EPA/635/R-22/191b
External Review Draft
www.epa.gov/iris
IRIS Toxicological Review of Hexavalent Chromium [Cr(VI)]
Supplemental Information
[CASRN 18540-29-9]
October 2022
Integrated Risk Information System
Center for Public Health and Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
Washington, DC
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Supplemental Information—Hexavalent Chromium
DISCLAIMER
This document is an external review draft for review purposes only. This information is
distributed solely for the purpose of public comment 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.
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—Hexavalent Chromium
CONTENTS
CONTENTS iii
APPENDIX A. SYSTEMATIC REVIEW PROTOCOL FOR HEXAVALENT CHROMIUM A-l
APPENDIX B. SUMMARY OF OTHER AGENCY CONCLUSIONS B-l
APPENDIX C. INFORMATION IN SUPPORT OF HAZARD IDENTIFICATION AND DOSE-RESPONSE
ANALYSIS C-l
C.l. PHARMACOKINETICS C-l
C.1.1. Absorption C-l
C.l.2. Distribution C-2
C.l.3. Metabolism C-10
C.l.4. Excretion C-14
C.l.5. Physiologically Based Pharmacokinetic Models C-15
C.l.6. Literature Overview of Studies Identified as ADME C-35
C.2. SUPPORTING EVIDENCE FOR SPECIFIC HEALTH EFFECTS C-48
C.2.1. Respiratory Effects C-48
C.2.2. Gastrointestinal Effects C-59
C.2.3. Hepatic Effects C-65
C.2.4. Hematological Effects C-73
C.2.5. Immune Effects C-75
C.2.6. Male Reproductive Effects C-97
C.2.7. Female Reproductive Effects C-103
C.2.8. Developmental Effects C-109
C.3. SUPPORTING EVIDENCE FOR CARCINOGENIC MODE OF ACTION C-112
C.3.1. Meta-analysis of Cr(VI) and Cancer of the Gl Tract C-112
C.3.2. Mechanistic Evidence Organized by the 10 Key Characteristics of Carcinogens C-140
C.3.3. Gene Expression Studies Relevant to Gastrointestinal Cancer Cell Signaling
Pathways C-227
C.3.4. Toxicogenomic Studies C-240
C.3.5. Susceptible Populations C-264
C.4. SUPPORTING EVIDENCE FOR EXPOSURE TO THE GENERAL POPULATION C-271
C.4.1. Drinking Water Data from the Third Unregulated Contaminant Monitoring Rule....C-271
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C.4.2. Local Data of Air, Soil, and Dust Cr(VI) Concentrations C-273
APPENDIX D. DOSE-RESPONSE MODELING D-l
D.l. BENCHMARK DOSE MODELING SUMMARY FOR NONCANCER ENDPOINTS D-3
D.l.l. Evaluation of Model Fit and Model Selection D-6
D.l.2. Calculation of Regional Deposited Dose Ratios (RDDR) D-18
D.2. BENCHMARK DOSE MODELING SUMMARY FOR CANCER ENDPOINTS D-22
D.2.1. Cancer Data for Dose Response Modeling D-22
D.2.2. Evaluation of Model Fit and Model Selection D-25
D.3. ALTERNATIVE APPROACHES FOR CANCER AND NONCANCER DOSE-RESPONSE
ASSESSMENT D-26
D.3.1. Noncancer Oral Dose-response Applying Default BW3/4Scaling Approaches D-26
D.3.2. Order of Uncertainty Factor Applications D-28
D.3.3. Uncertainty Assessment of Low-dose Extrapolation Method for Oral Cancer
Dose-response D-31
D.4. EXCLUSION OF HUMAN STUDIES FOR EXPOSURE-RESPONSE D-33
D.5. INDIVIDUAL-LEVEL ANALYSIS OF NEOPLASTIC AND NONNEOPLASTIC LESIONS IN MICE
FROM NTP (2008) D-38
D.6. PROBABILITY DISTRIBUTIONS OF HUMAN EQUIVALENT DOSE FOR CANCER AND
NONCANCER PODS DERIVED FROM TOXICOKINETIC MODELING D-42
D.6.1. Noncancer Model Outputs D-42
D.6.2. Cancer Model Outputs D-55
APPENDIX E. SAS Code for Life-Table Analysis E-l
APPENDIX F. QUALITY ASSURANCE FOR THE IRIS TOXICOLOGICAL REVIEW OF HEXAVALENT
CHROMIUM F-l
APPENDIX G. RESPONSE TO EXTERNAL COMMENTS G-l
REFERENCES FOR APPENDICES R-l
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Supplemental Information—Hexavalent Chromium
TABLES
Table B-l. Noncancer inhalation assessments by other national and international health
agencies and associations (in reverse chronological order) B-l
Table B-2. Cancer inhalation assessments by other national and international health agencies (in
reverse chronological order) B-2
Table B-3. Oral assessments by other national and international health agencies (in reverse
chronological order) B-4
Table C-l. Concentrations of chromium in erythrocytes and plasma (ng Cr/g) following ingestion
of sodium dichromate dihydrate in drinking water (male F334 rats) C-5
Table C-2. Ratio of erythrocytes:plasma concentrations following ingestion of sodium
dichromate dihydrate in drinking water (male F334 rats) C-5
Table C-3. Concentrations of chromium in erythrocytes and plasma (ng Cr/g) following ingestion
of sodium dichromate dihydrate in drinking water (female B6C3F1 mice) C-6
Table C-4. Ratio of erythrocytes:plasma concentrations following ingestion of sodium
dichromate dihydrate in drinking water (female B6C3F1 mice) C-6
Table C-5. Chromium in tissues (ng/g wet tissue or ng/mL blood) of mice and rats after ingesting
K2Cr07 in drinking water (8 mg Cr(VI)/kg-day) for 4 or 8 weeks C-7
Table C-6. Summary of oral and inhalation data from O'Flaherty and Radike (1991) C-9
Table C-7. Summary of oral and inhalation control group data from O'Flaherty and Radike (1991) C-10
Table C-8. The pH of the mouse, rat, and human gastrointestinal tract C-12
Table C-9. Selected studies of Cr(VI) reduction capacities C-13
Table C-10. Uncertainties and potential impacts of alternative dose metrics for rodent-to-human
extrapolation C-18
Table C-ll. Final human physiological parameters for dose-response modeling and rodent-to-
human extrapolation C-20
Table C-12. Normalized sensitivity coefficients of human gastric model parameters with respect
to pyloric flux dose metric C-23
Table C-13. Human equivalent dose (mg/kg-day) outputs of 20,000 Monte Carlo simulations of
varying baseline pH populations using the BW3/4-adjusted Cr(VI) dose escaping
stomach reduction C-25
Table C-14. Final mouse PBPK parameters for dose-response modeling and rodent-to-human
extrapolation C-26
Table C-15. Normalized sensitivity coefficients of mouse gastric model parameters with respect
to pyloric flux dose metric C-28
Table C-16. Lifetime average daily internal doses for the mouse during the NTP (2008) 2-year
bioassay of sodium dichromate dihydrate C-29
Table C-17. Average daily internal doses for the female mouse (F0 dams) during the NTP (1997)
bioassay C-29
Table C-18. Final rat PBPK parameters for dose-response modeling and rodent-to-human
extrapolation C-30
Table C-19. Normalized sensitivity coefficients of rat gastric model parameters with respect to
average daily dose escaping stomach reduction C-31
Table C-20. Lifetime average daily internal doses for the rat during the NTP (2008) 2-year
bioassay of sodium dichromate dihydrate (pH = 4.38) C-33
Table C-21. Lifetime average daily internal doses for the rat during the NTP (2007f) 90-day
bioassay of sodium dichromate dihydrate (pH = 4.38) C-33
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Table C-22. Comparison of internal-dose points of departure based on external-dose BMD
modeling and internal-dose BMD modeling C-34
Table C-23. In vivo Cr(VI) pharmacokinetic studies C-37
Table C-24. In vitro and ex vivo Cr(VI) studies primarily focused on pharmacokinetics in the Gl
tract and blood C-41
Table C-25. In vitro studies primarily examining distribution and reduction mechanisms C-42
Table C-26. Human biomonitoring and biomarker studies C-43
Table C-27. Gastric emptying rates for rats, mice, and humans expressed as half-emptying time
(T1/2) and transit time (KLSD). Vehicle indicated in parentheses if known C-45
Table C-28. Time-weighted average daily doses in mice for the NTP (2008) 2-year bioassay of
sodium dichromate dihydrate. Doses in mg/kg-day Cr(VI) C-46
Table C-29. Time-weighted average daily doses in rats for the NTP (2008) 2-year bioassay of
sodium dichromate dihydrate. Doses in mg/kg-day Cr(VI) C-47
Table C-30. Time-weighted average daily doses in rats for the NTP (2008) 2-year bioassay of
sodium dichromate dihydrate at different time periods. Doses in mg/kg-day
Cr(VI) C-47
Table C-31. Mechanistic studies prioritized for informing potential Cr(VI)-induced respiratory
toxicity C-49
Table C-32. Experimental animal studies providing apical evidence of toxic effects of ingested
Cr(VI) in the Gl tract C-59
Table C-33. Supporting mechanistic studies prioritized for informing Cr(VI)-induced Gl tract
toxicity C-62
Table C-34. Mechanistic studies prioritized for informing potential Cr(VI)-induced hepatic toxicity C-65
Table C-35. Mechanistic studies prioritized for informing potential Cr(VI)-induced hematological
effects C-74
Table C-36. Data summary tables for immunological outcomes included in the immune effects
animal evidence synthesis C-76
Table C-37. Mechanistic studies prioritized for informing potential Cr(VI)-induced immune
toxicity C-90
Table C-38. Summary of cytokine levels measured following Cr(VI) exposure C-95
Table C-39. Mechanistic studies prioritized for informing potential Cr(VI)-induced male
reproductive toxicity C-98
Table C-40. Mechanistic studies prioritized for informing potential Cr(VI)-induced female
reproductive toxicity C-104
Table C-41. Mechanistic studies prioritized for informing potential Cr(VI)-induced developmental
toxicity C-109
Table C-42. PECO for screening occupational studies relevant to Cr(VI) C-113
Table C-43. Occupational group-specific criteria for rating certainty of exposure to Cr(VI) C-116
Table C-44. Comparison of studies included in meta-analyses or that met PECO, with search
phase, study evaluation rating, and rationale for exclusion in EPA meta-analysis C-121
Table C-45. Summary effect estimates from random effects meta-analysis, by cancer site and
occupational group, where four or more estimates are included C-133
Table C-46. Mechanistic studies informing the intracellular reduction of Cr(VI) and reactivity of
Cr species with DNA and proteins C-141
Table C-47. Chromosomal mutation studies in humans exposed to Cr(VI) via inhalation
(evaluated in HAWC) C-147
Table C-48. Supporting genotoxicity studies in lung tissues and cells following Cr(VI) exposures C-164
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Table C-49. Supporting genotoxicity studies in humans and animals exposed to Cr(VI) via
inhalation or intratracheal instillation C-172
Table C-50. Supporting genotoxicity studies in gastrointestinal tract tissues and cells following
Cr(VI) exposures C-187
Table C-51. Supporting genotoxicity studies in animals exposed via the oral route to Cr(VI) C-190
Table C-52. Genotoxicity studies in animals exposed to Cr(VI) via i.p. injection C-191
Table C-53. In vitro genotoxicity studies in human cells C-199
Table C-54. Mechanistic studies relevant to altered DNA repair or genomic instability induced by
Cr(VI) exposure C-202
Table C-55. Studies of epigenetic alterations in humans, experimental animals, and human cells
in vitro exposed to Cr(VI) C-205
Table C-56. Evidence in human studies prioritized for informing potential Cr(VI)-induced
oxidative stress C-210
Table C-57. In vitro studies of Cr(VI)-induced oxidative stress C-221
Table C-58. Mechanistic studies informing Cr(VI)-induced cellular immortalization C-223
Table C-59. Mechanistic studies relevant to Cr(VI)-induced cell death, cell proliferation, and
changes in cellular energetics C-225
Table C-60. Gene expression studies in humans exposed to Cr(VI) C-228
Table C-61. Gene expression corresponding to positive results of Cr(VI) assays performed in vivo
(rats) or in vitro (human cells or TOX21 HTS assays). Direction of change
(measuring mRNA or protein): ^ (upregulated or activated); 4/ (downregulated
or inhibited); A (protein posttranslational modification or change of intracellular
localization) C-234
Table C-62. Summary of considered toxicogenomic studies for Cr(VI) overall confidence
classification C-241
Table C-63. Evaluation of the information available with microarray data using MIAME sections C-245
Table C-64. Evaluation of the DNA microarray experiments in Kopec et al. (2012b; 2012a) using
criteria outlined by Bourdon-Lacombe et al. (2015) C-245
Table C-65. Studies of genetic polymorphisms in humans occupationally exposed to Cr(VI) C-265
Table C-66. Statistical summary of UCMR3 chromium (VI) concentrations in large public water
systems (PWS) C-271
Table C-67. Summary of UCMR3 chromium (VI) concentration data (in ng/L) grouped by EPA
region C-272
Table C-68. Summary of UCMR3 Cr(VI) data for 20 large public water systems with the highest
mean concentrations C-272
Table C-69. Cr(VI) concentrations in ambient PMio (ng/m3) at monitoring sites in Midlothian,
Texas containing three cement manufacturing facilities and a steel mill (ATSDR,
2016) C-273
Table C-70. Cr(VI) concentrations in air measured at monitoring sites in Portland Oregon
reporting elevated metals concentrations (Oregon DEQ, 2016b) C-274
Table C-71. Cr(VI) concentrations (mean ± SD in ng/m3) in ambient PMio measured in urban and
suburban New Jersey (Huang et al., 2014) C-274
Table C-72. Cr(VI) Mean concentration in air districts with chromium plating and anodizing
facilities for the year 2005. Data from the California Air Resources Board C-275
Table C-73. Estimated environmental concentrations of chromium in selected locations within
the United States C-275
Table D-l. Noncancer endpoints selected for dose-response modeling for Cr(VI) (oral) from NTP
(2008) D-3
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Table D-2. Noncancer endpoints selected for dose-response modeling for Cr(VI) (oral) from NTP
(2007f) D-5
Table D-3. Noncancer endpoints selected for dose-response modeling for Cr(VI) (oral) from NTP
(1997) D-6
Table D-4. Noncancer endpoints selected for dose-response modeling for Cr(VI) (inhalation) D-6
Table D-5. BMD model results for diffuse epithelial hyperplasia in female mice from NTP (2008)
(no high doses omitted) D-8
Table D-6. Modeling alternatives for diffuse epithelial hyperplasia in mice from NTP (2008) D-8
Table D-7. RfDs for modeling alternatives of diffuse epithelial hyperplasia in mice from NTP
(2008) D-10
Table D-8. BMD model results for chronic liver inflammation in female rats from NTP (2008) D-ll
Table D-9. RfDs for modeling alternatives of chronic liver inflammation in female rats from NTP
(2008) D-12
Table D-10. BMD model results for fatty change in liver of female rats from NTP (2008) D-13
Table D-ll. BMD results for histiocytosis in male rats at 90 days from Glaser et al. (1990) D-14
Table D-12. BMD results for total protein in BALF in male rats at 90 days from Glaser et al. (1990).... D-14
Table D-13. BMD results for LDH in BALF in male rats at 90 days from Glaser et al. (1990) D-16
Table D-14. BMD results for albumen in BALF male rats at 90 days from Glaser et al. (1990) D-17
Table D-15. Calculation of RDDR for Glaser et al. (1985) and Glaser et al. (1990) using default
MMAD parameters D-19
Table D-16. Human equivalent concentrations of Cr(VI) in the 90-day inhalation study in rats by
Glaser etal. (1990) D-19
Table D-17. RDDR calculations under different human physiological activity for respiratory
effects D-20
Table D-18. RDDR calculations under different human ages and physiological activity for
systemic effects D-21
Table D-19. Data of neoplastic lesions in rats and mice (NTP, 2008) D-23
Table D-20. NTP historical control data for animals fed the NTP-2000 diet, from studies of all
routes and vehicles of administration (incidence, %, mean % ± standard
deviation %)a D-24
Table D-21. Summary of derivation of points of departure following oral exposure for effects
outside of the gastrointestinal tract (default approach) D-26
Table D-22. Effects and corresponding derivation of candidate values from PODS applying BW'/4
scaling D-27
Table D-23. Summary of derivation of points of departure following oral exposure using
alternative uncertainty factor process D-29
Table D-24. Effects and corresponding derivation of candidate values using alternative
uncertainty factor process D-29
Table D-25. Overview of studies excluded for exposure-response analysis of upper respiratory
tract (nasal) effects in humans D-33
Table D-26. Overview of studies excluded for exposure-response analysis of lung cancer in
humans based on screening studies for adequate exposure-response data3 D-35
Table D-27. Overview of studies excluded for exposure-response analysis of lung cancer in
humans based on screening the most recent analyses D-36
Table D-28. Overview of studies excluded for exposure-response analysis of lung cancer in
humans D-37
Table D-29. Individual-level overview of neoplastic and nonneoplastic lesions in male mice from
NTP (2008) D-38
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Table D-30. Individual-level overview of neoplastic and nonneoplastic lesions in female mice
from NTP (2008) D-39
Table D-31. Summary of neoplastic and nonneoplastic lesions in mice from NTP (2008) D-41
FIGURES
Figure C-l. Comparison of mean tissue concentrations in mice (n = 3) following 182 days of
either Cr(VI) or Cr(lll) oral exposure C-2
Figure C-2. Ratio of RBC:plasma concentration as a function of Cr(VI) drinking water
concentration (1 ppm = 1 mg/L) for male F334 rats and female B6C3F1 mice
using data from NTP (2008) C-4
Figure C-3. Mean concentration of total chromium in Gl tract tissues of mice and rats following
exposure to 180 mg/L Cr(VI) in drinking water for 90 days [approximately 31.9
and 20.5 mg/kg-day Cr(VI) for mice and rats, respectively] C-8
Figure C-4. Reduction of Cr(VI) in samples of human gastric juice (fasted subjects) using data
from Proctor et al. (2012) C-ll
Figure C-5. Urinary rates of excretion by human volunteers administered a glass of drinking
water containing 2.5-5.0 mg Cr(VI) at day 2 C-14
Figure C-6. Urinary excretion rate of a human volunteer ingesting a glass of drinking water with
Cr(VI) repeatedly throughout the day (0.8 mg Cr(VI) daily) for 17 days C-15
Figure C-7. Schematic of the gastric model and parameters for Cr(VI) C-20
Figure C-8. Time profiles of the average daily oral Cr(VI) dose (left) and gastric pH, reducing
capacity (/10), and gastric emptying rate KLSD (right) in the human C-22
Figure C-9. (a) Percent Cr(VI) escaping stomach reduction (and being emptied to the small
intestine) as a function of oral Cr(VI) dose for different values of baseline fasted-
state stomach pH (human), (b) Pyloric flux as a function of oral dose for the
human C-23
Figure C-10. Monte Carlo analysis (20,000 iterations) of the human equivalent dose at selected
values of the internal dose C-24
Figure C-ll. Distribution of the average daily oral Cr(VI) dose in the mouse C-27
Figure C-12. (a) Percent Cr(VI) escaping stomach reduction (and being emptied to the small
intestine) as a function of oral Cr(VI) dose for different values of baseline
stomach pH (mouse); (b) pyloric flux for the mouse using standard assumption
at PHS = 4.5 C-28
Figure C-13. Ad libitum drinking water assumptions applying data from the rat (Spiteri, 1982) C-31
Figure C-14. (a) Percent Cr(VI) escaping stomach reduction (and being emptied to the small
intestine) as a function of oral Cr(VI) dose for different values of baseline
stomach pH (rat); (b) dose escaping stomach reduction for the rat using
standard assumption at PHS = 4.38 C-32
Figure C-15. Forest plot displaying summary measures for esophageal cancer risk from studies
reporting odds ratios C-135
Figure C-16. Forest plot displaying summary measures for esophageal cancer risk from studies
reporting standardized mortality or incidence ratios C-136
Figure C-17. Forest plot displaying summary measures for stomach cancer risk from studies
reporting odds ratios C-137
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Figure C-18. Forest plot displaying summary measures for stomach cancer risk from studies
reporting standardized mortality or incidence ratios C-138
Figure C-19. Forest plot displaying summary measures for colon cancer from studies reporting
standardized mortality or incidence ratios C-139
Figure C-20. Forest plot displaying summary measures for rectal cancer risk from studies
reporting standardized mortality or incidence ratios C-140
Figure C-21. Overview of selected studies evaluating mutagenic markers in the gastrointestinal
tract of mice following ad libitum drinking water exposure C-184
Figure C-22. Overview of the NTP (2007f) genetic toxicology (ad libitum drinking water
exposure). Full circle of a pie chart represents 2 years C-185
Figure C-23. Overview of selected studies evaluating mutagenic markers (but finding no effect)
following ad libitum drinking water exposure (left) and oral gavage (right) C-186
Figure C-24. Overview of the Thompson et al. (2015a) study evaluating mutagenic markers in
rats (but finding no effect) following ad libitum drinking water exposure C-186
Figure C-25. KEGG pathways of gene expression changes in rats exposed to Cr(VI) via ingestion.
Red = activated or increased expression; turquoise = inactivated or decreased
expression; green = no data or no change detected C-230
Figure C-26. KEGG pathways of gene expression changes in human cells exposed to Cr(VI) in
vitro C-231
Figure C-27. KEGG pathways of gene expression changes in cells exposed to Cr(VI) reported by
ToxCast/Tox21 HTS assays C-232
Figure C-28. Design of microarray experiments conducted by Kopec et al. (2012b; 2012a) C-244
Figure C-29. Signal intensity boxplots for 8-day exposure mouse data (duodenum, top dose
excluded) C-248
Figure C-30. Signal intensity boxplots for 8-day exposure mouse data (duodenum, top 4 dose
groups) C-249
Figure C-31. Signal intensity boxplots for 90-day exposure mouse data (duodenum) C-250
Figure C-32. Signal intensity boxplots for 90-day exposure mouse data (duodenum) C-251
Figure C-33. Principal component analysis of 8-day exposure data for mice and duodenal tissues.
C-252
Figure C-34. Principal component analysis of 90-day exposure data for mice and duodenal
tissues C-253
Figure C-35. Hierarchical clustering of microarrays from duodenum, jejunum, and palate tissues
from mice exposed to SDD for 7 days and 90 days C-255
Figure D-l. Overview of the process for deriving candidate, organ-specific, and overall RfDs
(process also applicable to RfCs) D-2
Figure D-2. Alternative process for calculating the human equivalent dose for Cr(VI) D-28
Figure D-3. Dose-response data for tumors and diffuse epithelial hyperplasia of the mouse small
intestine (SI) and tumors of the rat oral cavity D-31
Figure D-4. Model outputs and distribution for rat (M) liver ALT (3 months) (NTP, 2008) D-42
Figure D-5. Model outputs and distribution for rat (F) liver ALT (90 days) (NTP, 2007f) D-43
Figure D-6. Model outputs and distribution for rat (M) liver ALT (90 days) (NTP, 2007f) D-44
Figure D-7. Model outputs and distribution for mouse (M) hyperplasia (NTP, 2008) D-45
Figure D-8. Model outputs and distribution for rat (M) liver ALT (12 months) (NTP, 2008) D-46
Figure D-9. Model outputs and distribution for mouse (F) hyperplasia (NTP, 2008) D-47
Figure D-10. Model outputs and distribution for mouse (F) liver chronic inflammation (2 years)
(NTP, 2008) D-48
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Figure D-ll. Model outputs and distribution for rat (F) liver chronic inflammation (2 years) (NTP,
2008) D-49
Figure D-12. Model outputs and distribution for mouse (F) Decreased F1 postnatal growth (NTP,
1997) D-50
Figure D-13. Model outputs and distribution for rat (M) decreased Hb at 22 days (NTP, 2008) D-51
Figure D-14. Model outputs and distribution for rat (F) decreased Hb at 90 days (NTP, 2007f) D-52
Figure D-15. Model outputs and distribution for rat (M) decreased Hb at 90 days (NTP, 2007f) D-52
Figure D-16. Model outputs and distribution for rat (M) decreased Hb at 12 months (NTP, 2008) D-53
Figure D-17. Model outputs and distribution for rat (F) decreased Hb at 23 days (NTP, 2007f) D-53
Figure D-18. Model outputs and distribution for rat (M) decreased Hb at 23 days (NTP, 2007f) D-54
Figure D-19. Model outputs and distribution for adenomas or carcinomas in the female mouse
small intestine (NTP, 2008) D-55
Figure D-20. Model outputs and distribution for adenomas or carcinomas in the male mouse
small intestine (NTP, 2008) D-56
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Supplemental Information—Hexavalent Chromium
ABBREVIATIONS
ADAF
age-dependent adjustment factors
i.v.
intravenous
ADME
absorption, distribution, metabolism,
IRIS
Integrated Risk Information System
and excretion
LCso
median lethal concentration
AIC
Akaike's information criterion
LD50
median lethal dose
ALT
alanine aminotransferase
LDH
lactate dehydrogenase
ALP
alkaline phosphatase
LOAEL
lowest-observed-adverse-effect level
Asc
ascorbate
MCH
mean cell hemoglobin
AST
aspartate aminotransferase
MCHC
mean cell hemoglobin concentration
ATSDR
Agency for Toxic Substances and Disease
MCV
mean cell (corpuscular) volume
Registry
MEF
maximal expiratory flow
BAL
bronchoalveolar lavage
MMAD
mass median aerodynamic diameter
BALF
bronchoalveolar lavage fluid
MN
micronuclei
BMD
benchmark dose
MOA
mode of action
BMDL
benchmark dose lower confidence limit
MTD
maximum tolerated dose
BMDS
Benchmark Dose Software
NCI
National Cancer Institute
BMI
body mass index
NOAEL
no-observed-adverse-effect level
BMR
benchmark response
NTP
National Toxicology Program
BMDC
bone marrow-derived stem cell
NZW
New Zealand White (rabbit breed)
BW
body weight
ORD
Office of Research and Development
CA
chromosomal aberration
OSHA
Occupational Safety and Health
CASRN
Chemical Abstracts Service Registry Number
Administration
CHO
Chinese hamster ovary (cell line cells)
PBPK
physiologically based pharmacokinetic
CPHEA
Center for Public Health and
PDC
potassium dichromate
Environmental Assessment
PND
postnatal day
CL
confidence limit
POD
point of departure
CNS
central nervous system
POD [AD J]
duration-adjusted POD
Cr(III)
trivalent chromium
POD [HED]
human equivalent dose POD
Cr(IV)
tetravalent chromium
POD [HEC]
human equivalent concentration POD
Cr(Vj
pentavalent chromium
RBC
red blood cell, also known as erythrocyte
Cr(VI)
hexavalent chromium
RD
relative deviation
DAF
dosimetric adjustment factor
RfC
inhalation reference concentration
DLCO
diffusing capacity of carbon monoxide
RfD
oral reference dose
DNA
deoxyribonucleic acid
RDDR
regional deposited dose ratio
ELF
epithelial lining fluid
RNA
ribonucleic acid
EPA
Environmental Protection Agency
SCE
sister chromatid exchange
ER
extra risk
SD
standard deviation
FDA
Food and Drug Administration
SDH
sorbitol dehydrogenase
FEV1.0
forced expiratory volume of 1 second
SE
standard error
FVC
forced vital capacity
SSD
sodium dichromate dihydrate
GD
gestation day
PK
pharmacokinetics
GGT
y-glutamyl transferase
TSCATS
Toxic Substances Control Act Test
GI
gastrointestinal
Submissions
GLP
good laboratory practices
TWA
time-weighted average
GSD
geometric standard deviation
UF
uncertainty factor
GSH
glutathione
UFa
animal-to-human uncertainty factor
GST
glutathione-S-transferase
UFh
human variation uncertainty factor
Hb
hemoglobin
UFl
LOAEL-to-NOAEL uncertainty factor
HEC
human equivalent concentration
UFs
subchronic-to-chronic uncertainly factor
HED
human equivalent dose
UFd
database deficiencies uncertainty factor
HERO
Health and Environmental Research Online
WOS
Web of Science
i.p.
intraperitoneal
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—Hexavalent Chromium
APPENDIX A. SYSTEMATIC REVIEW PROTOCOL FOR
HEXAVALENT CHROMIUM
1 The systematic review protocol for the IRIS Toxicological Assessment of Hexavalent
2 Chromium, developed in 2019 prior to the current draft fU.S. EPA. 2019b). has been updated to
3 reflect refinements to the systematic review procedures implemented in this draft The updated
4 version can be found on the IRIS website:
5 https://cfpub.epa.gov/ncea/iris drafts/recordisplay.cfm?deid=343950.
This document is a draft for review purposes only and does not constitute Agency policy.
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APPENDIX B. SUMMARY OF OTHER AGENCY
CONCLUSIONS
Table B-l. Noncancer inhalation assessments by other national and
international health agencies and associations (in reverse chronological
order)
Reference
Value
(Hg/m3)
Time
adjustment
Chemical note
Endpoints/Basis
ACGIH (2017)
0.2
8-h TWA
Cr(VI) inhalable
particulate
matter
Lung cancer; sinonasal cancer; respiratory
tract irritation; asthma. Based on Lindberg
and Hedenstierna (1983), with support from
other studies (including analyses of
Baltimore cohort data bv Gibb et al. (2000a,
b) and Park et al. (2004).
Texas Commission
on Environmental
Quality (TCEQ)
(2014)
0.0043
Lifetime/chronic
Particulate
compounds
Excess lung cancer mortality risk of 1 x 10~5,
using risk value derived from Gibb et al.
(2000b) and Crump et al. (2003).
0.066
Lifetime/chronic
Particulate
compounds
Respiratory effect (increased relative lung
weight after 90 d of exposure) in rats (Glaser
et al., 1985).
0.39
Acute
Particulate
compounds
Respiratory effect (increased relative lung
weight after 30 d of exposure) in rats (Glaser
et al., 1990).
International
Programme on
Chemical Safety
(IPCS) (2013)
0.03
Lifetime/chronic
Cr(VI) salts
Respiratory effects in rats (Glaser et al.,
1990).
0.005
Lifetime/chronic
Chromium
trioxide,
chromic acid
Upper respiratory effects in humans
(Lindberg and Hedenstierna, 1983).
National Institute
for Occupational
Safety and Health
(NIOSH) (2013)
0.2
8-h TWA, 40-h
work week
All Cr(VI)
compounds
Lung cancer and nonmalignant respiratory
effects. Based on analysis of Baltimore
cohort data bv Park et al. (2004).
Agency for Toxic
Substances and
Disease Registry
(ATSDR) (2012)
0.005
Chronic
Dissolved
aerosols and
mists
Upper respiratory effects (nasal
irritation/ulceration, mucosal atrophy, and
decreases in spirometric parameters), based
on Lindberg and Hedenstierna (1983).
N/A
Chronic
Particulates
Insufficient data
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Supplemental Information—Hexavalent Chromium
Reference
Value
(Hg/m3)
Time
adjustment
Chemical note
Endpoints/Basis
0.005
Intermediate
Dissolved
aerosols and
mists
Upper respiratory effects (nasal
irritation/ulceration, mucosal atrophy, and
decreases in spirometric parameters), based
on Lindberg and Hedenstierna (1983).
0.3
Intermediate
Particulates
Respiratory tract (lung) and other effects.
Based on quantitative analysis of rat studies
(Glaser et al. (1990; 1985)) performed bv
Malsch et al. (1994).
California EPA
(2008)
0.2
Chronic
Soluble
compounds
Respiratory effect (bronchoalveolar
hyperplasia) in rats (Glaser et al., 1990).
0.002
Chronic
Chromic
trioxide (as
chromic acid
mist)
Respiratory effects in humans (Lindberg and
Hedenstierna, 1983).
Occupational Safety
and Health
Administration
(OSHA) (2006b)
5
8-h TWA
All Cr(VI)
compounds
Lung cancer and nasal tissue damage. Based
on quantitative analysis of Baltimore cohort
data bv Gibb et al. f2000a, bl and Luippold
et al. (2003).
Dutch National
Institute for Public
Health and the
Environment
(RIVM) (2001)
0.0025
Chronic
Inhalable dust
Excess lifetime lung cancer risk of 1 x 10"4,
based on analysis of human occupational
studies by the 1987 and 1994 World Health
Organization air quality guidelines.15
U.S. EPA IRIS (1998)
0.008
Lifetime/chronic
Chromic acid
mists/dissolved
chromium
aerosols
Effects in the nasal cavity. Based on
Lindberg and Hedenstierna (1983).
0.1
Lifetime/chronic
Cr(VI)
particulates
Respiratory effects. Based on quantitative
analysis of rat studies (Glaser et al., 1990;
Glaser et al., 1985) performed bv Malsch et
al. (1994).
N/A = not applicable; TWA = time-weighted average.
aSelected values from states known by U.S. EPA to have derived independent values; most states typically adopt
values from U.S. EPA.
bRisk value rationale and studies unchanged in WHO (2000).
Table B-2. Cancer inhalation assessments by other national and international
health agencies (in reverse chronological order)
Reference
Risk factor (ng/m3) 1
Rationale
Texas Commission on
Environmental Quality
(TCEQ) (2014)
Unit risk factor: 2.28 x 10"3
(particulate compounds)
Linearly extrapolated lung cancer risk based on a
weighted average of Gibb et al. (2000b) and Crump
et al. (2003) (human occupational cohorts).
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Supplemental Information—Hexavalent Chromium
International Programme on
Chemical Safetv (IPCS) (2013)
Occupational exposure risk:
6 x 10"3
Linearly extrapolated lung cancer risk based on
Gibb et al. (2000b).
Environmental exposure risk:
4 x 10"2
International Agency for
Research on Cancer (IARC)
(2012).
Carcinogenic to humans
(Group l)b
Lung cancer, based on multiple evidence streams.
Positive associations between Cr(VI) exposure and
cancer of the nose and nasal sinuses in humans
also cited.
National Toxicology Program
(NTP) (2011)
Known to be human
carcinogen15
Cancers of the lung and sinonasal cavity, based on
studies in humans.
World Health Organization
(2000)
4 x 10"2
Linearly extrapolated lung cancer risk based on
multiple human occupational studies.
U.S. EPA IRIS (1998)
Inhalation unit risk: 1.2 x 10"2
Linearly extrapolated lung cancer risk based on
Mancuso (1997,1975) (human occupational
cohort).
California Department of
Health Services (CDHS)
(1985)
Inhalation potency: 0.15°
Linearly extrapolated lung cancer risk based on
Mancuso (1975).
aSelected values from states known by U.S. EPA to have derived independent values; most states typically adopt
values from U.S. EPA.
bAgency does not derive a quantitative risk factor.
cAs part of an updated evaluation of the science for the public health goal (PHG), California EPA (2011) calculated a
slope of 0.16 (iJg/m3)"1 (with a 95% upper confidence of 0.35) using Gibb et al. (2000b), and a lower bound slope
of 0.01 (ng/m3)"1 using Luippold et al. (2003).
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—Hexavalent Chromium
Table B-3. Oral assessments by other national and international health
agencies (in reverse chronological order)
Reference
Risk value or limit
Rationale13
Food Safety Commission of
Japan (2019)
Tolerable daily intake: 1.1 x 10"3 mg/kg-d
Cancer precursor, mouse small
intestine hyperplasia
Health Canada (2016)
Maximum acceptable concentration:
50 ng/L
Cancer precursor, mouse small
intestine hyperplasia
Texas Commission on
Environmental Quality
(TCEQ) (2016)
RfD: 3.1 x 10"3 mg/kg-d
Cancer precursor, mouse small
intestine hyperplasia
International Programme
on Chemical Safety (IPCS)
(2013)
Tolerable daily intake: 9 x 10"4 mg/kg-d
Mouse small intestine noncancer
effects
Agency for Toxic Substances
and Disease Registry
(ATSDR) (2012)
Chronic MRL: 9 x 10"4 mg/kg-d
Mouse small intestine noncancer
effects
Intermediate MRL: 5 x 10"3 mg/kg-d
Hematological effects (rat data at 22 d)
California EPA (2011)
Cancer PHG: 0.02 ng/L
1 x 10"6 cancer risk using OSF of
0.5 (mg/kg-d)"1 (mouse small intestine
tumors)
Noncancer PHG: 2 ng/L
Liver noncancer effects (rats)
California Department of
Public Health (2014; 2013)
Proposed MCL: 10 ng/L
fsee California State Water Board (2022)
fact sheet]
Cancer risk fsee California EPA (2011)1
NewJersev DEP (2009)
Soil remediation criterion: 1 ppm soil
concentration
1 x 10"6 cancer risk using OSF of 0.5
(mg/kg-d)"1 (mouse small intestine
tumors)
U.S. EPA/OPP (2008a, b)
OSF: 0.791 (mg/kg-d)"1
Upper-bound cancer risk estimate
(mouse small intestine tumors;
mutagenic MOA determined)
Assessments based on science or rules published prior to 2008 National Toxicology Program study
U.S. Food and Drug
Administration (2013)
Allowable level in bottled water: 0.1 mg/L
(or 100 ng/L) total chromium
Not specified
U.S. Environmental
Protection Agency [Federal
Reaister (2010)1
MCL: 100 ng/L (total chromium)
Allergic dermatitis0
World Health Organization
(2003)
50 ng/L
Provisional value (nonspecific)
Dutch National Institute for
Public Health and the
Environment (RIVM) (2001)
5 x 10"3 mg/kg-d
Provisional noncancer effects, based
on no-effect level frats; MacKenzie et
al. (1958)1
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Reference
Risk value or limit
Rationale13
U.S. EPA/IRIS (1998)
RfD: 3 x 10"3 mg/kg-d
No effect level for noncancer effects
(rats; (MacKenzie et al., 1958)1
MCL = maximum contaminant level; MRL = minimal risk level; OSF = oral slope factor; PHG = public health goal.
aSelected values from states known by U.S. EPA to have derived independent values; most states typically adopt
values from U.S. EPA (based on unspeciated total chromium).
bAII values based on mouse data from NTP (2008), unless otherwise noted.
cBased on rule promulgated in 1991 (National Primary and Secondary Drinking Water Regulations, 56 FR 3526,
1-30-91 and 54 FR 22062, 5-22-89).
In addition to the (mostly) quantitative assessments above, a qualitative assessment was
performed by the Dutch National Institute for Public Health and the Environment (RIVM) on
irreversible human health hazards from occupational inhalation exposure to Cr(VI) compounds
(Den Braver-Sewradi etal.. 2021: Hessel etal.. 2021: Palmen etal.. 20181. Categorization was
restricted to irreversible adverse health effects (likely, possible, insufficient evidence, and unlikely),
and focused primarily on inhalation risk (but recognizing that low incidental oral exposure may
occur in occupational settings). Health effects that were determined likely in humans were lung
cancer, nose and nasal sinus cancer, nasal effects (irritation, ulcerations and perforation of the
septum), chronic lung diseases, respiratory allergy, and allergic contact dermatitis. Stomach cancer
was categorized as a possible human health effect from inhalation. Health effects where there was
insufficient evidence in humans were immune effects (besides the dermal/respiratory allergies) and
reproductive effects (development, fertility, and lactation). Health effects that were determined to
be unlikely to occur in humans were larynx cancer, intestinal cancer, gastrointestinal effects,
hematological effects, hepatic effects, renal effects, neurological, cardiovascular effects, and dental
effects.
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—Hexavalent Chromium
APPENDIX C. INFORMATION IN SUPPORT OF
HAZARD IDENTIFICATION AND DOSE-RESPONSE
ANALYSIS
C.l. PHARMACOKINETICS
C.l.l. Absorption
Water soluble Cr(VI) compounds are rapidly absorbed into cells and tissues in the body via
phosphate and sulfate anion transport due to the structural similarity of the tetrahedral
configuration of the chromate (C1-O42") or dichromate (C1-2O72") anion to that of phosphate (HPO42")
and sulfate (SO42") anions fAlexander and Aaseth. 1995: Wetterhahn etal.. 19891. while Cr(III)
compounds are absorbed slowly by passive diffusion (Eastmond et al.. 20081. In the
gastrointestinal (GI) tract following oral ingestion, systemic uptake of Cr(VI) competes with the
rapid extracellular reduction to Cr(III) by gastric juices (Proctor etal.. 2012: De Flora et al.. 19971.
Studies listed in Appendix C.l.6 that administered Cr(VI) and Cr(III) to different treatment groups
have observed higher urinary blood, and tissue chromium in the groups exposed to Cr(VI). This
was also observed by separate NTP bioassays of Cr(VI) and Cr(III), which found the body burdens
of rats and mice exposed to Cr(VI) in drinking water were significantly higher than those exposed
to comparable levels of Cr(III) in feed (Collins etal.. 20101. Figure C-l illustrates the difference in
chromium concentrations of selected systemic tissues between the Cr(VI) and Cr(III) studies.
Despite the estimated daily dose of Cr(III) being threefold higher than that of Cr(VI), chromium
tissue concentrations were over tenfold higher for the Cr(VI) group. Because Cr(VI) is more readily
absorbed into the GI tract than Cr(III), this is also evidence that systemic absorption of Cr(VI) can
occur in rodents following chronic oral exposure, despite reduction of Cr(VI) to Cr(III) by gastric
juice fCollins etal.. 20101.
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—Hexavalent Chromium
60
50
ta
aa
©
« 40
E
c
o
ra 30
20
10
RBC
Kidney
l Cr{Vi) study * Cr(il!) study ¦ Cr(Vi) study HCr(lll) study ~ Cr(Vi) study HCr(ilE) study
Figure C-l. Comparison of mean tissue concentrations in mice (n = 3)
following 182 days of either Cr(VI) or Cr(III) oral exposure. Groups compared
are the 516 mg/L SDD group and the 2000 mg/L sodium picolinate group. These
correspond to approximately 10 mg/kg-day Cr(VI), and 30 mg/kg-day Cr(III)
respectively (average over study period weeks 14-51). These are a subset of data
from the NTP studies Collins et al. (2010). Data were collected after a 2-day wash-
out period, and therefore concentrations are lower than what would have been
measured during ongoing exposure.
Although fewer Cr(VI) pharmacokinetic studies are available for the inhalation route than
for the oral route (see Appendix C.1.6), there is evidence that indicates inhaled Cr(VI) is absorbed
systemically. The study in rats by Cohen etal. f 19971 of inhaled soluble (potassium chromate) and
insoluble (barium chromate) Cr(VI) observed absorption of both forms of Cr(VI). Elevated
chromium in this study was observed in lung components and systemic tissues (kidney, liver,
spleen), with higher levels in groups exposed to the soluble form of Cr(VI). Occupational studies in
humans who may have been exposed primarily via inhalation have measured elevated chromium in
multiple biomarkers such as red blood cells and urine (Appendix C.1.6). 0'Flaherty and Radike
Q9911 exposed rats to Cr(VI) or Cr(III) at concentrations of 200 |ig/m3 via aerosol inhalation (6
hours/day) and detected elevated chromium in all measured tissues and excreta relative to
controls (Table C-6).
C.1.2. Distribution
Upon systemic absorption, Cr(VI) circulates in plasma, where it is absorbed into red blood
cells (RBCs), white blood cells, and other systemic tissues. Both the uptake and reduction of Cr(VI)
by RBCs has been estimated to be rapid fDevov etal.. 20161. Uptake to RBCs is facilitated by
This document is a draft for review purposes only and does not constitute Agency policy.
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9
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12
13
14
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21
22
23
24
25
26
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31
32
33
34
35
Supplemental Information—Hexavalent Chromium
nonspecific anion transport channels, including the band-3 anion exchanger protein, an anion
carrier system of the red blood cell membrane fButtner etal.. 1988: Ottenwaelder etal.. 1988:
Ottenwalder etal.. 1987: Buttner and Beversmann. 19851. In humans, genetic polymorphisms in
the band-3 protein have been shown to be associated with increased accumulation of Cr(VI) in red
blood cells (Ou etal.. 20081.
Because irreversible binding to hemoglobin occurs, and Cr(III) exhibits a lower rate of
transport through cellular membranes than Cr(VI), Cr(III) remains trapped in RBCs over the
remaining life of the cells. Supporting evidence is provided by the studies presented in
Appendix C.1.6. This property has been exploited for diagnostic purposes whereby hexavalent
radiolabeled chromium-51 has been used to label and determine the survival time of RBCs in
humans (Gray and Sterling. 1950). Measured in vivo chromium concentration in plasma has been
observed to rapidly decrease to background levels after exposure to Cr(VI) has ceased, while in vivo
chromium concentration in RBCs decreases more gradually (as chromium-containing RBCs are
replaced over time).
Because chromium in the system varies with uptake of Cr(III) [both from diet and from
Cr(VI) reduction in the lumen], chromium concentration in RBCs may be normalized by
concentration in plasma to evaluate systemic distribution. Although it is noted in Kirman et al.
(2012) that the RBC:plasma ratios are generally equal to or less than 1 for low concentrations (and
exceed 1 at 60-180 mg/L), evaluating the data for ratios greater than 1 to assess absorption and
distribution may not be informative. For example, the RBC:plasma ratios are greater than 1 for
some of the control groups for rats and mice analyzed in the NTP (2008) Cr(VI) study (Tables C-2
and C-4). Instead, comparisons against control or Cr(III)-exposed groups are more appropriate.
Despite the complications from the 48-hour washout period,1 a comparison of the NTP f20081
RBC:plasma ratio data for dosed animals against control groups and comparison with groups from
the NTP (2007f) Cr(III) study can indicate systemic uptake of Cr(VI). A similar analysis using
concentration data for plasma and RBCs in the Kirman etal. (2012) study could not be performed
because concentrations are below the method detection limits for the control groups and low
concentration groups. For that dataset, RBC:plasma ratios are not informative until Cr(VI) drinking
water concentrations >20 mg/L in both species, and they cannot be compared to controls.
The RBC:plasma ratio analysis of NTP f20081 data are provided in Figure C-2 and Tables C-l
through C-4. Analysis of the NTP (2007f) Cr(III) data are not presented, but those data indicate
RBC:plasma ratios <1 for all Cr(III) dietary exposure groups, with no dose-dependent increase. For
rats exposed to Cr(VI) in drinking water, the RBC:plasma ratio increases by approximately 90-
225% above controls at 20 mg/L Cr(VI) drinking water concentration. For mice, the ratio increases
by approximately 40-100% above controls at 20 mg/L Cr(VI). Because this increase in relative
1 After two days without Cr(VI) exposure, chromium concentration in the plasma will decrease more rapidly
than concentration in RBCs. At the same time, chromium will enter plasma from the tissues, which may
counteract some of the washout.
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—Hexavalent Chromium
1 RBC concentration was not observed in rodents exposed to Cr(III), Cr(VI) concentrations at or
2 above 20 mg/L Cr(VI) in drinking water (equivalent to approximately2 0.88 mg/kg-day in rats and
3 1.5 mg/kg-day in mice) likely result in systemic Cr(VI) absorption beyond the liver (where
4 extensive reduction is expected to occur during the first-pass effect). More extensive systemic
5 distribution likely occurs as dose increases, as more Cr(VI) could escape reduction in the stomach,
6 small intestine, and liver.
7.0
6.0
5.0
4.0
F 3.0 --
ro
cu
E
1/1
_ro
Q.
Ci
CD
C£L
E
2
E
o
u
cu
DO
TO
i_
QJ
>
<
2.0 -
1.0
0.0
20 40 60 80 100 120 140 160
Cr(VI) drinking water concentration (ppm)
180 200
Figure C-2. Ratio of RBC:plasma concentration as a function of Cr(VI) drinking
water concentration (1 ppm = 1 mg/L) for male F334 rats and female B6C3F1
mice using data from NTP (2008).
2These are time-weighted average daily doses estimated from NTP f20081 drinking water consumption data
during the first 53 weeks of exposure.
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—Hexavalent Chromium
Table C-l. Concentrations of chromium in erythrocytes and plasma (ng Cr/g)
following ingestion of sodium dichromate dihydrate in drinking water (male
F334 rats)
Cr(VI) concentrations
0 mg/L
5 mg/L
20 mg/L
60 mg/L
180 mg/L
Erythrocytes
Day
Cr/g
Cr/g
tig Cr/g
tig Cr/g
tig Cr/g
6
0.044
0.051
0.126
0.252
0.391
13
0.051
0.036
0.203
0.504
0.899
182
0.05
0.054
0.208
0.591
0.997
371
0.055
0.064
0.16
0.526
0.693
Plasma
6
0.052
0.068
0.079
0.087
0.109
13
0.054
0.048
0.079
0.103
0.146
182
0.063
0.064
0.081
0.099
0.146
371
0.054
0.062
0.071
0.11
0.146
Data from NTP (2008). Time-weighted average daily doses for each exposure group are not listed, since they vary
with time over the lifespan of the rodent (and will be different at days 6,13,182, and 371).
Table C-2. Ratio of erythrocytes:plasma concentrations following ingestion of
sodium dichromate dihydrate in drinking water (male F334 rats)
Cr(VI)
0 mg/L
5 mg/L
20 mg/L
60 mg/L
180 mg/L
Day
Ratio
Ratio
Ratio
Ratio
Ratio
6
0.846
0.750
-11.4
1.59
88.5
2.90
242
3.59
324
13
0.944
0.750
-20.6
2.57
172
4.89
418
6.16
552
182
0.794
0.844
6.31
2.57
224
5.97
652
6.83
760
371
1.02
1.03
1.35
2.25
121
4.78
369
4.75
366
TWA:
0.888
0.867
-2.36
2.46
177
5.29
495
6.06
582
TWA = time-weighted average values.
For the chromium picolinate studies (NTP, 2007f), the RBC/plasma ratio did not increase as a function of dose for
rats (data not shown).
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—Hexavalent Chromium
Table C-3. Concentrations of chromium in erythrocytes and plasma (ng Cr/g)
following ingestion of sodium dichromate dihydrate in drinking water (female
B6C3F1 mice)
Cr(VI) concentrations
0 mg/L
5 mg/L
20 mg/L
60 mg/L
180 mg/L
Erythrocytes
Day
Cr/g
Cr/g
tig Cr/g
tig Cr/g
tig Cr/g
6
0.04
0.056
0.108
0.26
0.374
13
0.043
0.042
0.341
0.747
1.19
182
0.058
0.079
0.194
0.719
1.561
371
0.036
0.042
0.094
0.34
0.795
Plasma
6
0.064
0.075
0.111
0.15
0.213
13
0.034
0.038
0.133
0.204
0.311
182
0.051
0.07
0.116
0.167
0.253
371
0.065
0.086
0.118
0.15
0.209
Data from NTP (2008). Time-weighted average daily doses for each exposure group are not listed, since they vary
with time over the lifespan of the rodent (and will be different at days 6,13,182, and 371).
Table C-4. Ratio of erythrocytes:plasma concentrations following ingestion of
sodium dichromate dihydrate in drinking water (female B6C3F1 mice)
Cr(VI)
0 mg/L
5 mg/L
20 mg/L
60 mg/L
180 mg/L
Day
Ratio
Ratio
Ratio
Ratio
Ratio
6
0.625
0.747
19.5
0.973
55.7
1.73
177
1.76
181
13
1.26
1.11
-12.6
2.56
103
3.66
190
3.83
203
182
1.14
1.13
-0.764
1.67
47.1
4.31
279
6.17
443
371
0.554
0.488
-11.8
0.797
43.8
2.27
309
3.80
587
TWA:
1.01
0.950
-5.53
1.64
63.3
3.57
255
4.90
387
TWA = time-weighted average values.
For the chromium picolinate studies (NTP, 2007f), the RBC/plasma ratio did not increase as a function of dose for
mice (data not shown).
1 Twenty-one-day data from NTP f2007f) in rats, mice, and guinea pigs at 1, 3,10, 30,100,
2 and 300 mg/L Cr(VI) in drinking water showed increased chromium tissue concentrations
3 (including in the rat femur) beginning at 10-30 mg/L. Although dose (mg/kg-day) data are not
4 provided, evaluation of other dose data from National Toxicology Program studies for rats and mice
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1 at 21 days indicates that the dose for rats and mice at 10 mg/L Cr(VI) would be greater than 1
2 mg/kg-day (young growing mice will intake more water on a mg/kg basis).
3 Studies in rats and mice orally dosed with Cr(VI) have measured total chromium in
4 essentially all tissues, with highest concentrations in kidney, liver, spleen, and bone (Table C-5).
5 Additionally, total chromium concentrations in the small intestine following oral exposure have
6 been measured to be highest in the duodenum (the proximal small intestine) and lowest in the
7 ileum (the distal small intestine) (Figure C-3). This could be an indication that as Cr(VI) in drinking
8 water traverses the small intestine, it is reduced to Cr(III) in the lumen over time.
Table C-5. Chromium in tissues (|ig/g wet tissue or (ig/mL blood) of mice and
rats after ingesting K2Cr07 in drinking water (8 mg Cr(VI)/kg-day) for 4 or
8 weeks
Tissue
Controls
4-week exposure
8-week exposure
Mice
Liver
0.22 ±0.14
10.92 ± 5.48
13.83 ± 6.06
Kidney
0.24 ±0.14
3.77 ±0.99
4.72 ±0.68
Spleen
0.53 ±0.38
5.04 ± 1.45
10.09 ± 2.50
Femur
0.90 ± 0.48
7.43 ± 1.03
12.55 ±2.99
Lung
0.24 ±0.12
0.99 ±0.10
1.08 ±0.26
Heart
0.32 ±0.15
0.80 ±0.23
1.02 ± 0.20
Muscle
0.32 ±0.23
1.12 ±0.37
0.60 ±0.25
Blood
0.14 ±0.05
0.71 ±0.07
0.42 ± 0.04
Rats
Liver
0.19 ±0.14
3.32 ±0.93
3.59 ±0.73
Kidney
0.34 ±0.20
8.62 ± 2.40
9.49 ±4.38
Spleen
0.43 ± 0.20
3.65 ± 1.87
4.38 ±0.84
Femur
1.00 ± 0.46
1.85 ± 0.46
1.78 ±0.99
Lung
0.39 ±0.43
1.10 ±0.38
0.67 ±0.24
Heart
0.38 ±0.22
0.52 ±0.12
1.05 ±0.19
Muscle
0.24 ±0.14
0.19 ±0.10
0.17 ±0.10
Blood
0.19 ±0.17
0.73 ±0.15
0.58 ±0.13
Source: Kargacin et al. (1993).
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Figure C-3. Mean concentration of total chromium in GI tract tissues of mice
and rats following exposure to 180 mg/L Cr(VI) in drinking water for 90 days
[approximately 31.9 and 20.5 mg/kg-day Cr(VI) for mice and rats,
respectively]. Data from Kirman et al. (2012).
0'Flaherty and Radike (19911 exposed rats to Cr(VI) or Cr(III) at concentrations of 200
[ig/m3 via aerosol inhalation (6 hours/day) or 12.9 mg/L via drinking water ingestion (ad libitum)
for 40 days (with an additional 20-day recovery period of no exposure). These concentrations are
within the ranges used by some Cr(VI) toxicological studies fNTP f20081 range: 5-180 mg/L Cr(VI)
via drinking water; Glaser etal. (1985) range: 25-200 |ig/m3 via inhalation). Measured chromium
concentrations in the blood and lungs were higher in rats exposed to Cr(VI) via inhalation, while
chromium concentrations in the liver and intestine were higher in rats exposed to Cr(VI) via
drinking water. As a result, the severities of toxicological effects induced by Cr(VI) at both portal-
of-entry tissues and systemic tissues may differ by exposure route.
For tissues outside the portals of entry and for urine, Cr(VI)-exposed groups exhibited
higher chromium levels than Cr(III)-exposed groups (which is consistent with higher systemic
absorption of Cr(VI)). For tissues at or near the portals-of-entry (lung for inhalation, intestine for
oral ingestion), chromium concentrations were comparable or higher for Cr(III) groups when
compared to Cr(VI) groups. This could indicate higher localized clearance of Cr(VI) from portal
tissues into blood via absorption. Chromium excretion in feces following oral ingestion of either
Cr(VI) or Cr(III) was comparable (fecal chromium can be due to both elimination of systemic
chromium and the passing of unabsorbed chromium). All exposure groups (either Cr(VI) or Cr(III))
exhibited higher chromium concentrations than control groups (see Tables C-6 and C-7).
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Table C-6. Summary of oral and inhalation data from O'Flahertv and Radike
T19911
Study day
Lung
HgCr/g
Liver
Hg Cr/g
Intestine
Hg Cr/g
Kidney
Hg Cr/g
Muscle
Hg Cr/g
Blood
ng Cr/g
Urine
Hg Cr/d
Feces
mg Cr/d
Inhalation Cr(VI) (200 |ig/m3 6 h/d)
2
1.95
nd
1.10
nd
nd
42.5
0.520
nd
5
5.10
0.060
1.12
0.217
nd
58.4
0.207
nd
10
7.53
0.062
1.37
0.237
nd
73.8
0.266
0.018
20
13.3
0.066
2.36
0.310
0.047
72.8
0.135
0.048
40
24.3
0.089
3.24
0.580
0.054
75.7
0.047
0.082
60
13.0
0.038
0.820
0.137
0.027
39.8
0.012
nd
Ingestion Cr
VI) (12.9 mg/Lad libitum
2
nd
0.209
15.5
0.249
nd
9.00
0.622
0.997
5
nd
0.372
22.7
0.588
nd
11.8
1.79
0.835
10
nd
0.585
14.4
1.60
nd
18.5
2.01
0.949
20
1.17
1.18
29.0
1.71
0.077
48.9
3.08
0.977
40
0.650
1.50
6.80
1.90
0.103
58.3
2.19
1.51
60
0.450
0.509
0.830
0.634
0.070
11.3
0.217
nd
Inhalation Cr(lll) (200 |ig/mB 6 h/d)
2
3.43
nd
3.57
nd
nd
61.5
0.215
0.028
5
8.43
nd
4.19
nd
nd
64.8
0.101
0.035
10
17.1
nd
25.6
nd
nd
23.4
0.084
0.016
20
35.4
nd
39.4
nd
nd
12.0
0.032
0.032
40
63.7
nd
4.80
nd
nd
105.7
0.002
0.074
60
42.9
nd
0.840
nd
nd
89.0
0.001
nd
Ingestion Cr(lll) (12.9 mg/Lad libitum
2
nd
0.042
18.3
nd
nd
2.48
0.227
0.821
5
nd
trace
17.2
nd
nd
3.11
0.065
0.729
10
nd
0.034
20.6
nd
nd
16.8
0.040
1.20
20
nd
nd
26.8
nd
nd
5.60
0.075
1.07
40
nd
nd
7.15
nd
nd
4.72
0.017
1.12
60
nd
trace
0.830
nd
nd
5.52
nd
nd
Mean values (N = 6); nd = nondetect.
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Table C-7. Summary of oral and inhalation control group data from O'Flahertv
and Radike (1991)
Study day
Lung
HgCr/g
Liver
Hg Cr/g
Intestine
Hg Cr/g
Kidney
Hg Cr/g
Muscle
Hg Cr/g
Blood
ng Cr/g
Urine
Hg Cr/d
Feces
mg Cr/d
Inhalation control group
2
nd
0.036
1.13
nd
nd
nd
0.042
nd
5
nd
0.041
0.64
nd
nd
nd
0.001
nd
10
nd
nd
0.83
nd
nd
nd
nd
nd
20
nd
nd
1.08
nd
nd
nd
nd
0.02
40
nd
0.041
1.08
nd
nd
nd
nd
nd
60
nd
0.032
0.84
nd
nd
nd
nd
nd
Ingestion control group
2
nd
nd
0.65
1.58
trace
1.5
0.017
nd
5
nd
nd
0.83
nd
trace
1.6
nd
0.002
10
nd
nd
0.56
nd
nd
4.2
0.003
nd
20
nd
nd
0.85
nd
trace
3.4
nd
0.013
40
nd
0.035
0.68
nd
trace
6.8
0.01
nd
60
nd
0.032
0.72
nd
0.038
2.5
nd
nd
Mean values (N = 6); nd = nondetect.
C.1.3. Metabolism
Cr(VI) reduces to Cr(III) in the GI tract and in RBCs. Reduction takes place in the GI tract
tissue and liver following oral exposure (due to the first-pass effect) and in pulmonary tissues
following inhalation exposure. Extracellular reduction in gastric juice and in pulmonary fluids is
also possible. Extracellular reduction in the lung is likely to be less effective than reduction in the
GI tract, due to higher pH and lower reducing capacity. In blood, plasma reduces Cr(VI) poorly
relative to RBCs (Corbettetal.. 19981. Intracellular reduction of Cr(VI) (which occurs after Cr(VI)
enters the cells of a susceptible tissue) is a potential pathway for metabolic activation. Reactive
intermediaries and reactive oxygen species (ROS) are generated as Cr(VI) is intracellularly reduced
to Cr(III).
Extracellular reduction in the stomach is expected to impact the systemic uptake of
unreduced Cr(VI) and the exposure of the digestive tract epithelium. Stomach reduction may be a
major source for interspecies and interindividual differences due to the strong dependence on
gastrophysiology and pH. Figure C-4 illustrates the rate of reduction in human gastric juice under
different pH conditions. At higher values of pH, Cr(VI) reduction occurs slowly.
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Figure C-4. Reduction of Cr(VI) in samples of human gastric juice (fasted
subjects) using data from Proctor et al. (2012). Lines indicate model results by
Schlosser and Sasso (2014). (Left) 2:1 dilution of stomach contents, multiple
initial Cr(VI) concentrations. (Right) 10:1 dilution of stomach contents, initial Cr(VI)
concentration approximately 0.1 mg/L.
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The basal values of pH for humans and rodents in the fasted state are approximately 1.3 and
4, respectively (Table C-8). Under these conditions, humans would reduce Cr(VI) more effectively
than rodents. This pattern, however, is reversed during the fed state. Human gastric juice pH rises
to a peak of about 6, and then decreases to baseline within 2 hours (Mudie etal.. 20101. Rodent
gastric juice pH decreases during the fed state, but the dynamics are not well characterized.
Table C-8. The pH of the mouse, rat, and human gastrointestinal tract
Section
Female Balb/c mice
Female Wistar rats
Human3
Fed (n = 8)
Fasted
[n = 7)
Fed(n = 5)
Fasted (n = 5)
Fed
Fasted
Mean
SD
Mean
SD
Mean
SD
Mean
SD
Stomach
2.98
0.3
4.04
0.2
3.20
1.0
3.90
1.0
4.9
1.3
Duodenum
4.87
0.3
4.74
0.3
5.00
0.3
5.89
0.3
5.4
6.0
Jejunum
4.82
0.2
5.01
0.3
5.10
0.3
6.13
0.3
5.4-6.0
6.2-6.4
Ileum
4.81
0.3
5.24
0.2
5.94
0.4
5.93
0.4
6.6-7.4
Caecum
4.44
0.2
4.63
0.4
5.90
0.4
6.58
0.4
6.4
Proximal colon
4.69
0.3
5.02
0.3
5.51
0.5
6.23
0.4
6.8
Distal colon
4.44
0.3
4.72
0.2
5.77
0.5
5.88
0.5
Adapted from Mcconnell et al. (2008) and Parrott et al. (2009).
Fed-state pH values for humans represent time-weighted average values during the fed state, and not
peak/maximum values occurring during a meal.
Fed-state pH values for rodents were obtained from animals that had not undergone an overnight fast, thus pH
does not represent minimum values occurring during a meal,
standard deviations not available; summary data reviewed in Parrott et al. (2009).
Fed-state reduction kinetics have greater uncertainties, as the gastric juice will be
heterogeneous and the pH fluctuation temporary. Secretion of additional gastric juices and
enzymes responsible for meal digestion occurs, and various ingested food components may have
different effects on reduction rate. Therefore, diet could result in high interindividual variability of
fed-state reduction kinetics in the human population. This variability is apparent in ex vivo data by
Kirman etal. (20161 (see U.S. EPA (2021bll. In general, gastric juice in the fed state is believed to
have a greater capacity3 for Cr(VI) reduction (because dietary contents such as ascorbate and
secreted gastric juices may act as reducing agents). Table C-9 contains a summary of estimated
Cr(VI) reducing capacities for various tissues and fluids in mice, rats, and humans. As previously
noted in the absorption section, the extent of Cr(VI) reduction by components of the respiratory
system is complicated by airway geometries and localized particle deposition.
3Reduction capacity is the total amount of Cr(VI] that can be reduced (as t->co) and is a function of how much
reducing agent (components capable of reducing Cr(VI]] is contained in gastric juice. This differs from the
reduction rate (how fast Cr(VI] can reduce per unit of time], which is a function of stomach pH.
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Table C-9. Selected studies of Cr(VI) reduction capacities
Reference
Media (species)
Findings
Estimates of bodily fluid reduction capacity (ex vivo)a
Proctor et al. (2012)
Stomach contents (rat)
Study estimate: 15.7 ng/mL
Stomach contents (mouse)
Study estimate: 16.6 ng/mL
Kirman et al. (2013)
Gastric fluid (human)
Study estimate: 20 ng/mL [based on a mean of
7 ng/mL (fasted) from this study and a median
of 30 ue/mL (fed) from De Flora et al. (1987a)l
Schlosser and Sasso
(2014)
Gastric fluid reanalysis (rat, mouse,
human)
Reanalvsis of data bv Proctor et al. (2012) and
Kirman et al. (2013). Rat: 4/18 ue/mL
(fast/slow pool). Mouse: (3/31 ng/mL
fast/slow pool). Human: 10 ng/mL (fasted-
state kinetics).
De Flora et al. (2016)
Gastric fluid (human)
Colorimetric method: 10.2 ± 2.39 ng/mL
(premeal) and 20.4 ± 2.61 ng/mL (post-meal)
Mutagenicity assay: 13.3 ± 1.91 ng/mL
(premeal) and 25.6 ± 2.89 ng/mL (post-meal)
Kirman et al. (2016)
Gastric fluid (human)
Fasted state: 2.6 ± 2.8 and 12 ± 18 ng/mL for
fast and slow pools, respectively. Fed state:
0.68 ± 0.76 and 27 ± 28 ng/mLfor fast and slow
pools.
Gastric fluid reanalysis (rat, mouse,
human)
Mouse: 6.1/27 ng/mL (fast/slow pool).
Rat: 7.1/73 ng/mL (fast/slow pool).
De Flora et al. (1987a)
Gastric fluid (human)
8.3 ± 4.3 ng/mL (fasting), 31.4 ± 6.7 ng/mL
(fed)
Petrilli and De Flora
(1982)
Saliva (human)
1.4 ± 0.2 Hg/mL
Petrilli et al. (1986)
Epithelial lining fluid (human)
23.7 ± 15.9 |jg/mL
Estimates of cellular or organ reduction capacity3
De Flora et al. (1997)
Intestinal bacteria (human fecal)
3.8 ± 1.7 ng/109 bacteria (elimination via feces)
Liver (human)
2.2 ± 0.9 ng/g liver homogenate
Blood (human)
52.1 ± 5.9 ng/mL intact whole blood
Red blood cells (human)
63.4 ± 8.1 ng/mL RBC lysate soluble fraction
Petrilli et al. (1986)
Pulmonary alveolar macrophages
(human)
4.4 ± 3.9 ng/106 PAM S9 fraction
De Flora et al. (1987a)
Peripheral lung parenchyma (human)
200 ± 70 ng/g lung S12 fraction
Capellmann and Bolt
(1992)
Plasma (human)
0.48-0.63 nmol/mL [at intubation of 1.5
nmol/mL Cr(VI)]
Upreti et al. (2005)
Intestinal epithelial cells and gut
bacteria (rat)
Most Cr(VI) at 10 mg/L completely reduced by
bacteria in 6 h. Complete reduction by some
cells can take 24 h.
deduction capacities represent the mass of Cr(VI) that can be reduced by a tissue or fluid, per unit mass or volume
of the media.
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C.1.4. Excretion
Following oral ingestion, Cr(VI) and its metabolite Cr(III) are primarily eliminated via
urinary excretion (Figures C-5 and C-6). Due to poor GI tract absorption of Cr(III), a significant
amount of reduced chromium is eliminated in feces without being absorbed. Urinary excretion is
also a primary pathway for elimination following inhalation exposure. Intratracheal studies in
rodents have observed elevated urinary chromium, and biomonitoring studies in humans in
occupations where inhalation exposure could occur have also detected elevated chromium (see
Appendix C.1.6). Following chronic, low-dose oral exposure to Cr(VI), most systemic chromium is
likely in the trivalent form. Site-specific clearance of Cr(VI) by reduction to Cr(III) in tissues such as
the GI tract, liver, and blood is likely to be greater than systemic clearance of Cr(VI) in urine at low
doses. Variability in urinary clearance rates of Cr(VI) between individuals and across species likely
does not have a significant impact on toxicity under chronic low-dose exposure scenarios (since
most, if not all, systemic chromium will have been reduced to Cr(III)).
Intravenous studies have indicated a significant percentage of chromium could be excreted
via biliary excretion and fecal elimination; however, these elimination pathways are minor
following oral ingestion (due to reduction in the stomach and liver; see Appendix C.1.6).
Intravenous injection of Cr(VI) leads to high systemic concentrations that are not observed
following oral exposure, and thus some distribution or metabolic mechanisms (i.e., RBC uptake and
reduction) may become saturated.
0.2
0.15
ro
"D
i35
E
t 01
ro
0.05
a Subject HI (2.5 mg)
x Subject H4 (5 mg)
~ Subject HS (5 mg)
o Subject H1Q (5 mg)
8 10 12 14 16 18
Time (days)
Figure C-5. Urinary rates of excretion by human volunteers administered a
glass of drinking water containing 2.5-5.0 mg Cr(VI) at day 2. Data from
Kerger et al. (1996).
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0,08
0,07
0.06
™ 0,05
T3
O
0.04
0,03
0,02
0,01
• Urinary data
¦ losing starts
~ ,'V dosing stops
~
10
15
Time (days)
20
25
Figure C-6. Urinary excretion rate of a human volunteer ingesting a glass of
drinking water with Cr(VI) repeatedly throughout the day (0.8 mg Cr(VI)
daily) for 17 days. Data from Paustenbach etal. (1996).
C.1.5. Physiologically Based Pharmacokinetic Models
A description of the available physiologically based pharmacokinetic (PBPK) models for
Cr(VI) is available in Section 3.1.2 of the toxicological review. The PBPK model code used in this
assessment (in R/MCsim) is available for download in HERO (U.S. EPA. 2022b).
Significant uncertainties exist that may be difficult to fully characterize using PBPK models.
The stomach of rodents and humans will dynamically fluctuate between the fed and fasted states.
This affects reaction dynamics in multiple ways. As noted in Table C-8, glandular stomach pH is
decreased for the rodent during the fed state, while the opposite is true for humans. In addition to
pH effects, gastric emptying is delayed in the fed state to digest food, and the volume of contents in
the lumen will be increased. Gastric juice induced by food consumption may also have different
reducing capacities (and ingested food itself could impact reduction kinetics). MacKenzie et al.
(1959) measured absorption in fed and fasted rats following a single oral dose and observed rats in
the fasted state exhibited higher tissue and urinary chromium levels than rats in the fed state. This
would be consistent with more efficient Cr(VI) reduction in the fed rat than in the fasted rat Thus,
it has been demonstrated that Cr(VI) reduction in the rodent may be affected by fed status in vivo.
In addition to daily pH fluctuations, interindividual and life stage variability of stomach pH
in the human population is significant Hypochlorhydria (low stomach acid) is exhibited by an
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unknown fraction of the population,4 leading to a consistently high stomach pH fKalantzi etal..
2006: Feldman and Barnett. 1991: Christiansen. 19681. Among adults without hypochlorhydria,
5% of men can exhibit basal pH exceeding 5, and 5% of women can exhibit basal pH exceeding 6.8
(Feldman and Barnett. 1991). That Cr(VI) reduction will be decreased for individuals with high
stomach pH is expected, although the reduction rates are uncertain. Gastric juice reduction data
were obtained from adults with naturally low stomach pH or stomach pH elevated by proton pump
inhibitors. The gastric juice of those with high pH may be chemically or biologically different.
Neonates, infants, and young toddlers generally have neutral stomach pH for the first 20-30
months, which then lowers to the normal adult range of 1-2 fNeal-Kluever etal.. 2019: Bai etal..
2016).
C.l.5.1. Application of pharmacokinetic models for dose-response assessment
A previous PBPK application of the Kirmanetal. (2013) model by Thompson etal. (2014)
defined the internal dose as the average lifetime daily milligrams Cr(VI) absorbed per liter small
intestine segment for the duodenum, jejunum, and ileum individually. This metric was applied to
the NTP 2-year bioassay, and dose-response modeling was performed on pooled data (male and
female mice, duodenum, jejunum, and ileum). Thompson et al. (2014) excluded jejunum tissue
from the analysis of hyperplasia. Because of uncertainties in site-specific absorption for the human,
the study authors applied total small intestinal absorption (per L small intestine) as the human
dose metric for extrapolation.
Site-specific absorption in the rodent small intestine, however, is uncertain. Ingested
drinking water does not evenly distribute in the small intestine lumen, but instead forms multiple
discrete pockets of water that vary with time (Mudie etal.. 2014). Motility in the intestine is highly
variable, and the intestine secretes enzymes that can impact reduction rates. At the microscopic
level, data for Cr(VI) indicates uptake might not occur uniformly in GI tract epithelial cells
(Thompson et al.. 2015a). The well-mixed compartment assumption is likely an inaccurate
description of the system, particularly for distal regions of the intestine.
An alternative to the absorption dose metric is pyloric flux. Pyloric flux was defined by
Thompson etal. f20141 to be average daily mg Cr(VI) emptied from the stomach to the small
intestine, per liter small intestine. This estimate requires only the stomach portion of the
gastrointestinal tract PBPK model. Fewer parameters are required to simulate pharmacokinetics in
the stomach, and many of these parameters (such as gastric volume and emptying rate) are well
characterized in rodents and humans. The full whole-body PBPK model by Kirman etal. (2017)
contains approximately 100 PBPK parameters, and many of the fitted chemical-specific parameters
have high uncertainty due to the constant presence of background Cr(III) and reduced Cr(III) in all
40ne estimate is that less than 1% of the adult population might exhibit hypochlorhydria, whereas 10-20% of
the elderly population might exhibit this condition (Russell et al.. 1993).
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Cr(VI) pharmacokinetics studies. The stomach-only model applied in this assessment (Figure C-7)
contains approximately 20 parameters.
Furthermore, the data underlying the ex vivo reduction model were generated under batch
reaction conditions, which is similar to the stomach compartment. There is added uncertainty
when extrapolating ex vivo data to the complex and dynamic intestinal compartments (which may
contain different reducing agents). Uncertainties and the possible implications of these and other
candidate internal dose metrics are outlined in Table C-10.
For this assessment, a hybrid PBPK-BW3/4 scaling approach was used for effects in the small
intestine and systemic effects. The hybrid approach applied BW3/4 scaling to the mg/kg-day Cr(VI)
escaping stomach reduction and entering the small intestine. Because the volume of the small
intestine (like other tissues) varies between species by allometry, interspecies scaling by BW3/4is
numerically similar to scaling by small intestinal volume.
For effects in the oral mucosa, multiple dose metrics were explored. For example, the
concentration of Cr(VI) ingested, scaled by the exposed oral surface areas, can be used as a dose
metric. However, without such surface area data for rats, and without an oral cavity
pharmacokinetic or pharmacodynamic model, it was not possible to develop these alternative dose
metrics. In the absence of an adequately developed theory or information to develop and
characterize an oral portal-of-entry dosimetric adjustment factor, application of BW3/4 scaling is
recommended (U.S. EPA. 2011b. 2005).
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Table C-10. Uncertainties and potential impacts of alternative dose metrics for
rodent-to-human extrapolation
Dose metric
Added uncertainty
Extrapolation notes
Site-specific absorption
Daily mg Cr(VI) absorbed
in a small intestine (SI)
segment, per LSI segment
• Small intestine lumen not
well mixed.
• Fluctuations in intestinal
motility and secretions not
modeled.
• Cellular uptake in
epithelium not uniformly
distributed.
• High variability and
uncertainty for absorption
of Cr(VI)/reduced Cr,
perfusion of Cr(lll)/ Cr(VI)
from systemic plasma,
absorption of background
Cr(lll).
• Differences in relative
lengths of small intestinal
segments between
rodents and human
preclude direct
comparisons.
Human equivalent dose (HED) estimates: Similar
to pyloric flux, since rapid Gl uptake is assumed in
all species, and human absorption is still
normalized by total SI volume.
Variability assessment: Difficulty in assessing
interindividual variability site-specific absorption
fractions. Inconsistent dose metric basis between
humans and rodents, since only total Cr(VI)
absorption in whole intestine can be estimated by
current human PBPK models.
Pyloric flux
Daily mg Cr(VI) emptying
from the stomach to the
SI, per liter SI
• Absorption not modeled
(assumes 100% absorption
in all species).
• Reduction in small
intestine neglected.
HED estimates: Slightly higher than small
intestine absorption dose metric, since this
metric assumes 100% absorption for the rodent.
Variability assessment: Can only assess stomach
reduction variability.
Cr(VI) lumen
concentration
mg Cr(VI) in SI lumen, per
liter SI lumen
• Estimates of Cr(VI)
concentration in lumen
contents not well
characterized.
HED estimates: Similar to pyloric flux dose
metric, since it normalizes the Cr(VI) mass by
intestinal lumen volume (which will scale similarly
as intestinal tissue volume).
Variability assessment: Difficult to assess
variability.
BW3/4-adjusted
unreduced Cr(VI) dose
Daily mg Cr(VI) emptying
from the stomach, per kg
BW, multiplied by
(BWa/BWh)0'25
• Does not incorporate
volume of gastrointestinal
tissue, a site of observed
toxicity.
HED estimates: 10-20% lower than pyloric flux.
Normalizing unreduced Cr(VI) by a BW3/4
adjustment has a similar impact on HED as
normalizing to intestinal volumes.
Variability assessment: Can assess only stomach
reduction variability.
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Dose metric
Added uncertainty
Extrapolation notes
Stomach absorption
mg Cr(VI) absorbed in
stomach tissue, per liter
stomach tissue
• Estimates of Cr(VI)
stomach absorption not
well characterized.
• Intestinal dose metric still
applied for rodent.
HED estimates: Similar to pyloric flux due to pH
dependence.
Variability assessment: Difficult to assess
absorption variability. Would lead to different
dose metric basis between humans and rodents.
BW3/4 scaling
Daily mg/kg Cr(VI)
ingested, multiplied by
(BWa/BWh)0'25
• Does not correct for
species differences in
Cr(VI) reduction.
HED estimates: For extrapolations in the low-
dose region, would result in lower HEDs than all
other approaches. For extrapolations in the
high-dose region, would result in slightly lower
(~20% lower) HEDs than methods listed above
(due to high percentage of dose escaping for
human model at high doses).
Variability assessment: Cannot directly assess
inter-individual variability in pharmacokinetics.
Cr(VI) ingested
concentration
Parts per million (mg/L)
Cr(VI) ingested
• Does not correct for
species differences in
Cr(VI) reduction, tissue
uptake, or tissue exposure
duration.
• May require additional
scaling to account for
species differences in
epithelial surface area and
exposure time.
HED estimates: Would result in higher HEDs than
most other approaches for both oral and
intestinal tumors.
Feasible only for oral mucosa, prior to
mixing/dilution/reduction by gastric and
intestinal contents.
Variability assessment: Cannot directly assess
interindividual variability in pharmacokinetics.
BW3/4 scaling, adjusted for
target tissue volumes
Daily mg/kg Cr(VI)
ingested, multiplied by:
(BWa/BWh)0-25 X Va/Vh
(Va and Vh represent tissue
volume as % total body
volume)
• Does not correct for
species differences in
Cr(VI) reduction or tissue
uptake.
• Must assume steady-state
tissue delivery and
clearance.
HED estimates: Difference from alternative
approaches depends on organ site. Would be
representative of local tissue dose. Feasible only
for oral mucosa, prior to
mixing/dilution/reduction by gastric and
intestinal contents.
Variability assessment: Cannot directly assess
interindividual variability in pharmacokinetics.
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Supplemental Information—Hexavalent Chromium
Cr(VI)
ingestion
Saliva,
food &
water
PHS: Stomach pH
VSL: Stomach lumen volume
Cr(VI) "> Cr(lll)
RORAL
Reducing agent
formation
v
Reducing agent loss
Reducing
Cr(VI)
agent out
out
Gastric emptying
to small intestine
Figure C-7. Schematic of the gastric model and parameters for Cr(VI).
Parameter values and units defined in Tables C-ll (humans), C-13 (mice), and C-16
(rats).
C.l.5.2. PBPK model assumptions for the human
Table C-ll. Final human physiological parameters for dose-response
modeling and rodent-to-human extrapolation
Parameter
code variable
Parameter
value
Parameter source and notes
BW (kg)
80
Body weight. This value is chosen to maintain consistency for comparison with
default approaches (such as BW3/4 scaling (U.S. EPA, 2011b, 2005)).
VSLC (L/kg075)
9.92 x 10"3
(baseline),
2.02 x 10"3
(fasted)
Stomach lumen volume or stomach contents volume (scaled by BW3/4).
Baseline value (0.24 L for a 70-kg human) is based on ICRP (2006, 2002)
reference values for mass of stomach contents (average of adult male and
female). Fasted-state value (0.049 L for a 70-kg human, applied in the morning)
is based on the mean value measured bv Grimm et al. (2018): this is also the
default fasted value in GastroPlus (version 9.0) software. Lognormal coefficient
of variance of 0.1 applied for Monte Carlo simulations (based on GastroPlus
defaults).
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Supplemental Information—Hexavalent Chromium
Parameter
code variable
Parameter
value
Parameter source and notes
PHS
1.3 (baseline),
4.9 (fed spike)
Gastric pH. Varies based on fed status (Mudie et al., 2010; Parrott et al., 2009).
Mav be chronically elevated (>4) in some individuals (Kalantzi et al., 2006;
Feldman and Barnett, 1991; Christiansen, 1968). Values of 1.3 and 4.9 obtained
from Parrott et al. (2009), and decaying exponential function (e_09302t) following
spike during meals estimated bv digitizing data from Dressman et al. (1990).
For Monte Carlo simulations, the spikes were assumed to begin up to 10 min
after the breakfast/lunch/dinner oral doses and up to 30 min before (uniform
distribution). Lognormal coefficient of variance of 0.12 applied to baseline for
Monte Carlo simulations (based on GastroPlus defaults).
KLSD (h-1)
1.39
(baseline),
2.63 (fasted)
Gastric emptying rate (1st order). Based on standard reference value of half-
emptving time of noncaloric liauids in adults (30 min) bv ICRP (2006, 2002).
Fasted-state value based on fasted half-emptying time for water of 15.8 min
Mudie et al. (2014). Lognormal coefficient of variance of 0.2 applied for Monte
Carlo simulations (based on GastroPlus defaults).
RORAL (L/h)
Calculated
(see text)
= 0.33
(baseline)
= 0.129
(fasted)
Sum of drinking water/food/saliva/GI fluid introduction into gastric
compartment. This value is not set but calculated on the basis of steady-state
volume of stomach contents and stomach emptying rate (see text). As a
comparison, the default Kirman et al. (2017) values for the human are 0.13-
0.56 L/h (varving with drinking rate). ICRP (2006, 2002) estimates the average
daily generation of saliva and gastric juice in adults to be 0.133 L/h (which is
approximately equal to the fasted-state RORAL). Thus, the model assumes,
during a baseline 1-h ingestion event, an adult might consume approximately
0.2 L of food and/or drinking water such that the total introduction of contents
to the stomach is 0.33 L.
VSIC (fraction)
8.77e-3
Volume of small intestine tissue used for internal dose scaling (fraction of body
weight). Used for pyloric flux estimates only. Value for a 70-kg human (~0.62 L)
unchanged from Kirman et al. (2012) and Kirman et al. (2017). This is consistent
with the ICRP (2006, 2002) value for mass of intestine wall (0.65 kg for adult
males, 0.60 kg for adult females).
CRE01 (mg/L)
10.0 (fasted)
20.0 (fed)
Reducing capacity of human gastric juice assuming a single pool of reducing
agent according to the model bv Schlosser and Sasso (2014). Data from De
Flora et al. (2016) were used to derive fasted/fed-state values and to estimate a
lognormal distribution for Monte Carlo analyses (lognormal coefficient of
variance of 0.5). Model set fed-state values lasting 2 h for the 3 meals
(breakfast/lunch/dinner), beginning at the time of the spikes in gastric pH.
For additional kinetic parameters used in the model, see Schlosser and Sasso (2014).
GastroPlus default values used or cited alongside gastric PK parameters because they have been found consistent
with values identified by literature screening and also provided estimates of population variability.
1 The human PBPK model was run assuming the periodic bolus exposure profile for a period
2 of time until the internal dose metric reached steady-state (7 weeks). This was done to prevent an
3 underestimation of the internal dose, which could result from assuming continuous mg/kg-day
4 exposure (less reducing agent depletion occurs if the dose is spread evenly over 24 hours). These
5 drinking water assumptions are consistent with human surveys fU.S. EPA. 2019a: Barrai etal..
6 20091.
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In addition, a change in gastric volume and gastric emptying from baseline was
incorporated to account for an early morning fasted state, and a pH spike above baseline was
incorporated to account for the fed state. This special fasted state was applied only in the morning,
and the parameters only needed to be set shortly (1 hour) before the first ingestion because steady-
state in the gastric reducing agent mass balance was achieved quickly. These model assumptions
are illustrated in Figure C-8.
Figure C-8. Time profiles of the average daily oral Cr(VI) dose (left) and gastric
pH, reducing capacity (/10), and gastric emptying rate KLSD (right) in the
human. Exposure to Cr(VI) was assumed to occur via six discrete drinking water
events of varying magnitude, occurring daily. Gastric emptying was elevated for
3 hours in the morning beginning 1 hour prior to the first daily drinking event to
simulate a morning fasted status. Gastric volume was also reduced to the fasted-
state value during this time (not shown). Gastric pH was spiked to a value of 4.9
(which decreased exponentially) near the three other large drinking water events
(to simulate breakfast, lunch, and dinner fed status). Elevation of the reducing
capacity (lasting 2 hours) also occurred at the time of the spikes in pH. For Monte
Carlo simulations, a uniform distribution was applied to the timing of the pH and
reducing capacity spikes.
Local sensitivity analyses were performed on selected model parameters at a lower dose
level and an upper dose level. The sensitivity was characterized by the finite difference method,
and the sensitivity coefficients represent the ratios of the relative change in the response variable
(internal dose) to the relative change in the independent variable (parameter). For the human
model, the sensitivity of the internal dose to kinetic parameters was greater in the low-dose region.
This is also illustrated by Figure C-9 for the stomach pH parameter.
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Supplemental Information—Hexavalent Chromium
Table C-12. Normalized sensitivity coefficients of human gastric model
parameters with respect to pyloric flux dose metric
Parameter
Sensitivity
coefficient at
0.04 mg/kg-d
Sensitivity
coefficient at
0.4 mg/kg-d
CRE01 (reducing capacity of fast binary reaction, mg/L)
-1.2694
-0.7297
KLSD (gastric emptying rate, h"1)
0.7661
-0.0129
VSLC (baseline stomach lumen volume, fraction of BW)
-0.2226
-0.5593
VSLCFAST (fasted-state stomach lumen volume, fraction of BW)
-0.3550
-0.1289
K (rate constant for fast binary reaction, L/mg-h)
-1.1920
-0.0409
PHS (baseline)
0.2197
0.0143
PHSF (fed-state spike)3
5.1534
0.2461
Note: This model analysis incorporated only two pH spikes (lunch and dinner) and held CRE01 constant (no fed-
state increase to 20 mg/L).
aTo avoid simulation artifacts caused by TSPIKE and ingestion time occurring at same time, the values of TSPIKE
were set to 5 minutes prior to water ingestion events.
Figure C-9. (a) Percent Cr(VI) escaping stomach reduction (and being emptied
to the small intestine) as a function of oral Cr(VI) dose for different values of
baseline fasted-state stomach pH (human), (b) Pyloric flux as a function of oral
dose for the human. The pH spike was set to begin 10 minutes prior to Cr(VI)
ingestion for the three meals in this example (for human equivalent dose
calculations, this is a random variable).
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Figure C-10. Monte Carlo analysis (20,000 iterations) of the human equivalent
dose at selected values of the internal dose. Model assumes three daily spikes in
pH during the three large ingestion events and elevated gastric emptying/reduced
gastric volume during early morning ingestion event All simulations assume
lognormal distributions for the baseline and fasted parameters, with coefficient of
variance (CV) of 10% for stomach volume, 12% for baseline pH, 50% for fed and
fasted reduction capacities, and 20% for stomach emptying. A uniform distribution
was applied to the timing of each pH spike to allow for the oral dose to occur up to
30 minutes after the start of a large meal (pH spike), and up to 10 minutes before.
All other parameters held constant (Left) Human equivalent dose (HED) at pyloric
flux 4 mg/L-d. (Right) Human equivalent dose (HED) at pyloric flux 0.1 mg/L-day.
To evaluate the potential impact of pharmacokinetic susceptibility on adult populations
with high stomach pH, simulations were run using altered assumptions for baseline and fed-state
pH (see Table C-13). These simulations included estimating the HED for low-dose and high-dose
internal dose PODs. Standard default population simulations assumed a mean baseline pH of 1.3
and a fed spike of 4.9. The PHS = 4 population assumed a mean baseline pH of 4 and a fed spike pH
of 4.9. For all simulations, the baseline pH had a lognormal distribution with a coefficient of
variance of 0.12.
Although a fed-state pH spike was maintained for the high pH population, some uncertainty
exists regarding the daily pH profile in response to meals. The study in healthy elderly subjects by
Russell etal. (1993) observed that for individuals with high baseline pH, some exhibited minimal
pH change with meals, while others exhibited a decrease in pH with meals.
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Table C-13. Human equivalent dose (mg/kg-day) outputs of 20,000 Monte
Carlo simulations of varying baseline pH populations using the BW3/4-adjusted
Cr(VI) dose escaping stomach reduction
Internal dose
POD (mg/kg-d)
Model
assumption
Mean HED
(mg/kg-d)
SD (mg/kg-d)
Lowest 1% HED
(mg/kg-d)
0.03
Default
0.328
0.0942
0.171
PHS = 4
0.220
0.102
0.0596
0.001
Default
0.0320
0.00945
0.0165
PHS = 4
0.0178
0.0179
0.00204
0.000732
Default
0.0237
0.00708
0.0121
PHS = 4
0.00943
0.00404
0.00269
At high internal dose (which is most relevant for cancer extrapolation), the mean value for
the HED of the pH = 4 population is approximately 33% lower than the HED of the default pH = 1.3
population. At low internal dose (which is most relevant for noncancer extrapolation), the mean
value for the HED of the pH = 4 population is approximately 44% lower than the default. The value
of the lowest 1% for the default assumption (0.0165 mg/kg-day), however, is still slightly lower
than the mean value of the pH = 4 population (0.0178 mg/kg-day), meaning the pharmacokinetic
approach is protective for the average of that group.
For values lower than 0.001 mg/kg-day (i.e., 0.000732 mg/kg-day), the mean HED of the
pH = 4 population (0.00943 mg/kg-day) is 22% less than the lowest 1% HED of the pH = 1.3
population (0.0121 mg/kg-day). This is because at very low doses, the model is more sensitive to
differences in pH. However, all internal-dose PODs for this assessment (which are used to derive
human equivalent doses) are higher than 0.001 mg/kg-day. As a result, the pharmacokinetic
approach (which uses the lowest 1% value) is protective of the pH = 4 population.
The pharmacokinetics results for all PODs can be compared to BW3/4 scaling without
pharmacokinetic adjustment for interspecies Cr(VI) reduction (see Appendix D.3). By not
accounting for extracellular Cr(VI) reduction in either the rodent (gastric pH = 4.5) or the human
(gastric pH = 1.3), the default scaling approach technically applies to the most sensitive population
in terms of pharmacokinetics (i.e., a human population in which gastric pH = 4.5 and gastric juice
reduction capacity is equivalent to that of the rodent). However, this does not consider the extreme
case in which human pH is significantly higher than that assumed for the rodent (pH >> 4.5).
Applying BW3/4 adjustment in accordance with fU.S. EPA. 2011b. 20051 and applying an
intraspecies uncertainty factor (UFh) of 3 (rather than 10, because the default approach implicitly
accounts for the most sensitive pharmacokinetic population) is protective of the population that
has high pharmacokinetic susceptibility. As noted in Appendix D.3, this specifically applies to the
low-dose region, for which the model is most sensitive to gastric pH. At high doses, for which the
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Supplemental Information—Hexavalent Chromium
1 model is more sensitive to gastric reducing capacity, the lower 1% predictions from Monte Carlo
2 simulations using the pharmacokinetic model are more health protective than BW3/4 scaling.
3 Appendix D.3 contains a table of the RfD derivation using default approaches (no gastric
4 reduction adjustment) and with UFh = 3.
C.l.5.3. PBPK model assumptions for the mouse
Table C-14. Final mouse PBPK parameters for dose-response modeling and
rodent-to-human extrapolation
Parameter
code variable
Parameter
value
Notes
BW (g)
50
Body weight. The time-weighted average body weight of mice in the NTP 2008
bioassays. Additional study-specific values of rodent body weight were used
when necessary.
VSLC (L/kg0 75)
0.00696
Volume of the stomach lumen contents (scaled bv BW3/4). Based on Mcconnell
et al. (2008) "comfortablv full" volume (0.37 mL in 18-22g mice). For a 50 g
mouse, this equates to a stomach volume of 0.736 mL.
PHS
4.5
Gastric pH. Value unchanged from Kirman et al. (2012) and Kirman et al. (2017)
since reduction data in mice are available only for pH 4.5 (and thus, confidence is
highest for the mouse reduction rate at that pH). This parameter can vary with
both fed status and stomach region (forestomach vs. glandular stomach)
(Beaslev et al.. 2015: Kohl et al., 2013; Mcconnell et al., 2008; Browning et al.,
1983). The reduction model used in this assessment bv Schlosser and Sasso
(2014) performs well for the available data of Cr(VI) reduction in rodent gastric
juices.
KLSD (h1)
4.33
Gastric emptying rate (1st order). Value changed from default value of 9.4 h"1 by
Kirman et al. (2012) Kirman et al. (2017). Based on the default fed-state
GastroPlus stomach transit time of 19.2 min. This is consistent with the
literature, which estimates a half-emptying time for liquids in mice of
approximately 10 min (Roda et al., 2010; Mivasaka et al., 2004; Bennink et al.,
2003; Svmonds et al., 2002) (see Table C-27). This parameter can varv based on
fed status and gastric and dietary contents.
RORAL (mL/h)
3.2
(calculated)
Sum of drinking water/food/saliva/GI fluid introduction into gastric
compartment. This value is not set but calculated on the basis of steady-state
volume of stomach contents and stomach emptying rate (see text). As a
comparison, the value of RORAL bv Kirman et al. (2017) for the NTP (2008) data
ranges from 0.65 to 6.2 mL/h (varving with drinking rate). In Kirman et al.
(2017), this parameter was the sum of multiple individually defined rates that
had high uncertainty and variability. The value for the gastric fluid (acid)
production component defined in the Kirman et al. (2017; 2012) models was a
central estimate bv Thompson et al. (2011a) based on (Tibbitts, 2003; Wang et
al., 2000; Friis-Hansen et al., 1998; Ito and Schofield, 1974). Those data varied
significantly with time, fed status, and other factors, and the exact source of the
Thompson et al. (2011a) estimate could not be determined. The saliva secretion
rate component defined in Kirman et al. (2017; 2012) was based on a model bv
Timchalk et al. (2001), although it was not a measured parameter (it was instead
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Supplemental Information—Hexavalent Chromium
Parameter
code variable
Parameter
value
Notes
calibrated to lead pharmacokinetic data). Values defined in Kirman et al. (2017;
2012) for the food and water intake component of RORAL were studv specific.
VSIC (fraction)
0.0393
Volume of small intestine (fraction of body weight). Used for pyloric flux
estimates onlv. Value unchanged from Kirman et al. (2012), which is based on
fractional tissue volumes of the duodenum, jejunum, and ileum measured in that
studv. Value is consistent with Brown et al. (1997) (which estimates it to be 2-4
% of body weight).
For additional kinetic parameters used in the model, see Schlosser and Sasso (2014).
Time (lir)
Figure C-ll. Distribution of the average daily oral Cr(VI) dose in the mouse.
Exposure to Cr(VI) was assumed to occur ad libitum in drinking water according to
observed circadian drinking water data fYuan. 19931.
1 PBPK simulations were run assuming standard adult rodent physiology (Table C-14), with
2 circadian drinking water pattern (Figure C-ll), until steady-state was achieved (7 weeks). This
3 was done to prevent an underestimation of the internal dose, which could result from assuming
4 continuous mg/kg-day exposure (less reducing agent depletion occurs if the dose is spread evenly
5 over 24 hours).
6 Local sensitivity analyses were performed on selected model parameters at a lower dose
7 level and an upper dose level using the finite difference method. For the rodent model, dose region
8 had less effect on model sensitivity (Table C-15). However, the rodent model was very sensitive to
9 changes in pH (Figure C-12), since the kinetic function of rate vs. pH by Schlosser and Sasso (2014)
10 is steep in the region around pH 4.5. Ex vivo rodent kinetic data are available only at pH = 4.5
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1 (mice) and pH = 4.38 (rats) Proctor etal. f20121. The kinetic model by Schlosser and Sasso T20141
2 adequately fits the rodent ex vivo data at these values of pH. Because the true value of the rodent
3 whole stomach pH (glandular stomach + forestomach) during the NTP (2008) 2-year bioassay is
4 uncertain, and because no ex vivo data are available for rodent kinetics at low pH, the model will be
5 run only atpH = 4.5 (mice) and pH = 4.38 (rats) when used for the dose-response assessment.
6 These values are fair approximations for the model since they fall within the range observed in
7 rodents, but they are not without uncertainty (Beaslev etal.. 2015: Kohl etal.. 2013: Mcconnell et
8 al.. 2008: Browning et al.. 19831.
Table C-15. Normalized sensitivity coefficients of mouse gastric model
parameters with respect to pyloric flux dose metric
Parameter
Sensitivity
coefficient at 0.302
mg/kg-d
Sensitivity
coefficient at 8.89
mg/kg-d
CRE01 (reducing capacity of fast binary reaction, mg/L)
-0.5083
-0.3009
CRE02 (reducing capacity of slow binary reaction, mg/L)
-0.3576
-0.6615
KLSD (gastric emptying rate, h"1)
0.8101
0.3231
VSLC (stomach lumen volume, fraction of BW)
-0.0301
-0.3243
K (rate constant for fast binary reaction, L/mg-h)
-0.5173
-0.1001
KS (rate constant for slow binary reaction, L/mg-h)
-0.3582
-0.5428
KVF (rate constant for slowest binary reaction, L/mg-h)
-0.0031
-0.0077
PHS (stomach pH)
7.8453
6.0116
Figure C-12. (a) Percent Cr(VI) escaping stomach reduction (and being
emptied to the small intestine) as a function of oral Cr(VI) dose for different
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Supplemental Information—Hexavalent Chromium
values of baseline stomach pH (mouse); (b) pyloric flux for the mouse using
standard assumption at PHS = 4.5.
1 Because the internal dose is very close to linear (Figure C-12), benchmark dose modeling
2 can be performed on the basis of the external oral dose, and PBPK model adjustments can be done
3 in subsequent steps. Table C-16 below lists the predicted internal doses for the fNTP. 20081 2-year
4 drinking water bioassay. Table C-17 lists average daily internal doses for the female mouse (F0
5 dams) during the NTP (19971 bioassay.
