United States
Environmental Protection
1=1 m m Agency
EPA/690/R-l 5/015F
Final
9-29-2015
Provisional Peer-Reviewed Toxicity Values for
Soluble Tungsten Compounds
(Various CASRNs)
Superfund Health Risk Technical Support Center
National Center for Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, OH 45268
-------
AUTHORS, CONTRIBUTORS, AND REVIEWERS
CHEMICAL MANAGER
J. Phillip Kaiser, PhD, DABT
National Center for Environmental Assessment, Cincinnati, OH
DRAFT DOCUMENT PREPARED BY
SRC, Inc.
7502 Round Pond Road
North Syracuse, NY 13212
PRIMARY INTERNAL REVIEWERS
Paul G. Reinhart, PhD, DABT
National Center for Environmental Assessment, Research Triangle Park, NC
This document was externally peer reviewed under contract to:
Eastern Research Group, Inc.
110 Hartwell Avenue
Lexington, MA 02421-3136
Questions regarding the contents of this document may be directed to the U.S. EPA Office of
Research and Development's National Center for Environmental Assessment, Superfund Health
Risk Technical Support Center (513-569-7300).
li
Soluble Tungsten Compounds
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TABLE OF CONTENTS
COMMONLY USED ABBREVIATIONS AND ACRONYMS iv
BACKGROUND 2
DISCLAIMERS 2
QUESTIONS REGARDING PPRTVs 2
INTRODUCTION 3
REVIEW OF POTENTIALLY RELEVANT DATA (NONCANCER AND CANCER) 6
HUMAN STUDIES 15
Oral Exposures 15
Inhalation Exposures 15
ANIMAL STUDIES 15
Oral Exposures 15
Inhalation Exposures 32
OTHER DATA (SHORT-TERM TESTS, OTHER EXAMINATIONS) 32
Genotoxicity 32
Supporting Human Studies 32
Other Animal Toxicity Studies 33
Absorption, Distribution, Metabolism, and Elimination (ADME) Studies 33
Mode-of-Action/Mechanism/Therapeutic Action Studies 34
DERIVATION 01 PROVISIONAL VALUES 34
DERIVATION OF ORAL REFERENCE DOSES 36
Derivation of Subchronic Provisional RfD (Subchronic p-RfD) 36
Derivation of Chronic Provisional RfD (Chronic p-RfD) 43
DERIVATION OF INHALATION REFERENCE CONCENTRATIONS 45
CANCER WEIGHT-OF-EVIDENCE DESCRIPTOR 45
DERIVATION OF PROVISIONAL CANCER POTENCY VALUES 46
Derivation of Provisional Oral Slope Factor (p-OSF) 46
Derivation of Provisional Inhalation Unit Risk (p-IUR) 46
APPENDIX A. SCREENING PROVISIONAL VALUES 47
APPENDIX B. DATA TABLES 48
APPENDIX C. SUMMARIES OF SUPPORTING DATA 61
APPENDIX D. BENCHMARK DOSE MODELING RESULTS 74
APPENDIX E. REFERENCES 108
in
Soluble Tungsten Compounds
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COMMONLY USED ABBREVIATIONS AND ACRONYMS
a2u-g
alpha 2u-globulin
MN
micronuclei
ACGIH
American Conference of Governmental
MNPCE
micronucleated polychromatic
Industrial Hygienists
erythrocyte
AIC
Akaike's information criterion
MOA
mode of action
ALD
approximate lethal dosage
MTD
maximum tolerated dose
ALT
alanine aminotransferase
NAG
N-acetyl-P-D-glucosaminidase
AST
aspartate aminotransferase
NCEA
National Center for Environmental
atm
atmosphere
Assessment
ATSDR
Agency for Toxic Substances and
NCI
National Cancer Institute
Disease Registry
NOAEL
no-observed-adverse-effect level
BMD
benchmark dose
NTP
National Toxicology Program
BMDL
benchmark dose lower confidence limit
NZW
New Zealand White (rabbit breed)
BMDS
Benchmark Dose Software
OCT
ornithine carbamoyl transferase
BMR
benchmark response
ORD
Office of Research and Development
BUN
blood urea nitrogen
PBPK
physiologically based pharmacokinetic
BW
body weight
PCNA
proliferating cell nuclear antigen
CA
chromosomal aberration
PND
postnatal day
CAS
Chemical Abstracts Service
POD
point of departure
CASRN
Chemical Abstracts Service Registry
PODadj
duration-adjusted POD
Number
QSAR
quantitative structure-activity
CBI
covalent binding index
relationship
CHO
Chinese hamster ovary (cell line cells)
RBC
red blood cell
CL
confidence limit
RDS
replicative DNA synthesis
CNS
central nervous system
RfC
inhalation reference concentration
CPN
chronic progressive nephropathy
RfD
oral reference dose
CYP450
cytochrome P450
RGDR
regional gas dose ratio
DAF
dosimetric adjustment factor
RNA
ribonucleic acid
DEN
diethylnitrosamine
SAR
structure activity relationship
DMSO
dimethylsulfoxide
SCE
sister chromatid exchange
DNA
deoxyribonucleic acid
SD
standard deviation
EPA
Environmental Protection Agency
SDH
sorbitol dehydrogenase
FDA
Food and Drug Administration
SE
standard error
FEVi
forced expiratory volume of 1 second
SGOT
glutamic oxaloacetic transaminase, also
GD
gestation day
known as AST
GDH
glutamate dehydrogenase
SGPT
glutamic pyruvic transaminase, also
GGT
y-glutamyl transferase
known as ALT
GSH
glutathione
SSD
systemic scleroderma
GST
glutathione-S-transferase
TCA
trichloroacetic acid
Hb/g-A
animal blood-gas partition coefficient
TCE
trichloroethylene
Hb/g-H
human blood-gas partition coefficient
TWA
time-weighted average
HEC
human equivalent concentration
UF
uncertainty factor
HED
human equivalent dose
UFa
interspecies uncertainty factor
i.p.
intraperitoneal
UFh
intraspecies uncertainty factor
IRIS
Integrated Risk Information System
UFs
subchronic-to-chronic uncertainty factor
IVF
in vitro fertilization
UFd
database uncertainty factor
LC50
median lethal concentration
U.S.
United States of America
LD50
median lethal dose
WBC
white blood cell
LOAEL
lowest-observed-adverse-effect level
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PROVISIONAL PEER-REVIEWED TOXICITY VALUES FOR
SOLUBLE TUNGSTEN COMPOUNDS
(Various CASRNs)
BACKGROUND
A Provisional Peer-Reviewed Toxicity Value (PPRTV) is defined as a toxicity value
derived for use in the Superfund Program. PPRTVs are derived after a review of the relevant
scientific literature using established Agency guidance on human health toxicity value
derivations. All PPRTV assessments receive internal review by a standing panel of National
Center for Environment Assessment (NCEA) scientists and an independent external peer review
by three scientific experts.
The purpose of this document is to provide support for the hazard and dose-response
assessment pertaining to chronic and subchronic exposures to substances of concern, to present
the major conclusions reached in the hazard identification and derivation of the PPRTVs, and to
characterize the overall confidence in these conclusions and toxicity values. It is not intended to
be a comprehensive treatise on the chemical or toxicological nature of this substance.
The PPRTV review process provides needed toxicity values in a quick turnaround
timeframe while maintaining scientific quality. PPRTV assessments are updated approximately
on a 5-year cycle for new data or methodologies that might impact the toxicity values or
characterization of potential for adverse human health effects and are revised as appropriate. It is
important to utilize the PPRTV database (http://hhpprtv.ornl.gov) to obtain the current
information available. When a final Integrated Risk Information System (IRIS) assessment is
made publicly available on the Internet (http://www.epa.gov/iris). the respective PPRTVs are
removed from the database.
DISCLAIMERS
The PPRTV document provides toxicity values and information about the adverse effects
of the chemical and the evidence on which the value is based, including the strengths and
limitations of the data. All users are advised to review the information provided in this
document to ensure that the PPRTV used is appropriate for the types of exposures and
circumstances at the site in question and the risk management decision that would be supported
by the risk assessment.
Other U.S. Environmental Protection Agency (EPA) programs or external parties who
may choose to use PPRTVs are advised that Superfund resources will not generally be used to
respond to challenges, if any, of PPRTVs used in a context outside of the Superfund program.
This document has been reviewed in accordance with U.S. EPA policy and approved for
publication. Mention of trade names or commercial products does not constitute endorsement or
recommendation for use.
QUESTIONS REGARDING PPRTVs
Questions regarding the contents and appropriate use of this PPRTV assessment should
be directed to the EPA Office of Research and Development's National Center for
Environmental Assessment, Superfund Health Risk Technical Support Center (513-569-7300).
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INTRODUCTION
Tungsten is a metallic element that increases the hardness, toughness, elasticity, and
strength of steel and other metal alloys (HSDB, 2009b). Tungsten is used in electron lamps and
filaments for incandescent lamps (HSDB. 2009b). It is also used in the preparation of green and
blue pigments (HSDB. 2009b). The U.S. military has used tungsten as a replacement for lead
and depleted uranium in munitions since the late 1990s (Osterburg et ai. 2014). Tungsten has
very low solubility in water and is not volatile (ECB. 2000). The chemical symbol for tungsten
is W.
Sodium tungstate is a chemical intermediate for metallic tungsten and tungsten
compounds (HSDB. 2009a). Sodium tungstate is used as a flame retardant textile treatment
agent (HSDB, 2009a). In addition, sodium tungstate is a reagent for biological products (HSDB,
2009a). Sodium tungstate has high solubility in water and is not volatile (HSDB, 2009a). The
empirical formula for sodium tungstate is Na2C>4W (see Figure 1).
O
Na+ O-r0 Na+
O
Figure 1. Sodium Tungstate Structure
Sodium tungsten dihydrate is used in the fireproofing and waterproofing of fabrics
(O'Neil. 2006). The compound is also a catalyst in oxidation reactions and a corrosion inhibitor
in steel (O'Neil. 2006). Sodium tungstate dihydrate has high solubility in water (O'Neil. 2006).
The empirical formula for sodium tungstate dihydrate is Na2W04*2H20 (see Figure 2).
l-k
l"k
O O
Na+ O-W-O Na+
Figure 2. Sodium Tungstate Dihydrate Structure
Because soluble compounds of tungsten (i.e., sodium tungstate dihydrate and sodium
tungstate) are expected to ionize in the blood (Nlclnturf et aL 2011; Nlclntuif et ai. 2008). the
systemic toxicities of the chemicals would be due to elemental tungsten (and not the particular
salt), thus the toxicities of the soluble compounds of tungsten would be expected to be similar on
a molar basis. Therefore, although the subchronic and chronic provisional oral reference doses
(p-RfDs) presented below are derived based on doses for elemental tungsten, the values are
applicable for soluble tungsten compounds (e.g., sodium tungstate dihydrate and sodium
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Soluble Tungsten Compounds
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tungstate). A table of physicochemical properties for sodium tungstate and sodium tungstate
dihydrate are provided below (see Table 1). A summary of available toxicity values for soluble
tungsten compounds from the U.S. EPA and other agencies/organizations is provided in Table 2.
Table 1. Physicochemical Properties of Sodium Tungstate (CASRN 13472-45-2) and
Sodium Tungstate Dihydrate (CASRN 10213-10-2)
Property (unit)
13472-45-23
10213-10-2b
Boiling point (°C at 760 mmHg)
ND
ND
Melting point (°C)
698
Decomposes at 100°C
Density (g/cm3 at 20 °C)
4.18
3.25°
Vapor pressure (mmHg at 2,327°C)
ND
ND
pH (unitless)
ND
ND
Solubility in water (g/L at 20°C)
742°
909
Relative vapor density (air = 1)
ND
ND
Molecular weight (g/mol)
293.82
329.85d
•'HSDB (2009a).
hQ'Neil (2006).
°Lide (2005a): Lide (2005b)
dSigma-Aldrich (2014).
ND = no data.
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Soluble Tungsten Compounds
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Table 2. Summary of Available Toxicity Values for Soluble Tungsten Compounds
(Various CASRNs)
Source/Parametera'b
Value
Notes
Reference
Noncancer
ACGIH (TLV-TWA)
Soluble compounds of tungsten
1 mg W/m3
Basis: central nervous system
impairment; pulmonary fibrosis
ACGIH (2013): ACGIH
(2001)
ACGIH (TLV-STEL)
Soluble compounds of tungsten
3 mg W/m3
Basis: central nervous system
impairment; pulmonary fibrosis
ACGIH (2013); ACGIH
(2001)
ATSDR (MRLs)
Tungsten
NV
NA
ATSDR (2015)
Cal/EPA
NV
NA
Cal/EPA (2015b):
Cal/EPA (2015a)
NIOSH (REL-TWA)
Soluble compounds of tungsten
1 mg W/m3
NA
NIOSH (2015)
NIOSH (REL-STEL)
Soluble compounds of tungsten
3 mg W/m3
NA
NIOSH (2015)
OSHA (PEL)
Soluble compounds of tungsten
1 mg W/m3
Construction industry
OSHA (2011): OSHA
(2006)
IRIS
NV
NA
U.S. EPA (2015)
DWSHA
NV
NA
U.S. EPA (2012a)
HEAST
NV
NA
U.S. EPA (2011a)
CARA HEEP
NV
NA
U.S. EPA (1994)
WHO
NV
NA
WHO (2015)
Cancer
IRIS
NV
NA
U.S. EPA (2015)
HEAST
NV
NA
U.S. EPA (2011a)
IARC
NV
NA
IARC (2015)
NTP
NV
NA
NTP (2014)
Cal/EPA
NV
NA
Cal/EPA (2015b):
Cal/EPA (2015a)
5
Soluble Tungsten Compounds
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Table 2. Summary of Available Toxicity Values for Soluble Tungsten Compounds
(Various CASRNs)
Source/Parametera'b
Value
Notes
Reference
DWSHA
NV
NA
U.S. EPA (2012a)
ACGIH (WOE)
Tungsten
NV
Sufficient data were not available to
recommend carcinogenicity notations
ACGIH (2013); ACGIH
(2001)
aSources: ACGIH = American Conference of Governmental Industrial Hygienists; ATSDR = Agency for Toxic
Substances and Disease Registry; Cal/EPA = California Environmental Protection Agency; CARA = Chemical
Assessments and Related Activities; DWSHA = Drinking Water Standards and Health Advisories;
HEAST = Health Effects Assessment Summary Tables; HEEP = Health and Environmental Effects Profile;
IARC = International Agency for Research on Cancer; IRIS = Integrated Risk Information System; NIOSH = The
National Institute for Occupational Safety and Health; NTP = National Toxicology Program; OSHA = Occupational
Safety and Health Administration; WHO = World Health Organization.
Parameters: MRL = minimum risk level; PEL = permissible exposure limit; REL-STEL = recommended exposure
limit-short-term exposure limit; REL-TWA = recommended exposure limit-time weighted average;
TLV-STEL = threshold limit value-short-term exposure limit; TLV-TWA = threshold limit value-time-weighted
average; WOE = cancer weight of evidence.
NA = not applicable; NV = not available
Literature searches were conducted on sources published from 1900 through August 2015
for studies relevant to the derivation of provisional toxicity values for tungsten and sodium
tungstate (CASRN 7440-33-7, 13472-45-2, and 10213-10-2). Searches were conducted using
U.S. EPA's Health and Environmental Research Online (HERO) database of scientific literature.
The following databases were searched: PubMed, TOXLINE (including TSCATS1), and Web of
Science. The following databases were searched outside of HERO for health-related values:
ACGIH, ATSDR, Cal/EPA, U.S. EPA IRIS, U.S. EPA HEAST, U.S. EPA Office of Water
(OW), U.S. EPA TSCATS2/TSCATS8e, NIOSH, NTP, and OSHA.
REVIEW OF POTENTIALLY RELEVANT DATA (NONCANCER AND CANCER)
Tables 3 A and 3B provide an overview of the relevant database for tungsten and sodium
tungstate and include all potentially relevant repeated-dose short-term-, subchronic-, and
chronic-duration studies. Principal studies are identified. The phrase "statistical significance,"
used throughout the document, indicates ap-value of < 0.05, unless otherwise noted.
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Soluble Tungsten Compounds
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Table 3A. Summary of Potentially Relevant Noncancer Data for
Soluble Tungsten Compounds (Various CASRNs)
Number of
Male/Female, Strain,
Species, Study Type,
BMDL/
Reference
Category
Study Duration
Dosimetry3
Critical Effects
NOAEL3
BMCL3
LOAEL3
(comments)
Notesb
Human
1. Oral"
ND
2. Inhalation3
ND
Animal
1. Oral3
Short-term
22-24 M/22-24 F,
0, 1.3,39, 78, 125
Reduced immune
78
40 for
125 (immune
Osterburg et
PR
C57BL6 mouse,
in drinking water
response to
decreased
suppression)
al. (2014)
sodium tungstate
Staphylococcal
number of
dihydrate in drinking
ADD: 0, 1.3,39,
enterotoxin B (decreased
cytotoxic
water, 28 d
78, 125
number of activated
T cells in
helper and cytotoxic T
spleen
cells in spleen, decreased
in situ production of
interferon [IFN]-y)
Short-term
4 M/0 F, Wistar rat,
0, 5, 50 ppm in
No effects observed.
6.6
NDr
NDr
Chatteriee et
PR
sodium tungstate
diet
al. (1973)
dihydrate in diet, 28 d
ADD: 0, 0.65, 6.6
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Soluble Tungsten Compounds
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Table 3A. Summary of Potentially Relevant Noncancer Data for
Soluble Tungsten Compounds (Various CASRNs)
Category
Number of
Male/Female, Strain,
Species, Study Type,
Study Duration
Dosimetry3
Critical Effects
NOAEL"
BMDL/
BMCLa
LOAELa
Reference
(comments)
Notesb
Subchronic
10 M/10 F, S-D rat,
sodium tungstate
dihydrate in
deionized water via
gavage, 90 d
0, 6, 47, 78,125
ADD: 0,6,47, 78,
125
Goblet cell metaplasia
and inflammation of
glandular stomach
(both sexes). Also,
renal tubular lesions
(both sexes) and
decreased body weight
(males only) in
high-dose group.
47
2.3 for goblet
cell metaplasia
in males
78 (stomach
lesions)
USACHPPM
(2007a):
USACHPPM
(2007b)
NPR, PS
The results
from this
study were
published in
a
peer-review
ed report by
McCain et
al. (2015).
Subchronic
5 M/0 F, C57BL/6J
mouse, sodium
tungstate dihydrate in
drinking water, 16 wk
0, 15, 200,
1,000 mg/L in
drinking water
ADD: 0, 4, 49,
250
Decreased body weight
and bone marrow
cellularity in tibiae and
femora.
49
78 for
decreased bone
marrow
cell count
250 (decreased
body weight,
decreased bone
marrow
cellularity)
Kelly et al.
(2013)
PR
Subchronic
5 M/6 F, unspecified
strain rat, as sodium
tungstate in diet, 70 d
0,0.1, 0.5, 2.0% in
diet
ADD: 0, 91, 455,
1,820 (M)
0, 102, 510, 2,040
(F)
Decreased body weight
and mortality.
NDr
NDr
91
(decreased body
weight)
Kinard and
Van de Erve
(1941)
Sodium
tungstate
produced
deaths within
30 d at 0.5 or
2% W in diet.
PR
8
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Table 3A. Summary of Potentially Relevant Noncancer Data for
Soluble Tungsten Compounds (Various CASRNs)
Category
Number of
Male/Female, Strain,
Species, Study Type,
Study Duration
Dosimetry3
Critical Effects
NOAEL"
BMDL/
BMCLa
LOAELa
Reference
(comments)
Notesb
Subchronic
5 M/5 F, mixed
Wistar albino and
Minnesota piebald rat,
tungsten metal
powder in diet, 70 d
Note: This is the only
available study on the
repeated oral dose
toxicity of tungsten
metal.
0, 2, 5, 10% in diet
ADD: 0, 1,325,
3,450, 6,550 (M)
0, 1,450, 4,000,
7,325 (F)
Decreased body-weight
gain in females.
4,000
NDr
7,325 (decreased
body-weight
gain)
Kinard and
Van de Erve
(1943)
Tungsten
metal at up to
10% W in diet
produced no
deaths after
70 d.
PR
Subchronic
10 M/0 F, S-D rat,
sodium tungstate in
drinking water, 19 wk
0, 100, 200 ppm in
drinking water
ADD: 0, 13.9,
27.8
No effects observed.
27.8
NDr
NDr
Luo et al.
(1983)
PR
Chronic
10 M/0 F, S-D rat,
sodium tungstate in
drinking water, 30 wk
0, 100 ppm in
drinking water
ADD: 0, 11.9
No effects observed.
11.9
NDr
NDr
Luo et al.
(1983)
PR
Chronic
37 M/35 F (exposed)
52 M/52 F (control),
Long-Evans rat,
sodium tungstate in
drinking water,
lifetime
0, 5 ppm in
drinking water
ADD: 0, 0.6 (M)
0, 0.7 (F)
No effects observed.
0.7
NDr
NDr
Schroeder and
Mitotic iter
(1975b)
PR
Chronic
54 M/54 F, white
Swiss mouse, sodium
tungstate in drinking
water, lifetime
0, 5 ppm in
drinking water
ADD: 0, 1 (M)
0, 1 (F)
No effects observed.
1
NDr
NDr
Schroeder and
Mitcliener
(1975a)
PR
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Table 3A. Summary of Potentially Relevant Noncancer Data for
Soluble Tungsten Compounds (Various CASRNs)
Category
Number of
Male/Female, Strain,
Species, Study Type,
Study Duration
Dosimetry3
Critical Effects
NOAEL"
BMDL/
BMCLa
LOAELa
Reference
(comments)
Notesb
Reproductive/
12-16 M/12-16 F,
0, 1.3,39, 78, 125
Systemic: Reduced
78
Data not
125 (immune
Osterburg et
PR
developmental
C57BL6 mouse,
sodium tungstate
dihydrate in drinking
water, 19 wk (12 wk
premating, 1 wk
mating, 3 wk
gestation, and 3 wk
post-parturition); F1
offspring exposed for
an additional 90 d
in drinking water
ADD: 0, 1.3,39,
78, 125
immune response to
Staphylococcal
enterotoxin B in
F0 animals at PNW 3
(decreased number of
activated helper and
cytotoxic T cells in
spleen, decreased in situ
production of IFN-y) and
F1 animals at
approximately
PNWs 15-16 (decreased
number of activated
helper and cytotoxic T
cells in spleen).
amenable to
BMD modeling
suppression)
al. (2014)
Reproductive: No effects
observed.
125
(reproductive)
NDr
NDr
(reproductive)
Developmental: No
standard developmental
endpoints were reported.
Impaired immune
function was observed at
PNWs 15-16; however,
effects could have been
due to the subchronic,
postweaning exposure
rather than
developmental exposure.
NDr
(developmental)
NDr
NDr
(developmental)
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Soluble Tungsten Compounds
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Table 3A. Summary of Potentially Relevant Noncancer Data for
Soluble Tungsten Compounds (Various CASRNs)
Number of
Male/Female, Strain,
Species, Study Type,
BMDL/
Reference
Category
Study Duration
Dosimetry3
Critical Effects
NOAEL"
BMCLa
LOAELa
(comments)
Notesb
Reproductive/
40 M/40 F, S-D rat,
0, 3, 39, 78
Systemic: Lack of
78
NDr
NDr
Mclnturf et al.
PR
developmental
sodium tungstate
clinical signs of toxicity,
(2011);
dihydrate in deionized
ADD: 0, 3, 39, 78
body-weight effects,
Mclnturf et al.
water via gavage, 70 d
clear dose-related
(2008)
(14 d premating, 14 d
histopathological
mating, 22 d
changes, or clear
gestation, 20 d
dose-related changes in
post-parturition)
neurobehavioral tests of
F0 dams (pup retrieval,
open field behavior,
acoustic startle/prepulse
inhibition, Morris water
maze).
Reproductive: No effects
78
NDr
NDr
observed.
(reproductive)
(reproductive)
Developmental: No
78
NDr
NDr
effects observed.
(developmental)
(developmental)
Reproductive/
15 M/24 F, Wistar rat,
0, 1 mg/mL in
Systemic: Body weight
NDr
NDr
160 (decreased
Ballester et al.
PR
developmental
sodium tungstate in
drinking water
decreased in females of
body weight)
(2007);
drinking water, 12 wk
the exposed group,
Ballester et al.
and mate with
ADD: 0, 147 (M)
compared with controls.
(2005)
untreated animals
0, 160 (F)
Reproductive: No effects
160
NDr
NDr
observed.
(reproductive)
(reproductive)
11
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Table 3A. Summary of Potentially Relevant Noncancer Data for
Soluble Tungsten Compounds (Various CASRNs)
Number of
Male/Female, Strain,
Species, Study Type,
BMDL/
Reference
Category
Study Duration
Dosimetry3
Critical Effects
NOAEL3
BMCL3
LOAEL3
(comments)
Notesb
2. Inhalation3
ND
aDosimetry: Doses are equivalent tungsten doses in units of mg W/kg-day (doses for sodium tungstate x 62.6%). Tungsten accounts for 62.6% of total molecular weight
of Na2WC>4 (sodium tungstate). Presented BMDL/BMCL values were determined by the EPA for the purposes of this PPRTV assessment. Values are presented as
adjusted daily dose (ADD, in mg/kg-day) for oral noncancer effects.
bNotes: PS = principal study; PR = peer reviewed; NPR = not peer reviewed.
Treatment/exposure duration (unless otherwise noted): short-term = repeated exposure for >24 hours <30 days (U.S. EPA. 2002): long-term (subchronic) = repeated
exposure for >30 days <10% lifespan for humans (more than 30 days up to approximately 90 days in typically used laboratory animal species) (U.S. EPA. 2002):
chronic = repeated exposure for >10% lifespan for humans (more than approximately 90 days to 2 years in typically used laboratory animal species) (U.S. EPA. 2002).
BMR = bench mark response; ER = extra risk; F = female; FEL = frank effect level; M = male; NA = not applicable; ND = no data; NDr = not determined;
PNW = postnatal week; RD = relative deviation; S-D = Sprague-Dawley.
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Table 3B. Summary of Potentially Relevant Cancer Data for
Soluble Tungsten Compounds (Various CASRNs)
Category
Number of Male/Female,
Strain, Species, Study
Type, Study Duration
Dosimetry3
Critical Effects
BMDL/
BMCLa
Reference
(comments)
Notesb
Human
1. Oral (mg/kg-d)a
ND
2. Inhalation (mg/m3)a
ND
Animal
1. Oral (mg/kg-d)a
Carcinogenicity
(tumor promotion)
10 M/0 F, S-D rat, sodium
tungstate in drinking water,
19 wk
0, 100, 200 ppm
HED: 0, 3.33, 6.67
± known carcinogen
NSEE
No effects observed.
NDr
Luoetal. (1983)
(No evidence for
tumor promotion
by tungstate was
found.)
PR
Carcinogenicity
(tumor promotion)
10 M/0 F, S-D rat, sodium
tungstate in drinking water,
30 wk
0, 100 ppm
HED: 0, 2.86
± known carcinogen
NSEE
No effects observed.
NDr
Luoetal. (1983)
(No evidence for
tumor promotion
by tungstate was
found.)
Carcinogenicity
37 M/35 F (exposed)
52 M/52 F (control),
Long-Evans rat, sodium
tungstate in drinking water,
lifetime
0, 5 ppm
HED: 0, 0.1 (M),
0, 0.2 (F)
No effects observed.
NDr
Schroeder and
Mitcliener (1975b)
PR
Carcinogenicity
54 M/54 F, white Swiss
mouse, sodium tungstate in
drinking water, lifetime
0, 5 ppm
HED: 0, 0.1 (M),
0,0.1 (F)
No effects observed.
NDr
Schroeder and
PR
Mitchener (1975a)
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Table 3B. Summary of Potentially Relevant Cancer Data for
Soluble Tungsten Compounds (Various CASRNs)
Category
Number of Male/Female,
Strain, Species, Study
Type, Study Duration
Dosimetry3
Critical Effects
BMDL/
BMCLa
Reference
(comments)
Notesb
Carcinogenicity
(tumor promotion)
0 M/10-24 F, S-D rat,
sodium tungstate in
drinking water, sacrificed
125 or 198 d after injection
with known carcinogen,
NMU
0, 0, 150 ppm
sodium tungstate
HED: 0, 0 + NMU,
4.8 +NMU
After 125 d, the group exposed to
tungstate + NMU had statistically significantly
increased incidence of mammary carcinomas,
compared with the NMU-only group. After
198 d, both the NMU-only and
tungstate + NMU groups had >90% incidence
of mammary carcinomas. In both subgroups,
the first palpable mass was observed on D 56
in the tungstate + NMU group. In the
NMU-only group, the first palpable mass was
observed on D 71 and D 85 in the 125- and
198-d subgroups, respectively. No mammary
carcinomas were observed in the unexposed
control group. Tungstate-only exposure not
tested.
NDr
Wei et al. (1985)
(Limited evidence
for tumor
promotion by
tungstate was
found.)
PR
2. Inhalation (mg/m3)a
ND
dosimetry: Doses are equivalent tungsten doses in units of mg W/kg-day (doses for sodium tungstate x 62.6%). Tungsten accounts for 62.6% of total molecular
weight of Na2WC>4 (sodium tungstate). Presented BMDL/BMCL values were determined by the EPA for the purposes of this PPRTV assessment.
bNotes: PS = principal study; PR = peer reviewed; NPR = not peer reviewed.
ND = no data; NDr = not determined.
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HUMAN STUDIES
Oral Exposures
No studies have been identified.
Inhalation Exposures
No studies have been identified.
