United States
Environmental Protection
1=1 m m Agency
EPA/690/R-13/01 OF
Final
4-04-2013
Provisional Peer-Reviewed Toxicity Values for
Technical Grade Dinitrotoluene
(CASRN 25321-14-6)
Superfund Health Risk Technical Support Center
National Center for Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, OH 45268

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AUTHORS, CONTRIBUTORS, AND REVIEWERS
CHEMICAL MANAGER
Zhongyu (June) Yan, PhD
National Center for Environmental Assessment, Cincinnati, OH
Q. Jay Zhao, PhD, DABT
National Center for Environmental Assessment, Cincinnati, OH
PRIMARY INTERNAL REVIEWERS
Ghazi Dannan, PhD
National Center for Environmental Assessment, Washington, DC
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).
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TABLE OF CONTENTS
COMMONLY USED ABBREVIATIONS	iii
BACKGROUND	1
DISCLAIMERS	1
QUESTIONS REGARDING PPRTVs	1
INTRODUCTION	2
REVIEW OF POTENTIALLY RELEVANT DATA (CANCER AND NONCANCER)	7
HUMAN STUDIES	17
Oral Exposures	17
Inhalation Exposures	17
ANIMAL STUDIES	23
Oral Exposures	23
Inhalation Exposures	33
Other Data (Other Examinations)	34
DERIVATION OF PROVISIONAL REFERENCE DOSES	37
DERIVATION OF ORAL REFERENCE DOSES	38
Derivation of Subchronic Provisional RfD (Subchronic p-RfD)	38
Derivation of Chronic Provisional RfD (Chronic p-RfD)	38
DERIVATION OF INHALATION REFERENCE CONCENTRATIONS	38
CANCER WEIGHT-OF-EVIDENCE DESCRIPTOR	38
MODE-OF-ACTION DISCI SSION	40
DERIVATION OF PROVISIONAL CANCER POTENCY VALUES	41
Derivation of Provisional Oral Slope Factor (p-OSF)	41
Derivation of Provisional Inhalation Unit Risk (p-IUR)	41
APPENDIX A. PROVISIONAL SCREENING VALUES	42
APPENDIX B. DATA TABLES	51
APPENDIX C. BMD MODELING RESULTS	75
APPENDIX D. BENCHMARK DOSE CALCULATIONS FOR THE SCREENING
p-OSF	84
APPENDIX E. REFERENCES	107
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COMMONLY USED ABBREVIATIONS
BMC
benchmark concentration
BMCL
benchmark concentration lower bound 95% confidence interval
BMD
benchmark dose
BMDL
benchmark dose lower bound 95% confidence interval
HEC
human equivalent concentration
HED
human equivalent dose
IUR
inhalation unit risk
LOAEL
lowest-observed-adverse-effect level
LOAELadj
LOAEL adjusted to continuous exposure duration
LOAELhec
LOAEL adjusted for dosimetric differences across species to a human
NOAEL
no-ob served-adverse-effect level
NOAELadj
NOAEL adjusted to continuous exposure duration
NOAELhec
NOAEL adjusted for dosimetric differences across species to a human
NOEL
no-ob served-effect level
OSF
oral slope factor
p-IUR
provisional inhalation unit risk
POD
point of departure
p-OSF
provisional oral slope factor
p-RfC
provisional reference concentration (inhalation)
p-RfD
provisional reference dose (oral)
RfC
reference concentration (inhalation)
RfD
reference dose (oral)
UF
uncertainty factor
UFa
animal-to-human uncertainty factor
UFC
composite uncertainty factor
UFd
incomplete-to-complete database uncertainty factor
UFh
interhuman uncertainty factor
UFl
LOAEL-to-NOAEL uncertainty factor
UFS
subchronic-to-chronic uncertainty factor
WOE
weight of evidence
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PROVISIONAL PEER-REVIEWED TOXICITY VALUES FOR
TECHNICAL GRADE DINITROTOLUENE (CASRN 25321-14-6)
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 flittp://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.eov/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.
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
Technical grade dinitrotoluene (tgDNT), CAS No. 25321-14-6, is a mixture of
dinitrotoluene (DNT) isomers with the molecular formula C7H6N2O4 (MA1. 2011). tgDNT
comprises predominantly 2,4-DNT and 2,6-DNT (approximated as 76% and 19%, respectively).
The remaining 5% is a combination of the four other DNT isomers: 2,3-, 2,5-, 3,4-, and
3,5-DNT. tgDNT may also contain trace amounts of trinitrotoluene, cresols, mononitrobenzene,
and mononitrotoluenes and is used in the production of toluene diisocyanate, which is used in the
preparation of polyurethane products and in the manufacture of explosives (NLM. 2011).
Table 1 provides physicochemical properties of tgDNT.
Table 1. Physicochemical Properties of tgDNT (CASRN 25321-14-6)a
Property (unit)
Value
Boiling point (°C)
250
Melting point (°C)
ND
Density (g/cm3 at 71°C)
1.32
Vapor pressure (mm Hg at 25°C)
3.97 x 1(T4
pH (unitless)
ND
Solubility in water (mg/L at 22°C)
2.7 x 102
Relative vapor density (air =1)
6.27
Molecular weight (g/mol)
182.134
aNLM ("20ID.
ND = no data.
IRIS has developed assessments for 2,4-DNT (approximately 98% 2,4-DNT and 2%
2,6-DNT; U.S. EPA. 1993) and for a 2,4-/2,6-DNT mixture (various compositions of DNTs; U.S.
EPA. 1990). There is also a PPRTV assessment for 2,6-DNT (approximately 99% 2,6-DNT;
U.S. EPA, 2013). Table 2 provides a summary of available toxicity values from the
U.S. Environmental Protection Agency (U.S. EPA) and other agencies/organizations for tgDNT,
the 2,4-DNT and 2,6-DNT isomers, and the 2,4-/2,6-DNT mixture. For the purpose of this
PPRTV, only the toxicity of tgDNT (approximated as 76% 2,4-DNT and 19% 2,6-DNT) is
evaluated.
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Table 2. Summary of Available Toxicity Values for tgDNT (CASRN 25321-14-6), 2,4-DNT (CASRN 121-14-2),
2,6-DNT (CASRN 606-20-2), and 2,4-/2,6-DNT Mixture (no CASRN)a
Source/
Parameterb'c
tgDNT Value
(approximately
76% 2,4-DNT
and 19%
2,6-DNT)
2,4-DNT Value
(approximately
98% 2,4-DNT
and 2%
2,6-DNT)
2,6-DNT Value
(approximately
99% 2,6-DNT)
2,4-/2,6-DNT
Mixture Value
(various
compositions of
DNTs)
Notes
Reference
Date
Accessed
Cancer
IRIS/OSF
NV
NV
NV
6.8 x 10 1 per
mg/kg-d
IRIS entry is for 2,4-/2,6-DNT mixture with no
CASRN; principal study used rats dosed with a
mixture of 98% 2,4-DNT and 2% 2,6-DNT to
determine OSF
U.S. EPA
(1990)
9-13-2012
IRIS/drinking
water unit risk
NV
NV
NV
1.9 x 10~5 per
Hg/L
IRIS entry is for 2,4-/2,6-DNT mixture with no
CASRN; principal study used rats dosed with a
mixture of 98% 2,4-DNT and 2% 2,6-DNT to
determine OSF
U.S. EPA
(1990)
9-13-2012
HEAST
NV
NV
NV
NV
None
U.S. EPA
(2003)
9-13-2012
IARC/cancer
WOE
NV
NV
NV
NV
Group 2B—Possibly carcinogenic to humans
for 2,4- and 2,6-DNT
IARC (1996)
9-13-2012
NTP
NV
NV
NV
NV
None
NTP (2011)
9-13-2012
Cal EPA/unit risk
NV
8.9 x 1(T5 per
Hg/m3
NV
NV
Data source was RCHAS-S
Cal EPA
(2009)
9-13-2012
Cal EPA/OSF
NV
3.1 x 10 1 per
mg/kg-d
NV
NV
Data source was RCHAS-S
Cal EPA
(2009)
9-13-2012
ACGIH (cited in
NLM. 2011)
NV
NV
NV
NV
Group A3—Confirmed animal carcinogen with
unknown relevance to humans for tgDNT, 2,4-
and 2,6-DNT
NLM (2011)
9-13-2012
Drinking Water/
cancer risk health
advisory
5 x 1CT3 mg/L
5 x 10~3 mg/L
5 x 10~3 mg/L
NV
None
U.S. EPA
(2011a)
9-13-2012
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Table 2. Summary of Available Toxicity Values for tgDNT (CASRN 25321-14-6), 2,4-DNT (CASRN 121-14-2),
2,6-DNT (CASRN 606-20-2), and 2,4-/2,6-DNT Mixture (no CASRN)a
Source/
Parameterb'c
tgDNT Value
