroplating workers in Bulgaria and 18 control subjects gave blood samples to analyze for the frequency of micronuclei. The workers were split into two groups based on levels of exposure. Mean air chromium (total) concentrations were 43 and 83 μg/m

3 in the low- and high-exposure groups, 

respectively. Duration of employment ranged from 4 to 25 years with mean durations of 10.44 and 11.63 years in the low- and high-exposure groups, respectively. Mean chromium concentrations in erythrocytes and urine of the low exposure group were 4.31 and 3.97 μg/L, respectively. The mean chromium concentrations in erythrocytes and urine of the highexposure group were 8.4 and 5.0 μg/L, respectively.

+ 0.043 an

0.083 mchromiu

Micronuclei Occupational - 

Chromium plating

(chemical not specified) 

Human peripheral lymphocytes and buccal mucosal cells 

Blood samples and buccal mucosal cells from 15 Bulgarian chromium platers occupationally exposed were taken.Exposure was estimated with personal air samplers and in urine samples. Control subjects were matched with exposed individuals. Duration of exposure ranged from 2 to >20 years; mean duration of exposure was not reported. Mean air concentration of total chromium was 0.0075 mg chromium/m

3

in the low-exposure group and 0.0249 mg chromium/m

3 in the 

high-exposure group. Mean concentrations of chromium in urine were 18.63 (low) and 104.22 μg/L (high). 

+ Positive 

reported combin(0.0075 0.0249 chromiu

Micronuclei Occupational - 

Chromium plating (chromic acid) 

Human buccal and nasal cells 

Sixteen exposed Italian electroplating factory workers and 27 unexposed control subjects gave samples of exfoliated buccal and nasal swabs. Duration of exposure ranged from 0.5 

to 23 years with a mean duration of 8 years. Urine samples were collected at the end of work days to determine chromium 

exposure. Urinary chromium concentrations ranged from 2.5 to 88 μg/g creatinine; the mean urinary chromium concentration was not reported. Chromium levels in air were not determined. 

– NS 

a+ = positive; – = negative bAll exposure levels associated with positive results, highest exposure level for negative results; NS = not specified. cThe exposure level of 8.1 μg chromium/mm

3 is as reported by Deng et al., (1988); however, this appears to be a reporting error, as thi

equivalent to 8,100,000 mg chromium/m

3

. 

1 
150 DRAFT - DO NOT CITE OR QUOTE 

1 4.4.2. Intracellular reduction 

2 The mutagenic effects of hexavalent chromium are contingent upon its reduction within 

3 the cell. Extracellularly, soluble hexavalent chromium exists as a chromate oxyanion. The 

4 tetrahedral arrangement of the oxygen groups makes it structurally similar to phosphate and 

5 sulfate, allowing it to easily be taken up by the non-specific phosphate/sulfate anionic 

6 transporters and cross the cell membrane (Zhitkovich, 2005). This method of cellular uptake 

7 also allows an accumulation of chromium in the cell at concentrations much higher than that 

8 found extracellularly (Zhang et al., 2002). Chromium in its hexavalent state is 

9 thermodynamically stable in pure water, and is not reactive with DNA at physiological 

10 concentrations. However, hexavalent chromium is a strong oxidizer, and once inside the cell it 

11 can undergo rapid reduction. This is most often mediated by the non-enzymatic reductants 

12 ascorbate (vitamin C) and low molecular weight thiols including glutathione and cysteine. Other 

13 potential reductants include cytochrome P450 reductase, NAD(P)H-dependent flavoenzymes, 

14 and mitochondrial electron transport complexes (O’Brien et al., 2003; Sugden and Stearns, 2000; 

15 Standeven and Wetterhahn, 1989). 

16 The hexavalent chromium-reductant substrate complexes that are formed upon 

17 intracellular interaction of hexavalent chromium with these reductants are considered the first 

18 step in the reduction process, although the actual mechanisms of how these reactions proceed are 

19 unknown (Levina and Lay, 2005). There are two theorized pathways for the intracellular 

20 reduction of hexavalent chromium. When reductants are present in abundance, the process can 

21 occur with a two electron reduction to tetravalent chromium, immediately followed by a one 

22 electron reduction to trivalent chromium. If lower levels of reductants are available, the first step

23 of this process will occur as two distinct one electron transfers, producing the intermediates 

24 pentavalent and tetravalent chromium, and ultimately trivalent chromium (O’Brien et al., 2003). 

25 Either process can produce oxidative states of chromium localized within the cell that are able to 

26 damage DNA directly, forming DNA adducts and subsequent DNA breakage. These chromium 

27 species can also indirectly cause genetic damage via associated radical species derived from the 

28 reductants that can be involved in secondary DNA damage (Sugden and Stearns, 2000) and 

29 disruption of DNA replication. 

30 

31 Final reduction product: Trivalent chromium. 

32 Trivalent chromium is the ultimate product of hexavalent chromium reduction within the 

33 cell. It contains six coordination sites, allowing it to form stable complexes with amino acids, 
151 DRAFT - DO NOT CITE OR QUOTE 

1 proteins, RNA, and DNA. In vitro studies of the kinetics of chromium−DNA binding have 

2 shown that most of the DNA binding occurs within 1 hour of incubation (Quievryn et al., 2003). 

3 When hexavalent chromium is reduced by ascorbate or cysteine in the presence of the trivalent 

4 chromium chelator EDTA, the mutagenic response is all but eliminated and very little 

5 chromium−DNA binding is detected, indicating that the trivalent state is the most DNA reactive 

6 of all the valence states of chromium (O’Brien et al., 2003; Quievryn et al., 2003; Zhitkovich et 

7 al., 2001). Several types of chromium−DNA adducts have been detected following the 

8 intracellular reduction of hexavalent to trivalent chromium. 

9 DNA−peptide/amino acid ligand−trivalent chromium crosslinks. Trivalent chromium 

10 can form ternary DNA crosslinks with glutathione, ascorbate, cysteine, and histidine. Although 

11 the ascorbate−trivalent chromium−DNA adducts are recovered less frequently in vitro due to the 

12 low concentrations of vitamin C present in commonly used tissue culture media (Zhitkovich, 

13 2005), these adducts have been shown to be the most mutagenic of all the ternary adducts 

14 (Quievryn et al., 2003). These ternary adducts form by the attachment of trivalent chromium (in 

15 a binary complex with the ligand) to phosphate groups in DNA (Zhitkovich et al., 1995), 

16 primarily through coordinate covalent binding or electrostatic/ionic interactions (O’Brien et al., 

17 2003) (Figure 4-1). They have been detected in vitro in CHO cells following exposure to 

18 hexavalent chromium, and account for up to 50% of all chromium−DNA adducts. The ternary 

19 adducts have been found to cause mutagenic and replication-blocking lesions in human 

20 fibroblasts in vitro (Quievryn et al., 2003; Voitkun et al., 1998). 

21 

22 

23 

24 

25 

26 

27 

28 

29 

30 

31 

32 

33 
152 DRAFT - DO NOT CITE OR QUOTE 

1 

2 

3 

4 

5 

6 

7 

8 

9 

10 

11 

12 

13 

14 

15 Source: Zhitkovich (2005). 

16 

17 Figure 4-1. Ternary DNA adduct formation by chromium. Hexavalent chromium, 

18 when reduced intracellularly to trivalent chromium, can form ternary DNA crosslinks 

19 with the peptide or amino acid ligand (L) involved in the reduction. Here, chromium(III) 

20 directly coordinates to the 5’-phosphate in the DNA backbone and forms a hydrogen 

21 bond with the N-7 of deoxyguanosine. 

22 

23 DNA−trivalent chromium crosslinks. Reduction of hexavalent chromium in vitro 

24 produces a large proportion of binary trivalent chromium−DNA adducts, but these have not been 

25 detected in vivo. It has been theorized that the formation of the ternary adducts described above 

26 occurs far more frequently due to the high concentration of ligands capable of complexing with 

27 trivalent chromium before it can bind to DNA (Zhitkovich, 2005). In addition, these adducts 

28 have been found to be less mutagenic than the ternary adducts in vitro (Quievryn et al., 2003; 

29 Zhitkovich et al., 2001). 

30 DNA−protein crosslinks. These bulky lesions have been detected in hexavalent 

31 chromium-treated cells in vitro in Chinese hamster ovary cells (Costa, 1991) and in vivo in chick 

32 embryos (Hamilton and Wetterhahn, 1986). They are not detected in the presence of the 

33 trivalent chromium chelator EDTA, indicating that trivalent chromium is the species involved in 

34 their formation (Miller and Costa, 1989). It has been recently shown that the mechanism 

35 forming DNA−protein crosslinks induced by hexavalent chromium requires intracellular 

OH

O

O

Cr(III)

O

N

N

NH2

N

NH

O

O

O P O

O

O

O P O L

L

L L

O

H

H

DNA

DNA

7

OH

OH

O

O

Cr(III)

O

N

N

NH2

N

NH

O

N

N

NH2

N

NH

O

O

O P O

O

O

O P O L

L

L L

O

H

H

DNA

DNA

7

OH
153 DRAFT - DO NOT CITE OR QUOTE 

1 reduction to trivalent chromium, formation of DNA−trivalent chromium adducts, and subsequent 

2 capture of proteins by the DNA bound to trivalent chromium (Macfie et al., 2010). Tests for the 

3 mutagenicity of these crosslinks have proved inconclusive (reviewed in Macfie et al., 2010), but 

4 the bulkiness of these lesions indicates potential for genotoxicity resulting from replication fork 

5 stalling (Costa, 1991). 

6 DNA−DNA crosslinks. These inter- or intra-strand DNA crosslinks are likely formed by 

7 oligomers of trivalent chromium. They have been detected following hexavalent chromium 

8 exposure, although only when the reductants are ascorbate or cysteine, and not glutathione 

9 (Zhitkovich, 2005). However, these adducts have only been detected in vitro and are not 

10 expected to form in significant amounts in vivo; the high intracellular concentrations of ligands 

11 available to form complexes with trivalent chromium make it unlikely that these oligomers 

12 would have a chance to form (Salnikow and Zhitkovich, 2008). 

13 Repair of chromium−DNA adducts. Repair processes have been shown to be effectively 

14 carried out by nucleotide excision repair (NER), a DNA repair mechanism responsible for 

15 removal of bulky DNA lesions. Exposing NER-deficient human cells to hexavalent chromium 

16 was shown to induce apoptosis and clonogenic cell death. The most efficient substrates for this 

17 repair process are lesions that create major distortions in the DNA structure. Chromium−DNA 

18 adducts do not create major helix distortions, but their bulkiness makes them adequate substrates 

19 for NER, although they are less efficiently removed than optimal NER substrates such as UV 

20 light-induced lesions (Reynolds et al., 2004). 

21 Another closely related repair mechanism, mismatch repair (MMR), is responsible for the 

22 correction of errors in DNA replication. MMR enzymes recognize misincorporated bases during 

23 DNA replication and homologous recombination, and repair single base mispairings and small 

24 insertions or deletions. However, MMR has also been shown to be a causative factor in many of 

25 the toxic and genotoxic effects of hexavalent chromium, when processing the repair of the bulky 

26 lesions formed by chromium lead to the formation of DNA double-strand breaks (Peterson-Roth 

27 et al., 2005). In this study, mouse and human cell lines deficient in MMR exposed to hexavalent 

28 chromium had greatly increased clonogenic survival due to a diminished apoptotic response as 

29 compared to MMR-proficient cells. The apoptotic response in the MMR-proficient cells was 

30 preceded by a significant induction of DNA double-strand breaks, indicated by an increased 

31 formation of gamma-H2AX foci. These discrete foci form when phosphorylation of this histone 

32 H2A variant occurs in response to DNA double-strand breaks, and can be visualized and 

33 quantified by immunofluorescence. This increase in gamma-H2AX foci was not detected at 
154 DRAFT - DO NOT CITE OR QUOTE 

1 significant levels until 6 hours post-exposure to hexavalent chromium, suggesting that the DNA 

2 double-strand breaks were not induced directly by hexavalent chromium, but rather from 

3 processing of the damaged DNA. These foci also co-localized with cyclin B1 staining, 

4 indicating the breaks occurred in the G2 phase of the cell cycle and providing evidence that 

5 passage through S phase, where MMR would be taking place, was necessary for the induction of 

6 this damage (Salnikow and Zhitkovich, 2008). The mechanism of this toxic response mediated 

7 by MMR proteins is unknown, but has been theorized to involve the futile repair of damaged 

8 bases or the initiation of a stress response (Peterson-Roth et al., 2005). 

9 As mentioned above, apoptosis, or programmed cell death, has been observed in cells 

10 exposed to hexavalent chromium as a response to extensive DNA damage that cannot be 

11 adequately repaired by the cell. Ye et al. (1999) found hexavalent chromium induced apoptosis 

12 in human lung epithelial cells exposed to doses ranging from 75 to 300 μM in vitro; the authors 

13 theorized that this response involved reactive oxygen species formed both directly during the 

14 process of hexavalent chromium reduction and indirectly through the induction of p53. Flores 

15 and Perez (1999), using doses close to the IC50 values, observed apoptosis concurrent with DNA 

16 interstrand crosslinks and DNA single-strand breaks in murine keratinocytes transformed with 

17 the H-ras oncogene. These studies indicate that multiple mechanisms induced by hexavalent 

18 chromium exposure, including oxidative stress and DNA binding, can lead to cell death. 

19 However, no in vivo studies have been able to confirm the extent of apoptosis following 

20 hexavalent chromium exposure. In addition, several studies specifically measuring genotoxicity 

21 in vitro and in vivo have observed positive results at doses that did not elicit cytotoxicity (see 

22 previous section). 

23 An interesting addendum to the effects involving DNA repair mechanisms is the finding 

24 that hexavalent chromium, after intracellular reduction to the +3 oxidation state, can interfere 

25 with normal DNA replication and transcription processes. Intracellular trivalent chromium has 

26 been shown to inhibit the enzymatic activity of DNA polymerases, simultaneously increasing the 

27 rate of replication and the processivity of the DNA polymerase, thereby decreasing its fidelity 

28 and causing more frequent errors, with a dose-dependent increase in mutation frequency in vitro 

29 (Snow, 1991). There can also be replication arrest as a result of the bulky chromium−DNA 

30 lesions, creating a physical obstruction to the progression of DNA polymerases (Bridgewater et 

31 al., 1998). These effects were recently confirmed in a study utilizing the DNA synthesome, an in 

32 vitro DNA replication model system that is fully competent to carry out all phases of the DNA 

33 replication process mediated by human cells (Dai et al., 2009). This study found a reduction of 
155 DRAFT - DO NOT CITE OR QUOTE 

1 the fidelity and an inhibition of DNA synthesis that led to a dose-dependent increase in mutation 

2 frequency following intracellular exposure to trivalent chromium. Gene transcription has also 

3 been recently shown to be affected by exposure to hexavalent chromium in vitro via epigenetic 

4 mechanisms. Sun et al. (2009) found alterations in the levels of histone methylation in human 

5 lung A549 cells exposed to hexavalent chromium, indicating the capability of these exposures to 

6 lead directly to changes in gene expression. Thus, hexavalent chromium can lead to the 

7 disruption of DNA synthesis and gene transcription at multiple levels, corresponding to an 

8 observable, dose-dependent increase in mutation frequency in human cells. 

9 

10 Reduction intermediates: Pentavalent and tetravalent chromium. 

11 Depending on the reductant involved and the concentration of hexavalent chromium 

12 present, various amounts of the unstable intermediates pentavalent and tetravalent chromium can 

13 be generated prior to reduction to the final stable oxidative +3 state. At lower levels of 

14 hexavalent chromium exposure, intracellular concentrations of these reductants are sufficient to 

15 complete the reduction of hexavalent chromium to its trivalent state. However, at higher 

16 hexavalent chromium exposures, these levels are depleted, resulting in a higher yield of 

17 pentavalent chromium from the one-electron reducing thiols, glutathione and cysteine, as well as 

18 tetravalent chromium from the two-electron donating ascorbate. While pentavalent and 

19 tetravalent chromium can be short-lived states of chromium within the cell, they are DNA 

20 reactive and can participate in redox reactions, forming free radical species that can also damage 

21 DNA (Stearns and Wetterhahn, 1994). 

22 Redox cycling of the chromium ions can occur intracellularly when they are formed 

23 during reduction of hexavalent chromium. The process of hexavalent chromium reduction by 

24 glutathione is accompanied by the reduction of molecular oxygen, yielding superoxide radicals. 

25 Reduction by glutathione has been shown to involve the formation of glutathione-derived thiyl 

26 radicals that can directly damage DNA or react with other thiols to also generate superoxide 

27 radicals. These radical species will react with hydrogen peroxide to produce hydroxyl radicals 

28 via Haber-Weiss reactions (Shi et al., 1999). Both hydrogen peroxide and superoxide radicals 

29 can participate in redox reactions involving both the pentavalent and tetravalent transition states 

30 of chromium that can generate hydroxyl radicals via Fenton and Haber-Weiss reactions (Shi et 

31 al., 1999). Hydroxyl radicals can directly react with genetic material, forming DNA−protein 

32 crosslinks and DNA adducts with proteins and amino acids, damaging DNA bases, and 

33 producing DNA single- and double-strand breaks (reviewed in Kasprzak, 1996). 
156 DRAFT - DO NOT CITE OR QUOTE 

1 Although less frequent than the low molecular weight non-enzymatic reductants, 

2 reduction of hexavalent chromium can also occur by NAD(P)H-dependent flavoenzymes, 

3 including glutathione reductase, lipoyl dehydrogenase, and ferredoxin-NADP+ oxidoreductase 

4 (Shi and Dalal, 1990). These enzymes catalyze a one-electron reduction that can result in the 

5 formation of stable pentavalent chromium−NADPH complexes that can react with hydrogen 

6 peroxide to generate hydroxyl radicals (Shi et al., 1999). The ability to form complexes with 

7 biological ligands allows stabilization of pentavalent, but not tetravalent, chromium 

8 intermediates (Levina and Lay, 2005). These pentavalent chromium−NADPH complexes have 

9 been shown to form in vitro in E. coli (Shi et al., 1991) and in vivo in mice (Liu et al., 1995). 

10 Two other important non-enzymatic reducers of hexavalent chromium are ascorbate and 

11 cysteine. Ascorbate and cysteine are present at lower concentrations intracellularly than 

12 glutathione, but they have kinetically faster rates of hexavalent chromium reduction. Ascorbate 

13 has been shown to yield pentavalent and tetravalent chromium and radical species when the 

14 intracellular ratio of ascorbate to chromium is less than 3:1 (Stearns and Wetterhahn, 1994). The 

15 precise nature of the radical species relevant to DNA damage is not known, however, and the 

16 degree of damage attributable to oxidative mechanisms is the subject of much debate. One study 

17 found an increase in mutations and replication-blocking DNA lesions in human fibroblasts 

18 resulting from the ascorbate-driven reduction of hexavalent chromium, but concluded that the 

19 mechanism responsible did not involve oxidative radicals, in part because the DNA damage 

20 anticipated by species including hydroxyl radicals and pentavalent chromium-peroxo complexes, 

21 namely abasic sites and strand breaks, was not observed (Quievryn et al., 2003). This study also 

22 found that no mutagenesis occurred in the presence of a trivalent chromium chelator, indicating 

23 the involvement of trivalent chromium−DNA adducts (see previous section). Similarly, studies 

24 of the DNA damage resulting from the intracellular reduction of hexavalent chromium by 

25 cysteine have shown that, while the intermediate species pentavalent and tetravalent chromium 

26 and thiyl radicals were formed, they were not responsible for DNA damage; rather, the trivalent 

27 chromium−DNA adducts were found to be the mutagenic species (Zhitkovich et al., 2001). The 

28 same group also found an elimination of mutagenicity when glutathione reduction of hexavalent 

29 chromium occurred in the presence of phosphate ions that led to the sequestration of trivalent 

30 chromium, preventing its binding to DNA (Guttmann et al., 2008). 

