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www.epa.gov/iris
&EPA
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-l
APPENDIX B. BENCHMARK DOSE CALCULATIONS B-l
APPENDIX B-l. 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.
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 (ug/g wet tissue or ug/mL blood) of mice and rats after
ingesting K^Q^Oyin 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 B6C3Fi mice exposed to sodium
dichromate dihydrate in drinking water for 3 months [[[ 53
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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 B6C3Fi 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 B6C3Fi 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
B6C3Fi 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 inD. 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 B6C3Fi mice exposed to sodium dichromate dihydrate in drinking
water for 2 years (NTP, 2008) 187
Table 5-2. Summary of BMDio and BMDLio 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
B6C3Fi 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/kg-
day 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
3P-A5-HSH 3p-A5-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
IX
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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
15 deleterious effects during a lifetime. The inhalation RfC (expressed in units of mg/m3) 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
31 plausible upper bound on the estimate of risk per ug/m3 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.
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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
r\
19 chromium(VI) oxide, chromic acid, and selected salts of the chromate (CrC>4 ") and dichromate
r\
20 (C^O? ") 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
CAS
Number
Synonyms
(ChemID
Plus,
2008)
Structure
(ChemID
Plus,
2008)
Molecular
weight
Molecular
formula
Form
Stability/
reactivity
Melting
point
Density
Water
solubility
Other
solubility
Chromium(VI) oxide"
1333-82-0
Chromium oxide;
hexavalent chromium
oxide; chromic
trioxide; chromic
anhydride; chromic
acid anhydride (Anger
etal.,2005)
99.994 (Lide, 2008)
CrO3 (ChemID Plus,
2008)
Dark red, deliquescent
bipyramidal prismatic
crystals, flakes, or
granular powder
(O'Neil, 2006)
Deliquescent;
decomposition begins
above 198°C (Anger et
al., 2005); powerful
oxidizer (O'Neil, 2006)
197°C (Lide, 2008)
2.7 g/cm3 (Lide, 2008)
169g/100gH2O at
25°C (Lide, 2008)
Soluble in alcohol and
mineral acids (Lewis,
2007)
Chromic acida'b
7738-94-5 (H2CrO4);
13530-68-2 (H2Cr2O7)
Chromic(VI) acid; chromium
hydroxide oxide; dichromic acid
(H2Cr2O7)
118.010 (H2CrO4) (Lide, 2008);
218.001 (H2Cr2O7) (ChemID Plus)
H2CrO4;H2Cr2O7 (ChemID Plus,
2008)
Exists only as an aqueous solution
(Lide, 2008); yellow to orange-red
(Anger etal.,2005)
Strong oxidizing agent (Anger et al.,
2005)
Not applicable
Not applicable
Not applicable
Not applicable
Sodium chromate
7775-11-3
Sodium chromate(VI);
chromium disodium
oxide; disodium
chromate; rachromate;
chromic acid, disodium
salt; chromate of soda
161.974 (Lide, 2008)
Na2CrO4 (ChemID
Plus, 2008)
Yellow, orthorhombic
crystals (Anger et al.,
2005)
Hygroscopic (Anger et
al., 2005)
794°C (Lide, 2008)
2.72 g/cm3 (Lide, 2008)
87.6g/100gH2Oat
25°C (Lide, 2008)
Slightly soluble in
ethanol (Lide, 2008)
Sodium dichromate
10588-01-9
Sodium dichromate(VI);
sodium bichromate;
dichromic acid, disodium
salt; bichromate of soda
261.968 (Lide, 2008)
Na2Cr2O7 (ChemID Plus,
2008)
Light brown to orange-
red plates (Anger et al.,
2005)
Strongly hygroscopic;
decomposes above 400°C
(Lide, 2008); strong
oxidizing agent (Anger et
al., 2005)
357°C (Lide, 2008)
2.52 g/cm3 (Anger et al.,
2005)
187g/100gH2Oat25°C
(Lide, 2008)
Not available
Sodium dichromate, dihydrate
7789-12-0
Dichromic acid, disodium salt,
dihydrate
297.999 (Lide, 2008)
Na2Cr2O7«2H2O (ChemID Plus,
2008)
Orange-red, monoclinic,
translucent needles (Anger et al.,
2005)
Very hygroscopic, deliquesces
in air; Decomposes above 85°C;
strong oxidizing agent in acid
solution (Lide, 2008; Anger et
al., 2005)
Decomposes prior to melting
(Lide, 2008)
2.35 g/cm3 (Lide, 2008)
272.9 g/100 g H2O (73.18 wt%)
at 20°C (Anger et al., 2005)
Soluble in acetic acid (Lide,
2008)
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Table 2-1. Physical properties of selected hexavalent chromium compounds
Name
CAS
Number
Synonyms
(ChemID
Plus,
2008)
Structure
(ChemID
Plus,
2008)
Molecular
weight
Molecular
formula
Form
Stability/
reactivity
Melting
point
Density
Potassium chromate
7789-00-6
Potassium
chromate(VI);
bipotassium chromate;
dipotassium chromate;
chromate of potash;
tarapacaite;
chromic acid,
dipotassium salt
194.191 (Lide, 2008)
K2CrO4 (ChemID Plus,
2008)
Lemon yellow prisms
(Anger et al., 2005)
Non-hygroscopic
(Anger et al., 2005)
974°C (Lide, 2008)
2.73 g/cm3 (Lide,
2008)
Potassium dichromate
7778-50-9
Potassium dichromate(VI);
bichromate of potash;
potassium bichromate;
dipotassium bichromate;
dipotassium dichromate;
dipotassium dichromium
heptaoxide; lopezite;
dichromic acid,
dipotassium salt
294. 185 (Lide, 2008)
K2Cr2O7 (ChemID Plus,
2008)
Tabular or prismatic, bright
orange-red triclinic crystals
(Anger etal., 2005)
Non-hygroscopic;
Decomposes at 500°C
(Anger et al., 2005; Lide,
2008)
398°C (Lide, 2008)
2.68 g/cm3 (Lide, 2008)
Calcium chromate
13765-19-0
Calcium
chromate(VI);
calcium chrome
yellow; calcium
monochromate;
gelbin; yellow
ultramarine; chromic
acid, calcium salt
156.07 (Lide, 2008)
CaCrO4 (ChemID
Plus, 2008)
Yellow monoclinic
or rhombic crystals
(O'Neil, 2006)
Decomposes at
1,000°C (Lide,
2008); oxidizing
agent (Lewis, 2007)
Decomposes prior to
melting (Lide, 2008)
3. 12 g/cm3 (Anger et
al., 2005)
Ammonium dichromate
7789-09-5
Ammonium bichromate;
ammonium dichromate(VI);
diammonium dichromate;
chromic acid, diammonium
salt
252.065 (Lide, 2008)
(NH4)2Cr2O7 (ChemID Plus,
2008)
Large, bright, orange -red
crystals (Anger et al., 2005)
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)
Decomposes prior to melting
(Lide, 2008)
2. 155 g/cm3 (Lide, 2008)
Zinc chromate
13530-65-9
Zinc chromate(VI)
hydroxide; buttercup
yellow; chromic
acid, zinc salt; zinc
chrome yellow; zinc
teraoxychromate
181.403 (Lide,
2008)
ZnCrO4 (ChemID
Plus, 2008)
Yellow prisms
(Lide, 2008)
Not available
316°C(Lide, 2008)
3.40 g/cm3 (Lide,
2008)
Lead chromate
7758-97-6
Lead chromate(VI);
phoenicochroite;
plumbous
chromate; chromic
acid, lead salt;
chrome yellow
(O'Neil, 2006)
323.2 (Lide, 2008)
PbCrO4 (ChemID
Plus, 2008)
Yellow-orange
monoclinic crystals
(Lide, 2008)
Not available
844°C (Lide, 2008)
6. 12 g/cm3 (Lide,
2008)
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Table 2-1. Physical properties of selected hexavalent chromium compounds
Name
Water
solubility
Other
solubility
Potassium chromate
65.0g/100gH2Oat
25°C (Lide, 2008)
Insoluble in alcohol
(O'Neil, 2006)
Potassium dichromate
15.1g/100gH2Oat25°C
(Lide, 2008)
Insoluble in alcohol
(Lewis, 2007)
Calcium chromate
4.5g/100gH2O
(4.3 wt%) at 0°C
(Anger etal., 2005)
Soluble in dilute
acids; practically
insoluble in alcohol
(O'Neil, 2006)
Ammonium dichromate
35.6g/100gH2Oat20°C
(Lide, 2008)
Soluble in alcohol (Lewis,
2007)
Zinc chromate
3.08g/100gH2O
(Lide, 2008)
Dissolves readily in
acids (Anger et al.,
2005); insoluble in
acetone (Lide, 2008)
Lead chromate
0.000017 g/100 g
H2O at 20°C (Lide,
2008)
Insoluble in acetic
acid; soluble in
solutions of fixed
alkali hydroxides;
soluble in dilute
nitric acid (O'Neil,
2006)
aChromic acid is formed in aqueous solution when chromium(VI) oxide is dissolved in water; it cannot be isolated as a pure compound out of solution (Anger et
al., 2005; Page and Loar, 2004). The term, chromic acid, is sometimes used in reference to chromium(VI) oxide; however, it should be noted that there is a
structural difference between the anhydrous substance chromium(VI) oxide and the aqueous chromic acid that forms when the oxide is dissolved in water.
bChromic acid exists in solution as both H2CrO4 and H2Cr2O7 (Anger etal., 2005; Page and Loar, 2004; Cotton etal., 1999). H2CrO4isthe main species in basic
solutions (pH > 6) while H2Cr2O7 is the main species in strongly acidic solutions (pH
-------
3. TOXICOKINETICS
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 inter-
28 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
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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 K2Cr2O?) 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 K2Cr2O? 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 CrCb), hexavalent chromium (as K2Cr2O?), or K2Cr2O? in orange juice
9 resulted in 0.13, 6.9, and 0.6% absorption, respectively, across the GI tract (Kerger et al., 1996).
10 K2Cr2O? 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 K2CrC>4 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 K2Cr2O?/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 K2CrC>4) 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 jimol hexavalent chromium per day
24 (approximately 7 |ig/day as Na251CrC>4) 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 jig hexavalent chromium (as
26 Na251CrC>4) 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,
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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 ug hexavalent
18 chromium/mL. Intestinal bacteria, cultured from human feces from three volunteers, were
19 observed to reduce 3.8 ug hexavalent chromium/109 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 Na2CrC>4) occurred at
23 maximal rates of 40-60 ug/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
29 51CrCl3 orNa251CrC>4 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
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1 reducing agents aid in gastric reduction of hexavalent chromium, as intrajejunal intubation of
2 Na251CrC>4 alone resulted in 25% absorption, while Na251CrC>4 pre-incubated with HC1 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)
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Initial phase
Second phase
Initial transport capacity (CrO427erythrocyte/min)
22.7s
10.4 min
3.1 x 108
6.9s
10.1 min
2.5 x 108
Whole blood kinetics
Vmax (umol/mL/min)
Michaelis constant (Km) (mM/1 blood)
2.8
20.9
3.0
14.1
Source: Wiegandetal. (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). K^C^O? 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 ug hexavalent chromium/L. Hexavalent chromium was
8 detected in spiked plasma at concentrations of 2,000 and 10,000 ug hexavalent chromium/L, but
9 not at 1,000 ug 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 ug 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 K2Cr2O4in drinking water for 1 year (Table 3-2). Hexavalent chromium drinking water
19 concentrations ranged from 0.45 to 1 1.2 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
drinking water for 1 year
n
K2Cr2O4
concentration
Liver
(ng/g)
Kidney
Og/g)
Bone
(ng/g)
Spleen
(ng/g)
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(mg/L)
controls
0.45
2.2
4.5
7.7
11.2
male
0
0.02 ±
0.002
0.08 ±
0.017
0.15 ±
0.04
0.70 ±
0.04
1.22±
0.06
female
0
0.08 ±
0.007
0.17 ±
0.03
0.47 ±
0.06
0.55 ±
0.06
1.62±
0.14
male
0
0.14 ±
0.007
0.29 ±
0.02
0.45 ±
0.17
3.30 ±
0.03
4.40 ±
0.36
female
0.25 ±
0.02
0.39 ±
0.04
0.48 ±
0.07
1.09 ±
0.13
2.39 ±
0.09
3.98±
0.32
male
0
0.58 ±
0.04
1.27 ±
0.06
2.14 ±
0.25
3.43 ±
0.83
3.84±
0.49
female
0.72 ±
0.8
0.76 ±
0.04
1.48 ±
0.04
2.44 ±
0.25
5.10 ±
0.35
6.06 ±
0.58
male
0
0.95
0.68 ±
0.18
3.41 ±
0.44
5.24 ±
0.20
9.91 ±
0.83
female
0
0.91 ±
0.11
1.14±
0.1
4.48 ±
0.71
4.73 ±
0.8
11. 1±
0.86
1
2
3
4
5
6
7
8
9
10
11
12
Source: MacKenzie et al. (1959).
Kargacin et al. (1993) examined the species differences in distribution of chromium in
male mice and rats exposed to 8 mg hexavalent chromium/kg-day as K^C^O? in drinking water
for 4 or 8 weeks. Regardless of duration, chromium accumulated primarily in the spleen, liver,
and kidney of mice and rats (1-2 orders of magnitude higher than controls), with mouse liver and
kidney burdens being about two- to fourfold higher than rats (Table 3-3). Chromium
accumulation in bone was also significantly higher (four- and sevenfold higher at 4 and 8 weeks)
in mice than rats. The reason for the higher accumulation of chromium in mouse liver is
unknown, but may result from greater reduction of hexavalent chromium in the rat gut prior to
uptake from the GI tract. Alternatively, the mouse liver may have a higher hexavalent chromium
reduction capacity than rats, causing more trivalent chromium to be sequestered in hepatocytes.
13
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Table 3-3. Chromium in tissues (ug/g wet tissue or ug/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
Kidney
Spleen
Femur
Lung
Heart
Muscle
Blood
0.22 ±0.14
0.24 ±0.14
0.53 ±0.38
0.90 ±0.48
0.24 ±0.12
0.32 ±0.15
0.32 ±0.23
0.14 ±0.05
10.92 ±5.48
3.77 ±0.99
5.04 ±1.45
7.43 ± 1.03
0.99 ±0.10
0.80 ±0.23
1.12±0.37
0.71 ±0.07
13. 83 ±6.06
4.72 ±0.68
10.09 ±2.50
12.55 ±2.99
1.08 ±0.26
1.02 ±0.20
0.60 ±0.25
0.42 ±0.04
Rats
Liver
Kidney
Spleen
Femur
Lung
Heart
Muscle
Blood
0.19±0.14
0.34 ±0.20
0.43 ±0.20
1.00 ±0.46
0.39 ±0.43
0.38 ±0.22
0.24 ±0.14
0.19±0.17
3.32 ±0.93
8.62 ±2.40
3.65 ±1.87
1.85 ±0.46
1.10±0.38
0.52 ±0.12
0.19±0.10
0.73 ±015
3.59 ±0.73
9.49 ±4.38
4.38 ±0.84
1.78 ±0.99
0.67 ±0.24
1.05 ±0.19
0.17±0.10
0.58 ±0.13
Source: Kargacinetal. (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.
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1 Hexavalent chromium is capable of crossing the placenta, as pregnant mice given a single
2 intravenous injection of 10 mg hexavalent chromium/kg (as Na251CrC>4) on gestational day (GD)
3 13 exhibited total embryo chromium levels that were 12% of maternal blood levels (Danielsson
4 et al., 1982). Intraperitoneal injection of 10 mg trivalent chromium/kg (as 51CrCl3) in pregnant
5 mice on GD 8 resulted in approximately equal 51Cr activity in the embryo and maternal blood
6 (lijima 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
23 depletion agent (Aaseth et al., 1982). Incubation of human erythrocytes with K^Q^O? 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
26 97% of [51Cr] 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,
30 14, 9, and 2% of [51Cr] 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
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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), Na2CrC>4, 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 K^C^O? 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 K2C2O?) 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 TICb, 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 uM [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
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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 C>2 tension of only 1 mm Hg exists there. Secondly, the insensitivity to C>2 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 Na2CrO4to determine reduction kinetics. Using a series of P450 inhibitors and
9 TICb, the authors showed that hexavalent chromium reduction was mediated by flavoproteins,
10 NADP(H)-dependent P450 reductase, and cytochrome bs. 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
•max
Km
Inhibition by O2
Inhibition by T1C13
Observation
10.4-10.7
1.04-1.68
26-37%
96-100%
Inhibition by P450 inhibitors
Carbon monoxide
Piperonyl butoxide
Aminopyrine
None
None
None
18
19
20
21
22
Source: Myers and Myers (1998).
Proteoliposomes composed of recombinant human P450 reductase and cytochrome bs
were used to verify that electrons from NADP(H) could be transferred to cytochrome bs during
the reduction of hexavalent chromium (Jannetto et al., 2001). Markedly less hexavalent
chromium reduction occurred in proteoliposomes devoid of cytochrome bs. Further, hexavalent
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1 chromium reduction in proteoliposomes was almost identical to human liver microsomes when
2 corrected for the cytochrome bs 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 K^C^O?) appears to have a slower elimination
14 rate (half-life approximately 40 hours) than ingested trivalent chromium (as CrC^-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 K^C^O?) 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 K^C^O?) 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 Na2CrC>4, 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 |ig/L of
31 hexavalent chromium in breast milk, respectively.
32 In rats gavaged with a single dose of Na251CrC>4, approximately 99% of the administered
33 dose was eliminated in the feces, while 0.8% was eliminated in the urine, both within 4 days
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1 (Sayato et al., 1980). Approximately 81% and 2.17% of 7 ug hexavalent chromium/day (as
2 Na251CrC>4), administered to rats for 3 days, was eliminated in the feces and urine, respectively
3 (Febeletal., 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
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1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
O 'Flaherty Model (O 'Flaherty et al, 2001; O 'Flaherty, 1996, 1993)
The O'Flaherty rat and human model is composed of 10 tissue compartments, including
lung, GI tract, liver, kidney, bone, plasma, erythrocytes, urine, and well- and poorly-perfused
tissues (Figure 3-1). The model parameters, values, and definitions are shown in Table 3-7. The
lung compartment treats an inhaled dose as a dose deposited to the total lung; it does not
distinguish particle-dependent deposition into the various levels of the lower respiratory tract.
As such, it is well suited to simulate intratracheal instillation exposures. Inhaled doses are first
deposited into the bioavailable pool A of the lung. From there, doses can be absorbed into the
plasma, transferred to the GI tract (mucocilliary clearance), or transferred to pool B, which
represents the non-bioavailable portion of the dose. Chromium in pool B is transferred out of the
lung to the GI tract. First-order rate constants govern transfer of chromium from the lung. The
model does not discriminate between free trivalent chromium and trivalent chromium-organic
ligand complexes in the GI tract.
INHALATION
EXPOSURE
POOLB
±
LUNG
POOL A
Cr(VIH.Cr(lll)
WELL-PERFUSED
<
s
tn
F'OORLY-PERFUSED
BONE
>
v>
9 50
13
LIVER
KIDNEY
ORAL
EXPOSURE
FECAL EXCRETION
URINARY
EXCRETION
URINARY PATH
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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.
5
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Table 3-7. Chemical-specific parameters in the rat and human chromium
models
Parameter"
Rat
Cr(III)
Cr(VI)
Human
Cr(III)
Cr(VI)
Definition
Absorption
KGI
KLU
KMUCOA
KMUCOB
KLUAB
FRLUNG
FRTRGI
0.01
0.2
0.8
0.025
1.2
NA
NA
0.04
2.0
0.8
0.025
1.2
NA
NA
0.25
NA
NA
NA
NA
0.3
0.7
2.5
NA
NA
NA
NA
0.3
0.7
First-order rate constant for absorption from the GI tract
(Da1)
First-order rate constant for absorption from the bioavailable
lung pool (pool A) (Da"1)
First-order rate constant for mucociliary clearance from pool
A to the GI tract (Da"1)
First-order rate constant for mucociliary clearance from the
nonbioavailable lung pool (pool B) to the GI tract (Da"1)
First-order rate constant for transfer from pool A to pool B
(Da"1)
Fraction of inhaled chromium absorbed to blood
Fraction of inhaled chromium transferred to GI tract
Distribution
CR
KINRBC
KDIN
LDIN
WDIN
PDIN
BDIN
CR
KOUTRBC
KDOUT
LDOUT
WDOUT
PDOUT
BDOUT
5.0
0.0003
0.007
0.0001
0.0001
0.0001
0.0001
NA
0.0003
0.001
0.0003
0.001
0.003
0.003
15.0
1.5
1.5
1.5
1.5
0.01
0.01
NA
10.0
10.0
10.0
10.0
10.0
10.0
NAb
12.0
3.0
3.0
3.0
3.0
NAb
5.0
12.0
3.0
3.0
3.0
3.0
NAb
NAb
NA
30.0
30.0
30.0
30.0
NAb
15.0
NA
30.0
30.0
30.0
30.0
NAb
Relative clearance of chromium into mineralizing bone (liters
of blood plasma cleared per liter of new bone formed)
Clearance from plasma to red cell (L/Da)
Clearance from plasma to kidney (L/Da)
Clearance from plasma to liver (L/Da)
Clearance from plasma to other well-perfused tissues (L/Da)
Clearance from plasma to poorly -perfused tissues (L/Da)
Clearance from plasma to bone (L/Da)
Fraction deposition from blood to forming bone
Clearance from red cell to plasma (L/Da)
Clearance from kidney to plasma (L/Da)
Clearance from liver to plasma (L/Da)
Clearance from other well-perfused tissues to plasma (L/Da)
Clearance from poorly perfused tissues to plasma (L/Da)
Clearance from bone to plasma (L/Da)
Excretion
KFX
QEC
CLEARC
MAXC
KMC
FB
FI
1.5
0.065
NA
NA
NA
0.0
0.0
1.5
0.065
NA
NA
NA
0.0
0.0
14.0
NAC
12.0
0.008
0.0008
NA
NA
14.0
NAC
12.0
0.008
0.0008
NA
NA
First-order rate constant for loss of chromium from intestinal
tract contents to the feces (Da"1)
Excretion clearance from the plasma (urinary clearance)
(L/kg/Da)
Parameter in expression for clearance from blood plasma to
urine (L/day)
Parameter in expression for clearance from blood plasma to
urine (mg/day)
Parameter in expression for clearance from blood plasma to
urine (mg/L)
Fraction of body burden secreted in the bile
Fraction of body burden excreted via the GI tract
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Table 3-7. Chemical-specific parameters in the rat and human chromium
models
Parameter"
Rat
Cr(III)
Cr(VI)
Human
Cr(III)
Cr(VI)
Definition
Reduction
KREDRC
KREDBP
KREDKL
KREDGI
KRED
NA
NA
NA
NA
NA
0.7
NA
NA
10.0
0.5
NA
NA
NA
NA
NA
7.0
0.2
500.0
100.0
5.0
First-order rate constant for reduction of Cr(VI) to Cr(III) in
the red cell (Da"1)
First-order rate constant for reduction of Cr(VI) to Cr(III) in
blood plasma (Da"1)
First-order rate constant for reduction of Cr(VI) to Cr(III) in
kidney (Da"1)
First-order rate constant for reduction of Cr(VI) to Cr(III) in
GI tract contents (Da"1)
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
KHOLD
FR
0.7
0.05
0.10
0.7
0.05
0.10
NA
NA
NA
NA
NA
NA
Fraction of urinary chromium not excreted immediately; that
is, temporarily held in pool
First-order rate constant for excretion from the retained urine
pool (Da"1)
Fraction of chromium in retained urine that is associated with
the kidney
"Parameter names are those for human model in cases where the reported rat and human parameter names were not
identical.