A number of epidemiology studies have associated occupational exposure to dusts
containing tungsten (and tungsten compounds such as tungsten carbide and tungsten oxides) and
other metals in the hard metal alloy industry with pulmonary fibrosis, memory and sensory
deficits, and increased mortality due to lung cancer (ATSDR. 2005). However, respiratory and
neurological effects in hard metal alloy workers have been attributed to cobalt, rather than
tungsten (ATSDR. 2005).
Cross-sectional surveys of the U.S. general population have examined possible
associations between levels of metals (including tungsten) in urine or blood and serum thyroid
levels (Yorita Christensen. 2012). cardiovascular and cerebrovascular disease (Agarwal et ai.
2011). and various medical conditions (Mendv et al. 2012). In these studies, however, the
subjects' route of exposure is unknown. The studies are described in Table C-2 in Appendix C.
ANIMAL STUDIES
Oral Exposures
Overview of Animal Oral Exposure Studies
Noncancer endpoints evaluated in animals repeatedly exposed orally to sodium tungstate
include: (1) comprehensive endpoints as per U.S. EPA Health Effects Testing Guideline Office
of Prevention, Pesticides and Toxic Substances (OPPTS) 870.3100 in Sprague-Dawley (S-D)
rats exposed by gavage for 90 days (USACHPPM. 2007a. b); (2) neurological, systemic, and
reproductive endpoints as per U.S. EPA Testing Guideline OPPTS 870.3650 in S-D rats exposed
by gavage for 70 days before mating, during mating and gestation, and during early postnatal
periods (Mclntutf et al, 2011; Mclnturf et al, 2008); (3) hematological and spleen cell endpoints
in response to bacterial toxin injection in C57BL6 mice following 28-day exposure or about
19 weeks of exposure starting 90 days before mating and continuing through gestation and
weaning (Osterburg et al. 2014); (4) body weight and bone marrow eellularity endpoints in
C57BL/6J mice exposed via drinking water for 16 weeks (Kelly et al.. 2013); (5) body weight
and histology of esophagus and forestomach in S-D rats exposed via drinking water for 19 or
30 weeks (Luo et al.. 1983); (6) body weight and reproductive endpoints in Wistar rats exposed
via drinking water for 12 weeks (Ball ester et al.. 2007; Ball ester et al.. 2005); (7) body weight in
Wistar rats exposed via the diet for 28 days (Chatteriee et al.. 1973); and (8) body weight in rats
and mice exposed via drinking water for life (Schroeder and Nlitchener. 1975a. b).
Comprehensive oral noncancer toxicity assays of sodium tungstate in chronically exposed
animals are not available.
Decreased body weight, increased incidence of lesions in the kidney and glandular
stomach, decreased bone marrow cellularity, and decreased immune responses are the most
clearly identified effects noted in the subchronic-duration studies. In the 90-day gavage rat
study, biologically significantly decreased body weight (12-15%) occurred in males, but not
females, exposed to 125 mg W/kg-day (USACHPPM. 2007a. b). Increased incidences of kidney
lesions (cortical tubule regeneration) were also found in male and female rats at
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125 mg W/kg-day (USACUPPNl. 2007a. b). Increased incidences of stomach lesions
(inflammation and goblet cell metaplasia) occurred in male and female rats at 78 and
125 mg/kg-day, but not at 47 mg W/kg-day (USACUPPNl. 2007a. b). The 70-day gavage rat
study, in which 78 mg W/kg-day was the highest dose tested, found no exposure-related effects
on reproductive performance or histology of various organ tissues in F0 males or females, no
exposure-related histological tissue changes in F1 offspring at Postnatal Day (PND) 20 and 70,
and no biologically significant changes in neurobehavioral endpoints in F1 offspring at PND 4
and 7 (Mclnturf et al.. 201 1; Mclnturf et al.. 2008). No effects on reproductive endpoints were
found in C57BL6 mice following 19 weeks of exposure at doses up to 125 mg W/kg-day
(Osterburg et al. 2014). Similarly, no effects on reproductive endpoints were found in male or
female Wistar rats following 12 weeks of exposure at 147 or 160 mg W/kg-day, respectively,
although statistically significantly decreased body-weight gains were reported for both sexes
(Ball ester et al. 2007; Ball ester et al. 2005). Decreased responses to bacterial infection
(decreased numbers of activated (CD71+) helper and cytotoxic T cells in the spleen) were found
in F0- and F1 -generation mice at 125 mg W/kg-day, but not at 78 mg W/kg-day (Osterburg et al..
2014). The lowest-observed-adverse-effect level (LOAEL) and no-observed-adverse-effect level
(NOAEL) for decreased splenic response to injection with a bacterial toxic in adult C57BL6
mice after 28 days of exposure are also 125 and 78 mg W/kg-day, respectively (Osterburg et al..
2014). In the 16-week drinking water mouse study, 250 and 49 mg W/kg-day are the LOAEL
and NOAEL, respectively, for decreased body weight and decreased total bone marrow
eellularity (Kelly et al.. 2013).
Studies of similar noncancer endpoints in animals repeatedly exposed orally to tungsten
metal are not available. However, an early series of studies found that sodium tungstate was
more potent than tungsten metal in causing mortality and body-weight changes in rats (Kinard
and Van de Erve. 1943. 1941). Deaths occurred in groups of rats within 30 or 7 days of exposure
to 0.5 or 2% tungsten as sodium tungstate in the diet, respectively (Kinard and Van de Erve,
1941). In contrast, no deaths occurred in groups of rats exposed to 2, 5, or 10% tungsten as
tungsten metal in the diet for 70 days (Kinard and Van de Erve. 1943).
In the only available chronic-duration/carcinogenicity animal studies (Schroeder and
Nlitchener. 1975a. b), the study authors reported no evidence for carcinogenic responses in rats
or mice exposed to sodium tungstate in drinking water for life at a concentration of 5 ppm
tungsten (estimated human equivalent doses [HEDs] of 0.1-0.2 mg W/kg-day). Limitations of
these studies include inadequate reporting of histological findings for nonneoplastic lesions,
inclusion of only one exposure level, and absence of an exposure level close to a maximum
tolerated dose (MTD). Body weights of exposed mice and rats were within 10% of the mean for
nonexposed control animals in these studies. No evidence of sodium tungstate's tumor
promotion capability was found in one rat assay of tumors initiated by A-nitrososarcosine ethyl
ester (NSEE) (l.uo et al.. 1983). In another rat assay, tumors appeared earlier in rats exposed to
nitroso-A-methylurea (NMU) followed by sodium tungstate in drinking water, compared with
rats exposed to NMU alone (Wei et al.. 1985).
Short-Term-Duration Studies
Osterburg et al. (2014)
Groups of male and female C57BL6 mice (22-24/sex/group) were given water ad libitum
containing sodium tungstate dihydrate for 28 days. Sodium tungstate dihydrate was added to the
water bottles at levels calculated to administer ingested doses of approximately 2, 62.5, 125, or
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200 mg/kg-day sodium tungstate based on an estimated water consumption of
4.5 mL/mouse-day. Water consumption was measured daily, and water bottles were changed
2-3 times weekly. Body weights were measured weekly, and quantities of tungstate were
adjusted appropriately in water bottles. Control animals were allowed ad libitum access to
filtered water. Equivalent tungsten doses are calculated to be 0, 1.3, 39, 78, or 125 mg W/kg-day
(tungsten accounts for 62.6% of total molecular weight of Na2WC>4). The mice were 8-12 weeks
old when supplied by Charles River Laboratories, Wilmington, Massachusetts and acclimated for
7 days before the start of treatment. At the start of testing, the mice weighed between 19 and
22 g. A low-molybdenum diet was available to all mice ad libitum.
Following the 28-day exposure, mice were injected intraperitoneally (i.p.) with either
sterile saline or 20 |ig Staphylococcal enterotoxin B (SEB) to evaluate the adaptive immune
response (11-12/sex/group per treatment). Twenty-four hours later, the mice were sacrificed.
Blood and spleen tissue samples were collected and stained with lymphocyte and/or myeloid
immunophenotyping panels and analyzed by flow cytometry. In situ cytokine production by
splenic T cells was also measured.
No exposure-related effects on survival or clinical signs of toxicity were reported. No
exposure-related changes were observed in body weight or standard hematological endpoints
(e.g., counts of white blood cell [WBC] and red blood cell [RBC], hemoglobin concentration
[Hb], hematocrit [Hct]). No alterations in immunophenotypes of blood cells were reported. In
the spleen, there was no exposure-related difference in the number of helper or cytotoxic T cells
following SEB infection. However, the number of activated (CD71+) helper and cytotoxic T
cells in response to SEB infection were significantly decreased by 43 and 66%, respectively, in
mice exposed to 125 mg W/kg-day, compared with controls (see Table B-2). Levels of CD71-
and CY71+ helper and cytotoxic T cells did not differ among exposure groups in mice injected
with saline (instead of SEB). Additionally, in situ interferon gamma (IFN-y) production was
significantly reduced by about 55% in isolated spleen cells harvested from mice exposed to
125 mg W/kg-day following the SEB challenge, compared with the control (see Table B-2).
Again, no exposure-related effects were observed in mice injected with saline. Taken together,
these data indicate NOAEL and LOAEL values of 78 and 125 mg W/kg-day, respectively, for
immune suppression.
A second group of male and female C57BL6 mice (number not specified) were given
water ad libitum containing 0, 20, or 200 mg tungstate/kg-day (0, 12.5, or 125 mg W/kg-day) for
28 days. After the 28-day exposure, mice were challenged in a delayed-type hypersensitivity
Type IV (Type IV DTH) experiment using 4-hydroxy-3-nitrophenylacetic acid active acid
(NP-O-Su) as a secondary antigen. During the sensitization phase, tungstate exposure continued.
Ten days later, the mice were challenged with an NP-O-Su injection into the right hind foot pad;
the left hind foot pad received a saline injection for comparison. The extent of footpad swelling
was measured with a dial gauge 24 hours postinjection. The Type IV DTH experiment was
repeated using doses of 0, 0.2, 2, or 200 mg tungstate/kg-day (0, 0.1, 1.3, or 125 mg W/kg-day).
In the first DTH experiment, foot-pad swelling was significantly decreased by -30% in
mice exposed to 12.5 or 125 mg W/kg-day, compared with controls. In the second DTH
experiment, mice exposed to 125 mg W/kg-day also showed a significant decrease in swelling
(~70%> compared with controls). No significant, exposure-related changes were observed in
foot-pad swelling in mice exposed to 0.1 or 1.3 mg W/kg-day. The adversity of the diminished
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response is uncertain, and could be viewed as a positive action of tungstate (potentially
therapeutic) in allergically sensitized individuals. Therefore, aNOAEL/LOAEL determination is
not made for the Type IV DTH studies.
Chatterjee etal. (1973)
Groups of male Wistar rats (4/group) were fed ad libitum with basal diet containing 0, 5,
or 50 ppm tungsten (W) as sodium tungstate dihydrate for 28 days. Rats weighed 60-80 g when
they were obtained from CIBA Pharmaceuticals (Bombay, India). Treatment began after a
one-week observation period (age of rats unspecified). Rats were weighed biweekly. No
exposure-related effects on body-weight gain were reported (no other toxicity endpoints were
evaluated). Using the reported starting body-weight range and body-weight-gain data and a
calculated food consumption rate of 0.01 kg/day (based on the allometric equation for laboratory
mammals reported by U.S. EPA (1988): food consumption = 0.056 x (BW0 6611) where body
weight was 0.084 kg and 0.081 kg for the 5 and 50 ppm groups, respectively), daily tungsten
intakes were calculated to be 0, 0.65, or 6.6 mg W/kg-day. A NOAEL of 6.6 mg W/kg-day is
determined for lack of body-weight effects in male rats.
Subchronic-Duration Studies
USACHPPM(2007a): USACHPPM (2007b)
The U.S. Army Center for Health Promotion and Preventative Medicine (USCHPPM)
conducted a 90-day oral toxicity study in rats in accordance with EPA Health Effects Testing
Guidelines OPPTS 870.3100. The results of this study were later published in a peer-reviewed
paper by McCain et al. (2015). Groups of 5-week-old S-D rats (10/sex/group) were administered
10, 75, 125, or 200 mg/kg-day sodium tungstate (Na2WC>4) as sodium tungstate dihydrate in
water via gavage 7 days/week. Equivalent tungsten doses are calculated to be 6, 47, 78, or
125 mg W/kg-day (tungsten accounts for 62.6% of total molecular weight of Na2WC>4). Control
animals were given deionized water via gavage at the same volume per body weight as all other
dose groups (1 mL/kg). At the start of testing, the rats weighed between 199 and 230 g and were
randomly allocated to treatment groups based on body weight. Gavage doses were adjusted
weekly in accordance with body-weight changes.
Clinical examinations were performed prior to treatment and once weekly during
treatment, and the animals were observed daily for clinical signs of toxicity. Body weights and
feeder weights were measured on Days -3,-1,0 (day of first dose), and 7, and weekly
thereafter. Ophthalmic examinations were performed before treatment and within a week of
scheduled necropsy. Urinalysis was performed in eight rats/sex/group within 2 weeks of
necropsy (volume, color, appearance, pH, specific gravity, glucose, bilirubin, urobilinogen,
ketone, blood, protein, nitrite, and leukocytes). Following the 90-day exposure period, blood
samples were collected for hematology, blood coagulation, and clinical chemistry.
Hematological endpoints included WBC count and differential, RBC count, hemoglobin,
hematocrit, mean cell volume, mean cell hemoglobin, mean cell hemoglobin concentration, RBC
distribution width, platelets, and mean platelet volume. Blood coagulation was evaluated using
average prothrombin time and average activated prothrombin time. Clinical chemistry endpoints
included alkaline phosphatase, alanine aminotransferase (ALT), aspartate aminotransferase
(AST), blood urea nitrogen (BUN), calcium, cholesterol, creatinine kinase, creatinine, glucose
(nonfasting), lactate dehydrogenase, total bilirubin, total protein, triglycerides, sodium,
potassium, and chlorine.
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At sacrifice, a complete necropsy to detect gross pathological lesions was conducted on
all animals. Terminal body weight and organ weights for the brain, heart, liver, kidneys, spleen,
adrenals, thymus, epididymides/uterus, and testes/ovaries were recorded. The following tissues
were harvested and embedded in paraffin for histopathological evaluation in the 0, 78-, and
125-mg W/kg-day groups: brain, pituitary, thyroid w/parathyroid, thymus, lungs, trachea, heart,
bone marrow, salivary gland, liver, spleen, kidney, adrenal, pancreas, gonads, uterus, aorta,
esophagus, stomach, duodenum, jejunum, ileum, caecum, colon, urinary bladder, lymph node,
peripheral nerve, thigh musculature, eye, spinal cord (three levels), exorbital lachrymal gland,
and all gross lesions. Sections from select target tissues (kidney, stomach, and epididymides)
were also examined in the 6- and 47-mg W/kg-day groups.
No exposure-related clinical signs of toxicity were observed. Body weight was
biologically significantly decreased by 12-15% in males in the 125-mg W/kg-day group over the
last 3 weeks of the study, compared with controls. Reduced body weight was accompanied by
statistically significant reductions in food consumption in this group (quantitative data were not
reported by the study authors). No exposure-related changes in body weight or food
consumption were observed in lower-dose males and females. No ophthalmic abnormalities
were observed in any group. No significant, dose-related changes were observed in urinalysis,
clinical chemistry, or hematological endpoints. Decreased absolute liver, heart, testes, and
epididymis weights in high-dose males and increased kidney and spleen weights in high-dose
females were reported to have occurred, but no statistically significant dose-related changes in
organ-to-body weight ratios were reported. Pairwise analysis was not conducted, because
analysis of variance (ANOVA) did not indicate significant effects of dose on any organ:body
weight ratio.
Exposure-related histological changes were observed in the stomach, kidneys, and
epididymides. Statistically significantly increased incidences of goblet cell metaplasia and
subacute inflammation were observed in the glandular stomach of males and females in the
78- and 125-mg W/kg-day groups (see Table B-l). Subacute inflammation was characterized by
the presence of eosinophils and a few mononuclear cells throughout the submucosa. The study
authors considered goblet cell metaplasia and subacute inflammation to be nonadverse and
related to a physiologic effect of gavage administration. However, this explanation is not
consistent with the observed absence of lesions in control groups and the dose-related increases
in treated groups. The glandular stomach lesions are considered biologically significant,
dose-related, and compound-related effects for this assessment (see Table B-l). Supporting the
biological significance of these lesions is the understanding that goblet cells are not part of the
normal mammalian gastric epithelium, and that metaplasia (replacement of normal cells with
columnar absorptive cells and goblet cells of intestinal morphology) is a common histological
change associated with repeated inflammation of the gastric mucosa (Liu and Crawford. 2005).
Increased incidences of mild-to-severe regeneration of renal cortical tubules were observed in
males and females in the 125-mg W/kg-day group (see Table B-l). Affected tubules exhibited
abundant pale basophilic cytoplasm and closely packed, typically basally located, nuclei with a
moderate degree of anisokaryosis and scattered karyomegaly. Tubular dilation was observed in
the more severely affected rats. Minimal regeneration of renal cortical tubules was observed in
all dose groups (incidences were not reported by the study authors). Group mean and individual
animal severity scores were not provided. Chronic progressive nephropathy (basophilic tubules
with thickened basement membranes) was observed in all dose groups, but with no apparent
dose-response relationship (incidence data were not reported by the study authors). Luminal
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cellular debris was observed in the epididymides of 3/10 male rats in the 125-mg W/kg-day
group, an incidence that is not significantly elevated over the control incidence of 0/10
(see Table B-l). There were no testicular lesions that explained the presence of degenerated
cells in the lumen of the epididymis; therefore, the biological significance is uncertain. The
remaining histological findings were considered by the study authors to be incidental or
spontaneous, rather than exposure related (lesion type, location, and incidence and severity data
were not provided in the available report).
A LOAEL of 78 mg W/kg-day and a NOAEL of 47 mg W/kg-day are identified in male
and female S-D rats for significantly increased incidence of rats with goblet cell metaplasia and
inflammation in the glandular stomach, compared with controls. Additional effects found in the
highest dose group (125 mg W/kg-day) were decreased body weight in males and significantly
increased incidences of male and female rats with mild to severe renal cortical tubule
regeneration.
Kelly et al. (2013)
Groups of male C57BL/6J mice (5/dose/sacrifice) were given water ad libitum containing
0, 15, 200, or 1,000 mg/L tungsten as sodium tungstate dihydrate for 1, 4, 8, 12, or 16 weeks.
Mice were 4-weeks-old when purchased from Jackson Labs (Bar Harbor, Maine). Treatment
began after a 1-week acclimation period. Body weight was measured weekly, and daily water
consumption was monitored, but quantitative data were not reported. Using reference male
mouse body weight (0.0316 kg) and water consumption (0.00782 L/day) values for the
subchronic-duration studies (U.S. EPA. 1988). the estimated tungsten intakes in the 15, 200, and
1,000 mg/L groups are 4, 49, and 250 mg W/kg-day, respectively. At all interim and terminal
sacrifices, blood was collected for hematology (RBC count, WBC count and differential,
hematocrit, hemoglobin, platelets), tibiae weight and length was measured, and bone marrow
was harvested from both tibiae and femora. The total number of tibial and femoral bone marrow
cells per animal was quantified, and two million bone marrow cells/animal were used to quantify
B cell developmental fractions A-F using flow cytometry. Fractions A, B, C/C', D, E, and F
represent pre-pro-B cells, early pro-B, late pro- and large pre-B cells, small pre-B cells,
immature B cells, and mature B cells, respectively. Clonogenicity (activity) of bone marrow
precursor cells was assessed with the pre-B colony forming unit (CFU) assay. At the terminal
sacrifice, serum AST and ALT levels were also measured.
Body weight was statistically significantly lower in animals in the 250-mg W/kg-day
group by approximately 15%, compared with controls (data presented graphically). No
exposure-related statistically significant changes were observed in tibia weight or length or
serum AST or ALT enzyme levels.
After one week of exposure, peripheral WBC counts were statistically significantly
decreased in the 4, 49, and 250-mg W/kg-day groups by approximately 10, 25, or 50%,
compared with controls (data presented graphically). White cell differential counts showed that
lymphocytes were significantly decreased in the 49 and 250-mg W/kg-day groups, and
granulocytes and monocytes were significantly decreased in the 250-mg W/kg-day group. No
statistically significant, dose-related findings were observed in WBC counts at any other time
point. No exposure-related changes were observed in erythrocyte or platelet counts or
hemoglobin or hematocrit levels at any time-point.
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Total bone marrow cellularity was statistically significantly decreased by 20% in mice in
the 250-mg W/kg-day group at 16 weeks, but not in lower dosage groups or in any dose groups
at earlier time points (see Table B-3). After 16 weeks, all tungsten-exposed groups demonstrated
a statistically significant 83-117% increase in the percentage of late pro- and large pre-B cells in
the bone marrow, compared with control values (Fraction C/C'; see Table B-3). This effect was
not observed at the earlier time points. When normalized to total bone marrow cellularity, the
number of late pro- and large pre-B cells at 16 weeks was statistically significantly increased in
the 4- and 49-mg W/kg-day groups by 88 and 75%, respectively, compared with controls. The
50% increase in the 250-mg W/kg group was not statistically significant (see Table B-3). There
were also statistically significant increases (45—98% compared with control) in the percentages
of mature B cells (Fraction F) in the 49- or 250-mg/kg-day groups at early time points (1, 4, or
8 weeks), but these changes were not observed at 12 or 16 weeks. When normalized to total
bone marrow cellularity, the number of mature B cells was not statistically significantly altered
in any dosage group at any time point, compared with control. No exposure-related changes
were observed in the percentage or number of pre-pro-B, early pro-B, small pre-B, or immature
B cells (Fractions A, B, D, or E) at any time point.
B cell developmental profiles were also examined in separate groups of mice exposed to
4 mg W/kg-day for 4 weeks, followed by 4- or 8-week unexposed recovery periods. However,
the lack of consistent statistically significant effects of 4 weeks of exposure on any B cell
fractions makes the presented results from these groups difficult to interpret.
No exposure-related changes were found in the number of B lymphoid precursors at any
time point. Clonogenicity of bone marrow progenitor cells into lineage-specific precursors
ex vivo was statistically significantly increased in the CFU-pre-B assay using cells from mice
exposed for 16 weeks at 250 mg W/kg-day (113% increase), compared with controls
(see Table B-3). The adversity of this effect is uncertain, especially because peripheral WBC
counts were not elevated. Statistically significant effects on clonogenicity of bone marrow
progenitor cells were not observed with cells from mice in the lower exposure groups.
In nonadherent bone marrow cells, deoxyribonucleic acid (DNA) damage assessed by the
Comet assay was increased compared with control values, but findings across time points and
dosages were not consistent with a monotonic response with increasing dose or duration
(see Table B-4). Statistically significant increases were observed at 1, 4, 12, and 16 weeks in the
4-mg W/kg-day group; at 1, 4, and 8 weeks in the 49-mg W/kg-day group; and at 4 and 12 weeks
in the 250-mg W/kg-day group. The amount of DNA damage did not increase with increasing
dose; across time points, the magnitude of damage was higher in cells from 4-mg W/kg-day mice
than cells from 250-mg/kg-day mice. In CD 19+ B cells, DNA damage was statistically
significantly increased in the 4-mg W/kg-day group at 1 and 4 weeks and the 49-mg W/kg-day
group at 4 weeks, but decreased (not statistically significant) at 1 and 4 weeks in the
250-mg/kg-day group (see Table B-4; DNA damage in CD19+ B cells was not assessed at later
time-points). Immunoblot staining for yH2AX (another assay for DNA damage) in
CD 19+ B cells from exposed mice after 1 week of exposure was not statistically significantly
elevated compared with control values.
In summary, the results from this study indicate that 250 and 49 mg W/kg-day are the
LOAEL and NOAEL, respectively, for decreased body weight and decreased total bone marrow
cellularity in male C57BL/6J mice exposed by drinking water for 16 weeks. Persistent,
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exposure-related changes in blood cell counts were not observed. Increased percentages of late
pro- and large pre-B cells (Fraction C/C') were observed in all exposed groups from this study
[i.e., Kelly et al. (2013)1. after 16 weeks of exposure, but the adversity of this change is
uncertain. Kelly et al. (2013) noted that late pro- and large pre-B cells are particularly sensitive
to DNA damaging agents and hypothesized that an increased percentage of B cells in this
developmental stage may increase the probability of DNA damaging events in B cells and
potentially increase the probability for leukemia. However, a clear concordance was not
observed between DNA damage and percentages of late pro-and large pre-B cells. For example,
in nonadherent bone marrow cells isolated from mice exposed to the highest dose, DNA damage
was not increased, compared with control values at 1 or 16 weeks, but 16 weeks of exposure to
each of the dosage levels caused increased percentages of late pro-and large pre-B cells.
Kinard and Van de Erve (1941)
Groups of rats (strain unspecified; 37-days-old; 5-6/sex/group) were caged separately
and fed ad libitum with ground dog chow containing 0, 0.1, 0.5, or 2.0% tungsten as sodium
tungstate for up to 70 days. Food consumption was determined from the food remaining in the
feeders after each feeding, but the study authors noted that significant food wastage made this
measurement unreliable. Body weight was measured at 9-12-day intervals. Sodium tungstate
caused 100% mortality in the 2.0% group (within the first week of exposure) and 50 and 33%
mortality of males and females, respectively, in the 0.5% group (deaths occurred between
Days 17 and 29 of exposure). No mortalities were observed in the 0.1% group or control groups
(see Table B-5). Body weight and body-weight gain were reduced 9-11 and 12—16%,
respectively, in the 0.1% group compared with controls (see Table B-5). Absolute body-weight
data were not reported for other groups; however, body-weight gain in surviving rats from the
0.5% group was decreased by 114-131%), compared with controls (see Table B-5). In surviving
rats, apparent food consumption was decreased compared with controls (-17 and -4% in males
and females, respectively, from the 0.1% group; -53 and —28% in males and females,
respectively, from the 0.5% group, although again, the researchers did not consider these data to
be reliable. Using reference rat body weight (0.235 kg for males and 0.173 kg for females) and
food consumption (0.021 kg/kg BW-day for males and 0.017 kg/kg BW-day for females) values
for the subchronic-duration studies (U.S. EPA. 1988). the estimated tungsten intakes in the 0,
0.1, 0.5, and 2% groups were calculated to be 0, 91, 455, and 1,820 mg W/kg-day for males and
0, 102, 510, and 2,040 mg W/kg-day for females. A LOAEL of 91 mg W/kg-day is identified
for reduced body weight in males. A NOAEL was not identified.
Additional groups of rats were fed diets containing 0.1, 0.5, or 3.96% tungsten
equivalents of tungsten trioxide or 0.5, 2.0, or 5.0% tungsten equivalents of
ammoni um-/Mungstate. This allowed a comparison of toxicity among three soluble compounds
of tungsten. Mortalities for tungsten trioxide exposed groups of increasing dose were 0, 73%
(average of 80 and 66% for males and females), and 100%. Mortalities for
ammonium-p-tungstate exposed groups were 0, 80, and 100%. Tungsten trioxide at the lowest
tested concentration (0.1% W) decreased body weight by 6.3 and 7.4% in surviving males and
females after 70 days. Ammonium-/Mungstate at the lowest tested concentration (0.5% W)
decreased body weight by 3.9 and 5.3% in surviving males and females.
Considering body-weight data, the toxicity of the three tungsten compounds increased in
the order: ammonium-p-tungstate < tungsten trioxide < sodium tungstate, which follows the
order of increasing solubility. At the lowest tested concentrations, body-weight decreases were
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3.9-5% for am m on i um -/Mungstate (0.5% W), 6.3-7.4%) for tungsten trioxide (0.1%> W), and
9-11% for sodium tungstate (0.1%>W). At the 0.5% W concentration, a different order of
potency to produce mortality was observed: am m on i urn -/Mungstate (no mortalities) < sodium
tungstate (50% males, 33% females) < tungsten trioxide (80% males, 66% females). At the next
tested concentration (2% W), am m on i urn -/Mungstate produced 80% mortalities, and sodium
tungstate produced 100% mortalities. Tungsten trioxide was not tested at 2% W.
Kinard and Van de Erve (1943)
Groups of 10 rats (mixed Wistar albino and Minnesota piebald strains; 38-days-old;
5/sex/group) were caged separately and fed ad libitum with ground dog chow containing 0, 2, 5,
or 10%) tungsten metal powder for 70 days. Food consumption was determined from the food
remaining in the feeders after each feeding. Weight gains were recorded at 10-day intervals.
Animals were sacrificed after 70 days, and their gastrointestinal tracts were examined grossly.
During the dosing period, each male rat fed 2, 5, or 10% tungsten consumed an average of 21,
54.5, or 104 g of tungsten, respectively. Female rats, in the same period, consumed an average
of 14.8, 41, or 75 g of tungsten. During the 70-day period, weight gains of male rats fed 2, 5, or
10%) tungsten were 94, 113, or 108%> of those of male rats fed the control diet, while the weight
gains of female rats were 104, 97, or 85% of control weight gains, respectively (see Table B-6).