Table C-16. Lifetime average daily internal doses for the mouse during the
NTP (2008) 2-year bioassay of sodium dichromate dihydrate
Cr(VI) (mg/L)
TWA dose
(mg/kg-d)
Dose escaping (mg/kg-d)
Pyloric flux (mg/L-d)
Females
5
0.302
0.0463
1.18
20
1.18
0.197
5.00
60
3.24
0.636
16.2
180
8.89
2.31
58.7
Males
5
0.450
0.0700
1.78
10
0.914
0.149
3.79
30
2.40
0.443
11.3
90
5.70
1.29
32.9
TWA dose: Time-weighted average daily dose.
Table C-17. Average daily internal doses for the female mouse
(F0 dams) during the NTP (1997) bioassay
TWA dose (mg/kg-d)
Dose escaping (mg/kg-d)
11.6
3.09
24.4
8.61
50.6
24.8
BW = 24 g.
C. 1.5.4. PBPK m odel assumptions for the rat
6 Table C-18 outlines the kinetic parameters used for a standard rat. For additional kinetic
7 parameters used in the model, see Schlosser and Sasso (20141.
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Supplemental Information—Hexavalent Chromium
Table C-18. Final rat PBPK parameters for dose-response modeling and
rodent-to-human extrapolation
Parameter
code
variable
Parameter
value
Notes
BW (g)
450/395
(males)
260/215
(females)
Body weight (2-yr/12-mo). The time weighted average body weights of male and
female rats in the NTP 2008 bioassays. Additional study-specific values of rodent
body weight were used when necessary.
VSLC (L/kg0 75)
0.0125
Volume of the stomach lumen contents (scaled bv BW3/4). Based on Mcconnell et
al. (2008) "comfortablv full" volume (3.38 mL for 160-190 g rats). For a 260-g rat,
this yields a stomach volume of 4.55 mL For a 450-g rat, it yields 6.87 mL
PHS
4.38
Gastric pH. Value unchanged from Kirman et al. (2017), since reduction data in
rats are available only for pH 4.38 (and thus, confidence is highest for the rat
reduction rate at that pH). This parameter can vary with both fed status and
stomach region (forestomach vs. glandular stomach) (Beaslev et al., 2015; Kohl et
al., 2013; Mcconnell et al., 2008; Browning et al., 1983). The reduction model
used in this assessment by Schlosser and Sasso (2014) performs well for the
available data ofCr(VI) reduction in rodent gastric juices.
KLSD (h-1)
2.77
Gastric emptying rate (1st order). Changed from default value of 2.4 h"1 defined
bv Kirman et al. (2012) and Kirman et al. (2017). Based on the default fed-state
GastroPlus stomach transit time of 30 min. This is consistent with the literature,
which estimates a half-emptying time for liquids in rats of approximately 15 min
(Scamignato et al.. 1984: Purdon and Bass, 1973). This parameter can varv on the
basis of fed status and gastric and dietary contents.
RORAL (mL/h)
12-19
(calculated)
Sum of drinking water/food/saliva/GI fluid introduction into gastric compartment.
This value is not set but calculated on the basis of the steady-state volume of
stomach contents and stomach emptying rate (see text). As a comparison, the
default value calculated bv Kirman et al. (2017) for the NTP (2008) studv is 4-33
mL/h (varving with drinking rate). In Kirman et al. (2017), this parameter is the
sum of multiple individually defined rates that had high uncertainty and variability.
The value for the gastric fluid (acid) production component defined in the Kirman
et al. (2017; 2012) models was a central estimate bv Thompson et al. (2011a)
based on (Runfola et al., 2003; Tibbitts, 2003; Kitamura et al., 1999; Takeuchi et
al., 1998; Kuwahara et al., 1990; Wallmark et al., 1985). Those data varied
significantly with time, fed status, and other factors, and the exact source of the
Thompson et al. (2011a) could not be determined. The saliva secretion rate
component defined in Kirman et al. (2017; 2012) was based on a model bv
Timchalk et al. (2001), although it was not a measured parameter (it was instead
calibrated to lead pharmacokinetic data). Values defined in Kirman et al. (2017;
2012) for the food and water intake component of RORAL were studv specific.
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Supplemental Information—Hexavalent Chromium
14
13
^ 12
•3 11
"O
"5 10
o
0
1 9
1
0| u u u,u u u u Ul U u—_
0 5 10 15 20
Time (hours)
Figure C-13. Ad libitum drinking water assumptions applying data from the rat
(Spiteri. 1982).
1 PBPK simulations were run assuming standard adult rodent physiology, with circadian
2 drinking water pattern (see Figure C-13), until steady-state was achieved (7 weeks). This was done
3 to prevent an underestimation of the internal dose, which could result from assuming continuous
4 mg/kg-day exposure (less reducing agent depletion occurs if the dose is spread evenly over
5 24 hours).
6 Local sensitivity analyses were performed on selected model parameters at a lower dose
7 level and an upper dose level using the finite difference method (see Table C-19).
Table C-19. Normalized sensitivity coefficients of rat gastric model parameters
with respect to average daily dose escaping stomach reduction
Parameter
Sensitivity coefficient at
0.2 mg/kg-d
Sensitivity coefficient at
7.13 mg/kg-d
CRE01 (reducing capacity of fast binary reaction, mg/L)
-0.7410
-0.4692
CRE02 (reducing capacity of slow binary reaction, mg/L)
-0.2142
-0.6868
KLSD (gastric emptying rate, h"1)
0.8916
0.1877
VSLC (stomach lumen volume, fraction of BW)
-0.0410
-0.6081
K (rate constant for fast binary reaction, L/mg-h)
-0.7010
-0.0683
KS (rate constant for slow binary reaction, L/mg-h)
-0.2138
-0.4880
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Supplemental Information—Hexavalent Chromium
Parameter
Sensitivity coefficient at
0.2 mg/kg-d
Sensitivity coefficient at
7.13 mg/kg-d
KVF (rate constant for slowest binary reaction, L/mg-h)
-0.0046
-0.0206
PHS (stomach pH)
8.3698
5.2725
Figure C-14. (a) Percent Cr(VI) escaping stomach reduction (and being
emptied to the small intestine) as a function of oral Cr(VI) dose for different
values of baseline stomach pH (rat); (b) dose escaping stomach reduction for
the rat using standard assumption at PHS = 4.38.
1 Because the internal dose is very close to linear (Figure C-14), benchmark dose modeling
2 can be performed on the basis of the external oral dose, and PBPK model adjustments can be done
3 in subsequent steps. Table C-20 lists the predicted internal doses for the (NTP. 20081 2-year
4 drinking water bioassay. This table includes values calculated at the 1-year timepoint for males.
5 Additionally, BMD modeling was performed on the basis of internal dose to evaluate the difference
6 between PODs derived from internal-dose and external-dose BMD modeling (difference was 1.2%
7 for liver ALT).
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Supplemental Information—Hexavalent Chromium
Table C-20. Lifetime average daily internal doses for the rat during the NTP
(2008) 2-year bioassay of sodium dichromate dihydrate (pH = 4.38)
Cr(VI)
concentration
TWA dose
(mg/kg-d) at
2 years
Cr(VI) dose
escaping
stomach
reduction
(mg/kg-d) at
2 years
TWA dose
at 1 year
(mg/kg-d)
Cr(VI) dose escaping
stomach reduction
at 1 year
(mg/kg-d)
TWA dose
at 90 days
(mg/kg-d)
Cr(VI) dose
escaping
stomach
reduction at
90 days
(mg/kg-d)
Females
5
0.248
0.0195
0.0294
N/A
N/A
N/A
20
0.961
0.0881
1.14
N/A
N/A
N/A
60
2.60
0.339
3.01
N/A
N/A
N/A
180
7.13
1.66
8.28
N/A
N/A
N/A
Males
5
0.200
0.0156
0.237
0.0187
0.401
0.0325
20
0.796
0.0721
0.938
0.0875
1.58
0.165
60
2.10
0.264
2.49
0.336
4.16
0.699
180
6.07
1.40
7.19
1.79
11.7
3.66
TWA BW at 2 years: 450 g (males), 260 g (females). TWA BW at 1 year: 395 g (males), 215 g (females). TWA BW at
90 days: 246 g (males). No relevant dose-response 1-year data for female rats. Oral doses assumed the circadian
rat drinking water profile (Spiteri, 1982).
Table C-21. Lifetime average daily internal doses for the rat during the NTP
(2007f) 90-day bioassay of sodium dichromate dihydrate (pH = 4.38)
Cr(VI) concentration
TWA dose
(mg/kg-d) at 90 days
Cr(VI) dose escaping stomach reduction
(mg/kg-d) at 90 days
Females
0
0
0
21.8
1.74
0.181
43.6
3.49
0.500
87.2
6.28
1.26
174.5
11.5
3.33
349
21.3
9.00
Males
0
0
0
21.8
1.74
0.188
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1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
Supplemental Information—Hexavalent Chromium
Cr(VI) concentration
TWA dose
(mg/kg-d) at 90 days
Cr(VI) dose escaping stomach reduction
(mg/kg-d) at 90 days
43.6
3.14
0.446
87.2
5.93
1.22
174.5
11.2
3.38
349
20.9
9.22
BWa = 0.160 kg (females), 0.232 kg (males).
Table C-22. Comparison of internal-dose points of departure based on
external-dose BMD modeling and internal-dose BMD modeling
Species/
Sex
Data set
BMR
Internal3 BMD
mg/kg-d
Internal3 BMDL
mg/kg-d (linear
model)b
Internal dose derived
from external BMDLird
(exponential 2 model)b
% diff.
Rat/M
Liver ALT (NTP,
2008)
1RD
0.214
0.166
0.168
1.2
aDose escaping stomach reduction in rodent (mg/kg-d) estimated by pharmacokinetic modeling.
bData were amenable to BMD modeling with the highest dose omitted. Note: the time weighted average daily
doses used in this example were slightly (<5%) different than the final calculated values used elsewhere in this
assessment. As a result, the value deviates slightly from the final internal dose POD presented elsewhere.
C.l.5.5. General PBPKmodel considerations
Model estimates are based on physiological parameters near the standard reference values
in each species. Chromium ingestion can be associated with water intake (which increases volume
of the stomach contents and potentially dilutes reducing agent) and food intake (which increases
gastric juice production and volume and alters pH and gastric emptying).
Simulations in the human and the rodents assume RORAL (total gastric contents rate into
stomach, L/h) is equal to KLSD (gastric emptying rate, h"1) multiplied by VSL (gastric contents
volume, L). The Kirman etal. (2017) model instead calculates gastric contents volume as a function
of RORAL and KLSD. For rats and humans, the model produces reasonable values for stomach
contents volume, but for mice, the stomach volume is outside the range measured by Mcconnell et
al. f2008I Since the individual-level components of the RORAL parameter (gastric juice
production, saliva production, and time-varying water and food ingestion) have higher uncertainty
than stomach volume (which is a single, measurable parameter), this assessment defines a value for
VSL rather than for RORAL.
Previously, in Kirman etal. (2012) and Sasso and Schlosser (2015). a mathematical
discrepancy existed since the chromium concentration was determined by the volume of the
stomach lumen, while the reducing agent concentration was determined by volume of stomach
contents (which was a function of RORAL and gastric emptying). The volumetric basis for Cr(VI)
and reducing agent concentrations should be the same because they coexist in the same reaction
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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
36
Supplemental Information—Hexavalent Chromium
volume. If RORAL, gastric contents volume, and gastric emptying are related by a mass balance
equation, the volumetric basis for concentration calculation is the same for Cr(VI) and the reducing
agent, and the discrepancy is resolved. Simulating gastric kinetics using physiology that is not
harmonized (i.e., with the discrepancy between gastric lumen volume and steady-state gastric
contents volume) leads to high internal doses in all species (i.e., >20-70% of the dose escaping
reduction). This is because the mass balance of the gastric contents consistently produces a volume
significantly lower than the stomach lumen volume. The rate of reduction is dependent on the
chromium concentration, and the predicted chromium concentration may be overdiluted if
chromium mass is divided by lumen volume instead of gastric contents volume.
If most of the Cr(VI) that escapes the stomach reduction is assumed to be absorbed into the
system (which is reasonable given the high pH and surface area in the small intestine, and rapid
uptake of Cr(VI)), the modeling results in this assessment agree with in vivo pharmacokinetic
studies. Studies in rodents fFebel etal.. 2001: Thomann etal.. 19941 have estimated that
approximately 10% of an ingested Cr(VI) dose might ultimately be absorbed into the system as
Cr(VI) when compared to Cr(III) (which is absorbed less readily). In humans, the Cr(VI) absorbed
following oral ingestion has been estimated to be lower fFinlev etal.. 1997: Kerger etal.. 1997:
Kerger etal.. 1996: Paustenbach etal.. 1996). An in vitro Cr(VI) bioaccessibility study estimated a
significant percentage of Cr(VI) may be bioaccessible in humans at pH>3, even at low doses, but
bioaccessibility decreases sharply at lower values of pH (Wang etal.. 2022).
C.1.6. Literature Overview of Studies Identified as ADME
Table C-23 presents a summary of studies that contain primary in vivo pharmacokinetic
data in rats, mice, and humans following Cr(VI) exposure. These tables indicate whether studies
contained concurrent data for Cr(III) exposure, as these data are informative in directly assessing
differences between Cr(VI) and Cr(III) kinetics.
Table C-24 presents a summary of studies that contain in vitro or ex vivo data related to
absorption and/or reduction in the GI tract or blood. These studies primarily focus on quantitative
analysis of kinetics. Tables C-23 and C-24 also indicate whether a study has been used
quantitatively or qualitatively in the development of any previously published PBPK model.
Table C-25 presents a summary of studies related to the distribution and reduction of Cr(VI)
in a variety of systems. These studies differ from those in Table C-24 in that the experiments
primarily focused on mechanisms by modifying the enzymes or transport carriers in the systems
tested. Tables C-23 to C-25 include only those studies pertaining primarily to Cr(VI)
pharmacokinetics and do not include studies that primarily address Cr(VI) toxicity.
Table C-26 presents a summary of studies related to human biomonitoring of Cr(VI) in
industrial or volunteer populations that focus primarily on data on biomarkers of exposure as
opposed to human health effects. These differ from the human studies in Table C-23 in that the
exposure profiles are not controlled or may be difficult to estimate.
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Supplemental Information—Hexavalent Chromium
All tables in this section are slightly modified from those released in September 2014 due to
a rescreening of articles from the literature search, addition of new studies, and public comments.
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Supplemental Information—Hexavalent Chromium
Table C-23. In vivo Cr(VI) pharmacokinetic studies
Reference
Species
Tissue matrices and notes
Cr(lll)
control3
Intravenous (IV) injection
Cavalleri et al. (1985)
Rat
Bile, whole blood, and plasma. 2-h time-course data.
N
Cikrt and Bencko (1979)
Rat
Total body burden, urine, feces, liver, kidneys, plasma, and Gl
tract wall. 24-h time-course data.
Y
Marouani et al. (2012)
Mouse
Fetus, placenta, liver, kidney, serum. Injection to pregnant mice
at day 13 or 16 of gestation. Spot sample 1-h after injection.
Y
Liu et al. (1994)
Liu et al. (1996)
Mouse
Blood, liver, heart, spleen, kidney, and lung. Kinetics of
pentavalent chromium (Cr V) following Cr VI reduction. 60-min
time-course data.
N
Norseth et al. (1982)
Rat
Bile and liver. 2-h time-course data.
Y
Merritt etal. (1989)
Hamster
Urine, plasma, RBC, kidney, spleen, liver, and lung. Monthly or
weekly injections. 5-wk postexposure time-course data.
N
Richelmi et al. (1984)
Rat
Blood. In vivo Cr VI measurement of reduction and capacity.
Spot sample at 1-min postexposure.
N
Intraperitoneal (IP) injection
Afolaranmi and Grant
(2013)
Rat
Liver, kidney, heart, brain, lung, spleen, testes, blood, urine, and
feces. Effect of ascorbic acid. Spot sample 24 h postexposure.
N
Balakin et al. (1981)
Rat
Liver, whole body (excluding liver), wall of cecum, chime of
cecum, urine, and feces. Spot sample 30 min postexposure.
This is a chelation study that included a Cr Vl-only group.
Y
Brvson and Goodall
(1983)
Mouse
Total body burden, urine, and feces. 21-d time-course data.
Y
Bulikowski et al. (1999)
Rat
Skin. Injections over 30 d. Micronutrient interaction study with
Cr Vl-only groups.
N
Devov et al. (2019)
Rat
Plasma, RBC, and urine. Single IV injection. Multiple doses and
time-course data (hourly, daily, to 90 d for some groups).
Y
Doker et al. (2010)
Mouse
Liver, kidney, brain, lung, heart, and testis. Effect on other
essential metals analyzed. Spot sample at 12 h postexposure.
N
Manzo et al. (1983)
Rat
Bile, plasma, liver, urine, feces, stomach, small intestine, and
large intestine. Detection in Gl tissues postexposure. 2-h time-
course data.
Y
Ogawa et al. (1976)
Mouse
Urine, feces, and whole body. Spot sample data at 48 h
postexposure.
Y
Sankaramanivel et al.
(2006)
Rat
Bone (vertebrae, femur, and calvaria). IP injections once per d
for 5 d.
N
Suzuki (1988b)
Rat
Plasma, whole blood. 60-min time-course data.
N
Ueno et al. (1995)
Mouse
Liver. Total Cr and pentavalent (Cr V). 12-h time-course data.
N
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Supplemental Information—Hexavalent Chromium
Reference
Species
Tissue matrices and notes
Cr(lll)
control3
Minigaliveva et al.
(2014)
Rat
Liver, kidney, spleen, and brain. Injection 3 times per wk (less
than 7 wk). Spot sample at end of study.
N
Yamamoto et al. (1981)
Mouse,
rabbit
Urine, feces, blood, and liver. Single IP (50 or 200 nmol/kg),
time-course data over undetermined length (at least 7 d).
N
Subcutaneous injection
Mutti et al. (1979)
Rat
Urine, spleen, liver, renal cortex, renal medulla, lung, and bone.
48-h (single exposure) and 12-wk (repeated exposure) time-
course data.
N
Pereira et al. (1999)
Mouse
Liver, kidney, and spleen. Multiple injections (once per wk for
varying number of weeks). Spot sample at 1 wk after last
exposure.
N
Yamaguchi et al. (1983)
Rat
Urine, feces, lung, liver, kidney, brain, heart, spleen, testis,
muscle, hair, and blood. 30-d time-course data.
Y
Dermal
Corbett et al. (1997)
Human
Urine, RBC, and plasma. 4-d time-course data.
Oral
Collins et al. (2010)
(National Toxicology
Program studies)
NTP(2008)
NTP (2007f)
Rat,
mouse,
Guinea
Pig
Urine, feces, erythrocytes, plasma, liver, kidney, glandular
stomach, and forestomach (2-yr study). Multiple studies. Blood,
kidney, and femur (21-d study in rats only). No mouse urinary
data for chronic Cr III study. Chronic Cr Ill/Cr VI data at multiple
sacrifice times (after 2-d washout period). Time-course (2-d)
gavage data (urine/feces only) for Cr III only. Guinea pig data
only at 21 d.
Y
Donaldson and Barreras
(1966)
Human,
rat
Urine, feces. Oral dose and perfusion to the small intestine
(bypassing stomach reduction) to assess Cr VI reduction and
absorption.
Y
Iranmanesh et al.
(2013)
Rat
Liver, kidney, intestine, spleen, and testicle. Drinking water
exposure for 60 d. Spot sample after 7-d washout period. This
is a chelation study that included a Cr Vl-only group.
N
Finlev et al. (1997)
Finlev et al. (1996)
Kerger et al. (1997)
Kerger et al. (1996)
Paustenbach et al.
(1996)
Human
Human pharmacokinetic volunteer studies. Urine, plasma, and
RBC. Multiple exposure scenarios (i.e., single and repeated
doses). Time-course data over multiple days before, during, and
after exposure.
Y
Kirman et al. (2012)
Rat,
mouse
Oral cavity, stomach, duodenum, jejunum, ileum, plasma, red
blood cell, and liver. Spot sample at end of 90-d exposure
period.
N
Saxena et al. (1990)
Rat,
mouse
Oral (drinking water) study in pregnant rodents. Maternal
blood, placenta, and fetus.
N
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Supplemental Information—Hexavalent Chromium
Reference
Species
Tissue matrices and notes
Cr(lll)
control3
Sutherland et al. (2000)
Rat
Bone, kidney, liver, and testes. Exposure for 44 wk, with spot
samples 4-6 d postexposure (no time-course data).
N
Thomann et al. (1994)
Rat
Blood, liver, kidney, spleen, bone, and total carcass. 6-wk
exposure followed by 140 d postexposure. Time-course data of
pre- and postexposure periods.
N
Wang et al. (2015)
Rat
Heart, kidney, spleen, liver, lung, brain, stomach, testis, and
duodenum. Spot sample at end of 4-wk exposure period (after
overnight starvation).
N
Witmer et al. (1989)
Rat
Blood, kidney, spleen, liver, lung, brain, and testes. Spot sample
at end of 7- and 14-d exposure periods (24 h after last
treatment).
N
Yawets et al. (1984)
Rat
Liver. Single dose, spot sample.
N
Intratracheal
Bragt and van Dura
(1983)
Rat
Urine, feces, blood, heart, lungs, spleen, kidneys, liver, pancreas,
testes, and bone marrow (femur).
50-d postexposure time-course data for whole-body retention
and blood. 10-d time-course data for urine and feces. Spot
sample data for other tissues at 50 d postexposure. 3 different
Cr VI formulations.
N
Edel and Sabbioni
(1985)
Rat
Lung, trachea, kidney, liver, spleen, pancreas, epididymis, testes,
brain, heart, thymus, femur, skin, fat, muscle, stomach, small
intestine, large intestine, blood, plasma, RBC, lung lavage, urine,
and feces. Spot sample in tissues at 24 h postexposure. 7-d
time-course data of excretion.
Y
Perrault et al. (1995)
Sheep
Bronchoalveolar lavages (BAL), lung. Exposure and analysis of
particulate forms. 30-d time-course data for BAL; spot sample
for lung at day 30.
Y
Gao et al. (1993)
Rat
Blood, plasma, urine, and lymphocytes. 72-h time-course data.
Y
Vanoirbeek et al. (2003)
Rat
Lung, liver, plasma, RBC, and urine. Spot tissue samples at 2 and
7 d postexposure. 7-d time-course data of urinary excretion.
Y
Wiegand et al. (1988)
Wiegand et al. (1987)
Wiegand et al. (1984a)
Rabbit
Blood, plasma, RBC, liver, kidneys, urine, lung, and trachea. 4-h
postexposure time-course data.
Y
Song et al. (2014)
Rat
Blood, plasma, RBC, and lung. Once-per-wk exposure for 28 d.
Spot sample after overnight fast.
Inhalation
Antonini et al. (2010)
Rat
Lung, heart, kidney, liver, spleen, and brain. Exposure to
welding fume at 1, 4, 25,105 d.
N
Cohen et al. (1997)
Rat
Lung (and lung fluids/subcompartments), liver, kidney, and
spleen. Exposure for 5 h/d, 5 d/wk. Spot samples at 2 or 4 wk
(24 h postexposure)
N
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Supplemental Information—Hexavalent Chromium
Reference
Species
Tissue matrices and notes
Cr(lll)
control3
Kalliomaki et al. (1983;
1982)
Rat
Blood, liver, kidneys, stomach, spleen, and lung. Welding arc
fumes (with chromium concentration measurement). Exposures
vary in h per d or number of days exposed. Spot samples at 24
h postexposure. 106-d time-course data for elimination study.
N
Suzuki et al. (1984)
Rat
Lung, whole blood, plasma, RBC, kidney, spleen, heart, liver, and
testis. Aerosolized Cr III and Cr VI. Exposure for 2 or 6 h. 7-d
time-course data.
Y
Multiple routes
Coogan et al. (1991b)
Rat
Red blood cells, and white blood cells. Oral and IV injection.
Spot samples at 1 h, 24 h, and 7 d postexposure.
N
Febel etal. (2001)
Rat
Oral and intrajejunal injection. Urine, feces, jejunum, liver,
portae, hepatica, and cava caudalis. Spot sample data (at 60
min for intrajejunal injection, and 3 d for oral exposure).
Y
Kargacin et al. (1993)
Rat,
mouse
Oral and IP injection. Single and repeated exposures. Liver,
kidney, spleen, femur, lung, heart, muscle, and blood. Spot
sample data at 4 and 8 wk for chronic drinking water, 4 and 14 d
for repeated IP injections. Spot 24/72 h data for single IP
exposures.
N
MacKenzie et al. (1959)
Rat
Oral and injection into intestine. Stomach, intestine, blood,
liver, kidney, spleen, urine, and feces. Spot samples 1, 7, and 14
d postexposure after single oral dose. Spot sample 4 h after
intestinal injection and stomach tube experiments.
Y
Mivai (1980)
Mivai et al. (1980)
Rat,
mouse
Inhalation, intratracheal. Lung, plasma, RBC, spleen, kidney,
duodenum, testes, urine, and feces. Long-term (30+ d) time-
course data.
Y
O'Flahertv and Radike
(1991)
Rat
Oral and inhalation. Lung, liver, intestine, kidney, muscle, blood,
urine, and feces. Exposure for 40 d, with time-course data over
60 d.
Y
Sayato et al. (1980)
Rat
Oral gavage and IV injection. Blood, brain, skull, thyroid, lung,
heart, liver, spleen, pancreas, kidney, adrenal, stomach,
intestine, bone, muscle, testis, urine, and feces. 30-d time-
course data of feces/urine and body retention. 5-d time-course
data for tissues.
Y
Susa et al. (1988)
Mouse
Oral and IP injection. Liver, kidney, spleen, testes, urine and
feces. Spot sample 24 h postexposure. 3-d time-course data for
urine and feces. This is a chelation study that included Cr VI-
only groups.
N
aNotes (yes/no) if study also collected data for Cr III kinetics.
bNotes (yes/no) whether data from a study were used qualitatively or quantitatively in a published PBPK model.
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Supplemental Information—Hexavalent Chromium
Table C-24. In vitro and ex vivo Cr(VI) studies primarily focused on
pharmacokinetics in the GI tract and blood
Reference
Species
Test system
Notes
Gastric systems
De Flora et al.
(1987a)
Human
Gastric juice
Hourly gastric juice samples via nasogastric tube. Cr VI
reduction capacity estimated for fed and fasted humans.
Circadian effects also observed.
De Flora et al. (1997)
Human
Intestinal
bacteria, gastric
juice
Reduction and mutagenic activity of Cr VI analyzed at 60 min.
Reducing capacities derived for intestine and other tissues
(blood, RBC, lung fluids/bacteria, saliva).
De Flora et al. (2016)
Human
Gastric juice
Reduction and mutagenic activity of Cr VI analyzed at 60 min.
Donaldson and
Barreras (1966)
Human,
rat
Gastric juice;
intestinal rings
Binding of Cr VI and Cr III by gastric juice (at low and high pH),
and uptake by intestinal rings observed.
Gammelgaard et al.
(1999)
Rat
Artificial gastric
juice; small
intestine
lst-order reduction rate half-life derived; permeability
parameters through rat jejunum derived.
Kirman et al. (2013)
Human
Gastric juice
(fasted)
2nd-order reduction kinetics for human gastric juice derived.
pH-dependent model derived.
Kirman et al. (2016)
Human
Gastric juice
(multiple types)
Revised 2nd-order reduction kinetics and pH model. Analysis
of fed, fasted, and proton pump inhibitor (PPI) gastric
samples.
Proctor et al. (2012)
Rat,
mouse
Gastric juice and
contents
2nd-order reduction kinetics derived. Reduction capacities
estimated for both species.
Shrivastava et al.
(2003)
Rat
Crypt, mid and
upper villus,
intestinal loop
Cr VI reduction in various tissue types. Capacity and time
needed to reduce Cr VI analyzed.
Skowronski et al.
(2001)
N/A
Artificial gastric
juice
Oral bioaccessibility study. Examined Cr VI reduction in a
simulated soil matrix/gastric juice environment.
Wang et al. (2022)
Human
Artificial gastric
juice and tissue
In vitro model of intestinal injury. Examined Cr VI reduction
and injury as a function of dose and gastric pH.
Reduction or uptake in red blood cells
Aaseth et al. (1982)
Human
RBC
Reduction rate of Cr VI in RBC, and trapping of reduced Cr III
observed.
Afolaranmi et al.
(2010)
Human
Plasma, RBC,
whole blood
Distribution into different blood components (RBC and
plasma) observed.
Alexander and
Aaseth (1995)
Human,
rat
Human RBC, rat
liver cells
Cellular uptake and reduction analyzed. Effect of pH and
anion carrier inhibitors observed.
Beversmann et al.
(1984)
Human
RBC
RBC permeability and reduction analyzed.
Branca et al. (1989)
Human
Human RBC
Reduction of Cr VI in RBC observed.
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Supplemental Information—Hexavalent Chromium
Reference
Species
Test system
Notes
Coogan et al. (1991b)
Human,
rat
RBC, WBC,
whole blood
Uptake kinetics, and distribution in cells examined.
Corbett et al. (1998)
Human
Plasma, blood
Reduction in plasma quantified in fed/fasted individuals.
Devoy et al. (2016)
Human
Plasma, RBC,
whole blood
Uptake and retention by RBCs for different Cr VI and Cr III
species.
Kortenkamp et al.
(1987)
Human
RBC
Cellular uptake rates analyzed.
Richelmi et al. (1984)
Rat
RBC, plasma
Reduction of Cr VI in RBC and plasma observed.
Sakurai et al. (1999)
Rat
Blood
Reduction and fate in blood (focus on pentavalent, Cr V).
Wiegand et al. (1985)
Human,
rat
RBC
Uptake into RBC analyzed.
aNotes (yes/no) whether data from a study were used qualitatively or quantitatively in a published PBPK model.
Table C-25. In vitro studies primarily examining distribution and reduction
mechanisms
Human
Rat
Liver
Jannetto et al. (2001)
Aiyar et al. (1992)
Lewalter and Korallus (1989)
Alexander et al. (1982)
Levina et al. (2007)
Alexander et al. (1986)
Mvers and Mvers (1998)
Arillo et al. (1987)
Pratt and Mvers (1993)
De Flora etal. (1985)
Garcia and Jennette (1981)
Gruber and Jennette (1978)
Gunaratnam and Grant (2001)
Mikalsen et al. (1989)
Mikalsen et al. (1991)
Ohta etal. (1980)
Rossi and Wetterhahn (1989)
Rossi et al. (1988)
Standeven and Wetterhahn (1991a)
Ueno et al. (1990)
Wiegand and Bolt (1985)
Wiegand et al. (1986b)
Lung
Harris et al. (2005)
De Flora etal. (1985)
Krawic et al. (2017)
Standeven and Wetterhahn (1992)
Luczak et al. (2016)
Suzuki (1988a)
Levina et al. (2007)
Suzuki and Fukuda (1990)
Petrilli et al. (1986)
Petruzzelli et al. (1989)
Wong et al. (2012)
RBC
Buttnerand Beversmann (1985)
Buttner etal. (1988)
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Supplemental Information—Hexavalent Chromium
Human
Rat
Ormos and Manvai (1974)
Ormos and Manvai (1977)
Ottenwalder et al. (1987)
Ottenwaelder et al. (1988)
Wiegand et al. (1984b)
Wiegand and Ottenwaelder (1985)
Wiegand et al. (1986a)
Other
Arslan et al. (1987) (thymocytes)
Berndt (1976) (kidney)
Debetto et al. (1988) (thvmocvtes)
Liu et al. (1997) (skin)
Mertz et al. (1969) (embrvo)
Standeven and Wetterhahn (1991a) (kidnev)
Miscellaneous systems
Denniston and Uveki (1987), Ortega et al. (2005), Sehlmever et al. (1990), Sognier et al. (1991): Chinese hamster
ovary
Dillon et al. (2002): Chinese hamster lung
Kitagawa et al. (1982): Bovine RBCs.
Krepkiy et al. (2003): Rabbit liver metallothionein
Merritt et al. (1984): Rabbit blood
O'Brien et al. (1992): Glutathione and other thiols (not specific to a particular tissue or species).
Wei et al. (2016): HeLa cells and MCF-7 cells.
Wada et al. (1983): Dog liver.
Table C-26. Human biomonitoring and biomarker studies
Reference
Biomarker and industry/exposure notes
Bertram et al. (2014)
Urine/Welding (controlled experiment)
Black et al. (2015)
Urine/House dust (remediation study)
Caglieri et al. (2006)
Goldoni et al. (2006)
Goldoni et al. (2010)
Exhaled breath, plasma, RBCs, urine/Chrome plating
Cena et al. (2015)
Lung deposition (via deposition sampler)/Welding
Chang et al. (2006)
Whole blood/Residents living near electroplating factories
Coniglio et al. (1990)
Urine/Review
Gargas et al. (1994)
Urine/Human volunteer study of ingested chromite ore processing residue in
soil
Goldoni et al. (2008)
Exhaled breath, pulmonary tissues/Lung cancer patients
Kalahasthi et al. (2006)
Plasma/Chrome plating (Cr(VI) and Cr(lll) workers)
Lukanova et al. (1996)
Lymphocytes, RBCs, urine/Chrome plating
Mignini et al. (2009)
Urine blood/Leather working
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Supplemental Information—Hexavalent Chromium
Reference
Biomarker and industry/exposure notes
Mignini et al. (2004)
Miksche and Lewalter (1995)
RBCs, plasma, urine, whole blood/Review of multiple studies and workshop
proceedings containing some original data
Minoia and Cavalleri (1988)
Plasma, RBCs, urine/Dichromate-producing factory (multiple job categories)
Minoia et al. (1983)
Urine/Workers exposed to Cr(VI) and Cr(lll)
Muttamara and Leong (2004)
Blood, urine/Chromium alloy factory
Nomivama et al. (1980)
Urine/Population from geographic areas of known chromium pollution
Ohta and Inui (1992)
Lung tissue (autopsy)/Chromate factory
Pierre et al. (2008)
Urine/Chrome plating
Martin Remv et al. (2021)
Urine/Chrome plating
Santonen et al. (2022)
Urine, RBC, exhaled breath condensate, dermal samples/Multiple industries
Siogren et al. (1983)
Welinder et al. (1983)
Urine/Stainless steel welding
Verdonck et al. (2021)
Viegas et al. (2022)
Urine, RBC, blood, exhaled breath condensate/Multiple industries
Verschoor et al. (1988)
Urine/Chrome plating
Zhao et al. (2020)
Urine/Residential exposure
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Supplemental Information—Hexavalent Chromium
Table C-27. Gastric emptying rates for rats, mice, and humans expressed as
half-emptying time (T1/2) and transit time (KLSD). Vehicle indicated in
parentheses if known.
T1/2 (minutes)
KLSDa (h1)
Reference
Rat
17
2.4
Kirman et al. (2012)
15 (fed)
2.77
GastroPlus defaults
7.5 (fasted)
5.55
77 (liquid/semisolid)
0.54
Qualls-Creekmore et al. (2010)
118 (solids)
0.35
Enck and Wienbeck (1989)
1.1 fasted (liquid)
38
Takashima et al. (2013)
62 fed, 9 fastedb (liquid)
0.67, 4.6
Poulakos and Kent (1973)
119-138 (solid)
0.30-0.35
Schoonians et al. (2002)
21-27 (semisolid)
1.5-2
Purdon and Bass (1973)
4.95 fasted (liquid)
8.4
Kataoka et al. (2012)
16.5 (liquid)
2.52
Scarpienato et al. (1984)
Mouse
4.4
9.4
Kirman et al. (2012)
9.6 (fed)
4.33
GastroPlus defaults
2.4 (fasted)
17.3
30.6
1.36
Inada et al. (2004)
16-17 fed (semisolid)
2.60
Roda et al. (2010)
2 fasted (semisolid)
20.8
9-11 (liquids)
3.78-4.62
Svmonds et al. (2002)
158 (solids)
0.26
20 (nonnutrient liquid)
2.08
Svmonds et al. (2008)
36 (nutrient liquid)
1.16
91 (solids)
0.46
Choi et al. (2007)
30.6 (semisolid)
1.36
Osinski et al. (2002)
10 (nonnutrient liquid)
4.2
Miyasaka et al. (2004)
90 min (young mice);
58-67 min (old mice);
pharmaceuticals
0.46 (young);
0.62-0.72 (old)
De Smet et al. (2006)
28 (solids, 19-38)
1.49 (1.09-2.19)
Bennink et al. (2003)
15 (liquids, 11-19)
2.77 (2.19-3.78)
Human
35
1.2
Kirman et al. (2013)
13 (liquid, fasted)
3.20
Mudie et al. (2014)
Fasted
2.63; 3.47; 0.55
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Supplemental Information—Hexavalent Chromium
Ti/2 (minutes)
KLSDa (h1)
Reference
15.8 (water); 12 (saline);
75 (glucose)
Mudie et al. (2010) (review article; see citation
for further details of individual studies)
Fed
44 ± 15, 40 ± 13, 32 ± 7, 48 ± 9,
76 ± 6 (liquids); 105 ± 21 (solids)
0.55-1.30 (liquids); 0.40
(solids)
30
1.39
ICRP (2006, 2002)
30 (fed)
1.39
GastroPlus defaults
7.5 (fasted)
5.55
aKLSD = Ioge(2)/T1/2 x 60.
bPoulakos and Kent (1973) values from gastric emptying equation (l-exp(-t/tau), tau = 13 minutes fasted, 89
minutes fed, derived assuming 90% emptying at 30 minutes for the fasted state, 74% emptying at 120 minutes for
the fed state).
1 Time-weigh ted average daily doses differ from doses presented in the evidence tables of the
2 April 2014 preliminary materials document (U.S. EPA. 2014b 1 because those values were based on
3 the average of three lifestages (and not weekly/monthly time-course data). Round-off error
4 occurred at the low doses due to lack of significant figures reported in NTP lifestage summary data.
5 Time-weighted average daily doses for mice and rats are presented in Tables C-28 and C-29,
6 respectively. Lifetime average daily internal doses for the rat during the NTP 2-year bioassay (at
7 different data collection times) are presented in Table C-30.
Table C-28. Time-weighted average daily doses in mice for the NTP (2008)
2-year bioassay of sodium dichromate dihydrate. Doses in mg/kg-day Cr(VI).
Original average daily dose
(mg/kg-d)
Time-weighted average daily
dose (mg/kg-d)
Percent difference
Female mice
0.38
0.302
20
1.4
1.18
15
3.1
3.24
4
8.7
8.89
2
Male mice
0.38
0.450
18
0.91
0.914
0.4
2.4
2.40
<0.1
5.9
5.70
3
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Supplemental Information—Hexavalent Chromium
Table C-29. Time-weighted average daily doses in rats for the NTP (2008) 2-
year bioassay of sodium dichromate dihydrate. Doses in mg/kg-day Cr(VI).
Original average daily dose
(mg/kg-d)
Time-weighted average daily
dose (mg/kg-d)
Percent difference
Female rats
0.24
0.248
3
0.94
0.961
2
2.4
2.60
8
7
7.13
2
Male rats
0.21
0.200
4
0.77
0.796
1
2.1
2.10
<0.1
5.9
6.07
3
Table C-30. Time-weighted average daily doses in rats for the NTP f20081 2-
year bioassay of sodium dichromate dihydrate at different time periods. Doses
in mg/kg-day Cr(VI).
Cr(VI)
concentration
TWA dose at 2 years
(mg/kg-d)
TWA dose at 1 year
(mg/kg-d)
TWA dose at 90 days
(mg/kg-d)
Females
5
0.248
0.0294
-
20
0.961
1.14
-
60
2.60
3.01
-
180
7.13
8.28
-
Males
5
0.200
0.237
0.401
20
0.796
0.938
1.58
60
2.10
2.49
4.16
180
6.07
7.19
11.7
TWA BW at 2 years: 450 g (males) and 260 g (females). TWA BW at 1 year: 395 g (males) and 215 g (females). No
dose-response data for female rats at 1 year for this assessment.
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1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
Supplemental Information—Hexavalent Chromium
C.2. SUPPORTING EVIDENCE FOR SPECIFIC HEALTH EFFECTS
C.2.1. Respiratory Effects
C.2.1.1. Mechanistic studies relevant to noncancer respiratory toxicity
Mechanistic evidence investigating the biological pathways involved in respiratory toxicity
following the inhalation of Cr(VI) is summarized in Table C-31. Studies identified in preliminary
title and abstract screening as "mechanistic" were further screened and tagged as "inhalation" if
they were studies of humans or animals exposed via inhalation or intratracheal instillation and
conducted in lung tissues or cells or in cells derived from lung tissues. Studies of systemic toxicity
following inhalation exposures are summarized in Appendix C.3.2. A total of 255 potentially
relevant respiratory mechanistic studies were identified. A prioritization strategy was used to
identify the evidence most informative to chronic human exposures:
• Studies of respiratory organs and tissues from humans with quantified inhalation exposure
to Cr(VI)
• Experimental animal (mammalian) studies of respiratory organs and tissues exposed to
Cr(VI) via inhalation or intratracheal instillation
• In vitro studies in human primary or immortalized cells derived from respiratory tissues
• Any outcome measured in lung tissues except for those relevant to genotoxicity (see
Appendix C.3.2.2)
Forty-one studies meeting these prioritization criteria were identified; these studies
focused primarily on oxidative stress, apoptosis, and cellular toxicity of the lung. Mechanistic
evidence relevant to Cr(VI)-induced genotoxicity is reviewed in Appendix C.3.2.2.
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Supplemental Information—Hexavalent Chromium
Table C-31. Mechanistic studies prioritized for informing potential Cr(VI)-induced respiratory toxicity
System
Exposure3
Results
Comments
Reference
Oxidative stress
Exposed: lead chromate
pigment factory workers
(n = 22)
Referents: office workers
from chromate factory
(n = 16)
Mean (SD) duration of work
among chromate pigment
workers = 9.7 (20.5)* yr.
Chromium measured in urine,
blood, and air; air sampling for
200 min at flow rate 2-3 L/min;
urine and blood measured with
flameless atomic absorption
spectrophotometer.
Chromium in air ranged from
below LOD (0.0005 mg/m3
among office workers to 0.5150
mg/m3 in high-exposure area of
factory (pulverizing process);
mean (SD) chromium among
exposed group in blood:
6.75 (3.30) ng/L; in urine:
12.97 (16.31) (ng/g creatinine).
In blood and sputum:
No difference in 8-OHdG
adducts (in respiratory
epithelial and white
blood cells) between
exposed and control
groups, or with duration
of employment among
exposed groups
Chromium levels in blood (which are a marker of
recent exposure) were similar between exposed
and control groups; this suggests that exposure
misclassification might be contributing to the
null effects reported in the study
The authors also suggest urinary chromium
reflects chromium in reduced form, which might
not reflect genotoxicity in blood cells
No adjustment for supplements/vitamins or diet
*SD appears incorrect
Kim et al.
(1999)
Rat, Sprague-Dawley
0.25 mg/kg Na2Cr2C>7 (0.09 mg
Cr(VI)/kg) per day via
intratracheal instillation, 3 d.
1" 8-OHdG adducts
([32P] postlabeling) were
detected in lung, but not
liver
1" DNA-protein
crosslinks
1" DNA fragmentation
No measure of cytotoxicity
Izzotti et al.
(1998)
Rat, Sprague-Dawley, male
0.18 or 0.9 mg/m3 Na2CrC>4
solution mist inhalation, whole-
body exposures in 1 m3
volumetric inhalation chambers
for 1, 2, or 3 wk
1" 8-OHdG in lung only
at 1 wk (only stat. sig. for
0.18 mg/m3)
4/ 8-OHdG repair 1-3 wk
Cr levels confirmed in inhalation chambers with
personal air samplers and measured in whole
blood and urine
Indicates Cr(VI) exposure both increases
oxidative DNA damage and inhibits repair of
these lesions
Maeng et al.
(2003)
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Supplemental Information—Hexavalent Chromium
System
Exposure3
Results
Comments
Reference
Cr levels in blood and urine
increased with dose and
duration.
Rat, Sprague-Dawley, male
0.063 or 0.630 mg Cr/kg (as
K2Cr2C>7) via intratracheal
instillation, lx/wk, 4 wk
-t 8-OHdG
-t NF-kB; 4, CC16 in
club cells
Weekly instillations allow recovery period, which
could underestimate the responses, but
significant effects were still reported.
Also T* relative lung weight, T* albumin and
total protein level in BALF
Zhao et al.
(2014)
Apo ptosis
Exposed: Chromium workers
diagnosed with lung cancer
(n = 67 males)
Referent: male controls with
lung cancer but without
known exposure to
chromium (n = 104)
Mean exposure time 16.7 ± 10.0
(SD) yr (range 1-41 yr).
Total and hexavalent Cr
measured in soil and air samples
taken "in the vicinity of the
workplace" using atomic
absorption spectrometry.
Mean values of Cr(VI) in air of
smelting plants was 0.019-0.03
mg/m3. Soil chromium was 137
mg/kg.
In lung cancer tissues
(preserved in paraffin
blocks):
4/ survivin protein levels
(anti-apoptotic)
T* p53 protein levels
(pro-apoptotic)
The information regarding potential exposure is
sparse. Observed differences in the type of lung
cancer between exposed and referent could
impact results. No information on smoking,
which may be important to consider given all
participants had lung cancer.
Halasova et al.
(2010)
Rat, Sprague-Dawley, male
0.25 mg/kg Na2Cr2C>7 per day via
intratracheal instillation, 3 d.
T* apoptosis in bronchial
epithelium and lung
parenchyma
T* 13/18 apoptosis-
related genes (cDNA
array analysis) in lung
Exposures to Cr(VI) alone. TUNEL analysis, used
to measure apoptosis, is a sensitive method of
detection.
State another lab saw no lung cancer after
similar treatment for 30 mo, so predict
apoptosis is protective post-genotoxicity
D'Agostini et al.
(2002)
Lung cellular responses
Exposed: Electroplaters
(n = 42 females)
Referent: Jail wardens,
frequency matched on age,
BMI, alcohol, and smoking
(n = 43 females)
Cr(VI) in plasma measured using
atomic absorption
spectrophotometry.
Total Cr was not different
between exposed and referent
T* cytotoxicity in
exfoliated buccal and
nasal mucosa
Workers performed bright plating that has lower
potential for Cr(VI) exposure, and state that
there was good compliance with PPE usage. This
might account for the low plasma Cr(VI) levels
and similarity between exposed and referent.
Co-exposure to cobalt occurred, although levels
Wultsch et al.
(2017)
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Supplemental Information—Hexavalent Chromium
System
Exposure3
Results
Comments
Reference
(means of 0.44 and 0.41 ng/L,
respectively).
were not different between exposed and
referent.
High prevalence of smoking (frequency matched
between exposed and referent), which could
affect results.
Rat, F-344, male
360 ng/m3 K2C1-O4 via inhalation,
5 h/d, 5 d/wk, 2 or 4 wk.
1" total recoverable
cells, neutrophils (PMN),
and monocytes at 2 and
4 wk in BALF; decline at
4 wk compared to 2 wk
4/ % PAM in BALF; no
change in total PAM
levels
No changes in cell
viability (80-90%) among
exposure groups
Moderately informative: shorter exposure
period but results generally support similar
findings from chronic duration studies from
same group.
Ex vivo PAMs (exposed in vivo to foCrCU):
• Spontaneous: T* H2O2, no changes in
superoxide anion
• LPS-inducible: T* NO, 4^ IL-1 and TNFa,
-t IL-6
Cohen et al.
(1998)
Rat, Long-Evans hooded,
male
2 ng CaCrC>4 (insoluble) or
2 ng CrC>3 (soluble) via
intratracheal instillation, 9 h.
In vivo exposure: no
effect on cell viability
In vitro exposure: 4'
viability
Less informative: short exposure period; trypan
blue dye exclusion is a less sensitive measure to
determine cell viability
Galvin and
Oberg (1984)
Rat, Sprague-Dawley, male
and female
0.01, 0.05, 0.25 mg/kg Na2Cr2C>7-
2HzO, 5x/wk, or 0.05, 0.25, 1.25
mg/kg, lx/wk via intratracheal
instillation, 30 wk.
Tumors that appeared to
arise from tissues with
cellular inflammatory
foci involving alveolar
macrophages,
proliferation of
bronchiolar epithelium
or alveolar type II cells,
and chronic
inflammatory thickening
of alveolar septa. The
other main type of
nontumor lesion was
severe damage to the
bronchioloalveolar
region with alveolar
Steinhoff et al.
(1986)
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Reference
atelectasis and
subsequent confluent
fibrosis.
Rat, Sprague-Dawley, male
0, 0.063 and 0.630 mg Cr/kg (as
K2Cr2C>7) via intratracheal
instillation, lx/, 4 wk
1" relative lung weight
1" albumin and total
protein level in BALF
-t NF-kB; 4, CC16 in
club cells
More informative: weekly instillations allow
recovery period, which could underestimate the
responses, but significant effects were still
reported.
Also -t 8-OHdG
Zhao et al.
(2014)
In vitro studies of oxidative stress, cellular toxicity, and death in primary and immortalized human lung cells
HLF fetal human lung
fibroblasts
L-41 human epithelial-like
cells
1, 2, 5, 10, 15, 20, 25, and 30 nM
K2Cr207, 2, 24 or 48 h
1" cytotoxicity (MTT
assay), dose- and
duration dependent
(significant >20 nM);
cytotoxicity recovered
<5 nM after 24 h
-t ROS (DCFH-DA) at 2 h
1" antioxidant enzymes
(glutathione peroxidase,
glutathione reductase,
catalase) 1-5 nM
Oxidative stress and antioxidant enzymes
induced at mildly toxic nM concentrations
Asatiani et al.
(2011; 2010)
H460 human lung epithelial
cells
10-50 nM l\la2Cr207, 12 h
-t ROS
1" apoptosis; abrogated
by antioxidants MnTBAP,
catalase, DPI, or ROT, in
cells transfected with
antioxidant enzymes
SOD or GPx, or by
specific caspase
inhibitors
4/ Bcl-2; abrogated by
MnTBAP
Cr(VI) induces apoptosis by downregulating Bcl-2
via superoxide anion-mediated ubiquitin-
proteasomal degradation and mitochondrial
caspase-9 activation
Azad et al.
(2008)
Human lung epithelial cells
1" Src family kinases
(SFK) -> 1" JNK
SFK activation was not completely reliant on ROS
signaling
Barchowsky
(2006)
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Reference
BEAS-2B human bronchial
epithelial cells
0.3 (nontoxic) or 1.8 (toxic) nM
Cr(VI), 48 h
Cytotoxic signaling
pathways: glycolysis
regulation (GSK3beta,
p70S6K), oxidative stress
and inflammation (JNK,
MTF-1), and protein
degradation (UBC)
Bruno et al.
(2016)
A549 human lung
adenocarcinoma cells
BEAS-2B human bronchial
epithelial cells
0.5,1, and 2 nM Cr(VI)
(compound not reported), 3, 8,
or 24 h
In BEAS-2B:
1" cytotoxicity (>1 nM;
MTT)
4/ glutathione (3 h only)
'Y lipid peroxidation
(TBARS)
'Y heme oxygenase-1
(HO-1)
A549:
'Y lipid peroxidation
(TBARS)
BEAS-2B cell line more sensitive to Cr(VI) effects
than A549 cell line; polymorphisms for GST
genes might be responsible for differing cellular
responses to Cr(VI)
Caglieri et al.
(2008)
HLF human lung fibroblasts
(LL-24 cell line)
3, 6, and 9 nM Na2CrC>4, 24 h
'Y cytotoxicity, duration
and dose dependent
(stat. sig. >6 nM)
'Y apoptosis
-t p53 (4-6 fold)
Pretreatment with 1 mM ascorbate or 20 nM
tocopherol had no ameliorative effects
Also 'Y Cr-DNA adducts
Carlisle et al.
(2000a)
A549 (human lung
adenocarcinoma) and
BEAS2B human bronchial
epithelial cells
0.1, 0.5, 1.0 and 10 nM Na2CrC>4,
0.5,1, and 4 h
*Y apoptosis at 10 nM
(caspase-3 activity and
morphology)
Oxidative role in DNA damage decreased with
time at lower Cr(VI) concentrations and
increased with time at higher concentrations
A549 more sensitive than BEAS2B
Also 'Y oxidative DNA damage (Fpg-modified
comet assay)
Cavallo et al.
(2010)
BEAS-2B human bronchial
epithelial cells
1 nM Cr(VI), 48 h
'Y glycolysis
4/ respiration
Cr(VI) caused shift to fermentative metabolism
Cerveira et al.
(2014)
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4/ protein levels of
P-Fl-ATPase
-t GAPDH
Human non-small cell lung
carcinoma CL3 cells
10-80 nM K2Cr207, 1-12 h
1" JNK
-t MAPK11-14 (P38)
-t MAPK3, MAPK1
(ERK1/2)
Activation increased with dose and duration
Use of multiple oxidants and antioxidants shows
activation of these redox-initiated pathways do
not clearly correlate with Cr(VI)-induced
cytotoxicity
Chuang et al.
(2000)
BEAS-2B human bronchial
epithelial cells transformed
by chronic Cr(VI) exposure
Na2Cr2C>7
In Cr(VI)-transformed
cells:
1" metabolic adaptation
and antioxidant defense,
ATP production and
mitochondrial proton
leak via SIRT3
1" mitophagy proteins
Pinkl and PRKN (Parkin),
though mitophagy was
suppressed
SIRT3 upregulation by Cr(VI) suppresses
mitophagy; knockdown of SIRT3 suppressed cell
proliferation
NRF2 constitutively activated in
Cr(VI)-transformed cells
Clementino et
al. (2019)
BEAS-2B human bronchial
epithelial cells
5-20 nM Na2Cr207
-t NOTCH1 (Notchl)
-t CDKN1A (P21)
4/ FBP1
FBP1, involved in gluconeogenesis, is lost in
Cr(VI)-transformed cells
Reintroduction of FBP1 caused "I^ROS and
^apoptosis
Dai et al.
(2017a)
LL 24 human lung cells and
A549 human lung
adenocarcinoma cells
5-200 nM Cr(VI)
1" heme oxygenase gene
(only in LL 24 cells)
No effect on catalase,
GST, glutathione
reductase, Cu/Zn- and
Mn-SODs, GPx,
NAD(P)H:quinone
oxidoreductase, or IL-8
gene expression
RT-PCR and northern blot gene (RNA) expression
analyses
Authors conclude heme oxygenase is
responsible for Cr(VI)-induced stress responses
and not intracellular increases in glutathione and
ROS
Dubrovskaya
and
Wetterhahn
(1998)
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BEAS-2B human bronchial
epithelial cells
MOLT-4 lymphoblastic
leukemia cell line
0.5, 3, 6, 9, and 200 nM K2Cr2C>7,
4,12, or 24 h
1" apoptosis (PI; TUNEL
flow), dose and time
dependent
p53 at 0.5 |aM (12 h)
and 3 nM (4 h) in MOLT-
4 but not BEAS-2B cells
Inhibition of caspase-3,
-8 and -9 did not reduce
apoptosis
Cr(VI) induces apoptosis that could involve p53
in MOLT-4 cells but not in BEAS-2B; apoptosis
did not involve caspases 3, 8, or 9 in these cells
Gambelunghe
etal. (2006)
A549 human lung
adenocarcinoma cells
0.2 nM K2Q2O7, 6, 12, or 24 h
1" endoplasmic
reticulum (ER) stress via
-t GRP78 and p-PERKis
associated with "T*
apoptosis and autophagy
4/ mitochondrial
membrane potential
(MMP) at 6-12 h but not
24 h
Inhibiting ER stress (4PBA) reduced apoptosis
and autophagy
Suppressing apoptosis (Z-VAD-FMK) also
suppressed autophagy
Inhibiting autophagy (3-MA) increased apoptosis
Authors surmise Cr(VI)-induced autophagy
rescues 4' MMP at 24 h via phagocytosing
damaged mitochondria and then inhibiting
apoptosis
Ge et al. (2019)
A549 human lung
adenocarcinoma cells
10-500 nM NazCrzCb, 1 or 16 h
-t 8-OHdG
4/ OGGl mRNA, dose
dependent (RT-PCR and
RNase protection assay);
not affected by adding
H2O2
No effect on hAPE or
GAPDH
Authors conclude Cr(VI)-induced oxidative DNA
damage could be due partly to a reduced
capacity to repair endogenous and Cr(VI)-
induced 8-OHdG lesions
Also 1" DNA strand breaks, dose dependent
(comet assay) that were 10x higher with FAPY
Hodges et al.
(2002; 2001)
A549 human lung
adenocarcinoma cells
12.5-800 nM Cr(VI)
1" ROS and NF-kB, dose
dependent
Effects abrogated by
catalase, SOD, or
D-mannitol
No change in 8-OHdG
levels or hoggl
expression
Possible that <800 nM doses of Cr(VI) are
sufficient to induce ROS and NF-kB but too low
to induce oxidative DNA lesions
Kim et al.
(2003)
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A549 human lung
adenocarcinoma cells
5-80 nM NazCrzCb, 2 h
1" cytotoxicity >5 nM,
dose dependent
Cr(VI) + 1 mM ascorbate
-t ROS
Cr(VI) + glutathione
4, ROS
Ascorbate (max intracellular 80 nM) might
promote Cr(VI)-induced oxidative stress by
reducing intracellular Cr(VI) and stabilizing Cr(VI)
and Cr(IV)
Martin et al.
(2006)
Primary human bronchial
epithelial cells
BEAS-2B human bronchial
epithelial cells
25 and 50 nM Na2CrC>4, 3 or 6 h
Irreversible inhibition of
thioredoxin reductase
(TrxR)
Oxidation of protein
thiols thioredoxins (Trx)
and peroxiredoxins (Prx);
scavenging peroxynitrite
(MnTBAP) or adding
ascorbate did not
abrogate these effects
Inhibition of aconitase,
electron transport
complexes 1 and II
Cr(VI) oxidizes and inhibits mitochondrial and
cellular thioredoxins and peroxiredoxins
involved in cell survival and redox signaling,
leading to increased sensitivity to ROS damage
and decreased survival
Myers et al.
(2011; 2010;
2009; 2008)
A549 human lung
adenocarcinoma cells
10 nM Cr(VI)
-t ROS and JNK
activation at
subcytotoxic levels
1" Src family kinases
(Fyn, Lck) at levels that
did not induce ROS
O'Hara et al.
(2003)
BEAS-2B human bronchial
epithelial cells
SAECs (human small airway
epithelial cells)
0.2, 2.0, 20, and 200 nM
K2Cr2C>7,1, 2, 6, or 48 h
1" cytotoxicity (MTT
assay) at 0.2 nM (20%) in
BEAS-2B, 20 nM in SAEC,
dose dependent
In SAECs:
1" cellular
phosphoprotein
-t IL-6, IL-8
(precytotoxic, at 0.2 and
2.0 nM, respectively)
Cytotoxicity associated with inflammation and
immune response via protein phosphorylation
and cytokine signaling
Pascal and
Tessier (2004)
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Null for TNF-a
A549 human lung
adenocarcinoma cells
0.13, 0.67, 3.38, 16.9, and
84.57 nM CrC>3 or K2Cr2C>7
1" cytotoxicity >3.38 nM
(colony formation assay),
dose dependent
Cytotoxicity induced at nM concentrations
Popper et al.
(1993)
Primary human lung IMR90
fibroblasts
H460 human lung epithelial
cells
0.2-8 nM K2Cr04,3 h
-t DNA DSB with
ascorbate caused by
aberrant mismatch
repair
1" cytotoxicity and
apoptosis with
ascorbate; effects
reversed by suppressing
DNA mismatch repair
but p53 status had no
effect
1^1" cytotoxicity and cell
cycle delay in cells
deficient in oxidative
DNA damage repair
(XRCC1 knockdown);
effects reversed by
ascorbate
By restoring intracellular ascorbate to
physiological levels via DHA (max intracellular
0.9 mM), it was shown ascorbate can suppress
Cr(VI)-induced oxidative damage but promotes
Cr-DNA lesions that are either repaired by
mismatch repair, independently of p53, or lead
to cytotoxicity and apoptosis
Chromosomal aberrations not affected by XRCC1
status
Reynolds et al.
(2012; 2007;
2007)
A549 human lung
adenocarcinoma cells
1-20 nM Na2Cr207, 4 or 12 h
1" cytotoxicity with dose
(stat. sig. at 20 nM) at
4 h
4/ specific activity and
level of urokinase-type
plasminogen activator
(uPA) activity
1" uPA receptor protein
Cr(VI) inhalation leads to a net loss of urokinase-
type plasminogen activator activity that has
been shown to promote pulmonary fibrosis
Shumilla and
Barchowskv
(1999)
A549 human lung
adenocarcinoma cells
0.05, 0.1, 0.2, 0.4, 0.8, 1.6,3.2,
6.4, and 12.8 nM K2Cr207, 24 h
1" cell proliferation
<0.2 nM (A549 cells)
1" cytotoxicity >3.2 nM
Cr(VI)-induced autophagy is correlated with
transcription factor HMGA2 expressed in lung
cancer patients
Yang et al.
(2017)
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1" autophagosomes; this
effect was blocked by
silencing HMGA2
1" expression of LC3II,
Atgl2-Atg5, Atg4, AtglO,
HMGA1 and HMGA2
proteins
4/ expression of p62
BALF: bronchoalveolar lavage fluid.
ICP-AES: inductively coupled argon plasma atomic emission spectroscopy.
MMA-SS: manual metal arc-stainless steel.
PAM: pulmonary alveolar macrophages.
aPotassium dichromate units conversion: Cr(VI) = 0.353 x K2Cr2C>7; potassium chromate units conversion: Cr(VI) = 0.268 x foCrCU; sodium dichromate dihydrate
units conversion: Cr(VI) = 0.349 x Na2Cr20?2H20 (usually denoted as Na2Cr2C>7 because study authors frequently list the salt as the chemical compound even if
concentration or dose is based on the dihydrate); chromium trioxide units conversion: Cr(VI) = 0.52 x CrC>3.
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C.2.2. Gastrointestinal Effects
C.2.2.1. Apical outcomes relevant to toxicity of the gastrointestinal tract
1 The results relevant to GI tract toxicity from the four high confidence animal studies
2 synthesized in Section 3.2.2.2 of the toxicological review (Thompson etal.. 2012c: Thompson etal..
3 2011b: NTP. 2008. 2007f) are summarized in Table C-32. In addition to these four studies are other
4 reports that continued to evaluate the same tissues from these studies, and a fifth study (Thompson
5 etal.. 2015b) that was evaluated only for genotoxicity endpoints but also reported evidence of
6 hyperplasia and Cr accumulation in GI tissues following drinking water exposures.
Table C-32. Experimental animal studies providing apical evidence of toxic
effects of ingested Cr(VI) in the GI tract
System
Exposure3
Results
Reference
Mouse
(B6C3F1),
male and
female
0, 22, 44, 87, 174, 349
mg/L Cr(VI)
0, 3.1, 5.3, 9.1, 15.7, 27.9
mg/kg-d Cr(VI)
90 d
Diffuse epithelial hyperplasia of the duodenum
(>3.1 mg Cr(VI)/kg-d)
Duodenal villi short, thick, and blunted, with
cytoplasmic vacuolization in the epithelial cells lining
the villi tips (all doses, not quantitatively measured)
NTP (2007f)
Mouse,
BALB/c,
C57BL/6, and
B6C3F1, male
(strain
comparison
study)
0, 22, 44, 87 mg/L Cr(VI)
0, 2.8, 5.2, 8.7 mg/kg-d
Cr(VI)
90 d
Diffuse epithelial hyperplasia of the duodenum
(>2.8 mg Cr(VI)/kg-d)
Rat, F344/N,
male and
female
0, 22, 44, 87, 174, 349
mg/L Cr(VI)
0, 1.7, 3.5, 5.9, 11.2, 20.9
mg/kg-d Cr(VI)
90 d
Epithelial hyperplasia, squamous metaplasia, and
ulcers in the glandular stomach (20.9 mg/kg-d)
Rat (F344/N),
male
0, 5, 20, 60, or 180 mg/L
Cr(VI)
0.200, 0.760,2.10,6.07
mg/kg-d Cr(VI)
2 yr
No observed GI hyperplasia/metaplasia or stomach
ulcers
No salivary gland atrophy
NTP (2008)
Rat (F344/N),
female
0, 5, 20, 60, or 180 mg/L
Cr(VI)
0.248, 0.961,2.60,7.13
mg/kg-d Cr(VI)
2 yr
No observed GI hyperplasia/metaplasia or stomach
ulcers
Mild salivary gland atrophy at highest dose (>7.13 mg
Cr(VI)/kg-d)
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Mouse
(B6C3F1),
male
0, 5, 10, 30, or 90 mg/L
Cr(VI)
0.450, 0.914, 2.40, or
5.70 mg/kg-d Cr(VI)
2 yr
Diffuse epithelial hyperplasia of the duodenum
(>0.45 mg Cr(VI)/kg-d)
Focal epithelial hyperplasia >2.40 mg/kg-d, not
statistically significant
Short, broad, and blunt duodenal villi (no overt
damage, necrosis, or degeneration indicative of
atrophy)
Mouse,
(B6C3F1),
female
0, 5, 20, 60, or 180 mg/L
Cr(VI)
0.302, 1.18, 3.24, or 8.89
mg/kg-d Cr(VI)
2 yr
Diffuse epithelial hyperplasia of the duodenum
(>0.3 mg Cr(VI)/kg-d) and jejunum (8.89 mg/kg-d)
Focal epithelial hyperplasia >3.24 mg/kg-d, not
statistically significant
Short, broad, and blunt duodenal villi (no overt
damage, necrosis, or degeneration indicative of
atrophy)
Mouse,
B6C3F1
female
Oral, drinking
water
0,0.1, 1.4, 4.9, 20.9, 59.3,
and 181 mg/LCr(VI)
0,0.024, 0.32, 1.1, 4.6,
11.6, or 31.1 mg/kg-d
Cr(VI)
7 d (n = 5) or 90 d (n = 10)
7-d:
Duodenal hyperplasia (no statistically significant
change), villous atrophy (no statistically significant
change), and cytoplasmic vacuolization (statistically
significant at 31.1 mg/kg), with no change in crypt
apoptosis indices, mitotic activity, or increases in
karyorrhectic nuclei in crypts
90-d:
Diffuse epithelial hyperplasia of the small intestine
(>11.6 mg Cr(VI)/kg-d)
Villous atrophy in duodenum and jejunum (31.1
mg/kg-d)
Apoptosis in duodenal villi (31.1 mg/kg-d)
Cytoplasmic vacuolization in duodenum and jejunum
(>4.6 mg Cr(VI)/kg-d
Thompson et al.
(2011b)
Rat, Fischer
344/N female
Oral, drinking
water
0, 0.1, 1.4, 20.9, 59.3, and
181 mg/L Cr(VI)
0, 0.015, 0.21, 2.9, 7.2,
20.5 mg/kg-d Cr(VI)
7 d (n = 5) or 90 d (n = 10)
Diffuse epithelial hyperplasia of the small intestine
(>7.2 mg Cr(VI)/kg-d), villous cytotoxicity (>7.2 mg
Cr(VI)/kg-d)
Apoptosis in duodenal villi (>7.2 mg Cr(VI)/kg-d) (no
atrophy or vacuolization)
7 d: No statistically significant changes in GSH/GSSG
in oral mucosa or small intestine except in jejunum at
20.5 mg/kg-d and at 0.015 mg/kg-d in the oral
mucosa. Note: sample size is 5 for 7-d data.
90 d: 4/ GSH/GSSG in oral mucosa and jejunum
(>2.9 mg/kg-d) and in plasma (>7.2 mg/kg-d), dose-
dependent, statistically significant. No changes in
duodenum, or signs of lipid peroxidation (8-
isoprostane) in any tissues.
Thompson et al.
(2012c)
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Mouse,
B6C3F1
Oral, drinking
water
0,0.1, 1.4, 4.9, 20.9, 59.3,
and 181 mg/LCr(VI)
0,0.024, 0.32, 1.1, 4.6,
11.6, or 31.1 mg/kg-d
Cr(VI)
7 and 90 d
In scraped duodenal epithelium:
T* crypt enterocyte proliferation, dose dependent
T* villus cytotoxicity (disruption of cellular
arrangement, desquamation, nuclear atypia,
blunting)
T* crypt enterocyte proliferation, dose dependent
No effect on mitotic/apoptotic indices in crypt
compartment
7 d:
T* aberrant nuclei at villi tips but not in crypts
(>11.6 mg/kg-d)
90 d:
T* aberrant nuclei at villi tips but not in crypts
(>4.6 mg/kg-d)
O'Brien et al.
(2013)
Continued
analysis of tissues
from Thompson
et al. (2011b)
Mouse
(B6C3F1) and
rat (F344),
female
Oral, drinking
water
0 and 180 mg/LCr(VI)
0 and 31.1 mg/kg-d Cr(VI)
90 d
In duodenal villi and crypts:
X-ray fluorescence (spectro)microscopy (n-XRF) was
used to image the Cr content in the villus and crypt
regions of duodena. Cr(VI) was detected in crypts,
slightly above detection limits, and was >30x higher
in villi.
Villous blunting and crypt hyperplasia in the
duodenum (lengthening of the crypt compartment
by ~2-fold)
1.5-fold increase in the number of crypt enterocytes
No aberrant foci indicative of transformation
Thompson et al.
(2015a)
Continued
analysis of tissues
from Thompson
et al. (2011b)
Mouse,
B6C3F1
Oral, drinking
water
0,1.4, 21, and 180 mg/L
Cr(VI)
0, 0.32, 4.6, and 31.1
mg/kg-d Cr(VI)
7 d
21 and 180 mg/L Cr(VI) significantly increased the
number of crypt enterocytes
Synchrotron-based X-ray fluorescence (XRF)
microscopy revealed the presence of strong Cr
fluorescence in duodenal villi, but negligible Cr
fluorescence in the crypt compartment
No effect on aberrant villous foci, and X-ray
fluorescence detection of Cr(VI)
Thompson et al.
(2015b)
aPotassium dichromate units conversion: Cr(VI) = 0.353 x K2Cr2C>7; potassium chromate units conversion:
Cr(VI) = 0.268 x K2&O4; sodium dichromate dihydrate units conversion: Cr(VI) = 0.349 x Na2Cr20?2H20 (usually
denoted as Na2Cr207 because study authors frequently list the salt as the chemical compound even if
concentration or dose is based on the dihydrate); chromium trioxide units conversion: Cr(VI) = 0.52 x CrC>3.
C.2.2.2. Mechanistic endpoints relevant to toxicity of the gastrointestinal tract
1 Studies examining mechanistic endpoints relevant to interpretations of toxic effects in the
2 GI tract are summarized in Table C-33. Studies identified in preliminary title and abstract screening
3 as "mechanistic" were further screened and tagged as "GI" if conducted in GI tissues or cells. Only
4 studies conducted in vivo in animals or in vitro in human cells from the GI tract are prioritized for
5 consideration here:
6 • Studies of gastrointestinal organs and tissues from humans with quantified exposure to
7 Cr(VI)
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1 • Experimental animal studies of gastrointestinal tissues (except liver; these studies are
2 summarized in Appendix C.2.3) using quantified oral (drinking water, gavage, diet),
3 inhalation, or intratracheal instillation exposures
4 • In vitro studies in human primary or immortalized cells derived from gastrointestinal
5 tissues
6 • Mechanistic endpoints relevant to interpretations of gastrointestinal toxicity in humans
7 except for genotoxicity studies (see Appendix C.3.2.2) (apical outcomes synthesized for
8 noncancer hazard identification have been summarized above in Appendix C.2.2.1)
9 Ten studies in experimental animals and four studies in Gl-derived cells in vitro were
10 identified. No human exposure studies of toxicity of the GI tract were identified (studies in exposed
11 workers reporting genotoxic endpoints in buccal cells are summarized in Appendix C.3.2.2).
Table C-33. Supporting mechanistic studies prioritized for informing Cr(VI)-
induced GI tract toxicity
System
Exposure3
Results
Reference
Mouse,
B6C3F1
female
Oral, drinking
water
0,0.1, 1.4, 4.9, 20.9, 59.3,
and 181 mg/LCr(VI)
0,0.024, 0.32, 1.1, 4.6,
11.6, or 31.1 mg/kg-d
Cr(VI)
7 d (n = 5) or 90 d (n = 10)
7 d: No change in crypt apoptosis indices, mitotic
activity, or increases in karyorrhectic nuclei in crypts
4/ GSH/GSSG in oral (>11.6 mg/kg-d) and duodenal
(>4.6 mg/kg-d) epithelium; no change in plasma.
Note: sample size is only 5 for the 7-d group, and
some observed changes occurred at slightly lower
doses but were not statistically significant.
90-d:
4/ GSH/GSSG in duodenum and jejunum (>1.1
mg/kg-d) and in plasma (>11.6 mg/kg-d)
No statistically significant increases in protein
carbonyls or 8-OHdG levels in any tissues
Some altered cytokines and chemokines
Thompson et al.
(2011b)
Rat, Fischer
344/N female
Oral, drinking
water
0, 0.1, 1.4, 20.9, 59.3, and
181 mg/L Cr(VI)
0, 0.015, 0.21, 2.9, 7.2,
20.5 mg/kg-d Cr(VI)
7 d (n = 5) or 90 d (n = 10)
7 d: No statistically significant changes in GSH/GSSG
in oral mucosa or small intestine except in jejunum at
20.5 mg/kg-d and at 0.015 mg/kg-d in the oral
mucosa. Note: sample size is 5 for 7-d data.
90 d: 4/ GSH/GSSG in oral mucosa and jejunum
(>2.9 mg/kg-d) and in plasma (>7.2 mg/kg-d), dose
dependent, statistically significant. No changes in
duodenum, or signs of lipid peroxidation
(8-isoprostane) in any tissues.
Thompson et al.
(2012c)
Mouse,
B6C3F1
Oral, drinking
water
0,0.1, 1.4, 4.9, 20.9, 59.3,
and 181 mg/LCr(VI)
0,0.024, 0.32, 1.1, 4.6,
11.6, or 31.1 mg/kg-d
Cr(VI)
7 and 90 d
In scraped duodenal epithelium:
No effect on mitotic/apoptotic indices in crypt
compartment
7 d:
1" aberrant nuclei at villi tips but not in crypts
(>11.6 mg/kg-d)
90 d:
1" aberrant nuclei at villi tips but not in crypts
(>4.6 mg/kg-d)
O'Brien et al.
(2013)
Continued
analysis of tissues
from Thompson
et al. (2011b)
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F344 rats and
B6C3F1 mice
Oral, drinking
water
0, 0.1,1.4, 4.9 (mice
only), 20.9, 59.3, and
181 mg/L Cr(VI), 90
0, 0.015, 0.21, 2.9, 7.2,
20.5 mg/kg-d Cr(VI) (rats)
0,0.1, 1.4, 4.9, 20.9, 59.3,
and 181 mg/LCr(VI)
(mice)
Dose-dependent decreases in Fe levels in the
duodenum, liver, serum, and bone marrow
Considered in hematological effects; not in Gl effects
synthesis. This assessment determined that
evidence indicates Cr(VI) is likely to cause
hematological effects based on iron-deficient
anemia-like observations in rodents (see Section
3.2.5 of the toxicological review). Such observations
were made in some of the studies cited in this table
(including NTP (2008, 2007f)). This table does not list
the observed hematological effects or effects related
to iron homeostasis. See Section 3.2.5 of the
toxicological review for a synthesis of hematological
effects, or click the HAWC link for a summarv of
selected datasets.
Suh etal. (2014)
Continued
analysis of tissues
from Thompson
et al. (2011b) and
Thompson et al.
(2012c)
Mouse, SKH-1
hairless,
female
Oral, drinking
water
0, 5, and 20 mg/L Cr(VI)
1.20 and 4.82 mg
Cr(VI)/kg-d
9 mo
No effect on oxidative 8-OHdG adducts in
forestomach, glandular stomach, duodenal cells, lung
or skin
No measure of cytotoxicity
No changes in body weight
De Flora et al.
(2008)
Mouse,
C57BL/6J
Oral, drinking
water
0, 0.019, 0.19, 1.9 mg/L
Cr(VI)
150 d
2 animals per dose group
In proximal and distal sections of Gl tract:
Histopathology: no effects on villous
atrophy/blunting or inflammation; slight enterocyte
hypertrophy and crypt hyperplasia
Immunohistochemistry: no effect on Ki67
Sanchez-Martin
et al. (2015)
Rat, Wistar
Oral, drinking
water
0, 87, 174, 262, 349,
436 mg/L Cr(VI)
0, 1.7, 3.5, 5.2, 7.0,
8.7 mg/kg-d
60 d
Stomach:
4/ p53 protein (>87 mg/L) and mRNA (>174 mg/L)
T* c-Myc protein and mRNA (>174 mg/L)
T* galectin-1 protein (>174 mg/L) and mRNA
(>87 mg/L)
4/ RKIP protein and mRNA (>262 mg/L)
4/ Rho-GDIa protein and mRNA (>262 mg/L)
Colon:
4/ p53 protein and mRNA (>262 mg/L)
T* c-Myc protein (>262 mg/L) and mRNA (>87 mg/L)
T* galectin-1 protein (>349 mg/L) and mRNA (>174
mg/L)
4/ RKIP protein (>436 mg/L) and mRNA (>349 mg/L)
4/ Rho-GDIa protein (>262 mg/L) and mRNA
(>349 mg/L)
Tsao et al. (2011)
Rat, Sprague-
Dawley male
Intragastric
injection
1.77 mmol/kg Cr(VI); bile
sampling every 40 min
Alpha-(4-pyridyl l-oxide)-N-tert-butylnitrone (POBN)
carbon-centered radical adduct in bile of rats
exposed to Cr(VI)
Kadiiska et al.
(1998)
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Rat
Oral gavage
530 mg/kg-d Cr(VI), 3 d
106 mg/kg-d Cr(VI), 30 d
Note: The administered
gavage potassium
dichromate doses (1,500
mg/kg and 300 mg/kg)
are higher than the LDso
for rats listed in MSDS
(130 mg/kg)
Intestinal epithelial cells, 3-d exposure:
4/ glucose-6-phosphate dehydrogenase, glutathione
peroxidase, glutathione reductase, glutathione-S-
transferase, superoxide dismutase and catalase
4/ glutathione and total thiols
T* lipid peroxidation
Intestinal epithelial cells, 30-d exposure:
T* superoxide dismutase, glutathione peroxidase
Null glucose-6-phosphate dehydrogenase,
glutathione reductase and catalase
4/ glutathione-S-transferase
Sengupta et al.
(1990)
Rat, Wistar,
female
i.p. injection
8.8 mg/kg Cr(VI)
Single dose, 48 h
Type 2 cystatins were induced in kidneys and
submandibular acini salivary glands. Not detected in
parotid or sublingual glands, or in trachea, lung,
stomach, small intestine, large intestine, spleen,
liver, or pancreas.
Cohen et al.
(1993)
In vitro human primary and immortalized Gl cells or gastric fluid
Caco-2 human
colorectal
adenocarcin-
oma cells
0.1, 0.3, 1, 3,10, 30,
100 nM Cr(VI)
Increase in 8-OHdG at nontoxic and cytotoxic
concentrations
No change in p53, annexin-V (apoptosis markers),
LC3B (autophagy marker)
Translocation of ATF6 to nucleus (ER stress response
marker)
Thompson et al.
(2012a)
Human wild-
type HCT116
colon cancer
cells
30 nM Cr(VI)
(formulation and
compound uncertain)
Upregulated p53, p21CIPl/WAFl, ATM, DNA-PK,
ATR, AKT and p38 (upstream p53 kinases)
T* apoptosis involves DNA-PK-mediated p53
activation and increased PUMA concurrent with loss
of p21
Note: chemical formulation preparation information
not provided. The only information given is the
chemical was 30 nM Cr(VI) and it was "a gift from
Professor Naresh Dalai, Department of Chemistry,
Florida State University." The true dose is therefore
unclear (it is possible it is 1/3 this value if the
concentration is in units of the parent chromate
compound)
Hill et al. (2008b;
2008a)
Human gastric
cancer SGC-
7901 cells
3.53 nM Cr(VI)
Oxidative stress, apoptosis and necrosis all increased
when the Unconventional prefoldin RPB5 Interacting
protein (URI) is knocked down
Luo et al. (2016)
aPotassium dichromate units conversion: Cr(VI) = 0.353 x K2Cr2C>7; potassium chromate units conversion:
Cr(VI) = 0.268 x K2&O4; sodium dichromate dihydrate units conversion: Cr(VI) = 0.349 x Na2Cr20?2H20 (usually
denoted as Na2Cr207 because study authors frequently list the salt as the chemical compound even if
concentration or dose is based on the dihydrate); chromium trioxide units conversion: Cr(VI) = 0.52 x CrC>3.
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C.2.3. Hepatic Effects
C.2.3.1. Mechanistic studies relevant to hepatic toxicity
1 A large body of mechanistic information exists (125 studies) to inform the potential
2 hepatotoxicity of Cr(VI). Therefore, studies more informative for chronic human exposure were
3 prioritized:
4 • Studies of the liver or liver enzymes from humans with quantified exposure to Cr(VI)
5 • Experimental animal studies of the liver or liver enzymes using quantified oral (drinking
6 water, gavage, diet), inhalation, or intratracheal instillation exposure to Cr(VI)
7 • In vitro studies in human primary or immortalized cells derived from liver
8 • Mechanistic endpoints relevant to interpretations of hepatic health effects in humans,
9 including genotoxicity tests in liver tissues
10 This prioritization strategy identified 49 relevant studies. These include mammalian
11 studies of the liver or liver enzymes that focused on exposure routes more relevant to humans (oral
12 drinking water, gavage, and diet; inhalation) and repeat dose studies of longer duration (>28 days).
13 Shorter duration studies, however, also provided some supporting information, and in vitro studies
14 in human liver primary cells or cell lines provided insight into biological plausibility and human
15 relevance of the observed mechanisms. These studies, summarized in Table C-34, primarily
16 reported evidence of Cr(VI)-induced oxidative and endoplasmic reticulum stress, mitochondrial
17 dysfunction, inflammation, apoptosis, DNA damage, and cell proliferation.
Table C-34. Mechanistic studies prioritized for informing potential Cr(VI)-
induced hepatic toxicity
System
Route
Exposure3
Results
Comments
Reference
Oxidative and endoplasmic reticulum stress
Mouse, ICR
male
Oral feed
1 and 4 mg/kg/
K2Cr207-d, 36-d
repeat dose
Confirmation by
detection of T* Cr
content in liver
1" hepatic lipid
peroxidation and
MDA
1" GSH levels
1" CAT and GPx
activity and mRNA
-t Ho-1, Atf6, CHOP
gene expression
Jin etal. (2014)
Rat, Wistar
male
Oral
gavage
30 mg/kg/
K2Cr207-d, 28-d
repeat dose
1" hepatic lipid
peroxidation
4/ SOD, CAT, and GST
activity
1" Atfl (MAPK stress
response pathway)
Navva et al. (2017)
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Reference
Mouse,
C57BL/J5,
male and
female
Oral
drinking
water
55-5500 ng/L
Na2Cr207, 5 mo,
repeat dose
2 animals per
dose group
1" GCLC (glutamyl-
cysteine ligase
catalytic subunit)
Null NRF2 (NF-E2-
related factor 2)
1" GCLC but the
mRNA expression
was down
For this study,
n = 2 males and
2 females
Sanchez-Martin et al.
(2015)
Rat, Sprague-
Dawley,
female
Oral
gavage
2.5 and 10 mg/kg-
d Na2Cr207; 30,
60, 90, and 120 d
1" hepatic
mitochondrial and
microsome
peroxidation with
concurrent excretion
of lipid metabolites
MDA, FA, ACT, and
ACON
(n) not given,
concerns with results
interpretation
2002:
4 animals/group
Bagchi et al. (2002b;
1997b; 1995a), Stohs
et al. (2001)
Rat, Sprague-
Dawley,
female
Oral
gavage
25 mg/kg
Na2Cr207
(reported as 0.5
LDso), 48 h
1" hepatic
mitochondrial and
microsome
peroxidation with
concurrent excretion
of lipid metabolites
1995b n = 4-6
animals per group
Bagchi et al. (1995b)
Mouse,
C57BL/6NTac
and N12 p53-
deficient
C57BL/6TSG-
p53, female
Oral
gavage
2000&2002: 0.50
LDso, 0.10 LD50,
0.01 LDso.
2001: 0.50 LDso
reported as
95 mg/kg
Na2Cr207 after
24 h; 24 h, 48 h,
and time course
up to 96 h,
respectively
1" hepatic
cytochrome C
(reported as SOA
production)
1" hepatic lipid
peroxidation
Dosing and (n) not
given (2000&2002)
Bagchi et al. (2002a;
2001; 2000a)
Rat, albino
Oral
gavage
50 mg/kg-d
K2Cr207, 20 d
repeat dose
1" liver triglycerides
and phospholipids
Uninformative
factors expected to
decrease confidence
in mechanistic
reporting
Kumar and Rana (1982)
Rat, Sprague-
Dawley (SD),
male and
female
Oral
gavage
9 mg/kg and 17.5
mg/kg K2Cr207,
7 d
4/ free radical
scavenging capacity
(benzoic acid
hydroxylation
method)
4/ GSH
Dose-dependent
decreases
Zhong et al. (2017c)
Rat, Wistar,
female
Oral
drinking
water
5 and 20 mg/L
K2Q2O7,15 d
Null results CYP2E1
activity
4/ GSH
(at both doses)
Ma et al. (2015)
Rat, Sprague-
Dawley, male
i.p.
2.5, 5.0, 7.5, and
10 mg/kg-d
K2Cr207, 5 d
-t ROS, MDA
1" SOD, CAT activity
Results dose
dependent
Patlolla et al. (2009b)
Mouse, ddY,
male
i.p.
20 mg/kg K2Cr207,
single dose,
1" lipid peroxidation
(TBARS)
Susa et al. (1989)
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Reference
reports at 24 and
48 h
Rat, Sprague-
Dawley, male
i.p.
10-40 mg/kg
Na2Cr2C>7, single
dose
T* GSH 20 mg/kg
Standeven and
Wetterhahn (1991b)
Mouse, Swiss
albino, male
i.p.
1 mg/kg CrOs,
single dose,
reports at 5-8 wk
T* SOD, peroxidase,
CAT, lipid
peroxidation,
ascorbic acid content
in liver tissue
Mice from live
animal supply farm,
"around" 48 mice
range from 15 to
25 g body weight.
Increases were not
time dependent
Acharva et al. (2004a)
Rat, Wistar,
male
i.p.
20 mg/kg body
weight
of K2Cr2C>7, single
dose; 3-min, 3-h,
24-h time course
-t SOD at 24 h
Null for changes in
CAT, lipid
peroxidation
(TBARS), CYP450
Tagliari et al. (2004)
Rat, Wistar,
male
i.p.
20 mg/kg foCQCb,
single IP dose,
24 h
T* lipid peroxidation,
GSH level and GPx-1
activity; no change in
GR activity
4/ TrxR-1 activity
Kotvzova et al. (2015)
Mouse,
BALB/c
i.p.
0 or 400 nmol
K2Cr2C>7 (20.8 mg
Cr(VI)/kg), single
dose
In liver:
T* lipid peroxidation
(p < 0.05)
T* heme oxygenase
(p < 0.001)
4/ GSH-peroxidase
activity (p < 0.1);
slight but
nonsignificant
reduction in GSH
levels
Significantly
decreased %PCEs
(PCE/NCE
ratio = 0.64 ± 0.14)
(p<0.01)
Also T* micronucleus
frequency in bone
marrow cells
(p < 0.001)
Wronska-Nofer et al.
(1999)
HepG2 cells
(human
hepatocytes)
In vitro
5,10, 20, 40 nM
K2Cr207
-t SOD, Nrf2, Keapl
mRNA at 10 nM
4/ SOD, Nrf2, Keapl
mRNA over 10 nM
In a separate study X.
Zhong shows SOD
activity decrease
starting at 1 nM but
in L-02 (human fetal)
cells
Zhong et al. (2017a)
HepG2 cells
(human
hepatocytes)
In vitro
3-25 nM K2Cr207
T* ROS production
and MDA> 12.5 nM
Patlolla et al. (2009a)
Hep3B cells
(human
hepatocytes)
In vitro
20 nM K2Cr207
T* levels of SOD, GR,
NO, CAT, MDA
Zeng et al. (2013)
-02, human
etal
lepatocytes
i vitro
T* Endoplasmic
reticulum stress and
mitochondrial
damage, and
apoptosis; effects
reversed by
Liang et al. (2019)
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Reference
antioxidant
treatment
L-02 cells
(human fetal
liver)
In vitro
Various (Yuan 1-
32 nM; Xiao 2012
4-32 nM Xei 4
HM for caspase
3/beclin, Ca+2
and ROS; Xiao
2014 25 nM
typically used for
experiments (65—
75% survival
rate); Zhang 2016
10 nM for ROS,
MRCC, p53); all
units in Cr(VI)
(parent
compound was
K2Cr207)
T* ROS production
(Zhang 2016; Yuan
2012; Xiao 2012)
-t CHOP, PERK, IRE1
mRNA and protein
(ER stress 6-10 nM,
Zhang 2017)
4/ SOD, TRx, and GHS
(Zhong 2017a, 8 and
16 nM dose
dependent)
Doses not overtly
cytotoxic, could be
some decline in
viability. Zhang
2016, Xiao 2012, Yi
2016, Zhong 2017a
and Yuan 2015
measured ROS with
DCF
Zhang et al. (2017);
Zhong et al. (2017c); Yi
et al. (2016); Zhang et
al. (2016); Xiao et al.
(2012a); Xiao et al.
(2012b); Yuan et al.
(2012)
Mitochondrial dysfunction
Rat, Sprague-
Dawley,
female
Drinking
water
10 mg/kg-d and
2.5 mg/kg-d
Na2Cr2C>7,
respectively; 90 d
and 120 d
T* hepatic
mitochondrial and
microsome
peroxidation with
concurrent excretion
of lipid metabolites
This is the same
study/effect listed
above under
oxidative stress
Bagchi et al. (1997b;
1995a)
Mouse, female
C57BL/6NTac
N12 p53-
deficient
C57BL/6TSG-
p53
Assume
by gavage
2000&2002: 0.50
LDso, 0.10 LD50,
0.01 LDso.
2001: 0.50 LDso
reported as 95
mg/kg Na2Cr2C>7
after 24 h. 24 h,
24 h, and time
course up to 96 h
respectively
T* hepatic
cytochrome C
(reported as SOA
production)
T* hepatic lipid
peroxidation
Dosing and (n) not
given (2000&2002).
LDso (2001) not
consistent with LDso
reported in 1995b.
Bagchi et al. (2002a;
2001; 2000a)
Mouse, ICR,
male
Feed
1 and 4 mg/kg/
K2Cr207-d, 36 d
repeat dose
T* cytochrome C
Jin et al. (2014)
Mouse, Swiss
albino, male
Gavage
0, 25, 50, and 100
mg/kg K2Cr2C>7
single dose, 24 h
T* cytochrome C
(50&100 mg/kg)
Wang et al. (2010b)
L-02 human
fetal
hepatocytes
In vitro
Various (Yuan 1-
32 nM; Xiao 2012
4-32 nM Cr(VI);
Xei 4 nM for
caspase 3/beclin,
Ca+2 and ROS;
Xiao 2014 25 nM
typically used for
experiments (65—
75% survival
rate); Yi 2015 2-
4/ ATP production
(Yuan 2012, Xie
2014; Xiao 2014);
4/ mitochondrial
respiratory chain
complex (MRCC) 1
and II activity (25 nM
Xiao 2014/ Zhang
2015, Zhong 2017a)
Xiao 2014 strong CC
between mito ETC
dysfunction and
apoptosis
Yi et al. (2017); Zhong
et al. (2017c); Zhang et
al. (2016); Xiao et al.
(2014); Xie et al.
(2014); Xiao et al.
(2012a); Xiao et al.
(2012b); Yuan et al.
(2012)
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Route
Exposure3
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Comments
Reference
16 nM Cr(VI) for
mitochondrial
effects; Zhang
2016 10 nM 24 h
2x for 4 wk - ROS,
MRCC, p53),
Zhong 2017a
8&16 nM; all
units in Cr(VI)
(parent
compound was
K2Cr207)
4, MMP, ATP dose
dependent (1-4 nM,
Xie 2014)
T* VDAC expression
(protein&mRNA,
accelerates
movement of Ca2+
from ER to IMM;
Yuan 2012, Yi 2017),
Ca2+ effects
HepG2 human
hepatocytes
In vitro
5, 10, 20, 40 nM
K2Cr207
T* mtDNA copy
number, mt mass,
NDUFA1, Foxol,
Sirtl, Aktl, Crebl,
ATP50 and ATP3J
gene expression at
10 nM
4/ mtDNA copy
number, mt mass,
NDUFA1, Foxol,
AKT1, Crebl, MAPK2,
Pten, ATP50 and
ATP3J gene
expression over
10 nM
Zhong et al. (2017a)
L-02 human
fetal
hepatocytes
In vitro
1-4 nM K2Cr207,
24 h
4, ETFDH, CoQIO,
ATP production, SOD,
Bcl-2
T* ROS, caspase-3,
caspase-9, MDA
(lipid peroxidation),
mitochondrial
membrane
depolarization and
permeability
transition pore
(MPTP) opening,
Ca2+, Cyt c release,
Bax
Cr(VI) induces CoQIO
deficiency (essential
for cellular
respiration and
metabolism); effects
reversed by
pretreatment with
CoQIO
Zhong et al. (2017b)
Inflammation
Mouse, ICR,
male
Feed
1 and 4
mg/kg/K2Cr2C>7-d,
36 d repeat dose
-t Ho-1
Jin et al. (2014)
Rat, Wistar,
male
Gavage
30mg/kg/K2Cr2O7-
d, 28 d repeat
dose
T* serum levels of
ALT, AST, and ALP
T* TNFa, MAPK gene
expression
Navva et al. (2017)
Rat, Sprague-
Dawley (SD),
male and
female
Gavage
9mg/kg and
17.5mg/kg
K2Cr207, 7 d
T* serum levels of
ALT, AST (17.5
mg/kg)
Zhong et al. (2017c)
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Route
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Comments
Reference
L-02, human
fetal
hepatocytes
In vitro
4 and 8uM
K2Cr207 (85-80%
viability Yi 2017);
2-32uM Yi 2016;
Zhong 2017a
8-15 nM
1" ALT, AST leakage
1" TNFa, IL-ip, LBT4
-t Nf-kB p65 (Yi
2016, 16 nM)
All dose dependent
Yi et al. (2017); Zhong
et al. (2017c); Yi et al.
(2016)
Apo ptosis
Mouse, ICR,
male
Feed
1 and 4
mg/kg/K2Cr2C>7-d,
36 d repeat dose
1" Caspase 3, 7, 9
1" cytochrome C
ER stress response
Jin et al. (2014)
Rat, Wistar,
male
Gavage
30mg/kg/K2Cr2C>7-
d, 28-d repeat
dose
4/ Bcl-2
Navva et al. (2017)
Mouse, Swiss
albino, male
Gavage
0, 25, 50, and 100
mg/kg K2Cr2C>7
single dose, 24 h
1" cytochrome C,
p53, Casp 3
4/ Bcl-2
(100 mg/kg)
Wang et al. (2010b)
HepG2 cells
(human
hepatocytes)
In vitro
5,10, 20, 40 nM
K2Cr207
No significant change
in cell viability at
10 nM
4/ Significant (20%)
decline in cell
viability at 40 nM
Zhong et al. (2017a)
HepG2 cells
(human
hepatocytes)
In vitro
3-25 nM K2Cr207
4/ Significant (20%)
decline in cell
viability at 25 nM
Patlolla et al. (2009a)
Hep3B cells
(human
hepatocytes)
In vitro
2.5-100 nM
K2Cr207
4/ cell viability at
10 nm (10%), 20 nM
(20%)
1" caspase activity 20
HM
Zeng et al. (2013)
L-02 human
fetal
hepatocytes
In vitro
1" autophagosomes,
LC3-II, and protein
degradation;
4/ p62/SQSTMl
Autophagy
associated with ROS-
AKT-mTOR pathway
Autophagy blocked
by antioxidants
Inhibition of
autophagy induced
apoptosis
Liang et al. (2018)
L-02 human
fetal
hepatocytes
In vitro
Various (Yuan 1-
32 nM; Xiao 2012
4-32 nM; Xei
4 nM for caspase
3/beclin, Ca+2
and ROS; Xiao
2014 25 nM
typically used for
experiments (65—
75% survival rate
12h); Yi 2015 2-
16 nM for
-t p53 (Zhang 2016)
1" Caspase 3 (25 nM
Xiao 2014, Xie 2014
activity 1-4 nM 24 h;
Xiao 2012 dose
dependent)
1" apoptosis (25 nM,
Xiao 2014; sig at
8 nM in Yuan 2012)
1" Beclin-1 mRNA (1-
4 nM, Xie 2014)
Yuan et al. (2012); Xiao
et al. (2012a); Xiao et
al. (2012b); Xie et al.
(2014); Xiao et al.
(2014); Zhang et al.
(2016); Zhong et al.
(2017c), Zhang et al.
(2017)
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Reference
mitochondrial
effects; Zhang
2016 10 nM 24 h
2x wk for 4 wk);
all units in Cr(VI)
(parent
compound was
K2Cr207)
4/ Bcl-2,1" Bax&cyto
C (Zhang 2017 dose
dependent 6-10 nM)
L-02 human
fetal
hepatocytes
In vitro
0, 5,10,15 nM
Cr(VI)
1" Clusterin (CLU),
dose dependent
Overexpression of
CLU can counteract
Cr(VI)-induced MRCC
I inhibition,
preventing apoptosis
Xiaoetal. (2019)
DNA damage
Rat, Wistar,
male
Gavage
30 mg/kg/
K2Cr207-d, 28-d
repeat dose
4, OGG-1
-t GADD45
Navva et al. (2017)
Mouse,
C57BL/J5
Drinking
water
Na2Cr207; dose
range 55-5500
M-g/L, 5 mo,
repeat dose
2 animals per
dose group
t p73
-t P-yH2AX positive
(no dose
dependence)
For this study
n = 2 males and
2 females
Sanchez-Martin et al.
(2015)
Rat, Sprague-
Dawley,
female
Gavage
25mg/kg
Na2Cr2C>7
(reported as 0.5
LDso), single dose
-t DNASSBs in
hepatic tissue
(n) not given
Bagchi et al. (1995b),
Stohs et al. (2001)
Mouse, female
C57BL/6NTac
N12 p53-
deficient
C57BL/6TSG-
p53
Assume
by gavage
2000&2002: 0.50
LD50, 0.10 LD50,
0.01 LDso.
2001: 0.50
LDso = 95 mg/kg
Na2Cr2C>7, single
dose (?), 48 h,
24 h, up to 96 h
time course,
respectively
-t DNA
fragmentation in
hepatic tissue
Dosing and (n) not
given (2000&2002),
DNA fragmentation
measured by %
600-nm absorbance
in supernatant
(2000). DNA
fragmentation by
electrophoresis
(2001)
Bagchi et al. (2002a;
2001; 2000a)
Rat, Fischer
344, male
Drinking
water
100 and 200 mg/L
K2Cr2C>7, 3 wk
1" hepatic DPCs
Quantitative analysis
performed but not
presented, results
not visually
convincing
Coogan et al. (1991a)
Rat, Wistar,
female
Drinking
water
5 and 20 mg/L
K2Cr207,15 d
Null results for 06-
MeG adducts
Ma et al. (2015)
Mouse, Swiss
albino, female
Drinking
water
5 and 10 mg/L
Na2Cr2C>7and 10
mg/L K2Cr2C>7,
18 d (duration of
pregnancy)
Null results for
hepatic MN in
fetuses
De Flora et al. (2006)
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—Hexavalent Chromium
System
Route
Exposure3
Results
Comments
Reference
Mouse,
C56BL/6 Big
Blue, female
Intra-
tracheal
instilla-
tion
6.75 mg/kg
K2Cr207, 28 d,
single dose
1" mutation
frequency in liver,
but only compared
to pooled controls
(p = 0.043; not
statistically
significant compared
to concurrent liver
controls (p = 0.085)
MF higher in lung
than in liver or
kidney
Cheng et al. (2000)
Rat, Sprague-
Dawley, male
Intra-
tracheal
instilla-
tion
0.25 mg/kg
Na2Cr2C>7, 3 d
No effect on DNA-
protein crosslinks,
DNA fragmentation,
8-OHdG levels, or
gene expression,
including those
associated with
apoptosis, or various
forms of DNA
alterations in liver
tissue
D'Agostini et al. (2002);
Izzotti et al. (2002;
1998)
Mouse, BDF1,
female
i.p.