Exchanges between blood plasma and cortical and trabecular bone are simulated as functions of bone formation
and resorption rates.
QE = CLEAR -- - —
KM + CBP 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
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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.
7 For example, urinary elimination is 0.7% of GFR at 1 x 10~4 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 (Langard et al., 1978) and chronic Na2CrO4 or CrCb 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 K^C^CVday 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.
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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
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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 7957J
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
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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.
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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 Anti epidemic 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 exposure-
32 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
Year
Early 1965 a
Village (km from alloy plant)
Jinchanbao
(1.4)
Nuer River
Village
(1.5)
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
Yangxing
(3.0)
Shilitai
(3.5)
Wenjiatun
(5.0)
Later in 1 965 a'b
n wells sampled a>b
Cr+6(mg/L)a
0.001
0.001 -0.05
0.05-0.1
0.01-0.5
0.5-<1.0
1.0-<5.0
>5.0
maximum (mg/L)a>b
average (mg/L)b
1966 c
1967 b
1972 b
1974
1979 c
123
170
50
21
33
Number of wells (%)
73 (59)
35 (28)
7 (6)
8 (7)
0 (0)
0 (0)
0 (0)
0.4
0.031
7 (4)
1 (1)
5 (3)
27 (16)
17 (10)
76 (45)
37 (22)
20.0
2.6
10.5d
0.06-4.33
14 (28)
16 (32)
5(10)
12 (24)
2 (4)
1 (2)
0 (0)
<5
0.18
2(10)
19 (90)
0 (0)
0 (0)
0 (0)
0 (0)
0 (0)
0.05
0.02
0.002 - 20.0
O.05
O.05
O.05
0.01- 0.05 c
0.001-0.03
27 (82)
6 (18)
0 (0)
0 (0)
0 (0)
0 (0)
0 (0)
0.05
0.004
O.05
0.003 - 0.004
1
2
o
J
4
5
6
7
Cr+6: hexavalent chromium.
aAs reported by Beaumont et al. (2008)
bAs reported by Kerger et al. (2009)
°As 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.
A mortality study was described first by Zhang and Li in 1980 in an unpublished report
for the JinZhou health department, and later published in a Chinese journal (Zhang and Li,
1987). Mortality records for the period 1970-1978 were obtained from local police stations for
the five villages along the Nuer River, the district surrounding the ferrochromium alloy plant
(Tanghezi), and three other areas to the west (Yaotanghezi) and north (North Thanghezi, North
Nuer River) of the plant. Tanghezi and the other three areas were not affected by the
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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 area-
31 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
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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 "age-
21 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
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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.
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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 1978, based
on analyses by Beaumont et al. (2008) and Kerger et al. (2009)
Area (population or person-years)3
Rate per 100,000 person-years
All Cancer
Age-adjusted
rate
Age-adjustment
influence1"
Stomach Cancer
Crude rate
Estimated age-
adjusted rate b
Lung Cancer
Crude rate
Estimated age-
adjusted rateb
Areas in Contamination Zone
Jinchanbao (2900)
Nuer River Village (2800)
Shilitai (2600)
Yangxing(llOO)
Wenjiatun(1700)
Group average (~98,700)c
83.6
71.9
93.0
76.8
91.1
81.3
0.97
0.98
0.94
0.94
0.94
36.7
28.0
55.2
36.5
27.7
34.9
35.5
missing b
51.7
34.5
26.0
35.3
13.2
15.0
missing
21.4
20.8
17.1
12.8
14.7
missing
20.2
19.5
16.9
Comparison areas
TangHeZi (17,500)
North TangHeZi (3600)
North Nuer River (5800)
Yao TangHeZi (1500)
Group average - all (-252,500) e
Group average - without TangHeZi
(96,826) f
71.3
81.8
71.8
61.3
72.1
73.7
0.86
0.84
1.05
0.90
16.9
26.4 d
30.5
26.6
28.6
14.5
22.1
31.9
23.8
19.4
21.4
8.8
7.6
20.0
9.7
18.3
7.4
8.0
17.9
14.7
aArea population figures are based on approximate 1975 data from Beaumont et al. (2008); group values are total person-years for the combined area.
bAs calculated by Beaumont et al. (2008). Nuer River Village stomach cancer rate was not included in the primary analysis by Beaumont et al. (2008) because
it was missing in the original (1980) report; an additional analysis used a rate of 28.0 as reported by Zhang and Li (1987).
"Beaumont et al. (2008) estimate was 98,458 and Kerger et al. (2009) estimate was 98,850.
dBeaumont et al. (2008) report this value as 26.14 in Table 2, but based on the calculation of the estimated age-adjusted rate it appears that a value close to 26.3
was used; Kerger et al. (2009) report this value as 26.4.
"Beaumont et al. (2008) estimate was 252,277 and Kerger et al. (2009) estimate was 253,282.
fAs reported by Kerger et al. (2009).
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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
Comparison Group a
All 4 areasb
Excluding TangHeZi c
Liaoning province13
All Cancers
Risk ratio
1.13
1.10
1.23
(95% CI)
(0.86, 1.46)
(0.80,1.51)
(0.97, 1.53)
Stomach Cancer
Risk ratio
1.82
1.22
1.69
(95% CI)
(1.11,2.91)
(0.74, 2.01)
(1.12,2.44)
Lung Cancer
Risk ratio
1.15
1.76
1.78
(95% CI)
(0.62, 2.07)
(0.78, 3.98)
(1.03, 2.87)
"TangHeZi, North TangHeZi, North Nuer River, and Yao TangHeZi.
bReported by Beaumont et al. (2008).
"Reported 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
36
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1 Kings County/San Bernadino County, California (Fryzek et al, 2001)
1 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.
33
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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-observed-
11 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.
33
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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 B6C3Fi mice, and 3)
6 three strains of male mice (B6C3Fi, 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 no-
27 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
42 DRAFT - DO NOT CITE OR QUOTE
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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.
27
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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
parameter
Time on
treatment
Treatment group (mg hexavalent chromium/kg-day)
0
1.7
3.5
5.9
11.2
20.9
Males
Hct (percent)
Hgb
(g/dL)
MCV (fL)
MCH (pg)
Erythrocyte
count (106/uL)
Platelet count
(106/uL)
23 Days
3 Months
23 Days
3 Months
23 Days
3 Months
23 Days
3 Months
23 Days
3 Months
23 Days
3 Months
48.0±0.5a
45.7 ±0.2
15.9 ±0.1
15.3 ±0.1
61.1 ±0.5
51.8±0.1
20.1 ±0.2
17.3 ±0.1
7.94 ±
0.10
8.88 ±
0.05
745.2 ±
22.2
618.6 ±
20.0
44.7±0.7b
(93.1)
45.2 ±0.4
(98.9)
14.2±0.2b
(89.3)
15.2±0.1
(99.3)
53.6±0.6b
(87.7)
50.3±0.2b
(97.1)
16.9±0.2b
(84.0)
16.9±0.1b
(97.7)
8.38±0.11
(105.5)
9.04 ± 0.09C
(101.8)
1,065.3 ±
67. 9b
(143)
736.1 ±11. 5
(119)
39.8±0.8b
(82.9)
45.2 ±0.3
(98.9)
12.0±0.3b
(75.5)
15.0±0.1
(98.0)
48.0±0.4b
(78.6)
49.0±0.1b
(94.6)
17.2±0.7b
(85.6)
16.2±0.1b
(93.6)
7.13±0.35C
(89.8)
9.25 ± 0.07b
(104.2)
2,768.6 ±
328.5b
(372)
604.3 ± 24.5
(98)
36.2±1.0b
(75.4)
44.8 ±0.7
(98.0)
10.9±0.3b
(68.6)
14.4±0.2b
(94.1)
46.4±0.6b
(75.9)
44.4±1.0b
(85.7)
18.2 ±0.4
(90.5)
14.2±0.4b
(82.1)
6.0 ± 0.28b
(75.6)
10.15±0.22b
(114.3)
3,504.7 ±
235.0b
(470)
909.8 ±119.1b
(147)
34.4±0.5b
(71.7)
42.9 ± 0.4b
(93.9)
10.3±0.3b
(64.8)
13.3±0.2b
(86.9)
46.2±0.3b
(75.6)
39.7±0.5b
(76.6)
19.7 ±0.3
(98.1)
12.3±0.2b
(71.1)
5.25±0.19b
(66.1)
10.87 ± 0.07b
(122.4)
4,226.0 ±
204.5b
(567)
1,743. 1±
178.0b
(282)
32.3±l.lb
(67.3)
36.9±0.8b
(80.7)
9.2±0.3b
(57.9)
10.9±0.3b
(71.2)
46.4±0.5b
(75.9)
36.0±0.4b
(69.5)
20.7 ±0.6
(103.0)
13.0±0.5b
(75.1)
4.54±0.33b
(57.2)
8.52±0.45b
(95.9)
4,688.8 ±
242.7b
(629)
5,123.0 ±
638.9b
(828)
Females
Hct (percent)
Hgb
(g/dL)
MCV (fL)
MCH (pg)
23 Days
3 Months
23 Days
3 Months
23 Days
3 Months
23 Days
3 Months
48.0±0.4a
44.6 ±0.4
15.9 ±0.1
15.2 ±0.1
61.1 ±0.4
53.3 ±0.1
20.4 ±0.1
18.4 ±0.1
46.6 ±0.9
(97.1)
45.2 ±0.1
(101.3)
14.7±0.3b
(92.5)
15.4±0.1
(101.3)
53.9±0.5b
(88.2)
53. 3 ±0.1
(100)
17.3 ±0.2
(84.8)
17.9±0.1b
(97.3)
42.9±0.8b
(89.4)
44.1 ±0.3
(98.9)
13.0±0.3b
(81.8)
14.9 ±0.1
(98.0)
48.8±0.5b
(79.9)
52.4±0.2b
(98.3)
18.0 ±0.3
(88.2)
17.8±0.1b
(96.7)
39.2±0.7b
(81.7)
42.9±0.2b
(96.2)
11.8±0.3b
(74.2)
14.3±0.1b
(94.1)
46.6±0.6b
(76.3)
50.5±0.3b
(94.7)
18.9 ±0.7
(92.6)
16.9±0.1b
(91.8)
37.2±0.7b
(79.6)
42.6±0.5b
(95.5)
10.9±0.2b
(68.6)
14.1±0.2b
(92.8)
45.7±0.4b
(74.8)
48.0±0.9b
(90.1)
21.0 ±0.6
(102.9)
15.9±0.4b
(86.4)
33.4±0.6b
(69.6)
38.3±0.5b
(85.9)
9.7±0.2b
(61.0)
12.0±0.2b
(78.9)
46.5±0.5b
(76.1)
40.0 ± 0.7b
(75.0)
23.1 ±0.5
(113.2)
12.5±0.3b
(67.9)
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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
parameter
Erythrocyte
count (106/uL)
Platelet count
(106/uL)
Time on
treatment
23 Days
3 Months
23 Days
3 Months
Treatment group (mg hexavalent chromium/kg-day)
0
7.82 ±
0.09
8.30 ±
0.06
611.5 ±
43.7
588.9 ±
17.1
1.7
8.52 ±0.14
(109.0)
8.60 ± 0.05b
(103.6)
1,156.3 ±
76.4b
(189)
605. 8 ±17.1
(103)
3.5
7.22 ±0.19
(92.3)
8.40±0.04C
(101.2)
2808.8 ±
198.5b
(459)
574.8 ±21. 3
(98)
5.9
6.32±0.36b
(80.8)
8.47±0.04C
(102.0)
3295.0 ±
349.7b
(539)
528.2 ±14.1
(90)
11.2
5.27±0.23b
(67.4)
8.93±0.11b
(107.6)
4,318.4 ±
234.9b
(706)
619.3 ±55.4
(105)
20.9
4.21±0.16C
(53.8)
9.62±0.10b
(115.9)
5,132.8 ±
247.0b
(839)
1,524.9 ±
193.3b
(259)
"Values 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).
bSignificantly different (p < 0.01) from the control group by Dunn's or Shirley's test.
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>l 1.2 mg hexavalent chromium/kg-
14 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,
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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
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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
parameter
Time on
treatment
Treatment group (mg hexavalent chromium/kg-day)
0
1.7
3.5
5.9
11.2
20.9
Males
ALT (IU/L)
SDH (IU/L)
Bile acids (umol/L)
Cholesterol (mg/dL)
Triglycerides
(mg/dL)
Creatine kinase
(IU/L)
3 Months
3 Months
3 Months
3 Months
3 Months
3 Months
98±6a
31±2
22.0 ±2.2
89 ±2
170 ±9
214 ±26
274 ± 30C
(280)
55±5C
(177)
24.0 ±3.4
(109)
95 ±2
(107)
169 ±8
(99)
286 ± 32
(134)
461 ± 102C
(470)
110±24C
(355)
34.5 ±7.0
(157)
86 ±4
(97)
172 ± 15
(101)
291 ±36
(136)
447±121C
(456)
102 ± 24C
(329)
32.6 ±5.3
(148)
65±2C
(73)
170 ± 13
(100)
364 ± 23C
(170)
740±81C
(755)
173 ± 20C
(558)
45.3±2.8C
(206)
86±3b
(97)
164 ± 12
(96)
413±16C
(193)
191±17C
(195)
59±6C
(190)
28.1±2.0C
(128)
71±2C
(80)
98±8C
(57)
374 ± 44C
(175)
Females
ALT (IU/L)
SDH (IU/L)
Bile acids (umol/L)
Cholesterol (mg/dL)
Triglycerides
(mg/dL)
Creatine kinase
(IU/L)
3 Months
3 Months
3 Months
3 Months
3 Months
3 Months
64±5a
22 ±2
19.7 ±2.5
95 ±2
139 ±18
197 ±23
437 ± 68C
(683)
101 ± 17C
(459)
50.4±6.0C
(256)
111±4
(117)
116 ±10
(93)
311±94
(158)
218±27C
(340)
65 ± 10C
(295)
39.9±4.3C
(203)
94 ±2
(99)
98 ±9
(70)
265 ± 23
(135)
245 ± 30C
(383)
81±13C
(368)
35.3 ±3.5
(179)
87 ±2
(92)
81±4C
(58)
296 ± 24C
(150)
246 ± 37C
(384)
96 ± 20C
(436)
45.3±5.6C
(230)
83±2b
(87)
76±7C
(55)
359±23C
(182)
248 ± 22C
(387)
103 ± 12C
(468)
38.7±3.2b
(196)
79±2C
(83)
59±6C
(42)
432 ± 48C
(219)
1
2
3
4
5
6
7
"Values 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).
bSignificantly different (p < 0.05) from the control group by Dunn's or Shirley's test.
Significantly different (p < 0.01) from the control group by Dunn's or Shirley's test.
Source: NTP (2007).
Changes in organ weights in rats exposed to sodium dichromate dihydrate in drinking
water for 3 months are summarized in Table 4-6 (NTP, 2007). Treatment-related effects were
generally observed at doses >11.2 mg hexavalent chromium/kg-day. In males, decreases were
observed in absolute and relative liver weights and in absolute and relative spleen weights; in
females, relative right kidney weights and relative spleen weights were increased. Changes in
weights of other organs were considered by NTP (2007) to be secondary to changes in body
weight rather than due to adverse effects of treatment.
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Table 4-6. Selected organ weights in male and female F344/N rats exposed
to sodium dichromate dihydrate in drinking water for 3 months
Organ
Treatment group (mg hexavalent chromium/kg-day)
0
1.7
3.5
5.9
11.2
20.9
Males
Liver, absolute weight
Liver, relative weightd
Spleen, absolute weight
Spleen, relative weightd
10.89 ±0.42a
32.91 ±0.65
0.64 ± 0.02
1.94 ±0.03
10.30 ±0.28
31. 91 ±0.61
0.60 ±0.01
1.85 ±0.03
11.45 ±0.38
33. 98 ±0.75
0.62 ±0.02
1.83 ±0.04
10.51 ±0.18
31. 90 ±0.54
0.60 ±0.02
1.81±0.05C
9.20±0.17b
29.15 ±
0.53C
0.53±0.01b
1.69±0.02b
8.88±0.18b
29.80 ±0.35b
0.60±0.01b
2.00 ±0.03
Females
Right kidney, relative weight
Spleen, relative weightd
3.34±0.09a
2.12 ±0.05
3.32 ±0.04
2.04 ±0.03
3.55 ±0.05
2.16 ±0.05
3.55 ±0.07
2.22 ±0.03
3.58±0.10C
2.25±0.05C
3.63±0.09C
2.39±0.03C
aValues are means ± SE; n = 10 rats/group.
bSignificantly different (p < 0.01) from the control group by Williams's or Dunnett's test.
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).
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 dose-
19 dependent increase in the incidence of histiocytic cellular infiltration of pancreatic lymph nodes
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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-to-
6 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/kg-
9 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.
15
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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
Tissue (lesion type)
Treatment Group (mg hexavalent chromium/kg-day)
0
1.7
3.5
5.9
11.2
20.9
Males
Duodenum (histiocytic cellular
infiltration)
Stomach, glandular (ulcer)
Stomach, glandular (focal
regenerative hyperplasia)
Stomach, glandular (focal
squamous hyperplasia)
Pancreatic lymph node (ectasia)
Pancreatic lymph node (lymphoid
hyperplasia)
Pancreatic lymph node (histiocytic
cellular infiltration)
0/10a
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
5/10b
(1.0)
7/10c
(1.1)
0/10
0/10
0/10
0/10
0/10
2/10
(1.0)
9/10c
(1.2)
0/10
0/10
0/10
0/10
3/10
(1.0)
4/10b
(1.0)
8/10c
(1.4)
0/10
0/10
1/10
(2.0)
1/10
(1.0)
3/10
(1.0)
5/10b
(1.0)
7/10c
(1.4)
8/10c
(3.0)
10/10C
(2.2)
7/10c
(2.6)
10/10C
(1.7)
6/10c
(2.7)
9/10c
(1.9)
Females
Duodenum (histiocytic cellular
infiltration)
Stomach, glandular (ulcer)
Stomach, glandular (focal
regenerative hyperplasia)
Stomach, glandular (focal
squamous hyperplasia)
Pancreatic lymph node (ectasia)
Pancreatic lymph node (lymphoid
hyperplasia)
Pancreatic lymph node (histiocytic
cellular infiltration)
Liver (histiocytic cellular
infiltration)
Liver (chronic focal inflammation)
Bone marrow (hyperplasia)
0/10a
0/10
0/10
0/10
0/10
0/10
4/10
(1.0)
0/10
3/10
(1.0)
0/10
1/10C
(1.0)
0/10
0/10
0/10
0/10
0/10
8/10
(1.4)
3/10
(1.3)
5/10
(1.0)
0/10
5/10c
(1.0)
0/10
0/10
0/10
0/10
2/10
(1.5)
7/10
(1.7)
6/10c
(1.0)
2/10
(1.0)
0/10
7/10c
(1.4)
0/10
0/10
0/10
0/10
0/10
7/10
(1.3)
6/10c
(1.0)
7/10
(1.0)
0/10
8/10c
(1.6)
0/10
0/10
0/10
1/10
(1.0)
0/10
7/10
(1.7)
9/10c
(1.2)
2/10
(1.0)
0/10
10/10C
(1.7)
10/10C
(3.5)
10/10C
(2.0)
10/10C
(2.4)
10/10C
(1.8)
10/10C
(2.1)
9/10b
(1.9)
8/10c
(1.0)
10/10C
(1.0)
4/10b
(1.0)
"Number of animals with lesion/number of animals examined; parenthesis indicate average severity grade, with
l=minimal; 2=mild; 3=moderate; 4=severe.
bSignificantly different (p < 0.05) from the control group by the Fisher exact test.
Significantly different (p < 0.01) from the control group by the Fisher exact test.
Source: NTP (2007).