(approximately
76% 2,4-DNT
and 19%
2,6-DNT)
2,4-DNT Value
(approximately
98% 2,4-DNT
and 2%
2,6-DNT)
2,6-DNT Value
(approximately
99% 2,6-DNT)
2,4-/2,6-DNT
Mixture Value
(various
compositions of
DNTs)
Notes
Reference
Date
Accessed
Health effect
assessment
2.3 x lCT1 per
mg/kg-dd and
2.1 x lO^per
mg/kg-de
6.8 x 10 1 per
mg/kg-df
NV
NV
dBased on a 104-wk study in rats with
increased incidence of liver tumors in males;
"Based on a 104-wk study in rats with
increased incidence of liver tumors in females;
fBased on a 2-yr study in rats with increased
incidence of combined mammary/hepatic
tumors;
U.S. EPA
(1987)
2-6-2013
PPRTV
NV
NV
1.5 x 10° per
mg/kg-d
NV
Based on a BMDL10Hed of 0.25 from a 52-wk
study in rats with increased incidence of liver
tumors in males
U.S. EPA
(2013)
4-3-2013
Noncancer
ACGIH/TLV
0.2 mg/m3
NV
NV
NV
NA
NLM (2011)
9-13-2012
ATSDR/acute
oral MRL
NV
5 x 10~2
mg/kg-d
NV
NV
Toxicological profile for 2,4-DNT; based on
neurotoxicity in dogs
ATSDR
(1998)
11-21-2012
ATSDR/chronic
or intermediate-
duration oral
MRL
NV
2 x 10~3
mg/kg-d8
4 x 1() 3 mg/kg-dh
NV
8Chronic oral MRL for 2,4-DNT; based on
neurotoxicity, Heinz bodies, and biliary tract
hyperplasia in dogs; hIntermediate-duration
oral MRLfor 2,6-DNTbased on hematological
effects of splenic extramedullary
erythropoiesis and lymphoid depletion in dogs
ATSDR
(1998)
11-21-2012
Cal EPA/REL
NV
NV
NV
NV
NA
Cal EPA
(2012a. b)
8-1-2012
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Table 2. Summary of Available Toxicity Values for tgDNT (CASRN 25321-14-6), 2,4-DNT (CASRN 121-14-2),
2,6-DNT (CASRN 606-20-2), and 2,4-/2,6-DNT Mixture (no CASRN)a
Source/
Parameterb'c
tgDNT Value
(approximately
76% 2,4-DNT
and 19%
2,6-DNT)
2,4-DNT Value
(approximately
98% 2,4-DNT
and 2%
2,6-DNT)
2,6-DNT Value
(approximately
99% 2,6-DNT)
2,4-/2,6-DNT
Mixture Value
(various
compositions of
DNTs)
Notes
Reference
Date
Accessed
Drinking water
NV
2 x 1(T3
mg/kg-d (1-d
Health advisory)
1 x 1CT1 mg/L
(Drinking water
equivalent level)
1 x 10° mg/L (1-
and 10-d Health
advisory for a
10-kg child)
1 x 10 3 mg/kg-d
(1-d Health
advisory)
4 x 10 2 mg/L
(Drinking water
equivalent level)
4 x 1CT1 and
4 x l(T2 mg/L (1-
and 10-d Health
advisory for a
10-kg child)
NV
NA
U.S. EPA
(201 la)
2-6-2013
NIOSH/REL
1.5 mg/m3
NV
NV
NV
TWA for 10-hr workday; document specifies
CASRN for tgDNT but notes that various
isomers of DNT exist
NIOSH
9-13-2012
(2007)
OSH A/PEL
1.5 mg/m3
NV
NV
NV
TWA for 8-hr workday
OSHA
9-13-2012
(2006)
IRIS/Oral RfD
NV
2 x 1(T3
mg/kg-d
NV
NV
Based on a 2-yr study in dogs dosed with 98
2,4-DNT and 2% 2,6-DNT; critical effect of
CNS neurotoxicity, Heinz bodies in
erythrocytes, and hyperplasia of biliary tract
U.S. EPA
9-13-2012
(1993)
IRIS/Inhalation
RfC
NV
NV
NV
NV
None
U.S. EPA
(1990)
9-13-2012
HEAST/
subchronic Oral
RfD
NV
2 x icr3
mg/kg-d
NV
NV
Based on a 2-yr study in dogs dosed with a
mixture of 98% 2,4-DNT and 2% 2,6-DNT;
critical effect of CNS neurotoxicity, Heinz
bodies in erythrocytes, and hyperplasia of
biliary tract
U.S. EPA
(2003)
9-13-2012
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Table 2. Summary of Available Toxicity Values for tgDNT (CASRN 25321-14-6), 2,4-DNT (CASRN 121-14-2),
2,6-DNT (CASRN 606-20-2), and 2,4-/2,6-DNT Mixture (no CASRN)a
Source/
Parameterb'c
tgDNT Value
(approximately
76% 2,4-DNT
and 19%
2,6-DNT)
2,4-DNT Value
(approximately
98% 2,4-DNT
and 2%
2,6-DNT)
2,6-DNT Value
(approximately
99% 2,6-DNT)
2,4-/2,6-DNT
Mixture Value
(various
compositions of
DNTs)
Notes
Reference
Date
Accessed
Health effects
assessment
NV
NV
NV
NV
NA
U.S. EPA
(1987)
2-6-2013
PPRTV
NV
NV
3 x 10 3 mg/kg-d
(screening
subchronic
p-RfD)
3 x 1() 4 mg/kg-d
(screening
chronic p-RfD)
NV
Based on a LOAELhed of 3 mg/kg-d for
splenic extramedullary hematopoiesis in male
and female dogs in a 13-wk oral study
U.S. EPA
4-3-2013
(2013)
CARA HEEP
NV
NV
NV
NV
None
U.S. EPA
(1994)
9-13-2012
WHO
NV
NV
NV
NV
None
WHO (2012)
8-1-2012
aNo information was available from any source for 2,3-, 2,5-, 3,4-, and 3,5-DNT.
bSources: Integrated Risk Information System (IRIS); Health Effects Assessment Summary Tables (HEAST); International Agency for Research on Cancer (IARC);
National Toxicology Program (NTP); California Environmental Protection Agency (Cal EPA); American Conference of Governmental Industrial Hygienists (ACGIH);
Agency for Toxic Substances and Disease Registry (ATSDR); National Institute for Occupational Safety and Health (NIOSH); Occupational Safety and Health
Administration (OSHA); Chemical Assessments and Related Activities (CARA); Health and Environmental Effects Profile (HEEP); World Health Organization
(WHO).
Parameters: weight of evidence (WOE); reference dose (RfD); reference concentration (RfC); oral slope factor (OSF); minimum risk level (MRL); time-weighted
average (TWA); reference exposure level (REL); permissible exposure limit (PEL); Reproductive and Cancer Hazard Assessment Section (RCHAS).
d ''See notes column for corresponding information.
NA = not applicable; NV = not available.
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Literature searches were conducted on sources published from 1900 through
July 10, 2012 for studies relevant to the derivation of provisional toxicity values for tgDNT. The
following databases were searched by chemical name, synonyms, or CAS No.: ACGIH,
ANEUPL, AT SDR, BIO SIS, Cal EPA, CCRIS, CD AT, ChemlDplus, CIS, CRISP, DART,
EMIC, EPIDEM, ETICBACK, FEDRIP, GENE-TOX, HAPAB, HERO, HMTC, HSDB, IARC,
INCHEM IPCS, IP A, ITER, IUCLID, LactMed, NIOSH, NTIS, NTP, OSHA, OPP/RED,
PESTAB, PPBIB, PPRTV, PubMed (toxicology subset), RISKLINE, RTECS, TOXLINE, TRI,
U.S. EPA IRIS, U.S. EPA HEAST, U.S. EPA HEEP, U.S. EPA OW, and U.S. EPA
TSCATS/TSCATS2. The following databases were searched for relevant health information:
ACGIH, AT SDR, Cal EPA, U.S. EPA IRIS, U.S. EPA HEAST, U.S. EPA HEEP, U.S. EPA
OW, U.S. EPA TSCATS/TSCATS2, NIOSH, NTP, OSHA, and RTECS.
REVIEW OF POTENTIALLY RELEVANT DATA
(CANCER AND NONCANCER)
Table 3 provides an overview of the relevant database for tgDNT and includes potentially
relevant repeated long-term-, subchronic-, and chronic-duration studies. The phrase "statistical
significance," used throughout the document, indicates ap-value of <0.05.
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Table 3. Summary of Potentially Relevant Data for tgDNT (CASRN 25321-14-6)
Category
Number of
Male/Female, Strain,
Species, Study Type,
Study Duration
Dosimetry3
Critical Effects
NOAEL
BMDL/
BMCL
LOAEL
Reference
Comments
Notes
Human
1. Oral
Acuteb
ND
Short-term0
ND
Long-termd
ND
Chronic"
ND
2. Inhalation
Acuteb
ND
Short-term0
ND
Long-termd
154/0 workers,
occupational survey,
12 mo, mixed isomers
of DNT (unknown
composition of DNT)
NV
Complaints such as
unpleasant taste,
weakness, headache,
loss of appetite,
dizziness, nausea,
insomnia, pain in
extremities,
vomiting, and
numbness and
tinging; clinical signs
included low-grade
anemia, and cyanosis
NV
DU
NV
McGee et al.
(1942)