25 mg/kg
Na2Cr2C>7- acute;
12.5 mg/kg -
subchronic, single
injection for acute
(1-14 d) or every
4 wk for 128 d
1" changes in ploidy
in acute group
N ranged from 3 to 5
per group. All
regions of liver
Garrison et al. (1990)
Rat, Sprague-
Dawley, male
i.p.
20 or 50 mg/kg-d
Na2Cr2C>7
lh: DNA-DNA and
DNA-protein
crosslinks in liver,
lung and kidney
1" DNA strand breaks
in liver
36-40 h: DNA-
protein crosslinks in
lung and kidney
Tsapakos et al. (1981),
Tsapakos et al. (1983)
Mouse, albino
male
i.p.
0 or 20 mg
Cr(VI)/kg, single
dose
DNA damage (comet
assay), 15 min
postinjection (all
back to control levels
at 3 h):
1" liver, kidney
No increases in
spleen, lung, brain
Same pattern as
Cr(V) complexes
Cytotoxicity not
reported
DNA damage
reduced with
deferoxamine
Ueno et al. (2001)
Mouse
i.p.
80 mg/kg K2CrC>4
DNA damage (comet
assay) in liver, lung,
kidney, spleen, and
bone marrow
Sasaki et al. (1997)
Hep3B cells
(human
hepatocytes)
In vitro
20 nM K2Cr207
1" DNA damage (30%
comet cells) in p53-
deficient Hep3B cells
Zeng et al. (2013)
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Supplemental Information—Hexavalent Chromium
System
Route
Exposure3
Results
Comments
Reference
when caspase-3 was
blocked
HepG2 cells
(human
hepatocytes)
In vitro
3-25 nM K2Cr207
1" DNA damage
(comet assay), dose-
dependent
Patlolla et al. (2009a)
Cell proliferation
Mouse,
C57BL/J5
Drinking
water
Na2Cr2C>7; dose
range 55-5,500
M-g/L, 5 mo,
repeat dose
2 animals per
dose group
4/ pl6 and pl9
For this study
n = 2 males and
2 females
Sanchez-Martin et al.
(2015)
Mouse,
C57BL/J5
Drinking
water
(in vitro
study)
10 nM K2Cr207for
24 h 2x wk for
4 wk, 5 mo,
repeat dose
1" senescence
Cr(VI) concentration
was chosen
according to the
Cr(VI) values
recorded in the
blood circulation of
exposed workers
Zhang et al. (2016)
i.p. = intraperitoneal injection.
aPotassium dichromate units conversion: Cr(VI) = 0.353 x K2Cr2C>7; potassium chromate units conversion:
Cr(VI) = 0.268 x K2Cr04; sodium dichromate dihydrate units conversion: Cr(VI) = 0.349 x Na2Cr20?2H20 (usually
denoted as Na2Cr2C>7 because study authors frequently list the salt as the chemical compound even if
concentration or dose is based on the dihydrate); chromium trioxide units conversion: Cr(VI) = 0.52 x CrC>3.
C.2.4. Hematological Effects
C.2.4.1. Mechanistic studies relevant to hematological effects
1 Mechanistic evidence indicating the biological pathways involved in hematological toxicity
2 following Cr(VI) exposure is summarized in Table C-35. Studies identified in preliminary title and
3 abstract screening as "mechanistic" were further screened and tagged as "hematology" if involving
4 red blood cells (erythrocytes) or reporting other endpoints relevant to hematological toxicity
5 (e.g., measures of hemoglobin levels). Studies were prioritized for consideration in the synthesis of
6 mechanistic evidence for hematological effects if they were conducted in mammalian species:
7 • Studies in humans with quantified oral or inhalation exposure to Cr(VI)
8 • Studies in experimental animals with quantified oral (drinking water, gavage, diet),
9 inhalation, or intratracheal instillation exposure to Cr(VI)
10 • In vitro studies in human primary erythrocytes
11 • Mechanistic endpoints relevant to interpretations of hematological effects in humans
12 Twelve hematological studies were identified to include in the mechanistic syntheses,
13 including four drinking water exposure studies in rats and mice, one i.p. injection study in mice, and
14 seven investigations using human primary erythrocytes.
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Supplemental Information—Hexavalent Chromium
Table C-35. Mechanistic studies prioritized for informing potential Cr(VI)-
induced hematological effects
System/Route
Exposure3
Results/Comments
Reference
Rat, Wistar, male
700 mg/L K2Q2O7
(67 mg/kg) in
drinking water,
14 d
In plasma: T* IL-ip, TNF-a, 8-iso-PGF(2a),
and creatinine
In plasma and urine: T* ll-dehydro-TXB2
Markers indicating arachidonic acid
peroxidation
Mitrov et al. (2014)
Rat, Wistar females,
GD 9-21
Oral, drinking water
0, 50,100, 200, and
400 mg/L K2Q2O7
In pregnant rats:
4/ RBC counts, hemoglobin, hematocrit,
and MCV levels at 200 and 400 mg/L
Samuel et al. (2012)
F344 rats and B6C3F1
mice
Oral, drinking water
0, 0.1, 1.4, 4.9,
20.9, 59.3, and 181
mg/L Cr(VI), 90 d
0, 0.015, 0.21, 2.9,
7.2, 20.5 mg/kg-d
Cr(VI) (rats)
0, 0.024,0.32, 1.1,
4.6, 11.6, or 31.1
mg/kg-d Cr(VI)
(mice)
Dose-dependent decreases in Fe levels in
the duodenum, liver, serum, and bone
marrow
Induction of divalent metal transporter 1
and transferrin receptor 1 in duodenum
1" Cr RBC:plasma ratios in rats >20.9 mg/L
Suh et al. (2014)
Rat, Sprague-Dawley
Oral, drinking water
0, 30,100, and 300
mg/L K2Cr207 (0,
10.6, 35.4, and
106.1 mg/L Cr(VI))
0, 2.49, 7.57, 21.41
mg/kg-d Cr(VI)
4 wk
Mean body weight gain, mean water
consumption, clinical chemistry
determinations, and oxidative stress levels
in plasma
Mild anemic effects and T* plasma
malondialdehyde (MDA) levels correlated
with 4/ global DNA methylation at 35.4
and 106.1 mg/L
4/ plasma glutathione peroxidase (GSH-Px)
activity (all exposed groups)
No effect on pl6 methylation or plasma 8-
OHdG levels
Wang et al. (2015)
Mouse, Swiss
Intraperitoneal
injection
4 mg/kg-d K2Cr207,
5 d/wk, 2 wk
4/ Hemoglobin, hematocrit, and RBC
counts
Echinocytic transformation
Leucopenia after 2 wk
Rav and Sarkar (2012)
Human, primary
erythrocytes
0, 0.1, 0.5,1.0,2.5,
and 5 mM K2Cr207,
lh
1" erythrocyte hemolysis and protein
carbonyl content, dose-dependent
1" lipid peroxidation (MDA levels)
4/ total SH content, NO levels
1" SOD and glutathione S-transferase
4/ catalase, G6PD, glutathione peroxidase,
glutathione reductase, and thioredoxin
reductase
Ahmad et al. (2011)
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Supplemental Information—Hexavalent Chromium
System/Route
Exposure3
Results/Comments
Reference
Human, primary
erythrocytes
0 or 8 mM
Na2Cr2C>7, 0, 2, and
4 h
1" lipid peroxidation (TBARS) >2 h
No hemolysis, but observed echinocytic
transformation of RBCs
4/ GSH levels and GSSG-R activity
No effect on catalase, GSH-Px, or SOD
activities
1" methemoglobin (hemoglobin oxidation)
and 4/ NADH-methemoglobin reductase
activity in RBCs
Fernandes et al. (1999)
Human, primary
erythrocytes
5-25 ng Cr(VI)/L
blood
4/ glutathione reductase
No effect on other erythrocyte enzymes
Koutras et al. (1964)
Human, primary
erythrocytes
0, 1, 10, or 20 nM
Cr(VI), 48 h
Evidence of eryptosis (apoptotic-like death
of erythrocytes): T* intracellular Ca2+, 4/
ATP, 4/ cell volume, T* annexin-V
(phosphatidylserine) binding
1" hemolysis
No effect on ceramide formation
(inconsistent with eryptosis)
Lupescu et al. (2012)
Human, primary
erythrocytes and
mitochondria from
placenta tissue
0.05, 0.5, 1, 5
Hg/mL K2Cr2C>7
1" lipid peroxidation level (TBARS)
(decreased with coadministration of
estrogen metabolite 4-OHE2)
4/ SOD and GST activity
4/ nitric oxide levels in blood
Sawicka et al. (2017;
2017)
Human, primary
erythrocytes
0, 1.25, 2.5, 5, 10,
20, 40, 80, and 160
HM Cr(VI), 48 h
1" hemolysis, dose dependent
Evidence of eryptosis: T* intracellular Ca2+,
4/ ATP, 4/ cell volume, T* annexin-V
(phosphatidylserine) binding
Blocking Ca influx lessened cell volume
reduction
1" ROS; incubation with NAC did lower ROS
levels but did not affect annexin-V binding
Zhang et al. (2014)
aPotassium dichromate units conversion: Cr(VI) = 0.353 x K2Cr2C>7; potassium chromate units conversion:
Cr(VI) = 0.268 x K2&O4; Sodium dichromate dihydrate units conversion: Cr(VI) = 0.349 x Na2Cr20?2H20 (usually
denoted as Na2Cr207 because study authors frequently list the salt as the chemical compound even if
concentration or dose is based on the dihydrate); chromium trioxide units conversion: Cr(VI) = 0.52 x CrC>3.
C.2.5. Immune Effects
C.2.5.1. Immune toxicity evidence tables
1 The immune evidence from experimental animals synthesized in Section 3.2.6 of the
2 toxicological review is summarized in Table C-36. These studies were identified using the main
3 PECO criteria in Appendix A and screened for outcomes that inform Cr(VI)-induced immune
4 toxicity. The evidence is organized by the immune toxicity endpoints identified in the World Health
5 Organization's Guidance for Immunotoxicity Risk Assessment for Chemicals (IPCS. 20121.
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—Hexavalent Chromium
Table C-36. Data summary tables for immunological outcomes included in the
immune effects animal evidence synthesis
Reference
Species
(strain)
Exposure
design
Dose3
Exposure
route
Endpoint
Results
Host resistance
Cohen et
al. (2006)
Rats (male,
F344)
Short-term
(5 d)
118.57 Hg/m3
for 5 h/d for 5
consecutive d
Inhalation
Pathogen
clearance
Decreased 72 h
postinfection, but not 24 or
48 h postinfection and only
in the high-dose group.
Effect observed in both
soluble and insoluble forms
of Cr(VI), but the effect was
not correlated with
chromium lung burden.
Cohen et
al. (2010)
Rats (male,
F344)
Short-term
(5 d)
118.57 Hg/m3
for 5 h/d for 5
consecutive d
Inhalation
Pathogen
clearance
Decreased 72 h
postinfection, but not 24 or
48 h postinfection and only
in the high-dose group.
Effect observed in both
soluble and insoluble forms
of Cr(VI), but the effect was
not correlated with
chromium lung burden.
Antibody responses
NTP (2005)
Mice
(female,
B6C3F1)
Short-term
(28 d)
15.6, 31.3, 62.5,
125, 250 mg/L
SDD
Drinking
water
IgM AFC/106 cells
Increased ~30% for 31.3
and 62.5 mg/L
Not reproducible in second
assay.
IgM AFC/spleen
34% incr. for 62.5 mg/L
dose only.
Not reproducible in second
assay.
NTP
(2006b)
Rats
(female,
Sprague-
Dawley)
Short-term
(28 d)
14.3, 57.3, 172,
516 mg/L SDD
Drinking
water
IgM AFC/106 cells
No effect.
IgM AFC/spleen
No effect.
NTP
(2006a)
Rats
(female,
F344)
Short-term
(28 d)
14.3, 57.3, 172,
516 mg/L SDD
Drinking
water
IgM AFC/106 cells
66% incr. at 57.3 mg/L dose
only.
IgM AFC/spleen
62% incr. at 57.3 mg/L dose
only.
Glaser et al.
(1985)
Rats (male,
WistarTNO-
W 74)
Short-term
(28 d)
0.025, 0.050,
0.10 mg/m3
Inhalation
# spleen cells
necessary for lysis
of 50% hemolysis
SRBCs
No effect.
Subchronic
(90 d)
0.025, 0.050,
0.10, 0.20
mg/m3
# spleen cells
necessary for lysis
of 50% hemolysis
SRBCs
Increased response for
0.050 mg/m3, 0.050
mg/m3 + 2-mo recovery
and 0.20 mg/m3 groups.
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Supplemental Information—Hexavalent Chromium
Reference
Species
(strain)
Exposure
design
Dose3
Exposure
route
Endpoint
Results
Ex vivo WBC function
NTP (2005)
Mice
(female,
BC3F1)
Short-term
(28 d)
15.6, 31.3, 62.5,
125, 250 mg/L
SDD
Drinking
water
MLR
No effect.
NK cell activity
No effect.
Spleen cell
proliferation
No effect on anti-CD3
spleen cell proliferation.
NTP
(2006b)
Rats
(female,
Sprague-
Dawley)
Short-term
(28 d)
14.3, 57.3, 172,
516 mg/L SDD
Drinking
water
NK cell activity
No effect.
Spleen cell
proliferation
No effect on anti-CD3
spleen cell proliferation.
NTP
(2006a)
Rats
(female,
F344)
Short-term
(28 d)
14.3, 57.3, 172,
516 mg/L SDD
Drinking
water
NK cell activity
No effect.
Spleen cell
proliferation
No effect on anti-CD3
spleen cell proliferation.
Glaser et al.
Rats (male,
WistarTNO-
W 74)
Short-term
(28 d) &
subchronic
(90 d)
Short-term
(0.050 mg/m3),
subchronic
(0.025, 0.050,
0.20 mg/m3)
Inhalation
Phagocytosis
For both exposure
regimens, phagocytosis
significantly increased at
lower Cr(VI) levels (up to
0.050 mg/m3). Following
subchronic exposure to
0.20 mg/m3, phagocytosis
decreased significantly. In
both instances, the
investigators verified
cellular viability prior to
initiating the assay.
(1985)
Subchronic
(90 d)
0.20 mg/m3
Spleen cell
proliferation
Compared to control, ConA
stimulated T cell
proliferative response (30
|jg/mL, not 15 ng/mL ConA)
was elevated in rats
exposed to Cr(VI).
Shrivastava
Mice (Swiss)
Short-term
&
subchronic
(3, 6, 9 wk)
14.8 mg/kg
Drinking
water
Phagocytosis
Compared to week 0,
phagocytosis of spleen
macrophages was
significantly reduced to
36 ± 7% at the 9-wk
timepoint.
et al.
(2005b)
Spleen cell
proliferation
Compared to week 0, ConA
stimulated T cell
proliferative response was
increased two-fold in mice
exposed to Cr(VI), but the
investigators did not
analyze the findings
statistically.
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Supplemental Information—Hexavalent Chromium
Reference
Species
(strain)
Exposure
design
Dose3
Exposure
route
Endpoint
Results
Snvder and
Valle
(1991)
Rat (F344)
Short-term
(3 or 10 wk)
100, 200 m/L
Drinking
water
Spleen cell
proliferation
Compared to control,
proliferative response to
ConA was elevated at 100
mg/L and decreased at 200
mg/L in splenocytes
isolated from rats exposed
to chromium in drinking
water.
Response to LPS was
increased at 100 mg/L and
similar to control at 200
mg/L (3-wk exposure) in
splenocytes isolated from
rats exposed to chromium
in drinking water. Nodose-
related pattern apparent.
MLR
Chromium (100 mg/L) had
no effect on thymidine
uptake from rats exposed
for 10 wk unless
splenocytes were cultured
in the presence of 0.1 mg/L
chromate; investigators did
not analyze findings
statistically.
Cohen et
al. (1998)
Rat (F344)
Short-term
(28 d)
360 |Jg/m3
Inhalation
Reactive oxygen
species
Nitric oxide
Potassium chromate had
no effect on O2- or H2O2
production in the presence
or absence of IFN-y at 4 wk,
but increased opsonized
zymosan-stimulated O2-
and decreased H2O2
production in the presence
IFN-y.
Chromium had no effect on
LPS-stimulated nitric oxide
production at 4 wk, but
reduced IFN-g-stimulated
production at 4 wk.
Mitogen-
stimulated
cytokine
production (LPS)
by pulmonary
alveolar
macrophages
exposed in vivo
for 4 wk
Decreased IL-1, TNFa
Nonstatistically significant
increase in IL-6.
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Supplemental Information—Hexavalent Chromium
Reference
Species
(strain)
Exposure
design
Dose3
Exposure
route
Endpoint
Results
Johansson
et al.
(1986)
Rabbit
(strain not
specified)
Chronic
0.9 ± 0.4 mg/m3
Inhalation
Phagocytosis
No effect.
Note: Study outcome could
have been affected by the
3-d gap between exposure
to chromium and
evaluation of effects on
phagocytosis.
Karaulov et
al. (2019)
Rat (Wistar)
Chronic
20 mg/kg-d
Drinking
water
Mitogen-
stimulated
cytokine
production
(ConA) by
splenocytes
exposed in vivo
for 45, 90, or 135
d
Increased IL-4 (days 45, 90,
and 135) and decreased IL-
6 (day 135).
No effect on IL-10 and IFNy.
Immune organ pathology
NTP (2005)
Mice
(female,
B6C3F1)
Short-term
(28 d)
15.6, 31.3, 62.5,
125, 250 mg/L
SDD
Drinking
water
Gross spleen and
thymus lesions
No effect.
NTP
(2006b)
Rats
(female,
Sprague-
Dawley)
Short-term
(28 d)
14.3, 57.3, 172,
516 mg/L SDD
Drinking
water
Gross spleen and
thymus lesions
No effect.
NTP
(2006a)
Rats
(female,
F344)
Short-term
(28 d)
14.3, 57.3, 172,
516 mg/L SDD
Drinking
water
Gross spleen and
thymus lesions
No effect.
NTP (2007f)
Rats (male
and female,
F344)
Subchronic
(3 mo)
62.5, 125, 250,
500, and
1,000 mg/L SDD
Drinking
water
Gross spleen and
thymus lesions
Histopathology on
spleen, thymus,
lymph nodes
(mandibular,
mesenteric and
pancreatic)
No effect.
Note: Although the effect is
unlikely to be due to
immunotoxicity, histiocytic
cellular infiltration of
pancreatic lymph nodes
was reported in male (^125
mg/L) and female (1,000
mg/L) rats.
Mice (male
and female,
B6C3F1)
Subchronic
(3 mo)
62.5, 125, 250,
500, and
1,000 mg/L SDD
No effect.
Note: Although the effect is
unlikely to be due to
immunotoxicity, histiocytic
cellular infiltration of
mesenteric lymph nodes
was reported in male and
female mice exposed to
125 mg/L or more.
Mice (male,
BALB/c)
Subchronic
(3 mo)
62.5, 125 and
250 mg/L SDD
No effect.
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Supplemental Information—Hexavalent Chromium
Reference
Species
(strain)
Exposure
design
Dose3
Exposure
route
Endpoint
Results
Mice (male,
am3-
C57BL/6)
Subchronic
(3 mo)
62.5, 125 and
250 mg/LSDD
No effect.
Note: Although the effect is
unlikely to be due to
immunotoxicity, histiocytic
cellular infiltration of
mesenteric lymph nodes
was reported in male mice
exposed to 250 mg/L.
NTP (2008)
Rat (male
and female,
F344/N)
2-yr (day
22, 6 and
12 mo)
14.3, 57.3, 172,
or 516 mg/L
SDD
Drinking
water
Gross spleen and
thymus lesions
Histopathology on
spleen, thymus,
lymph nodes
(mandibular and
mesenteric)
No effect.
Note: Although the effect is
unlikely to be due to
immunotoxicity, histiocytic
cellular infiltration of
mesenteric and pancreatic
lymph nodes was reported
in male and female rats
exposed to 57.3 mg/L or
greater.
Mice (male
and female,
B6C3F1)
2-yr (day
22, 6 and
12 mo)
Male and
female rats -
14.3, 57.3, 172,
516; Male mice
-14.3, 28.6,
85.7, or 257.4
mg/LSDD;
Female mice -
14.3, 57.3, 172,
or 516 mg/L
SDD
No effect.
Note: Although the effect is
unlikely to be due to
immunotoxicity, histiocytic
cellular infiltration of the
mesenteric lymph nodes of
all exposed groups of males
and females and of the
pancreatic lymph nodes of
85.7 and 257.4 mg/L males
and 172 and 516 mg/L
females.
Karaulov et
Rats (male,
Wistar)
Chronic
(135 d)
20/mg/kg-d
Drinking
water
Histopathology of
spleen, thymus,
lymph nodes
Compared to control,
structural changes
including decreased
reticular epitheliocytes and
associations with T cells
that could lead to
functional impairment of
the central immune
system, data not reported
for other timepoints.
al. (2019)
Compared to control,
structural changes
structural effects including
increased B-zone and a
decrease in the T-zone
were observed in spleens
across all timepoints.
Lymph node size was
increased and was
attributed to changes in
cellular elements.
This document is a draft for review purposes only and does not constitute Agency policy.
C-80 DRAFT—DO NOT CITE OR QUOTE
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Supplemental Information—Hexavalent Chromium
Reference
Species
(strain)
Exposure
design
Dose3
Exposure
route
Endpoint
Results
NTP (1996)
Mice
(female,
BALBC)
Subchronic
(90 d)
15, 50, 100, 400
mg/L PDC
Oral diet
Gross spleen and
thymus lesions
No effect.
Glaser et al.
(1986)
Rats (male,
WistarTNO-
W 74)
Chronic
(18 mo
exposure +
12 mo
recovery)
0.025, 0.050,
0.010 mg/m3
Cr(VI)
Inhalation
Histopathology of
spleen
No effect.
Note: Animals were
evaluated only after the full
30-mo study duration
(i.e., including the 12-mo
recovery period).
Immunoglobulin levels
NTP (2005)
Mice
(female,
B6C3F1)
Short-term
(28 d)
15.6, 31.3, 62.5,
125, 250 mg/L
SDD
Drinking
water
Antigen-specific
IgM
No effect on serum titers of
antigen-specific IgM
(SRBC).
NTP
(2006a)
Rats
(female,
F344)
Short-term
(28 d)
14.3, 57.3, 172,
516 mg/L SDD
Drinking
water
Antigen-specific
IgM
No effect on serum titers of
antigen-specific IgM (KLH).
NTP
(2006b)
Rats
(female,
Sprague-
Dawley)
Short-term
(28 d)
14.3, 57.3, 172,
516 mg/L SDD
Drinking
water
Antigen-specific
IgM
No effect on serum titers of
antigen-specific IgM
(SRBC).
Glaser et al.
(1985)
Rats (male,
WistarTNO-
W 74)
Short-term
(28 d)
0.025, 0.050,
0.10 mg/m3
Inhalation
Total serum Ig
Total serum Ig data not
shown or mentioned in the
results.
Subchronic
(90 d)
0.025, 0.050,
0.10, 0.20
mg/m3
Dose-responsive increase in
total serum Ig, significant at
concentrations
>0.025 mg/m3, peaked at
0.10 mg/m3, and declined
to control levels at 0.20
mg/m3.
Glaser et al.
(1986)
Rats (male,
WistarTNO-
W 74)
Chronic
(18 mo
exposure +
12 mo
recovery)
Sodium
dichromate -
0.025, 0.050,
0.10 mg/m3
Inhalation
Total serum Ig
According to the
investigators, total serum Ig
levels decreased in all
sodium dichromate
exposure groups and for all
timepoints (monthly for
first 6 mo, every 3 mo
thereafter), but observed
effects were not significant;
data not shown.
Glaser et al.
(1990)
Rats (male,
albino
Wistar)
Short-term
(30 d)
0.050, 0.10,
0.20, 0.40
mg/m3
Inhalation
Total serum Ig
No effect on total serum Ig
levels; data not shown.
Subchronic
(90 d)
No effect on total serum Ig
levels; data not shown.
This document is a draft for review purposes only and does not constitute Agency policy.
C-81 DRAFT—DO NOT CITE OR QUOTE
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Supplemental Information—Hexavalent Chromium
Reference
Species
(strain)
Exposure
design
Dose3
Exposure
route
Endpoint
Results
Subchronic
+ recovery
(90 d + 30-d
recovery)
No effect on total serum Ig
levels; data not shown.
Immune organ weight
NTP (2005)
Mice
(female,
B6C3F1)
Short-term
(28 d)
15.6, 31.3, 62.5,
125, 250 mg/L
SDD
Drinking
water
Absolute and
relative spleen
and thymus
weight
Nonreplicated decrease in
relative spleen weight (31.3
mg/L).
No effect on relative
thymus weight.
Note: Since significant
changes in body weight
were reported, absolute
weights are not reliable.
NTP
(2006b)
Rats
(female,
Sprague-
Dawley)
Short-term
(28 d)
14.3, 57.3, 172,
516 mg/L SDD
Drinking
water
Absolute spleen,
thymus, and
lymph node
weight
No effect (spleen, thymus).
Protocol indicates lymph
node weight was collected,
but data were not
reported.
NTP
(2006a)
Rats
(female,
F344)
Short-term
(28 d)
14.3, 57.3, 172,
516 mg/L SDD
Drinking
water
Absolute spleen,
thymus, and
lymph node
weight
No effect (spleen, thymus).
Protocol indicates lymph
node weight was collected,
but data were not
reported.
NTP (2007f)
Rats (male
and female,
F344/N)
Subchronic
(3 mo)
62.5, 125, 250,
500, and
1,000 mg/L SDD
Drinking
water
Absolute and
relative spleen
and thymus
weight
Males - Relative spleen
weights of 250 and 500
mg/L significantly less than
control. Thymus weight
unaffected.
Females - Relative spleen
weights of 500 and 1,000
mg/L significantly less than
control. Thymus weight
unaffected.
This document is a draft for review purposes only and does not constitute Agency policy.
C-82 DRAFT—DO NOT CITE OR QUOTE
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Supplemental Information—Hexavalent Chromium
Reference
Species
(strain)
Exposure
design
Dose3
Exposure
route
Endpoint
Results
Mice (male
and female,
B6C3F1)
Subchronic
(3 mo)
62.5, 125, 250,
500, and
1,000 mg/LSDD
Males - No effect on
absolute spleen or thymus
weight. Increased relative
spleen and thymus weight
(500 mg/L and 1,000 mg/L).
Females - No effect on
spleen weight. Absolute
thymus weight increased
for single dose group.
Relative thymus weight
increased for 125, 250, 500,
and 1,000 mg/L dose
groups.
NOTE: Effects on organ
weight were attributed to
reduced body weights of
the mice.
Mice (male,
B6C3F1)
Subchronic
(3 mo)
62.5,125, and
250 mg/LSDD
Absolute thymus weight
decreased (250 mg/L),
considered treatment
related.
Spleen weight unaffected.
Mice (male,
BALB/c)
Subchronic
(3 mo)
62.5, 125, and
250 mg/LSDD
No effect on spleen or
thymus weight.
Mice (male,
am3-
C57BL/6)
Subchronic
(3 mo)
62.5, 125, and
250 mg/LSDD
Significant decrease in
absolute thymus weight
and relative spleen weights
(250 mg/L)
NOTE: Effects on organ
weight were attributed to
reduced body weights of
the mice.
Karaulov et
Rats (male,
Wistar)
Chronic
(135 d)
20/mg/kg-d
Drinking
water
Absolute spleen
and thymus
weight
Absolute spleen and
thymus weight decreased
in rats exposed to
chromium in drinking water
for up to 135 d.
al. (2019)
Shrivastava
Mice (male,
Swiss)
Short-term
&
subchronic
(3, 6, 9 wk)
14.8 mg/kg
Drinking
water
Relative spleen
weight
Compared to week 0,
relative spleen weight
decreased gradually and
achieved statistical
significance at the 9-wk
timepoint.
et al.
(2005b)
This document is a draft for review purposes only and does not constitute Agency policy.
C-83 DRAFT—DO NOT CITE OR QUOTE
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Supplemental Information—Hexavalent Chromium
Reference
Species
(strain)
Exposure
design
Dose3
Exposure
route
Endpoint
Results
Jin et al.
(2016)
Mouse
(male, ICR)
Short-term
50 mg/L for 7 d
or 200 mg/L for
21 d
Drinking
water
Relative spleen
weight
Compared to control,
relative spleen weight was
significantly increased
following exposure to 50
mg/L potassium
dichromate on day 7.
Compared to control,
relative spleen weight was
increased following
exposure to 50 mg/L
potassium dichromate for
21 d, but the effect was not
significant.
Glaser et al.
(1985)
Rats (male,
WistarTNO-
W 74)
Short-term
(28 d)
0.025, 0.050,
0.10 mg/m3
Inhalation
Relative spleen
weight
Compared to control,
relative spleen weight
increased for
concentrations
(>0.050 mg/m3).
Subchronic
(90 d)
0.025, 0.050,
0.10, 0.20
mg/m3
Relative spleen
weight
Compared to control,
relative spleen weight
increased for
concentrations
(>0.050 mg/m3).
Glaser et al.
(1986)
Rats (male,
WistarTNO-
W 74)
Chronic
(18 mo
exposure +
12 mo
recovery)
Sodium
dichromate -
0.025, 0.050,
0.10 mg/m3
Inhalation
Spleen weight
No effect on spleen weight
(relative or absolute not
specified).
Note: animals were
evaluated only after the full
30-mo study duration
(i.e., including the 12-mo
recovery period).
Kim et al.
(2004)
Rats (male,
Sprague-
Dawley)
Subchronic
(13 wk)
0.2, 0.5, 1.25
mg/m3
Inhalation
Relative spleen
weight
No effect on relative spleen
weight.
WBC counts (spleen cells)
NTP (2005)
Mice
(female,
BC3F1)
Short-term
(28 d)
15.6, 31.3, 62.5,
125, 250 mg/L
SDD
Drinking
water
Total WBCs
Absolute and
relative splenic
phenotypic
analysis
No effect on total WBC
counts.
No effect on splenic
absolute or relative levels B
cells (ig+), T cells (CD3+),
T helper cells (CD4+/CD8),
T cytotoxic cells
(CD4-/CD8+), immature T
cells (CD4+/CD8+), and
monocytes (Mac-3+ cells).
This document is a draft for review purposes only and does not constitute Agency policy.
C-84 DRAFT—DO NOT CITE OR QUOTE
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Supplemental Information—Hexavalent Chromium
Reference
Species
(strain)
Exposure
design
Dose3
Exposure
route
Endpoint
Results
NTP
(2006b)
Rats
(female,
Sprague-
Dawley)
Short-term
(28 d)
14.3, 57.3, 172,
516 mg/LSDD
Drinking
water
Total WBCs
Absolute and
relative splenic
phenotypic
analysis
No effect on total WBC
counts.
No effect on splenic
absolute number of B cells
(CD45+), T cells (CD5+),
T helper cells (CD4+/CD5+),
T cytotoxic cells
(CD8+/CD5+), and NK cells
(CD8+).
Percent macrophages
increased in low and high
dose Cr(VI) groups, no
other subpopulations
affected.
NTP
(2006a)
Rats
(female,
F344)
Short-term
(28 d)
14.3, 57.3, 172,
516 mg/LSDD
Drinking
water
Total WBCs
Absolute and
relative splenic
phenotypic
analysis
No effect on total WBC
counts.
No effect on splenic
absolute and relative
numbers of B cells (CD45+),
T cells (CD4+/CD5+), T
helper cells (CD4+), and
T cytotoxic cells
(CD8+/CD5+).
Absolute number of
macrophages (HIS36+)
increased at low dose.
Increased NK cells (~40%
change, single dose level
172 mg/L) and
macrophages (~35%
change, single dose level
14.3 mg/L).
This document is a draft for review purposes only and does not constitute Agency policy.
C-85 DRAFT—DO NOT CITE OR QUOTE
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Supplemental Information—Hexavalent Chromium
Reference
Species
(strain)
Exposure
design
Dose3
Exposure
route
Endpoint
Results
Karaulov et
al. (2019)
Rats (male,
Wistar)
Chronic
(135 d)
20/mg/kg-d
Drinking
water
Total WBCs
Absolute and
relative splenic
phenotypic
analysis
No effect on WBC counts
after 90 d exposure.
Decreased absolute
number splenic karyocytes
and myeloid cells.
Timepoint-specific effects
on absolute number splenic
plasma cells.
Absolute number of CD3+
cells decreased on days 90
and 135.
Relative number of CD3+
cells unaffected.
Absolute number of CD4+
cells decreased on days 90
and 135.
Relative number of CD4+
cells decreased on day 45.
Absolute and relative
number of CD8+ cells
decreased on day 90.
Absolute number of
thymocytes decreased, but
a dose-response was not
evident.
Increased absolute number
bone marrow myeloid cells,
lymphocytes, neutrophils,
and karyocytes at the 135-d
timepoint.
WBC (hematology)
NTP (2005)
Mice
(female,
B6C3F1)
Short-term
(28 d)
15.6, 31.3, 62.5,
125, 250 mg/L
SDD
Drinking
water
Hematology
No effect.
NTP
(2006b)
Rats
(female,
Sprague-
Dawley)
Short-term
(28 d)
14.3, 57.3, 172,
516 mg/L SDD
Drinking
water
Hematology
No effect.
NTP
(2006a)
Rats
(female,
F344)
Short-term
(28 d)
14.3, 57.3, 172,
516 mg/L SDD
Drinking
water
Hematology
No effect.
NTP (2007f)
Mice (male
and female,
B6C3F1)
Subchronic
(3 mo)
62.5, 125, 250,
500, and
1,000 mg/L SDD
Drinking
water
Hematology
No effect, either sex.
Mice (male,
BALB/c)
Subchronic
(3 mo)
62.5,125 and
250 mg/L SDD
No effect.
This document is a draft for review purposes only and does not constitute Agency policy.
C-86 DRAFT—DO NOT CITE OR QUOTE
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Supplemental Information—Hexavalent Chromium
Reference
Species
(strain)
Exposure
design
Dose3
Exposure
route
Endpoint
Results
Mice (male,
am3-
C57BL/6)
Subchronic
(3 mo)
62.5, 125 and
250 mg/LSDD
No effect.
NTP (2007f)
Rats (male
and female,
F344/N)
Subchronic
(3 mo)
62.5, 125, 250,
500, and
1,000 mg/LSDD
Drinking
water
Hematology
Elevated WBC and
lymphocytes in males and
females, primarily in high
dose groups (500 and 1,000
mg/L).
Increased neutrophil and
monocyte counts (at higher
exposures in males and
females) were reported to
be an inflammatory
response associated with
lesions observed
histopathologically
(e.g., gastric lesions) and
not believed to fully
account for increased
leukocyte numbers.
NTP (2008)
Rat (male
and female
F344/N)
2-yr (day
22, 6 and
12 mo)
14.3, 57.3, 172,
or 516 mg/L
SDD
Drinking
water
Hematology
Increased WBC, neutrophils
and eosinophils,
sporadically with time and
generally in higher dose
groups.
Mice (male
and female,
B6C3F1)
2-yr (day
22, 6 and
12 mo)
Male and
female mice -
14.3, 57.3, 172,
516; Male mice
-14.3, 28.6,
85.7, or 257.4
mg/LSDD;
Female mice -
14.3, 57.3, 172,
or 516 mg/L
SDD
Increased WBC, monocytes
and basophils, but only on
day 22 in the higher dose
groups.
Neutrophils increased on
day 22 in top two dose
groups and at 12 mo for
top dose group.
Lymphocytes increased for
day 22 (14.3 mg/L-516
mg/L).
Shrivastava
Mice (Swiss)
Short-term
&
subchronic
(3, 6, 9 wk)
14.8 mg/kg
Drinking
water
Hematology
WBC decreased
significantly at the 3-wk
timepoint. Compared to
week 0, the relative
number of lymphocytes,
granulocytes, and
monocytes decreased
significantly at all
timepoints.
et al.
(2005a)
NTP (1996)
Mice
(female,
BALBC)
Subchronic
(90 d)
15, 50, 100, 400
mg/L PDC
Oral diet
Hematology
No effect.
This document is a draft for review purposes only and does not constitute Agency policy.
C-87 DRAFT—DO NOT CITE OR QUOTE
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Supplemental Information—Hexavalent Chromium
Reference
Species
(strain)
Exposure
design
Dose3
Exposure
route
Endpoint
Results
Krim et al.
(2013)
Rat (male,
albino
Wistar)
Short-term
(30 d)
15 mg/kg PDC
Oral
gavage
Hematology
No effect.
Glaser et al.
(1986)
Rats (male,
Wistar TNO-
W 74)
Chronic
(18 mo
exposure +
12 mo
recovery)
Sodium
dichromate -
0.025, 0.050,
0.10 mg/m3
Inhalation
Hematology
No effect on total WBC
counts observed in all
sodium dichromate
exposure groups and for all
timepoints (monthly for
first 6 mo, every 3 mo
thereafter).
Glaser et al.
(1985)
Rats (male,
Wistar TNO-
W 74)
Short-term
(28 d) &
subchronic
(90 d)
Short-term
(0.025, 0.050,
0.10 mg/m3) &
Subchronic
(0.025, 0.050,
0.10, 0.20
mg/m3 CrOs)
Inhalation
Hematology
No effect.
Glaser et al.
(1990)
Rats (male,
Wistar
BOR:WISW)
Short-term
(30 d) &
subchronic
(90 d)
0.050, 0.10,
0.20, 0.40
mg/m3 CrOs
Inhalation
Hematology
Elevated blood WBCs
(0.050-0.40 mg/m3) at 30 d
and 90 d, effect lost after
30-d recovery period
(following 90 d of
exposure).
Kim et al.
(2004)
Rats (male,
Sprague-
Dawley)
Subchronic
(13 wk)
0.2, 0.5, 1.25
mg/m3
Inhalation
Hematology
No effect on total WBC
counts.
SRBC = sheep red blood cell; KLH = keyhole limpet hemocyanin; MLR = mixed lymphocyte reaction; NK = natural
killer; ConA = concanavalin A; LPS = liposaccharide.
aPotassium dichromate units conversion: Cr(VI) = 0.353 x K2Cr2C>7; potassium chromate units conversion:
Cr(VI) = 0.268 x faCrCU; sodium dichromate dihydrate units conversion: Cr(VI) = 0.349 x Na2Cr2C>72H20 (usually
denoted as Na2Cr2C>7 because study authors frequently list the salt as the chemical compound even if
concentration or dose is based on the dihydrate); chromium trioxide units conversion: Cr(VI) = 0.52 x CrC>3.
C.2.5.2. Mechanistic studies relevant to immunotoxicity
1 Studies initially tagged as "mechanistic" in the preliminary title and abstract screening were
2 further screened and tagged "immune" if they reported any immunotoxicological outcome. A large
3 body of mechanistic information (329 studies) exists to inform the potential immunotoxicity of
4 Cr(VI). Within this evidence base, studies were tagged with immune-related categories if they
5 reported relevant outcomes: "chronic inflammation" (39 studies) or "immune suppression"
6 (34 studies) if relevant to cancer (reviewed in Section 3.2.3 of the toxicological review) and
7 "cytokines" if a study reported cytokine measures (28 studies). In addition, studies tagged as
8 "dermal" in "potentially relevant supplemental material" were rescreened to identify allergic
9 sensitization (68 studies) or immune stimulation (61 studies) outcomes that also appeared to
10 involve nondermal exposures to Cr(VI).
This document is a draft for review purposes only and does not constitute Agency policy.
C-88 DRAFT—DO NOT CITE OR QUOTE
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Supplemental Information—Hexavalent Chromium
1 Subsequent prioritization of the immune-relevant studies that are more informative for
2 chronic human exposure was conducted to identify mammalian studies of the immune system that
3 focused on exposure routes more relevant to humans (oral drinking water and inhalation) for
4 durations ranging from short-term to chronic. In addition, supporting information in vitro studies
5 in human and animal primary lymphocytes and cell lines provided insight into biological
6 plausibility and human relevance of the observed mechanisms. These prioritization criteria are as
7 follows:
8 • Studies in humans with quantified oral or inhalation exposure to Cr(VI)
9 • Studies in experimental animals with quantified oral (drinking water, gavage, diet),
10 inhalation, or intratracheal instillation exposure to Cr(VI)
11 • Ex vivo assays performed on immune-relevant cells exposed in vivo
12 • In vitro studies in primary or immortalized mammalian cells derived from immune organ or
13 tissues
14 • Mechanistic endpoints relevant to interpretations of immune health effects in humans and
15 animals
16 Fourteen studies were identified that primarily reported evidence of Cr(VI)-induced
17 alterations in cell differentiation or activation, effector cell function, cell proliferation, and cell-cell
18 communication; these studies are summarized in Table C-37. In addition, 21 studies reporting
19 cytokine measures were prioritized; these studies are summarized in Table C-38.
This document is a draft for review purposes only and does not constitute Agency policy.
C-89 DRAFT—DO NOT CITE OR QUOTE
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Supplemental Information—Hexavalent Chromium
Table C-37. Mechanistic studies prioritized for informing potential Cr(VI)-induced immune toxicity
System
Route
Exposure3
Results
Comments
Reference
Effects on immune cell differentiation or activation
Human
monocyte
derived
dendritic cells
(MoDC)
Human
monocyte
derived
dendritic
cells (MoDC)
25, 50, 75, 100 nM
K2Cr207, 48 h
1" CD86 (dose dependence with significance
at 100 nM); no change in CD83
100 nM K2Cr2C>7 considered
nontoxic dose when cells were
75% viable
Toebak et al.
(2006)
Mouse
splenocytes
from male and
female
C57BL/6
In vitro
0, 2, 5 nM K2Cr207, 24 h
4/ activation of T cells stimulated with anti-
CD3 and anti-CD28 (4/ CD69 at both doses and
4, CD25 at 5 jiM)
Significant 4^ CD4+T cell
viability at 5 nM, but not 2 nM
Dai et al. (2017b)
Effects on immune effector function of specific cell types
Mouse
RAW264.7
macrophages
In vitro
50 ng/mL welding fumes
(250 |jg/mL), 3 or 6 h
4/ phagocytosis following exposure to Ni WF
(50 ng/mL) at 3 and 6 h timepoints, but not by
other welding fumes
4/ number live cells and
percentage viable cells for all
welding fumes (250 ng/mL) at
24 h, but only Ni-Cu WF
caused a reduction in live cells
at 50 ng/mL; GMA-MS = Cr(VI)
not detected; GMA-
SS = 2,600 ± 120 ng/g Cr(VI);
Ni-Cu WF = 422 ± 35 ng/g
Cr(VI)
Badding et al.
(2014)
Human
primary
lymphocytes
In vitro
K2Cr207, 7 d
4/ IgG production at 0.1-10 nM w/80%
reduction at 2 nm by lymphocytes stimulated
with PWM
Effects correlated with Cr
content in cells
Borella and
Bargellini (1993)
Mouse
(BALB/cABOM)
primary
peritoneal
macrophages
In vitro
0.313-40 nM, 18 h
(random migration) or
2.5 nM and 10 jiM, 24h
(phagocytosis) Na2CrC>4
No changes in random migration
(chemokinesis) up to 2.5 nM, but 4^ random
migration in concentrations at >5 nM for 18 h
in "stimulated" macrophages
4/ phagocytosis in resting macrophages at
^2.5 nM, but not at lower concentrations
Viability not affected by 2.5
and 1.25 nM Cr(VI) during 28 d
of exposure. 5 nM showed
decreased viability after 48h.
Chemokinesis studies carried
out using stimulated macs, but
stimuli not specified.
Christensen et al.
(1992)
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—Hexavalent Chromium
System
Route
Exposure3
Results
Comments
Reference
Mouse
splenocytes
from male and
female
C57BL/6
In vitro
0, 2, 5 nM K2Cr207, 24 h
4/ production cell surface expression of
CD107a (indicates degranulation by CD8+
T cells)
Significant 4^ CD8+T cell
viability at 5 nM and 2 nM
Dai etal. (2017b)
Bovine
alveolar
macrophages
In vitro
10-1000 ng/mL M MA-
SS, MIG-SS, MMA-MS,
MMA-CI, MIG-MS
welding fumes, or
K2Cr04,18 h
4/ phagocytosis by 50% at 0.018 ng/mL K2CrC>4
Welding fumes with higher Cr(VI) content
decreased phagocytosis more potently than
fumes containing less Cr(VI)
Inhibited phagocytosis at
concentration ~10x less than
the LCso (i.e., 1.59 |Jg/mL)
Hooftman et al.
(1988)
Human PMBCs
from shoe,
leather, and
hide industry
workers
Ex vivo/In
vitro
PBMCs collected from
exposed humans
exposed Cr(VI) in vitro to
10"5 mg/L, 1 h
4/ percent phagocytosis, phagocytosis index
and percent killing by PMNs collected from
exposed workers and treated with Cr(VI) ex
vivo
Mignini et al.
(2009)
Effects on immune cell proliferation
Human
primary
lymphocytes
In vitro
0.1,1,10,100 nM
Cr(VI), 48 h
4/ anti-CD3 proliferation at all concentrations
4/ anti-CD3/anti-CD28 proliferation at 10 and
100 nM
Cr(VI) test substance reported
as CrC>3 as source, given as ion
concentration. Resting and
CD3 activated lymphocytes
showed decreased viability (to
~80%) at 1 nM, with drop after
10 nM.
Akbar et al. (2011)
Human
primary
lymphocytes
In vitro
K2Cr207, 4 d
1" proliferation by PHA-stimulated cells at
10"8-10-6 mol/L (4 d)
4/ proliferation by PHA-stimulated cells at
10"6-2.5 x 10"6 mol/L (4 d)
Biphasic pattern; effects
correlated with Cr content in
cells
Borella and
Bargellini (1993)
Mouse
splenocytes
from male and
female
C57BL/6
In vitro
0, 2, 5 nM K2Cr207, 96 h
4/ proliferation by anti-CD3/anti-CD28
stimulated CD4+ T cells at 2 and 5 nM and
CD8+ cells at 5 nM
Significant 4^ CD4+T cell
viability at 5 nM, but not 2 nM.
Dai etal. (2017b)
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Route
Exposure3
Results
Comments
Reference
Rat
splenocytes,
Fischer 344
(splenocytes
from Sprague-
Dawley rats
served as
stimulator cells
in the mixed
lymphocyte
cultures)
In vitro
LPS/ConA assay: 0.01-
100 mg/mL K2CrC>4, cells
cultured "up to" 72 h
Mixed lymphocyte
response (MLR):
In vivo/ex vivo - 100
mg/L for 10 wk followed
by 0.1 mg/L for 5 d of
culture
In vitro-0.1 mg/L
K2Cr04, 5 d
4/ mitogen stimulated proliferation by
T lymphocyte (ConA) and B lymphocytes (LPS)
cultures 0.1 mg/L and lower, no effect at
higher doses
1" MLR in cells exposed in vivo and in vitro (no
statistics)
1" or no effect on MLR at 0.1 mg/L in vitro
(statistics provided for only one of two
experiments)
Snvder and Valle
(1991)
Cross-sectional
study in Italy
of 20 exposed
and 24
unexposed
workers
Ex vivo/In
vitro
PBMCs collected from
exposed workers
treated with additional
Cr(VI) ex vivo
No effect on ConA-stimulated proliferation in
PBMCs collected from unexposed workers in
the presence of Cr(VI) administered ex vivo
No effect on ConA-stimulated proliferation in
PBMCs isolated from exposed workers and
treated with Cr(VI) ex vivo
Nonsignificant biphasic response in Con-A
stimulated proliferation in PBMCs collected
from unexposed HLA-B8-DR3-negative
subjects treated Cr(VI) ex vivo
No effect on Con-A stimulated proliferation in
PBMCs collected from exposed HLA-B8-DR3-
positive subjects treated Cr(VI) ex vivo
The effect of Cr(VI) exposure ex vivo on
proliferation of lymphocytes collected from
HLA-B8-DR3-negative and -positive subjects
stimulated by ConA was investigated, but
comparisons between exposed and
unexposed subjects in the presence and
absence of Cr(VI) were not reported.
4/ ConA-stimulated proliferation in PBMCs
collected from exposed HLA-B8-DR3-negative
group treated with Cr(VI) in vitro in the
absence of the monocytic/macrophage
component.
Mignini et al.
(2004)
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Route
Exposure3
Results
Comments
Reference
The effect of Cr(VI) exposure ex vivo on
proliferation of lymphocytes collected from
HLA-B8-DR3-negative subjects stimulated by
ConA in the absence of the
monocytic/macrophagic component was
investigated, but comparisons between
exposed and unexposed subjects in the
presence and absence of Cr(VI) were not
reported.
Cross-sectional
study in Italy
of 40 exposed
tannery
workers and
44 controls
Ex vivo/In
vitro
Lymphocytes collected
from exposed workers
treated with additional
Cr(VI) ex vivo
1" ConA- and PHA-stimulated proliferation in
PBMCs collected from workers exposed to
high concentration of Cr(VI) (Group B) ex vivo
No effect on LPS-stimulated proliferation in
PBMCs collected from unexposed workers
treated with low concentration of Cr(VI) ex
vivo
1" ConA- and PHA-stimulated proliferation in
PBMCs collected from unexposed workers
treated with 10~5 mg/mL Cr(VI) in vitro
4/ ConA- and PHA-stimulated proliferation in
PBMCs collected from unexposed workers
treated with 10~2 mg/mL Cr(VI) in vitro
4/ LPS-stimulated proliferation in PBMCs
collected from unexposed workers treated
with 10~2 mg/mL or 10"5 mg/mL Cr(VI) in vitro
The effect of Cr(VI) exposure in vitro on
proliferation of lymphocytes collected from
exposed workers stimulated by ConA, PHA,
and LPS was investigated, but comparisons
between exposed and unexposed workers in
the presence and absence of Cr(VI) were not
reported.
Mignini et al.
(2009)
Effects on communication between immune cells
Human
peripheral
blood
mononuclear
Ex vivo/In
vitro
PBMCs collected from
exposed humans
exposed to Cr(VI) in vitro
to 10"5 mg/L, 1 h
No change in ICAM-1, VCAM, and ELAM-1E-
selectin levels
Mignini et al.
(2009)
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Reference
cells from
shoe, leather,
and hide
industry
workers
Human
peripheral
blood
lymphocytes
In vitro
588 Hg/mL, 0.5 h
No effect on E-rosetting
Data not shown
Bravo et al. (1990)
Cross-sectional
study in China
of 56 workers
exposed to
potassium
dichromate
and 50
unexposed
individuals
living 20 km
from factory
In vivo
14.4 ± 18.1 ng/m3
C3 (g/L) - Exposed: 1.20 ± 0.24;
Unexposed: 0.91 ± 0.13
C4 (g/L) - Exposed: 0.32 ± 0.07;
Unexposed: 0.23 ± 0.05
Qian et al. (2013),
low
Mouse splenic
T cells
In vitro
2 or 5 (xM, 24 h
Decreased anti-CD3/CD28-induced secretion
of IL-2, IL-4, and IL-10 in splenocytes treated
with 2 or 5 |xM Cr(VI)
Dai et al. (2017b)
See Table C-38 for effects on cytokine levels following Cr(VI) exposure.
aPotassium dichromate units conversion: Cr(VI) = 0.353 x K2Cr2C>7; potassium chromate units conversion: Cr(VI) = 0.268 x K2Cr04; sodium dichromate dihydrate
units conversion: Cr(VI) = 0.349 x Na2Cr2C>72H20 (usually denoted as Na2Cr2C>7 because study authors frequently list the salt as the chemical compound even if
concentration or dose is based on the dihydrate); chromium trioxide units conversion: Cr(VI) = 0.52 x CrC>3.
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Table C-38. Summary of cytokine levels measured following Cr(VI) exposure
Reference
Study design
Cytokines
Cytokines measured in blood, serum, and plasma
Kuo and Wu
(2002)
Blood collected from Cr(VI)-exposed
workers
-t IL-6 and IL-8
4/ TNF-a (NS)
No effect on IL-2, IL-4, IL-10, or IFN-y
Sazakli et al.
(2014)
Blood collected from people exposed to
Cr(VI) in drinking water
T* IL-12, dose dependent
No effect on IL-6, IL-8, or IL-10
Snvder et al.
(1996)
Blood collected from people exposed to
Cr(VI) environmentally in Hudson
County, New Jersey
4, IL-6
Qian et al. (2013)
Serum collected from Cr(VI)-exposed
workers
4, IL-6, IL-10, IL-17A, IFN-y, and IFN-y/IL-4
No effect on IL-2 or TNF-a
Mignini et al.
(2009)
Plasma collected from Cr(VI)-exposed
workers
-t IL-2 and IL-6
4/ IL-12
No change in IL-ip, IL-4, TNF-a, or IFN-y
Mitrov et al.
(2014)
Plasma collected from rats exposed to
Cr(VI)
-t IL-ip and TNF-a
Jin et al. (2016)
Serum from LPS-stimulated mice
exposed to Cr(VI)
-t IL-6 and TNF-a
Thompson et al.
(2012c)
Plasma from Cr(VI)-exposed rats
4/ IL-12 and CXCL10 (IP-10)
No effect on IL-la, IL-ip, IL-2, IL-4, IL-5, IL-6, IL-10,
IL-13, 1L-17, 1L-18, TNF-a, IFN-y, CCL5, CXCL1,
Eotaxin, G-CSF, GM-CSF, MCP-1, or MlP-la
Thompson et al.
(2011b)
Plasma from Cr(VI)-exposed mice
"Few cytokines exhibited significant changes" but
no specific data; tested IL-la, IL-ip, IL-2, IL-4, IL-5,
IL-6, IL-7, IL-9, IL-10, IL-12, IL-13, IL-15, IL-17, TNF-a,
IFN-y, CXCL1, CCL5, CXCL10, G-CSF, GM-CSF, MCP-
1, and MlP-la
Cytokines measured in BALF
Cohen et al.
(2010)
BALF from Cr(VI)-exposed rats
No effect on TNF-a, MIP-2, MCP-1, IL-6, IL-10, or IL-
12
Cytokines secreted by MoDC
Reutter et al.
(1997)
Human MoDC exposed to Cr(VI) in vitro
-t IL-ip
Toebak et al.
(2006)
Human MoDC exposed to Cr(VI) in vitro
No effect on IL-8, CCL5, CCL17, CCL18, CCL20, and
CCL22
Cytokines secretion by stimulated PBMCs, lymphocytes, splenocytes, and macrophages
Akbar et al.
(2011)
Stimulated (anti-CD3 or anti-CD3/anti-
CD28) primary human lymphocytes
4/ IL-2 and IFN-y
Ban et al. (2010)
ConA-stimulated lymph nodes collected
from mice
4/ IL-4 (NS), IL-5 (NS), and IL-13 (NS)
-t IFN-y (NS)
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Reference
Study design
Cytokines
Cohen et al.
(1998)
Pulmonary macrophages collected from
Cr(VI) exposed rats, stimulated with LPS
ex vivo
4, IL-1, TNF-a, and IL-6 (NS)
Dai et al. (2017b)
Stimulated (anti-CD3/anti-CD28) splenic
lymphocytes collected from Cr(VI)-
exposed mice
4, IL-2, IL-4, and IL-10
Kativar et al.
(2008)
PHAand LPS-stimulated PBMCs
collected from exposed workers
T* PHA-stimulated IL-2 (NS) production
T* PHA-stimulated IL-6 production
No effect on LPS-stimulated TNF-a production
Karaulov et al.
(2019)
Mitogen-stimulated (ConA) splenocytes
collected from rats
-t IL-4 and IL-10 (NS)
4/ IL-6
No effect on INF-y
Cytokines secretion by unstimulated PBMCs
Lindemann et al.
(2008)
PBMCs collected from chromium
sensitized workers and exposed to Cr(VI)
in vitro
-t IL-4, IL-10, and IFN-y
No effect on IL-2 or IL-12
Cytokines secretion by peritoneal macrophages
Christensen et al.
(1992)
Newcastle disease virus infected mouse
peritoneal macrophages exposed to
Cr(VI) in vitro
4/ IFN-a/p
Jin et al. (2016)
Mouse peritoneal macrophages
-t IL-la, IL-ip, IL-6, and TNF-a
Cytokines secretion by cell cultures
Adam et al.
(2017)
TPA stimulated THP-1 cells
-t IL-ip
Badding et al.
(2014)
RAW264.7 cells exposed to Cr(VI)
-t TNF-a (NS)
No effect on IL-6 or IL-ip
Ban et al. (2010)
Spleens collected from mice
4/ IL-4, IL-5, IL-13, and IFN-y
Jin et al. (2016)
Serum from LPS-stimulated RAW264.7
cells exposed to Cr(VI)
-t IL-6 and TNF-a
Cytokines secreted by HaCaT cultures
Wang et al.
(2010a)
Human HaCaT cells treated with Cr(VI)
-t IL-la and TNF-a
Lee et al. (2014)
Human HaCaT cells treated with Cr(VI)
-t IL-la and TNF-a
Cytokines secreted by duodenum
Thompson et al.
(2012c)
Duodenum from Cr(VI)-exposed rats
-t IL-la
4/ IL-4
-t IL-6 (60 mg/LSDD)
No effect on IL-ip, IL-2, IL-5, IL-10, IL-12, IL-13, IL-
17, IL-18, TNF-a, IFN-y, CCL5, CXCL1, CXCL10,
Eotaxin, G-CSF, GM-CSF, MCP-1, or MlP-la
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Reference
Study design
Cytokines
Thompson et al.
(2011b)
Duodenum from Cr(VI)-exposed mice
4/ IL-ip and TNF-a, dose-dependent trends
For all other cytokines, no specific data were
reported, other than "Several cytokines were
significantly altered—generally beginning at 60
mg/LSDD"; tested IL-la, IL-2, IL-4, IL-5, IL-6, IL-7, IL-
9, IL-10, 1L-12, 1L-13, IL-15, IL-17, IFN-y, CXCL1,
CCL5, CXCL10, G-CSF, GM-CSF, MCP-1, and MlP-la
Cytokines secreted by oral mucosa
Thompson et al.
(2012c)
Oral mucosa from Cr(VI)-exposed rats
No effect on IL-la, IL-ip, IL-2, IL-4, IL-5, IL-6, IL-10,
IL-12, 1L-13, IL-17, 1L-18, TNF-a, IFN-y, CCL5, CXCL1,
CXCL10, Eotaxin, G-CSF, GM-CSF, MCP-1, or MlP-la
Thompson et al.
(2011b)
Oral mucosa from Cr(VI)-exposed mice
"Significant differences from control animals were
generally limited to the highest treatment dose,"
but no specific data; tested IL-la, IL-ip, IL-2, IL-4,
IL-5, IL-6, IL-7, IL-9, IL-10, IL-12, IL-13, IL-15, IL-17,
TNF-a, IFN-y, CXCL1, CCL5, CXCL10, G-CSF, GM-CSF,
MCP-1, and MlP-la
NS = not statistically significant, BALF = bronchoalveolar lavage fluid, ConA = concanavalin A; HaCaT
cells = immortalized human keratinocytes; LPS = lipopolysaccharide; MoDC = monocyte-derived dendritic cell;
PBMC = peripheral blood mononuclear cell; TPA = 12-0-tetradecanoylphorbol-13-acetate.
C.2.6. Male Reproductive Effects
C.2.6.1. Mechanistic studies relevant to male reproductive toxicity
1 Mechanistic evidence indicating the biological pathways involved in male reproductive
2 toxicity following Cr(VI) exposure is summarized in Table C-39. Studies identified in preliminary
3 title and abstract screening as "mechanistic" were further screened and tagged as "reproductive" if
4 they involved reproductive tissues or cells; 49 studies were identified. Studies were prioritized for
5 consideration in the synthesis of mechanistic evidence for male reproductive effects if they were
6 conducted in mammalian species:
7 • Studies in humans with quantified oral or inhalation exposure to Cr(VI)
8 • Studies in experimental animals with quantified oral (drinking water, gavage, diet),
9 inhalation, or intratracheal instillation, or injection exposure to Cr(VI)
10 • In vitro studies in primary or immortalized mammalian cells derived from male
11 reproductive tissues (i.e., Leydig, Sertoli, male germ cells)
12 • Mechanistic endpoints relevant to interpretations of male reproductive health effects in
13 humans
14 A total of 25 reproductive studies were identified to include in the male reproductive
15 mechanistic synthesis. Several of the included oral exposure animal toxicological studies in that
16 section were identified as also reporting mechanistically relevant data, as well as i.p. injection
17 studies that did not meet PECO criteria but were reviewed as being potentially relevant for
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1 mechanistic analysis. In vitro studies that evaluated Leydig, Sertoli, or male germ cells were also
2 considered for mechanistic evidence.
Table C-39. Mechanistic studies prioritized for informing potential Cr(VI)-
induced male reproductive toxicity
System
Route
Exposure3
Results
Reference
Oxidative stress
Mouse, male
(strain not
reported)
Oral (not
specified)
5 mg/kg-d
K2Cr207, 30- or
60-d
4/ serum antioxidant enzymes (CAT, SOD,
GPx)
T* serum MDA
Rasool et al.
(2014)
Rat, Sprague-
Dawley, male
Oral
(inferred to
be gavage)
10 mg/kg-d
[form of Cr(VI)
not reported],
13-d
4/ testicular and epididymal CAT, SOD, GST,
glutathione
T* testicular and epididymal MDA
Kim et al.
(2012)
Monkey, bonnet,
male
Oral
(drinking
water)
100, 200, 400
mg/L K2Cr207,
180-d
4/ testicular SOD, CAT, GPx, GR, G-6-PDH, y-
GT, and vitamins A, C, E
T* testicular GST and reduced glutathione
T* testicular H2O2 and OH-
Aruldhas et al.
(2005)
Monkey, bonnet,
male
Oral
(drinking
water)
50, 100, 200,
400 mg/L
K2Cr207, 6-mo
4/ SOD, and GDH in seminal plasma and
sperm
T* H2O2 in seminal plasma and sperm
Subramanian
et al. (2006)
Rat, Wistar Fl,
male
Oral
(drinking
water)
50-200 mg/L
K2Cr207, GD 9-
14 or GD 15-21;
Fl animals
evaluated on
PND30
T* lipid peroxidation, H2O2, OH- in Sertoli cells
4/ SOD, CAT, GPx, GSR, GST, and GSH in
Sertoli cells
Shobana et al.
(2020)
Rat, Wistar, male
Oral
(gavage)
3.5 mg/kg-d
Cr(VI), 8-wk
T* testicular MDA, GSSG, NO
4/ testicular GSH, SOD, CAT, carnitine
Mitigated by cotreatment with antioxidant
Bashandv et
al. (2021)
Mouse, Swiss
albino, male
i.p.
injection
1 mg/kg CrOs,
single injection
4/ testicular SOD, CAT, peroxidase
T* testicular lipid peroxidation potential
Acharva et al.
(2006)
Rat, Wistar, male
i.p.
injection
1-2 mg/kg-d
K2Cr207,15-d
4/ testicular CAT
T* testicular metallothionein
T* testicular MDA, O2-
Marouani et
al. (2015a)
Rat, Wistar, male
i.p.
injection
10 mg/kg-d
Na2Cr207,10-d
4/ testicular SOD, CAT, GPx
T* testicular MDA
Mitigated by cotreatment with antioxidant
Hfaiedh et al.
(2014)
Rat, Wistar, male
i.p.
injection
2mg/kg-d,
K2Q2O7, 21-d
T* testicular indicators of lipid peroxidation
(TBARS and H2O2) with significant effect
decrease with antioxidant pretreatment
4/ testicular GSH and activity antioxidant,
phosphatase, and aminotransferase mitigated
by antioxidant pretreatment
El-Demerdash
et al. (2019)
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Route
Exposure3
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Reference
Mouse, Swiss
albino, male
i.p.
injection
10 mg/kg CrC>3,
single dose with
evaluation
5,6,7, and 8 wk
after treatment
(control 5 wk
only)
1" testicular indicators of lipid peroxidation
(TBARS)
Acharva et al.
(2004b)
Cultured mouse
Leydig cells
(TM3), Sertoli
cells (TM4), and
spermatogonia!
stem cells
In vitro
3.125-50 nM
Cr(VI)
1" ROS after 4 h
4/ mRNA expression of antioxidant enzymes
(Sod, Cat, Gpxl, Gsta4) after 24 h
1" mRNA expression of Gstal at all doses in
somatic cells and low doses in germ cells after
24 h
Das et al.
(2015)
Cultured mouse
spermatogonia!
stem cells (C18-4)
In vitro
5-75 nM Cr(VI)
1" ROS after 24 h
Lv et al. (2018)
Cell cycle regulation and apoptosis in somatic and germ cells
Rat, Wistar, male
Oral
(gavage)
3.5 mg/kg-d
Cr(VI), 8-wk
1" p53 expression in spermatogenic cells
4/ DNA content of spermatogenic cells
Mitigated by cotreatment with antioxidant
(melatonin)
Bashandv et
al. (2021)
Rat, Wistar, male
i.p.
injection
1-2 mg/kg-d
K2Cr207,15-d
1" BAX and DNA fragments in testis
Marouani et
al. (2015a)
Mouse, ICR, male
i.p.
injection
16.2 mg/kg-d
Cr(VI), 1-wk
1" BAX and DNA fragments (y-H2AX) in testis
Qualitative histopathology showing
degenerative changes in seminiferous tubules
and germ cells; Cr(VI) treated males also had
decreased litter sizes
Mitigated by cotreatment with antioxidant
(melatonin)
Lv et al. (2018)
Rat, Wistar, male
i.p.
injection
2mg/kg-d,
K2Cr207, 21-d
Qualitative histopathology showed
degeneration of spermatogenic cells in testes
and moderate atrophy
El-Demerdash
et al. (2019)
Mouse, Swiss
albino, male
i.p.
injection
10 mg/kg CrC>3,
single dose with
evaluation
5,6,7, and 8 wk
after treatment
(control 5 wk
only)
4/ sperm count at all timepoints
1" sperm abnormalities at all timepoints
Acharva et al.
(2004b)
Rabbit, ITRC
colony, male
i.p.
injection
2mg/kg-d,
K2Cr2C>7,
evaluation at 3
and 6 wk 72 h
after last
injection
Qualitative histological analysis, progressive
testicular interstitial edema, no
spermatocytes in seminiferous tubules
Behari et al.
(1978)
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Route
Exposure3
Results
Reference
Cultured mouse
Leydig cells
(TM3), Sertoli
cells (TM4), and
spermatogonia!
stem cells
In vitro
3.125-50 nM
Cr(VI)
1" TUNEL-positive cells
4/ mitochondrial membrane potential
1" biomarkers of intrinsic apoptosis
(e.g., cleavage of caspases 3 and 9,
4, BCL2/BAX ratio)
4/ biomarkers of extrinsic apoptosis (Fas,
caspase 8) in somatic cells
Mitigated by cotreatment with antioxidant
(N-acetyl-L-cysteine)
Das et al.
(2015)
Cultured mouse
spermatogonia!
stem cells (C18-4)
In vitro
5-75 nM Cr(VI)
1" TUNEL-positive cells
-t DNA fragments (y-H2AX)
1" chromatin condensation
1" biomarkers of intrinsic apoptosis
(e.g., cleavage of caspases 3 and 9, T* BAX,
4/ BCL-2)
Mitigated by cotreatment with antioxidant
(melatonin)
No effect on biomarkers of extrinsic apoptosis
(caspase 8)
(after 24 h)
Lv et al. (2018)
Primary coculture
of rat (Wistar)
Sertoli cells and
germ cells
In vitro
0.5, 1, 10, 100
Hg/L Cr(VI)
4/ late spermatocytes and round spermatids
1" cells with alterations in meiotic prophase
1" asynapsis and fragmented synaptonemal
complexes
Geoffrov-
Siraudin et al.
(2010)
Altered steroidogenesis and effects on the hypothalamic-pituitary-gonadal axis
Rat, Sprague-
Dawley Fl, male
Oral
(gavage)
3-12 mg/kg-d
Cr(VI), GD 12-
21; Fl animals
evaluated on
PND21
Biphasic effect on testosterone (1" at low
dose, 4/ at high dose)
Biphasic mRNA and protein expression of LHR
(1" at low dose, 4^ at high dose)
1" low dose expression of FSHR (mRNA only),
SCARB1, LIF, PDGFA (no change at high dose)
4/ high dose expression of IGF1, CYP17A1
(protein only), HSD17B3 (mRNA only), StAR
(protein only, not significant)
No change in mRNA or protein expression of
CYP11A1, insulin-like-3 hormone, NR5A1,
SOX9, AMH, DHH
Zheng et al.
(2018)
Rat, Wistar, male
Oral
(gavage)
3.5 mg/kg-d
Cr(VI)
4/ plasma testosterone, LH
-t FSH
Mitigated by cotreatment with antioxidant
Bashandy et
al. (2021)
Rabbit, New
Zealand white,
male
Oral
(gavage)
3.6 mg-kg/d
Cr(VI), 10-wk
4/ plasma testosterone
Yousef et al.
(2006)
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Route
Exposure3
Results
Reference
Rat, Wistar Fl,
male
Oral
(drinking
water)
50-200 mg/L
K2Q2O7, GD 9-
14, Fl animals
evaluated on
PND60
4/ serum testosterone, prolactin
1" serum estrogen, LH, FSH
4/ protein expression of AR, LHR, PRLR, and
ERa in Leydig cells
1" protein expression of ERP in Leydig cells
4/ protein expression of StAR, CYP11A1,
3PHSD, CYP17A1,17PHSD, 5a reductase,
aromatase in Leydig cells
4/ specific activities of 3PHSD and 17PHSD in
Leydig cells
4/ protein expression of AR, FSHR, ERa, ERP,
and 5a reductase in Sertoli cells
Navin et al.
(2021)
Rat, Wistar Fl,
male
Oral
(drinking
water)
50-200 mg/L
K2Q2O7, GD 9-
14 or GD 15-21,
Fl animals
evaluated on
PND30
4/ serum testosterone, prolactin
1" serum estrogen, LH, FSH
4/ mRNA and protein expression of AR and
FSHR in Sertoli cells
4/ protein expression of transcriptional
regulators of Fshr (USF-1, USF-2, GATA-1,
c-jun, c-fos) and Ar (Sp-1, ARA54, CBP, SRC-1)
in Sertoli cells
1" protein expression of cyclin D1 and p53
(inhibitors of Ar expression)
4/ mRNA expression of Ar and Fshr in Sertoli
cells
Shobana et al.
(2020)
Rat, Wistar Fl,
male
Oral
(drinking
water)
50-200 mg/L
K2Cr207, GD 9-
14; Fl animals
evaluated on
PND 120
4/ testosterone in serum and testicular
interstitial fluid
4/ serum FSH and LH
4/ gene and protein expression of AR and
FSHR in Sertoli cells
Kumar et al.
(2017)
Rat, Wistar, male
Oral
(drinking
water)
K2Q2O7, 500
mg/L in drinking
water
[estimated to
be 73.05
mg/kg-d Cr(VI)],
30-d
4/ serum prolactin (60% of control)
No change in serum LH accumulation of Cr in
target tissues (pituitary, hypothalamus, liver).
30% reduction in water intake and 11.6%
reduction in BW. Study also includes in vitro
study in primary anterior pituitary cells (see
later in table).
Quinteros et
al. (2007)
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Supplemental Information—Hexavalent Chromium
System
Route
Exposure3
Results
Reference
Rat, Wistar, male
Oral
(drinking
water)
K2Q2O7, 200
mg/L in drinking
water
[estimated to
be 11.6 mg/kg-
d Cr(VI)], 30-d
1" Lipid peroxidation in pituitary and
hypothalamus; no change in liver.
1" SOD activity in pituitary only
1" CAT activity in liver only
1" glutathione reductase activity in
hypothalamus only
No changes in GPx activity
1" in HO-1 mRNA expression in hypothalamus
and pituitary only
1" MT-3 in hypothalamus and MT-1 in
anterior pituitary
Accumulation of Cr in target tissues (pituitary,
hypothalamus, liver). No significant change in
water consumption or BW. Did not measure
if oxidative effects impacted downstream
hormones.
Nudler et al.
(2009)
Rat, Wistar, male
i.p.
injection
2mg/kg-d,
K2Q2O7, 21-d
4/ serum testosterone
-t serum FSH
Mitigated by cotreatment with antioxidant
El-Demerdash
et al. (2019)
Rat, Wistar, male
i.p.
injection
1-2 mg/kg-d
K2Q2O7,15-d
4/ serum testosterone and LH
-t serum FSH
Marouani et
al. (2012)
Rat, Wistar, male
i.p.
injection
10 mg/kg-d
Na2Cr207,10-d
4/ serum testosterone
Mitigated by cotreatment with antioxidant
Hfaiedh et al.
(2014)
Cultured mouse
Leydig cells (TM3)
and Sertoli cells
(TM4)
In vitro
6.25-25 nM
Cr(VI)
4/ testosterone secretion by TM3 cells
4/ mRNA expression of steroidogenic
enzymes (Cypllal, Hsd3bl, Cypl7al,
Cypl9al) in TM3 cells
4/ mRNA expression of Fshr, Ar in TM4 cells
1" mRNA expression of Star in TM3 cells
Das et al.
(2015)
Primary anterior
pituitary cells
from male Wistar
rat
In vitro
K2Q2O7, 0.1-10
HM up to 72 h
4/ prolactin at 0.1 nM at 72 h, 1 and 10 nM at
48 h and72h
No change in LH
1" Caspase 3 and 10 nM [cytotoxic, prevented
pretreatment with an antioxidant (NAC)]
Same study that showed decreased prolactin
and no change in LH in vivo (see earlier in
table). Cell viability significantly reduced after
24 h at 10 nM (~65%); 1 nM after 72 h.
Quinteros et
al. (2007)
Primary anterior
pituitary cells
from male Wistar
rat
In vitro
K2Q2O7, 10 nM
for 72 h
Mechanisms involved in apoptosis include
decreased CAT, GPx, increased p53 and Bax
Data not fully reviewed because cytotoxic
concentration was used, as demonstrated in
Quinteros et al. (2007)
Quinteros et
al. (2008)
Effects on Sertoli cells and the blood-testis barrier
Rat, Wistar Fl,
male
Oral
(drinking
water)
50-200 mg/L
K2Q2O7, GD 9-
14 or GD 15-21,
Fl animals
4/ secretory products of Sertoli cells (inhibin,
androgen binding protein, transferrin, lactate,
pyruvate, retinoic acid)
Shobana et al.
(2020)
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Route
Exposure3
Results
Reference
evaluated on
PND30
4/ mRNAand protein expression of tight
junction proteins (claudin-11 and occludin) in
Sertoli cells
Rat, Wistar Fl,
male
Oral
(drinking
water)
50-200 mg/L
K2Cr207, GD 9-
14, Fl animals
evaluated on
PND 120
4/ mRNA and protein expression of tight
junction proteins (claudin-11 and occludin) in
Sertoli cells
Kumar et al.
(2017)
Rat, Druckrey,
male
i.p.
injection
2 mg/kg-d
K2Cr207,15-d
Leakage of Sertoli cell tight junctions and
adverse effects on late-stage spermatids
Murthv et al.
(1991)b
Mouse Sertoli
cells (TM3)
In vitro
6.25-25 nM
Cr(VI)
4/ mRNA expression of tight junction
signaling molecules (tight junction protein 1,
vimentin, occludin)
Das et al.
(2015)
Primary coculture
of rat (Sprague-
Dawley) Sertoli
cells and germ
cells
In vitro
10 ng/L Cr(VI)
4/ gap junction signaling and derealization of
connexin 43 from the membrane to the
cytoplasm after 8 d; no effects on adherin or
tight junction proteins (claudin-11 and N-
cadherin)
1" transepithelial resistance
Carette et al.
(2013)
aPotassium dichromate units conversion: Cr(VI) = 0.353 x K2Cr2C>7; potassium chromate units conversion:
Cr(VI) = 0.268 x foCrCU; sodium dichromate dihydrate units conversion: Cr(VI) = 0.349 x Na2Cr20?2H20 (usually
denoted as Na2Cr207 because study authors frequently list the salt as the chemical compound even if
concentration or dose is based on the dihydrate); chromium trioxide units conversion: Cr(VI) = 0.52 x CrC>3.
bNote: Scientific integrity is a concern due to evidence of self-plagiarism within this research group.
C.2.7. Female Reproductive Effects
C.2.7.1. Mechanistic studies relevant to female reproductive toxicity
1 Mechanistic evidence indicating the biological pathways involved in female reproductive
2 toxicity following Cr(VI) exposure is summarized in Table C-40. Studies identified in preliminary
3 title and abstract screening as "mechanistic" were further screened and tagged as "reproductive" if
4 they involved reproductive tissues or cells. Studies were prioritized for consideration in the
5 synthesis of mechanistic evidence for female reproductive effects if they were conducted in
6 mammalian species:
7 • Studies in humans with quantified oral or inhalation exposure to Cr(VI)
8 • Studies in experimental animals with quantified oral (drinking water, gavage, diet),
9 inhalation, or intratracheal instillation, or injection exposure to Cr(VI)
10 • In vitro studies in primary or immortalized mammalian cells derived from female
11 reproductive tissues (e.g., thecal and granulosa cells)
12 • Mechanistic endpoints relevant to interpretations of female reproductive health effects in
13 humans
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Supplemental Information—Hexavalent Chromium
1 A total of 12 reproductive studies were identified to include in the female reproductive
2 mechanistic synthesis. Several of the included oral exposure animal toxicological studies in that
3 section were identified as also reporting mechanistically relevant data, as well as i.p. injection
4 studies that did not meet PECO criteria but were reviewed as being potentially relevant for
5 mechanistic analysis. In vitro studies conducted in relevant cell types, such as thecal and granulosa
6 cells, were also considered for mechanistic evidence.
Table C-40. Mechanistic studies prioritized for informing potential Cr(VI)-
induced female reproductive toxicity
System
Route
Exposure3
Results
Reference
Altered steroidogenesis
Rat, lactating
Sprague-Dawley
Oral
(drinking
water)
50, 100, 200 mg/L
K2Cr207, PNDs 1-21;
F1 animals evaluated
on PNDs 25, 45, 65
For Fl:
4/ FSH receptor gene expression in ovary
4/ E2, T, P4 (dose dependent, in hormone
section of animal tox)
1" FSH (not dose dependent)
Mitigated by cotreatment with vitamin C
Stanlev et al.
(2013)
Rat, lactating
Sprague-Dawley
Oral
(drinking
water)
5, 10, 25, 50, 100,
and 200 mg/L
K2Cr207, PNDs 1-21;
F1 animals evaluated
on PNDs 25, 45, 65
For Fl:
4, E2, T, P4 (50 mg/L, PND 25)
1" time to puberty (50 mg/L)
Cotreatment with estradiol restored the
ovarian protein expression of several
antioxidant enzymes (Gpxl, catalase,
Prdx3, and Txn2)
Stanlev et al.
(2014)
Rat, lactating
Sprague-Dawley
Oral
(drinking
water)
50 mg/L K2Cr207,
PNDs 1-21; F1
animals evaluated on
PND25
For Fl:
4/ ovarian expression of steroidogenic
acute regulator protein (StAR),
3p-hydroxysteroid dehydrogenase, and
aromatase
1" genes involved in the metabolic
clearance of estradiol (Cyplal, Cyplbl,
UDP-glucuronosyltransferases, Sultlal,
NAD(P)H quinone oxidoreductase 1)
Mitigated by cotreatment with
resveratrol
Banu et al.
(2016)
Rat, Wistar,
female, GDs 9-
21; female pups
PND65
Oral
(drinking
water)
Group 1: 50,100,
200, and 400 mg/L
K2Cr207, GDs 9-21; F1
animals evaluated on
PND0
Group 2: 200 mg/L
K2Cr207, GDs 9-PND
65; F1 animals
evaluated on PNDs 0,
3, 7, 18, 45, 65
For Fl:
4/ serum progesterone, estradiol,
testosterone, prolactin, growth hormone
-t serum LH and FSH
Samuel et al.
(2012)
Primary rat
granulosa cells
In vitro
10 nM K2Cr207,12 or
24 h
4/ FSH receptor protein expression
Pretreatment with vitamin C mitigated
Stanlev et al.
(2013)
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Supplemental Information—Hexavalent Chromium
System
Route
Exposure3
Results
Reference
Primary Sprague-
Dawley rat
granulosa cells
(immature rats,
23-25 d old);
immortalized rat
granulosa cells
In vitro
10 nM K2Cr207,12 or
24 h
4/ ErP and FSH receptor gene expression
Pretreatment with vitamin C mitigated
Stanlev et al.
(2011)
Immortalized rat
granulosa cells
In vitro
12.5 nM K2Cr207,
12 and 24 h
4/ gene expression of FSH receptor, LH
receptor, Era, ErP, StAR, SF-1 (24 h only),
and 17p-hydroxysteroid dehydrogenases
4/ cell proliferation 50%
Banu et al.
(2008)
Oxidative stress
Rat, lactating
Sprague-Dawley
Oral
(drinking
water)
50, 100, 200 mg/L
K2Cr207 (2013)
5, 10, 25, 50, 100,
and 200 mg/L K2Cr207
(2014)
PND1-21; F1 animals
evaluated on PND 25
(2014) or PNDs 25,
45, 65 (2013)
For Fl:
4/ ovarian SOD, catalase, glutathione
peroxidase, and glutathione reductase
activity (100 mg/L 2013; 50 mg/L, 2014)
4/ ovarian protein expression of GPxl,
Txn2, Prdx3, CAT expression (2014)
1" ovarian protein expression of
glutathione-S-transferase (2013)
1" ovarian LPO, H202 (dose dependent
2013; 50 mg/L, 2014)
Mitigated by cotreatment with VitC
(2013) or EDA (2014)
Stanley et al.
(2014; 2013)
Rat, strain not
reported
(assume
Sprague-Dawley)
Oral
(drinking
water)
25 mg/L K2Cr207, GDs
9.5-14.5; F1 animals
evaluated on PND 1
For Fl:
1" p53/SOD2 protein colocalization in the
ovary; p53 has been demonstrated to
reduce SOD2 antioxidant activity
Sivakumar et
al. (2014)
Rat, lactating
Sprague-Dawley
Oral
(drinking
water)
50 mg/L K2Cr207, PND
1-21; F1 animals
evaluated on PND 25
For Fl:
4/ ovarian protein expression of catalase,
glutathione peroxidase (GPxl),
peroxiredoxin (PRDX) 3, and thioredoxin
(TXN).
1" ovarian protein expression of SOD1
and SOD2
1" oxidative damage in ovary (LPO, H202)
Oxidative damage mitigated by
cotreatment with resveratrol
Banu et al.
(2016)
Mouse, Swiss
albino, female
Oral
(gavage)
5 and 10 mg
K2Cr207/kg, 30-d
1" Lipid peroxidation in ovary (MDA)
4/ ovarian SOD and CAT activity, and 4^
levels of vitamin C and glutathione (dose-
dependent)
Mitigated by cotreatment with vitamin E
Rao et al.
(2009)
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Supplemental Information—Hexavalent Chromium
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Route
Exposure3
Results
Reference
Rat, Wistar,
female, GD 9-21;
female pups PND
65
Oral
(drinking
water)
Group 1: 50,100,
200, and 400 mg/L
K2Q2O7, GDs 9-21; F1
animals evaluated on
PND 0
Group 2: 200 mg/L
K2Q2O7, GD9-PND
65; F1 animals
evaluated on PNDs 0,
3, 7, 18, 45, 65
For Fl:
4/ ovarian SOD, CAT, GPx activity
4/ ovarian ascorbic acid
T* ovarian LPO and H2O2 at all ages
Samuel et al.
(2012)
Rat, Wistar,
female
i.p.
injection
1 and 2 mg
K2Cr207/kg, 15-d
T* Superoxide anion in uterus (as
measured by cytochrome C and
iodonitrotetrazolium reduction)
4/ CAT activity in uterus
T* lipid peroxidation in uterus
4/ metallothionine
All dose dependent
Marouani et
al. (2015b)
Primary rat
granulosa and
theca cells;
immortalized rat
granulosa cells
In vitro
10 nM K2Cr207, 12 h
and 24 h
4/ intracellular vitamin C levels
4/ SOD1, SOD2, CAT, GLRX1, GST Ml,
GSTM2, GSTA, GR, TXN1, TXN2, TXNRD2,
and PRDX3 gene expression (time
dependent)
4/ GR, GST, GPx, SOD, CAT activity
t H2O2, LPO
Immortalized GCs showed similar effect.
Cell viability not reported. Vitamin C
failed to mitigate CrVI effects on GSTM1,
GSTM2, TXN1, and TXN2 in TCs
Stanlev et al.
(2013)
Apo ptosis
Rat, lactating
Sprague-Dawley
Oral
(drinking
water)
50 mg/L K2Cr207,
PNDs 1-21; F1
animals evaluated on
PND 25
For Fl:
T* follicular cell apoptosis (TUNEL)
T* ovarian protein expression of
cytochrome C, caspase-3
4/ ovarian protein expression of Bcl-2,
Bcl-XL, HIF-la
Mitigated by cotreatment with
resveratrol
Banu et al.
(2016)
Rat, pregnant
Sprague-Dawley
Oral
(drinking
water)
25 mg/L K2Cr207, GDs
9.5-14.5; F1 animals
evaluated on GDs
15.5 and 17.5, PNDs
1, 4, 25
For Fl:
T* germ cell apoptosis (TUNEL)
Banu et al.
(2015)
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System
Route
Exposure3
Results
Reference
Rat, lactating
Sprague-Dawley
Oral
(drinking
water)
5, 10, 25, 50, 100,
and 200 mg/L K2Cr207
(2014)
50, 100, 200 mg/L
K2Q2O7 (2013)
PNDs 1-21; F1
animals evaluated on
PND25 (2014) or
PNDs 25, 45, 65
(2013)
For Fl:
T* dose-dependent follicular (granulosa)
cell apoptosis (TUNEL) and atretic %
T* ovarian protein expression of caspase-
3 (50 mg/L, 2014)
4/ ovarian protein expression of Bcl-2,
Bcl2ll (50 mg/L, 2014)
granulosa and theca cells with 50 mg/L
were 50% positive PND 25 (2013). 5 mg/L
were 30% positive PDN 25 (2014)
Stanley et al.
(2014; 2013)
Rat, pregnant
Sprague-Dawley
Oral
(drinking
water)
10 mg/L K2Cr207, GDs
9.5-14.5; F1 animals
evaluated on PND 1
For Fl:
T* germ cell apoptosis (TUNEL)
T* ovarian protein expression of acetyl-
p53, cleaved caspase-3, BAX, PUMA
4/ ovarian protein expression of Bcl-2,
Bcl-XL, p-AKT
Effects other than p-AKT were
exacerbated by SIRT1 inhibitor
Sivakumar et
al. (2022)
Rat, strain not
reported
(assume
Sprague-Dawley)
Oral
(drinking
water)
25 mg/L K2Cr207, GDs
9.5-14.5; F1 animals
evaluated on PND 1
For Fl:
T* germ cell apoptosis (TUNEL)
T* ovarian protein expression of BAX,
caspase 3, p53, p27
4/ ovarian protein expression of p-AKT,
p-ERK, and XIAP
Sivakumar et
al. (2014)
Rat, Wistar,
female
i.p.
injection
1 and 2 mg
K2Cr207/kg, 15-d
4/ relative ovary/uterine weight (with
decreased bw; 40% and 137% of controls,
dose dependent)
T* apoptotic cells and protein expression
of Bax in uterus
Uterine Bcl-2 was not detected in control
or Cr(VI) treatment groups
Apoptosis was characterized by
chromatin condensation, detected by
borated toluidine blue staining; Bax/Bcl-2
by immunostaining
Marouani et
al. (2015b)
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System
Route
Exposure3
Results
Reference
Primary Sprague-
Dawley rat
granulosa cells
(immature rats,
22-25 d old)
In vitro
10 nM K2Cr207,12 or
24 h
1" apoptosis
1" translocation of cytochrome C from
mitochondria to cytosol, T* cleaved
caspase-3 and PARP (important terminal
events in apoptosis)
1" Bax, t-Bad
4/ Bcl-2, Be I-XL, pBad-112/136, Hsp-70,
Hsp-90
1" p-ERK, p-JNK; 4^ p-AKT; no change in
p-p38 (indicates suppression of AKT
pathway but activation of ERK1/2
pathway)
1" p53 (total and phosphorylated at
specific serine sites); higher in
mitochondria compared to cytosol,
suggesting translocation to the
mitochondria
4/ apoptosis after cotreatment with
ERK1/2 and JNK inhibitor
4/ p53 activity after cotreatment with
ERK1/2 inhibitor; no effect of JNK
inhibitor
1" p-ERK in mitochondria and nucleus
Mitigated by pretreatment with
vitamin C
Banu et al.
(2011)
Primary Sprague-
Dawley rat
granulosa cells
(immature rats,
23-25 d old);
immortalized rat
granulosa cells
In vitro
10 nM K2Cr207,12 or
24 h
Cell cycle arrest at G1 phase (decreased
cell population at S and G2-M phases)
4/ protein expression of cyclin-
dependent kinases 1, 2, 4, 6 in both cell
types; cyclins D2&3, E2, Bl; PCNA
1" protein expression of pl5, pl6, p27
Results time dependent
Mitigated by pretreatment with
vitamin C
Stanlev et al.
(2011)
Ovarian extracellular matrix
Rat, pregnant
Sprague-Dawley
Oral
(drinking
water)
25 mg/L K2Cr2C>7, GDs
9.5-14.5; F1 animals
evaluated on GDs
15.5 and 17.5, PNDs
1, 4, 25
For Fl:
1" ovarian protein expression of Xpnpep2
and 4/ collagen (Coll, Col3, Col4) in
fetuses
4/ ovarian protein expression of Xpnpep2
and 1" collagen (Coll, Col3, Col4) in pups
at PNDs 1, 4, and 25
Protein expression of Xpnpep2 and
collagens measured using
immunohistochemistry
Banu et al.
(2015)
aPotassium dichromate units conversion: Cr(VI) = 0.353 x K2Cr2C>7; potassium chromate units conversion:
Cr(VI) = 0.268 x faCrCU; sodium dichromate dihydrate units conversion: Cr(VI) = 0.349 x Na2Cr20?2H20 (usually
denoted as Na2Cr207 because study authors frequently list the salt as the chemical compound even if
concentration or dose is based on the dihydrate); chromium trioxide units conversion: Cr(VI) = 0.52 x CrC>3.
This document is a draft for review purposes only and does not constitute Agency policy.
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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
Supplemental Information—Hexavalent Chromium
C.2.8. Developmental Effects
C.2.8.1. Mechanistic studies relevant to developmental toxicity
Mechanistic evidence indicating the biological pathways involved in developmental toxicity
following Cr(VI) exposure is summarized in Table C-41. Studies identified in preliminary title and
abstract screening as "mechanistic" were further screened and tagged as "developmental" if they
involved embryonic development or survival. The following studies were prioritized:
• Studies in humans with quantified oral or inhalation exposure to Cr(VI)
• Studies in experimental animals with quantified oral (drinking water, gavage, diet),
inhalation, or intratracheal instillation, or injection exposure to Cr(VI)
• In vitro studies in primary or immortalized mammalian cells derived from tissues relevant
to mammalian development, including embryonic and placental tissues and cells and cells
involved in organ development (e.g., osteoblasts)
• Mechanistic endpoints relevant to interpretations of effects on human development,
including genotoxicity tests relevant to fetal development (e.g., rodent dominant lethal test)
A total of 14 developmental studies were identified to include in the developmental toxicity
mechanistic synthesis. Studies were prioritized for consideration in the synthesis of mechanistic
evidence for developmental effects if they were conducted in mammalian species. Several of the
included oral exposure animal toxicological studies in that section were identified as also reporting
mechanistically relevant data, as well as i.p. injection studies that did not meet PECO criteria but
were reviewed as being potentially relevant for mechanistic analysis. In vitro studies conducted in
relevant cell types derived from tissues relevant to mammalian development were also considered
for mechanistic evidence. In vitro studies in human trophoblasts or mitochondria isolated from
human placentas were considered as potentially relevant to effects in the placenta, and studies in
osteoblasts were also considered as potentially relevant for the evaluation of skeletal effects.
Effects are also expected to be more likely in in vitro embryonic studies compared to in vivo
studies, as the in vitro studies incubated sperm or blastocytes directly with potassium dichromate.
Table C-41. Mechanistic studies prioritized for informing potential Cr(VI)-
induced developmental toxicity
System
Route
Exposure3
Results
Reference
Fetal genotoxicity
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—Hexavalent Chromium
System
Route
Exposure3
Results
Reference
Mouse, pregnant
Swiss albino
Oral
(drinking
water) or
i.p.
injection
Drinking water
study: 5 and 10
mg/L K2Cr2C>7,
duration of
pregnancy
i.p. study: 50
mg/kg Na2Cr2C>7
or K2Cr2C>7, single
dose on GD 17
Euthanasia on
GD 18
1" significant increase in micronucleated
polychromatic erythrocytes in maternal
bone marrow, fetal liver, and fetal
peripheral blood after i.p. injection.
No effects after oral dosing.
De Flora et al.
(2006)
In vitro evaluations of embryo development
Dub:(ICR) mouse
blastocysts from
day 4 of gestation
with 6 d of
exposure or
embryos from day
8 for 24 h
In vitro
0.25-2 nM
K2Cr207
1" blastocyst (1 and 2 nM) and embryo (all
concentrations) SCEs
No effects on embryo hatching, attachment
of trophoblast outgrowth
4/ blastocyst inner cell masses
4/ embryo development including crown-
rump length
liiima et al.
(1983)
Sperm and
untreated oocytes
from BDF1 mice
In vitro
3.125, 6.25, 12.5,
25, or 50 nM
K2Cr207
4/ acrosome reaction (12.5 nM+)
1" time to expanded and hatching blastocyst
stage
4/ blastocyst ICM and TE cell proliferation
4/ ICM-TE expression sox2, pou5fl, klf4 all
cone; cdx2 at 12.5 nM; eomes and krt8 at 25
HM (all pluripotent marker genes)
Sperm viability was significantly decreased
at 6.25 nM
Yoisungnern et
al. (2015)
Balb/c mouse
embryos at 2-cell
stage
In vitro
and
CaCrC>4 at 0.02-
2.0 ng/L (20, 2
and 0.2 nM and
40, 4, and 0.4
\iM,
respectively)
4/ blastocyst maturation after 3 d of culture
with both salts; K2Cr2C>7 arresting all at 4-cell
stage at high dose
4/ hatching, both salts
4/ implantation CaCrC>4
Jacauet and
Drave (1982)
Mechanisms affecting bone development
Rat, Sprague-
Dawley, male
i.p.
injection
60 ng/kg
foC^Cb, single
dose 48 h
1" TSH, effects on follicle morphology
including atrophy
4/ free T4, T3, follicle size
Pretreatment (i.p.) with ascorbic acid
inhibits effects on hormones, treatment
with mixture produces nonstatistically
significant effects on hormones and
morphology
Qureshi and
Mahmood
(2010)
Immortalized rat
osteoblasts (FFC
cells)
In vitro
0.1-100 nM
Cr(VI) oxide
4/ cell viability (measured as ALP activity as
a marker of cytotoxicity)
Mitigated by vitamin C; not by vitamins B2
and E
Nine and Grant
(1999)
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—Hexavalent Chromium
System
Route
Exposure3
Results
Reference
Immortalized rat
osteoblasts (FFC
cells)
In vitro
0.1, 0.5, 1.0 nM
Cr(VI) oxide
4/ protein synthesis at 0.1 nM, 4^ DNA, RNA
synthesis at all doses
No change in collagen synthesis
4/ production of collagen fibers, mitigated
by ascorbic acid
Lower doses suppressed collagenase activity
(measured by L-leucine release) more than
high doses (up to 100 nM)
Nine et al.
(2002)
Immortalized rat
osteoblasts (FFC
cells)
In vitro
0.1-100 nM
Cr(VI) oxide
4/ cell viability (measured as ALP activity as
a marker of cytotoxicity), partially mitigated
by pretreatment to deplete GSH.
No change in GSH content
4/ glutathione reductase activity after 48 h
at 0.1-1 nM Cr(VI)
Ning and Grant
(2000)
Mechanisms affecting insulin regulation
Wistar rats,
exposed via
drinking water
from GDs 9-14; F1
males evaluated
on PND59
Oral
(drinking
water)
50, 100, or 200
mg/L K2Cr207,
GDs 9-14.
Euthanasia on
PND60
4/ insulin receptor protein, IRS-1, and p-IRS-
l^632 in liver and gastrocnemius muscle
T* AktSer473 and no change in AKT in liver
4/ Akt and nonmonotonic effect on AktSer473
in gastrocnemius muscle
-t GLUT 2 in liver
4/ GLUT 4 in gastrocnemius muscle
T* PPARy expression
Shobana et al.
(2017)
Oxidative stress and apoptosis in the placenta
Timed pregnant
Sprague-Dawley
rats
Oral
(drinking
water)
50 mg/L K2Cr207,
GDs 9.5-14.5.
Euthanasia on
GD 18.5
T* hypertrophy, basal zone thickness,
pyknotic nuclei (not quantitated)
Hemorrhagic lesions observed w/treatment
T* apoptosis (TUNEL) in various regions/cell
types (Al%)
T* Casp-3 in yolk sac and metrial gland
(maternal compartment), nondetectable in
basal and NS in labyrinth zones (fetal)
-t AIF, Bax, ATM, p53, NOXA, PUMA, p27 all
areas (Casp-3 indep)
4/ Bcl-2, Be I-XL, XIAP
Banu et al.
(2017a)
(appears to be
the same
experiment as
Banu et al.
(2017b))
Timed pregnant
Sprague-Dawley
rats
Oral
(drinking
water)
50 mg/L K2Cr207,
GDs 9.5-14.5.
Euthanasia on
GD 18.5
4/ fetal weight, cytokeratin (TC marker),
Cyclin D1 in metrial gland, basal and
labyrinth zones
4/ markers for TGCs in basal and labyrinth
zones, glycogen cells in basal zone, syncytial
trophoblast in labyrinth zone
T* marker for uterine NK cells in labyrinth
zone
t LPO, H2O2
4/ Gpx, SOD activity in whole extracts
4/ Prdx3, Txn2 mitochondrial expression all
areas samples
Banu et al.
(2017b)
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—Hexavalent Chromium
System
Route
Exposure3
Results
Reference
Human placental
tissues
Ex vivo
0.02 to 1.2 mg/L
Cr detected in
placental tissue
Placenta from male birth (results from
higher Cr concentrations):
1" CytoC, Casp-3, apoptosis inducing factor
(AIF), vBAX, and p53, Bcl-2, Bcl-XL
4/ XIAP (x-linked apoptosis inhibitor)
Placenta from female birth (results from
higher Cr concentrations):
-t CytoC, Casp-3, AIF, BAX, Bcl-2, Bcl-XL
Null p53, XIAP
Banu et al.