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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 B6C3Fi 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/kg-
24 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
51 DRAFT - DO NOT CITE OR QUOTE
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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
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Table 4-8. Selected organ weights in male and female B6C3Fi mice exposed
to sodium dichromate dihydrate in drinking water for 3 months
Organ
Treatment Group (mg hexavalent chromium/kg-day)
0
3.1
5.3
9.1
15.7
27.9
Males
Right kidney, absolute weight
Right kidney, relative weight"1
Liver, absolute weight
0.28±0.01a
7.25 ±0.11
1.60 ±0.08
0.28 ±0.01
7.68 ±0.29
1.54 ±0.05
0.26 ±0.01
7.43 ±0.35
1.50 ±0.05
0.26±0.01b
7.75 ±0.20
1.40±0.05b
0.24±0.01C
7.76 ±0.30
1.33±0.06C
0.26±0.01C
8.18±0.07C
1.34±0.04C
Females
Liver, relative weight"1
1.15±0.03a
1.14 ±0.04
1.06 ±0.02
1.11 ±0.04
1.04±0.02b
0.99±0.02C
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
"Values are means ± SE; n = 10 mice/group.
bSignificantly different (p < 0.05) from the control group by Williams's or Dunnett's test.
'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).
Gross and microscopic examinations of male and female mice exposed to sodium
dichromate dihydrate in drinking water for 3 months showed nonneoplastic lesions of the
duodenum and mesenteric lymph nodes (NTP, 2007); incidence data are summarized in
Table 4-9. In the duodenum, a dose-related increase in the incidence of minimal-to-mild
histiocytic cellular infiltration was observed in males and females in all treatment groups and in
the incidence of minimal-to-mild epithelial hyperplasia at> 5.3 mg hexavalent chromium/kg-
day; a slight dose-related increase in severity was observed. The duodenum had short, thick
duodenal villi, elongated crypts with diffuse hyperplasia, and hyperplastic epithelial cells with
swollen, vaculolated cytoplasm and increased numbers of "mitotic figures" (incidence data not
reported). NTP (2007) stated that duodenal lesions were indicative of regenerative hyperplasia
subsequent to epithelial cell injury. Minimal histiocytic cellular infiltration, morphologically
similar to that observed in rats (discussed above), was observed in mesenteric lymph nodes in
male and female mice at>5.3 mg hexavalent chromium/kg-day.
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Table 4-9. Incidence of nonneoplastic lesions observed in male and female
B6C3Fi mice exposed to sodium dichromate dihydrate in drinking water for
3 months
Tissue (lesion type)
Treatment group (mg hexavalent chromium/kg-day)
0
3.1
5.3
9.1
15.7
27.9
Males
Duodenum (histiocytic cellular
infiltration)
Duodenum (epithelial hyperplasia)
Mesenteric lymph node (histiocytic
cellular infiltration)
0/10a
0/10
0/10
4/10b
(1.0)
0/10
0/9
5/10c
(1.0)
8/10c
(1.3)
4/9b
(1.0)
10/10C
(1.3)
10/10C
(1.8)
6/8c
(1.0)
10/10C
(1.7)
10/10C
(2.1)
3/8
(2.0)
10/10C
(1.9)
10/10C
(1.8)
8/10c
(1.3)
Females
Duodenum (histiocytic cellular
infiltration)
Duodenum (epithelial hyperplasia)
Mesenteric lymph node (histiocytic
cellular infiltration)
0/10a
0/10
0/10
7/10c
(1.0)
0/10
0/10
8/9c
(1.3)
9/9c
(1.1)
6/10c
(1.0)
10/10C
(1.3)
10/10C
(1.1)
6/10c
(1.0)
10/10C
(1.4)
10/10C
(1.5)
4/9b
(1.3)
10/10C
(1.7)
10/10C
(1.4)
9/10c
(1.1)
"Number of animals with lesion/number of animals examined; parenthesis indicate average severity grade, with
l=minimal; 2=mild; 3=moderate; 4=severe.
bSignificantly different (p < 0.05) from the control group by the Fisher exact test.
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 B6C3Fi 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 (B6C3Fi,
15 BALB/c, and am3-C57EL/6) on the effects of exposure to sodium dichromate dihydrate in
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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 B6C3Fi 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 B6C3Fi, 10 male BALB/c, and 5 male am3-C57EL/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 aw3-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 B6C3Fi 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 B6C3Fi, 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 B6C3Fi 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.
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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 B6C3Fi or aw3-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 B6C3Fi, BALB/c, and aw3-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 B6C3Fi 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 aw3-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 B6C3Fi 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-C57EL/6 mice; in B6C3Fi 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 B6C3Fi 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-C57EL/6 mice at 8.7 mg hexavalent chromium/kg-
27 day; NTP (2007) stated that this change was related to decreased body weight.
28
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Table 4-10. Hematological effects in male B6C3Fl9 BALB/c, and
a/wJ-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
B6C3Fi mice
MCV (fL)
MCH (pg)
47.7 ± 0.2a
15.3 ±0.1
46.6 ± 0.2b
(97.7)
14.9±0.1b
(93.1)
46.4 ±0.2
(97.3)
14.7±0.1b
(96.1)
44.7 ±0.1
(93.7)
14.2±0.0b
(92.8)
BALB/c mice
MCV (fL)
MCH (pg)
44.8±0.2a
15.0 ±0.1
43.8±0.2b
(97.8)
14.5±0.1b
(96.7)
42.9 ±0.2
(95.8)
14.2±0.1b
(94.7)
42.6 ±0.2
(95.1)
14.0±0.1b
(93.3)
ajw3-C57BL/6 mice
MCV (fL)
MCH (pg)
45.8 ± 0.2 a
14.4 ±0.1
44.2 ±0.4
(96.5)
14.1±0.1b
(97.9)
43.7±0.3b
(95.4)
13.8±0.1b
(95.8)
40.5 ±0.3
(88.4)
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 B6C3F! 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).
bSignificantly 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 B6C3Fi, BALB/c, and aw3-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, dose-
7 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 B6C3Fi 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
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increased at 8.7 mg hexavalent chromium/kg-day in am3-C57EL/6 mice, but not in B6C3Fi or
BALB/c mice.
Table 4-11. Incidence of nonneoplastic lesions observed in male B6C3Fi,
BALB/c, and amJ-C57BL/6 mice exposed to sodium dichromate dihydrate in
drinking water for 3 months
Tissue (lesion type)
Treatment Group (mg hexavalent chromium/kg-day)
0
2.8
5.2
8.7
B6C3Fj mice
Duodenum (histiocytic cellular
infiltration)
Duodenum (epithelial hyperplasia)
Liver (glycogen depletion)
Pancreas (secretory depletion)
0/10a
0/10
1/10
(1.0)
0/10
8/10c
(1.0)
4/10b
(1.0)
2/10
(1.5)
2/10
(1.0)
10/10C
(1.4)
10/10C
(1.1)
9/10c
(1.4)
7/10c
(1.0)
10/10C
(2.0)
10/10C
(1.6)
10/10C
(2.2)
9/10c
(1.0)
BALB/c mice
Duodenum (histiocytic cellular
infiltration)
Duodenum (epithelial hyperplasia)
Pancreas (secretory depletion)
0/10a
0/10
0/10
4/10b
(1.0)
2/10
(1.0)
6/10c
(1.0)
8/10c
(1.8)
10/10C
(1.1)
9/10c
(1.3)
10/10C
(1.7)
10/10C
(1.4)
10/10C
(1.5)
a»i5-C57BL/6 mice
Duodenum (histiocytic cellular
infiltration)
Duodenum (epithelial hyperplasia)
Liver (glycogen depletion)
Pancreas (secretory depletion)
Mesenteric lymph node (histiocytic
cellular infiltration)
0/5a
0/5
0/5
0/5
0/5
2/5
(1.0)
5/5c
(1.0)
4/5b
(2.0)
3/5
(1.0)
0/5
5/5c
(1.4)
5/5c
(1.2)
5/5c
(1.6)
4/5b
(1.0)
0/5
4/5b
(1.8)
5/5c
(1.8)
5/5c
(3.8)
5/5c
(1.6)
4/5b
(1.5)
4
5
6
7
"Number of animals with lesion/number of animals examined; parenthesis indicate average severity grade, with
l=minimal; 2=mild; 3=moderate; 4=severe.
bSignificantly different (p < 0.01) from the control group by Fisher exact test.
Significantly different (p < 0.01) from the control group by Fisher exact test.
Source: NTP (2007).
In conclusion, the comparative 3-month drinking water study on sodium dichromate
dihydrate in male B6C3Fi, BALB/c, and am3-C57BL/6 mice showed similar effects in the
3 strains (NTP, 2007). A LOAEL of 2.8 mg hexavalent chromium/kg-day was identified based
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1 on histopathological changes in the duodenum in B6C3Fi mice (histiocytic cellular infiltration
2 and epithelial hyperplasia), BALB/c mice (histiocytic cellular infiltration), and am3-C57EL/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 etal, 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,
59 DRAFT - DO NOT CITE OR QUOTE
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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 etal, 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
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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 andNoreen, 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 etal, 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
61 DRAFT - DO NOT CITE OR QUOTE
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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 chromium-
20 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 Vyskociletal, 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,
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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, p2-microglobulin,
6 p-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 p2-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
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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
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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
parameter
MCV (fL)
Hct (percent)
Hgb (g/dL)
Time on
treatment
Day 22
Month 3
Month 6
Month 12
Day 22
Month 3
Month 6
Month 12
Day 22
Month 3
Month 6
Month 12
Treatment group (mg hexavalent chromium/kg-day)
0
59.5 ± 0.4 a
48.6 ±0.2
49.8 ±0.1
52.6 ±0.2
46.0 ±1.1
45.3 ±0.4
45.9 ±0.4
47.6 ±0.5
15.5 ±0.3
15.1 ±0.1
15.2 ±0.1
15.8 ±0.2
0.21
58.6 ±0.5
(98.5)
48.3 ±0.2
(99.4)
49.5 ±0.1
(99.4)
52.4 ±0.2
(99.6)
44.4 ±0.4
(96.5)
44.5 ±0.3
(98.2)
45.7 ±0.5
(99.6)
46.6 ±0.4
(97.9)
15.1 ±0.2
(97.4)
14.9 ±0.1
(98.7)
15.2 ±0.2
(100)
15.4 ±0.2
(97.5)
0.77
54.9±0.5C
(92.3)
47.3±0.2C
(97.3)
48.6±0.1C
(97.6)
51.9 ±0.3
(98.7)
43.2±0.6b
(93.9)
44.5 ±0.4
(98.2)
45.5 ±0.4
(99.1)
47.4 ±0.5
(99.6)
14.2±0.2C
(91.6)
14.9 ±0.2
(98.7)
15.0 ±0.2
(98.7)
15.6 ±0.2
(98.7)
2.1
47.4 ± 0.4C
(80.0)
45.7±0.2C
(94.0)
47.8±0.2C
(96.0)
51.4±0.3C
(97.7)
38.7±0.6C
(84.1)
44.1 ±0.5
(97.4)
45.5 ±0.5
(99.1)
47.7 ±0.4
(100.2)
12.0±0.3C
(77.4)
14.6±0.2b
(96.7)
14.9 ±0.1
(98.0)
15.6 ±0.2
(98.7)
5.9
45.0±0.7C
(75.6)
39.2±0.6C
(80.7)
45.4±0.5C
(91.2)
49.9±0.2C
(94.9)
33.5±0.8C
(72.8)
41.0±0.5C
(90.5)
45.0 ±0.3
(98.0)
47.3 ±0.4
(99.4)
10.1±0.2C
(65.2)
12.9±0.2C
(85.4)
14.5±0.1C
(95.4)
15.3±0.1b
(96.8)
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Table 4-12. Hematological effects in male F344/N rats exposed to sodium
dichromate dihydrate in drinking water for up to 12 months
Hematological
parameter
MCH (pg)
MCHC (g/dL)
Erythrocyte count
(106/uL)
Time on
treatment
Day 22
Month 3
Month 6
Month 12
Day 22
Months
Month 6
Month 12
Day 22
Month 3
Month 6
Month 12
Treatment group (mg hexavalent chromium/kg-day)
0
19.8 ±0.1
16.2 ±0.1
16.3 ±0.1
17.0 ±0.1
33.3 ±0.1
33.4 ±0.1
32.7 ±0.1
32.3 ±0.2
7.80 ±0.13
9.28 ±0.05
9.34 ±0.06
9.27 ±0.10
0.21
19.5 ±0.2
(98.5)
16.2 ±0.1
(100)
16.1 ±0.1
(98.8)
16.8 ±0.1
(98.8)
33.3 ±0.1
(100)
33.5 ±0.2
(100.3)
32.5 ±0.1
(99.4)
32.1 ±0.3
(99.4)
7.74 ±0.15
(99.2)
9.24 ± 0.06
(99.6)
9.43 ±0.08
(101.0)
9. 17 ±0.07
(98.9)
0.77
17.7±0.2C
(89.4)
15.7±0.0C
(96.9)
15.7±0.1C
(96.3)
16.6±0.1b
(97.6)
32.2 ±0.2
(96.7)
33.2 ±0.1
(99.4)
32.3±0.1b
(98.8)
32.0 ±0.2
(99.1)
8.06 ±0.16
(103.3)
9.46 ±0.11
(101.9)
9.54 ±0.11
(102.1)
9.40 ±0.12
(101.4)
2.1
14.8±0.2C
(74.7)
15.0±0.1C
(92.6)
15.3±0.1C
(93.9)
16.2±0.1C
(95.3)
31.2±0.2C
(93.7)
32.7±0.1C
(97.9)
32.1±0.1C
(98.2)
31.6±0.2b
(97.8)
8.10 ±0.14
(103.8)
9.75±0.11C
(105.1)
9.71±0.08C
(104.0)
9.61 ±0.11
(103.7)
5.9
16.3±0.5C
(82.3)
11.9±0.3C
(73.5)
14.3±0.2C
(87.7)
15.7±0.1C
(92.4)
36.2 ±0.8
(108.7)
30.2±0.3C
(90.4)
31.6±0.2C
(96.6)
31.5±0.2b
(97.5)
6.21 ± 0.13 c
(79.6)
10.93 ±0.16C
(117.7)
10.15 ±0.13C
(108.7)
9.74±0.08C
(105.1)
1
2
3
4
5
6
7
"Values 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).
bSignificantly different (p < 0.05) from the control group by Dunn's or Shirley's test.
Significantly different (p < 0.01) from the control group by Dunn's or Shirley's test.
Source: NTP (2007).
Results of clinical chemistry analyses in special study male rats (clinical chemistry was
not assessed in female rats) show that exposure to sodium dichromate dihydrate in drinking
water produced dose-dependent increases in serum ALT activity (NTP, 2008). Significant
increases in serum ALT activity were observed at 4 days and at 6 months in rats treated with
>2.1 mg hexavalent chromium/kg-day and at 22 days and 3 and 12 months at >0.77 mg
hexavalent chromium/kg-day (Table 4-13). Serum ALT enzyme activity reached maximum
increases (approximately 170-260% of control values) in rats treated for 3-12 months at daily
doses of >2.1 mg hexavalent chromium/kg-day. In rats treated for 12 months with 2.1 and
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1
2
3
4
5
6
7
8
9
10
5.9 mg hexavalent chromium/kg-day, serum SDH activity was 164 and 173% of control values,
respectively; however, no increases in SDH activity were observed at other doses or time points.
No increases in serum AP activity were observed in any treatment group throughout the 12-
month treatment period. Increased serum ALT activity is consistent with histopathological
findings of minimal chronic inflammation of the liver observed in core study animals (discussed
below); however, because other clinical chemistry markers of hepatic damage were not observed,
NTP (2008) suggested that increased serum ALT activity may reflect enzyme induction rather
than hepatocellular damage. Changes in other clinical chemistry outcomes were generally <5%
compared to controls and did not exhibit dose- or duration-dependence.
Table 4-13. Serum ALT activity in male F344/N rats exposed to sodium
dichromate dihydrate in drinking water for up to 12 months
Time on treatment
Day 4
Day 22
Months
Month 6
Month 12
Treatment group (mg hexavalent chromium/kg-day)
0
54±2a
45 ± 1
82 ±4
122 ± 15
102 ±6
0.21
53 ±2
(98)
46 ± 1
(102)
82 ± 12
(100)
114±9
(93)
107 ±8
(105)
0.77
60 ±3
(113)
58±2C
(129)
135±18b
(165)
150 ±12
(123)
135 ± 10b
(132)
2.1
68±lc
(126)
75±3C
(167)
176±13C
(215)
238 ±2C
(195)
261±23C
(256)
5.9
70±2C
(130)
73±4C
(162)
216±21C
(263)
210±12C
(172)
223 ± 15C
(219)
11
12
13
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.
bSignificantly different (p < 0.05) from the control group by Dunn's or Shirley's test.
Significantly different (p < 0.01) from the control group by Dunn's or Shirley's test.
Source: NTP (2008).
Gross and microscopic examinations of core study rats exposed to sodium dichromate
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;
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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/kg-
20 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
68 DRAFT - DO NOT CITE OR QUOTE
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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.
10
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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
Tissue (lesion type)
Treatment group (mg hexavalent chromium/kg-day)
0
0.21
0.77
2.1
5.9
Males
Liver (histiocytic cellular infiltration)
Liver (chronic inflammation)
Liver (basophilic focus)
Small intestine, duodenum (histiocytic
cellular infiltration)
Lymph node, mesenteric (histiocytic
cellular infiltration)
Lymph node, mesenteric (hemorrhage)
Tissue (lesion type)
l/50a
(1.0)
19/50
(1.1)
22/50
0/48
13/49
(2.0)
2/49
(1.5)
0/50
25/50
(1.2)
28/50
0/48
11/50
(1.5)
7/50
(1.1)
2/49
(1.0)
21/49
(1.3)
29/49b
6/47b
(1.2)
30/49
(1.9)
9/49b
(1.3)
5/50
(1.4)
28/50b
(1.1)
32/50b
36/46c
(1.1)
39/50c
(2.1)
8/50b
(1.1)
34/49c
(1.4)
26/49
(1.3)
30/49
47/48c
(1.5)
41/49 c
(2.1)
17/49C
(1.3)
Treatment group (mg hexavalent chromium/kg-day)
0
0.24
0.94
2.4
7.0
Females
Liver (histiocytic cellular infiltration)
Liver (chronic inflammation)
Liver (fatty change)
Liver (clear cell focus)
Small intestine, duodenum (histiocytic
cellular infiltration)
Lymph node, mesenteric (histiocytic
cellular infiltration)
Lymph node, mesenteric (hemorrhage)
Lymph node, pancreatic (histiocytic
cellular infiltration)
Salivary gland (atrophy)
l/50a
(1.0)
12/50
(1.3)
3/50
(3.3)
7/50
0/46
21/50
(1.7)
11/50
(1.1)
17/29
(2.0)
9/50
(1.3)
5/50
(1.0)
21/50b
(1.2)
7/50
(3.6)
5/50
0/49
18/50
(1.4)
13/50
(1.3)
20/36
(1.9)
7/50
(1.4)
21/50C
(1.3)
28/50c
(1.3)
10/50b
(2.5)
7/50
1/48
(1.0)
27/50
(1.5)
16/50
(1.3)
23/30
(2.6)
10/50
(1.2)
42/50c
(2.0)
35/50c
(1.6)
13/50C
(2.5)
20/50 c
30/46C
(1.0)
36/50c
(2.0)
14/50
(1.1)
32/34c
(2.8)
17/50b
(1.4)
47/50c
(2.6)
39/50c
(2.1)
16/50C
(2.8)
7/50
47/50c
(1.2)
42/50c
(2.4)
2 1/50 b
(1.3)
27/33
(3.0)
17/50
(2.1)
"Number of animals with lesion/number of animals examined; parenthesis indicate average severity grade, with
l=minimal; 2=mild; 3=moderate; 4=severe.
bSignificantly different (p < 0.05) from the control group by the Poly-3 test.
Significantly different (p < 0.01) from the control group by the Poly-3 test.
Source: NTP (2008).
70
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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 dose-
19 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.
21
71 DRAFT - DO NOT CITE OR QUOTE
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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
Neoplasm type
Treatment group (mg hexavalent chromium/kg-day)
0
0.21
0.77
2.1
5.9
Males
Oral mucosa, squamous cell papilloma
Overall rate3 'b
0/50
(0%)
0/50
(0%)
0/49
(0%)
0/50
(0%)
1/49
(2%)
Oral mucosa, squamous cell carcinoma
Overall rate3
Adjusted rate0
0/50
(0%)
0%
p< 0.001
0/50
(0%)
0%
0/49
(0%)
0%
0/50
(0%)
0%
6/49
(12%) [543]
13.5%
p = 0.015
Tongue, squamous cell papilloma
Overall rate3-13
0/50
(0%)
0/50
(0%)
0/49
(0%)
0/50
(0%)
1/49
(2%)
Tongue, squamous cell carcinoma
Overall rate3
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 rate3
Adjusted rate0
Neoplasm type
0/50
(0%)
0%
p< 0.001
1/50
(2%) [729T]
2.4%
0/49
(0%)
0%
0/50
(0%)
0%
7/49
(14.5%) [543]
15.7%
p = 0.007
Treatment group (mg hexavalent chromium/kg-day)
0
0.24
0.94
2.4
7.0
Females
Oral mucosa, squamous cell carcinoma
Overall rate3
Adjusted rate0
0/50
(0%)
0%
p< 0.001
0/50
(0%)
0%
0/50
(0%)
0%
2/50
(4%) [646]
4.6%
11/50
(22%) [506]
23.9%
p< 0.001
Tongue, squamous cell papilloma
Overall rate3-13
1/50
(2%)
1/50
(2%)
0/50
(0%)
0/50
(0%)
0/50
(0%)
Tongue, squamous cell carcinoma
Overall rate3-13
0/50
(0%)
0/50
(0%)
0/50
(0%)
1/50
(2%)
0/50
(0%)
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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
Neoplasm type
Treatment group (mg hexavalent chromium/kg-day)
0
0.24
0.94
2.4
7.0
Females
Oral mucosa or tongue, squamous cell papilloma or carcinoma
Overall rate3
Adjusted rate0
1/50
(2%) [618]
2.2%
p< 0.001
1/50
(2%) [729T]
2.3%
0/50
(0%)
0%
2/50
(4%) [646]
4.6%
11/50
(22%) [506]
23.9%
p = 0.002
"Overall rate: number of animals with lesion/number of animals examined; parenthesis are the percent of animals
examined with lesion; brackets are days to first incidence; T: observed at terminal sacrifice. />-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.