However, the absolute body-weight data is not available for evaluation. The study authors
attributed the different weight gains by male and female rats fed diets containing 10% tungsten
to reduced food consumption in the female rats. No exudation of blood into the mucous
membranes of the small or large intestines was found in rats fed tungsten for 70 days. These
results show that oral exposure to tungsten metal in the diet is markedly less toxic than
water-soluble compounds of tungsten, such as sodium tungstate. No mortalities occurred in rats
fed diets containing up to 10% tungsten as tungsten metal for up to 70 days, whereas deaths
occurred within 30 days of exposure in rats fed diets containing 0.5 or 2% tungsten as sodium
tungstate. Using tungsten consumption and body-weight data from Kinard and Van de Erve
(19411 tungsten metal doses were calculated to be approximately 0, 1,325, 3,450, or
6,550 mg W/kg-day and 0, 1,450, 4,000, or 7,325 mg W/kg-day for males and females,
respectively. A LOAEL of 7,325 mg W/kg-day and a NOAEL of 4,000 mg W/kg-day are
identified in female rats for decreased body-weight gain compared with controls.
Luo etal. (1983)
Male weanling inbred S-D rats were given demineralized drinking water ad libitum
containing 0, 100, or 200 ppm tungsten as sodium tungstate for 19 weeks (10/group). All rats
were fed ad libitum a nutritionally adequate semipurified diet containing 0.064 ppm of tungsten.
Body weights were monitored throughout the exposure period. At sacrifice, the esophagus and
forestomach were removed and fixed in 10% formalin for histopathological examinations. The
average body weight of 100 ppm tungsten-fed rats was similar to controls from Weeks 0-19;
body-weight data for rats given 200 ppm for 19 weeks were not reported. Using reference male
weanling S-D rat body weight (0.267 kg) and water consumption (0.037 L/day) values for the
subchroni c-durati on studies (U.S. EPA, 1988), daily intake of tungsten in the 100- and 200-ppm
groups was estimated to be 13.9 and 27.8 mg W/kg-day. No histopathological alterations were
observed in any of control or tungsten-only exposed rats. NOAELs of 27.8 mg W/kg-day for a
lack of observed effects in male rats are identified for 19-week exposures.
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Chronic-Duration/Carcinogenicity Studies
Luo etal. (1983)
Male weanling inbred S-D rats were given demineralized drinking water ad libitum
containing 0 or 100 ppm for 30 weeks (10/group). All rats were fed ad libitum a nutritionally
adequate semipurified diet containing 0.064 ppm of tungsten. Body weights were monitored
throughout the exposure period. At sacrifice, the esophagus and forestomach were removed and
fixed in 10% formalin for histopathological examinations. Body-weight data for rats given
100 ppm for 30 weeks were not reported. Using reference male weanling S-D rat body weight
(0.523 kg) and water consumption (0.062 L/day) values for the chronic-duration studies (U.S.
EPA, 1988). daily intake of tungsten in the 100-ppm groups was estimated to be
11.9 mg W/kg-day. No histopathological alterations were observed in any of control or
tungsten-only exposed rats. NOAELs of 11.9 mg W/kg-day for a lack of observed effects in
male rats are identified for 30-week exposures, respectively.
Schroeder andMitchemr (1975b)
A group of 72 Long-Evans rats (37 males/35 females) was given 5 ppm of tungsten for
life as sodium tungstate in deionized drinking water to which essential elements manganese,
cobalt, copper, zinc, and molybdenum were added in amounts similar to those in commercial
diets. A control group (52 males/52 females) received the same basal drinking water without
tungsten. The diet fed was low in trace metals. The animals were weighed at weekly intervals
for an unspecified number of weeks, at monthly intervals for the remaining weeks of the first
year, and then at 3-month intervals. Urine was collected from 12 rats/sex/group for analysis of
urinary protein, pH, and glucose when rats were 162-days-old. Blood samples were collected
from 12 rats/sex/group for analysis of serum cholesterol, glucose, and uric acid levels at various
time-points throughout the study, starting when rats were 90-days-old. At 20 months of age, a
pneumonia epidemic led to the death of-30% of the animals in both the control and
tungsten-exposed groups (no significant difference in the number of deaths among groups). The
remaining animals (26 control males, 24 control female, 25 exposed males, and 20 exposed
females) were necropsied at natural death. At necropsy, the animals were weighed and examined
for gross pathological changes. The heart, lung, kidney, liver, spleen, and all tumors were fixed
for microscopic examination. The longevity of rats was calculated based on the mean lifespan of
the last five surviving animals.
A statistically significant increase in body weight from controls was first observed at
180 days in males and 360 days in females. Body weight in males remained significantly higher
than controls to 540 days; in females at 540 days, body weight was slightly, but not significantly,
increased (see Table B-9). However, these findings are not considered biologically significant
because observed increases in body weight were <10% different from controls. At necropsy
following natural death, no significant differences in body weight or heart weight were reported
in tungsten-fed rats compared with controls (it is unclear whether any other organs were
weighed). The longevity of tungsten-fed females was comparable with female controls.
However, the longevity of tungsten-fed males was statistically significantly decreased by 13%
compared with male controls (see Table B-9). Urinary parameters of tungsten-fed animals were
unremarkable. Intermittently during the study, fasting serum cholesterol and glucose levels were
significantly different from controls; however, no trends were apparent. There were no
exposure-related increases in total gross or malignant tumor incidences (see Table B-9). It is not
clear whether the heart, lung, kidney, liver, and spleen were assessed for nonneoplastic
histological changes.
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Using reference Long-Evans rat body weight (0.472 kg for males and 0.344 kg for
females) and water consumption (0.057 L/day for males and 0.046 L/day for females) values for
the chronic-duration studies (U.S. EPA. 1988). estimated daily tungsten intakes were 0.6 and
0.7 mg W/kg-day for males and females, respectively. Based on these estimates, HEDs were
calculated to be 0.1 and 0.2 mg W/kg-day for males and females, respectively, using a DAF of
0.24 for rats based on body weight to 3/4 power scaling (U.S. EPA, 2011b). Although a
significant decrease in longevity was observed in male rats, based on the lifespan of the final five
surviving rats; the occurrence of a pneumonia epidemic 20 months into the study decreases
confidence that this was an exposure-related effect. A NOAEL of 0.7 mg W/kg-day is identified
for female rats, respectively. No evidence for a carcinogenic response to tungsten was found.
Schroeder andMitchemr (1975a)
Using methods similar to those with rats (Schroeder and Mitchener. 1975b). white Swiss
mice, 54 of each sex, were given 0 (control) or 5 ppm of tungsten for life as sodium tungstate in
their drinking water. Body weight was monitored, and at natural death, gross and microscopic
examinations were performed on major organs. The body weights of tungsten-fed mice at
540 days of exposure were comparable with controls (see Table B-9). The longevity of
tungsten-fed male mice was decreased by 15%, compared with male controls; however, this
finding was not statistically significant. The longevity of tungsten-fed females was comparable
with female controls (see Table B-9). Exposure to tungsten was reported to be not "significantly
tumorigenic"; however, tumor incidence data were not reported. Using reference mouse body
weight (0.0317 kg for males and 0.0288 kg for females) and water consumption (0.0078 L/day
for males and 0.0073 L/day for females ) values for the chronic-duration studies (U.S. EPA.
1988). estimated daily tungsten intakes in males and females were 1 mg W/kg-day. Based on
these estimates, HEDs were calculated to be 0.1 mg W/kg-day for males and females, using a
DAF of 0.14 for mice based on body weight to 3/4 power scaling (U.S. EPA. 2011b). A
NOAEL of 1 mg W/kg-day for a lack of observed effects is identified for male and female mice.
No evidence for a carcinogenic response to tungsten was found.
Reproductive/Developmental Studies
Osterbure et al. (2014)
In a one-generation study, groups of male and female C57BL6 mice (12-16/sex/group)
were given water ad libitum containing sodium tungstate dihydrate for 12 weeks prior to mating,
through 1 week mating, 3 weeks gestation, and 3 weeks postparturition (19 weeks total). The F1
generation was exposed for an additional 90 days. Sodium tungstate dihydrate was added to the
water bottles at levels calculated to administer ingested doses of approximately 2, 62.5, 125, or
200 mg sodium tungstate/kg-day based on an estimated water consumption of
4.5 mL/mouse-day. Water consumption was measured daily, and water bottles were changed
2-3 times weekly. Body weights were measured weekly, and quantities of tungstate were
adjusted appropriately in water bottles. Control animals were allowed ad libitum access to
filtered water. Equivalent tungsten doses are calculated to be 0, 1.3, 39, 78, or 125 mg W/kg-day
(tungsten accounts for 62.6% of total molecular weight of Na2WC>4). Animals were
8-12-weeks-old when supplied by Charles River Laboratories, Wilmington, Massachusetts.
Mice were acclimated for 7 days prior to the start of treatment. At the start of testing, the mice
weighed between 15 and 18 g. A low-molybdenum diet was available to all mice ad libitum.
Mice were housed individually during the course of the study and pair mated for breeding. After
confirmation of the pregnancy, males were removed.
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The number of live births, litter size, and sex ratio were recorded for each litter. F0
animals were sacrificed at weaning (Study Week 19), while F1 offspring (12-16/group) were
sacrificed after the additional 90-day exposure (approximately Study Weeks 31-32).
Twenty-four hours prior to sacrifice, the mice were injected intraperitoneally with either sterile
saline or 20 |ig SEB to evaluate the adaptive immune response (6-8/sex/group per treatment).
At sacrifice, blood and spleen tissue samples were collected and stained with lymphocyte and/or
myeloid immunophenotyping panels and analyzed by flow cytometry. Complete blood counts
and hematological parameters were evaluated. In situ cytokine production by splenic T cells was
also measured.
No exposure-related changes were observed in body weight. No significant, dose-related
changes in the number of live births, litter size, or sex ratio of the pups were observed; mating
success was not reported. With two exceptions (monocyte percentage and RBC distribution
width), there were no statistical differences in hematological endpoints between F0 and
F1 animals within the same group; therefore, the hematological data for F0 and F1 animals were
combined for analysis. The only statistically significant, dose-related hematological finding was
a decrease in the percentage of monocytes in F0 and F1 animals (combined) exposed to "higher
concentrations." A pair-wise analysis was not reported, and F0 and F1 animal data and statistics
were not reported individually. No alterations in immunophenotypes of blood cells were
reported. In the spleen, there was no exposure-related difference in the number of helper or
cytotoxic T cells following SEB infection in either F0 or F1 animals. However, the number of
activated (CD71+) helper and cytotoxic T cells in response to SEB infection in F0 and F1 mice
exposed to 125 mg W/kg-day was significantly decreased by 60-80%, compared with respective
controls (see Table B-2). Levels of CD71- and CY71+ helper and cytotoxic T cells did not
differ among exposure groups in mice injected with saline (instead of SEB). Additionally, in situ
IFN-y production was significantly reduced by 47% in isolated spleen cells harvested from
F0 mice exposed to 125 mg W/kg-day following the SEB challenge, compared with control
(see Table B-2). Again, no exposure-related effects were observed in F0 mice injected with
saline. Cytokine production was not significantly altered in F1 mice (see Table B-2).
In summary, a reproductive NOAEL of 125 mg W/kg-day is identified based on lack of
litter effects. A developmental NOAEL/LOAEL determination cannot be made. Standard
developmental endpoints were not assessed, and the observed immune suppression at Postnatal
Weeks (PNWs) 15-16 may be attributable to the 90-day postweaning exposure to tungsten rather
than the developmental exposure. A systemic NOAEL of 78 mg W/kg-day and a LOAEL of
125 mg W/kg-day are identified for immune suppression in F0 and F1 animals, consistent with
the 28-day study reported above.
Mclnturf et al. (2011); Mclnturfetal (2008)
Groups of S-D rats (40/sex/group) were administered 0, 5, 62.5, or 125 mg/kg-day
sodium tungstate (Na2WC>4) as sodium tungstate dihydrate in deionized water via gavage
7 days/week for 70 days (including 14 days premating, 14 days mating, 22 days gestation, and
through PND 20). Rats were 8-weeks-old when they were obtained from Charles River
Laboratories (Wilmington, MA), and dosing began after a 2-week quarantine period. The rats
were single-housed except during the mating period, observed daily for clinical signs of toxicity,
and weighed weekly. During gestation, pregnant dams were weighed on Gestation Days (GDs)
1, 10, 15, and 20. Gavage doses were adjusted weekly based on body weight. Equivalent
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tungsten doses are calculated to be 3, 39, or 78 mg W/kg-day (tungsten accounts for 62.6% of
total molecular weight of Na2WC>4).
At parturition (PND 0), gestation length was recorded. On PND 1, the number of pups in
each litter was recorded and all pups were inspected for external malformations. On PND 4,
litters were culled to eight pups (four/sex when possible) and weighed. Individual pup weights
were measured starting on PND 5 and weekly thereafter until PND 70. Forty F0 males and
twenty F0 females per group were necropsied on the last day of treatment (PND 20), and
two pups/sex/litter were sacrificed on PND 20 and 70. In 20 male F0 and 10 female F0
rats/group and one pup/sex/litter per sacrifice, various organs (reported as "heart, spleen, kidney,
liver, lungs, brain, testes, ovaries, thymus, bone, gastrointestinal tract, etc.") were removed and
postfixed in 4% paraformaldehyde for histological evaluation. In the remaining 20 male F0 and
10 female F0 rats/group and one pup/sex/litter per sacrifice, sodium tungstate concentrations
were measured in the heart, spleen, kidney, lungs, liver, gastrointestinal tract, brain, femur,
blood, and thymus. Tungsten levels were also quantified in mammary secretions of all dams
once between PNDs 10 and 14 (see Table C-3 for toxicokinetic data).
Both pups (number not specified) and dams (20/group) were assessed for neurobehavior
during the postnatal period; however, results were only reported for rats in the 0, 3, and
78-mg W/kg-day groups (it is unclear whether or not the rats in the 39-mg W/kg-day group were
evaluated for neurobehavior). Pups (number not specified) were assessed for early, reflexive
behaviors with the righting reflex on PND 4 and separation distress (as measured by ultrasonic
vocalization frequency) on PND 7. Dams (20/group) were assessed for instinctual maternal
responses using maternal retrieval of pups on PND 2 (latency to retrieve three pups moved to the
opposite side of the home cage), open-field behavior on PND 27 (7 days posttreatment), acoustic
startle/prepulse inhibition on PND 28 (8 days posttreatment), and the Morris water maze on
PNDs 35-88 (15-18 days posttreatment).
No mating or fertility indices were provided in the study report; however, the study
authors stated that sodium tungstate had "no apparent effect on mating success." No
dose-related changes were observed in gestational weight gain or the number of pups per litter.
Mean group gestational length was significantly increased by 2% in high-dose dams; however,
this slight increase to 22.08 days from 21.55 days in controls is not considered to be biologically
significant. A reproductive NOAEL of 78 mg W/kg-day is identified for the lack of effects on
reproductive performance (mating success) in male and female S-D rats.
No dose-related effects were observed for incidence of external malformations or
histological lesions in PND 20 or 70 pups. Pup weight and postnatal growth were not altered
with gestational and lactational exposure to sodium tungstate. However, pups demonstrated a
dose-related increase in the number of ultrasonic distress vocalizations when separated from their
dams for 60 seconds on PND 7 (see Table B-7). This finding may suggest increased stress and
anxiety in pups exposed to tungsten during gestation and lactation. The number of vocalizations
was statistically significantly increased by 76% in the 78-mg W/kg-day group, compared with
controls. The 18% increase observed in the 3-mg W/kg-day group was not statistically
significant. Data for the 39-mg W/kg-day group were not reported; it is unclear whether or not
this group was evaluated for pup neurobehavior. Tungsten exposure did not alter righting reflex
in pups on PND 4 (quantitative data not reported by study authors). The highest dose,
78 mg W/kg-day, is a developmental NOAEL for offspring postnatal growth, external
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malformations in offspring at birth, histological lesions in tissues of offspring at PNDs 20 and
70, and righting reflex on PND 4. Although a statistically significant (76%) increase in the
number of distress vocalizations was observed when PND 7 pups were separated from their dams
in high-dose pups, compared with control pups, the biological significance of this effect is not
certain.
No dose-related changes in body weight or clinical signs of toxicity were reported in
male or female F0 rats. The report stated that histiocytic inflammation from minimal to mild
with cardiomyocyte degeneration and necrosis was observed in "several" F0 animals in the
78-mg W/kg-day group on PND 20. According to the method section, histopathological
examinations were conducted in 10 F0 animals/sex/group; however, a data table in the report
states that "two animals of five" in the high-dose group displayed minimal or mild myocarditis
with cardiomyocyte degeneration and necrosis. Therefore, due to reporting inconsistencies and
ambiguities, as well as the apparently small number of animals examined, it is unclear whether
or not cardiac lesions were exposure related. No other significant exposure-related lesions were
observed (incidence data were not provided by the study authors). In dams, no dose-related
effects were observed for maternal retrieval, acoustic startle/prepulse inhibition, or water maze
training and acquisition. Altered open-field behaviors were observed in low- and high-dose
dams on PND 27; however, findings did not demonstrate a consistent dose-related pattern
(see Table B-7). Compared with controls, low-dose females demonstrated a significant increase
in distance traveled and time spent ambulating, while time spent engaged in stereotypic
behaviors was significantly decreased. High-dose females spent significantly less time resting
than controls; however, no significant differences were observed in distance travelled or time
spent ambulating. Instead, significantly more time was spent engaged in stereotypic behaviors,
compared with controls. Findings from the 39-mg W/kg-day group may provide insight into the
seemingly contradictory results; however, it is unclear whether or not the dams from the
mid-dose group were evaluated in neurobehavioral assays. A NOAEL of 78 mg W/kg-day is
identified for systemic effects in F0 males and females exposed to sodium tungstate for 70 days
for lack of clinical signs of toxicity, body-weight effects, clear dose-related histopathological
changes, or clear dose-related changes in neurobehavioral tests of dams (maternal retrieval of
pups on PND 2, open-field behavior on PND 27 [7 days postexposure], acoustic startle/prepulse
inhibition on PND 28, and the Morris water maze 15-18 days postexposure).
Ballester et al. (2005)
Groups of male Wistar rats (15/group) were given drinking water containing 0 or
2 mg/mL of sodium tungstate for 12 weeks. Equivalent tungsten doses are calculated to be
1 mg W/mL (tungsten accounts for 62.6% of total molecular weight of Na2WC>4). Body weight
and blood sugar levels were measured regularly during the 12-week exposure period. Sexual
function was measured after 10 weeks of exposure. Exposed and control animals were placed in
a cage overnight with one untreated female animal, and removed the next morning. Nightly
pairings continued for up to 9 nights until the presence of a vaginal tag and/or spermatozoa were
observed. After assessment of mating success, all males were sacrificed. Serum was collected
and glucose, insulin, FSH, LH, and testosterone levels were measured. Testes were fixed for
histological and ultrastructural evaluation. Expression levels of testicular insulin receptors were
evaluated using Western blot analysis.
Body-weight gain was significantly decreased by 17% in exposed males, compared with
controls (see Table B-8). Before treatment began, the average body weight for control and
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treated animals was 200 g. The body-weight gain for the control group was 234.1 g and 195.2 g
for rats receiving 1 mg W/mL. Based on these data, the terminal body weight was 434.1 g for
the control group and 395.2 g for rats receiving 1 mg W/mL. Thus, terminal body weight was
decreased by 9% (not biologically significant) compared to controls.
No exposure-related changes were observed in serum glucose or insulin levels. No
exposure-related changes were observed in the mating index, serum hormone levels, or testicular
histology or ultrastructure. No changes in testicular insulin receptor expression were reported.
Using reference male Wistar rat body weight (0.217 kg) and water consumption (0.032 L/day)
values for the subchronic-duration studies (U.S. EPA. 1988). daily tungsten intakes were
estimated to be 147 mg W/kg-day. A stand-alone reproductive NOAEL of 147 mg W/kg-day is
identified for male rats. A systemic NOAEL of 147 mg W/kg-day is identified based on a lack
of statistically and/or biologically significant effects.
Ballester et al. (2007)
Groups of female Wistar rats (24/group) were given drinking water containing 0 or
2 mg/mL of sodium tungstate for 12 weeks. Equivalent tungsten doses are calculated to be
1 mg W/mL (tungsten accounts for 62.6% of total molecular weight of Na2WC>4). During the
exposure period, body weight, food and water intake, and glycemia were measured regularly.
Sexual function was measured after 10 weeks of exposure. Exposed and control animals were
placed in a cage overnight with one untreated male animal, and removed the next morning.
Nightly pairings continued for up to 9 nights until the presence of a vaginal plug and/or
spermatozoa were observed. Females that successfully mated were allowed to deliver, and the
fertility index (number of parturitions/number of matings) and the litter sizes were recorded.
After assessment of reproductive function, all females were sacrificed. Serum was collected and
glucose, insulin, ALT, follicle stimulating hormone (FSH), luteinizing hormone (LH), and
progesterone levels were measured. Ovaries were fixed for histological evaluation. Expression
levels of ovarian and uterine estrogen, progesterone, follicle-stimulating hormone, lutenizing
hormone, prolactin, insulin, and GLUT 3 hexose transporters were evaluated using Western blot
analysis and real-time polymerase chain reaction.
Body-weight gain was significantly decreased by 41% in exposed females, compared
with controls (see Table B-8). Before treatment began, the average body weight for control and
treated animals was 200 g. The body-weight gain for the control group was 77.2 g and 45.7 g for
rats receiving 1 mg W/mL. Based on these data, the terminal body weight was 277.2 g for the
control group and 245.7 g for rats receiving 1 mg W/mL. Thus, terminal body weight was
decreased by 11% (biologically significant) compared to controls.
No exposure-related changes were observed in food or water intake in females. No
exposure-related changes were observed in serum glucose, insulin, or ALT levels. No
exposure-related changes were observed in the mating or fertility indices, litter size, serum
hormone levels, or ovarian histology. Expression levels of ovarian and uterine estrogen,
progesterone, FSH, LH, prolactin, insulin, and GLUT 3 hexose transporters were not altered with
exposure. Using reference female Wistar rat body weight (0.156 kg) and water consumption
(0.025 L/day) values for the subchroni c-durati on studies (U.S. EPA. 1988). daily tungsten
intakes were estimated to be 160 mg W/kg-day. A reproductive NOAEL of 160 mg W/kg-day is
identified for female rats. A systemic LOAEL of 160 mg W/kg-day is identified for decreased
body weight.
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Tumor-Promotion Studies
Luo etal. (1983)
The effect of tungsten on NSEE-induced esophageal and forestomach carcinogenesis was
investigated in male S-D rats. Male weanling rats were given demineralized drinking water ad
libitum containing 0 or 100 ppm tungsten as sodium tungstate for 19 weeks (10/group) or
30 weeks (10/group). Another group of 10 rats were exposed to 200 ppm tungsten for 19 weeks.
Other groups (20-41/group) were exposed to NSEE alone via gavage twice weekly between
Weeks 4 and 8 (8 doses of NSEE) or between Weeks 4 and 12 (16 doses of NSEE)
(see Table B-10 for more details). Additional groups of rats (15-22/group) were exposed to
100 ppm tungsten for 19 weeks plus 16 doses of NSEE, 200 ppm tungsten for 19 weeks plus
8 doses of NSEE, or 100 ppm tungsten for 30 weeks plus 16 doses of NSEE (see Table B-10 for
more details). All rats were fed ad libitum a nutritionally adequate semipurified diet containing
0.064 ppm of tungsten and 0.026 ppm molybdenum. Body weights were monitored throughout
the exposure period. At sacrifice, the esophagus and forestomach were removed and fixed in
10% formalin for histopathological examinations. The changes of epithelial cells were divided
into four stages: Stage I, hyperplastic lesions (hyperkeratosis, simple hyperplasia, and papillary
hyperplasia); Stage II, precancerous lesions (marked endophytic growth of epithelium, marked
dysplasia, papilloma, and papillomatosis); Stage III, early carcinoma (malignant changes of basal
cells or papilloma, carcinoma in situ, and early infiltrative carcinoma); and Stage IV, advanced
carcinoma (carcinoma with extensive invasions).
Body weights, reported only for control, 100 ppm tungsten-treated, and NSEE-treated
groups up to 19 weeks, were comparable through 13 weeks. Reduced growth was observed in
NSEE-treated rats from 13-20 weeks, while the average body weight of 100 ppm
tungsten-treated rats was similar to controls during this period.
Using reference male S-D rat body weight (0.267 kg) and water consumption
(0.037 L/day) values for the chronic-duration studies (U.S. EPA. 19881 daily intake of tungsten
in the 100 and 200 ppm groups was estimated to be 13.9 and 27.8 mg W/kg-day, respectively.
Using these estimates, HEDs were calculated to be 3.33 and 6.67 mg W/kg-day, respectively,
using a DAF of 0.24 for rats based on body weight to 3/4 power scaling (U.S. EPA. 2011b).
No nonneoplastic or neoplastic histopathological lesions in the esophagus or forestomach
were observed in any of the tungsten-only exposed rats (13.9 or 27.8 mg W/kg-day for 19 weeks
or 11.9 mg W/kg-day for 30 weeks). In rats exposed to 16 doses of NSEE alone, increased
incidences of hyperplastic and precancerous lesions in the esophagus and forestomach were
found, as well as increased incidences of early or late carcinomas. Rats exposed to eight doses
of NSEE alone also had increased incidences of esophageal hyperplastic or precancerous lesions
(compared with nonexposed controls), but no esophageal carcinomas. Forestomach lesion
incidence data were not reported for this group; however, they were reportedly "similar" to
esophageal lesions. Incidences of rats with hyperplastic lesions, precancerous lesions, or
carcinomas in esophagus or forestomach were not significantly different between groups
exposed to 13.9 mg W/kg-day plus NSEE and comparable groups exposed to NSEE alone.
Incidences for esophageal or forestomach carcinomas were not significantly different between
groups exposed to 27.8 mg W/kg-day (for 19 weeks) plus eight doses of NSEE and groups
exposed to eight doses of NSEE alone. The results suggest that tungsten did not display a
tumor-promoting activity under the conditions of this experiment. However, the incidence of
rats with precancerous esophageal lesions in the 27.8 mg W/kg-day plus eight doses of NSEE
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group was significantly elevated, compared with incidence in the group exposed to eight doses of
NSEE alone. Neoplastic and nonneoplastic tumor incidences for all groups are reported in
Table B-10.
I-up et al. (1983) also reported that supplemental molybdenum (2 or 200 ppm in diet)
significantly inhibited NSEE-induced carcinogenesis, and that exposure to supplemental tungsten
alone (11.9 mg W/kg-day in diet for 30 weeks) produced markedly decreased liver
concentrations of molybdenum (98% decreased compared with control). Based on these
findings, Luo et al. (1983) hypothesized that exposure to tungsten at 23.8 mg W/kg-day
increased the incidence of precancerous lesions induced by eight doses of NSEE (from 4/31 with
NSEE alone to 22/22 with tungsten + NSEE; see Table B-10) by counteracting the cancer
inhibiting action of molybdenum.
Wei et al. (1985)
The effect of tungsten on NNMU-induced mammary carcinogenesis was investigated in
35-day-old virgin S-D rats. Three groups of female rats (Groups 1-3), one of which was used as
a control (Group 1), were given ad libitum a nutritionally adequate semipurified diet containing
0.026 ppm of molybdenum and 0.064 ppm toluene and deionized drinking water. In a 4th group,
150 ppm of sodium tungstate was added to the drinking water. After 15 days, all noncontrol
animals (Groups 2-4) received a single intravenous injection of 5 mg/100 g body weight NMU,
however, it is unclear from the study report the duration that the rats received tungsten in the
diet. One week after administration of carcinogen NMU, 10 ppm of molybdenum was added to
the drinking water in Group 3. Rats were palpated for mammary tumors twice weekly and
weighed weekly. Animals were sacrificed on Day 125 or 198 after NMU administration
(10-24/group/sacrifice), and all mammary tumors were removed for histological evaluation.
There were no significant differences in body weight among the groups. None of the control rats
developed mammary tumors. NMU alone produced mammary carcinomas, with 97.8% being
adenocarcinomas or papillary carcinomas and 2.2% being fibroadenomas. After 125 days, the
mammary cancer incidence in Group 4 (sodium tungstate + NMU; 192%) was statistically
significantly higher than that in Group 2 (NMU alone, 50.0%) or Group 3
(NMU + molybdenum; 45.5%). After 198 days, the mammary cancer incidence in Group 3
(NMU+ molybdenum; 1.5 and 50.0%) was statistically significantly lower than in Group 2
(NMU alone; 2.0 and 90.5%) or Group 4 (NMU+ sodium tungstate; 2.6 and 95.1%). Only
histologically confirmed carcinomas were included in the statistical evaluation (see Table B-l 1).
The first palpable mammary tumor was found in the tungsten-supplemented rats (both 125- and
198-day subgroups) only 56 days after the injection of NMU, whereas in both unsupplemented
and molybdenum-supplemented rats, the first palpable mammary tumors were observed 71 and
85 days after NMU treatment in the 125- and 198-day subgroups, respectively. Most carcinomas
were highly aggressive, but nonmetastatic. This study shows an inhibitory effect of
molybdenum on NMU-induced mammary carcinogenesis and a promoting effect of tungsten, an
antagonist of molybdenum, on tumor growth.
Using reference female S-D rat body weight (0.338 kg) and water consumption
(0.045 L/day) values for the chronic-duration studies (U.S. EPA. 1988). daily intake of tungsten
was estimated to be 20 mg W/kg-day. Using this estimate and a DAF of 0.24 for rats based on
body weight to 3/4 power scaling (U.S. EPA. 201 lb), the HED was calculated to be
4.8 mg W/kg-day.
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Inhalation Exposures
No studies have been identified.
OTHER DATA (SHORT-TERM TESTS, OTHER EXAMINATIONS)
Genotoxicity
Sodium tungstate has been tested in a number of short-term-duration genotoxicity tests
(see Table C-l in Appendix C for more details). The results were predominantly negative,
although positive findings were noted in a few assays. Genotoxicity studies for tungsten metal
were not identified. Sodium tungstate did not induce mutations in Salmonella typhimurium or
Escherichia coli [Covance Laboratories (2004a) as cited in Jackson et al. (2013)1. but was
weakly positive for inducing reverse mutations at trp5 and ilvl in Saccharomyces cerevisiae
(Singh. 1983). Sodium tungstate induced mutations in Plasmodium fischeri (Ulitzur and Barak.