(2018)
Human
trophoblastic cell
line BeWo
In vitro
5,15, 30 nM
K2Cr207 for 12
and 24 h
-t GPX1 mRNA with 5 mM Cr(VI) treatment
after 12 h, dose-dependent; decreased after
24 h
4/ GPXl and SOD1 expression, 15 and 30
HM, 12 and 24 h
4/ Catalase and SOD2
mRNA, 5,15, and 30 nM, after 12 and 24 h,
dose-dependent
4, PRDX3 and TXN2, 5 jiM,
after 24 h only
4/ PRDX3
and TXN2 mRNA, 15 and 30 jiM, 12 and 24 h
Banu et al.
(2018)
Primary human
erythrocytes and
mitochondria from
placenta tissue
In vitro
0.05, 0.5, 1, 5
|jg/mL K2Q2O7
1" lipid peroxidation level (TBARS);
decreased with coadministration of
estrogen metabolite 4-OHE2
4/ SOD and GST activity; SOD increased with
coadministration of estrogen metabolite 4-
OHE2; GST increased with coadministration
of estrogen metabolite 16a-OHEl
4/ nitric oxide levels in blood; estrogen
metabolites caused further reduction
Sawicka et al.
(2017; 2017)
aPotassium dichromate units conversion: Cr(VI) = 0.353 x K2Cr2C>7; potassium chromate units conversion:
Cr(VI) = 0.268 x K2&O4; sodium dichromate dihydrate units conversion: Cr(VI) = 0.349 x Na2Cr20?2H20 (usually
denoted as Na2Cr207 because study authors frequently list the salt as the chemical compound even if
concentration or dose is based on the dihydrate); chromium trioxide units conversion: Cr(VI) = 0.52 x CrC>3.
C.3. SUPPORTING EVIDENCE FOR CARCINOGENIC MODE OF ACTION
C.3.1. Meta-analysis of Cr(VI) and Cancer of the GI Tract
1 This section describes the methods for the review and meta-analysis of GI cancer risk
2 reported by occupational studies of workers with inhalation exposure to Cr(VI) (toxicological
3 review, Section 3.2.1). Occupational studies that analyzed cancer risks related to Cr(VI) exposure
4 were identified as part of the overall assessment search strategy process as described in the Cr(VI)
5 Protocol fU.S. EPA. 2019bl This search strategy, which was conditioned on terms for Cr(VI),
6 identified 35 potentially relevant citations. Since these searches only identified references that
7 mentioned chromium or related terms in the title or abstract, an additional search strategy was
8 developed to identify studies of occupational groups with routine exposure to Cr(VI). Our list of
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occupational groups with potential substantial exposure to Cr(VI) included those in categories I or
II identified by the Occupational Safety and Health Administration (OSHA; see Table C-42) (Shaw
Environmental. 20061. Group I industries are "primary industry sectors where the majority of
occupational exposures occur to hexavalent chromium" while Group II industries "represent
industries with limited potential for occupational exposure to hexavalent chromium; consequently,
fewer data were available on occupational exposures and controls for these industries." This search
resulted in 2,341 references.
Titles and abstracts for the second set of the references were screened by seven individuals
using Distiller imposing a rule that each study be screened by two reviewers; conflicts were
resolved by discussion. Screening decisions were guided by a PECO (population, exposure,
comparator, outcome) statement designed to capture studies examining associations of cancers of
the GI tract with Cr(VI)-exposed occupations (Table C-42). For our initial screening stage, we
included all cancer sites along the digestive tract Different studies used different naming
conventions, partially due to the use of differing International Classification of Disease (ICD) coding
versions.
Table C-42. PECO for screening occupational studies relevant to Cr(VI)
PECO
Element
Evidence
Population
Human including epidemiological studies, case-control studies, cohort/prospective studies,
follow-up studies, occupational mortality studies
Exposure
Industries including any in group 1 or group II. Include analyses of cancer in relation to occupation
(e.g., stomach cancer and occupation in Sweden).
Group 1
Group II
Chromate or chromium
production, ferrochrome
production
Chromium dye production
Chromated copper arsenate
producers
Chromium catalyst users
Chromium catalyst production
Chromium dioxide producers
Chromium metal production
Chromium sulfate producers
Chromium plating, chrome
plating, electroplating
Leather work and tanning, tanners
Stainless steel production
Portland cement work
Welding, Stainless steel (carbon
steel welding low prevalence of
exposure to generally low levels)
Producers of refractory brick
Chromium pigment production
Nonferrous superalloy producers and users
Paint and coatings production
Producers of precase concrete products
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Supplemental Information—Hexavalent Chromium
PECO
Element
Evidence
Printing ink producers
Textile dying
Plastic colorant producers and
users
Producers of colored glass
Plating mixture production
Printing - if working with pigments
Grinders, polishers (stainless
steel)
Aircraft manufacturing; aerospace
Wood preserving
Brick masons, bricklayers
Painters - if in industry like
shipbuilding, automobile
manufacture; painting metals
Metal casting, cutting
Steel and iron foundry workers
Steel mills
Comparator
Analyses of mortality due to cancer or incidence of cancer and associations with occupational
groupings (industries; professions)
Outcome
Gastrointestinal tract cancers (incidence, prevalence, mortality)3
Specific GI cancers identified by ICD-10, -9, -8 or -7 codes, including:
Oral cavity [ICD 140-149 (includes cancers of the mouth, lip, tongue, gum, or oropharynx)]
Esophagus (ICD 150)
Stomach (ICD 151)
Small intestine [ICD 152 (includes the duodenum)]
Colon (ICD 153)
Rectum [ICD 154 (includes the rectosigmoid junction and anus)
aAs noted above, nomenclature for cancer sites varied across studies. Some of the alternative designations
included: buccal cavity, oral cavity; salivary glands; pharynx; hypopharynx; cardia, corpus, gastric, gastric cardia;
bowel, intestine, large intestine; colorectal; digestive tract, digestive system, digestive organs (and peritoneum),
gastrointestinal tract.
1 A total of 199 references were identified during title and abstract screening, and these
2 underwent full-text screening by three reviewers who resolved conflicts via discussion. Of these
3 199 references, 97 references were retained; the majority (93) were uniquely identified references.
4 A snowball search was conducted by cross-checking the reference lists identified using the two
5 search strategies with the studies included in the three recent meta-analyses, which resulted in
6 identification of an additional 20 references. In total, 35 references from the previous literature
7 searches, 93 references from the subsequent occupationally focused search, and 20 references from
8 the snowball search of the reference lists in the three most recent meta-analyses were included in
9 this review. Of these, 21 studies were not included because they were earlier follow-ups, the
10 cohorts were not exposed to Cr(VI), or they did not contain results for site-specific GI tract cancers.
This document is a draft for review purposes only and does not constitute Agency policy.
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C. 3.1.1. Study evaluation criteria
Studies were evaluated with respect to population selection; exposure and outcome
evaluation; confounding; analysis; selective reporting; sensitivity; and overall confidence, following
the framework outlined in the IRIS Handbook (U.S. EPA. 2020bl Criteria were developed for the
exposure domain to identify those studies that used exposure assessment definitions that identified
groups with higher certainty and prevalence of exposure to Cr(VI) fU.S. EPA. 2019bl For the
evaluation of selected outcomes, we had higher confidence in studies of cancer incidence compared
to mortality. Cancer deaths ascertained from death certificates were considered a valid outcome
ascertainment method, acknowledging the potential for misdiagnosis of the underlying cause of
death and subsequent underascertainment, particularly for cancers with longer survival periods.
We had greater confidence in cancer incidence and mortality coding for stomach cancer compared
with other sites in the gastrointestinal tract because the probability that the diagnosis on the death
certificate is the same as that in the hospital medical records is higher fPercv etal.. 1990: Percy et
al„ 19811.
With a few exceptions, most of the studies compared cancer incidence or mortality in an
occupational group to that in the country or other geographical region in which the facilities were
located, by calculating a standardized incidence ratio (SIR) or standardized mortality ratio (SMR).
This study design can be subject to the healthy worker effect, a type of selection bias that results in
an underestimate of the relative risk because individuals in the workforce are a healthier
population than a general population that might be used for comparison. This limitation, and
reduction in study sensitivity, is not generally present in studies using internal comparisons.
Greater confidence was given to studies that considered major confounders of the association
between Cr(VI) exposure and GI tract cancers. Risk factors for these cancers vary by site, but
generally include sex, age, race/ethnicity, and geographic region. Individual-level information on
other risk factors, such as smoking and alcohol consumption that could be risk factors for certain GI
tract cancers and that might differ between the occupational groups and comparison populations,
was not usually available to adjust the SMRs or SIRs, but the magnitude of bias likely differed across
the occupational categories. Other risk factors such as obesity, H. pylori infection, dietary factors
and family history of such cancers, likely were not differentially associated with chromium-exposed
occupations or jobs and thus any differences would be expected to be random. Appropriate
analysis methods were prioritized and largely included standardized ratios for mortality or
incidence of cancer or relative risk estimates for comparisons of exposure groups within the study
population; in a few studies, odds ratios were estimated for case-control study designs.
C.3.1.2. Evaluation of exposure to Cr(VI)
For the purposes of this meta-analysis, only occupational studies were considered, and
studies were evaluated with respect to certainty of exposure to hexavalent chromium.
Occupational groups were identified after inventorying the database of references, and specific
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Supplemental Information—Hexavalent Chromium
criteria developed for "good," "adequate," and "deficient" ratings for decreasing certainty of
exposure within each one. Many of the identified studies were registry based, with occupation
inferred based on a standardized set of occupation or industry codes. In the absence of further
information on potential for Cr(VI) exposure, the certainty of exposure for these studies was
deemed "deficient."
Since the focus of this meta-analysis was occupational exposure to Cr(VI), criteria to
evaluate the certainty of exposure to Cr(VI) were developed specific to occupational groups.
Exposure certainty was rated as "good," "adequate," or "deficient" using the guidelines in
Table C-43. Potential bias in exposure assignments, and other domains of risk of bias and
sensitivity, were evaluated using the methods described in the IRIS Handbook (U.S. EPA. 2020b).
The results of the study evaluations with domain-specific ratings and overall confidence ("high,"
"medium," or "low") are available in HAWC for the cancer mortality studies with comparisons to
external populations and studies with comparisons within the target study population and are
shown in Table C-44.
Table C-43. Occupational group-specific criteria for rating certainty of
exposure to Cr(VI)
Occupation group
Potential
coexposures
Good
Adequate
Deficient
Brick masons/stone
masons/tile setters/brick
layers/cement or concrete
workers
The main source ofCr(VI)
exposure in this group
comes from exposure to
Portland cement
(production or use).
Asbestos, cement
dust, silica,
fiberglass, talc,
solvents, asphalt
(US DHHS, 1990:
Pedersen and
Portland cement
production, exposure
assigned using task-
related data from job
histories and other
industrial hygiene
evidence
Cement production,
exposure assigned
using task-related
data from job
histories
Cohort studies of
bricklayers or case-
control studies,
where occupation
was assigned on the
basis of standard
codes for industry/
occupation
Sieber, 1988; Seta
etal., 1988)
Chromate production,
ferrochromium industry
The main source ofCr(VI)
exposure in this group
comes from exposure to
chromate and related
compounds (production or
use).
Asbestos, nickel,
acid and alkali
mists, nitrogen
oxides, cyanide,
solvents
(IARC, 1990)
Cohort studies of
chromate workers,
including chromate
production,
ferrochromium
industry, with
categories based on
tasks involving direct
exposure to Cr(VI)
Cohort studies of
chromate workers,
including chromate
production,
ferrochromium
industry, or case-
control studies, with
categories based on
(1) ever employment
or duration of
employment, or (2)
standard codes for
industry/occupation.
Cohort studies of
chromate workers,
including chromate
production, ferro-
chromium industry,
or case-control
studies, where the
exposure assessment
description was not
sufficient to
determine the
prevalence or
frequency of
exposure to Cr(VI).
Building
construction/carpenters/
wood workers
Asbestos, silica,
wood dust,
formaldehyde,
wood
Cohort studies of
construction workers,
carpenters, or
woodworkers with
categories based on
Cohort studies of
construction workers,
carpenters or
woodworkers with
categories based on
Cohort studies of
construction workers,
carpenters or
woodworkers, or
case-control studies,
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Supplemental Information—Hexavalent Chromium
Occupation group
Potential
coexposures
Good
Adequate
Deficient
The main source ofCr(VI)
exposure in this group
comes from exposure to
refractory brick or Portland
cement (construction,
building) and from wood
treated with chromated
copper arsenate (CCA).
preservatives,
solvents
(Robinson et al.,
1996)
tasks in Portland
cement mixing or
wood preservation or
working with treated
wood
tasks in cement
mixing (nonspecific)
or broader wood
working categories.
where occupation
was assigned on the
basis of standard
codes for
industry/occupation
Automotive workers
The main source ofCr(VI)
exposure in this group
comes from exposure to
metalwork (e.g., welding)
and to automotive paint.
Solvents, welding
fumes, asbestos in
brakes and
clutches, metal
welding fluids
(Gibel et al., 1985)
(OSHA, 2006a)
Cohort studies with
task-specific
exposure
assignments based
on job histories,
specifically spray
painting, welding, or
metal cutting (see
criteria for painting,
welding or metal
work) with
supplemental
industrial hygiene
evidence
Cohort studies with
task-specific
exposure
assignments based
on job histories,
specifically spray
painting, welding, or
metal cutting (see
criteria for painting,
welding or metal
work, but with no
supplemental
information
Cohort studies of
automotive workers,
or case-control
studies, where
occupation was
assigned on the basis
of standard codes for
industry/occupation
Aircraft manufacturing
workers
The main source ofCr(VI)
exposure in this group
comes from exposure to
metalwork (e.g., welding)
and to aircraft paint.
Solvents, heavy
metal salts,
welding fumes,
epoxy resins,
asbestos, other
fibers, ionizing
radiation
(Lipworth et al.,
2011; Costa et al.,
1989)
Cohort studies with
task-specific
exposure
assignments based
on job histories,
specifically spray
painting, welding, or
metal cutting (see
criteria for painting,
welding or metal
work), with
supplemental
industrial hygiene
evidence; sprayers
and hosemen
Cohort studies with
task-specific
exposure
assignments based
on job histories,
specifically spray
painting, welding, or
metal cutting (see
criteria for painting,
welding or metal
work, but with no
supplemental
information
Cohort studies of
aircraft
manufacturing
workers or case-
control studies,
where occupation
was assigned on the
basis of standard
codes for
industry/occupation
Painter/paint
product/paint, and coating
manufacturers
The main source ofCr(VI)
exposure in this group
comes from exposure to
plaster and chromium-
based pigments (usually
used in marine,
automotive, aircraft, etc.
paints).
Solvents,
pigments,
aromatic azo
dyes, PAHs, resins
(IARC, 2010)
Cohort studies with
task-specific
exposure
assignments based
on job histories;
spray painting or
coating in the
marine, automotive
or aircraft
manufacturing
industries, with
supplemental
industrial hygiene
evidence
Cohort studies with
task-specific
exposure
assignments based
on job histories;
spray painting or
coating in the
marine, automotive
or aircraft
manufacturing
industries, but with
no supplemental
information
Cohort studies of
painters, plasterers,
or paint
manufacturing
workers, or case-
control studies,
where occupation
was assigned on the
basis of standard
codes for
industry/occupation
Printers
Solvents, dyes,
lead salts
Cohort studies with
task-specific
exposure
Cohort studies with
task-specific
exposure
Cohort studies of
printing workers or
case-control studies,
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Supplemental Information—Hexavalent Chromium
Occupation group
Potential
coexposures
Good
Adequate
Deficient
The main source ofCr(VI)
exposure in this group
comes from exposure to
chromium-based pigments
in ink.
(Lvnge et al.,
1995)
assignments based
on job histories;
photoengravers,
press operators, with
supplemental
industrial hygiene
evidence
assignments based
on job histories;
photoengravers,
press operators, but
with no supplemental
information
where occupation
was assigned on the
basis of standard
codes for
industry/occupation
Textiles
The main source ofCr(VI)
exposure in this group
comes from exposure to
chromium-based pigments
in fabric dyes.
Solvents, textile
dusts and fibers,
formaldehyde,
dyes
(IARC, 1998)
Cohort studies with
task-specific
exposure
assignments based
on job histories
(e.g., textile dying),
with supplemental
industrial hygiene
evidence
Cohort studies with
task-specific
exposure
assignments based
on job histories
(e.g., textile dying),
but with no
supplemental
information
Cohort studies of
textile workers or
case-control studies,
where occupation
was assigned on the
basis of standard
codes for
industry/occupation
Welder/metal fumes
The main source ofCr(VI)
exposure in this group
comes from welding on
stainless steel, and
intensity of exposure varies
by specific welding
technique. For welding,
highest exposure during
shielded metal arc welding,
less for gas metal arc
welding and tungsten inert
aas weldina fPesch et al.,
2018j.
Nickel and other
metals, arsenic
asbestos,
formaldehyde,
silica dust
(IARC, 1990)
(IARC, 2018)
Cohort studies with
task-specific
exposure
assignments based
on job histories;
stainless steel
welding: shielded
metal arc welding, or
stainless steel
welding: unspecified
technique but with
monitoring data or
other Cr(VI)-specific
information
Cohort studies with
task-specific
exposure
assignments based
on job histories;
stainless steel
welding (unspecified
technique)
Cohort studies with
task-specific exposure
assignments based on
job histories; gas
metal arc welding,
tungsten inert gas
welding; or cohort
studies of welders or
case-control studies,
where occupation
was assigned on the
basis of standard
codes for
industry/occupation
Tanners
The main source ofCr(VI)
exposure in this group
comes from the "two bath"
tanning process which uses
hexavalent chromium salts
as the tanning material
(Stern, 2003).
Benzidine-based
azo dyes,
aromatic organic
solvents,
formaldehyde,
airborne leather
dust
(IARC, 1981)
Work processes
involving leather
tanning and cohort
description supports
that at least 50% of
cohort first employed
as leather tanners
when two-bath
process was still used
(pre 1940s in United
States) and before
mechanization was
introduced.
Work processes
involving leather
tanning and cohort
description supports
that a large portion
of cohort first
employed as leather
tanners when two-
bath process was still
used (pre 1940s in
United States) and
before
mechanization was
introduced
Work processes
involving leather
tanning and cohort
description supports
that most of the
cohort (>70%) first
employed as leather
tanners when one
bath process was
used (post 1940s in
United States); or
occupation was
assigned based on
standard codes for
industry/occupation
Metal Workers
The main source ofCr(VI)
exposure in this group
comes from work with
chrome plating, stainless
steel and steel alloys (tasks
include: plating, melting,
Nickel
(electroplating),
polynuclear
aromatic
hydrocarbons,
silica, carbon
monoxide, nickel,
phenol,
Cohort studies
analyzing stainless
steel categories/tasks
with some
monitoring data or
industrial hygiene
documentation.
Stainless steel
Cohort studies
involving steel
foundries with
subgroup analyses.
Cohort studies
analyzing stainless
steel categories with
Iron or steel
foundries; If
occupation was
assigned on the basis
of standard codes for
industry/occupation
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Occupation group
Potential
coexposures
Good
Adequate
Deficient
pouring, cutting, grinding
and welding operations).
formaldehyde,
isocyanates,
amines
(IARC, 1990)
machining,
production of
stainless steel
products (grinding,
polishing) (based on
job histories),
stainless steel
production (based on
job histories), steel
foundries (by work
area/task)
no or minimal
monitoring data.
The meta-analyses focused on the studies considered to be "medium" or "high" overall
confidence for which EPA had greater certainty in the exposure assessment for Cr(VI) and minimal
concern for other sources of bias. These studies reported a variety of effect estimates, including
standardized incidence or mortality ratios, standardized risk ratios, odds ratios, and proportionate
mortality ratios. Studies that calculated proportionate mortality ratios were not included. In some
instances, multiple risk estimates were reported—for example, for men or women separately, for
exposure or occupational subgroups, or by latency period. A priori, we selected risk estimates
(1) that were adjusted for potential confounders including age, sex, time period, and geographic
region; (2) for the longest latency period; (3) from the most recent follow-up of a specific study
cohort; and (4) for the most highly exposed subgroup of the study population. A comparison of the
studies included in the three most recent meta-analyses and this analysis, with our rationale for
decisions to exclude, are in Table C-44. The table indicates the citations included in our meta-
analysis and those in the three most recent meta-analyses. The studies included in each meta-
analysis comprised an overlapping but different set of studies reflecting the various time periods
used for the literature searches, the inclusion criteria, and the results of the evaluations of study
"quality" used in the studies. In this analysis, the primary reason for considering a study "low"
confidence was that exposure to Cr(VI) in the population was too uncertain.
When reviewing the studies captured by our literature search and evaluation of studies,
some cancer sites or groupings were difficult to reconcile across studies due to differences in ICD
codes included, for example, or changes in coding practices and diagnostic naming conventions
over time and across geographical sites. Consequently, determining whether common cancer sites
were contained within some of the groupings was difficult Further, in some cases, the number of
studies for a given cancer site was small enough (and heterogenous enough) that a meta-analysis
seemed unlikely to yield useful information. Consequently, we performed quantitative meta-
analysis to derive summary risk estimates for a subset of GI tract cancers by site: esophagus,
stomach, rectum, and colon. For each of these four sites, more studies were available to include in a
summary effect estimate, and these studies used relatively consistent definitions for these specific
cancer sites.
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Supplemental Information—Hexavalent Chromium
1 Separate meta-analyses were performed to obtain summary estimates from studies
2 reporting odds ratios (stomach cancer, esophageal cancer), and from studies reporting SMR, SIR, or
3 SRR estimates (all four sites). All analyses were performed using the "metafor" package in R, with a
4 random effects model. This package was also used to generate forest plots. The potential for
5 publication bias was evaluated using the Egger's test (Egger etal.. 19971 for funnel plot asymmetry.
6 The 12 statistic value is used to represent the percentage of variation
7 across studies that is due to heterogeneity rather than chance.
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Supplemental Information—Hexavalent Chromium
Table C-44. Comparison of studies included in meta-analyses or that met PECO, with search phase, study
evaluation rating, and rationale for exclusion in EPA meta-analysis
All included
EPA
included
Welling
included
Deng
included
Suh
included
Search
Overall
rating
Rationale for exclusion
Andersson et al. (2010)
X
PECO_l
Medium
Birk et al. (2006)
X
X
X
PECO_l
Medium
Davies et al. (1991)
X
X
X
PECO_l
Medium
Franchini et al. (1983)
X
X
X
PECO_l
Medium
Gibbet al. (2015)
X
X
PECO_l
Medium
Haves et al. (1989)
X
X
X
X
PECO_l
Medium
Huvinen and Pukkala
X
X
PECO_l
Medium
(2013)
Huvinen and Pukkala
X
X
PECO_l
Medium
(2016)
Koh et al. (2013)
X
X
PECO_l
Medium
Korallus et al. (1993)
X
X
X
X
PECO_l
Medium
Langard et al. (1990)
X
X
X
PECO_l
Medium
Rafnsson et al. (1997)
X
PECO_l
Medium
Rosenman and Stanburv
X
X
PECO_l
Medium
(1996)
Silverstein et al. (1981)
X
X
X
PECO_l
Medium
Sorahan and Harrington
X
X
X
PECO_l
Medium
(2000)
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Supplemental Information—Hexavalent Chromium
All included
EPA
included
Welling
included
Deng
included
Suh
included
Search
Overall
rating
Rationale for exclusion
Sorahan et al. (1987)
X
X
X
PECO_l
Medium
Haves et al. (1979)
X
PECO_l
Medium
Kano et al. (1993)
X
X
X
PECO_l
Medium
Becker (1999)
X
X
X
PECO_2
Medium
Boice et al. (1999)
X
X
X
PECO_2
Medium
Dalager et al. (1980)
X
X
PECO_2
Medium
Danielsen et al. (1996)
X
PECO_2
Medium
Delzell et al. (2003)
X
PECO_2
Medium
Edling et al. (1986)
X
X
PECO_2
Medium
Garabrant and Wegman
X
X
PECO_2
Medium
(1984)
Garabrant et al. (1988)
X
PECO_2
Medium
Hansen et al. (1996)
X
PECO_2
Medium
lala et al. (2006)
X
X
X
PECO_2
Medium
Jakobsson et al. (1993)
X
X
X
PECO_2
Medium
Jakobsson et al. (1997)
X
X
X
PECO_2
Medium
Kaerlev et al. (2000)
X
PECO_2
Medium
Kusiak et al. (1993)
X
PECO_2
Medium
Lipworth et al. (2011)
X
X
X
PECO_2
Medium
Lvnge et al. (1995)
X
PECO_2
Medium
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Supplemental Information—Hexavalent Chromium
All included
EPA
included
Welling
included
Deng
included
Suh
included
Search
Overall
rating
Rationale for exclusion
Mikoczv and Hagmar
X
X
PECO_2
Medium
(2005)
Montanaro et al. (1997)
X
X
X
X
PECO_2
Medium
Morgan et al. (1981)
X
PECO_2
Medium
Moulin et al. (1990)
X
X
X
X
PECO_2
Medium
Moulin et al. (1993a)
X
X
X
X
PECO_2
Medium
Park et al. (2005)
X
X
PECO_2
Medium
Polednak (1981)
X
PECO_2
Medium
Ramanakumar et al. (2008)
X
PECO_2
Medium
Santibanez et al. (2008)
X
PECO_2
Medium
Sciannameo et al. (2019)
X
PECO_2
Medium
Siogren et al. (1987)
X
PECO_2
Medium
Sorahan et al. (1994)
X
X
X
PECO_2
Medium
Tarvainen et al. (2008)
X
PECO_2
Medium
Vevalkin and Gerein (2006)
X
PECO_2
Medium
Xu et al. (1996)
X
X
PECO_2
Medium
Olsen et al. (1988)
X
PECO_2
Medium
Simonato et al. (1991)
X
X
X
X
PECO_2
Medium
Axelsson et al. (1980)
X
X
Snowball ID
Medium
Costantini et al. (1989)
X
X
X
Snowball ID
Medium
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Supplemental Information—Hexavalent Chromium
All included
EPA
included
Welling
included
Deng
included
Suh
included
Search
Overall
rating
Rationale for exclusion
Dabetal. (2011)
X
X
Snowball ID
Medium
Hara et al. (2010)
X
X
X
Snowball ID
Medium
Horiguchi et al. (1990)
X
X
X
X
Snowball ID
Medium
Pippard et al. (1985)
X
X
X
X
Snowball ID
Medium
Smailvte et al. (2004)
X
X
X
Snowball ID
Medium
Deschamps et al. (1995)
X
X
X
Snowball ID
Medium
Aragones et al. (2002)
PECO_l
Low
Low confidence due to exposure assessment, which was
based on self-reported occupation at one timepoint.
Concern that occupation at one point in time does not
represent etiologically relevant time window.
Guberan et al. (1989)
X
X
PECO_l
Low
Low confidence related to nonspecific exposure
definition.
Koh et al. (2011)
X
PECO_l
Low
Main limitation is uncertain potential for exposure
(highest likelihood for production and maintenance, but
duration unknown and use of last held job could
introduce misclassification) and low numbers of cases.
Parent et al. (1998)
X
PECO_l
Low
Low confidence due to the nonspecific nature of the
exposure assignments.
Satoh et al. (1981)
X
X
PECO_l
Low
Although potential for chromium exposure seems clear,
there is little information to inform potential for
selection bias or outcome ascertainment, and low
number of cases (n = 11).
Sweenev et al. (1985)
X
PECO_l
Low
Main limitations are uncertain potential for chromium
exposure and low number of deaths for certain cancer
sites.
Walrath et al. (1987)
X
PECO_l
Low
Main limitation is unclear potential for chromium
exposure.
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—Hexavalent Chromium
All included
EPA
included
Welling
included
Deng
included
Suh
included
Search
Overall
rating
Rationale for exclusion
Andielkovich et al. (1992)
PECO_2
Low
Low confidence study due to lack of information on
likelihood of Cr(VI) exposure.
Andersen et al. (1999)
PECO_2
Low
Low confidence study due to lack of information on
potential for Cr(VI) exposure, lack of consideration of
latency.
Bertazzi and Zocchetti
PECO_2
Low
Main limitation is lack of certainty regarding potential for
chromium exposure.
(1980)
Bethwaite et al. (1990)
PECO_2
Low
Low confidence study due to lack of certainty regarding
Cr exposure.
Bouchardv et al. (2002)
PECO_2
Low
Main limitation is lack of certainty for occupation in
general and for chromium exposure potential.
Brown et al. (2002)
PECO_2
Low
Main limitation is the lack of certainty regarding
chromium exposure, and potential healthy worker
effect.
Brownson et al. (1989)
PECO_2
Low
Main limitation is lack of certainty regarding exposure
(and occupation only at time of diagnosis).
Bulbulvan et al. (1999)
PECO_2
Low
Main limitation is lack of certainty regarding chromium
exposure.
Chiazze et al. (1980)
PECO_2
Low
Main limitations are lack of certainty regarding
chromium exposure, and uncertainty due to missing
data. Further limitations are small sample size and use
of PMR analysis.
Chow et al. (1994)
PECO_2
Low
Main limitation is the lack of certainty regarding
chromium exposure and potential healthy worker effect.
Chow et al. (1995)
PECO_2
Low
Main limitation is the lack of certainty regarding
chromium exposure and potential healthy worker effect.
Cocco et al. (1998)
PECO_2
Low
Main limitation is lack of certainty regarding chromium
exposure.
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Supplemental Information—Hexavalent Chromium
All included
EPA
included
Welling
included
Deng
included
Suh
included
Search
Overall
rating
Rationale for exclusion
Costa et al. (1989)
PECO_2
Low
Main limitation is lack of specificity about which workers
might be exposed to chromium and inclusion of short-
term workers, lack of information on longest held or
usual occupational group.
Danielsen et al. (1993)
PECO_2
Low
Low confidence study, given short time period (1977
onward) that stainless steel was in use during the overall
study period from 1940-1979.
Divine and Barron (1986)
PECO_2
Low
Low confidence primarily due to uncertainties in
exposure domain. Type of welding metal was not
reported so there is low certainty about the extent of
exposure to chromium in the industry.
Dubrow and Wegman
PECO_2
Low
Low confidence due to uncertainties in the exposure
domain due to likely misclassification in exposure
assignments; usual occupation on death certificate and
broad exposure categories.
(1984)
Dubrow and Gute (1988)
PECO_2
Low
Primary limitation is the nonspecific nature of the
exposure assignments and low sensitivity.
Engel et al. (2002)
PECO_2
Low
Although the greater specificity in the incident cancer
ascertainment is a strength, the nonspecific nature of
the exposure assignments based on occupational and
industry codes constrained any conclusions regarding
any associations with Cr(VI).
Finkelstein and Verma
PECO_2
Low
Exposure based on membership in the bricklayers union
is nonspecific with large uncertainties in the prevalence,
frequency and intensity of exposure to Cr(VI).
(2005)
Golka et al. (2012)
PECO_2
Low
In addition to the nonspecific occupational and exposure
group definitions for Cr(VI), the numbers of cases in the
chromium VI relevant groups was small.
Greene et al. (1979)
PECO_2
Low
The lack of specificity in the exposure assignments is the
major limitation, and the number of deaths was small.
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Supplemental Information—Hexavalent Chromium
All included
EPA
included
Welling
included
Deng
included
Suh
included
Search
Overall
rating
Rationale for exclusion
Huebner et al. (1992)
PECO_2
Low
Although design and analysis are appropriate, main
limitation is uncertain potential for chromium exposure.
Jansson et al. (2015)
PECO_2
Low
Low confidence study due to lack of information on
potential for Cr(VI) exposure and lack of consideration of
latency.
Ji and Hemminki (2006)
PECO_2
Low
Low confidence study due to lack of information on
potential for Cr(VI) exposure.
Kaerlev et al. (2002)
PECO_2
Low
Main limitation is unclear potential for chromium
exposure.
Kane et al. (1997)
PECO_2
Low
Main limitation is unclear potential for chromium
exposure.
Keller and Howe (1993)
PECO_2
Low
Main limitation is unclear potential for chromium
exposure.
Kraus et al. (1982)
PECO_2
Low
Certainty regarding chromium exposure is low and it is
unclear how census data were used to calculate
expected number of deaths.
Lindsav et al. (1993)
PECO_2
Low
Main limitation is lack of certainty regarding chromium
exposure.
Macleod et al. (2017)
PECO_2
Low
Low confidence study due to lack of certainty regarding
chromium exposure.
Malker and Gemne (1987)
PECO_2
Low
Main limitation is lack of certainty regarding chromium
exposure.
Matanoski et al. (1986)
PECO_2
Low
Main limitation is lack of information on potential for
chromium study.
Mcmillan and Pethvbridge
PECO_2
Low
Low numbers of deaths, uncertain potential for
chromium exposure, and questionable statistical
analysis.
(1983)
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Supplemental Information—Hexavalent Chromium
All included
EPA
included
Welling
included
Deng
included
Suh
included
Search
Overall
rating
Rationale for exclusion
Melkild etal. (1989)
PECO_2
Low
Main limitation is small sample size and uncertainty
regarding chromium exposure.
Minder and Beerporizek
X
PECO_2
Low
Main limitation is lack of certainty for chromium
exposure potential.
(1992)
Park et al. (1994)
PECO_2
Low
Low confidence due to the nonspecific nature of the
exposure assignments.
Pukkala et al. (2009)
X
PECO_2
Low
Low confidence study due to lack of information on
potential for Cr(VI) exposure, lack of consideration of
latency.
Richiardi et al. (2012)
PECO_2
Low
Main limitation is unclear potential for chromium
exposure.
Robinson et al. (1995)
X
PECO_2
Low
Main limitation is unclear potential for chromium
exposure.
Sale and Alterman (2005)
X
PECO_2
Low
Main limitation is unclear potential for chromium
exposure.
Silverstein et al. (1985)
PECO_2
Low
Main limitation is unclear potential for chromium
exposure. The welding conducted at the plant was not
likely to have involved exposure to Cr(VI).
Siodahl et al. (2007)
X
PECO_2
Low
The large size of the cohort, almost complete
ascertainment, number of cancer cases, and analysis of
cancer incidence is a strength, allowing for analyses of
relatively rare cancer types. The nonspecific nature of
the exposure definition, however, reduced certainty that
prevalence of Cr(VI) exposure was adequate.
Stellman and Garfinkel
PECO_2
Low
Main limitation is unclear potential for chromium
exposure.
(1984)
Stern (2003)
X
PECO_2
Low
Main limitation is low potential for chromium exposure
during study period.
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—Hexavalent Chromium
All included
EPA
included
Welling
included
Deng
included
Suh
included
Search
Overall
rating
Rationale for exclusion
Sun et al. (2002)
PECO_2
Low
Main limitation is unclear potential for chromium
exposure.
Urbaneia Arrue et al.
PECO_2
Low
Main limitation is unclear potential for chromium
exposure.
(1995)
Wang et al. (1999)
PECO_2
Low
Main limitation is unclear potential for chromium
exposure.
Westberg et al. (2013)
PECO_2
Low
Main limitation is unclear potential for chromium
exposure.
Yuan et al. (2011)
PECO_2
Low
Main limitation is lack of information on case and control
selection and use of a single biomarker measurement of
total chromium of unclear timing after diagnosis.
Ahn et al. (2006)
X
Snowball ID
Low
There is some likelihood of Cr(VI) exposure in certain
process areas, but industrial hygiene measures indicate
levels could be fairly low. Combined with rather short
follow-up and low numbers of cases, it may be difficult
to infer cancer associations with Cr(VI).
Amandus (1986)
X
Snowball ID
Low
Main limitation is uncertainty regarding likelihood of
Cr(VI) exposure.
Blair (1980)
X
Snowball ID
Low
Low confidence study due to lack of certainty regarding
Cr exposure.
Gonzalez et al. (1991)
X
Snowball ID
Low
Exposure definitions were not specific to Cr(VI).
Jarvholm et al. (1982)
X
Snowball ID
Low
Main limitations are small sample and unclear potential
for chromium exposure.
Kneller et al. (1990)
X
Snowball ID
Low
Main limitation is lack of uncertainty for chromium
exposure potential.
Krstev et al. (2005)
X
Snowball ID
Low
Main limitation is lack of certainty regarding potential for
chromium exposure.
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Supplemental Information—Hexavalent Chromium
All included
EPA
included
Welling
included
Deng
included
Suh
included
Search
Overall
rating
Rationale for exclusion
Mallin et al. (1989)
X
Snowball ID
Low
Main limitation is uncertain potential for chromium
exposure.
Mcdowall (1984)
X
Snowball ID
Low
Main limitation is lack of information on potential for
chromium exposure. Classification by tasks within this
cohort of cement workers allowed adequate exposure
contrast for dust exposure, but whether the exposures
were to Portland cement is unclear. Therefore there is
less certainty about exposure to Cr(VI).
Santibanez et al. (2012)
X
Snowball ID
Low
Main limitation is unclear potential for chromium
exposure.
Stern et al. (2001)
X
Snowball ID
Low
Main limitation is unclear potential for chromium
exposure.
Becker et al. (1991)
X
PECO_l
Exclude
Earlier studv of the cohort reported bv Becker (1999).
Gibb et al. (2000b)
X
PECO_l
Exclude
Earlier studv of the cohort reported bv Gibb et al. (2015).
Luippold et al. (2003)
X
X
PECO_l
Exclude
No Gl tract cancer results.
Park et al. (2004)
X
PECO_l
Exclude
Lung cancer only.
Proctor et al. (2016)
X
PECO_l
Exclude
Lung cancer only.
Rafnsson and
PECO_l
Exclude
Earlier studv of the cohort reported bv Rafnsson et al.
Johannesdottir (1986)
(1997).
Sorahan et al. (1998)
X
PECO_l
Exclude
No analyses for Gl tract cancer.
Steenland et al. (1991)
X
PECO_l
Exclude
Cohort was not exposed to Cr(VI).
Steenland (2002)
X
PECO_l
Exclude
Cohort was not exposed to Cr(VI).
Takahashi and Okubo
X
PECO_l
Exclude
Earlier studv of the cohort reported bv Hara et al. (2010).
(1990)
Moulin (1995)
X
PECO_2
Exclude
Letter to the editor focused on lung cancer.
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—Hexavalent Chromium
All included
EPA
included
Welling
included
Deng
included
Suh
included
Search
Overall
rating
Rationale for exclusion
Becker et al. (1985)
PECO_2
Exclude
Earlier studv of the cohort reported bv Becker (1999).
Delzell et al. (1993)
PECO_2
Exclude
Earlier studv of the cohort reported bv Delzell et al.
(2003).
laia et al. (2002)
PECO_2
Exclude
In Italian. Same analvses as laia et al. (2006).
Mastrangelo et al. (2002)
PECO_2
Exclude
Meta-analysis.
Mikoczv et al. (1994)
X
PECO_2
Exclude
Earlier studv of the cohort reported bv Mikoczv and
Hagmar (2005).
Moulin et al. (2000)
X
PECO_2
Exclude
Focus of the study is on lung cancer.
Sorahan and Cooke (1989)
PECO_2
Exclude
Earlier studv of the cohort reported bv Sorahan et al.
(1994).
Stern et al. (1987)
PECO_2
Exclude
Earlier studv of the cohort reported bv Stern (2003).
Svensson et al. (1989)
PECO_2
Exclude
Earlier studv of the cohort reported bv Jakobsson et al.
(1997).
Vevalkin and Milvutin
PECO_2
Exclude
Earlier studv of the cohort reported by Vevalkin and
(2003)
Gerein (2006).
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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
36
37
Supplemental Information—Hexavalent Chromium
C.3.1.3. Results
As shown in Table 3-13 in the toxicological review, the summary effect estimates showed
small increases in risk for each cancer site associated with Cr(VI) exposure, although only the
estimate for rectal cancer was statistically significant Few studies reported odds ratios, but in each
case (esophagus and stomach), summary effect estimates based on these studies were somewhat
higher compared with summary estimates based on other relative risk measures (although neither
odds ratio-based estimate was statistically significant). No evidence of funnel plot asymmetry was
found based on Egger's regression test, indicating that publication bias was not likely to be present.
Summary effect estimates were also derived for each cancer site, stratified by occupational
grouping (see Table C-45 and Figures C-15 to C-20). This separation by occupational grouping did
show some expected patterns for colon cancer risk estimates (see Figure C-19) in that the
occupations with a higher certainty of exposure to Cr(VI) (i.e., ferrochromium, chromate
production, stainless-steel workers, chromium pigment-exposed workers) showed higher summary
effect estimates. Inconsistencies remained among the studies overall, however, and the results for
cancer of the rectum did not show a similar pattern of risk (see Figure C-20).
All risks were either slightly above or close to the null (RRs ranging from 1.01 to 1.45) with
the exception of stomach cancer among tannery workers [relative risk (RR) of 0.72], For example,
when looking at stomach cancer, there was a (nonsignificant) decreased risk for tannery workers,
and a (nonsignificant) increased risk for those working with metal coatings and metal platers (RRs
of 0.72 and 1.26, respectively). Risks for other occupational groups were close to the null, ranging
from 1.01 to 1.10. Similarly, variation within occupational groups occurred—the group
"ferrochromium, chromate production, stainless-steel workers," had modestly elevated risks for
esophageal and colon cancer (RRs of 1.22 and 1.26), while risks were very close to 1 for stomach or
rectal cancer (RRs of 1.01 and 1.04). Looking across cancer sites, for the occupational groups with
four or more estimates, those with a higher certainty of exposure prevalence (i.e., ferrochromium,
chromate production and stainless-steel workers, and chromium pigment exposed workers) had
higher relative risk estimates for esophageal and colon cancers but not stomach or rectal cancers.
The number of studies within another category with more certainty in the probability of Cr(VI)
exposure, "estimated or measured chromium exposure," was too small to calculate a summary
estimate. For esophageal cancer, the two studies in this category indicated elevated, but not
significant, effect estimates. For colon cancer, this category included two analyses within one study
of chromate production workers with exposure prior to and after work process changes that
reduced Cr(VI) concentrations. Effect estimates are not consistent with what would be expected,
however, since higher risk was observed for the post-change workers. A small number of colon
cancer cases contributed to the effect estimates (pre-change n = 7, post-change n = 4), and there
was evidence of bias from the healthy worker effect with consequent impacts on sensitivity.
Heterogeneity in effect estimates (magnitude and direction) also was evident within occupational
groups for a specific cancer site, as shown in the forestplots (Figures C-15 to C-20).
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Supplemental Information—Hexavalent Chromium
Table C-45. Summary effect estimates from random effects meta-analysis, by
cancer site and occupational group, where four or more estimates are
included
Cancer
site
Occupational group
Number of
individual effect
estimates
Summary effect estimate
(95% confidence interval)
I2
Esophagus
Ferrochromium, chromate
production, stainless-steel
workers3
6
1.22 (0.90, 1.64)
0
Chromium pigment-exposed
workers
5
1.42 (0.87, 2.32)
10.6
Stomach
Ferrochromium, chromate
production, stainless steel
workers
13
1.01 (0.75, 1.35)
49.9
Welders
5
1.10 (0.76, 1.60)
19.7
Tannery workers
6
0.79 (0.56, 1.12)
12.7
Portland cement workers,
masons
4
1.02 (0.65, 1.61)
59.0
Chromium pigment-exposed
workers
6
1.07 (0.80, 1.42)
0
Metal coatings, metal platers
6
1.26 (0.81, 1.98)
54.8
Colon
Ferrochromium, chromate
production, stainless steel
workers
4
1.26 (0.82, 1.91)
44.0
Portland cement workers,
masons
4
0.88 (0.61, 1.27)
0
Chromium pigment-exposed
workers
4
1.45 (0.68, 3.09)
41.7
Rectum
Ferrochromium, chromate
production, stainless steel
workers3
10
1.04 (0.78, 1.38)
0
Welders
5
1.28 (0.69, 2.41)
39.2
Tannery workers
4
1.32 (0.80, 2.21)
25.3
Chromium pigment-exposed
workers
4
1.11 (0.63, 1.98)
16.7
aWarning displayed during estimation of the summary estimate indicates that "Ratio of largest to smallest
sampling variance extremely large. May not be able to obtain stable results."
1 These results could be due to misclassification and heterogeneity of Cr(VI) exposure among
2 and within the included studies. Although this analysis included studies that analyzed associations
3 among occupational groups or subgroups with greater certainty of exposure to Cr(VI), variation in
4 the prevalence, frequency, and magnitude of exposure is likely within the exposure groups, which
5 could decrease the ability to detect an association if it existed. Other factors that could contribute
6 to the observed heterogeneity of risk estimates include presence of coexposures and bias due to the
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use of occupational cohorts. Cancer risk in these industries is likely affected by prevalent exposures
to other carcinogens in addition to Cr(VI), which would vary both within and across occupational
groupings. As noted in Appendix Table C-43, two industry groupings with higher certainty of Cr(VI)
prevalence, ferrochromium, chromate production, and stainless-steel workers, and chromium
pigment-exposed workers, had occupational settings characterized by different coexposures, which
argues against a strong common confounder. In some cases, authors did attempt to adjust for
coexposures or restrict the study population to minimize their effect. Most of the studies estimated
relative risk using SMRs, which also are subject to a bias toward the null due to the healthy worker
effect The summary effect estimates for esophageal and stomach cancers calculated using odds
ratios from the few case-control studies were not subject to this bias and indicated a higher risk.
These odds ratio estimates are based on very few studies, however, and are highly uncertain.
Previous meta-analyses reported summary effect estimates for stomach cancer that ranged
between 0.93 fDeng etal.. 20191 to 1.27 fWelling et al.. 20151. A statistically significant increase in
risk of stomach cancer was reported from two of the previous five estimates (Welling etal.. 2015:
Cole and Rodu. 20051. This assessment's finding of no increased risk (summary RR of 1.01) is
within the range of these previous estimates. Two of the five previous meta-analyses included
estimates for cancers of the esophagus, colon, and rectum (Deng etal.. 2019: Gatto etal.. 20101.
This assessment's summary estimate of 1.08 for esophageal cancer was not significantly elevated,
and was slightly less than that from Gatto etal. (20101. The effect estimate for colon cancer of 1.10
(95% CI: 0.97,1.25), was close to the estimate reported by Deng etal. f20191. Finally, this
assessment's estimate of rectal cancer risk was significantly elevated, and very similar to those
previously reported (1.18, 95% CI: 1.01,1.37), compared with 1.17 f Gatto etal.. 20101 and 1.14
fDengetal.. 20191.
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Supplemental Information—Hexavalent Chromium
Study Weight; Estimate 135* CI]
Estimated exposure toichromium compounds
Santibanez.2008 517089 i—B-j-i 5147* 0.5210,16,1,881
Metal coating si, metal platers
Ramanakumar 2008 730020
¦ 1 48.53* 4.2011.07,16.511
Summary Effect Estimate 100.00* 1.4310.19,11.091
0,14 1 7.39
Odds Ratio
Figure C-15. Forest plot displaying summary measures for esophageal cancer
risk from studies reporting odds ratios.
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Supplemental Information—Hexavalent Chromium
Study
Weight; Estimate {95% CI]
Lipworth 2011 1235276
Estimated or measured Chromium exposure
i—1—i
19.15% 0.94 [0.64, 1.37]
Boice 1999 699183
5.67% 1.04 [0.52, 2.08]
Montaro 1997 5030179
Tannery workers
1.42% 0.85 [0.21, 3.40]
Morgan 1981 5025858
Chromium pigment exposed workers
l k 1
9.22% 1.06 [0.62, 1.83]
Lynge 1995 5025817
1.42% 3.77 [0.94, 15.07]
Lynge 1995 5025817
0.71% 1.16 [0.16, 8.23]
Kano 1993 50275
1.42% 2.20 [0.55, 8.80]
Deschamps 1995 77671
2.13% 1.48 [0.48, 4.59]
Rafnsson 1997 1232193
Portland cement workers, Masons
2.13% 1.15 [0.37, 3.57]
Koh 2013 1509959
1.42% 2.35 [0.59, 9.40]
Dab 2011 2662181
7.80% 0.76 [0.42, 1.37]
Garabrant 1988 702055
Aircraft and automotive workers
9.93% 1.14 [0.68, 1.92]
Simonato 1991 1258204
Welders
2.84% 0.57 [0.21, 1.52]
Moulin 1993 1258210
2.84% 0.78 [0.29, 2.08]
Becker 1999 1795224
2.13% 1.21 [0.39, 3.75]
Moulin 1993 758628
Ferrochromium, Chromate production, Stainless Steel workers
7.80% 1.00 [0.55, 1.81]
Huvinen 2013 2968072
2.84% 0.86 [0.32, 2.29]
Gibb 2015 2966034
i ; ¦ i
12.77% 1.28 [0.81, 2.03]
Davies 1991 758627
6.38% 1.62 [0.84, 3.11]
Summary Effect Estimate
1 1 1 1 1 1
0.14 0.37 1 2.72 7.39 20.09
Relative Risk
100.00% 1.08 [0.92, 1.28]
Figure C-16. Forest plot displaying summary measures for esophageal cancer
risk from studies reporting standardized mortality or incidence ratios.
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Supplemental Information—Hexavalent Chromium
Study
Weight; Estimate [95% CI]
Edlirig 1986 1260383
Tanneryworkers
1—"i—1
33.00% 0.82 [0.32, 2.12;
Garabrant 1984 5025733
7.33% 0.94 [0.11,7.97;
Xu 1996 1012261
Metal coatings, metal platers
i—¦—1
32.60% 2.40 [0.92, 6.25;
Ramanakumar 2008 730020
1 h-B 1
27.07% 1.50 [0.52, 4.35;
Summary Effect Estimate
4^
100.00% 1.38 [0.77, 2.49;
0.05 0.37 1 2.72 20.09
Odds Ratio
Figure C-17. Forest plot displaying summary measures for stomach cancer
risk from studies reporting odds ratios.
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Supplemental Information—Hexavalent Chromium
Study
Weight; Estimate [95% CI]
Andersson 2010 1673891
Lipworth 2011 1235276
Boice 1999 699183
Kusiak 1993 1032279
Pippard 1985 6553359
Montaro 1997 5030179
Mikoczy 2005 5029788
iaia 2006 5029632
Ediing 1986 1260383
Costaritini 1989 1235919
Morgan 1981 5025858
Lynge 1995 5025817
Lynge 1995 5025817
Kano 1993 50275
Hayes 1989 14024
Deschamps 1995 77671
Sorahari 2000 1230938
Sorahan 1987 1514540
Sorahari 1987 1514540
Horiguchi 1990 1916855
Hara 20101258239
Franchini 1983 1231037
Rafnsson 1997 1232193
Koh 20131509959
Jakobsson 1993 5029443
Dab 2011 2662181
Smailtye 2004 3080195
Smailtye 2004 3080194
Garabrant 1988 702057
Delzell 2003 5029430
Sjogren 1987 1233957
Simonato 1991 1258206
Moulin 1993 1258210
Danielsen 1996 3081487
Becker 1999 1795227
Park 2005 5025867
Moulin 1993 758628
Moulin 19901260384
Langard 1990 1516040
Korallus 1993 1234647
Korallus 1993 1234647
Jakobsson 1997 1792177
Huvinen 2013 2968072
Gibb 2015 2966034
Davies 1991 758627
Davies 1991 758627
Birk 2006 1233708
Axelsson 1980 14268
Pulp arid paper
Estimated or measure*! chromium exposure
Gold miners
Tanneryiworkers
I ¦—i 1
Chromium pigment exposed workers
I—> 1
Metal coatings, metal platers
Portland cement Workers, Masons
Painting and varnish, Woodworkers
Aircraft and automotive workers
I ¦ I :
Welders
I—i— 1
Ferrochromium, Chromate production, Stainless Steel workers
: I ¦ 1
4.01% 1,11 [0.79, 1.56]
3.73% 0.72(0.49, 1.(
2.57% 1.04 [0.58, 1.{
4.95% 1.52(1.25, 1.«
0.75%
0.52 [0.13,
2.08]
2.44%
0.79 [0.43,
1.47]
2.91%
0.91 [0.54,
1.54]
0.40%
0.39 [0.05,
2.77]
1.78%
1.50 [0.67,
3.34]
1.78%
0.43 [0.19,
0.96]
4.01% 1.01 [0.72, 1.42]
0.75% 1.04 [0.26, 4.16]
0.40% 0.32 [0.05, 2.27]
2.14% 1.20 [0.60, 2.40]
0.75% 2.14 [0.54, 8.56]
0.75% 1.52 [0.38, 6.08]
2.69% 1.68 [0.95, 2.96]
3.46% 1.86 [1.21, 2.85]
1.32% 0.82 [0.31, 2.18]
1.06% 1.23 [0.40, 3.81]
2.91% 0.67 [0.40, 1.13]
0.40% 3.33 [0.47, 23.66]
3.46% 1.08 [0.70, 1.66]
3.09% 1.70 [1.04, 2.77]
2.80% 0.85 [0.49, 1.46]
1.06% 0.38 [0.12, 1.18]
3.25% 0.79 [0.50, 1.25]
0.75% 0.50 [0.13, 2.00]
2.30% 0.40 [0.21, 0.77]
3.57% 1.21 [0.80, 1.82]
1.06% 1.67 [0.54, 5.18]
3.73% 0.96 [0.65, 1.41]
1.78% 2.09 [0.94, 4.65]
1.06% 1.03 [0.33, 3.19]
1.56% 0.65 [0.27, 1.55]
0.75%
5.96 (1.49, 23.83]
1.97%
0.92 [0.44, 1.93]
1.32%
2.75 [1.03, 7.33]
1.97%
1.40 [0.67, 2.94]
2.69%
1.92 [1.09, 3.38]
1.32%
0.63 [0.24, 1.68]
2.14%
0.80 [0.40, 1.60]
2.69%
0.80 [0.45, 1.41]
1.97%
0.48 [0.23, 1.01]
0.75%
0.66 [0.17, 2.64]
3.32%
0.73 [0.47, 1.14]
0.75%
0.50 [0.13, 2.00]
2.91%
1.03 [0.61, 1.74]
Summary Effect Estimate
100.00% 1.01 [0.89, 1.15]
0.14 1 7.39
Relative Risk
Figure C-18. Forest plot displaying summary measures for stomach cancer
risk from studies reporting standardized mortality or incidence ratios.
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Supplemental Information—Hexavalent Chromium
Study
Weight; Estimate {95% CI]
Lipworth 2011 1235276
Estimated or measured chromium exposure
K*
40.24% 1.15 [0.94, 1.41]
Boice 1999 699183
1—?—1
9.75% 1.02 [0.68. 1.53]
Mikoczy 2005 5029788
Tannery jworkers
9.75% 1.00 [0.66, 1.50]
laia 2006 5029632
0.42% 1.29 [0.18. 9.16]
Lynge 1995 5025817
Chromium pigment exposed workers
0.85% 0.68 [0.17, 2.72]
Lynge 1995 5025817
1.70% 0.83 [0.31, 2.21]
Kano 1993 50275
0.85% 2.30 [0.58, 9.20]
Deschamps 1995 77671
1.70% 3.08 [1.16, 8.21]
Hara 2010 1258239
Metal coatings, metal platers
1.27% 0.55 [0.18, 1.71]
Rafnsson 1997 1232193
Portland cement Workers, Masons
2.12% 0.56 [0.23, 1.35]
Koh 2013 1509959
0.85% 0.84 [0.21, 3.36]
Jakobssort 1993 5029443
3.81% 1.05 [0.55, 2.02]
Dab 2011 2662181
5.09% 0.93 [0.53. 1.64]
Danielsert 1996 3081487
Welders
1.70% 1.21 [0.45, 3.22]
Becker 1999 1795225
0.85% 0.34 [0.09, 1.37]
Jakobssort 1997 1792177
Ferrochromium, Chromate production, Stainless Steel workers
5.09% 1.40 [0.80, 2.47]
Huvinen 2013 2968072
H--—1
9.32% 1.30 [0.86, 1.97]
Davies 1991 758627
i ¦ 1
1.70% 2.63 [0.99, 7.01]
Davies 1991 758627
2.97% 0.62 [0.30, 1.30]
Summary Effect Estimate
i i i i i i i
0.05 0.14 0.37 1 2.72 7.39 20.09
Relative Risk
100.00% 1.10 [0.97, 1.25]
Figure C-19. Forest plot displaying summary measures for colon cancer from
studies reporting standardized mortality or incidence ratios.
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Study
Weight; Estimate {95% CI]
Lipworth 2011 1235276
Boice 1999 699183
Pippard 1985 6553359
Montaro 1997 5030179
Mikoczy 2005 5029788
laia 2006 5029632
Morgan 1981 5025858
Lyrtge 1995 5025817
Lyrtge 1995 5025817
Sorahart 2000 1230938
Horiguchi 1990 1916855
Hara 2010 1258239
Rafnssort 1997 1232193
Koh 2013 1509959
Jakobsson 1993 5029443
Garabrant 1988 702056
Sjogren 1987 1233956
Simonato 1991 1258205
Moulin 1993 1258210
Danielsen 1996 3081487
Becker 1999 1795226
Moulin 1993 758628
Langard 1990 1516040
Jakobsson 1997 1792177
Huvinen 2013 2968072
Gibb 2015 2966034
Davies 1991 758627
Birk 2006 1233707
Axelsson 1980 14268
Estimated or measured chromium exposure
Tannery jworkers
t-
Chromium pigment exposed workers
Metal coatingsj, metal platers
Portland cement yvorkers, Masons
Aircraft and automotive workers
Welders
Ferrochromium, Chromate production, Stainless Steel workers
6.36% 0.65 [0.37, 1.14]
3.46% 1.08 [0.49, 2.40]
1.23% 1.27 [0.32, 5.08]
3.98% 2.06 [0.98, 4.32]
8.05% 0.98 [0.60, 1.60]
0.62% 1.75 [0.25, 12.42]
11.61% 1.39 [0.95, 2.04]
1.23% 0.84 [0.21, 3.36]
1.23% 0.51 [0.13, 2.04]
3.46% 1.54 [0.69, 3.43]
0.62% 4.28 [0.60, 30.38]
2.93% 1.31 [0.55, 3.15]
2.38% 1.06 [0.40, 2.82]
4.48% 3.05 [1.53, 6.10]
3.98% 1.31 [0.62, 2.75]
7.65% 1.04 [0.63, 1.73]
1.23% 4.31 [1.08, 17.23]
3.98% 0.68 [0.32, 1.43]
0.62% 0.66 [0.09, 4.69]
2.38% 1.82 [0.68, 4.85]
2.38% 1.24 [0.46, 3.29]
1.81% 0.81 [0.26, 2.51]
1.23% 0.80 [0.20, 3.20]
4.97% 1.40 [0.73, 2.69]
7.23% 1.06 [0.63, 1.79]
1.04
1.02
1.23% 1.02
1.23% 0.50 [0.13, 2.00]
0.50, 2.18]
'0.51. 2.04]
:0,26, 4.08]
Summary Effect Estimate
~~i i r~
0.14 1 7.39
Relative Risk
100.00% 1.18 [1.01, 1.37]
Figure C-20. Forest plot displaying summary measures for rectal cancer risk
from studies reporting standardized mortality or incidence ratios.
C.3.2. Mechanistic Evidence Organized by the 10 Key Characteristics of Carcinogens
The hazard identification of cancers of the lung and GI tract include an analysis of whether a
mutagenic mode of action (MOA) could be involved in Cr(VI)-induced carcinogenesis. Because a
large and diverse set of mechanistic studies was identified that has potential relevance for
informing Cr(VI)-induced carcinogenicity in the GI tract and lung, several prioritization factors have
been considered to identify the most informative evidence for the MOA analysis for cancer of the GI
tract and lung following Cr(VI) exposures.
The first phase of the identification and screening of literature pertinent to the MOA
analysis is described in Appendix B.l. Mechanistically relevant studies are not included in the
initial PECO criteria, which are intended to identify studies in humans and animals reporting apical
health effects data that will be evaluated for reporting quality, risk of bias, and sensitivity. Instead,
studies reporting mechanistic data are initially screened and categorized to provide a clearer view
of the proposed biological pathways and processes involved in the toxicity of the chemical and to
identify critical research gaps. The initial broad literature search for Cr(VI) identified 1,522 Cr(VI)
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mechanistic studies, which were screened for relevance and sorted into groups primarily based on
the 10 key characteristics (KCs) of carcinogens fSmith etal.. 20161. These studies, summarized in
Appendix Sections C.3.3-C.3.12, were generally prioritized if they measured mechanistically
relevant biomarkers in humans exposed to Cr(VI) or were experimental studies conducted in
mammals exposed to Cr(VI) or in human primary cells or cell lines.
Because of the importance of determining whether Cr(VI) is mutagenic, it was determined
that the evidence that could be most informative for the mutagenic potential of Cr(VI) would be
subject to study evaluation for reporting, risk of bias, and sensitivity. This includes test systems in
animals that measure mutations (e.g., transgenic rodent assays) and structural and numerical
chromosomal aberrations (e.g., the micronucleus assay). The studies identified as most informative
for mutagenic risk and evaluated in HAWC are summarized separately below for the GI tract and
the lung. All other evidence for genotoxicity and other characteristics of carcinogens are
summarized and synthesized as supporting evidence for biological pathways and processes related
to carcinogenesis.
C.3.2.1. Electrophilicity andDNA reactivity (KC#1)
Studies informing the ability of Cr(VI), the reductive intermediates Cr(V) and Cr(IV), and the
final form Cr(III) to bind DNA and proteins, forming adducts and crosslinks, are summarized in
Table C-46, followed by a summary of the major findings.
Table C-46. Mechanistic studies informing the intracellular reduction of Cr(VI)
and reactivity of Cr species with DNA and proteins
Study findings
Reference
Formation and stabilization of intracellular Cr species and reactive oxygen species
Cr(V) complexes characterized by elemental analyses, electrospray mass spectrometry
(ESMS), and electron paramagnetic resonance (EPR) spectroscopy
Bartholomews et al.
(2013)
Reduction of Cr(VI) generates Cr(V), superoxide and hydroxyl radicals in purified human
cytochrome b(5) and NADPH:P450 reductase in reconstituted proteoliposomes (PLs)
Borthirv et al. (2007)
Two Cr(V) electron spin resonance (ESR) signals, g = 1.979 (nonthiol dependent) and
1.985 (thiol-dependent) in human bronchial epithelial cells (BEAS-2B)
Signals blocked by suppressing NAD(P)H
Borthirv et al. (2008)
ESR spectroscopy and electrospray mass spectrometry measured long-lived Cr(V)
complexes formed by reduction of Cr(VI) with p-bromobenzenethiol (RSH)
Levina et al. (2010)
Cr-DNA adducts in acellular/in vitro test systems
Cr(VI) reduction by glutathione produces 2 Cr(V) complexes and glutathione thiyl radical,
correlated with Cr-DNA adduct formation; no DNA strand breaks
Cr(VI) reduction by H2O2 produces hydroxyl radical, DNA strand breaks, and 8-OHdG
adducts with no Cr(V) generation
Aivar et al. (1991,
1990; 1989)
Cr(VI) showed weak complexation with DNA at high molar ratios of CrC>42" to nucleotides
(r > 1) but not at low molar ratios (r = 1:20 to r = 1:1).
Arakawa et al. (2000)
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Supplemental Information—Hexavalent Chromium
Study findings
Reference
Calf thymus DNA and defined DNA polynucleotides
Borges and
Wetterhahn (1989)
DNA-chromium adducts cause guanine-specific arrests of DNA replication in calf thymus
DNA using mammalian polymerases alpha and beta
Bridgewater et al.
(1998)
Low, nonphysiological levels of ascorbate lead to GSH reduction of Cr(VI) that produce
weakly mutagenic glutathione-Cr(lll)-DNA adducts and no oxidative damage in human
fibroblasts
Guttmann et al.
(2008)
T* ATM activation by Cr(VI) in ascorbate-deficient cells; no ATM activation when
ascorbate levels are restored in human lung H460 cells and normal human lung
fibroblasts
Luczak et al. (2016)
DNA-protein crosslinks formed in human lung A549 cells in 3 steps: Cr(VI) reduction to
Cr(lll), Cr(lll)-DNA binding, and capture of proteins by DNA-bound Cr(lll)
Macfie et al. (2010)
Interstrand DNA crosslinks formed in XPA-null (GM04312), FANCD2-null (GM16633), and
FANCD2-complemented (GM16634) human fibroblasts with ascorbate (1.3% of total
adducts, dose-dependent) and glutathione (<1%, sublinear)
Absence of FANCD2 and XPF-ERCC1 endonuclease produced no hypersensitivity to Cr(VI)
with restored ascorbate levels
Authors interpreted as evidence that DNA crosslinks are more commonly formed in vitro
Morse et al. (2013)
T* ascorbate-Cr(lll)-DNA crosslinks in human lung A549 cells with restored ascorbate
levels (25% of total Cr-DNA adducts)
Ascorbate-Cr(lll)-DNA crosslinks inhibited by Mg2+ ions suggests predominant binding of
ascorbate-Cr(lll) to DNA through phosphate oxygen
Quievrvn et al. (2002)
Cr-DNA adducts, and not oxidative strand breaks, responsible for mutation and
replication fork stalling in SV40-immortalized human HF/SV fibroblasts
Ternary adducts more mutagenic than binary
Mutation spectra equally deletions and point mutations targeting G/C
Quievryn et al. (2003)
Reduction of Cr(VI) by ascorbate produced stable adducts in supercoiled cJ)X174 DNA that
could be disrupted only by phosphate treatment at high concentrations of ascorbate (1
mM) and not at lower concentrations of ascorbate (0.2 mM)
Quievrvn et al. (2006)
Cr(lll) forms adducts with DNA via formation of Cr-protein crosslinks with amino acids in
intact cell cultures.
Salnikow et al. (1992)
Fanconi anemia cells (hypersensitive to DNA crosslinking agents) are hypersensitive to
Cr(VI)-induced apoptosis, clonogenic lethality, and DNA DSBs (gH2AXfoci).
Vilcheck et al. (2006;
2002)
In human fibroblasts, ternary adducts glutathione-Cr(lll)-DNA and histidine-Cr(lll)-DNA
are more mutagenic than cysteine-Cr(lll)-DNA; binary Cr-DNA adducts were weakly
mutagenic
Voitkun et al. (1998)
Cr(VI) reduction by cysteine forms Cr-DNA and Cys-Cr-DNA adducts and interstrand DNA-
DNA crosslinks that increase with Cr(VI) concentration but did not produce DNA breaks or
oxidative DNA damage
Zhitkovich et al.
(2000)
In human fibroblasts, binding of Cr(lll) and the formation of Cr(lll)-DNA adducts induces
structural distortions of DNA
Ascorbate-Cr(lll)-DNA and cysteine-Cr(lll)-DNA adducts were found to be 31-fold and 5.3-
fold more mutagenic than the binary Cr(lll)-DNA adducts, respectively
Zhitkovich et al.
(2001)
Cysteine-dependent Cr(VI) reduction led to Cr-DNA adducts (54%), cysteine-Cr-DNA
adducts (45%), and interstrand DNA crosslinks (1%)
Zhitkovich et al.
(2002)
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Study findings
Reference
Cr(lll)-DNA binding:
Zhou etal. (2016)
• To backbone phosphates through reversible electrostatic interactions
• To nucleobases with the preference G>C>T~A, generating stable crosslinks
resistant to dissociation by EDTA; this binding is slow due to slow ligand
exchange in Cr(lll) complexes
In vivo test systems
Exposed: Four human adult volunteers
Referents: Preingestion background DNA-protein crosslink levels for each individual
served as the controls
Ingestion of a single bolus dose of 5,000 ng Cr(VI) as K2Cr2C>7 alone (Cr(VI) or reduced to
Cr(lll) with orange juice; approximately equivalent to 71 ng Cr(VI)/kg, assuming a body
weight of 70 kg.
Blood samples were collected at 0, 60,120,180, and 240 min after ingestion.
Kuvkendall et al.
(1996)
• No significant changes in DNA protein cross-linking after ingestion
• Very small sample size limits confidence in the results
• The only known ingestion study in humans; all other human studies evaluate
inhalation in occupational cohorts
Rat, Fischer 344, male, exposed to 100 or 200 mg/L K2Cr2C>7 in drinking water, 3 or 6 wk
Coogan et al. (1991a)
• T* DNA-protein crosslinks in liver; not in splenic lymphocytes
• No cytotoxicity detected
Rat, exposed to 20 mg/kg by i.p. injection, 40 h
Cudo and Wetterhahn
• T* Cr binding to DNA, nonhistone proteins, and cytoplasmic RNA-protein fraction
in liver
(1985)
DNA reactivity of Cr species
Formation of Cr(V) and free radicals generated by these species is considered to play an
important role in Cr(VI)-induced DNA damage. Cr(V) intermediates have been shown to induce
direct oxidative DNA damage through abstraction of H atoms at the deoxyribose sugar moiety,
resulting in the generation of abasic sites (Sugden and Wetterhahn. 19971. Cr(V) can also induce
oxidative damage indirectly through the generation of reactive oxygen species, causing oxidative
damage at dG sites and formation of 8-hydroxy-2'-deoxyguanosine (8-OHdG) residues, presumably
via production of hydroxyl radicals generated through a Fenton-like reaction (i.e.,
Cr(V)+H202->Cr(VI) + -OH + 0H-) (reviewed in Levina and Lay (20051 and Sugden and Stearns
("200011.
Cr(IV) is the major transient form of intracellular reduction of Cr(VI) in cells with
physiological levels of ascorbate. An in vitro study using synthetic compounds of Cr(VI) reduction
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intermediates showed significantly increased mutation frequencies in cells exposed to Cr(IV)
compared to Cr(V) fWakeman et al.. 20171. In the presence of hydrogen peroxide, Cr(IV) is a more
potent Fenton-like reagent than Cr(V) and generates hydroxyl radicals, which has been shown to
cause DNA strand breaks and oxidative damage at dG positions that are preventable by hydroxyl
radical scavengers (Luo etal.. 19961. In addition, this process generates Cr(V), leading to further
oxidative DNA damage.
Cr(III) is a thermodynamically stable species produced by the reduction of Cr(VI) through
the intermediary species Cr(V) and Cr(IV), which transiently exist in variable amounts during the
intracellular reduction of Cr(VI). The interaction of Cr(III) with DNA is responsible for the
formation of DNA lesions, the most common of which are the binary Cr(III)-DNA adducts (Floro and
Wetterhahn. 19841. Two different forms of Cr(III)-DNA adducts have been suggested by a study
investigating the reation of DNAzyme Cel3d with CrCl3. The results showed that Cr(III) first binds
to the DNA phosphate backbone through weak electrostatic interactions, then slowly coordinates
with all four nucleobases, forming highly stable DNA interstrand crosslinks (Zhou etal.. 20161. A
more recent study concluded instead that Cr(III) is coordinated with N7 of dG as a [Cr(H20)s]+
complex located within the major groove of the DNA double helix structure without the direct
participation of neighboring bases of phosphate groups (Brown etal.. 2020). but also supported the
formation of interstrand crosslinks. It is likely that the existing evidence of the reactions of Cr(III)
complexes with DNA do not provide a full model of all possible Cr-DNA interactions that occur
during Cr(VI) reductions with variable amounts of intracellular reducers.
Binary Cr(III)-DNA adducts can further conjugate proteins and form DNA-protein cross-
links (DPCs). The DPCs represent ternary protein-Cr(III)-DNA adducts generated by a rate-limiting
reaction of binary Cr(III)-DNA adducts with proteins. The formation of DPCs in cultured cells
exposed to Cr(VI) is decreased by the depletion of glutathione and is facilitated by the restoration of
physiological levels of ascorbate (Macfie etal.. 2010). Overall, the biological significance of the
DPCs is still incompletely understood. In addition to their genotoxic potential, some studies
demonstrated their ability to inhibit specific gene expression fMacfie etal.. 20101.
Other ternary adducts have been identified in cells exposed to Cr(VI), including ascorbate-
Cr(III)-DNA, glutathione-Cr(III)-DNA, cysteine-Cr(III)-DNA, and histidine-Cr(III)-DNA. Ascorbate-
Cr(III)-DNA adducts were detected in Cr(VI)-treated human A549 lung cancer cells with restored
ascorbate levels, accounting for approximately 6% of the total DNA-bound chromium fOuievrvn et
al.. 2002). In addition, the binding of Cr(III) and the formation of Cr(III)-DNA adducts induces
structural distortions in DNA (Zhitkovich et al.. 2001).
Biological effects of Cr-DNA interactions
Binary Cr(III)-DNA adducts formed by the reaction of Cr(III) aqua complexes and DNA are
reportedly weakly mutagenic lesions, with a considerably lower mutagenic potential when
compared to any ternary ligand-Cr-DNA adduct (Ouievryn etal.. 2003). Indeed, ascorbate-Cr(III)-
DNA and cysteine-Cr(III)-DNA adducts were found to be 31-fold and 5.3-fold more mutagenic than
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the binary Cr(III)-DNA adducts, respectively fHolmes et al.. 2008: Zhitkovich et al.. 20011.
Consequently, ascorbate appears to be the most important intracellular reducer of Cr(VI) that
forms highly mutagenic DNA adducts. The ternary adducts glutathione-Cr(III)-DNA and histidine-
Cr(III)-DNA were also found to be mutagenic, and their mutagenicity exceeded that of cysteine-
Cr(III)-DNA (Voitkun et al.. 19981. Ternary adducts are also more genotoxic than binary Cr(III)-
DNA adducts, demonstrated through more prominent DNA replication fork stalling by ternary
adducts in comparison to binary adducts (e.g., fOuievryn etal.. 2003: Snow and Xu. 19911.
Under lower, non-physiological levels of ascorbate, reduction of Cr(VI) by glutathione in
vitro produced mutagenic glutathione-Cr(III)-DNA adducts fGuttmann et al.. 20081. This finding
implies that lesions produced at physiological concentrations of GSH in ascorbate-depleted cells are
less mutagenic and suggests that studies employing standard cell cultures with low intracellular
ascorbate could have underestimated the mutagenicity of Cr(VI). Taken together, studies
performed under non-physiological, low ascorbate levels favored the production of Cr(V) and a
lower amount of highly mutagenic ternary species, which did not accurately reflect the genotoxic
and mutagenic effects of Cr(VI) in vivo fOuievryn etal.. 20061.
Cells with restored ascorbate levels display considerably different cell signaling responses
to Cr(VI) than in ascorbate-depleted cells. As previously discussed, reduction of Cr(VI) by
glutathione in vitro and in cells with depleted ascorbate leads to an appreciable formation of Cr(V),
which can act as an oxidant fOuievryn et al.. 20031. while reduction of Cr(VI) by ascorbate is a low
oxidant generating process fWong etal.. 20121. Treatment with Cr(VI) also induces double-strand
breaks in cells with restored ascorbate; however, these are formed selectively in euchromatin and
their signaling is dependent on ATR rather than on ATM kinase fDelougherv etal.. 20151.
C.3.2.2. Genotoxicity (KC#2)
In vivo studies
Inhalation route of exposure
Mutagenic MO A studies
Studies considered most relevant to a mutagenic MOA analysis for lung cancer are studies of
occupationally or environmentally exposed humans or studies in experimental animals exposed via
inhalation or intratracheal instillation and include measures of gene mutation (prior to
tumorigenesis), micronuclei induction, and chromosomal aberrations. Occupational studies were
considered only if they included a comparison or referent population exposed to Cr(VI) at lower
levels (or no exposure/exposure below detection limits) or for shorter periods of time. Animal
studies were considered if they included a concurrent control group exposed to vehicle-only
treatment or an untreated control.
Twenty-nine studies in humans occupationally exposed and one study in transgenic mice
were identified. These were evaluated in HAWC using criteria specific to the mutational assay used
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Supplemental Information—Hexavalent Chromium
1 in the study to judge the outcome ascertainment domain. The overall confidence judgments and
2 summaries of the study findings can be found in the Cr(VI) Toxicological Review in Section 3.2.3
3 Cancer—Mechanistic Evidence; more extensive summaries of the human studies are in Table C-47
4 below. Human studies reporting other outcomes informative to genotoxicity are summarized in the
5 following sections.
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Supplemental Information—Hexavalent Chromium
Table C-47. Chromosomal mutation studies in humans exposed to Cr(VI) via inhalation (evaluated in HAWC)
Study overview
Exposure3
Results
Comments
Reference
Chromosomal aberrations
Cross-sectional study,
South India.
Exposed: n = 72 (n = 36
directly exposed via
work in a tannery,
n = 36 indirectly
exposed via residence in
proximity to tanneries)
Referent: n = 36
unexposed controls
("normal and healthy
individuals who had not
exposed themselves to
any kind of chemicals or
radiation")
Assessment: Exposure to Cr(VI) inferred based
on occupation and residence.
In addition, Cr was measured in urine and air
samples (unclear where air samples were
collected)
Levels: There was a gradient in levels of both,
there were detectable chromium levels in both
air and urine for "controls"
Direct exposure (n = 36) (mean ± SD):
Total Cr in air (1 mg/m3): 0.101 ± 0.003
Cr(VI) in air (0.001 mg/m3): 0.021 ± 0.003
Cr content in urine: 2.11 ± 1.01
Indirect exposure (n = 36):
Total Cr in air (1 mg/m3): 0.089 ± 0.003
Cr(VI) in air (0.001 mg/m3): 0.013 ± 0.005
Cr content in urine: 1.81 ± 0.88
Controls (n = 36):
Total Cr in air (1 mg/m3): 0.014 ± 0.004
Cr(VI) in air (0.001 mg/m3): 0.006 ± 0.001
Cr content in urine: 0.54 ± 0.39
Duration: Directly exposed subjects were
"selected based on the duration of their
exposure (0-5; 6-10; 11-15; 16-20; 21-25
years) and were known to be exposed to Cr(VI)
for a minimum of 8 h/day," while indirect
exposure was inferred from residence of at
least 30 yr duration, "in and around the
tanneries."
T* chromosomal
aberrations in DE
group compared to
IE group and
controls
Also observed T*
mean tail length for
comet assay in DE
group compared to
IE group and
controls and T* MN
among directly
exposed subjects
compared to
indirectly exposed &
controls; further
elevated in those
with longer duration
of exposure
Low confidence. There is evidence of a
gradient of chromium exposure across
the three study groups, but inference is
limited by small sample size and lack of
description.
Some of the controls also had detectable
chromium in urine, suggesting this is not
really a true "control" group.
Concerns with chromosomal aberrations
assay—culture of 72 h may have missed
first in vitro cell division.
Very limited evaluated of confounders.
Balachandar et al.
(2010)
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Study overview
Exposure3
Results
Comments
Reference
Cross-sectional study,
Bulgaria.
Exposed: Chromium
plate workers (n = 15)
Referents 1: age,
gender, smoking-
matched controls
(n = 15)
Referents 2: individuals
of similar age from
unpolluted rural region
(n = 8)
Assessment: Blood samples and buccal
mucosal cells taken from exposed group;
exposure to Cr(VI) inferred based on
occupation. Also measured Crwith personal
air samplers and in urine samples.
Levels: There was a gradient of chromium in air
and urine across groups, although there was
detectable Cr in urine of rural controls.
Mean air concentration of total chromium was
0.0075 mg Cr/m3 in the low-exposure group
(n = 4) and 0.0249 mg Cr/m3 in the high-
exposure group (n = 7). (4 workers in the
exposed group temporarily discontinued
exposures and were considered separately.)
Mean concentrations of Cr in urine were 18.63
Hg/L (low) and 104.22 ng/L (high).
Results reported for combined groups
(0.0075 and 0.0249 mg Cr/m3).
Duration: Duration of exposure ranged from 2
to >20 yr; mean duration of exposure was not
reported.
In exposed workers
compared to
referent 1:
Buccal cells:
No difference in
frequencies of
chromosomal
aberrations or SCEs
Study also reported
significantly
increased MN in
buccal cells and
lymphocytes in
referent 1 compared
to referent 2
Low confidence. Although exposed and
unexposed workers were matched on
age, sex, and smoking habit, the two
unexposed (worker and rural) groups
were combined, resulting in lower
confidence in comparability of exposed
and unexposed group comparisons.
Inference is further limited by small
sample size and lack of description.
Similar proportion of centromere-
positive and -negative micronuclei
indicate both clastogenic and aneugenic
effects occurring.
Benova et al.
(2002)
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Supplemental Information—Hexavalent Chromium
Study overview
Exposure3
Results
Comments
Reference
Cross-sectional study of
workers at a single
facility in China.
Exposed: n = 7
electroplating workers
exposed to chromium
Referent: n = 10 office
workers
Note: also included n = 7
electroplating workers
exposed to nickel
Assessment: Exposure to Cr(VI) inferred based
on occupation. Also measured Cr in personal
air samples from work room, hair, and stool
samples.
Levels: Authors note that there seemed to be
little cross-contamination of nickel and
chromium in respective work areas based on
air samples, but stool samples showed similar
levels of both compounds between exposure
groups. Hair levels of chromium were higher
in chromium compared with nickel workers.
The mean chromium (total) air concentration
(by random air collection) was 8.1 ng/mm3, the
mean chromium concentration in stool
samples was 8.5 ng/g stool, and the mean
chromium concentration in hair was 35.68
Mg/g-
(The exposure level of 8.1 ng 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.)
Duration: Mean duration of occupational
exposure was 12.8 yr.
1" chromosomal
aberrations in
chromium workers
compared to nickel
workers & controls
1" SCE in chromium
& nickel workers
compared to
controls
Low confidence. Although controls were
age and sex matched to exposed subjects
and were stated to have similar
socioeconomic status, the sample size is
quite small and the analysis limited. Also
unclear how well differentiated
chromium exposure is by group-
analyses of chromium in hair suggest a
delineation with controls, but no
information on stool samples that
showed similarities between nickel and
chromium workers.
Deng et al. (1988)
Cross-sectional study,
Slovak Republic.
Exposed: n = 73 male
welders
Referent: n = 71 male
controls (administrative
officers and hospital
employees)
Assessment: Exposure to Cr(VI) inferred based
on occupation. Also measured Cr in blood.
Levels: Exposed workers had average values
about twice as high as referent group (stated
to be significantly different). Mean ± SE was
0.07 ± 0.04 vs. 0.03 ± 0.007 nmol/L
Duration: Mean ± SD duration of occupational
exposure was 10.2 ± 1.7 yr.
No differences in
CAs, CTAs, and CSAs
between exposed
and control groups
1" CAs in individuals
with Gln/GIn
genotype compared
to Arg/GIn or
Arg/Arg genotypes
in XRCC1 Arg299Gln;
more pronounced in
Cr-exposed workers
Medium confidence. Main limitations
are related to lack of description
(e.g., for participant selection) and lack
of evaluation of confounders aside from
smoking.
Halasova et al.
(2012)
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Study overview
Exposure3
Results
Comments
Reference
Cross-sectional study,
Slovak Republic.
Exposed: n = 39 male
welders
Referent: n = 31 male
controls (source not
given)
Assessment: Exposure to Cr(VI) inferred based
on occupation. Also measured Cr in blood.
Levels: Exposed workers had average values
about twice as high as referent group.
Mean ± SE was 0.07 ± 0.04 vs. 0.03 ± 0.007
Hmol/L.
Duration: Mean ± SD duration of occupational
exposure was 10.2 ± 1.7 yr.
No significant
differences in
frequencies of CTAs
between exposed
and control groups;
only minor
differences in CAs
between groups
1" CSAs in exposed
compared to control
groups
1" CAs & CTAs in
individuals with
Gln/GIn genotype
compared to
Arg/GIn or Arg/Arg
genotypes in XRCC1
Arg299Gln
Low confidence. Main limitations are
related to sample size, unclear
differentiation between exposure
groups, and lack of description (e.g., for
participant selection).
Halasova et al.
(2008)
Cross-sectional study,
Finland.
Exposed: n = 23 male
welders
Referent: n = 22 male
office employees at a
printing company
Assessment: Exposure to Cr(VI) inferred based
on occupation. Welders were chosen due to
"exposure to MMA/SS welding fumes with
little or no exposure to other agents in their
occupational history."
Also measured total Cr in urine.
Levels: Urine levels are not discussed in text
(table shows values ranging from 0.20 to 1.55
Hmol/L).
Duration: Welders likely had Cr(VI) exposure
due to history of manual metal arc welding for
at least 4 yr and most for much longer
(mean ± SD = 21 ± 10 yr).
No significant
differences
(frequency of
chromosome
aberrations or SCEs)
Low confidence. Although Cr(VI)
exposure seems likely to occur among
these welders, the analysis is limited by
small sample size when stratifying by
smoking (found to be related to the
outcome).
Husgafvel-
Pursiainen et al.
(1982)
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Study overview
Exposure3
Results
Comments
Reference
Cross-sectional study,
Japan.
Exposed: n = 51 male
stainless steel welders
Referent: n = 33 male
office or research
workers in the same
factory
Assessment: Exposure to Cr(VI) inferred based
on occupation.
Total Cr also measured in urine samples.
Levels: Mean urinary Cr was 9.8 and 4.2 ng/L
among exposed and referent group,
respectively.
Duration: Welders had been "constantly
engaged" in stainless steel welding for 5-20 yr
(mean 12 yr) and thus are presumed to have
high potential for Cr(VI) exposure.
1" chromosomal
aberrations and
SCEs in welder
compared to
controls
Low confidence. The main limitations
are related to the outcome evaluation
and to poorly described and reported
data analysis and lack of consideration of
potential confounders.
Koshi et al. (1984)
Cross-sectional study,
Sweden.
Exposed: n = 24 stainless
steel welders from six
industries
Referent: n = 24
matched referents who
"had no occupational
(or other) experience
with the handling of
stainless steel (or other
known
mutagenic/carcinogenic
agents)"
Assessment: Exposure to Cr(VI) inferred based
on occupation. Also measured total Cr in air
(welders only) and urine (all).
Levels: Mean urinary Cr was 47 and 1.5
Hmol/mol creatinine among exposed and
referent group, respectively. Mean air Cr level
81 ng/m3.
Duration: Welders were selected for their
"long and intense" welding on stainless steel
(mean work duration of 19 yr).
No significant
differences
(frequency of breaks
or fragments; gaps
and isogaps;
interchanges,
dicentrics, rings, and
markers; structural
aberrations,
hyperdiploidy; SCEs)
Low confidence. Main limitations are
related to outcome ascertainment and
statistical analysis, as well as limited
description of results.
Littorin et al.
(1983)
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Study overview
Exposure3
Results
Comments
Reference
Cross-sectional study,
South Korea.
Exposed: n = 51 male
chrome plating and
buffing workers
Referent: n = 31 male
office workers from
"industrial areas" in
South Korea
Assessment: Exposure to Cr(VI) inferred based
on occupation.
Also measured Cr measured in air samples
(total and VI), blood, and end-shift urine
samples (See Table 1).
Levels: Concentrations in blood and urine were
significantly higher in exposed workers,
indicating adequate delineation between
groups. For example, the geometric mean
blood level of Cr was 0.9 and 0.2 |Jg/dL in
exposed and referent workers, respectively.
Differently, while air measures were higher for
exposed workers the difference was not
statistically significant.
Duration: Mean duration of occupational
exposure was 9.1 yr (range: 1 mo to 40 yr).
1" frequency of
chromatid
exchange;
chromosome/chro-
matid breaks and
exchanges; and of
translocations, with
higher blood Cr
1" frequency of
translocations in
exposed compared
with unexposed.
Low confidence. Main limitations are
related to lack of description for analysis
and results reporting.
Maeng et al.
(2004)
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Study overview
Exposure3
Results
Comments
Reference
Cross-sectional study,
Italy.
Exposed: n = 38 male
plating factory workers
(two groups from
factories using nickel
and chromium for bright
plating, and two groups
from factories using
only chromium for hard
plating)
Referent: n = 35
"healthy male sanitary
workers" not known to
have chromium
exposure
Note: Analysis of SCEs
only included n = 21
workers from factories
using only chromium,
and n = 14 "healthy
blood donors" with
similar selection as
unexposed worker
control group
Assessment: Exposure to Cr(VI) inferred based
on occupation. Exposed group was stratified
based upon coexposure to nickel ("bright"
plating, vs. "hard" plating).
Also measured Cr in urine.
Levels: Urinary Cr levels were lowest in
controls (mean ± SD = 1.9 ± 1.4 ng/g crt),
intermediate in bright plating (6.1 ± 2.8 |jg/g
crt), and highest in hard plating groups
(10.0 ± 7.5 ng/g crt), indicating adequate
delineation between groups.
Duration: Mean (SD) yr of exposure: bright
plating = 9 (11); hard plating = 7 (3)
1" frequency of total
aberrations,
chromosome-type
aberrations in all
exposed. Also T*
chromatid-type
aberrations in bright
platers.
1" SCEs for some
worker compared to
blood donors.
Low confidence. Main limitations are
related to outcome ascertainment, small
sample size for certain analyses, and lack
of description (e.g., for participant
selection and statistical analysis).
Sarto et al. (1982)
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Study overview
Exposure3
Results
Comments
Reference
Micronuclei
Cross-sectional study,
South India.
Exposed: n = 72 (n = 36
directly exposed via
work in a tannery,
n = 36 indirectly
exposed via residence in
proximity to tanneries)
Referent: n = 36
unexposed controls
("normal and healthy
individuals who had not
exposed themselves to
any kind of chemicals or
radiation")
Assessment: Exposure to Cr(VI) inferred based
on occupation and residence. In addition, Cr
was measured in urine and air samples
(unclear where air samples were collected)
Levels: There was a gradient in levels of both
urine and air, there were detectable chromium
levels in both air and urine for "controls."
Direct exposure (n = 36) (mean ± SD):
Total Cr in air (1 mg/m3): 0.101 ± 0.003
Cr(VI) in air (0.001 mg/m3): 0.021 ± 0.003
Cr content in urine: 2.11 ± 1.01
Indirect exposure (n = 36):
Total Cr in air (1 mg/m3): 0.089 ± 0.003
Cr(VI) in air (0.001 mg/m3): 0.013 ± 0.005
Cr content in urine: 1.81 ± 0.88
Controls (n=36):
Total Cr in air (1 mg/m3): 0.014 ± 0.004
Cr(VI) in air (0.001 mg/m3): 0.006 ± 0.001
Cr content in urine: 0.54 ± 0.39
Duration: Directly exposed subjects were
"selected based on the duration of their
exposure (0-5; 6-10; 11-15; 16-20; 21-25
years) and were known to be exposed to Cr(VI)
for a minimum of 8 h/day" while indirect
exposure was inferred from residence of at
least 30 year's duration, "in and around the
tanneries."
T* micronuclei
peripheral
lymphocytes among
directly exposed
subjects compared
to indirectly
exposed & controls;
and further elevated
in those with longer
duration of
exposure
Low confidence. There is evidence of a
gradient of chromium exposure across
the three study groups, but inference is
limited by small sample size and lack of
description.
Some controls also had detectable
chromium in urine, suggesting this is not
really a true "control" group.
Very limited evaluation of confounders.
Balachandar et al.
(2010)
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Supplemental Information—Hexavalent Chromium
Study overview
Exposure3
Results
Comments
Reference
Cross-sectional study,
Bulgaria.
Exposed: Chromium
plate workers (n = 15)
Referents 1: age,
gender, smoking-
matched controls
(n = 15)
Referents 2: individuals
of similar age from
unpolluted rural region
(n = 8)
Assessment: Blood samples and buccal
mucosal cells taken from exposed group;
exposure to Cr(VI) inferred based on
occupation. Also measured Cr with personal
air samplers and in urine samples.
Levels: There was a gradient of chromium in air
and urine across groups, although there was
detectable Cr in urine of rural controls.
Mean air concentration of total chromium was
0.0075 mg Cr/m3 in the low-exposure group
(n = 4) and 0.0249 mg Cr/m3 in the high-
exposure group (n = 7). (4 workers in the
exposed group temporarily discontinued
exposures and were considered separately.)
Mean concentrations of Cr in urine were 18.63
Hg/L (low) and 104.22 ng/L (high).
Results reported for combined groups
(0.0075 and 0.0249 mg Cr/m3).
Duration: Duration of exposure ranged from 2
to >20 yrs; mean duration of exposure was not
reported.
1" micronuclei per
peripheral blood
leukocytes (PBLs) &
1" overall number of
PBLs with
micronuclei in
exposed workers
compared to
controls
1" micronuclei in
buccal cells in
exposed workers
compared to
controls
No significant
difference between
proportion of C+
and C- micronuclei
in buccal or PBLs in
exposed workers
compared to
controls
Low confidence. Positive results
reported for combined groups (0.0075
and 0.0249 mg chromium/m3).
Although exposed and unexposed
workers were matched on age, sex, and
smoking habit, the two unexposed
(worker and rural) groups were
combined, resulting in lower confidence
in comparability of exposed and
unexposed group comparisons.
Inference is further limited by small
sample size and lack of description.
Benova et al.
(2002)
Cross-sectional study,
India.
Exposed: n = 102 male
welders
Referent: n = 102 male
controls selected from
the general population
"with no history of
exposure to welding
fumes or any known
physical or chemical
agent in the workplace,
but belonged to the
same age group and
socio-economic status
as the welders."
Assessment: Exposure to Cr(VI) inferred based
on occupation. Welders used shielded metal
arc welding and were working with stainless
steel electrodes.
Also measured Cr in blood for a sample (~50%)
of subjects.
Levels: Welders had much higher chromium
compared with controls, indicating delineation
of exposure. Mean Cr was 151.65 and 17.86
Hg/L in exposed and referent, respectively.
DNA damage was measured by comet assay in
all 204 subjects; frequency of micronuclei was
measured in 58 welders and 53 controls.
Duration: The duration of exposure varied
widely (range: 1-24 yr). (Overall mean not
presented)
In buccal cells of
exposed welders
compared to
referent:
1" micronuclei
(p < 0.001);
correlated with
duration of work
(p = 0.0001), age
(p = 0.007), and Cr
level in blood
Low confidence. Limitations related to
outcome evaluation, such as the use of
outdated methods no longer
recommended, could lead to inaccurate
scoring. A description/details on
participant selection (e.g., concern for
potential selection bias) is lacking.
Study also reported T* mean comet tail
length in whole blood cells (p < 0.001).
Danadevi et al.
(2004)
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Supplemental Information—Hexavalent Chromium
Study overview
Exposure3
Results
Comments
Reference
Cross-sectional study,
Egypt.
Exposed: n = 41 male
electroplating workers
exposed to chromium
and nickel
Referent: n = 41 male
administrative workers
at the same facility
Assessment: Exposure to Cr(VI) inferred based
on occupation. Also measured Cr (and nickel)
in serum.
Levels: Serum Cr significantly higher in exposed
compared with controls. Mean Cr was 3.30
and 0.23 ng/L in exposed and referent,
respectively.
Duration: Exposed workers were required to
have worked in electroplating section at least
2 yr, but most worked for considerably longer
with mean ± SD = 26.68 ± 11.21 yr.
In buccal cells of
exposed
electroplaters
compared to
referent:
1" micronucleus
induction (p < 0.001)
1" serum Cr
correlates with T*
micronuclei
(p < 0.05)
Medium confidence. Exposed and
unexposed groups are delineated,
although limited description of methods
(e.g., participant selection) and known
coexposure to nickel could limit
inference.
Study also reported T* serum 8-OHdG.
El Saftv et al.
(2018)
Cross-sectional study,
China.
Exposed: n = 87 workers
from a single factory in
China, who had
"occupational exposure
to chromate from
different work sections"
Referent: n = 30 working
in administrative offices
without chromate
exposure
Assessment: Exposure to Cr(VI) inferred based
on occupation. Also measured total Cr in air
samples and in blood.
Levels: Authors state "The concentration of Cr
in the air and blood of subjects in the exposure
group were significantly higher than the
control group (p < 0.001)," which increases
confidence in delineation of exposure groups.
Geometric Mean ± SD of Cr in blood was
8.5 ± 1.3 ng/L in exposed vs. 4.1 ± 1.4 ng/L in
referent group, while median (IQR) of air
concentrations were 15.5 (19.0) vs. 0.2 (0.4)
mg/m3.
Duration: Median duration of employment
was 5 yr in both exposed and referent.
1" MN in peripheral
lymphocytes in
exposed workers
compared with
referent
Medium confidence. Main limitations
are related to lack of description
(e.g., for participant selection).
Study also reported T* hypermethylation
of CpG sites and 8-OHdG adducts.
Hu et al. (2018)
Related studies:
Li et al. (2014a;
2014b)
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Supplemental Information—Hexavalent Chromium
Study overview
Exposure3
Results
Comments
Reference
Exposed 1: male welders
working in areas
without collective
protections (n = 27)
Exposed 2: male welders
working in locations
with smoke extraction
systems (n = 33)
Referents: office
workers with no history
of occupational
exposure to welding
fumes or other
physical/chemical agent
in workplace (n = 30)
Assessment: Exposure to Cr(VI) inferred based
on occupation.
Also measured total Cr in blood and urine.
Levels: Cr levels in blood and urine were higher
among both groups of welders compared with
controls (means 129 to 145, compared with 92
Hg/L), and urinary chromium was higher
among welders working without smoke
extraction systems.
Duration: Welders exposed for 0.5-45 yr
1" mean BN % in
lymphocytes of
welder compared to
controls
Low confidence. Main limitations are
related to lack of description (e.g., for
participant selection, analysis), unknown
contribution of Cr(VI) to Cr exposure
(states that <5% of welding was done on
stainless steel, which raises concern that
total Cr measured in blood and urine
may be attributed to Cr(lll) exposure)
and known coexposures to other metals.
larmarcovai et al.
(2005)
Cross-sectional study,
China.
Exposed: n = 29
"healthy" chrome
platers employed for at
least one yr at two
facilities
Referent: n = 29 subjects
"randomly selected
from the healthy
workers in the same
enterprises and been
engaged in public
security, support
services, or
administration work for
more than one yr, and
had no specific
chromate exposure
history"
Assessment: Exposure to Cr(VI) inferred based
on occupation. Also measured Cr in blood.
Levels: Blood Cr levels were significantly higher
among exposed compared with unexposed
workers, indicating adequate delineation
between groups. Mean (range) values were
15.2 (2.1, 42) in exposed vs. 4.6 (0.2, 28) in
referent group.
Duration: Chrome platers had been employed
for at least one yr.
1" micronuclei
frequencies in
peripheral
lymphocytes of Cr-
exposed workers
compared to
controls, but no
correlation between
blood Cr
concentration and
micronuclei
Low confidence. Limitations are the
limited and poorly described statistical
analysis and limited description (e.g., for
participant selection). Small sample size.
Inconsistent results could indicate the
influence of other occupational hazards
on micronuclei concentrations.
Linaing et al.
(2016)
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Supplemental Information—Hexavalent Chromium
Study overview
Exposure3
Results
Comments
Reference
Cross-sectional study,
Sweden.
Exposed: n = 24 stainless
steel welders from six
industries
Referent: n = 24
matched referents who
"had no occupational
(or other) experience
with the handling of
stainless steel (or other
known
mutagenic/carcinogenic
agents)"
Assessment: Exposure to Cr(VI) inferred based
on occupation. Also measured total Cr in air
(welders only) and urine (all).
Levels: Mean urinary Cr was 47 and 1.5
Hmol/mol creatinine among exposed and
referent group, respectively. Mean air Cr level
81 ng/m3.
Duration: Welders were selected for their
"long and intense" welding on stainless steel
(mean work duration of 19 yr).
No significant
differences in
micronuclei
between exposed
and referent groups
Uninformative (for micronucleus only).
Main limitations are primarily due to
extended culture times and the lack of a
measure of cell replication, which could
result in bias toward the null. Other
limitations are related to outcome
ascertainment and statistical analysis, as
well as limited description of results.
Littorin et al.
(1983)
Cross-sectional study,
China.
Exposed: n = 120
chromate-exposed
workers working at a
chromate production
facility
Referent: n = 97
unexposed workers at
same facility ("without
contact history of
harmful substances")
Assessment: Exposure to Cr(VI) inferred based
on occupation. Also measured Cr in whole
blood.
Levels: Cr levels were significantly higher
among exposed compared with controls,
indicating delineation of exposure. Median
(interquartile range) of Cr in whole blood was
2.81 (3.86) and 0.99 (1.21) ng/L in exposed and
referent groups, respectively.