31 The ability of these intermediate chromium species to generate damaging free radicals is 

32 not in doubt, however, and there is evidence of reactive oxygen species generated by pentavalent 

33 chromium causing DNA damage. A decrease in DNA strand breaks was observed when 
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1 hexavalent chromium reduction with glutathione occurred in the presence of free radical 

2 scavengers (Kortenkamp et al., 1990). In addition, DNA double-strand breaks in subcellular 

3 systems were observed when ascorbate-mediated reduction of hexavalent chromium generated 

4 hydroxyl radicals via a Fenton-like reaction (Shi et al., 1994). 

5 In an attempt to explain these conflicting results, it has been theorized that the 

6 responsible free radicals may be chromium-based and not oxygen-based radicals. This is due to 

7 the observation that the mutational spectra observed by chromium-induced radicals differs from 

8 that expected by damage due to reactive oxygen species that are generated following exposure to 

9 hydrogen peroxide, X-rays, or ionizing radiation (Sugden and Stearns, 2000). Hexavalent 

10 chromium has been shown to induce the formation of 8-oxo-deoxyguanosine adducts (8-oxo-dG) 

11 that are known to be induced by oxidative damage (Sander et al., 2005), but these lesions have 

12 also been shown to be induced directly by pentavalent chromium, with the subsequent addition 

13 of molecular oxygen (Sugden and Martin, 2002). In addition, the oxididant-sensitive dyes used 

14 to detect reactive oxygen species intracellularly can also be oxidized directly by pentavalent 

15 chromium and chromium-based radicals (O’Brien et al., 2003). Therefore, the induction of 

16 mutagenic lesions by the intracellular reduction of hexavalent chromium could be attributed to 

17 non-oxygen-dependent mechanisms. 

18 Pentavalent chromium has been detected using electron paramagnetic resonance (EPR) 

19 spectroscopy following intraperitoneal administration of hexavalent chromium in vivo, both in 

20 the liver and red blood cells of chick embryos (Liebross and Wetterhahn, 1992), and in mouse 

21 liver and blood (Liu et al., 1994). In in vitro, levels of DNA strand breaks were found to 

22 correlate with increasing levels of pentavalent chromium in Chinese hamster V79 cells 

23 (Sugiyama et al., 1989). Another in vitro study in human leukemic T-lymphocyte MOLT4 cells 

24 detected pentavalent chromium species and hydroxyl radicals with EPR following exposure to 

25 hexavalent chromium (Mattagajasingh et al., 2008). The same study also observed a dose26 dependent increase in protein carbonyls and malondialdehyde (MDA) generated via protein 

27 oxidation and lipid peroxidation, respectively, although the lipid peroxidation only occurred 

28 significantly at much higher exposures of chromate (≥100 μM) compared with the protein 

29 oxidation, which was significant as low as 10 μM. Tetravalent chromium has been more 

30 difficult to observe due to its unstable nature compared to pentavalent chromium, but this species 

31 was shown to induce mitotic recombination in the somatic wing spot assay in Drosophila (Katz 

32 et al., 2001). Both species caused an induction of NF-kB, a nuclear transcription factor involved 

33 in the cellular response to oxidative damage, in cultured Jurkat cells. This activation was 
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1 enhanced by hydrogen peroxide and eliminated when catalase was added to decompose 

2 hydrogen peroxide, indicating that hydroxyl radicals may have had a role (Shi et al., 1999). 

3 In summary, there are many potential mechanisms involved in the genotoxicity of 

4 hexavalent chromium when reduced intracellularly. Intermediate valence states can react 

5 directly and indirectly through coordinate complexes with DNA as well as form radical species, 

6 and the final reduction product, trivalent chromium, can form various damaging DNA adducts. 

7 Additionally, significant evidence points to the aberrant processing of DNA mismatches induced 

8 by chromium−DNA adducts, leading to apoptosis of the damaged cells, or further promotion of 

9 these mutagenic lesions as the DNA double-strand breaks generated are substrates for error10 prone repair processes such as non-homologous end joining. 

11 

12 4.5. SYNTHESIS OF MAJOR NONCANCER EFFECTS - ORAL 

13 In humans, several case reports have been published on clinical signs and symptoms in 

14 individuals following acute accidental or intentional ingestion of high doses (fatal or near fatal) 

15 of hexavalent chromium compounds, including chromic acid, potassium dichromate, and 

16 ammonium dichromate. Clinical presentation of patients following acute, high-dose exposure 

17 was similar, regardless of the specific hexavalent chromium compound ingested, and included 

18 the following: abdominal pain, nausea, and vomiting; hematemesis and bloody diarrhea; caustic 

19 burns of mouth, pharynx, esophagus, stomach, and duodenum and GI hemorrhage; anemia, 

20 decreased blood Hgb, abnormal erythrocytes, and intravascular hemolysis; hepatotoxicity 

21 (hepatomegaly, jaundice, elevated blood bilirubin, and liver enzyme activities); renal failure 

22 (oliguria and anuria); cyanosis; and metabolic acidosis, hypotension, and shock. Findings on 

23 tissue biopsies included hepatic fatty degeneration and necrosis and renal tubular degeneration 

24 and necrosis. 

25 Information on chronic human health effects resulting from exposure to hexavalent 

26 chromium comes from several studies of human populations unknowingly consuming food or 

27 drinking water contaminated with hexavalent chromium over some extended time period. These 

28 studies have been primarily focused on cancer. However, the noncancer effects that have been 

29 recorded are consistent with the GI effects observed following acute exposures to hexavalent 

30 chromium and have included oral ulcers, diarrhea, abdominal pain, dyspepsia, stomach pain, and 

31 vomiting (JinZhou Antiepidemic Station, 1979). 

32 Table 4-25 presents a summary of studies of the noncancer effects of hexavalent 

33 chromium exposure from repeated-dose oral toxicity studies in experimental animals. The most 
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1 sensitive targets of toxicity identified in these studies included the blood, liver, and GI tract. The 

2 effects seen in these target organs are more specifically discussed below. 

3 In regard to hematological effects, NTP (2007) observed microcytic, hypochromic 

4 anemia (i.e., decreased Hct, Hgb, MCV, and MCH) at a dose of 1.7 mg/kg-day of hexavalent 

5 chromium in both male and female F344/N rats in a three-month (subchronic) study. In this 

6 same study, NTP (2007) also saw histopathological changes (i.e., histiocytic cellular infiltration) 

7 in the pancreatic lymph nodes in male F344/N rats at 1.7 mg/kg-day of hexavalent chromium. 

8 Finally, in a chronic (two-year) study, NTP (2008) observed histopathological changes (i.e., 

9 histiocytic cellular infiltration) in the mesenteric lymph nodes in male F344/N rats at 0.77 

10 mg/kg-day of hexavalent chromium and male and female B6C3F1 mice at 0.38 mg/kg-day of 

11 hexavalent chromium. 

12 In the NTP (2007) subchronic study referenced above, liver effects were also observed at 

13 1.7 mg/kg-day of hexavalent chromium and included increased serum liver enzyme activities 

14 (i.e., ALT and SDH) in both males and females and increased bile acids in females. In their two15 year bioassay, NTP (2008) found an increased incidence of chronic inflammation of the liver at 

16 0.24 mg/kg-day of hexavalent chromium in female F344/N rats and increased incidences of 

17 histopathological changes to the liver (i.e., basophilic foci) at 0.77 mg/kg-day of hexavalent 

18 chromium in male F344/N rats. In this same bioassay, increased incidences of histopathological 

19 changes to the liver (i.e., histiocytic cellular infiltration) were seen at 0.38 mg/kg-day of 

20 hexavalent chromium in female B6C3F1 mice. 

21 Effects of hexavalent chromium ingestion on the GI tract have been primarily observed in 

22 the small intestine (duodenum). In a three-month study, NTP (2007) saw histopathological 

23 changes to the duodenum in male F344/N rats at 1.7 mg/kg-day of hexavalent chromium, in male 

24 and female B6C3F1 mice at 5.3 mg/kg-day of hexavalent chromium, and in male BALB/c and 

25 am3-C57BL/6 mice at 2.8 mg/kg-day of hexavalent chromium. These changes included 

26 epithelial hyperplasia and histiocytic cellular infiltration. In their two-year study, NTP (2008) 

27 also found increased incidences of histopathological changes to the duodenum in male F344/N 

28 rats at 0.77 mg/kg-day of hexavalent chromium and in male and female B6C3F1 mice at 0.38 

29 mg/kg-day of hexavalent chromium. Similar to that observed in the subchronic study, these 

30 changes in the duodenum included epithelial hyperplasia and histiocytic cellular infiltration. 

31 Animal studies also provide evidence that oral exposure to hexavalent chromium 

32 compounds produces reproductive effects, including histopathological changes to reproductive 

33 organs in males (Aruldhas et al., 2006, 2005, 2004; Chowdhury and Mitra, 1995; Li et al., 2001; 
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1 Zahid et al., 1990) and females (Murthy et al., 1996); alterations in sperm, including decreased 

2 count, decreased motility, and abnormal morphology (Subramanian et al., 2006; Yousef et al., 

3 2006; Li et al., 2001; Zahid et al., 1990); decreased plasma testosterone levels (Yousef et al., 

4 2006; Chowdhury and Mitra, 1995); increased estrous cycle length (Kanojia et al., 1998, 1996; 

5 Murthy et al., 1996); changes in mating behavior and decreased fertility in males (Bataineh et al., 

6 1997); and adverse reproductive outcomes, including decreased numbers of live fetuses and 

7 implantations, and increased numbers of resorptions and pre- and postimplantation losses 

8 (Bataineh et al., 2007; Elsaieed and Nada, 2002; Elbetieha and Al-Hamood, 1997; Junaid et al., 

9 1996a, b, 1995; Kanojia et al., 1998, 1996; Trivedi et al., 1989). These studies are summarized 

10 in Table 4-25. 

11 Developmental effects observed in animal studies have included decreased fetal weight 

12 and length (Elsaieed and Nada, 2002; Kanojia et al., 1998; Junaid et al., 1996a, b, 1995; Trivedi 

13 et al., 1989); external (subdermal hemorrhage and tail malformations) and skeletal abnormalities 

14 (decreased ossification) (Elsaieed and Nada, 2002; Junaid et al., 1996a, b, 1995; Kanojia et al., 

15 1998, 1996; Trivedi et al., 1989); and delayed sexual maturation and function in female offspring 

16 (Banu et al., 2008; Al-Hamood et al., 1998). These effects were seen at hexavalent chromium 

17 doses ranging from about 2 to 100 mg/kg-day. 

18 In contrast to results of the above studies on reproductive toxicity, reproductive effects 

19 were not observed in dietary exposure studies conducted by NTP that investigated the potential 

20 effects of hexavalent chromium on male reproductive organs in rats and mice (NTP, 1996a,b) 

21 and on reproductive outcomes in a continuous breeding study in mice (NTP, 1997). The reason 

22 for the inconsistent results between the NTP studies and the other reproductive toxicity studies of 

23 hexavalent chromium are not readily apparent, as daily dose ranges evaluated in the NTP studies 

24 overlapped with those used in the other studies showing hexavalent chromium-induced 

25 reproductive effects. 

26 
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Table 4-25. Observed effects and corresponding NOAELs and LOAELs for subchronic, chronic, anreproductive toxicity studies following oral exposure to hexavalent chromium 

Species Sex Exposure level1 Exposure duration NOAEL (mg/kg-day) LOAEL (mg/kg-day) Effects at the NOAEL/LOAEL 

Subchronic Studies 

F344/N rat 

F, M 0, 1.7, 3.5, 5.9, 

11.2, or 20.9 mg/kg-day via drinking water 

3 months F: ND 

M: ND 

1.7 1.7 

F: Microcytic, hypochromic anemia (decreaseMCV, MCH), increased serum liver enzyme (ALT and SDH) and bile acids, and histopath

changes to the duodenum (histiocytic cellular infiltration) M: Microcytic, hypochromic anemia (decreasHgb, MCV, MCH), increased serum liver enzactivities (ALT and SDH), and histopathologchanges to pancreatic lymph nodes (histiocyt

infiltration) 

B6C3F1

mouse 

F, M 0, 3.1, 5.3, 9.1, 

15.7,or 27.9 mg/kg-day via drinking water 

3 months F: ND 

M: ND 

3.1 3.1 

Histopathological changes (histiocytic cellulainfiltration) in the duodenum 

B6C3F1, 

BALB/c, and am3- C57BL/6 

mouse 

M 0, 2.8, 5.2, or 8.7 

mg/kg-day via drinking water 

3 months ND 2.8 Histopathological changes in the duodenum i

mice (histiocytic cellular infiltration and epithhyperplasia), BALB/c mice (histiocytic cellulinfiltration), and am3-C57BL/6 mice (epithel

hyperplasia) 

Wistar rat M 0, 73.05 mg/kgday via drinking 

water 

30 days ND ND Decreased serum prolactin levels. Data not a

for estimation of a NOAEL or LOAEL. 

Wistar rat M 0, 20 mg/L in 

drinking water 

10 weeks ND ND Liver histopathologic changes. Doses in mg 

chromium/kg-day could not be estimated. 

Wistar rat M 0, 1.5 mg/kg-day 

via drinking water 

22 weeks ND 1.5 Changes in serum enzymes; liver triglyceride

glycogen and cholesterol; liver histopathologchanges. 

Swiss mouse 

M 0, 177, 265, 353, 

530, or 706 mg/L in drinking water

8 weeks ND ND Liver histopathologic changes. Doses in mg 

chromium/kg-day could not be estimated. 
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Table 4-25. Observed effects and corresponding NOAELs and LOAELs for subchronic, chronic, anreproductive toxicity studies following oral exposure to hexavalent chromium 

Species Sex Exposure level1 Exposure duration NOAEL (mg/kg-day) LOAEL (mg/kg-day) Effects at the NOAEL/LOAEL 

Wistar rat F 0, 1.4 mg/kg-day 

via drinking water 

22 weeks ND 1.4 Changes in liver weight; serum enzyme level

triglycerides, glucose; liver glycogen; liver histopathology. 

Wistar rat F, M F: 0, 1.76-2.47 

mg/kg-day via drinking water M: 0, 1.4-2.18 mg/kg-day via drinking water 

6 months ND ND Changes in urinary markers of renal function

histopathologic examination of the kidney. 

Chronic Studies 

F344/N rat 

F, M F: 0.24, 0.94, 2.4 

or 7.0 mg/kg-day via drinking water M: 0.21, 0.77, 2.1, or 5.9 mg/kg-day via drinking water 

2 years F: ND 

M: 0.21 

0.24 0.77 

F: Increased incidence of chronic inflammatio

liver M: Increased incidences of nonneoplastic histopathological changes to the liver (basophduodenum (histiocytic cellular infiltrate), and mesenteric lymph nodes (histiocytic cellular i

and hemorrhage) 

B6C3F1

mouse 

F, M F: 0.38, 1.4, 3.1 

or 8.7 mg/kg-day via drinking water M: 0.38, 0.91, 2.4, or 5.9 mg/kg-day via drinking water 

2 years F: ND 

M: ND 

0.38 0.38 

F: Increased incidences of histopathological cthe duodenum (diffuse epithelial hyperplasia)mesenteric lymph nodes (histiocytic cellular infiltration), liver (histiocytic cellular infiltratpancreas (depletion of cytoplasmic zymogen M: Increased incidences of histopathological 

the duodenum (diffuse epithelial hyperplasia) mesenteric lymph nodes (histiocytic cellular infiltration) 
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Table 4-25. Observed effects and corresponding NOAELs and LOAELs for subchronic, chronic, anreproductive toxicity studies following oral exposure to hexavalent chromium 

Species Sex Exposure level1 Exposure duration NOAEL (mg/kg-day) LOAEL (mg/kg-day) Effects at the NOAEL/LOAEL 

Dog Not 

specified 

0, 0.45, 2.25, 4.5, 6.75, 11.2 mg/L in drinking water

4 years ND ND No effects were observed. Doses in mg hexa

chromium/kg-day could not be estimated. 

SpragueDawley rat 

F, M 0.05 to 2.8 

mg/kg-day via drinking water 

1 year 2.4-2.8 ND No adverse effects observed at the highest do

Reproductive/Developmental Studies 

Bonnet monkey 

M 0, 1.0, 2.1, 4.1, 

and 8.3 mg/kgday via drinking water 

180 days ND 2.1 Reversible changes to male reproductive orga

including disruption of spermatogenesis, effesperm count and velocity, and histopathologicchanges 

Charles Foster rat 

M 0, 20, 40, or 

60 mg/kg-day via 

gavage 

90 days ND 20 Decreased serum testosterone levels and loss 

HSH activity in testes 

Wistar rat M 0, 5.2 or 

10.4 mg/kg-day via gavage 

6 days ND 5.2 Decreased sperm counts and histopathologica

to the testes 

BALB/c mouse 

M 0, 6.4, 12.7, or 

25.5 mg/kg-day via gavage 

35 days ND 6.4 Increased percentage of degenerated tubules, 

undergenerated tubules without spermatogonabnormal sperm, and reduced number of sper

New Zealand White rabbit 

M 0 or 3.6 mg/kgday via gavage 10 weeks ND 3.6 Decreased testes and epididymis weight and d

sperm output 
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Table 4-25. Observed effects and corresponding NOAELs and LOAELs for subchronic, chronic, anreproductive toxicity studies following oral exposure to hexavalent chromium 

Species Sex Exposure level1 Exposure duration NOAEL (mg/kg-day) LOAEL (mg/kg-day) Effects at the NOAEL/LOAEL 

SpragueDawley rat 

F, M F: 0, 0.25, 1.1, 

2.5, or 9.5 mg/kg-day via the diet M: 0, 0.35, 1.1, 2.1, or 8.5 mg/kg-day via the diet 

9 weeks F: 2.5 

M: 2.1 

F: 9.5 

M: 8.5 

F: Slight erythrocyte microcytosis M: Slight erythrocyte microcytosis 

BALB/c mouse 

F, M F: 0, 1.8, 5.6, 

12.0, 48.4 mg/kg-day via the diet M: 0, 1.1, 3.5, 7.4, or 32.5 mg/kg-day via the diet 

9 weeks F: 1.8 

M: 3.5 

F: 5.6 

M: 7.4 

F: Histopathological changes to the liver (cyt

vacuolization) M: Histopathological changes to the liver (cyvacuolization) 

BALB/c mouse 

F 0, 7.9, 16.1, or 

37.1 mg/kg-day via the diet (F1

generation) 

Continuous 

breeding study 

ND 7.9 Erythrocyte microcytosis (slight decrease in M

the F1 generation 

Druckrey rat 

F 0, 70, 127, or 

170 mg/kg-day via drinking water 

3 months ND 70 Dam: Increased pre- and post-implantation lo

Offspring: Decreased fetal weight and externskeletal abnormalities 

Swiss mouse 

F 0, 63, 119, or 

174 mg/kg-day via drinking water 

GDs 6 though 14 ND 63 Dam: Decreased fertility 

Offspring: Decreased fetal body weight and dskeletal development 
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Table 4-25. Observed effects and corresponding NOAELs and LOAELs for subchronic, chronic, anreproductive toxicity studies following oral exposure to hexavalent chromium 

Species Sex Exposure level1 Exposure duration NOAEL (mg/kg-day) LOAEL (mg/kg-day) Effects at the NOAEL/LOAEL 

Wistar rat F 0 or 7.9 mg/kgday via drinking 

water 

GD 6 through 15 ND 7.9 Dam: Increased pre-implantation loss/litter, p

implantation loss/litter, resorptions/litter, and fetuses/litter and decreased live fetuses/litter Offspring: Decreased fetal weight and increaswith fetal abnormalities or malformations incvisceral and skeletal changes. 