PR
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Table 3. Summary of Potentially Relevant Data for tgDNT (CASRN 25321-14-6)
Category
Number of
Male/Female, Strain,
Species, Study Type,
Study Duration
Dosimetry3
Critical Effects
NOAEL
BMDL/
BMCL
LOAEL
Reference
Comments
Notes
Long-termd
714 workers (sex not
reported), occupational
survey, 3 yr, mixed
isomers of DNT
(unknown composition
of DNT)
<1 mg/m3
Fewer complaints of
unpleasant taste,
weakness, headache,
loss of appetite,
dizziness, nausea,
insomnia, pain in
extremities,
vomiting, and
numbness and
tinging; reduced
incidences of
low-grade anemia,
and cyanosis
compared to McGee
et al. (1942) studv
NV
DU
NV
McGee et al.
A follow-up
study to McGee
et al. (1942)
PR
(1947)
30/0 workers,
occupational survey,
exposure duration
(varies), mixed isomers
of DNT (unknown
composition of DNT)
and coexposed to
toluene diamine (TDA)
undetectable-
0.23 mg/m3
(personal sample);
undetectable-
042 mg/m3 (area
sample)
Significant reduction
in sperm count
NC
NC
NC
Alirenliolz
(1980)

NPR
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Table 3. Summary of Potentially Relevant Data for tgDNT (CASRN 25321-14-6)
Category
Number of
Male/Female, Strain,
Species, Study Type,
Study Duration
Dosimetry3
Critical Effects
NOAEL
BMDL/
BMCL
LOAEL
Reference
Comments
Notes
Long-termd
50/0 workers (nonsemen
study) and 41/0 workers
(semen study);
occupational survey,
exposure duration
(varies), mixed isomers
of DNT (approximately
80% 2,4-DNT and 20%
2,6-DNT) and
coexposed to TDA
0.00929-0.318
mg/m3
(time-weighted
average, TWA)
No significant
difference in serum
enzymes, sperm
volume, sperm
counts and
morphological
changes in workers
in exposed group,
and spontaneous
abortions in their
wives
0.0739
mg/m3
(mean,
TWA)
DU
NV
Alirenliolz and
Mever (1982)

NPR

203/0 workers,
occupational survey,
>6 mo, coexposure to
DNT and TDA, mixed
isomers of DNT
(unknown composition
of DNT)
<1.5 mg/m3
(Permissible
Exposure Limit,
PEL)
No significant
differences in work
history, medical
history, physical
examination,
reproductive history,
fertility, laboratory
serum (follicle-
stimulating
hormone), mean
sperm count, and
sperm morphology in
exposed group
NV
DU
NV
Hamill et al.
(1982)
Investigated
only male
reproductive and
fertility
endpoints
PR

208/10 workers,
occupational study, no
specified exposure
duration, DNT/TDA,
mixed isomers of DNT
(unknown composition
of DNT)
NV
No significant
associations found
for male
reproductive toxicity
NV
DU
NV
Levine (1983)
Exposure-related
information on
fertility among
female
employees was
insufficient to
analyze
NPR
Reproductive
study
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Table 3. Summary of Potentially Relevant Data for tgDNT (CASRN 25321-14-6)
Category
Number of
Male/Female, Strain,
Species, Study Type,
Study Duration
Dosimetry3
Critical Effects
NOAEL
BMDL/
BMCL
LOAEL
Reference
Comments
Notes
Long-termd
586 workers (sex not
reported), retrospective
survey, DNT/TDA
mixed isomers of DNT
(unknown composition
of DNT)

No significant
difference was found
between the fertility
of workers exposed
to DNT in the three
U.S. chemical plants
and the fertility of
unexposed workers
NV
DU
NV
Levine et al.