Duration: Mean (SD) yr of exposure in
chromate group = 14.57 (5.85).
1" MN frequency
ratio in lymphocytes
of exposed; results
of exposure-SNP
interaction on MN
presented as well
Medium confidence. Main limitations
are related to lack of description
(e.g., for participant selection and
statistical analysis).
Long et al. (2019)
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Supplemental Information—Hexavalent Chromium
Study overview
Exposure3
Results
Comments
Reference
Cross-sectional study,
Portugal.
Exposed 1: n = 5
stainless steel welders
exposed to Cr(VI)
Exposed 2: n = 33
tannery workers
exposed to Cr(lll)
Referent: n = 20-30
unexposed controls
("not known to be
exposed to either
environmental or
occupational
carcinogens")
Assessment: Exposure to Cr(VI) inferred based
on occupation.
Also measured Cr in plasma and in urine (mid-
shift for welders).
Levels: Urinary and plasma chromium levels
were higher in both exposed groups compared
with controls. For example, mean ± SD levels
in plasma were 2.43 ± 2.11 in tanners,
1.55 ± 0.67 in welders, and 0.41 ± 0.11 ng/L.
Duration: Not reported
1" micronuclei in
lymphocytes among
tanners compared
to control group;
(there was also a
marginal increase in
the welders group,
but not statistically
significant)
Low confidence. Main limitation is small
number of welders, lack of description
for participant selection, analysis, and
confounders.
Study also reported T* formation of DNA
protein crosslinks in welders compared
to controls.
Medeiros et al.
(2003)
Cross-sectional study,
Italy.
Exposed: n = 17 tannery
finishing workers with
potential exposure to
Cr(VI)
Referent (2 groups):
n = 21 and n = 17
workers "from different
industries"
Note: also evaluated
n = 21 tannery workers
with potential exposure
to Cr(lll)
Assessment: Exposure to Cr(VI) inferred based
on occupation. State that tannery finishing
workers had potential for exposure to Cr(VI)
but with no supporting description or
evidence. Although unclear from the text,
workers might have been from several
different tanneries with differing potential for
exposure to Cr(VI) containing dyes.
Levels: Not reported
Duration: Not reported
No significant
associations
Low confidence. Main limitation is
unclear potential for Cr(VI) exposure for
tannery finishing workers.
Migliore et al.
(1991)
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Supplemental Information—Hexavalent Chromium
Study overview
Exposure3
Results
Comments
Reference
Cross-sectional study,
India.
Exposed: n = 100 male
electroplaters exposed
to Cr(VI) and nickel.
Group II: exposed <10
yr, n = 50; Group III:
exposed for >10 yr,
n = 50
Referent: n = 50
unexposed controls
(Group 1)
Assessment: Exposure to Cr(VI) inferred based
on occupation. Also measured Cr in plasma.
Levels: Concentrations were significantly
higher in exposed (and higher for longer
exposed) workers compared with unexposed.
Mean + SD levels for longer exposed and
shorter exposed workers were 2.9 + 0.8 and
1.7 + 0.55 ng/L, respectively, while values for
referent were 0.55 + 0.08 ng/L.
Duration: Group II exposed 1-9 yr; Group III
exposed 10-25 yr.
In buccal cells of
Group II compared
to Group 1, and in
Group III compared
to Group II:
1" micronucleus
frequency (p < 0.05),
correlated with Cr
levels in plasma
(p< 0.01)
Low confidence. Main limitations are
related to outcome ascertainment,
limited statistical analysis, and lack of
description (e.g., for participant
selection). Coexposure to nickel is also a
concern.
Study also reported T* nuclear anomalies
(karyorrhexis, karyolysis, pyknosis)
(p < 0.05).
Qavvum et al.
(2012)
Cross-sectional study,
India.
Exposed: n = 66 welders
Referent: n = 60 controls
("selected from the
general population with
no history of
occupational exposure
to welding fumes or any
known physical or
chemical agent in the
workplace, but
belonged to the same
age group and socio-
economic status as the
welders")
Assessment: Exposure to Cr(VI) inferred based
on occupation. State that welders were
engaged in SMA welding, working with
electrodes containing 20% chromium.
Levels: Not reported.
Duration: Duration of welding ranged from 5
to 20 yr.
In buccal cells of
exposed welders
compared to
referent:
'Y micronucleus
frequency and mean
comet tail length
(DNA damage) that
increased with
duration of work
(p < 0.05)
Medium confidence. The overall sample
size is adequate but might not be
sufficient for analyses stratified by
smoking and alcohol consumption (and
might need to consider both
simultaneously). Potential for chromium
exposure seems high given occupational
context, but lack of measurements in
environmental or biological media are
lacking.
Sudha et al.
(2011)
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Supplemental Information—Hexavalent Chromium
Study overview
Exposure3
Results
Comments
Reference
Cross-sectional study,
Bulgaria.
Exposed 1: n = 30 male
workers at a hydraulic
machinery plant. Of
these, n = 16 had low
level exposure to
chromium (various
occupations, did not
work close to
electroplating tanks),
while n = 14 had higher
exposure to chromium
due to work as
electroplaters
Exposed 2: n = 10
hospitalized
electroplaters from
different plants were
recruited from an
occupational health
clinic
Referent 1: n = 5 male
administrative workers
from the hydraulic
machinery plant
Referent 2: n = 13
administrative workers
(workplace not
described)
Assessment: Exposure to Cr(VI) inferred based
on occupation. The workers were split into
two groups based on levels of exposure.
Also measured Cr in air, erythrocytes, and
urine for exposed workers only.
Levels: Mean air chromium (total)
concentrations were 43 and 83 ng/m3 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 ng/L, respectively.
The mean chromium concentrations in
erythrocytes and urine of the high-exposure
group were 8.4 and 5.0 ng/L, respectively.
Duration: Duration of employment ranged
from 4 to 25 yr with mean durations of 10.44
and 11.63 yr in the low- and high-exposure
groups, respectively.
1" MN and
binucleated cells
carrying
MN in lymphocytes
of exposed
compared to
control; also found
correlations of Cr
measured in air,
erythrocytes and
urine, with higher
MN.
Low confidence. Limitations are due to
small sample size, questionable pooling
of various exposed and control groups,
lack of consideration of confounding, and
limited description of analysis.
Vaglenov et al.
(1999)
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Supplemental Information—Hexavalent Chromium
Study overview
Exposure3
Results
Comments
Reference
Cross-sectional study,
Austria.
Exposed: n = 22 bright
chrome plating workers
exposed to chromium
and cobalt
Referent: n = 22 jail
wardens
Assessment: Exposure to Cr(VI) inferred based
on occupation. Welders used mainly TIG
process (95%) with smaller proportions of
electric arc and very little autogenous welding.
Also measured Cr in whole blood.
Levels: Blood levels were higher in welders
compared with controls. Mean + SD levels for
exposed workers at the beginning and end of
the work week were 1.4 + 0.9 and 2.3 + 1.5
Hg/L, respectively, while values for referent
were 0.2 + 0.2 ng/L.
Duration: All welders worked 8 h/d, 3 wk
before and during the collection of the
samples.
In exfoliated cells of
exposed chrome
platers compared to
referent:
Buccal cells: T*
micronucleus
frequency by 23%
that was not quite
statistically
significant
(p = 0.516)
Nasal cells: T*
micronucleus
frequency by 97%
(p = 0.005)
1" nuclear
anomalies in both
cell types
Low confidence. Limitations are due to
small sample size and presence of
coexposures, which precluded more
detailed analysis to separate effects.
Wultsch et al.
(2014)
Cross-sectional study,
China
Exposed: n = 79
chromate production
workers
Referent: n = 112
peasant volunteers
without occupational
chromate exposures
Assessment: Exposure to Cr(VI) inferred based
on occupation. Also measured Cr in blood,
urine, and air.
Levels: Concentrations were higher in all media
among exposed (mean (range); air: 13.01
(1.03-56.60) ng/m3; blood: 9.19 (1.17-51.88)
Hg/L; urine: 17.03 (2.78-97.23) ng/g)
creatinine compared to controls (air: 0.073
(0.023-0.235) ng/m3; blood: 3.44 (0.25-22.51)
Hg/L; urine: 1.42 (0.39-26.82) ng/g.
Duration: Mean (SE) yr of work among
chromate group = 14.89 (8.65).
-t MN in
binucleated cells
among exposed
compared to control
group. Moderate
correlations (0.353-
0.517) between Cr
in blood, urine, air,
and MN
Low confidence. Limitations include
unclear recruitment processes (leaving
potential for selection bias), potential
exposure to chromium in control group
reducing sensitivity, and limited analysis
(including unclear approach to address
confounding).
Xiaohua et al.
(2012)
The following studies were found to be uninformative due to critical deficiencies in the exposure or outcome domain: Cid et al. (1991), Coelho et al. (2013),
Hilali et al. (2008), Sarto et al. (1990), Sellappa et al. (2010), and Wultsch et al. (2017).
aPotassium dichromate units conversion: Cr(VI) = 0.353 x K2Cr2C>7; potassium chromate units conversion: Cr(VI) = 0.268 x foCrCU; sodium dichromate dihydrate
units conversion: Cr(VI) = 0.349 x Na2Cr20?2H20 (usually denoted as Na2Cr2C>7 because study authors frequently list the salt as the chemical compound even if
concentration or dose is based on the dihydrate); chromium trioxide units conversion: Cr(VI) = 0.52 x CrC>3.
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Supplemental Information—Hexavalent Chromium
Supporting genotoxicity studies in lung tissue
In addition to the studies measuring gene and chromosomal mutation summarized above,
other mechanistic evidence investigating genotoxicity specific to lung tissues following exposures
to Cr(VI) was identified in preliminary title and abstract screening. These studies were tagged as
"mechanistic" and further screened and tagged as "inhalation" and "cancer" if they were
epidemiological studies of humans exposed to Cr(VI) via inhalation or studies conducted in lung
tissues or cells that were relevant to carcinogenic processes. Four additional genotoxicity studies of
lung tumor tissue in occupationally exposed humans were identified. Genotoxicity evidence from in
vitro studies conducted in human primary or immortalized lung cells examining genotoxicity
endpoints relevant to lung cancer are also summarized below. The evidence is summarized in
Table C-48.
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Supplemental Information—Hexavalent Chromium
Table C-48. Supporting genotoxicity studies in lung tissues and cells following Cr(VI) exposures
Study overview
Exposure3
Results
Comments
Reference
Gene mutation or gene expression in tumor tissue
20 lung tumor & normal
tissue samples from 19
individuals undergoing
surgery for lung cancer
or at autopsy
Assessment: Based on occupation
Levels: Not reported
Duration: Male workers exposed to chromate
for average 21.7 ± 9.1 (8-38) yr
P53 mutations found in 4
(20%) of 20 chromate-
exposed lung samples
4/ occurrence of p53
mutations in chromate
exposed workers
Key differences in chromate
exposed workers: no G-to-T
transversions; 50% point
mutations had changes in
AT base pairs; 50% of those
with point mutations had
double missense mutations
P53 mutations in
chromate-exposed
workers with lung cancer;
the pattern of p53
mutations in lung cancer
patients exposed to
chromate differed from
that of common lung
cancers in 3 respects.
No adjustments for
potential confounders; no
information on smoking
provided; small sample
size; limited information
on selection.
Kondo et al. (1997)
Exposed 1: exocrine
pancreatic cancer cases
with K-ras mutated
tumors (n = 83)
Exposed 2: exocrine
pancreatic cases without
K-ras mutated tumors
(n = 24)
Assessment: Finnish job-exposure matrix
(Finjem): Inhalation exposure to chromium
dust or fumes from welding, smelting,
grinding, or related processing of steel or
other materials containing chromium
(including metallic chromium, Cr(lll), Cr(VI),
and other chromium compounds)
Industrial hygiene evaluation: inhalation and
dermal exposure to Cr(lll) and Cr(VI)
Levels: Not reported
Duration: Not reported
T* OR of K-ras codon 12
mutated pancreatic cancer
with inhalation exposure to
chromium
T* proportion of glycine to
valine mutations (G-to-T
transversions) with
inhalation exposure to
chromium
PCR-RFLP analysis of
formalin-fixed and
paraffin-embedded tumor
specimens for point
mutations at codon 12 of
the K-ras gene.
Very few individuals
actually exposed to Cr;
wide confidence intervals
indicate model instability.
Alguacil et al. (2003)
Exposed: Chromium
workers diagnosed with
lung cancer (n = 67
males)
Assessment: Total and hexavalent Cr
measured in soil and air samples taken "in the
vicinity of the workplace" using atomic
absorption spectrometry
In lung cancer tissues
(preserved in paraffin
blocks):
4/ surviving (anti-apoptotic)
T* p53 (pro-apoptotic)
The information regarding
potential exposure is
sparse. There were also
differences in the type of
lung cancer between
Halasova et al. (2010)
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Supplemental Information—Hexavalent Chromium
Study overview
Exposure3
Results
Comments
Reference
Referent: male controls
with lung cancer but
without known exposure
to chromium (n = 104)
Levels: Mean values ofCr(VI) in air of smelting
plants was 0.019-0.03 mg/m3. Soil chromium
had a value of 137 mg/kg.
Duration: Mean exposure time 16.7 ± 10.0
(SD) yr (range 1-41 yr)
exposed and referent
which may impact results.
No information on
smoking, which could be
important to consider
given all participants had
lung cancer.
In addition, P53 detection
by IHC is nonspecific and
will include nonfunctional
P53 protein.
Exposed: lung cancer
specimens from ex-
chromate workers
(n = 19)
Referents 1: lung cancer
specimens from
individuals never
exposed to chromate,
silica, or other
occupational compounds
(n = 52)
Referents 2: lung cancer
specimens from
nonasbestos
pneumoconiosis (n = 63)
Assessment: Based on occupational history
Levels: Not reported
Duration: Not reported
In lung cancer tissues
(squamous cell carcinomas)
from chromate-exposed
patients compared to
nonexposed or
pneumoconiosis patients:
1" cyclin D1 expression
(p < 0.001)
No difference in bcl-2 or p53
expression
No assessment of
exposure; reliance on
work history alone.
Minimal details on
case/control selection.
No consideration of
confounders, except
smoking status.
Katabami et al. (2000)
Mouse, transgenic
C57BL/6 Big Blue® mice
Intratracheal instillation (single
administration): 0,1.7, 3.4, or 6.8 mg/kg
Cr(VI)
Measured mutation frequency in lung at 1, 2,
or 4 wk postexposure
Significantly increased
mutation frequency at all
doses; increased with dose
and duration posttreatment
Mutation spectrum:
increased frequency of G:C
to T:Atransversions,
associated with oxidative
damage
Preliminary experiment
identified doses >6.75
mg/kg were lethal.
Potentially underpowered
with 4 mice per dose
group.
Positive control not
concurrently tested with
Cr(VI)-treated group.
Cheng et al. (2000; 1998)
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Supplemental Information—Hexavalent Chromium
Study overview
Exposure3
Results
Comments
Reference
Inconsistent/low numbers
of PFUs scored per animal.
Spontaneous mutations
primarily G:C to A:T
transitions.
In vitro genotoxicity in primary and immortalized human lung cells
A549 (human lung
adenocarcinoma)
10 nM K2Cr04, 1-24 h
Distribution of bulky DNA
adducts and oxidative DNA
damage and mutational
signature of p53 mutations
following exposure to Cr(lll),
Cr(VI), and Cr(V).
Arakawa et al. (2012)
HLF human lung
fibroblasts (LL-24 cell
line)
3, 6, and 9 nM Na2CrC>4, 24 h
1" Cr-DNA adducts
Pretreatment with ImM
ascorbate or 20 nM
tocopherol had no
ameliorative effects.
Also 1" cytotoxicity,
duration- and dose-
dependent (stat. sig.
>6 nM).
1" apoptosis
1" p53 (4- to 6-fold)
Carlisle et al. (2000a)
A549 (human lung
adenocarcinoma) and
BEAS2B (human
bronchial epithelial) cells
0.1, 0.5,1.0, and 10 nM Na2CrC>4, 0.5,1, and 4
h
1" oxidative DNA damage
(Fpg-modified comet assay)
Oxidative role in DNA
damage decreased with
time at lower Cr(VI)
concentrations and
increased with time at
higher concentrations.
A549 more sensitive than
BEAS2B.
Also 1" apoptosis at 10
HM (caspase-3 activity
and morphology).
Cavallo et al. (2010)
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Study overview
Exposure3
Results
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Reference
H460 human lung
epithelial cells, IMR90
normal human lung
fibroblasts, and normal
mouse embryonic
fibroblasts
0, 5,10,15, and 20 nM K2Cr04
DNA damage response to
Cr(VI)-induced DNA double-
strand breaks
(phosphorylation of yH2AX)
dependent on ATR kinase
and not ATM in ascorbate-
restored cells
DNA DSBs only formed in
euchromatin
Involvement of ATR and
DSBs forming in actively
transcribed regions
increases the probability
that Cr(VI) can generate
carcinogenic mutations.
Delougherv et al. (2015)
Human bronchial
epithelial cells and IMR-
90 embryonic lung
fibroblasts
K2Cr04, 25-200 jiM, 1-12 h
1" DNA-protein crosslinks,
dose-dependent, persistent
at 12 h
Fornace et al. (1981)
A549 human lung
adenocarcinoma cells
10-500 nM Na2Cr207, 1 or 16 h
1" DNA strand breaks, dose-
dependent (comet assay)
that were 10x higher with
FAPY
-t 8-OHdG
Authors conclude that
Cr(VI)-induced oxidative
DNA damage might partly
be due to a reduced
capacity to repair
endogenous and Cr(VI)-
induced 8-OHdG lesions.
Also 4, OGG1 mRNA,
dose-dependent (RT-PCR
and RNase protection
assay); not affected by
adding H202.
No effect on hAPE or
GAPDH.
Hodges et al. (2002;
2001)
HeLa and human lung
bronchial epithelial cells
0.25 nM Na2Cr04, 30 d, or 10 nM, 16 or 48 h
1" chromosomal aberrations
with acute or chronic
exposures
Chromosomal instability
caused in part by
suppressed activation of
BubRl and expression of
Emil, causing activation
of APC/C, following
nocodazole-induced
mitotic arrest activation.
Hu et al. (2011)
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Supplemental Information—Hexavalent Chromium
Study overview
Exposure3
Results
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Reference
Human U20S
osteosarcoma cells,
Werner syndrome (WS)
skin fibroblasts
(AG03141), WI-38 fetal
lung fibroblasts,
telomerase-immortalized
cell lines (hTERT
GM01604, (hTERT
AMIE15010, AG03141,
hTERT BJ skin fibroblasts)
0-4 nM Cr(VI), 6-48 h
T* yH2AX foci in S-phase
T* WRN colocalization at
yH2AX foci
T* telomere defects
exacerbated by lack of
telomerase
Lack of WRN slowed Cr(VI)-
induced DNA DSB repair
Cr(VI) induces DNA DSBs
and stalled replication
forks; WRN helicase plays
a role in the cellular
recovery from Cr(VI)-
induced replicative stress.
Liu et al. (2010a, 2009)
A549 (human lung
adenocarcinoma) and
BEAS2B (human
bronchial epithelial) cells
0, 0.5, 1, 2, 3, 5 nM Cr(VI), 2-72 h
4, Gene 33 (Mig6, ERRFI1),
dose- and time-dependent
(>1 nM, 24 h); reversed by
NAC
-t DNA DSBs (yH2AX), dose-
dependent (>2 nM)
Suppression of Gene 33
increases DNA damage
(yH2AX, micronuclei) and
cell transformation
Cr(VI) suppresses Gene
33, inhibiting the Cr(VI)-
induced DNA damage
response mediated in part
by Gene 33-induced cell
signaling pathways.
Park et al. (2016)
Human lung epithelial
A549 and colon HCT116
(MLH-/-) and DLD1
(MSH6-/-) cells
1-20 nM K2Cr04, 3-12 h
T* survival, 4, apoptosis in
mismatch repair (MMR)-
deficient cells
-t DNA DSBs (yH2AX) and
apoptosis in MMR-
proficient cells
yH2AX foci occur in G2, but
no G2 cell cycle arrest
No p53 induction in either
cell type at subtoxic levels
MMR responsive to Cr-
DNA adducts, not
oxidative damage or
crosslinks.
In MMR+ cells, apoptosis
induced by Cr-DNA
adducts independently of
p53.
Peterson-Roth et al.
(2005); Zhitkovich et al.
(2006)
S-9 fraction from
pulmonary alveolar
macrophages or S-12
fraction of peripheral
10-30 ng sodium dichromate dihydrate per
plate
4/ mutagenicity in the Ames
assay when Cr(VI) was
preincubated with lung
fractions
Petrilli et al. (1986), De
Flora et al. (1987b)
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Supplemental Information—Hexavalent Chromium
Study overview
Exposure3
Results
Comments
Reference
lung parenchyma of
human patients
Primary human lung
IMR90 fibroblasts, H460
human lung epithelial
cells, and XPA- and XPF-
human fibroblasts
1-5 nM K2Cr04, 3 h
Cr-DNA adducts are
substrate for nucleotide
excision repair (NER)
1" mutagenicity of these
adducts and T* apoptosis
with NER deficiency
NER efficiently removes
Cr-DNA adducts.
Reynolds et al. (2004)
Human colon HCT116
(MLH1-/-) and DLD1
(MSH6-/-), lung epithelial
H460, and lung fibroblast
IMR90 cell lines
2-10 nmol/L K2Cr04,3 h
Ternary ascorbate-Cr-DNA
adducts are substrate for
error-prone mismatch
repair (MMR) MSH2-MSH6
dimer, leading to T* DNA
DSBs and T* apoptosis
Cells deficient in MMR have
higher survival and lower
DNA DSBs
Colon cells deficient in
MMR have increased
survival following Cr(VI)
exposures, increasing
probability of clonal
selection of these cells.
Reynolds et al. (2009)
Primary human lung
IMR90 fibroblasts
H460 human lung
epithelial cells
0.2-8 nM K2Cr04,3 h
1" DNA DSB with ascorbate
caused by aberrant
mismatch repair
1" cytotoxicity and
apoptosis with ascorbate;
effects reversed by
suppressing DNA mismatch
repair but p53 status had no
effect
1^1" cytotoxicity and cell
cycle delay in cells deficient
in oxidative DNA damage
repair (XRCC1 knockdown);
effects reversed by
ascorbate
Chromosomal aberrations
not affected by XRCC1
status
By restoring intracellular
ascorbate to physiological
levels via DHA (max
intracellular 0.9 mM), it
was shown that ascorbate
can suppress Cr(VI)-
induced oxidative damage
but promotes Cr-DNA
lesions that are either
repaired by mismatch
repair, independently of
p53, or lead to
cytotoxicity and
apoptosis.
Reynolds et al. (2012;
2007; 2007)
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Supplemental Information—Hexavalent Chromium
Study overview
Exposure3
Results
Comments
Reference
Primary human bronchial
epithelial cells; p53+ and
p53- H358
bronchoalveolar
carcinoma isogenic cells
200 nM K2Cr04,2 h
1" DNA strand breaks
1" apoptosis in p53+ cells
Apoptosis mediated by p53-
upregulated modulator of
apoptosis (PUMA), BAX,
cytochrome C and caspase-3
Russo et al. (2005)
Primary human bronchial
fibroblasts (PHBFs)
1-10 nM Na2Cr04, 24 h
Relative survival of 74%
(1 nM), 57% (2.5 nM), 13%
(5 nM) and 0% (10 jiM)
Chromosomal damage in
18% (1 nM) and 33%
(2.5 nM) of metaphases
Wise J Petal. (2002)
Human SV40
transformed fibroblasts,
Werner syndrome
fibroblasts, primary
human lung IMR90
fibroblasts, and human
colon HCT116 MLH1-/-
and MLH1+ cells
0-30 nM K2Cr04,3 h
1" nuclear relocalization of
WRN in response to Cr(VI)
4/ cell survival, T* DNA DSBs
and 4/ RAD51foci in cells
lacking WRN
4/ DNA DSBs in cells lacking
mismatch repair
Error-prone mismatch
repair of Cr-DNA adducts
generates DNA DSBs and
repair of persistent DNA
DSBs is dependent on
WRN helicase.
Zecevic et al. (2009)
aPotassium dichromate units conversion: Cr(VI) = 0.353 x K2Cr2C>7; potassium chromate units conversion: Cr(VI) = 0.268 x foCrCU; sodium dichromate dihydrate
units conversion: Cr(VI) = 0.349 x Na2Cr20?2H20 (usually denoted as Na2Cr207 because study authors frequently list the salt as the chemical compound even if
concentration or dose is based on the dihydrate); chromium trioxide units conversion: Cr(VI) = 0.52 x CrC>3.
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Supplemental Information—Hexavalent Chromium
1 Supporting inhalation exposure genotoxicity studies
2 Another set of genotoxicity studies was identified that was informative for interpretations
3 of genotoxic risk in humans but did not specifically measure genotoxicity in lung tissues. These
4 studies were also identified in preliminary title and abstract screening as "mechanistic" and were
5 further screened and tagged as "inhalation," "cancer," and "genotoxicity" if they were
6 epidemiological studies of humans or experimental animal studies exposed to Cr(VI) via inhalation
7 that measured genotoxicity endpoints. After removal of endpoints already considered that
8 reported gene and chromosomal mutation measures and studies specific to lung tissues (see
9 above), 29 genotoxicity studies of humans occupationally exposed and 1 study in animals exposed
10 via intratracheal instillation were identified. The evidence is summarized in Table C-49.
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Supplemental Information—Hexavalent Chromium
Table C-49. Supporting genotoxicity studies in humans and animals exposed to Cr(VI) via inhalation or
intratracheal instillation
Study overview
Exposure3
Results
Comments
Reference
DNA strand breaks
Exposed 1: directly
exposed (DE) to Cr(VI) for
>8h/d in tannery industry
(n = 36)
Exposed 2: indirectly
exposed (IE) to Cr(l) for
>30 yr based on residence
near tannery industry
(n = 36)
Referents: age-matched
controls, unexposed to
chemicals or radiation
(n = 36)
Assessment: Exposure to Cr(VI) inferred
based on occupation and residence.
In addition, Cr was measured in urine and air
samples (unclear where air samples were
collected).
Levels: There was a gradient in levels of both,
there were detectable chromium levels in
both air and urine for "controls."
Direct exposure (n = 36) (mean ± SD):
Total Cr in air (1 mg/m3): 0.101 ± 0.003
Cr(VI) in air (0.001 mg/m3): 0.021 ± 0.003
Cr content in urine: 2.11 ± 1.01
Indirect exposure (n = 36):
Total Cr in air (1 mg/m3): 0.089 ± 0.003
Cr(VI) in air (0.001 mg/m3): 0.013 ± 0.005
Cr content in urine: 1.81 ± 0.88
Controls (n = 36):
Total Cr in air (1 mg/m3): 0.014 ± 0.004
Cr(VI) in air (0.001 mg/m3): 0.006 ± 0.001
Cr content in urine: 0.54 ± 0.39
Duration: Directly exposed subjects were
"selected based on the duration of their
exposure (0-5; 6-10; 11-15; 16-20; 21-25
years) and were known to be exposed to
Cr(VI) for a minimum of 8 h/day" while
indirect exposure was inferred from
residence of at least 30 year's duration, "in
and around the tanneries."
T* mean tail length for
comet assay in DE group
compared to IE group and
controls
Some of the controls also
had detectable chromium
in urine, suggesting this is
not really a true "control"
group.
Very limited evaluation of
confounders.
Small sample size.
Study also reported T*
CAs & MN in DE group
compared to IE group and
controls.
Balachandar et al.
(2010)
Exposed: male welders
(n = 102)
Referents: male general
population controls
Assessment: Blood samples from 51 welders
& 49 controls, selected randomly, on 4th day
of the work week. Cr and Ni content
measured with ICP-MS.
T* DNA mean tail length in
welders
Limitations are related to
outcome evaluation, such
as the use of outdated
methods no longer
Danadevi et al. (2004)
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Supplemental Information—Hexavalent Chromium
Study overview
Exposure3
Results
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Reference
(n = 102), age and SES-
matched to exposed
Levels: Welders had higher Cr and Ni
compared to controls [(Cr, 151.65 versus
17.86 mg/L; Ni, 132.39 versus 16.91 mg/L;
p< 0.001)].
Duration: The duration of exposure varied
widely (range: 1-24 yr). (Overall mean not
presented).
recommended, which
could lead to inaccurate
scoring. Also a lack of
description/details on
participant selection
(e.g., concern for
potential selection bias).
Comet assay conducted in
all subjects, but
micronucleus test
conducted only in 58
welders and 53 controls,
selected randomly from
population (study
reported T* MN in
welders compared to
controls and with
increased duration of
welding work).
Exposed: Chrome-plating
workers (n = 19)
Referents 1: hospital
workers (n = 18)
Referents 2: university
personnel (n = 20)
Assessment: Total Cr measured in urine,
erythrocytes, and lymphocytes using graphite
furnace atomic absorption.
Levels: Total Cr was higher in exposed
workers compared with hospital workers (see
Table 3; for example, postshift mean urine
levels were 7.31 [SD = 4.33] in exposed vs.
0.12 [SD = 0.07] ng/g crt in referent).
Duration: Mean (SD) yr of exposure among
chrome-plating workers = 6.3 (4.3).
In peripheral blood
lymphocytes:
T* comet tail moment
correlated with Cr
lymphocyte concentrations
Null apoptotic nuclei
Did not exclude smokers
(high prevalence)
although did present
results stratified by
smoking (small numbers).
Unclear whether exposure
was to Cr(VI) specifically
(possible with chrome
plating workers but
measured total Cr in
urine). State that
previous air monitoring
for total chromium
showed levels of 0.4 to
5.6 ng/m3, which is fairly
low.
The comet assay is an
insensitive method for
measuring apoptosis.
Gambelunghe et al.
(2003)
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Supplemental Information—Hexavalent Chromium
Study overview
Exposure3
Results
Comments
Reference
Exposed: chromium
exposed workers (n = 10)
Referents: nonexposed
workers (n = 10)
Assessment: Urine and blood samples were
taken from workers at the end of a work
week.
Levels: Chromium concentrations in the
factory ranged from 0.001 to 0.055 mg
Cr(VI)/m3 (obtained from personal and area
samplers). Mean chromium concentrations
in urine (5.97 ng/g creatinine), whole blood
(5.5 ng/L), plasma (2.8 ng/L), and
lymphocytes (1.01 ng/1010 cells) of exposed
workers were significantly higher than in
nonexposed workers.
Duration: The mean duration of exposure was
15 yr.
No difference in DNA strand
breaks (alkaline elution
assay) between groups
Very small sample and
low exposure levels,
which probably limited
power.
Study also reported no
increased incidence in
8-OHdG.
Gao et al. (1994)
Exposed 1: male welders
working in areas without
collective protections
(n = 27)
Exposed 2: male welders
working in locations with
smoke extraction systems
(n = 33)
Referents: office workers
with no history of
occupational exposure to
welding fumes or other
physical/chemical agent
in workplace (n = 30)
Assessment: Exposure to Cr(VI) inferred
based on occupation. Also measured total Cr
in blood and urine.
Levels: Cr levels in blood and urine were
higher among both groups of welders
compared with controls (means 129 to 145,
compared with 92 ng/L), and urinary
chromium was higher among welders
working without smoke extraction systems.
Duration: Welders exposed for 0.5-45 yr.
-t OTMx2 distribution
(measure of DNA damage)
in welders at the end of the
work week compared to
beginning
1" DNA strand breaks at end
of work week in welders
Main limitations are lack
of description (e.g., for
participant selection,
analysis), unknown
contribution of Cr(VI) to
Cr exposure, and known
coexposures to other
metals.
Study also reported T*
frequency of
chromosomal damage in
welders.
larmarcovai et al. (2005)
Exposed: welders (n = 93)
Referents: general
population controls with
no history of occupational
exposure to welding
fumes; age and SES-
matched to exposed
group (n = 60)
Assessment: Exposure determined by
occupation.
Levels: Not reported.
Duration: 5-15 yr.
1" DNA mean tail length in
welders compared to
controls
Study was not included due
to a critically deficient rating
in the exposure domain
when evaluated in HAWC
for the micronucleus
frequency endpoint.
Study also reported T*
frequency of MN in
welders compared to
controls and in welders
with increased duration of
work.
Sellappa et al. (2010)
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Study overview
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Results
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Reference
Exposed: male welders
(n = 66)
Referents: male
individuals from the
general population with
no history of occupational
exposure to welding
fumes or other
physical/chemical
exposure in workplace;
age and SES-matched to
welders (n = 60)
Assessment: Exposure determined by
occupation.
Levels: Not reported.
Duration: Duration of welding ranged from 5-
20 yr.
1" DNA mean tail length in
welders compared to
controls, and in welders
with increased duration of
work
Study also reported T*
frequency of MN in
welders compared to
controls and in welders
with increased duration of
work.
Sudha et al. (2011)
Exposed: individuals
(n = 115; 29 female, 86
male) with exposure to
sodium dichromate for at
least 6 mo
Referents: healthy
volunteers (n = 60; 15
female, 45 male) in the
same city without
chromate exposure
history.
Assessment: Air-Cr concentration collected
with point dust sampler and measured with
electrothermal atomic absorption
spectrometry. Personal air samples collected
through full-shift (8h) sampling to calculate
cumulative dose postshift blood samples
collected; chromium measured with ICP-MS.
Levels: Mean (SD) chromium in blood of
exposed workers = 12.45 (20.28) ng/L.'T*
accumulation of Cr in peripheral red blood
cells in chromate-exposed workers.
Duration: Mean (SD) yr of employment
among exposed group: 12.86 (6.02); range:
1-33.
1" urinary 8-hydroxy-2-
deoxyguanosine, DNA
strand breaks and global
DNA hypomethylation in
chromate-exposed workers
Urinary 8-hydroxy-2'-
deoxyguanosine, DNA
strand breaks and global
DNA hypomethylation.
No adjustment for diet or
other nonfolate
supplements.
4/ serum folate in
chromate-exposed
workers.
Wang et al. (2012)
Exposed: electroplating
workers (n = 157)
Referents: individuals
without exposure to
chromium or known
physical/chemical
genotoxic agents (n = 93)
Assessment: Air-Cr and blood Cr determined
by graphite furnace atomic absorption
spectrophotometer.
Levels: median (range) Cr in erythrocytes
(Hg/L) among exposed: 4.41 (0.93-14.98);
among controls: 1.54(0.14-4.58). Median
(range) short-term concentrations of Cr in air:
0.060 (0.016-0.531) mg/m3.
Duration: Median (min-max) yr of exposure
among exposed group: 5.3 (0.5-23).
1" 8-OHdG adducts among
exposed compared to
referents
1" Olive tail moment, tail
length, & tail DNA% among
exposed compared to
referents
Limited adjustment for
confounders (including
diet).
Potential coexposures to
other metals in the
workplace.
Zhang et al. (2011)
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Study overview
Exposure3
Results
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Reference
Rat, Wistar
Intratracheal instillation, 1.3 and 2.5 mg/kg
Na2Cr207, 24 h.
1" DNA strand breaks in
peripheral lymphocytes
Fluorometric analysis of
DNA unwinding (FADU)
assay.
Gaoetal. (1992)
DNA-protein crosslinks
Exposed 1: Full-time
tannery workers, directly
involved in chromium
tanning or finishing
process (n = 33)
Exposed 2: Full-time
manual metal arc
stainless steel welders
(n = 5)
Referents: Control
individuals with no known
exposure to
environmental or
occupational carcinogens
(n = 30)
Assessment: Exposure to Cr(VI) inferred
based on occupation. Also measured Cr in
plasma and in urine (mid-shift for welders).
Levels: Urinary and plasma chromium levels
were higher in both exposed groups
compared with controls. For example,
mean ± SD levels in plasma were 2.43 ± 2.11
in tanners, 1.55 ± 0.67 in welders, and
0.41 ± 0.11 ng/L.
Duration: Not reported.
1" DNA-protein crosslinks in
tannery workers & welders
compared to controls
Main limitation is small
number of welders, lack
of description for
participant selection,
analysis, and confounders.
Study also reported "MVIN
in tannery workers &
welders compared to
controls.
Medeiros et al. (2003)
Exposed: residents living
near Hudson County, New
Jersey chromium-
containing landfills and
with urinary chromium
>0.5 ng/L (n = 33)
Referents: individuals
living in noncontaminated
areas (n = 49)
Assessment: No description of exposure
assessment protocol.
Levels: Based on recruitment, exposed group
had urinary chromium >0.5 ng/L.
Duration: Not reported.
1" DNA-protein crosslinks in
exposed compared to
controls, after adjustments
for covariates
Control for the covariates
(age, gender, race,
smoking, weight)
increases confidence in
results.
Unclear whether
chromium measures were
also assessed in the
control population and
whether unexposed
status was confirmed.
Taiolietal. (1995)
Exposed: male stainless
steel welders working in
open environment (n = 5)
Referents: age-matched
male control blood
Assessment: Based on occupation. Welders
worked in stainless steel industry using
acetylene flame method in open environment
without protective masks over nose or
mouth.
Levels: Not reported.
1" DNA-protein crosslinks in
lymphocytes of welders
4/ excess of glutathione
over cysteine in welders
Comparisons of reduction
rates and extent of DNA
damage and DNA-protein
adducts to levels of
intracellular reductants
glutathione and cysteine.
Quievrvn et al. (2001)
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Supplemental Information—Hexavalent Chromium
Study overview
Exposure3
Results
Comments
Reference
samples obtained from
local blood center (n = 22)
Duration: Not reported.
Small sample size limits
confidence in results.
Exposed: Chrome-platers
from metallurgic plant
(n = 14)
Referents: residents of
the same town, not living
in vicinity of the factory
and not known to be
exposed to chromium or
other metals (n = 12) and
additional unexposed
individuals living in
nearby coastal town
(n = 6)
Assessment: Personal breathing sampling
pump with sampling flow of 21 min-1 for all
workers over the course of one 8-h shift;
collection using Millipore filters; analyzed
with atomic absorption flame method for
total chromium.
11 workers also fitted with pumps with
medium range flow (1.21 min"1); collection
with 5-mm PVC filters; analyzed with visible
absorption spectrophotometer for Cr(VI),
with portion of each sample analyzed for
total chromium by flame atomic absorption.
Blood samples collected post work shift;
analyzed with flameless atomic absorption
spectrometry.
Urine samples collected pre & post work shift
Levels: Ambient levels of total chromium in
chrome-plating plant ranged from 0.009 to
0.327 mg/m3 (median = 0.041 mg/m3) as
measured with Millipore filters and from
0.008 to 0.19 mg/m3 (median = 0.027 mg/m3)
measured by Higitest filters. Cr(VI) levels in
ambient air ranged from 0.0005 to 0.13
mg/m3 (median = 0.003 mg/m3).
Duration: Workers had been continually
employed at metallurgic plant for 8-h work
shifts for 1.5-15 yr (mean: 9.5 ± 4.0).
1" chromium in pre-&
postshift urine,
erythrocytes, and
lymphocytes elevated in
exposed compared to
referents
No difference in DNA-
protein crosslinks between
exposed and referents;
however, there were +
associations between DNA
protein crosslinks and
chromium in erythrocytes at
low and moderate
exposures with saturation
at higher exposure levels
Small sample size limits
confidence.
No consideration of
covariates.
Potential confounding by
other occupational
exposures.
Zhitkovich et al. (1996)
Exposed: railroad arc
welders (n = 21)
Referents: unexposed
controls (office workers,
supervisors, janitors,
laboratory technicians)
(n = 26)
Assessment: Chromium and nickel measured
in blood of controls and welders with atomic
absorption.
Levels: No difference in nickel levels between
groups; small but not statistically significant
difference in chromium between groups
(numbers not provided).
1" DNA-protein cross-links
in welders compared to
controls
Unclear how an effect
detected was when there
was no overall/
meaningful difference in
chromium or nickel
between groups - could
possibly be due to an
unmeasured confounder.
Costa et al. (1993)
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Supplemental Information—Hexavalent Chromium
Study overview
Exposure3
Results
Comments
Reference
Duration: Welders had been exposed full
time to welding fumes for at least 6 mo, but
not stainless-steel welding.
The exposed group did
not actually experience
high levels of Cr exposure,
which might have limited
power to detect effects.
Small sample size limits
confidence.
Sister chromatid exchange
Exposed: Chromium plate
workers (n = 15)
Referents 1: age, gender,
smoking-matched
controls (n = 15)
Referents 2: individuals of
similar age from
unpolluted rural region
(n = 8)
Assessment: Blood samples and buccal
mucosal cells taken from exposed group;
exposure was estimated with personal air
samplers and in urine samples.
Levels: 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 ng/L (low) and 104.22 ng/L (high)
Duration: Duration of exposure ranged from
2 to >20 yr; mean duration of exposure was
not reported.
No difference in SCE/cell
between exposed and
controls
Although exposed and
unexposed workers were
matched on age, sex, and
smoking habit, the two
unexposed(worker and
rural) groups were
combined, resulting in
lower confidence in
comparability of exposed
and unexposed group
comparisons. Inference is
further limited by small
sample size and lack of
description.
Study also reported T*
micronuclei in peripheral
lymphocytes and buccal
cells in workers compared
to controls.
Benova et al. (2002)
Exposed: chromium
electroplating workers
(n = 7)
Referents: age and sex-
matched nonexposed
office employees (n = 10)
Assessment: Air samples from the
electroplating room were collected, along
with stool and hair samples to determine
exposure.
Levels: The mean chromium (total) air
concentration (by random air collection) was
8.1 ng/mm3, the mean chromium
concentration in stool samples was 8.5 ng/g
stool, and the mean chromium concentration
in hair was 35.68 ng/g. The valence of
T* chromosomal
aberrations and sister
chromatid exchanges (SCE)
in exposed group
Although controls were
age and sex matched to
exposed subjects and
were stated to have
similar socioeconomic
status, the sample size is
quite small and the
analysis limited. Also
unclear how well
differentiated chromium
exposure is by group-
Deng et al. (1988)
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Supplemental Information—Hexavalent Chromium
Study overview
Exposure3
Results
Comments
Reference
chromium that workers were exposed to was
unspecified.
Duration: Mean employment period of
12.8 yr among exposed groups.
analyses of chromium in
hair suggest delineation
with controls, but no
information on stool
samples, which showed
similarities between nickel
and chromium workers.
Also reports coexposure
to nickel.
Exposed: male stainless
steel welders (n = 23)
Referents: men employed
in office of printing
company (n = 22)
Assessment: Urine sampling at end workday
to evaluate chromium concentration.
Levels: Urinary chromium levels ranged from
0.20 to 1.55 nmole/L.
Duration: Welders had been employed in
manual metal arc (MMA) welding for at least
4 yr; mean (SD) length of employment = 21
(10). Welders worked in poorly ventilated
areas.
No differences in SCE
between exposure groups
Although Cr(VI) exposure
seems likely to occur
among these welders, the
analysis is limited by small
sample size when
stratifying by smoking
(found to be related to
the outcome).
Study also reported no
differences in CA between
exposure groups.
Husgafvel-Pursiainen et
al. (1982)
Exposed: male stainless
steel welders (survey 1
n = 17; survey 2 & 3
n = 44)
Referents: male office
workers (survey 1 n = 6;
survey 2 n = 7; survey 3
n = 20)
Assessment: Classification based on
occupation. Spot urine samples collected
during workday; analyzed with direct
flameless atomic absorption spectrometer.
Levels: Mean urinary Cr was 9.8 and 4.2 ng/L
among exposed and referent group,
respectively.
Duration: Stainless steel welders employed
for 5-20 yr (mean 12.1).
No differences in sister
chromatid exchanges (SCE)
in exposed compared to
controls
The main limitations are
related to the outcome
evaluation and to poorly
described and reported
data analysis and lack of
consideration of potential
confounders.
Study also reported T*
chromosomal aberrations
in exposed compared to
controls.
Koshi et al. (1984)
Exposed 1: chromium
exposed electroplating
male workers (n = 14)
Exposed 2: nickel-
chromium exposed
Assessment: Urine and blood samples
collected; analyzed with atomic absorption
spectrophometry.
Levels: Cr workers had the highest blood Cr
(11.39 Ig/L) and urine Cr concentrations (14.7
Ig/g creatinine).
1" sister chromatid
exchanges and high
frequency cells in Cr & Ni-Cr
groups
Small sample size limits
confidence.
Observed synergistic
effect with smoking.
Lai et al. (1998)
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Supplemental Information—Hexavalent Chromium
Study overview
Exposure3
Results
Comments
Reference
electroplating male
workers (n = 34)
Referents: male
administrative workers
free of exposure to heavy
metals and solvents
(n = 43)
Duration: At least 6 mo of electroplating
experience at the start of the study. Mean
(SD) yr of work among chromium
workers = 6.6 (5.8); among nickel-chromium
workers = 3.7 (4.6).
Exposed: manual metal
arc stainless steel welders
(n = 24)
Referents: matched
controls (n = 24)
Assessment: Exposure to Cr(VI) inferred
based on occupation. Also measured total Cr
in air (welders only) and urine (all).
Levels: Mean urinary Cr was 47 and 1.5
Hmol/mol creatinine among exposed and
referent group, respectively. Mean air Cr
level 81 ng/m3.
Duration: Welders were selected for their
"long and intense" welding on stainless steel
(mean work duration of 19 yr).
No difference in cytogenetic
effects (i.e., chromosomal
aberrations, sister
chromatid exchanges, or
micronuclei) between
groups
Main limitations are
related to outcome
ascertainment and
statistical analysis and to
limited description of
results.
Littorin et al. (1983)
Exposed: male chromium
platers (n = 12)
Referents: none
Assessment: Venous blood and urine sample
were collected over a 5-yr period.
Levels: Urinary chromium concentrations
ranged from 1.2 to 57.0 ng/g with a mean
urinary chromium concentration of 17.9 ng/g
creatinine.
Duration: Employment duration ranged from
6.6 to 25.1 yr, with mean employment
duration of 15.5 yr.
No association between
urinary Cr and sister
chromatid exchanges
Small sample size and no
control group used in
study limits exposure
comparisons and power
for analysis; limited
adjustment for
confounders.
Nagava et al. (1991)
Exposed: male chromium
platers (n = 44)
Referents: male controls
unexposed to Cr or other
harmful agents (n = 47)
(further grouping by
smoking within exposed
and referents)
Assessment: Urinary collected during working
hours; analyzed with direct flameless atomic
absorption spectrophotometer.
Levels: Mean among all chromium
platers = 0.25 nmol/L.
Duration: Duration of employment: 0.5-30.7
yr [mean (SD): 13.8 (8.7)].
No association between
urinary Cr and sister
chromatid exchanges
Limited adjustment of
confounders: considered
stratification only by
smoking status.
Nagava et al. (1989)
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Supplemental Information—Hexavalent Chromium
Study overview
Exposure3
Results
Comments
Reference
Exposed: male chromium
platers (n = 24)
Referents: sex, age,
smoking-matched office
worker controls,
unexposed to Cr (n = 24)
Assessment: Urine samples analyzed with
direct flameless atomic absorption
spectrophotometer.
Levels: The mean (SD) concentration of
chromium in the urine was 13.1 (16.7) ng/L.
Duration: Duration of employment ranged
from 0.5 to 30.5 yr with a mean employment
of 11.6 yr.
No difference in SCEs
between exposed and
unexposed groups; no
association between urinary
Cr and sister chromatid
exchanges among exposed
Authors suggest that null
results could be due to
low exposures.
Consideration of smoking
but minimal other
confounders.
Nagava(1986)
Exposed: male welders
(n = 39)
Referents: unexposed
men (n = 18)
Assessment: Chromium in urine samples
(time of day unspecified) from workers
analyzed with atomic absorption
spectrometry.
Levels: Mean (SD) chromium among
welders = 28.4 (19.8) ng/L
Duration: Authors state that employees had
been employed since 1983 (paper published
in 1991); ~7—8 yr (?).
4/ sister chromatid
exchange frequency in
welders compared to
controls
Only considered age and
smoking as potential
covariates.
Authors note some
concern with alkaline
filter elution that might
impact validity of results.
Podd et al. (1991)
Exposed: male chromium
platers (n = 38)
Referents: male sanitary
workers unexposed to
ionizing radiation for at
least 5 yr & no mutagenic
drugs (n = 35)
Assessment: Exposure to Cr(VI) inferred
based on occupation. Exposed group was
stratified based upon coexposure to nickel
("bright" plating, vs. "hard" plating). Also
measured Cr in urine.
Levels: Urinary Cr levels were lowest in
controls (mean ± SD = 1.9 ± 1.4 ng/g crt),
intermediate in bright plating (6.1 ± 2.8 ng/g
crt), and highest in hard plating groups
(10.0 ± 7.5 ng/g crt), indicating adequate
delineation between groups.
Duration: Mean (SD) yr of exposure: bright
plating = 9 (11); hard plating = 7 (3).
Association between urinary
Cr and sister chromatid
exchanges
Main limitations are
related to outcome
ascertainment, small
sample size for certain
analyses, and lack of
description (e.g., for
participant selection and
statistical analysis).
Study also reported T*
chromosomal aberrations
and sister chromatid
exchanges in exposed
groups.
Sarto et al. (1982)
Exposed: chromium
platers (n = 12)
Referents: controls
(n = 10)
Assessment: Based on occupation.
Levels: Cr(VI) exposure levels and blood
concentrations were not reported.
Duration: Exposure durations ranged from 0.5
to 18 yr (mean exposure duration was not
reported).
1" sister chromatid
exchanges in exposed group
Very small sample size; no
consideration of
confounders; no exposure
information on
participants.
Stella et al. (1982)
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Supplemental Information—Hexavalent Chromium
Study overview
Exposure3
Results
Comments
Reference
Results supported by in
vitro findings (human
lymphocytes cultured &
treated with Cr(VI) and
Cr(lll).
Exposed: chromium
workers (n = 35)
Referents: age and
gender-matched controls
(n = 35)
Assessment: Based on occupation.
Levels: Not reported.
Duration: Exposure duration ranged from 2 to
14 yr with a mean (SD) of 6.5 (4.2) yr.
1" sister chromatid
exchanges in exposed
group; association with
work duration; synergy with
smoking
1" high frequency cells in
exposed group; synergy
with smoking
No quantitative
assessment of exposure;
exposure based on work
only; limited sample size.
Only adjusted for
smoking, no other
confounding incorporated
into Cr analysis.
Wu et al. (2000)
Exposed: chromium
platers (n = 35)
Referents: healthy
subjects with no history
of disease or previous
exposure to chromium or
other metals (n = 35)
Assessment: Personal exposure monitoring
for 8 h working shift (1.71/min) on only 10
individuals in the exposed group.
Blood and urine samples collected at end of
shift and analyzed with atomic absorption
spectrophotometry.
Levels: Individual time-weighted average
range: 0.049-1.130 mg/m3.
Duration: The mean duration of employment
was 6.5 yr.
1" sister chromatid
exchange and percent high
frequency cells in exposed
group compared to controls
Personal air sampling only
obtained for n = 10
individuals in the exposed
group; SCE analysis
conducted based on work
group rather than
measured exposure level.
Unable to draw
conclusions about effect
of genotype due to small
sample size.
Wu et al. (2001)
Exposed: male welders
(n = 39)
Referents: matched
controls not substantially
exposed to carcinogens
(n = 39)
Assessment: Venous blood samples analyzed
with atomic absorption spectrometry.
Levels: Mean (SD) concentration of chromium
in exposed group erythrocytes: 4.3 (7.0) ng/L.
Duration: Not reported.
1" sister chromatid
exchange and DNA single
strand breaks in exposed
compared to controls
Only considered smoking
status, no other
covariates.
Possible confounding by
coexposure to other toxic
metals, such as nickel,
which was also measured
in this study.
Werfel et al. (1998)
aPotassium dichromate units conversion: Cr(VI) = 0.353 x K2Cr2C>7; potassium chromate units conversion: Cr(VI) = 0.268 x K2Cr04; sodium dichromate dihydrate
units conversion: Cr(VI) = 0.349 x Na2Cr20?2H20 (usually denoted as Na2Cr207 because study authors frequently list the salt as the chemical compound even if
concentration or dose is based on the dihydrate); chromium trioxide units conversion: Cr(VI) = 0.52 x CrC>3.
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1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
Supplemental Information—Hexavalent Chromium
Oral route of exposure
Mutagenic MOA studies
Studies considered most relevant to a mutagenic MOA analysis for cancer of the GI tract are
studies that measure gene mutation (prior to tumorigenesis), micro nuclei induction, and
chromosomal aberrations following oral exposures in experimental animals. This includes gavage
exposures with the acknowledgment that this dosing regimen condenses the exposure time,
inhibiting gastric reduction and potentially increasing Cr(VI) exposure. Human studies of
occupationally exposed workers that tested GI tissues (i.e., buccal cells from the oral cavity) were
also considered. Although these subjects were exposed via inhalation, this route of exposure is
presumed to be relevant to tissues in the oral cavity given exposure when breathing and via
mucociliary clearance.
No oral exposure studies in humans meeting these criteria were identified, but eight studies
reporting occupational measures of mutagenic biomarkers in buccal cells were identified; these
studies have already been summarized with the mutagenic MOA studies for inhalation exposures in
the preceding section. Eighteen studies in animals exposed via drinking water, diet, or gavage were
identified; some the findings reported in these studies are visualized in Figures C-21 to C-24. These
studies were evaluated in HAWC; the evaluations and the study findings are summarized in Tables
3-18 and 3-19 in Section 3.2.3.3 of the toxicological review.
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Supplemental Information—Hexavalent Chromium
40
35
30
25
Cr(VI) 20
i
15
10
5
0
mg/kg-d „
Aoki et al. (2019)
Male transgenic mice
28d 90d
Maximum:
0.7 mg/kg-d
40
35
30
25
20
15
10
5
0
Thompson et al. (2015)
Female B6C3FJ mice
_ Maximum:
0.45 mg/kg-d
ooooo
oooo
Mutation frequency
Duodenum Duodenum
No effect (5/group) No effect (4/group)
OOOOO
Multiple markers
Duodenum crypt
No effect (5/group)
O'Brien et al. (2013)
Female B6C3FJ mice
90d
"T" MN in duodenum villi but not crypt
(p<0.05, 5/group)
Figure C-21. Overview of selected studies evaluating mutagenic markers in the
gastrointestinal tract of mice following ad libitum drinking water exposure.
Full circle of a pie chart represents 2 years. Bar chart represents the maximum dose
range or the dose where an effect is first observed (whichever is lower). Full or
empty circles represent sample size per group (darkened if an effect was observed).
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—Hexavalent Chromium
IK
NTP {2007) genetic toxicology studies
(peripheral blood)
40
35
30
Cr(VI) 25
mg/kg-d 20
15
10
NTP (2007) study 1
Maximum dose:
27.9 mg/kg-d
Male B6C3F!:
OOOOO no effect (5/group)
ooooo
Female B6C3Ft:
no effect (5/group)
40
35
30
25
20
15
10
5
0
1
OOOOO
NTP (2007) study 2
maximum dose;
8,7 mg/kg-d
Male BALB/c:
no effect (5/group)
Male B6C3Ft:
equivocal
(p=0.031, 5/group)
Male am3-C57BL/6:
f %MN NCEs
(p<0.001, 10/group)
Figure C-22. Overview of the NTP f2007fl genetic toxicology (ad libitum
drinking water exposure). Full circle of a pie chart represents 2 years. Bar chart
represents the maximum dose range or the dose where an effect is first observed
(whichever is lower). Full or empty circles represent sample size per group
(darkened or shaded if an effect was observed).
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Supplemental Information—Hexavalent Chromium
20d
28d
90d
Typical sample
sizes: 5-10
ooooo
ooooo
(no effects)
Typical doses: up
to ~30 mg/kg-d
40
35
30
25
20
15
10
5
0
ooo
Aoki et al. (2019)
Thompson et al. (2015)
O'Brien et al. (2013)
OOOOO
ooooo
Drinking water
De Flora et al. (2008)
No effects (up to 165
mg/kg-d, n=10/group)
Gavage with short-term
follow-up (2 days)
De Flora et al. (2008)
BDF1 mice
Gavage
OOOOO
ooooo
No effects (up to 17.7
mg/kg, n=10/group) 10
5
o
t
Shindo et al. (1989)
MS/Ae and CD-I mice
Gavage and IP
No effects (up to 85.7 mg/kg,
n=3/group) OOO
Figure C-23. Overview of selected studies evaluating mutagenic markers (but
finding no effect) following ad libitum drinking water exposure (left) and oral
gavage (right). Full circle of a pie chart represents 2 years. Bar chart represents
the maximum dose range. Empty circles represent sample size per group.
Thompson et al. (2015)
Male transgenic rat
28d
/J \ Ad libitum drinking
/ I \ water exposure
40
35
30
25
20
15
10
0
I
5 —^— 1 dose group:
11 mg/kg-d
OOOOO
No effect in oral cavity
(5/group)
Figure C-24. Overview of the Thompson et al. (2015a) study evaluating
mutagenic markers in rats (but finding no effect) following ad libitum
drinking water exposure. Full circle of a pie chart represents 2 years. Bar chart
represents the maximum dose range. Empty circles represent sample size per
group.
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Supplemental Information—Hexavalent Chromium
1 Supporting genotoxicity studies in gastrointestinal tract tissue
2 In addition to the studies measuring gene and chromosomal mutation identified above,
3 mechanistic evidence of genotoxicity in GI tract tissues or in cells isolated from the GI tract were
4 identified in the preliminary title and abstract screening. These studies were tagged as
5 "mechanistic" and further screened and tagged as "GI tract" and "cancer" if they were studies in
6 humans or animals conducted in GI tissues or cells that were relevant to carcinogenic processes.
7 Seven genotoxicity studies of GI tissues in experimental animals and 10 studies in cells derived
8 from GI tissues were identified; no human studies were identified. This evidence is summarized in
9 Table C-50.
Table C-50. Supporting genotoxicity studies in gastrointestinal tract tissues
and cells following Cr(VI) exposures
System
Exposure3
Results
Reference
Mouse, B6C3F1,
female
Oral, drinking
water
0, 0.1, 1.4, 4.9, 20.9,
59.3, and 181 mg/L
Cr(VI)
0, 0.024, 0.32, 1.1, 4.6,
11.6, or 31.1 mg/kg-d
Cr(VI)
7 d (n = 5) or 90 d
(n = 10)
7 and 90 d:
No increases in 8-OHdG adducts in any tissues
Thompson et al.
(2011b)
Mouse, B6C3F1 &
rat, F344, female
Oral, drinking
water
0 and 180 mg/LCr(VI)
0 and 31.1 mg/kg-d
Cr(VI)
13 wk
yH2AX elevated in duodenal villi but not crypts
No aberrant foci indicative of transformation
Thompson et al.
(2015a)
Continued analysis
of tissues from
Thompson et al.
(2011b)
Mouse, B6C3F1
Oral, drinking
water
0, 1.4, 21, and 180 mg/L
Cr(VI)
0,0.32, 4.6, and 31.1
mg/kg-d Cr(VI)
7 d
No effect on yH2AX foci or on micronucleus
induction in crypt enterocytes
Thompson et al.
(2015b)
Mouse, SKH-1
hairless, female
Oral, drinking
water
0, 5, and 20 mg/L Cr(VI)
1.20 and 4.82 mg
Cr(VI)/kg-d
9 mo
No effect on DNA-protein crosslinks or
oxidative 8-OHdG adducts in forestomach,
glandular stomach, duodenal cells, lung or skin
No measure of cytotoxicity
De Flora et al.
(2008)
Mouse, C57BL/6J
Oral, drinking
water
0,0.019, 0.19, 1.9 mg/L
Cr(VI)
150 d
2 animals per dose
group
In proximal and distal sections of GI tract:
Immunohistochemistry: 1.5-fold increase in
yH2AX in distal sections
Sanchez-Martin et
al. (2015)
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Supplemental Information—Hexavalent Chromium
System
Exposure3
Results
Reference
Rat
Oral gavage
530 mg/kg-d Cr(VI), 3 d
106 mg/kg-d Cr(VI), 30 d
Note: The administered
gavage potassium
dichromate doses (1,500
mg/kg and 300 mg/kg)
are higher than the LDso
for rats listed in MSDS
(130 mg/kg)
Intestinal epithelial cells, 3-d exposure:
4/ glucose-6-phosphate dehydrogenase,
glutathione peroxidase, glutathione reductase,
glutathione-S-transferase, superoxide
dismutase and catalase
4/ glutathione and total thiols
1" lipid peroxidation
Intestinal epithelial cells, 30-d exposure:
1" superoxide dismutase, glutathione
peroxidase
Null glucose-6-phosphate dehydrogenase,
glutathione reductase and catalase
4/ glutathione-S-transferase
Sengupta et al.
(1990)
Mouse, ddY, 4 per
group
Oral gavage
0 or 85.7 mg/kg Cr(VI)
Single dose
p.o.: 1" DNA damage (comet assay) in stomach,
colon, and lung (also in brain, liver, kidney,
bladder, but not in bone marrow) in cells
collected 8 h after treatment
Effects subsided at 24 h in all dose groups
No clinical or microscopic signs of cytotoxicity
Sekihashi et al.
(2001)
In vitro human primary and immortalized Gl cells or gastric fluid
Human primary
lymphocytes and
gastric mucosal
cells
177 nM or 0.57 mM
Cr(VI)
1" DNA damage (comet assay) (p < 0.001)
Btasiak et al. (1999),
Trzeciak et al.
(2000)
Pre- and post-meal
gastric fluid
samples from
healthy volunteers
(n = 8)
0.021 mg/L Cr(VI)
4/ mutagenicity of Cr(VI) (assessed via Ames
reversion test) as a function of time in human
gastric juice
De Flora et al.
(2016)
Human gastric
cancer SGC-7901
cells
3.53 nM Cr(VI)
DNA damage (comet assay, yH2AX), oxidative
stress, apoptosis and necrosis all increased
when the Unconventional prefoldin RPB5
Interacting protein (URI) is knocked down
Luoetal. (2016)
Human primary
gastric and nasal
mucosa cells
0.087-0.349 nmoles/mL
Cr(VI)
1" DNA damage (comet assay) and cytotoxicity,
equal sensitivity in human and rat primary
gastric and nasal mucosal cells
Pool-Zobel et al.
(1994)
Human lung
epithelial A549
and colon HCT116
(MLH-/-) and DLD1
(MSH6-/-) cells
1-20 nM K2Cr04, 3-12 h
1" survival, 4^ apoptosis in mismatch repair
(MMR)-deficient cells
1" DNA DSBs (yH2AX) and apoptosis in MMR-
proficient cells
yH2AX foci occur in G2, but no G2 cell cycle
arrest
No p53 induction in either cell type at subtoxic
levels
MMR responsive to Cr-DNA adducts, not
oxidative damage or crosslinks
In MMR+ cells, apoptosis induced by Cr-DNA
adducts independently of p53
Peterson-Roth et al.
(2005); Zhitkovich
et al. (2006)
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Supplemental Information—Hexavalent Chromium
System
Exposure3
Results
Reference
Human colon
HCT116 (MLH1-/-)
and DLD1
(MSH6-/-), lung
epithelial H460,
and lung fibroblast
IMR90 cell lines
2-10 nmol/L K2Cr04,3 h
Ternary ascorbate-Cr-DNA adducts are
substrate for error-prone mismatch repair
(MMR) MSH2-MSH6 dimer, leading to ^ DNA
DSBs and T* apoptosis
Cells deficient in MMR have higher survival and
lower DNA DSBs
Colon cells deficient in MMR have increased
survival following Cr(VI) exposures, increasing
probability of clonal selection of these cells
Reynolds et al.
(2009)
Caco-2 human
colorectal
adenocarcinoma
cells
0.1, 0.3, 1, 3, 10, 30, 100
HM Cr(VI)
Increase in 8-OHdG at nontoxic and cytotoxic
concentrations, increase in yH2AX only at
cytotoxic concentrations (24 h)
No change in p53, annexin-V (apoptosis
markers), LC3B (autophagy marker)
Translocation of ATF6 to nucleus (ER stress
response marker)
Thompson et al.
(2012a)
Human SV40
transformed
fibroblasts,
Werner syndrome
fibroblasts,
primary human
lung IMR90
fibroblasts, and
and human colon
HCT116 MLH1-/-
and MLH1+ cells
0-30 nM K2Cr04,3 h
1" nuclear relocalization of WRN in response to
Cr(VI)
4/ cell survival, T* DNA DSBs and 4' RAD51 foci
in cells lacking WRN
4/ DNA DSBs in cells lacking mismatch repair
Error-prone mismatch repair of Cr-DNA adducts
generates DNA DSBs and repair of persistent
DNA DSBs is dependent on WRN helicase
Zecevic et al. (2009)
aPotassium dichromate units conversion: Cr(VI) = 0.353 x K2Cr2C>7; potassium chromate units conversion:
Cr(VI) = 0.268 x foCrCU; sodium dichromate dihydrate units conversion: Cr(VI) = 0.349 x Na2Cr2C>72H20 (usually
denoted as Na2Cr207 because study authors frequently list the salt as the chemical compound even if
concentration or dose is based on the dihydrate); chromium trioxide units conversion: Cr(VI) = 0.52 x CrC>3.
1 Supporting oral exposure genotoxicity studies
2 Besides the studies identified above that reported gene or chromosomal mutations or
3 measured genotoxicity endpoints directly in GI tissues, a small set of in vivo experimental animal
4 studies was identified that measured genotoxicity in tissues other than the GI tract following oral
5 exposures to Cr(VI). These studies identified in the preliminary title and abstract screening as
6 "mechanistic" were further screened and tagged as "oral exposure, "cancer," and "genotoxicity" if
7 they were in vivo oral exposure studies that measured genotoxicity endpoints. After removal of
8 endpoints already considered (see above), five genotoxicity studies in experimental animals were
9 identified; no human studies were identified. This evidence is summarized in Table C-51.
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Supplemental Information—Hexavalent Chromium
Table C-51. Supporting genotoxicity studies in animals exposed via the oral
route to Cr(VI)
System
Exposure3
Results
Comments
Reference
Rat, Fischer
344
Oral-drinking water, 0,
0.35, 1.77, 7.07 mg
Cr(VI)/L, 48 h
Comparison to single
gavage doses (20 mL/kg)
at same concentrations
No increase in
unscheduled DNA
synthesis in hepatocytes
collected from the rat
livers and analyzed in the
in vivo-in vitro hepatocyte
DNA repair assay
No measure of
cytotoxicity
RDS not determined
Mirsalis et al.
(1996)
Rat, Sprague-
Dawley
Oral-drinking water, 0,
10.6, 35.4, 106.1 mg/L
Cr(VI)
0, 2.49, 7.57, 21.41
mg/kg-d Cr(VI)
4 wk
In plasma: no change in
8-OHdG levels
1" MDA at two high
doses
4/ GSH-Px
4/ global DNA
methylation at two
high doses
No change in P16
methylation
Wang et al. (2015)
Mouse, ddY, 4
per group
Oral gavage, 0 or 85.7
mg/kg Cr(VI)
Single dose
Also i.p.: 0 or 32.1 mg
Cr(VI)/kg
p.o.: 1" DNA damage
(comet assay) in stomach,
colon, liver, kidney,
bladder, lung, and brain,
but not in bone marrow in
cells collected 8 h after
treatment
i.p.: 1" DNA damage
(comet assay) in stomach,
colon, and bladder (but
not in liver, kidney, lung,
brain, or bone marrow) at
8 h
Effects subsided at 24
h in all dose groups
No clinical or
microscopic signs of
cytotoxicity
Sekihashi et al.
(2001)
Mouse, Swiss
albino
Oral gavage, 0, 0.21,
0.42, 0.84, 1.68, 3.37,
6.7, 13.5, or 26.9 mg/kg
Cr(VI)
Single dose
1" DNA strand breaks
(comet assay) in
leukocytes at 24, 48, 72,
and 96 h and 1 and 2 wk
posttreatment
Dose-response from 0.59-
9.5 mg/kg. Peak response
at 48 h. No cytotoxicity
detected (trypan blue).
Dana Devi et al.
(2001)
Mouse, Swiss
albino
Oral gavage, 0, 8.8,17.7,
and 35.4 mg/kg Cr(VI)
Single dose or lx/d, 5 d
1" DNA damage (comet
assay) in lymphocytes
(statistically significant);
increasing with dose
Wang et al. (2006)
aPotassium dichromate units conversion: Cr(VI) = 0.353 x K2Cr2C>7; potassium chromate units conversion:
Cr(VI) = 0.268 x K2Cr04; sodium dichromate dihydrate units conversion: Cr(VI) = 0.349 x Na2Cr20?2H20 (usually
denoted as Na2Cr2C>7 because study authors frequently list the salt as the chemical compound even if
concentration or dose is based on the dihydrate); chromium trioxide units conversion: Cr(VI) = 0.52 x CrC>3.
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1 Injection studies
2 Supporting evidence of the genotoxic effects of Cr(VI) is provided by studies investigating
3 mechanisms of genotoxic effects by more direct routes of administration in vivo,
4 e.g., intraperitoneal (i.p.) injection. Twenty-three studies, summarized in Table C-52, were
5 identified that measured genotoxic endpoints in animals exposed to Cr(VI) via i.p. injection.
Table C-52. Genotoxicity studies in animals exposed to Cr(VI) via i.p. injection
System
Exposure3
Results
Comments
Reference
Gene and chromosome mutation
Mouse, CD-I,
male
i.p., 0, 20, 30,
40, and 50
mg/kg foCrCM
(0, 5.4, 10.6,
14.1, or 17.7
mg Cr(VI)/kg),
single dose
1" micronuclei in peripheral
blood reticulocytes
Awogi et al. (1992)
Mouse, BDFi,
male
Mouse, Swiss
albino,
pregnant
females
i.p., 0 or 50
mg/kg K2Cr2C>7,
24 h
1" micronuclei in bone marrow
of males or dams (p < 0.001) and
in peripheral blood and liver of
fetuses (p < 0.001)
No effect on
PCE/NCE ratios (no
cytotoxicity)
No effect on fetus
body weights
De Flora et al. (2006)
Mouse, MS and
ddY
i.p., 0, 12.5, 25,
or 50 mg/kg
K2Cr04 (0, 4.4,
8.8, or 17.7 mg
Cr(VI)/kg),
single dose
1" micronuclei in bone marrow
at 17.7 mg Cr(VI)/kg; statistically
significant trend
Cytotoxicity not
reported
Havashi et al. (1982)
Mouse, ddY,
male
i.p., 40 mg/kg
K2Cr04(14.1
mg Cr(VI)/kg),
single dose
In peripheral blood reticulocytes
sampled at 0, 24, 48, and 72 h
and hepatocytes at 5 d post-
partial hepatectomy:
1" micronucleus frequency
Igarashi and Shimada
(1997)
Mouse, Slc:ddY
i.p., 0, 30, 40,
and 50 mg/kg
K2Cr04 (0,
10.6,14.1, or
17.7 mg
Cr(VI)/kg),
lx/d,2 d
1" micronucleus frequency in
bone marrow cells; statistically
significant dose-response
%PCEs decreased at
two highest doses
Itoh and Shimada
(1996)
Mouse, lacZ
transgenic
(Muta Mouse)
i.p., 40 mg/kg
K2Cr04 (0 or
14.1 mg
Cr(VI)/kg),
lx/d, 2 d, or
single dose
sampled on d 1
and d 7
1" micronucleus frequency in
peripheral blood reticulocytes
1" mutant frequency in liver at
Id
1" mutant frequency in bone
marrow at 7 d
7 d postinjection is
too long to detect
MN in bone marrow
Cytotoxicity not
reported
Itoh and Shimada
(1997), Itoh and
Shimada (1998)
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System
Exposure3
Results
Comments
Reference
Mouse,
C57BL/6J/BOM,
female, mated
to T-stock male
i.p., 0, 10 or 20
mg/kg foCrCM
(0, 2.7, or 5.4
mg Cr(VI)/kg)
+ mouse spot test in offspring
Decline in number of
surviving offspring
with dose
Knudsen(1980)
Rat, white
outbred, male
i.p., 0 or 14 mg
K2Cr207/kg-
body mass,
single dose
(4.9 mg/kg
Cr(VI), 24 h
Rodent dominant lethal test:
dominant lethal mutation
frequency of 0.665 by comparing
the number of live fetuses in the
Cr(VI) treatment group to the
control group
Micronucleus test in bone
marrow
Also exposed via
gavage; was
evaluated in HAWC
for male repro and
mutagenic outcomes
Marat et al. (2018)
Rat, Wistar
i.p. 21 mg
K2Cr207/kg-
body mass,
single dose
(4.9 mg/kg
Cr(VI), 48 h
1" chromosomal aberrations in
the bone marrow but not in
lymphocytes unless dose
reached toxic levels
Newton and Lilly
(1986)
Mouse, CBA x
C57BI/6J hybrid
male
i.p., 0, 0.5,1.0,
2.0, 10, or 20
mg/kg K2Cr2C>7
(0, 0.18, 0.35,
0.70, 3.5, or
7.1 mg
Cr(VI)/kg),
single dose
1.p., 0,1.0, or
2.0 mg/kg
K2Cr207 (0,
0.35, 0.70 mg
Cr(VI)/kg),
lx/d, 21 d
Rodent dominant lethal test
Single dose: Statistically
significant decrease in embryo
survival at 7.1 mg Cr(VI)/kg
Repeat dose: Statistically
significant decrease in embryo
survival at 0.7 mg Cr(VI)/kg
Paschin et al. (1982)
i.p., 0, 1, 5, or
10 mg/kg
K2Cr207 (0.35,
1.77, or 3.54
mg Cr(VI)/kg),
single dose
1" micronucleus frequency in
bone marrow at 24, 48, or 72 h;
peak at 48 h
No measure of
cytotoxicity
Mouse, BALB/c,
C57BL/6, and
DBA/2
i.p., K2CrC>4
1" micronucleus frequency in
PCEs in all mouse strains
Sato et al. (1990)
Mouse, MS/Ae
and CD-I, male
i.p., 0, 10, 20,
40, or 80
mg/kg K2CrC>4
(0,3.5,7.1,
14.1, or 28.3
mg Cr(VI)/kg),
single dose
1" micronuclei in bone marrow
cells, dose-response
%PCEs decreased at
highest dose
Shindo et al. (1989)
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System
Exposure3
Results
Comments
Reference
Mouse, ddY,
CD-I, BDF1, and
ms, male
i.p., 0, 15, 30,
or 60 mg/kg
K2C1-O4, single
dose, 24 h
T* micronucleus frequency in
PCEs in all mouse strains
The Collaborative
Study Group for the
Micronucleus Test
(1988)
Mouse, NMRI
i.p., 0, 12.12,
24.25, or 48.5
mg/kg foCrCM
(0, 3.2, 6.49, or
13.0 mg
Cr(VI)/kg), 2
doses 24 h
apart
T* micronuclei in bone marrow
at 13 mg Cr(VI)/kg; statistically
significant trend
Cytotoxicity not
reported
Wild (1978)
Mouse, B6C3F1,
male,
8-10/group
i.p., 0, 0.51,
5.1, and 51.0
Hg Na2CrOVd,
4 wk
(5.5 x 10"5,
0.055, 0.55
mg/kg Cr(VI))
No significant increase in
micronucleated erythrocytes
(PCEs or NCEs) per 1,000 cells
analyzed from peripheral blood
collected at the end of the
treatment period.
Witt et al. (2000)
Mouse, BALB/c
i.p., 0 or 400
Hmol K2Cr2C>7
(20.8 mg
Cr(VI)/kg),
single dose
T* micronucleus frequency in
bone marrow cells (p < 0.001)
Significantly decreased %PCEs
(PCE/NCE ratio = 0.64 ±0.14)
(p < 0.01)
In liver:
T* lipid peroxidation
(p < 0.05)
T* heme oxygenase
(p < 0.001)
4/ GSH-peroxidase
activity (p < 0.1);
slight but
nonsignificant
reduction in GSH
levels
Wronska-Nofer et al.
(1999)
DNA damage
Mouse, BDF1,
female
i.p., 25mg/kg
Na2Cr2C>7-
acute;
12.5mg/kg-
subchronic,
single injection
for acute (1-14
d) or every 4
wk for 128 d
T* changes in ploidy in acute
group
N ranged from 3 to 5
per group. All
regions of liver
Garrison et al. (1990)
Rat, Sprague-
Dawley, male
i.p., 0, 2.5, 5.0,
7.5, and 10
mg/kg-d
K2Cr2C>7, 5 d
In peripheral blood lymphocytes:
T* DNA damage (comet assay)
In liver: ^ ROS,
MDA, SOD, CAT
activity
Patlolla et al. (2009b)
Mouse
i.p., l<2CrC>4
DNA damage (comet assay) in
liver, lung, kidney, spleen, and
bone marrow
Sasaki et al. (1997)
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System
Exposure3
Results
Comments
Reference
Rat, Sprague-
Dawley, male
i.p., 20 or 50
mg/kg-d
1 h: DNA-DNA and DNA-protein
crosslinks in liver, lung and
kidney
'Y DNA strand breaks in liver
36-40 h: DNA-protein crosslinks
in lung and kidney
Tsapakos et al.
(1981), Tsapakos et
al. (1983)
Mouse, albino
male
i.p., 0 or 20 mg
Cr(VI)/kg,
single dose
DNA damage (comet assay), 15
min post-injection (all back to
control levels at 3 h):
'Y liver, kidney
No increases in spleen, lung,
brain
Same pattern as
Cr(V) complexes
Cytotoxicity not
reported
DNA damage
reduced with
deferoxamine
Ueno et al. (2001)
aPotassium dichromate units conversion: Cr(VI) = 0.353 x K2Cr2C>7; potassium chromate units conversion:
Cr(VI) = 0.268 x foCrCU; sodium dichromate dihydrate units conversion: Cr(VI) = 0.349 x Na2Cr20?2H20 (usually
denoted as Na2Cr2C>7 because study authors frequently list the salt as the chemical compound even if
concentration or dose is based on the dihydrate); chromium trioxide units conversion: Cr(VI) = 0.52 x CrC>3.
Summary of supporting in vivo genotoxicity evidence
Human evidence
In addition to the studies of gene and chromosome mutation, other types of genotoxicity
studies conducted among humans exposed occupationally or environmentally to Cr(VI) are
considered supporting evidence for the ability of Cr(VI) to cause genetic damage in exposed
workers. These are biomonitoring DNA damage assays conducted on peripheral blood that
measure DNA strand breaks, adducts, crosslinks, or other DNA damage and repair-related
endpoints (e.g., sister chromatid exchange). These studies are summarized in Table C-49. They did
not undergo formal study evaluation unless they included other endpoints that met the mutagenic
prioritization criteria.
DNA damage in exposed humans
Seven of eight studies of exposed chromium industry workers detected significant increases
in DNA strand breaks in peripheral blood using the comet assay; seven of these studies also
confirmed exposures by detecting higher Cr levels in air and/or biomarkers compared to referents
(Wang etal.. 2012: Sudha etal.. 2011: Zhang etal.. 2011: Balachandar etal.. 2010: Iarmarcovai etal..
2005: Danadevi etal.. 2004: Gambelunghe etal.. 2003: Gao etal.. 19941. These tests provide
supporting evidence for increased genetic damage following Cr(VI) exposure, though they do not
anticipate the proportion of DNA strand breaks that could lead to mutation. Five studies evaluated
DNA-protein crosslinks, which are considered biomarkers for the genotoxic effects of Cr(VI)
exposure in humans (Zhitkovich. 20051. Four of these studies documented increases among
exposed groups compared to controls (Medeiros etal.. 2003: Ouievrvn etal.. 2001: Taioli etal..
1995: Costa etal.. 19931. The fifth study did not document clear differences between exposed and
controls but did identify positive associations between DNA-protein crosslinks and chromium in
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erythrocytes at low and medium exposure levels, with a saturation of crosslink incidence at higher
levels fZhitkovich etal.. 19961. Fifteen studies evaluated sister chromatid exchange (SCE).
Elevated levels of SCEs following exposures are indicative of increased DNA repair and are
considered biomarkers of exposure to potential genotoxic agents but do not correlate well with
mutation frequency (Eastmond. 2014). Among these, six studies documented increased SCEs per
cell among exposed groups of welders (Werfel etal.. 19981 or electroplating workers fWu etal..
2001: Wu etal.. 2000: Lai etal.. 1998: Deng etal.. 1988: Stella etal.. 19821 compared to control
groups. Similarly, one study documented an association between urinary chromium and SCE fSarto
etal.. 19821. Seven studies did not observe impacts on SCEs, either through comparing exposed
and control groups (Benova etal.. 2002: Nagava. 1986: Koshi etal.. 1984: Littorin etal.. 1983:
Husgafvel-Pursiainen etal.. 19821 and/or through evaluating the association with urinary
chromium directly (Nagava etal.. 1991: Nagava etal.. 1989: Nagava. 19861. One study documented
a decrease in SCE frequency among welders compared to controls, though the authors noted
concerns with the alkaline filter elution that may have impacted the validity of the results fPopp et
al.. 19911.
Target tissue analyses of genotoxicity
A small number of studies conducting analyses of genotoxicity in human gastric fluid or
primary human GI or lung cells were also identified. In a gastric reduction capacity experiment
using pre- and post-meal gastric fluid samples from healthy volunteers (n = 8), higher reducing
capacity and significantly decreased mutagenicity (evaluated by the Ames assay) were observed in
post-meal samples compared to pre-meal samples. A 70% total Cr(VI) reduction was observed
within 1 minute with a 98% reduction by 30 minutes fDe Flora et al.. 20161. Because gastric
emptying occurs in vivo (reduction and emptying are competitive processes), a fraction of ingested
Cr(VI) will empty to the small intestine prior to reduction (see Section 3.1 of the toxicological
review and Appendix C.l). In a study of lung reduction capacity by the same group, the S-9 fraction
from pulmonary alveolar macrophages (PAM) isolated from the lung of human subjects (n = 47)
was capable of lowering Cr(VI)-induced mutagenicity in the Ames assay by approximately 25%
when preincubated for 1 hour prior to plating (Petrilli etal.. 19861. Similar results were obtained
by the S-12 fractions of peripheral lung parenchyma isolated from healthy subjects and from
patients with lung cancer on the mutagenicity of Cr(VI) in the Ames assay; samples from smokers
had a significantly higher ability to reduce Cr(VI) (De Flora et al.. 1987b).
Pool-Zobel etal. (1994) performed the comet assay for measuring DNA strand breaks on
human mucosal cells from macroscopically healthy tissues of patients collected during biopsy
treated with 0.087-0.349 [imoles/mL Cr(VI) in vitro. The results showed genotoxicity occurring at
non-cytotoxic doses, with responses in the cells from humans paralleling those of cells from SD rats
(see DNA damage section in synthesis of animal genotoxicity evidence). Similarly, a separate group
reported statistically significant increases in DNA damage using the comet assay in two studies of
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human primary gastric mucosal cells exposed to concentrations >177 |iM Cr(VI), which underwent
repair within an hour f Trzeciak et al.. 2000: Btasiaketal.. 19991.
Tumor senotvpins
The study of mutations occurring in oncogenes or tumor suppressor genes in tumor tissues
can help identify chemical-specific driver mutations that could be key for tumor progression, as
well as signature mutations that can potentially establish a causal association between chemical
exposure and tumors. One study, Alguacil etal. (20031. evaluated mutations in the KRAS oncogene
in tumor tissues, comparing pancreatic cancer cases with and without KRAS mutated tumors in
individuals with inhalation exposure to chromium (ascertained using occupational history and a
job-exposure matrix). The exposed workers with pancreatic tumors had increased odds of KRAS
mutations in these tumors. Study authors also documented an increased proportion of G-to-T
transversions with inhalation exposure to chromium. However, very few individuals were
identified as having occupational chromium exposure, resulting in wide confidence intervals
around the effect estimates (Alguacil etal.. 20031. In addition, because pancreatic tumors have not
been associated with occupational Cr(VI) exposure, and nearly 100% of pancreatic tumors
(pancreatic ductal adenocarcinomas) have mutations in the KRAS gene (Waters and Per. 2018). this
evidence may have little biological relevance to Cr(VI)-induced cancer.
Three studies evaluated p53 mutations among chromate factory workers with lung cancer,
comparing cases with and without chromium exposure. Kondo etal. f 19971 identified fewer p53
mutations among chromate workers. Yet, study authors also identified specific patterns of p53
mutations among lung cancer cases with prior chromate exposure, including double missense
mutations. However, lack of adjustment for confounding and small sample size limit confidence in
these findings (Kondo etal.. 1997). Similarly, Katabami et al. (2000) detected an upregulation in
cyclin D1 protein expression but no differences in p53 or bcl-2 protein expression in lung cancer
tissues from chromate-exposed patients compared to non-exposed or pneumoconiosis lung cancer
patients, though this study also had a small sample size and only considered confounding due to
smoking status. Cyclin D1 is involved in the regulation of cell cycle progression and is elevated in a
number of human cancers (Alao. 2007). and when paired with the absence of a protective p53-
induced apoptotic response, may indicate a factor in Cr(VI)-induced cancer development. The third
study, Halasova etal. (2010). determined that expression of the apoptosis inhibitor survivin protein
was decreased, concomitant with an increase in pro-apoptotic p53 levels, in former chromium
workers with lung cancer compared to control lung cancer patients. However, little information
was given regarding the potential exposures of these workers, and no information on confounders
including smoking status was included. Although this finding is not surprising given these
interconnected pathways of cell fate determination, the potential for co-exposures and
co-morbidities precludes the ability to draw conclusions from these findings.
Overall, specific driver mutations or mutational signatures considered to be specific to
Cr(VI) exposure have not been identified in exposed humans. However, there is evidence that
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critical human cancer effector pathways are directly and indirectly impacted after Cr(VI) exposure.
Cr-DNA adducts, well established to occur in controlled conditions in cell cultures and acellular test
systems in vitro (see Section 3.2.3.4 of the toxicological review and Appendix C.3.2.1 for a broader
discussion of Cr-DNA adduct formation), could potentially provide additional support connecting
exposure to genotoxic chemicals with effect. However, due to their transient nature, they do not
appear to have the potential to be used as biomarkers of genotoxicity following Cr(VI) exposure in
humans; accordingly, no evidence of the recovery of Cr-DNA adducts has been identified in Cr(VI)-
exposed humans or animals.
Animal evidence
DNA damage
Genotoxicity endpoints that did not meet the mutagenicity prioritization criteria have also
been reported in animal studies. These include measures of DNA damage that may not reflect
actual mutation frequency, as well as studies using less relevant routes of exposure (i.e., i.p.
injection studies).
Only one animal study was identified that reported DNA damage measures following direct
exposure to the lung. Gao etal. (1992) exposed Wistar rats to 0.45 and 0.87 mg/kg Cr(VI) via
intratracheal instillation and detected a significant increase of DNA strand breaks in peripheral
lymphocytes after 24 hours. Several drinking water exposure studies were identified that reported
mostly negative findings for DNA damage. Thompson et al. (2015b; 2015a) conducted
immunohistochemical staining for phosphorylated histone H2AX (yH2AX), a marker of DNA
double-strand breaks, in the intestinal villi and crypts of mice after oral exposure.
Immunohistochemical grading reported moderate staining in the crypts that was not treatment-
related, and moderate staining in the villi after exposure to 31 mg/kg Cr(VI)-day (high dose) after
13 weeks (Thompson etal.. 2015a). A 7-day follow-up study by the same group also reported no
treatment-related increase in yH2AX foci in the crypts, although these results may have biased
toward the null due to the 24 hour recovery period given the potentially rapid disappearance of
yH2AX (Thompson et al.. 2015b). Another group reported a 1.5-fold increase in yH2AX in the
'distal section' of the GI tract in C57BL/6J mice exposed to up to 1.9 mg/L Cr(VI) in drinking water
for 150 days, although the low number of animals studied (2/group) make these findings less
informative (Sanchez-Martin etal.. 2015). A separate genotoxicity study reported no evidence of
DNA-protein crosslinks in GI tissues (forestomach, glandular stomach, and duodenum) of female
SKH-1 mice after 9 months of low dose oral exposure to 1.20 and 4.82 mg Cr(VI)/kg-day through
drinking water fDe Flora etal.. 20081.
Three studies in mice administering Cr(VI) via gavage reported significant, dose-dependent
increases in DNA damage, measured by the comet assay, in multiple tissues, including lymphocytes
(Wang etal.. 2006). leukocytes (Dana Devi etal.. 2001). stomach, colon, liver, kidney, bladder, lung,
and brain (Sekihashi etal.. 2001). Single, bolus gavage doses greatly condense the exposure time,
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inhibiting gastric reduction (ad libitum drinking water exposures are distributed over a 24-hour
period, whereas gavage occurs over a very short period). This difference in pharmacokinetics could
potentially explain the difference in genotoxicity results between gavage and drinking water
observations. The only tissue Sekihashi etal. (20011 tested that did not find an increase in DNA
damage was the bone marrow, and no indications of cytotoxicity were observed in the animals,
indicating that Cr(VI) did not reach the bone marrow at sufficient concentrations to induce DNA
damage (Dana Devi etal.. 2001: Sekihashi etal.. 20011.
Similarly, studies in rats and mice uniformly indicate Cr(VI) can cause gene and
chromosomal mutations and DNA damage when injected intraperitoneally (i.p.); these are
summarized in Table C-52. While less informative for GI tract cancers, intraperitoneal dosing
experiments are considered supplemental to oral dosing studies in providing mechanistic evidence
to inform mutagenic and genotoxic effects. Dosing via i.p. injection results in higher systemic tissue
concentrations of Cr(VI) compared to oral and inhalation exposure because this route bypasses
Cr(VI) reduction mechanisms that would otherwise dampen systemic Cr(VI) distribution and
absorption (see Section 3.1 of the toxicological review and Appendix C.l). Systemic effects are
more likely following i.p. injection compared to oral exposure. However, some mechanistic studies
aim to examine the effects of Cr(VI) on target tissues, irrespective of route, and i.p. injections may
be the only feasible method to expose some systemic target organs to carefully controlled and
consistent concentrations of Cr(VI).
Although in vitro studies of human cells were prioritized over other mammalian cells, Pool-
Zobel etal. (19941 compared responses from both human and rat cells. This study performed the
comet assay for measuring DNA strand breaks on human and rat gastric mucosal cells from
macroscopically healthy tissues of patients collected during biopsy or from Sprague-Dawley rats
treated with 0.087-0.349 [imoles/mL Cr(VI) in vitro. The results showed genotoxicity occurring at
non-cytotoxic doses, with responses in the cells from SD rats paralleling those from human cells,
providing some evidence of species concordance for genotoxicity induced by Cr(VI).
Signature mutations
Other investigations of specific Cr(VI)-induced mutations that may be relevant to GI
carcinogenesis have been reported. An analysis of the specific types of point mutations induced by
a chemical can determine whether, compared to spontaneous mutations, certain mutations are
more associated with exposures, i.e., signature mutations. Chemical-specific mutational signatures
can potentially establish an association between chemical exposure and mutation, as well as
lending mechanistic insight into the types of DNA damage most associated with the specific
mutation. In addition to analyzing mutation frequency, two studies examined specific types of
point mutations in the mouse small intestine after 28 or 90 days of exposure. G:C to T:A
transversions, mutations that frequently result from the DNA damage associated with oxidative
stress, were observed to occur at a slightly higher frequency (11%) in the lung of the Cr(VI)-treated
transgenic mice (6.75 mg/kg, intratracheal instillation) (Cheng etal. (2000: 199811. consistent with
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Supplemental Information—Hexavalent Chromium
in vitro findings by this group fLiu etal.. 19991. The G:C to T:A transversions correlated with
glutathione levels, presumably because the antioxidant is reducing higher levels of intracellular
Cr(VI) and thus increasing reactive oxygen species generation.
In another study in transgenic mice, an increase in G:C to T:A transversions was not
observed in mutations recovered from the duodenum in animals exposed to Cr(VI) in drinking
water (Aoki etal.. 2019). This study did, however, detect a higher rate of A:T to T :A transversions
in the Cr(VI)-exposed animals at 28 days that was not detectable at 90 days; the significance of this
mutation in relation to Cr(VI) is not known, but it indicates a potential signature mutation that
could be investigated further. The Cheng et al. (2000; 19981 study reported a higher frequency of
all mutation types in Cr(VI)-exposed animal lung tissue compared to controls, whereas the Aoki et
al. (2019) study did not detect an increase in mutations over background in the duodenum.
Although the study did not conduct additional testing to determine whether this difference is
attributable to a lack of oxidative DNA damage (and subsequent G:C to T:A transversions) in the
animals in the Aoki etal. (2019) study, it is possible that mutations related to oxidative damage are
more likely to be induced in a single high intratracheal instillation exposure (6.75 mg/kg Cr(VI)) in
Cheng et al. (2000; 19981. compared to a longer, lower dose exposure period (up to 0.7 mg/kg-d for
28 days or 0.45 mg/kg-d for 90 days, drinking water) used by Aoki etal. (2019). Some consistency
in results is noted by the finding that both studies reported that a high proportion of spontaneous
mutations were G:C to A:T transitions. Overall, there is not enough evidence to conclude that there
may be a signature mutation associated with Cr(VI) exposure.
In vitro studies
In vitro investigations of the mechanisms of genotoxicity induced by Cr(VI) can provide
support to observations in vivo. In general, if a study was conducted only in human primary cells or
cell lines derived from a specific tissue (e.g., lung, GI tissues, liver), the genotoxicity evidence is
summarized in those sections and not repeated here.
Table C-53. In vitro genotoxicity studies in human cells
System
Exposure3
Results
Comments
Reference
Gene and chromosome mutation
WIL2-NS human
B lymphoblastoid
cells
0, 0.01, 0.10,
1.00, 10, 100,
and 1000 nM
K2Cr207, 48 h
1" micronuclei, all
concentrations (p < 0.001)
1" necrotic cells >100 nM
4/ nuclear division index
Folate deficiency
increased DNA damage
Alimba et al.
(2016)
Primary human
lymphocytes
from four donors
0, 0.001, 0.01,
0.1, and 0.25
Hg/ml_
K2Cr207, 48 h
1" chromosomal aberrations,
all concentrations (p < 0.05)
1" micronuclei, all
concentrations (p < 0.05)
Significant increases in
chromosomal
mutations occurring at
noncytotoxic
concentrations
Botta et al. (1996)
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Supplemental Information—Hexavalent Chromium
System
Exposure3
Results
Comments
Reference
(0, 0.35, 3.54,
35.4, and 88.4
ng/mL Cr(VI))
4/ mitotic index with dose;
cytotoxic dose (50% decrease)
estimated to be 0.15 ng/mL
TK6 human
lymphoblastoid
cells
5 nM K2Cr2C>7,
5 h
"Hotspot" mutations at the
hprt gene (6-thioguanine
resistant):
C:G->A:T transversion, bp 243
(4.5%)
A:T->T:A transversion, bp 247
(2%)
G:C->A:T transition, bp 289
(2.5%)
C:G->T:A transition, bp 312
(4%)
Hprt bp 243 is hotspot
for H2O2 (G:C->C:G
transversion) and BaP
Hprt bp 247 is hotspot
for X-rays (A:T bp
deletion)
Overall, little overlap
between Cr(VI)
mutation spectra and
that of oxidative DNA
damaging agents
Chen et al. (1994a,
b)
HeLa cells
1,10, and 100
HM Na2Cr2C>7;
1, 2.5, 8, 24, or
48 h
Mutation spectra: Single-base
substitutions at G/C
predominant
More transversions and fewer
transitions compared to
spontaneous
Intracellular Cr(lll)
inhibits DNA synthesis
and replication fidelity
by inhibiting DNA
synthesome
polymerases a, 5, and £
Dai et al. (2009)
Human dermal
fibroblasts
1-6 or 200 nM
Na2CrC>4, 6 h
1" DNA DSBs (neutral comet
assay; yH2AX foci) only in
PCNA-positive cells that were
ATM+/+
Low cytotoxicity and ROS
generation detected previously
Cr(VI) exposure
generates S-phase
dependent DNA DSBs
that activate ATM
kinase
Ha et al. (2004)
HeLa and human
lung bronchial
epithelial cells
0.25 nM
Na2CrC>4, 30 d,
or 10 nM, 16
or 48 h
1" chromosomal aberrations
with acute or chronic
exposures
Chromosomal
instability caused in
part by suppressed
activation of BubRl
and expression of Emil,
causing activation of
APC/C, following
nocodazole-induced
mitotic arrest
activation
Hu et al. (2011)
DNA damage
TK6 human
lymphoblastoid
cells
0.2-1 mM
CrCU and
Na2CrC>4
1" DNA strand breaks (comet
assay); associated with
oxidized base damage as
measured by FPG and Endolll
addition
Cr(VI) delayed IR-
induced DNA damage
repair
El-Yamani et al.
(2011)
Human
fibroblast strains
CRL 1187,
XP12BE
(CRL1223) and
0, 2, 5, 50 nM
K2Cr04, 4 h
1" DNA single-strand breaks
induced in cells both deficient
and proficient in excision
repair
Other repair
mechanisms involved
in repair of DNASSBs
Fornace (1982)
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System
Exposure3
Results
Comments
Reference
XP25RO (CRL
1261)
H460 human
lung epithelial
cells, IMR90
normal human
lung fibroblasts,
and normal
mouse
embryonic
fibroblasts
0, 5, 10, 15,
and 20 nM
K2Cr04
DNA damage response to
Cr(VI)-induced DNA double-
strand breaks (yH2AX foci)
dependent on ATR kinase and
not ATM in ascorbate-restored
cells
DNA DSBs only formed in
euchromatin
Involvement of ATR
and DSBs forming in
actively transcribed
regions increases the
probability that Cr(VI)
can generate
carcinogenic mutations
Delougherv et al.
(2015)
Human U20S
osteosarcoma
cells, Werner
syndrome skin
fibroblasts
(AG03141),
WI-38 fetal lung
fibroblasts,
telomerase-
immortalized cell
lines (hTERT
GM01604,
(hTERT
AMIE15010,
AG03141, hTERT
BJ skin
fibroblasts)
0-4 nM Cr(VI),
6-48 h
T* yH2AX foci in S-phase
T* WRN colocalization at
yH2AX foci
T* telomere defects
exacerbated by lack of
telomerase
Lack of WRN slowed Cr(VI)-
induced DNA DSB repair
Cr(VI) induces DNA
DSBs and stalled
replication forks; WRN
helicase plays a role in
the cellular recovery
from Cr(VI)-induced
replicative stress
Liu et al. (2010a,
2009)
Wild-type and
pol zeta mutated
D2781Nand
L2618M human
B-cell leukemia
cell line
NazCrzCband
KBrOs
Increased sensitivity to DNA
damage (micronuclei, SCE) in
cells with weaker variants of
DNA polymerase zeta
Increased susceptibility
to Cr(VI)-induced
mutations in variants of
DNA replication
enzymes
Suzuki et al. (2018)
Human TK6
lymphoblastoid,
HeLa cervical
carcinoma
epithelial, and
293T kidney
epithelial cells
1-2000 ng/L
K2Cr04, 10
min-14 d
Cytotoxicity > 373 ng K2Cr04/L
(=100 ng Cr/L) with survival
rate of 50%, 17%, and 10% for
HeLa cells, 293T and TK6 cells,
respectively
Trace amounts (>9.8 ng/L) of
Cr(VI) initiate DNA damage
response and genotoxicity that
increases with time and dose
Primary Cr(VI)-induced
DNA damage response
pathways are error-
free HR and error-
prone TLS pathways
Tian etal. (2016)
Human SV40
transformed
fibroblasts,
Werner
syndrome
fibroblasts,
primary human
lung IMR90
0-30 nM
K2Cr04,3 h
T* nuclear relocalization of
WRN in response to Cr(VI)
4/ cell survival, T* DNA DSBs
and 4/ RAD51foci in cells
lacking WRN
4/ DNA DSBs in cells lacking
mismatch repair
Error-prone mismatch
repair of Cr-DNA
adducts generates DNA
DSBs and repair of
persistent DNA DSBs is
dependent on WRN
helicase
Zecevic et al.