SpragueDawley rat 

F 0 or 35 mg/kgday via drinking 

water 

GDs 1–3 or 4–6 ND 35 Dam: Impaired implantation, increased resorp

decreased number of viable fetuses 

ITRCBred mouse 

F 0, 48, 98, or 239 

mg/kg-day via drinking water 

Entire gestational 

period 

Dam: 48 

Offspring: ND

Dam: 98 

Offspring: 48

Dam: Decreased body weight gain and increaresorptions and postimplantation loss Offspring: Decreased fetal length and weight 

Swiss mouse 

F 0, 53, 101, or 

152 mg/kg-day via drinking water 

GD 14 though 19 Dam: 53 

Offspring: ND

Dam: 101 

Offspring: 53

Dam: Decreased body weight gain and reduceof implantation sites 

Swiss mouse 

F 0, 53, 101, or 

152 mg/kg-day via drinking water 

GD 14 though 19 Dam: 53 

Offspring: ND

Dam: 101 

Offspring: 53

Dam: Decreased body weight gain Offspring: Reduced fetal weight and length aincreased incidence of reduced caudal ossific

1 Unless otherwise noted, dose or concentration expressed as hexavalent chromium. 

F = female; M = male; ND = not determined 
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1 

2 Based on a review of the NOAELs and LOAELs in Table 4-25, the most sensitive 

3 hexavalent chromium-induced effects in rats were increased incidence of chronic inflammation 

4 of the liver in females; and increased incidences of nonneoplastic histopathological changes to 

5 the liver (basophilic foci), duodenum (histiocytic cellular infiltrate), and mesenteric lymph nodes 

6 (histiocytic cellular infiltrate and hemorrhage) in males. In mice, the most sensitive hexavalent 

7 chromium-induced effects were increased incidences of histopathological changes to the 

8 duodenum (diffuse epithelial hyperplasia), mesenteric lymph nodes (histiocytic cellular 

9 infiltration), liver (histiocytic cellular infiltration), and pancreas (depletion of cytoplasmic 

10 zymogen granules) in females; and increased incidences of histopathological changes to the 

11 duodenum (diffuse epithelial hyperplasia) and mesenteric lymph nodes (histiocytic cellular 

12 infiltration) in males. All of these effects were observed in the two-year chronic study by NTP 

13 (2008), and in general, occurred at lower doses than the reproductive or developmental effects. 

14 

15 4.6. EVALUATION OF CARCINOGENICITY 

16 4.6.1. Summary of Overall Weight-of-Evidence 

17 Under the U.S. EPA Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005a), 

18 hexavalent chromium is “likely to be carcinogenic to humans” via the oral route of exposure 

19 based on a statistically significant increase in the incidence of tumors of the oral mucosa and 

20 tongue of rats and of the small intestine of mice; and evidence of an association between oral 

21 exposure to hexavalent chromium and stomach cancer in humans. Additionally, available 

22 evidence indicates that chromium interacts with DNA, resulting in DNA damage and 

23 mutagenesis. Thus, hexavalent chromium is proposed to induce carcinogenicity via a mutagenic 

24 mode of action. 

25 

26 4.6.2. Synthesis of Human, Animal, and Other Supporting Evidence

27 Human studies in which health outcomes (primarily cancer) were evaluated among 

28 populations who resided near sources of industrial waste containing hexavalent chromium 

29 compounds provide some evidence of possible associations between oral exposure to hexavalent 

30 chromium and cancer. These epidemiological studies evaluated populations in Liaoning 

31 Province, China (Kerger et al., 2009; Beaumont et al., 2008; Zhang and Li, 1997, 1987), Kings 

32 County/San Bernadino County, California (Fryzek et al., 2001), Nebraska (Bednar and Kies, 

33 1991), and Glasgow, UK (Eizaguirre-Garcia et al., 2000, 1999) that unknowingly were exposed 
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1 to hexavalent chromium over some time period. Of these studies, the most detailed analyses 

2 were of data collected from the Jinzou area of Liaoning Province, China, where groundwater, 

3 surface water, and agricultural soils were contaminated with chromium derived from hexavalent 

4 chromium production (e.g., 0.001–20 mg chromium/L in residential well water). This study 

5 found evidence of an excess risk of mortality from stomach cancer from 1970-1978 in residents 

6 of the area, relative to the reference populations (four other areas in Liaoning Province, and the 

7 total population of the province) (Beaumont et al., 2008). The association with stomach cancer 

8 mortality was weaker when an urban area was excluded from the reference population (Kerger et 

9 al., 2009). However, there was little difference between stomach cancer rates in urban compared 

10 to rural areas during this period; indicating no sound rationale for excluding this urban area from 

11 the reference group. Studies of chromium-exposed populations in California and Nebraska 

12 (Fryzek et al., 2001; Bednar and Kies, 1991) found no significant correlation between cancer 

13 mortality and drinking water concentration, and the study of the population in Glasgow 

14 (Eizaguirre-Garcia et al., 2000, 1999) found no correlation between leukemia risk and distance 

15 from a former chromium processing facility (where elevated soil concentrations for hexavalent 

16 chromium were measured). Interpretation of the findings from these three studies is limited by 

17 the analysis of all cancer mortality (rather than individual cancer types) in the case of the 

18 California and Nebraska studies and leukemia only in the case of the Glasgow study. 

19 Evidence of carcinogenicity in animals was provided by the NTP (2008) bioassay 

20 conducted in rats and mice. In this study, exposure of F344/N rats to sodium dichromate 

21 dihydrate in drinking water for 2 years resulted in a statistically significant increase in the 

22 incidence of squamous epithelial papillomas and carcinomas of the oral mucosa and tongue 

23 (noted by NTP as rare when compared with historical controls) at the highest exposure level 

24 (average daily doses of 5.9 and 7.0 mg hexavalent chromium/kg-day in males and females, 

25 respectively), but not at the three lower exposure levels. NTP (2008) also exposed B6C3F1 mice 

26 to sodium dichromate dihydrate in drinking water for 2 years and reported statistically significant 

27 increases in the incidence of adenomas and carcinomas of the small intestine in males and 

28 females at doses ≥2.4 and ≥3.1 mg hexavalent chromium/kg-day, respectively. 

29 As discussed in detail in Section 4.6.3, hexavalent chromium is proposed to induce 

30 carcinogenicity via a mutagenic mode of action. The key precursor events leading to 

31 mutagenicity have been identified in animals and these events are anticipated to occur in humans 

32 and progress to tumors. 
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1 The “likely to be carcinogenic to humans” descriptor is appropriate when the weight of 

2 the evidence is adequate to demonstrate carcinogenic potential to humans but does not reach the 

3 weight of evidence for the descriptor “carcinogenic to humans”. The database supports this 

4 descriptor for hexavalent chromium exposure via the oral route. On the other hand, available 

5 evidence to support the descriptor of “carcinogenic to humans” was also considered. 

6 The “carcinogenic to humans” descriptor indicates strong evidence of human 

7 carcinogenicity, and can be characterized by different combinations of evidence. One line of 

8 evidence indicates this descriptor is appropriate when there is convincing epidemiologic 

9 evidence of a causal association between human exposure and cancer (U.S. EPA, 2005a). This is 

10 not the case for exposure to hexavalent chromium via ingestion. A moderately elevated risk of 

11 stomach cancer mortality was seen in Jinzou (Liaoning Province, China), but this risk has not 

12 been established (or examined) in other populations exposed to drinking water contaminated 

13 with hexavalent chromium. The epidemiologic data are not sufficient to establish a causal 

14 association between exposure to hexavalent chromium by ingestion and cancer. 

15 A second line of evidence under which this descriptor may be appropriate involves a 

16 lesser weight of epidemiologic evidence that is strengthened by other information, including 

17 strong evidence of an association between human exposure and either cancer or the key events of 

18 the MOA and extensive evidence of carcinogenicity in animals (U.S. EPA, 2005a). As discussed 

19 above, the epidemiologic evidence for the oral route of hexavalent chromium exposure is not 

20 considered strong. In addition, extensive evidence of the carcinogenicity of hexavalent 

21 chromium in animals via ingestion does not exist. Only one multiple dose chronic oral 

22 carcinogenicity study of hexavalent chromium in animals is available (i.e., the two-year bioassay 

23 in rodents conducted by NTP [2008]). Taken together, these considerations do not provide a 

24 basis for the characterization of hexavalent chromium as “carcinogenic to humans” via oral 

25 exposure. Therefore, EPA concluded that, based on the available information, the descriptor 

26 “likely to be carcinogenic to humans” is the most appropriate descriptor for the carcinogenic 

27 potential of hexavalent chromium via ingestion. 

28 

29 4.6.3. Mode of Action Information 

30 4.6.3.1. Hypothesized Mode of Action 

31 The hypothesized mode of action for carcinogenicity induced by hexavalent chromium is 

32 via mutagenesis. The hypothesis is that carcinogenicity can be induced directly by reduced 

33 forms of chromium interacting with DNA to form adducts and crosslinks that can lead to DNA 
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1 breaks and mutations, and indirectly by free radical species generated during the reduction 

2 process that can also lead to DNA breakage and mutagenesis. 

3 

4 Key events 

5 Hexavalent chromium is readily taken up by cells through sulfate transporters, due to the 

6 structural similarity of hexavalent chromium to the tetrahedral sulfate and phosphate anions 

7 (Bridges and Zalups, 2005). Once inside the cell, hexavalent chromium quickly undergoes a 

8 series of reduction reactions to yield pentavalent, tetravalent, and ultimately the 

9 thermodynamically stable trivalent chromium. Many potential enzymes as well as non10 enzymatic cellular reductants capable of reducing hexavalent chromium exist within the cell. 

11 These reductants include glutathione, ascorbate, cysteine, lipoic acid, NAD(P)H, fructose, and 

12 ribose (reviewed in McCarroll et al., 2009). Following this intracellular reduction, several 

13 possible mechanisms leading to mutagenicity can occur. 

14 Hexavalent chromium itself does not interact directly with DNA. However, the products 

15 of its reduction within the cell (pentavalent, tetravalent, and trivalent chromium) have all been 

16 shown to be DNA reactive (O’Brien et al., 2003). Hexavalent chromium is reduced by 

17 glutathione to yield pentavalent chromium and thiyl radicals, which can react with other thiol 

18 molecules to produce superoxide radicals. Both pentavalent and tetravalent chromium can 

19 participate in Fenton reactions, generating hydroxyl radicals (Salnikow and Zhitkovich, 2008; 

20 Volko et al., 2006). All of these species can cause DNA single- and double-strand breaks, base 

21 modifications, and lipid peroxidation, which may lead to mutations if not adequately repaired. 

22 Trivalent chromium is the ultimate product of the intracellular reduction of hexavalent 

23 chromium. Trivalent chromium is capable of interacting directly with DNA, forming stable 

24 coordination complexes with nucleic acids and peptides (Salnikow and Zhitkovich, 2008). In 

25 particular, trivalent chromium is capable of forming ternary complexes with DNA and an 

26 intracellular reducer, such as ascorbate, glutathione, or cysteine (Salnikow et al., 1992; 

27 Zhitkovich et al., 1996), as well as crosslinking DNA and proteins, and forming intrastrand 

28 DNA-DNA crosslinks (Voitkun et al., 1998; Zhitkovich, 2005). These chromium-DNA 

29 complexes, as well as DNA-protein and DNA-DNA crosslinks, all have the capability of causing 

30 DNA single- and double-strand breaks, which, if not adequately repaired, could lead to cell 

31 death, or if misrepaired, could result in mutation.

32 Thus, once inside the cell, hexavalent chromium, through reduction to its pentavalent, 

33 tetravalent, and trivalent forms, is capable of inducing a wide range of mutagenic and genotoxic 
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1 damage, including the formation of DNA adducts, DNA-protein and DNA-DNA crosslinks, 

2 mutations, DNA single and double-strand breaks, abasic sites, oxidized DNA bases, 

3 chromosomal aberrations, sister chromatid exchanges, and micronuclei. 

4 

5 4.6.3.2. Experimental Support for the Hypothesized Mode of Action 

6 Strength, consistency, and specificity of association 

7 A large database of experimental data exists on the mutagenic activity of hexavalent 

8 chromium compounds (these results are summarized in Section 4.5.1 and in the corresponding 

9 tables). In vitro, positive results were found in the majority of tests performed on hexavalent 

10 chromium compounds in bacterial test systems (see Table 4-21). Similarly, in yeast (S. 

11 cerevisiae and S. pombe), all available studies described positive results for the detection of gene 

12 mutations, mitotic gene conversion, and mitotic crossing over. 

13 In mammalian cell lines and primary cells, all studies using whole cells in vitro yielded 

14 positive results (Table 4-22). Evidence of mutation induction was shown at the tk locus in the 

15 mouse lymphoma assay, as well as at the hgprt locus in Chinese hamster ovary cells (V79 and 

16 AT3-2). In human cells, chromosome aberrations, DNA damage, and DNA-DNA and DNA17 protein crosslinks were detected in primary cultures and established cell lines originating from 

18 target organs, including the gastric mucosa, bronchial epithelium, and fibroblasts from the 

19 bronchial tubes and lung. Chromosome aberrations, sister chromatid exchanges, and DNA 

20 damage were observed in primary human dermal fibroblasts and lymphocytes as well as 

21 bronchial fibroblasts and epithelial cells. Chromosome aberrations and DNA damage were 

22 found in mouse carcinogenic cell lines, and sister chromatid exchanges were detected in mouse 

23 blastocysts. In rats, DNA damage and unscheduled DNA synthesis were observed in rat gastric 

24 mucosal cells and hepatocytes as well as in primary lymphocytes, and transformation was 

25 observed in rat liver epithelial cells upon exposure to hexavalent chromium. A number of 

26 studies have been performed using cultured Chinese hamster ovary cells, showing chromosomal 

27 aberrations and sister chromatid exchanges as well as DNA damage, DNA-protein crosslinks, 

28 and induced DNA methylation, and three studies showed induced transformation in cultured 

29 Syrian hamster embryo cells. 

30 In vivo, most studies of the mutagenicity of hexavalent chromium compounds have 

31 yielded positive results (Table 4-23). Somatic and germ cell mutations were detected in 3-day32 old Drosophila melanogaster larvae fed potassium chromate, potassium dichromate, or calcium 

33 chromate (Kaya et al., 2002; Spano et al., 2001; Amrani et al., 1999; Graf and Wurgler, 1996; 
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1 Zimmering et al., 1985). A number of in vivo oral exposure studies of the mutagenicity of 

2 hexavalent chromium in mice and rats are available, with slightly differing results depending on 

3 the method used. In the two studies in rats, Coogan et al. (1991b) found DNA-protein crosslinks 

4 in liver and not in splenic lymphocytes following 3 or 6 week exposures of 100 or 200 mg/L in 

5 drinking water, but Mirsalis et al. (1996) did not find any evidence of DNA repair via 

6 unscheduled DNA synthesis in rat hepatocytes following 48-hour exposures of up to 20 mg/L in 

7 drinking water or a single gavage dose of 20 mL/kg at the same concentration. In other studies 

8 of mice exposed via gavage, DNA damage as measured by the comet assay was found in 

9 peripheral leukocytes (including isolated lymphocytes), stomach, colon, liver, kidney, bladder, 

10 lung, and brain (Wang et al., 2006; Devi et al., 2001; Sekihashi et al., 2001), but neither DNA 

11 damage nor micronuclei were found in bone marrow (De Flora et al., 2006; Sekihashi et al., 

12 2001; Shindo et al., 1989). Similarly, in studies of mice exposed via drinking water, De Flora et 

13 al. (2008, 2006) reported negative results for the detection of micronuclei in the bone marrow of 

14 pregnant Swiss albino mice and in the fetal polychromatic erythrocytes after exposures up to 20 

15 mg/L and also in adult BDF1 mice following 500 mg/L exposure for 210 days. 

16 Interestingly, NTP (2007) investigated micronuclei induction in male mouse bone 

17 marrow following a three-month drinking water exposure and found differing results depending 

18 on the strain of mouse used. In one phase of the study, results were negative in B6C3F1 mice 

19 exposed to doses as high as 349 mg/L, while in another phase, following exposures of 0, 21.8, 

20 43.6, or 87.2 mg/L hexavalent chromium, results were negative in BALB/c mice, equivocal in 

21 B6C3F1 mice, and significantly positive at ≥ 43.6 mg/L exposures in am3-C57BL/6 mice, with a 

22 statistically significant positive trend starting at 21.8 mg/L. 

23 Somatic and germ cell mutations were detected in Drosophila melanogaster treated 

24 intraperitoneally with chromic acid or potassium dichromate (Rodriguez-Arnaiz and Martinez, 

25 1986) or with sodium dichromate via filter paper (Rasmuson, 1985). Following parenteral 

26 exposure in mice, DNA damage was detected in the stomach, colon, bladder, lung, brain, liver, 

27 and kidney (Sekihashi et al., 2001; Ueno et al., 2001; Amlacher and Rudolph, 1981); mutations 

28 were found in the liver of transgenic mice (Itoh and Shimada, 1998; 1997), in the germ cells of 

29 hybrid male mice (Paschin et al., 1982), and in the offspring of exposed female mice (Knudsen, 

30 1980); and micronuclei were increased in bone marrow and polychromatic erythrocytes (De 

31 Flora et al., 2006; Wronska-Nofer et al., 1999; Itoh and Shimada, 1996; Hayashi et al., 1982; 

32 Paschin and Toropzev, 1982; Wild, 1978), as well as in the liver and peripheral blood of mice 

33 exposed prenatally (De Flora et al., 2006). In rats exposed parenterally, DNA damage was 
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1 detected in leukocytes (Patlolla and Tchounwou, 2006), and DNA-protein crosslinks were found 

2 in lung, liver, and kidney (Tsapakos et al., 1983). Mutations were observed in the lung and 

3 kidney from transgenic mice exposed intratracheally to hexavalent chromium (Cheng et al., 

4 2000); DNA-protein crosslinks and DNA fragmentation and adducts were found in the lung of 

5 rats similarly exposed (Izzotti et al., 1998), while in rats exposed via inhalation, chromosomal 

6 aberrations and sister chromatid exchanges were observed in peripheral lymphocytes (Koshi et 

7 al., 1987). 

8 In addition to the in vivo evidence in animals for the genotoxicity of hexavalent 

9 chromium, several studies are available in humans (Table 4-24). In the only mutagenicity study 

10 following oral doses, DNA-protein crosslinks were not detected in peripheral lymphocytes up to 

11 4 hours after the 4 volunteers were given 71 μg hexavalent chromium/kg (Kuykendall et al., 

12 1996). Another study (Gao et al., 1994) failed to detect DNA damage in peripheral lymphocytes 

of workers inhalationally exposed to 0.001-0.055 mg/m313 . However, several studies of 

14 occupational exposures via inhalation provide evidence of significant levels of chromium15 induced DNA damage (Gambelunghe et al., 2003), and the formation of micronuclei (Benova et 

16 al., 2002; Vaglenov et al., 1999), chromosomal aberrations (Deng et al., 1988; Sarto et al., 1982), 

17 and sister chromatid exchanges (Wu et al., 2001, 2000; Deng et al., 1988; Sarto et al., 1982; 

18 Stella et al., 1982) in peripheral lymphocytes and/or buccal mucosal cells. These studies 

19 detected genotoxicity in workers exposed to mean air concentrations as low as 0.0075 and 

0.0249 mg/m320 (Benova et al., 2002). In addition, three studies found negative results for 

21 micronuclei and sister chromatid exchange, but the exposure concentrations were not reported 

22 (Nagaya et al., 1991, 1986; Sarto et al., 1990). 

23 

24 Dose-response concordance and temporal relationship 

25 As noted above, hexavalent chromium is hypothesized to induce carcinogenicity via a 

26 mutagenic mode of action. The initial key events in the hypothesized mutagenic mode of action 

27 are the capability of the hexavalent form of chromium to pass through the cell membrane and, 

28 once inside, to be reduced to pentavalent, tetravalent, and trivalent chromium. 