0 Adjusted rate: Poly-3 estimated neoplasm incidence (expressed as percent of animals with neoplasm) adjusted for
intercurrent mortality. />-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).
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
Neoplasm type
Treatment group (mg hexavalent chromium/kg-day)
0
0.21
0.77
2.1
5.9
Males
Pancreatic acinar adenoma
Benign pheochromocytoma (adrenal medulla)
Neoplasm type
1/50^
6/49^
2/50
13/50C
6/49c
14/49 c
2/50
5/50
2/49
4/49
Treatment group (mg hexavalent chromium/kg-day)
0
0.24
0.94
2.4
7.0
Females
Mononuclar cell leukemia
8/50a'b
18/50C
13/50
7/50
11/50
"Number of animals with lesion/number of animals examined.
bNot statistically significant for positive trend (p > 0.05) by the Poly-3 test.
Significantly different from controls by the Poly-3 test (p < 0.05).
Source: NTP (2008).
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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/kg-
8 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 B6C3Fi 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
74 DRAFT - DO NOT CITE OR QUOTE
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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.
33
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Table 4-17. Hematological effects in female B6C3Fi mice exposed to sodium
dichromate dihydrate in drinking water for up to 12 months
Hematological
parameter
MCV (fL)
MCH (pg)
Erythrocyte
count (106/uL)
Time on
treatment
Day 22
Month 3
Month 6
Month 12
Day 22
Month 3
Month 6
Month 12
Day 22
Month 3
Month 6
Month 12
Treatment group (mg hexavalent chromium/kg-day)
0
48.8±0.2a
47.2 ±0.1
45.8 ±0.2
46.9 ±0.3
16.4 ±0.1
15.8 ±0.0
15.3 ±0.1
15.5 ±0.1
10.25 ±0.15
10.10 ±0.16
10.56 ±0.15
9.58 ±0.10
0.38
48.3±0.1b
(90.0)
46.9 ±0.3
(99.4)
45.5 ±0.3
(99.3)
46.9 ±0.3
(100)
16.2±0.0b
(98.8)
15.7±0.1
(99.4)
15.2±0.1
(99.3)
15.7 ±0.2
(101.3)
10.20 ±0.08
(99.5)
10.66 ±0.13b
(105.5)
10.81 ±0.10
(102.4)
9.72 ±0.09
(101.4)
1.4
47.8±0.2C
(98.0)
46.7 ±0.1
(98.9)
45.1±0.2b
(98.5)
46.3 ±0.3
(98.7)
15.9±0.1C
(97.0)
15.6±0.0C
(98.7)
15.1 ±0.1
(98.7)
15.5 ±0.1
(100)
10.47 ±0.19
(102.1)
10.55 ±0.17b
(104.5)
10.60 ±0.13
(100.4)
9.77 ±0.10
(102.0)
3.1
47.0 ± 0.2C
(96.3)
45.1±0.2C
(95.6)
44.6 ± 0.2C
(97.4)
45.2±0.2C
(96.4)
15.7±0.1C
(95.7)
14.9±0.1C
(88.6)
14.9±0.1C
(97.4)
15.1±0.1b
(97.4)
10.77 ±0.13b
(105.1)
10.95 ±0.10C
(108.4)
10.77 ±0.20
(102.0)
9.95±0.13b
(103.9)
8.7
46.8±0.2C
(95.9)
43.7±0.3C
(92.6)
42.8±0.3C
(93.4)
43.9±0.5C
(93.6)
15.5±0.1C
(94.5)
14.3±0.1C
(90.5)
14.1±0.1C
(92.2)
14.4±0.2C
(92.9)
10.61 ±0.13b
(103.5)
11.55 ±0.16C
(114.4)
11.50±0.20C
(108.9)
10.30 ±0.21C
(107.5)
"Values 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).
bSignificantly different (p < 0.05) from the control group by Dunn's or Shirley's test.
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
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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.
21
77 DRAFT - DO NOT CITE OR QUOTE
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Table 4-18. Incidence of nonneoplastic lesions observed in male and female
B6C3Fi mice exposed to sodium dichromate dihydrate in drinking water for
2 years
Tissue (lesion type)
Treatment group (mg hexavalent chromium/kg-day)
0
0.38
0.91
2.4
5.9
Males
Small intestine, duodenum (diffuse
hyperplasia)
Small intestine, duodenum (histiocytic
cellular infiltration)
Lymph node, mesenteric (histiocytic
cellular infiltration)
Lymph node, pancreatic (histiocytic
cellular infiltration)
Pancreas (cytoplasmic alteration)
Tissue (lesion type)
0/50a
0/50
14/47
(1.2)
0/5
0/49
11/50C
(2.0)
2/50
(1.0)
38/49c
(1.1)
2/13
(1.0)
1/49
(3.0)
18/50C
(1.6)
4/50
(1.0)
31/49C
(1.2)
2/10
(1.0)
1/50
(3.0)
42/50c
(2.1)
37/50c
(1.2)
32/49c
(1.5)
5/8b
(1.4)
9/49°
(2.1)
32/50c
(2.1)
35/50c
(1.7)
42/46c
(2.5)
12/16b
(2.3)
8/48c
(2.6)
Treatment group (mg hexavalent chromium/kg-day)
0
0.38
1.4
3.1
8.7
Females
Small intestine, duodenum (diffuse
hyperplasia)
Small intestine, duodenum (histiocytic
cellular infiltration)
Small intestine, jejunum (diffuse
hyperplasia)
Small intestine, jejunum (histiocytic
cellular infiltration)
Liver (histiocytic cellular infiltration)
Liver (chronic inflammation)
Lymph node, mesenteric (histiocytic
cellular infiltration)
Lymph node, pancreatic (histiocytic
cellular infiltration)
Pancreas (cytoplasmic alteration)
0/50a
0/50
0/50
0/50
2/49
(1.0)
16/49
(1.1)
3/46
(1.0)
0/14
0/48
16/50C
(1.6)
0/50
2/50
(2.0)
0/50
15/50C
(1.1)
21/50
(1.1)
29/48c
(1.3)
1/12
(1.0)
6/50b
(2.5)
35/50c
(1.7)
4/50
(1.3)
1/50
(1.0)
0/50
23/50c
(1.0)
22/50
(1.10
26/46c
(1.1)
2/15
(1.5)
6/49b
(2.0)
31/50C
(1.6)
33/50c
(1.2)
0/50
2/50
(1.0)
32/50c
(1.0)
27/50c
(1.1)
40/50C
(1.9)
7/14c
(1.9)
14/50C
(2.4)
42/50c
(2.2)
40/50C
(2.0)
8/50c
(1.9)
8/50c
(1.6)
45/50c
(1.9)
24/50
(1.0)
42/50c
(2.7)
8/13c
(2.5)
32/50c
(2.6)
"Number of animals with lesion/number of animals examined; parenthesis indicate average severity grade, with
l=minimal; 2=mild; 3=moderate; 4=severe.
bSignificantly different (p < 0.05) from the control group by the Poly-3 test.
Significantly different (p < 0.01) from the control group by the Poly-3 test.
Source: NTP (2008).
Incidence data for neoplastic lesions of the small intestine in male and female mice
exposed to sodium dichromate dihydrate in drinking water for 2 years are summarized in
78
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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 B6C3Fi mice exposed to sodium dichromate dihydrate in
drinking water for 2 years
Tissue and lesion type
Treatment group (mg hexavalent chromium/kg-day)
0
0.38
0.91
2.4
5.9
Males
Duodenum, adenoma
Overall rate3-13
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 rate3
Adjusted rate0
1/50
(2%) [665]
2.2%
p< 0.001
0/50
(0%)
0%
1/50
(2%) [729T]
2.3%
5/50
(10%) [729T]
10.8%
15/50
(30%) [451]
32.9%
p< 0.001
Duodenum, carcinoma
Overall rate3
Adjusted rate0
0/50
(0%)
0%
/?< 0.011
0/50
(0%)
0%
0/50
(0%)
0%
2/50
(4%) [729T]
4.3%
3/50
(6%) [729T]
6.8%
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Table 4-19. Incidence of neoplastic lesions observed in the small intestine of
male and female B6C3Fi mice exposed to sodium dichromate dihydrate in
drinking water for 2 years
Jejunum, adenoma
Overall rate3
Adjusted rate0
Tissue and lesion type
0/50
(0%)
0%
p = 0.002
0/50
(0%)
0%
0/50
(0%)
0%
0/50
(0%)
0%
3/50
(6%) [714]
6.8%
Treatment group (mg hexavalent chromium/kg-day)
0
0.38
0.91
2.4
5.9
Males
Jejunum, multiple carcinoma
Overall rate3-13
0/50
1/50
0/50
0/50
0/50
Jejunum, all carcinoma (includes multiple)
Overall rate3-13
0/50
2/50
0/50
1/50
2/50
All small intestine"1, adenoma
Overall rate3
Adjusted rate0
1/50
(2%) [665]
2.2%
p< 0.001
1/50
(2%) [729T]
2.3%
1/50
(2%) [729T]
2.3%
5/50
(10%) [729T]
10.8%
17/50
(34%) [451]
37.2%
p< 0.001
All small intestine"1, carcinoma
Overall rate3
Adjusted rate0
0/50
(0%)
0%
p = 0.014
2/50
(4%) [729T]
4.5%
1/50
(2%) [729T]
2.3%
3/50
(6%) [729T]
6.5%
5/50
(10%) [729T]
11.4%
p = 0.028
All small intestine"1, adenoma or carcinoma
Overall rate3
Adjusted rate0
Tissue and lesion type
1/50
(2%) [665]
2.2%
p< 0.001
3/50
(6%) [729T]
6.8%
2/50
(4%) [729T]
4.6%
7/50
(14%) [729T]
15.1%
p = 0.032
20/50
(40%) [451]
43.8%
p< 0.001
Treatment group (mg hexavalent chromium/kg-day)
0
0.38
1.4
3.1
8.7
Females
Duodenum, multiple adenoma
Overall rate3-13
0/50
(0%)
0/50
(0%)
0/50
(0%)
1/50
(2%)
6/50
(12%)
p<0.05
Duodenum, all adenoma (includes multiple)
Overall rate3
Adjusted rate0
0/50
(0%)
0%
p< 0.001
0/50
(0%)
0%
2/50
(4%) [729T]
4.2%
13/50
(25%) [729T]
27.8%
p< 0.001
12/50
(24%) [693]
25.2%
p< 0.001
Duodenum, carcinoma
Overall rate3
Adjusted rate0
0/50
(0%)
0%
p< 0.001
0/50
(0%)
0%
0/50
(0%)
0%
1/50
(2%) [729T]
2.1%
6/50
(12%) [625]
12.6%
p = 0.019
Jejunum, multiple adenomas
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Table 4-19. Incidence of neoplastic lesions observed in the small intestine of
male and female B6C3Fi mice exposed to sodium dichromate dihydrate in
drinking water for 2 years
Overall rate3-13
Tissue and lesion type
0/50
(0%)
0/50
(0%)
0/50
(0%)
0/50
(0%)
1/50
(2%)
Treatment group (mg hexavalent chromium/kg-day)
0
0.38
1.4
3.1
8.7
Females
Jejunum, all adenomas (including multiple)
Overall rate3
Adjusted rate0
0/50
(0%)
0%
p = 0.002
1/50
(2%) [729T]
2.2%
0/50
(0%)
0%
2/50
(4%) [729T]
4.3%
5/50
(10%) [729T]
10.6%
p = 0.035
Jejunum, carcinoma
Overall rate3-13
1/50
(2%)
0/50
(0%)
2/50
(4%)
2/50
(4%)
1/50
(2%)
All small intestine"1, adenoma
Overall rate3
Adjusted rate0
0/50
(0%)
0%
p< 0.001
1/50
(2%) [729T]
2.2%
2/50
(4%) [729T]
4.2%
15/50
(30%) [729T]
32.0%
p< 0.001
16/50
(32%) [693]
33.7%
p< 0.001
All small intestine"1, carcinoma
Overall rate3
Adjusted rate0
1/50
(2%) [729T]
2.2%
p< 0.001
0/50
(0%)
0%
2/50
(4%) [729T]
4.2%
3/50
(6%) [729T]
6.4%
7/50
(14%) [625]
14.7%
p = 0.037
All small intestine"1, adenoma or carcinoma
Overall rate3
Adjusted rate0
1/50
(2%) [729T]
2.2%
p< 0.001
1/50
(2%) [729T]
2.2%
4/50
(8%) [729T]
8.3%
17/50
(34%) [729T]
36.3%
p< 0.001
22/50
(44%) [625]
45.9%
p< 0.001
"Overall rate: number of animals with lesion/number of animals examined; parenthenesis are the percent of animals
examined with lesion; brackets indicate the days to first incidence; T: observed at terminal sacrifice. />-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.
°Adjusted rate: Poly-3 estimated neoplasm incidence (expressed as % of animals with neoplasm) adjusted for
intercurrent mortality. />-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.
dDuodenum, jejunum, or ileum.
Source: NTP (2008).
In conclusion, the NTP (2008) 2-year toxicology and carcinogenicity study on sodium
dichromate dihydrate identified a LOAEL for noncancer effects of 0.38 mg hexavalent
chromium/kg-day in male and female B6C3Fi 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 B6C3Fi 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 B6C3Fi mice based on
16 increased incidences of neoplasms of the small intestine.
17
18 Borneffetal., 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
1 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
12 drinking water consumption for primates (0.09 x body weight0'7945; 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 high-
19 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 high-
29 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/kg-
29 day).
30
31 Chowdhury andMitra, 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
8 and 3p-A5-hydroxysteroid dehydrogenase (3P-A5-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
19 groups, respectively. In all treatment groups, 3P-A5-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 Ley dig 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
2 of 3P-A5-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
6 testosterone levels and loss of 3P-A5-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,/? < 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 Lietal.,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 Zahidetal, 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 B6C3Fi 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 x 2 contingency chi-
24 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 Murthyetal, 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 B6C3Fi 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 Yousefetal, 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
1 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 high-
27 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 (Fo) 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, FO
5 breeding pairs were separated and continued on study diets; litters born during the post-
6 separation period (Fi animals) were reared with the FO dams until weaning (PND 21). Dam and
7 pup weights and dam food consumption were monitored during the lactational period. Upon
8 weaning, FO 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 FI animals, animals were maintained
13 on the same study diets as their parents. During post-lactational exposure of the FI 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 FI 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 FI adults (time from separation to terminal sacrifice not
23 reported) were the same as those described above for FO 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 FO 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/kg-
9 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 FO 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 treatment-
16 related effects. In the FO 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 FI 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 FI 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 FI 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 FI 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 FI 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 Fl 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 treatment-
7 related effects. In the FI 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 FI 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 FI 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 FO mice and 37.1 mg hexavalent chromium/kg-day in FI 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 Kcmojia et al, 1998
1 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 post-
25 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 Junaidetal, 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 treatment-
24 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 low-
8 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 ElsaieedandNada, 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, pre-
26 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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1 laboratory mammals (0.10 x body weight0'7377; 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. I/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 pre-
13 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 Junaidetal, 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 treatment-
8 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 etal, 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 high-
16 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-HamoodetaL, 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
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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, NO AELs and/or LO AELs,
32 expressed in mg hexavalent chromium/kg-day, could not be derived from this study.
33
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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
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Table 4-20. Evidence of mutagenicity of hexavalent chromium compounds in experimental
systems
Chemical
Ammonium
chromate
Calcium chromate
Chromic acid
Potassium chromate
Potassium
dichromate
Sodium chromate
Sodium dichromate
Sodium dichromate
dihydrate
In vitro studies
(non-mammalian cells)
DNA
damage
ND
ND
ND
•
•
ND
ND
ND
Mutations
•
•
•
•
•
•
•
•
In vitro studies
(mammalian cells)
DNA
damage
ND
ND
ND
•
•
•
•
ND
Mutations
ND
ND
ND
•
•
ND
ND
•
Chromosomal
damage
ND
•
•
•
•
•
•
ND
In vivo studies
(D. melanogaster or mammals)
DNA
damage
ND
ND
ND
• (M)
• (M)
ND
•(M)
ND
Mutations
ND
•(D)
• (D)
•(D)
•(M)
• (D)
ND
• (D)
ND
Chromosomal
damage
ND
ND
ND
•(M)
• (M)
ND
ND
•(M)
• = positive results
ND = no data identified for this review
(D) = study in D. melanogaster
(M) = study in laboratory mammal
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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-
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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 B6C3Fi 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
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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-C57EL/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
23 exposed to chromium fumes for 1 week (1.84 mg/m3) or 2 months (0.55 mg/m3) (Koshi et al., 1987).
24
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Table 4-21. In vitro genotoxicity studies of hexavalent chromium in non-mammalian cells
Endpoint
Reverse mutations
Reverse mutations
Reverse mutations
Reverse mutations
Reverse mutations
Reverse mutations
Reverse mutations
Reverse mutations
Reverse mutations
Reverse mutations
Reverse mutations
Reverse mutations
Reverse mutations
Reverse mutations
Reverse mutations
Reverse mutations
Reverse mutations
Reverse mutations
Reverse mutations
Chemical form
Ammonium chromate
Ammonium chromate
Ammonium chromate
Calcium chromate
Calcium chromate
Calcium chromate
Calcium chromate
Calcium chromate
Calcium chromate
Chromic acid
Chromic acid
Chromium trioxide
Chromium trioxide
Chromium trioxide
Potassium chromate
Potassium chromate
Potassium chromate
Potassium chromate
Potassium chromate
Test system
S. typhimurium TA97,
TA1538, TA98, TA100
S. typhimurium TA1537
S. typhimurium TA1535
S. typhimurium TA97,
TA1538, TA98, TA100
S. typhimurium TA1537
S. typhimurium TA1535
S. typhimurium TA98
S. typhimurium TA100,
TA1535, TA1537, TA1538
E. coli WP2 uvrA
S. typhimurium TA102,
TA2638
E. coli, WP2/pKM101, WP2
uvrA/pKMlOl
S. typhimurium TA97,
TA1538, TA98, TA100
S. typhimurium TA1537
S. typhimurium TA1535
S. typhimurium TA102
S. typhimurium TA97,
TA1538, TA98, TA100
S. typhimurium TA1537
S. typhimurium TA1535
E. coli Hs30R
Results3
Without
activation
+
±
-
+
±
-
-
-
-
+
+
+
±
-
+
+
±
-
+
With
activation
NS
NS
-
NS
NS
-
±
-
±
ND
ND
NS
NS
-
ND
NS
NS
-
ND
Reference
DeFlora et al, 1984
DeFlora et al, 1984
DeFlora et al., 1984
DeFlora et al., 1984
DeFlora et al., 1984
DeFlora et al., 1984
Dunkeletal., 1984
Dunkeletal., 1984
Dunkeletal., 1984
Watanabe et al., 1998
Watanabe et al., 1998
DeFlora et al., 1984
DeFlora et al., 1984
DeFlora et al., 1984
Marzin and Phi, 1985
DeFlora et al., 1984
DeFlora et al., 1984
DeFlora et al., 1984
Nakamuro et al., 1978
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Table 4-21. In vitro genotoxicity studies of hexavalent chromium in non-mammalian cells
Endpoint
Reverse mutations
Reverse mutations
Reverse mutations
Reverse mutations
Reverse mutations
Reverse mutations
Reverse mutations
Reverse mutations
Reverse mutations
Reverse mutations
Reverse mutations
Reverse mutations
Reverse mutations
Reverse mutations
Reverse mutations
Reverse mutations
Reverse mutations
Reverse mutations
Reverse mutations
Reverse mutations
Chemical form
Potassium chromate
Potassium chromate
Potassium chromate
Potassium dichromate
Potassium dichromate
Potassium dichromate
Potassium dichromate
Potassium dichromate
Potassium dichromate
Potassium dichromate
Potassium dichromate
Potassium dichromate
Potassium dichromate
Sodium chromate
Sodium dichromate
Sodium dichromate
Sodium dichromate
Sodium dichromate
Sodium dichromate
Sodium dichromate
Test system
E. coli Wp2 her- try-,
B/rWP2
E. coli Wp2(try-)
E. co// WP2uvrA, CM571
S. typhimurium TA97, TA98,
TA100, TA1535, TA1537
S. typhimurium TA102
S. typhimurium TA100
E. coli Wp2 her- try-,
B/rWP2
E. coli HsSOR
E. coli WP2, WP2uvrA,
CM571
E. co// WP2uvrA, CM571
S. cerevisiae D7
S. typhimurium TA98
S. typhimurium TA1538
E. coli Wp2(try-)
S. typhimurium TA102,
TA2638
S. typhimurium TA102
S. typhimurium TA100
S. typhimurium TA97
S. typhimurium TA1537,
TA1538, TA98, TA100
S. typhimurium TA1535
Results3
Without
activation
+ (Wp2 her)
+
+
+
+
+
+ (Wp2 her)
+
+
+
+
±
-
+
+
+
+
+
±
-
With
activation
ND
ND
ND
+
ND
+
ND
ND
ND
ND
ND
-
-
ND
ND
+
-
NS
NS
-
Reference
Kanematsuetal., 1980
Venitt and Levy, 1974
SeoandLee, 1993
Zeigeretal., 1992
MarzinandPhi, 1985
Venieretal., 1982
Kanematsuetal., 1980
Nakamuro et al., 1978
Nishioka, 1975
SeoandLee, 1993
Sing, 1983
Venieretal., 1982
Venieretal., 1982
Venitt and Levy, 1974
Watanabe et al., 1998
Bennicellietal., 1983
DeFlora, 1978
DeFlora et al., 1984
DeFlora et al., 1984
DeFlora et al., 1984
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Table 4-21. In vitro genotoxicity studies of hexavalent chromium in non-mammalian cells
Endpoint
Reverse mutations
Reverse mutations
Reverse mutations
Reverse mutations
Induction of SOS
response
Induction of SOS
response
Induction of SOS
response
Induction of SOS
response
Induction of SOS
response
Mutations
Mutations
Mutations
Mutations
Mutations
Mutations
Mutations
Chemical form
Sodium dichromate
Sodium dichromate
dihydrate
Sodium dichromate
dihydrate
Sodium dichromate
dihydrate
Chromic acid
Potassium chromate
Potassium chromate
Potassium dichromate
potassium dichromate
Ammonium chromate
Ammonium chromate
Calcium chromate
Chromic acid
Chromic acid
Potassium chromate
Potassium chromate
Test system
E. coli, WP2/pKM101, WP2
uvrA/pKMlOl
S. typhimurium TA102,
TA2638a
S. typhimurium TA100, TA98
E. coli, WP2 uvrA/pKMlOl
E. co// AB 1157, GC2375,
UA4202, PQ30
E. coli PQ37, PQ35
E. coli AB1157, GC2375,
UA4202, PQ30
E. co// AB 1157, GC2375,
UA4202, PQ30
E. coli PQ37, PQ35
S. typhimurium TA1978
(rec+), TA1538 (rec - )
B. subtilis
S. typhimurium TA97, TA98,
TA100
S. typhimurium TA1978
(rec+), TA1538 (rec - )
B. subtilis
S. typhimurium TA98,
TA100, TA1537
S. typhimurium TA100
Results3
Without
activation
+
+
+
+
+
+
+
+
+
+
+
-
+
+
+
+
With
activation
ND
-
+
+
ND
-
ND
ND
-
ND
ND
-
ND
ND
ND
ND
Reference
Watanabe et al., 1998
Ryden et al., 2000
NTP, 2007
NTP, 2007
Llagostera et al., 1986
Olivier and Marzin, 1987
Llagostera et al., 1986
Llagostera et al., 1986
Olivier and Marzin, 1987
Gentile etal., 1981
Gentile etal., 1981
Bramsetal., 1987
Gentile etal., 1981
Gentile etal., 1981
Arlauskasetal., 1985
Arlauskasetal., 1985
122
DRAFT - DO NOT CITE OR QUOTE
-------
Table 4-21. In vitro genotoxicity studies of hexavalent chromium in non-mammalian cells