(2009)
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Supplemental Information—Hexavalent Chromium
System
Exposure3
Results
Comments
Reference
fibroblasts, and
human colon
HCT116 MLH1-/-
and MLH1+ cells
GM03714A,
GM0131B, and
GM0922B
human
lymphoblastic
cell lines
l<2CrC>4 and
51Cr042"
0, 20, 50, 100,
150, and 200
HM, 3, 6, or 12
h
Differences in cytotoxicity and
DNA damage in response to
Cr(VI) due to differences in
rate of uptake of Cr(VI) among
3 individual cell lines
Zhang et al. (2002)
aPotassium dichromate units conversion: Cr(VI) = 0.353 x K2Cr2C>7; potassium chromate units conversion:
Cr(VI) = 0.268 x faCrCU; sodium dichromate dihydrate units conversion: Cr(VI) = 0.349 x Na2Cr20?2H20 (usually
denoted as Na2Cr207 because study authors frequently list the salt as the chemical compound even if
concentration or dose is based on the dihydrate); chromium trioxide units conversion: Cr(VI) = 0.52 x CrC>3.
C.3.2.3. Alters DNA repair or causes genomic instability (KC#3)
1 Mechanistic studies relevant to detecting Cr(VI)-induced suppression of DNA repair
2 processes (except for those caused by Cr(VI)-induced epigenetic modifications) or genomic
3 instability resulting from Cr(VI) exposure have been summarized in Table C-54.
Table C-54. Mechanistic studies relevant to altered DNA repair or genomic
instability induced by Cr(VI) exposure
Study overview
Exposure3
Results
Comments
Reference
Effects on DNA repair
Exposed: chromate
workers (n = 87)
Referents:
employees with no
direct contact with
chromium
products
(e.g., managers,
officers, support
crew) (n = 30)
Exclusions: cancer,
cardiovascular
disease, kidney
disease,
pulmonary disease
Workers exposed to
chromate by
inhalation for ~5.0 yr
(IQR: 3.0-10.0 yr)
Postshift fasting
blood samples
collected;
measurement with
ICP-MS. Mean (SD)
blood Cr in exposed
group: 14.5 (33)
ng/mL
ELISA of DNA repair-related
genes POLBeta, ASCC3, BRCC3,
and XRCC2
XRCC2 and BRCC3 protein levels
were statistically associated
with miR-3940-5p levels
Main limitations are
related to lack of
description (e.g., for
participant
selection)
Li et al. (2014b)
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Supplemental Information—Hexavalent Chromium
Study overview
Exposure3
Results
Comments
Reference
Exposed: females
working in the
chromium
industry;
subgroups based
on years of
contact with
chromium (1-2; 3-
5; 7-10; 15+)
(n = 66)
Referents: females
with no contact
with the
chromium industry
(n = 15)
Exposure based on
years working in the
chromium industry
(1-2; 3-5; 7-10;
15+) yr
4/ DNA repair synthesis in
lymphocytes in exposed group;
nonlinear relationship with
duration of contact with
chromium
Limited sample size
within each exposed
group when
analyzed by duration
(1-2 yr: n = 13; 3-5
yr: n = 15; 7-10 yr:
n = 21; 15+ yr:
n = 17)
Rudnvkh and
Zasukhina (1985)
hTERT
immortalized
clonal cell line
derived from
human bronchial
fibroblasts
(WTHBF-6)
0.1-0.3 ng/cm2 zinc
chromate, 24, 72,
and 120 h
After 120 h, but not 24 h, Cr(VI)
induced dose-dependent
decreases in nuclear Rad51,
inhibition of the nuclear import
of Rad51C and BRCA2,
inhibition of Rad51
nucleofilaments, and complete
blocking of homologous
recombination repair (HR)
Prolonged exposure
to Cr(VI) suppresses
HR, increasing
reliance on error-
prone DNA DSB
repair pathways and
the potential for
mutation
Browning et al.
(2017; 2016)
WTHBF-6 human
bronchial
fibroblasts
0.1, 0.15, and 0.2
Hg/cm2 zinc
chromate (0.12,
0.18, and 0.24 ppm),
24, 48, 72, 96, and
120 h
Time-dependent increases in
DNA damage and DNA DSB
signaling, decreases in Rad51
foci formation
Cr(VI)-induced suppression of
E2F1 transcription factor for
Rad51 is involved
Qin et al. (2014),
Speer et al.
(2021)
Aneuploidy and genomic instability
Primary human
fibroblasts
0, 2, 20, and 40 ng/L
(0.01, 0.102, and
0.205 nM) K2Cr04,
24 h
Using 24 color M-FISH:
T* chromosomal aberrations
(structural and numerical),
dose-dependent
Simple and complex aneuploidy
was observed at all doses,
dose-dependent
Slowly resolved with
time up to 30 d
postexposure
Figgitt et al.
(2010)
BJ normal human
foreskin
fibroblasts, hTERT
+ and -
0.04, 0.4, and 4 mM
Cr (VI) (K2Cr207), 24
h
In hTERT-deficient cells, 30 d
postexposure:
Persistent induction of
dicentrics, nucleoplasms
bridges, micronuclei and
aneuploidy
4/ clonogenic survival
T* (5-gal staining and apoptosis
Cr(VI) induced
persistent genomic
instability
Telomerase-positive
cells were not
affected except for
persistent
tetraploidy
Glaviano et al.
(2006)
Human MRC-5
cells
0, 0.25, 0.5,1, 2, and
4 nM K2Cr207, 30 h
T* kinetochore-positive
micronuclei
Authors determined
aneuploidy caused
by malsegregation at
Giierci et al.
(2000), Seoane et
al. (2002; 2001,
1999)
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Supplemental Information—Hexavalent Chromium
Study overview
Exposure3
Results
Comments
Reference
anaphase and not by
nondisjunction
WTHBF-6 human
bronchial
fibroblasts
0.5 and 1 ng/cm2
lead chromate
24 h exposure: no effect
120 h exposure: T* aneuploidy;
associated with centrosome
amplification
Lead oxide had no
effect
Holmes et al.
(2006)
WTHBF-6 human
bronchial
fibroblasts
0.1, 0.15, and 0.2
Hg/cm2 zinc
chromate (0.12,
0.18, and 0.24 ppm),
24, 72, and 120 h
1" centrosome amplification
1" aneuploidy
Premature centriole
disengagement in S and G2, and
premature centrosome
separation in interphase
Martino et al.
(2015)
Primary human
skin fibroblasts
0.01-100 nM
Na2CrC>4 and 0.001-
10 nM CaCrC>4
1" aberrant mitotic spindles
and cell division patterns, dose-
dependent
Niis and Kirsch-
Volders (1986)
Primary human
peripheral blood
lymphocytes
0.00476 nM and
0.00952 nM K2Cr207
1" aneuploidy, dose-dependent
1" SCEs, dose-dependent
No change in cell cycle
proliferative index
Aneuploidy and DNA
repair initiated at
very low subtoxic
concentrations
Rao et al. (1999)
BEAS-2B human
bronchial
epithelial cells
1 nM K2Cr207
1" aneuploidy
Subclones induced tumors
when injected into nude mice
No microsatellite instability in
aneuploid cells; DNA MMR and
MLH1 expression was
unaffected
Rodrigues et al.
(2009)
WTHBF-6 human
bronchial
fibroblasts
0.5 and 1 ng/cm2
lead chromate; 72,
96, and 120 h
1" spindle assembly checkpoint
bypass (centromere spreading,
premature centromere division
and anaphase, and 4, MAD2
levels)
No effect with lead
glutamate
Wise et al. (2006)
Human primary
and immortalized
urothelial cells
expressing hTERT
(hTUl cells)
1-5 nM NaCr04
1" aneuploidy and
chromosomal damage in
chronic (not acute) incubations
in primary and hTERT-
immortalized human urothelial
cells, dose- and time-
dependent
Solid-stain
chromosomal
analysis could be
prone to false
positive
Wise et al. (2016)
aPotassium dichromate units conversion: Cr(VI) = 0.353 x K2Cr2C>7; potassium chromate units conversion:
Cr(VI) = 0.268 x foCrCU; sodium dichromate dihydrate units conversion: Cr(VI) = 0.349 x Na2Cr20?2H20 (usually
denoted as Na2Cr207 because study authors frequently list the salt as the chemical compound even if
concentration or dose is based on the dihydrate); chromium trioxide units conversion: Cr(VI) = 0.52 x CrC>3.
C.3.2.4. Induces epigenetic alterations (KC#4)
1 Seven studies in humans occupationally exposed to Cr(VI) were identified that evaluated
2 epigenetic alterations in relation to Cr(VI) exposure and mechanistic or apical outcomes, including
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changes in microRNA levels, global methylation changes, and the methylation of specific genes. The
study findings are summarized in Table C-55.
Table C-55. Studies of epigenetic alterations in humans, experimental animals,
and human cells in vitro exposed to Cr(VI)
Study overview
Exposure3
Results
Comments
Reference
Exposed: lung tumor
samples from
chromate workers with
lung cancer during
surgery or autopsy
(n = 36)
Referents: lung tumor
samples from lung
cancer patients
without chromate
exposure (n = 25)
Exposure intensity
ascertained based on
work period in
chromate industry.
Mean (range) of
exposure to
chromate =
22.61 (12-38) yr
1" methylation of CpG
sites at APC, MGMT,
and hMLHl genes in
chromate lung cancer
cases compared to lung
cancer referents
Limited description
of selection; no
consideration of
confounders; no
confirmation of lack
of exposure in
referent group.
AN et al. (2011)
Exposed: factory
workers with
occupational exposure
to chromate (n = 87)
Referents:
administrative workers
from the same factory,
without chromate
exposure (n = 30)
Exclusions: skin
infection; cancer;
cardiovascular disease;
kidney disease;
pulmonary disease;
history of allergy,
asthma, or allergic
rhinitis
Air samples collected
at 10 locations for 8 h
during regular working
hours (flow rate:
lL/min); measurement
with atomic absorption
spectrometry. Median
(IQR) air chromium in
exposed group = 15.5
(19.0) ng/m3; referent
group = 0.2 (0.4) ng/m3
Peripheral venous
blood collected after
work shift; chromium
measured by ICP-MS.
Mean (IQR) blood
chromium in exposed
group = 6.4 (7.2) ng/L;
referent
group = 3.9(1.5) ng/L
1" methylation of CpG
sites at DNA repair
genes (MGMT, HOGG1,
XRCC1, ERCC3, and
RAD51) in exposed
groups
Main limitations are
related to lack of
description (e.g., for
participant
selection).
Simultaneous in vitro
work demonstrated
hypermethylation in
human bronchial
epithelial 16HBE cells
treated with Cr(VI).
Hu et al. (2018)
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Study overview
Exposure3
Results
Comments
Reference
Exposed: lung cancer
cases with chromate
exposure (at surgery or
autopsy)
(n = 23 patients;
n = 30 lung cancer
tumors)
Referents: lung cancer
cases with no
chromate exposure
(n = 38)
Chromate exposures
for average (SD)
22.9 (6.9) yr
1" methylation of pl6
gene in chromate lung
cancer compared to
lung cancer referents,
but nonsignificant
(p = 0.528)
1" methylation of pl6
gene with duration of
chromium exposure in
chromate lung cancer
cases (p = 0.064)
Chromate lung cancer
with methylation of
pl6 gene had reduced
expression of pl6
protein (0.076)
Methylation-specific
PCR and immuno-
histochemistry of
pl6 (tumor
suppressor gene).
Smoking affected
methylation of pl6
gene in referent lung
cancer cases only.
No confirmation of
lack of chromate
exposure in
referents.
Small sample sizes,
especially for some
of the subanalyses
based on duration of
exposure.
Analyses based on
samples - some
people contributed
multiple samples to
the analysis; these
would not be
independent.
No consideration of
confounders.
Kondo et al. (2006)
Cross-sectional study,
China.
Exposed: n = 87
workers at a chromate
production facility
exposed to chromate
Referent: n = 30
workers from same
facility, but unexposed
to any chromium
products
Exposure to Cr(VI)
inferred based on
occupation.
Also measured total Cr
in blood. Blood
chromium levels were
significantly higher in
exposed compared
with control subjects.
Mean ± SD levels in
blood were 14.5 ± 33
and 4.4 ± 1.9 ng/mL in
exposed and referent
groups, refer to air
monitoring (using
cellulose filter) as
showing all samples
<50 ng/m3, but data
not shown.
The exposed group was
divided by the median
into two subgroups for
high and low exposure.
4/ miR-3940-5p
expression associated
with Cr blood level,
after adjusting for work
duration, gender, age,
smoking, drinking, and
BMI
4/ miR-3940-5p & miR-
590-5p in exposed
group
Main limitations are
related to lack of
description (e.g., for
participant
selection).
Li et al. (2014b)
Exposed: chromate
workers with lung
cancer (n = 26 patients,
n = 35 tumors)
Chromate workers
exposed to chromate
for mean (SD)
22.9 (7.3) yr
4/ expression of
hMLHl and hMSH2
proteins in chromate
lung cancer
Several samples
taken from the same
patients-these are
Takahashi et al.
(2005)
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Supplemental Information—Hexavalent Chromium
Study overview
Exposure3
Results
Comments
Reference
Referents: lung cancer
cases without
chromate exposure
(n = 26 patients, n = 26
tumors)
In chromate lung
cancer group, 4,
expression of nMLHl in
lung cancers with MSI
at 3 or more loci
not statistically
independent.
No adjustment for
covariates, though
authors noted no
significant
differences in age,
Brinkman score,
cancer stage, etc. in
the evaluated
characteristics.
An additional sub-
analysis looked at
methylation of MLH1
among chromate
lung samples, but it
was only conducted
among 8 samples. 5
of 8 had methylation
at hMLHl gene, and
4 of those 5 also had
repression of hMLHl
protein.
Cross-sectional study,
China.
Exposed: n = 29
"healthy" chrome
platers employed for at
least 1 yr at two
facilities
Referent: n = 29
subjects "randomly
selected from the
healthy workers in the
same enterprises and
been engaged in public
security, support
services, or
administration work
for more than one yr,
and had no specific
chromate exposure
history."
Exposure to Cr(VI)
inferred based on
occupation. Chrome
platers had been
employed for at least
lyr.
Also measured Cr in
blood; values were
significantly higher
among exposed
compared with
unexposed workers,
indicating adequate
delineation between
groups. Mean (range)
values were 15.2 (2.1,
42) in exposed vs. 4.6
(0.2, 28) in referent
group.
4/ methylation of
mitochondrial genes
(MT-TF, MT-RNR1) in
chromium-exposed
workers compared to
controls
No difference in
methylation level of
LINE-1 or in mtDNA
copy number between
groups
Limitations are the
limited and poorly
described statistical
analysis, and limited
description (e.g., for
participant
selection). Small
sample size.
Inconsistent results
might indicate the
influence of other
occupational hazards
on micronuclei
concentrations.
Linaing et al.
(2016)
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Supplemental Information—Hexavalent Chromium
Study overview
Exposure3
Results
Comments
Reference
Exposed: individuals
(n = 115; 29 female, 86
male) with exposure to
sodium dichromate for
at least 6 mo
Referents: healthy
volunteers (n = 60; 15
female, 45 male) in the
same city without
chromate exposure
history
Exclusions: medical
history of liver or renal
disease, hypertension,
diabetes,
cardiovascular disease,
or pregnancy
Air-Cr concentration
collected with point
dust sampler and
measured with
electrothermal atomic
absorption
spectrometry.
Personal air samples
collected through full
shift (8 h) sampling to
calculate cumulative
dose
Postshift blood
samples collected;
chromium measured
with ICP-MS; mean
(SD) chromium in
blood of exposed
workers = 12.45
(20.28) ng/L
1" accumulation of Cr
in peripheral red blood
cells
Global DNA
hypomethylation in
chromate-exposed
workers
1" urinary 8-hydroxy-
2-deoxyguanosine,
DNA strand breaks.
No adjustment for
diet or other
nonfolate
supplements.
4/ serum folate in
chromate-exposed
workers.
Wang et al. (2012)
Rat, Sprague-Dawley
0,10.6, 35.4,106.1
mg/L Cr(VI)
0, 2.49, 7.57, 21.41
mg/kg-d Cr(VI) in
drinking water, 4 wk
Mild anemic effects
and increased plasma
malondialdehyde
(MDA) levels occurred
in rats exposed to 100
mg/L or 300 mg/L
Plasma glutathione
peroxidase (GSH-Px)
activity decreased in all
exposed groups
Global DNA
methylation, pl6
methylation
No change in 8-OHdG
levels
Mean body weight
gain, mean water
consumption, clinical
chemistry
determinations, and
oxidative stress
levels in plasma.
Wang et al. (2015)
In vitro, 16HBE human
bronchial epithelial
cells
0, 0.8,1.6,3.1,6.2,
12.5, 25.0, 50.0 and
100.0 nM Cr2072"; 12,
24 or 48 h
1" toxicity (>12.5 nM)
and DNA damage
(comet) (all
concentrations), dose-
dependent
4/ pl6 expression and
hypermethylation of
pl6 CpGl, CpG31, and
CpG32 that correlated
with toxicity and DNA
damage
1" p53 expression
without CpG
methylation (>5 nM)
Hu et al. (2016a)
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Supplemental Information—Hexavalent Chromium
Study overview
Exposure3
Results
Comments
Reference
In vitro, 16HBE human
bronchial epithelial
cells
0, 2, 5, and 10 nM
Na2Cr04, 24 h
miR-3940-5p, which
normally suppresses
XRCC2 and inhibits HR,
is downregulated by
Cr(VI), enhancing DNA
DSB repair
Follow-up study to Li
et al. (2014b).
Li et al. (2016)
Interpreting the
effects of one
dysregulated miRNA
is difficult.
aPotassium dichromate units conversion: Cr(VI) = 0.353 x K2Cr2C>7; potassium chromate units conversion:
Cr(VI) = 0.268 x faCrCU; sodium dichromate dihydrate units conversion: Cr(VI) = 0.349 x Na2Cr20?2H20 (usually
denoted as Na2Cr207 because study authors frequently list the salt as the chemical compound even if
concentration or dose is based on the dihydrate); chromium trioxide units conversion: Cr(VI) = 0.52 x CrC>3.
C.3.2.5. Induces oxidative stress (KC#5)
1 Table C-56 summarizes studies of markers of systemic oxidative stress measured in urine
2 and blood in humans occupationally exposed to Cr(VI). Twenty-three studies were identified.
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Supplemental Information—Hexavalent Chromium
1 Human studies of systemic oxidative stress
Table C-56. Evidence in human studies prioritized for informing potential Cr(VI)-induced oxidative stress
System
Exposure3
Results
Comments
Reference
Exposed: workers exposed to
chromium from chemical,
building, and metal industries
(n = 40)
Referents: age- and sex-
matched individuals, unexposed
to Cr, living away from
incinerators, industries, energy
plants, etc. (n = 40)
Assessment: Urinary chromium
evaluated from Saturday morning
spot samples at end of the work
week; assessment with
electrothermic atomization-atomic
absorption spectrometry.
Levels: Mean (SD) U-Cr (ng/g
creatinine) was 0.62 (0.50) among
workers and 0.30 (0.13) among
controls.
Duration: No information provided
about duration of Cr exposure.
In red blood cells of
exposed individuals:
4, GSH
4/ GSH/GSSG ratio
In plasma:
4/ plasma acid ascorbic
levels
4/ total plasma
antioxidant capacity
4/ TRAP
Null effects on GSSG,
DHA, lipoperoxidation
(TBA-RM), total thiol
levels
Systemic increases in oxidative
stress with chromium exposure.
De Mattia et al.
(2004)
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Exposure3
Results
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Reference
Exposed 1: Cement workers in
building construction (n = 22
males)
Exposed 2: Tannery workers
(n = 20 males)
Referent: "normal healthy"
volunteers (n = 23 males)
Assessment: Blood and urine total Cr
measured using inductively coupled
optical emission spectrometry
Levels: Highest blood and urine Cr in
tannery workers, followed by
cement workers, then referents.
Group 1 (control) n = 23
Blood: 3.81 ±5.57 ng/L
Urine: 6.27 ± 5.31 ng/L
Group II (cement) n=22
Blood: 15.27 ± 2.61 ng/L
Urine: 17.22 ± 3.33 ng/L
Group III (tannerv) n = 20
1" plasma
malondialdehyde
4/ total thiol
1" p53 protein
Unclear if exposure was to Cr(VI)
specifically, although more likely for
cement workers compared with
tannery workers (as described in the
discussion section); however,
separating effects is impossible,
given total Cr was measured in
blood and urine. Poor working
conditions (e.g., lack of PPE) and co-
exposures limit ability to attribute
effects to chromium. The
population also included
adolescents (minimum age 14 yr),
which may affect comparability to
other studies that only included
adults.
Elhosarv et al.
(2014)
Blood: 18.90 ± 1.88 ng/L
Urine: 20.84 ± 1.67 ng/L
Duration: State that "Cement and
tannery workers were usually
exposed to chromium 8 h daily for a
duration ranged from 1 month to 40
years."
Cross-sectional study, Egypt.
Exposed: n = 41 male
electroplating workers exposed
to chromium and nickel
Referent: n = 41 male
administrative workers at the
same facility
Assessment: Exposure to Cr(VI)
inferred based on occupation. Also
measured Cr (and nickel) in serum.
Levels: Serum Cr significantly higher
in exposed compared with controls.
Mean Cr was 3.30 and 0.23 ng/L in
exposed and referent, respectively.
Duration: Exposed workers were
required to have worked in
electroplating section at least 2 yr,
but most worked for considerably
longer with
mean ± SD = 26.68 ± 11.21 yr.
1" 8-OHdG adducts in
serum
Exposed and unexposed groups are
delineated, although limited
description of methods
(e.g., participant selection) and
known coexposure to nickel could
limit inference.
Results correlated with increased
micronuclei in buccal cells.
El Saftv et al. (2018)
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Exposure3
Results
Comments
Reference
Exposed: workers from
bichromate plant with mixed Cr
exposure (n = 10)
Referents: workers from
bichromate plant with no Cr
exposure (n = 10), age and
alcohol consumption matched
to exposed group
Assessment: Urine and blood
samples collected at the end of the
work week; analyzed with
electrothermal atomic absorption
spectrophotometer.
Levels: Mean (SD) Cr in whole blood,
plasma, and urine of exposed
workers: 5.5 (1.2) ng/L, 2.8 (0.4)
Hg/L, 5.9 (1.1) ng/g creatinine,
respectively. Mean (SD) Cr in whole
blood, plasma, and urine of
referents: 0.7 (0.1) ng/L, 0.7 (0.1)
Hg/L, 0.7 (0.1) ng/g creatinine,
respectively.
Duration: No information on
duration of exposure
No difference in 8-OHdG
adducts (lymphocytes
and urine) or DNA strand
breaks (lymphocytes)
between exposed and
referents
Did not appear to control for key
covariates - presents unadjusted
results; very small sample size also
limits confidence in results.
Faux et al. (1994)
Exposed: chromium-exposed
workers (n = 10)
Referents: nonexposed workers
(n = 10)
Assessment: Urine and blood
samples taken from workers at the
end of a workweek.
Levels: Chromium concentrations in
the factory ranged from 0.001 to
0.055 mg Cr(VI)/m3 (obtained from
personal and area samplers). Mean
chromium concentrations in urine
(5.97 ng/g creatinine), whole blood
(5.5 ng/L), plasma (2.8 ng/L), and
lymphocytes (1.01 ng/1010 cells) of
exposed workers were significantly
higher than in nonexposed workers.
Duration: The mean duration of
exposure was 15 yr
No difference in 8-OHdG
adducts or DNA strand
breaks (lymphocytes)
between exposed and
referents
Also null for DNA strand breaks;
authors theorize null findings due to
low exposure levels or insensitive
measures used (very small sample +
low exposure levels - probably very
limited power).
Gao et al. (1994)
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Exposure3
Results
Comments
Reference
Exposed 1: Full-time tannery
workers (n = 33)
Exposed 2: Full- or part-time
stainless steel welders (n = 16)
Referents: individuals
unexposed to known
environmental or occupational
carcinogens (n = 30)
Assessment: Spot urine and venous
blood samples collected from all
subjects on the last day of the work
week. Analyzed with graphite
furnace atomic absorption
spectrophotometer.
Levels: Mean (SD) not reported
Duration: No information on
duration of exposure
1" lipid peroxidation
products (MDA) in urine
of welders and tanners
4/ thiol antioxidants
(glutathione) in
lymphocytes of welders
Cr levels in plasma correlated with
urinary MDA in welders, not
tanners, who are primarily exposed
to Cr(lll).
Goulart et al.
(2005)
Exposed: Polishers working with
chromium-tanned leather
(n = 34)
Referents: Individuals not
employed in industry, free of
acute or chronic disease
(n = 104)
Assessment: Chromium measured in
air at tannery 1978-1990
Levels: Workstation concentrations
ranged from mean (SD):
0.023 ± 0.009 mg Cr/m3to
0.11 ±0.07 mg Cr/m3
Duration: Workers exposed to
chromium for 3-16 yr
1" lipid peroxidation
(TBARS) & 4, Se in
plasma in exposed group
Exposure assessment methods likely
underestimate actual exposure
value; TBARS results potentially
confounded by other occupational
exposures.
Gromadzinska et al.
(1996)
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Exposure3
Results
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Reference
Cross-sectional study, China.
Exposed: n = 87 workers from a
single factory in China, who had
"occupational exposure to
chromate from different work
sections"
Referent: n = 30 working in
administrative offices without
chromate exposure.
Assessment: Exposure to Cr(VI)
inferred based on occupation;
median duration of employment was
5 yr in both exposed and referent.
Also measured total Cr in air samples
and in blood.
Levels: Authors state 'The
concentration of Cr in the air and
blood of subjects in the exposure
group were significantly higher than
the control group (p < 0.001)," which
increases confidence in delineation
of exposure groups. Geometric
mean ± SD of Cr in blood was
8.5 ± 1.3 ng/L in exposed vs.
4.1 ± 1.4 ng/L in referent group,
while median (IQR) of air
concentrations were 15.5 (19.0) vs.
0.2 (0.4) mg/m3.
Duration: Workers had been in the
same work section for at least 3 mo
and in the factory for at least 1 yr.
Median (IQR) yr of working among
the Cr group = 5.0 (7.0).
1" hypermethylation of
CpG sites (in RNA
isolated from whole
blood), serum 8-OHdG,
and MN in peripheral
blood lymphocytes in
exposed workers
compared with referent
Main limitations are related to lack
of description (e.g., for participant
selection).
Hu etal. (2018)
Related studies: Li
et al. (2014a;
2014b)
Exposed: male chrome-plating
workers (n = 25)
Referents: unexposed males
(administrators and others)
(n = 28)
Assessment: Chromium measured in
whole blood, urine, and air; blood
and urine measured with graphite
furnace atomic absorption.
Levels: Mean (SD) concentrations for
exposed group: air = 65 (23.6)
Hg/m3; blood = 5.98 (3.17) |Jg/L;
urine = 5.25 (3.03) ng/g creatinine.
Duration: Chrome-plating factory
workers had been exposed for 1-12
yr [mean (SD): 5.9 (3.5) yr].
1" Malondialdehyde
measured in blood and
urine
A strength of this study was that
chromium was measured in both air
and biological samples.
Limited adjustment for
confounders.
Huang et al. (1999)
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Exposure3
Results
Comments
Reference
Exposed: chrome-plating
workers (n = 50)
Referents: administrative
workers, age and SES matched
to exposed (n = 50)
Assessment: Chromium in urine
samples measured with flameless
atomic absorption
spectrophotometer with graphite
furnace.
Levels: Mean (SD) in exposed group:
10.42 (8.34 ng/g creatinine).
Duration: Chrome plating workers
had been exposed to chromium for
15-20 yr.
1" Plasma lipid
peroxidation
4/ Erythrocyte
antioxidant enzymes
This study is one of the only studies
that adjusted for diet in
investigating antioxidant enzymes.
High variation of urinary chromium
among exposed individuals.
Kalahasthi et al.
(2006)
Exposed: lead chromate
pigment factory workers
(n = 22)
Referents: office workers from
chromate factory (n = 16)
Assessment: Chromium measured in
urine, blood, and air; air sampling for
200 min at flow rate of 2-3 L/min;
urine and blood measured with
flameless atomic absorption
spectrophotometer.
Levels: Chromium in air ranged from
below LOD (0.0005 mg/m3 among
office workers to 0.5150 mg/m3 in
high exposure area of factory
(pulverizing process); mean (SD)
chromium among exposed group in
blood: 6.75 (3.30) ng/L; in urine:
12.97 (16.31) (ng/g creatinine).
Duration: Mean (SD) duration of
work among chromate pigment
workers = 9.7 (20.5)* yr.
In blood and sputum:
No difference in 8-OHdG
adducts (in respiratory
epithelial and white
blood cells) between
exposed and control
groups, or with duration
of employment among
exposed groups
Chromium levels in blood (which
are a marker of recent exposure)
were similar between exposed and
control groups; this suggests that
exposure misclassification could be
contributing to the null effects
reported in the study.
The authors also suggest that
urinary chromium reflects
chromium in reduced form, which
might not reflect genotoxicity in
blood cells.
No adjustment for
supplements/vitamins or diet.
*SD appears incorrect
Kim et al. (1999)
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Exposure3
Results
Comments
Reference
Exposed: workers from
electroplating plants (3
chromium; 1 nickel-chromium; 2
mixed) (n = 50)
Referents: office workers with
no previous exposure to
chromium (n = 20)
Assessment: Urine samples obtained
at end of work shift; analyzed with
atomic absorption
spectrophotometry. Air chromium
measured with personal sampling.
Levels: Mean (SD) urinary chromium
among exposed = 5.72 (7.65) ng/g
creatinine.
Duration: Electroplaters had been
employed for mean (SD): 75.6 (73.1)
mo.
T* urinary 8-OHdG
adducts among exposed
group
High degree of variation in urinary
chromium levels among exposed
group.
Did adjust for some dietary factors
(betel nut, alcohol), but this could
have been more extensive; no
adjustment for supplements/
vitamins.
Did not account for coexposures to
other metals encountered in the
factories, especially the mixed
plants
Kuo et al. (2003)
Cross-sectional study, Korea.
Exposed: n = 51 male chrome
plating and buffing workers
Referent: n = 31 male office
workers from "industrial areas"
in South Korea.
Assessment: Exposure to Cr(VI)
inferred based on occupation. Also
measured Cr measured in air
samples (total and VI), blood, and
end-shift urine samples (See
Table 1).
Levels: Concentrations in blood and
urine were significantly higher in
exposed workers, indicating
adequate delineation between
groups. For example, the geometric
mean blood level of Cr was 0.9 and
0.2 ng/dL in exposed and referent
workers, respectively. Differently,
while air measures were higher for
exposed workers the difference was
not statistically significant.
Duration: Mean duration of
occupational exposure was 9.1 yr
(range: 1 mo-40 yr).
1" lipid peroxidation
(TBARS) in plasma
1" frequency of
chromatid exchange,
chromosome/chromatid
breaks and exchanges,
and of translocations,
correlated with higher
blood Cr
1" frequency of
translocations in exposed
compared with
unexposed
Main limitations are related to lack
of description for analysis and
results reporting.
Maeng et al. (2004)
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Exposure3
Results
Comments
Reference
Exposed: Electroplaters (n = 90),
evenly split among near bath
workers, degreasers, and
washers
Referent: Quality control
personnel in same facilities
(n = 30)
Assessment: Air samples (locations
not specified) used to measure Cr(VI)
using spectrophotometer.
Levels: Median Cr(VI) exposure level
was highest in near bath
(0.38 mg/m3) followed by degreasers
(0.20 mg/m3) and washers (0.05
mg/m3); levels were below the LOD
for referent workers.
Duration: Median (IQR) working yr
among exposed = 4.5 (4.2).
T* serum
malondialdehyde
Cr(VI) was measured in air samples,
which lends confidence that
exposure was occurring and at
significantly higher levels in exposed
workers vs. referents.
Mozafari et al.
(2016)
Exposed: Electroplaters (n = 105
males)
Referent: office workers (n = 125
males)
Assessment: Air samples from
personal breathing zones used to
measure Cr(VI) using UV-visible
spectrophotometer (also measured
total Cr); values combined with
duration of employment to estimate
cumulative exposure. Total Cr was
measured in urine, hair, and
fingernails using graphite furnace
atomic absorption
spectrophotometry.
Levels: Total and Cr(VI) in air were
higher in exposed workers (see Table
2); for example, the geometric mean
daily cumulative Cr(VI) was 155.6
(GSD = 3.3) in exposed vs. 4.8
(GSD = 1.9) |Jg/m3 in referents. Total
chromium in biosamples was also
significantly higher.
Duration: Mean (SD) working yr
among exposed group = 9.4 (5.6).
T* urinary 8-OHdG
T* urinary
malondialdehyde
The sample size is larger compared
with other similar studies, and
Cr(VI) was measured in air samples,
which lends confidence that
exposure was occurring and at
significantly higher levels in
electroplaters vs. referents.
Pan et al. (2017)
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Exposure3
Results
Comments
Reference
Exposed (direct): cement
production unit factory workers
(n = 60)
Exposed (indirect):
administrative workers in
cement production factory
(n = 28)
Referents: healthy individuals
from nearby city (n = 30)
Assessment: Serum chromium
measured with platform partitionate
varian graphite furnace.
Levels: Mean (SE) serum chromium
in direct exposed group: 5.2 (0.4)
Mg/L
Duration: mean (SE) yr of
employment direct
exposed = 4.7 (0.08); indirect
exposed = 4.5 (0.17).
4, TTM & TAC
No difference in TBARS
or NO (indicators of lipid
peroxidation)
No evaluation of air chromium
levels; very limited consideration of
covariates.
Pournourmohamm
adi et al. (2008)
Exposed: individuals (n = 115; 29
female, 86 male) with exposure
to sodium dichromate for at
least 6 mo
Referents: healthy volunteers
(n = 60; 15 female, 45 male) in
the same city without chromate
exposure history
Assessment: Air-Cr concentration
collected with point dust sampler
and measured with electrothermal
atomic absorption spectrometry.
Personal air samples collected
through full shift (8h) sampling to
calculate cumulative dose. Postshift
blood samples collected; chromium
measured with ICP-MS.
Levels: Mean (SD) chromium in
blood of exposed
workers = 12.45 (20.28) ng/L.
Duration: Mean (SD) yr of
employment among exposed group:
12.86 (6.02); range: 1-33.
-t urinary 8-OHdG, DNA
strand breaks and global
DNA hypomethylation in
chromate exposed
workers
1" accumulation of Cr in
peripheral red blood cells
& 4/ serum folate in
chromate-exposed
workers
No adjustment for diet or other
nonfolate supplements.
Wang et al. (2012)
Exposed: chromium platers
(n = 35)
Referents: healthy subjects with
no history of disease or previous
exposure to chromium or other
metals (n = 35)
Assessment: Personal exposure
monitoring for 8-h working shift
(1.71/min) on only 10 individuals in
the exposed group.
Blood and urine samples collected at
end of shift and analyzed with
atomic absorption
spectrophotometry.
Levels: Individual time-weighted
average range: 0.049-1.130 mg/m3.
Duration: The mean duration of
employment was 6.5 yr.
Significantly lower SOD
levels in Cr workers
(6.86 ±0.80 U/mgHb)
compared to controls
(7.16 ±0.53 U/mg Hb)
(p<0.01)
Also 1" sister chromatid exchange
and percent high frequency cells in
exposed group compared to
controls.
Personal air sampling only obtained
for n = 10 individuals in the exposed
group; SCE analysis conducted
based on work group rather than
measured exposure level.
Unable to draw conclusions about
effect of genotype due to small
sample size.
Wu et al. (2001)
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Exposure3
Results
Comments
Reference
Cross-sectional study, Austria.
Exposed: n = 22 bright chrome
plating workers exposed to
chromium and cobalt
Referent: n = 22 jail wardens
Assessment: Exposure to Cr(VI)
inferred based on occupation.
Welders used mainly TIG process
(95%) with smaller proportions of
electric arc and very little
autogenous welding.
Also measured Cr in whole blood;
levels were higher in welders
compared with controls.
Levels: Mean + SD levels for exposed
workers at the beginning and end of
the work week were 1.4 + 0.9 and
2.3 + 1.5 ng/L, respectively, while
values for referent were 0.2 + 0.2
M-g/L.
Duration: All workers worked for 8 h
per day 3 wk before and during the
sample collection
No changes (slight but
not statistically
significant) in plasma
malondialdehyde,
oxidized low density
lipoprotein, and total
antioxidant capacity
(TEAC) (biochemical
parameters of redox
status)
Limitations are due to small sample
size and presence of coexposures,
which precluded more detailed
analysis to separate effects.
"I^MN and rates of Nbuds in buccal
and nasal mucosal cells.
Wultsch et al.
(2014)
Exposed: n = 319 living in
villages with historic Cr
contamination
Referents: n = 307 living in
villages without historic Cr
contamination
Assessment: Cr measured in
groundwater (7-m or 8-m deep
wells), soil (field surface), and air (24
h/d for 5 d in both exposed and
unexposed villages).
Levels[median (min, Ql, Q3, max):
Groundwater mg/L exposed: 0.002
(0.002, 0.002, 1.1, 2.5), n = 13;
unexposed: 0.002 (0.002, 0.002,
0.002, 0.002), n = 18
Soil mg/kg exposed: 69.5 (48.7, 59.1,
93.9, 417.1), n = 45; unexposed: 29.2
(20.1, 26.4, 30.4, 41.11), n = 30
Air ng/m3 exposed: 19.3 (10.1,13.7,
28.4, 82.9), n = 15; unexposed: 13.12
(5.0, 10.9, 16.8, 18.7), n = 15
Duration: Mean (SD) yr of residence:
45 (13).
In serum of exposed
group:
-t MDA (p< 0.001)
1" Catalase activity
(p < 0.001)
^GSH-Px activity
(p < 0.001)
-t 8-OHdG (p = 0.008)
4/ SOD activity
(p < 0.001)
Systemic increases in oxidative
stress observed with increasing
chromium exposure.
Xu et al. (2018)
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Supplemental Information—Hexavalent Chromium
System
Exposure3
Results
Comments
Reference
Exposed: Electroplaters
(n = 117) at one of five different
metal factories
Referent: office workers (n = 45)
Assessment: Total Cr in urine
measured using graphite atomic
absorption spectrophotometry.
Levels: Urine Cr was higher in
exposed compared to referent
(mean [SD] of 0.74 [0.53] vs. 0.34
[0.18] ng/g creatinine, respectively).
Duration: individuals with <9 yr of
exposure: mean (SD) = 8 (2);
individuals with >10 yr of
exposure = 10 (8).
T* urinary 8-OHdG
Unclear if exposure was to Cr(VI)
specifically (possible with
electroplaters but seem to have
measured total Cr in urine). Also,
while difference in mean urine Cr
was significant, the levels seem
somewhat low. Coexposures with
nickel, did not exclude smokers
(high prevalence), and significantly
higher alcohol consumption among
exposed workers may affect results.
Yazar and Yildirim
(2018)
Exposed: Electroplaters at 7
workshops in Tehran (n = 30
males)
Referent: Age- and sex-matched
dairy production workers (n = 30
males)
Assessment: Blood Cr levels
measured using flameless atomic
absorption spectrometer.
Levels: Blood Cr higher in exposed
vs. referent (mean [SD] = 5.97 [1.74]
vs. 4.22 [0.08] ng/mL), increased
from 4.42 ng/L to 10.6 Hg/L.
Duration: Work duration 1-10 yr.
1" lipid peroxidation
4/ plasma antioxidant
capacity
4/ plasma total thiol (SH
groups)
Unclear if exposure was to Cr(VI)
specifically (possible with
electroplaters). Also, while
difference in mean blood Cr was
significant, the levels were more
similar than expected between
exposed and referent.
Zendehdel et al.
(2014)
Exposed: electroplating workers
(n = 157)
Referents: individuals without
exposure to chromium or
known physical/chemical
genotoxic agents (n = 93)
Assessment: Air-Cr and blood Cr
determined by graphite furnace
atomic absorption
spectrophotometer.
Levels: median (range) Cr in
erythrocytes (ng/l) among exposed:
4.41 (0.93-14.98); among controls:
1.54 (0.14-4.58). Median (range)
short-term concentrations of Cr in
air: 0.060 (0.016-0.531) mg/m3.
Duration: Median (min-max) yr of
exposure among exposed group: 5.3
(0.5-23).
T* urinary 8-OHdG
adducts among exposed
compared to referents
1" DNA damage
(measured by the comet
assay) in lymphocytes
among exposed
compared to referents
Limited adjustment for confounders
(including diet).
Potential coexposures to other
metals in the workplace.
Zhang et al. (2011)
aPotassium dichromate units conversion: Cr(VI) = 0.353 x K2Cr2C>7; potassium chromate units conversion: Cr(VI) = 0.268 x foCrCU; sodium dichromate dihydrate
units conversion: Cr(VI) = 0.349 x Na2Cr20?2H20 (usually denoted as Na2Cr207 because study authors frequently list the salt as the chemical compound even if
concentration or dose is based on the dihydrate); chromium trioxide units conversion: Cr(VI) = 0.52 x CrC>3.
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Supplemental Information—Hexavalent Chromium
1 Human in vitro studies of oxidative stress
2 Table C-57 summarizes in vitro studies of markers of oxidative stress in response to Cr(VI)
3 exposure. Because all in vivo animal studies of oxidative stress following Cr(VI) exposures focusing
4 on organ- or tissue-specific oxidative stress are already categorized within the health effect section
5 for supporting evidence relevant to the study (i.e., respiratory, GI, hepatic, hematological, male or
6 female reproductive, developmental), they have not been repeated here. In vitro studies of
7 oxidative stress induced by Cr(VI) were included if they were conducted in human primary cells or
8 immortalized human cell lines and not already summarized in another health effect section.
Table C-57. In vitro studies of Cr(VI)-induced oxidative stress
System
Exposure3
Results
Reference
Human chronic
myelogenous
leukemic (CML)
K562 cells,
promyelocytic
leukemic HL-60
cells, and normal
human peripheral
blood
mononuclear
(HPBM) cells
12.5 and 25 nM
Na2Cr2C>7, 24 or
48 h
4/ cytochrome c CT* SOD)
1" hydroxyl radical
1" intracellular 2,7-DCFD fluorescence
1" DNA fragmentation
No apoptosis (TUNEL) in HPBM; T* apoptosis in
K562 at low dose but necrosis at high dose
Human cultured leukemic cells more sensitive
than primary cells
Bagchi et al. (2001;
2000b)
Primary human
lymphocytes
0, 50, 100, 200,
600, and 1000
HM K2Cr207, 1 h
1" DNA strand breaks (comet) (>400 nM;
p< 0.001)
DNA damage T* with Endo III and 4^ with catalase
(p < 0.001), indicating oxidative lesions
Slight reduction in cell viability (trypan blue
exclusion) (viability at top dose was 84.7%)
Blasiak and Kowalik
(2000)
Human umbilical
vein endothelial
cells (HUVECs)
1-20 mM
K2Cr207
1" stress response/ inflammatory pathways (JNK,
p38 MAPK, NLRP3, ICAM-1, VCAM-1, TNF-a, IL-1|J)
1" intracellular ROS
1" apoptosis induced by mitochondrial (intrinsic)
pathway
Cao et al. (2019)
Whole human
blood
0.01-40.0 ng
K2Cr2C>7/mL
1" glutathione peroxidase
4/ SOD, GSH
4/ ferric-reducing ability of plasma (FRAP)
Dlugosz et al. (2012)
Primary human
lymphocytes and
erythrocytes
K2Cr207
4/ GSH, -t GSSG and ROS
Husain and Mahmood
(2017)
Primary human
lymphocytes
1-100 nM
Na2Cr2C>7,1 h
1" standard and FPG-modified comet assay DNA
strand breaks (>100 nM)
-t 8-OHdG (>10 nM)
Significant interindividual variation in comet and
FPG-comet DNA damage correlated with OGG1
polymorphisms
Lee et al. (2005, 2004)
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1
2
3
4
5
6
7
8
9
10
11
12
Supplemental Information—Hexavalent Chromium
System
Exposure3
Results
Reference
Primary human
fibroblasts
0.5-500 nM
Cr(VI)
4/ O2 consumption, dose-dependent (20-500 nM)
1" standard and FPG-modified comet assay DNA
strand breaks (0.5-3 nM)
Attributed to affected mitochondrial function and
glucose catabolism
Liu et al. (2010b)
Human leukemicT-
lymphocyte MOLT4
cells
0-200 nM
K2CrC>4, 2 h
4/ multiple antioxidants, dose-dependent
(p< 0.01 at 10 nM)
1" DNA-protein crosslinks (25 nM)
-t ROS (DCFH-DA)
1" DNA-protein crosslinks and protein carbonyls (2
h) and MDA (4 h), dose-dependent
ESR showed reaction of Cr(VI) with NADPH,
glutathione reductase or H2C>2-generated Cr(V)
and OH radicals
Pretreatment with antioxidants reduced protein
carbonyl, MDA and DPC formation but not with
catalase inhibitor or riboflavin pretreatments
Mattagajasingh et al.
(2008; 1997, 1996,
1995)
Human diploid
fibroblasts
0, 0.2, 0.5, 1, 2,
3, 5 nM K2Cr2C>7
1" DNA strand breaks >0.5 nM
Inhibition of excision repair did not prevent repair
of breaks
Scavenging superoxide (SOD) or H2O2 (catalase)
but not hydroxyl radicals (Kl) reduced DNA
damage; reduced glutathione potentiated damage
Snvder (1988)
aPotassium dichromate units conversion: Cr(VI) = 0.353 x K2Cr2C>7; potassium chromate units conversion:
Cr(VI) = 0.268 x K2&O4; sodium dichromate dihydrate units conversion: Cr(VI) = 0.349 x Na2Cr20?2H20 (usually
denoted as Na2Cr2C>7 because study authors frequently list the salt as the chemical compound even if
concentration or dose is based on the dihydrate).
C.3.2.6. Induces chronic inflammation (KC#6)
Mechanistic studies relevant to immunomodulation (including immune stimulation) are
summarized in Appendix C.2.5.
C.3.2.7. Immunosuppression (KC#7)
Mechanistic studies relevant to immunomodulation (including immune suppression) are
summarized in Appendix C.2.5. The evaluation of evidence for effects of Cr(VI) on the immune
system, presented in Section 3.2.6 of the toxicological review, suggests that Cr(VI) could have
immunomodulatory effects that can suppress (as well as stimulate) the immune system. This
immunosuppressive effect was primarily determined from a limited number of host resistance
assays, and the significance for Cr(VI)-induced carcinogenesis is not currently known.
C.3.2.8. Modulation of receptor-mediated effects (KC#8)
No evidence exists that Cr(VI) itself has receptor binding activity, although indirectly it can
initiate cell signaling cascades involving receptor-mediated pathways (summarized in Appendix
C.3.3) and can affect the expression of sex hormone cell receptors (summarized in Appendix C.2.6
and C.2.7).
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Supplemental Information—Hexavalent Chromium
C.3.2.9. Causes immortalization (KC#9)
1 Enabling replicative immortality is a hallmark of cancer and may be informed by studies
2 that indicate inhibition of senescence induced by Cr(VI) exposure. Mechanistic studies reporting
3 endpoints relevant to senescence are summarized in Table C-58.
Table C-58. Mechanistic studies informing Cr(VI)-induced cellular
immortalization
System
Exposure3
Results
Comments
Reference
Exposed: male
welders (n = 75)
and sandblasters
(n = 5) from
shipyard industry
Referents: subjects
from exposed
group with
chromium blood
levels >2 ng/L who
underwent
intervention to
reduce exposure
for 5 mo (n = 9)
Shipyard industry
welders with mean
(range) yr working in
industry:
18.5 (2-35).
Chromium measured in
blood and urine with
atomic absorption
spectrometer; mean
(range) Cr levels in first
sampling period:
blood = 0.91 (0.1-6.1)
Hg/L; urine = 1.33 (0.1-
50.2) ng/L
Cr levels in
blood and
urine
associated
with ApoJ/CLU
glycoprotein
levels in serum
Authors conclude the
upregulation of
Apolipoprotein J/Clusterin
glycoprotein that promotes
cellular senescence by Cr(VI)
is induced by oxidative
stress.
Findings differ from earlier
studv bv this group (Katsiki
etal., 2004).
Small sample size for the
intervention arm of the
study.
Alexopoulos et al.
(2008)
Exposed: male
workers (n = 55
welders; n = 10
sandblasters;
n = 15 other) (total
n = 80)
Referents:
nonexposed males
of the same age
range (n = 30)
Blood and urine
samples collected;
analyzed with graphite
furnace atomic
absorption
spectroscopy
Higher Cr(VI) in blood
(llx) and urine (57x) in
welders compared to
controls
4/ serum
ApoJ/CLU in
exposed;
dose-
dependent
decrease
based on level
of exposure
and duration
of exposure
Reduced biomarker of cell
survival and senescence
Apolipoprotein J/Clusterin
Findings differ from later
study by this group
(Alexopoulos et al., 2008)
Did not appear to adjust for
covariates.
Did not provide sample size
for subgroup analyses by
duration of exposure -
difficult to assess confidence
in these results.
Katsiki et al. (2004)
L-02 human fetal
hepatocytes
0, 5, 10, 15 nM Cr(VI)
T* Clusterin
(CLU), dose-
dependent
Overexpression of CLU can
counteract Cr(VI)-induced
MRCC 1 inhibition, enhancing
survival.
Xiao et al. (2019)
aPotassium dichromate units conversion: Cr(VI) = 0.353 x K2Cr2C>7; potassium chromate units conversion:
Cr(VI) = 0.268 x faCrCU; sodium dichromate dihydrate units conversion: Cr(VI) = 0.349 x Na2Cr20?2H20 (usually
denoted as Na2Cr2C>7 because study authors frequently list the salt as the chemical compound even if
concentration or dose is based on the dihydrate); chromium trioxide units conversion: Cr(VI) = 0.52 x CrC>3.
C. 3.2.10. Alters cell proliferation, celldeath, or nutrient supply (KC#10)
4 Table C-59 summarizes human, animal, and in vitro studies of markers of cell proliferation,
5 cell death, or changes in cellular nutrient supply in response to Cr(VI) exposure. Human
6 occupational and in vivo animal studies and in vitro studies using human primary or immortalized
7 cell lines relevant to cell proliferation and death following Cr(VI) exposures using organ-specific
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Supplemental Information—Hexavalent Chromium
1 test systems or markers from these systems have already been categorized within the
2 corresponding health effect sections for supporting evidence. Human or animal in vivo studies
3 were included here if they measured any systemic markers of cell proliferation or death or were
4 not previously summarized elsewhere. Similarly, in vitro studies were included if they were
5 conducted in human primary cells or immortalized human cell lines that have not already been
6 summarized in another health effect section. An exception has been made for studies of Cr(VI)-
7 induced changes in cellular energetics. These tables include all identified mammalian in vivo and
8 human in vitro studies that pertain to cellular nutrient supply, regardless of whether they were also
9 reported elsewhere.
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Supplemental Information—Hexavalent Chromium
Table C-59. Mechanistic studies relevant to Cr(VI)-induced cell death, cell
proliferation, and changes in cellular energetics
System
Exposure
Results
Comments
Reference
Cell cycle progression
Human lung
fibroblasts (HLFs)
1 nM Na2Cr204, 24 h
T* Gl/S and G2/M arrest
Gl/S checkpoint bypass
involves Aktl
T* cell survival and T*
mutation frequency (but 4,
DNA DSBs and CAs) with
inhibition of protein
tyrosine phosphatase (PTP),
mediated by polo-like
kinase 1 (Plkl)
No change in apoptosis
Plkl mediates cell
cycle checkpoint
bypass and mitotic
progression leading to
increased survival of
cells with Cr(VI)-
induced DNA damage
Kost et al.
(2012); Chun et
al. (2010); Bae
et al. (2009a);
Lai et al. (2009)
Cell death
Exposed: Chrome-
plating workers
(n = 19)
Referents 1: hospital
workers (n = 18)
Referents 2:
university personnel
(n = 20)
Total Cr measured in
urine, erythrocytes,
and lymphocytes
using graphite
furnace atomic
absorption
Total Cr was higher in
exposed workers
compared with
hospital workers (see
Table 3; for example,
postshift mean urine
levels were 7.31
[SD = 4.33] in exposed
vs. 0.12 [SD = 0.07]
|jg/g crt in referent).
In peripheral blood
lymphocytes:
No change in apoptosis
(nuclear fluorescence
measured by FACS flow
cytometry)
T* DNA damage (measured
by the comet assay)
Did not exclude
smokers (high
prevalence) although
did present results
stratified by smoking
(small numbers).
Unclear if exposure
was to Cr(VI)
specifically (possible
with chromeplating
workers, but
measured total Cr in
urine). State that
previous air
monitoring for total
chromium showed
levels of 0.4 to 5.6
|jg/m3, which is fairly
low.
Gambelunghe
et al. (2003)
HLF fetal human lung
fibroblasts
L-41 human
epithelial-like cells
1, 2, 5, 10, 15, 20, 25,
and 30 nM K2Cr2C>7, 2,
24 or 48 h
T* cytotoxicity (MTT assay),
dose- and duration-
dependent (significant >20
HM); <5 nM cytotoxicity
recovered after 24 h
Toxicity at 20 nM due to
apoptosis (morphology,
caspase-3, DNA
fragmentation)
-t ROS (DCFH-DA) at 2 h
T* antioxidant enzymes
(glutathione peroxidase,
glutathione reductase,
catalase) 1-5 nM
Oxidative stress and
antioxidant enzymes
induced at mildly
toxic nM
concentrations.
Asatiani et al.
(2011; 2010;
2004)
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Supplemental Information—Hexavalent Chromium
System
Exposure
Results
Comments
Reference
Human chronic
myelogenous
leukemic (CML) K562
cells, promyelocytic
leukemic HL-60 cells,
and normal human
peripheral blood
mononuclear (HPBM)
cells
12.5 and 25 nM
Na2Cr2C>7, 24 or 48 h
4/ cytochrome c CT* SOD)
1" hydroxyl radical
1" intracellular 2,7-DCFD
fluorescence
1" DNA fragmentation
No apoptosis (TUNEL) in
HPBM; 1" apoptosis in K562
at low dose but necrosis at
high dose
Human cultured
leukemic cells more
sensitive than primary
cells.
Bagchi et al.
(2001; 2000b)
Human umbilical vein
endothelial cells
(HUVECs)
1-20 mM K2Cr207
1" stress response/
inflammatory pathways
(JNK, p38 MAPK, NLRP3,
ICAM-1, VCAM-1, TNF-a, IL-
lb)
1" intracellular ROS
1" apoptosis
Apoptosis induced by
mitochondrial
(intrinsic) pathway.
Cao et al.
(2019)
HLF human lung
fibroblasts (LL-24 cell
line)
3, 6, and 9 nM
Na2Cr04, 24 h
1" cytotoxicity, duration-
and dose-dependent (stat.
sig. >6 nM)
1" apoptosis
1" p53 (4- to 6-fold)
1" Cr-DNA adducts
Pretreatment with 1
mM ascorbate or 20
HM tocopherol had
no ameliorative
effects.
Carlisle et al.
(2000a)
HeLa cells
1,10, and 100 nM
Na2Cr2C>7; 1, 2.5, 8,
24, or 48 h
Intracellular Cr(lll) inhibits
DNA synthesis and
replication fidelity by
inhibiting DNA synthesome
polymerases a, 5, and £
Dai et al. (2009)
Human lymphoma
U937 cells lacking
functional p53 gene
20 nM Cr(VI), 24 h
1" mitochondria-dependent
apoptotic pathway changes
(intracellular Ca2+, DNA
fragmentation, caspase-3,
low mitochondrial
membrane potential
(MMP), and nuclear
morphology)
1" hydroxyl and superoxide
anion radicals (measured by
ESR-spin trapping)
Apoptosis inhibited by NAC
DNA fragmentation
suppressed by inhibiting
intracellular Ca2+ and
calpain
No increases in Fas or JNK
Authors conclude
Ca(2+)-calpain- and
mitochondria-
caspase-dependent
pathways play
significant roles in the
Cr(VI)-induced
apoptosis via the
G2/M cell cycle
checkpoint.
Havashi et al.
(2004)
Normal human
foreskin (BJ)
fibroblasts
immortalized by
human telomerase
(BJ-hTERT)
0-6, 9, or 12 nM
Na2Cr04, 24 h
1" cell cycle arrest
4/ clonogenic survival, dose-
dependent
1" % apoptotic cells with
dose
Pritchard et al.
(2001)
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Supplemental Information—Hexavalent Chromium
System
Exposure
Results
Comments
Reference
Primary human
lymphocytes
Cr(VI) complexes
sodium bis(2-ethyl-2-
hydroxybutyrato)oxoc
hromate(V),
Na[Cr(V)0(ehba)(2)]
and sodium bis(2-
hydroxy-2-
methylbutyrato)oxoc
hromate(V),
Na[Cr(V)0(hmba)(2)]
'Y apoptosis and ROS and 4^
cell viability with Cr(V) and
Cr(VI)
Cr(V)-induced apoptosis
partially reversed with
antioxidants
Cr(V) and Cr(VI) activate Src-
family protein tyrosine
kinases leading to caspase-3
activation
Cr(VI)-induced
apoptosis partially
induced by ROS
generated by Cr(V)
intermediates via
SFKs.
Vasant et al.
(2003; 2001)
Cellular energetics
BEAS-2B human
bronchial epithelial
cells
1 nM Cr(VI), 48 h
'Y glycolysis
4/ respiration
4/ protein levels of (5-F1-
ATPase
-t GAPDH
Cr(VI) caused shift to
fermentative
metabolism.
Cerveira et al.
(2014)
BEAS-2B human
bronchial epithelial
cells
5-20 nM Na2Cr207
-t NOTCH1 (Notchl)
-t CDKN1A (P21)
4, FBP1
FBP1, involved in
gluconeogenesis, is
lost in Cr(VI)-
transformed cells.
Reintroduction of
FBP1 caused "I^ROS
and ^apoptosis.
Dai et al.
(2017a)
L-02 human fetal
hepatocytes
4-32 nM
4/ mitochondrial respiratory
chain complex (MRCC) 1 and
II activity (25 nM)
Cr(VI)-induced MRCC 1
inhibition activates
caspase-3; process
dependent on ROS.
Xiao et al.
(2012a; 2012b)
L-02 human fetal
hepatocytes
0, 2, 8, 32 nM Cr(VI),
24 h
'Y voltage-dependent anion
channel 1 (VDAC1)
expression, ROS, and
apoptosis with 4^ ATP (32
M-M)
Effects reversed with NAC
pretreatment or blocking
VDAC1
Cr(VI)-induced
apoptosis and
decreased ATP
mediated by ROS and
VDAC1.
Yuan et al.
(2012)
aPotassium dichromate units conversion: Cr(VI) = 0.353 x K2Cr2C>7; potassium chromate units conversion:
Cr(VI) = 0.268 x K2&O4; sodium dichromate dihydrate units conversion: Cr(VI) = 0.349 x Na2Cr20?2H20 (usually
denoted as Na2Cr207 because study authors frequently list the salt as the chemical compound even if
concentration or dose is based on the dihydrate).
C.3.3. Gene Expression Studies Relevant to Gastrointestinal Cancer Cell Signaling Pathways
1 Mechanistic evidence investigating the cell signaling pathways involved in carcinogenesis
2 following exposure to Cr(VI) is summarized in Table C-61. Studies identified in preliminary title
3 and abstract screening as "mechanistic" were further screened and tagged as "cell signaling" if they
4 reported relevant gene expression data. Studies were prioritized if they were (a) oral, inhalation,
5 or intratracheal instillation exposures in vivo, or (b) in vitro exposures in human cells. Two studies
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Supplemental Information—Hexavalent Chromium
1 in humans, two in vivo studies in rats, and 84 in vitro studies in human cells were identified. This
2 does not include studies reporting toxicogenomic data, which are summarized in Appendix C.3.4.
3 The human studies, presented in Table C-60, measured increases in p53 expression in the
4 peripheral blood of chromium-exposed workers compared to unexposed workers. Although these
5 studies were not formally evaluated for risk of bias and sensitivity, the potential for coexposures
6 among these workers (Elhosarv etal.. 2014) or lack of Cr measures in exposed workers (Hanaoka
7 etal.. 19971 precludes certainty regarding the potential association between increased p53
8 expression and Cr(VI) exposure specifically.
Table C-60. Gene expression studies in humans exposed to Cr(VI)
System
Exposure
Results
Reference
Exposed 1: Cement
workers in building
construction (n = 22
males)
Exposed 2: Tannery
workers (n = 20
males)
Referent: "normal
healthy" volunteers
(n = 23 males)
Blood and urine total Cr measured using inductively
coupled optical emission spectrometry
Highest blood and urine Cr in tannery workers,
followed by cement workers, then referents.
Total chromium levels (ng/L) mean ± SD:
Referent (n = 23):
Cr content in blood: 3.81 ± 5.57
Cr content in urine: 6.27 ± 5.31
Cement (n = 22):
Cr content in blood: 15.27 ± 2.61
Cr content in urine: 17.22 ± 3.33
Tannery (n = 20):
Cr content in blood: 18.90 ± 1.88
Cr content in urine: 20.84 ± 1.67
State that "Cement and tannery workers were usually
exposed to chromium 8 h daily for a duration ranged
from 1 month to 40 years."
Unclear if exposure was to Cr(VI) specifically, although
more likely for cement workers compared with
tannery workers (as described in the discussion
section); however, separating effects is impossible,
given total Cr was measured in blood and urine. Poor
working conditions (e.g., lack of PPE) and coexposures
limit ability to attribute effects to chromium. The
population also included adolescents (minimum age
14 yr), which could affect comparability.
1" p53 protein
expression
(detected by
immuno-
cytochemistry)
in peripheral
blood of
tannery and
cement
workers
Elhosarv et al.
(2014)
Exposed: chromate
plant workers
(n = 31 males)
Referents:
volunteers without
occupational
chemical exposures
(n = 10)
Duration of exposure in workers = 0-23 yr
No assessment of Cr levels in workers or referents
1" serum p53
protein
expression
(detected by
ELISA) in serum
of chromium
workers
Hanaoka et al.
(1997)
9 All other studies were reviewed for effects relevant to the KEGG (Kyoto Encyclopedia of
10 Genes and Genomes, https: //www.genome,ip /kegg /pathwav.html) "Pathways in cancer" maps for
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humans fhttps: //www.genome.ip/pathwav/hsa052001. Table C-61 summarizes the reference gene
IDs and direction of change for each. KEGG pathways are publicly available, manually drawn, and
curated pathway maps, based on evidence from recognized evidence-based relationships among
genes involved in cancer-related processes. Data from the two rat oral studies or from the 90 in
vitro studies in human cells specific to Cr(VI) were then overlayed onto the cancer pathway KEGG
maps for rats and humans, respectively, creating two maps (Figures C-25 and C-26). A third map
(Figure C-27) was created using gene expression changes reported by ToxCast/Tox21 high
throughput screening (HTS) assays in human cells exposed to Cr(VI) in vitro. Genes are color
coded: red = activation or increased expression, turquoise = inactivation or decreased expression,
violet = discordant changes showing both activation and inactivation among different studies, and
yellow = proteins that were modified or have changed localization. Green indicates a gene whose
expression (or activity of its products) was not tested or not found to have changed.
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Supplemental Information—Hexavalent Chromium
Figure C-25. KEGG pathways of gene expression changes in rats exposed to CrfVI) via ingestion. Red = activated or
increased expression; turquoise = inactivated or decreased expression; green = no data or no change detected.
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Supplemental Information—Hexavalent Chromium
Figure C-26. KEGG pathways of gene expression changes in human cells exposed to Cr(VI) in vitro. Red = activated
or increased expression; turquoise = inactivated or decreased expression; violet = discordant results from different
studies; yellow = proteins that were modified or have changed localization; green = no data or no change detected.
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Supplemental Information—Hexavalent Chromium
Figure C-27. KEGG pathways of gene expression changes in cells exposed to Cr(VI) reported by ToxCast/Tox21
HTS assays. Red = activated or increased expression; turquoise = inactivated or decreased expression; green = no data or
no change detected.
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C.3.3.1. Cell signaling pathways
Tissue-specific in vivo animal evidence
The oncogene c-Myc was found to show a dose-dependent increase (protein and mRNA) in
the stomach and colon of male Wistar rats after 60 days of exposure to Cr(VI) in drinking water,
supporting increased cell proliferation in these tissues fTsao etal.. 20111. The same study also
observed decreased stomach and colon expression of the tumor-suppressor p53, MAPK inhibitor
RKIP, and Rho-GDIa, which is involved in the Rho-regulated pathways for metastasis/cytoskeleton
reorganization. Down-regulation of RKIP led to the activation of MEK/ERK signaling pathway in
the rat stomach and colon. Activation of the ERK/MAPK signaling pathway promotes cell
proliferation, tumor cell invasion, and angiogenesis and inhibits apoptosis fGuo etal.. 20201. Tsao
etal. (20111 also reported increased galectin-1. Galectins are associated with gastric cancer cell
motility in response to integrin signaling, and galectin-1 is overexpressed in gastric tumor cells and
digestive cancers fWu etal.. 2018: Kim etal.. 20101. In a separate study, Ki-67—a nuclear protein
associated with cellular proliferation, malignant metastasis, and tumor growth (Li etal.. 20151—
showed non-dose-dependent increases in transcript expression in the duodena of mice after oral
exposure through drinking water at 11.6 and 31 mg/kg Cr(VI)-day (Rager etal.. 2017: Kopec etal..
2012a).
In vitro human evidence
In vitro studies in various human cell types demonstrated the role of several processes
relevant to the cancer development that include (1) activation of MAPK signal pathway
extracellular signal-regulated kinase (ERK), Jun kinase (JNK/SAPK), and p38 MAPK involved in cell
proliferation; (2) changes involving DNA damage checkpoint/DNA repair components (e.g., ATM,
ATR, XRCC1, RAD17, RAD51, TP53 and DNA-PK); (3) changes in the expression of genes involved in
the reactive oxygen species homeostasis (e.g., NFE2L2, NOX, SOD1, SOD2, CAT, GSR); (4) changes in
apoptosis-regulating genes (BCL2, MCL1, BBC3, BAX, CASP3, CASP9); and (5) changes suggesting
tissue remodeling and epithelial-mesenchymal transition (SNAI2, ZEB1, PLAUR, CDH1, KLF8) and
pathways with pleiotropic roles in cancer (NOTCH, HIF-la, PI3K/Akt).
The effects of chromium exposure were shown to be dependent on cell context and
exposure level/time. For instance, exposure to Cr(VI) resulted in considerably different changes in
nuclear binding of transcription factors AP-1, NF-kB, SP1, and YB-1 in human MDA-MB-435 breast
cancer cells in comparison with ratH4IIE hepatoma cells (Kaltreider etal.. 19991. Exposure
level/time dependence was shown on transcriptional activity of NF-kB: At low exposure levels of
20 [iM for 2 hours, Cr(VI) exposure inhibited both basal and TNF-a-stimulated NF-KB-driven
transcriptional activity in human A549 lung carcinoma cells. This inhibition occurred through the
interaction of NF-kB with transcriptional coactivators (Shumilla etal.. 19991. In contrast,
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Supplemental Information—Hexavalent Chromium
exposures of HepG2 cells to potassium dichromate at 10 [J.M for 48 hours significantly increased
transcription from the NF-kB response element fTullv etal.. 20001.
Discordant changes in the expression or activity of certain genes were observed between
experiments in cells exposed to cytotoxic levels of Cr(VI) and cells transformed by Cr(VI). This can
be exemplified by the expression of BCL2 gene, a founding member of the BCL2 gene family of
apoptosis regulators. In immortalized human hepatocytes exposed to cytotoxic levels of Cr(VI),
decreased expression of the anti-apoptotic BCL2 gene led to increased apoptosis (Zhong etal..
2017b), while in Cr(VI)-transformed BEAS-2B cells, the BCL2 gene was upregulated, contributing to
an apoptosis-resistant phenotype that is consistent with the malignant properties of transformed
cells (Medan etal.. 2012). These results exemplify the complexity of molecular changes induced by
exposure of cells to Cr(VI) and their dependence on exposure level and cellular context.
Table C-61. Gene expression corresponding to positive results of Cr(VI) assays
performed in vivo (rats) or in vitro (human cells or TOX21 HTS assays).
Direction of change (measuring mRNA or protein): t (upregulated or activated); I
(downregulated or inhibited); A (protein posttranslational modification or change of
intracellular localization).
Study
Gene symbol
KEGG ID
Rat in vivo studies (Rattus norvegicus)
Bagchi et al. (1997a)
PrkcaT
24680
Tsaoetal. (2011)
Tp53^
Arhgdia^
Pebpl^
MycT
LgalslT
24842
360678
29542
24577
56646
Human in vitro studies (Homo sapiens)
Abreu et al. (2018)
HSPA1A (Hsp72)l
Hsp90a -l
Adam et al. (2017)
NLRP3t
IL1B (IL-lb)T
Akbar et al. (2011)
IL-2 down^
3558
Antonios et al. (2009)
CD86t
Asatiani et al. (2004)
CAT (Catalase)T
SOD1 (Cu,Zn-SOD)T
Bae et al. (2009b)
FGR A
ABL1 (ABL) A
25
Barchowskv (2006)
Lckt
Fynt
Browning and Wise (2017)
Rad51c A
BRCA2A
675
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Supplemental Information—Hexavalent Chromium
Study
Gene symbol
KEGG ID
Cammarota et al. (2006)
MMP2^
TIMP1 (TIMP)T
4313
Carlisle et al. (2000a; 2000b)
P53t
7157
Castorina et al. (2008)
ERBB2t (24h+)
ERBB3t (24h+)
2064
Cervak et al. (2004)
TP53t
CDKN1A (P21)T
MAPK3, MAPK1 (ERKl/2)t
7157
1026
5594, 5595
Chuang et al. (2000)
jnkT
MAPK11-14 (P38)T
MAPK3, MAPK1 (ERKl/2)t
5599
5594, 5595
Chuang and Yang (2001)
MAPK3, MAPK1 (ERKl/2)t
JUNt
5594, 5595
3725
Chun et al. (2010)
Plklt
Clementino et al. (2019)
SIRT3t
Pinklt
PRKN (Parkin)T
Curtis et al. (2007)
ILlaT
3552
Dai et al. (2017a)
NOTCH1 (Notchl)T
CDKN1A (P21)T
FBPl^
4851
1026
Delougherv et al. (2015)
ATRt
Ding et al. (2013)
CDHl^
VIM t
FNl^
CTNNB1 (P-catenin -A)
SNAI2 (Slug)T
Zeblt
KLF8t
999
2335
1499
Dubrovskava and Wetterhahn (1998)
HOt
Gambelunghe et al. (2006)
TP53t
CASP3t
CASP8t
CASP9t
7157
836
841
842
Ganapathv et al. (2017)
BCI2T
KRAS (Ras)t
596
3845
Havashi et al. (2004)
CAPN1 (calpain)T
He et al. (2013)
IGF1R (IGF-IR)T
IRS it
hifiaT
RELA (NFkB)t
3480
3091
5970
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Supplemental Information—Hexavalent Chromium
Study
Gene symbol
KEGG ID
CXCL8 (IL-8)T
3576
Hill etal. (2008a)
TP53t
CDKN1A (P21)T
atmT
PRKDC (DNA-PK)T
atrT
AKT1 (AKT)T
MAPK11-14 (P38 MAPK)T
7157
1026
207
Hill etal. (2008b)
TP53t
CDKN1A (P21)>L-
PUMAt
BAX T
PRKDC (DNA-PK)T
7157
1026
27113
581
Hodges et al. (2004)
JUNt
JNKt
3725
5599
Hu et al. (2018)
MGMT^
XRCCl^
OGG! (HOGGl)^
RAD51^
5888
Kaczmarek et al. (2007)
hifiaT
3091
Kaltreider et al. (1999)
FOS, JUN (APl)t
NFkBt
spiT
YBXl (YBl)t
2353,3725
5970
6667
Kim et al. (2003)
RELA (NFkB)T
5970
Kost et al. (2012)
PTP^
Lai et al. (2009)
CDKN1B (P27) A
RBI A
1027
5925
Li et al. (2016)
XRCC2t
Liu et al. (2009)
WRN A
Lozsekova et al. (2002)
VCL (Vinculin)l
TLN1 (Talin)l
CDH1 (E-cadherin)l
DSP (Desmoplaktin)l
999
Lu etal. (2018b)
STK11 (LKBl)^
Maiumder et al. (2003)
SLC30A1 (Zn-Tl)^
Medan et al. (2012)
BCL2t
596
Mvers et al. (2011)
TXN (TRx) A
TXNRD1 (TrxR)^
7296
Nemec and Barchowskv (2009)
statiT
VEGFA^
6772
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Supplemental Information—Hexavalent Chromium
Study
Gene symbol
KEGG ID
spiT
6667
Nemec et al. (2010)
Fynt
statiT
irfiT
6772
O'Hara et al. (2003)
MAPK8 (JNK)T
Fynt
LckT
5599
O'Hara etal. (2004)
BmxT
PTK2 (Fak)T
PTK2B (Pyk2)T
Fynt
STAT5A, STAT5B (Stat5)T
Aplt
5747
6776, 6777
2353, 3725
O'Hara etal. (2005)
STAT3t
LckT
6774
O'Hara etal. (2007)
Lckt
STAT3t
IL-6t
6774
3569
Pritchard et al. (2000)
ICAMlt
Reynolds and Zhitkovich (2007)
TP53t
7157
Rizzi et al. (2014)
MAPK3, MAPK1 (ERKl/2)t
5594, 5595
Russo et al. (2005)
BBC3 (PUMA) t
PMAIP1 (NOXA)t
BAX A
CYCS A
CASP3t
27113
5366
581
54205
836
Shumilla et al. (1999)
RELA(NFkB)^
5970
Shumilla and Barchowskv (1999)
PLAU (uPA)^
PLAUR (uPAR)T
Park et al. (2015)
TP53t
CDKN1A (P21)T
7157
1026
Park et al. (2016)
ERFFIl^
Tessier and Pascal (2006)
MAPK11-14 (P38)T
MAPK8 (SAPK/JNK)t
MAPK3, MAPK1 (ERKl/2)t
5599
5594, 5595
Tullv et al. (2000)
TP53t
FOSt
RELA (NFkB)T
AHRt (inferred)
GADD45t
HSPA1A (HSP70)T
7157
2353
5970
1647
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Supplemental Information—Hexavalent Chromium
Study
Gene symbol
KEGG ID
Vasant et al. (2003)
Lck (p56lck)T
FYN (pS^T
Ly(p56/53ly)T
CASP3t
836
Vilcheck et al. (2006)
FANCD2T
Wakeman et al. (2005)
MAPK11-14 (P38)T
Wakeman and Xu (2006)
atmT
SMC1A (SMCl)t
atrT
RAD17t
Wang et al. (2019)
RELA (NFkB, p65) T
IL-6t
HIF1A (HIF-la)T
5970
3569
3091
Xla et al. (2011)
BTD^
Xiao et al. (2012b)
MRCCI^
HSP1A1(HSP70)^
HSP90AB1(HSP90)^
3326
Xiao et al. (2012a)
MRCC1, 2i
BUB1B (BuBRl)^
CDC25A (CDC25)^
Yang et al. (2017)
MAP1LC3A (LC3Il)T
Atgl2-Atg5t
Atg4t
AtgioT
HMGAlt
HMGA2t
SQSTM1 (p62)^
Yi et al. (2016)
STIMlT
MAPK3, MAPK1 (ERKl/2)t
RELA (NFkB)T
Ca2+t
5594, 5595
5970
C00076
Yi et al. (2017)
VDAClT
Zeng et al. (2013)
S0D1 (SOD)^
GSR(GR)^
catI
NO^
Zhang et al. (2016)
TP53t
BCL2^
MCL1 (Mcl-l)^
CDK2^
CCNE1 (Cyclin E)^
7157
596
1017
898
Zhang et al. (2017)
PI3K/Akt^
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Supplemental Information—Hexavalent Chromium
Study
Gene symbol
KEGG ID
ER stress
Mito dysfunction
Zhong et al. (2017b)
ETFDh4
SOD^
CASP3t
CASP9t
BCL2^
Ca2+t
CYCS A
836
842
596
C00076
54205
Zhong et al. (2017a)
SODlT
SOD2t
KEAPlt
NFE2L2 (NRF2) t
PPARGC1A (PGC-la)t
NRFlt
tfamT
sirtiT
foxoiT
AKTlt
CREBlt
9817
4780
2308
207
Zuoetal. (2012)
RELA (NFkB)T
JUNt
apiT
PTGS2 (COX2)T
5970
3725
5743
Tox21 Assays, Assay ID: DTXSID6032061 (sodium dichromate dihydrate)
TOX21_TR_LU C_GH3_Antago n ist
THRB^
TOX21_SSH_3T3_GU3_Antagonist
GU3^
2737
TOX21_p53_BLA_p2_ch2
TOX21_p53_BLA_p2_ratio
TOX21_p53_BLA_p3_ch2
TOX21_p53_BLA_p3_ratio
TOX21_p53_BLA_p5_ch2
TOX21_p53_BLA_p5_ratio
TP53t
7157
TOX21_GR_BLA_Antagonist_ch2
TOX21_GR_BLA_Antagonist_ratio
NR3C1^
TOX21_CAR_Antagonist
NR1I3^
TOX21_Aromatase_lnhibition
CYP19A1^
TOX21_RORg_LUC_CHO_Antagonist**
RORC^
TOX21_PR_BLA_Antagonist_ratio**
PGR^
TOX21_H2AX_HTRF_CHO_Agonist_ratio**
H2AFXt
TOX21_ERR_Antagonist**
ESRRA^
TOX21_ERb_BLA_Antagonist_ratio**
ESR2^
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12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
Supplemental Information—Hexavalent Chromium
Study
Gene symbol
KEGG ID
TOX21_ARE_BLA_agonist_ratio**
NFE2L2T
4780
TOX21_AR_LUC_MDAKB2_Antagonist_0.5nM_R1881**
AR^
367
C.3.4. Toxicogenomic Studies
Several studies of Cr(VI) exposure measuring toxicogenomic or cell signaling changes were
identified in the evidence base. Given the complexity of these studies and comprehensive
applicability of the evidence reported, an extra level of review and analysis was applied to these
studies.
C. 3.4.1. Prioritization of studies for consideration
Full-text screening of 39 mechanistic studies identified as reporting toxicogenomic data was
performed; these studies are summarized in Table C-62. Studies were prioritized on the basis of
relevance for providing mechanistic insight for Cr(VI)-mediated carcinogenesis in the lung or GI
tract Of these 39 studies, 13 studies were identified that fit these criteria. A further targeted
evaluation of these 13 studies was conducted to determine relevance; 8 were prioritized for
evaluation in HAWC (the preliminary evaluations of the 5 studies that were not evaluated in HAWC
can be found in Appendix C.3.4.3). Of these eight studies selected for evaluation in HAWC, five used
the same microarray dataset, so only one evaluation was necessary for these five (details below); an
independent analysis using this dataset was also conducted by EPA. In addition to this evaluation
(Kopec et al.. 2012b). one study in humans occupationally exposed to Cr(VI) (Hu etal.. 2017). one
additional in vivo animal study (Chappell etal.. 2019). and one in vitro study (Huang etal.. 2017)
were evaluated for risk of bias and sensitivity in HAWC.
Two of the included studies, Kopec et al. (2012b: 2012a). generated microarray datasets
from tissues collected in female B6C3F1 mice and F344 rat duodenal and jejunal epithelia following
7 and 90 days of exposure to 0.3-520 mg/L (as sodium dichromate dihydrate, SDD) in drinking
water, bioassays originally reported by Thompson et al. (2012c: 2011b). Five additional studies
reported analyses using the same datasets: four from the same research group (Rager etal.. 2017:
Thompson etal.. 2016: Suh etal.. 2014: Thompson etal.. 2012bl and one analysis conducted
independently by EPA fMezencev andAuerbach. 20211. Five of these studies were included in the
synthesis of toxicogenomic data analysis; one, Suh etal. (2014). was not included because the scope
was restricted to genes involved in iron homeostasis. One study evaluation, pertaining to (1) the
quality of the animal study that generated the microarray data, and (2) the quality and usability of
the generated microarray, was deemed sufficient to determine confidence in this original dataset,
and this could apply to all studies using this dataset. The essential details of this evaluation can be
found in the HAWC database under Kopec et al. f2012bl.
The analysis by EPA, described in Mezencev andAuerbach f20211. provides mechanistic
insight interpretable toward human relevance of the NTP 2-year rodent bioassays and suggests the
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1 existence of potentially vulnerable subgroups. As a part of the independent analysis of this dataset
2 by Mezencev andAuerbach f2021I an evaluation of the microarray data was conducted; these
3 details are described in the following section.
Table C-62. Summary of considered toxicogenomic studies for Cr(VI) overall
confidence classification
Author (year)
Species (strain)
Exposure
design
Exposure
route
Inclusion
Microarray
Hu etal. (2017)
Human study of
chromate production
workers in China
Cohort
Occupational
Yes, evaluation in HAWC
M
Kopec et al.
(2012b)a
Rat (F344/N), mouse
(B6C3F1)
Subchronic
Drinking water
Yes, evaluation in HAWC
Chappell et al.
(2019)
Mouse (B6C3F1)
Subchronic
Drinking water
Yes, evaluation in HAWC
M
Huang et al. (2017)
Human (BEAS-2B
human lung epithelial
cell line)
—
In vitro
Yes, evaluation in HAWC
Kopec et al.
(2012a)
Rat (F344/N), mouse
(B6C3F1)
Subchronic
Drinking water
Yes, but not evaluated,
same dataset as Kopec et
al. (2012b)
Thompson et al.
(2012b)
Rat (F344/N), mouse
(B6C3F1)
Subchronic
Drinking water
Yes, but not evaluated,
same dataset as Kopec et
al. (2012b)
Thompson et al.
(2016)
Rat (F344/N), mouse
(B6C3F1)
Subchronic
Drinking water
Yes, but not evaluated,
same dataset as Kopec et
al. (2012b)
Rager et al. (2017)
Mouse (B6C3F1)
Subchronic
Drinking water
Yes, but not evaluated,
same dataset as Kopec et
al. (2012b)
Mezencev and
Auerbach (2021)
Mouse (B6C3F1)
Subchronic
Drinking water
Yes, but not evaluated,
same dataset as Kopec et
al. (2012b)
Sanchez-Martin et
al. (2015)
Mouse (C57BL/6J)
Subchronic
Drinking water
No, targeted evaluation
below
Izzotti et al. (2002)
Rat (Sprague-Dawley)
Short-term
Intratracheal
instillation
Yes, targeted evaluation
below
Lu etal. (2018a)
Human (BEAS-2B)
-
In vitro
Yes, targeted evaluation
below
Clancv et al. (2012)
Human (BEAS-2B)
-
In vitro
Yes, targeted evaluation
below
Chen et al. (2002)
Human (BEAS-2B)
-
In vitro
Yes, targeted evaluation
below
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Supplemental Information—Hexavalent Chromium
Author (year)
Species (strain)
Exposure
design
Exposure
route
Inclusion
Microarray
Suh et al. (2014)
Rat (F344/N), female;
mouse (B6C3F1),
female
Subchronic
Drinking water
No, limited scope
D'Agostini et al.
Rat (Sprague-Dawley)
Short-term
Intratracheal
No, study limited in scope
(2002)
instillation
to a subset of genes; same
data as Izzotti et al. (2002)
Izzotti et al. (2004)
Rat (Sprague-Dawley)
Short-term
Intratracheal
instillation
No, same data as Izzotti et
al. (2002)
Madeiczvk et al.
(2015)
Rat
Acute
Injection-i.p.
No, limited scope
Kumar et al. (2013)
Mouse (Swiss albino)
Acute
Injection-i.p.
No, limited scope
Hamilton et al.
Chick embryo
Acute
Injection-i.p.
No, limited scope, and
(1998)
model system less relevant
to intestinal or respiratory
carcinogenesis
Pritchard et al.
(2005)
Human (fibroblasts
with ectopic
expression of h-TERT)
—
In vitro
No, limited scope
Andrew et al.
(2003)
Human (BEAS-2B)
-
In vitro
No, limited scope
Joseph et al. (2008)
Human (skin
fibroblasts)
In vitro
No, model system less
relevant to intestinal or
respiratory carcinogenesis
Sun et al. (2011)
Human (BEAS-2B)
-
In vitro
No, limited scope
Gavin et al. (2007)
Human (peripheral
blood mononuclear
cells)
—
In vitro
No, limited scope
Lei et al. (2008)
Rat (lung epithelial
cells)
-
In vitro
No, limited scope
Guo et al. (2013a)
Human (skin
fibroblasts)
In vitro
No, model system less
relevant to intestinal or
respiratory carcinogenesis
Vaquero et al.
(2013)
Human (Alexander
hepatoma cells)
-
In vitro
No, limited scope
Guo et al. (2013b)
Acellular protein
binding
-
In vitro
No, limited scope
Ovesen et al.
(2014)
Mouse (Hepa-lclc7)
-
In vitro
No, limited scope
Lou et al. (2015)
Human (B
lymphoblastoid cells)
-
In vitro
No, limited scope
Johnson et al.
(2016)
Yeast (Saccharomyces
cerevisiae)
In vitro
No, limited scope, and
model system less relevant
to intestinal or respiratory
carcinogenesis
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Supplemental Information—Hexavalent Chromium
Author (year)
Species (strain)
Exposure
design
Exposure
route
Inclusion
Microarray
Luczak et al. (2016)
Human (H460 lung
carcinoma cell line)
--
In vitro
No, limited scope
Bruno et al. (2016)
Human (BEAS-2B)
--
In vitro
No, limited scope
Hu etal. (2016b)
Human (16HBE
bronchial epithelial
cell line)
—
In vitro
No, limited scope
Park et al. (2017)
Human (BEAS-2B)
--
In vitro
No, limited scope
Chen et al. (2019)
Human (16HBE)
--
In vitro
No, limited scope
Hu etal. (2019)
Human (16HBE)
--
In vitro
No, limited scope
Wu et al. (2012)
Human (BEAS-2B)
--
In vitro
No, limited scope
High (H), medium (M), low (L), or uninformative (U).
aThis study used animals from Thompson et al. (2011b) and Thompson et al. (2012c). Additional included analyses
using the same dataset: Kopec et al. (2012a), Thompson et al. (2012b), Thompson et al. (2016), and Rager et al.
(2017). Suh et al. (2014) used same dataset but the analysis was limited in scope.
bStudy was high confidence for all reported endpoints except for qPCR, which was determined to be
uninformative.
C.3.4.2. Analysis of data reported by Kopec et al. f2012b: 2012a)
Several identified studies used the microarray dataset generated by Kopec et al. (2012b;
2012a) from tissues collected in female B6C3F1 mice and F344 rat duodenal and jejunal epithelia
following 7 and 90 days of exposure to 0.3-520 mg/L (as sodium dichromate dihydrate, SDD) in
drinking water, bioassays originally reported by Thompson et al. (2012c: 2011b). The exposure
levels and tissues were selected based on previously reported significant occurrence of tumors of
the small intestines in mice following chronic exposure to Cr(VI) in drinking water (NTP. 2008).
Description of the studies and dataset
Mice B6C3F1 were continuously exposed to drinking water containing SDD at target
concentrations 0 (control), 0.3, 4,14, 60,170, and 520 mg/L SDD until study termination at days 8
or 91, when the animals were euthanized and specimens of intestinal tissues (duodenum, jejunum)
and oral mucosa (palate) were collected for gene expression analysis (Kopec etal.. 2012a:
Thompson etal.. 2011b). Tissue collection, isolation of RNA, design and implementation of
microarray experiment, and the processing of microarray data have been described in detail fKopec
etal.. 2012a). The dataset "T ranscriptomic data to assess hexavalent chromium mode of action in
mice and rats" is deposited in the Gene Expression Omnibus (GEO)
(https://www.ncbi.nlm.nih.gov/geo/) as a SuperSeries GSE87262. This dataset consists of
394 microarrays from the platforms Agilent-014868 Whole Mouse Genome Microarray 4x44K and
Agilent-014879 Whole Rat Genome Microarray 4x44K. The mouse subset of the data was deposited
under the accession number GSE87259 and includes 214 microarrays. The data are available in the
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—Hexavalent Chromium
functional genomics data repository GEO supporting MIAME-compliant data submissions in the
form of raw data (.gpr files) and normalized data (normalized following a referenced
semiparametric approach).
Evaluation of microarrav experiment and generated micro array data
A summary of the micro array study design performed by Kopec et al. (2012b; 2012a) can be
found in Figure C-28. An evaluation of the microarray data reporting quality was conducted using
the Minimum Information About a Microarray Experiment (MIAME) fBrazma etal.. 20011 (Table C-
63). An evaluation focusing on study design and implementation and on the quality and usability
of preprocessed expression data for their reanalysis was also conducted using criteria developed by
Bourdon-Lacombe etal. (2015) (Table C-64). Additional criteria for DNA micro arrays presented by
this group were not applied, as this evaluation is not focusing on the reported results of the
microarray study.
Experimental design: direct comparison with
complete balanced block
CTlx TRlx TRlx CTlx CT2x TR2x TR2x CT2x CT3 x TR3x TR3x CT3x
V V
R G
V V
R G
V V
R G
V V
R G
v V
R G
V V
R G
R G
R G
R G
R G
R G
R G
CTlx, CT2x, CT3x: biological replicates for control animals for a given exposure level x
TRlx, TR2x, TR3x: biological replicates for exposed animals for a given exposure level x
2 exposure levels per 3 slides -> 9 slides for 6 exposure levels (one tissue, one timepoint)
2 timepoints x 3 tissue types ->36 microarrays per each time point and tissue type
Biological replicates of the same exposure groups are hybridized on separate slides
Technical replicates (dye swaps): every microarray is dye swapped and contains the
same biological samples (not different samples of the same exposure groups)
3
Figure C-28. Design of microarray experiments conducted by Kopec et al.
(2012b: 2012a). Multiplexing of the treatment-control pairs were performed on
the same chips, indicating a limited influence of interchip differences in the
comparative expression analysis.
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Supplemental Information—Hexavalent Chromium
Table C-63. Evaluation of the information available with microarray data
using MIAME sections
MIAME section
Evaluation of the available information
Part 1: Experimental design
Information provided in sufficient detail. Dose-response type of experiment [0.1,
1.4, 5, 20, 50,180 mg/L Cr(VI)) in drinking water continuously] with two timepoints
(8 and 91 d). Other experimental variables: 3 tissue types (duodenum, jejunum,
palate epithelium). Three biological replicates per exposure level/tissue/timepoint.
Part2: Array design
Information available due to the commercial nature of microarray platform.
Commercial microarray Agilent-014868 Whole Mouse Genome Microarray 4x44K
(www.agilent.com). Designed to represent all known genes in the mouse genome
and their resulting transcripts, the microarray comprises 41,534 60-mer
oligonucleotide probes representing over 41,000 mouse genes and transcripts.
Part 3: Samples
Information provided in sufficient detail.
Organism: Mus musculus strain B6C3F1; sex = female; 6-7 wk old at exposure.
Sample: RNA (isolation and evaluation of purity and integrity reported).
Labeling: Following manufacturer's protocol.
Part 4: Hybridizations
Information provided in sufficient detail. Hybridization was performed following
manufacturer's protocol (Agilent Manual: G4140-90050 v. 5.0.1). Information on
the dye swap and hybridization design reported adequately (see Figure C-28).
Part 5: Measurements
Reported sufficiently. Original scans: not available (these are usually not provided).
Raw data provided. Normalized data provided as a gene expression matrix.
Normalization approach (a semi parametric) was reported and properly referenced.
Part 6: Normalization
controls
Included in microarray design.
Table C-64. Evaluation of the DNA microarray experiments in Kopec et al.
(2012b: 2012a) using criteria outlined by Bourdon-Lacomhe et al. (2015)
Criterion
Status for Kopec et al. (2012b; 2012a)
Mandatory or important criteria important in evaluating the overall quality of toxicogenomics experiments
Control animals were handled alongside treated
animals using identical procedures (e.g., controls in
oral gavage experiments received vehicle only) and
at similar times.
True. No additional variable (including time) was
identified between exposure groups and corresponding
controls.
(Equivalent to confounding/variable control and exposure
domains in the study evaluation in HAWC.)
A minimum of three biological replicates (animals)
were used per group.
True for all exposures/tissues/timepoints with single
exception for mice-duodenum-90-d-1.4 mg/L Cr(VI)
exposure and control groups (2 replicates available only).
This deficiency affects only 1 of 18 tissue-exposure groups
from 90-d mouse study. The impact is limited.
If temporality is considered, time-matched controls
were used.
True. Two timepoints have separate time-matched
controls. In fact, each exposure group even has separate
unexposed controls.
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Supplemental Information—Hexavalent Chromium
Criterion
Status for Kopec et al. (2012b: 2012a)
The appropriate animal model and tissue was used,
and a rationale is given for the doses selected.
True. The study used the same mouse strain and exposure
levels as previous NTP bioassav (NTP, 2008, 2007f) and
focused on the tissues (duodenum, jejunum) in which the
NTP study detected pathological changes of interest.
(The same as the exposure design domain in the study
evaluation in HAWC).
If dose-response is considered for risk modeling
(including estimation of the BMD), a minimum of
three doses plus control was used; ideally, at least
one of these doses should be near the NOAEL.
True. Six doses plus control were employed. LOAELfor
duodenal epithelial hyperplasia in female mice was at 5
mg/L Cr(VI) exposure in 2-yr NTP bioassay (38% cumulative
incidence). The evaluated study included much lower
exposures [1.4 mg/L and 0.1 mg/L Cr(VI), and shorter
time].
Tests to assess various toxicities
(e.g., histopathology, biomarkers of disease) were
done using the same biological samples.
Partially true. Animals from the same study and exposed
under the same conditions were used for histopathology
evaluation and other phenotypic assays of target tissues.
Criteria that are required or should be considered in DNA microarray methodologies
RNA A260/A280 ratios are reported and are above
1.8 to indicate sample purity or are consistent across
samples.
Partially true. Determination of the purity of RNA by
A260/A280 has been indicated in the text, but the values
have not been reported.
This reviewer's experience is that these values are
frequently determined and used to assess the quality of
RNA preparations, but they are usually not reported,
because of irrelevance of their actual values with respect
to publication (if >1.8, the RNA is used for downstream
experiments; If not, RNA is isolated again). This reviewer
considers the fact that the ratio has been determined and
used to assess the purity of RNA as sufficient even if its
value is not reported.
The integrity of RNA was assessed [common
strategies include an RNA integrity number (RIN), an
RNA quality indicator (RQI) or 28s:18s ratio] to
ensure minimal RNA degradation or consistency
across samples.
True. Determination of the RNA integrity was performed
using denatured gel electrophoresis. This is an older and
more laborious, but less expensive method than using a
lab-on-a chip (e.g., Agilent Bioanalyzer), which provides a
specific RIN score. This reviewer considers the fact that
the integrity of RNA was verified by denatured
electrophoresis as sufficient.
When multiple microarrays are necessary and the
experiment was run over different days, the samples
were randomized across the slides/days to avoid
confounding effects (often referred to as a block
design). Note: not always specified in the methods.
Of three biological replicates for given exposure level, one
exposure and one control specimen were always
hybridized on the same microarray slide. Three replicates
were spread across three different slides. This design
ensures that even if each of these 3 slides is processed on
a different day, the confounding due to batch effect can be
eliminated if the data are analyzed with consideration for
pairing of specimens on the same slides. The information
on timing of microarray processing was not provided;
nevertheless, the batch effect can be identified through
data analysis (if present) and under some circumstances it
can be also corrected.
Generally, gene annotation and data quality are
more robust when commercially produced
microarray platforms are used.
True. Commercial microarray platform has been used.
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Supplemental Information—Hexavalent Chromium
Criterion
Status for Kopec et al. (2012b: 2012a)
Species appropriate microarrays were used
(i.e., mouse arrays for mouse samples).
True. Mouse microarray Agilent-014868 Whole Mouse
Genome Microarray 4x44K has been used.
Labeling and hybridization were done according to
manufacturer protocol. Any deviations are
reported.
True. Manufacturer's protocol has been reportedly
followed (Agilent Manual: G4140-90050 v. 5.0.1), and no
deviation was indicated.
When cohybridizations of treated and control
samples are done (use of different fluorophores for
control and treated samples), dye-swapping
experiments were done, or that dye bias was
assessed statistically is indicated.
True. Dye-swapping was performed (see Figure C-28;
green and red colors represent Cy3 and Cy5 dyes).
Scanner specific quality control software was used
to test microarray quality.
True. GenePix Pro 6.0 software was used for data
collected by GenePix 4000B scanner. All data has
reportedly passed quality control. The results of quality
control assessment were not presented (which is not
unusual in the field).
Data quality was assessed (through MA plots, heat
maps, boxplots, scatterplots, signal to noise ratio,
etc.).
Partially true. Heatmaps for duodenal and jejunal data for
8-d and 90-d timepoints with hierarchical clustering on
specimens was provided [Figures 6 (8 d) and 8 (91 d),
(Kopec et al., 2012b)l. This is not an unsupervised analysis
and only differentially expressed genes were analyzed.
This reviewer does not consider this criterion to be "a hard
criterion." Data quality plots can be usually re-created
when needed and assessed by study evaluator.
In the case that outliers are identified, a minimum of
three replicates per group remain and a justification
for removal has been provided.
Partially true. In one specific tissue/exposure
combination, only 2 replicates are available. An
explanation for the missing replicates was not provided,
but it is not certain that the replicates represented outliers
(it could have been a technical failure affecting 2 specific
microarrays). Other than that, removal of other
microarrays was not identified.
The data were preprocessed (e.g., background
subtracted and log transformed) and normalized
(i.e., adjusted to remove technical variations
between arrays) prior to statistical analysis.
True. Preprocessed data were submitted to the GEO
repository.
Data files were made available through an open
access public database such as Gene Expression
Omnibus (GEO), Chemical Effects in Biological
Systems (CEBS) or ArrayExpress).
True. See GEO https://www.ncbi.nlm.nih.gov/geo/);
SuperSeries GSE87262.
1 Distribution of normalized expression intensities [from GEO)
2 The dataset for the mouse small intestine reported by Kopec et al. f2012b: 2012a) was
3 further analyzed. Distributions of normalized expression intensities were retrieved using the
4 GE02Rtool (Figures C-29 through C-32). The distributions demonstrate that the values submitted
5 by the study authors are median-centered and therefore cross-comparable.
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—Hexavalent Chromium
GSE87259/GPL7202, selected samples
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-------
Supplemental Information—Hexavalent Chromium
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Figure C-30. Signal intensity boxplots for 8-day exposure mouse data
(duodenum, top 4 dose groups). The image includes top 4 exposure groups and
corresponding controls. Note: 1 mg/L SDD = 0.349 mg/L CrfVI}.
GSES7259 GPL7202, selected samples
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This document is a draft for review purposes only and does not constitute Agency policy,
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Supplemental Information—Hexavalent Chromium
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for 4-60 mg/L SDD. Note: 1 mg/L SDD = 0.349 mg/L Cr(VI).
GSE87262, selected samples
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Supplemental Information—Hexavalent Chromium
GSE87259/GPL7202, selected samples
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(duodenum). The image includes all exposure groups and corresponding controls
except for 4 mg/L SDD exposed and control groups. Note: 1 mg/L SDD = 0.349
mg/L Cr(VI).