PR
(1985)
156/0 and 301/0 (two
cohorts from two
different plants),
occupational cohort
study, exposure at least
30 d during 1950s for
the first cohort;
exposure for >30 d
during the 1940s and
1950s for the second
cohort. 76% 2,4-DNT,
19% 2,6-DNT, and 5%
other isomers for the
first cohort; 98%
2,4-DNT and 1%
2,6-DNT for the second
cohort
NV
For total workers in
both plants, no
significant increases
in death from any
specific types of
cancer were
observed. Excess
mortality from
ischemic heart
disease (IHD) at both
plants with
standardized
mortality ratios
(SMRs) of 13 land
143 (95% confidence
intervals [CI]s:
65-234, and
107-187,
respectively)
NV
NU
NA
Levine et al.
(1986)
Additional
analyses
revealed al5-yr
latent period and
suggested a
relationship
between
duration and
intensity of
exposure
PR
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Table 3. Summary of Potentially Relevant Data for tgDNT (CASRN 25321-14-6)
Category
Number of
Male/Female, Strain,
Species, Study Type,
Study Duration
Dosimetry3
Critical Effects
NOAEL
BMDL/
BMCL
LOAEL
Reference
Comments
Notes
Long-termd
4989 exposed (M)/5636
unexposed workers (M),
occupational
cardiovascular mortality
study, participants
exposed for >5 mo,
(unknown composition
of DNT)
NV
No significant
association for an
increased risk of
either IHD or
cerebrovascular
disease
NV
NU
NA
Stavner et al.
(1992)

PR
4989 exposed (M)/7436
unexposed (M),
occupational
carcinogenicity study,
participants exposed for
>5 mo, (unknown
composition of DNT)
NV
Excess of
hepatobiliary cancer
in exposed workers;
standardized
mortality ratio
(SMR) of 2.67 (95%
confidence interval
[CI]: 0.98-5.83)
when compared with
U.S. population;
standard rate ratio
(SRR) of 3.88 (95%
CI: 1.04-14.41)
when compared with
unexposed group
NV
NU
NV
Stavner et al.
(1993)
Failed to
demonstrate an
exposure-
response
relationship
between
duration of
exposure and
hepatobiliary
cancer mortality;
limited by small
number of
participants with
long exposure
durations.
PR
The study
subjects were
selected from
the second
plant
examined by
Levi tie et al.
(1986)
Chronic6
500 workers (sex not
reported), occupational
carcinogenicity study,
exposed for 7 to 37 yr,
30% tgDNT, which
consisted of 75%
2,4-DNT and 20%
2,6-DNT.
NV
High exposure to
DNT related to
urothelial cancer
NV
NU
NV
Bruiting et al.

PR
Retrospective
study
(1999)
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Table 3. Summary of Potentially Relevant Data for tgDNT (CASRN 25321-14-6)
Category
Number of
Male/Female, Strain,
Species, Study Type,
Study Duration
Dosimetry"
Critical Effects
NOAEL
BMDL/
BMCL
LOAEL
Reference
Comments
Notes
Chronic"
180 workers exposed
for 7 to 37 yr (sex not
reported), 30% tgDNT,
which consisted of 75%
2,4-DNT and 20%
2,6-DNT
NV
A straight
dose-dependent
pathological (tubular
and/or glomerular
damage) pattern;
nephrotoxic effect
toward the proximal
tubule under the
exposure conditions
NV
NU
NV
Bruning et al.

PR
(2001)
Three case studies(sex
not reported); >7 yr,
exposure to DNT and
possible exposure to
nitrobenzene, mixed
isomers of DNT
(unknown composition
of DNT)

High exposure to
DNT associated with
human urothelial
cancer
NV
NU
NV
Hartli et al.
(2005)

PR
Animal
1. Oral
Subchronic
10/10, albino Fischer
344 (F344), rat, diet,
4 wk
M: 0,31.9,61.9,
or 134
F: 0, 32.0, 63.6, or
120
Increases in
hematological
parameters,
including
methemoglobin
(MetHb),
reticulocytes, and
Heinz bodies in
males
NDr
NV
31.9
CUT (1983)

NPR
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Table 3. Summary of Potentially Relevant Data for tgDNT (CASRN 25321-14-6)
Category
Number of
Male/Female, Strain,
Species, Study Type,
Study Duration
Dosimetry"
Critical Effects
NOAEL
BMDL/
BMCL
LOAEL
Reference
Comments
Notes
Chronic
10/10 (Study initial
130/130), F344, rat,
diet, 26 wk
M: 0,3.47,13.6,
or 34.6
F: 0,3.22,13.9,
or 34.9
Increased absolute
and relative liver
weights and
increased
hepatotoxicity in
males
3.47
2.16
based on
increased
incidence of
hepatocyte
necrosis in
males
13.6
CUT f1982a)

NPR, PS

10/10, F344, rat, diet,
52 wk
M: 0,3.47, 13.9,
or 34.9
F: 0, 3.46, 13.9, or
35.1
Increased absolute
and relative liver
weight and
hepatotoxicity in
males
NDr
NU
3.47
CUT f 1982a)

NPR
20/20, F344, rat, diet,
55 wk
M: 34.9
F: 35.1
Hepatotoxicity in
both males and
females
NDr
NV
NDr
CUT f 1982a)
All surviving
high dose rats
were terminated
at 55 wk due to
severe toxicity.
NPR
20/20, F344, rat, diet,
78 wk
M: 0, 3.49, or
14.0
F: 0, 3.45, or 14.0
Increased relative
liver weight and
hepatotoxicity in
females
NDr
NU
3.45
CUT f 1982a)

NPR
75-87/84-87, F344,
rat, diet, 104 wk
M: 0,3.51, or
14.0
F: 0,3.46, or 14.0
Increased absolute
and relative liver
weights and
hepatotoxicity in
females
NDr
0.363
based on
increased
incidence of
hepatocyte
necrosis in
males
3.46
CUT f1982a)

NPR, PS

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Table 3. Summary of Potentially Relevant Data for tgDNT (CASRN 25321-14-6)
Category
Number of
Male/Female, Strain,
Species, Study Type,
Study Duration
Dosimetry"
Critical Effects
NOAEL
BMDL/
BMCL
LOAEL
Reference
Comments
Notes
Chronic
28/0, F344, rat, diet,
52 wk
0,35
Decreased body
weight, increased
absolute and relative
liver weights,
accompanied by
pathological findings
in the liver and bile
duct
NDr
NU
35
Leonard et al.