Endpoint
Mutations
Mutations
Mutations
Mutations
Mutations
Mutations
Mutations
Mutations
Mutations
Mutations
Mutations
Mutations
Frame shift mutations
Frame shift mutation
Frame shift mutation
Frame shift mutations
Frame shift mutations
Frame shift mutations
Chemical form
Potassium chromate
Potassium chromate
Potassium chromate
Potassium dichromate
Potassium dichromate
Potassium dichromate
Potassium dichromate
Potassium dichromate
Sodium dichromate
Potassium dichromate
Potassium dichromate
Sodium dichromate
Calcium chromate
Potassium chromate
Potassium chromate
Potassium dichromate
Potassium dichromate
Sodium dichromate
Test system
E. coli WP2 uvrA pKm 101
B. subtilis HI!
S. typhimurium TA1535,
TA1538
S. typhimurium TA 1535
pSK1002
S. typhimurium TA100,
TA1025, TA98
S. typhimurium TA1978
(rec+), TA1538 (rec - )
E. coli WP2uvrA
B. subtilis
B. subtilis
B. subtilis NIG45, NIG17
B. subtilis HI!
S. typhimurium TA1978
(rec+),
TA1538 (rec - )
S. typhimurium TA98,
TA1537
S. typhimurium TA 1537
E. coli 343/358, 7415, 7435,
7477
S. typhimurium TA97a, TA98
S. typhimurium TA100,
TA1537, TA1538
S. typhimurium TA97,
TA1978
Results3
Without
activation
+
+
-
+
+
+
+
+
+
+
+
+
+
+
+
+
-
+
With
activation
ND
ND
ND
+
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
+
ND
ND
Reference
Arlauskasetal., 1985
Nishioka, 1975
Arlauskasetal., 1985
Yamamotoetal, 2002
LeCurieuxetal., 1993
Gentile etal., 1981
Venieretal., 1987
Gentile etal., 1981
Gentile etal., 1981
Matsui, 1980
Nishioka, 1975
Gentile etal., 1981
Haworth et al., 1983
LaVelle, 1986
LaVelle, 1986
Tagliari etal., 2004
Kanematsuetal., 1980
Bennicellietal., 1983
123
DRAFT - DO NOT CITE OR QUOTE
-------
Table 4-21. In vitro genotoxicity studies of hexavalent chromium in non-mammalian cells
Endpoint
Frame shift mutations
Frame shift mutations,
base pair substitutions
Frame shift mutations,
base pair substitutions
Frame shift mutations,
base pair substitutions
Frame shift mutations,
base pair substitutions
Frame shift mutations,
base pair substitutions
Frame shift mutations,
base pair substitutions
Frame shift mutations,
base substitutions
Frame shift mutations,
base pair substitutions
Frame shift mutations,
base pair substitutions
Base pair substitutions
Base pair substitutions
Base pair substitutions
Base pair substitutions
Base pair substitutions
Base pair substitutions
Chemical form
Sodium dichromate
Calcium chromate
Calcium chromate
Chromic acid
Chromic acid
Potassium chromate
Potassium chromate
Potassium dichromate
Sodium dichromate
Sodium dichromate
Ammonium chromate
Ammonium chromate
Calcium chromate
Calcium chromate
Calcium chromate
Chromic acid
Test system
S. typhimurium TA1537,
TA1538
S. typhimurium TA1537,
TA98, TA100
S. typhimurium TA1535
S. typhimurium TA1537,
TA98, TA100
S. typhimurium TA1535
S. typhimurium TA1537,
TA98, TA100
S. typhimurium TA1535
S. typhimurium TA98 TA100,
TA1535, TA1538
S. typhimurium TA1537,
TA98, TA100
S. typhimurium TA1535
S. typhimurium TA1537,
TA1538, TA98, TA100
S. typhimurium TA1535
S. typhimurium TA100,
TA1535
S. typhimurium TA1537,
TA1538, TA98, TA100
S. typhimurium TA1535
S. typhimurium TA1537,
TA1538, TA98, TA100
Results3
Without
activation
-
+
-
+
-
+
-
+
+
-
±
-
+
±
-
±
With
activation
ND
+
-
+
-
+
-
+
+
-
NS
-
ND
NS
-
NS
Reference
Bennicellietal., 1983
Petrilli and De Flora, 1977
Petrilli and De Flora, 1977
Petrilli and De Flora, 1977
Petrilli and De Flora, 1977
Petrilli and De Flora, 1977
Petrilli and De Flora, 1977
Bianchietal., 1983
Petrilli and De Flora, 1977
Petrilli and De Flora, 1977
DeFlora et al., 1984;
DeFlora, 1981
DeFlora, 1981
Haworth et al., 1983
DeFlora et al., 1984;
DeFlora, 1981
DeFlora, 1981
DeFlora et al., 1984;
DeFlora, 1981
124
DRAFT - DO NOT CITE OR QUOTE
-------
Table 4-21. In vitro genotoxicity studies of hexavalent chromium in non-mammalian cells
Endpoint
Base pair substitutions
Base pair substitutions
Base pair substitutions
Base pair substitutions
Base pair substitutions
Base pair substitutions
Base pair substitutions
Base pair substitutions
Base pair substitutions
Reverse mutation,
induction of gene
conversion
Forward mutation
Mitotic cross-over
Mitotic gene
conversions
Mitotic gene conversion,
point reverse mutation
Mitotic gene conversion
at trp5 locus, reverse
mutation of ilvl-92 allele
Chemical form
Chromic acid
Potassium chromate
Potassium chromate
Potassium dichromate
Potassium dichromate
Sodium dichromate
Sodium dichromate
Sodium dichromate
Sodium dichromate
Potassium dichromate
Potassium dichromate
Chromic acid
Chromic acid
Sodium chromate
Chromic acid
Test system
S. typhimurium TA1537,
TA1538, TA98, TA100
S. typhimurium TA1537,
TA1538, TA98, TA100
S. typhimurium TA1535
S. typhimurium TA100,
TA102
S. typhimurium TA100,
TA1535
S. typhimurium TA100,
TA102, TA92
S. typhimurium TA1537,
TA1538, TA98, TA100
S. typhimurium TA1535
S. typhimurium TA1535
S. cerevisiae D7
Schizosaccharomyces pombe
972, h-
S. cerevisiae D7
S. cerevisiae D7
S. cerevisiae D7
S. cerevisiae D7
Results3
Without
activation
±
±
-
+
-
+
±
-
-
+
±
+
+
+
+
With
activation
NS
NS
-
+
ND
ND
NS
-
ND
ND
ND
ND
ND
ND
ND
Reference
DeFlora et al., 1984;
DeFlora, 1981
DeFlora et al., 1984;
DeFlora, 1981
DeFlora, 1981
Tagriarietal.,2004
Kanematsuetal., 1980
Bennicellietal., 1983
DeFlora, 1981, DeFlora et
al., 1984
DeFlora, 1981
Bennicellietal., 1983
Kharab and Singh, 1985
Bonatti et al., 1976
Fukunagaetal., 1982
Singh, 1983; Fukunaga et
al., 1982
Bronzetti and Galli, 1989
Vashishat and Vasudeva,
1987
125
DRAFT - DO NOT CITE OR QUOTE
-------
Table 4-21. In vitro genotoxicity studies of hexavalent chromium in non-mammalian cells
Endpoint
Mitotic gene conversion
at trp5 locus, reverse
mutation of ilvl-92 allele
Induction of disomic and
diploid spores
umu gene expression
DNA damage
DNA-protein crosslinks
DNA polymerase arrest
Chemical form
Potassium dichromate
Potassium dichromate
Potassium dichromate
Potassium dichromate
Potassium chromate
Sodium dichromate
Test system
S. cerevisiae D7
S. cerevisiae D1S13
S. typhimurium TA1535
E. coli PQ37
E. coli DNA
PSV2neo-based plasmid
DNA
Results3
Without
activation
+
+
±
+
-
-
With
activation
ND
ND
-
ND
ND
+
Reference
Vashishat and Vasudeva,
1987
Soraetal, 1986
Nakamuraetal., 1987
LeCurieuxetal., 1993
Fornace etal., 1981
Bridgewater et al., 1998,
1994
a+ = positive, ± = equivocal or weakly positive, - = negative, ND = no data; NS = not specified.
126
DRAFT - DO NOT CITE OR QUOTE
-------
Table 4-22. In vitro genotoxicity studies of hexavalent chromium in mammalian cells
Endpoint
DNA damage
DNA damage
DNA damage
DNA damage
DNA damage
DNA adducts, [32P] post-
labeling
DNA fragmentation
DNA fragmentation
DNA fragmentation
DNA fragmentation
DNA strand breaks
DNA strand breaks
DNA strand breaks
DNA strand breaks
Chemical
form
Potassium
dichromate
Potassium
dichromate
Potassium
dichromate
Potassium
dichromate
Sodium
dichromate
Potassium
chromate
Potassium
chromate
Potassium
chromate
Potassium
chromate
sodium
chromate
Potassium
dichromate
Sodium
dichromate
Sodium
dichromate
Potassium
chromate
Test system
Human lymphocytes
Human gastric mucosa
Human peripheral blood lymphocytes
Human lymphocytes, human
lymphoblastoid TK-6 cells
Human gastric mucosa cells, Rat gastric
mucosa cells
Calf thymus DNA
Human bronchial epithelial cells
Human embryonic lung fibroblasts (IMR-
90)
Mouse L1210 leukemia cells
Chinese hamster ovary cells
Vero kidney fibroblsts, Pam 212
keratinocytes
Rat primary lymphocytes
Rat hepatocytes
Human lymphocytes
Results"
Without
activation
+
+
+
+
+
"
+
+
+
+
+
+
+
+
With
activation
ND
ND
ND
ND
ND
(+lmM
H202)
ND
ND
ND
ND
ND
ND
ND
ND
Reference
Blasiak and Kowalik, 2000
Trzeciak et al., 2000
Trzeciak et al., 2000
Cemeli et al., 2003
Pool-Zobeletal., 1994
Adams etal., 1996
Fornace etal., 1981
Fornace etal., 1981
Fornace etal., 1981
Blankenship et al., 1997
Flores and Perez, 1999
Gealy et al., 2007
Gaoetal., 1993
Depault etal., 2006
127
DRAFT - DO NOT CITE OR QUOTE
-------
Table 4-22. In vitro genotoxicity studies of hexavalent chromium in mammalian cells
Endpoint
DNA strand breaks
DNA strand breaks
DNA strand breaks
DNA strand breaks
DNA-DNA crosslinks
DNA-protein crosslinks
DNA-protein crosslinks
DNA-protein crosslinks
DNA-protein crosslinks
DNA-protein crosslinks
Induced DNA methylation
Unscheduled DNA
synthesis
DNA synthesis inhibition
DNA synthesis inhibition
Chemical
form
Potassium
chromate
Potassium
chromate
Sodium
dichromate
Potassium
dichromate
Sodium
chromate
Potassium
chromate
Potassium
chromate
Potassium
chromate
Potassium
chromate
Sodium
chromate
Potassium
chromate
Sodium
dichromate
Potassium
chromate
Potassium
dichromate
Test system
Human fibroblast
Bacteriophage I DNA
Rat primary lymphocytes
Human lymphocytes, human gastric
mucosa cells
Human lung fibroblasts
Human embryonic lung fibroblasts (IMR-
90)
Human fibroblast
Chinese hamster cells (V79-UL)
Mouse L1210 leukemia cells
Human HL-60 cells
Chinese hamster V79 cells (hpr\gpt+
transgenic cell line G12)
Rat hepatocytes
HeLa S3 cells
Mouse L cells
Results"
Without
activation
+
+
+
+
+
+
+
+
+
+
+ (T)
+ (T)
+
+
With
activation
ND
+
(+lmM
H202)
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
Reference
Fornace 1982
Adams etal, 1996
Eliaetal, 1994
Blasiaketal., 1999
Xuetal., 1996
Fornace etal., 1981
Fornace, 1982
Merk et al., 2000
Fornace etal., 1981
Capellmann et al., 1995
Klein et al., 2002
Gaoetal., 1993
Heil and Reifferscheid, 1992
Nishio and Uyeki, 1985
128
DRAFT - DO NOT CITE OR QUOTE
-------
Table 4-22. In vitro genotoxicity studies of hexavalent chromium in mammalian cells
Endpoint
DNA polymerase arrest
Mutations at the HGPRT
locus
Mutations at the HGPRT
locus
Forward mutation
Forward mutation
Forward mutation
Forward mutation
Morphological
transformation
Morphological
transformation
Cell transformation
Transformations
Chromosomal damage
Chromosomal damage
Chromosomal damage
Chromosomal damage
Chemical
form
Sodium
chromate
Potassium
dichromate
Potassium
dichromate
Calcium
chromate
Calcium
chromate
Calcium
chromate
Calcium
chromate
Calcium
chromate
Sodium
chromate
dihydrate
Calcium
chromate
Potassium
chromate
Calcium
chromate
Chromic acid
Potassium
chromate
Potassium
dichromate
Test system
Human lung fibroblasts
Chinese hamster ovary cells (ATS -2)
Chinese hamster cells (V79)
Mouse lymphoma cells (L5178Y tk+/tk")
Mouse lymphoma cells (L5178Y tk+)
Mouse lymphoma cells (L5178Y tk+)
Mouse lymphoma cells (L5178Y tk+)
Syrian hamster embryo cells
Syrian hamster cells
Balb/3T3, Syrian hamster embryo,
R-MuLV-RE cells
Rat liver epithelial cells
Chinese hamster ovary cells
Chinese hamster ovary cells
Chinese hamster ovary cells
Chinese hamster ovary cells
Results"
Without
activation
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
With
activation
ND
ND
ND
+
+
+
+
ND
ND
ND
ND
ND
ND
ND
ND
Reference
Xuetal., 1996
Paschin et al., 1983
Paschin et al., 1983
McGregor etal, 1987
Mitchell etal, 1988
Myhr and Caspary, 1988
Oberlyetal., 1982
Eliasetal., 1991
DiPaolo and Casto, 1979
Dunkeletal., 1981
Briggs and Briggs, 1988
Levis and Majone, 1979
Levis and Majone, 1979
Levis and Majone, 1979
Seoane and Dulout, 1999
129
DRAFT - DO NOT CITE OR QUOTE
-------
Table 4-22. In vitro genotoxicity studies of hexavalent chromium in mammalian cells
Endpoint
Chromosomal damage
Chromosomal damage
Chromosomal damage
Chromosomal damage
Chromosomal damage
Chromosome aberrations
Chromosome aberrations
Chromosome aberrations
Chromosome aberrations
Chromosome aberrations
Chromosome aberrations
Chromosome aberrations
Chromosome aberrations
Chromosome aberrations
Chromosome aberrations
Chromosome aberrations
Chemical
form
Potassium
dichromate
Potassium
dichromate
Sodium
chromate
Sodium
dichromate
Sodium
dichromate
Calcium
chromate
Calcium
chromate
Chromic acid
Chromic acid
Chromic acid
Potassium
chromate
Potassium
chromate
Potassium
chromate
Potassium
chromate
Potassium
chromate
Potassium
dichromate
Test system
Chinese hamster ovary cells
Chinese hamster ovary cells
Chinese hamster ovary cells
Chinese hamster ovary cells
Chinese hamster ovary cells
Chinese hamster lung DON cells
Chinese hamster ovary cells (C3H10T1/2)
BHK and Chinese hamster ovary cells
Mouse mammary FM3 A carcinoma cells
Chinese hamster lung DON cells
Human fibroblasts
Chinese hamster lung DON cells
Chinese hamster ovary cells
BHK and Chinese hamster ovary cells
Mouse mammary FM3 A carcinoma cells
Human fibroblasts
Results"
Without
activation
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
With
activation
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
Reference
Levis and Majone, 1979
Majone and Levis, 1979
Levis and Majone, 1979
Levis and Majone, 1979
Majone and Levis, 1979
Koshi and Iwaski 1983;
Koshi 1979
Senetal., 1987
Bianchi et al., 1980
Umeda and Nishmura, 1979
Koshi and Iwaski 1983;
Koshi 1979
MacRaeetal., 1979
Koshi and Iwaski 1983;
Koshi 1979
MacRaeetal., 1979
Bianchi et al., 1980
Umeda and Nishmura, 1979
MacRaeetal., 1979
130
DRAFT - DO NOT CITE OR QUOTE
-------
Table 4-22. In vitro genotoxicity studies of hexavalent chromium in mammalian cells
Endpoint
Chromosome aberrations
Chromosome aberrations
Chromosome aberrations
Chromosome aberrations
Chromosome aberrations
Chromosome aberrations
Chromosome aberrations
Chromosome aberrations
Chromosome aberrations
Chromosome and
chromatid aberrations
Sister chromatid
exchanges
Sister chromatid
exchangse
Sister chromatid
exchanges
Sister chromatid
exchanges
Chemical
form
Potassium
dichromate
Potassium
dichromate
Potassium
dichromate
Sodium
chromate
Sodium
chromate
Sodium
chromate
Sodium
chromate
Sodium
chromate
Sodium
dichromate
Potassium
dichromate
Calcium
chromate
Calcium
chromate
Calcium
chromate
Chromic acid
Test system
Chinese hamster ovary cells
BHK and Chinese hamster ovary cells
Mouse mammary FM3 A carcinoma cells
Human primary bronchial fibroblasts
Human bronchial fibroblasts (WTHBF-
6 cells)
Human bronchial epithelial cells (BEP2D
cells)
Chinese hamster ovary cells
Chinese hamster ovary cells (AA8
(parental), EM9 (XRCC1 mutant), and
H9T3
BHK and Chinese hamster ovary cells
Human lymphocytes
Human lymphocytes
Chinese hamster lung DON cells
Chinese hamster ovary cells
Chinese hamster ovary cells
Results"
Without
activation
+
+
+
+
+
+
+
+
+
+
+
+
+
+
With
activation
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
Reference
MacRaeetal., 1979
Bianchi et al., 1980
Umeda and Nishmura, 1979
Wise etal., 2004, 2002
Holmes et al., 2006
Wiseetal.,2006a
Blankenship et al., 1997
Grlickova-Duzevik, 2006
Bianchi et al., 1980
Imreh and Radulescu, 1982
Gomez-Arroyo etal., 1981
Koshi and Iwaski 1983;
Koshi 1979
Levis and Majone, 1979
Levis and Majone, 1979
131
DRAFT - DO NOT CITE OR QUOTE
-------
Table 4-22. In vitro genotoxicity studies of hexavalent chromium in mammalian cells
Endpoint
Sister chromatid
exchanges
Sister chromatid
exchanges
Sister chromatid
exchanges
Sister chromatid
exchanges
Sister chromatid
exchanges
Sister chromatid
exchanges
Sister chromatid
exchanges
Sister chromatid
exchanges
Sister chromatid
exchanges
Sister chromatid
exchanges
Sister chromatid
exchanges
Sister chromatid
exchanges
Sister chromatid
exchanges
Sister chromatid
exchanges
Sister chromatid
exchanges
Chemical
form
Chromic acid
Chromic acid
Chromic acid
Potassium
chromate
Potassium
chromate
Potassium
chromate
Potassium
chromate
Potassium
chromate
Potassium
chromate
Potassium
dichromate
Potassium
dichromate
Potassium
dichromate
Potassium
dichromate
Potassium
dichromate
Potassium
dichromate
Test system
Chinese hamster cells DON
Chinese hamster lung DON cells
BHK and Chinese hamster ovary cells
Human fibroblasts
Chinese hamster lung DON cells
Chinese hamster ovary cells
Chinese hamster ovary cells
Chinese hamster cells DON
BHK and CHO cells
Human lymphocytes
Human lymphocytes
Human fibroblasts
Chinese hamster ovary cells
Chinese hamster ovary cells
Chinese hamster ovary cells
Results"
Without
activation
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
With
activation
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
Reference
Ohnoetal., 1982
Koshi and Iwaski 1983;
Koshi 1979
Bianchi et al., 1980
MacRae et al, 1979
Koshi and Iwaski 1983;
Koshi 1979
Levis and Majone, 1979
MacRae etal., 1979
Ohnoetal., 1982
Bianchi et al., 1980
Gomez-Arroyo etal., 1981
Imreh and Radulescu, 1982
MacRae etal., 1979
Levis and Majone, 1981
Levis and Majone, 1979
Majone and Levis, 1979
132
DRAFT - DO NOT CITE OR QUOTE
-------
Table 4-22. In vitro genotoxicity studies of hexavalent chromium in mammalian cells
Endpoint
Sister chromatid
exchanges
Sister chromatid
exchanges
Sister chromatid
exchanges
Sister chromatid
exchanges
Sister chromatid
exchanges
Sister chromatid
exchanges
Sister chromatid
exchanges
Sister chromatid
exchanges
Sister chromatid
exchanges
Disruption of mitosis
Chemical
form
Potassium
dichromate
Potassium
dichromate
Potassium
dichromate
Potassium
dichromate
Sodium
chromate
Sodium
chromate
Sodium
dichromate
Sodium
dichromate
Sodium
dichromate
Sodium
chromate
Test system
Chinese hamster ovary cells
Chinese hamster cells DON
BHK and Chinese hamster ovary cells
Mouse blastocysts
Chinese hamster ovary cells
BHK and Chinese hamster ovary cells
Chinese hamster ovary cells
Chinese hamster ovary cells
BHK and Chinese hamster ovary cells
Human bronchial fibroblasts (WTHBF-6
cells)
Results"
Without
activation
+
+
+
+
+
+
+
+
+
+
With
activation
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
Reference
MacRaeetal., 1979
Ohnoetal., 1982
Bianchi et al., 1980
lijimaetal., 1983
Levis and Majone, 1979
Bianchi et al., 1980
Levis and Majone, 1979
Majone and Levis, 1979
Bianchi et al., 1980
Wiseetal.,2006b
1 + = positive, ± = equivocal or weakly positive, - = negative, (T) = toxicity, ND = no data.