29 The available studies show that hexavalent chromium induces tumors in the stomach of 

30 humans (Beaumont et al., 2008) and in the tongue, oral mucosa, and intestines of rodents (NTP, 

31 2008). Studies of a cohort in Liaoning Province, China, exposed to 0.001–20 mg chromium/L in 

32 residential well water (Beaumont et al., 2008; Zhang and Li, 1997, 1987) reported an excess risk 

33 of mortality from stomach cancer in residents of the area. NTP (2008) reported a statistically 
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1 significant increase in the incidence of tumors of the oral mucosa and tongue in rats exposed to 

2 hexavalent chromium for two years in drinking water at average daily doses of 5.9 and 7.0 

3 mg/kg-day for males and females, respectively, and tumors of the small intestine in mice 

4 exposed to average daily doses of ≥2.4 and 3.1 mg/kg-day in males and females, respectively. 

5 Correlating these data with mutagenicity testing by establishing temporal and dose and/or site 

6 concordance can be difficult, as in vivo assays designed to detect mutagenicity are conducted 

7 within a relatively short time after the exposure period has ended, and tend to rely mainly on 

8 cells from tissues such as bone marrow and/or blood that are actively replicating and therefore 

9 sensitive to mutagenic agents. There is evidence, however, that hexavalent chromium can 

10 accumulate and induce mutagenicity in tissues at the site of entry and systemically, at doses 

11 relevant to human exposures. 

12 Following drinking water exposures, only one animal study has directly investigated 

13 target tissue genotoxicity (De Flora et al., 2008). With regard to dose, the De Flora et al. (2008) 

14 study tested levels (5 and 20 mg/L, or 1.2 and 4.82 mg/kg-day of hexavalent chromium) that 

15 were just below those leading to murine intestinal (duodenum, jejunum, and ileum) tumors in the 

16 two-year NTP study (30 and 50 mg/L for males and females, respectively). Negative results 

17 were reported for DNA-protein crosslinks and DNA adducts when measuring the forestomach, 

18 glandular stomach, and duodenum of mice exposed to hexavalent chromium for 9 months via 

19 drinking water. However, the shorter study duration makes a direct comparison of these results 

20 to the duodenal tumors reported in the chronic NTP bioassay infeasible. 

21 Other studies have shown evidence of in vivo genotoxicity in non-target tissues at early 

22 time points following exposure. In three studies that used the comet assay to detect DNA 

23 damage following oral gavage exposures in mice, Devi et al. (2001) found evidence of DNA 

24 damage in leukocytes that peaked at 48 hours post-exposure, Wang et al. (2006) detected DNA 

25 damage in lymphocytes after 1-day or 5-day consecutive exposures, and Seikihashi et al. (2001) 

26 detected DNA damage in stomach, colon, liver, kidney, bladder, lung, and brain within 8 hours 

27 of dosing that subsided by 24 hours post-treatment. 

28 Devi et al. (2001) found positive dose-dependent results at >10-fold lower doses (0.21, 

29 0.42, 0.84, 1.68, and 3.37 mg hexavalent chromium/kg). In fact, many of the positive in vivo 

30 mutagenicity studies found a positive trend with dose, including oral exposures (Devi et al., 

31 2001; Wang et al., 2006) and parenteral exposures (Paschin and Toropzev, 1982; Knudsen, 1980; 

32 Itoh and Shimada, 1996; Wild, 1978; Shindo et al., 1989; Hayashi et al., 1982) and rats (Patlolla 

33 et al., 2008) in mice. 
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1 Therefore, the detection of DNA damage, a key event for the mutagenic mode of action, 

2 following oral exposure to hexavalent chromium, that exhibits dose dependence and that is 

3 observed at time points prior to tumor development, strengthens the causal nature of this 

4 association. Although DNA-protein crosslinks and DNA adducts were not detected in target 

5 tissues following drinking water exposure in mice (De Flora et al., 2008), the lack of these 

6 findings did not preclude the observation of mutations in other tissues and organs, considered to 

7 be early events following hexavalent chromium exposure leading to carcinogenesis. 

8 

9 Biological plausibility and coherence 

10 Mutagenicity as a mode of action for carcinogenicity in humans is a biologically 

11 plausible mechanism for tumor induction. Hexavalent chromium has been shown to be 

12 mutagenic in vitro and in vivo, across species and tissue types. Human studies have shown 

13 induction of DNA damage, chromosomal aberrations, and micronucleus induction following 

14 exposure to hexavalent chromium, and in vivo animal studies show that hexavalent chromium 

15 induces DNA damage in rat blood, bone marrow, lung, liver, and kidney, and in mouse blood, 

16 lung, liver, kidney, bladder, colon, and brain. Exposures that induced a mutagenic response in 

17 these studies included doses within the range causing tumors in rats and mice in a chronic 

18 exposure bioassay (NTP, 2008). 

19 Only one study examined tumor target tissue for evidence of mutagenicity (De Flora et 

20 al., 2008). De Flora et al. (2008) found negative results for DNA-protein crosslinks and DNA 

21 adducts in the duodenum in mice following drinking water exposures. Other available drinking 

22 water exposure studies of hexavalent chromium that measured mutagenicity in mice failed to 

23 show evidence of micronucleus induction in the blood or bone marrow (De Flora et al., 2008, 

24 2006; NTP, 2007; Mirsalis et al., 1996). 

25 It has been proposed that the positive results for DNA damage found in mice following 

26 oral gavage exposures (Wang et al., 2006; Devi et al., 2001; Sekihashi et al., 2001) were the 

27 result of overwhelming the reductive capacity of the gastrointestinal tract in mice, allowing the 

28 accumulation and subsequent absorption of hexavalent chromium. This proposal would indicate 

29 that the comparatively lower concentrations of hexavalent chromium administered in the 

30 drinking water studies (De Flora et al., 2008; 2006) are effectively reduced to trivalent chromium 

31 when ingested, thereby inhibiting cellular uptake and subsequent DNA damage. 

32 While this is a plausible explanation for these results, which are unusual in that they 

33 represent the only component of the hexavalent chromium mutagenicity database that does not 
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1 show overwhelmingly positive results, there are inconsistencies with this explanation. For 

2 example, although the doses administered in De Flora et al. (2008) were lower than those in 

3 Wang et al. (2006) and Sekihashi et al. (2001), Devi et al. (2001) found positive results at doses 

4 approximately 6-fold lower than the lowest dose used by De Flora et al. (2008). 

5 In addition, genetic differences have been implicated in predicting the severity of 

6 genotoxic responses to hexavalent chromium exposure. In the three-month NTP bioassay 

7 (2007), three different strains of mice (B6C3F1, BALB/c, and am3-C57BL/6) were exposed to 

8 hexavalent chromium in drinking water at concentrations of 21.8, 43.6, or 87.2 mg/L, and found 

9 different results for micronucleus induction in polychromatic erythrocytes among strains. The 

10 BALB/c mice showed no micronucleus induction, and the B6C3F1 mice were positive only at the 

11 highest dose of 87.2 mg/L. However, the am3-C57BL/6 mice responded with an overall positive 

12 trend, with the two highest doses statistically significant, and the lowest dose nearly so. Based 

13 on the expected reduction capacity of an average 50 g mouse, it does not appear that the 

14 reductive capacities were overwhelmed in the NTP bioassay. The average rate of hexavalent 

chromium exposure for all three strains of mice was estimated to have been 2.9 x 10-2 15 mg/hour at 

16 the highest dose (NTP, 2007). This rate is within the estimated reductive capacity of the mouse 

gastrointestinal tract of 4.4 x 10-2 17 mg/hour that is based on an estimated 0.33 mL/hour rate of 

18 drinking water consumption. However, the micronucleus results could reflect minor differences 

19 in the capacities of these three strains of mice to reduce hexavalent chromium extracellularly, 

20 since the exact reductive capacity of each mouse strain used is unknown. 

21 The transgenic am3-C57BL/6 mouse contains multiple copies of the ΦX174 am3 allele, 

22 which is sensitive to A/T base-pair substitution mutagens. Finding positive results in this strain 

23 is consistent with DNA damage due to oxidative and/or crosslinking mechanisms. This suggests 

24 that interindividual differences in the capacity and fidelity of DNA repair processes could 

25 determine susceptibility to ingested hexavalent chromium. In keeping with this, one DNA repair 

26 pathway important in resolving mismatched bases during DNA replication, mismatch repair 

27 (MMR), has recently been implicated in the genotoxic responses to hexavalent chromium 

28 exposure. 

29 It has been shown that the processing of chromium-DNA adducts by the mismatch repair 

30 (MMR) pathway is responsible for turning these lesions into frank DNA double-strand breaks 

31 (Peterson-Roth et al., 2005). This study found that cells deficient in MMR were not subject to 

32 the same toxic responses to hexavalent chromium as were cells with these repair processes intact. 

33 This loss of MMR function leads to an unstable mutator phenotype, in which replication errors, 
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1 particularly those occurring in simple nucleotide repeat sequences known as microsatellites, are 

2 not corrected, leading to an increase in mutation frequency (Loeb et al., 2008). Further, these 

3 effects would be exacerbated by the physical and chemical interference with DNA replication 

4 that occurs when trivalent chromium is present intracellularly (Eastmond et al., 2008). 

5 There are several forms of cancer that exhibit microsatellite instability. For example, 

6 microsatellite instability has been implicated as the cause of the majority of cases of hereditary 

7 nonpolyposis colorectal cancer due to the inactivation of genes involved in the MMR pathway. 

8 In an epidemiological study of chromate-exposed workers, microsatellite instability was reported 

9 to occur in 79% of hexavalent chromium-induced lung tumors compared to only 15% in the non10 chromate lung cancer group (Hirose et al., 2002). The same group also reported finding 

11 increased DNA methylation in the promoter region of the tumor suppressor gene p16 and the 

12 MMR gene hMLH1 in human lung cancers in these chromate-exposed workers, indicating that 

13 chromium can induce epigenetic effects (Kondo et al., 2006; Takahashi et al., 2005). These 

14 findings reflect a loss of functional MMR capability that could be mechanistically involved in 

15 chromate-induced lung cancer. 

16 It was found that all four proteins responsible for MMR function were required for the 

17 processing of chromium-DNA adducts into DNA double-strand breaks (Peterson-Roth et al., 

18 2005). The genes involved in MMR are known to be highly polymorphic in humans (Goode et 

19 al., 2002), and given spontaneous background rates of mutation in human cells, it would not be 

20 unexpected to find small populations of cells that have acquired mutations in one of these four 

21 MMR genes. An inactivating mutation in any one of these would result in a growth advantage to 

22 cells exposed to hexavalent chromium, allowing them to evade apoptotic responses to these 

23 genotoxic lesions, as well as incurring further microsatellite instability, leading to a mutator 

24 phenotype. Thus, a selective advantage upon chronic exposure to even low levels of hexavalent 

25 chromium could translate into a clonal expansion of these MMR-deficient cells, leading to 

26 further evasion of cell death and increasing mutation frequencies, resulting in a state of genomic 

27 instability. 

28 In addition, it is of note that among the available oral exposure studies in mice, all studies 

29 that investigated DNA damage or micronucleus induction in bone marrow cells found negative 

30 results, including the study by Sekihashi et al. (2001), which found DNA damage in every tissue 

31 examined (liver, kidney, lung, brain, stomach, colon, and bladder) except for the bone marrow. 

32 The reason for the negative findings in these assays is unknown. 

33 
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1 Bioavailability

2 As noted above, there is uncertainty surrounding the ability of hexavalent chromium to 

3 induce mutagenicity and carcinogenicity in humans considering the potential for reduced 

4 bioavailablity. Intrinsic to the mutagenic and carcinogenic processes of hexavalent chromium is 

5 its ability to reach relevant tissues prior to being reduced to pentavalent, tetravalent, and trivalent 

6 chromium. When hexavalent chromium is reduced to the trivalent form extracellularly, this 

7 reduction process effectively detoxifies hexavalent chromium, since trivalent chromium is nearly 

8 impermeable to the cell. 

9 Quantitative studies of GI absorption of hexavalent chromium in humans have estimated 

10 that as much as 10% of an ingested dose of 5 mg is absorbed (Kuykendall et al., 1996), 

11 indicating that not all hexavalent chromium is reduced by the gastric juices of the stomach. In 

12 rats and mice, daily oral doses of 8 mg hexavalent chromium/day for 8 weeks resulted in 

13 absorption and accumulation of chromium in the bone, spleen, liver, and kidney (Kargacin et al., 

14 1993); rats given 0.138 μmol hexavalent chromium/day for 3 days exhibited GI absorption of 

15 about 16% (Febel et al., 2001); and the absorption of 4–10% of a single daily dose of 57 μg 

hexavalent chromium (as Na51 16 CrO4) was observed in rats, regardless of fasting state (MacKenzie 

17 et al., 1959). Distribution studies have shown that hexavalent chromium, once absorbed, 

18 distributes to nearly all tissues, particularly concentrating in the kidney, liver, bone, and red 

19 blood cells. Thus, at oral doses within human exposure ranges, hexavalent chromium was not 

20 completely reduced by the GI tract, making available some portion of ingested hexavalent 

21 chromium to be absorbed directly by the mucosal cells of the GI tract, or to be distributed to 

22 other tissues throughout the body. 

23 However, based on an understanding of chromium chemistry, as well as in vitro and in 

24 vivo studies conducted by De Flora et al. (1997, 2008), the reduction of at least some portion of 

25 ingested hexavalent chromium to trivalent chromium likely occurs in the GI tract (see Section 3). 

26 No data are currently available on the capacity of the rodent stomach to reduce hexavalent 

27 chromium. However, based on in vitro measurements De Flora et al. (1997) estimated that the 

28 reductive capacity of the human GI tract is sufficiently large to effectively reduce even high 

29 doses of ingested hexavalent chromium to the less toxic trivalent form. Given this assertion, it is 

30 appropriate to ask whether the observed effects at the doses employed in the NTP (2008) study 

31 resulted from an exceedance of the reductive capacity of the rodent GI tract. This is important 

32 because if the effects observed only occurred due to the reductive capacity of the rodent GI tract 
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1 being exceeded, these results may be less relevant to human risk at the lower doses that humans 

2 are more likely to be exposed. 

3 In discussing the results of the NTP (2008) study, the original NTP investigators, Stout et 

4 al. (2009), specifically addressed this extracellular reduction issue. Qualitatively, Stout et al. 

5 (2009) noted that, in the two-year NTP study, the observed increases in neoplasms of the small 

6 intestine of mice and the toxicity to the erythron, histiocytic infiltration, and uptake of hexavalent

7 chromium into the tissues of rats and mice suggested that, under the conditions of this study, at 

8 least a portion of the administered hexvalent chromium was not reduced in the stomach. 

9 Moreover, Stout et al. (2009) also pointed out the significant disparity in the oral toxicity and 

10 carcinogenicity of hexavalent chromium versus trivalent chromium in rodents, including the 

11 absence of increases in neoplasms or nonneoplastic lesions of the small intestine in rats or mice 

12 exposed to chromium picolinate monohydrate, a trivalent chromium compound tested in an 

13 earlier NTP bioassay. Stout et al. (2009) believe that these data provide additional evidence that 

14 hexavalent chromium is not completely reduced in the stomach and is responsible for the 

15 observed effects. 

16 In addressing the De Flora et al. (2008) suggestion that increases in neoplasms of the 

17 small intestine observed in mice are the result of a saturation of the gastric reduction capacity, 

18 Stout et al. (2009) took a more quantitative approach. Stout et al. (2009) postulated that if the 

19 threshold mechanism proposed by De Flora et al. (2008) actually existed, the dose that saturated 

20 the reduction capacity would likely represent an inflection point on a sublinear dose-response 

21 curve, with doses above the inflection point demonstrating an increasing rate of response per unit 

22 dose. To test this hypothesis, Stout et al. (2009) evaluated tissue concentration and mouse small 

23 intestine neoplasm data for linearity and found that data that were statistically nonlinear were 

24 supralinear (i.e., exhibited a decreasing rate of response per unit dose), which does not support 

25 the presence of a reduction threshold. 

26 Finally, De Flora et al. (1997) estimated the reductive capacity of human gastric juice to 

27 be about 84 to 88 mg of hexavalent chromium per day. Similar data are not available for the 

28 reductive capacity of mouse gastric juice. However, Stout et al. (2009) assumed that hexavalent 

29 chromium reduction is equally effective in mice and humans and that gastric secretion scales 

across species by body weight3/4 30 . Then, they estimated the reductive capacity of the gastric juice 

31 from a 50-g mouse to be approximately 0.4 mg/day (8 mg/kg-day). Stout et al. (2009) then 

32 pointed out that this value is greater than all of the male mouse doses and is nearly equivalent to 

33 the average daily dose of hexavalent chromium in the high-dose group of female mice in the 
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1 NTP (2008) study. Therefore, Stout et al. (2009) concluded from their analysis that the 

2 neoplasms in the small intestine of mice occurred at dose levels that did not exceed the estimated 

3 hexavalent chromium reduction capacity of the gastric juices in mice. 

4 

5 4.6.3.3. Other Possible Modes of Action 

6 It has been proposed that cellular proliferation subsequent to cytotoxicity may be 

7 involved in the carcinogenicity of hexavalent chromium. Evidence of diffuse duodenal 

8 hyperplasia in mice in all exposure groups was observed in the 3-month NTP (2007) study. The 

9 sites where hyperplasia was observed correlated with the site of tumors observed in the two-year 

10 bioassay (NTP, 2008). However, there is no evidence that this hyperplasia occurred prior to 

11 mutagenicity. Temporal evidence exists of mutagenicity occurring within 24 hours of exposure 

12 (see above), making it unlikely that this mutagenesis was the result of regenerative proliferation. 

13 In addition, several mutagenicity studies specifically measuring cytotoxicity reported positive 

14 findings at doses below those inducing toxicity, including in vivo rodent studies by Itoh and 

15 Shimada (1996), Sekihashi et al. (2001), Devi et al. (2001), NTP (2007), and Coogan et al. 

16 (1991b). Therefore, although hyperplasia is involved in the carcinogenic process, a mode of 

17 action involving cytotoxicity and hyperplasia is not regarded as an initial causative event. 

18 

19 4.6.3.4. Conclusions About the Hypothesized Mode of Action 

20 As noted above, hexavalent chromium is hypothesized to be carcinogenic by a mutagenic 

21 mode of action. The key events in the hypothesized mutagenic mode of action are the uptake of 

22 hexavalent chromium into the cell followed by intracellular reduction to pentavalent, tetravalent, 

23 and trivalent chromium. These reduced forms of hexavalent chromium and the free radicals that 

24 are formed during the reduction process are capable of directly interacting with cellular 

25 components, giving rise to mutagenicity (including DNA adduct formation, DNA damage, gene 

26 mutations, chromosomal aberrations, and micronuclei formation). Considering the database, 

27 there is evidence that hexavalent chromium can accumulate and induce mutagenicity in various 

28 tissues throughout the body at doses relevant to human exposures and, for oral exposures, within 

29 the reductive capacity of the gastrointestinal tract. 

30 

31 1. Is the hypothesized mode of action sufficiently supported in the test animals?

32 The experimental evidence that hexavalent chromium is mutagenic, as presented in 

33 Section 4.5.1, includes multiple adverse genetic effects including DNA adduct formation, DNA 
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1 damage, gene mutations, chromosomal aberrations, and the formation of micronuclei. In 

2 addition to the evidence supporting a mutagenic mode of action in test animals, alternative or 

3 additional hypothesized modes of action for hexavalent chromium carcinogenicity have not been 

4 demonstrated. 

5 

6 2. Is the hypothesized mode of action relevant to humans?

7 Mutagenicity is a well-established cause of carcinogenicity. The evidence discussed 

8 above demonstrates that hexavalent chromium is a mutagen in bacteria, yeast, cultured rodent 

9 and human cells, fruit flies, mice, and rats, supporting the presumption that it could also be a 

10 mutagen in humans. Moreover, several studies of exposed workers provide direct evidence of 

11 DNA damage by hexavalent chromium. In conclusion, the weight of evidence supports a 

12 mutagenic mode of action for hexavalent chromium carcinogenicity. 

13 

14 3. Which populations or lifestages can be particularly susceptible to the hypothesized mode of 

15 action?