Principal component analysis
Principal component analysis was performed to visualize multidimensional gene expression
data and identify relationships among specimens. This analysis was executed using BMDExpress
2.20.0148 BETA fSciome. 20181 separately for 8-day and 91-day mouse duodenum gene expression
data. The data were normalized and log2-transformed. This method reduces high-dimensionality
of microarray data (41,268 signal values per micro array) onto 2-dimensional space with
orthogonal variables PCI and PC2 that capture the maximum amount of variance. The 8-day
exposure duodenal data show separation for three highest exposure levels along PC2 (Figure C-33).
The 90-day data show separation only for two highest exposure groups (combined) and 4.61
mg/kg-day group from all other groups (Figure C-34). The results suggest separation of microarray
data by exposure, which is more pronounced in the 8-day than in the 90-day dataset and for higher
but not for lower exposure levels.
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Supplemental Information—Hexavalent Chromium
PCI Vs. PC2
-320,000 -300,000 -280,000 -260,000 -240,000 -220,000 -200,000 -180,000 -160,000 -140,000
-120,000 -100,000
PC1
-80,000 -60,000 -40,000 -20,000
20,000 40,000 60,000 80,000 100,000
0.0 • 0.028 0.38 - 1.15 A 4.89 13.05 30.29
Figure C-33. Principal component analysis of 8-day exposure data for mice and duodenal tissues. Exposure levels
[mg/kg-day Cr(VI)] are color-coded.
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PCI Vs. PC2
-280.000 -260.000 -240,000 -220,000 -200.000 -180.000 -160,000 -140.000 -120.000 -100.000
PC1
3,000 -60,000 -40,000 -20.000
20.000 40.000 60,000 80,000
0.0 0.024 0.31 A 1.08 4.61 • 11.52 - 30.96
Figure C-34. Principal component analysis of 90-day exposure data for mice and duodenal tissues. Exposure levels
[mg/kg-day Cr(VI)] are color-coded.
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Hierarchical clustering
Hierarchical clustering was performed with the GENE-E tool (Broad Institute) for all mouse-
related data with GEO accession number GSE87259 (Figure C-35). Data used were all signal
intensities normalized by the study authors; distance metrics were 1- Pearson correlation
coefficient; the linkage method was average. Separation between 8-day and 90-day data was forced
through their separate analysis by hierarchical clustering.
The result of this unsupervised clustering displays clear separation of overall gene
expression of palate specimens from duodenum and jejunum for both 8-day and 91-day exposures,
which is consistent with expected biological differences. Duodenum specimens for 8-day exposure
to 520 mg/L clearly separate from all other duodenum and jejunum specimens. Duodenum
specimens [8 day/20-60 mg/L Cr(VI)] and jejunum specimens [8 day/20-180 mg/L Cr(VI)] cluster
together but separately from those exposed to 0.1-5 mg/L Cr(VI) for 8 days. Low exposures
[0.3 mg/L and 1.4 mg/L Cr(VI)] tend to cluster together with vehicle controls. In 90-day data,
duodenum and jejunum specimens from mice exposed to the highest concentrations of Cr(VI)
(60 mg/L and 180 mg/L) form a well-defined cluster with separation between duodenum and
jejunum specimens.
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Figure C-35. Hierarchical clustering of microarrays from duodenum, jejunum, and palate tissues from mice
exposed to SDD for 7 days and 90 days. This visualization cannot provide adequate resolution and serves only for
illustrative purposes. Text color coding: Green-exposed, gray-controls. Colored squares: red - duodenum, beige -
jejunum, blue - palate; yellow - 8 days, violet - 91 days. Expression color coding: blue-low, red-high. For a higher
resolution image, see fU.S. EPA. 2022al.
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The study that generated microarray dataset GSE87259 does not substantiate major
concerns with respect to the risk of bias. The only potential issue, which has been identified, is
apparently incomplete outcome data due to the discrepancy between the number of mice reportedly
allocated to the gene expression study and the number of mice needed to produce the dataset
GSE87259. This discrepancy is of possibly little significance, because the number of allocated mice
has been reported in an article that was not actually reporting microarray data generation,
processing, or interpretation (Thompson etal.. 2011b). The study authors could have refined the
design of the microarray study and eventually processed less mouse tissue for gene expression
analysis than originally planned. Issues specific to reporting and design of the microarray
experiment were of little significance with respect to the quality and usability of data for
toxicogenomic analysis. The results of the principal component analysis of normalized data
supplied by study authors demonstrated that the microarrays are cross-comparable among
different dose levels for a given tissue type and exposure time, which supports their use for
transcriptomics BMD determination and for analysis of gene expression differences between
exposed and control animals within the same tissue type.
In addition, the expression data were found to be similar for jejunum and duodenum based
on the results of unsupervised hierarchical clustering. This clustering presents relationships among
specimens by a tree whose branch lengths reflect the degree of similarity in the overall gene
expression between specimens. Moreover, the jejunum and duodenum were found to differ
considerably from palate tissue with respect to overall gene expression. This finding is consistent
with expectations based on biological differences and supports the quality of microarray data
through biological plausibility. Furthermore, duodenum specimens [8-day, 20-60 mg/L Cr(VI)] and
jejunum specimens [8-day, 20-180 mg/L Cr(VI)] were shown to cluster together but separately
from the same specimens isolated from mice exposed to 0.1-5 mg/L Cr(VI). This finding supports
the existence of dose dependence of overall expression data and implies the existence of differences
between low and high exposure groups. Interestingly, the low exposures 0.1 mg/L and 1.4 mg/L
Cr(VI) tend to cluster together with vehicle controls. Thus, the result of hierarchical clustering
shows consistency with biological expectations (support for quality of microarray data) and
identifies meaningful natural classes among specimens.
Another issue not addressed by this evaluation is related to the use of single-channel data
from two-color microarrays used in accordance with a two-color protocol and with cohybridization
of Cy-3 and Cy-5 labeled specimens. During a discussion with collaborators, a concern was raised
that Cy-3 only data were used, but Cy-5 data were excluded from further analysis. The study
authors argued in their report that Cy-5 data can be unreliable due to sensitivity of this dye to
ambient ozone. This issue has been recognized by the scientific community and the means for its
remediation are available from the microarray supplier (Agilent). Most likely, these means have not
been used by the study authors and they have decided to disregard affected Cy-5 data after the
experiment was completed. Therefore, it is unlikely that this approach does not represent a
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selective reporting that increases the risk of bias. Although some concerns could remain with
respect to the data processing, separate channel analysis for two-channel microarrays has been
explored and recommended by other investigators (Smyth and Altman. 2013).
This evaluation did not address the raw gene expression data and their preprocessing due to
time and resource limitations. Nevertheless, a collaborator was able to process raw data using a
code supplied by the study author and demonstrate reproducibility of the raw data processing
through independent generation of the same normalized data as supplied by study authors to the
GEO [personal communication],
C.3.4.3. Targeted study evaluations
The following five studies were prioritized for relevance for providing mechanistic evidence
informative to Cr(VI)-mediated carcinogenesis in the lung or GI tract, but preliminary targeted
evaluations determined that full study evaluations in HAWC were not warranted. Because these
have not been included in the HAWC database, the limited preliminary evaluations have been
provided below.
Lu etal. (2018a)
A full study evaluation to judge the potential risk of bias is not warranted. The source of
BEAS-2B cells was not reported, and the description of transformation of cells is very limited,
missing any narrative on how the cell culture changed during the 6-month incubation, whether the
cell growth was evaluated in the process, or how often cultures had to be subcultured, which are all
good practices to report for development of new cells by long-term exposures. Small, medium, and
large colonies were reportedly used for implantation in the animal study, but only a large colony
from the soft agar assay has been reportedly isolated and maintained as a cell culture, indicating
inconsistency in reporting. The growth of tumors seems to be too high for 6-day time after
implantation. The concentrations of Cr(VI) at which transformation of cells was achieved were
comparable to those used in similar studies, equivalent to 0.037 mg/L and 0.074 mg/L of sodium
dichromate dihydrate [0.013 and 0.026 mg/L Cr(VI)].
Sanchez-Martin etal. (2015)
This study examines changes in (1) histopathology, (2) IHC markers of proliferation (Ki-67)
and DNA damage (p-yH2AX), and (3) expression of selected genes by qPCR in the liver and in the
proximal (PSI) and distal (DSI) "sections of gastrointestinal tract" of the C57BL/6 J mice exposed to
Cr(VI) in drinking water. Mice were exposed to 0,19,191, and 1,919 |ig/L Cr(VI) for 60 days and
subsequently to the same concentration of Cr(VI) in drinking water and 0-125 mg/kg/day B[a]P for
90 days.
This summary addresses the gene expression analysis reported in the study by Sanchez-
Martin etal. (2015). Gene expression changes reported in this study are of little informative value
due to the reasons indicated below. Even though the study suggests interesting patterns of gene
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expression with discordant expression changes across anatomical sites and exposure levels, an
evaluation is not justified because of considerable reporting deficiencies and the high risk of bias.
• Changes in gene expression are reported only in the form of a heat map. Information about
the color coding of expression changes in the heat map is incomplete. No expression values
and no statistical tests for significance of their differences are reported.
• The study is not a whole-genome ("omics") study, and it deals only with expression of
selected genes with limited justification for their selection.
• The sample size appears to be 4 animals per exposure level (2 animals of each sex). This
design allows identification of only differentially expressed genes that do not show
substantial sex differences in response to the Cr(VI) exposure.
• The study uses qPCR for the evaluation of expression of selected genes in the proximal (PSI)
and distal (DSI) "sections of the gastrointestinal tract" These sections are not sufficiently
characterized, which allows ambiguous interpretation. The "proximal" and "distal" are
indicated to reflect position of the section relative to the stomach, but this does not add
sufficient information to ascertain whether only the small intestine or also the colon were
examined and which specific segments of these anatomical structures were sampled for
downstream analysis.
• The study employs GAPDH as an endogenous control for normalization of the gene
expression. The choice of GAPDH is not justified and its invariant expression in the liver and
intestine across all exposures has not been demonstrated. There is a lack of confidence for
appropriateness of the use of GAPDH as an endogenous control in this study.
• Primers used in the qPCR studies are not reported. Although this information is mentioned
as being provided in the supplemental data, the information on the sequences and origin of
primers (references, software used for their design, experimental validation of primers) is
missing.
• The authors indicate the use of the AACt method for calculation of gene expression from the
qPCR data. Since no information is given on the validation of primers and amplification
efficacy for the target genes and an endogenous control, the use of AACt method is not
supported and this method might not be appropriate in this study.
Clancy etal. (2012)
The source of BEAS-2B cells is reported; description of transformation of cells is sufficiently
reported; growth media and exposure conditions were properly reported; exposure was performed
at minimally cytotoxic concentration (0.5 |iM) of potassium chromate [0.1 |iM Cr(VI)], which does
not seem to have been determined in this study but is consistent with other reports. The form of
Cr(VI) and its source are reported (potassium chromate, Sigma). The cells have been altered by
Cr(VI)-mediated transformation (morphology, growth pattern in soft agar), and so sensitivity is not
an issue. Methods for mRNA analysis are succinctly described and refer to manufacturers'
protocols. qPCR validation relied on the GAPDH gene as an internal control, which is a frequent
practice in the field, but not appropriate without justification (the justification has not been
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provided in this report). Differentially expressed genes were selected based on t-test p-value of 0.05
and a fold-change cut-off of 1.50. The lack of proper qPCR validation does not invalidate a
microarray study using systems biology approaches.
Chen etal. f20021
Sources of BEAS-2B and MEF cells were provided; media composition was reported; sources
of vectors pCR-FLAG-IKK, pCR-FLAG-IKK-KM, pcDNA3-myc-IAPl, and pEGFPluc were indicated.
The Cr(VI) compound used for this study, however, was not specified. Exposure levels of Cr(VI)
were adequately described. For assays other than cytotoxicity/viability, conditions were adequately
selected to avoid convolution of the study outcomes with cell death. Likewise, exposure conditions
(concentrations, times) were chosen well with respect to sensitivity of outcome detection, as
evidenced by demonstrated differences between Cr(VI)-exposed and solvent control cells.
The microarray study employed (1) an old expression array design, (2) only a fold change-
based identification of differentially expressed genes, and (3) an unknown number of biological or
technical replicates. RT-PCR was used instead of qPCR for validation of selected genes identified by
microarray analysis, and endogenous control 7S RNA was used without justification. RT-PCR primer
design software, sequences, annealing sites, and amplicon lengths were reported. Reverse
transcription conditions were reported but the reverse transcriptase used in the reaction was not
described. RT-PCR conditions were reported.
Methods used in this study complemented each other and, in this way, compensated for the
identified deficiencies of individual experiments. For instance, deficiencies of microarray
experimental design and analysis were compensated by validation RT-PCR and demonstrated IAP-
mediated inhibition of cell death in cells exposed to Cr(VI). The somewhat surprising lack of
specification of Cr(VI) compound used in this study can be perceived as a critical deficiency
rendering most of the study uninformative (at least experiments that employed Cr(VI)-exposure).
Izzottietal. f20021
Izzotti etal. f20021 analyzed gene expression in Sprague-Dawley rats intratracheally
exposed to sodium dichromate5 at the dose of 0.25 mg/kg [0.09 mg/kg Cr(VI)] body weight for 3
days and sodium chloride control, using in-house radioactively labeled cDNA microarrays that
probed expression of 216 genes tested in duplicates and 5 house-keeping genes. Gene expression
was examined in lungs and livers of SDD-exposed and NaCl-exposed (control) groups. Genes were
considered differentially expressed if the fold change exceeded 2. This study identified 56 genes
overexpressed in lungs of Cr(VI)-exposed rats, which included glutathione metabolism-related
5As with many studies, the compound may be referred to as "sodium dichromate" (NazCnOy), when the
compound is administered in an aqueous solution and the mass units are based on sodium dichromate
dihydrate (NazCnOy 2H2O). Unless otherwise noted, the conversion factor for sodium dichromate dihydrate
(0.349) was used to convert Cr(VI) units for studies labeled as either sodium dichromate or sodium
dichromate dihydrate.
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genes, membrane channels/transporters, cell signaling molecules, cell cycle-related molecules,
stress response/protein folding-related genes, as well as DNA synthesis/DNA repair and apoptosis-
related genes. These expression data are consistent with generation of reactive oxygen species, cell
proliferation, and inhibition of apoptosis. Protein misfolding-related genes are likely reflecting
oxidative protein damage and increased protein synthesis. The study found no changes in gene
expression in livers of Cr(VI)-exposed mice relative to control animals, which indicated no
significant systemic effects after intratracheal exposure. Although these study results support
findings of other toxicogenomic and non-omic mechanistic studies, the study likely provides an
incomplete picture of molecular changes induced by Cr(VI) exposure. This is because (1) it
evaluated expression of a limited range of genes using in-house produced microarrays, and (2) the
dose used in this study [0.09 mg/kg Cr(VI)] failed to induce lung tumors as in other studies in
Sprague-Dawley rats exposed 5 times per week over 30 months (Steinhoffetal.. 1986).
C.3.4.4. Toxicogenomic analyses
Toxicogenomic analyses of genome-wide changes in gene or protein expression in response
to Cr(VI) exposure can help inform carcinogenic signaling pathways relevant to lung and GI cancer.
Four studies were fully evaluated in HAWC: one human study (Hu etal.. 2017). two in vivo animal
studies (Chappell etal.. 2019: Kopec etal.. 2012b). and one in vitro study using the human BEAS-2B
cell line (Huang et al.. 2017)). with one evaluation, Kopec et al. (2012b). representing five studies
that used the same microarray dataset (see details below). An independent analysis of the
published in vivo toxicogenomic data by Kopec et al. f2012bl was conducted by Mezencev and
Auerbach f20211 and is described below.
In vivo studies
One medium confidence toxicogenomic study was identified in humans. Hu etal. (2017)
performed proteomic analysis of sera in male workers recruited from a chromate production facility
in China. Primary limitations of this study were the lack of description of participant selection and a
relatively small sample size. There were two stages of analysis; first, 25 exposed and 16 unexposed
workers underwent "proteomics technology and bioinformatics analysis," and second, 41 exposed
and 25 unexposed workers underwent a validation analysis to confirm findings from the first stage.
Sixteen significantly enriched pathways were identified related to innate immune system function,
extracellular matrix organization, platelet-related processes, and metabolism (Hu etal.. 2017).
Notably, the increased abundance of SHH, a gene that promotes tumor growth and metastasis if
overactivated, in the sera of Cr(VI) exposed workers is consistent with the potential role of SHH in
Cr(VI)-mediated carcinogenesis identified by other toxicogenomic studies fMezencev and Auerbach.
2021: Huang etal.. 2017).
Six of the eight in vivo toxicogenomic analyses in animals after oral exposure to Cr(VI) were
published by the ToxStrategies firm. A high confidence study, Kopec et al. (2012b). conducted an
analysis of gene expression in the oral mucosa and duodena in tissues collected from female Fischer
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344 rats and female B6C3F1 mice exposed to sodium dichromate dihydrate (SDD) in drinking water
as described in the original studies by Thompson et al. f2012c: 2011bl Because the same dataset
was used in four other studies published by this group repeated (Rager etal.. 2017: Thompson et al..
2016: Kopec etal.. 2012a: Thompson et al.. 2012b). this study evaluation (in HAWC) specific to the
original animal studies and the microarray dataset generation was not repeated.
Kopec etal. f2012al reported gene expression changes in mouse intestinal epithelia after
8 days or 91 days corresponding to oxidative stress, xenobiotic metabolism signaling, glutathione
metabolism, cell cycle progression, lipid metabolism, and immune response pathways. In addition,
canonical DNA repair pathways (i.e., NER, MMR, and BRCA1) were enriched for genes differentially
expressed in the duodena of mice exposed to SDD for 8 days; however this response was absent in
duodena of mice exposed for 90 days and in jejuna of mice exposed for both time periods (Kopec et
al.. 2012a). A subsequent publication using the same mouse dataset reported gene expression
changes indicating reduced apoptosis at day 91 and increased cell growth and proliferation at days
8 and 91 (Rager etal.. 2017). Cancer-related signaling identified from the 8-day exposure data
largely reflected increased expression of matrix metalloproteases (Mmp2, Mmp7, Mmp9, MmplO,
and Mmpl3). MmplO and Mmpl3 showed dose-dependent upregulation atday 91, which indicated
cell migration, tissue remodeling and angiogenesis. In the same study, a parallel analysis of
ToxCast/Tox21 and Comparative Toxicogenomics Database (CTD) data for Cr(VI) compounds
showed a variety of differences when comparing these in vitro results to the in vivo results for this
particular dataset; for example, some pathways associated with DNA damage (e.g., p53) were only
activated in vitro (Rager etal.. 2017). In a toxicogenomic study in duodena of rats exposed to SDD in
drinking water at concentrations up to 180 mg/L, functional analysis revealed enrichment of cell
cycle, DNA metabolism, DNA replication, and DNA repair pathways at day 8 but not day 91 (Kopec et
al.. 2012b). A comparative analysis of the same datasets for rats and mice showed a strong dose-
response relationship of the number of differentially expressed genes in the duodenum in both
species when total Cr tissue levels exceeded lOmg/kg, with a minimal transcriptomic response in
the oral mucosa evidenced by very few gene expression changes showing dose-responsive statistical
significance (Thompsonetal.. 2016).
Additional reports published by this group included a reanalysis of the mouse dataset,
limited to 7 of 23 gene categories, which suggested a higher similarity in Cr(VI) induced gene
expression changes in the mouse small intestine to expression changes induced by four
nonmutagenic carcinogens versus four mutagenic carcinogens (Thompson etal.. 2012b). The
comparison dataset represented gene expression in rat liver reported by Ellinger-Ziegelbauer et al.
f2005I The limited nature of the analysis (cross-species, cross-tissue and cross-platform
comparison of gene expression data for the chemical of interest using a single in vivo study
annotated for four mutagenic and four nonmutagenic carcinogens) make the results difficult to
interpret
Another medium confidence gene expression comparison study by the same group using a
new dataset reported significant overlap between DEGs induced by oral exposure Cr(VI) and two
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fungicides, captan and folpet, that also cause intestinal tumors in mice fChappell etal.. 20191.
Common pathways modulated by Cr(VI) and the higher concentrations of captan or folpet include
those involving HIF1, API, PPAR, mTOR 4, and Peroxisome (Chappell et al.. 2019). Although these
authors suggest the commonalities between two nonmutagenic compounds and Cr(VI) imply a
nonmutagenic MOA for Cr(VI)-induced mouse intestinal tumors, concordance among gene
expression across these three toxicants does not provide solid evidence for ruling out mechanisms
that are not shared by all these toxicants. The study was also limited by a single timepoint and
reporting inconsistencies for pathways found to be unique for duodena of Cr(VI) exposed mice.
An independent analysis of the 8- and 91-day B6C3F1 mouse data subset published by
ToxStrategies, Inc. (Rager etal.. 2017: Kopec etal.. 2012b: Kopec etal.. 2012a: Thompson etal..
2011b) that was deposited in the Gene Expression Omnibus implicated activation of oncogenic
signaling (MYC, MYCN, EGFR, ERBB2, TRIM24) and inhibition of tumor suppressors (CDKN2A,
STAT1), which support sustained cell proliferation in the duodenum fMezencev and Auerbach.
2021) (see Appendix C.3.4.2). Similarly, a parallel analysis of enrichment of the cancer "hallmark"
and oncogenic signature gene set collections from the Molecular Signatures Database (MSigDB)
identified multiple molecular changes in duodena of mice orally exposed to Cr(VI) known to be
relevant for carcinogenesis, including c-Myc targets, E2F targets, and alterations in G2M checkpoint
and DNA repair pathways. Gene sets enriched in the duodena of mice exposed for 8 days support
angiogenesis, impaired apoptosis, and epithelial-mesenchymal transition, which also represent
hallmarks of cancer. Enrichment of the cholesterol homeostasis gene set found for 8-day and 90-
day exposures at several exposure levels implies activation of cholesterol biosynthesis that is
associated with intestinal crypt hyperproliferation and tumorigenesis. Enriched gene sets from the
Oncogenic Signature collection imply oncogenic activation of KRAS, SRC, SHH, and PI3K/AKT/mTOR
signaling and inactivation of signaling mediated by tumor suppressors PTEN and RB (Mezencev and
Auerbach. 2021).
The analyses by Mezencev and Auerbach (2021) (see Appendix C.3.4.2) also indicate
oxidative stress in duodena of mice exposed to Cr(VI) for 91 days through inferred activation of the
NFE2L2 upstream regulator. This gene encodes a redox-sensitive transcription factor NRF2, which,
upon activation, accumulates in the nucleus where it regulates expression of genes involved in the
oxidative stress response (He etal.. 2020). In addition, a collection of 26 genes known to be
responsive to oxidative stress was also significantly enriched in duodena of mice exposed to Cr(VI)
for 91 days. This is in contrast with data after an 8-day exposure, which indicate that this collection
of genes was enriched in control mice. As a result, in mice exposed to Cr(VI), lower amounts of ROS
are inferred in duodena of mice exposed for 8 days, but higher amounts of ROS are inferred in
duodena of animals exposed for 91 days.
Taken together, the results support duodenal carcinogenicity of Cr(VI) ingested in drinking
water in mice through activation of oncogenic signaling, inactivation of signaling mediated by tumor
suppressors, sustained cell proliferation, oxidative stress, impaired apoptosis, and tissue
remodeling.
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A notable result of the analyses by Mezencev andAuerbach f20211 was the identification of
a potential role for the CFTR (cystic fibrosis transmembrane conductance regulator) in
carcinogenesis in mouse small intestines. Toxicogenomic analysis of Kopec etal. (2012b; 2012a)
datasets by Mezencev andAuerbach (2021) suggested inactivation of CFTR in mice exposed to
concentrations of Cr(VI) as low as 0.1 mg/L for 8 days. This inactivation does not appear to be
attributable to tissue damage, which was observed in these same animals following subchronic
exposure to Cr(VI) concentrations >60 mg/L (Thompson etal.. 2011b). Therefore, suppression of
CFTR activity might represent an early effect of Cr(VI) exposure that contributes to the carcinogenic
process. Considering the recently reported tumor-suppressor role of the CFTR gene in mouse and
human intestinal cancers (Than etal.. 2016). this finding expands the range of plausible mechanisms
that could be operative in Cr(VI)-mediated carcinogenesis of intestinal and possibly other tissues,
which include mutagenesis, inflammation, or cytotoxicity followed by regenerative proliferation in
the carcinogenic MOA of Cr(VI).
Another toxicogenomic study, a limited short-term intratracheal study in rats, was
identified. Izzotti etal. f20021 observed gene expression changes in the lung consistent with the
generation of reactive oxygen species, cell proliferation, and inhibition of apoptosis. The study
found no changes in gene expression in livers of Cr(VI)-exposed mice relative to control animals,
which indicated no significant systemic effects after 3 days of intratracheal exposure (Izzotti etal..
2002). The study was determined of limited value, however, due to low exposure levels and to its
limited range of genes evaluated by in-house produced microarrays of an old design and therefore
was not considered for evaluation in HAWC.
In vitro studies
Four toxicogenomic in vitro studies were also identified as particularly informative for
Cr(VI)-induced carcinogenicity and cellular transformation. All four studies were partially
evaluated (Appendix C.3.4.3), but only one, Huang etal. (2017). was fully evaluated in HAWC. This
study was found to be high confidence for all assays reported in the study, including cell
transformation, Western blotting, and an siRNA knockdown, but was determined to be
uninformative for qPCR findings due to reporting issues and lack of optimization for this assay.
Clancy etal. (2012) demonstrated transformation of bronchial epithelial BEAS-2B cells
exposed to 0.5 [J.M Cr(VI) for 4 weeks that coincided with differential expression of genes that
showed enrichment in several pathways related to cancer development These included cell
mobility and migration, TGF(3 receptor signaling, MAP kinase activity, regulation of apoptosis,
response to hypoxia, and pathways involved in pancreatic cancer and small-cell lung cancer (Clancy
etal.. 20121. Transformation of BEAS-2B cells using a similar study design (0.5 [J.M Cr(VI) for
4 weeks) was confirmed by a separate group that also demonstrated acquisition of a proliferative,
migratory, invasive, andtumorigenic phenotype by Cr(VI)-transformed BEAS-2B cells (Huangetal..
2017). In this study, Cr(VI)-mediated transformation was associated with activation of the
hedgehog (Hh) signaling pathway, which interplays with multiple oncogenic pathways, and Gli2-
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mediated inhibition of autophagy. Inhibition of autophagy by Hh signaling activation has been
found in the lung cancer cell lines, which support biological relevance of this mechanistic finding.
Likewise, a study by Lu etal. (2018a) demonstrated the ability of Cr(VI) to transform BEAS-2B cells
exposed to 0.125 [J.M or 0.25 [J.M of Cr(VI) for 6 months, which displayed tumorigenicity after
subcutaneous injection in nude mice. Proteomic analysis revealed downregulation of STK11
encoded by the tumor-suppressor gene LKB1, suggesting possible activation of Wnt/(3-catenin and
mTOR signaling pathways that are involved in the development of various cancers. A fourth study
demonstrated the importance of NF-kB activation for survival and transformation of cells exposed
to Cr(VI), with upregulation of transcriptional targets cIAPl and cIAP2 fChen etal.. 20021.
C.3.5. Susceptible Populations
C.3.5.1. Genetic polymorphisms
Summary of evidence in humans
Genetic polymorphisms can alter individual susceptibility to health effects of environmental
exposures, including chromium. Thirteen studies in humans were identified that evaluated genetic
polymorphisms in relation to chromium exposure and cancer-related outcomes (mechanistic or
apical). The study findings are summarized in Table C-65.
Seven studies evaluated genetic polymorphisms in relation to mechanistic outcomes
relevant to cancer (e.g., mutations, genome instability). Of these, one focused on micronuclei, with
interaction effects reported for some genes related to DNA repair and tumor suppression (XRCC3,
BRCA2, NBS1) (Long etal.. 2019). Two studies of the same study population reported increased
chromosomal aberrations among welders with polymorphisms of one gene that encodes DNA repair
enzymes (XRCC1) but not others (XPC, XPD, EPG, XRCC3, hOGGl) (Halasova etal.. 2012: Halasova et
al.. 2008). Similarly, polymorphisms in XRCC1 were also associated with increases in DNA strand
breaks among welders (Iarmarcovai et al.. 2005) and measures of DNA damage such as olive tail
moment, tail length, and tail DNA% among electroplating workers fZhang etal.. 20121. Finally, two
studies of electroplating workers from another study population evaluated potential differential
effects on sister chromatid exchange due to polymorphisms in genes related to detoxification
(GSTM1, GSTT1); interaction effects were detected for GSTT1 (Wu etal.. 2001) in one study but not
the other (Wu etal.. 2000).
Four studies evaluated genetic polymorphisms in the context of cancer. One study identified
an increased risk of lung cancer in individuals with certain polymorphisms in XPD (Sarlinova etal..
20151. which is involved in nucleotide excision repair. Three studies approached the question in a
different way, probing the frequency of certain gene variants in cancer cases. Polymorphisms in the
surfactant protein B gene were found to be more common in small-cell carcinomas from workers
exposed to Cr(VI) compared to referents (Ewis etal.. 2006). In another study, the odds of hMLHl
polymorphisms was found to be elevated in chromate-related lung cancer cases compared to
hospital-matched referents (Halasova etal.. 2016). Finally, one study evaluated microsatellite
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1 instability (operationalized as replication error (RER), defined as microsatellite instability at two or
2 more loci) among individuals with lung cancer; study authors report increased frequency of RER
3 among cases with chromate exposure compared to those without chromate exposure as well as an
4 association between duration of chromate exposure and lung cancer cases with RER compared to
5 those without RER (Hirose etal.. 2002).
6 Although it is difficult to draw conclusions based solely on the human evidence, the existing
7 data suggest that genetic polymorphisms may play a role in cancer susceptibility of individuals
8 exposed to Cr(VI), and the impact of polymorphisms relevant to DNA damage and detoxification
9 pathways in particular can provide important insight on the cancer MOA for Cr(VI).
Table C-65. Studies of genetic polymorphisms in humans occupationally
exposed to Cr(VI)
Study overview
Exposu re
Resu Its
Comments
Reference
Cases: workers in
chromate factory
who developed
lung cancer (n = 31)
Additional case
groups: samples
from lung
adenocarcinoma
(n = 38) and
squamous cell
carcinoma (n = 46)
from individuals
never employed in
chromate-related
industries
Controls 1: workers
in chromate factory
who did not
develop lung cancer
(n = 26)
Controls 2:
randomly selected
healthy individuals
(n = 89)
Mean (SD) yr of chromate
exposure in the workplace:
cases = 22.8 (6.56) yr;
controls = 20.1 (7.71) yr
1" SP-B gene
variants in
chromate case
group & in
chromate small cell
carcinoma
compared to
referent small cell
carcinoma
SNP genotyping of
Surfactant protein B
gene.
No evaluation for
potential confounding.
Ewis et al.
(2006)
Cross-sectional
study, Slovak
Republic.
Exposed: n = 73
male welders
Referent: n = 71
male controls
(administrative
officers and
hospital employees)
Exposure to Cr(VI) inferred
based on occupation.
Mean ± SD duration of
occupational exposure was
10.2 ± 1.7 yr.
Also measured Cr in blood.
Exposed workers had average
values about twice as high as
referent group (stated to be
significantly different).
1" Cas in individuals
with Gln/GIn
genotype compared
to Arg/GIn or
Arg/Arg genotypes
in XRCC1
Arg299Gln; more
pronounced in Cr-
exposed workers
Main limitations are
related to lack of
description (e.g., for
participant selection)
and lack of evaluation
of confounders aside
from smoking.
SNP genotyping of
genes encoding DNA
Halasova et al.
(2012)
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Study overview
Exposu re
Resu Its
Comments
Reference
Mean ± SE was 0.07 ± 0.04 vs.
0.03 ± 0.007 nmol/L.
repair enzymes (XRCC1,
XPC, hOGGl).
Cross-sectional
study, Slovak
Republic.
Exposed: n = 39
male welders
Referent: n = 31
male controls
(source not given)
Exposure to Cr(VI) inferred
based on occupation.
Mean ± SD duration of
occupational exposure was
10.2 ± 1.7 yr.
Also measured Cr in blood.
Exposed workers had average
values about twice as high as
referent group. Mean ± SE
was 0.07 ± 0.04 vs.
0.03 ± 0.007 nmol/L.
1" Cas & CTAs in
individuals with
Gln/GIn genotype
compared to
Arg/GIn or Arg/Arg
genotypes in XRCC1
Arg299Gln
Main limitations are
related to sample size,
unclear differentiation
between exposure
groups, and lack of
description (e.g., for
participant selection).
SNP genotyping of
genes encoding DNA
repair enzymes (XRCC1
and XRCC3).
Halasova et al.
(2008)
Cases: chromium-
exposed lung
cancer patients
(n = 45)
Controls: hospital
patients with no
previous malignant
disease in medical
records or family
history; matched on
age, gender, and
ethnicity (n = 61)
Mean(SD) exposure time in
cases: 9.3 (1.7) yr
1" odds of hMLHl
polymorphisms in
lung cancer cases
SNP genotyping of DNA
repair genes XRCC3,
hMLHl, and hMSH2.
No detailed
information on
exposure/occupational
history nor were
exposure levels
quantified; no
consideration of
confounders.
Halasova et al.
(2016)
Exposed: chromate
workers with lung
cancer (n = 28;
n = 38 tumors)
Referents: lung
cancer patients
without chromium
exposure (n = 26;
n = 26 tumors)
Chromate workers exposed to
chromium for mean (SD)
22.9 (6.9) yr
1" frequency of RER
in lung cancers with
chromate exposure
compared to
without chromate
exposure
^duration of
chromate exposure
in chromate lung
cancer cases with
RER compared to
those without RER
-t MSI with -t
duration of
chromate exposure
No difference in
frequency of LOH in
tumors with or
without chromate
exposure
Multiple samples taken
from some chromate
exposed patients—
these would not be
statistically
independent.
No consideration of
confounders.
Hirose et al.
(2002)
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Study overview
Exposu re
Resu Its
Comments
Reference
Cross-sectional
study, France.
Exposed: n = 60
male welders from
36 workshops in
the "building trade"
Referent: n = 30
office workers
recruited from
"general or
administration
services" without
history of
occupational
exposure to
welding fume or
other physical or
chemical agents
Exposure to Cr(VI) inferred
based on occupation. State
that <5% of welding was done
on stainless steel, which raises
concern that total Cr
measured in blood and urine
may be attributed to Cr(lll)
exposure.
Also measured total Cr in
blood and urine. Cr levels in
blood and urine were higher
among both groups of welders
compared with controls
(means 129 to 145, compared
with 92 ng/L), and urinary
chromium was higher among
welders working without
smoke extraction systems.
SNP genotyping of
DNA repair genes,
XRCC1 and XRCC3
XRCC1 variant allele
coding Gin amino
acid at position 399
was associated with
a higher number of
DNA strand breaks
Main limitations are
related to lack of
description (e.g., for
participant selection,
analysis), unknown
contribution of Cr(VI) to
Cr exposure and known
co-exposures to other
metals.
1" mean BN % in
lymphocytes of welder
compared to controls.
larmarcovai et
al. (2005)
Cross-sectional
study, China.
Exposed: n = 120
chromate exposed
workers working at
a chromate
production facility
Referent: n = 97
unexposed workers
at same facility
("without contact
history of harmful
substances")
Exposure to Cr(VI) inferred
based on occupation.
Also measured Cr in whole
blood. Cr levels were
significantly higher among
exposed compared with
controls, indicating delineation
of exposure. Median
(interquartile range) of Cr in
whole blood was 2.81 (3.86)
and 0.99 (1.21) ng/L in
exposed and referent groups,
respectively.
Interaction
between
micronuclei
frequency and SNPs
in the following
genes: XRCC3,
BRCA2, NBS1
Main limitations are
related to lack of
description (e.g., for
participant selection
and statistical analysis)
SNP genotyping of
XRCC3, BRCA2, NBS1.
Long et al.
(2019)
Exposed: workers in
a chromate factory
(n = 141)
Referents: farmers
from area approx.
90 miles from
chromate factory
(n = 54)
Full shift (8 h) personal
exposure sample taken; flow
2.1 min"1. Median (IQR) of air
Cr(VI) in workers = 17.8 (39.5)
Hg/m3; in referents = 0.06
(0.12) ng/m3
Blood samples collected;
analyzed with graphite furnace
atomic absorption with
Zeeman background
correction; Median (IQR) of Cr
in workers = 6.0 (7.86); 2.64
(2.11)
1" accumulation of
Cr in RBCs per air
Cr(VI) exposure
among wild type
Band 3 Memphis
genotype
SNP genotyping of
genes involved in anion
transport proteins.
No major concerns with
study quality, except
for minimal
information on
participant selection.
Qu et al. (2008)
Cases: chromium-
exposed lung
cancer patients
(n = 50)
Controls:
Individuals with no
Mean (SD) exposure time in
cases: 9.3 (1.7) yr
1" risk of lung
cancer with the
following
genotypes: XPD
Lys/Gln+XPC
Lys/GIn and XPD
SNP genotyping of
XPC(rs2228001), XPD
(rsl3181,)
XRCCl(rs25487), and
hOGGl (rsl052123).
Sarlinova et al.
(2015)
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Study overview
Exposu re
Resu Its
Comments
Reference
previous malignant
disease in medical
records or family
history; age,
gender, & ethnicity
matched to cases
(n = 69)
Lys/Gln+XPC
Gln/GIn
No quantitative
assessment of
exposure; no
adjustment for missing
data.
Exposed 1:
residents of areas
contaminated with
Cr(VI),
asymptomatic with
regard to dermal
irritation (n = 108)
Exposed 2:
residents of areas
contaminated with
Cr(VI), reporting
dermal irritation
(n = 38)
Referents:
asymptomatic
residents of area
with no history of
Cr(VI)
contamination
(n = 148)
Mean (SD) residing at
contaminated site (among
exposed group):
24.17 (15.23) yr
1" OR dermal
irritation in GSTM1
null genotype when
comparing exposed
symptomatic
individuals to
exposed
asymptomatic
individuals
1" OR dermal
irritation in GSTT1
null genotype when
comparing exposed
symptomatic
individuals to
control
asymptomatic
individuals
SNP genotyping of
genes (GSTT1, GSTM1,
NQOl and hOGGl)
involved in Cr(VI)
reduction and fate in
cell.
Only adjusted for
smoking, not other
confounders.
Sharma et al.
(2012)
Exposed: chromium
workers (n = 35)
Referents: age and
gender matched
controls (n = 35)
Exposure duration ranged
from 2 to 14 yr with a mean
(SD) of 6.5 (4.2) yr.
1" sister chromatid
exchanges in
exposed group;
association with
work duration;
synergy with
smoking
1" high frequency
cells in exposed
groups; synergy
with smoking
SNP genotyping for
GSTM1 and Tl.
Limited sample size.
Only adjusted for
smoking, not other
confounders.
Wu et al. (2000)
Exposed: chromium
platers (n = 35)
Referents: healthy
subjects with no
history of disease
or previous
exposure to
chromium or other
metals (n = 35)
The mean duration of
employment was 6.5 yr.
Personal exposure monitoring
for 8-h working shift
(1.71/min); blood and urine
samples collected at end of
shift and analyzed with atomic
absorption spectrophotometry
Individual time-weighted
average range: 0.049-1.130
mg/m3
1" sister chromatid
exchange and
percent high
frequency cells in
exposed group
compared to
controls
SNP genotyping for
GSTM1 and Tl.
Personal air sampling
only obtained for n = 10
individuals in the
exposed group.
Unable to draw
conclusions about
effect of genotype due
to small sample size.
Wu et al. (2001)
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Study overview
Exposu re
Resu Its
Comments
Reference
Exposed:
Air-Cr determined by graphite
1" chromium
Polymorphisms in
Zhang et al.
electroplating
furnace atomic absorption
concentrations in
XRCC1 and Arg399Gln
(2012)
workers (n = 157)
spectrophotometer
erythrocytes among
associated with
Referents:
exposed compared
Cr-induced DNA
individuals without
to referents
damage
exposure to
1" Olive tail
SNP genotyping for
chromium or
moment, tail
DNA repair genes:
known
length, & tail DNA%
XRCC1 Arg399Gln,
physical/chemical
among exposed
XRCC lArg 194T rp, E RCC
genotoxic agents
compared to
1C8092A, ERCC5
(n = 93)
referents
Hisll04Asp, ERCC6
Gly399Asp,
GSTPlllel05Val, OGG1
Ser326Cys, XPC
Lys939Gln,
XPDLys751Gln.
Limited adjustment for
confounders (including
diet).
Potential coexposures
to other metals in the
workplace.
discrepancy between table and text in the original publication. Values from text noted above; values from table
reported as 23.8 (7.0) years.
Carriers of the cystic fibrosis mutant allele
Cystic fibrosis is an inherited autosomal recessive disorder caused by inactivating mutations
in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which codes for the CFTR
anion channel protein. CFTR regulates the secretion of chloride and bicarbonate. Loss of CFTR
function causes abnormal mucus production, which affects every organ in the body, particularly the
lung and GI tract (De Boeck. 2020). Cystic fibrosis patients have a higher risk of developing
colorectal cancer (Miller etal.. 2020: Scott etal.. 20201. Tumor suppressor status of the CFTR gene
has been suggested based on the results of epidemiological, clinical, and experimental studies
(reviewed in Zhang etal. f201811. In a mouse model with an intestinal-specific CFTR gene knock-
out, Than et al. (20161 demonstrated that CFTR-deficient mice have a significantly increased risk of
development of tumors in the colon and small intestines. In addition, the loss of CFTR activity was
shown to enhance intestinal tumorigenesis in ApcMin mice that carry mutated tumor-suppressor
gene adenomatous polyposis coli (APC). These findings demonstrate that impairment of CFTR leads
to tumorigenesis in the mouse small intestine.
The analyses by Mezencev and Auerbach f20211 fsee C.3.13.2) of the toxicogenomic data
reported in Kopec et al. (2012b; 2012a) from mice exposed to Cr(VI) have identified a potential role
for CFTR in the carcinogenic effects of Cr(VI). These data indicate that CFTR was inactivated in mice
exposed to Cr(VI) levels as low as 0.1 mg/L in drinking water for 8 days. This inactivation does not
appear to be attributable to tissue damage, which was observed in these animals following
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subchronic exposure to Cr(VI) concentrations >60 mg/L fThompson etal.. 2011bl. Therefore,
suppression of CFTR activity might represent an effect of Cr(VI) exposure that contributes to the
carcinogenic process.
Tumorigenicity of impaired CFTR activity in animal models supports the relevance of the
Cr(VI)-mediated inactivation of CFTR for the development of small intestinal tumors in mice
exposed to Cr(VI) in drinking water. These findings indicate the identification of vulnerable groups,
such as APC mutation carriers and carriers of the mutated CFTR allele, that can be more sensitive to
the Cr(VI)-mediated carcinogenicity. This reasoning likely extends to humans, because (1) CFTR
reportedly acts as a tumor-suppressor in human colon fThan etal.. 20161 and (2) germline
mutations in the APC gene or its regulatory sequences are known to cause familial adenomatous
polyposis (FAP) in humans. FAP is associated with high risk of colon cancer and increased risk of
cancers at other sites, including the duodenum, thyroid gland, and stomach (Tasperson etal.. 2017:
Leoz etal.. 20151.
In the United States alone, more than 10 million people are carriers of a mutated CFTR allele
that confers an approximate 50% reduction in CFTR expression levels. Although these individuals
do not develop cystic fibrosis, the deficit in CFTR function has been shown to lead to an increased
risk for several conditions associated with the disease, including colorectal cancer (OR = 1.44,
95% CI: 1.01-2.05) (Miller etal.. 2020). CFTR suppression induced by low Cr(VI) exposures in
drinking water can be expected to occur in all exposed populations, but a more significant effect
would be expected in humans already producing low levels of this protein. Moreover, enhancement
of tumorigenicity of the APC mutations by CFTR inactivation implies that carriers of these mutations
might be more susceptible to the tumorigenicity induced by events that inactivate CFTR, including
Cr(VI) exposure. Based on the analogy with the ApcMin mice study, humans affected by germline
APC mutations can be reasonably expected to be more vulnerable to carcinogenicity mediated by
Cr(VI) or other toxicants that can inactivate CFTR.
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C.4. SUPPORTING EVIDENCE FOR EXPOSURE TO THE GENERAL
POPULATION
C.4.1. Drinking Water Data from the Third Unregulated Contaminant Monitoring Rule
Cr(VI) was among 30 contaminants selected for monitoring at public water systems (PWS)
for the Third Unregulated Contaminant Monitoring Rule (UCMR3) between 2013 and 2015. A PWS
is a network of pipes and conveyances constructed to provide water for human consumption (U.S.
EPA. 2006a. b). Small systems, serving 10,000 or fewer people, account for more than 97% of the
total number of PWSs, while large systems, serving more than 10,000 people, account for the
remaining 3% fU.S. EPA. 2006a. b). A majority of the U.S. population is served by large PWSs (nearly
90% fU.S. EPA. 2006a. b)), and all of them (approximately 4,200) were tested under UCMR3. For
small water systems, approximately 800 systems were randomly selected and used as a
representative sample (U.S. EPA. 2012b). Small water systems were omitted from analyses
presented in this section. Although most of the public water systems in the United States have
reported Cr(VI) concentrations below 1 |ig/L, the highest concentrations have approached the MCL
(for total chromium) of 100 |ig/L. This is 50 times lower than the lowest concentration used in the
NTP f20081 bioassay (5 mg/L = 5,000 |J.g/L). When converting to dose, the lowest doses in rats and
mice were 0.2 mg/kg-day and 0.3 mg/kg-day, respectively. By BW3/4 scaling,6 this would adjust to
0.057 mg/kg-day human equivalent dose for rats and 0.05 mg/kg-day for mice. A standard 70-kg
reference human ingesting 2 liters of water/day at 100 |ig/L (0.05 mg/L) would ingest a Cr(VI) dose
of 0.0029 mg/kg-day. Therefore, the lowest NTP doses are approximately 20 times higher than a
potential human drinking water dose at 100 |ig/L. This is only an illustrative comparison and does
not account for differences in Cr(VI) reduction.
Table C-66. Statistical summary of UCMR3 chromium (VI) concentrations in
large public water systems (PWS)
Parameter (units)
Statistic3
Total number of facilities reporting
3,927
Number of facilities >MRL
3,573
Number of measurements
45,712
Average PWS mean (ng/L)
0.485
Maximum PWS mean (ng/L)
42.31
Maximum measured value (ng/L)
97.38
25th %tile of PWS means (ng/L)
0.0413
50th %tile of PWS means (ng/L)
0.0963
75th %tile of PWS means (ng/L)
0.229
6Assuming rat BW of 0.45 kg, mouse BW of 0.05 kg (based on study-specific data), and human BW of 70 kg.
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Parameter (units)
Statistic3
95th %tile of PWS means (ng/L)
1.87
Standard deviation of PWS means (ng/L)
1.84
aData below the minimum reporting level (MRL, 0.03 ng/L) are included as % the MRL in calculations. Data are from
the posted January 2017 release of the EPA Third Unregulated Contaminant Monitoring Rule (UCMR3) (U.S. EPA,
2014c). Only data collected for large PWSs were used for statistical analysis. Statistics performed on the mean
PWS values (each PWS had multiple facilities that collected multiple samples).
Table C-67. Summary of UCMR3 chromium (VI) concentration data (in |ig/L)
grouped by EPA region
Region
Count
Mean
Max
Percentiles
25th
50th
75th
95th
1
237
0.131
3.80
0.0359
0.0647
0.128
0.420
2
351
0.281
23.0
0.0432
0.0829
0.239
0.709
3
282
0.165
1.47
0.0502
0.0899
0.189
0.513
4
905
0.124
2.42
0.0364
0.0692
0.133
0.365
5
748
0.206
3.31
0.0265
0.126
0.199
0.751
6
432
0.521
42.3
0.0238
0.0561
0.157
1.77
7
132
0.693
3.16
0.0475
0.277
1.19
2.35
8
162
0.273
1.99
0.0444
0.151
0.381
0.898
9
519
2.050
30.5
0.126
0.586
1.96
8.89
10
159
0.230
1.42
0.0719
0.142
0.274
0.750
Data below the minimum reporting level (MRL, 0.03 ng/L) are included as % the MRL in calculations. Data are from
the posted January 2017 release of the EPA Third Unregulated Contaminant Monitoring Rule (UCMR3) (U.S. EPA,
2014c). Only data for large PWSs were used for statistical analysis.
Table C-68. Summary of UCMR3 Cr(VI) data for 20 large public water systems
with the highest mean concentrations
PWSID
Location
PWSID Name
Mean
(Hg/L)
Max.
(Hg/L)
n
OK1020801
OK
Norman
42.3
97.4
80
CA2410005
CA
City of Los Banos
30.5
38.0
8
AZ0407154
AZ
Town of Buckeye Sundance
28.8
33.0
8
AZ0407056
AZ
AZ American Water Co. - Paradise Valley
28.0
30.1
4
AZ0408020
AZ
Kingman Municipal Water
25.6
79.0
24
AZ0407500
AZ
City of Surprise - Mountain Vista
23.9
39.0
16
PR0004074
PR
Guanica Urbano
23.0
26.3
11
CA1010018
CA
City of Kerman
19.4
31.0
16
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PWSID
Location
PWSID Name
Mean
(Hg/L)
Max.
(Hg/L)
n
AZ0407078
AZ
Valencia Water Co. - Town Division
18.9
22.0
15
CA5010017
CA
City of Patterson
18.2
22.0
12
CA5710006
CA
City of Woodland
17.7
26.0
22
CA5710009
CA
University of California - Davis
17.5
47.0
16
OK2001412
OK
Moore
17.5
54.0
47
OK2000922
OK
Mustang
15.7
29.9
12
CA3310007
CA
City of Coachella
15.6
19.0
16
AZ0407695
AZ
AZ American Water Co. - Agua Fria
15.0
62.0
56
AZ0407094
AZ
Goodyear Water Department
14.4
27.0
20
CA5710001
CA
City of Davis
14.0
41.0
32
CA3310020
CA
Indio Water Authority
13.0
19.0
20
AZ0407025
AZ
City of Phoenix
12.8
54.0
80
Total n =
515
Data below the minimum reporting level (MRL, 0.03 ng/L) are included as % the MRL in calculations. Data are from
the posted January 2017 release of the EPA Third Unregulated Contaminant Monitoring Rule (UCMR3) (U.S. EPA.
2014c). Only data collected for large PWSs were used for statistical analysis.
C.4.2. Local Data of Air, Soil, and Dust Cr(VI) Concentrations
1 Because Cr(VI) is classified as a hazardous air pollutant under the Clean Air Act, data for air,
2 dust, and soil are available from state and local environmental departments. Tables C-69 through C-
3 73 list datasets from publicly available sources that were found by screening national, state, and
4 local environmental department websites. These datasets are not from EPA sources, and values are
5 subject to change. Readers are advised to consult the citations and the state websites for the raw
6 data, and detailed information related to data collection and interpretation. This is not an
7 exhaustive summary of all air, dust, and soil Cr(VI) and total chromium (Cr(VI)+Cr(III))
8 concentrations in the United States.
Table C-69. Cr(VI) concentrations in ambient PMio (ng/m3) at monitoring sites
in Midlothian, Texas containing three cement manufacturing facilities and a
steel mill fATSDR.20161
Location
Mean (confidence interval)
Jaycee Park
0.016 (0.0094-0.024)
Old Fort Worth Road
0.055 (0.029-0.086)
Tayman Drive
0.018 (0.0097-0.035)
Wyatt Road
0.07 (0.037-0.12)
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Location
Mean (confidence interval)
JA Vitovsky
0.021 (max)3
Midlothian HS
0.039 (max)3
Mountain Peak Elementary
0.039 (max)3
aMaximum value reported only (descriptive statistics not generated by TCEQ because of the small number of
observations).
Table C-70. Cr(VI) concentrations in air measured at monitoring sites in
Portland Oregon reporting elevated metals concentrations fOregon DEO.
2016hl
Location
Date
Mean ± SDa
(ng/m3)
Min
Max
Metal finishing site (Southeast Portland)
Milwaukie Johnson Creek
April-Sept 2016
0.321 ±0.239
0.047
1.16
SE Harney Dr.
April-Dec 2016
0.121 ±0.118
0.038
1.01
SE 45th Ave & SE Harney
March 2016-March 2017
0.0707 ± 0.0501
0.035
0.44
Glass producer site (Northeast Portland)
Daycare Center
March 2016-Feb 2017
0.201 ±0.332
0.037
3.63
Winterhaven Elementary
March-Sept 2016
0.0759 ± 0.0604
0.037
0.695
Powell & SE 22nd
March 2017
0.147 ±0.247
0.036
3.1
Haig & SE 20th
March 2017
0.129 ±0.316
0.038
2.88
Reed College
May-Sept 2016
0.095 ± 0.0374
0.038
0.209
Glass producer site (North Portland)
Tubman School
March-Aug 2016
0.0625 ± 0.0338
0.037
0.222
Portland North Coast Electric
March-July 2016
0.0993 ±0.112
0.036
0.655
Portland Water Bureau East
March-Aug 2016
0.118 ±0.0979
0.038
0.6
Portland Water Bureau West
March-July 2016
0.102 ±0.0568
0.04
0.271
aAverage daily value as reported by Oregon Department of Environmental Quality, applying the Kaplan-Meir
method for nondetects.
Table C-71. Cr(VI) concentrations (mean ± SD in ng/m3) in ambient PMio
measured in urban and suburban New Jersey fHuang et al.. 20141
Location
Soluble Cr(VI)
Total Cr(VI)
Summer
Winter
Summer
Winter
Meadowlands
0.3 ±0.16
0.11 ±0.04
1.25 ±0.58
1.32 ±0.56
Elizabeth
0.21 ±0.13
0.19 ±0.09
1.56 ±0.48
1.41 ±0.56
Rahway
0.33 ±0.36
0.14 ±0.07
0.99 ±0.76
1.05 ±0.36
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Supplemental Information—Hexavalent Chromium
Location
Soluble Cr(VI)
Total Cr(VI)
Summer
Winter
Summer
Winter
Piscataway3
0.2 ±0.18
0.03 ±0.01
0.86 ±0.5
0.94 ±0.49
Suburban (all other locations urban).
Table C-72. Cr(VI) Mean concentration in air districts with chromium plating
and anodizing facilities for the year 2005. Data from the California Air Resources
Board.
District
Monitoring site
Mean concentration
(ng/m3)
South Coast Air Quality Management
District
Azusa-803 Loren Ave.
0.08
Burbank - W. Palm Ave.
0.113
North Long Beach
0.10
San Diego County Air Pollution Control
District
Chula Vista
0.038
El Cajon-Redwood Avenue
0.048
Ventura County Air Pollution Control
District
Simi Valley-Cochran Street
0.05
Bay Area Air Quality Management
District
Fremont-Chapel Way
0.05
San Francisco-Arkansas Street
0.11
San Joaquin Valley Air Pollution Control
District
Fresno-lst Street
0.063
Stockton-Hazelton Street
0.12
Sacramento Metropolitan Air Quality
Management District
Roseville-N Sunrise Blvd
0.058
Adapted from CARB (2006).
Table C-73. Estimated environmental concentrations of chromium in selected
locations within the United States
Media and location
Units3
Mean
Max.
Reference
Ambient air, Barrio Logan San
ng/m3
0.42
22.0
Residential areas near facilities potentially
Diego CA
emitting Cr(VI) from California EPA (CalEPA,
2004, 2003) (Mav 2001-Mav 2002)
Ambient air, Portland OR glass
ng/m3
N/A
3.63
Elevated metals site data from Oregon DEQ
and metal sites
(Oregon DEQ, 2016b). See Table 5
Ambient PMio, Deer Park and
ng/m3
0.1
0.4b
24-h average data from TCEQ (2006-2013)
Karnack, Texas
(TCEQ, 2017)
Ambient PMio; soluble+
ng/m3
1.17
1.56
Urban and suburban areas of New Jersey
insoluble Cr(VI), New Jersey
(Huang et al., 2014)
Ambient PMio; soluble Cr(VI),
ng/m3
0.189
0.33
New Jersey
Surface soil, Portland OR glass
mg/kg Cr(VI)
N/A
3.0
Data from Oregon DEQ (Oregon DEQ,
and metal sites
mg/kg total
chromium
N/A
63
2016a, c)
mg/kg Cr(lll)
19.5
130
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Supplemental Information—Hexavalent Chromium
Media and location
Units3
Mean
Max.
Reference
Background (bulk soil),
Montana
mg/kg Cr(VI)
N/Ac
1.2
Data from Montana DEQ (Hvdrometrics,
2013)
House dust, New Jersey
Mg/g
4.6
56.6
Background house dust in NJ (Stern et al.,
2010) (ue/m2are surface loading units)
Hg/m2
10
169.3
aUnits of Cr(VI) unless otherwise noted.
bMaximum highest and second-highest hourly measurements are 1.9 and 0.7 ng/m3, respectively.
c88% of values below the limit of detection (<0.29 mg/kg).
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APPENDIX D. DOSE-RESPONSE MODELING
This appendix provides technical detail on dose-response evaluation and determination of
points of departure (PODs) for relevant toxicological endpoints. Figure D-l provides an overview of
the process of RfD/RfC derivation. The endpoints were modeled using EPA's Benchmark Dose
Software (BMDS, Version 3.2). Sections D.l (noncancer) and D.2 (cancer) describe the common
practices used in evaluating the model fit and selecting the appropriate model for determining the
POD, as outlined in the Benchmark Dose Technical Guidance Document (U.S. EPA. 2012a). Logfiles of
BMD model outputs are contained in U.S. EPA (2021a).
Some statistical models (Gamma, dichotomous Hill, Weibull, and log-logistic) were run with
constrained slope or power parameters (>1) fU.S. EPA. 2012al. As noted in the Benchmark Dose
Software (BMDS) version 3.2 user guide (U.S. EPA. 2020a). some models with unrestricted
coefficients can give complicated shapes, in particular for high-degree polynomial models (which
produce unrealistic "wavy" results with negative response rates). Although Bayesian model
averaging is an available feature of BMDS 3.2, only frequentist models were run in this assessment.
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—Hexavalent Chromium
Evidence integration results
Organ/system 1
Organ/system 2
Organ/system x
Hazards not meeting
criteria for dose-
response modeling:
no cRfDs derived
Organ/system hazards meeting criteria for dose-response modeling
(i.e., "evidence indicates" or above, depending on database)
I
Organ/System 1
I 1
Organ/System 2 Organ/System 3
Select based on:
a) Study confidence
y b) Most sensitive endpoint
c) Clustering of values or
d) Combination of factors
Organ-specific RfD 1 osRfD2 osRfD3
I
Overall RfD
Select among the osRfDs based on:
a) Prior considerations used to select studies and data for dose-response
b) Consideration of overall toxicity
c} Study confidence
d) Confidence in each value and strength of dose-response analyses
e) Direct graphical comparison of PODs and toxicity values
Figure D-l. Overview of the process for deriving candidate, organ-specific, and
overall RfDs (process also applicable to RfCs).
This document is a draft for review purposes only and does not constitute Agency policy.
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D.l. BENCHMARK DOSE MODELING SUMMARY FOR NONCANCER
ENDPOINTS
For this assessment, dose-response modeling of endpoints for the oral route of exposure
was performed based on the time-weighted average daily dose of Cr(VI), in mg/kg-day. This value
could then be converted to an internal rodent dose, depending on the tissue or endpoint. The time-
weighted average was calculated based on time-course dose data available through the data
collection time for each endpoint. For example, for endpoints measured at 12 months in the NTP
(20081 study, the time-weigh ted average daily dose over 12 months was applied, as opposed to the
average daily dose over the full 2-year bioassay.
For dose-response modeling of endpoints for the inhalation route, inhaled concentration
was used. Adjustments for respiratory-tract particle dosimetry and 24-hour/day time conversion
were performed during the interspecies extrapolation step.
The noncancer endpoints selected for dose-response modeling are presented in Tables D-l
through D-3 (oral) and Table D-4 (inhalation). For each endpoint, the exposure doses and data used
for the modeling are presented.
Table D-l. Noncancer endpoints selected for dose-response modeling for
Cr(VI) (oral) from NTP r200m
Species/sex
endpoint
Doses and effect
data
Mouse/Male
Diffuse epithelial
hyperplasia
(duodenum) at lifetime
Cr(VI) mg/L
0
5
10
30
90
TWA dose mg/kg-d
0
0.450
0.914
2.40
5.70
Incidence /Total
0/39
11/43
18/45
42/48
32/40
Mouse/Female
Diffuse epithelial
hyperplasia
(duodenum) at lifetime
Chronic inflammation
(liver) at lifetime
Cr(VI) mg/L
0
5
20
60
180
TWA dose mg/kg-d
0
0.302
1.18
3.24
8.89
Incidence /Total
0/42
16/42
35/48
31/42
42/48
Incidence/Total
16/49
21/50
22/50
27/50
24/50
Rat/Female
Fatty change (liver) at
lifetime
Chronic inflammation
(liver) at lifetime
Cr(VI) mg/L
0
5
20
60
180
TWA dose mg/kg-d
0
0.248
0.961
2.60
7.13
Incidence /Total
3/50
7/50
10/50
13/50
16/50
Incidence /Total
12/50
21/50
28/50
35/50
39/50
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Supplemental Information—Hexavalent Chromium
Species/sex
endpoint
Doses and effect
data
Rat/Male
Cr(VI) mg/L
0
5
20
60
180
TWA dose mg/kg-d
(lifetime)
0
0.200
0.796
2.10
6.07
TWA dose mg/kg-d
(12 mo)
0
0.237
0.938
2.49
7.19
TWA dose mg/kg-d
(3 mo)
0
0.401
1.58
4.16
11.7
Chronic Inflammation
(liver) at lifetime
Incidence/Total
19/50
25/50
21/49
28/50a
26/49
ALT (liver) at 12 mo.
IU/L ± SE, n = 10/group
102 ±6
107 ±8
135 ± 10
261 ±23
223 ± 15
ALT (liver) at 3 mo.
IU/L ± SE, n = 10/group
82 ±4
82 ± 12
135 ± 18
176 ± 13
216 ±21
Rat/Male
N
10
10
10
8
10
TWA dose mg/kg-d
(12 mo)
0
0.237
0.938
2.49
7.19
RBC (106/nL,
mean ± SE)
9.27 ±0.10
9.17 ±0.07
9.4 ±0.12
9.61 ±0.11
9.74 ±0.08
MCV (fL, mean ± SE)
52.6 ±0.2
52.4 ±0.2
51.9 ±0.3
51.4 ±0.3
49.9 ±0.2
Hematological changes
at 12 mo.
MCH (pg, mean ± SE)
17 ±0.1
16.8 ±0.1
16.6 ±0.1
16.2 ±0.1
15.7 ±0.1
MCHC (mean ±SE)
32.3 ±0.2
32.1 ±0.3
32.0 ±0.2
31.6 ±0.2
31.5 ±0.2
Hgb (mean ± SE, g/dL)
15.8 ±0.2
15.4 ±0.2
15.6 ±0.2
15.6 ±0.2
15.3 ±0.1
Hematological changes
at 90d.
N
10
10
10
10
10
TWA Dose mg/kg-d (90
d)
0
0.401
1.58
4.16
11.7
Hgb (mean ± SE, g/dL)
15.1 ±0.1
14.9 ±0.1
14.9 ± 0.2
14.6 ±0.2
12.9 ±0.2
Hematological changes
at 22d.
N
10
10
10
10
10
TWA Dose mg/kg-d (22
d)
0
0.634
2.49
6.67
17.7
Hgb (mean ± SE, g/dL)
15.5 ± 0.3
15.1 ±0.2
14.2 ± 0.2
12.0 ±0.3
10.1 ±0.2
aRevised estimates for time weighted average daily doses calculated from NTP data. These may differ from the
average doses presented elsewhere in this toxicological review, which are typically rounded or based on averages
of fewer timepoints.
This document is a draft for review purposes only and does not constitute Agency policy.
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Table D-2. Noncancer endpoints selected for dose-response modeling for
Cr(VI) (oral) from NTP r2007fi
Species/Sex
endpoint
Doses and effect data
Rat/Female
Liver changes at 90
d
N
10
10
10
10
10
10
Cr(VI) mg/L
0
20
40
90
170
350
TWA dose mg/kg-da
0
1.7
3.5
5.9
11.2
20.9
ALT (mean ±SE, IU/L)
64 ±5
437 ± 68
218 ± 27
245 ± 30
246 ± 37
248 ± 22
Rat/Male
Liver changes at 90
d
N
8
10
10
10
10
10
Cr(VI) mg/L
0
20
40
90
170
350
TWA dose mg/kg-da
0
1.7
3.5
5.9
11.2
20.9
ALT (mean ±SE, IU/L)
98 ±6
274 ± 30
461 ± 10
2
447 ±121
740 ± 81
191 ± 17
Rat/Male
Hematological
changes at 90 d
N
10
10
10
10
10
10
TWA dose mg/kg-da
0
1.7
3.5
5.9
11.2
20.9
Hgb (mean ± SE, g/dL)
15.3 ±0.1
15.2 ±0.1
15.0 ±
0.1
14.4 ± 0.2
13.3 ±
0.2
10.9 ± 0.3
Rat/Male
Hematological
changes at 23 d
N
10
10
10
10
10
10
TWA dose mg/kg-d
0
2.92
5.55
10.3
18.3
30.6
Hgb (mean ± SE, g/dL)
15.9 ±0.1
14.2 ± 0.2
12.0 ±
0.3
10.9 ±0.3
10.3 ±
0.3
9.2 ±0.3
Rat/Female
Hematological
changes at 90 d
N
10
10
10
10
10
10
TWA dose mg/kg-da
0
1.7
3.5
5.9
11.2
20.9
Hgb (mean ± SE, g/dL)
15.2 ±0.1
15.4 ±0.1
14.9 ±
0.1
14.3 ±0.1
14.1 ±
0.2
12.0 ± 0.2
Rat/Female
Hematological
changes at 23 d
N
10
9
8
9
10
9
TWA dose mg/kg-d
0
2.97
5.56
9.83
17.7
30.9
Hgb (mean ± SE, g/dL)
15.9 ±0.1
14.7 ± 0.3
13.0 ±
0.3
11.8 ±0.3
10.9 ±
0.2
9.7 ±0.2
aThese are the values for both males and females at 14 weeks provided by NTP (2007f). Alternatively, slightly
different doses in mg/kg-d may be estimated from the NTP data: 1.74, 3.14, 5.93,11.2, 20.9 for males, and 1.74,
3.49, 6.28,11.5, 21.3 for females. For this assessment, the average value was applied to both male and female
rats at 14 weeks. For data at 23 days, NTP did not provide time weighted average doses, so they were estimated
from raw data. Sex-specific doses at 23 days are listed because they differ greatly at high drinking water
concentration.
This document is a draft for review purposes only and does not constitute Agency policy.
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Table D-3. Noncancer endpoints selected for dose-response modeling for
Cr(VI) (oral) from NTP T19971
Species/Sex
endpoint
Doses and effect data
Mouse/Female
TWA dose mg/kg-d
0
11.6
24.4
50.6
F1 male pups PND14
Pup weight g ± SE
(N litters)
7.95 ±0.50
(15)
7.69 ±0.36
(13)
7.51 ±0.48
(12)
6.93 ±0.27
(16)
F1 male pups PND21
9.38 ±0.64
(15)
8.52 ±0.59
(14)
8.66 ±0.63
(12)
7.94 ±0.34
(16)
F1 female pups PND14
7.71 ±0.38
(15)
7.85 ±0.36
(15)
8.05 ±0.53
(13)
7.04 ±0.33
(18)
F1 female pups PND21
9.03 ±0.55
(15)
8.77 ±0.55
(16)
9.01 ±0.68
(13)
8.17 ±0.42
(18)
TWA dose is for the female F0 (maternal) generation.
Table D-4. Noncancer endpoints selected for dose-response modeling for
Cr(VI) (inhalation)
Species/Sex endpoint
Doses and effect data
Glaser et al. (1990) (n = 10/group)
Concentration
(mg/m3 Cr(VI))
0
0.054
0.109
0.204
0.403
90 d, no recovery
Lactate dehydrogenase
(LDH) in BAL fluid
(U/L) mean ± SD
29 ±5
34 ±3
31 ±4
63 ± 11
83 ± 17
Albumin in BALF
(mg/L) mean ± SD
77 ± 13
115 ± 23
86 ± 13
117 ± 20
184 ± 59
Total protein in BALF
(mg/L) mean ± SD
226 ± 30
396 ± 79
326 ± 35
703 ±178
975 ± 246
Histiocytosis
Incidence
2/10
9/10
10/10
9/10
10/10
Note: Nominal/target inhalation concentrations were replaced with the reported mean concentrations measured in
the studies.
D.l.l. Evaluation of Model Fit and Model Selection
Basic statistical background and guidance on choosing a model structure for the data being
analyzed, fitting models, comparing models, and calculating confidence limits to derive a BMDL to
use as a POD are outlined in EPA's Benchmark Dose Technical Guidance fU.S. EPA. 2012al. Sections
2.3.9 and 2.5. Empirical models that provide the best fit to the dose-response data are typically used
in the absence of data to support development of a biologically based model. Although these models
are empirical, parameters are typically constrained on some of them for the purposes of
strengthening the biological plausibility of the results (i.e., many toxic effects exhibit a monotonic
dose-response), and to prevent imprecise BMDs/BMDLs resulting from steeply supralinear models
[fU.S. EPA. 2012a) §2.3.3.3], Consistent with EPA's Benchmark Dose Technical Guidance fU.S. EPA.
2012a), initial runs of the log-probit model did not constrain the slope parameter, whereas initial
runs of the gamma, dichotomous Hill, Weibull, and log-logistic models constrained their slope or
power parameters to be >1. As noted in Benchmark Dose Software (BMDS) Version 3.2 User Guide
This document is a draft for review purposes only and does not constitute Agency policy.
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1 fU.S. EPA. 2020al. some models with unrestricted coefficients can give more complicated shapes, in
2 particular high-degree polynomial models (which produce unrealistic "wavy" results with negative
3 response rates).
4 For each candidate endpoint/study the following steps were taken:
5 1) Goodness-of-fit was assessed for all models [(U.S. EPA. 2012a) §2.3.5],
6 a. Models having a goodness-of-fit p-value of less than 0.1 were rejected.7
7 b. Models not adequately describing the dose-response relationship (especially in the low-
8 dose region) were rejected on the basis of examining the dose group-scaled residuals8
9 and graphs of models and data.
10 The models that remained (after rejecting those that did not meet the recommended default
11 statistical criteria for adequacy and fail in visual inspection of model fit) were used for
12 determining the BMDL. The default selection criteria are listed below [(U.S. EPA. 2012a)
13 §2.3.9]:
14 2) If the BMDL estimates from the remaining models were sufficiently close (generally defined
15 as being within threefold, as in the case of this assessment), it was assumed there was no
16 particular influence of the individual models on the estimates. In this case, the model with
17 the lowest AIC was chosen.
18 3) If the BMDL estimates from the remaining models were not sufficiently close, it was
19 assumed there was some model dependence (i.e., model uncertainty) of the estimate. In this
20 case, if there was no clear remaining biological or statistical basis on which to choose among
21 them, the lowest BMDL was selected as a reasonable conservative estimate fU.S. EPA
22 ("2012al Section 2.3.91.
23 4) In some cases, modeling attempts did not yield useful results. When this occurred, the
24 NOAEL (or LOAEL) was used as a candidate POD.
25 Logfiles of BMD model outputs are contained in U.S. EPA (2021a).
D.l.1.1. Modeling issues related to diffuse epithelial hyperplasia in mice
26 Benchmark dose modeling did not result in useful results for diffuse epithelial hyperplasia in
27 female mice from NTP f20081. Using BMDS (v 3.2), three models fit the full dataset adequately
28 (based on goodness-of-fit p-value >0.10): dichotomous Hill, log-logistic, and log-probit However,
7For the goodness-of-fit test and a p-value of a, the critical value is the 1- a percentile of the distribution
at the appropriate degrees of freedom. Models are rejected if there are large values of corresponding to
p-values less than 0.1, the limiting probability of a Type I error (false positive) selected for this purpose.
8Scaled residuals reported by BMDS for dichotomous responses are defined as (Observed - Expected)/SE,
where "Expected" is the predicted number of responders and SE equals the estimated standard error of that
predicted number. For dichotomous models, the estimated standard error is equal to V[n x p x (1 -p)], where n
is the sample size and p is the model-predicted probability of response. Model fit is considered questionable if
the scaled residual value for any dose group, particularly the control or low dose group, is greater than 2 or
less than -2.
This document is a draft for review purposes only and does not constitute Agency policy.
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the log-probit model yielded a very low BMDL (150 times lower than the lowest nonzero dose of
0.302 mg/kg-day). Because the model fit was adequate compared to the other two models, it could
not be excluded from model selection. The residuals for the log-probit result were sufficiently low,
and its AIC was between that of the other two models (see below). Changing model parameter
restrictions did not resolve the issue. It was concluded, based on the criteria outlined above in
Section D.l.l, that there was too much uncertainty in the BMD estimate to use these model results
for determining the POD.
Table D-5. BMD model results for diffuse epithelial hyperplasia in female mice
from NTP (2008) (no high doses omitted)
Model
BMR
BMD
mg/kg-d
BMDL
mg/kg-d
Goodness-of-fit
p-value
AIC
Log-logistic
10% ER
0.0722
0.0530
0.1145
205.07
Log-probit
10% ER
0.0199
0.00199
0.3043
204.80
Dichotomous Hill
10% ER
0.0561
0.0268
0.4132
204.08
The lowest dose for female mice is 0.302 mg/kg-d.
The primary reason for the high uncertainty on the BMD estimate is that the response rate
(38%) at the lowest dose was much higher than the BMR of 10% ER (the control group had 0%
response). In addition, the data are supralinear and plateau at the three high doses (as the incidence
approaches 100%).
Dropping high doses can address the supralinear shape and high-dose effect, to achieve
adequate model fit in the response region of interest. In this case, dropping the highest dose does
not resolve the issue because the three high doses exhibit a flat response. However, omitting the
two highest doses can achieve an optimal model fit within the set of models run (see below).
Table D-6. Modeling alternatives for diffuse epithelial hyperplasia in mice from
NTP r2oom
Doses
BMD
BMDL
Species/Sex
Model
dropped
BMR
mg/kg-d
mg/kg-d
Mice/M
Quanta l-linear
l
10% ER
0.148
0.121
Mice/F
Log-logistic
0
10% ER
0.0722
0.0530
Dichotomous Hill
0
10% ER
0.0561
0.0268
Log-probit
0
10% ER
0.0199
0.00199
Quantal-linear
2
10% ER
0.0852
0.0672
LOAEL
-
-
LOAEL = 0.302
LOAEL/10 = 0.0302
The lowest dose for female mice is 0.302 mg/kg-d.
This document is a draft for review purposes only and does not constitute Agency policy.
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Other approaches to address the modeling issues for this dataset include increasing the BMR
to be closer to the lowest observed response rate (which would decrease the uncertainty on the
BMD) or attempting alternative modeling (such as Bayesian model averaging). Other statistical
issues can arise when implementing these approaches (e.g., an additional uncertainty adjustment
would be needed when increasing the BMR).
As shown in the table above, the LOAEL divided by a UFl = 10 (the LOAEL-to-NOAEL
uncertainty factor) produces a reasonable result when compared to the alternative BMDLs. The
value (0.0302 mg/kg-day) is within the bounds of the alternatives (significantly higher than log-
probit, 13% higher than dichotomous Hill, and 43% lower than log-logistic).
Because the response rate is high at the lowest dose, and there are no data near the true
10% response rate, there is high uncertainty in estimating the lower 95% confidence limit on the
BMDio.
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—Hexavalent Chromium
Table D-7. RfDs for modeling alternatives of diffuse epithelial hyperplasia in mice from NTP (2008)
Species/Sex
Model
Doses
dropped
BMR
BMDLor
LOAEL
mg/kg-d
Internal
dose
mg/kg-d
TWA BW
kg
BW3/4
adjust
PODhed
mg/kg-d
Composite
UF
RfD
mg/kg-d
Mice/M
Quanta l-linear
l
10% ER
0.121
0.0182
0.05
2.88 x 10"3
0.0443
10
4.43 x 10"3
Mice/F
Log-logistic
0
10% ER
0.0530
0.00792
0.05
1.25 x 10"3
0.0204
10
2.04 x 10"3
Dichotomous
Hill
0
10% ER
0.0268
0.00400
0.05
6.32 x 10"4
0.0106
10
1.06 x 10"3
Log-probit
0
10% ER
0.00199
0.000296
0.05
4.68 x 10"5
7.95 x 10"4
10
7.95 x 10"5
Quanta l-linear
2
10% ER
0.0672
0.0101
0.05
1.60e x 10"3
0.0258
10
2.58 x 10"3
LOAEL
-
-
0.302
0.0463
0.05
7.32 x 10"3
0.0911
100
9.11 x 10"4
Mean and median value of log-logistic, log-probit, and dichotomous Hill results (with 0 dosses dropped) is 1.06 x 10 3 mg/kg-d.
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—Hexavalent Chromium
D.1.1.2. Modeling issues related to chronic liver inflammation in female rats
1 An issue similar to that described above for hyperplasia also applied to data for chronic liver
2 inflammation in female rats. Three adequately fitting models produced very different results, with
3 one of them producing a BMDL that was over 75 times lower than the lowest dose.
Table D-8. BMD model results for chronic liver inflammation in female rats
from NTP f200Rl
Model
BMR
BMD
mg/kg-d
BMDL
mg/kg-d
Goodness-of-fit
p-value
AIC
Log-logistic
10% ER
0.232
0.142
0.3871
312.44
Log-probit
10% ER
0.0546
0.00325
0.943
311.63
Dichotomous Hill
10% ER
0.107
0.0424
0.8962
311.73
The lowest dose in female rats was 0.248 mg/kg-d.
4 As with female mouse hyperplasia, there was too much uncertainty in the BMD estimate to
5 use these model results for determining the POD. As a result, it was determined that this dataset
6 was not amenable to BMD modeling, and the lowest dose was chosen as the LOAEL (greater than
7 10% extra risk from control occurred at this level).
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—Hexavalent Chromium
Table D-9. RfDs for modeling alternatives of chronic liver inflammation in female rats from NTP (2008)
Model
BMR
BMDL or LOAEL
mg/kg-d
Internal dose
mg/kg-d
TWA BW
kg
bw3/4
adjust
PODhed
mg/kg-d
Composite
UF
RfD
mg/kg-d
Log-logistic
10% ER
0.142
0.0109
0.260
2.60 x 10"3
0.0402
10
4.02 x 10"3
Log-probit
10% ER
0.00325
2.43 x 10"4
0.260
5.80 x 10"5
9.97 x 10"4
10
9.97 x 10"5
Dichotomous Hill
10% ER
0.0424
3.20 x 10"3
0.260
7.64 x 10"4
0.0128
10
1.28 x 10"3
LOAEL
--
0.248
0.0195
0.260
4.66 x 10"3
0.0669
100
6.69 x 10"4
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—Hexavalent Chromium
D.l.1.3. Modeling issues related to liver fatty changes in female rats
1 As shown in the table below, all models achieved an adequate fit. Dichotomous Hill and log-
2 probit results were significantly different than the others. The model fits were adequate compared
3 to the other models, and they could not be excluded from model selection. The log-probit BMDL was
4 over 130 times lower than the lowest dose.
Table D-10. BMD model results for fatty change in liver of female rats from NTP
f200Rl
Model
BMR
BMD
mg/kg-d
BMDL
mg/kg-d
Goodness-of-
fit p-value
AIC
Dichotomous Hill
10% ER
0.426
0.0117
0.911
239.410
Log-logistic
10% ER
1.953
1.105
0.394
240.375
Multistage Degrees 1-4 and Gamma,
Weibull
10% ER
2.300
1.414
0.335
240.843
Logistic
10% ER
3.480
2.532
0.205
242.244
Log-probit
10% ER
0.342
0.00182
0.995
239.237
Probit
10% ER
3.325
2.387
0.217
242.074
The lowest dose in female rats was 0.248 mg/kg-d.
5 There was too much uncertainty in the BMD estimate to use these model results for
6 determining the POD. The lowest dose was chosen as the NOAEL (less than 10% extra risk from
7 control occurred at the lowest dose).
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—Hexavalent Chromium
D.l.1.4. Modeling issues related to lower respiratory effects in male rats at 90 days
1 The following 90-day datasets in male rats from Glaser etal. (19901 were determined not to
2 be amenable for BMD modeling:
3 • Histiocytosis: the only adequately-fitting model did not produce a useable result; parameter
4 hit bound
5 • Total protein, albumen, and LDH in BALF: all models had goodness of fitp-value < 0.1
Table D-ll. BMD results for histiocytosis in male rats at 90 days from Glaser et
al. fl9901
Model
Restriction
Risk type
BMD
BMDL
BMDU
P-
Value
AIC
BMDS
recommend-
ation notes
Dichotomous
Hill
Restricted
Extra Risk
0.000613
-
0.0387232
0.3535
31.4
BMD computation
failed
Log-probit
Unrestricted
Extra Risk
2.61 x 10"5
-
Infinity
0.3696
31.4
Log-logistic
Restricted
Extra Risk
8.57 x 10"4
1.91 x 10"4
0.0161718
0.4778
29.5
BMD/BMDL
ratio > 3
BMD 10x lower
than lowest
nonzero dose
BMDL 10x lower
than lowest
nonzero dose
Gamma
Restricted
Extra Risk
4.89 x 10"3
3.00 x 10"3
0.0147435
0.0122
33.0
Goodness of fit
p-value <0.1
Goodness of fit
p-value <0.1
Multistage
Degree 4
Restricted
Extra Risk
4.89 x 10"3
3.00 x 10"3
0.009323
0.0122
33.0
Multistage
Degree 3
Restricted
Extra Risk
4.89 x 10"3
3.00 x 10"3
0.009323
0.0122
33.0
Multistage
Degree 2
Restricted
Extra Risk
4.89 x 10"3
3.00 x 10"3
0.009323
0.0122
33.0
Multistage
Degree 1
Restricted
Extra Risk
4.89 x 10"3
3.00 x 10"3
0.0089504
0.0122
33.0
Weibull
Restricted
Extra Risk
4.89 x 10"3
3.00 x 10"3
0.0120185
0.0122
33.0
Logistic
Unrestricted
Extra Risk
9.65 x 10"3
5.97 x 10"3
0.015877
0.0011
35.9
Probit
Unrestricted
Extra Risk
1.21 x 10"2
8.19 x 10"3
0.019674
0.0078
37.6
Table D-12. BMD results for total protein in BALF in male rats at 90 days from
Glaser et al. (1990)
Model
Restriction
Risk type
BMRF
BMD
BMDL
BMDU
Test 4
p-Value
BMDS
recommendation
notes
Hill (NCV -
normal)
Restricted
Std. Dev.
1
0.1801
-
0.1862
<0.0001
BMD computation
failed
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—Hexavalent Chromium
Model
Restriction
Risk type
BMRF
BMD
BMDL
BMDU
Test 4
p-Value
BMDS
recommendation
notes
Exponential 2
(NCV - normal)
Restricted
Std. Dev.
1
0.0646
0.0471
0.0894
<0.0001
Goodness of fit
p-value < 0.1
Exponential 3
(NCV - normal)
Restricted
Std. Dev.
1
0.0646
0.0471
0.0894
<0.0001
Exponential 4
(NCV - normal)
Restricted
Std. Dev.
1
0.0181
0.0094
0.0334
<0.0001
Exponential 5
(NCV - normal)
Restricted
Std. Dev.
1
0.0180
0.0094
0.0365
<0.0001
Polynomial
Degree 4 (NCV-
normal)
Restricted
Std. Dev.
1
0.0250
0.0173
0.0389
<0.0001
Polynomial
Degree 3 (NCV-
normal)
Restricted
Std. Dev.
1
0.0250
0.0173
0.0389
<0.0001
Polynomial
Degree 2 (NCV-
normal)
Restricted
Std. Dev.
1
0.0250
0.0173
0.0389
<0.0001
Power
(NCV - normal)
Restricted
Std. Dev.
1
0.0250
0.0173
0.0406
<0.0001
Linear
(NCV - normal)
Unrestricted
Std. Dev.
1
0.0250
0.0173
0.0370
<0.0001
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—Hexavalent Chromium
Table D-13. BMD results for LDH in BALF in male rats at 90 days from Glaser etal. (1990)
Model
Restriction
Risk type
BMRF
BMD
BMDL
BMDU
Test 4
p-Value
AIC
BMDS
recommendation
notes
Power
(NCV - normal)
Restricted
Std. Dev.
1
Failed
-
Infinity
<0.0001
369.1135
BMD computation
failed
Exponential 3 (NCV
- normal)
Restricted
Std. Dev.
1
0.0554
0.0420
0.0803
<0.0001
373.2741
Goodness of fit
p-value <0.1
Exponential 5 (NCV
- normal)
Restricted
Std. Dev.
1
0.1789
0.1243
0.1832
0.0724
343.1427
Hill (NCV - normal)
Restricted
Std. Dev.
1
0.1548
0.1225
0.1580
0.0204
345.2719
Polynomial Degree
3 (NCV - normal)
Restricted
Std. Dev.
1
0.0464
0.0300
0.0474
<0.0001
374.5440
Polynomial Degree
2 (NCV - normal)
Restricted
Std. Dev.
1
0.0487
0.0326
0.0497
<0.0001
371.2904
Linear
(NCV - normal)
Unrestricted
Std. Dev.
1
0.0375
0.0282
0.0512
<0.0001
371.7154
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—Hexavalent Chromium
Table D-14. BMD results for albumen in BALF male rats at 90 days from Glaser etal. (1990)
Model
Restriction
Risk Type
BMRF
BMD
BMDL
BMDU
Test 4
p-Value
AIC
BMDS
recommendation
notes
Exponential 2
(NCV - normal)
Restricted
Std. Dev.
1
0.1093
0.0842
0.1484
<0.0001
481.45
Goodness of fit
p-value < 0.1
Exponential 3
(NCV - normal)
Restricted
Std. Dev.
1
0.2113
0.0864
0.3101
<0.0001
482.93
Exponential 4
(NCV - normal)
Restricted
Std. Dev.
1
0.0822
0.0640
0.1101
<0.0001
485.12
Exponential 5
(NCV - normal)
Restricted
Std. Dev.
1
0.2239
0.1611
0.2647
<0.0001
484.98
Hill (NCV-normal)
Restricted
Std. Dev.
1
0.2057
0.1468
0.2229
<0.0001
481.81
Polynomial Degree
4 (NCV-normal)
Restricted
Std. Dev.
1
0.1653
0.0818
0.2777
<0.0001
481.14
Polynomial Degree
3 (NCV-normal)
Restricted
Std. Dev.
1
0.1695
0.0811
0.2698
<0.0001
481.60
Polynomial Degree
2 (NCV-normal)
Restricted
Std. Dev.
1
0.1593
0.0686
0.2343
<0.0001
483.10
Power
(NCV - normal)
Restricted
Std. Dev.
1
0.0822
0.0578
0.3883
<0.0001
483.12
Linear
(NCV - normal)
Unrestricted
Std. Dev.
1
0.0822
0.0578
0.1254
<0.0001
483.12
This document is a draft for review purposes only and does not constitute Agency policy.
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D.1.2. Calculation of Regional Deposited Dose Ratios (RDDR)
Fractional depositions in the pulmonary region (Fpu), the tracheobronchial region (Ftb), and
the extrathoracic region (Fet) for rats and humans were calculated using the Multi-Path Particle
Dosimetry (MPPD) model, a computational model that can be used for estimating airway particle
deposition and clearance (ARA (200911. Logfiles of MPPD outputs are contained in U.S. EPA
(2021a). Note: For this assessment, ARA MPPD Version 2.11 was applied. ARA MPPD Version 3.04,
and then subsequently EPA MPPD Version 1.01 have since been released. However, they do not
have the ability to save or load model runs, or the ability to run batch simulations; therefore,
version 2.11 results were maintained due to documentation and QA/QC capabilities. Versions ARA
3.04 and EPA 1.01 were tested using identical inputs as those specified below for Version ARA 2.11,
and differences between the older and newer models were negligible.9
For the MPPD model runs, the Yeh-Schum 5-lobe model was used for the human and the
asymmetric multiple path model was used for the rat Both models were run under nasal breathing
scenarios with the inhalability adjustment selected 'on'.
The human parameters used in the model for calculating Fr (fractional deposition in
respiratory tract region r) and in the subsequent calculation of the human equivalent concentration
at each rodent concentration were as follows: breathing frequency, 12 per minute (default); tidal
volume, 625 mL (default); ventilation rate Ve , 7.5 L/minute (calculated); functional residual
capacity, 3,300 mL (default); and upper respiratory tract volume, 50 mL (default). The parameters
used for the rat were breathing frequency, 102 per minute (default); tidal volume, 2.1 mL (default);
Ve, 0.214 L/minute (calculated); functional residual capacity, 4 mL (default); and upper respiratory
tract volume, 0.42 mL (default). All other parameters were also set to the default MPPD software
values. The density of sodium dichromate is 2.52 g/cm3. The aerosol Cr(VI) concentration was
converted to aerosol sodium dichromate concentration by molecular weight conversion (see
Table D-17). Mass median aerodynamic diameter (MMAD) and geometric standard deviation (GSD)
varied slightly with concentration.
RDDR was calculated using the following equation:
RDDR =
r [SAr)A [Ve)h (Fr)H
For the human, regional-specific surface areas for lung regions (used as normalizing
factors) were 200 cm2 for extrathoracic (ET), 3,200 cm2 for tracheobronchial (TB), and 54 m2 for
pulmonary (PU) fU.S. EPA. 19941. For the rat, lung surface areas were 15 cm2 for ET, 22.5 cm2 for
TB, and 0.34 m2 for PU CU.S. EPA. 19941.
differences in Fr and RDDRr between ARA v.2.11 and EPA v.1.01 were less than 10%
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—Hexavalent Chromium
Table D-15. Calculation of RDDR for Glaser et al. (1985) and Glaser et al.
(1990) using default MMAD parameters
Concentration as
reported
[mg/m3 Cr(VI)]
Aerosol
concentration3
MMAD ± GSD
(pm)
Fr
Rat
Fr
Human
RDDRC
TB
PU
TB
PU
TB
PU
Glaser et al. (1990)
54
136.0
0.28 ± 1.63
0.0277
0.1355
0.0664
0.1348
1.69
4.56
109
274.6
0.28 ± 1.63
0.0277
0.1355
0.0664
0.1348
1.69
4.56
204
513.9
0.39 ± 1.72
0.0244
0.1117
0.0585
0.1191
1.69
4.25
403
1015
0.39 ± 1.72
0.0244
0.1117
0.0585
0.1191
1.69
4.25
Glaser et al. (1985)b
52
131
0.20 ± 1.5
0.0334
0.1663
0.0781
0.1619
1.74
4.65
aAerosol concentration = Cr(VI) concentration -f 0.39696 by molar mass conversion (sodium dichromate
MW = 261.97 g/mol and contains 2 moles of Cr; Cr MW = 51.996 g/mol).
bGlaser et al. (1985) reported MMAD ± GSD (0.20 ± 1.5 nm) for all exposure groups. Analysis of Glaser et al. (1990)
found that aerosol concentration did not impact fractional deposition, and thus only one RDDR calculation was
performed for Glaser et al. (1985).
Table D-16. Human equivalent concentrations of Cr(VI) in the 90-day
inhalation study in rats by Glaser et al. f19901
Concentration as
reported
[mg/m3 Cr(VI)]
Continuous
exposure
adjustment Factor3
RDDRb
Human equivalent
concentration (mg/m3)
TB
Pulmonary
TB
Pulmonary
54
0.917
1.69
4.56
83.5
225.5
109
0.917
1.69
4.56
168.5
455.2
204
0.917
1.69
4.25
316.5
794.8
403
0.917
1.69
4.25
625.3
1570
Continuous exposure adjustment factor = (22/24) x (7/7); animals were exposed to Cr(VI) 22 hours per day and 7
days per week.
bPlease refer to Table D-17.
cHuman equivalent concentration = concentration as reported x continuous exposure adjustment factor x RDDR.
1 As shown in the tables above, the change in RDDR as a function of concentration and the
2 differences in particle size reported by Glaser etal. (19901 are negligible. The values of RDDR were
3 the same for the tracheobronchial region, and within 7% for the pulmonary region. As a result,
4 dose-response modeling does not need to be performed on the human equivalent concentrations
5 and can instead be performed on reported external concentrations. Conversion to a human
6 equivalent concentration can be done after calculating an external point of departure.
7 Furthermore, the RDDR estimated using particle sizes reported by Glaser etal. (1985) differs by
This document is a draft for review purposes only and does not constitute Agency policy.
D-19 DRAFT—DO NOT CITE OR QUOTE
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Supplemental Information—Hexavalent Chromium
1 less than 3%. As a result, the same RDDR values would be applied to extrapolations for both
2 studies.
3 Since RDDR is a strong function of age and physical activity (due to differences in breathing
4 rate, tidal volume, and surface area), MPPD (version 2.11) was run in batch mode for the adult
5 (Ye/Schum 5-lobe, uniform expansion) under varying degrees of physical activity. Values for
6 breathing rate and tidal volume under different physical activities were obtained from U.S. EPA
7 (~2011al
Table D-17. RDDR calculations under different human physiological activity
for respiratory effects
Human
activity
Breathing rate
(min-1)
Tidal volume
(mL)
VE L/min
(calculated)
Fr
Human
RDDR
TB
PU
TB
PU
TB+PU
MMAD: Adult Yeh/Schum 5-lobe, uniform expansion
Breathing rate/tidal volumes for adult male (U.S. EPA, 2011a)
Resting 1
12
750
9
0.0657
0.1514
1.4258
3.3799
2.8369
Resting 2
12
500
6
0.0664
0.1096
2.1161
7.0034
5.2491
Resting 3
15
500
7.5
0.062
0.0977
1.8130
6.2851
4.6279
Average RDDR (resting):
1.785
5.556
4.2380
Light work 1
17
1670
28.39
0.0588
0.1472
0.5050
1.1020
0.9478
Light work 2
16
1250
20
0.0599
0.1508
0.7037
1.5270
1.3154
Average RDDR (light work):
0.6044
1.3145
1.1316
Average RDDR (resting & light work):
1.1947
3.4353
2.6848
Heavy work
21
2030
42.63
0.0578
0.1285
0.3422
0.8407
0.6979
Maximal work
40
3050
122
0.0598
0.0806
0.1156
0.4684
0.3236
MMAD default
12
625
7.5
0.0664
0.1348
1.6929
4.5553
3.6733
Human respiratory parameters (tidal volume and breathing rate) obtained from U.S. EPA (2011a).
Aerosol parameters: MMAD (0.28 ± 1.63 nm), concentration 136 mg/m3, and density 2.52 g/cm3.
Inhalation parameters: Inhalability adjustment 'on'.
RDDR calculations (see Table D-17: rat Ftb 0.0277, rat Fpu 0.1355, rat Ve 0.214 L/minute (calculated).
Surface areas (rat): 15 cm2 for ET, 22.5 cm2 for TB, and 0.34 m2 for PU.
Surface areas (adult male human): 200 cm2 for ET, 3200 cm2 for TB, and 54 m2 for PU (U.S. EPA, 1994).
Calculation performed using total fractional deposition in the TB and PU regions and using total surface area (with
PU and TB units resolved).
Note: aerosol concentration in mg/m3 had no impact on predicted fractional lung depositions. Results for Fr of the
human TB and PU regions were identical if aerosol concentration was set to 1 or 136 mg/m3. For consistency, the
value 136 mg/m3 (corresponding to the lowest Cr(VI) concentration in Glaser et al., (1990)) was applied.
8 For systemic effects (i.e., nonrespiratory-tract organ weights), the total fractional deposition
9 is applied, and RDDR uses species body weight as the normalizing factor:
10 RDDRjot = x -^a x Zi2I]a
[BW)a [Ve)h [Ftot)h
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—Hexavalent Chromium
1 The current assessment does not apply RDDRtot to any endpoints.
Table D-18. RDDR calculations under different human ages and physiological
activity for systemic effects
Human
activity
Breathing rate
(min-1)
Tidal volume
(mL)
VE L/min
(calculated)
Ftot
Human
RDDRtot3
MMAD: Adult Yeh/Schum 5-lobe, uniform expansion
Breathing rate/tidal volumes for adult male (U.S. EPA, 2011a)
Resting 1
12
750
9
0.2752
2.7579
Resting 2
12
500
6
0.231
4.9285
Resting 3
15
500
7.5
0.2173
4.1914
Average RDDR (resting):
3.9593
Light work 1
17
1670
28.39
0.2966
0.8112
Light work 2
16
1250
20
0.2871
1.1896
Average RDDR (light work):
1.0004
Average RDDR (resting & light work):
2.4798
Heavy work
21
2030
42.63
0.3007
0.5329
Maximal work
40
3050
122
0.3632
0.1542
MMAD default
12
625
7.5
0.2576
3.5357
Human respiratory parameters (tidal volume and breathing rate) obtained from U.S. EPA (2011a).
Aerosol parameters: MMAD (0.28 ± 1.63 nm), concentration 136 mg/m3, and density 2.52 g/cm3.
Inhalation parameters: Inhalability adjustment 'on'.
RDDR calculations: rat Ftot 0.228, rat Ve 0.214 L/minute (calculated).
Body weight (rat): 0.5 kg.
Body weight (adult male human): 70 kg.
Calculation performed using total fractional deposition in the ET, TB, and PU regions, and using species body
weight as the normalization factor.
This document is a draft for review purposes only and does not constitute Agency policy.
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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
Supplemental Information—Hexavalent Chromium
D.2. BENCHMARK DOSE MODELING SUMMARY FOR CANCER ENDPOINTS
D.2.1. Cancer Data for Dose Response Modeling
For this assessment, dose-response modeling of endpoints for the oral route was performed
on the basis of time-weighted average daily dose of Cr(VI), in mg/kg-day. This value could then be
converted to an internal rodent dose, depending on the tissue or endpoint. The time-weighted
average was calculated based on time-course dose data available through the data collection time
for each endpoint.
The cancer endpoints selected for dose-response modeling are based on the data presented
in Table D-19. For reference, historical control data from the National Toxicology Program
encompassing the time period of the sodium dichromate dihydrate bioassays are presented in
Table D-20. These were not used to make adjustments to the dose-response modeling data.
Datasets modeled were:
• Male mice bearing adenomas or carcinomas of the small intestine (duodenum, jejunum, or
ileum)
• Female mice bearing adenomas or carcinomas of the small intestine (duodenum, jejunum,
or ileum)
• Male rats bearing squamous cell carcinoma or papilloma (oral mucosa or tongue)
• Female rats bearing squamous cell carcinoma or papilloma (oral mucosa or tongue)
For each endpoint, the exposure doses and data used for the modeling are presented. The
sample sizes were adjusted to be based on the number of animals surviving longer than one year.
The incidences were based on the number of tumor-bearing animals. For example, a mouse with
two tumors in the duodenum and one tumor in the jejunum is counted only once, and a rat with
both a squamous cell carcinoma in the tongue and a squamous cell papilloma in the oral mucosa is
counted once.
This document is a draft for review purposes only and does not constitute Agency policy.