133
DRAFT - DO NOT CITE OR QUOTE
-------
Table 4-23. In vivo genotoxicity studies of hexavalent chromium in D. melanogaster and laboratory
mammals
Endpoint
DNA damage,
Comet assay
DNA damage,
Comet assay
DNA damage,
Comet assay
DNA damage,
Comet assay
DNA damage,
Comet assay
Chemical
Potassium
chromate
Potassium
chromate
Potassium
dichromate
Potassium
dichromate
Potassium
dichromate
Test system
DNA damage in
stomach, colon,
bladder, lung,
brain; but not in
liver, kidney or
bone marrow
DNA damage in
stomach, colon,
liver, kidney,
bladder, lung, and
brain; but not in
bone marrow
DNA damage in
peripheral
lymphocytes
Mouse leukocytes
DNA damage in
liver and kidney,
but not in spleen,
lung, or brain
Test conditions
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
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.
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).
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 (0 021 042 084 168 337
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.
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.
Results"
+
+
+
+
+
Doseb
32. 1 mg hexavalent
chromium/kg
(intraperitoneal)
85.7 mg hexavalent
chromium/kg
(gavage)
8.8 mg hexavalent
chromium/kg
(gavage)
0.21 mg hexavalent
chromium/kg-body
weight
(gavage)
20 mg hexavalent
chromium/kg
(intraperitoneal
injection)
Reference
Sekihashi et
al., 2001
Sekihashi et
al., 2001
Wang et al.,
2006
Devietal.,
2001
Ueno et al.,
2001
134
DRAFT - DO NOT CITE OR QUOTE
-------
Table 4-23. In vivo genotoxicity studies of hexavalent chromium in D. melanogaster and laboratory
mammals
Endpoint
DNA damage,
Comet assay
DNA
alterations
DNA-protein
crosslinks
DNA-protein
crosslinks
Chemical
Potassium
dichromate
Sodium
dichromate
dihydrate
Potassium
chromate
Sodium
dichromate
dihydrate
Test system
Rat leukocytes
DNA-protein
crosslinks, DNA
fragmentation, and
DNA adducts in
lung, but not liver
DNA-protein
crosslinks detected
in liver but not
splenic
lymphocytes
DNA-protein
crosslinks detected
in lung, liver, and
kidney nuclei
Test conditions
Parenteral exposure: Sprasue-Dawlev 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.
Intratracheal exposure: Sprasue-Dawlev 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.
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.
Parenteral exposure: Male Sprasue-Dawlev rats
were given a single intraperitoneal injection of 20
or 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 whole
lung were used for analysis.
Results"
+
+
+
+
Doseb
0.88 mg hexavalent
chromium/kg-day
(intraperitoneal
injection)
0.09 mg hexavalent
chromium/kg
(intratracheal
instillation)
100 hexavalent
chromium/L
(drinking water)
7 mg hexavalent
chromium/kg
(intraperitoneal
injection)
Reference
Patlolla and
Tchounwou,
2006
Izzotti et al.,
1998
Coogan et al.,
1991b
Tsapakos et
al., 1983
135
DRAFT - DO NOT CITE OR QUOTE
-------
Table 4-23. In vivo genotoxicity studies of hexavalent chromium in D. melanogaster and laboratory
mammals
Endpoint
Oxidative
DNA damage,
DNA protein
crosslinks
Suppressed
nuclear DNA
synthesis
Unscheduled
DNA synthesis
Mutation
Chemical
Sodium
dichromate
dihydrate
Potassium
dichromate
Potassium
dichromate
Potassium
chromate
Test system
Mouse
forestomach,
glandular stomach,
and duodenum
cells
Mouse tubular
renal cells
Rat hepatocytes
Mutations in liver,
but not bone
marrow cells
Test conditions
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 B6C3FJ 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-oxo-
2'deoxyguanosine) were measured in forestomach,
glandular stomach, and duodenum 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.
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.
Parenteral exposure: Male lacZ transsenic
Muta™ 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).
Results"
_
+
_
+
Doseb
20 mg hexavalent
chromium/L
(drinking water)
NS
20 mg hexavalent
chromium/L
(drinking water)
14.1 mg hexavalent
chromium/kg
(intraperitoneal
injection)
Reference
De Flora et
al., 2008
Amlacher and
Rudolph,
1981
Mirsalis et
al., 1996
Itoh and
Shimada,
1998, 1997
136
DRAFT - DO NOT CITE OR QUOTE
-------
Table 4-23. In vivo genotoxicity studies of hexavalent chromium in D. melanogaster and laboratory
mammals
Endpoint
Mutation
Mutations
Gene mutation
Gene mutation
Gene mutation
(wing somatic
mutation)
Gene mutation
(white-ivory
eye spot test)
Chemical
Potassium
chromate
Potassium
dichromate
Calcium
chromate
Chromic acid
Chromium oxide
Chromium oxide
Test system
Mouse offspring
Mouse (C57BL/
6 Big Blue) lung
and kidney tissue,
but not in liver
tissue
D. melanogaster
D. melanogaster
D. melanogaster
D. melanogaster
Test conditions
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.
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 in
producing reactive intermediates during
intracellular reduction of chromium (VI).
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.
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.
2-3 -Day-old larvae were fed potassium chromate
or chromium(VI) oxide for 3 days at
concentrations of 0, 1, or 5 mM.
2-3 -Day-old larvae were fed potassium chromate
or chromium(VI) oxide for 2 days at
concentrations of 0, 1, or 5 mM.
Results"
+
+
+
+
+
Doseb
2.7 mg hexavalent
chromium/kg
(intraperitoneal
injection)
6.75 mg hexavalent
chromium/kg
(intratracheal
instillation)
500 ppm
(in diet)
100 ppm
(intraperitoneal
injection)
ImM
(in diet)
5mM
(in diet)
Reference
Knudsen,
1980
Cheng etal.,
2000
Zimmering et
al., 1985
Rodriguez-
Arnaiz and
Martinez,
1986
Graf and
Wurgler,
1996
Graf and
Wurgler,
1996
137
DRAFT - DO NOT CITE OR QUOTE
-------
Table 4-23. In vivo genotoxicity studies of hexavalent chromium in D. melanogaster and laboratory
mammals
Endpoint
Gene mutation
Gene mutation
(wing somatic
mutation)
Gene mutation
Gene mutation
(white-ivory
eye spot test)
Gene mutation
Gene mutation
Gene mutation
Chemical
Potassium
chromate
Potassium
chromate
Potassium
chromate
Potassium
chromate
Potassium
dichromate
Potassium
dichromate
Potassium
dichromate
Test system
D. melanogaster
D. melanogaster
D. melanogaster
D. melanogaster
D. melanogaster
D. melanogaster
D. melanogaster
Test conditions
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/flr3) and inversion
heterozygous (mwh/TM3) flies were used.
2-3 -Day-old larvae were fed potassium chromate
or chromium(VI) oxide for 3 days at
concentrations of 0, 1 or 5 mM.
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 balancer-
heterozygous wings from adult flies were then
examined in the wing somatic mutation and
recombination test (SMART).
2-3 -Day-old larvae were fed potassium chromate
or chromium(VI) oxide for 2 days at
concentrations of 0, 1 or 5 mM.
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.
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/flr3) and inversion
heterozygous (mwh/TM3) flies were used.
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.
Results"
+
+
+
+
+
+
Doseb
0.1 mM
(in diet)
ImM
(in diet)
0.5 mM (48 hours)
(in diet)
5 mM (6 hours)
(in diet)
5mM
(in diet)
0.5 mM
(in diet)
0.1 mM
(in diet)
100 ppm
(intraperitoneal
injection)
Reference
Amranietal.,
1999
Graf and
Wurgler,
1996
Spano et al.,
2001
Graf and
Wurgler,
1996
Kayaetal.,
2002
Amranietal.,
1999
Rodriguez-
Arnaiz and
Martinez,
1986
138
DRAFT - DO NOT CITE OR QUOTE
-------
Table 4-23. In vivo genotoxicity studies of hexavalent chromium in D. melanogaster and laboratory
mammals
Endpoint
Gene mutation
Dominant
lethality
Chromosome
aberrations,
sister
chromatid
exchange
Micronuclei
Chemical
Sodium
dichromate
Potassium
dichromate
Chromium
fumes; no further
information was
given and
chromium
valence state was
not specified
Potassium
chromate
Test system
D. melanogaster
Mouse
Chromosome
aberrations and
sister chromatid
exchange in rat
peripheral
lymphocytes, but
not in bone
marrow cells
Mouse bone
marrow cells
Test conditions
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.
Parenteral exposure: CBA x C57B1/6J 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 post-
implantation loss.
Inhalation exposure: Sprasue-Dawlev 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/m3 for
1 week (5 hour/day, 5 day/week) or 0.55 mg/m3
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.
Oral exposure: Male MS/Ae and CD-I 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).
Results"
+
+
+
+
_
Doseb
2.34 mM
7.1 mg hexavalent
chromium/kg
(acute
intraperitoneal
injection)
0.7 mg hexavalent
chromium/kg
(repeated
intraperitoneal
injection)
1.84 mg/m3 (1-
week inhalation
exposure)
0.55 mg/m3 (2-
month inhalation
exposure)
113. Img
hexavalent
chromium/kg
(gavage)
Reference
Rasmuson,
1985
Paschin et al.,
1982
Koshi et al.,
1987
Shindo etal.,
1989
139
DRAFT - DO NOT CITE OR QUOTE
-------
Table 4-23. In vivo genotoxicity studies of hexavalent chromium in D. melanogaster and laboratory
mammals
Endpoint
Micronuclei
Micronuclei
Micronuclei
Micronuclei
Micronuclei
Micronuclei
Micronuclei
Chemical
Potassium
chromate
Potassium
chromate
Potassium
chromate
Potassium
chromate
Potassium
chromate
Potassium
dichromate
Potassium
dichromate
Test system
Mouse bone
marrow cells
Mouse peripheral
red blood cells
Mouse bone
marrow cells
MS and ddY
mouse bone
marrow cells
Mouse bone
marrow
Mouse (BDFO
bone marrow cells
and peripheral
blood cells
Mouse (BDFj)
bone marrow cells
Test conditions
Parenteral exposure: Male MS/Ae and CD-I 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).
Parenteral exposure: Male lacZ transsenic
Muta™ 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).
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, or
17.7 mg hexavalent chromium/kg).
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).
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).
Oral exposure: BDF1 male mice were
administered potassium dichromate in drinking
water at 0, 10, or 20 mg hexavalent chromium/L
for 20 days.
Oral exposure: BDF1 male mice were given a
single doses of potassium dichromate by gavage
of 0 or 50 mg hexavalent chromium/kg.
Results"
+
+
+
+
+
Doseb
14.1 mg hexavalent
chromium/kg
(intraperitoneal
injection)
14.1 mg hexavalent
chromium/kg
(intraperitoneal
injection)
10.6 mg hexavalent
chromium/kg
(intraperitoneal
injection)
17.7 mg hexavalent
chromium/kg
(intraperitoneal
injection)
13 mg hexavalent
chromium/kg
(intraperitoneal
injection)
20 mg hexavalent
chromium/L
(drinking water)
50 mg hexavalent
chromium/kg
(gavage)
Reference
Shindo etal.,
1989
Itoh and
Shimada,
1997
Itoh and
Shimada,
1996
Hayashi et
al., 1982
Wild, 1978
De Flora et
al., 2006
De Flora et
al., 2006
140
DRAFT - DO NOT CITE OR QUOTE
-------
Table 4-23. In vivo genotoxicity studies of hexavalent chromium in D. melanogaster and laboratory
mammals
Endpoint
Micronuclei
Micronuclei
Micronuclei
Micronuclei
Micronuclei
Chemical
Potassium
dichromate
Potassium
dichromate
Potassium
dichromate
Potassium
dichromate
Potassium
dichromate
Test system
Mouse bone
marrow cells
Mouse (Swiss)
bone marrow —
dams; fetal liver
cells and
peripheral blood
cells
Mouse (BDFO
bone marrow cells
Mouse bone
marrow
Mouse bone
marrow
Test conditions
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.
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.
Parenteral exposure: BDF1 male mice were given
single intraperitoneal doses of potassium
dichromate of 0 or 50 mg hexavalent
chromium/kg.
Parenteral exposure: Balb C mice were
administered potassium dichromate as a single
intraperitoneal injection at a dose of 0 or 400 umol
(20.8 mg hexavalent chromium/kg).
Parenteral exposure: CBA x C57B1/6J 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.
Results"
+
+
+
Doseb
20 mg hexavalent
chromium/L
(drinking water)
10 mg hexavalent
chromium/L
(drinking water)
50 mg hexavalent
chromium/kg
(intraperitoneal
injection)
20.8 mg hexavalent
chromium/kg
(intraperitoneal
injection)
0.35 mg hexavalent
chromium/kg
(intraperitoneal
injection)
Reference
Mirsalis et
al., 1996
De Flora et
al., 2006
De Flora et
al., 2006
Wronska-
Noferetal.,
1999
Paschin and
Toropzev,
1982
141
DRAFT - DO NOT CITE OR QUOTE
-------
Table 4-23. In vivo genotoxicity studies of hexavalent chromium in D. melanogaster and laboratory
mammals
Endpoint
Micronuclei
Micronuclei
Micronuclei
Micronuclei
Chemical
Potassium
dichromate
Sodium
dichromate
dihydrate
Sodium
dichromate
dihydrate
Sodium
dichromate
dihydrate
Test system
Mouse (Swiss):
bone marrow-
dams; fetal liver
and peripheral
blood cells
Mouse (B6C3FO
peripheral red
blood cells
Mouse (B6C3FJ,
BALB/c or
am3-C51BL/6)
peripheral red
blood cells
Mouse (BDFj)
bone marrow or
peripheral blood
cells
Test conditions
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.
Oral exposure: B6C3Fi (10/sex/sroup) 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.
Oral exposure: B6C3F, (5/sroup), BALB/c
(5/group) and am3-C5TBL/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.
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.
Results"
+
_
±
-
+
_
Doseb
50 mg hexavalent
chromium/kg
(intraperitoneal
injection)
349 mg hexavalent
chromium/L
(drinking water)
(drinking water)
87.2 mg hexavalent
chromium/L
(BsCsFO
87.2 mg hexavalent
chromium/L
(BALB/c)
21.8 mg hexavalent
chromium/L
(am3-C5TBL/6)
500 mg hexavalent
chromium/L
(drinking water)
Reference
De Flora et
al., 2006
NTP, 2007
NTP, 2007
De Flora et
al., 2006
142
DRAFT - DO NOT CITE OR QUOTE
-------
Table 4-23. In vivo genotoxicity studies of hexavalent chromium in D. melanogaster and laboratory
mammals
Endpoint
Micronuclei
Micronuclei
Chemical
Sodium
dichromate
dihydrate
Sodium
dichromate
dihydrate
Test system
Mouse (Swiss)
bone marrow-
dams; fetal liver
and peripheral
blood cells
Mouse (Swiss):
bone marrow-
dams; fetal liver
and peripheral
blood cells
Test conditions
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.
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.
Results"
+
Doseb
10 mg hexavalent
chromium/L
(drinking water)
50 mg hexavalent
chromium/kg
(intraperitoneal
injection)
Reference
De Flora et
al., 2006
De Flora et
al., 2006
a+ = positive, ± = equivocal or weakly positive, - = negative, (T) = toxicity.
bLowest effective dose for positive results, highest dose tested for negative results, NS = not specified.
143
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
18 exposed to 0.001-0.055 mg hexavalent chromium/m3 (Gao et al., 1994) or in volunteers
19 ingesting single oral doses of 71 jig 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
22 hexavalent chromium/m3 (Wu et al., 2001). However, chromosome aberrations and sister
23 chromatid exchanges in peripheral lymphocytes from chrome platers were not observed at lower
24 exposure levels (0.0075 and 0.0249 mg chromium[total]/m3) (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
29 0.043-0.083 mg chromium(total)/m3 (Vaglenov et al., 1999) and 0.0075-0.0249 mg
30 chromium(total)/m3 (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
32 chromium(total)/m3, although chromosome aberrations and sister chromatid exchanges were not
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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
8 chromium(total)/m3 (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
12 chromium/m3(Wuetal.,2001).
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Table 4-24. In vivo genotoxicity studies in humans exposed to hexavalent chromium
Endpoint
DNA strand
breaks
DNA strand
breaks,
hydroxylation of
deoxyquanosine
DNA-protein
crosslinks
Exposure type
(chemical form)
Occupational -
Chromium plating
(chromic acid)
Occupational -
Production of
dichromate
(included
exposure to
chromic acid,
potassium
dichromate and
sodium
dichromate)
Experimental oral
exposure -
(potassium
dichromate)
Cell type
Human
peripheral
lymphocytes
Human
peripheral
lymphocytes
Human
peripheral
lymphocytes
Test conditions
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 pre-
and 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 ug/g creatinine (pre-shift) and 7.31 ug/g creatinine (post-
shift). Mean erythrocyte and lymphocyte concentrations in the
exposed workers were 4.94 ug/L and 50.3 ug/1012cells,
respectively. Air concentrations of chromium were not
reported.
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/m3 (obtained from personal and area samplers).
Mean chromium concentrations in urine (5.97 ug/g creatinine),
whole blood (5.5 ug/1), plasma (2.8 ug/L), and lymphocytes
(1.01 ug/1010 cells) of exposed workers were significantly
higher than in non-exposed workers.
Four adult volunteers ingested a single bolus dose of 5,000 ug
hexavalent chromium as potassium dichromate (approximately
equivalent to 71 ug 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.
Results3
+
-
-
Exposure levelb
NS
0.001-0.055 mg
hexavalent
chromium/m3
(measured
exposure range)
71 ug hexavalent
chromium/kg
Reference
Gambelunghe
etal.,2003
Gao etal.,
1994
Kuykendall et
al., 1996
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Table 4-24. In vivo genotoxicity studies in humans exposed to hexavalent chromium
Endpoint
Chromosome
aberrations,
sister chromatic!
exchanges
Sister chromatic!
exchanges
Chromosomal
aberrations,
sister chromatic!
exchanges
Sister chromatic!
exchanges
Sister chromatic!
exchanges
Exposure type
(chemical form)
Occupational -
Chromium
electroplating
(chemical not
specified)
Occupational -
Chromium
electroplating
(chemical not
specified)
Occupational -
Chromium plating
(chromic acid)
Occupational -
Chromium plating
(chromic acid
fumes)
Occupational -
Chromium
electroplating
(chemical not
specified)
Cell type
Human
peripheral
lymphocytes
Human
whole blood
cells
Human
peripheral
lymphocytes
Human
peripheral
lymphocytes
Human
peripheral
lymphocytes
Test conditions
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 ug/mm3, the mean chromium concentration in stool
samples was 8.5 ug/g stool, and the mean chromium
concentration in hair was 35.68 ug/g. The valence of
chromium that workers were exposed to was unspecified.
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/m3. The mean urinary chromium
concentration of the exposed workers was 3.67 ug/g creatinine.
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.
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.
Thirty -five chromium electroplating factory workers in Taiwan
and 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.