1988) and induced SOS DNA repair in E. coli (Rossman et al. 1991; Rossman et al. 1984). In
mammalian cells, sodium tungstate did not induce mutations in mouse lymphoma cells
(L5 178Y TK±) [Covance Laboratories (2004b) as cited in Jackson et al. (2013)1; chromosomal
aberrations (CAs) in Chinese hamster ovary (CHO) cells [Covance Laboratories (2003) as cited
in Jackson et al. (2013)1. Syrian hamster embryo cells (Larramendv et al.. 1981). or human
purified lymphocytes (Larramendv et al.. 1981); or sister chromatid exchange (SCEs) in human
whole-blood cultures (Larramendv et al.. 1981). Sodium tungstate reportedly induced mutations
in Chinese hamster V79 lung cells (Zelikoff et al.. 1986). but only an abstract report of this study
is available, precluding independent evaluation. In in vivo animal tests, sodium tungstate did not
induce bone marrow micronuclei (MN) in mice [Covance Laboratories (2004c) as cited in
Jackson et al. (2013)1 and equivocal findings were reported for DNA damage (assayed by the
Comet assay and yH2AX immunoblotting) in bone marrow of orally exposed mice (Kelly et al..
2013).
Supporting Human Studies
Cross-sectional surveys examining possible associations between several medical
conditions and concentrations of tungsten and other metals in urine or blood collected from the
general U.S. population reported the following findings (the route of exposure in these studies is
unknown; see Table C-2 in Appendix C for more details):
• an association between urinary tungsten concentration and serum levels of thyroid
stimulating hormone (TSH), but not with levels of other thyroid hormones (Yorita
Christensen. 2012);
• an association between elevated urinary tungsten concentration and cardiovascular and
cerebrovascular disease (Agarwal et al.. 2011); and
• an association between elevated urinary tungsten concentration and asthma, but not with
other medical conditions, including coronary heart disease, heart attack, congestive heart
failure, stroke, or thyroid problems (Mendy et al.. 2012).
Epidemiology studies of hard alloy workers exposed to dusts containing mixtures of
tungsten and other metals reported increased risks for respiratory and neurologic effects, but the
results are confounded by exposure to mixtures of metals and have been attributed to cobalt
(ATSDR. 2005).
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Other Animal Toxicity Studies
A number of other animal toxicity studies were identified that had designs of limited
usefulness for PTV development, used exposure routes or durations not suitable for PTV
development, or were inadequately reported. Reported findings in these studies include
(see Table C-2 in Appendix C for more details):
• no carcinogenic response within 12 months of intratracheal instillation of tungsten into
guinea pigs (Schepers, 1971);
• pre- and postimplantation losses and delayed fetal ossification following 8-month
exposures of female rats to 0.005 mg/kg-day of unspecified tungsten compound for up to
8 months before and during pregnancy [Nadeenko and Lenchenko (1977), and Nadeenko
et al. (1977, 1978) as cited in AT SDR (2005)1;
• histological lesions in the gastrointestinal tract, liver, and kidney in rats exposed for a
"subacute" duration to doses of 10 or 100 mg/kg sodium tungstate (Nadeenko. 1966); and
• impaired conditioned behavior responses without brain lesions in rats exposed to 0.05
and 0.5 mg/kg-day sodium tungstate for 7 months and decreased blood cholinesterase
activity and unspecified brain lesions in rabbits exposed to 0.05 or 5 mg/kg for 8 months
(Nadeenko. 1966).
Reported acute lethality values for sodium tungstate include: oral LD50 values ranging
from 240 mg/kg for mice to 1,190 mg/kg for rats (Nadeenko. 1966) and 1,453 mg/kg for rats
[Huntingdon Life Sciences (1999a) as cited in Jackson et al. (2013)1; a rat 4-hour median lethal
concentration (LC50) value >5.01 mg/L [Huntingdon Life Sciences (1999a) as cited in Jackson et
al. (2013)1; and a rat dermal LD50 value >2,000 mg/kg [Huntingdon Life Sciences (1999c,d,e) as
cited in Jackson et al. (2013)1.
Other studies examining the respiratory tract in rats after intratracheal instillation of
single doses of suspended dusts of tungsten metal, tungsten carbide + carbide dusts, or tungsten
carbide + cobalt dusts have reported:
• pulmonary fibrosis in rats exposed to tungsten carbide + cobalt dusts, but not in rats
exposed to tungsten metal or tungsten carbide + carbon dusts (Delahant. 1955; Schepers.
1955); and
• pulmonary fibrosis in rats exposed to intratracheal instillation of tungsten metal
(Mezentseva, 1967).
Absorption, Distribution, Metabolism, and Elimination (ADME) Studies
Tungstate was widely distributed among tissues in the body of rats exposed to aqueous
solutions of sodium tungstate by gavage for 7 months at doses of 5 or 125 mg/kg-day starting
from 14 days before mating through PND 20 (Nlclnturf et al.. 2008). Tungstate was detected in
all examined tissues in exposed animals at the end of exposure (including the brain in adults and
offspring, the placenta, and dam milk secretions). By PND 70, tungstate concentrations in
tissues were below detection limits (Nlclnturf et al.. 2008). In mice exposed to sodium tungstate
in drinking water for 16 weeks, elemental tungsten accumulation in bone was shown to: (1) be
rapid (peaking after 1 week of exposure); (2) increase with increasing exposure level; and
(3) clear from bone less rapidly than it accumulated (Kelly et al.. 2013). An early study showing
no marked differences in tissue distribution in rats exposed to different forms of tungsten was
likely due to lack of proper analytical methods available at that time (Kinard and Aull. 1945).
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Mode-of-Action/Mechanism/Therapeutic Action Studies
In mechanistic studies, dietary exposure of rats to sodium tungstate altered ascorbic acid
metabolism in rats exposed for 28 days (Chatteriee et aL 1973) and depleted xanthine oxidase
activity in the lungs of rats exposed for 3 weeks (Rodell et aL 1987). A number of studies have
reported that orally administered sodium tungstate corrected hyperglycemia in insulin- and
noninsulin-dependent rats (Oliveira et aL, 2014; Ballester et aL 2007; Ball ester et aL 2005;
Fernandez-Alvarez et aL 2004; Munoz et aL 2001; l.e Lamer et al.. 2000; Rodrieuez-Gallardo
et al.. 2000; Barbera et al.. 1997).
DERIVATION OF PROVISIONAL VALUES
Data are inadequate for derivation of reference values for tungsten metal. Early studies
indicated that repeated oral exposure of tungsten metal (70 days at dietary concentrations up to
10%) is markedly less toxic (lethal) to rats than sodium tungstate or other compounds of tungsten
(tungsten trioxide and ammonium tungstate) (Kinard and Van de Erve, 1943, 1941). However,
these studies examined a limited number of endpoints and did not conduct histological
examination of tissues, and thus, do not provide an adequate basis for deriving a subchronic
p-RfD for tungsten metal. No other studies that examined pertinent endpoints in animals
repeatedly exposed orally to tungsten metal were located.
Tables 4 and 5 present a summary of noncancer reference and cancer values,
respectively, for sodium tungstate. IRIS data are indicated in the table, if available.
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TABLE 4. Summary of Noncancer Reference Values for
Soluble Tungsten Compounds (Various CASRNs)
Toxicity Type
(units)
Species/Sex
Critical Effect
p-Reference Value
POD
Method
POD
UFc
Principal Study
Subchronic
p-RfD
Rat/males
Glandular stomach goblet cell
metaplasia
8 x 1CT3 mg W/kg-d
BMD
BMDLio = 2.3 mg W/kg-d
300
USACHPPM
(2007a):
USACHPPM
(2007b)
Chronic p-RfD
Rat/males
Glandular stomach goblet cell
metaplasia
8 x l(T4 mg W/kg-d
BMD
BMDLio = 2.3 mg W/kg-d
3,000
USACHPPM
(2007a):
USACHPPM
(2007b)
Subchronic and
chronic p-RfC
Not derived due to inadequate data for soluble tungsten compounds.
Table 5. Summary of Cancer Values for Soluble Tungsten Compounds (Various CASRNs)
Toxicity Type
Species/Sex
Tumor Type
Cancer Value
Principal Study
p-OSF or p-IUR
Not derived due to inadequate data to assess the carcinogenicity of tungsten and soluble compounds.
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DERIVATION OF ORAL REFERENCE DOSES
Derivation of Subchronic Provisional RfD (Subchronic p-RfD)
The 90-day gavage study of S-D rats is selected as the principal study for deriving a
subchronic p-RfD for soluble tungsten compounds (USACHPPM. 2007a. b). The results of this
study were later published in a peer-reviewed paper by McCain et al. (2015). Increased
incidence of glandular stomach goblet cell metaplasia is selected as the critical effect.
Justification of the Critical Effect
Increased incidence of glandular stomach cell metaplasia is selected as the critical effect
for deriving the subchronic p-RfD for soluble tungsten compounds. Several other endpoints
associated with tungsten exposure were considered for identification of critical effect however
the biological significance and/or relevance of these effects are questionable. For example,
cortical tubule regeneration was observed in the kidneys of male and female rats following
90 days of gavage exposure (USACHPPM. 2007a. b), however this lesion is indicative of an
adaptive repair response rather than an actual pathological effect. A potential
immunosuppressive effect was noted in mice challenged with bacterial antigen following 28 days
of tungsten exposure (Osterburg et al.. 2014). The study authors reported decreased percentages
of 'activated' splenic CD71+ helper and cytotoxic T cells, however the total number of T cells in
the spleen did not differ from control mice bringing into question the functional relevance of the
observed effect. Therefore, glandular stomach goblet cell metaplasia in male rats (USACHPPM.
2007a. b) was considered as a potential critical effect in lieu of these aforementioned effects.
In addition to the qualitative considerations for identification of a critical effect, glandular
stomach goblet cell metaplasia represents the most sensitive LOAEL among all considered
effects; the LOAEL (78 mg W/kg-day; NOAEL of 46 mg W/kg-day) for this effect in the 90-day
gavage S-D rat study by (USACHPPM. 2007a. b) is lower than LOAELs for other effects
observed in other studies of animals orally exposed to sodium tungstate for subchronic durations
(see Table 3 A). These LOAELs are:
• 125 mg W/kg-day (and NOAEL of 78 mg W/kg-day) for decreased body weight in male
rats and increased incidence of male and female rats with mild to severe cortical tubule
regeneration in the kidneys (USACHPPM, 2007a. b);
• 250 mg W/kg-day (and NOAEL of 49 mg W/kg-day) for decreased body weight and
decreased bone marrow eellularity in male C57BL/6J mice exposed for 16 weeks (Kelly
et al.. 2013).
• 125 mg W/kg-day (and NOAEL of 78 mg W/kg-day) for decreased splenic response to
bacterial toxin injection (decreased percentage splenic CD71+ helper and cytotoxic
T cells 24 hours after injection with SEB) in C57BL/6J mice exposed for 28 days (adult
mice) or 90 days before mating, followed by gestation, birth, and weaning (F0 and
F1 mice) (Osterburg et al.. 2014); and
• 160 mg W/kg-day for decreased body weight in female rats (Ballester et al.. 2007;
Ball ester et al.. 2005).
The authors of the principal study interpreted the glandular stomach lesions to be a
"nonspecific response to some physiologic effect of gavage administration, as opposed to a
manifestation of systemic toxicity," but for this assessment, these lesions are considered to be
biologically significant, dose-related, and compound-related effects, as evidenced by their
absence in the control and lower-dose groups, which also received gavage treatment, and their
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dose-related pattern of occurrence. Supporting the biological significance of these lesions to
humans is the understanding that goblet cells are not part of the normal mammalian gastric
epithelium, and that metaplasia (replacement of normal cells with columnar absorptive cells and
goblet cells of intestinal morphology) is a histological change associated with repeated
inflammation of the gastric mucosa, which could occur due to chemical exposure (Liu and
Crawford, 2005). Furthermore, because it is unclear if the pathology of the glandular stomach
was examined in rats from the available drinking water studies for tungsten or in any other
toxicity study (e.g., dietary studies in rats or mice), it is not possible to determine whether the
observed lesions are due in part to bolus dosing (i.e., gavage) in the principal study
(USACHPPM. 2007a. b). Whereas glandular stomach goblet cell metaplasia could be
considered an irritant effect due to gavage treatment of sodium tungstate, the available data do
not support this conclusion. Irritation of the gastrointestinal tract is often coupled with decreased
food consumption and a subsequent reduction in body weight. Whereas body weight and food
consumption were statistically and/or biologically significantly reduced in males at
125 mg W/kg-day in the gavage study (USACHPPM. 2007a. b), the incidence of glandular
stomach goblet cell metaplasia was statistically significantly increased at 78 mg W/kg-day at
which no significant alterations in body weight and food consumption were observed
[see Table 6 and Table B-l in this document and Tables 3 and 4 from (McCain et al.. 2015)1.
Also, body weight and food consumption were not reduced in females at >75 mg W/kg-day,
where the incidence of glandular stomach goblet cell metaplasia was statistically significantly
increased in females. These data suggest that although a tungsten-induced irritant effect (i.e.,
reduced body weight and food consumption) may be occurring in male rats at 125 mg W/kg-day,
it is most likely unrelated to the development of glandular stomach goblet cell metaplasia at
75 mg W/kg-day (USACHPPM, 2007a. b). In the absence of more definitive information,
glandular stomach goblet cell metaplasia is considered a valid endpoint for human health risk
assessment.
Selection of stomach lesions in S-D rats as the critical effect has some uncertainty,
because the occurrence of these lesions was not mentioned in the available reports of a
reproductive study for a duration equivalent to subchronic study (70 days, including 14 days
premating, 14 days mating, 22 days gestation, and 20 days postparturition)in S-D rats exposed by
gavage to sodium tungstate in water (Mclnturf et al„ 2011; Mclnturf et al.. 2008). or in any other
study. However, the histological examinations of tissues in the F0 rats in this study were
inconsistently and ambiguously reported in the published reports. Only the 2011 report
discussed the histology data, noting in the methods section that "various organ tissues (heart,
spleen, kidney, liver, lungs, brain, testes ovaries, thymus, bone, gastrointestinal tract, etc.,)" from
10 F0 animals/sex/group were prepared for histological examination, but noting in a data table
[see Table 2, p. 136 of Mclnturf et al. (2011)1 that in the high-dose (75 mg W/kg-day) F0 group
"two animals of five displayed myocarditis with cardiomyocyte degeneration and necrosis." In
concluding statements, Mclnturf et al. (2011) noted that pathological examinations showed no
treatment-related histopathological lesions in any organs except the heart, but incidence data that
would allow independent review were not included. In the absence of more complete reporting
of incidence data for the histological findings, it is uncertain whether or not the F0 animals
exposed to 78 mg W/kg-day in this study showed glandular stomach lesions similar to those
reported in the 90-day toxicity study reported by USACHPPM (2007a) and USACHPPM
(2007b). Furthermore, the development of glandular stomach lesions in rats may require
treatment with sodium tungstate for at least 90 days as was done in the studies by USACHPPM
(2007a) and USACHPPM (2007c). In the reproductive study by Mclnturf et al. (2011) and
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Mclnturf et al. (2008). F0 rats were treated for a total of only 70 days, which may not be
sufficient time for the development of glandular stomach lesions. Kinard and Van de Erve
(1943) grossly examined the gastrointestinal tract of rats treated for 70 days with tungsten metal.
It is unclear if the glandular stomach was specifically examined in the study and the study
authors only ambiguously reported that "No extravasations of blood were observed in the
gastrointestinal track."
Support for deriving a subchronic p-RfD for soluble tungsten compounds based on
glandular stomach lesions in rats comes from the identification of 78 mg W/kg-day as a NOAEL
for reproductive effects in F0 S-D rats and systemic and neurobehavioral effects in F1 rat
offspring following a total of 70 days of gavage exposure before mating, during mating and
gestation, and during early postnatal periods (Mclnturf et al.. 2011; Mclnturf et al.. 2008). This
study did not include an exposure level that identified LOAELs for evaluated endpoints in F0- or
F1-generation rats. Similarly, a reproductive study of C57BL/6J mice exposed to doses as high
as 125 mg W/kg-day in drinking water for a total of 90 days before mating, followed by
exposure during gestation, birth, and weaning, found no statistically significant effects on F0
body weight or reproductive performance (the number of live births, litter size, and sex ratio of
offspring) (Osterbure et al.. 2014). These observations indicate that reproductive and
developmental effects will not occur at doses lower than the rat LOAEL for glandular stomach
lesions, 78 mg W/kg-day.
A number of other effects have been observed at dose levels below 78 mg W/kg-day in
studies of animals subchronically exposed to sodium tungstate, but these effects are of uncertain
biological significance and not suitable to serve as critical effects for the subchronic p-RfD.
• Mice exposed for 16 weeks to dose levels of 4, 49, or 250 mg W/kg-day had a
statistically significantly greater percentage of bone marrow B cells in late pro-/large
B cell developmental stages (C/C), compared with control values (Kelly et al.. 2013).
The change did not steadily increase in magnitude with increasing dose level and was not
apparent at earlier time points (Weeks 1, 4, 8, or 12). At 16 weeks, mean percentages of
bone marrow B cells in the C/C' fraction for the control, low-, medium-, and high-dose
groups were 0.06, 0.11, 0.11, and 0.13, respectively (see Table B-3).
• Statistically significant changes in an index of DNA damage (increased tail moment in
the Comet assay) in nonadherent bone marrow cells also was reported for mice in the
4- and 49-mg W/kg-day dose groups at most time points (Weeks 1, 4, 8, 12, and 16), but
statistically significant changes were not observed at most time points in the
250-mg W/kg-day group (Kelly et al.. 2013) (see Table B-4). The magnitude of the
significant increases in tail moment (compared with control values) ranged from
26-151%, but the data do not provide clear evidence for increased damage with
increasing dose level or duration of exposure (see Table B-4). Similar Comet assay
results were found with CD 19+ B cells isolated from bone marrow of mice in Weeks 1
and 4 (Kelly et al.. 2013) (see Table B-4).
• In mice allergically sensitized to 4-hydroxy-3-nitrophenylacetic acid active ester
(NP-O-Su) and exposed to sodium tungstate in drinking water for 28 days before
sensitization, the response to a challenge injection of NP-O-SU was decreased in mice
exposed to 12.5 or 125 mg W/kg-day (but not to 1.3 or 0.1 mg W/kg-day), compared with
sensitized mice without exposure to sodium tungstate (Osterbure et al.. 2014). Swelling
at the site of challenge injection was decreased by about 30% in groups exposed to 12.5
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or 125 mg W/kg-day, compared with swelling in sensitized mice without tungstate
exposure. The biological significance of the diminished response is uncertain, and could
be viewed as a positive action of tungstate (potentially therapeutic) in allergically
sensitized individuals.
Justification of the Principal Study
The 90-day gavage study of rats was selected as the principal study, because:
1. it was an adequately designed, conducted, and reported study of subchronic toxicity in
rodents, and
2. it identified a LOAEL for glandular stomach lesions in rats that was lower than LOAELs
for other effects (described in the previous section) identified in other adequately
designed and conducted studies of subchronic duration oral toxicity in rats (Nlclnturf et
al.. 2011; Mclnturt*et al.. 2008) and mice (Osterburg et aL 2014; Kelly et al.. 2013).
Approach for Deriving the Subchronic p-RfD
Data sets for the most sensitive endpoint from the principal study, glandular stomach
lesions (goblet cell metaplasia in male and female rats), were selected to derive potential PODs
via benchmark dose (BMD) modeling (see Table 6). For comparative purposes, other potential
sensitive data sets were selected for BMD modeling, including kidney, body weight, and immune
system endpoints (see Table 6). BMDs and benchmark dose lower confidence limits (BMDLs)
from the best fitting models for the selected dichotomous-variables data sets are presented in
Table 7. Also presented in Table 7 are HED BMDLs, converted from the animal BMDLs using
U.S. EPA (2011b) recommended body weight314 scaling factors (DAFs) for systemic effects.
The body weight374 scaling factor was not applied to the stomach lesion-based BMDLs, because
allometric scaling has not been extensively evaluated with portal-of-entry effects and models to
predict differences in deposited mass per glandular stomach surface area across species have not
been developed for soluble tungsten compounds (U.S. EPA. 2011b).
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Table 6. Data for Sensitive Endpoints in Rodents Exposed Orally to
Sodium Tungstate for Subchronic Durations
Endpoint
Dose (mg W/kg-d)
USACHPPM (2007a): USACHPPM f2007b)—Rat. eavaee. 90 d
Glandular stomach goblet cell
metaplasia
0
6
47
78
125
Male
0/10
1/10
4/10
8/9
8/10
Female
0/10
0/10
4/10
8/10
10/10
Mild to severe renal cortical tubule
regeneration
0
6
47
78
125
Male
0/10
0/10
0/10
1/9
10/10
Female
0/10
0/10
0/10
1/10
8/10
Male terminal BW (g)
Mean ± SD («)
553 ±31 (10)
571 ±49
(10)
548 ±38 (10)
535 ±67
(10)
486 ± 52
(9)
Kellv et al. (2013)—Mouse, drinking water. 16 wk
Total bone marrow cell count (106)
0
4
49
250
Mean ± SD («)
118.89 ±9.51
(5)
121.02 ± 10.
44 (5)
122.12 ± 12.20
(5)
95.55 ± 11.97
(5)
Osterbury et al. (2014)—Mouse, drinking water. 28 d. 19 wk (F0), or 32 wk (Fl)
% CD71+ helper T cells (mean± SD)
0
1.3
39
78
125
28 d (n = 12/group)
4.85 ± 4.26
NR
3.61 ±2.29
3.54 ± 1.39
2.76 ± 1.77
19 wk (F0, 6/group)
6.21 ±0.96
6.41 ± 1.52
4.71 ±4.51
4.61 ±3.18
2.28 ± 1.00
32 wk (Fl, 6/group)
7.20 ± 1.86
4.13 ± 1.06
3.14 ±0.93
4.96 ± 1.20
2.85 ± 1.30
% CD71+ cytotoxic T cells (mean± SD)
0
1.3
39
78
125
28 d (12/group)
12.87 ±7.10
NR
8.60 ±6.79
5.75 ±2.88
4.44 ±4.92
19 wk (F0, 6/group)
7.98 ± 1.20
6.81 ±2.82
5.75 ±4.14
2.31 ±5.14
1.58 ±0.56
32 wk (Fl, 6/group)
6.33 ± 1.20
5.37 ±0.71
3.29 ±0.61
5.77 ± 1.20
2.52 ±0.61
NR = not reported; SD = standard deviation.
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Table 7. BMD and BMDL Values from Best Fitting Models for Sensitive,
Biologically Relevant Endpoints in Rodents Exposed Orally to
Sodium Tungstate for Subchronic Durations"
Endpoint
Best Fitting Model
BMD
(mg W/kg-d)
BMDL
(mg W/kg-d)
HEDb BMDL
(mg W/kg-d)
USACHPPM (2007a); USACHPPM (2007b)—Data sets: rat. 90 d. savase
Glandular stomach
goblet cell
metaplasia
Male—LogLogistic
Female—Multistage 2-degree
Os
cK
r--
II II
o o
BMDLio — 2.3
BMDLio = 8.1
NDr;
portal-of-entry
effect
Glandular stomach
subacute
inflammation
Male—not amenable to modeling
Female—LogLogistic
NDr
BMDio = 43
NDr
BMDLio = 26
NDr;
portal-of-entry
effect
Renal cortical
tubule
regeneration
Male—Weibull
Female—Gamma
BMDio = 77.5
BMDio = 75.9
BMDLio = 61.6
BMDLio = 58.6
BMDLio = 14.8
BMDLio = 14.1
Terminal body
weight, male rat
Male—Polynomial 2-degree,
constant variance
BMDio = 108.2
BMDLio = 91.9
BMDLio = 22.1
Kellv et al. (2013)—Data set: mouse. 16 wk. drinking water
Total bone
marrow cell count
Male—Polynomial 3-degree,
constant variance
BMDisd — 183.7
BMDLisd — 77.8
BMDLisd — 10.9
Osterburg et al. (2014)—Data sets: mouse, 28 d, 19 wk (TO), or 32 wk (Fl)
% CD71+
cytotoxic T cells
28 d—Hill, nonconstant variance
19 wk (F0)—not amenable to
modeling
32 wk (Fl)—not amenable to
modeling
BMDisd — 49.1
NDr
NDr
BMDLisd — 39.9
NDr
NDr
BMDLisd — 5.6
NDr
NDr
% CD71+ helper
T cells
28 d—Exponential (2),
nonconstant variance
19 wk (F0)—not amenable to
modeling
32 wk (Fl)—not amenable to
modeling
BMDisd = 333.6
NDr
NDr
BMDLisd = 118.4
NDr
NDr
BMDLisd = 16.6
NDr
NDr
aModeling results are described in more detail in Appendix D.
' HEDs were calculated using species-specific D AFs based on the animal:human BW"4 ratio recommended by U.S.
EPA (2011b): mouse:human ratio = 0.14; rat:human ratio = 0.24.
NDr = not determined.
The BMDLio value of 2.3 mg W/kg-day for glandular stomach goblet cell metaplasia in
male rats was selected as the POD. This selection is consistent with the selection of stomach
lesions as the critical effect based on a comparison of animal LOAELs, and with the BMDL for
male stomach lesions being lower than HED BMDLs for other sensitive endpoints (see Table 7).
For systemic effects, because soluble compounds of tungsten (i.e., sodium tungstate dihydrate
and sodium tungstate) are expected to ionize in the blood (Nlclnturf et aL 2011; Mclnturf et aL
2008). the toxicities of the chemicals would be due to tungsten (and not the particular salt), thus
the toxicities of the soluble compounds of tungsten would be expected to be similar on a molar
basis. Therefore, although the subchronic p-RfD presented below is derived based on doses for
41
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tungsten, the value is applicable for sodium tungstate dihydrate and sodium tungstate (i.e.,
soluble tungsten compounds).
The subchronic p-RfD for soluble tungsten compounds, based on the BMDLio of
2.3 mg W/kg-day for goblet cell metaplasia in the glandular stomach of male rats, is derived as
follows:
Subchronic p-RfD = BMDLio UFc
= 2.3 mg W/kg-day ^ 300
= 8 x 10"3 mg W/kg-day
Table 8 summarizes the UFs for the subchronic p-RfD for soluble tungsten compounds.
Table 8. Uncertainty Factors for the Subchronic p-RfD for
Soluble Tungsten Compounds
UF
Value
Justification
UFa
10
A UFa of 10 is applied to account for uncertainty in extrapolating from animals to humans, in the
absence of information to assess species differences in toxicokinetic and toxicodynamic
characteristics of soluble tungsten compounds, and in the absence of a rationale to support use of
HED for a POD based on portal-of-entry effects.
UFh
10
A UFh of 10 is applied to account for human variability in susceptibility, in the absence of
information to assess toxicokinetic and toxicodynamic variability of soluble tungsten compounds
in humans.
UFd
3
A UFd of 3 is applied because the database contains several adequately designed
subchronic-duration animal toxicity studies of sodium tungstate that collectively identify
glandular stomach lesions as a critical effect occurring at dose levels that do not cause
reproductive, developmental, or immune svstem effects COsterburg et al.. 2014; Kellv et al..
2013; Mclnturf et al.. 2011; Mclnturf et al.. 2008; USACHPPM. 2007a. b). However, there is
indication of neurotoxicity effects CNadeenko. 1966). which was not comprehensively
investigated.
UFl
1
A UFl of 1 is applied because the POD is a BMDL.
UFS
1
A UFS of 1 is applied because the POD comes from a subchronic-duration study of rats.
UFC
300
Composite Uncertainty Factor = UFA x UFH x UFD x UFL x UFS.
The confidence in the subchronic p-RfD for soluble tungsten compounds is medium as
explained in Table 9 below.
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Table 9. Confidence Descriptors for the Subchronic p-RfD for Soluble Tungsten
Compounds
Confidence
Categories
Designation
Discussion
Confidence in
study
H
Confidence in the orincroal studv (TJSACHPPM. 2007a. M is high because the
study design and conduct was in accordance with EPA Health Effects Testing
Guidelines for a study of 90-d oral toxicity in rodents. In addition, the results
were published in a peer-reviewed paper (McCain et al.. 2015).
Confidence in
database
M
Confidence in the subchronic-duration oral exposure database is medium,
because the database contains one adequately designed subchronic-duration
animal toxicity study that identifies glandular stomach lesions as a critical effect
and other subchronic-duration studies indicating that reproductive,
developmental, or immune system effects occur at doses higher than those
causing stomach lesions (Osterburg et al.. 2014; Kellv et al.. 2013; Mclnturf et
al.. 2011; Mclnturf et al.. 2008; USACHPPM. 2007a. M. However, there is
limited information regarding neurotoxicity (Nadeenko. 1966). which was not
comprehensively investigated.
Confidence in
subchronic p-RfDa
M
The overall confidence in the subchronic p-RfD is medium.
aThe overall confidence cannot be greater than the lowest entry in the table.
H = high; M = medium.
Derivation of Chronic Provisional RfD (Chronic p-RfD)
There are four studies in which rodents were chronically treated with sodium tungstate
via drinking water (Osterburg et aL 2014; I .up et aL 1983; Schroeder and Mitchener. 1975a. b).