D-22 DRAFT—DO NOT CITE OR QUOTE
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Supplemental Information—Hexavalent Chromium
Table D-19. Data of neoplastic lesions in rats and mice (NTP. 2008)
Tumor type and species/sex
Administered mg/L, mg/kg-d Cr(VI)a and
incidence/total
Male B6C3F1 mice
0 mg/L
5
10
30
90
0 mg/kg-d
0.450
0.914
2.40
5.70
Adenomas (duodenum)
1/50
0/50
1/50
5/50
15/50*
Carcinomas (duodenum)
0/50
0/50
0/50
2/50
3/50
Adenomas or carcinomas
(duodenum, jejunum, or ileum)
Incidence/Total
1/50
3/50
2/50
7/50*
20/50*
Incidence/Total (adj)b
1/50
3/49
2/49
7/50*
20/50*
Animals dead prior to day 365
0
1
1
0
0
Female B6C3F1 mice
0 mg/L
5
20
60
180
0 mg/kg-d
0.302
1.18
3.24
8.89
Adenomas (duodenum)
0/50
0/50
2/50
13/50*
12/50*
Carcinomas (duodenum)
0/50
0/50
0/50
1/50
6/50*
Adenomas or carcinomas
(duodenum, jejunum, or ileum)
Incidence/Total
1/50
1/50
4/50
17/50*
22/50*
Incidence/Total (adj)b
1/49
1/50
4/49
17/50*
22/49*
Animals dead prior to day 365
1
0
1
0
1
Male F344 rats
0 mg/L
5
20
60
180
0 mg/kg-d
0.200
0.796
2.10
6.07
Squamous cell carcinoma (oral mucosa)
0/50
0/50
0/49
0/50
6/49*
Squamous cell papilloma (oral mucosa)
0/50
0/50
0/49
0/50
1/49
Squamous cell carcinoma (tongue)
0/49
1/50
0/47
0/49
0/48
Squamous cell papilloma (tongue)
0/49
0/50
0/47
0/49
1/48
Squamous cell carcinoma or
papilloma (oral mucosa or
tongue)
Incidence/Total
0/50
1/50
0/49
0/50
7/49*
Incidence/Total (adj)b
0/50
1/47
0/47
0/50
7/49*
Animals dead prior to day 365
0
3
2
0
0
Female F344 rats
0 mg/L
5
20
60
180
0 mg/kg-d
0.248
0.961
2.60
7.13
Squamous cell carcinoma (oral mucosa)
0/50
0/50
0/50
2/50
11/50*
Squamous cell carcinoma (tongue)
0/45
0/49
0/48
1/48
0/48
Squamous cell papilloma (tongue)
1/45
1/49
0/48
0/48
0/48
Squamous cell carcinoma (oral
mucosa or tongue)
Incidence/Total
1/50
1/50
0/50
2/50
11/50*
Incidence/Total (adj)b
1/50
1/50
0/50
2/50
11/50*
Animals dead prior to day 365
0
0
0
0
0
aTime-weighted average daily doses calculated from NTP water consumption data.
bTumor incidences adjusted based on the number of animals surviving beyond 365 days. First tumor onset: 451
days for intestinal tumors in mice, and 506 days for oral tumors in rats (both occurring at the highest doses).
* Denotes significant difference from the control group reported by NTP (2008) using the Poly-3 test (p < 0.05).
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—Hexavalent Chromium
Table D-20. NTP historical control data for animals fed the NTP-2000 diet,
from studies of all routes and vehicles of administration (incidence, %,
mean % ± standard deviation %)a
Male B6C3F1 mice
Female B6C3F1 mice
Male F344/N rats
Female F344/N rats
Adenomas
Duodenum
8/1499 (0.53%)
3/1598 (0.19%)
-
-
0.55% ± 1.20%
0.19% ± 0.56%
Jejunum
1/1499 (0.07%)
0/1598 (0.00%)
--
--
0.07% ± 0.36%
0.00% ± 0.00%
Ileum
-
-
-
-
SI unspecified
9/1499 (0.60%)
3/1598 (0.19%)
-
-
0.62% ± 1.20%
0.19% ± 0.56%
Carcinomas
Duodenum
3/1499 (0.20%)
1/1598 (0.06%)
-
-
0.21% ± 0.79%
0.06% ± 0.35%
Jejunum
25/1499 (1.67%)
4/1598 (0.25%)
1/1449 (0.07%)
0/1350 (0.00%)
1.69% ± 1.83%
0.23% ± 0.57%
0.07% ± 0.37%
0.00% ± 0.00%
Ileum
2/1499 (0.13%)
2/1598 (0.13%)
-
-
0.14% ± 0.50%
0.13% ± 0.48%
SI unspecified
30/1499 (2.00%)
7/1598 (0.44%)
1/1449 (0.07%)
0/1350 (0.00%)
2.03% ± 1.81%
0.42% ± 0.70%
0.07% ± 0.37%
0.00% ± 0.00%
Adenomas or carcinomas
SI unspecified
38/1499 (2.54%)
10/1598 (0.63%)
1/1449 (0.07%)
0/1350 (0.00%)
2.59% ± 2.26%
0.61% ± 0.90%
0.07% ± 0.37%
0.00% ± 0.00%
Male B6C3F1 mice
Female B6C3F1 mice
Male F344/N rats
Female F344/N rats
Squamous cell carcinomas
Oral mucosa
1/1499 (0.07%)
2/1598 (0.13%)
5/1449 (0.35%)
5/1350 (0.37%)
0.07% ± 0.36%
0.13% ± 0.48%
0.32% ± 0.66%
0.38% ± 0.72%
Tongue
1/1499 (0.07%)
4/1598 (0.25%)
0/1449 (0.00%)
2/1350 (0.15%)
0.07% ± 0.36%
0.26% ± 0.63%
0.00% ± 0.00%
0.15% ± 0.52%
Oral cavityb
2/1499 (0.13%)
6/1598 (0.38%)
5/1449 (0.35%)
7/1350 (0.52%)
0.14% ± 0.50%
0.39% ± 0.71%
0.32% ± 0.66%
0.54% ± 0.95%
Squamous cell papillomas
Oral mucosa
1/1499 (0.07%)
0/1598 (0.00%)
1/1449 (0.07%)
2/1350 (0.15%)
0.03% ± 0.18%
0.00% ± 0.00%
0.07% ± 0.37%
0.15% ± 0.52%
Tongue
1/1499 (0.07%)
2/1598 (0.13%)
4/1449 (0.28%)
5/1350 (0.37%)
0.07% ± 0.36%
0.13% ± 0.48%
0.25% ± 0.60%
0.38% ± 0.91%
Oral cavityb
2/1499 (0.13%)
2/1598 (0.13%)
5/1449 (0.35%)
7/1350 (0.52%)
0.10% ± 0.40%
0.13% ± 0.48%
0.32% ± 0.66%
0.54% ± 0.95%
Squamous cell carcinomas or papillomas squamous
Tongue
2/1499 (0.13%)
6/1598 (0.38%)
4/1449 (0.28%)
7/1350 (0.52%)
0.14% ± 0.50%
0.39% ± 0.87%
0.25% ± 0.60%
0.54% ± 1.10%
Oral cavityb
4/1499 (0.27%)
8/1598 (0.50%)
10/1449 (0.69%)
14/1350 (1.04%)
0.24% ± 0.59%
0.52% ± 0.91%
0.64% ± 0.78%
1.08% ± 1.58%
aMarch 2007 historical control reports for F344/N rats and B6C3F1 mice (NTP, 2007a, b, c, d). Control data encompass
chronic studies that include the NTP sodium dichromate dihydrate study. Denominator is number of animals necropsied.
bOral mucosa, tongue, pharynx, tooth, gingiva. Note: for oral cavity, papillomas include both papillomas squamous and papillomas.
This document is a draft for review purposes only and does not constitute Agency policy.
D-24 DRAFT—DO NOT CITE OR QUOTE
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Supplemental Information—Hexavalent Chromium
D.2.2. Evaluation of Model Fit and Model Selection
1 Following EPA's Benchmark Dose Technical Guidance (U.S. EPA. 2012a) Sections 2.3.9 and
2 2.5 and EPA's Choosing Appropriate Stage of a Multistage Model for Cancer Modeling fU.S. EPA.
3 2014a"):
4 1) All orders of the Multistage model up to two less than the number of dose groups were fit
5 (e.g., up to model order k-2 if there are k dose groups).
6 a. If all parameter (y, pi, ..., (3k-2) estimates were positive, the model with the lowest AIC
7 was chosen as the best-fitting model if at least one of the models provides an adequate
8 fit to the data. Consistent with EPA's guidance when there is an a priori reason to prefer
9 a specific model(s) [(U.S. EPA. 2012a) §2.3.5 and §2.3.9], Multistage models having a
10 goodness-of-fitp-value of less than 0.05 were rejected.
11 b. Otherwise (i.e., if any parameter is estimated to be zero and is thus at a boundary), the
12 following procedure (2) was followed:
13 2) Model fits of orders 1 and 2 (linear and quadratic, respectively) were examined for
14 adequate fit The linear model parameters (y, pi), and the quadratic model parameters (y,
15 (31, (32) were examined.
16 a. If only one of the models exhibited adequate fit, that model was chosen.
17 b. If both models exhibited adequate fit:
18 i) The model with the lowest AIC was chosen if all of the parameters (y, pi,and p2)
19 were positive.
20 ii) Otherwise, the model with the lower BMDL (more health protective) was chosen. If
21 the BMD/BMDL ratio is larger than 3, the matter was referred to EPA statisticians
22 and health assessors for a decision.
23 Logfiles of BMD model outputs are contained in U.S. EPA (2021a).
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—Hexavalent Chromium
D.3. ALTERNATIVE APPROACHES FOR CANCER AND NONCANCER DOSE-
RESPONSE ASSESSMENT
D.3.1. Noncancer Oral Dose-response Applying Default BW3/4 Scaling Approaches
1 As a comparison against the pharmacokinetic method, RfDs were calculated using default
2 BW3/4 scaling. However, this comparison applies UFh = 3 (removing the pharmacokinetic portion of
3 the intraindividual variability). By not accounting for Cr(VI) reduction in either the rodent (gastric
4 pH = 4.5) or the human (gastric pH = 1.3), the default scaling approach focuses on a sensitive
5 population in terms of pharmacokinetics (i.e., a human population where baseline gastric pH = 4.5,
6 and gastric juice reduction capacity is equivalent to that of the rodent). All uncertainty factors are
7 described in Section 4. Study-specific body weights (and not default animal body weights) are used
8 in order to make a direct comparison of default and PBPK methods (which relied on study-specific
9 body weight).
Table D-21. Summary of derivation of points of departure following oral
exposure for effects outside of the gastrointestinal tract (default approach)
Species/
Sex
Model
BMR
BMD
mg/kg-d
BMDL
mg/kg-d
TWA BW (kg)
PODhed
mg/kg-da
Diffuse epithelial hyperplasia of the duodenum at 2 vears (NTP, 2008)
Mice/M
Quanta 1-
linearb
10
0.148
0.121
0.05
0.0191
Mice/F
LOAEL
-
-
0.302
0.05
0.0478
Changes in the liver enzvme alanine aminotransferase (ALT) (NTP, 2008)
Rat/M 12 mo.
Exp2b
1RD
1.83
1.56
0.395
0.414
Rat/M 3 mo.
NOAEL
-
-
1.58
0.246
0.372
Changes in the liver enzvme alanine aminotransferase (ALT) at 90 d (NTP, 2007f)
Rat/M
LOAEL
-
-
1.74
0.232
0.404
Rat/F
LOAEL
-
-
1.74
0.160
0.368
Chronic inflammation at 2 vears (NTP, 2008)
Rat/F
LOAEL
-
-
0.248
0.260
0.0592
Mice/F
Log-logistic
10% ER
3.70
1.33
0.05
0.210
Liver fattv change at 2 vears (NTP, 2008)
Rat/F
NOAEL
-
-
0.248
0.260
0.0592
Decreased offspring growth (NTP, 1997)
Mouse/F
NOAEL
-
-
11.6
0.024
1.53
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—Hexavalent Chromium
Species/
BMD
BMDL
PODhed
Sex
Model
BMR
mg/kg-d
mg/kg-d
TWA BW (kg)
mg/kg-da
Decreased hemoglobin (Hb) (NTP, 2008)
Rat/M (22 d)
Exp-4
1SD
1.07
0.816
0.138
0.166
aBW3/4 scaling adjustment: mg/kg-day multiplied by (BWa/80)1/4). Animal BW set to study/sex-specific time
weighted average values for hybrid PBPK modeling/BW3/4 scaling approach to maintain consistency with bioassay
PBPK simulation.
bData were amenable to BMD modeling with the highest dose omitted.
Table D-22. Effects and corresponding derivation of candidate values from
PODS applying BW% scaling
Endpoint and
reference
PODhed
(mg/kg-d)
POD
Type
UFa
UFh
UFl
UFs
UFd
Composite
UF
Candidate
value (mg/kg-d)
Gastrointestinal
Mouse (M) hyperplasia
(NTP, 2008)
0.0191
BMDLio
3
3
1
1
1
10
1.91 x 10"3
Mouse (F) hyperplasia
(NTP, 2008)
0.0478
LOAEL
3
3
10
1
1
100
4.78 x 10"4
Liver
Rat (M) liver ALT (12
mo) (NTP, 2008)
0.414
BMDLird
3
3
1
1
1
10
0.04143
Rat (M) liver ALT (3 mo)
(NTP, 2008)
0.372
NOAEL
3
3
1
3
1
30
0.01243
Rat (M) liver ALT (90 d)
(NTP, 2007f)
0.404
LOAEL
3
3
10
3
1
300
1.35 x 10"3a
Rat (F) liver ALT (90 d)
(NTP, 2007f)
0.368
LOAEL
3
3
10
3
1
300
1.23 x 10"3a
Rat (F) liver chronic
inflammation (2 yr)
(NTP, 2008)
0.0592
LOAEL
3
3
10
1
1
100
5.92 x 10"4
Mouse (F) liver chronic
inflammation (2 yr)
(NTP, 2008)
0.210
BMDLio
3
3
1
1
1
10
0.0210a
Rat (F) liver fatty
change (2 vr) (NTP,
2008)
0.0592
NOAEL
3
3
1
1
1
10
5.9 x 10"3
Developmental
Mouse (F) decreased
offspring growth (NTP,
1997)
1.53
NOAEL
3
3
1
1
1
10
0.153a
Hematological
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—Hexavalent Chromium
Endpoint and
reference
PODhed
(mg/kg-d)
POD
Type
UFa
UFh
UFl
UFS
UFd
Composite
UF
Candidate
value (mg/kg-d)
Rat (M) decreased Hb
(22 d) (NTP, 2008)
0.166
BMDLisd
3
3
1
1
1
10
0.0166a
aDenotes values that are higher than RfDs derived from pharmacokinetic modeling.
RfDs derived from the pharmacokinetic modeling are more health-protective than BW3/4 scaling at high doses. This
is because at high doses, the model is less sensitive to gastric pH and more sensitive to gastric reducing capacity.
The assumed human variability in gastric reducing capacity is very high, causing the lower 1% prediction to
ultimately produce a value lower than BW3/4 scaling. At low doses, the model is more sensitive to gastric pH. The
BW3/4 method is essentially assuming that the human gastric pH > 4 (whereas the pharmacokinetic model
assumes the human gastric pH = 1.3). As a result, the effectiveness of human gastric reduction when compared
to the rodent has a stronger impact on the model at low doses and produces less health-protective RfDs.
D.3.2. Order of Uncertainty Factor Applications
1 An alternative uncertainty factor approach applies some uncertainty factors that represent
2 uncertainties on the internal rodent dose (specifically UFl and UFa) to the rodent internal dose
3 prior to calculation of the human equivalent dose. The remaining uncertainty factors are then
4 applied after HED calculation to estimate the candidate RfDs. This process is outlined in Figure D-2.
5 Because of nonlinearities in the human gastric pharmacokinetics, this ultimately leads to slightly
6 different RfDs. Tables D-23 and D-24 illustrate what some of the PODs and RfDs would be using
7 this approach (with special focus on those leading to the final organ-specific chronic values; not all
8 endpoints were evaluated).
Rodent
Dose Response Data
Control
#/#
Low
#/#
Mid
#/#
High
#/#
Dose response
modeling
Rodent external dose BMDL
or LOAEl/NOAEl
/ Human \
I gastric model J
Solve daily oral dose
pioducing the equivalent
interna! dose in humans
Apply remaining
uncertainty factors
Internal dose POD in
mg/kg-d Cr(VI)
escaping stomach
reduction
BW3«
scaling
Apply uncertainty
factorsUF,, UFA
BMDL or LOAEL/NOAEL in
mg/kg-d Cr(VI) escaping
stomach reduction
Figure D-2. Alternative process for calculating the human equivalent dose for
Cr(VI). Uncertainty factors UFl and UFa are applied to the internal rodent dose
prior to animal-to-human extrapolation.
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—Hexavalent Chromium
Table D-23. Summary of derivation of points of departure following oral
exposure using alternative uncertainty factor process
Species/
Sex
Model
BMR
BMD
mg/kg-d
BMDL
mg/kg-d
Internal
dose3
mg/kg-d
TWA
BW
(kg)
bw3/4
adjust13
UFa,
UFl
Internal
dose POD
PODhed
(mg/kg-d)c
Diffuse epithelial hyperplasia of the duodenum at 2 vears (NTP, 2008)
Mice/M
Quantal
lineard
10%
ER
0.148
0.121
0.0182
0.05
2.88 x 10"3
3,1
9.60 x 10"4
0.0158
Mice/F
LOAEL
-
-
0.302
0.0463
0.05
7.32 x 10"3
3, 10
2.44 x 10"4
4.13 x 10"3
Changes in the liver enzvme alanine aminotransferase (ALT) at 90 d (NTP, 2007f)
Rat/M
LOAEL
-
-
1.74
0.188
0.232
0.0436
3, 10
1.45 x 10"3
0.0234
Rat/F
LOAEL
-
-
1.74
0.181
0.160
0.0383
3, 10
1.28 x 10"3
0.0209
Chronic inflammation at 2 vears (NTP, 2008)
Rat/F
LOAEL
-
-
0.248
0.0195
0.260
4.66 x 10"3
3, 10
1.55 x 10"4
2.64 x 10"3
Mice/F
Log-
logistic
10%
ER
3.70
1.33
0.225
0.05
0.0356
3,1
0.0119
0.116
Liver fattv change at 2 vears (NTP, 2008)
Rat/F
NOAEL
-
-
0.248
0.0195
0.260
4.66 x 10"3
3,1
1.55 x 10"3
0.0250
Decreased offspring growth (NTP, 1997)
Mouse/F
NOAEL
-
-
11.6
3.09
0.024
0.407
3,1
0.136
0.354
aDose escaping stomach reduction in rodent (mg/kg-d) estimated by PBPK modeling.
bBW3/4 scaling adjustment: mg/kg-d multiplied by (BWa/80)1/4. Animal BW set to study/sex-specific time weighted
average values for hybrid PBPK modeling/BW3/4 scaling approach to maintain consistency with bioassay PBPK
simulation.
cPODhed in mg/kg-d Cr(VI) oral dose ingested by humans (lower 1% value of 20,000 Monte Carlo PBPK simulations
needed to achieve the internal dose POD). See Appendix C.1.5 for details.
dData were amenable to BMD modeling with the highest dose omitted.
Table D-24. Effects and corresponding derivation of candidate values using
alternative uncertainty factor process
Endpoint and
reference
PODhed
(mg/kg-d)
POD
Type
UFa
UFh
UFl
UFS
UFd
Composite
UFa
Candidate
value (mg/kg-d)
Digestive tract tissues
Mouse (M)
hvoerplasia (NTP,
2008)
0.0158
BMDLio
[3]
3
[1]
1
1
3[10]
5.27 x 10"3
Mouse (F)
hvoerplasia (NTP,
2008)
4.13 x 10"3
LOAEL
[3]
3
[10]
1
1
3 [100]
1.38 x 10"3
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—Hexavalent Chromium
Endpoint and
reference
PODhed
(mg/kg-d)
POD
Type
UFa
UFh
UFl
UFS
UFd
Composite
UFa
Candidate
value (mg/kg-d)
Liver
Rat (M) liver ALT
(90 d) (NTP, 2007f)
0.0234
LOAEL
[3]
3
[10]
3
1
10 [300]
2.34 x 10"3
Rat (F) liver ALT
(90 d) (NTP, 2007f)
0.0209
LOAEL
[3]
3
[10]
3
1
10 [300]
2.09 x 10"3
Rat (F) liver chronic
inflammation (2 yr)
(NTP, 2008)
2.64 x 10"3
LOAEL
[3]
3
[1]
1
1
3 [10]
8.80 x 10"4
Mouse (F) liver
chronic
inflammation (2 yr)
(NTP, 2008)
0.116
BMDLio
[3]
3
[1]
1
1
3 [10]
0.0387
Rat (F) liver fatty
change (2 vr) (NTP,
2008)
0.0250
NOAEL
[3]
3
[1]
1
1
3 [10]
8.33 x 10"3
Developmental
Mouse (F)
Decreased F1
postnatal growth
(NTP, 1997)
0.354
NOAEL
[3]
3
[1]
1
1
3 [10]
0.118
aUFA and UFl have been applied to the internal rodent dose prior to calculation of the PODhed. The composite UF
applied to the PODhed reflects those applied after calculation of the PODhed (UFh, UFd). The values in [brackets]
indicate the product of all the uncertainty factors that have been applied in all steps.
This document is a draft for review purposes only and does not constitute Agency policy.
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1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
Supplemental Information—Hexavalent Chromium
D.3.3. Uncertainty Assessment of Low-dose Extrapolation Method for Oral Cancer Dose-
response
Because a mutagenic mode of action for Cr(VI) carcinogenicity (see Section 3.2.3) is
"sufficiently supported in (laboratory) animals" and "relevant to humans," EPA used a linear low
dose extrapolation from the POD in accordance with Guidelines for Carcinogen Risk Assessment (U.S.
EPA. 20051. However, multiple modes of action for tumor formation in the mouse small intestine
could be occurring in parallel, and presenting different approaches may shed light on uncertainties
in the assessment (U.S. EPA. 2005). For comparative purposes, a nonlinear estimate is provided
using a reference value approach based on one of the other modes of action outlined in Section
3.2.3 (inflammatory hyperplasia being a key event or precursor to tumor development).
The dose-response relationships for diffuse epithelial hyperplasia in the small intestine of
male and female mice from the chronic NTP (2008) bioassay were more sensitive than the dose-
responses for adenomas and carcinomas in the same tissue (Figure D-3). The nonlinear dose-
response approach would assume the noncancer organ-specific reference dose for gastrointestinal
toxicity (based on hyperplasia dose-response presented in Section 4.1) is protective of tumors in
the small intestine: 9 x 10"4mg/kg-day.
1.00
0.90
<11
u
0.80
c
u
0.40
ro
i
Ll_
0.30
0.20
0.10
0.00
O SI tumors (mice M+F)
X Oral tumors (rats M+F)
« SI hyperplasia (mice M+F)
4 6
Cr(VI) mg/kg-d
i—
8
10
Figure D-3. Dose-response data for tumors and diffuse epithelial hyperplasia
of the mouse small intestine (SI) and tumors of the rat oral cavity.
Applying the lifetime OSF for small intestinal tumors of 0.5 risk per mg/kg-day, the oral
dose for 1/10,000 risk would be 0.0001/0.5 = 2 x 10-4 mg/kg-day. The nonlinear, RfD-based
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—Hexavalent Chromium
1 estimate (9 x 10"4 mg/kg-day) is 4.5x higher. Based on the OSF, there would be a 4.5/10,000
2 increased cancer risk at the dose estimated using the nonlinear, RfD-based approach.
3 Tumors of the rat oral cavity did not have a proposed mode of action, and the dose-response
4 for these tumors was less sensitive than that for tumors of the small intestine in mice (see
5 Sections 3.2.3 and 4.1). The adult-based OSF for oral tumors is 0.1 risk per mg/kg-day (see
6 Section 4.3.3), and the ADAF-adjusted lifetime OSF10 would be 0.17 risk per mg/kg-day. For this
7 tumor type, the oral dose for 1/10,000 risk would be 5.9 x 10~4 mg/kg-day. The RfD-based
8 estimate would be 1.5 x higher than this dose. Applying that OSF, there would be approximately a
9 1.5/10,000 increased cancer risk at the dose estimated using the RfD-based approach.
10ADAF calculation: 10 x 0.1 x 2/70 + 3 x 0.1 x 14/70 + 1 x 0.1 x 54/70 = 0.166 (see Section 4.3.4).
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—Hexavalent Chromium
DA. EXCLUSION OF HUMAN STUDIES FOR EXPOSURE-RESPONSE
Table D-25. Overview of studies excluded for exposure-response analysis of
upper respiratory tract (nasal) effects in humans
Study
Population or industry
Reason(s) for exclusion
Armienta-Hernandez and
General population & chromate
production
Air data and nasal effects data not
contained in study, and source of data not
cited.
Rodriguez-Castillo (1995)
Bloomfield and Blum (1928)
Electroplating
Cannot determine accuracy or precision of
air concentration measurements.
Ceballos et al. (2019) (related
Paint stripping/aircraft refinishing
Air concentration data not representative
of inhaled dose due to full face mask use
by exposed workers.
study:
Ceballos et al. (2017))
Elhosarv et al. (2014)
Cement and tannery facilities
No air concentration data. Cannot
determine if exposure was to Cr(VI) or
Cr(lll).
Fagliano et al. (1997)
Residential (soil)
No air concentration data. Cannot
determine if exposure was to Cr(VI) or
Cr(lll).
Gomes (1972)
Electroplating
Relationship between air concentration
and outcome cannot be estimated from
presented data.
Horiguchi et al. (1990)
Electroplating
No air measurements.
Kitamura et al. (2003)
Electroplating
Did not include the preferred nasal
outcome measurements.
Kleinfeld and Rosso (1965)
Electroplating
Relationship between air concentration
and outcome cannot be estimated from
presented data. Cannot determine
accuracy or precision of air concentration
measurements.
Korallus et al. (1982)
Chromate production
No air measurements.
Lee and Goh (1988)
Electroplating
No air measurements.
Lin et al. (1994)
Electroplating
Measurement only for total chromium in
air, hexavalent chromium preferred.
Lucas and Kramkowski (1975)
Electroplating
Single exposure group.
Lucas(1976)
Painting/varnishing
Single exposure group, coexposures, did
not include the preferred nasal outcome
measurements.
Machle and Gregorius (1948)
Chromate production
Relationship between air concentration
and outcome unable to be estimated from
results as they are presented.
Mancuso (1951)
Chromate production
Measurement only for total chromium in
air, hexavalent chromium preferred.
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—Hexavalent Chromium
Study
Population or industry
Reason(s) for exclusion
PHS (1953)
Chromate production
Relationship between air concentration
and outcome cannot be estimated from
presented data.
Rovle (1975b)
Electroplating
Relationship between air concentration
and outcome cannot be estimated from
presented data.
Singhal et al. (2015)
Chromate production and
electroplating
No air measurements.
Sorahan et al. (1998) (related:
Ni-Cr platers
Relationship between air concentration
and outcome cannot be estimated from
presented data.
Sorahan et al. (1987))
Vigliani and Zurlo (1955)
Chromate production and
electroplating
No description of methods.
Wang et al. (1994)
Ferrochromium production
No air measurements.
Yuan et al. (2016)
Children in school near
electroplating plants
Did not include the preferred nasal
outcome measurements.
Note: Some studies excluded for consideration of nasal dose-response assessment were still included in the IRIS
assessment for other hazards. For some institutional references (e.g., NIOSH reports), the primary investigators
or report editors are listed as the authors.
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—Hexavalent Chromium
Table D-26. Overview of studies excluded for exposure-response analysis of
lung cancer in humans based on screening studies for adequate exposure-
response data3
Reference
Reason for exclusion
Ahn and Jeong (2014)
Not an occupational study of chromium exposure and cancer; purpose was
not to estimate a measure of relative risk.
Alderson et al. (1981)
Exposure assignments were based on tasks/ job title, not chromium
measurements. No air sampling was described.
Alexander et al. (1996)
Cumulative exposure estimated using approach with high likelihood of
exposure misclassification and lack of confidence in its representation of
exposure to individual participants. Median follow-up for most of the cohort
was less than 10 yr and median age at end of study was 42 yr, which reduced
the ability to ascertain cancer deaths.
Armienta-Hernandez and
Rodriguez-Castillo (1995)
No air data.
Becker et al. (1985)
Group-level exposure assignments were based on tasks/job title, not
chromium measurements. No air sampling was described.
Beveridge et al. (2010)
Group-level exposure assignments were based on job title, not chromium
measurements. No air sampling was described.
Bidstrup (1951)
Chromium exposures were not individually assigned; no measures of
association provided. No air sampling was described.
Bidstrup and Case (1956)
Exposure assignments were based on tasks/ job title, not chromium
measurements. No air sampling was described.
Blot et al. (2000)
Exposure metrics were not based on air measurements.
Boffetta et al. (2010)
Not an occupational study of chromium exposure and cancer; purpose was
not estimating a measure of relative risk.
Brown et al. (2004)
No effect estimates were reported for lung cancer and chromium exposure.
Chatham-Stephens et al. (2013)
Not an epidemiological study. No outcome measurements. Risk assessment
was performed.
Cole and Rodu (2005)
Not an epidemiological study (meta-analysis).
Davies et al. (1991)
Group-level exposure assignments were based on job title, not chromium
measurements.
Franchini et al. (1983)
No air data.
Frentzel-Bevme (1983)
Group-level exposure assignments were based on job title, not chromium
measurements. No air sampling was described.
Girardi et al. (2015)
Exposure metrics were not based on air measurements.
Halasova et al. (2009)
Inadequate exposure information.
Hall et al. (2020)b
Group-level exposure assignments were based on tasks/job title, not
chromium measurements. No air sampling was described.
Haves et al. (1989)
Group-level exposure assignments were based on job title, not chromium
measurements. No air sampling was described.
Hill and Ferguson (1979)
Analysis of trends over time; no analyses of associations with exposure
metrics based on air measurements.
Johnson et al. (2011)
Ecological study with biomarker data and no air data.
Koh et al. (2013; 2011)
Inadequate exposure information.
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—Hexavalent Chromium
Reference
Reason for exclusion
Linos et al. (2011)
Ecological study with no air data.
Milatou-Smith et al. (1997)
Siogren et al. (1987)
Group-level exposure assignments were based on job tasks, not chromium
measurements. No air sampling was described.
Moulin et al. (1993b)
Group-level exposure assignments were based on job tasks, not chromium
measurements. No air sampling was described.
Moulin et al. (1993a)
No chromium measurements.
Moulin et al. (1990)
No chromium measurements.
NJ DEP (2008)
Relationship between air concentration and outcome cannot be estimated
from presented data.
Pesch et al. (2019)
Exposures were based on tasks/ job title and air concentrations that were not
from this study population/location.
Rafnsson et al. (1997)
Group-level exposure assignments were based on job tasks and duration of
job, not chromium measurements.
Rosenman and Stanburv (1996)
Group-level exposure assignments were based on occupation, not chromium
measurements. No air sampling was described.
Rovle (1975a)
Inadequate exposure information. This article is part 1 of 2 articles. Air
sampling was described in part 2, and concentrations were reported as
exceeding certain values, but measured concentrations were not reported.
Shixiong (1994)
Categorical control data.
Sorahan and Harrington (2000)
Group-level exposure assignments were based on occupation, not chromium
measurements. No air sampling was described.
Sorahan et al. (1987)
Group-level exposure assignments were based on occupation, not chromium
measurements. No air sampling was described.
Sorahan et al. (1998)
Group-level exposure assignments were based on occupation, not chromium
measurements. No air sampling was described.
Tavlor (1966)
No chromium measurements.
van Wiingaarden et al. (2004)
Not an epidemiological study (meta-analysis).
TO MA (1987)
No chromium measurements.
Yang et al. (2013)
Not an epidemiological study (review).
Zhivin et al. (2013)
Exposure assignments were qualitative; based on time and numeric score for
level, not chromium measurements.
aThese studies were obtained via title/abstract screening and backward bibliography searches. Studies were
excluded from consideration after full-text screening based on the rationale provided. In HERO (click here), these
studies contain multiple inclusion/exclusion tags due to their potential relevance to other health effects. All were
excluded from consideration for the lung cancer exposure-response,
laryngeal cancer (respiratory tract outside of the lung).
Table D-27. Overview of studies excluded for exposure-response analysis of
lung cancer in humans based on screening the most recent analyses
Reference
Reason for exclusion
Mancuso (1997)
Mancuso and Hueper(1951)
Crump et al. (2003)
Luippold et al. (2003)
Painesville Ohio cohort studies superseded bv Proctor et al. (2016)
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—Hexavalent Chromium
Reference
Reason for exclusion
Haves et al. (1979)
Braver et al. (1985)
Park et al. (2004)
Park and Stavner (2006)
Baltimore Maryland cohort studies superseded by Gibb et al.,
(2020; 2015; 2000b)
Korallus et al. (1982)
Korallus et al. (1993)
German cohort studies superseded bv Birk et al. (2006)
Pastides et al. (1994)
Castle Havne, North Carolina cohort superseded bv Luippold et al.
(2005)
Machle and Gregorius (1948)
Baltimore and Painesville cohort studies superseded bv Proctor et
al. (2016) and Gibb et al. (2020; 2015)
Table D-28. Overview of studies excluded for exposure-response analysis of
lung cancer in humans
Reference
Reason for exclusion
Luippold et al. (2005)
SMR analysis conducted where no slope or standard error were
produced or could be calculated based on published data.
AEI (2002)
Note: These studies had passed the initial full-text screening (despite inadequacies in exposure data) because they
contained quantitative analyses that warranted further review for consideration. Studies were excluded from
consideration after review of the quantitative methods and their utility for the exposure-response assessment.
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—Hexavalent Chromium
D.5. INDIVIDUAL-LEVEL ANALYSIS OF NEOPLASTIC AND
NONNEOPLASTIC LESIONS IN MICE FROM NTP (2008)
Table D-29. Individual-level overview of neoplastic and nonneoplastic lesions
in male mice from NTP (2008)
Tumors
Hyperplasia
ID
Cr(VI) (mg/L)
Duod
Jej
II
Duod
Jej
II
11
0
A
-
-
-
-
-
55
5
C (multi)
-
-
-
64
5
w
A
--
81
5
--
C
--
DE
LT
--
105
10
-
-
C
DE
LT
CY
140
10
A
-
-
-
-
-
155
30
A
-
-
DE
-
-
161
30
A, C
--
--
DE
LT
--
162
30
-
c
-
DE
-
-
165
30
A
-
-
DE
LT
-
167
30
A
-
-
DE
-
-
172
30
C
--
--
DE
LT
173
30
A
--
--
DE
--
--
202
90
-
c
-
DE
DE
-
203
90
-
A
-
-
-
No eval
205
90
C
-
-
DE
-
-
206
90
A
--
--
DE
--
--
211
90
-
c
-
DE
-
-
214
90
A(multi)
-
-
DE
-
-
215
90
A, C
A
-
DE
-
-
217
90
A
--
--
--
--
--
218
90
A (multi)
-
-
-
-
-
219
90
A(multi)
-
-
DE
-
LT
222
90
A
-
-
DE
-
-
223
90
A
-
-
DE, FE
-
-
227
90
A
No eval
-
-
No eval
-
234
90
--
A
--
DE
--
--
235
90
A (multi)
-
-
DE
LT
-
238
90
A (multi)
-
-
DE
-
-
240
90
A
--
--
--
No eval
No eval
242
90
A, C
-
-
--
245
90
A
-
-
-
-
-
249
90
A (multi)
-
-
DE
--
--
Duod = duodenum, Jej = jejunum, II = ileum; A = adenoma, C = carcinoma, LT = lymphoid tissue hyperplasia,
DE = diffuse epithelial hyperplasia, FE = focal epithelial hyperplasia, CY = cyst. Shaded rows correspond to
exposed animals with no observed intestinal hyperplasia.
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—Hexavalent Chromium
Table D-30. Individual-level overview of neoplastic and nonneoplastic lesions
in female mice from NTP (2008)
Tumors
Hyperplasia
ID
Cr(VI) (mg/L)
Duod
Jej
II
Duod
Jej
II
268
0
--
c
--
--
-
--
317
5
--
A
--
--
--
--
351
20
--
C
--
DE
LT
--
371
20
A
--
--
DE, FE
DE
--
379
20
A
--
--
DE
--
--
380
20
--
c
--
DE
--
--
408
60
A
--
--
DE
--
--
411
60
A
--
--
--
--
--
412
60
A
--
--
DE
LT
--
413
60
A(multi)
--
--
DE
--
--
415
60
A
--
--
DE, CY
LT
--
416
60
--
A
--
--
--
--
420
60
--
A
--
DE, FE
--
--
421
60
A
C
--
DE
--
--
423
60
A
--
--
DE
--
--
427
60
A
--
w /¦
--
^ /¦
--
428
60
A
--
-
--
-
--
431
60
A
--
-
DE
-
--
438
60
C
--
-
DE
-
--
439
60
--
C
-
DE
-
--
440
60
A
--
-
DE
-
--
446
60
A
--
-
DE
-
--
450
60
A
--
-
--
-
--
451
180
A
--
-
DE
-
--
452
180
A(multi)
--
-
DE
-
--
454
180
--
A
-
DE
DE
--
455
180
A(multi)
A
-
DE
DE
458
180
A
--
-
DE
--
--
459
180
C
--
-
--
--
--
461
180
--
A
-
DE
DE
--
466
180
--
C
-
DE, LT
--
--
470
180
C
--
-
DE
--
--
472
180
--
A
-
DE
DE
--
474
180
c
--
-
DE
DE
--
475
180
A(multi)
--
-
DE
--
--
486
180
--
A(multi)
-
DE
--
--
This document is a draft for review purposes only and does not constitute Agency policy.
D-39 DRAFT—DO NOT CITE OR QUOTE
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Supplemental Information—Hexavalent Chromium
Tumors
Hyperplasia
ID
Cr(VI) (mg/L)
Duod
Jej
II
Duod
Jej
II
488
180
A(multi)
-
--
DE
-
--
489
180
A
--
--
DE
--
--
490
180
A
--
--
DE
--
--
492
180
C
--
--
DE
--
--
495
180
A(multi)
--
--
DE
--
--
496
180
A, C
--
--
DE
DE
--
497
180
A
--
--
DE
--
--
498
180
A(multi)
--
--
DE
--
--
499
180
C
--
--
[dilation]
--
--
Duod = duodenum, Jej = jejunum; II = ileum, LT = lymphoid tissue hyperplasia, DE = diffuse epithelial hyperplasia,
FE = focal epithelial hyperplasia, CY = cyst.
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—Hexavalent Chromium
Table D-31. Summary of neoplastic and nonneoplastic lesions in mice from
NTP f200m
Concentration
(mg/L)
Sex
Total # animals
with tumors in the
small intestine
# animals with tumors in the
small intestine and no
nonneoplastic lesions3 in the
small intestine
Animal IDs
0
M + F
2
2 (100%)
11, 268
5
M + F
4
3 (75%)
55, 64, 317
10
M
2
1 (50%)
140
20
F
4
0
30
M
7
0
60
F
17
5 (29.4%)
411, 416, 427, 428, 450
90
M
20
7 (35%)
203, 217, 218, 227, 240,
242, 245
180
F
22
2 (9.1%)
459, 499
All (excluding
control)
M + F
76
18 (23.7%)
Nonneoplastic lesions considered: lymphoid tissue hyperplasia, diffuse epithelial hyperplasia, focal epithelial
hyperplasia, cyst. Full individual-level datasets are available from NTP (2007e).
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—Hexavalent Chromium
D.6. PROBABILITY DISTRIBUTIONS OF HUMAN EQUIVALENT DOSE FOR
CANCER AND NONCANCER PODS DERIVED FROM TOXICOKINETIC
MODELING
D.6.1. Noncancer Model Outputs
CO
m
c
& M
effect = ALT (M rats, 3mo)
internal dose = 0,0389
1% = 0 191103294
5% = 0,224575355
25% = 0,287618225
50% = 0,344144
75% = 0,4138996
95% = 0,54371452:5
99% = 0,651395117999999
mean = 0,358805702715
SD = 0.0986748978985679
0,2 0.4 0,6 0,8
Human Equivalent Dose (mg/kg-d)
Figure D-4. Model outputs and distribution for rat (M) liver ALT (3 months)
fNTP. 20081.
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—Hexavalent Chromium
effect. = ALT (F rats, 90 clays)
internal dose = 0 0383
1% = 0.190428372
5% = 0.223410275
2:5% = 0.286987275
50% = 0.34304515
75% = 0.4119692
95% = 0.542653605
99% = 0 662561118
mean = 0.35789279697
SO = 0 099773638470065
0.2 0.4 0.6
Human Equivalent Dose (mg/kg-d)
0.8
Figure D-5. Model outputs and distribution for rat (F) liver ALT (90 days)
fNTP. 2007fl.
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—Hexavalent Chromium
effect = ALT (M rats, SO days)
internal dose = 0 0436
1% = 0 203400808
5% = 0 2384878
26% = 0.3018511
50% = 0,3610878
75% = 0.43379095
95% = 0.57163872
99% = 0.691671556
mean = 0.376436112105605
SD = 0 103640077739163
0,2 0.4 0.6
Human Equivalent Dose (mg/kg-d)
0.8
Figure D-6. Model outputs and distribution for rat (M) liver ALT (90 days)
fNTP. 2007fl
This document is a draft for review purposes only and does not constitute Agency policy.
D-44 DRAFT—DO NOT CITE OR QUOTE
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Supplemental Information—Hexavalent Chromium
lO
effect = duodenum hyperplasia (M mice)
internal dose = 0 00288
1% = 0.0442955848
5% = 0.0524186705
25% = 0.066970275
50% = 0.07984916
75% = 0 ...095 8703575
95% = 0.12708.256
99% = 0.154496666
mean = 0 083483330105
SO = 0 0233374428988372
0.05 0.10 0.15 0.20
Human Equivalent Dose (mg/kg-d)
0.25
Figure D-7. Model outputs and distribution for mouse (M) hyperplasia (NTP.
20081.
This document is a draft for review purposes only and does not constitute Agency policy.
D-45 DRAFT—DO NOT CITE OR QUOTE
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Supplemental Information—Hexavalent Chromium
effect = ALT (M rats. 12mo)
internal dose = 0.0451
1% = 0.204277786
5% = 0.23778671
25% = 0.304086825
50% = 0 36242845
75% = 0.434701775
95% = 0 568548025
99% = 0 680955527
mean = 0.37724788617
SO = 0.102271588409528
0.2 0.4 0.6 0.8
Human Equivalent Dose (mg/kg-d)
1.0
Figure D-8. Model outputs and distribution for rat (M) liver ALT (12 months)
fNTP. 2oom.
This document is a draft for review purposes only and does not constitute Agency policy.
D-46 DRAFT—DO NOT CITE OR QUOTE
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Supplemental Information—Hexavalent Chromium
effect = duodenum hyperplasia (F mice)
internal dose = 0 00732
1% = 0.0910873779
5% =0.10884108
25% = 0.138874675
50% = 0.1657233
75% = 0.1981843
95% = 0.2584384
99% = 0 308668257
mean = 0.172016876169
5D= 0.04634864419-64019
0.1 0.2 0.3 0.4
Human Equivalent Dose (mg/kg-d)
0.5
Figure D-9. Model outputs and distribution for mouse (F) hyperplasia (NTP.
20081.
This document is a draft for review purposes only and does not constitute Agency policy.
D-47 DRAFT—DO NOT CITE OR QUOTE
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Supplemental Information—Hexavalent Chromium
effect = liver irtflam (F mice)
internal dose = 0 0356
1% = 0...181763399
5% = 0.215539665
25% = 0 ..277742225
50% = 0.3336448
75% = 0.4026741
95% =0.53183048
93% = 0.650178387999939
mean = 0.34850284942
SD = 0.0987779500935221
0.2 0.4 0.6
Human Equivalent Dose (mg/kg-d)
0.8
Figure D-10. Model outputs and distribution for mouse (F) liver chronic
inflammation (2 years) (NTP. 2008).
This document is a draft for review purposes only and does not constitute Agency policy.
D-48 DRAFT—DO NOT CITE OR QUOTE
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Supplemental Information—Hexavalent Chromium
CM
effect = liver inflamfatty (F rats;
internal dose = 0.00466
1% = 0.066934497
5% = 0.078705274
25% = 0,. 1001134
50% = 0.1187845
75% = 0 14238395
95% = 0.185751495
99% = 0 22740899
mean = 0.1238829686705
SD= 0.033638618071271
0,05 0.10 0.15 0.20 0,25
Human Equivalent Dose (mg/kg-d)
0,30 0.35
Figure D-ll. Model outputs and distribution for rat (F) liver chronic
inflammation (2 years) (NTP. 2008).
This document is a draft for review purposes only and does not constitute Agency policy.
D-49 DRAFT—DO NOT CITE OR QUOTE
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Supplemental Information—Hexavalent Chromium
0.5
effect = Decr^ offspring gr. (mice)
internal dose = 0.407
1% = 0 70032483
5% = 0.76126823
25% = 0 87166235
50% = 0 96858215
75% = 1 08697425
95% = 1-30501375
99% = 148876274
mean = 0.99258860561
SD = 0..169433635452483
1.0 1.5 2.0
Human Equivalent Dose (mg/kg-d)
Figure D-12. Model outputs and distribution for mouse (F) Decreased F1
postnatal growth (NTP. 1997).
This document is a draft for review purposes only and does not constitute Agency policy.
D-50 DRAFT—DO NOT CITE OR QUOTE
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Supplemental Information—Hexavalent Chromium
to
m
I?
c oo
0}
O
OJ
effect = Deer. Hb (male rats, 22d)
internal dose = 0.0144
1% = 0.125369232
5% = 0.151925605
25% = 0.1981984
50% = 0.23965095
75% = Q.2890G9125
95% = 0.3747056^5
99% = 0.453968132
mean = 0.248716540307
SP= 0.06 9688783970485
0.2 0.4 0.6
Human Equivalent Dose (mg/kg-d)
0.8
Figure D-13. Model outputs and distribution for rat (M) decreased Hb at 22
days fNTP. 20081
This document is a draft for review purposes only and does not constitute Agency policy.
D-51 DRAFT—DO NOT CITE OR QUOTE
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Supplemental Information—Hexavalent Chromium
o
co
in
£
W
c
-------
Supplemental Information—Hexavalent Chromium
m
j=? CM
W
c
0)
Q
effect = Deer. Hb (male rats 12mo)
internal dose = 0.0891
1% = 0,288293974
5% = 0.326516395
25% = 0.403170975
50% = 0,4712605
75% = 0.554853975
95% = 0 ..713693.265
99% = 0.060138524999398
mean = 048932937878
SD = 0 120394455457457
0.2 0.4 0.6 0.8 1.0
Human Equivalent Dose (mg/kg-d)
1.2
Figure D-16. Model outputs and distribution for rat (M) decreased Hb at 12
months fNTP. 20081
o
CO
iq
CNJ*
o
m
J 2
o
O
d
effect = Deer Hb (female rats 23d. NTP 2007
internal dose = 0.0704
1% = 0.254302866
5% = 0.29363863
25% = 0 ..3646689
50% = 043020075
75% = 0.511624625
95% = 0.662630025
99% = 0.791418718
mean = 0.44754038062
SD = 0.115050363952225
0.2 0.4 0.6 0.8 1.0 1.2
Human Equivalent Dose (mg/kg-d)
1.4
Figure D-17. Model outputs and distribution for rat (F) decreased Hb at 23
days fNTP. 2007fi
This document is a draft for review purposes only and does not constitute Agency policy.
D-53 DRAFT—DO NOT CITE OR QUOTE
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Supplemental Information—Hexavalent Chromium
co
£
en CM
C
0)
O
effect = Deci Hb imale rats 23d, NTP 2007)
internal dose = 0-0722
1% = 0.259199173
5% = 0.296420185
25% = 0.3685601
50% = 0.4316499
75% = 0.512875875
95% = 0.665251295
99% = 0.797580424
mean = 0.450222213525
SQ = 0.11490737119805
0,2 0.4 0.6 0.8 1.0
Human Equivalent Dose (mg/kg-d)
1.2
Figure D-18. Model outputs and distribution for rat (M) decreased Hb at 23
days fNTP. 2007fi
This document is a draft for review purposes only and does not constitute Agency policy.
D-54 DRAFT—DO NOT CITE OR QUOTE
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Supplemental Information—Hexavalent Chromium
D.6.2. Cancer Model Outputs
CO -
>.
-M
-------
Supplemental Information—Hexavalent Chromium
effect = SI tumors (M mice)
internal dose = 0 0267
1% = 0...163225824
5% = 0.193513175
25% = 0.25083585
50% = 0.3029345
75% = 0.36782775
95% = 0.48419735
99% = 0.586314313
mean = 0 316391495565
SD = 0.0906471895624962
0.2 0.4 0.6
Human Equivalent Dose (mg/kg-d)
0.8
Figure D-20. Model outputs and distribution for adenomas or carcinomas in
the male mouse small intestine (NTP. 2008).
This document is a draft for review purposes only and does not constitute Agency policy.
D-56 DRAFT—DO NOT CITE OR QUOTE
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Supplemental Information—Hexavalent Chromium
APPENDIX E. SAS CODE FOR LIFE-TABLE ANALYSIS
1 The following pages contain the SAS programs for life-table analysis.
This document is a draft for review purposes only and does not constitute Agency policy.
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OPTIONS NODATE NONUMBER orientation=landscape linesize=max; *BT added 7/3/19;
/-,
This program calculates the risk of lung cancer from inhalation exposure to Or(VI),
using a lifetable approach based on BEIR IV. The basic exposure-response model is RR :::::: exp (beta
* CE5).
The basic code for the lifetable calculations were developed and provided to EPA
by Randall Smith at NIOSH. The code from NIOSH calculates the baseline risk (RO) and the exposed
risk (Rx)
from exposure to an exposure concentration of X Level using NIOSH Model 1: Rx :::::: RO * exp(COEF "k
X Level).
EPA h in 'in 'Mi HI' ¦ II i i II :
1) Th . I I 1 mi | in ' I '-yita I'll h ' I n updated
2) Th ,m ii I mI ii i n i I i in hi I n i i 1 all iln M I i 1 on mid-point
of year:
'I l in : mi r (age+ 0 1 I i | i i i i i, 11 1 i
3) An ,m ii l n h i. I - n idded to >1 nl u i i > i I I i i . I l I (Rx - RO) / ( 1 - RO)
3) A Im I" * n n !¦ !¦ ' ! to f inc1 i 11 | mi I I i. I I i iImi /ields an extra risk of 0.01
(1%) .
This is referred to as EC1%, which may then be used to calculate the unit risk: UR :::::: 0.01 /
EC1%
-,/
/* .\Beta Version.sas 19jan00, 26jul00, 25oct01, 06dec05, 30novl8
E x p e r i in e n t a 1 v e r s i o i i
title "Lifetable calculation of lung cancer risk";
title2 "under a non-linear relative rate model";
j -k (.
I Compute excess risk by the BEIR IV method using SAS datasteps. I
e cause-
e rate
ef*X)]
(with Lag)
Coef de : of exposure and
hO is t ?=0) .
(Except j.u.l luui, liilisli ,i.lu .l u 11 c l i. Li 11 ui. age.)
A few simple models of f(Ooef*Xl are . ii | 111 1 as
ill 1 I I w. Mi 1111 I i ii 1 in 1 I 111 I | l. f ied with
i I i i i I in i work i I 1111: I i ii 1 11111 I
¦ .1 HI I 11. LIB \ I I ' " ,1 II I I I I ¦ l .1 I i 11 1 I I I I l .SAS) .
i a -
:rf
(1988) .
+USER-SUPPLIED ASSIGNMENTS:
re assigned using "%LET" state-
ST X, DURATION, LASTAGE.
ow.
puting risk are defined
is entered as a life-table in
j.. 11 a j.. v j.. a u a i. 1.11 i. r i e a a i. a s l e p
This document is a draft for review purposes only and does not constitute Agency policy.
E-2 DRAFT-DO NOT CITE OR QUOTE
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Supplemental Information—Hexavalent Chromium
NOTES: +
Ix Datastep "EX RISK" is where the desired risks are computed.
below.)
¦ Linear Rel. Rate
k is over the
at a younger age.
+ SAS Programmer: Randall Smith
| The Nat'l Inst, for
| 2 6 j u ].. 2 0 0 0 F 2 3 j u 12 0 0.1
+ Modifications :
I 2 6 j u ].. 0 0 Fi>
al Safety & Health
ir 18nov2 018
: 3 iuio:.i
i defirri i
model
exposui
sk) ,
3 0 n o v 18
1 the
i n a (
. 3 s tt.
March 2019: BT (SRC) Added rnaxro CON
runs macro BEIR4 until the EXPOSURE
extra risk::::::0 . 01 (the point of departing i. rujjj .
,|ju;,l!j. us is i. i.:..ed . |
I w In i c. l'i i t e r a t i v e 1 v
.'.ON corresponds to an
Miac.ro CONVE R4 works with one value for the exposure
variable > i.e., when the data C Levels includes one record.)
i i ih nev
macro CONVERGE BEIR4
: 1. OW) .
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—Hexavalent Chromium
i. i
i Go I
1 =•
2 => Linear RelatDv
3 -
4 =
0 ::::::
C u in u 1 a t i 'v e e x id o s u r e id a r a in e t e r:
A S S G NME N T S (M a c. r o v a r i a b 1 e s)
f.
, v- i~> r-> o ill - o o f" f" o r< +• q ; |
'ate |
mined
Del ow
V %Let Model
V %Let COEF
Lag or delay between exposure and effect: "k/ %Let Lag
1;
0.001298;
5;
A n e e x d o s 11 r e b e n i i i s :
*/ %Let Agelst_x = 16;
*/ %Let Duration = 85;
(Y/N)? "k/ %Let EnvAdj = Yes;
o f
V %Let LastAge =85;
V
PART II. USER-SUPPLIED ASSIGNMENTS (Datesets AllCause, Cause, X Levels ): */
data AllCause (label="Unexposeds' age-spec mortalty rates (all)"
drop=Lx rename=(BLx=Lx) );
age (Age)
Label Age
BLx
Lx
CndPrDth
qi
AllCause
'Age at start of year (Age=i)"
'Number alive at start of year"
'Number alive at end of year"
'Pr[Death before age i+1 | alive at age i]
'Pr[Survive to age i+1 | Alive at age i]"
'Age-spec mortality rate (all causes)";
if _n_=l then input age //// 61 BLx 6; /* ////
input Lx 66;
CndPrDth = (BLx - Lx)/BLx;
qi = 1-CndPrDth;
if qi <= 0 then AllCause = le+50;
else AllCause = - log(qi);
if age < &LastAge then output; else STOP;
BLx=Lx;
age+1;
retain age BLx;
skip next 4 lines
cards;
This document is a draft for review purposes only and does not constitute Agency policy.
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0 = Life-table starting age. (Required: Values must begin 4 lines down!)
The following are 2017 Life-table values of US population
starting at birth and ending at age 85.
(Source: Nat.Vital Statistics Reports 2019 Vol 68 No 7, Table 1,
https://www.cdc.gov/nchs/data/nvsr/nvsr68/nvsr68_07-50 8.pdf)
100000 99422 99384 99360 99341 99326 99312 99299 99288 99278
99268 99259 99249 99236 99217 99191 99158 99116 99066 99006
98937 98858 98770 98674 98573 98466 98355 98241 98122 97999
97872 97740 97603 97461 97314 97163 97006 96843 96674 96501
96321 96135 95939 95732 95511 95275 95023 94753 94461 94144
93797 93419 93008 92560 92070 91538 90963 90345 89684 88978
88226 87424 86570 85664 84706 83696 82632 81507 80315 79048
77697 76265 74715 73064 71296 69418 67402 65245 62933 60462
57839 55053 52123 49035 45771 42382
*run;*BT 7/3/19 added Run statement here;
data CAUSE (label="Unexposeds' age-cause-spec mortalty rates");
/ * +
I Specify unexposeds' age-specific mortality rates (per year) |
I from specific cause. I
_i -k /
label Age = "Age"
Rate_e5 = "Age,cause-specific rate per 100,000"
Rate = "Age,cause-specific rate per individual";
if _n_ = 1 then input age /* input starting age */
///; /* III => skip next 3 lines */
input Rate_e5 @@;
Rate = Rate_e5 * le-5; /* Convert to rate per individual */
if age <= 4
then DO; output; age+1; END;
else DO i = 0,1,2,3,4; /* */
if age < &LastAge /* Fill out into yearly intervals from */
then output; /* inputted five year intervals after age 4*/
age+1; /* */
END;
cards;
0 = Start age of cause-specific rate (Required: Rates begin 3 lines down!)
The following are 2017 ICD10 = 113, (C33-C34) death rates per 100,000 for US pop'n
starting at birth.
For ages 5 and above, each rate holds for the age thru age+4 years.
Source: CDC Wonder, https://wonder.cdc.gov/ucd-icdlO.html
0 0.038 0.038 0.038 0.038 0.010 0.019 0.033 0.045 0.120 0.382 1.074 3.131 8.506 24.321 54.508
87.599 131.875 198.108 265.763 309.625
*run; *BT 7/3/19 added Run statement here;
data X_LEVELS (label= "Exposure levels (e.g., concentrations)" );
/ * +
I Specify environmental exposure levels I
I and update label for the variable, XLevel, if necessary: |
+ */
/* +
| BT 3/8/19: Add maxro CONVERGE_BEIR4 which iteratively runs macro |
| BEIR4 until the EXPOSURE_CONCENTRATION corresponds to extra_risk=0.01|
I The intent was to make as few changes to BEIR4 as possible. The data |
I X_LEVELS and variable XLevel are retained but the initial value of |
| XLevel is provided in the call to macro CONVERGE_BEIR4 (the value |
I of Xlevel in the cards statement is not used in the calculations. |
_i -k /
input XLevel @@;
label XLevel= "Cr(VI) exposure (jag Cr (VI)/m3) ";
cards;
1
This document is a draft for review purposes only and does not constitute Agency policy.
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%Macro BEIR4;
/* March 2019 - BT (SRC): Macro BEIR4 is now called by macro CONVERGE BEIR4.
¦alculations and printed results in a rnacrc
) 1 e a p p 1 i c a t i o i i s o f t h e a 1 g o r i t h in.
/* PART III. Perform calculations:
data EX_RISK (label = "Estimated excess risks [Method=BEIR IV]"
/*keep :::::: XLevel Rx ex risk RskRatio RO extra Risk */
rename= (Rx=Risk));
/">
Calc
in w<
worl.
< and excess risk fo
:il by BEIR IV method
and work.Cause to d
ich exposure concentration I
.ng information in I
1 ( : I < : I < : I < :ll i I. >< >pllla tioi'l : |
^ ^ */
format hi F15.8; *BT 7/.'i/rn: kmcii mki i orrnat statement;
length XLevel 8.;
label Age = "Age at start of year (i)"
XTime = "Exposure duration midway between i & i+1"
XDose = "CE5(adj) (pg Cr(VI)/m3-yrs)"
RO = "Cumulative Risk of lung cancer (unexposed) (RO)"
Rx = "Cumulative risk of lung cancer (exposed) (Re)"
Ex_Risk = "Excess risk (Rx-Ro)"
RskRatio = "Ratio of risks (Rx/Ro)"
hi = "Lung Cancer hazard (unexposed) (hi)"
hix = "Lung Cancer hazard (exposed) (hei)"
hstari = "All cause hazard (unexposed) (h*i)"
hstarix = "All cause hazard (exposed) (he*i)"
qi = "Probability of surviving year i assuming alive at start (unexposed) (qi)
S_li = "Probability of surviving to end of year i (unexposed) (Sl,i)"
S_lix = "Probability of surviving to end of yeari (exposed) (Sel,i)";
3/19: Calculation of unexposed's risk (following DO LOOP) could be omitted from the
i
require further changes to BEIR4(?).
*e . g . r %if i=l ido; */
if _n_=l then DO;
/* Calculate unexposed?s risk (RO) to be retained
/"k based on equation 2A-21 (pg. 131) of BEIR IV:
/* Initialize:
V S li
1;
RO
0;
DO pointer = 1 to min(n_all,n_cause) until (age>=&LastAge-l);
set allcause (keep=age AllCause rename=(AllCause=hstari))
point=pointer nobs=n_all;
set cause (keep=age Rate rename=(age=ageCause Rate=hi))
point=pointer nobs=n_cause;
if Age NE AgeCause then
put "** WARNING: Age values in datasets ALLCAUSE and CAUSE don't conform **"
/ 013 "Rates misaligned on age could give incorrect results'
/ @13 Pointer=
+2 "Age(ALLCAUSE)=" Age +2 "Age(CAUSE)=" AgeCause /;
qi = exp(-hstari);
RO = RO + ( hi/hstari
S_li = S_li * qi;
S li
END;
END;
(1-qi) );
/* End of 5 if n =1 then DO; 5 stmt */
retain RO;
This document is a draft for review purposes only and does not constitute Agency policy.
E-6 DRAFT-DO NOT CITE OR QUOTE
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Supplemental Information—Hexavalent Chromium
/* Calculate exposed?s risk (Rx, renamed to Risk) for each exposure level */
/* ultimately based on equation 2A-22 (pg. 132) of BE1R IV */
/* but re-expressed in a form similar to equation 2A--21: */
-J;' BT 3/20/19. This version of CONVERGE RETR'1 will work when there is one concentration in data
set x levels - i.e., one value for xL:vci .
The R" 1 ""i" f"r ¦ icvcl " i mmmted out;
DO p l 111 I i 11 ... I
'k set 1 1 point::::::pointR n I ii i /"' I 1 I : determines when to end the loop. Nobs is
set ai 1111 l I m ion, so the Cm i n I is a u I il I it first run through loop - just one
recor 1 m 1 n variable (XR I > in 'mi :et ; I I '/
/* BT 3/20/19: added the next lint to set the exposure concentration :::::: current value of
^exposure cone. */
xlevel = &exposure_conc;
/* Initialize
k/ S lix
1; Rx
0;S li=l; R0=0;
DO pointer = 1 to min(n_all,n_cause) until (age>=&LastAge-l);
set allcause (keep=age AllCause rename=(AllCause=hstari))
point=pointer nobs=n_all;
set cause (keep=Rate rename=(Rate=hi))
point=pointer nobs=n_cause;
XTime
min( max(0,(age+0.5-&Agelst_x-&Lag))
, ^Duration - 0.5 );
if UpCase ('
/* Occ.upat:
&EnvAdj")
onal to Ei
= "YES"
vironrnei
1 Conversion
then XDose
V
L/0.52
L/10 0 0
ar */
(L) per dav */
.)) */
/* 3Onov2 018 ( 'ELSE') */
XLevel
* 385, 10 /*
* 20/'.' /*
C o i i v e r t i i i g ¦ t a (C r 0 3) '
Converting /m3 to ug,
* XTime;
ERSE if UpCase("&EnvAdj") = "NO"
then XDose = XRevel*XTime;
else DO; put //"Macro variable ENVADJ incorrectly specified."
/"It should be either YES or NO. Value specified is: &ENVADJ"
/;
STOP;
END;
hix=.;
exp(&COEF*XDose); else
(1 + &COEF*XDose); else
&COEF*XDose; else
(1 + XDose)**&COEF; else
iser-defined model goes here. */
END;
hstarix = hstari /* hi=backgrd rate is included in hstari
+ (hix - hi); /* so that adding in the excess
/ "k from exposure (hix-hi) gives the
/* total rate of the exposed.
if
SModel
= 1 then
hix =
hi
if
SModel
= 2 then
hix =
hi
if
SModel
= 3 then
hix =
hi
if
SModel
= 4 then
hix =
hi
if
SModel
= 0 then
DO;
hix =
-99999; /'
' Code
fo
qix = exp (-hstarix);
Rx = Rx + ( hix/hstarix * S_lix
S_lix = S_lix * qix;
( 1-qix ) );
qi = exp(-hstari);
RO = RO + ( hi/hstari * S_li
S_li = S_li * qi;
output;
(1-qi) );
END;
This document is a draft for review purposes only and does not constitute Agency policy.
E-7 DRAFT-DO NOT CITE OR QUOTE
-------
Supplemental Information—Hexavalent Chromium
Ex_Risk = Rx - RO;* Rx :::::: risk in exposed population;
RskRatio = Rx / RO; "k RO :::::: from cancer;
Extra_risk = Ex_Risk/(1-RO);
/* BT 3/20/19 added:*/
call symput(,Extra_Riskm',Extra_Risk) ;
1 I 1 l I L 9 i | I i Mi n i I l11
I i i i I I i 3k 'I i ii I iii i Ijx Risk) ; */
Eiff_E _Fisk = a) > _n \ _t ai j~t-Extra_Risk) ;
call symput('Delta_Ex_Risk',Diff_Ex_Risk);
output;
END; "k corresponds to X Levels;
STOP;
run;
%Mend BEIR4;
BT: March 2019: i. >a i a im< iters for the convergence that are used
in the modified v<:i::i<>n of the BEIR4 macro.
%macro Converge_BEIR4 (init_exposure_conc=, ex_risk_target=, conv_criterion=, max_iteration=);
%Let Delta_Ex_Risk = 1; * initial high value to mak<
(i . e . r mce)
/* BT 4/15/19: added next line to avoid error during compiling of BEIR4*/
%Let Extra_Riskm = 1;
%Let i=l; "k first time through loop;
%Do %Until (%sysevalf(&Delta_Ex_risk < &conv_criterion) OR %sysevalf(&i >
&max_iteration)) ;
"k first time through loop, set expsosure conc::::::init exposure cone;
%If &i=l %Then
%Do;
%Let exposure_conc=&init_exposure_conc;
%End;
%If &i>l %Then
%Do;
data tempBEIRCONVERGE;
*BEIR4 has run at least once. Adjust exposure cone
Extra Riskin is created in BE1R4 (=Extra Risk) ;
NumLoops=&i;
thisExposureConc=&exposure_conc;
/* BT 4/15/19: replaced all of the co with the same code that we used
111 I I |( : IIK :: :< > < '-Ode . "k /
numvar= _ _ t;
denvar=&Extra_Ris km;
thisexposureconc = thisexposureconc * (numvar/denvar);
^update the concentration;
call symput('exposure_conc',thisexposureconc);
output;
Run;
%End; "^Corresponds to If i>l statement;
%BEIR4;
%Let i=%eval(&i+l);
%End;
This document is a draft for review purposes only and does not constitute Agency policy.
E-8 DRAFT-DO NOT CITE OR QUOTE
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Supplemental Information—Hexavalent Chromium
stop;
run;
"k rename variables to enable overwriting the values of S 11 and S lix in ex risk with the values
in newSRCData;
"k Data file ternpSRCData has -85 while the ex Risk file has age 0-84, with last two records
both having .;
Data ternpSRCData; Set newSRuuara{rename=(SRC_Age=age SRC_S_li=S_li SRC_S_lix=S_lix));
if age=&LastAge then age=%sysevalf(&Lastage-l); Else age=age;
Run;
"k there are duplicate values for age in both ex risk and ternpSRCData
which may produce too many records, if that happens, then we use two set-
statements;
Data ex_risk; merge ex_risk ternpSRCData; By Age; Run;
/* BT 7/5/19: End of code that was added to rnerg : for unexposed risk
(S li and S lix) t< > i n<: ic::i of the output, bv age;
*/
*BT 7/3/19: made the the h m i the following Proc Print procedure:
- commented m I I i| I option and add' I split, uniform and width= options
- included all i i >1 i.o the format c;
proc print data=ex_risk 1 I il I 1 n ])S split=,/, wi< lth=FTTLL;
format age F4. Xdos- hi hstari Ell. hi hstari> ji Ell. qix Ell.
S_li Ell. S_lix Ell. R0 E Fi k Ell. E _h J- Ell. ;
label Age = "Age at start of year li)"
XDose = "CE5(adj) (ug Cr (VI) /m3-yrs) "
R0 = "Cumulative Risk of lung cancer (unexposed) (R0)"
Risk = "Cumulative risk of lung cancer (exposed) (Re)"
Ex_Risk = "Excess risk/[Rx-Ro]/ /(Ex_Risk)"
hi = "Lung Cancer hazard (unexposed) (hi)"
hix = "Lung Cancer hazard (exposed) (hei)"
hstari = "All cause hazard (unexposed) (h*i)"
hstarix = "All cause hazard (exposed) (he*i)"
qi = "Probability of surviving year i assuming alive at start (unexposed) (qi)"
qix = "Probability of surviving year i assuming alive at start (exposed) (qei)"
S_li = "Probability of surviving to end of year i (unexposed) (Sl,i)"
S_lix = "Probability of surviving to end of yeari (exposed) (Sel,i)";
Var Age Xdose hi hstari hix hstarix qi qix S_li S_lix R0 Risk Extra_risk; *BT
7/3/19: Var statement added;
label Extra_risk="Extra Risk (Re a€" R0)\(1 a€" R0)";
run;
%End; *end of the If statement that tests if convergence was met;
%Mend Converge_BEIR4;
j -k (.
I II ii h ' h 1 " I '1 i 1:1 i 111 i i mi <:< >nvim-:i ,i:. i m . inc user should alsol
I review parameters and
-------
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Supplemental Information—Hexavalent Chromium
feLet EC IPercent
&exposure_conc;
Rei
convergei
%If %sysevalf(&Delta_Ex_risk < &conv_criterion) %then %do;
title5 "based on beta=&COEF, Concentration=&EC_lPercent, and LastAge=&LastAge";
data
_null_;
pointer=l;
set allcause
/* Modi fi e d 2 6-j u1v-00 */
inter nobs=n_all;
nter nobs=n_cause;
winter nobs=n all;
(keep=age
rename=(age=ageallO)) point=po
set cause (keep=age
rename=(age=ageCs0)) point=poi
pointer=n_all;
set allcause (keep=age
rename=(age=agealll)) point=po
pointer=n_cause;
set cause (keep=age
rename=(age=ageCs1)) point=pointer nobs=n_cause
Tmp = sum(min(AgeAl11,AgeCsl, C&Lastage-l) ) ,1) ;
file PRINT;
if ageallO NE ageCsO then DO;
put /"ERROR: The initial age for all-caus
/'
es rate differs from the"
initial age for the cause-specific rate.";
END;
else DO;
put
/
//
/
//
/
/
/
/
Values of macro variables used in this computation:
''Macro_Var" @29 "Description"
"Value"
'' &Model
617
@17
@17
''MODEL"
/
@3
"SCoef "
@17
"COEF"
11
@3
"&Lag "
@17
"LAG"
11
@3
"SAgelst x"
@17
"AGE1ST X
/
@3
"SDuration"
@17
"DURATION
/
@3
"SEnvAdj"
@17
"ENVADJ"
/
/
/
@3
//
// @3
// " —
/"The
// ;
@17
@25
" @2S
@2S
@2S
@2S
@2S
@2S
@2S
@2S
@2S
@2S
@2S
@2S
@2S
@29 "
1 = Loglinear Relative Rate,
2 = Linear Relative Rate,
3 = Linear Absolute Rate,
4 = 'Power' Relative Rate,
0 = User defined.
Exposure parameter estimate"
Exposure Lag "
Age exposure begins"
Duration of exposure"
Adjust dose from intermittent"
occupational exposures to "
continuous environmental exposures'
@10 "&EC_1 Per cent" @25 " (\iq Cr(VI)/m3); Rx = " @39 "&Extra_Riskm"
ageallO " up to age " Tmp "."
EC1% =
risks are calculated from age
if agealll NE ageCsl then
put /"WARNING: The last age for the all-causes rates differs from"
/" the last age for the cause-specific rates, suggesting"
/" the possibility that the rates weren't entered as desired."
/;
END;
Stop;
run;
/* BT 7/5/19: Start of code that was added to merge unexposed risk
(S li and S lix) to the ic::i <>i i n<: output, by age;
Data newSRCData(keep=SRC_age SRC_S_li SRC_S_lix);
set ex_Risk;
SRC_age=0; SRC_S_li=l; SRC_S_lix=l;
output;
do obsnum=l to last-1;
set ex_Risk point=obsnum nobs=last;
if _error_ then abort;
SRC_age=age+l; SRC_S_li=S_li; SRC_S_lix=S_lix;
output;
end;
This document is a draft for review purposes only and does not constitute Agency policy.
E-9 DRAFT-DO NOT CITE OR QUOTE
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Supplemental Information—Hexavalent Chromium
Values of macro variables used in this computation:
Value Macro_Var Description
1 MODEL 1 = Loglinear Relative Rate,
2 = Linear Relative Rate,
3 = Linear Absolute Rate,
4 = 'Power' Relative Rate,
0 = User defined.
0.001298 COEF Exposure parameter estimate
5 LAG Exposure Lag
16 AGE1ST_X Age exposure begins
85 DURATION Duration of exposure
Yes ENVADJ Adjust dose from intermittent
occupational exposures to
continuous environmental exposures
EC1% = 1.1795769661 (f/ml); Rx = 0.0099999947
The risks are calculated from age 0 up to age 85 .
This document is a draft for review purposes only and does not constitute Agency policy.
E-ll DRAFT-DO NOT CITE OR QUOTE
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Supplemental Information—Hexavalent Chromium
Age at
start of
year (i)
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
CElO(adj)
(f\cc-yrs)
0.0000E+00
0.0000E+00
0.0000E+00
0.0000E+00
0.0000E+00
0.0000E+00
0.0000E+00
0.0000E+00
0.0000E+00
0.0000E+00
0.0000E+00
0.0000E+00
0.0000E+00
0.0000E+00
0.0000E+00
0.0000E+00
0.0000E+00
0.0000E+00
0.0000E+00
0.0000E+00
0.0000E+00
1.7939E+00
Lung Cancer
hazard
(unexposed)
(hi)
0.0000E+00
3.8000E-07
3.8000E-07
3.8000E-07
3.8000E-07
1.0000E-07
1.0000E-07
1.0000E-07
1.0000E-07
1.0000E-07
1.9000E-07
1.9000E-07
1.9000E-07
1.9000E-07
1.9000E-07
3.3000E-07
3.3000E-07
3.3000E-07
3.3000E-07
3.3000E-07
4.5000E-07
4.5000E-07
All cause
hazard
(unexposed)
(h*i)
5.7968E-03
3.8228E-04
2.4152E-04
1.9124E-04
1.5101E-04
1.4096E-04
1.3091E-04
1.1078E-04
1.0072E-04
1.0073E-04
9.0668E-05
1.0075E-04
1.3099E-04
1.9148E-04
2.6209E-04
3.3275E-04
4.2366E-04
5.0459E-04
6.0584E-04
6.9717E-04
7.9881E-04
8.9056E-04
Lung Cancer
hazard
(exposed)
(hei)
0.0000E+00
3.8000E-07
3.8000E-07
3.8000E-07
3.8000E-07
1.0000E-07
1.0000E-07
1.0000E-07
1.0000E-07
1.0000E-07
1.9000E-07
1.9000E-07
1.9000E-07
1.9000E-07
1.9000E-07
3.3000E-07
3.3000E-07
3.3000E-07
3.3000E-07
3.3000E-07
4.5000E-07
4.5105E-07
All cause
hazard
(exposed)
(he*i)
5.7968E-03
3.8228E-04
2.4152E-04
1.9124E-04
1.5101E-04
1.4096E-04
1.3091E-04
1.1078E-04
1.0072E-04
1.0073E-04
9.0668E-05
1.0075E-04
1.3099E-04
1.9148E-04
2.6209E-04
3.3275E-04
4.2366E-04
5.0459E-04
6.0584E-04
6.9717E-04
7.9881E-04
8.9056E-04
Probability of
surviving
year i
assuming
alive at start
(unexposed)
(qi)
i 9.9422E-01 :
; 9.9962E-01 i
; 9.9976E-01 |
i 9.9981E-01 s
; 9.9985E-01 i
9.9986E-01
i 9.9987E-01 |
9.9989E-01 j
9.9990E-01 |
! 9.9990E-01 j
: 9.9991E-01 i
i 9.9990E-01 :
i 9.9987E-01 ;
; 9.9981E-01 s
; 9.9974E-01 ;
i 9.9967E-01 ;
| 9.9958E-01 s
; 9.9950E-01 :
i 9.9939E-01 ;
i 9.9930E-01 j
; 9.9920E-01 |
! 9.99111 01 ;
Probability
of surviving
year i
assuming
alive at start
(exposed)
(qei)
9.9422E-01
9.9962E-01
9.9976E-01
9.9981E-01
9.9985E-01
9.9986E-01
9.9987E-01
9.9989E-01
9.9990E-01
9.9990E-01
9.9991E-01
9.9990E-01
9.9987E-01
9.9981E-01
9.9974E-01
9.9967E-01
9.9958E-01
9.9950E-01
9.9939E-01
9.9930E-01
9.9920E-01
9.9911E-01
Probability of
surviving to
end of year i
(unexposed)
(Sl,i)
1.0000E+00
9.9422E-01
9.9384E-01
9.9360E-01
9.9341E-01
9.9326E-01
9.9312E-01
9.9299E-01
9.9288E-01
9.9278E-01
9.9268E-01
9.9259E-01
9.9249E-01
9.9236E-01
9.9217E-01
9.9191E-01
9.9158E-01
9.9116E-01
9.9066E-01
9.9006E-01
9.8937E-01
9.8858E-01
Probability
of surviving
to end of
yeari
(exposed)
(Sel,i)
1.0000E+00
9.9422E-01
9.9384E-01
9.9360E-01
9.9341E-01
9.9326E-01
9.9312E-01
9.9299E-01
9.9288E-01
9.9278E-01
9.9268E-01
9.9259E-01
9.9249E-01
9.9236E-01
9.9217E-01
9.9191E-01
9.9158E-01
9.9116E-01
9.9066E-01
9.9006E-01
9.8937E-01
9.8858E-01
Cumulative
Risk of lung
cancer
(unexposed)
(R0)
0.0000E+00
3.7773E-07
7.5534E-07
1.1329E-06
1.5103E-06
1.6097E-06
1.7090E-06
1.8083E-06
1.9075E-06
2.0068E-06
2.1954E-06
2.3840E-06
2.5726E-06
2.7611E-06
2.9496E-06
3.2769E-06
3.6040E-06
3.9310E-06
4.2578E-06
4.5844E-06
5.0295E-06
5.4741E-06
Cumulative
risk of lung
cancer
(exposed)
(Re)
0.0000E+00
3.7773E-07
7.5534E-07
1.1329E-06
1.5103E-06
1.6097E-06
1.7090E-06
1.8083E-06
1.9075E-06
2.0068E-06
2.1954E-06
2.3840E-06
2.5726E-06
2.7611E-06
2.9496E-06
3.2769E-06
3.6040E-06
3.9310E-06
4.2578E-06
4.5844E-06
5.0295E-06
5.4752E-06
Extra Risk (Re-
R0)\(1-
R0)
This document is a draft for review purposes only and does not constitute Agency policy.
E-12
DRAFT-DO NOT CITE OR QUOTE
-------
Supplemental Information—Hexavalent Chromium
Age at
start of
year (i)
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
CElO(adj)
(f\cc-yrs)
5.3818E+00
8.9697E+00
1.2558E+01
1.6145E+01
1.9733E+01
2.3321E+01
2.6909E+01
3.0497E+01
3.4085E+01
3.7673E+01
4.1261E+01
4.4848E+01
4.8436E+01
5.2024E+01
5.5612E+01
5.9200E+01
6.2788E+01
6.6376E+01
6.9964E+01
7.3552E+01
7.7139E+01
8.0727E+01
Lung Cancer
hazard
(unexposed)
(hi)
4.5000E-07
4.5000E-07
4.5000E-07
1.2000E-06
1.2000E-06
1.2000E-06
1.2000E-06
1.2000E-06
3.8200E-06
3.8200E-06
3.8200E-06
3.8200E-06
3.8200E-06
1.0740E-05
1.0740E-05
1.0740E-05
1.0740E-05
1.0740E-05
3.1310E-05
3.1310E-05
3.1310E-05
3.1310E-05
All cause
hazard
(unexposed)
(h*i)
9.7243E-04
1.0241E-03
1.0861E-03
1.1279E-03
1.1597E-03
1.2120E-03
1.2543E-03
1.2968E-03
1.3496E-03
1.4027E-03
1.4559E-03
1.5094E-03
1.5529E-03
1.6171E-03
1.6817E-03
1.7466E-03
1.7911E-03
1.8670E-03
1.9329E-03
2.0409E-03
2.1600E-03
2.3112E-03
Lung Cancer
hazard
(exposed)
(hei)
4.5315E-07
4.5527E-07
4.5739E-07
1.2254E-06
1.2311E-06
1.2369E-06
1.2427E-06
1.2485E-06
3.9928E-06
4.0114E-06
4.0302E-06
4.0490E-06
4.0679E-06
1.1490E-05
1.1544E-05
1.1598E-05
1.1652E-05
1.1706E-05
3.4286E-05
3.4447E-05
3.4607E-05
3.4769E-05
All cause
hazard
(exposed)
(he*i)
9.7243E-04
1.0241E-03
1.0861E-03
1.1280E-03
1.1598E-03
1.2121E-03
1.2544E-03
1.2968E-03
1.3498E-03
1.4029E-03
1.4561E-03
1.5097E-03
1.5531E-03
1.6179E-03
1.6825E-03
1.7475E-03
1.7920E-03
1.8680E-03
1.9359E-03
2.0440E-03
2.1632E-03
2.3147E-03
Probability of
surviving
year i
assuming
alive at start
(unexposed)
(qi)
i 9.9903E-01 :
; 9.9898E-01 i
; 9.9891E-01 |
; 9.9887E-01 s
; 9.9884E-01 i
9.9879E-01 :
i 9.9875E-01 j
i 9.9870E-01 ;
i 9.9865E-01 ;
9.9860E-01 i
; 9.9855E-01 j
; 9.9849E-01 |
: 9.9845E-01 j
; 9.9838E-01 |
| 9.9832E-01 j
i 9.9825E-01 |
; 9.9821E-01 s
| 9.9813E-01 |
i 9.9807E-01 ;
9.9796E-01 j
i 9.9784E-01 |
9.9769E-01 ;
Probability
of surviving
year i
assuming
alive at start
(exposed)
(qei)
9.9903E-01
9.9898E-01
9.9891E-01
9.9887E-01
9.9884E-01
9.9879E-01
9.9875E-01
9.9870E-01
9.9865E-01
9.9860E-01
9.9854E-01
9.9849E-01
9.9845E-01
9.9838E-01
9.9832E-01
9.9825E-01
9.9821E-01
9.9813E-01
9.9807E-01
9.9796E-01
9.9784E-01
9.9769E-01
Probability of
surviving to
end of year i
(unexposed)
(Sl,i)
9.8770E-01
9.8674E-01
9.8573E-01
9.8466E-01
9.8355E-01
9.8241E-01
9.8122E-01
9.7999E-01
9.7872E-01
9.7740E-01
9.7603E-01
9.7461E-01
9.7314E-01
9.7163E-01
9.7006E-01
9.6843E-01
9.6674E-01
9.6501E-01
9.6321E-01
9.6135E-01
9.5939E-01
9.5732E-01
Probability
of surviving
to end of
yeari
(exposed)
(Sel,i)
9.8770E-01
9.8674E-01
9.8573E-01
9.8466E-01
9.8355E-01
9.8241E-01
9.8122E-01
9.7999E-01
9.7872E-01
9.7740E-01
9.7603E-01
9.7461E-01
9.7314E-01
9.7163E-01
9.7006E-01
9.6843E-01
9.6674E-01
9.6501E-01
9.6320E-01
9.6134E-01
9.5938E-01
9.5731E-01
Cumulative
Risk of lung
cancer
(unexposed)
(R0)
5.9184E-06
6.3622E-06
6.8055E-06
7.9865E-06
9.1660E-06
1.0344E-05
1.1521E-05
1.2696E-05
1.6432E-05
2.0163E-05
2.3889E-05
2.7609E-05
3.1324E-05
4.1751E-05
5.2160E-05
6.2552E-05
7.2926E-05
8.3280E-05
1.1341E-04
1.4348E-04
1.7348E-04
2.0342E-04
Cumulative
risk of lung
cancer
(exposed)
(Re)
5.9225E-06
6.3715E-06
6.8222E-06
8.0281E-06
9.2383E-06
1.0453E-05
1.1671E-05
1.2894E-05
1.6799E-05
2.0717E-05
2.4648E-05
2.8591E-05
3.2547E-05
4.3702E-05
5.4891E-05
6.6113E-05
7.7367E-05
8.8653E-05
1.2165E-04
1.5473E-04
1.8789E-04
2.2114E-04
Extra Risk (Re-
R0)\(1-
R0)
This document is a draft for review purposes only and does not constitute Agency policy.
E-13
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-------
Supplemental Information—Hexavalent Chromium
Age at
start of
year (i)
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
CElO(adj)
(f\cc-yrs)
8.4315E+01
8.7903E+01
9.1491E+01
9.5079E+01
9.8667E+01
1.0225E+02
1.0584E+02
1.0943E+02
1.1302E+02
1.1661E+02
1.2019E+02
1.2378E+02
1.2737E+02
1.3096E+02
1.3455E+02
1.3813E+02
1.4172E+02
1.4531E+02
1.4890E+02
1.5248E+02
1.5607E+02
1.5966E+02
Lung Cancer
hazard
(unexposed)
(hi)
3.1310E-05
8.5060E-05
8.5060E-05
8.5060E-05
8.5060E-05
8.5060E-05
2.4321E-04
2.4321E-04
2.4321E-04
2.4321E-04
2.4321E-04
5.4508E-04
5.4508E-04
5.4508E-04
5.4508E-04
5.4508E-04
8.7599E-04
8.7599E-04
8.7599E-04
8.7599E-04
8.7599E-04
1.3188E-03
All cause
hazard
(unexposed)
(h*i)
2.4740E-03
2.6485E-03
2.8455E-03
3.0865E-03
3.3615E-03
3.6927E-03
4.0381E-03
4.4092E-03
4.8284E-03
5.3079E-03
5.7950E-03
6.3014E-03
6.8172E-03
7.3433E-03
7.9032E-03
8.4874E-03
9.1319E-03
9.8165E-03
1.0521E-02
1.1246E-02
1.1995E-02
1.2794E-02
Lung Cancer
hazard
(exposed)
(hei)
3.4931E-05
9.5341E-05
9.5786E-05
9.6233E-05
9.6682E-05
9.7133E-05
2.7903E-04
2.8033E-04
2.8164E-04
2.8295E-04
2.8427E-04
6.4009E-04
6.4307E-04
6.4607E-04
6.4909E-04
6.5212E-04
1.0529E-03
1.0578E-03
1.0628E-03
1.0677E-03
1.0727E-03
1.6224E-03
All cause
hazard
(exposed)
(he*i)
2.4776E-03
2.6588E-03
2.8562E-03
3.0976E-03
3.3731E-03
3.7047E-03
4.0739E-03
4.4464E-03
4.8669E-03
5.3477E-03
5.8360E-03
6.3964E-03
6.9151E-03
7.4443E-03
8.0072E-03
8.5945E-03
9.3088E-03
9.9983E-03
1.0707E-02
1.1438E-02
1.2192E-02
1.3098E-02
Probability of
surviving
year i
assuming
alive at start
(unexposed)
(qi)
i 9.9753E-01 :
; 9.9736E-01 i
; 9.9716E-01 |
i 9.9692E-01 ;
; 9.9664E-01 ;
9.9631E-01 j
; 9.9597E-01 ;
: 9.9560E-01 j
; 9.9518E-01 i
i 9.9471E-01 :
; 9.9422E-01
; 9.9372E-01
i 9.9321E-01 ;
; 9.9268E-01 s
: 9.9213E-01 :
; 9.9155E-01 j
; 9.9091E-01 |
; 9.9023E-01 j
; 9.8953E-01 i
9.8882E-01 s
i 9.8808E-01
! 9.8729E-01 ;
Probability
of surviving
year i
assuming
alive at start
(exposed)
(qei)
9.9753E-01
9.9734E-01
9.9715E-01
9.9691E-01
9.9663E-01
9.9630E-01
9.9593E-01
9.9556E-01
9.9514E-01
9.9467E-01
9.9418E-01
9.9362E-01
9.9311E-01
9.9258E-01
9.9202E-01
9.9144E-01
9.9073E-01
9.9005E-01
9.8935E-01
9.8863E-01
9.8788E-01
9.8699E-01
Probability of
surviving to
end of year i
(unexposed)
(Sl,i)
9.5511E-01
9.5275E-01
9.5023E-01
9.4753E-01
9.4461E-01
9.4144E-01
9.3797E-01
9.3419E-01
9.3008E-01
9.2560E-01
9.2070E-01
9.1538E-01
9.0963E-01
9.0345E-01
8.9684E-01
8.8978E-01
8.8226E-01
8.7424E-01
8.6570E-01
8.5664E-01
8.4706E-01
8.3696E-01
Probability
of surviving
to end of
yeari
(exposed)
(Sel,i)
9.5509E-01
9.5273E-01
9.5020E-01
9.4749E-01
9.4456E-01
9.4138E-01
9.3790E-01
9.3408E-01
9.2994E-01
9.2542E-01
9.2049E-01
9.1513E-01
9.0930E-01
9.0303E-01
8.9633E-01
8.8919E-01
8.8158E-01
8.7341E-01
8.6472E-01
8.5551E-01
8.4578E-01
8.3553E-01
Cumulative
Risk of lung
cancer
(unexposed)
(R0)
2.3329E-04
3.1422E-04
3.9494E-04
4.7541E-04
5.5562E-04
6.3555E-04
8.6322E-04
1.0899E-03
1.3156E-03
1.5401E-03
1.7634E-03
2.2608E-03
2.7549E-03
3.2455E-03
3.7325E-03
4.2154E-03
4.9848E-03
5.7468E-03
6.5012E-03
7.2474E-03
7.9850E-03
9.0817E-03
Cumulative
risk of lung
cancer
(exposed)
(Re)
2.5446E-04
3.4517E-04
4.3606E-04
5.2710E-04
6.1826E-04
7.0953E-04
9.7070E-04
1.2320E-03
1.4932E-03
1.7544E-03
2.0153E-03
2.5992E-03
3.1819E-03
3.7632E-03
4.3427E-03
4.9200E-03
5.8439E-03
6.7633E-03
7.6773E-03
8.5856E-03
9.4873E-03
1.0834E-02
Extra Risk (Re-
R0)\(1-
R0)
This document is a draft for review purposes only and does not constitute Agency policy.
E-14
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Supplemental Information—Hexavalent Chromium
Age at
start of
year (i)
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
84
CElO(adj)
(f\cc-yrs)
1.6325E+02
1.6684E+02
1.7042E+02
1.7401E+02
1.7760E+02
1.8119E+02
1.8478E+02
1.8836E+02
1.9195E+02
1.9554E+02
1.9913E+02
2.0272E+02
2.0630E+02
2.0989E+02
2.1348E+02
2.1707E+02
2.2065E+02
2.2424E+02
2.2783E+02
2.2783E+02
Lung Cancer
hazard
(unexposed)
(hi)
1.3188E-03
1.3188E-03
1.3188E-03
1.3188E-03
1.9811E-03
1.9811E-03
1.9811E-03
1.9811E-03
1.9811E-03
2.6576E-03
2.6576E-03
2.6576E-03
2.6576E-03
2.6576E-03
3.0963E-03
3.0963E-03
3.0963E-03
3.0963E-03
3.0963E-03
3.0963E-03
All cause
hazard
(unexposed)
(h*i)
1.3708E-02
1.4733E-02
1.5901E-02
1.7239E-02
1.8603E-02
2.0533E-02
2.2345E-02
2.4496E-02
2.6694E-02
2.9472E-02
3.2525E-02
3.6079E-02
4.0056E-02
4.4352E-02
4.9367E-02
5.4690E-02
6.1072E-02
6.8884E-02
7.6927E-02
7.6927E-02
Lung Cancer
hazard
(exposed)
(hei)
1.6300E-03
1.6376E-03
1.6453E-03
1.6529E-03
2.4947E-03
2.5063E-03
2.5180E-03
2.5298E-03
2.5416E-03
3.4255E-03
3.4415E-03
3.4575E-03
3.4737E-03
3.4899E-03
4.0849E-03
4.1039E-03
4.1231E-03
4.1423E-03
4.1617E-03
4.1617E-03
All cause
hazard
(exposed)
(he*i)
1.4019E-02
1.5051E-02
1.6228E-02
1.7573E-02
1.9116E-02
2.1059E-02
2.2882E-02
2.5044E-02
2.7255E-02
3.0239E-02
3.3309E-02
3.6879E-02
4.0872E-02
4.5184E-02
5.0356E-02
5.5698E-02
6.2099E-02
6.9930E-02
7.7992E-02
7.7992E-02
Probability of
surviving
year i
assuming
alive at start
(unexposed)
(qi)
i 9.8639E-01 :
; 9.8538E-01 i
; 9.8422E-01 |
i 9.8291E-01 ;
; 9.8157E-01 ;
; 9.7968E-01 j
; 9.7790E-01
i 9.7580E-01 i
; 9.7366E-01 :
9.7096E-01 i
; 9.6800E-01 i
; 9.6456E-01 i
; 9.6074E-01 i
; 9.5662E-01 s
9.5183E-01 :
; 9.4678E-01 ;
; 9.4076E-01 s
9.3344E-01 s
; 9.2596E-01 |
: 9.2596E-01 ;
Probability
of surviving
year i
assuming
alive at start
(exposed)
(qei)
9.8608E-01
9.8506E-01
9.8390E-01
9.8258E-01
9.8107E-01
9.7916E-01
9.7738E-01
9.7527E-01
9.7311E-01
9.7021E-01
9.6724E-01
9.6379E-01
9.5995E-01
9.5582E-01
9.5089E-01
9.4582E-01
9.3979E-01
9.3246E-01
9.2497E-01
9.2497E-01
Probability of
surviving to
end of year i
(unexposed)
(Sl,i)
8.2632E-01
8.1507E-01
8.0315E-01
7.9048E-01
7.7697E-01
7.6265E-01
7.4715E-01
7.3064E-01
7.1296E-01
6.9418E-01
6.7402E-01
6.5245E-01
6.2933E-01
6.0462E-01
5.7839E-01
5.5053E-01
5.2123E-01
4.9035E-01
4.5771E-01
4.2382E-01
Probability
of surviving
to end of
yeari
(exposed)
(Sel,i)
8.2466E-01
8.1318E-01
8.0103E-01
7.8814E-01
7.7441E-01
7.5974E-01
7.4391E-01
7.2708E-01
7.0910E-01
6.9004E-01
6.6948E-01
6.4755E-01
6.2410E-01
5.9911E-01
5.7264E-01
5.4452E-01
5.1502E-01
4.8401E-01
4.5132E-01
4.1746E-01
Cumulative
Risk of lung
cancer
(unexposed)
(R0)
1.0164E-02
1.1231E-02
1.2282E-02
1.3315E-02
1.4840E-02
1.6336E-02
1.7799E-02
1.9229E-02
2.0623E-02
2.2441E-02
2.4204E-02
2.5907E-02
2.7546E-02
2.9118E-02
3.0865E-02
3.2524E-02
3.4090E-02
3.5557E-02
3.6921E-02
3.6921E-02
Cumulative
risk of lung
cancer
(exposed)
(Re)
1.2169E-02
Extra Risk (Re-
R0)\(1-
R0)
1.3491E-02
1.4798E-02
1.6089E-02
1.8003E-02
1.9887E-02
2.1739E-02
2.3556E-02
2.5333E-02
2.7662E-02
2.9928E-02
3.2126E-02
3.4250E-02
3.6294E-02
3.8576E-02
4.0749E-02
4.2808E-02
4.4745E-02
4.6552E-02
4.6552E-02 1 0.0099999947
This document is a draft for review purposes only and does not constitute Agency policy.
E-15 DRAFT-DO NOT CITE OR QUOTE
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Supplemental Information—Hexavalent Chromium
APPENDIX F. QUALITY ASSURANCE FOR THE IRIS
TOXICOLOGICAL REVIEW OF HEXAVALENT
CHROMIUM
This assessment is prepared under the auspices of the U.S. Environmental Protection
Agency's (EPA's) Integrated Risk Information System (IRIS) Program. The IRIS Program is housed
within the Office of Research and Development (ORD) in the Center for Public Health and
Environmental Assessment (CPHEA). EPA has an agency-wide quality assurance (QA) policy that is
outlined in the EPA Quality Manual for Environmental Programs (see CIO 2105-P-01.11 and follows
the specifications outlined in EPA Order CIO 2105.1.
As required by CIO 2105.1, ORD maintains a Quality Management Program, which is
documented in an internal Quality Management Plan (QMP). The latest version was developed in
2013 using Guidance for Developing Quality Systems for Environmental Programs fOA/G-11 An
NCEA/CPHEA-specific QMP was also developed in 2013 as an appendix to the ORD QMP. Quality
assurance for products developed within CPHEA is managed under the ORD QMP and applicable
appendices.
The IRIS Toxicological Review of Hexavalent Chromium is designated as Highly Influential
Scientific Information (HISA) and is classified as QA Category A. Category A designations require
reporting of all critical QA activities, including audits. The development of IRIS assessments is done
through a seven-step process. Documentation of this process is available on the IRIS website:
https://www.epa.gOv/iris/basic-information-about-integrated-risk-information-system#process.
Specific management of quality assurance within the IRIS Program is documented in a
Programmatic Quality Assurance Project Plan (PQAPP). A PQAPP is developed using the EPA
Guidance for Quality Assurance Project Plans (OA/G-5). and the latest approved version is dated
June 2022. All IRIS assessments follow the IRIS PQAPP, and all assessment leads and team members
are required to receive QA training on the IRIS PQAPP. During assessment development, additional
QAPPs may be applied for quality assurance management. They include:
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—Hexavalent Chromium
Title
Document number
Date
Program Quality Assurance Project
Plan (PQAPP) for the Integrated Risk
Information System (IRIS) Program
L-CPAD-0030729-QP-1-5
June 2022
An Umbrella Quality Assurance
Project Plan (QAPP) for Dosimetry
and Mechanism-Based Models
(PBPK)
L-CPAD-0032188-QP-1-2
January 2021
Quality Assurance Project Plan
(QAPP) for Enhancements to
Benchmark Dose Software (BMDS)
L-HEEAD-0032189-QP-1-2
October 2020
During assessment development, this project undergoes quality audits during assessment
development including:
Date
Type of audit
Major findings
Actions taken
Augusts 2018
Technical system audit
None
None
August 2019
Technical system audit
None
None
August 2020
Technical system audit
None
None
July 2021
Technical system audit
None
None
August 2022
Technical system audit
None
None
During Step 3 and Step 6 of the IRIS process, the IRIS toxicological review is subjected to
external reviews by other federal agency partners, including the Executive Offices of the White
House. Comments during these IRIS process steps are available in the docket EPA-HQ-ORD-2014-
0313 on http://www.regulations.gov.
During Step 4 [include this section AFTER Step 4] of assessment development, the IRIS
Toxicological Review of [chemical X] undergoes public comment from [insert date of public
comment]. Following this comment period, the toxicological review undergoes external peer review
by [SAB/NAS/contractor peer-review panel] on [insert date of ERD]. The peer-review report is
available on the [NAS/SAB website—include the URL], All public and peer-review comments are
available in the docket [insert chemical docket number—make sure that the ERD public comments are
available in the docket as well].
[Include this section AFTER Step 6] Prior to release (Step 7 of the IRIS process), the final
toxicological review is submitted to management and QA clearance. During this step the CPHEA QA
Director and QA Managers review the project QA documentation and ensure that EPA QA
requirements are met
This document is a draft for review purposes only and does not constitute Agency policy.
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Supplemental Information—Hexavalent Chromium
APPENDIX G. RESPONSE TO EXTERNAL
COMMENTS
1 [Template placeholder]
This document is a draft for review purposes only and does not constitute Agency policy.
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REFERENCES FOR APPENDICES
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This document is a draft for review purposes only and does not constitute Agency policy.
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This document is a draft for review purposes only and does not constitute Agency policy.
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This document is a draft for review purposes only and does not constitute Agency policy.
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This document is a draft for review purposes only and does not constitute Agency policy.
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This document is a draft for review purposes only and does not constitute Agency policy.
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