PR
(1987)

Developmental
Female, 22 control, 13,
7, 13,7, 13, 6 for
treatment groups,
respectively, F344 rat,
gavage, GDs 7-20
0, 14,35,37.5,
75, 100, or 150
Increased maternal
relative liver weight
(maternal effects);
increased resorption
at 150 mg/kg-d (fetal
effects)
Maternal:
37.5
Fetal:
100
NU
Maternal:75
Fetal: 150
Price et al.
(1985)
CUT (1982b)

PR
NPR
Reproductive
ND
Carcinogenic
28/0, F344 rat, diet,
52 wk
ADD: 0,35
HED: 0, 9.3
47% increase in
incidence of
hepatocellular
carcinomas
compared with
controls
NV
NV
NV
Leonard et al.
(1987)

PR
75-87/84-87, F344,
rat, diet, 104 wk
ADD: 0,3.51, or
14 (M); 0, 3.46,
or 14.03 (F)
HED: 0,0.922,
or 3.45 (M); 0,
0.851, or 3.23 (F)
Dose-dependent
increase in
hepatocellular
carcinomas and
neoplastic nodules,
mammary
fibroadenomas, and
subcutaneous
fibromas
NV
BMDLiohed:
0.224
NV
CUT (1982a)

NPR, PS

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Table 3. Summary of Potentially Relevant Data for tgDNT (CASRN 25321-14-6)