In a reproductive study, Osterburg et al. (2014) treated F0 mice for 19 weeks, and in a chronic
study, I.uo et al. (1983) treated male rats for 19 or 30 weeks. However, these studies did not
perform comprehensive evaluations. Osterburg et al. (2014) only focused on hematological and
immunological endpoints and I.uo et al. (1983) on esophageal and forestomach carcinogenesis.
Schroeder and Mitchener (1975b) and Schroeder and Mitchener (1975a) treated rats and mice
with sodium tungstate for lifetime durations, but these studies only tested one dose in drinking
water. Thus, in the absence of comprehensive studies of toxicity endpoints that tested multiple
doses in humans or animals chronically exposed to sodium tungstate by the oral route, a chronic
p-RfD for soluble tungsten compounds is derived from the same POD for the subchronic p-RfD.
Justification for selecting the critical effect and principal study is described in the previous
section of this document.
The chronic p-RfD for soluble tungsten compounds is derived as follows:
Chronic p-RfD = subchronic BMDLio ^ UFc
= 2.3 mg W/kg-day ^ 3,000
= 8 x 10"4 mg W/kg-day
Table 10 summarizes the UFs for the chronic p-RfD for soluble tungsten compounds.
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Table 10. Uncertainty Factors for the Chronic p-RfD for Soluble Tungsten Compounds
UF
Value
Justification
UFa
10
A UFa of 10 is applied to account for uncertainty in extrapolating from animals to humans, in the
absence of information to assess species differences in toxicokinetic and toxicodynamic
characteristics of soluble tungsten compounds, and in the absence of a rationale to support use of
HED for a POD based on portal-of-entry effects.
UFh
10
A UFh of 10 is applied to account for human variability in susceptibility, in the absence of
information to assess toxicokinetic and toxicodynamic variability of soluble tungsten compounds
in humans.
UFd
3
A UFd of 1 is applied because the database contains several adequately designed
subchronic-duration animal toxicity studies of sodium tungstate that collectively identity glandular
stomach lesions as a critical effect occurring at dose levels that do not cause reproductive,
developmental or immune svstem effects (Osterbure et al.. 2014; Kellv et al.. 2013; Mclnturf et
al.. 2011; Mclnturf et al.. 2008; USACHPPM. 2007a. b). Uncertainty associated with the absence
of chronic-duration toxicity data is accounted for in the UFS. However, there is indication of
neurotoxicity effects CNadeenko. 1966). which was not comprehensivelv investigated.
UFl
1
A UFl of 1 is applied because the POD is a BMDL.
UFS
10
A UFS of 10 is applied to account for uncertainty in deriving the chronic p-RfD from the
subchronic p-RfD.
UFC
3,000
Composite Uncertainty Factor = UFA x UFH x UFD x UFL x UFS.
The confidence in the chronic p-RfD for soluble tungsten compounds is low as explained
in Table 11 below.
Table 11. Confidence Descriptors for the Chronic p-RfD for Soluble Tungsten
Compounds
Confidence
Categories
Designation
Discussion
Confidence in
study
H
Confidence in the principal study is high because the study design and conduct
was in accordance with EPA Health Effects Testing Guidelines for a study of
90-d oral toxicity in rodents. In addition, the results were published in a
peer-reviewed paper McCain et al. (2015).
Confidence in
database
L
Confidence in the database is low, because it contains no comprehensive studies
of toxic endpoints in humans or animals orally exposed to tungstate for chronic
durations, and there is limited information rc sardine neurotoxicity I Nadecnko.
1966).
Confidence in
chronic p-RfDa
L
The overall confidence in the chronic p-RfD is low.
aThe overall confidence cannot be greater than the lowest entry in the table.
H = high; L = low.
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DERIVATION OF INHALATION REFERENCE CONCENTRATIONS
Human and animal data are inadequate to derive subchronic or chronic p-RfCs for
soluble tungsten compounds.
Epidemiology studies of hard alloy workers exposed to dusts containing mixtures of
tungsten and other metals reported increased risks for respiratory and neurologic effects, but the
results are confounded by exposure to mixtures of metals and have been attributed to cobalt
(ATSDR. 20051
CANCER WEIGHT-OF-EVIDENCE DESCRIPTOR
Table 12 identifies the cancer weight-of-evidence (WOE) descriptor for soluble tungsten
compounds.
Table 12. Cancer WOE Descriptor for Soluble Tungsten Compounds
(Various CASRNs)
Possible WOE Descriptor
Designation
Route of Entry (oral,
inhalation, or both)
Comments
"Carcinogenic to Humans "
NS
NA
No human data are available to support
this designation.
"Likely to Be Carcinogenic to
Humans "
NS
NA
No human data or adequate animal cancer
bioassays are available to support this
designation.
"Suggestive Evidence of
Carcinogenic Potential"
NS
NA
No human data or adequate animal cancer
bioassays are available to support this
designation.
"Inadequate Information to
Assess Carcinogenic
Potential"
Selected
NA
No human data or adequate animal
cancer bioassays are available.
"Not Likely to Be
Carcinogenic to Humans "
NS
NA
No human data or adequate animal cancer
bioassays are available to support this
designation.
NA = not applicable; NS = not selected.
No studies were located examining possible associations between exposure to tungsten,
sodium tungstate, or sodium tungstate dihydrate and increased risk of cancer in humans. Studies
in animals are inadequate to assess the carcinogenicity of tungsten or sodium tungstate.
Schroeder and Mitchener (1975a) and Schroeder and Mitchener (1975b) reported that no
evidence for carcinogenic responses was found in rats or mice exposed to sodium tungstate in
drinking water for life at a concentration of 5 ppm tungsten, but the studies are limited by
inclusion of only one exposure level and absence of an exposure level close to a maximum
tolerated dose. No evidence of sodium tungstate's tumor promotion capability was found in one
rat assay of tumors initiated by NSEE (Luo et ai, 1983). In another rat assay, tumors appeared
slightly earlier in rats exposed to NMU followed by sodium tungstate in drinking water,
compared with rats exposed to NMU alone (Wei et al.. 1985). Results from a number of
short-term-duration genotoxicity tests with sodium tungstate were predominately negative
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Soluble Tungsten Compounds
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(see Table C-l in Appendix C for more details). Genotoxicity studies for tungsten metal were
not identified.
DERIVATION OF PROVISIONAL CANCER POTENCY VALUES
Derivation of Provisional Oral Slope Factor (p-OSF)
Not derived due to inadequate data.
Derivation of Provisional Inhalation Unit Risk (p-IUR)
Not derived due to inadequate data.
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APPENDIX A. SCREENING PROVISIONAL VALUES
No screening values are presented.
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APPENDIX B. DATA TABLES
Table B-l. Histological Lesions Observed in Male and Female Sprague-Dawley Rats after
Exposure to Sodium Tungstate Dihydrate in Water for 90 Days by Gavage"
Parameter
Exposure Group, mg Sodium Tungstate/kg-d (mg W/kg-d)b
Male
0
10(6)
75 (47)
125 (78)
200 (125)
Kidney
Mild to severe cortical
tubule regeneration
0/10
0/10
0/10
1/9
10/10*
Glandular stomach
Subacute inflammation
0/10
2/10
1/10
5/9*
4/10
Goblet cell metaplasia
0/10
1/10
4/10
8/9*
8/10*
Epididymides
Luminal cellular debris
0/10
1/10
0/10
0/10
3/10
Female
0
10(6)
75 (47)
125 (78)
200 (125)
Kidney
Mild to severe cortical
tubule regeneration
0/10
0/10
0/10
1/10
8/10*
Glandular stomach
Subacute inflammation
0/10
0/10
1/10
8/10*
9/10*
Goblet cell metaplasia
0/10
0/10
4/10
8/10*
10/10*
aUSACHPPM (2007a): USACHPPM (2007b).
Equivalent tungsten doses were calculated based on molecular weights (tungsten accounts for 62.6% of total
molecular weight of Na2WC>4).
°Minimal renal cortical tubule regeneration was observed in all dose groups (incidence not reported). Group mean
and individual animal severity scores were not reported.
* Statistically significantly different from controls at p< 0.05, as calculated for this review (Fisher's Exact test,
two-tailed).
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Table B-2. Splenic Immune Responses in Male and Female C57BL6 Mice after Exposure
to Sodium Tungstate Dihydrate in Drinking Water for 28 Days or 1 Generation11
Parameterb
Exposure Group, mg Sodium Tungstate/kg-d (mg W/kg-d)c
28-d study
0
2 (1.3)
62.5 (39)
125 (78)
200 (125)
% CD71+ helper T cells'1
4.85 ± 1.23
NR
3.61 ±0.66
(-26%)
3.54 ±0.40
(-27%)
2.76 ±0.51*
(-43%)
% CD71+ cytotoxic T cells'1
12.87 ±2.05
NR
8.60 ± 1.96
(-33%)
5.75 ±0.83
(-55%)
4.44 ± 1.42*
(-66%)
IFN-y (pg/mL)e
6.98 ±0.77
NR
5.04 ± 1.10
(-28%)
3.16 ±0.62
(-55%)
2.44 ±0.73*
(-65%)
1-Generation study—F0 mice (12 wk premating + 7 wk mating, gestation, lactation)
0
2 (1.3)
62.5 (39)
125 (78)
200 (125)
% CD71+ helper T cells'1
6.21 ±0.39
6.41 ±0.62
(+3%)
4.71 ± 1.84
(-24%)
4.61 ± 1.30
(-26%)
2.28 ±0.41*
(-63%)
% CD71+ cytotoxic T cells'1
7.98 ±0.49
6.81 ± 1.15
(-15%)
5.75 ± 1.69
(-28%)
2.31 ± 2.10
(-71%)
1.58 ±0.23*
(-80%)
IFN-y (pg/mL)f
7.11 ± 1.7
10.15 ±2.66
(+43%)
3.89 ± 1.51
(-45%)
2.52 ± 1.22
(-65%)
3.74 ±2.9*
(-47%)
1-Generation study—F1 mice (exposure via F0 dam + direct exposure for 90 d postweaning)
0
2 (1.3)
62.5 (39)
125 (78)
200 (125)
% CD71+ helper T cells'1
7.20 ± 0.76
4.13 ±0.43
(-43%)
3.14 ±0.38
(-56%)
4.96 ±0.49
(-31%)
2.85 ±0.53*
(-60%)
% CD71+ cytotoxic T cells'1
6.33 ±0.49
5.37 ±0.29
(-15%)
3.29 ±0.25
(-48%)
5.77 ±0.45
(-9%)
2.52 ±0.25*
(-60%)
IFN-y (pg/mL)g
4.54 ± 1.22
4.46 ± 1.30
(-2%)
11.23 ±5.18
(+147%)
2.16 ± 0.14
(-52%)
3.10 ± 1.15
(-32%)
aOsterburg et al. (2014).
bValues are expressed as mean ± standard error of the mean (SEM) (% change compared with control); % change
control = [(treatment mean - control mean) + control mean] x 100; n= 12 mice/group for the 28-day study and
n = 6 mice/group for F0 and F1 mice in the 1-generation study. Distribution of sexes within each group was not
reported.
Equivalent tungsten doses were calculated based on molecular weights (tungsten accounts for 62.6% of total
molecular weight of Na2WC>4).
dOnly control and high-dose values were reported in the text; data for other dose groups were extracted from
Figures 4 and 5 in the primary report using Grablt! software.
eOnly control value was reported in the text; data for other dose groups were extracted from Figure 6 in the
primary report using Grablt! software.
fOnly control and high-dose values were reported in the text; data for other dose groups were extracted from
Figure 6 in the primary report using Grablt! software.
8Data for all dose groups were extracted from Figure 6 in the primary report using Grablt! software.
* Statistically significantly different from controls at p< 0.05, as reported by the study authors.
NR = not reported.
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Table B-3. Total Bone Marrow Cellularity, Specific Cell Fraction Percentages and
Counts, and Clonogenicity of Bone Marrow Precursors in Male C57BL/6 J Mice after
Exposure to Sodium Tungstate Dihydrate for 16 Weeks in Drinking Water"
Exposure Group, mg/L W in Water (average daily dose, mg W/kg-d)c
Parameterb
0
15(4)
200 (49)
1,000 (250)
Number of animals
5
5
5
5
Total bone marrow cell counts at
16 wk (106)
118.89 ±9.51
121.02 ± 10.44
(+2%)
122.12 + 12.20
(+3%)
95.55+ 11.97*
(-20%)
% of cells per
1 wk
fraction
(determined in
2 million bone
marrow cells per
time point)d
C/C' Fraction6
0.5 ±0.09
0.45 ±0.13
(-10%)
0.59 + 0.15
(+18%)
0.43 + 0.08
(-14%)
F Fractionf
6.08 ±0.84
7.4 ± 1.41
(+22%)
7.4+1.83
(+22%)
12.06 + 2.11*
(+98%)
4 wk
C/C' Fraction
0.25 ±0.06
0.26 ±0.06
(+4%)
0.36 + 0.12
(+44%)
0.36 + 0.08
(+44%)
F Fraction
4.57 ±0.63
5.83 ± 1.50
(+28%)
6.63 + 1.40*
(+45%)
4.9 + 0.79
(+7%)
8 wk
C/C' Fraction
0.08 ±0.03
0.12 ±0.05
(+50%)
0.11 + 0.04
(+38%)
0.12 + 0.05
(+50%)
F Fraction
5.24 ± 1.16
7.32±1.64
(+40%)
8.6+1.95*
(+64%)
8.62+ 1.96*
(+65%)
12 wk
C/C' Fraction
0.09 ±0.02
0.07 + 0.01
(-22%)
0.05 + 0.02
(-44%)
0.06 + 0.02
(-33%)
F Fraction
7.35 ± 1.20
8.86+1.18
(+21%)
7.05+1.15
(-4%)
9.01+ 1.58
(+23%)
16 wk
C/C' Fraction
0.06 ±0.02
0.11 + 0.01*
(+83%)
0.11 + 0.02*
(+83%)
0.13 + 0.02*
(+117%)
F Fraction
9.19 ± 1.62
9.04 + 0.43
(-2%)
9.64+1.48
(+5%)
9.57+ 1.31
(+4%)
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Table B-3. Total Bone Marrow Cellularity, Specific Cell Fraction Percentages and
Counts, and Clonogenicity of Bone Marrow Precursors in Male C57BL/6 J Mice after
Exposure to Sodium Tungstate Dihydrate for 16 Weeks in Drinking Water"
Parameterb
Exposure Group, mg/L W in Water (average daily dose, mg W/kg-d)c
0
15(4)
200 (49)
1,000 (250)
C/C' Fraction cell number normalized
to total bone marrow count at 16 wk
(106)g
0.08 ±0.02
0.15 ±0.03*
(+88%)
0.14 ±0.04*
(+75%)
0.12 ±0.03
(+50%)
Clonogenicity of precursors at 16 wk
(number of colonies)11
94.9 ±23.2
146.1 ±33.5
(+54%)
154.5 ±32.5
(+63%)
202.0 ±32.5*
(+113%)
"Kellv et al. (2013).
bValues are expressed as mean ± SD (% change compared with control); % change control = [(treatment
mean - control mean) + control mean] x 100.
0 Average daily doses (mg/kg-day) were calculated for this review using reference male mouse body weight
(0.0316 kg) and water consumption (0.00782 L/day) values for subchronic-duration studies (U.S. EPA. 1988).
dNo statistically significant differences were observed in the A (pre-pro B cells), B (early pro-B cells), D (small
pre-B cells), or E (immature B cells) fractions at any time point.
eC/C' fraction contains late pro-B cells and large pre-B cells.
fF fraction contains mature B cells.
gNo statistically significant differences were observed in C/C' fraction at earlier time points or the A, B, D, or E
fractions at any time point.
hColony number means and standard deviation values were extracted from Figure 6 in the primary report using
Grablt! software.
* Statistically significantly different from controls at p< 0.05, as reported by the study authors (ANOVA followed
by Newman-Keuls post hoc test).
51
Soluble Tungsten Compounds
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09-29-2015
Table B-4. DNA Damage in Male C57BL/6J Mice after Exposure to Sodium Tungstate
Dihydrate for 16 Weeks in Drinking Water"
Exposure Group, mg/L W in Water (average daily dose, mg W/kg-d)c
0
15(4)
200 (49)
1,000 (250)
Number of animals
5
5
6
5
Cell type
Exposure duration
Tail moment (Comet assay)b
Nonadherent bone
marrow cells
1 wk
62.6 ±5.8
78.6 ±4.9*
(+26%)
78.0 + 5.5*
(+26%)
53.0 + 4.9
(-15%)
4 wk
21.1 ± 1.9
36.4 ±2.6*
(+73%)
42.6 + 2.3*
(+102%)
35.1+ 1.9*
(+66%)
8 wk
10.7 ± 1.0
20.8 ± 1.6*
(+94%)
24.0+1.3*
(+124%)
10.4+1.0
(-3%)
12 wk
18.8 ±2.3
47.1±2.6*
(+151%)
25.3+ 1.6
(+35%)
31.2+ 1.3*
(+66%)
16 wk
17.2 ± 1.0
30.9 + 2.3*
(+80%)
26.6+ 1.6
(+55%)
22.7+1.0
(+32%)
CD19+B cells
(isolated from
bone marrow)d
1 wk
62.6 ±0.9
78.2 + 0.9*
(+25%)
62.5 + 0.5
(0%)
44.0+1.4
(-30%)
4 wk
66.2 ±0.9
84.2+ 1.4*
(+27%)
114.5+ 1.4*
(+73%)
59.5 + 0.9
(-10%)
aKellv et al. (2013).
bValues are expressed as mean ± SD (% change compared with control); % change control = [(treatment
mean - control mean) + control mean] x 100. Means and standard deviation values were extracted from Figure 7
in the primary report using Grablt! software.
0 Average daily doses (mg/kg-day) were calculated for this review using reference male mouse body weight
(0.0316 kg) and water consumption (0.00782 L/day) values for subchronic-duration studies (U.S. EPA. 19881.
isolated B cells were not evaluated for DNA damage at Weeks 8, 12, or 16.
* Statistically significantly increased from controls at p< 0.05, as reported by the study authors (ANOVA followed
by Newman-Keuls post hoc test).
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Table B-5. Mean Body Weight and Survival of Male and Female Rats after Exposure to
Sodium Tungstate for up to 70 Days in Diet3
Parameterb
Exposure Group, % W Equivalent in Diet (average daily dose, mg W/kg-d)c
Male
0
0.1% (91)
0.5% (455)
2.0% (1,820)
Number of animals
5-6
5-6
6
5
Terminal body weight (g)
285
260 (-9%)
NR
NA
Body-weight-gain (g)d
189
166 (-12%)
-26 (-114%)
NA
Survival6
100% (NR)
100% (NR)
50% (3/6)
0% (0/5)
Female
0
0.1% (102)
0.5% (510)
2.0% (2,040)
Number of animals
5-6
5-6
6
5
Terminal body weight (g)
188
168 (-11%)
NR
NA
Body-weight-gain (g)d
105
88 (-16%)
-33 (-131%)
NA
Survival6
100% (NR)
100% (NR)
33% (2/6)
0% (0/5)
aKinard and Van de Erve (1941).
bBody weight and weight gain expressed as mean (% change compared with control); % change
control = [(treatment mean - control mean) control mean] x 100
0 Average daily doses were calculated for this review using reference rat body weight (0.235 kg for males and
0.173 kg for females) and food consumption (0.021 kg/kg bw-day for males and 0.017 kg/kg bw-day for females)
values for the subchronic duration studies (U.S. EPA. 1988).
dBody-weight gain (Days 0-70) data were extracted from Chart 1 of the primary report using Grablt! software.
"Survival expressed as % survival (number surviving/total number). All deaths in the 2.0% occurred within the
first week. All deaths in the 0.5% group between Day 17 and 29 of exposure.
NR = not reported by study authors; NA = not applicable; all high-concentration rats died within 1 week.
Note: Statistics were not reported by study authors; data reporting is not adequate for statistical analysis.
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Table B-6. Mean Body-Weight Gain of Male and Female Rats after Exposure to
Powdered Tungsten Metal for 70 Days in Diet3
Parameterb
Exposure Group, % Powdered Tungsten Metal in Diet
(average daily dose, mg W/kg-d)c
Male
0
2.0% (1,325)
5% (3,450)
10% (6,550)
Number of animals
5
5
5
5
Body-weight gain (g)
183
172 (-6%)
207 (13%)
198 (8%)
Female
0
2.0% (1,450)
5% (4,000)
10% (7,325)
Number of animals
5
5
5
5
Body-weight gain (g)
110
114(4%)
107 (-3%)
93 (-15%)
aKinard and Van de Erve (1943).
bBody-weight gain expressed as mean (% change compared with control); % change control = [(treatment
mean - control mean) control mean] x 100.
0 Average daily doses (mg W/kg-day) were calculated for this review using tungsten consumption and body-weight
data reported by the study authors.
Note: Statistics were not reported by study authors; data reporting is not adequate for statistical analysis.
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Table B-7. Neurobehavior of Sprague-Dawley Rat Dams and Pups after Exposure to
Sodium Tungstate Dihydrate for 70 Days in Drinking Water
(14 Days Premating, 14 Days Mating, 22 Days Gestation, and through PND 20)a
Parameter13
Exposure Group, mg Sodium Tungstate/kg-d (mg W/kg-d)c
Dams
0
5(3)
62.5 (39)
125 (78)
Number of animals
20
20
20
20
Open-field behavior (7 d post exposure)
Distance travelled (cm)
5,521 ±689
8,640 ±421* (+56%)
NR
5,532 ± 293 (0%)
Resting time (s)
933 ±24
926 ± 27 (-1%)
NR
829 ±29* (-11%)
Ambulatory time (s)
131 ± 30
306 ± 13* (+134%)
NR
82 ± 4 (-37%)
Time in stereotypic movements (s)
735 ±26
568 ± 20* (-23%)
NR
889 ± 26* (+21%)
Pups
0
5(3)
62.5 (39)
125 (78)
Number of animals/litters
NR
NR
NR
NR
Separation distress at PND 7
Number of distress vocalizations
during 60-s removal of dam
19.5 ±3.2
23.1 ±3.8 (+18%)
NR
34.4 ±4.1* (+76%)
"Mclnturf et al. (2011): Mclnturf et al. (2008).
bValues expressed as mean ± SD (% change compared with control); % change control = [(treatment
mean - control mean) + control mean] x 100.
Equivalent tungsten doses were calculated based on molecular weights (tungsten accounts for 62.6% of total
molecular weight of Na2WC>4).
* Statistically significantly different from controls at p< 0.05, as calculated by study authors (ANOVA followed
by Tukey's post hoc comparisons).
NR = not reported by study authors; it is unclear whether or not neurobehavior was assessed in the mid-dose
group.
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Table B-8. Mean Body-Weight Gain of Male and Female Rats after Exposure to
Sodium Tungstate for 12 Weeks in Drinking Water3
Parameterb
Exposure Group, mg/L W in Water (average daily dose, mg W/kg-d)c
Male
0
1,248 (147)
Number of animals
15
15
Body-weight gain (g)
234.1 ±7.5
195.2 ± 8.6* (-17%)
Female
0
1,248 (160)
Number of animals
24
24
Body-weight gain (g)
77.2 ±3.0
45.7 ±3.5* (-41%)
aBallester et at (2007): Ballester et al. (2005).
bBody-weight gain expressed as mean ± SEM (% change compared with control); % change control = [(treatment
mean - control mean) control mean] x 100.
Equivalent tungsten doses (mg/L) were calculated based on molecular weights (tungsten accounts for 62.6% of
total molecular weight of Na2WC>4). Average daily dose were calculated for this review using reference Wistar
rat body weight and water consumption values for the subchronic duration studies (U.S. EPA. 1988).
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Table B-9. Mean Body Weight, Longevity, and Tumor Incidence of
Male and Female Long-Evans Rats and Swiss Mice after Lifetime Exposure to
Sodium Tungstate in Drinking Water"
Parameter
Exposure Group, ppm Tungsten Equivalent in Drinking
Water (average daily dose, mg W/kg-d)
[HED, mg W/kg-d]b
Male rats
0
5 (0.6) [0.1]
Initial number of animals
52
37
Body weight (g)°
Day 180
409.6 ±5.17
433.4 ±7.1* (+6%)
Day 360
484.5 ±6.3
529.1 ± 8.2* (+9%)
Day 540
501.3 ± 11.8
539.3 ±9.1* (+8%)
Longevity0"1
1,126 ± 18.2
983 ±7.3* (-13%)
Number of rats with tumors at necropsy6
4/26
4/25
Number of rats with malignant tumors at necropsy6
2/26
2/25
Female rats
0
5 (0.7) [0.2]
Initial number of animals
52
35
Body weight (g)°
Day 180
250.3 ±4.8
265.5 ± 3.9 (+6%)
Day 360
277.9 ±5.52
297 ± 6.3* (+7%)
Day 540
290.8 ±5.52
315.2 ±6.5 (+8%)
Longevity0"1
1,139 ±29.6
1,063 ± 22.8 (-7%)
Number of rats with tumors at necropsy6
17/24
13/20
Number of rats with malignant tumors at necropsy6
8/24
5/20
Male mice
0
5 (1) [0.1]
Initial Number of animals
54
54
Body weight (g)° Day 540
46.8 ± 1.33
43.5 ± 1.80 (-7%)
Longevity0"1
939 ±44.25
797 ± 7.5 (-15%)
Number of mice with tumors at necropsy
NR
NR
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Table B-9. Mean Body Weight, Longevity, and Tumor Incidence of
Male and Female Long-Evans Rats and Swiss Mice after Lifetime Exposure to
Sodium Tungstate in Drinking Water"
Parameter
Exposure Group, ppm Tungsten Equivalent in Drinking
Water (average daily dose, mg W/kg-d)
[HED, mg W/kg-d]b
Female mice
0
5 (1) [0.1]
Initial number of animals
54
54
Body weight (g)° Day 540
42.6 ± 1.32
40.0 ± 1.25 (-6%)
Longevity0"1
922 ± 28.44
945 ± 22.99 (+2%)
Number of mice with tumors at necropsy
NR
NR
aSchroeder and Mitchener (1975a): Schroeder and Mitchener (1975b).
bEstimated daily tungsten intakes were calculated using reference Long Evans rat body weight (0.472 kg for males
and 0.344 kg for females) and water consumption (0.057 L/day for males and 0.046 L/day for females) values for
the chronic duration studies (U.S. EPA. 1988) and reference mouse body weight (0.0317 kg for males and
0.0288 kg for females) and water consumption (0.0078 L/day for males and 0.0073 L/day for females) values for
the chronic duration studies (U.S. EPA. 1988). HEDs were calculated using species-specific DAFs based on the
animal:human BW' 1 ratio recommended by U.S. EPA (2011b): mouse:human ratio = 0.14; rat:human
ratio = 0.24.
°Values are expressed as mean ± SEM (% change compared with control); % change control = [(treatment
mean - control mean) control mean] x 100.
dLongevity was defined as the mean life span of the last five surviving animals per group.
eRats that died during a pneumonia epidemic (Month 20) were not included in necropsy data.
* Statistically significantly different from controls at p< 0.05, as calculated by study authors (Student's t test).
NR = not reported by study authors.
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Table B-10. Esophageal and Forestomach Lesions in Male Sprague-Dawley Rats after
Exposure to Sodium Tungstate for 19-30 Weeks in Drinking Water, with or without
iV-Nitrososarcosine Ethyl Ether (NSEE)a
Parameter
Exposure Group, ppm Tungsten (average daily dose, mg W/kg-d)
[HED, mg W/kg-d]b
19 wk
0
0 +
8 doses
NSEEC
0 +
16 doses
NSEEd
100 (13.9)
[3.33]
100 (13.9)
[3.33] +
16 doses
NSEEd
200
(27.8)
[6.67]
200 (27.8)
[6.67] +
8 doses
NSEEC
Esophagus
Hyperplasic lesions6
0/10
26/31
20/20
0/10
15/15
0/10
22/22
Precancerous lesionsf
0/10
4/31
20/20
0/10
15/15
0/10
22/221
Early carcinoma8
0/10
0/41
12/20
0/10
9/15
0/10
1/22
Late carcinoma11
0/10
0/41
4/20
0/10
3/15
0/10
0/22
Forestomach
Hyperplasic lesions
0/10
NR
20/20
0/10
15/15
NR
NR
Precancerous lesions
0/10
NR
20/20
0/10
15/15
NR
NR
Early carcinoma
0/10
NR
11/20
0/10
9/15
NR
NR
Late carcinoma
0/10
NR
3/20
0/10
3/15
NR
NR
30 wk
0
0 + 16 doses NSEEd
100 (11.9) [2.86]
100 (11.9) [2.86] +
16 doses NSEEd
Esophagus
Hyperplasic lesions
0/10
21/21
0/10
14/14
Precancerous lesions
0/10
21/21
0/10
14/14
Early carcinoma
0/10
6/21
0/10
2/14
Late carcinoma
0/10
13/21
0/10
10/14
Forestomach
Hyperplasic lesions
0/10
21/21
0/10
14/14
Precancerous lesions
0/10
21/21
0/10
14/14
Early carcinoma
0/10
4/21
0/10
3/14
Late carcinoma
0/10
17/21
0/10
11/14
aLuo et al. (1983).
bAverage daily doses (mg/kg-day) were calculated for this review using reference male weanling S-D rat body
weight and water consumption values for the subchronic and chronic duration studies CU.S. EPA. 19881. HEDs
were calculated using a D AF of 0.24 for rats based on the animal:human BW"4 ratio recommended by U.S. EPA
(2011b).
°1 mL/kg NSEE given by gavage twice weekly between Week 4 and 8.
dl mL/kg NSEE given by gavage twice weekly between Week 4 and 12.