16 The mutagenic mode of action is considered relevant to all populations and lifestages. 

17 According to EPA’s Supplemental Guidance (U.S. EPA, 2005b), there may be increased 

18 susceptibility to early-life exposures for carcinogens with a mutagenic mode of action. 

19 Therefore, because the weight of evidence supports a mutagenic mode of action for hexavalent 

20 chromium carcinogenicity and in the absence of chemical-specific data to evaluate differences in 

21 susceptibility, early-life susceptibility should be assumed and the age-dependent adjustment 

22 factors (ADAFs) should be applied, in accordance with the Supplemental Guidance. In addition, 

23 individuals with genetic polymorphisms conveying deficiencies in DNA repair capacity may 

24 have increased susceptibility to hexavalent chromium carcinogenicity. 

25 

26 4.6.3.5 Mutagenic Across All Routes of Exposure 

27 There is evidence that ingested hexavalent chromium can reach the systemic circulation 

28 and affect tissues beyond those at or near the site of entry.

29 Following inhalation exposures, hexavalent chromium has been shown to induce lung 

30 tumors in a number of human studies. In addition to hexavalent chromium activity in the lungs, 

31 evidence exists that hexavalent chromium is absorbed by the lung when inhaled and can then 

32 enter systemic circulation. Consistent with this, DNA damage, micronucleus induction, and 

33 sister chromatid exchanges have been observed in circulating peripheral lymphocytes from 
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workers exposed to inhalation concentrations as low as 7.5 and 24.9 μg/m31 (Benova et al., 2002), 

2 and for durations of 4 months to 14 years (Gambelunghe et al., 2003), 0.5 to 18 years (Stella et 

3 al., 1982), 2 to >20 years (Benova et al., 2002), or 4 to 25 years (Vaglenov et al., 1999). These 

4 mutagenicity studies indicate that, while tumor incidence following inhalation exposure to 

5 hexavalent chromium occurs primarily in the lungs, hexavalent chromium also has the capacity 

6 to damage DNA in other tissues at timepoints and concentrations relevant to human exposures. 

7 EPA has concluded that hexavalent chromium is carcinogenic by a mutagenic mode of 

8 action. Considering the available oral and inhalation evidence for mutagenicity and subsequent 

9 carcinogenicity and that these events are capable of occurring in all cells, this mode of action is 

10 applicable to all routes of exposure and tumor types. 

11 

12 4.7. SUSCEPTIBLE POPULATIONS AND LIFE STAGES 

13 4.7.1. Possible Childhood Susceptibility

14 No studies are available that address the possible adverse effects of hexavalent chromium 

15 in children. However, there is evidence that hexavalent chromium may act through a mutagenic 

16 mode of action. In accordance with the Supplemental Guidance (U.S. EPA, 2005b), the 

17 mutagenic mode of carcinogenic action for hexavalent chromium would indicate an increased 

18 carcinogenic susceptibility for early-life exposures. In addition, developmental toxicity also is of 

19 concern due to the mutagenicity of hexavalent chromium and the possibility for genetic damage 

20 to the germ cells of the F1 generation that could be transmitted to the F2 generation. The 

21 reproductive and developmental toxicity studies that have been conducted employing hexavalent 

22 chromium suggest that the developing fetus may be a target of toxicity, as well as male and 

23 female reproductive organs, which may result in a reduction in fertility. 

24 

25 4.7.2. Possible Gender Differences

26 The extent to which men and women differ in susceptibility to hexavalent chromium is 

27 unknown. However, animal data exist that imply a difference between males and females in 

28 their response to ingestion of hexavalent chromium. For example, in the NTP (2008) study, at 

29 the highest concentration administered (516 mg/L), female rats exhibited a higher incidence of 

30 tumors of the oral cavity than male rats (i.e., 11/48 (23%) versus 7/50 (14%), respectively). The 

31 biological significance of this finding at lower doses and for other species, including humans, is 

32 unknown. 

33 
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1 5. DOSE-RESPONSE ASSESSMENTS 

2 

3 

4 5.1. ORAL REFERENCE DOSE (RfD) 

5 5.1.1. Choice of Principal Study and Critical Effect—with Rationale and Justification 

6 Two types of studies are available that provide information on the toxicological effects of 

7 ingested chromium in humans. The first type of study provides evidence of acute human health 

8 effects in individuals who accidentally or intentionally ingested high (fatal or near-fatal) doses of 

9 hexavalent chromium. The second type of study provides evidence of chronic human health 

10 effects (primarily cancer) in populations exposed unintentionally to food or drinking water 

11 containing high levels of hexavalent chromium over an extended time period. Because both 

12 types of studies provide little information on dose-response relationships and because the second 

13 type of study is primarily concerned with cancer as an outcome, these available human data are 

14 not useful for quantifying the risk of noncancer effects resulting from chronic exposure to 

15 hexavalent chromium. 

16 In animals, the effects of subchronic oral exposure to hexavalent chromium have been 

17 evaluated in rats (NTP, 2007; Quinteros et al., 2007; Rafael et al., 2007; Acharya et al., 2001; 

18 Chopra et al., 1996; Vyskocil et al., 1993) and mice (NTP, 2007; Asmatullah and Noreen, 1999), 

19 and the effects of chronic oral exposure to hexavalent chromium have been evaluated in rats 

20 (NTP, 2008; MacKenzie et al., 1958), mice (NTP, 2008), and dogs (Anwar et al., 1961). In 

21 particular, the subchronic and chronic studies conducted by NTP (2008, 2007) provide the most 

22 useful dose-response data on the noncancer effects of oral hexavalent chromium exposure 

23 because of their comprehensive assessments of numerous toxicological endpoints at multiple 

24 dose levels. A number of other studies of reproductive and developmental toxicity of hexavalent 

25 chromium have been conducted in rats, mice and rabbits. These studies are summarized in Table 

26 4-25. 

27 Results from the NTP (2007) subchronic (i.e., 90-day) study identified several hexavalent 

28 chromium-induced noncancer effects, including hematological effects, hepatotoxicity, alterations 

29 in lipid metabolism, and histopathological changes in GI tissues, and pancreatic and mesenteric 

30 lymph nodes. The most sensitive hexavalent chromium-induced noncancer effects were 

31 microcytic, hypochromic anemia, increased serum liver enzyme activities, and histopathological 

32 changes to the duodenum and pancreatic lymph nodes in rats; and histopathological changes in 
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1 the duodenum in mice. In the two-year toxicology and carcinogenicity study by NTP (2008), the 

2 most sensitive noncancer effects identified were histopathological changes to the liver, 

3 duodenum, and mesenteric lymph nodes in rats; and in the duodenum, mesenteric lymph nodes, 

4 and liver in mice. LOAELs of 1.7—3.1 mg hexavalent chromium/kg-day were identified in the 

5 subchronic NTP (2007) study, and LOAELs of 0.24—0.77 mg hexavalent chromium/kg-day 

6 were identified in the chronic NTP (2008) study. 

7 Other subchronic and chronic oral exposure studies of hexavalent chromium compounds 

8 do not provide suitable data for identifying points of departure (PODs) for RfD derivation 

9 because comprehensive toxicological evaluations were not conducted in these studies. In 

10 addition, interpretation of results from these studies was compromised because of the small 

11 number of animals evaluated, the lack of a dose-response relationship, or inadequate reporting of 

12 results (see Table 4-25). Where LOAELs were identified based on examination of a limited set 

13 of endpoints (e.g., Acharya et al, 2001; Chopra et al, 1996), the LOAELs were higher than those 

14 identified in the chronic NTP (2008) bioassay. 

15 Studies of reproductive and developmental toxicity indicate that hexavalent chromium 

16 exposure can affect reproductive organs, increase pre- and postnatal implantation loss, and cause 

17 reduced fetal weight and fetal abnormalities. In general, the NOAELs or LOAELs associated 

18 with reproductive and developmental effects are higher than those identified in the subchronic 

19 and chronic toxicity studies summarized in Table 4-25. 

20 Thus, based on the comprehensive examination of endpoints and measurement of 

21 sensitive endpoints of toxicity, the bioassays by NTP (2008, 2007) were deemed the best 

22 candidates for use in deriving an oral RfD for hexavalent chromium. Specifically, five studies, 

23 three subchronic (i.e., one in rats and two in mice) (NTP, 2007) and two chronic (i.e., one in rats 

24 and one in mice) (NTP, 2008), were identified as candidate principal studies. The key results 

25 from these five studies are summarized below. 

26 

27 Subchronic Studies

28 NTP (2007) 90-day studies in rats and mice 

29 In F344/N rats, sodium dichromate dihydrate was administered in drinking water to 

30 groups of males and females at five different concentrations for 90 days. Based on average 

31 water consumption rates, the mean effective doses of hexavalent chromium were estimated by 
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1 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 

2 study identified a LOAEL in male and female rats of 1.7 mg hexavalent chromium/kg-day; a 

3 NOAEL was not identified because effects were observed at the lowest dose tested. This 

4 LOAEL was based on observations of microcytic, hypochromic anemia, increased serum liver 

5 enzyme activities, and histopathological changes to pancreatic lymph nodes (in males) and 

6 histopathological changes to the duodenum (in females) at daily doses ≥1.7 mg hexavalent 

7 chromium/kg-day. 

8 In B6C3F1 mice, groups of males and females were exposed to sodium dichromate 

9 dihydrate in drinking water for 90 days. Based on water consumption monitored throughout the 

10 study, NTP calculated average daily doses over the 90-day treatment duration of approximately 

11 0, 3.1, 5.3, 9.1, 15.7, and 27.9 mg hexavalent chromium/kg-day for both males and females. 

12 Based on histopathological changes (histiocytic cellular infiltration) in the duodenum in both 

13 sexes, a LOAEL of 3.1 mg hexavalent chromium/kg-day was identified for male and female 

14 mice; a NOAEL was not identified because the effects observed were at the lowest dose tested. 

15 In a comparative 90-day drinking water study in male B6C3F1, BALB/c, and am3-

16 C57BL/6 mice, groups of each strain were exposed to three different concentrations of sodium 

17 dichromate dihydrate. Based on water consumption and body weights monitored throughout the 

18 study, NTP calculated average daily doses over the 90-day treatment duration of approximately 

19 0, 2.8, 5.2, or 8.7 mg hexavalent chromium/kg-day for all strains. At the end of the study, 

20 similar effects were observed in all 3 strains. A LOAEL of 2.8 mg hexavalent chromium/kg-day 

21 was identified based on histopathological changes in the duodenum in B6C3F1 mice (histiocytic 

22 cellular infiltration and epithelial hyperplasia), BALB/c mice (histiocytic cellular infiltration), 

23 and am3-C57BL/6 mice (epithelial hyperplasia); a NOAEL was not identified because effects 

24 seen were at the lowest dose tested. 

25 

26 Chronic Studies

27 NTP (2008) two-year studies in rats and mice 

28 In F344/N rats, groups of 50 males and females were administered sodium dichromate 

29 dihydrate in drinking water at four different concentrations for two years. Based on measured 

30 water consumption rates and body weights in rats, NTP estimated that male rats received time31 weighted average doses of hexavalent chromium of 0.21, 0.77, 2.1, or 5.9 mg/kg-day, while 
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1 female rats received 0.24, 0.94, 2.4 or 7.0 mg/kg-day. This study identified NOAEL and 

2 LOAEL values for noncancer effects in male rats of 0.21 and 0.77 mg hexavalent chromium/kg3 day, respectively, based on increased incidences of nonneoplastic histopathological changes to 

4 the liver (basophilic foci), duodenum (histiocytic cellular infiltrate), and mesenteric lymph nodes 

5 (histiocytic cellular infiltrate and hemorrhage). In female rats, a LOAEL for noncancer effects of 

6 0.24 mg hexavalent chromium/kg-day was identified based on the increased incidence of chronic 

7 inflammation of the liver (observed in all treatment groups); a NOAEL was not identified 

8 because effects observed were at the lowest dose tested. 

9 In B6C3F1 mice, groups of 50 males and females were administered sodium dichromate 

10 dihydrate in drinking water at four different concentrations for two years. Based on measured 

11 amounts of water consumption and body weights in mice, NTP estimated that male mice 

12 received average doses of hexavalent chromium of 0.38, 0.91, 2.4, or 5.9 mg/kg-day, while 

13 female mice received 0.38, 1.4, 3.1 or 8.7 mg/kg-day. This study identified a LOAEL for 

14 noncancer effects of 0.38 mg hexavalent chromium/kg-day in both male and female B6C3F1

15 mice; a NOAEL value was not identified because effects seen were at the lowest dose 

16 administered. In males, the LOAEL was based on increased incidences of histopathological 

17 changes to the duodenum (diffuse epithelial hyperplasia) and mesenteric lymph nodes 

18 (histiocytic cellular infiltration); in females, the LOAEL was based on increased incidences of 

19 histopathological changes to the duodenum (diffuse epithelial hyperplasia), mesenteric lymph 

20 nodes (histiocytic cellular infiltration), liver (histiocytic cellular infiltration), and pancreas 

21 (depletion of cytoplasmic zymogen granules). 

22 The NTP (2008) study was of chronic duration (i.e., 2 years), involved the use of multiple 

23 dose groups, and included a comprehensive evaluation of multiple endpoints. Also, this bioassay 

24 used lower doses than the subchronic (90-day) studies also conducted by NTP (2007), and thus 

25 provided dose-response information at lower exposure levels than the 90-day studies. 

26 Additionally, the chronic NTP (2008) study was more sensitive, yielding lower LOAELs than 

27 the subchronic studies. Thus, the chronic NTP (2008) study was selected as the principal study. 

28 As indicated, NTP (2008) observed several hexavalent chromium-induced noncancer 

29 effects in their chronic studies in rats and mice. Based on a comparison of LOAELs in rats and 

30 mice (Table 4-25), the lowest LOAELs were observed for the following seven effects: 

31 
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1 1. Chronic liver inflammation in female rats, 

2 2. Histiocytic cellular infiltration in the liver of female mice, 

3 3. Diffuse epithelial hyperplasia in the duodenum of male mice, 

4 4. Diffuse epithelial hyperplasia in the duodenum of female mice, 

5 5. Histiocytic cellular infiltration in the mesenteric lymph nodes of male mice 

6 6. Histiocytic cellular infiltration in the mesenteric lymph nodes of female mice, 

7 7. Cytoplasmic cellular alteration of acinar epithelial cells in the pancreas of female mice. 

8 

9 All of these effects occurred at the lowest doses tested (i.e., 0.24 mg/kg-day in female 

10 rats and 0.38 mg/kg-day in male and female mice), and were considered as possible critical 

11 effects for derivation of the RfD for hexavalent chromium. The incidences of these seven effects 

12 across all treatment groups in NTP (2008) are shown below in Table 5-1. 

13 
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Table 5-1. Incidence Data for Lesions From All Treatment Groups of 

Female F344/N Rats and Male and Female B6C3F1 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 21/50a28/50b35/50b39/50b

Dose 

(mg hexavalent chromium/kg-day) 

0 0.38 0.91 2.4 5.9 

Male Mice 

Duodenum: Diffuse epithelial 

hyperplasia 0/50 11/50b

18/50b42/50b32/50a

Mesenteric lymph node: Histiocytic 

cellular infiltration 14/47 38/47b

31/49b32/49b42/46a

Dose 

(mghexavalent chromium/kg-day) 

0 0.38 1.4 3.1 8.7 

Female Mice 

Duodenum: Diffuse epithelial 

hyperplasia 0/50 16/50b

35/50b31/50b42/50b

Mesenteric lymph node: Histiocytic 

cellular infiltration 3/46 29/48b

26/46b40/50b42/50b

Liver: Histiocytic cellular infiltration 2/49 15/50b23/50b32/50b45/50b

Pancreas: Acinus, cytoplasmic 

alteration 0/48 6/50a

6/49a 14/50b32/50b

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 

2 5.1.2. Methods of Analysis—Including Models (PBPK, BMD, etc.) 

3 To determine the specific endpoint for use in derivation of the RfD, all available 

4 dichotomous models in the EPA’s Benchmark Dose Software (BMDS) were fit to the incidence 

5 data for the seven selected endpoints in female rats and male and female mice administered 

6 sodium dichromate dihydrate in drinking water for two years (NTP, 2008). The incidence data 

7 employed in the BMD modeling of these seven endpoints are shown in Table 5-1. To provide 

8 candidate PODs based on these endpoints, 10% extra risk was selected as the benchmark 

9 response (BMR) in accordance with U.S. EPA’s Benchmark Dose Technical Guidance (U.S. 
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1 EPA, 2000), which recommends selecting a response level near the lower range of detectable 

2 observations that also facilitates comparisons across endpoints. 

3 For chronic liver inflammation in female rats, the log-logistic model provided the best fit, 

4 yielding BMD10 and BMDL10 values of 0.22 and 0.14 mg hexavalent chromium/kg-day, 

5 respectively. For diffuse epithelial hyperplasia in the duodenum of male mice, the multistage 

6 and quantal linear models provided the best fit, yielding BMD10 and BMDL10 values of 0.16 and 

7 0.13 mg hexavalent chromium/kg-day, respectively. For diffuse epithelial hyperplasia in the 

8 duodenum of female mice, the best fit was provided by several models (i.e., gamma, multistage, 

9 quantal linear, and Weibull), yielding BMD10 and BMDL10 values of 0.12 and 0.09 mg 

10 hexavalent chromium/kg-day, respectively. For both histiocytic cellular infiltration in the liver 

11 and acinar cytoplasm alteration in the pancreas of female mice, the log-logistic model provided 

12 the best fit, yielding BMD10 and BMDL10 values of 0.17 and 0.12 mg hexavalent chromium/kg13 day, respectively, for the liver lesions and 0.68 and 0.52 mg hexavalent chromium/kg-day, 

14 respectively, for the pancreatic lesions. Finally, for the lesions of the mesenteric lymph nodes 

15 (i.e., histiocytic cellular infiltration) in male and female mice, none of the available dichotomous 

16 models in BMDS provided adequate fit to the data, even with the two highest doses dropped 

17 from the analysis; thus, data sets for these lesions were considered to be not suitable for BMD 

18 analysis. A summary of this BMD modeling information is presented in Table 5-2, and further 

19 details of this modeling are contained in Appendix B-1. 

20 
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Table 5-2. Summary of BMD10 and BMDL10 from the best fitting models 

for lesions of the liver, duodenum, mesenteric lymph nodes, and pancreas 

in female rats and male and female mice after exposure to sodium 

dichromate dihydrate in drinking water for 2 years (NTP, 2008) 

End point Species/sex Model 

Number 

of doses 

BMDa

(mg/kg-day) 

BMDLa

(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 infiltrationb

Mouse/male –— –— –— –— 

Duodenum: 

diffuse epithelial 

hyperplasia 

Mouse/female Gamma/Multistage/quantal 

linear/Weibull 3 0.12 0.09 

Mesenteric lymph 

node: histiocytic 

cellular infiltrationb

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 

aBMDs and BMDLs from dichotomous data are associated with a 10% extra risk; doses are in terms of mg 

hexavalent chromium/kg-day. 

bNone 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 

2 The lowest BMDL10 value of 0.09 mg hexavalent chromium/kg-day, based on the 

3 selection of the incidence of diffuse epithelial hyperplasia of the duodenum in female mice as the 

4 critical effect, was identified as the POD from which to derive the RfD for hexavalent chromium. 

5 

6 5.1.3. RfD Derivation—Including Application of Uncertainty Factors (UFs) 

7 The following uncertainty factors (UFs) were applied to the POD of 0.09 mg/kg-day, 

8 based on the incidence of diffuse epithelial hyperplasia of the duodenum in female mice from 

9 NTP (2008), to derive the RfD for hexavalent chromium. 

10 
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1 • An UF of 10 was used to account for uncertainty in extrapolating from laboratory 

2 animals to humans (i.e., interspecies variability) because information was 

3 unavailable to quantitatively assess toxicokinetic or toxicodynamic differences 

4 between animals and humans. 

5 

6 • An UF of 10 was used to account for variation in susceptibility among members of 

7 the human population (i.e., interindividual variability) because information is 

8 unavailable to predict potential variability in human susceptibility. 