Results3
+
+
+
+
+
Exposure levelb
8. lug
chromium/mm3c
5.99 mg
hexavalent
chromium/m3
NS
NS
NS
Reference
Deng et al.,
1988
Wu et al.,
2001
Sarto et al.,
1982
Stella etal.,
1982
Wu et al.,
2000
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Table 4-24. In vivo genotoxicity studies in humans exposed to hexavalent chromium
Endpoint
Chromosome
aberrations,
sister chromatic!
exchanges
Sister chromatic!
exchanges
Sister chromatic!
exchanges
Exposure type
(chemical form)
Occupational -
Chromium plating
(chemical not
specified)
Occupational -
Chromium plating
(chemical not
specified)
Occupational -
Chromium plating
(chemical not
specified)
Cell type
Human
peripheral
lymphocytes
and buccal
mucosal
cells
Human
peripheral
lymphocytes
Human
peripheral
lymphocytes
Test conditions
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/m3
in the low-exposure group and 0.0249 mg chromium/m3 in the
high-exposure group (number of workers in each exposure
group was not reported). Mean concentrations of chromium in
urine were 18.63 ug/L (low) and 104.22 ug/L (high)
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 ug/g
with a mean urinary chromium concentration of 17.9 ug/g
creatinine. Sister chromatid exchange frequency in
lymphocytes was determined in blood-urine paired samples.
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 1 1.6 years. Exposure concentrations were not
reported. The mean concentration of chromium in the urine
was 13.1 ug/L.
Results3
-
-
-
Exposure levelb
Results reported
for combined
groups (0.0075
and 0.0249 mg
chromium/m3)
NS
NS
Reference
Benova et al.,
2002
Nagayaetal.,
1991
Nagaya, 1986
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Table 4-24. In vivo genotoxicity studies in humans exposed to hexavalent chromium
Endpoint
Micronuclei
Micronuclei
Micronuclei
Exposure type
(chemical form)
Occupational -
Chromium
electroplating
(chemical not
specified)
Occupational -
Chromium plating
(chemical not
specified)
Occupational -
Chromium plating
(chromic acid)
Cell type
Human
peripheral
lymphocytes
Human
peripheral
lymphocytes
and buccal
mucosal
cells
Human
buccal and
nasal cells
Test conditions
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 ug/m3 in the low- and high-exposure groups,
respectively. Duration of employment ranged from 4 to
25 years with mean durations of 10.44 and 1 1.63 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 ug/L, respectively. The mean
chromium concentrations in erythrocytes and urine of the high-
exposure group were 8.4 and 5.0 ug/L, respectively.
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/m3
l~f o
in the low-exposure group and 0.0249 mg chromium/m in the
high-exposure group. Mean concentrations of chromium in
urine were 18.63 (low) and 104.22 ug/L (high).
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 ug/g creatinine; the mean urinary chromium
concentration was not reported. Chromium levels in air were
not determined.
Results3
+
+
-
Exposure levelb
0.043 and
0.083 mg
chromium/m3
Positive results
reported for
combined groups
(0.0075 and
0.0249 mg
chromium/m3)
NS
Reference
Vaglenov et
al., 1999
Benova et al.,
2002
Sarto et al.,
1990
a+ = positive; - = negative
bAll exposure levels associated with positive results, highest exposure level for negative results; NS = not specified.
°The exposure level of 8.1 ug chromium/mm3 is as reported by Deng et al., (1988); however, this appears to be a reporting error, as this concentration is
equivalent to 8,100,000 mg chromium/m3.
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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,
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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
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1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
DNA
\^
\
(
•^
O
0 = P-0
7
DH
\ / ^H
'r"\^VH
L v-l
L^^J
\ /^
OH
(,
0 = P— 0
ANH
NH
^AN,
Source: Zhitkovich (2005).
Figure 4-1. Ternary DNA adduct formation by chromium. Hexavalent chromium,
when reduced intracellularly to trivalent chromium, can form ternary DNA crosslinks
with the peptide or amino acid ligand (L) involved in the reduction. Here, chromium(III)
directly coordinates to the 5 '-phosphate in the DNA backbone and forms a hydrogen
bond with the N-7 of deoxyguanosine.
DNA- trivalent chromium crosslinks. Reduction of hexavalent chromium in vitro
produces a large proportion of binary trivalent chromium-DNA adducts, but these have not been
detected in vivo. It has been theorized that the formation of the ternary adducts described above
occurs far more frequently due to the high concentration of ligands capable of complexing with
trivalent chromium before it can
bind to DNA (Zhitkovich, 2005). In addition, these adducts
have been found to be less mutagenic than the ternary adducts in vitro (Quievryn et al., 2003;
Zhitkovich et al., 2001).
DNA-protein crosslinks.
These bulky lesions have been detected in hexavalent
chromium-treated cells in vitro in Chinese hamster ovary cells (Costa, 1991) and in vivo in chick
embryos (Hamilton and Wetterhahn, 1986). They are not detected in the presence of the
trivalent chromium chelator EDTA, indicating that trivalent chromium is the species involved in
their formation (Miller and Costa, 1989). It has been recently shown that the mechanism
forming DNA-protein crosslinks induced by hexavalent chromium requires intracellular
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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
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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 cyclinBl 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 jiM 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
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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).
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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 Dalai, 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-dep endent m echani sm s.
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 dose-
26 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 (>WO |iM) compared with the protein
29 oxidation, which was significant as low as 10 jiM. 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 error-
10 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 B6C3Fi 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 two-
15 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 B6C3Fi 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 B6C3Fi mice at 5.3 mg/kg-day of hexavalent chromium, and in male BALB/c and
25 am3-C57EL/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 B6C3Fi 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;
159 DRAFT - DO NOT CITE OR QUOTE
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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, and
reproductive 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
Reference
Subchronic Studies
F344/N
rat
B6C3FJ
mouse
B6C3F1;
BALB/c,
and am3-
C57BL/6
mouse
Wistar rat
Wistar rat
Wistar rat
Swiss
mouse
F, M
F, M
M
M
M
M
M
0, 1.7, 3.5, 5.9,
11.2, or 20.9
mg/kg-day via
drinking water
0,3.1,5.3,9.1,
15.7,or27.9
mg/kg-day via
drinking water
0,2.8, 5.2, or 8.7
mg/kg-day via
drinking water
0, 73.05 mg/kg-
day via drinking
water
0, 20 mg/L in
drinking water
0, 1.5 mg/kg-day
via drinking
water
0, 177, 265, 353,
530, or 706 mg/L
in drinking water
3 months
3 months
3 months
30 days
10 weeks
22 weeks
8 weeks
F:ND
M:ND
F:ND
M:ND
ND
ND
ND
ND
ND
1.7
1.7
3.1
3.1
2.8
ND
ND
1.5
ND
F: Microcytic, hypochromic anemia (decreased Hgb,
MCV, MCH), increased serum liver enzyme activities
(ALT and SDH) and bile acids, and histopathological
changes to the duodenum (histiocytic cellular
infiltration)
M: Microcytic, hypochromic anemia (decreased Hct,
Hgb, MCV, MCH), increased serum liver enzyme
activities (ALT and SDH), and histopathological
changes to pancreatic lymph nodes (histiocytic cellular
infiltration)
Histopathological changes (histiocytic cellular
infiltration) in the duodenum
Histopathological changes in the duodenum in B6C3FJ
mice (histiocytic cellular infiltration and epithelial
hyperplasia), BALB/c mice (histiocytic cellular
infiltration), and a/w3-C57BL/6 mice (epithelial
hyperplasia)
Decreased serum prolactin levels. Data not adequate
for estimation of a NOAEL or LOAEL.
Liver histopathologic changes. Doses in mg hexavalent
chromium/kg-day could not be estimated.
Changes in serum enzymes; liver triglycerides,
glycogen and cholesterol; liver histopathologic
changes.
Liver histopathologic changes. Doses in mg hexavalent
chromium/kg-day could not be estimated.
NTP (2007)
NTP (2007)
NTP (2007)
Quinteros et al.
(2007)
Rafael et al.
(2007)
Acharya et al.
(2001)
Asmatullah and
Noreen(1999)
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Table 4-25. Observed effects and corresponding NOAELs and LOAELs for subchronic, chronic, and
reproductive toxicity studies following oral exposure to hexavalent chromium
Species
Wistar rat
Wistar rat
Sex
F
F, M
Exposure level1
0, 1.4 mg/kg-day
via drinking
water
F: 0, 1.76-2.47
mg/kg-day via
drinking water
M:0, 1.4-2.18
mg/kg-day via
drinking water
Exposure
duration
22 weeks
6 months
NOAEL
(mg/kg-day)
ND
ND
LOAEL
(mg/kg-day)
1.4
ND
Effects at the NOAEL/LOAEL
Changes in liver weight; serum enzyme levels,
triglycerides, glucose; liver glycogen; liver
histopathology.
Changes in urinary markers of renal function. No
histopathologic examination of the kidney.
Reference
Chopra et al.
(1996)
Vyskocil et al.
(1993)
Chronic Studies
F344/N
rat
B6C3FJ
mouse
F, M
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
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
2 years
F:ND
M:0.21
F:ND
M:ND
0.24
0.77
0.38
0.38
F: Increased incidence of chronic inflammation of the
liver
M: Increased incidences of nonneoplastic
histopathological changes to the liver (basophilic foci),
duodenum (histiocytic cellular infiltrate), and
mesenteric lymph nodes (histiocytic cellular infiltrate
and hemorrhage)
F: Increased incidences of histopathological changes to
the duodenum (diffuse epithelial hyperplasia),
mesenteric lymph nodes (histiocytic cellular
infiltration), liver (histiocytic cellular infiltration), and
pancreas (depletion of cytoplasmic zymogen granules)
M: Increased incidences of histopathological changes to
the duodenum (diffuse epithelial hyperplasia) and
mesenteric lymph nodes (histiocytic cellular
infiltration)
NTP (2008)
NTP (2008)
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Table 4-25. Observed effects and corresponding NOAELs and LOAELs for subchronic, chronic, and
reproductive toxicity studies following oral exposure to hexavalent chromium
Species
Dog
Sprague-
Dawley
rat
Sex
Not
specified
F, M
Exposure level1
0, 0.45, 2.25, 4.5,
6.75, 11.2mg/L
in drinking water
0.05 to 2.8
mg/kg-day via
drinking water
Exposure
duration
4 years
1 year
NOAEL
(mg/kg-day)
ND
2.4-2.8
LOAEL
(mg/kg-day)
ND
ND
Effects at the NOAEL/LOAEL
No effects were observed. Doses in mg hexavalent
chromium/kg-day could not be estimated.
No adverse effects observed at the highest dose tested
Reference
Anwar et al.
(1961)
MacKenzie et
al. (1958)
Reproductive/Developmental Studies
Bonnet
monkey
Charles
Foster rat
Wistar rat
BALB/c
mouse
New
Zealand
White
rabbit
M
M
M
M
M
0, 1.0,2.1,4.1,
and 8.3 mg/kg-
day via drinking
water
0, 20, 40, or
60 mg/kg-day via
gavage
0,5. 2 or
10.4 mg/kg-day
via gavage
0, 6.4, 12.7, or
25.5 mg/kg-day
via gavage
0 or 3.6 mg/kg-
day via gavage
180 days
90 days
6 days
35 days
10 weeks
ND
ND
ND
ND
ND
2.1
20
5.2
6.4
3.6
Reversible changes to male reproductive organs,
including disruption of spermatogenesis, effects on
sperm count and velocity, and histopathological
changes
Decreased serum testosterone levels and loss of 3(3-A5-
HSH activity in testes
Decreased sperm counts and histopathological changes
to the testes
Increased percentage of degenerated tubules,
undergenerated tubules without spermatogonia,
abnormal sperm, and reduced number of spermatogonia
Decreased testes and epididymis weight and decreased
sperm output
Aruldhas et al.
(2006, 2005,
2004);
Subramanian et
al. (2006)
Chowdhury
and Mitra
(1995)
Lietal. (2001)
Zahid et al.
(1990)
Yousef etal.
(2006)
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Table 4-25. Observed effects and corresponding NOAELs and LOAELs for subchronic, chronic, and
reproductive toxicity studies following oral exposure to hexavalent chromium
Species
Sprague-
Dawley
rat
BALB/c
mouse
BALB/c
mouse
Druckrey
rat
Swiss
mouse
Sex
F, M
F, M
F
F
F
Exposure level1
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
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
0,7.9, 16.1, or
37.1 mg/kg-day
via the diet (Fi
generation)
0, 70, 127, or
170 mg/kg-day
via drinking
water
0,63, 119, or
174 mg/kg-day
via drinking
water
Exposure
duration
9 weeks
9 weeks
Continuous
breeding study
3 months
CDs 6 though 14
NOAEL
(mg/kg-day)
F:2.5
M:2.1
F: 1.8
M:3.5
ND
ND
ND
LOAEL
(mg/kg-day)
F:9.5
M:8.5
F: 5.6
M:7.4
7.9
70
63
Effects at the NOAEL/LOAEL
F: Slight erythrocyte microcytosis
M: Slight erythrocyte microcytosis
F: Histopathological changes to the liver (cytoplasmic
vacuolization)
M: Histopathological changes to the liver (cytoplasmic
vacuolization)
Erythrocyte microcytosis (slight decrease in MCH) in
the F! generation
Dam: Increased pre- and post-implantation losses
Offspring: Decreased fetal weight and external and
skeletal abnormalities
Dam: Decreased fertility
Offspring: Decreased fetal body weight and delays in
skeletal development
Reference
NTP (1996b)
NTP (1996a)
NTP (1997)
Kanojia et al.
(1998)
Junaid et al.
(1996a)
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Table 4-25. Observed effects and corresponding NOAELs and LOAELs for subchronic, chronic, and
reproductive toxicity studies following oral exposure to hexavalent chromium
Species
Wistar rat
Sprague-
Dawley
rat
ITRC-
Bred
mouse
Swiss
mouse
Swiss
mouse
Sex
F
F
F
F
F
Exposure level1
0 or 7.9 mg/kg-
day via drinking
water
0 or 35 mg/kg-
day via drinking
water
0,48, 98, or 239
mg/kg-day via
drinking water
0, 53, 101, or
152 mg/kg-day
via drinking
water
0, 53, 101, or
152 mg/kg-day
via drinking
water
Exposure
duration
GD 6 through 15
CDs 1-3 or 4-6
Entire gestational
period
GD 14 though 19
GD 14 though 19
NOAEL
(mg/kg-day)
ND
ND
Dam: 48
Offspring: ND
Dam: 53
Offspring: ND
Dam: 53
Offspring: ND
LOAEL
(mg/kg-day)
7.9
35
Dam: 98
Offspring: 48
Dam: 101
Offspring: 53
Dam: 101
Offspring: 53
Effects at the NOAEL/LOAEL
Dam: Increased pre-implantation loss/litter, post-
implantation loss/litter, resorptions/litter, and dead
fetuses/litter and decreased live fetuses/litter
Offspring: Decreased fetal weight and increased litters
with fetal abnormalities or malformations including
visceral and skeletal changes.
Dam: Impaired implantation, increased resorptions, and
decreased number of viable fetuses
Dam: Decreased body weight gain and increased
resorptions and postimplantation loss
Offspring: Decreased fetal length and weight
Dam: Decreased body weight gain and reduced number
of implantation sites
Dam: Decreased body weight gain
Offspring: Reduced fetal weight and length and
increased incidence of reduced caudal ossification
Reference
Elsaieed and
Nada (2002)
Bataineh et al.
(2007)
Trivedi et al.
(1989)
Junaid et al.
(1996b)
Junaid et al.
(1995)
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 non-
10 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 ATS-2). In human cells, chromosome aberrations, DNA damage, and DNA-DNA and DNA-
17 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-day-
32 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 BDFi 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 B6C3Fi 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 B6C3Fi mice, and significantly positive at> 43.6 mg/L exposures in aw3-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 jig hexavalent chromium/kg (Kuykendall et al.,
12 1996). Another study (Gao et al., 1994) failed to detect DNA damage in peripheral lymphocytes
13 of workers inhalationally exposed to 0.001-0.055 mg/m3. However, several studies of
14 occupational exposures via inhalation provide evidence of significant levels of chromium-
15 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
20 0.0249 mg/m3 (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 (B6C3Fi, BALB/c, and am3-C57EL/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 B6C3Fi 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
15 chromium exposure for all three strains of mice was estimated to have been 2.9 x 10"2 mg/hour at
16 the highest dose (NTP, 2007). This rate is within the estimated reductive capacity of the mouse
17 gastrointestinal tract of 4.4 x 10"2 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 aw3-C57BL/6 mouse contains multiple copies of the OX174 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 non-
10 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 p!6 and the
12 MMR gene hMLHl 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
1 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 (imol 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 jig
16 hexavalent chromium (as Na51CrC>4) 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
30 across species by body weight3 4. 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 I. 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 life stages 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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1 workers exposed to inhalation concentrations as low as 7.5 and 24.9 |ig/m3 (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 FI 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
o
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 B6C3Fi 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 B6C3Fi, 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 B6C3Fi 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 time-
31 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/kg-
3 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 B6C3Fi 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 B6C3Fi
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 B6C3F! Mice Exposed to Sodium
Bichromate 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
21/503
28/50b
35/50b
39/50b
Dose
(mg hexavalent chromium/kg-day)
0
0.38
0.91
2.4
5.9
Male Mice
Duodenum: Diffuse epithelial
hyperplasia
Mesenteric lymph node: Histiocytic
cellular infiltration
0/50
14/47
ll/50b
38/47b
18/50b
31/49b
42/50b
32/49b
32/503
42/46a
Dose
(mghexavalent chromium/kg-day)
0
0.38
1.4
3.1
8.7
Female Mice
Duodenum: Diffuse epithelial
hyperplasia
Mesenteric lymph node: Histiocytic
cellular infiltration
Liver: Histiocytic cellular infiltration
Pancreas: Acinus, cytoplasmic
alteration
0/50
3/46
2/49
0/48
16/50b
29/48b
15/50b
6/50a
35/50b
26/46b
23/50b
6/49a
31/50b
40/50b
32/50b
14/50b
42/50b
42/50b
45/50b
32/50b
1
2
3
4
5
6
7
""Significantly different (p<0.05) from the control group by Dunn's or Shirley's test.
bSignificantly different (p<0.01) from the control group by Dunn's or Shirley's test.
Source: NTP (2008)
5.1.2. Methods of Analysis—Including Models (PBPK, BMD, etc.)
To determine the specific endpoint for use in derivation of the RfD, all available
dichotomous models in the EPA's Benchmark Dose Software (BMDS) were fit to the incidence
data for the seven selected endpoints in female rats and male and female mice administered
sodium dichromate dihydrate in drinking water for two years (NTP, 2008). The incidence data
employed in the BMD modeling of these seven endpoints are shown in Table 5-1. To provide
candidate PODs based on these endpoints, 10% extra risk was selected as the benchmark
response (BMR) in accordance with U.S. EP'A''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 BMDio and BMDLio 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 BMDio and BMDLio 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 BMDio and BMDLio 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 BMDio and BMDLio values of 0.17 and 0.12 mg hexavalent chromium/kg-
13 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-l.
20
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Table 5-2. Summary of BMDio and BMDLio 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
Liver, chronic
inflammation
Duodenum:
diffuse epithelial
hyperplasia
Mesenteric lymph
node: histiocytic
cellular infiltration13
Duodenum:
diffuse epithelial
hyperplasia
Mesenteric lymph
node: histiocytic
cellular infiltration13
Liver: histiocytic
cellular infiltration
Pancreas: acinus,
cytoplasmic
alteration
Species/sex
Rat/female
Mouse/male
Mouse/male
Mouse/female
Mouse/female
Mouse/female
Mouse/female
Model
Log-logistic
1 -Degree polynomial
multistage/quanta! linear
Gamma/Multistage/quantal
linear/Weibull
Log-logistic
Log-logistic
Number
of doses
5
4
3
5
5
BMDa
(mg/kg-day)
0.22
0.16
0.12
0.17
0.68
BMDLa
(mg/kg-day)
0.14
0.13
0.09
0.12
0.52
1
2
3
4
5
6
7
8
9
10
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)
The lowest BMDLio value of 0.09 mg hexavalent chromium/kg-day, based on the
selection of the incidence of diffuse epithelial hyperplasia of the duodenum in female mice as the
critical effect, was identified as the POD from which to derive the RfD for hexavalent chromium.
5.1.3. RfD Derivation—Including Application of Uncertainty Factors (UFs)
The following uncertainty factors (UFs) were applied to the POD of 0.09 mg/kg-day,
based on the incidence of diffuse epithelial hyperplasia of the duodenum in female mice from
NTP (2008), to derive the RfD for hexavalent chromium.
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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 FO and FI generation
26 animals were exposed to hexavalent chromium in the diet, and the offspring of FI
27 animals were evaluated on PND 21.
28
29 For this assessment, the RfD of 0.0009 or 9 x 10"4 mg/kg-day for hexavalent chromium
30 was derived by dividing the BMDLio (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)
2 were applied to this POD to yield an oral RfD of 3 x 10~3 mg/kg-day.
4 5.2. UNCERTAINTIES IN THE ORAL REFERENCE DOSE
5 The following discussion identifies uncertainties associated with the RfD for
6 hexavalent chromium. As presented above, an RfD of 9 x 10~4 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 BMDLio 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 B6C3Fi 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 B6C3Fi 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/? < 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 (atp < 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
2
o
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5
6
7
the oral mucosa and tongue at the highest exposure levels (average daily doses of 5.9 and 7.0 mg
hexavalent chromium/kg-day in males and females, respectively), but not at the three lower
exposure levels. The incidences of squamous cell papillomas or carcinomas in the oral cavity of
male and female F344/N rats exposed to sodium dichromate dihydrate in drinking water for 2
years in the NTP (2008) study are presented in Table 5-3 (for male rats) and Table 5-4 (for
female rats).
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)
0
14.3
57.3
172
516
Estimated
daily intake of hexavalent chromium3
(mg/kg-day)
0
0.21
0.77
2.1
5.9
Incidence of squamous cell
papillomas or carcinomas1"
0/50 (0%)
1/50 (2%)
0/49 (0%)
0/50 (0%)
7/49 (14.5%)c
"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.
Statistically significantly elevated above control atp < 0.05 using Fisher's Exact Test.
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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)
0
14.3
57.3
172
516
Estimated
daily intake of hexavalent chromium3
(mg/kg-day)
0
0.24
0.94
2.4
7.0
Incidence of squamous cell
papillomas or carcinomas1"
1/50 (2%)
1/50 (2%)
0/50 (0%)
2/50 (4%)
11/50(22%)C
"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.