"Hyperplastic lesions defined by study authors as hyperkeratosis, simple hyperplasia, and papillary hyperplasia.
Precancerous lesions defined by study authors as marked endophytic growth of epithelium, marked dysplasia,
papilloma, and papillomatosis.
gEarly carcinoma defined by study authors as malignant changes of basal cells or papilloma, carcinoma in situ, and
early infiltrative carcinoma.
hAdvanced carcinoma defined by study authors as carcinoma with extensive invasions.
'Statistically significantly greater than rats treated with 8 doses of NSEE only at p< 0.05, as calculated for this
review (Fisher's Exact test).
NR = not reported by study authors.
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09-29-2015
Table B-ll. Mammary Carcinomas in Female Sprague-Dawley Rats after Exposure to
Tungsten for 125 or 198 Days in Drinking Water, with NMUa
Parameter
Exposure Group, ppm Tungsten (average daily dose, mg W/kg-d)
[HED, mg W/kg-d]b
0
0 +
NMUC
0 + NMUC +
10 ppm
Molybdenum
0 + NMUC +
150 ppm W
(20 mg W/kg-d)
[4.8 mg W/kg-d]
125 d
Number of animals
10
22
22
24
% animals with mammary carcinomas
0
50
45.5
79.2d
# of carcinomas/carcinoma-bearing rat
0
2
1.3
1.7
Day of 1st palpable mass
NA
71
71
56
198 d
Number of animals
10
21
20
23
% animals with mammary carcinomas
0
90.5
50.0°
95.7
# of carcinomas/carcinoma-bearing rat
0
2
1.5
2.6
Day of 1st palpable mass
NA
85
85
56
aWei et al. (1985)
bAverage daily doses (mg/kg-day) were calculated for this review using reference female S-D rat body weight
(0.338 kg) and water consumption (0.045 L/day) values for the chronic duration studies (U.S. EPA. 19881. HED
was calculated using a D AF of 0.24 for rats based on the animal:human BW1/4 ratio recommended by U.S. EPA
(2011b).
°5 mg/kg NMU was administered intravenously on Day 15.
Statistically significantly different than rats treated with NMU-only at p< 0.05, as calculated by the study authors.
NA = not applicable.
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09-29-2015
APPENDIX C. SUMMARIES OF SUPPORTING DATA
Table C-l. Summary of Soluble Tungsten Compounds Genotoxicity
Endpoint
Test System
Dose/
Concentration3
Resultsb
Comments
References
Without
Activation
With
Activation
Genotoxicity studies in prokaryotic organisms
Mutation
S. typhimurium,
E. coli
NR
ND
OECD 471 guidelines followed using sodium
tungstate as test material.
Covance Laboratories
(2004a) as cited in
Jackson ct al. (2013)
Reverse mutation
S. cerevisiae
0.1 M sodium
tungstate
(+)
ND
Study authors reported weak positive results for
conversion at trp 5 and reversion at ilv 1.
Singh (1983)
Mutation (bacterial
bioluminescence
test)
P. fischeri
25 mmol/L
sodium tungstate
+
ND
Ulitzur and Barak
(1988)
SOS repair
induction
E. coli WP2S (X)
0.005 M sodium
tungstate
+
ND
1 prophage induction was increased 5 times over
control.
Rossinan et al. (1991);
Rossinan et al. (1984)
Genotoxicity studies in nonmammalian eukaryotic organisms
No data
Genotoxicity studies in mammalian cells—in vitro
Mutation
Mouse lymphoma
cells (L5178YTK±)
NR
ND
OECD 476 guidelines followed using sodium
tungstate as test material.
Covance Laboratories
(2004b) as cited in
Jackson et al. (2013)
Mutation
Chinese hamster lung
cells (V79)
NR
+
ND
>three-fold increase above background.
Zelikoff et al. (1986)
(abstract only)
CAs
CHO
NR
ND
OECD 473 guidelines followed using sodium
tungstate as test material.
Covance Laboratories
(2003) as cited in
Jackson etal. (2013)
CAs
Syrian hamster
embryo cells
0.03 M sodium
tungstate
dihydrate
ND
Larramendv et al.
(1981)
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Table C-l. Summary of Soluble Tungsten Compounds Genotoxicity
Endpoint
Test System
Dose/
Concentration3
Resultsb
Comments
References
Without
Activation
With
Activation
CAs
Human purified
lymphocytes
0.03 M sodium
tungstate
dihydrate
ND
I arntmendv et al.
(1981)
SCE
Human whole blood
cultures
0.03 M sodium
tungstate
dihydrate
ND
I arntmendv et al.
(1981)
Genotoxicity studies in mammals—in vivo
Mouse bone
marrow MN test
Mouse
NR
OECD 474 guidelines followed using sodium
tungstate as test material.
Covance Laboratories
(2004c) as cited in
Jackson et al. (2013)
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Table C-l. Summary of Soluble Tungsten Compounds Genotoxicity
Endpoint
Test System
Dose/
Concentration3
Resultsb
Comments
References
Without With
Activation Activation
DNA damage
(Comet assay;
yH2AX
immunoblot)
4-wk-old mouse
(C57BL/6J,
5 males/group);
sodium tungstate
dihydrate in drinking
water for 1, 4, 8, 12,
or 16 wk
0, 4, 49,
250 mg W/kg-d
as sodium
tungstate
dihydrate
±
In nonadherent bone
marrow cells, DNA
damage assessed by the
Comet assay was
increased compared with
control, but findings
across time points and
dosages were not
consistent with a
monotonic response with
increasing dose and
duration.
In CD 19+B cells, DNA
damage was significantly
increased in the
4-mg W/kg-d group at
1 and 4 wk and the
49-mg W/kg-d group at
4 wk, but significantly
decreased at 1 and 4 wk
in the 250-mg/kg-d
group.
Significant increases in DNA damage in nonadherent
bone marrow cells were observed at 1, 4, 12, and
16 wk in the 4-mg W/kg-d group; at 1, 4, and 8 wk in
the 49-mg W/kg-d group; and 4 and 12 wk in the
250-mg W/kg-d group. The amount of DNA damage
did not increase with increasing dose; across time
points, the magnitude of damage was higher in cells
from 4-mg W/kg-d mice than 250-mg/kg-d mice.
DNA damage in CD 19+ B cells was not assessed at
later time-points (8 or 12 wk).
Immunoblot staining for yH2AX (another assay for
DNA damage) in CD 19+ B cells from exposed mice
after 1 wk of exposure was not significantly elevated,
compared with control values.
Kellvetal. (2013)
Genotoxicity studies in subcellular systems
ND
aLowest effective dose for positive results, highest dose tested for negative results.
b+ = positive; (+) = weak positive; - = negative; ± = equivocal.
ND = no data; NR = not reported.
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Table C-2. Other Studies
Test
Materials and Methods
Results
Conclusions
References
Supporting evidence—cancer effects in humans
ND
Supporting evidence—noncancer effects in humans
Cross-sectional survey
of U.S. population
examining possible
associations between
serum thyroid levels
and levels of metals in
blood and urine
Multiple linear regression
analysis of 2007-2008
NHANES data for urinary levels
of tungsten (and lead, cadmium,
mercury, barium, cobalt,
antimony, thallium, and
uranium) and serum levels of
T3, FT3, T4, FT4, and TSH.
Analysis also included serum
levels of cadmium, lead, and
mercury.
Models were adjusted for age,
sex, race-ethnicity, total serum
lipids, serum cotinine,
pregnancy and menopausal
status, and use of medications
thought to affect thyroid
function.
Significant adjusted betas (SE) for
change in natural log (ln)-transformed
serum thyroid hormone per unit of
tungsten in urine (|ig/L urine):
Model with urinary tungsten only:
In T3: -0.20 (p = 0.010)
In T4: -0.027 (p = 0.008).
Model with all metals measured in
urine and blood:
In TSH: +0.042 (p = 0.016).
Analysis included data for 1,587 adults
with no history of thyroid disease or use
of thyroid medications.
An association between increasing urinary
tungsten levels and increasing TSH levels
was indicated. Levels of other thyroid
hormones were not associated with urinary
tungsten levels when models included all
metals measured in urine and blood.
Route of exposure of subjects is unknown.
Yorita Christensen
(2012)
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Table C-2. Other Studies
Test
Materials and Methods
Results
Conclusions
References
Cross-sectional survey
of U.S. population
examining possible
associations between
incidence for CCVD
and levels of metals in
urine
Logistic regression analysis of
1999-2006 NHANES data for
urinary levels of tungsten (and
antimony, arsenic, barium,
beryllium, cadmium, cesium,
cobalt, lead, molybdenum,
platinum, thallium, and
uranium) and incidence of
CCVD as determined from
questionnaire asking about
history of stroke, angina, heart
attack, coronary heart disease,
and congestive heart failure.
Adjusted OR (95% CI) for CCVD by
log-transformed urinary levels of
tungsten (ng/mg creatinine):
OR = 1.78 (CI: 1.28-2.48).
(« = 573, CCVD; n = 4,462
non-CCVD)
Model was adjusted for age, sex, race,
education, hypertension, diabetes,
hyper-cholesterolemia, chronic kidney
disease, body mass index, C-reactive
protein, smoking status, and serum
cotinine.
An association between elevated urinary
concentrations of tungsten and
cardiovascular and cerebrovascular disease
was indicated.
Associations were also indicated with
elevated urinary concentrations of
antimony, cobalt, and cadmium, but not
arsenic, barium, beryllium, cesium, lead,
molybdenum, platinum, thallium, or
uranium.
Route of exposure of subjects is unknown.
Aearwal et al. (2011)
Cross-sectional survey
of U.S. population
examining possible
associations between
various medical
conditions and levels
of metals in urine
Logistic regression analysis of
2007-2008 NHANES data for
urinary levels of tungsten (and
antimony, barium, beryllium,
cadmium, cesium, cobalt, lead,
molybdenum, thallium,
platinum, and uranium) and the
following medical conditions
(determined by yes or no
answers to questions): asthma,
overweight, blood transfusions,
vision problems, arthritis, gout,
congestive heart failure,
coronary heart disease, angina,
heart attack, stroke, emphysema,
thyroid problem, chronic
bronchitis, liver condition, and
cancer.
Adjusted OR (95% CI) for asthma by
log-transformed urinary levels of
tungsten (|ig/g creatinine):
OR = 1.72 (CI: 1.15-2.59)
Study population included 922 male
and 935 female subjects.
Model was adjusted for age, sex,
race/ethnicity, education, family
income status, alcohol consumption,
smoking status, serum cotinine, and
other metals.
No significant associations were found
between urinary tungsten levels and
other medical conditions assessed.
An association between elevated urinary
levels of tungsten and asthma was
indicated, but not for any of the other
medical conditions assessed, including
coronary heart disease, heart attack,
congestive heart failure, stroke, or thyroid
problems.
Route of exposure of subjects is unknown.
Mendv et al. (2012)
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Table C-2. Other Studies
Test
Materials and Methods
Results
Conclusions
References
Supporting evidence—cancer in animals
Carcinogenicity other
than regular
oral/inhalation
exposure
Guinea pigs (unspecified sex
and number) were injected
intratracheally with either a
suspension or a solution of
tungsten. Animals were
examined after 12 mo for
pulmonary lesions.
Tungsten-induced pulmonary lesions
and proliferation of epithelial cells were
reported; however, no tumors were
induced.
No evidence for carcinogenicity using this
protocol (study design details were
limited).
Scheners (1971)
Supporting evidence—noncancer effects in animals
Reproductive/
developmental studies
12-15 female rats were orally
administered 0 or 0.005 mg/kg-d
of an unspecified tungsten
compound for up to 8 mo before
and during pregnancy.
Pre- and postimplantation losses and
delayed fetal ossification
Due to inadequate reporting of study design
and results, findings are difficult to
interpret. As such, reliable
NOAEL/LOAEL determinations cannot be
made.
Nadeenko and
Lenchenko (1977);
Nadeenko et al. (1977,
1978) as cited in
ATSDR (2005)
Neurotoxicity
Albino rats were orally exposed
to 0, 0.005, 0.05, or 0.5 mg/kg-d
of sodium tungstate for 7 mo.
Strain, sex, and animal number
were not reported.
Neurobehavioral testing
(conditioned reflexes to a light
and bell) was conducted at an
unspecified time point during
the study. Details on the
neurobehavioral protocol and
endpoints examined are limited.
Brains were examined
microscopically at 8 mo.
Impaired conditioned responses were
reported in rats from the 0.05- and
0.5-mg/kg-d groups. No brain lesions
were reported.
Due to inadequate reporting of study design
and results, findings are difficult to
interpret. As such, reliable
NOAEL/LOAEL determinations cannot be
made.
Nadeenko (1966)
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Table C-2. Other Studies
Test
Materials and Methods
Results
Conclusions
References
Neurotoxicity
Albino rabbits (7/group) were
exposed to 0, 0.005, 0.05, 0.5, or
5 mg/kg-d of sodium tungstate
for 8 mo. Strain and sex and
animal number were not
reported. Blood cholinesterase
activity was measured monthly
and brains were examined
microscopically at 8 mo.
Blood cholinesterase activity in rabbits
was significantly decreased in the
5-mg/kg-d group from 4 to 8 mo and in
the 0.5-mg/kg-d group at 8 mo.
Unspecified lesions were observed in
the cerebral cortex of rabbits in the
0.5 and 5-mg/kg-d groups. No
neurotoxic effects were reported for
lower dose groups.
Due to inadequate reporting of study design
and results, findings are difficult to
interpret. As such, reliable
NOAEL/LOAEL determinations cannot be
made.
Nadeenko (1966)
Short-term-duration
studies
Acute oral and inhalation
toxicities of sodium tungstate
were determined in rats (OECD
401 and 403 guideline studies,
respectively).
Oral LD5o = 1,453 mg/kg
Inhalation LC50 (4-hr) >5.01 mg/L
Huntingdon Life
Sciences (1998, 1999a)
as cited in Jackson et al.
(2013)
Short-term-duration
studies
Acute lethality of tungsten
trioxide, sodium tungstate, and
sodium phosphotungstate was
assessed in rats and mice. Acute
lethality of sodium tungstate
was also assessed in rabbits and
guinea pigs.
LD50 values for sodium tungstate:
Rat—1,190 mg/kg
Mouse—240 mg/kg
Rabbit—875 mg/kg
Guinea pig—1,152 mg/kg
LD50 values for tungsten trioxide:
Rat—NR
Mouse—840 mg/kg
LD50 values for sodium
phosphotungstate:
Rat—1,600 mg/kg
Mouse—700 mg/kg
Clinical signs of toxicity in rats and
mice following acute exposure to all
compounds included hunching,
decreased muscle tone, and paresis in
hind limbs.
Acute toxicity of sodium tungstate in
different species showed the following
ranking:
rat < guinea pig < rabbit < mouse.
Acute toxicity of the 3 tungsten compounds
ranked tungsten trioxide < sodium
phosphotungstate < sodium tungstate in
correlation with their relative solubilities.
Nadeenko (1966)
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Table C-2. Other Studies
Test
Materials and Methods
Results
Conclusions
References
Short-term-duration
studies
Subacute toxicity was assessed
in albino rats and rabbits (sex,
species, animal number, and
duration were not reported).
Animals were orally exposed to
10, 25, 50, or 100 mg sodium
tungstate/kg-d. General
condition, behavior, weight,
hematology, and blood
cholinesterase activity were
measured. Internal organs were
examined microscopically in the
10- and 100-mg/kg-d groups
only. It is unclear if a control
group was used.
All doses of sodium tungstate retarded
growth of rats and increased blood
cholinesterase activity. The following
histopathological changes were
observed in the gastrointestinal tract,
liver, and kidneys of animals in the
10- and 100-mg/kg-d groups: increase
in the vascular permeability with
hemorrhages, appearance of
degenerative-dystrophic changes, and a
moderate proliferative-cellular reaction.
Due to inadequate reporting of study design
and results, findings are difficult to
interpret. As such, reliable
NOAEL/LOAEL determinations cannot be
made.
Nadeenko (1966)
Short-term-duration
studies other than
oral/inhalation
Acute dermal toxicity of sodium
tungstate was determined in rats.
Dermal and eye irritation were
determined in rabbits and skin
sensitization was determined in
guinea pigs (OECD 402, 404,
405, and 406 guideline studies,
respectively).
Dermal LD50 > 2,000 mg/kg
Skin irritation and sensitization studies
were negative, but sodium tungstate
was classified as slightly irritating to
eyes.
Huntingdon Life
Sciences (1999b, c, d, e)
as cited in Jackson et al.
(2013)
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Table C-2. Other Studies
Test
Materials and Methods
Results
Conclusions
References
Short-term-duration
studies other than
oral/inhalation
A 10% suspension of dusts of
unknown particle size of
tungsten metal dust, tungsten
carbide and carbon (94:6 ratio),
or tungsten carbide and cobalt
(91:9 ratio) in isotonic saline
was injected intratracheally into
groups of six guinea pigs (age,
sex, and strain not reported). A
total of 150 mg of each dust was
injected in three equal doses at
weekly intervals. Animals were
sacrificed at 1, 4, 8, and 12 mo
and lungs were examined
microscopically.
(n= 1-3 animals/time point per
group).
No compound-related deaths were
reported. Large, circumscribed
pigmented lesions were observed
during gross examination of the lungs
of all animals exposed to tungsten
carbide and carbon. Beneath the
visceral pleura, widely distributed,
small, discrete foci of pigmentation
were occasionally observed. These
lesions were only observed at 1 and
4 mo following exposure to tungsten
metal dust.
Exposure to tungsten carbide and cobalt
resulted in several histopathological
changes at 1 mo (proliferation of
interstitial cells with thickening of
alveolar walls around massed tungsten
particles, inflamed mucosa of bronci
and bronchioles, and partial or complete
closure of some bronchioles). After
1 yr, residue lesions were observed
(persistent, focal, interstitial, cellular
infiltration in relation to retained
particles). Various degrees of
peribronchial, peribronchiolar, and
perivascular fibrocellular reactions
were observed. Slight atrophic
vesicular emphysema was also present.
The results suggest that inhalation of
tungsten carbide particles together with
cobalt may lead to pulmonary fibrosis and
other histopathological lung changes;
however, fibrosis was not observed in
animals exposed only to tungsten metal
dust or tungsten carbide and carbon dust.
Dclahant (1955):
Schcocrs (1955)
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Table C-2. Other Studies
Test
Materials and Methods
Results
Conclusions
References
Short-term-duration
studies other than
oral/inhalation
White rats were administered
single doses of metallic tungsten
via intratracheal injections of
50 mg of material suspended in
0.5 mL of physiologic saline.
Age, weight, sex, strain, and
animal numbers were not
reported. Rats were sacrificed
at 4, 6, or 8 mo after injection
and lungs were examined
microscopically. Other organs
were evaluated macroscopically.
Lungs showed infiltration by
macrophages, chiefly around
pulmonary blood vessels, and
thickening of the walls between the
alveoli at 4 mo. At 6 mo, large
numbers of round cells were seen
surrounding the tungsten particles
around the bronchi. Collagen fibers
had overgrown these foci by 8 mo. At
8 mo, the endothelium was swollen and
the walls of the vessels were thickened.
Macroscopic changes were not
observed in other organs at 4, 6, or
8 mo.
The results suggest that inhalation exposure
to tungsten may result in pulmonary
fibrosis.
Mezentseva (1967)
ADME
ADME
Male C57BL/6J mice (5/group)
were given water ad libitum
containing 0, 15, 200, or
1,000 mg/L as sodium tungstate
dihydrate for 16 wk (0, 4, 49, or
250 mg W/kg-d). Elemental
tungsten concentration in the
tibia bones was quantified at 1,
4, 8, 12, and 16 wk, using
inductively coupled plasma
mass spectrometry. An
additional group was given
15 mg/L (4 mg W/kg-d) for
4 wk, and tungsten bone
deposition was measured
following 4- and 8-wk recovery
periods.
Tungsten levels were significantly
elevated in tibiae by Wk 1 in all
exposure groups. Tungsten
concentration was elevated in a
dose-dependent manner, increased by
approximately 5-, 80-, and 450-fold in
the 4-, 49-, and 250-mg/kg-d groups
(data presented graphically). Tungsten
accumulation rates were significantly
lower during subsequent weeks
(p < 0.001), and levels at subsequent
time-points were similar to levels at
Wk 1. Following the 4- and 8-wk
recovery periods, tungsten levels in
bone were still significantly elevated
compared with controls; however,
levels were -50% less than in mice
exposed for 8 or 12 wk.
Tungsten accumulation in bone increased
with increasing dose level. Accumulation
in bone was rapid, peaking after 1 wk of
exposure. Tungsten was not cleared from
bone as rapidly as it accumulated.
Kellvetal. (2013)
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Table C-2. Other Studies
Test
Materials and Methods
Results
Conclusions
References
ADME
Male and female
Sprague-Dawley rats (40/group)
were administered 0, 5, or
125 mg/kg-d sodium tungstate
as sodium tungstate dihydrate
(0, 3, or 78 mg W/kg-d) in
deionized water via gavage
7 d/wk for 70 d (including 14 d
premating, 14 d mating, 22 d
gestation, and through PND 20).
Ten F0 rats/sex were sacrificed
on the last day of treatment
(PND 20) and 1 pup/sex/litter
was sacrificed on PND 20 and
70 for determination of
tungstate concentrations in
various tissues and organs by
inductively coupled plasma
mass spectrometry. Tungstate
levels were also quantified in
the mammary secretions of all
dams once between PND 10 and
14.
At PND 20, significantly increased
tungstate levels were observed in the
kidney, lungs, liver, gastrointestinal
tract, brain, and femur of pups and the
spleen, kidney, lung, liver, brain, femur,
blood, thymus, and testes of adults.
Tungstate levels were below detection
levels by PND 70 (50 d post exposure).
Tungstate concentrations in dam milk
secretions measured PND 10-14 were
0.005, 0.021, or 0.45 ppminthe 0-, 3-,
or 78-mg W/kg-d groups, respectively.
The measured concentration in the
high-dose group was statistically
significantly (p < 0.05) higher than
controls, but the low-dose group did not
differ significantly from controls.
Tungstate distributed widely in the body. It
crossed the blood brain barrier and the
placenta. Tungstate accumulation in milk
increased with increasing dose level.
Mclnturf etal. (2011):
Mclnturf et al. (2008)
ADME
Male and female rats
(unspecified strain; 2/sex/group)
were fed ground dog chow
containing 2 or 10% W as
tungsten metal; 10% W as
purified tungsten metal; 0.1% W
as tungsten oxide, 0.1% W as
sodium tungstate, or 0.5% W as
ammonium-p-tungstate for
100 d. Control groups were fed
only ground dog chow.
Tungsten was generally observed (in
trace amounts) in the blood, kidney, and
liver, regardless of administered
compound. In about 50% of the
animals, trace amounts of tungsten were
observed in the lung, muscles, and
testes; again, findings were not related
to administered compound. Tungsten
was generally not observed in the brain,
heart, or uterus (a single exception in
each case).
There were no marked differences among
the tissue distribution patterns of the
various tungsten compounds tested.
The later tissue distribution studies by
Kellv et al. (2013) and Mclnturf et al.
Kinard and Aull (1945)
(2008). usins inductively coupled plasma
mass spectrometry, provide more accurate
descriptions of tissue distribution than this
early study.
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Table C-2. Other Studies
Test
Materials and Methods
Results
Conclusions
References
Studies of Mode of Action/ Mechanism/ Therapeutic Action
Mode of action/
mechanistic
Male Wistar rats (4/group) were
fed basal diets containing 0, 5,
or 50 ppm tungsten as sodium
tungstate dihydrate for 28 d (0,
0.65, or 6.6 mg W/kg-d).
L-ascorbic acid metabolism was
evaluated.
Concentration of ascorbic acid was
significantly increased in the liver and
spleen of the 0.65-mg W/kg-d group,
but not the 6.6-mg W/kg-d group,
compared with control. Significantly
decreased urinary excretion of ascorbic
acid and glucuronic acid was observed
in both exposure groups, compared with
controls. Liver extracts from rats in
both exposure groups synthesized
significantly greater amounts of
L-ascorbic acid from L-gluconolactone,
2,3-dioxobulonic acid from
dehydroascorbic acid, and L-Xylulose
from sodium L-gulonate, compared
with livers extracts from control rats.
Conversion of D-glucuronolactone to
D-glucuronic acid by liver extracts was
not altered with dietary tungsten
exposure.
Dietary exposure to tungsten altered
ascorbic acid metabolism.
Giatteriee et al. (1973)
Mode of action/
mechanistic
Rats were fed basal or tungsten
supplemented diet (0.7 g sodium
tungstate/kg) for 3 wk.
Following treatment, lungs were
removed for quantification of
xanthine oxidase (XO) activity.
Tungsten-exposed rats had negligible
XO activities in isolated lungs. When
isolated lungs were exposed to
hyperoxia, XO-depleted lungs from
tungsten-exposed rats developed less
acute edematous injury during
perfusion with buffer or purified
neutrophil elastase than XO-replete
lungs from control rats.
The results indicate that supplemental
sodium tungstate in the diet depleted XO
activity in the lungs of rats.
Rodelletal. (1987)
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Table C-2. Other Studies
Test
Materials and Methods
Results
Conclusions
References
Mode of action/
mechanistic/
therapeutic action
Multiple studies reported that sodium tungstate corrected hyperglycemia in insulin- and noninsulin-dependent rat
models of diabetes when administered orally. Prevention and/or regeneration of pancreatic beta-cell populations in a
diabetic animals models have also been demonstrated (Oliveira et al.. 2014; Fernandez-Alvarez et al.. 2004).
Improved sexual and/or reproductive function in diabetic rat models was also reported following sodium tungstate
administration (Ballester et al.. 2007; Ballester et al.. 2005).
Oliveira et al. (2014);
Ballester et al. (2007);
Ballester et al. (2005);
Fernandez-Alvarez et al.
(2004); Miifioz et al.
(2001); Le Lamer et al.
(2000); Rodriguez-
Gallardo et al. (2000);
Barbera et al. (1997)
CCDV = cardiovascular and cerebrovascular disease; CI = confidence interval; FT3 = free triiodothyronine; FT4 = free thyroxine; ND = no data; NHANES = National
Health and Nutrition Examination Survey; NR = not reported; OR = odds ratio; T3 = triiodothyronine; T4 = thyroxine; XO = xanthine oxidase.
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APPENDIX D. BENCHMARK DOSE MODELING RESULTS
MODELING PROCEDURE FOR DICHOTOMOUS DATA
The benchmark dose (BMD) modeling of dichotomous data was conducted with the
U.S. EPA's Benchmark Dose Software (BMDS) (Version 2.2.2). For these data, all of the
dichotomous models (i.e., Gamma, Multistage, Logistic, Log-logistic, Probit, Log-probit, and
Weibull models) available within the software were fit using a default benchmark response
(BMR) of 10% extra risk based on the U.S. EPA's Benchmark Dose Technical Guidance
Document (U.S. EPA 2012b). Adequacy of model fit was judged based on the
X2 goodness-of-fit /rvalue (p > 0.1), magnitude of scaled residuals in the vicinity of the BMR,
and visual inspection of the model fit. Among all models providing adequate fit, the lowest
benchmark dose lower confidence limit (BMDL) was selected if the BMDLs estimated from
different models varied greater than three-fold; otherwise, the BMDL from the model with the
lowest Akaike's information criterion (AIC) was selected as a potential point of departure (POD)
from which to derive a provisional oral reference dose (p-RfD).
In addition, data from exposures much higher than the study
lowest-observed-adverse-effect level (LOAEL) do not provide reliable information regarding the
shape of the response curve at low doses. However, such exposures can have a strong effect on
the shape of the fitted model in the low-dose region of the dose-response curve in some cases.
Thus, if lack of fit is due to characteristics associated with dose-response data for high doses,
then the U.S. EPA's Benchmark Dose Technical Guidance Document allows for data to be
adjusted by eliminating high-dose groups (U.S. EPA 2012b).
MODELING PROCEDURE FOR CONTINUOUS DATA
The BMD modeling of continuous data was conducted with the U.S. EPA's BMDS
(Version 2.2.2). For these data, all continuous models available within the software were fit
using a default BMR of 1 standard deviation (SD) relative risk. For changes in body weight, a
BMR of 10% change relative to the control mean was also used. An adequate fit was judged
based on the goodness-of-fit /rvalue (p > 0.1), magnitude of the scaled residuals in the vicinity
of the BMR, and visual inspection of the model fit. In addition to these three criteria forjudging
adequacy of model fit, a determination was made as to whether the variance across dose groups
was constant. If a constant variance model was deemed appropriate based on the statistical test
provided in BMDS (i.e., Test 2), the final BMD results were estimated from a constant variance
model. If the test for homogeneity of variance was rejected (p < 0.1), the model was run again
while modeling the variance as a power function of the mean to account for this nonconstant
variance. If this nonconstant variance model did not adequately fit the variance data (i.e., Test 3;
p < 0.1), the data set was considered unsuitable for BMD modeling. Among all models
providing adequate fit, the lowest BMDL was selected if the BMDLs estimated from different
models varied greater than three-fold; otherwise, the BMDL from the model with the lowest AIC
was selected as a potential POD from which to derive a p-RfD.
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The following data sets were selected for BMD modeling:
• incidence data for goblet cell metaplasia and mild to severe renal cortical tubule
regeneration, and body-weight data for male and female rats exposed by gavage for
90 days OJSACHPPM. 2007a. b),
• total bone marrow cell counts in male C57BL/6J mice exposed to sodium tungstate in
drinking water for 16 weeks (Kelly et al.. 2013). and
• percentages of CD71+ helper and cytotoxic T cells in spleens of adult C57BL/6J mice
exposed for 28 days, for 19 weeks (F0 mice) and for 32 weeks (F1 mice) following
challenge with Staphylococcal enterotoxin B (Osterburg et al. 2014).