9 

10 • An UF was not needed to account for extrapolation from subchronic-to-chronic 

11 exposure because a chronic study was used to derive the chronic RfD. 

12 

13 • An UF for LOAEL to NOAEL extrapolation was not used because the current 

14 approach is to address this extrapolation as one of the considerations in selecting a 

15 BMR for BMD modeling. In this case, a BMR represented by a 10% extra risk of 

16 diffuse epithelial hyperplasia was selected under an assumption that it represents a 

17 minimal biologically significant change. 

18 

19 • An UF of 1 was used to account for database deficiencies. The toxicity of ingested 

20 hexavalent chromium has been extensively examined in a range of animal 

21 toxicology studies. The database for oral toxicity includes a chronic drinking water 

22 study in rats and mice, a chronic drinking water study in rats, a subchronic drinking 

23 water study in rats and mice, and a number of reproductive/developmental toxicity 

24 studies in monkeys, rabbits, rats, and mice. The reproductive toxicity database 

25 includes a continuous breeding study (NTP, 1997), in which F0 and F1 generation 

26 animals were exposed to hexavalent chromium in the diet, and the offspring of F1

27 animals were evaluated on PND 21. 

28 

 For this assessment, the RfD of 0.0009 or 9 x 10-4 29 mg/kg-day for hexavalent chromium 

30 was derived by dividing the BMDL10 (or POD) of 0.09 mg/kg-day by a composite uncertainty 

31 factor of 100 (10 for extrapolation from animals to humans and 10 for human variability). 

32 

33 5.1.4. Previous RfD Assessment 

34 The previous RfD assessment for hexavalent chromium was completed in September 

35 1998. The previous RfD was based on a NOAEL identified from a one-year drinking water 

36 study in rats in which animals were exposed to hexavalent chromium (as potassium chromate) at 

37 a dose of 2.5 mg/kg-day (MacKenzie et al., 1958). No toxicity was reported in these animals at 

38 this dose, resulting in identification of a NOAEL of 2.5 mg/kg-day, the only dose administered in 

39 the study, as the POD. A composite uncertainty factor of 300 (10 for interspecies extrapolation, 

40 10 for intraspecies extrapolation, and 3 for subchronic to chronic extrapolation) and a modifying 
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1 factor of 3 (to account for concerns raised by the epidemiology study of Zhang and Li, 1987) 

were applied to this POD to yield an oral RfD of 3 x 10-3 2 mg/kg-day. 

3 

4 5.2. UNCERTAINTIES IN THE ORAL REFERENCE DOSE 

5 The following discussion identifies uncertainties associated with the RfD for 

hexavalent chromium. As presented above, an RfD of 9 x 10-4 6 mg/kg-day was derived based on 

7 the incidence of diffuse epithelial hyperplasia of the duodenum in female mice from a two-year 

8 drinking water study (NTP, 2008). UFs were applied to the POD, a BMDL10 generated through 

9 BMD modeling. Factors accounting for uncertainties associated with a number of steps in the 

10 analyses were adopted to account for extrapolating from an animal bioassay to humans with 

11 varying susceptibilities. 

12 An adequate range of animal toxicology data is available for the hazard assessment of 

13 hexavalent chromium via ingestion, as described previously in Section 4. The database of oral 

14 toxicity studies includes a chronic drinking water study in rats and mice, a chronic drinking 

15 water study in rats, a subchronic drinking water study in rats and mice, and several 

16 reproductive/developmental toxicity studies in monkeys, rabbits, rats, and mice. Toxicity 

17 associated with oral exposure to hexavalent chromium is observed in the liver, GI tract, and 

18 reproductive organs, with the liver and GI tract being the most sensitive target organs. In 

19 addition to oral toxicity data, there are absorption, distribution, metabolism, and excretion 

20 studies, although information on internal or target organ dose of hexavalent chromium is not 

21 available. 

22 Consideration of the available dose-response data to determine an estimate of oral 

23 exposure that is likely to be without an appreciable risk of adverse health effects over a lifetime 

24 led to the selection of the two-year drinking water study in F344/N rats and B6C3F1 mice (NTP, 

25 2008) and increased incidence of diffuse epithelial hyperplasia in the duodenum of female mice 

26 as the principal study and critical effect, respectively, for deriving the RfD for hexavalent 

27 chromium. 

28 The selection of the BMD model for identifying the POD does not lead to significant 

29 uncertainties since benchmark effect levels were within the range of the experimental data. 

30 However, the selected models do not represent all possible models one might fit, and other 
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1 models could be selected to yield more extreme results, both higher and lower than those 

2 included in this assessment. 

3 Animal to human extrapolation yields further uncertainties. The effect and the 

4 magnitude of this effect associated with the dose at the POD in mice are extrapolated to humans. 

5 Pharmacokinetic models are useful to examine species differences in pharmacokinetic 

6 processing; however, dosimetric adjustment using pharmacokinetic modeling was not possible 

7 for the toxicity observed following oral exposure to hexavalent chromium. Information was 

8 unavailable to quantitatively assess toxicokinetic or toxicodynamic differences between animals 

9 and humans. Accordingly, a 10-fold UF was used to account for uncertainty in extrapolating 

10 from laboratory animals to humans in the derivation of the RfD. 

11 Heterogeneity among humans is another area of uncertainty. In the absence of 

12 hexavalent chromium-specific data on variation in human response, a factor of 10 was used in 

13 the derivation of the RfD. Human variation may be larger or smaller than this 10-fold factor; 

14 however, hexavalent chromium-specific data to examine the potential magnitude of over- or 

15 underestimation are unavailable. 

16 

17 5.3. ORAL CANCER ASSESSMENT 

18 5.3.1. Choice of Study/Data—with Rationale and Justification 

19 Several epidemiology studies have examined the association between oral exposure to 

20 environmental hexavalent chromium and cancer in populations that resided near sources of 

21 industrial waste containing hexavalent chromium compounds, including studies of populations in 

22 Liaoning Province, China (Kerger et al., 2009; Beaumont et al., 2008; Zhang and Li, 1997, 1987, 

23 1980), Kings County/San Bernadino County, California (Fryzek et al., 2001), Nebraska (Bednar 

24 and Kies, 1991), and Glasgow, UK (Eizaguirre-Garcia et al., 2000, 1999). The Liaoning 

25 Province studies provide some evidence of an excess risk of mortality from stomach cancer; 

26 however, because of various limitations, including limited characterization of exposure, the 

27 Liaoning Province studies are not considered adequate for dose-response analysis. 

28 The NTP rodent bioassay, in which F344/N rats and B6C3F1 mice were administered 

29 sodium dichromate dihydrate, a hexavalent chromium compound, in drinking water for two years 

30 (NTP, 2008), was selected as the basis for deriving the oral cancer slope factor for hexavalent 

31 chromium. This bioassay was selected for dose-response assessment because it is a well-
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1 conducted lifetime animal study of hexavalent chromium carcinogenicity via ingestion (see 

2 detailed summary of the study in Section 4.2.1.2). No other adequate studies of hexavalent 

3 chromium carcinogenicity by ingestion are available. 

4 

5 5.3.2. Dose-Response Data 

6 The dose-response data considered in the derivation of the cancer slope factor (CSF) for 

7 hexavalent chromium were the incidence of benign and malignant tumors in rat oral mucosa and 

8 mouse small intestine observed in the NTP (2008) bioassay. 

9 Incidence data for neoplastic lesions of the oral cavity in male and female rats exposed to 

10 sodium dichromate dihydrate in drinking water for 2 years are summarized in Table 4-15. 

11 Neoplasms observed in the oral cavity of treated rats were squamous cell carcinoma of the oral 

12 mucosa (both sexes), squamous cell papilloma of the oral mucosa (males only), squamous cell 

13 carcinoma of the tongue (both sexes), and squamous cell papilloma of the tongue (both sexes). 

14 The incidences of squamous cell carcinoma of the oral mucosa (13.6%) and of combined 

15 squamous cell papilloma or carcinoma (15.7%) of the oral mucosa were statistically significantly 

16 increased (at p < 0.05) in male rats treated with 5.9 mg/kg-day hexavalent chromium (the highest 

17 dose tested) compared with controls. The incidences of squamous cell carcinoma of the oral 

18 mucosa (23.9%) and of combined squamous cell carcinoma of the oral mucosa or tongue 

19 (23.9%) were statistically significantly increased (at p < 0.05) in female rats treated with 7.0 mg 

20 hexavalent chromium/kg-day (the highest dose tested) compared with controls. The incidences 

21 of other neoplastic lesions of the oral cavity were not statistically significantly increased in any 

22 treatment group in male or female rats compared with controls, although the incidence of 

23 squamous cell carcinoma of the oral mucosa in female rats in the penultimate (2.4 mg/kg-day) 

24 dose group (4.6%) exceeded that of historical controls (i.e., 0/300 in drinking water studies; 

25 5/1,400 (0.4%) by all routes of exposure). Other neoplasms observed in treated rats included 

26 pancreatic acinar adenomas and benign pheochromocytomas in males and mononuclear cell 

27 leukemias in females (see Table 4-16); however, the incidence of these neoplasms did not exhibit 

28 dose-dependence. Thus, NTP (2008) concluded that evidence of a relationship between 

29 neoplastic changes in tissues other than the oral cavity and exposure to sodium dichromate 

30 dihydrate was equivocal. In summary, exposure of rats to sodium dichromate dihydrate in 

31 drinking water for 2 years resulted in a significant increase in squamous epithelial neoplasms of 
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1 the oral mucosa and tongue at the highest exposure levels (average daily doses of 5.9 and 7.0 mg 

2 hexavalent chromium/kg-day in males and females, respectively), but not at the three lower 

3 exposure levels. The incidences of squamous cell papillomas or carcinomas in the oral cavity of 

4 male and female F344/N rats exposed to sodium dichromate dihydrate in drinking water for 2 

5 years in the NTP (2008) study are presented in Table 5-3 (for male rats) and Table 5-4 (for 

6 female rats). 

7 

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) 

Sodium dichromate dihydrate 

concentration 

(mg/L) 

Estimated 

daily intake of hexavalent chromiuma

(mg/kg-day) 

Incidence of squamous cell 

papillomas or carcinomasb 

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 drinking water intakes and mean body weights observed during 

the study. 

bNumber of animals with lesion/number of animals examined. 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 
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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 chromiuma

(mg/kg-day) 

Incidence of squamous cell 

papillomas or carcinomasb 

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 drinking water intakes and mean body weights observed during 

the study. 

bNumber of animals with lesion/number of animals examined. 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 

2 Also from the NTP (2008) study, incidence data for neoplastic lesions of the small 

3 intestine in male and female B6C3F1 mice exposed to sodium dichromate dihydrate in drinking 

4 water for 2 years are summarized in Table 4-19. In male mice, statistically significant increases 

5 (at p < 0.05) were observed in the incidences of adenomas or carcinomas combined in the small 

6 intestine (duodenum, jejunum, and ileum) at hexavalent chromium doses ≥2.4 mg/kg-day (i.e., at 

7 the two highest doses tested). Furthermore, significant positive trends were observed in the 

8 incidences of duodenal adenomas, duodenal carcinomas, jejunal adenomas, small intestine 

9 adenomas, small intestine carcinomas and small intestine adenomas or carcinomas combined in 

10 male mice. In female mice, statistically significant increases (at p < 0.05) were observed in the 

11 incidences of duodenal adenomas, small intestine adenomas, and small intestine adenomas or 

12 carcinomas combined at hexavalent chromium doses ≥3.1 mg/kg-day (i.e., at the two highest 

13 doses tested). Furthermore, significant positive trends were observed in the incidences of 

14 duodenal adenomas, duodenal carcinomas, jejunal adenomas, small intestine adenomas, and 

15 small intestine adenomas or carcinomas combined in female mice. No other statistically or 

16 biologically significant increases in neoplasms were observed in other tissues. 

17 In summary, exposure of B6C3F1 mice to sodium dichromate dihydrate in drinking water 

18 for 2 years resulted in statistically significant increases in the incidences of neoplasms of the 

19 small intestine in males and females at hexavalent chromium doses ≥2.4 and ≥3.1 mg/kg-day, 
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1 respectively. The incidences of adenomas and carcinomas combined in the small intestine of 

2 male and female B6C3F1 mice exposed to sodium dichromate dihydrate in drinking water for 2 

3 years are summarized in Tables 5-2 and 5-3, respectively. In evaluating the tumor incidences in 

4 rats and mice, the mouse was determined to be the most sensitive species because tumor 

5 incidences were statistically significantly elevated at lower doses and a greater response was 

6 exhibited by the mice at the two highest doses. Therefore, the mouse tumor incidence data were 

7 used as the basis for the oral CSF derived employing BMD modeling. 

8 

Table 5-5. Incidences of adenomas and carcinomas combined in the small 

intestine of male B6C3F1 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 

chromiuma

(mg/kg-day) 

Incidence of adenomas or 

carcinomasb 

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 weights observed during 

the study. 

bCalculated from reported percentages of mice with adenomas or carcinomas. Incidence estimates included all 

animals that were examined for intestinal tumors and survived for at least 451 days. In each of the control and 

first two dose groups, one animal died prior to day 451. In the high-dose group, two animals died prior to day 

451. None of these animals were found to have intestinal 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 
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Table 5-6. Incidences of adenomas and carcinomas combined in the small 

intestine of female B6C3F1 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 

chromiuma

(mg/kg-day) 

Incidence of adenomas or 

carcinomasb 

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 drinking water intakes and mean body weights observed during 

the study. 

bCalculated from reported percentages of mice with adenomas or carcinomas. Incidence estimates included all 

animals that were examined for intestinal tumors and survived for at least 451 days. In all of the dose groups 

except the low-dose group, one animal died prior to day 451. None of these animals were observed to have 

intestinal adenomas or carcinomas at the time of death. 

c

Statistically significantly elevated above control at p < 0.05 using Fisher’s Exact Test. 

1 

2 

3 5.3.3. Dose Adjustments and Extrapolation Method(s) 

4 The EPA Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005a) recommend that 

5 the method used to characterize and quantify cancer risk from a chemical is determined by what 

6 is known about the mode of action of the carcinogen and the shape of the cancer dose-response 

7 curve. The dose response is assumed to be linear in the low-dose range when evidence supports 

8 a mutagenic mode of action because of DNA reactivity, or if another mode of action that is 

9 anticipated to be linear is applicable. A linear low-dose extrapolation approach was used to 

10 estimate human carcinogenic risk associated with hexavalent chromium exposure due to the 

11 mutagenic mode of carcinogenic action of this chemical. 

12 In order to derive an oral CSF, BMD modeling was carried out using the EPA’s BMDS 

13 (U.S. EPA, 2007). The EPA’s BMDS offers several possible mathematical dose-response 

14 functions for use with dichotomous data including logistic, gamma, Weibull, quantal linear, 

15 probit, and multistage models. For this assessment, EPA relied on the results obtained from the 

16 multistage model only, as this is the model preferred by the Agency for conducting cancer dose17 response assessments. In applying the benchmark dose approach to the derivation of a CSF, the 

18 standard procedure is to calculate a lower 95% confidence bound on the dose corresponding to 
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1 the benchmark response (BMR), where the BMR is typically set at 10% extra risk. This lower 

2 confidence bound is referred to as the BMDL10. The CSF is then calculated by dividing the 

3 BMR by the BMDL10 and then converting this slope value to human equivalents. 

4 In estimating the CSF, the incidence of neoplasms in the small intestine of mice was 

5 employed, as this species was deemed to be more sensitive than the rat. Only animals that 

6 survived for at least 451 days, the time until appearance of the first tumor, were considered at 

7 risk for tumor development. Consequently, the incidence estimates included all animals that were 

8 examined for intestinal tumors and survived for at least 451 days (see Tables 5-5 and 5-6). The 

9 BMD modeling results for the incidence of neoplasms in the small intestine of male and female 

10 mice are shown in Appendix B-2. For male mice, the two-stage multistage model exhibited the 

best fit to the data yielding a slope of 0.09 (mg/kg-day)-1 11 . For female mice, the two-stage 

multistage model also exhibited the best fit to the data yielding a slope of 0.10 (mg/kg-day)-1 12 . 

13 In order to estimate an oral CSF, these slopes were converted to human equivalents. For 

14 this conversion, body weight to the 3⁄4 power scaling was used, where the time-weighted average 

15 male and female mouse body weights of controls (i.e., 50 and 53 grams, respectively) were 

16 employed, along with an assumed human body weight of 70 kg. The mouse body weights were 

17 taken from the NTP (2008) study report. The following equation was then used to convert the 

18 slopes derived from the BMD modeling to oral CSFs expressed in human equivalents: 

19 

Slope x (WH/WA)

0.25 20 = CSF, 

21 where 

22 WH = animal body weight (kg) 

23 WA = human body weight (kg) 

24 

25 Using the above equation, the CSFs resulting from the fitting of the two-stage multistage 

26 model in BMDS to the incidence of neoplasms in the small intestine of male or female mice 

were 0.5 and 0.6 (mg/kg-day)-1 27 , respectively, expressed in human equivalents. 

28 

29 5.3.4. Oral Slope Factor 

30 The CSF values based on the incidence of small intestine tumors in male and female mice 

are similar (i.e., 0.5 (mg/kg-day)-1 for males and 0.6 (mg/kg-day)-1 31 for females). Given the 
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1 poorer fit of the multistage model to the female mouse data, a CSF estimate based on the male 

2 mouse data was considered to be associated with less uncertainty. Therefore, the CSF of 0.5 

(mg/kg-day)-1 3 , based on the incidence of neoplasms in the small intestine of male mice, was 

4 selected as the most appropriate CSF for hexavalent chromium. 

5 

6 5.3.5. Application of Age-Dependent Adjustment Factors 

7 Because a mutagenic mode of action for hexavalent chromium carcinogenicity is 

8 sufficiently supported in laboratory animals and is relevant to humans (see Section 4.6.3.4), and 

9 in the absence of chemical-specific data to evaluate differences in age-specific susceptibility, 

10 increased early-life susceptibility to hexavalent chromium is assumed and age-dependent 

11 adjustment factors (ADAFs) should be applied, as appropriate, in accordance with the 

12 Supplemental Guidance for Assessing Susceptibility from Early-Life Exposure to Carcinogens 

(U.S. EPA, 2005b). The oral slope factor of 0.5 (mg/kg-day)-1 13 , calculated from data applicable 

14 to adult exposures, does not reflect presumed early-life susceptibility to this chemical. Example 

15 calculations for estimating cancer risks based on age at exposure are provided in Section 6 of the 

16 Supplemental Guidance for Assessing Susceptibility from Early-Life Exposure to Carcinogens 

17 (U.S. EPA, 2005b). 

18 The Supplemental Guidance for Assessing Susceptibility from Early-Life Exposure to 

19 Carcinogens establishes ADAFs for three specific age groups. The current ADAFs and their 

20 corresponding age groups are 10 for exposed individuals <2 years old, 3 for exposed individuals 

21 2 to <16 years old, and 1 for exposed individuals 16 years old and older (U.S. EPA, 2005b). The 

22 10-fold and 3-fold adjustments to the slope factor are to be combined with age-specific exposure 

23 estimates when estimating cancer risks from early life (<16 years of age) exposures to hexavalent 

24 chromium. 

25 To illustrate the use of the ADAFs established in the Supplemental Guidance for 

26 Assessing Susceptibility from Early-Life Exposure to Carcinogens (U.S. EPA, 2005b), sample 

27 calculations are presented for three exposure duration scenarios, including full lifetime, assuming 

28 the exposure rate to hexavalent chromium remains constant at an average daily dose of 0.0001 

29 mg hexavalent chromium/kg-day (Table 5-7). 

30 
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Table 5-7. Application of ADAFs for a 70-year exposure 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 

2 Note that the partial risk for each age group is the product of the values in columns 2–5 

3 (e.g., 10 × 0.5 × 0.0001 × 2/70 = 0.00001 for exposures from age 0 to <2 years), and the total 

4 risk is the sum of the partial risks. Thus, a 70-year risk estimate for a constant average daily 

dose of 0.0001 mg/kg-day starting at birth is 0.00008 or 8 × 10-5 5 . 