Number of









Male/Female, Strain,









Species, Study Type,



BMDL/




Category
Study Duration
Dosimetry"
Critical Effects
NOAEL
BMCL
LOAEL
Reference
Comments
Notes
2. Inhalation
Subchronic
ND
Chronic
ND
Developmental
ND
Reproductive
ND
Carcinogenic
ND
""Dosimetry: NOAEL, BMDL/BMCL, and LOAEL values are converted to an adjusted daily dose (ADD in mg/kg-d) for oral noncancer effects. Values are also presented as
a human equivalent dose (HED in mg/kg-d) for oral carcinogenic effects. All long-term exposure values (4 wk and longer) are converted from a discontinuous to a
continuous (weekly) exposure.
''Acute = exposure for <24 hr (U.S. EPA. 2002).
°Short-term = repeated exposure for >24 hr <30 d (U.S. EPA. 2002).
'Long-term = repeated exposure for >30 d <10% lifespan (based on 70-yr typical lifespan) (U.S. EPA. 2002).
"Chronic = repeated exposure for >10% lifespan (U.S. EPA. 2002).
Significant (increase/decrease/difference) means either statistically or biologically significant (increase/decrease/difference) in this document.
GD = gestation day, ND = no data, NA = not applicable, NV = not available, NDr = not determinable, NC = not calculated, NPR = not peer-reviewed, PR = peer-reviewed,
PS = principal study.
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HUMAN STUDIES
Oral Exposures
No studies were identified.
Inhalation Exposures
Relevant data are available from epidemiological studies on the effects in humans of
inhalation exposure to 2,4/2,6-DNT mixtures of various compositions. These effects have been
evaluated in several occupational studies of workers in DNT manufacturing plants in which
exposures were identified by the study authors to be predominantly via the inhalation route with
contributions from the dermal route. No details on the exposure concentrations to DNT are
given in these studies, and, therefore, they can only be used as qualitative descriptions of
symptoms reported upon exposure. Also, in some of the studies, the isomer composition of the
DNT mixture was not specified. The identified studies from occupational exposure to DNT have
examined the clinical long-term effects (McGee et al.. 1947; McGee et al.. 1942). the potential
for adverse reproductive effects (Levine et al, 1985; Levins, 1983; Ahrenholz and Meyer, 1982;
Ham ill et al.. 1982; Ahrenholz, 1980). adverse effects on the cardiovascular system and
carcinogenic risk (Harth et al.. 2005; Brunine et al.. 2001; Bruning et al. 1999; Stavner et al..
1993; Stavner et al.. 1992; Levine et al.. 1986).
Epidemiological Studies of General Toxicity
McGee et al. (1942) and McGee et al. (1947)
In an epidemiological study performed by McGee et al. (1942). 154 male workers in a
military plant that manufactured powder containing a DNT mixture (primarily 2,4-DNT) were
observed for 12 months. In the 12-month period, 96 individuals reported complaints of
unpleasant taste (62%), 78 reported weakness (51%), 76 reported headache (49%), 72 reported
loss of appetite (47%), 68 reported dizziness (44%), 57 reported nausea (37%), 57 reported
insomnia (37%), 40 reported pain in extremities (26%), 35 reported vomiting (23%), and
29 reported numbness and tinging (19%). Additionally, 84 individuals exhibited clinical signs of
sickness, which included pallor (36%), cyanosis (34%), and anemia (23%). These symptoms are
consistent with methemoglobinemia and disappeared 2 to 3 days after exposure to the powder
was terminated. The study authors also reported two instances of acute toxic hepatitis with
jaundice.
After an effort was initiated to reduce the exposure between 1942 and 1945, a follow-up
study by the same investigators in the same plant (McGee et al.. 1947) evaluated 714 workers
"3
(sex not reported) who were exposed to less than 1 mg/m DNT. Each of the individuals
received medical examinations at intervals of 2 to 4 weeks. From these examinations, the study
authors reported that signs and symptoms of illness were noticeably decreased when compared to
the signs and symptoms experienced by the 154 workers from the 12-month study in 1942. Only
13.2%) and 8.7% of men from the follow up study reported weakness and headaches,
respectively, while around 50% of the men from the 1942 study reported the same effects. The
reports of loss of appetite, nausea and vomiting, vertigo, pain, or tingling/numbing in the
extremities were also reduced in this follow-up study as compared to the initial study. Pallor was
rarely observed in the follow-up study, no hepatitis was observed, and a marked reduction in
cyanosis (8.7%) and anemia (10.2%) was reported as well. The study authors (McGee et al..
1947; McGee et al.. 1942) did not provide detailed DNT compositions or exposure data, and no
unexposed control groups were used as a basis for comparison.
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Epidemiological Studies of Reproductive Effects
Ahrenholz (1980) and Ahrenholz and Meyer (1982)
Ahrenholz (1980) reported on the potential for reproductive effects in male workers
exposed to mixed isomers of DNT (composition of DNT was not reported, and exposure
duration varied) and toluene diamine (TDA) in a TDA plant at Olin Chemical Company in
Brandenburg, KY. TDA was produced through catalytic hydrogenation of DNT. The study
authors conducted environmental and medical surveys in September 1979, and a follow-up
investigation was conducted in January 1980. During both the initial and follow-up surveys,
personal air samples were taken by mounting sample collection media in the operators' breathing
zones. Area air sampling was also conducted. Medical evaluations consisting of a detailed
questionnaire were used to acquire information on a range of potentially toxic effects. In
addition, tobacco and alcohol consumption and medical history were recorded. A reproductive
history was also elicited. The wives of workers were given a different, more detailed
reproductive questionnaire in an attempt to validate the information given by the workers
themselves. A physical examination with a special emphasis on the male reproductive system
and secondary sex characteristics was performed. Blood specimens were obtained for analyses
of blood urea nitrogen (BUN), creatinine, bilirubin, alkaline phosphatase, serum glutamic oxalic
transaminase (SGOT), serum glutamic pyruvic transaminase (SGPT), serum testosterone, serum
luteinizing hormone, and serum follicle stimulating hormone. A semen specimen was also
collected and analyzed for volume, sperm count, and morphologic pattern. The male workers
that participated in the surveys were divided into three exposure groups: (1) exposed,
(2) intermediate exposed (contact with DNT for one to several years on an intermittent basis but
with no exposure for the last 2 years), and (3) unexposed (no exposure during their employment).
The exposure groupings were determined by reviewing job descriptions and by discussing
exposures with the individuals, the company, and the local union representatives.
The study group consisted of 30 male workers (9 from the exposed group, 12 from the
intermediate exposed group, and 9 from the unexposed group). However, only seven total
personal samples and three area samples were collected with the concentration ranging from not
3	3
detectable to 0.23 mg/m (personal samples) and from not detectable to 0.42 mg/m (area
samples). The author stated that the concentrations of DNT in the plant were below the OSHA
"3
standard PEL of 1.5 mg/m over an 8-hour workday. Serum examinations of renal and hepatic
profiles indicated no significant difference between any of the tested groups. A slight increase in
miscarriages among the wives of the exposed workers was found but could not be conclusively
related to the exposures. There were no significant differences in congenital defects and total
pregnancies between the groups. However, sperm counts in the exposed group were
significantly lower compared to the unexposed group. No significant differences were reported
between unexposed and intermediate exposed groups or between intermediate exposed and
exposed groups. The study author concluded that the results were strongly suggestive of a
reproductive problem but more workers needed to be evaluated.
Ahrenholz and colleagues (Ahrenholz and Meyer, 1982) also conducted a similar study at
Olin Chemical Company, in Moundsville, WV where male workers were exposed to both DNT
and TDA. DNT was reported in an approximate 80:20 ratio of 2,4-DNT and 2,6-DNT. Fifty
males (in the nonsemen portion of the study) and 41 of these 50 workers (in the semen portion of
the study) were divided into three groups based on the same criteria in Ahrenholz (1980). The
exposure duration ranged from 3 to 27.5 years. Due to laboratory error in preparation of the
sampling media, personal exposure data were determined to be invalid. Seven DNT area
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3	3	3
samples ranged in concentration from 0.026 mg/m (0.00929 mg/m , TWA) to 0.89 mg/m
(0.318 mg/m3, TWA) with a mean of 0.207 mg/m3 (0.0739 mg/m3, TWA). The study authors
reported no significant difference between the exposed and unexposed groups in serum enzymes,
sperm volume, sperm counts, and morphological changes. The questionnaire data on the
employees' reproductive history did not show statistically significant differences in the number
of spontaneous abortions in wives of workers employed in the DNT area when compared with
non-DNT exposed workers in the plant. A NOAEL of 0.0739 mg/m is identified based on no
significant differences in serum enzymes, sperm volume, sperm counts and morphological
changes in workers in the exposed group, and spontaneous abortions in their wives.
The studies conducted by Ahrenholz (1980) and Ahrenholz and Mever (1982) were
limited by small sample size, limited exposure data, problematic grouping, coexposure with
TDA and other unknown chemicals, and unknown composition of DNT (Ahrenholz. 1980).
Hamill et at. (1982)