Statistically significantly elevated above control atp < 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 B6C3Fi 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 (atp < 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 (atp < 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 B6C3Fi 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 B6C3Fi 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 B6C3Fi mice exposed to sodium dichromate dihydrate in
drinking water for 2 years (NTP, 2008)
Sodium dichromate dihydrate
concentration
(mg/L)
0
14.3
28.6
85.7
257.4
Estimated
daily intake of hexavalent
chromium3
(mg/kg-day)
0
0.38
0.91
2.4
5.9
Incidence of adenomas or
carcinomas1"
1/49 (2%)
3/49(6.1%)
2/49(4.1%)
7/50 (14%)c
20/48 (41.7%)c
9
10
11
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.
Statistically significantly elevated above control atp < 0.05 using Fisher's Exact Test.
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Table 5-6. Incidences of adenomas and carcinomas combined in the small
intestine of female B6C3Fi mice exposed to sodium dichromate dihydrate
in drinking water for 2 years (NTP, 2008)
Sodium dichromate dihydrate
concentration
(mg/L)
0
14.3
57.3
172
516
Estimated
daily intake of hexavalent
chromium3
(mg/kg-day)
0
0.38
1.4
3.1
8.7
Incidence of adenomas or
carcinomas1"
1/49 (2%)
1/50 (2%)
4/49 (8.2%)
17/49 (34.7%)c
22/49 (44.9%)c
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.
Statistically significantly elevated above control atp < 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 dose-
17 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 BMDLio. The CSF is then calculated by dividing the
3 BMR by the BMDLio 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
11 best fit to the data yielding a slope of 0.09 (mg/kg-day)"1. For female mice, the two-stage
12 multistage model also exhibited the best fit to the data yielding a slope of 0.10 (mg/kg-day)"1.
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
20 Slope x (WH/WA)°-25 = 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
27 were 0.5 and 0.6 (mg/kg-day)"1, 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
31 are similar (i.e., 0.5 (mg/kg-day)"1 for males and 0.6 (mg/kg-day)"1 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
3 (mg/kg-day)"1, 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
13 (U.S. EPA, 2005b). The oral slope factor of 0.5 (mg/kg-day)"1, 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
0-<2 yrs
2-<16 yrs
>16yrs
ADAF
10
3
1
Slope Factor
(per mg/kg-d)
0.5
0.5
0.5
Average Daily Dose
(mg/kg-d)
0.0001
0.0001
0.0001
Duration
adjustment
2 yrs/70 yrs
14 yrs/70 yrs
54 yrs/70 yrs
Total risk
Partial risk
1 x 1Q-5
3 x 1Q-5
4 x 1Q-5
8 x 1Q-5
1
2
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4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
Note that the partial risk for each age group is the product of the values in columns 2-5
(e.g., 10 x 0.5 x 0.0001 x 2/70 = 0.00001 for exposures from age 0 to <2 years), and the total
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 x 10'5.
If calculating the cancer risk for a 30-year exposure to a constant average daily dose of
0.0001 mg hexavalent chromium/kg-day from ages 0 to 30 years, the duration adjustments would
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 x 10"5.
If calculating the cancer risk for a 30-year exposure to a constant average daily dose of
0.0001 mg hexavalent chromium/kg-day from ages 20 to 50 years, the duration adjustments
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 x 10'5.
5.3.6. Uncertainties in Cancer Risk Values
As in most risk assessments, extrapolation of data from experimental animals to estimate
potential lifetime cancer risks to human populations from exposure to hexavalent chromium
yields uncertainties. Some of these uncertainties can be evaluated for their quantitative impact
on the final result, while for others, only their qualitative impact can be assessed. The principal
uncertainties in the assessment of the cancer risk from exposure to hexavalent chromium are
summarized below in Table 5-8, and discussed in more detail in the following text.
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Table 5-8. Summary of uncertainties in the cancer risk assessment for
hexavalent chromium
Consideration/
approach
Low-dose
extrapolation
procedure
Cross-species
scaling
Statistical
uncertainty at
POD
Species/gender
combination
Human relevance
of rodent tumor
data
Human
population
variability in
metabolism and
response/
sensitive
subpopulations
Impact on oral slope
factor
Alternatives could J, or
t CSF by an unknown
extent
Alternatives could J, or
t CSF [e.g., sixfold |
(scaling by B W) or |
twofold (scaling by
BW2/3)]
| CSF 25% if MLE
(i.e., BMD10) used
rather than lower
bound (BMDL10) for
POD
Human risk could J, or
t, depending on
relative sensitivity
Lack of human
relevance of tumor
data would | CSF
Low-dose risk f or J, to
an unknown extent
Decision
Multistage model
used to determine
POD, linear low-
dose extrapolation
from POD
BW3/4 (default
approach)
BMDL (default
approach for
calculating
reasonable upper
bound CSF)
Male mouse tumors
(adenomas or
carcinomas of the
small intestine)
Tumors with
significant dose-
response considered
for estimating
potential human
cancer response
Considered
qualitatively
Justification
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.
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).
Size of bioassay results in sampling variability;
lower bound is 95% confidence interval on
administered dose.
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.
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.
No data are available to support the range of human
variability /sensitivity to hexavalent chromium.
2
3
4
5
Choice of low-dose extrapolation approach. The mode of action is a key consideration in
clarifying how risks should be estimated for low-dose exposure. A linear, low-dose
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.
9 Cross-species scaling. The default cross-species scaling factor (BW3 4) 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
O/Q 1
15 for default cross-species scaling factors (e.g., BW or BW ).
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 BMDLio for male mice is approximately 25% lower than the BMDio.
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
o
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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 (Eizaguirre-
9 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 B6C3Fi 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 B6C3Fi 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 BMDLio 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
15 the RfD. The RfD of 0.0009 or 9 x 10"4 mg/kg-day for hexavalent chromium was derived by
16 dividing the BMDLio (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
2 neoplasms in the small intestine of male and female mice was 0.5 (mg/kg-day)"1 and 0.6 (mg/kg-
3 day)"1, respectively, expressed in human equivalents. Because of the poorer fit of the multistage
4 model to the female mouse data, the cancer potency estimate of 0.5 (mg/kg-day)"1 based on the
5 male mouse data was selected as the CSF for hexavalent chromium.
6
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1 APPENDIX A. SUMMARY OF EXTERNAL PEER REVIEW AND PUBLIC
2 COMMENTS AND DISPOSITION
o
3
4
5
6 [page intentionally left blank]
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APPENDIX B. BENCHMARK DOSE CALCULATIONS
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APPENDIX B-l. Details of Benchmark Dose Analysis for the RfD
Table B-l.l Incidence data for nonneoplastic lesions from all treatment groups of
female F344/N rats and male and female B6C3Fi mice exposed to sodium
dichromate dihydrate in drinking water for 2 years (NTP, 2008)
Dose
(mg hexavalent chromium/kg-day)
0.24
0.94
2.4
7.0
Female Rats
Liver, chronic inflammation
Male Mice
Duodenum: Diffuse epithelial
hyperplasia
Mesenteric lymph node:
Histiocytic cellular infiltration
12/50
0
0/50
14/47
28/50b
35/50b
21/50a
Dose
(mg hexavalent chromium/kg-day)
0.38 0.91 2.4
ll/50b
38/47b
18/50b
31/49b
42/50b
32/49b
Dose
(mg hexavalent chromium/kg-day)
0.38 1.4 3.1
39/50b
5.9
32/50a
42/46a
8.7
Female Mice
Duodenum: Diffuse epithelial
hyperplasia
Mesenteric lymph node:
Histiocytic cellular infiltration
Liver: Histiocytic cellular
infiltration
Pancreas: Acinus, cytoplasmic
alteration
0/50
3/46
2/49
0/48
16/50b
29/48b
15/50b
6/50a
35/50b
26/46b
23/50b
6/49a
31/50b
40/50b
32/50b
14/50b
42/50b
42/50b
45/50b
32/50b
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 goodness-
of-fit statistic, only the log-logistic model provided an adequate fit (x^-value >0.1) to the data
(Table B-l.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-l.l).
B-2
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Table B-1.2. BMDio and BMDLio 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
Gamma3
Logistic
Log-logistic1"
Multi-stage0
Probit
Log-probitb
Quanta! linear
Weibulf
BMD10
(mg/kg-day)
0.51
0.84
0.22
0.51
0.88
0.89
0.51
0.51
BMDL10
(mg/kg-day)
0.37
0.65
0.14
0.37
0.70
0.61
0.37
0.37
x2 p-value
0.04
0.01
0.37
0.04
0.01
0.01
0.04
0.04
AIC
317.97
321.45
312.57
317.97
321.80
320.86
317.97
317.97
""Restrict power >1
bSlope restricted to >1
'Restrict 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
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Figure B-l.l. Predicted and observed incidence of chronic inflammation of the liver
in female rats exposed to sodium dichromium dihydrate in drinking water for 2
years*
Log-Logistic Model with 0.95 Confidence Level
0.9
0.8
0.7
"S 0.6
c 0.5
o
0.4
0.3
0.2
0.1
Log-Logistic
BMDL
BMD
3 4
dose
13:3404/082008
*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 (x2/'-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 (x2/'-value > 0.1).
Comparing across models, a better fit is generally indicated by a lower AIC (EPA, 2000b). As
assessed by AIC, the 1-degree polynomial multistage model provided the best fit to the data
(Figure B-1.2). Based on the multistage model, the BMD associated with a 10% extra risk was
0.16 mg hexavalent chromium/kg-day and its lower 95% confidence limit (BMDL) was 0.13 mg
hexavalent chromium/kg-day.
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Table B-1.3. BMDio and BMDLio 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
All doses
Gamma3
Logistic
Log-logistic13
Multi-stage0
Probit
Log-probitb
Quanta! linear
Weibulf
Highest dose dropped
Gamma3
Logistic
Log-logistic13
Multi-stage"1
Probit
Log-probitb
Quantal linear
Weibull3
BMD10
(mg/kg-day)
0.31
0.90
0.15
0.31
0.90
0.48
0.31
0.31
(four doses modeled)
0.22
0.47
0.26
0.16
0.45
0.28
0.16
0.22
BMDL10
(mg/kg-day)
0.25
0.74
0.12
0.25
0.76
0.36
0.25
0.25
0.14
0.39
0.15
0.13
0.37
0.23
0.13
0.14
x2 p-value
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.43
0.03
0.20
0.52
0.04
0.33
0.52
0.47
AIC
270.99
296.25
247.93
270.99
296.18
274.38
270.99
270.99
167.67
177.09
169.23
166.34
176.19
167.41
166.34
167.50
"Restrict power >1
bSlope restricted to >1
'Restrict 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
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Figure B-1.2. Predicted and observed incidence of diffuse epithelial hyperplasia in
the duodenum of male mice exposed to sodium dichromium dihydrate in drinking
water for 2 years*
Multistage Model with 0.95 Confidence Level
0.8
-a 0.6
-value >
0.1) to the full dataset (Table B-l .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 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 considered not suitable for
benchmark dose modeling.
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Table B-1.4. BMDio and BMDLio 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
All doses
Gamma3
Logistic
Log-logistic13
Multi-stage0
Probit
Log-probitb
Quanta! linear
Weibulf
BMD10
(mg/kg-day)
0.38
0.53
0.16
0.43
0.56
0.83
0.38
0.38
BMDL10
(mg/kg-day)
0.26
0.39
0.08
0.26
0.43
0.52
0.26
0.26
x2 p-value
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
AIC
285.94
286.38
284.48
287.88
286.35
289.36
285.94
285.94
Highest dose dropped (four doses modeled)
Gamma3
Logistic
Log-logistic13
Multi-stage11
Probit
Log-probitb
Quanta! linear
Weibulf
Two highest doses
Gamma3
Logistic
Log-logistic13
Multi-stage6
Probit
Log-probitb
Quanta! linear
Weibulf
0.47
0.61
0.21
0.47
0.63
1.24
0.47
0.47
0.24
0.35
0.08
0.24
0.37
0.56
0.24
0.24
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
258.50
259.04
256.81
258.50
259.08
261.28
258.50
258.50
dropped (three doses modeled)
0.11
0.17
0.05
0.11
0.17
0.17
0.11
0.11
0.07
0.12
0.03
0.07
0.12
0.11
0.07
0.07
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
187.77
189.97
183.77
187.77
190.12
190.37
187.77
187.77
"Restrict power >1
bSlope restricted to >1
'Restrict 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-7 DO NOT CITE OR QUOTE
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Diffuse Epithelial Hyperplasia of the Duodenum in Female Mice. As assessed by the chi-
square goodness-of-fit statistic, none of the models provided an adequate fit (x^-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
r\
provided an adequate fit (x />-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-l .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-8 DO NOT CITE OR QUOTE
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Table B-1.5. BMDio and BMDLio 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
All doses
Gamma3
Logistic
Log-logistic13
Multi-stage0
Probit
Log-probitb
Quanta! linear
Weibulf
BMD10
(mg/kg-day)
0.34
0.88
0.12
0.34
0.93
0.52
0.34
0.34
BMDL10
(mg/kg-day)
0.27
0.72
0.09
0.27
0.78
0.38
0.27
0.27
x2 p-value
0.00
0.00
0.04
0.00
0.00
0.00
0.00
0.00
AIC
275.34
293.17
245.54
275.34
294.03
279.54
275.34
275.34
Highest dose dropped (four doses modeled)
Gamma3
Logistic
Log-logistic13
Multi-stage"1
Probit
Log-probitb
Quanta! linear
Weibulf
Two highest doses
Gamma"
Logistic
Log-logistic13
Multi-stage"
Probit
Log-probitb
Quantal linear
Weibull3
0.20
0.55
0.11
0.20
0.54
0.29
0.20
0.20
0.16
0.46
0.08
0.16
0.45
0.24
0.16
0.16
0.00
0.00
0.04
0.00
0.00
0.00
0.00
0.00
213.41
236.10
200.07
213.41
235.61
220.04
213.41
213.41
dropped (three doses modeled)
0.12
0.34
0.12
0.12
0.32
0.20
0.12
0.12
0.09
0.27
0.06
0.09
0.26
0.16
0.09
0.09
0.87
0.00
1.00
0.87
0.00
0.48
0.87
0.87
126.06
141.77
127.77
126.06
140.65
127.17
126.06
126.06
aRestrictpower>l
bSlope restricted to >1
'Restrict 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-9 DO NOT CITE OR QUOTE
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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*
Multistage Model with 0.95 Confidence Level
I
c
o
'o
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
Multistage
BMDL BMD
0.2
0.4
0.6 0.8
dose
1.2
1.4
09:36 04/09 2008
*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 (%2
/7-value > 0.1) to the full dataset (Table B-l.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-10
DO NOT CITE OR QUOTE
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Table B-1.6. BMDio and BMDLio 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
All doses
Gamma3
Logistic
Log-logistic13
Multi-stage0
Probit
Log-probitb
Quanta! linear
Weibulf
BMD10
(mg/kg-day)
0.41
0.77
0.09
0.41
0.85
0.68
0.41
0.41
BMDL10
(mg/kg-day)
0.30
0.61
0.06
0.30
0.69
0.47
0.30
0.30
x2 p-value
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
AIC
282.46
290.18
263.55
282.46
291.41
285.85
282.46
282.46
Highest dose dropped (four doses modeled)
Gamma3 0.20 0.15 0.00 224.84
Logistic 0.40 0.33 0.00 230.81
Log-logistic13 0.07 0.05 0.00 215.19
Multi-stage11 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
Quanta! linear 0.20 0.15 0.00 224.84
Weibuir 0.20 0.15 0.00 224.84
Two highest doses dropped (three doses modeled)
Gamma3
Logistic
Log-logistic13
Multi-stage6
Probit
Log-probitb
Quanta! linear
Weibuir
0.14
0.31
0.07
0.14
0.30
0.21
0.14
0.14
0.10
0.24
0.04
0.10
0.23
0.15
0.10
0.10
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
172.32
178.99
164.47
172.32
178.74
178.11
172.32
172.32
"Restrict power >1
bSlope restricted to >1
'Restrict 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.
Restrict 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-11 DO NOT CITE OR QUOTE
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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 (x2/'-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. BMDio and BMDLio 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
Gamma3
Logistic
Log-logistic1"
Multi-stage0
Probit
Log-probitb
Quanta! linear
Weibulf
BMD10
(mg/kg-day)
0.35
0.85
0.17
0.35
0.88
0.62
0.35
0.35
BMDL10
(mg/kg-day)
0.28
0.70
0.12
0.28
0.75
0.48
0.28
0.28
x2 p-value
0.08
0.00
0.44
0.08
0.00
0.01
0.08
0.08
AIC
255.40
267.56
251.36
255.40
268.64
260.00
255.40
255.40
""Restrict power >1
bSlope restricted to >1
'Restrict 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-12 DO NOT CITE OR QUOTE
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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*
Log-Logistic Model with 0.95 Confidence Level
0.8
£ 0.6
o
I
c
o
0.2
Log-Logistic
3MDL
BMD
4 5
dose
11:1704/092008
*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 ofAcinar 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 (%2 p-
value >0.1) to the data (Table B-l .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-l.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-13
DO NOT CITE OR QUOTE
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Table B-1.8. BMDio and BMDLio 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
Gamma3
Logistic
Log-logistic1"
Multi-stage0
Probit
Log-probitb
Quanta! linear
Weibulf
BMD10
(mg/kg-day)
0.92
2.43
0.68
0.92
2.24
1.77
0.92
0.92
BMDL10
(mg/kg-day)
0.72
2.03
0.52
0.72
1.89
1.40
0.72
0.72
x2 p-value
0.13
0.09
0.19
0.13
0.11
0.11
0.13
0.13
AIC
206.82
211.78
205.22
206.82
210.99
209.99
206.82
206.82
"Restrict power >1
bSlope restricted to >1
'Restrict 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-14 DO NOT CITE OR QUOTE
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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*
Log-Logistic Model with 0.95 Confidence Level
0.8
0.7
0.6
0.5
< 0.4
c
.0
ro 0.3
0.2
0.1
Log-Logistic
BMDL
BMD
01 23456789
dose
11:41 04/092008
*BMDs and BMDLs indicated are associated with a 10% extra risk, and are in units of mg
hexavalent chromium/kg-day.
Source: ATSDR
B-15
DO NOT CITE OR QUOTE
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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):
Multistage Cancer Model with 0.95 Confidence Level
0.6
0.5
-o 0.4
8
-------
Dependent variable = Response
Independent variable = Dose
Total number of observations = 5
Total number of records with missing values
Total number of parameters in model = 3
Total number of specified parameters = 0
Degree of polynomial = 2
= 0
Maximum number of iterations = 250
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
Default Initial Parameter Values
Background = 0.0291151
Beta(l) = 0.0232273
Beta(2) = 0.0107072
Asymptotic Correlation Matrix of Parameter Estimates
Background Beta(l) Beta(2)
Background 1 -0.73 0.62
Beta(l) -0.73 1 -0.96
Beta(2) 0.62 -0.96 1
Variable
Background
Beta(1)
Beta(2)
Indicates that this value is not calculated.
95.0% Wald Confidence Interval
Lower Conf. Limit Upper Conf. Limit
Model
Full model
Fitted model
Reduced model
B-17
DO NOT CITE OR QUOTE
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Goodness of Fit
Dose
0.0000
0.3800
0.9100
2.4000
5.9000
Est. Prob.
0.0287
0.0391
0.0581
0.1374
0.4160
Expected
1.408
1.915
2.848
6.869
19.969
Observed
1.000
3.000
2.000
7.000
20.000
Size
49
49
49
50
48
Scaled
Residual
-0.349
0.800
-0.518
0.054
0.009
ChiA2 = 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-18 DO NOT CITE OR QUOTE
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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):
Multistage Cancer Model with 0.95 Confidence Level
0.6
0.5
I 0.4
o
-g
| 0.3
o
to
2 0.2
LJ_
0.1
0
Multistage Cancer
Linear extrapolation
BMD Lower Bound
14:57 03/20 2009
Source: NJDEP
Multistage Cancer Model. (Version: 1.7; Date: 05/16/2008)
Input Data File:
M:\ChromiumVI\ms c_FEMALE_MICE_INTESTINAL_TUMORS_NTP_2 0 0 8_Setting. (d)
Gnuplot Plotting File:
M:\ChromiumVI\msc_FEMALE_MICE_INTESTINAL_TUMORS_NTP_2008_Setting.plt
Fri Feb 05 09:54:51 2010
HMDS Model Run
The form of the probability function is:
P[response] = background + (1-background)*[1-EXP(
-betal*doseAl-beta2*doseA2)]
The parameter betas are restricted to be positive
Dependent variable = Response
Independent variable = Dose
B-19
DO NOT CITE OR QUOTE
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Total number of observations = 5
Total number of records with missing values
Total number of parameters in model = 3
Total number of specified parameters = 0
Degree of polynomial = 2
= 0
Maximum number of iterations = 250
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
Default Initial Parameter Values
Background = 0.0398439
Beta(l) = 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(l)
Background 1 -0.62
Beta(l) -0.62 1
Parameter Estimates
Variable
Background
Beta(1)
Beta(2)
- Indicates that this value is not calculated.
95.0% Wald Confidence Interval
Lower Conf. Limit Upper Conf. Limit
Model
Full model
Fitted model
Reduced model
B-20
DO NOT CITE OR QUOTE
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Goodness of Fit
Dose
0.0000
0.3800
1.4000
3.1000
8.7000
Est. Prob.
0.0141
0.0433
0.1176
0.2287
0.5050
Expected
0.690
2.166
5.761
11.208
24.746
Observed
1.000
1.000
4.000
17.000
22.000
Size
49
50
49
49
49
Scaled
Residual
0.376
-0.810
-0.781
1.970
-0.785
ChiA2 = 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
B-21 DO NOT CITE OR QUOTE
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