For the male rat stomach lesion incidence data, all models provided adequate fit by the
X2 goodness-of-fit criteria (see Table D-l). The LogLogistic model was selected as the model
with the lowest BMDL, because BMDLs from models with adequate fit ranged widely by about
seven-fold (see Table D-l).
Table D-l. Modeling Results for Incidence Data for Goblet Cell Metaplasia in
Glandular Stomach in Male Sprague-Dawley Rats Exposed to
Sodium Tungstate in Water by Gavage for 90 Days3
Model
DF
x2
x2
Goodness-of-Fit
/>-Valueb
Scaled
Residuals0
AIC
BMDio
(mg W/kg-d)
BMDLio
(mg W/kg-d)
Gammad
4
2.34
0.67
0.12
40.86
6.76
4.65
Logistic
3
5.49
0.14
-0.05
46.14
21.10
14.01
LogLogistic6'
3
2.51
0.47
0.32
43.00
7.72
2.34
LogProbit6
3
3.1
0.38
0.78
43.68
12.30
7.70
Multistage (l-degree)f
4
2.34
0.67
0.12
40.86
6.76
4.65
Multistage (2-degree)f
4
2.34
0.67
0.12
40.86
6.76
4.65
Multistage (3-degree)f
4
2.34
0.67
0.12
40.86
6.76
4.65
Multistage (4-degree)f
4
2.34
0.67
0.12
40.86
6.76
4.65
Probit
3
5.4
0.14
-0.04
46.38
20.22
13.94
Weibulld
4
2.34
0.67
0.12
40.86
6.76
4.65
aUSACHPPM (2007a): USACHPPM (2007b)
bValues <0.1 fail to meet conventional goodness-of-fit criteria.
°Scaled residuals for dose group near BMD.
dPower restricted to >1.
"Slope restricted to >1.
fBetas restricted to >0.
DF = degree(s) of freedom.
The BMDS output for the selected model (LogLogistic) follows.
75
Soluble Tungsten Compounds
-------
"O
= 1
Total number of observations = 5
Total number of records with missing values = 0
Maximum, number of iterations = 5 00
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
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User has chosen the log transformed model
Default Initial Parameter Values
background = 0
intercept = -4.65642
slope = 1.31441
Asymptotic Correlation Matrix of Parameter Estimates
the user,
intercept
slope
( *** The model parameter(s) -background
have been estimated at a boundary point, or have been specified by
and do not appear in the correlation matrix )
intercept
1
-0. 98
slope
-0.98
1
Parameter Estimates
Interval
Variable
Limit
background
intercept
slope
Estimate
-4.94902
1.34644
Std. Err.
95.0% Wald Confidence
Lower Conf. Limit Upper Conf.
* - Indicates that this value is not calculated.
Analysis of Deviance Table
Model
Full model
Fitted model
Reduced model
Log(likelihood) # Param's Deviance Test d.f. P-value
-18.1245 5
-19.5021 2 2.75535 3 0.4309
-33.4625 1 30.6761 4 <.0001
AIC: 43.0043
Goodness of Fit
Dose Est._Prob. Expected Observed Size
Scaled
Residual
0.0000
6.0000
47.0000
78.0000
125.0000
0.0000
0.0733
0.5585
0.7144
0. 8252
Chi^2 = 2.51 d.f. = 3
Benchmark Dose Computation
0.000 0.000 10 0.000
0.733 1.000 10 0.323
5.585 4.000 10 -1.009
6.430 8.000 9 1.159
8.252 8.000 10 -0.210
P-value = 0.4735
Specified effect
Risk Type
Confidence level
BMD
BMDL
0.1
Extra risk
0. 95
7.71959
2 .34372
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For the female rat stomach lesion incidence data, all models provided adequate fit to the
data. BMDLs ranged from 4-18 mg W/kg-day. The 1-degree multistage model was considered
an outlier based on poorer fit criteria than the other models, which all have sigmoidal shape
capabilities. The range of BMDLs from the other models (7-18 mg W/mg/kg-day) was
considered sufficiently close (within a three-fold range), so the model with the lowest AIC was
selected (Multistage 2-degree).
Table D-2. Modeling Results for Incidence Data for Goblet Cell Metaplasia in
Glandular Stomach of Female Sprague-Dawley Rats Exposed to
Sodium Tungstate in Water by Gavage for 90 Days3
Model
DF
x2
X2 Goodness of Fit
/>-Valucb
Scaled
Residuals0
AIC
BMDio
(mg W/kg-d)
BMDLio
(mg W/kg-d)
Gammad
3
0.18
0.98
0.12
27.74
29.59
11.29
Logistic
3
0.8
0.85
0.51
28.57
30.01
17.97
LogLogistic6
3
0.41
0.94
0.14
28.10
31.75
14.50
LogProbit6
3
0.27
0.97
0.14
27.87
32.13
14.27
Multistage (l-degree)f
4
3.61
0.46
-1.06
31.15
5.89
4.02
Multistage (2-degree)f
4
0.33
0.99
-0.31
26.06
19.94
8.14
Multistage (3-degree)f
3
0.12
0.99
-0.21
27.66
25.22
7.42
Multistage (4-degree)f
2
0.11
0.95
-0.24
29.65
24.32
6.76
Probit
3
0.47
0.93
0.44
28.09
28.87
16.78
Weibulld
3
0.11
0.99
-0.16
27.64
26.13
10.57
aUSACHPPM (7007a): USACHPPM f2007b)
bValues <0.1 fail to meet conventional goodness-of-fit criteria.
°Scaled residuals for dose group near BMD.
dPower restricted to >1.
"Slope restricted to >1.
fBetas restricted to >0.
The BMDS output for the selected model (Multistage 2-degree) follows.
78
Soluble Tungsten Compounds
-------
"O
'l-beta2*dose/s2) ]
The parameter betas are restricted to be positive
Dependent variable = Effect
Independent variable = Dose
Total number of observations = 5
Total number of records with missing values = 0
Total number of parameters in model = 3
Total number of specified parameters = 0
Degree of polynomial = 2
79
Soluble Tungsten Compounds
-------
FINAL
09-29-2015
Maximum number of iterations = 5 00
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
Default Initial Parameter Values
Background = 0
Beta(l) = 0
Beta(2) = 6.32551e+015
Asymptotic Correlation Matrix of Parameter Estimates
( *** The model parameter(s) -Background -Beta(l)
have been estimated at a boundary point, or have been specified by
the user, and do not appear in the correlation matrix )
Beta(2)
Beta (2)
Parameter Estimates
Interval
Variable
Limit
Background
Beta(1)
Beta(2)
Estimate
0
0
Std. Err.
0.000264865 *
* - Indicates that this value is not calculated.
95.0% Wald Confidence
Lower Conf. Limit Upper Conf.
Model
Full model
Fitted model
Reduced model
Analysis of Deviance Table
Log(likelihood) # Param's Deviance Test d.f. P-value
-11.7341 5
-12.0279 1 0.587544 4 0.9644
-34.2965 1 45.1247 4 <.0001
AIC: 26.0558
Goodness of Fit
Dose Est._Prob. Expected Observed Size
Scaled
Residual
0.0000
6.0000
47.0000
78.0000
125.0000
0.0000
0.0095
0.4429
0.8004
0.9841
0.000
0.095
4.429
8.004
9.841
0.000
0.000
4.000
8.000
10.000
10.000
10.000
10.000
10.000
10.000
0. 000
-0.310
-0.273
-0.003
0. 403
Chi^2 =0.33
d.f. = 4
P-value = 0.9876
Benchmark Dose Computation
Specified effect
Risk Type
Confidence level
0.1
Extra risk
0. 95
BMD
BMDL
19.9447
8.14146
BMDU
25.1421
Taken together, (8.14146, 25.1421) is a 90
interval for the BMD
two-sided confidence
80
Soluble Tungsten Compounds
-------
FINAL
09-29-2015
For the male rat renal cortical regeneration incidence data, all models, except for the
multistage 1- and 2-degree models, provided adequate fit to the data. BMDLs for models
providing adequate fit were sufficiently close (differed by less than two- to three-fold), so the
model with the lowest AIC was selected (Weibull).
Table D-3. Modeling Results for Incidence Data for Mild to Severe Cortical Tubule
Regeneration in Kidneys of Male Sprague-Dawley Rats Exposed to Sodium Tungstate in
Water by Gavage for 90 Days"
Model
DF
x2
X2 Goodness of Fit
/>-Valucb
Scaled
Residuals0
AIC
BMDio
(mg W/kg-d)
BMDLio
(mg W/kg-d)
Gammad
4
1.9
0.75
-0.91
11.29
67.19
57.00
Logistic
3
0
1
0
10.28
77.80
64.20
LogLogistice
4
0.02
1
-0.02
8.31
77.41
66.67
LogProbit6
3
0
1
0
10.28
77.68
66.53
Multistage (1-degree/
4
15.07
0.005
-0.62
30.57
16.60
10.35
Multistage (2-degree/
4
8.84
0.07
-1.40
22.04
36.09
25.26
Multistage (3-degree/
4
5.3
0.26
-1.00
16.60
48.71
36.48
Multistage (4-degree)f
4
3.06
0.55
-0.69
13.05
57.54
44.38
Probit
3
0
1
0
10.28
77.60
63.48
Weibulld
4
0
1
0
8.28
77.52
61.55
aUSACHPPM (2007a): USACHPPM (2007b)
bValues <0.1 fail to meet conventional goodness-of-fit criteria.
°Scaled residuals for dose group near BMD.
dPower restricted to >1.
"Slope restricted to >1.
fBetas restricted to >0.
The BMDS output for the selected model (Weibull) follows.
81
Soluble Tungsten Compounds
-------
FINAL
09-29-2015
Weibull Model, with 6MR of 10% Extra Risk for the BMD and 0,95 Lower < nt d e Limit for the BMDL
0.6
0.4 -
0.2
BMDL
0 20 40 80 30 100 120
dose
08:14 07/14 2014
Figure D-3. Weibull (Renal Cortical Tubule Regeneration, Male Rat)
Weibull Model using Weibull Model (Version: 2.16; Date: 2/28/2013)
Input Data File:
C:/USEPA/PTV/NaTungstate/corttubregen/male/wei_corttubregen_male_Wei-BMR10-Restrict.(d
)
Gnuplot Plotting File:
C:/USEPA/PTV/NaTungstate/corttubregen/male/wei_corttubregen_male_Wei-BMR10-Restrict.pi
t
Mon Jul 14 08:14:53 2014
BMDS Model Run
The form of the probability function is:
P[response] = background + (1-background)*[1-EXP(-slope*dose/spower)]
Dependent variable = Effect
Independent variable = Dose
Power parameter is restricted as power >= 1.000000
Total number of observations = 5
Total number of records with missing values = 0
Maximum number of iterations = 5 00
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
82
Soluble Tungsten Compounds
-------
FINAL
09-29-2015
the user,
Slope
Default Initial (and Specified) Parameter Values
Background = 0.0833333
Slope = 2.25391e-012
Power = 5.73553
Asymptotic Correlation Matrix of Parameter Estimates
( *** The model parameter(s) -Background -Power
have been estimated at a boundary point, or have been specified by
and do not appear in the correlation matrix )
Slope
1.$
Parameter Estimates
Interval
Variable
Limit
Background
Slope
1.#QNAN
Power
Estimate
0
1. 03115e-035
18
Std. Err.
NA
1.#QNAN
NA
95.0% Wald Confidence
Lower Conf. Limit Upper Conf.
1.#QNAN
NA - Indicates that this parameter has hit a bound
implied by some ineguality constraint and thus
has no standard error.
Model
Full model
Fitted model
Reduced model
Analysis of Deviance Table
Log(likelihood) # Param's Deviance Test d.f.
-3.13949 5
-3.13962 1 0.000258311 4
-26.0941 1 45.9092 4
P-value
<.0001
AIC: 8.27924
Goodness of Fit
Dose Est._Prob. Expected Observed Size
Scaled
Residual
0.0000
6.0000
47.0000
78.0000
125.0000
0.0000
0.0000
0.0000
0.1111
1.0000
Chi^2 = 0.00 d.f. = 4
Benchmark Dose Computation
0.000 0.000 10 0.000
0.000 0.000 10 0.000
0.000 0.000 10 -0.011
1.000 1.000 9 0.000
10.000 10.000 10 0.000
P-value = 1.0000
Specified effect
Risk Type
Confidence level
BMD
BMDL
0.1
Extra risk
0. 95
77.5191
61.5537
83
Soluble Tungsten Compounds
-------
FINAL
09-29-2015
For the female kidney lesion incidence data, all models, except for the multistage
1-degree model, provided adequate fit to the data. BMDLs for models providing adequate fit
were sufficiently close (differed by less than two- to three-fold), so the model with the lowest
AIC was selected (Gamma).
Table D-4. Modeling Results for Incidence Data for Mild to Severe Cortical Tubule
Regeneration in Kidneys of Female Sprague-Dawley Rats Exposed to Sodium Tungstate in
Water by Gavage for 90 Days"
Model
DF
x2
X2 Goodness of Fit
/>-Valucb
Scaled
Residuals0
AIC
BMDio
(mg W/kg-d)
BMDLio
(mg W/kg-d)
Gamma'1
4
0.07
1.00
-0.19
18.60
75.93
58.56
Logistic
3
0.11
0.99
0.16
20.70
80.08
58.45
LogLogistice
3
0.02
1.00
0.05
20.55
78.50
59.22
LogProbit6
3
0
1.00
0.01
20.51
78.07
59.72
Multistage (1-degree/
4
9.99
0.04
-0.53
31.27
23.08
13.85
Multistage (2-degree/
4
5.04
0.28
-1.13
25.25
43.96
29.41
Multistage (3-degree/
4
2.4
0.66
-0.78
21.76
57.03
41.00
Multistage (4-degree)f
4
0.94
0.92
-0.67
19.80
66.34
48.55
Probit
3
0.04
1.00
0.08
20.57
78.94
57.61
Weibulld
3
0.06
1.00
0.10
20.61
79.30
56.87
aUSACHPPM (2007a): USACHPPM (2007b)
bValues <0.1 fail to meet conventional goodness-of-fit criteria.
°Scaled residuals for dose group near BMD.
dPower restricted to >1.
"Slope restricted to >1.
fBetas restricted to >0.
The BMDS output for the selected model (Gamma) follows.
84
Soluble Tungsten Compounds
-------
FINAL
09-29-2015
Gamma Multi-Hit Model, with BMRof 10% Extra Risk for the BMD and 0.95 Lower Confidence Limit for the BMDL
Gamma Multi-Hit
1
8
6
0.4
0.2
0
BMDL
BMD
0
20
40
60
80
100
120
dose
09:21 07/14 2014
Figure D-4. Gamma (Renal Cortical Tubule Regeneration, Female Rat)
Gamma Model. (Version: 2.16; Date: 2/28/2013)
Input Data File:
C :\/USEPA/PTV/NaTungstate/corttubregen/female/gam_corttubregen_female_Gam-BMR10-Restric
t. (d)
Gnuplot Plotting File:
C :\/USEPA/PTV/NaTungstate/corttubregen/female/gam_corttubregen_female_Gam-BMR10-Restric
t.. pit
Mon Jul 14 09:21:29 2014
HMDS Model Run
The form of the probability function .is:
P [ response] = background-!- (1-background) *CumGamma [ si ope* dose, power] ,
where CumGamma(.) is the cummulative Gamma distribution function
Dependent variable = Effect
Independent variable = Dose
Power parameter is restricted as power >=1
Total number of observations = 5
Total number of records with missing values = 0
Maximum number of iterations = 5 00
Relative Function Convergence has been set to: le-008
85
Soluble Tungsten Compounds
-------
FINAL
09-29-2015
Parameter Convergence has been set to: le-008
Default Initial (and Specified) Parameter Values
Background = 0.0833333
Slope = 0.141027
Power = 15.4879
the user,
Slope
Asymptotic Correlation Matrix of Parameter Estimates
( *** The model parameter(s) -Background -Power
have been estimated at a boundary point, or have been specified by
and do not appear in the correlation matrix )
Slope
1
Parameter Estimates
Interval
Variable
Limit
Background
Slope
0.195348
Power
Estimate
0
0.168864
18
Std. Err.
NA
0.0135125
NA
95.0% Wald Confidence
Lower Conf. Limit Upper Conf.
0.14238
NA - Indicates that this parameter has hit a bound
implied by some ineguality constraint and thus
has no standard error.
Model
Full model
Fitted model
Reduced model
Analysis of Deviance Table
Log(likelihood) # Param's Deviance Test d.f. P-value
-8.25485 5
-8.30002 1 0.0903344 4 0.999
-23.5697 1 30.6296 4 <.0001
AIC:
Dose
Est. Prob.
18 . 6
Goodness of Fit
Expected Observed Size
Scaled
Residual
0.0000
6.0000
47.0000
78.0000
125.0000
0.0000
0.0000
0.0015
0.1192
0.7799
0.000
0.000
0.015
1.192
7.799
0.000
0.000
0.000
1.000
8.000
10
10
10
10
10
0. 000
-0.000
-0.121
-0.187
0.154
Chi^2 =0.07
d.f. = 4
P-value = 0.9993
Benchmark Dose Computation
Specified effect
Risk Type
Confidence level
0.1
Extra risk
0. 95
BMD
BMDL
75.9286
58 .5642
86
Soluble Tungsten Compounds
-------
FINAL
09-29-2015
For the male rat body-weight data, all constant variance models provided adequate fits to
the variance and means (see Table D-5). BMDLs for models providing adequate fit were
sufficiently close (differed by less than two- to three-fold), so the model with the lowest AIC was
selected (2-degree Polynomial).
Table D-5. Modeling Results for Terminal Body-Weight Data in Male Sprague Dawley
Rats Exposed to Sodium Tungstate in Water by Gavage for 90 Days"
Model
Test for
Significant
Difference
/>-Valucb
Variance
/>-Valuce
Means
/j-Value'
Scaled
Residuals'1
AIC
BMD io
(mg W/kg-d)
BMDLio
(mg W/kg-d)
Constant variance
Exponential (Model 2)e
0.005
0.15
0.44
0.77
433.63
100.78
69.57
Exponential (Model 3)e
0.005
0.15
0.66
-0.03
433.80
108.40
79.57
Exponential (Model 4)e
0.005
0.15
0.44
0.77
433.63
100.78
68.37
Exponential (Model 5)e
0.005
0.15
0.36
-0.03
435.80
108.40
79.57
Hill®
0.005
0.15
0.36
-0.03
435.80
108.57
79.71
Linearf
0.005
0.15
0.48
0.76
433.44
100.34
71.30
Polynomial (2-degree)f
0.005
0.15
0.84
-0.04
431.80
108.23
91.92
Polynomial (3-degree)f
0.005
0.15
0.66
-0.02
433.79
108.91
91.93
Polynomial (4-degree)f
0.005
0.15
0.66
-0.01
433.79
109.23
91.94
Power6
0.005
0.15
0.66
-0.03
433.79
108.67
79.80
aUSACHPPM (2007a): USACHPPM (2007b)
bValues >0.05 fail to meet conventional goodness-of-fit criteria.
°Values <0.10 fail to meet conventional goodness-of-fit criteria.
dScaled residuals for dose group near the BMD.
Tower restricted to >1.
Coefficients restricted to be negative.
The BMDS output for the selected model (2-degree polynomial) follows.
87
Soluble Tungsten Compounds
-------
FINAL
09-29-2015
Polynomial Model, with BMR of 0.1 Rel. Dev. for the BMD and 0.95 Lower Confidence Limit for the BMDL
620
Polynomial
600
580
560
540
520
500
480
460
440
BMDL
BMD
0
20
40
60
80
100
120
dose
09:40 07/14 2014
Figure D-5. Constant Variance Polynomial (2-Degree) (Body Weight, Male Rat)
Polynomial Model. (Version: 2.18; Date: 05/19/2014)
Input Data File:
C :\/USEPA/PTV/NaTungstate/terminalbdwt/ply_termbdwt_male_Ply2-ConstantVariance-BMRlStd-
RestrictUp.(d)
Gnuplot Plotting File:
C :\/USEPA/PTV/NaTungstate/terminalbdwt/ply_termbdwt_male_Ply2-ConstantVariance-BMRlStd-
RestrictUp.pit
Mon Jul 14 09:40:08 2014
BHDS Model Run
The form of the response function is:
Y[dose] = beta_0 + beta_l*dose + beta_2*doseA2 + ...
Dependent variable = Mean
Independent variable = Dose
rho is set to 0
The polynomial coefficients are restricted to be negative
A constant variance model is fit
Total number of dose groups = 5
Total number of records with missing values = 0
88
Soluble Tungsten Compounds
-------
FINAL
09-29-2015
Maximum number of iterations = 5 00
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
Default Initial Parameter Values
alpha = 1
rho = 0 Specified
beta_0 = 560.877
beta_l = 0
beta_2 = -0.00516693
Asymptotic Correlation Matrix of Parameter Estimates
the user,
alpha
beta_0
beta 2
( *** The model parameter(s) -rho -beta_l
have been estimated at a boundary point, or have been specified by
and do not appear in the correlation matrix )
alpha
1
-3e-008
-5.le-009
beta_0
-3e-008
1
-0.63
beta_2
-5.le-009
-0. 63
1
Interval
Variable
Limit
alpha
3051.3
beta_0
578 .357
beta_l
beta_2
0.00117138
Estimate
2185.79
561.581
-2 . 78654e-02 6
-0.00479406
-0.00708991
Parameter Estimates
Std. Err.
441.596
8 .55925
NA
-0.00249821
95.0% Wald Confidence
Lower Conf. Limit Upper Conf.
1320.28
544.805
NA - Indicates that this parameter has hit a bound
implied by some ineguality constraint and thus
has no standard error.
Table of Data and Estimated Values of Interest
Dose
Obs Mean
Est Mean
Obs Std Dev Est Std Dev Scaled Res.
0
6
47
78
125
10
10
10
10
9
553
571
548
535
486
5 62
561
551
532
487
31
49
38
67
52
46.
46.
46.
46.
46.
-0.58
0. 649
-0.202
0.175
-0.0432
Model Descriptions for likelihoods calculated
Model A1: Yij = Mu(i) + e(ij)
Var{e(ij)} = Sigma^2
Model A2: Yij = Mu(i) + e(ij)
Var{e(ij)} = Sigma(i)^2
89
Soluble Tungsten Compounds
-------
FINAL
09-29-2015
Model A3: Yij = Mu(i) + e(ij)
Var{e(ij)} = Sigma^2
Model A3 uses any fixed variance parameters that
were specified by the user
Model R: Yi = Mu + e(i)
Var{e(i)} = Sigma^2
Model
A1
A2
A3
fitted
R
Likelihoods of Interest
Log(likelihood) # Param's
-212.479311 6
-209.104822 10
-212.479311 6
-212.898456 3
-220.102427 2
Explanation of Tests
AIC
436.958622
438.209645
436.958622
431.796911
444.204855
Test 1: Do responses and/or variances differ among Dose levels?
(A2 vs. R)
Are Variances Homogeneous? (A1 vs A2)
Are variances adeguately modeled? (A2 vs. A3)
Does the Model for the Mean Fit? (A3 vs. fitted)
Test 2
Test 3
Test 4
(Note: When rho=0 the results of Test 3 and Test 2 will be the same.)
Tests of Interest
Test -2*log(Likelihood Ratio) Test df p-value
Test 1
Test 2
Test 3
Test 4
21.9952
6.746
6.746
0.838289
0.004925
0.1498
0.1498
0.8403
The p-value for Test 1 is less than .05. There appears to be a
difference between response and/or variances among the dose levels
It seems appropriate to model the data
The p-value for Test 2 is greater than .1. A homogeneous variance
model appears to be appropriate here
The p-value for Test 3 is greater than .1. The modeled variance appears
to be appropriate here
The p-value for Test 4 is greater than .1. The model chosen seems
to adeguately describe the data
Benchmark Dose Computation
Specified effect = 0.1
Risk Type = Relative deviation
Confidence level = 0.95
BMD = 108.232
BMDL
91.9183
90
Soluble Tungsten Compounds
-------
FINAL
09-29-2015
For the mouse total bone marrow cell count data, with constant variance model applied,
all of the models provided an adequate fit to the variance and all of the models except for the
Exponential (Model 5) provided an adequate fit to the means. BMDLs for models providing
adequate fit were sufficiently close (differed by less than two- to three-fold), so the model with
the lowest AIC was selected (3-degree Polynomial).
Table D-6. Modeling Results for Total Bone Marrow Cell Counts in Male C57BL/6J Mice
Exposed to Sodium Tungstate in Drinking Water for 16 Weeks"
Model
Test for Significant
Difference
/>-Valucb
Variance
p-Value0
Means
/j-Value'
Scaled
Residuals'1
AIC
BMDisd
(mg W/kg-d)
BMDLisd
(mg W/kg-d)
Constant variance
Exponential (Model 2)e
0.01
0.94
0.36
1.15
119.79
95.54
63.22
Exponential (Model 3)e
0.01
0.94
0.60
2.16 x 10-°8
120.04
226.89
73.53
Exponential (Model 4)e
0.01
0.94
0.36
1.15
119.79
95.54
49.41
Exponential (Model 5)e
0.01
0.94
NA
-2.3 x 10-°8
122.04
228.73
50.14
Hill®
0.01
0.94
0.60
-7.15 x 10-°6
120.04
228.80
50.36
Lineal
0.01
0.94
0.41
1.07
119.56
101.07
69.69
Polynomial (2-degree/
0.01
0.94
0.80
-0.02
118.21
157.24
76.72
Polynomial (3-degree)f
0.01
0.94
0.86
-0.003
118.06
183.72
77.75
Power6
0.01
0.94
0.60
-5.08 x 10-°8
120.04
229.80
77.97
"Kellv et al. (2013)
bValues >0.05 fail to meet conventional goodness-of-fit criteria.
°Values <0.10 fail to meet conventional goodness-of-fit criteria.
dScaled residuals for dose group near the BMD.
Tower restricted to >1.
Coefficients restricted to be negative.
NA = not applicable (BMDL computation failed or the BMD was higher than the highest dose tested).
The BMDS output for the selected model (3-degree polynomial) follows.
91
Soluble Tungsten Compounds
-------
FINAL
09-29-2015
Polynomial Model, with BMR of 1 Std Dev. for the BMD and 0.95 Lower Confidence Limit for the BMDL
Polynomial
140
130
120
110
100
90
80
BMDL
BMD
0
50
100
150
200
250
dose
10:54 07/14 2014
Figure D-6. Constant Variance Polynomial (3-Degree) (Total Bone Marrow Cell Counts,
Male Mice)
Polynomial Model. (Version: 2.18; Date: 05/19/2014)
Input Data File:
C:/USEPA/PTV/NaTungstate/bonemarrowcellct/ply_bonemarrowcellct_Ply3-ConstantVariance~B
MRlStd-RestrictUp.(d)
Gnuplot Plotting File:
C :./USEPA/PTV/NaTungstate/bonemarrowcellct/ply_bonemarrowcellct_Ply3-ConstantVariance-B
MRlStd-RestrictUp.pit
Mon Jul 14 10:54:58 2014
BMDS Model Run
The form of the response function is:
Y[dose] = beta_0 + beta_l*dose + beta_2*doseA2 + ...
Dependent variable = Mean
Independent variable = Dose
rho is set to 0
The polynomial coefficients are restricted to be negative
A constant variance model is fit
92
Soluble Tungsten Compounds
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FINAL
09-29-2015
Total number of dose groups = 4
Total number of records with missing values = 0
Maximum number of iterations = 5 00
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
Default Initial Parameter Values
alpha = 122.889
rho = 0 Specified
beta_0 = 118.89
beta_l = 0
beta_2 = -0.0124316
beta 3 = 0
Asymptotic Correlation Matrix of Parameter Estimates
the user,
alpha
beta_0
beta 3
( *** The model parameter(s) -rho -beta_l -beta_2
have been estimated at a boundary point, or have been specified by
and do not appear in the correlation matrix )
alpha beta_0 beta_3
1 2.5e-007 -1.8e-007
2.6e-007 1 -0.5
-1.8e-007 -0.5 1
Parameter Estimates
Interval
Variable
Limit
alpha
161.659
beta_0
125.804
beta_l
beta_2
beta_3
3.3099e-007
Estimate
99.8021
120.736
-0
-2 .74523e-024
-1. 61114e-006
-2 . 25987e-006
Std. Err.
31.5602
2.58593
NA
NA
-9.62 413e-007
95.0% Wald Confidence
Lower Conf. Limit Upper Conf.
37.9452
115.668
NA - Indicates that this parameter has hit a bound
implied by some ineguality constraint and thus
has no standard error.
Table of Data and Estimated Values of Interest
Dose N Obs Mean Est Mean Obs Std Dev Est Std Dev Scaled Res.
0
4
49
250
119
121
122
95.5
121
121
121
95 . 6
9.51
10.4
12.2
12
9. 99
9. 99
9. 99
9. 99
-0.413
0.0636
0.352
-0.00265
Model Descriptions for
Model A1: Yij =
Var{e(ij)} =
Model A2: Yij =
likelihoods calculated
Mu(i) + e(i j)
Sigma^2
Mu(i) + e(i j)
93
Soluble Tungsten Compounds
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FINAL
09-29-2015
Var{e(ij)} = Sigma(i)^2
Model A3: Yij = Mu(i) + e(ij)
Var{e(ij)} = Sigma^2
Model A3 uses any fixed variance parameters that
were specified by the user
Model R: Yi = Mu + e(i)
Var{e(i)} = Sigma^2
Likelihoods of Interest
Model
Log(likelihood)
# Param's
AIC
A1
-55.881351
5
121.762702
A2
-55.677704
8
127.355408
A3
-55.881351
5
121.762702
fitted
-56.031890
3
118.063780
R
-63.846659
2
131.693318
Explanation of Tests
Test 1
Test 2
Test 3
Test 4
(Note:
Tests of Interest
: Do responses and/or variances differ among Dose levels?