6 If calculating the cancer risk for a 30-year exposure to a constant average daily dose of 

7 0.0001 mg hexavalent chromium/kg-day from ages 0 to 30 years, the duration adjustments would 

8 be 2/70, 14/70, and 14/70, and the partial risks would be 0.00001, 0.00003, and 0.00001, 

resulting in a total risk estimate of 0.00005 or 5 × 10-5 9 . 

10 If calculating the cancer risk for a 30-year exposure to a constant average daily dose of 

11 0.0001 mg hexavalent chromium/kg-day from ages 20 to 50 years, the duration adjustments 

12 would be 0/70, 0/70, and 30/70, and the partial risks would be 0, 0, and 0.00002, resulting in a 

total risk estimate of 0.00002 or 2 × 10-5 13 . 

14 

15 5.3.6. Uncertainties in Cancer Risk Values 

16 As in most risk assessments, extrapolation of data from experimental animals to estimate 

17 potential lifetime cancer risks to human populations from exposure to hexavalent chromium 

18 yields uncertainties. Some of these uncertainties can be evaluated for their quantitative impact 

19 on the final result, while for others, only their qualitative impact can be assessed. The principal 

20 uncertainties in the assessment of the cancer risk from exposure to hexavalent chromium are 

21 summarized below in Table 5-8, and discussed in more detail in the following text. 
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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 determine 

POD, linear lowdose extrapolation 

from POD 

A linear-low-dose extrapolation approach was used 

to estimate human carcinogenic risk associated 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 

BW2/3)] 

BW3/4 (default 

approach) 

In the absence of hexavalent chromium-specific 

information on interspecies differences in 

toxicokinetics, the default scaling factor of BW3/4

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., BMD10) used 

rather than lower 

bound (BMDL10) 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 

3 Choice of low-dose extrapolation approach. The mode of action is a key consideration in 

4 clarifying how risks should be estimated for low-dose exposure. A linear, low-dose 

5 extrapolation approach was used to estimate human carcinogenic risk associated with hexavalent 
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1 chromium exposure consistent with a hypothesized mutagenic mode of carcinogenic action of 

2 hexavalent chromium (U.S. EPA, 2005a). 

3 The multistage model was used to model the tumor incidence data because this is the 

4 model preferred by the Agency for conducting cancer dose-response assessments; however, it is 

5 unknown how well this model or the linear low-dose extrapolation predicts low-dose risks for 

6 hexavalent chromium. The selected model does not represent all possible models one might fit, 

7 and other models could conceivably be selected to yield more extreme results consistent with the 

8 observed data, both higher and lower than those included in this assessment. 

Cross-species scaling. The default cross-species scaling factor (BW3/4 9 ) was applied to 

10 address toxicological equivalence of internal doses between rodent species and humans, 

11 consistent with the 2005 Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005a). 

12 Because it is unknown whether there are differences in the pharmacokinetic pathways in animals 

13 and humans following hexavalent chromium exposure, it is not possible to estimate the 

14 magnitude of the uncertainty in the use of this default beyond that associated with other choices 

for default cross-species scaling factors (e.g., BW2/3 or BW115 ). 

16 Statistical uncertainty at the POD. Measures of statistical uncertainty require assuming 

17 that the underlying model and associated assumptions are valid for the data under consideration. 

18 For the multistage model applied to the incidence of male mice GI tract tumors, there is a 

19 reasonably typical degree of uncertainty at the 10% extra risk level (the POD for linear low-dose 

20 extrapolation). That is, the BMDL10 for male mice is approximately 25% lower than the BMD10. 

21 Choice of species/gender. The oral CSF for hexavalent chromium was quantified using 

22 the tumor incidence data for mice, which were thought to be more sensitive than rats to the 

23 carcinogenicity of hexavalent chromium. While tumor responses in the mouse were higher than 

24 those of rats at a comparable dose level, suggesting greater sensitivity of the mouse, it is 

25 unknown whether this higher sensitivity would be maintained at lower exposures. 

26 Relevance to humans. The Guidelines for Carcinogen Risk Assessment (EPA, 2005a) 

27 state that site concordance is not a prerequisite for evaluating the implications of animal study 

28 results for humans. Chemicals that are mutagenic and cause tumors at multiple sites in animals 

29 are likely relevant to human carcinogenesis. Hexavalent chromium is thought to be carcinogenic 

30 through a mutagenic mode of action and is a multisite carcinogen in rodents. Considering all of 

31 the available information, the carcinogenicity observed in rodent studies is considered relevant to 
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1 human exposure. In addition, the concordance of the alimentary system tumors across rats and 

2 mice lends strength to the concern for human carcinogenic potential. 

3 Human population variability. The extent of inter-individual variability in response to 

4 hexavalent chromium is unknown. Although a mutagenic mode of action would indicate 

5 increased early-life susceptibility, the data exploring whether there is differential sensitivity to 

6 hexavalent chromium carcinogenicity across life stages is unavailable. This lack of 

7 understanding about potential differences in metabolism and susceptibility across exposed 

8 human populations thus represents a source of uncertainty. The uncertainties associated with this 

9 lack of data and knowledge about human variability can, at present, only be considered in 

10 qualitative terms; however, EPA has developed ADAFs to quantitatively account for some of the 

11 potential differences in age-dependent response to carcinogens with a mutagenic mode of action. 

12 ADAFs are to be applied to the CSF for hexavalent chromium when assessing cancer risks in 

13 exposed populations composed of individuals less than 16 years old (U.S. EPA, 2005b). More 

14 specific guidance in applying these ADAFs was provided in Section 5.3.5. 

15 

16 5.3.7. Previous Cancer Assessment 

17 The previous IRIS assessment for hexavalent chromium was posted to the IRIS database 

18 in 1998. In that assessment, EPA concluded that the oral carcinogenicity of hexavalent 

19 chromium could not be determined (and was thus classified as Group D) because no data were 

20 located in the available literature that suggested that hexavalent chromium is carcinogenic by the 

21 oral route of exposure. Therefore, no oral CSF was derived. 

22 
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1 6. MAJOR CONCLUSIONS IN THE CHARACTERIZATION OF HAZARD AND DOSE 

2 RESPONSE 

3 

4 

5 6.1. HUMAN HAZARD POTENTIAL 

6 Hexavalent chromium compounds are a group of substances that contain chromium in the 

7 hexavalent or +6 oxidation state. As a class, hexavalent chromium compounds are strong 

8 oxidizing agents, and thus it is rare to find hexavalent chromium naturally occurring in the 

9 environment because it is readily reduced to trivalent chromium (i.e., chromium in the +3 

10 oxidation state) by organic matter. However, hexavalent chromium compounds released to the 

11 environment by anthropogenic sources may persist in natural waters and soils that contain low 

12 amounts of reducing materials. Major uses or former uses of hexavalent chromium compounds 

13 include metal plating, manufacture of pigments and dyes, corrosion inhibitors, chemical 

14 synthesis, refractory production, leather tanning, and wood preservation. Individuals may be 

15 exposed to hexavalent chromium compounds through ingestion of drinking water or contact with 

16 soils or other media contaminated with these substances. 

17 Toxicokinetic studies in mice and rats have examined the absorption, distribution, 

18 metabolism, and elimination of hexavalent chromium compounds. Hexavalent chromium can be 

19 absorbed via oral, inhalation, or dermal routes of exposure in humans and laboratory animals. 

20 For this toxicological review, however, the focus is on the toxicokinetics of hexavalent 

21 chromium following ingestion. Once ingested, hexavalent chromium compounds can interact 

22 with endogenous fluids and other organic matter in the GI tract, resulting, to some extent, in the 

23 reduction of hexavalent chromium to trivalent chromium. This process, whereby hexavalent 

24 chromium is reduced to trivalent chromium in the GI tract, is termed “extracellular” reduction. 

25 The extent of absorption of ingested hexavalent chromium into the GI tissues appears to be 

26 determined by both the solubility of the hexavalent chromium compound ingested and how 

27 rapidly hexavalent chromium is reduced to trivalent chromium in the GI tract, as trivalent 

28 chromium does not diffuse readily across cell membranes. Hexavalent chromium can easily 

29 cross cell membranes due to its ability to use existing nonspecific sulfate and phosphate anion 

30 transport mechanisms. Reduced trivalent chromium, however, can form complexes with organic 

31 ligands, which allow it to pass more easily across cell membranes. 

32 Ingested hexavalent chromium is distributed throughout the body. Liver, kidney, and 

33 bone are the primary sites of chromium accumulation. Once inside the cell, hexavalent 
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1 chromium is reduced to trivalent chromium, either enzymatically or non-enzymatically. This 

2 process is called “intracellular” reduction to distinguish it from the extracellular process 

3 described above. This intracellular reduction yields such reactive intermediates as chromium(V) 

4 and chromium(IV). These reactive intermediates, along with oxygen radicals generated during 

5 this intracellular reduction, can indirectly damage DNA. In addition, trivalent chromium, the 

6 final product of the intracellular reduction of hexavalent chromium, can form adducts with a 

7 number of macromolecules, including DNA.

8 Hexavalent chromium is eliminated primarily in the urine as trivalent chromium. Biliary 

9 excretion of hexavalent chromium has been measured in animals following intravenous injection. 

10 However, this elimination route is relatively minor, with excreted hexavalent chromium or 

11 trivalent chromium-ligand complexes being readily reabsorbed from the gut. Chromium can also 

12 be eliminated in hair, nails, and breast milk. There does not appear to be a gender difference in 

13 the toxicokinetics of hexavalent chromium, and inter-individual variability in the pre-systemic 

14 reduction and subsequent absorption and elimination may be primarily driven by differences in 

15 gastric contents and intervals between meals. 

16 Two PBTK models have been developed for hexavalent and trivalent chromium in rats 

17 and humans (O’Flaherty et al., 2001; O’Flaherty, 1996, 1993). The inclusion of trivalent 

18 chromium in the model allows for the use of trivalent chromium exposure time course data to aid 

19 in parameterization of chromium elimination and to evaluate the ability of the model to predict 

20 elimination of hexavalent chromium as trivalent chromium. However, the limitations of the 

21 performance of the model against limited oral exposure data sets make it inadequate for use in 

22 deriving reference values. 

23 Two types of studies provide information on the toxicological effects in humans resulting 

24 from exposure to ingested hexavalent chromium. In the first type of study, acute human health 

25 effects have been observed following oral ingestion of hexavalent chromium in individuals 

26 accidentally or intentionally ingesting high (fatal or near-fatal) doses of hexavalent chromium. 

27 In the second type of study, chronic human health effects have been reported in human 

28 populations exposed unintentionally to elevated levels of hexavalent chromium in food or 

29 drinking water over an extended time period. 

30 In animals, the effects of subchronic oral exposure to hexavalent chromium have been 

31 evaluated in rats (NTP, 2007; Quinteros et al., 2007; Rafael et al., 2007; Acharya et al., 2001; 

32 Chopra et al., 1996; Vyskocil et al., 1993) and mice (NTP, 2007; Asmatullah and Noreen 1999;), 

33 and the effects of chronic oral exposure to hexavalent chromium have been evaluated in rats 
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1 (NTP, 2008, MacKenzie et al., 1958), mice (NTP, 2008), and dogs (Anwar et al., 1961). Results 

2 from the NTP (2007) subchronic study identified several hexavalent chromium-induced effects, 

3 including hematological effects, hepatotoxicity, alterations in lipid metabolism, and 

4 histopathological changes in GI tissues and pancreatic and mesenteric lymph nodes. The most 

5 sensitive hexavalent chromium-induced effects in rats were microcytic, hypochromic anemia, 

6 increased serum liver enzyme activities, and histopathological changes to the duodenum and 

7 pancreatic lymph nodes; in mice, the most sensitive effect was histopathological changes in the 

8 duodenum. The most sensitive noncancer effects in the NTP (2008) two-year toxicology and 

9 carcinogenicity study were histopathological changes to the liver, duodenum, and mesenteric 

10 lymph nodes in rats; and in the duodenum, mesenteric lymph nodes, and liver in mice. 

11 A number of animal studies have evaluated the reproductive/developmental toxicity of 

12 hexavalent chromium via the oral route of exposure. Collectively, these studies provide 

13 evidence that oral exposure to hexavalent chromium compounds produces reproductive effects, 

14 including histopathological changes to reproductive organs in males (Aruldhas et al., 2006, 2005, 

15 2004; Chowdhury and Mitra, 1995; Li et al., 2001; Zahid et al., 1990) and females (Murthy et al., 

16 1996); alterations in sperm, including decreased count, decreased motility, and abnormal 

17 morphology (Subramanian et al., 2006; Yousef et al., 2006; Li et al., 2001; Zahid et al., 1990); 

18 decreased plasma testosterone levels (Yousef et al., 2006; Chowdhury and Mitra, 1995); 

19 increased estrous cycle length (Kanojia et al., 1998, 1996; Murthy et al., 1996); changes in 

20 mating behavior and decreased fertility in males (Bataineh et al., 1997); and adverse 

21 reproductive outcomes, including decreased numbers of live fetuses and implantations, and 

22 increased numbers of resorptions and pre- and postimplantation losses (Bataineh et al., 2007; 

23 Elsaieed and Nada, 2002; Elbetieha and Al-Hamood, 1997; Junaid et al., 1996a, b, 1995; Kanojia 

24 et al., 1998, 1996; Trivedi et al., 1989). Developmental effects observed have included 

25 decreased fetal weight and length (Elsaieed and Nada, 2002; Kanojia et al., 1998; Junaid et al., 

26 1996a, b, 1995; Trivedi et al., 1989); external (subdermal hemorrhage and tail malformations) 

27 and skeletal abnormalities (decreased ossification) (Elsaieed and Nada, 2002; Junaid et al., 

28 1996a, b, 1995; Kanojia et al., 1998, 1996; Trivedi et al., 1989); and delayed sexual maturation 

29 and function in female offspring (Banu et al., 2008; Al-Hamood et al., 1998). In contrast to 

30 results of the above studies, effects were not observed in dietary exposure studies conducted by 

31 NTP that investigated the potential for hexavalent chromium to produce effects on male 

32 reproductive organs in rats and mice (NTP, 1996a,b) and on reproductive outcomes in a 

33 continuous breeding study in mice (NTP, 1997). The reasons for these inconsistent results are 
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1 not readily apparent, as daily dose ranges evaluated in the NTP studies overlapped with those 

2 used in other studies showing hexavalent chromium-induced adverse reproductive effects. 

3 Human studies of possible associations between oral exposure to hexavalent chromium 

4 and cancer are limited to a few epidemiology studies in which health outcomes (primarily 

5 cancer) were evaluated among populations who were exposed to drinking water contaminated 

6 with hexavalent chromium in Liaoning Province, China (Kerger et al., 2009, Beaumont et al., 

7 2008; Zhang and Li, 1997, 1987), Kings County/San Bernadino County, California (Fryzek et 

8 al., 2001; Bick et al., 1996), Nebraska (Bednar and Kies, 1991), and Glasgow, UK (Eizaguirre9 Garcia et al., 2000, 1999). Analyses of data collected from the Jinzou area of Liaoning Province, 

10 China, where groundwater, surface water, and agricultural soils were heavily contaminated with 

11 chromium derived from hexavalent chromium production (e.g., 0.001–20 mg chromium/L in 

12 residential well water), provide evidence of an excess risk of mortality from stomach cancer 

13 from 1970-1978 in residents of the area, relative to the reference populations in the province 

14 (four other areas in Lianoning Province, and the total population of the province) (Beaumont et 

15 al., 2008). The other epidemiologic studies did not find a significant correlation between 

16 hexavalent chromium concentrations in drinking water (or proximity to the source of hexavalent 

17 chromium soil contamination) and cancer. 

18 Exposure of rats to sodium dichromate dihydrate in drinking water for 2 years resulted in 

19 a significant increase in squamous epithelial neoplasms of the oral mucosa and tongue at the 

20 highest exposure level (average daily doses of 5.9 and 7.0 mg hexavalent chromium/kg-day in 

21 males and females, respectively), but not at the three lower exposure levels (NTP, 2008). 

22 Exposure of B6C3F1 mice to sodium dichromate dihydrate in drinking water for 2 years resulted 

23 in significant increases in the incidences of neoplasms of the small intestine in males and females 

24 at doses ≥ 2.4 and ≥ 3.1 mg hexavalent chromium/kg-day, respectively. NTP (2008) concluded 

25 that results from these studies provide clear evidence of carcinogenic activity of sodium 

26 dichromate dihydrate in male and female F344/N rats based on increased incidences of 

27 squamous cell neoplasms of the oral cavity and clear evidence of carcinogenic activity of sodium 

28 dichromate dihydrate in male and female B6C3F1 mice based on increased incidences of 

29 neoplasms of the small intestine. 

30 The potential mutagenicity of hexavalent chromium has been studied extensively. 

31 Although study results vary with specific test systems, experimental conditions, and hexavalent 

32 chromium compounds tested, results of in vitro and in vivo studies provide substantial evidence 

33 for mutagenic activity of hexavalent chromium compounds. The mutagenicity of hexavalent 
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1 chromium is mediated through the generation of highly reactive chromium intermediates (e.g., 

2 chromium(IV) and chromium(V)) and reactive oxygen species formed during the intracellular 

3 reduction of hexavalent chromium. Reactive chromium intermediates and oxygen species react 

4 with DNA, leading to oxidative DNA damage, chromium-DNA adducts, DNA strand breaks, and 

5 chromosomal aberrations (Wise et al., 2008). 

6 In in vitro test systems, hexavalent chromium compounds have mostly tested positive for 

7 gene mutations (including reverse mutations, frame shift mutations, and base pair substitutions) 

8 and DNA damage (including DNA-protein crosslinks) in bacterial cells (Salmonella 

9 typhimurium, Escherichia coli, Bacillus subtilis); for forward mutations and mitotic gene 

10 conversion in yeast (Saccharomyces cerevisiae); and for DNA damage (DNA strand breaks, 

11 fragmentation, DNA-protein crosslinks, DNA-DNA crosslinks), chromosomal damage (sister 

12 chromatid exchanges and chromosomal aberrations), and DNA synthesis inhibition in 

13 mammalian cell lines and primary cultures (including primary cultures of human gastric mucosal 

14 cells, respiratory tract cells, and lymphocytes). In in vivo test systems, hexavalent chromium 

15 compounds have tested positive for mutations in Drosophila melanogaster and for DNA damage 

16 (DNA-protein crosslinks, DNA strand breaks), mutations, chromosomal damage (sister 

17 chromatid exchanges, chromosomal aberrations, and micronuclei), and DNA synthesis inhibition 

18 in rats and mice. Thus, the mutagenic activity of hexavalent chromium has been demonstrated in 

19 numerous studies using both in vitro and in vivo experimental systems. Given the weight of the 

20 available evidence, hexavalent chromium is proposed to act through a mutagenic mode of 

21 carcinogenic action, and thus age-dependent adjustment factors (ADAFs) should be applied. 

22 Under the Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005a), hexavalent 

23 chromium is “likely to be carcinogenic to humans” via the oral route of exposure based on a 

24 statistically significant increase in the incidence of tumors of the oral mucosa and tongue of rats 

25 and of the small intestine of mice; and evidence of an association between oral exposure to 

26 hexavalent chromium and stomach cancer in humans. Additionally, available evidence indicates 

27 that chromium interacts with DNA, resulting in DNA damage and mutagenesis. Thus, 

28 hexavalent chromium is proposed to induce carcinogenicity via a mutagenic mode of action. 

29 
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1 6.2. DOSE RESPONSE 

2 6.2.1. Noncancer — Oral

3 NTP (2008), a 2-year animal bioassay that used multiple dose groups and included a 

4 comprehensive assessment of endpoints, was selected as the principal study for derivation of the 

5 RfD. Dose-response analysis using BMD methods was conducted for the following endpoints 

6 from this study: histopathological changes of the liver (chronic inflammation in female rats and 

7 histiocytic cellular infiltration in female mice), duodenum (diffuse epithelial hyperplasia in male 

8 and female mice), mesenteric lymph node (histiocytic cellular infiltration in male and female 

9 mice), and pancreas (cytoplasm cellular alteration of acinar epithelial cells in female mice). 