Hamill et al. (1982) conducted a study to determine the reproductive effects of
occupational DNT and/or TDA exposure among 203 male workers from a chemical complex in
Lake Charles, LA. Of the 203 employees in the cohort, 84 were exposed to DNT and/or TDA
(exposure level within the OSHA PEL of 1.5 mg/m within an 8-hour workday, detailed
exposure data and composition of DNT were not reported), and 119 were not exposed. Each
worker was subjected to a semen analysis for sperm count and morphology, blood testing (for
serum follicle-stimulating hormone [FSH] measurement), a urogenital examination, an
estimation of testicular volume, and an interview. Information was also acquired on
reproductive history, medical and surgical history, past and present work history, assessment of
exposure, exposure to other chemicals with potential reproductive toxicity, smoking habits,
alcohol consumption, and recent medication. Data were collected between January and
June 1981. Based on exposure history (intensity, frequency, and how recent exposure to DNT
and/or TDA occurred), the participants were classified into four groups: (1) none to minimal,
(2) low to high, (3) low to moderate, and (4) high. The duration was at least 6 months.
No significant differences were observed between the exposure groups with respect to
work history, medical history, or physical examination characteristics. Additionally, there were
no significant differences in reproductive histories, or decreases in fertility related to DNT and/or
TDA exposure. Finally, no significant differences were discovered in the laboratory findings
including serum FSH, mean sperm count, and sperm morphology. The study authors concluded
that both TDA and DNT did not present a detectable reproductive hazard to the workers.
However, the study is limited due to lack of detailed exposure data, exposure to mixed
chemicals, and unknown composition of DNT.
Levine (1983)
Levine (1983) investigated the effect of DNT exposure on the fertility of workers
occupationally exposed to a DNT mixture (composition not further specified; exposure duration
not reported) in a U.S. toluene diisocyanate (TDI) plant. DNT and TDA are intermediates in the
manufacturing of TDI. DNT was used to manufacture TDA in the plants and data were collected
in 1981. Due to the job rotation, it was assumed that there was a coexposure to both DNT and
TDA. A total of 208 male (166 white, 42 nonwhite) and 10 married female workers were
interviewed. Exposure-related fertility among female employees was insufficient to analyze.
The fertility analysis for male employees revealed that their wives exhibited no evidence of an
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abnormal aggregation of miscarriages, stillbirths, neonatal deaths, or birth defects. This study
revealed little to suggest that occupational exposure to DNT may have affected reproduction
adversely and is limited due to lack of comparison with unexposed male employees, lack of data
regarding quantitative exposure, exposure to mixed chemicals and other unknown chemicals, and
unknown composition of DNT.
Levine et al (1985)
Levine et al. (1985) investigated the effect of DNT exposure on the fertility of workers
occupationally exposed to a DNT mixture (composition not further specified) in three
U.S. chemical plants. The plants manufactured TDA, DNT, and/or TDI. Data were collected
between 1979 and 1981, several years after the exposure period (between 1973 and 1976). A
total of 586 workers (144 from Plant A, 207 from Plant B, and 235 from Plant C; sex not
reported) were interviewed. No significant difference was found between the fertility of workers
who were exposed to DNT in the three U.S. chemical plants and the fertility of unexposed
workers. The study is limited due to the lack of quantitative exposure data provided, exposure to
mixed and other unknown chemicals, and unknown composition of DNT.
Epidemiological Studies of Carcinogenicity or Cardiovascular Diseases
Levine et al (1986)
Levine et al. (1986) evaluated workers at two army ammunition plants to assess the
relationship between exposure to DNT and carcinogenicity. In the first plant, located in
Joliet, IL, tgDNT (approximately 76% 2,4-DNT, 19% 2,6-DNT, and 5% other isomers) was
manufactured and purified to at least 98% 2,4-DNT and about 1% 2,6-DNT. From the first
plant, a total of 156 men, who worked in the DNT production line for at least 30 days during the
1950s, participated in the study. At the second plant, located in Radford, VA, the purified DNT
(98%) 2,4-DNT and approximately 1% 2,6-DNT) was used in certain single-based propellant
formulations. This cohort consisted of 301 men who had worked for 30 days or more during the
1940s and 1950s in specific jobs that had potential for DNT exposure. Workers from both plants
were presumed white males and considered exposed to DNT via the inhalation and dermal
routes. The exposure levels from the first plant were judged by the study authors to be high.
Jobs at the second plant were categorized by plant technical personnel according to opportunity
for exposure: high, moderate, low or none. Cohort mortality was followed from enrollment
through the end of 1980. Numbers of observed and expected deaths were recorded for each
underlying cause and the standardized mortality ratio (SMR) was computed using mortality rates
of U.S. white males as the standard.
Of the 457 men in both plants, 164 had died compared to 127 expected deaths using
mortality rates of U.S. white males as the standard. The combined SMR of 129 (p = 0.001) for
all causes of death was significantly high, and it increased to an SMR of 140 (p = 0.00007) after
15 years elapsed since entry into the study. This increase in overall mortality was attributed to
increased death from disease of the circulatory system (SMR: 140,/? = 0.002) or due to death
from accidents, poisonings, and violence (SMR: 191, p = 0.0007). Death as a result of malignant
neoplasms was less than the expected mortality rate (SMR: 87); however, this decrease was not
significant. No significant increases in death from any specific types of cancer were observed.
Increased mortality from disease of the circulatory system was determined to be primarily based
on an increase in mortality from ischemic heart disease (IHD) (SMRs of 131 and 143 for the first
and the second plant, respectively; 95% confidence intervals of 65-234 and 107-187 for the first
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and the second plant, respectively). Deaths from IHD remained high even when compared with
expected numbers derived using mortality rates of the counties in which the plants were located.
The relationship between mortality from IHD to duration and intensity of DNT exposure
was analyzed at 15 years from entry into the study onward. The results are suggestive of a dose-
and duration-response relationship for DNT and mortality from IHD. The study authors
suggested that the increase in mortality from heart disease was a result of damage to the coronary
arteries from exposure to DNT. The study is limited due to lack of exposure data, exposure to
other unknown chemicals, and lack of unexposed controls.
Stavner et al. (1992)
Stayner et al. (1992) performed a retrospective cohort study using current and former
white male workers from a propellant production facility in Radford, VA. The study aimed to
determine the possible relationship between exposure to DNT (composition of DNT is not
reported) and the risk of death as a result of cardiovascular disease including ischemic heart IHD
and cerebrovascular disease. A total of 4989 workers with probable exposure to DNT and
5636 unexposed workers were selected for the study (workers' operations were rated concerning
the probability of exposure to DNT). All exposed and unexposed workers who participated in
the study had been employed for at least 5 months at the study plant between January 1949 and
January 1980. The difference in mortality between the cohorts (exposed and unexposed groups)
and the U.S. population was evaluated using SMRs. SMRs were also used to evaluate specific
causes of death in addition to standardized rate ratios (SRRs), which are ratios of observed
deaths in the exposed groups to the observed deaths in the unexposed groups. Death from all
causes (including cardiovascular and noncardiovascular causes) was similar for DNT-exposed
(SMR: 1.00) and unexposed (SMR: 0.99) groups when compared to the U.S. population.
Mortality from cerebrovascular disease in the DNT-exposed group was less than that in the
unexposed group (SMR: 0.95, SRR: 0.89). IHD mortality in the DNT-exposed group was
similar to that of the unexposed group (SMR: 0.98, SRR: 0.99). Hypertension without heart
disease (SMR: 1.17) and other myocardial degeneration (SMR: 1.41) were slightly elevated in
the DNT-exposed group. The study authors concluded that DNT exposure did not appear to be
associated with an increased risk of either IHD or cerebrovascular disease. The study authors
also concluded that potential biases related to the company's medical screening program for
workers exposed to DNT may have limited the ability to detect these effects. However, the study
authors did not provide exposure or DNT composition data, and the definition of DNT-exposed
groups was not clear.
Stavner et al. (1993)
Stavner et al. (1993) investigated the relationship between workers exposed to DNT and
cancer of the liver and biliary tract. The cohort was selected from the second plant examined by
Levine et al. (19861 but with more subjects who were exposed to DNT. A total of 4989 male
workers exposed to DNT and 7436 unexposed male workers were included in this investigation.
All the enrolled workers had worked at least 5 months at the study facility between January 1949
and January 1980. The vital status (i.e., whether dead or alive) of the workers at the end of 1982