(A2 vs. R)
: Are Variances Homogeneous? (A1 vs A2)
: Are variances adeguately modeled? (A2 vs. A3)
: Does the Model for the Mean Fit? (A3 vs. fitted)
When rho=0 the results of Test 3 and Test 2 will be the same.)
Test -2*log(Likelihood Ratio) Test df p-value
Test 1 16.3379 6 0.01205
Test 2 0.407294 3 0.9387
Test 3 0.407294 3 0.9387
Test 4 0.301078 2 0.8602
The p-value for Test 1 is less than .05. There appears to be a
difference between response and/or variances among the dose levels
It seems appropriate to model the data
The p-value for Test 2 is greater than .1. A homogeneous variance
model appears to be appropriate here
The p-value for Test 3 is greater than .1. The modeled variance appears
to be appropriate here
The p-value for Test 4 is greater than .1. The model chosen seems
to adeguately describe the data
Benchmark Dose Computation
Specified effect = 1
Risk Type = Estimated standard deviations from the control mean
Confidence level = 0.95
BMD = 183.715
BMDL = 77.7506
94
Soluble Tungsten Compounds
-------
FINAL
09-29-2015
The 28-day percentage CD71+ helper T cell data sets were amenable to BMD modeling
(see Tables D-7). With constant variance model applied, none of the models provided an
adequate fit to the variance. After applying the model for nonconstant variance, all models
provided an adequate fit to the means, though the BMDL computation failed for several models.
BMDLs for models providing adequate fit were sufficiently close (differed by less than two- to
three-fold), so the model with the lowest AIC was selected (Exponential [Models 2 and 3] and
Power all yielded the same results).
Table D-7. Modeling Results for Percentage CD71+ Helper T Cells in the Spleen of
Adult C57BL/6J Mice Exposed to Sodium Tungstate in Drinking Water for 28 Days"
Model
Test for
Significant
Difference
/>-Valucb
Variance
/>-Valuce
Means
/>-Valuce
Scaled
Residuals'1
AIC
BMDisd
(mg W/kg-d)
BMDLisd
(mg W/kg-d)
Constant variance
Exponential (Model 2)e
0.001
0.0005
0.84
0.03
144.35
183.65
74.18
Exponential (Model 3)e
0.001
0.0005
0.84
0.03
144.35
183.65
74.18
Exponential (Model 4)e
0.001
0.0005
0.61
0.00
146.27
NA
NA
Exponential (Model 5)e
0.001
0.0005
0.61
0.00
146.27
NA
NA
Hill6
0.001
0.0005
0.63
-0.18
146.24
342.58
0.00001
Lineal
0.001
0.0005
0.80
0.07
144.44
168.11
89.53
Polynomial (2-degree/
0.001
0.0005
0.80
0.07
144.44
168.11
89.53
Polynomial (3-degree/
0.001
0.0005
0.80
0.07
144.44
168.11
89.53
Power6
0.001
0.0005
0.80
0.07
144.44
168.11
89.53
Nonconstant variance
Exponential (Model 2)e
0.001
0.33
0.19
-0.05
133.68
333.62
118.36
Exponential (Model 3)e
0.001
0.33
0.19
-0.05
133.68
333.62
118.36
Exponential (Model 4)e
0.001
0.33
0.73
0
132.43
NA
NA
Exponential (Model 5)e
0.001
0.33
NA
0
134.32
NA
NA
Hill®
0.001
0.33
NA
0
134.32
NA
NA
Linear6
0.001
0.33
0.11
-0.17
134.69
266.99
130.94
Polynomial (2-degree/
0.001
0.33
0.11
-0.17
134.69
266.99
130.94
Polynomial (3-degree/
0.001
0.33
0.11
-0.17
134.69
266.99
130.94
Polynomial (4-degree)f
0.001
0.33
0.11
-0.17
134.69
266.99
130.94
Power6
0.001
0.33
0.19
-0.05
133.68
333.62
118.36
"Osterburg et al. (2014)
bValues >0.05 fail to meet conventional goodness-of-fit criteria.
°Values <0.10 fail to meet conventional goodness-of-fit criteria.
dScaled residuals for dose group near the BMD.
Tower restricted to >1.
Coefficients restricted to be negative.
NA = not applicable (BMDL computation failed or the BMD was higher than the highest dose tested).
95
Soluble Tungsten Compounds
-------
FINAL
09-29-2015
The BMDS output for the selected model follows.
Exponential Model 2. with BMR of 1 Std. Dev. for the BMD and 0.95 Lower Confidence Level for BMDL
8
Exponential
7
6
5
4
3
2
1
BMDL
BMD
0
50
100
150
200
250
300
350
dose
14:12 07/14 2014
Figure D-7. Nonconstant Variance Exponential Model 2 (Percentage CD71+ Helper
T Cells, Mice, 28 Days, Drinking Water)
Exponential Model. (Version: 1.9; Date: 01/29/2013)
Input Data File:
C:/USEPA/PTV/NaTungstate/Tcell/adult2 8day/exp_Tcell_adult_28day_Exp-ConstantVariance-B
MRlStd-Up.(d)
Gnuplot Plotting File:
Mon Jul 14 14:12:37 2014
BMDS Model Run
The form of the response function by Model:
Model 2
Model 3
Model 4
Model 5
Y[dose] = a * exp{sign * b * dose}
Y[dose] = a * exp{sign * (b * dose)Ad)
Y[dose] = a * [c-(c-l) * exp{-b * dose}]
Y[dose] = a * [c-(c-l) * exp{-(b * dose)Ad}]
Note: Y[dose] is the median response for exposure
sign = +1 for increasing trend in data;
sign = -1 for decreasing trend.
dose;
96
Soluble Tungsten Compounds
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FINAL
09-29-2015
Model 2 is nested within Models 3 and 4.
Model 3 is nested within Model 5.
Model 4 is nested within Model 5.
Dependent variable = Mean
Independent variable = Dose
Data are assumed to be distributed: normally
Variance Model: exp(lnalpha +rho *ln(Y[dose]))
The variance is to be modeled as Var(i) = exp(lalpha + log(mean(i)) * rho)
Total number of dose groups = 4
Total number of records with missing values = 0
Maximum number of iterations = 5 00
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
MLE solution provided: Exact
Initial Parameter Values
Variable Model 2
lnalpha -2.63791
rho 3.28816
a 2.81592
b 0.00413591
c 0
d 1
Parameter Estimates
Variable Model 2
lnalpha -2.85622
rho 3.47741
a 4.63872
b 0.
00409388
c
0
d
1
NC = No
Convergence
Table of Stats From Input
Data
Dose
N Obs Mean
Obs Std Dev
0
12 4.85
4.26
39
12 3.61
2.29
78
12 3.54
1.39
125
12 2.76
1.77
Estimated Values of
Interest
Dose
Est Mean Est Std
Scaled Residual
0
4.639 3.455
0.2118
39
3.954 2.618
-0.4555
78
3.371 1.983
0.2958
125
2.781 1.419
-0. 05053
Other models for which likelihoods are calculated:
Model A1: Yij = Mu(i) + e(ij)
97
Soluble Tungsten Compounds
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FINAL
09-29-2015
Var{e(ij)} = SigmaA2
Model A2: Yij
Var{e(ij)}
Model A3: Yij
Var{e(ij)}
Model R: Yij
Var{e(ij)}
Model
Mu(i) + e(i j)
Sigma(i)A2
Mu(i) + e(i j)
exp(lalpha + log(mean(i)) * rho)
Mu + e(i)
SigmaA2
Likelihoods of Interest
Log(likelihood) DF
A1
A2
A3
R
2
-69.00187
-60.04898
-61.15947
-70.97856
-62.84106
AIC
148.0037
136.098
134.3189
145.9571
133.6821
Additive constant for all log-likelihoods = -44.11. This constant added to the
above values gives the log-likelihood including the term that does not
depend on the model parameters.
Explanation of Tests
Does response and/or variances differ among Dose levels? (A2 vs. R)
Are Variances Homogeneous? (A2 vs. Al)
Are variances adeguately modeled? (A2 vs. A3)
Does Model 2 fit the data? (A3 vs. 2)
Tests of Interest
-2*log(Likelihood Ratio) D. F. p-value
Test
1:
Test
2 :
Test
3:
Test
4 :
Test
Test 1
Test 2
Test 3
Test 4
21.86
17. 91
2.221
3.363
0. 001284
0.00046
0.3294
0.1861
The p-value for Test 1 is less than .05. There appears to be a
difference between response and/or variances among the dose
levels, it seems appropriate to model the data.
The p-value for Test 2 is less than .1. A non-homogeneous
variance model appears to be appropriate.
The p-value for Test 3 is greater than .1.
variance appears to be appropriate here.
The p-value for Test 4 is greater than .1.
to adeguately describe the data.
The modeled
Model 2 seems
Benchmark Dose Computations:
Specified Effect = 1.000000
Risk Type = Estimated standard deviations from control
Confidence Level = 0.950000
BMD = 333.62
98
Soluble Tungsten Compounds
-------
FINAL
09-29-2015
BMDL = 118.363
The 19-week (F0 mice) percentage of CD71+ helper T cells data sets were not amenable
to modeling, because the available constant-variance and nonconstant-variance models did not
provide adequate fits to the variances (see Table D-8).
Table D-8. Modeling Results for Percentage of CD71+ Helper T Cells in the Spleen of
F0 C57BL/6J Exposed to Sodium Tungstate in Drinking Water for ~19 Weeks11
Model
Test for
Significant
Difference
/>-Valucb
Variance
p-Value0
Means
/j-Value'
Scaled
Residuals'1
AIC
BMDisd
(mg W/kg-d)
BMDLisd
(mg W/kg-d)
Constant variance
Exponential (Model 2)e
0.0001
0.0002
0.80
0.78
89.64
74.46
38.58
Exponential (Model 3)e
0.0001
0.0002
0.65
0.66
91.50
82.99
39.17
Exponential (Model 4)e
0.0001
0.0002
0.80
0.78
89.64
74.46
24.04
Exponential (Model 5)e
0.0001
0.0002
0.35
0.66
93.50
82.99
39.17
Hill6
0.0001
0.0002
0.40
0.58
93.34
84.80
52.13
Linear
0.0001
0.0002
0.87
0.65
89.36
81.07
52.18
Polynomial (2-degree/
0.0001
0.0002
0.72
0.51
91.29
87.38
52.42
Polynomial (3-degree/
0.0001
0.0002
0.74
0.42
91.23
90.30
52.64
Polynomial (4-degree)f
0.0001
0.0002
0.76
0.37
91.18
92.47
52.82
Power6
0.0001
0.0002
0.70
0.58
91.34
84.83
52.25
Nonconstant variance
Exponential (Model 2)e
0.0001
0.001
0.07
0.73
91.57
69.74
30.79
Exponential (Model 3)e
0.0001
0.001
0.07
0.73
91.57
69.74
30.79
Exponential (Model 4)e
0.0001
0.001
NA
0.68
85.25
4.75
0.77
Exponential (Model 5)e
0.0001
0.001
0.26
0.04
89.74
119.30
93.36
Hill6
0.0001
0.001
0.54
-0.51
87.71
95.10
82.91
Lineal
0.0001
0.001
0.09
0.79
90.88
87.08
53.80
Polynomial (2-degree/
0.0001
0.001
0.44
0.13
87.19
111.67
90.86
Polynomial (3-degree/
0.0001
0.001
0.67
0.08
86.04
117.50
95.67
99
Soluble Tungsten Compounds
-------
FINAL
09-29-2015
Polynomial (4-degree)f
0.0001
0.001
0.73
0.05
85.78
119.86
109.07
Power6
0.0001
0.001
0.53
0.04
87.75
120.77
95.89
"Osterburg et al. (2014)
bValues >0.05 fail to meet conventional goodness-of-fit criteria.
°Values <0.10 fail to meet conventional goodness-of-fit criteria.
dScaled residuals for dose group near the BMD.
Tower restricted to >1.
Coefficients restricted to be negative.
NA = not applicable (BMDL computation failed or the BMD was higher than the highest dose tested).
The 32-week (F1 mice) percentage of CD71+ helper T cells data sets were not amenable
to modeling, because the available constant-variance and nonconstant-variance models did not
provide adequate fits to the variances (see Table D-9).
Table D-9. Modeling Results for Percentage of CD71+ Helper T Cells in the
Spleen of F1 C57BL/6J Exposed to Sodium Tungstate in Drinking Water for ~32 Weeks"
Model
Test for
Significant
Difference
/>-Valucb
Variance
/>-Valuce
Means
/>-Valuce
Scaled
Residuals'1
AIC
BMDisd
(mg W/kg-d)
BMDLisd
(mg W/kg-d)
Constant variance
Exponential (Model 2)e
<0.0001
0.46
<0.0001
1.621
69.41
84.55
44.20
Exponential (Model 3)e
<0.0001
0.46
<0.0001
1.621
69.41
84.55
44.20
Exponential (Model 4)e
<0.0001
0.46
0.009
-3.70 x 10-°7
58.03
0.32
0.00
Exponential (Model 5)e
<0.0001
0.46
0.002
1.13 x 10-°7
60.03
0.56
0.00
Hill®
<0.0001
0.46
0.002
-1.72 x 10-°7
60.03
0.79
0.00
Lineal
<0.0001
0.46
<0.0001
1.47
69.64
93.61
57.54
Polynomial (2-degree/
<0.0001
0.46
<0.0001
1.47
69.64
93.61
57.54
Polynomial (3-degree/
<0.0001
0.46
<0.0001
1.47
69.64
93.61
57.54
Polynomial (4-degree)f
<0.0001
0.46
<0.0001
1.4
71.62
96.67
57.59
Power6
<0.0001
0.46
<0.0001
1.47
69.64
93.61
57.54
Nonconstant variance
Exponential (Model 2)e
<0.0001
0.66
<0.0001
-0.52
69.28
108.34
53.81
Exponential (Model 3)e
<0.0001
0.66
<0.0001
-0.52
69.28
108.34
53.81
Exponential (Model 4)e
<0.0001
0.66
0.0043
-0.01
59.54
0.36
0.003
Exponential (Model 5)e
<0.0001
0.66
0.0010
-0.01
61.54
0.49
0.003
Hill®
<0.0001
0.66
0.0010
0.13
61.54
0.82
1.25 x 10-13
Linearf
<0.0001
0.66
<0.0001
-0.40
69.27
111.00
66.71
Polynomial (2-degree/
<0.0001
0.66
<0.0001
-0.40
69.27
111.00
66.71
Polynomial (3-degree/
<0.0001
0.66
<0.0001
-0.28
71.19
113.29
67.15
100
Soluble Tungsten Compounds
-------
FINAL
09-29-2015
Polynomial (4-degree)f
<0.0001
0.66
<0.0001
-0.21
71.07
114.45
67.83
Power6
<0.0001
0.66
<0.0001
-0.40
69.27
111.00
66.71
aOsterburg et al. (2014)
bValues >0.05 fail to meet conventional goodness-of-fit criteria.
°Values <0.10 fail to meet conventional goodness-of-fit criteria.
dScaled residuals for dose group near the BMD.
Tower restricted to >1.
Coefficients restricted to be negative.
For the percentage CD71+ cytotoxic T cell data sets from the 28-day study were
amenable to BMD modeling (see Table D-10). With constant variance model applied, none of
the models provided an adequate fit to the variance. After applying the model for nonconstant
variance, all models provided an adequate fit to the means except for the Exponential Models 3
and 5. BMDLs for models providing adequate fit were sufficiently close (differed by less than
two- to three-fold), so the model with the lowest AIC was selected (Hill, with nonconstant
variance model applied).
Table D-10. Modeling Results for Percentage CD71+ Cytotoxic T Cells in the Spleen of
Adult C57BL/6J Mice Exposed to Sodium Tungstate in Drinking Water for 28 Days3
Model
Test for
Significant
Difference
/>-Valucb
Variance
/>-Valuce
Means
/>-Valuce
Scaled
Residuals'1
AIC
BMDisd
(mg W/kg-d)
BMDLisd
(mg W/kg-d)
Constant variance
Exponential (Model 2)e
0.0004
0.02
0.91
-0.23
216.74
59.78
33.10
Exponential (Model 3)e
0.0004
0.02
0.91
-0.23
216.74
59.78
33.10
Exponential (Model 4)e
0.0004
0.02
0.86
0.10
218.59
51.83
18.81
Exponential (Model 5)e
0.0004
0.02
NA
0.00
220.56
51.73
18.95
Hill®
0.0004
0.02
NA
0.00
220.56
51.01
12.18
Linearf
0.0004
0.02
0.54
-0.62
217.79
82.06
56.46
Polynomial (2-degree/
0.0004
0.02
0.54
-0.62
217.79
82.06
56.46
Polynomial (3-degree/
0.0004
0.02
0.54
-0.62
217.79
82.06
56.46
Power6
0.0004
0.02
0.54
-0.62
217.79
82.06
56.46
Nonconstant variance
Exponential (Model 2)e
0.0004
0.11
0.28
-0.32
215.32
83.12
41.32
Exponential (Model 3)e
0.0004
0.11
<0.0001
-1.90
235.08
15,535.20
31.77
Exponential (Model 4)e
0.0004
0.11
0.19
-0.05
216.55
71.93
27.36
Exponential (Model 5)e
0.0004
0.11
NA
-0.31
216.92
56.44
36.86
Hill6
0.0004
0.11
0.74
-0.31
214.92
49.07
39.87
Linearf
0.0004
0.11
0.12
0.36
217.06
106.04
66.23
Polynomial (2-degree/
0.0004
0.11
0.12
0.36
217.06
106.04
66.23
101
Soluble Tungsten Compounds
-------
FINAL
09-29-2015
Polynomial (3-degree/
0.0004
0.11
0.12
0.36
217.06
106.04
66.23
Power6
0.0004
0.11
0.12
0.36
217.06
106.04
66.23
aOsterburg et at. (2014)
bValues >0.05 fail to meet conventional goodness-of-fit criteria.
°Values <0.10 fail to meet conventional goodness-of-fit criteria.
dScaled residuals for dose group near the BMD.
Tower restricted to >1.
'Coefficients restricted to be negative.
NA = not applicable (BMDL computation failed or the BMD was higher than the highest dose tested).
The BMDS output for the selected model follows:
Hill Model, with BMR of 1 Std. Dev. for the BMD and 0.95 Lower Confidence Limit for the BMDL
18
16
14
12
10
BMDL
20
BMD
40
60
dose
30
100
120
15:05 07/16 2014
Figure D-8. Nonconstant Variance Hill (Percentage CD71+ Cytotoxic T Cells, Mice,
28 Days, Drinking Water)
Hill Model. (Version: 2.17; Date: 01/28/2013)
Input Data File:
C:/USEPA/PTV/NaTungstate/cytotoxic_Tcell/adult28day/hil_cytotox_Tcell_adult_2 8day_Hil-
ModelVariance-BMRlStd-Restrict.(d)
102
Soluble Tungsten Compounds
-------
FINAL
09-29-2015
Gnuplot Plotting File:
C:/USEPA/PTV/NaTungstate/cytotoxic_Tcell/adult2 8day/hil_cytotox_Tcell_adult_2 8day_Hil-
ModelVariance-BMRlStd-Restrict.pit
Wed Jul 16 15:05:32 2014
BMDS Model Run
The form of the response function is:
Y[dose] = intercept + v*dose^n/(k^n + dose^n)
Dependent variable = Mean
Independent variable = Dose
Power parameter restricted to be greater than 1
The variance is to be modeled as Var(i) = exp(lalpha + rho * ln(mean(i)))
Total number of dose groups = 4
Total number of records with missing values = 0
Maximum number of iterations = 5 00
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
Default Initial Parameter Values
lalpha = 3.47363
rho = 0
intercept = 12.87
v = -8.43
n = 11.8827
k = 38.4977
Asymptotic Correlation Matrix of Parameter Estimates
( *** The model parameter(s) -n
have been estimated at a boundary point, or have been specified by
the user,
lalpha
rho
intercept
and do not appear in the correlation matrix )
lalpha
1
-0. 98
0.35
-0.49
-0.15
rho
-0.98
1
-0.41
0.52
0.15
intercept
0.35
-0.41
1
-0. 94
-0.54
v
-0.49
0.52
-0.94
1
0.48
k
-0.15
0.15
-0.54
0.48
1
Interval
Variable
Limit
lalpha
3.23412
rho
2.60907
intercept
16.5982
v
2.29559
n
Estimate
0.612805
1.33432
12.4839
-7.46343
-11.9627
18
Parameter Estimates
Std. Err.
1.33743
0.65039
2.09917
95.0% Wald Confidence
Lower Conf. Limit Upper Conf.
-2.00851
0.0595841
8.36961
-2.96414
NA
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39.4424
2.17384
35.1817
43.703
NA - Indicates that this parameter has hit a bound
implied by some inequality constraint and thus
has
no standard error.
Table of Data and
Estimated Values of Interest
Dose
N Obs Mean Est Mean Obs Std Dev
Est Std Dev
0
12 12.9
12.5 7.1
7.32
39
12 8.6
9.13 6.79
5.94
78
12 5.75
5.02 2.88
3. 99
125
12 4.44
5.02 4.92
3. 99
Model Descriptions for
likelihoods calculated
Model A1
: Yij =
Mu(i) + e(i j)
Var{e(ij)} =
SigmaA2
Model A2
: Yij =
Mu(i) + e(i j)
Var{e(ij)} =
Sigma(i)A2
Model A3
: Yij =
Mu(i) + e(i j)
Var{e(ij)} =
exp(lalpha + rho*ln(Mu(i)))
Model A3 uses any
fixed variance parameters that
were
specified by
the user
Model R
: Yi =
Mu + e(i)
Var{e (i)} =
SigmaA2
Likelihoods of Interest
Model
Log(likelihood) # Param's
AIC
A1
-105.278932 5
220.557864
A2
-100.231462 8
216.462923
A3
-102.403795 6
216.807590
fitted
-102.458954 5
214.917909
R
-112.540270 2
229.080539
Explanation of Tests
Test 1:
Do responses
and/or variances differ among
Dose levels?
(A2 vs. R)
Test 2:
Are Variances
Homogeneous? (A1 vs A2)
Test 3:
Are variances
adequately modeled? (A2 vs. A3)
Test 4:
Does the Model for the Mean Fit? (A3 vs. fitted)
(Note:
When rho=0 the
results of Test 3 and Test 2
will be the
Tests of Interest
0.183
-0.309
0. 634
-0.504
Test
p-value
Test 1
Test 2
Test 3
Test 4
24.6176
10.0949
4.34467
0.110319
0.0004018
0.01778
0.1139
0.7398
The p-value for Test 1 is less than .05. There appears to be a
difference between response and/or variances among the dose levels
It seems appropriate to model the data
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The p-value for Test 2 is less than . 1. A non-homogeneous variance
model appears to be appropriate
The p-value for Test 3 is greater than .1. The modeled variance appears
to be appropriate here
The p-value for Test 4 is greater than .1. The model chosen seems
to adeguately describe the data
Benchmark Dose Computation
Specified effect = 1
Risk Type = Estimated standard deviations from the control mean
Confidence level = 0.95
BMD = 49.0739
BMDL = 39.8748
The percentage CD71+ cytotoxic T cell data sets from the 19-week (F0 mice) study were
not amenable to modeling, because the available constant-variance and nonconstant-variance
models did not provide adequate fits to the variances (see Table D-l 1).
Table D-ll. Modeling Results for Percentage CD71+ Cytotoxic T Cells in the Spleen of F0
C57BL/6J Mice Exposed to Sodium Tungstate in Drinking Water for 19 Weeks"
Model
Test for
Significant
Difference
/>-Valucb
Variance
p-Value0
Means
/>-Valuce
Scaled
Residuals'1
AIC
BMDisd
(mg W/kg-d)
BMDLisd
(mg W/kg-d)
Constant variance
Exponential (Model 2)e
<0.0001
<0.0001
0.72
0.79
102.85
42.25
22.76
Exponential (Model 3)e
<0.0001
<0.0001
0.62
0.33
104.45
52.56
23.77
Exponential (Model 4)e
<0.0001
<0.0001
0.72
0.79
102.85
42.25
19.26
Exponential (Model 5)e
<0.0001
<0.0001
0.50
0.0005
105.96
52.43
22.83
Hill6
<0.0001
<0.0001
0.50
9.06 x 10-°6
105.96
50.08
20.56
Linearf
<0.0001
<0.0001
0.68
-0.87
103.00
60.21
41.93
Polynomial (2-degree/
<0.0001
<0.0001
0.68
-0.87
103.00
60.21
41.93
Polynomial (3-degree/
<0.0001
<0.0001
0.68
-0.87
103.00
60.21
41.93
Polynomial (4-degree)f
<0.0001
<0.0001
0.68
-0.87
103.00
60.21
41.93
Power6
<0.0001
<0.0001
0.68
-0.87
103.00
60.21
41.93
Nonconstant variance
Exponential (Model 2)e
<0.0001
0.0004
0.02
0.78
104.72
38.97
19.32
Exponential (Model 3)e
<0.0001
0.0004
0.74
0.00
97.75
124.35
111.94
Exponential (Model 4)e
<0.0001
0.0004
0.03
0.66
104.10
0.96
0.13
Exponential (Model 5)e
<0.0001
0.0004
0.44
0.00
99.75
124.35
86.45
105
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Hill®
<0.0001
0.0004
0.02
0.31
104.96
39.75
NA
Linearf
<0.0001
0.0004
0.04
-0.86
103.57
72.04
NA
Polynomial (2-degree/
<0.0001
0.0004
0.16
0.55
100.37
119.54
94.66
Polynomial (3-degree/
<0.0001
0.0004
0.41
0.26
98.02
124.05
107.52
Polynomial (4-degree)f
<0.0001
0.0004
0.62
0.14
96.91
124.28
111.85
Power®
<0.0001
0.0004
0.90
0.00
95.75
124.67
115.50
aOsterburg et al. (2014)
bValues >0.05 fail to meet conventional goodness-of-fit criteria.
°Values <0.10 fail to meet conventional goodness-of-fit criteria.
dScaled residuals for dose group near the BMD.
Tower restricted to >1.
Coefficients restricted to be negative.
NA = not applicable (BMDL computation failed or the BMD was higher than the highest dose tested).
The percentage CD71+ cytotoxic T cell data sets from the 32-week (F1 mice) study were
not amenable to modeling because none of the constant or nonconstant variance models provided
adequate fits to the means (see Table D-12).
Table D-12. Modeling Results for Percentage CD71+ Cytotoxic T Cells in the Spleen of F1
C57BL/6J Mice Exposed to Sodium Tungstate in Drinking Water for 32 Weeks"
Model
Test for
Significant
Difference
/>-Valucb
Variance
/j-Value'
Means
/>-Valuce
Scaled
Residuals0
AIC
BMDisd
(mg W/kg-d)
BMDLisd
(mg W/kg-d)
Constant variance
Exponential (Model 2)e
<0.0001
0.21
<0.0001
3.13
56.09
62.07
37.18
Exponential (Model 3)e
<0.0001
0.21
<0.0001
3.13
56.09
62.07
37.18
Exponential (Model 4)e
<0.0001
0.21
<0.0001
3.13
56.09
62.07
0.46
Exponential (Model 5)e
<0.0001
0.21
<0.0001
3.13
58.09
62.07
0.42
Hill6
<0.0001
0.21
<0.0001
0.02
56.36
2.40
0.35
Lineal
<0.0001
0.21
<0.0001
3.02
55.82
68.19
46.07
Polynomial (2-degree/
<0.0001
0.21
<0.0001
2.77
57.60
78.97
46.70
Polynomial (3-degree/
<0.0001
0.21
<0.0001
2.41
56.73
89.78
49.71
Polynomial (4-degree)f
<0.0001
0.21
<0.0001
2.15
55.86
95.91
54.37
Power6
<0.0001
0.21
<0.0001
3.02
55.82
68.19
46.07
Nonconstant variance
Exponential (Model 2)e
<0.0001
0.66
<0.0001
-2.65
57.62
53.84
26.91
Exponential (Model 3)e
<0.0001
0.66
<0.0001
-0.00016
50.81
119.63
99.51
Exponential (Model 4)e
<0.0001
0.66
<0.0001
-0.30
55.28
1.61
0.51
Exponential (Model 5)e
<0.0001
0.66
<0.0001
-0.00016
52.81
119.63
98.90
Hill6
<0.0001
0.66
<0.0001
-0.35
55.12
1.76
0.39
106
Soluble Tungsten Compounds
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Linearf
<0.0001
0.66
<0.0001
2.95
57.78
66.90
41.52
Polynomial (2-degree/
<0.0001
0.66
<0.0001
2.58
54.60
94.70
80.35
Polynomial (3-degree/
<0.0001
0.66
<0.0001
-0.15
51.60
103.16
89.83
Polynomial (4-degree)f
<0.0001
0.66
<0.0001
-0.08
50.26
107.85
100.00
Power6
<0.0001
0.66
<0.0001
-8.65 x 10-°5
48.81
120.83
100.81
aOsterburg et al. (2014)
bValues >0.05 fail to meet conventional goodness-of-fit criteria.
°Values <0.10 fail to meet conventional goodness-of-fit criteria.
dScaled residuals for dose group near the BMD.
Tower restricted to >1.
Coefficients restricted to be negative.
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Soluble Tungsten Compounds
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