10 All available dichotomous models in the EPA’s BMDS were fit to the incidence data for 

11 the selected endpoints, using 10% extra risk as the BMR in accordance with U.S. EPA’s 

12 Benchmark Dose Technical Guidance (U.S. EPA, 2000). 

13 Based on the lowest BMDL10 value of 0.09 mg hexavalent chromium/kg-day, diffuse 

14 epithelial hyperplasia of the duodenum in female mice was selected as the POD for derivation of 

the RfD. The RfD of 0.0009 or 9 x 10-4 15 mg/kg-day for hexavalent chromium was derived by 

16 dividing the BMDL10 (or POD) of 0.09 mg/kg-day by a composite uncertainty factor of 100 (10 

17 for extrapolation from animals to humans and 10 for human variability). 

18 

19 6.2.2. Cancer — Oral

20 The mode of action is a key consideration in clarifying how risks should be estimated for 

21 low-dose exposure. A linear low-dose extrapolation approach was used to estimate human 

22 carcinogenic risk associated with hexavalent chromium exposures. This approach is supported 

23 by the evidence for genotoxicity and a mutagenic mode of action. 

24 The CSF for hexavalent chromium is based on tumor incidence data from the NTP (2008) 

25 animal bioassay. The incidence of neoplasms in the small intestine of mice was used to derive 

26 the CSF. Only animals that survived for at least 451 days, the time until appearance of the first 

27 tumor, were considered at risk for tumor development. 

28 BMD modeling was carried out using the multistage model in EPA’s BMDS (U.S. EPA, 

29 2007) to identify a POD. In applying the BMD approach to the derivation of a CSF, the lower 

30 95% confidence bound on the dose corresponding to the BMR (defined as 10% extra risk of 

31 small intestine tumors) was calculated. This lower confidence bound is referred to as the 

32 BMDL. The CSF was calculated by dividing the BMR by the BMDL, and then converting this 

33 CSF to human equivalents using body weight to the 3⁄4 power scaling. 
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1 The CSF resulting from the fitting of the multistage model in BMDS to the incidence of 

neoplasms in the small intestine of male and female mice was 0.5 (mg/kg-day)-1 2 and 0.6 (mg/kgday)-1 3 , respectively, expressed in human equivalents. Because of the poorer fit of the multistage 

model to the female mouse data, the cancer potency estimate of 0.5 (mg/kg-day)-1 4 based on the 

5 male mouse data was selected as the CSF for hexavalent chromium. 

6 
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A-1 DRAFT - DO NOT CITE OR QUOTE 

1 APPENDIX A. SUMMARY OF EXTERNAL PEER REVIEW AND PUBLIC 

2 COMMENTS AND DISPOSITION 

3 

4 

5 

6 [page intentionally left blank] 
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 for the RfD 

Table B-1.1 Incidence data for nonneoplastic lesions from all treatment groups of 

female F344/N rats and male and female B6C3F1 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 21/50a28/50b 35/50b39/50b

Dose

(mg hexavalent chromium/kg-day) 

0 0.38 0.91 2.4 5.9 

Male Mice 

Duodenum: Diffuse epithelial 

hyperplasia 0/50 11/50b

18/50b 42/50b32/50a

Mesenteric lymph node: 

Histiocytic cellular infiltration 14/47 38/47b

31/49b 32/49b42/46a

Dose 

(mg hexavalent chromium/kg-day) 

0 0.38 1.4 3.1 8.7 

Female Mice 

Duodenum: Diffuse epithelial 

hyperplasia 0/50 16/50b

35/50b 31/50b42/50b

Mesenteric lymph node: 

Histiocytic cellular infiltration 3/46 29/48b

26/46b 40/50b42/50b

Liver: Histiocytic cellular 

infiltration 2/49 15/50b

23/50b 32/50b45/50b

Pancreas: Acinus, cytoplasmic 

alteration 0/48 6/50a

6/49a 14/50b32/50b

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 provided an adequate fit (χ

2

p-value ≥0.1) to the data 

(Table B-1.2). Based on the log-logistic model, the 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). 
B-3 DO NOT CITE OR QUOTE 

Table B-1.2. BMD10 and BMDL10 values and goodness-of-fit statistics from models 

fit to incidence data for chronic inflammation of the liver in female rats exposed to 

sodium dichromium dihydrate in drinking water for 2 years 

Model 

BMD10 

(mg/kg-day) 

BMDL10 

(mg/kg-day) x2

 p-value AIC 

Gammaa 0.51 0.37 0.04 317.97 

Logistic 0.84 0.65 0.01 321.45 

Log-logisticb 0.22 0.14 0.37 312.57 

Multi-stagec 0.51 0.37 0.04 317.97 

Probit 0.88 0.70 0.01 321.80 

Log-probitb

 0.89 0.61 0.01 320.86 

Quantal linear 0.51 0.37 0.04 317.97 

Weibulla 0.51 0.37 0.04 317.97 

aRestrict power ≥1 

b

Slope restricted to >1 

cRestrict betas ≥0; lowest degree polynomial (up to n-2) with an adequate 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-4 DO NOT CITE OR QUOTE 

Figure B-1.1. Predicted and observed incidence of chronic inflammation of the liver 

in female rats exposed to sodium dichromium dihydrate 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 7

Fraction 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 Male Mice. As assessed by the chi-square 

goodness-of-fit statistic, none of the models provided an adequate fit (χ

2

p-value ≥ 0.1) to the full 

dataset (Table B-1.3). 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. After dropping the highest dose, the gamma, log-logistic, multistage, log-probit, 

quantal linear, and Weibull models provided adequate 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. 
B-5 DO NOT CITE OR QUOTE 

Table B-1.3. BMD10 and BMDL10 values and goodness-of-fit statistics from models 

fit to incidence data for diffuse epithelial hyperplasia in the duodenum in male mice 

exposed to sodium dichromium dihydrate in drinking water for 2 years 

Model 

BMD10 

(mg/kg-day) 

BMDL10 

(mg/kg-day) x2 p-value AIC 

All doses 

Gammaa 0.31 0.25 0.00 270.99 

Logistic 0.90 0.74 0.00 296.25 

Log-logisticb 0.15 0.12 0.00 247.93 

Multi-stagec 0.31 0.25 0.00 270.99 

Probit 0.90 0.76 0.00 296.18 

Log-probitb 0.48 0.36 0.00 274.38 

Quantal linear 0.31 0.25 0.00 270.99 

Weibulla 0.31 0.25 0.00 270.99 

Highest dose dropped (four doses modeled) 

Gammaa 0.22 0.14 0.43 167.67 

Logistic 0.47 0.39 0.03 177.09 

Log-logisticb 0.26 0.15 0.20 169.23 

Multi-staged

0.16 0.13 0.52 166.34 

Probit 0.45 0.37 0.04 176.19 

Log-probitb 0.28 0.23 0.33 167.41 

Quantal linear 0.16 0.13 0.52 166.34 

Weibulla 0.22 0.14 0.47 167.50 

aRestrict power ≥1 

b

Slope restricted to >1 

cRestrict betas ≥0; lowest degree polynomial (up to n-2) with an adequate fit is reported; no degree of polynomial 

provided a fit, a 3-degree polynomial is reported. 

dRestrict betas ≥0; lowest degree polynomial (up to n-2) with an adequate fit is reported; degree polynomial =1. 

AIC = Akaike information criterion; BMD = benchmark dose; BMDL = lower confidence limit (95%) on the 

benchmark dose 

Source: ATSDR 
B-6 DO NOT CITE OR QUOTE 

Figure B-1.2. Predicted and observed incidence of diffuse epithelial hyperplasia in 

the duodenum of male mice exposed to sodium dichromium dihydrate in drinking 

water for 2 years* 

0

0.2

0.4

0.6

0.8

1

0 0.5 1 1.5 2 2.5

Fraction Affected

dose

Multistage Model with 0.95 Confidence Level

15:45 04/08 2008

BMDL BMD

 

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 the models provided an adequate fit (χ

2

p-value ≥

0.1) to the full dataset (Table B-1.4). 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 lowdose region of the response curve. Dropping the highest dose did not result in adequately fitting 

models, nor did dropping the two highest doses. This dataset is considered not suitable for 

benchmark dose modeling. 
B-7 DO NOT CITE OR QUOTE 

Table B-1.4. BMD10 and BMDL10 values and goodness-of-fit statistics from models 

fit to incidence data for histiocytic cellular infiltration in mesenteric lymph nodes of 

male mice exposed to sodium dichromium dihydrate in drinking water for 2 years 

Model 

BMD10 

(mg/kg-day) 

BMDL10 

(mg/kg-day) x2 p-value AIC 

All doses 

 Gammaa 0.38 0.26 0.00 285.94 

 Logistic 0.53 0.39 0.00 286.38 

 Log-logisticb 0.16 0.08 0.00 284.48 

 Multi-stagec

 0.43 0.26 0.00 287.88 

 Probit 0.56 0.43 0.00 286.35 

 Log-probitb

 0.83 0.52 0.00 289.36 

 Quantal linear 0.38 0.26 0.00 285.94 

 Weibulla

 0.38 0.26 0.00 285.94 

Highest dose dropped (four doses modeled) 

 Gammaa 0.47 0.24 0.00 258.50 

 Logistic 0.61 0.35 0.00 259.04 

 Log-logisticb 0.21 0.08 0.00 256.81 

 Multi-staged

 0.47 0.24 0.00 258.50 

 Probit 0.63 0.37 0.00 259.08 

 Log-probitb

 1.24 0.56 0.00 261.28 

 Quantal linear 0.47 0.24 0.00 258.50 

 Weibulla 0.47 0.24 0.00 258.50 

Two highest doses dropped (three doses modeled) 

 Gammaa 0.11 0.07 0.00 187.77 

 Logistic 0.17 0.12 0.00 189.97 

 Log-logisticb 0.05 0.03 0.00 183.77 

 Multi-stagee 0.11 0.07 0.00 187.77 

 Probit 0.17 0.12 0.00 190.12 

 Log-probitb 0.17 0.11 0.00 190.37 

 Quantal linear 0.11 0.07 0.00 187.77 

 Weibulla

 0.11 0.07 0.00 187.77 

aRestrict power ≥1 

b

Slope restricted to >1 

cRestrict betas ≥0; lowest degree polynomial (up to n-2) with an adequate fit is reported; no degree of polynomial 

provided a fit, a 3-degree polynomial is reported. 

dRestrict betas ≥0; lowest degree polynomial (up to n-2) with an adequate fit is reported; no degree of polynomial 

provided a fit, a 2-degree polynomial is reported. 

eRestrict betas ≥0; lowest degree polynomial (up to n-2) with an adequate 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-8 DO NOT CITE OR QUOTE 

Diffuse Epithelial Hyperplasia of the Duodenum in Female Mice. As assessed by the chisquare goodness-of-fit statistic, none of the models provided an adequate fit (χ

2

p-value ≥0.1) to 

the data (Table B-1.5). 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. After dropping the highest dose, an adequate fit was still not achieved. 

After dropping the two highest doses, all of the models except for the logistic and probit models 

provided an adequate fit (χ

2

p-value ≥ 0.1) to the data. Comparing across models, a better fit is 

generally indicated by a lower AIC (EPA, 2000b). As assessed by AIC, the gamma, multistage, 

quantal linear, and Weibull models generated identical goodness of fit statistics and benchmark 

doses, as these models all took the form of a 1-degree polynomial multistage model which 

provides the best fit (Figure B-1.3). Based on these models, the BMD associated with a 10% 

extra risk was 0.12 mg hexavalent chromium/kg-day and its lower 95% confidence limit 

(BMDL) was 0.09 mg hexavalent chromium/kg-day. 
B-9 DO NOT CITE OR QUOTE 

Table B-1.5. BMD10 and BMDL10 values and goodness-of-fit statistics from models 

fit to incidence data for diffuse epithelial hyperplasia in the duodenum of female 

mice exposed to sodium dichromium dihydrate in drinking water for 2 years 

Model 

BMD10 

(mg/kg-day) 

BMDL10 

(mg/kg-day) x2

 p-value AIC 

All doses 

 Gammaa 0.34 0.27 0.00 275.34 

 Logistic 0.88 0.72 0.00 293.17 

 Log-logisticb

 0.12 0.09 0.04 245.54 

 Multi-stagec 0.34 0.27 0.00 275.34 

 Probit 0.93 0.78 0.00 294.03 

 Log-probitb 0.52 0.38 0.00 279.54 

 Quantal linear 0.34 0.27 0.00 275.34 

 Weibulla 0.34 0.27 0.00 275.34 

Highest dose dropped (four doses modeled) 

 Gammaa

 0.20 0.16 0.00 213.41 

 Logistic 0.55 0.46 0.00 236.10 

 Log-logisticb

 0.11 0.08 0.04 200.07 

 Multi-staged 0.20 0.16 0.00 213.41 

 Probit 0.54 0.45 0.00 235.61 

 Log-probitb

 0.29 0.24 0.00 220.04 

 Quantal linear 0.20 0.16 0.00 213.41 

 Weibulla

 0.20 0.16 0.00 213.41 

Two highest doses dropped (three doses modeled) 

 Gammaa 0.12 0.09 0.87 126.06 

 Logistic 0.34 0.27 0.00 141.77 

 Log-logisticb 0.12 0.06 1.00 127.77 

 Multi-stagee 0.12 0.09 0.87 126.06 

 Probit 0.32 0.26 0.00 140.65 

 Log-probitb 0.20 0.16 0.48 127.17 

 Quantal linear 0.12 0.09 0.87 126.06 

 Weibulla 0.12 0.09 0.87 126.06 

aRestrict power ≥1 

b

Slope restricted to >1 

cRestrict betas ≥0; lowest degree polynomial (up to n-2) with an adequate fit is reported; no degree of polynomial 

provided a fit, a 3-degree polynomial is reported. 

dRestrict betas ≥0; lowest degree polynomial (up to n-2) with an adequate fit is reported; no degree of polynomial 

provided a fit, a 2-degree polynomial is reported. 

eRestrict betas ≥0; lowest degree polynomial (up to n-2) with an adequate 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 dichromium 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 1.4

Fraction Affected

dose

Multistage Model with 0.95 Confidence Level

09:36 04/09 2008

BMDL BMD

 

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 of 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. Dropping 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. BMD10 and BMDL10 values and goodness-of-fit statistics from models 

fit to incidence data for histiocytic cellular infiltration in mesenteric lymph nodes 

of female mice exposed to sodium dichromium dihydrate in drinking water for 2 

years 

Model 

BMD10 

(mg/kg-day) 

BMDL10 

(mg/kg-day) x2 p-value AIC 

All doses 

 Gammaa 0.41 0.30 0.00 282.46 

 Logistic 0.77 0.61 0.00 290.18 

 Log-logisticb 0.09 0.06 0.00 263.55 

 Multi-stagec

 0.41 0.30 0.00 282.46 

 Probit 0.85 0.69 0.00 291.41 

 Log-probitb 0.68 0.47 0.00 285.85 

 Quantal linear 0.41 0.30 0.00 282.46 

 Weibulla

 0.41 0.30 0.00 282.46 

Highest dose dropped (four doses modeled) 

 Gammaa

 0.20 0.15 0.00 224.84 

 Logistic 0.40 0.33 0.00 230.81 

 Log-logisticb 0.07 0.05 0.00 215.19 

 Multi-staged

 0.20 0.15 0.00 224.84 

 Probit 0.40 0.34 0.00 230.85 

 Log-probitb

 0.37 0.24 0.00 231.76 

 Quantal linear 0.20 0.15 0.00 224.84 

 Weibulla

 0.20 0.15 0.00 224.84 

Two highest doses dropped (three doses modeled) 

 Gammaa 0.14 0.10 0.00 172.32 

 Logistic 0.31 0.24 0.00 178.99 

 Log-logisticb

 0.07 0.04 0.00 164.47 

 Multi-stagee

 0.14 0.10 0.00 172.32 

 Probit 0.30 0.23 0.00 178.74 

 Log-probitb

 0.21 0.15 0.00 178.11 

 Quantal linear 0.14 0.10 0.00 172.32 

 Weibulla 0.14 0.10 0.00 172.32 

aRestrict power ≥1 

b

Slope restricted to >1 

cRestrict betas ≥0; lowest degree polynomial (up to n-2) with an adequate fit is reported; no degree of polynomial 

provided a fit, a 3-degree polynomial is reported. 

dRestrict betas ≥0; lowest degree polynomial (up to n-2) with an adequate fit is reported; no degree of polynomial 

provided a fit, a 2-degree polynomial is reported. 

eRestrict betas ≥0; lowest degree polynomial (up to n-2) with an adequate 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 Female Mice. As assessed by the chi-square 

goodness-of-fit statistic, only the log-logistic model 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. BMD10 and BMDL10 values and goodness-of-fit statistics from models 

fit to incidence data for histiocytic cellular infiltration in the liver of female rats 

exposed to sodium dichromium dihydrate in drinking water for 2 years 

Model 

BMD10

(mg/kg-day) 

BMDL10

(mg/kg-day) x2 p-value AIC 

Gammaa 0.35 0.28 0.08 255.40 

Logistic 0.85 0.70 0.00 267.56 

Log-logisticb 0.17 0.12 0.44 251.36 

Multi-stagec

 0.35 0.28 0.08 255.40 

Probit 0.88 0.75 0.00 268.64 

Log-probitb

 0.62 0.48 0.01 260.00 

Quantal linear 0.35 0.28 0.08 255.40 

Weibulla 0.35 0.28 0.08 255.40 

aRestrict power ≥1 

b

Slope restricted to >1 

cRestrict betas ≥0; lowest degree polynomial (up to n-2) with an adequate 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 dichromium 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 9

Fraction 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 of the Pancreas in Female Mice. As 

assessed by the chi-square goodness-of-fit statistic, all of the models provide adequate fits (χ

2

pvalue ≥0.1) to the data (Table B-1.8). Comparing across models, 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 (Figure B-1.5). Based on the log-logistic model, the 

BMD associated with a 10% extra risk was 0.68 mg hexavalent 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. BMD10 and BMDL10 values and goodness-of-fit statistics from models 

fit to incidence data for pancreas: acinus, cytoplasmic alteration in female mice 

exposed to sodium dichromium dihydrate in drinking water for 2 years 

Model 

BMD10 

(mg/kg-day) 

BMDL10 

(mg/kg-day) x

2

 p-value AIC 

Gammaa 0.92 0.72 0.13 206.82 

Logistic 2.43 2.03 0.09 211.78 

Log-logisticb 0.68 0.52 0.19 205.22 

Multi-stagec 0.92 0.72 0.13 206.82 

Probit 2.24 1.89 0.11 210.99 

Log-probitb

 1.77 1.40 0.11 209.99 

Quantal linear 0.92 0.72 0.13 206.82 

Weibulla 0.92 0.72 0.13 206.82 

aRestrict power ≥1 

b

Slope restricted to >1 

cRestrict betas ≥0; lowest degree polynomial (up to n-2) with an adequate 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 dichromium 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 9

Fraction 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 for the Oral Slope Factor 

 The fit of the multistage model to the incidence of 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_2008_Setting.(d) 

 Gnuplot Plotting File: 

M:\ChromiumVI\msc_MALE_MICE_INTESTINAL_TUMORS_NTP_2008_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 positive 
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 Parameter 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 Estimates 

 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 Deviance Test d.f. P-value 

 Full model -77.3728 5 

 Fitted model -77.8649 3 0.984149 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 % two-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 of neoplasms in the small intestine of 

female 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_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 positive 

 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 Parameter 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 correlation 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 Deviance Test d.f. P-value 

 Full model -88.9774 5 

 Fitted model -91.8504 2 5.74595 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 % two-sided confidence 

interval for the BMD 

Multistage Cancer Slope Factor = 0.0973173