was collected and used to develop SMRs and SRRs to analyze the various relationships between
DNT exposure and mortality.
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Mortality for the study cohort was compared to the expected mortality for the
U.S. population. Death as a result of cancer was less than expected for the DNT exposed and
unexposed groups (SMRs of 0.84 and 0.78, respectively). An increase in hepatobiliary cancers
(defined as biliary, liver, and gall bladder cancers combined) was observed in the DNT-exposed
cohort as compared to the U.S. population (SMR: 2.67, 95% CI = 0.98-5.83) (but this was
identified as borderline statistically significant \p = 0.052]). When compared to unexposed
workers of the same facility, however, the SRR for hepatobiliary cancers in the DNT-exposed
group was significantly increased (SRR: 3.88, 95% CI = 1.04-14.41,/? = 0.04). No
exposure-response relationship was detected between the duration of exposure to DNT and
hepatobiliary cancer mortality.
According to the investigators, the study had several limitations, mainly that it was
originally designed to evaluate the risk associated with exposure to nitroglycerin (which was also
manufactured at the plant) rather than DNT. Other limitations included the small number of
hepatobiliary cancer cases (six), the small number of workers with a long exposure period to
DNT, and the lack of quantitative DNT exposure data. Worker exposure was classified
qualitatively, and those workers that were "probably" exposed were included in the exposed
group. Individuals in this group may have had minimal contact with DNT, as the group was
defined only by having contact with materials containing DNT as opposed to being exposed to
DNT directly. Therefore, inclusion of these individuals could have introduced bias by
preventing the detection of the true level of excess risk for hepatobiliary cancer following
exposure to DNT. Another limitation identified by the study authors was the possibility of
exposure to chemicals other than DNT. Nevertheless, the study authors concluded that the
excess in hepatobiliary cancer mortality observed among DNT-exposed workers in this study
added some support to the hypothesis that occupational exposure to DNT may be carcinogenic.
The authors noted that this study investigated more subjects, and hence, it has more statistical
power to detect an excess of hepatobiliary cancer than the Levine et al. (1986) study.
Bruningetal. (1999)
Bruning et al. (1999) performed a retrospective survey on underground miners who were
formerly exposed to an explosive (Donarit) containing 30% tgDNT, which consisted of
approximately 75% 2,4-DNT and 20% 2,6-DNT (the remaining 5% was unknown). The cohort
was selected from a mining area in Mansfeld, which is located in the former German Democratic
Republic (GDR). Health records were used to identify miners with former exposures to DNT
(n = 500) and their incidence of urogenital malignant diseases. Of these 500 subjects (sex not
reported), a group of 340 miners with available information on malignant urogenital tract disease
was asked to participate. Among the 340 miners, 183 gave their consent and were subjected to a
standard medical examination and a retrospective occupational exposure assessment. Additional
information was obtained on occupational histories, including exposures to any type of
hazardous chemicals, smoking histories, history of former kidney and renal diseases, as well as
history of cancers within families.
The study authors reported that the miners had been exposed through two routes:
inhalation of the smoke after explosions and skin contact with DNT-containing explosive sticks.
The exposures were ranked into low, medium, high, and very high exposure categories. Between
1984 and 1997, 14 cases of renal cell cancer and 6 cases of urothelial cancer were identified in
the group of 500 underground miners with former exposure to DNT. Exposure duration ranged
from 7-37 years, and latency periods ranged from 21-46 years. The incidences of urothelial and
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renal cancer in this group were 4.5 and 14.3 times higher than anticipated, respectively, on the
basis of the cancer registers of the GDR. The exposure categorization of the 14 renal cell cancer
cases revealed a distribution (i.e., number of workers in low, medium, high and very high
exposure categories) similar to that of the 183 exposed miners without cancer. However, the six
cases of urothelial cancer were predominantly confined to the high exposure category. The study
authors concluded that high exposure to DNT might be associated with urothelial tumor
formation. The study was limited due to the lack of quantitative exposure data and coexposure
to other unknown chemicals.
Bruninz et al (2001)
In another study, Brunine et al. (2001) investigated signs of subclinical renal damage in
the same subjects that were reported in Brunine et al. (19991 consisting of a group of 161 no
cancer miners and 19 cases with renal (n= 14) or urothelial cancer (n = 5), all of whom had been
exposed to explosives containing tgDNT [the same exposure duration and DNT composition
information as presented in Burning et al. (1999)1. The exposures were categorized
semiquantitatively, according to the type and duration of contact with DNT, into low, medium,
high, and very high. Evaluation of urinary protein excretion patterns indicated that there was a
straight dose-dependent relationship in the pathology of tubular and/or glomerular damage,
indicating that DNT-induced damage is directed toward the renal tubular system. In addition,
there was a dose-dependent increase in the biomarkers of alphal-microglobulin and glutathione
S-transferase alpha, indicating a nephrotoxic effect toward the proximal tubule under the
exposure conditions. By contrast, there was no similar change in glutathione S-transferase pi,
indicating no nephrotoxicity to the distal tubule. The study was limited due to the lack of
exposure data and coexposure to other unknown chemicals.
Earth et al. (2005)
In this case report, Harth et al. (2005) reported a cluster of three cases of urothelial cancer
(sex not reported) among a group of about approximately 60 workers who were exposed to
DNTs during manufacture of a DNT explosive (Donarit) at a factory in the former GDR. The
cases occurred within a period of 12 years (1990-2002) leading to a 15.9-fold higher incidence
of cancer of the urinary bladder than that of the federal state where the chemical factory was
located, even though no adjustments were made for age and smoking. From 1970 until 1974, the
production of DNT and nitrobenzene was located in one building, raising the possibility of
coexposure to nitrobenzene during this period. The exposure durations for the three cases were
longer than 7 years. The observation of the cluster of urothelial cancer in people highly exposed
to DNTs underlines the possibility of human carcinogenicity of DNTs, with the human
urothelium as a relevant target tissue. The study was limited due to the lack of exposure data,
unknown composition of DNT, and coexposure to other unknown chemicals.
ANIMAL STUDIES
Oral Exposures
The effects of oral exposure of animals to tgDNT have been evaluated in one subchronic
(CUT. 1983). two chronic, (Leonard et al.. 1987; CUT. 1982a) one developmental (Price et al..
1985; CUT. 1982b). and two carcinogenicity studies (Leonard et al.. 1987; CUT. 1982a).
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Subchronic Studies
cut a983)
In an unpublished, nonpeer-reviewed study, CUT (1983) administered tgDNT (consisting
of 76.49% 2,4-DNT, 18.83% 2,6-DNT, 2.43% 3,4-DNT, 1.54% 2,3-DNT, 0.65% 2,5-DNT, and
0.040%) 3,5-DNT) via diet to groups of 10 albino F344 rats/sex/group for 4 weeks. The nominal
doses were 0, 37.5, 75, or 150 mg/kg-day per group. Based on weekly tgDNT consumption
provided by the study authors, the adjusted daily doses (ADDs, calculated based on TWA doses)
are 0, 31.9, 61.9, or 134 mg/kg-day for males and 0, 32.0, 63.6, or 120 mg/kg-day for females.
Fresh diets were prepared weekly and adjusted based on body weight and food consumption
data. No certificate of good laboratory practice (GLP) was included in the study report.
The study authors observed animals for signs of morbidity and mortality twice daily and
recorded signs of gross toxicity and/or pharmacologic effects, food consumption, and individual
body weights weekly. Blood samples were collected from nonfasted females and males on
Days 27 and 28, respectively. Blood samples were analyzed for methemoglobin (MetHb),
reticulocytes, and Heinz bodies. After sacrifice, gross pathological examinations were
performed on the lungs, liver, spleen, kidneys, ovaries, and vagina. Organ weights were not
measured.
No treatment-related mortalities occurred during the study period. The only clinical signs
noted by the study authors were alopecia around the right eye of two females in the low-dose
group and urine stains on the fur of four high-dose females at Week 3 and two high-dose females
at Week 4. At Weeks 3 and 4, body weights of females treated with 120 mg/kg-day were
significantly decreased by 17% and 21%, respectively. All the high-dose treated males
experienced a significant reduction in mean body weight in a time-dependent manner compared
with controls (10%, 26%, 36%, and 38% reductions compared to controls at Weeks 1, 2, 3, and
4, respectively). In addition, body weights of the mid-dose males were significantly decreased
by 11% and 17% compared with controls at Weeks 3 and 4, respectively (see Table B. 1). Food
consumption was reduced in all treatment groups compared with controls in a dose-dependent
manner (data not shown). Therefore, the decreased body weights were likely related to the
reduction in food consumption in addition to tgDNT treatment.
Dose-dependent, significant increases in mean reticulocytes and Heinz bodies were
reported in all treated animals (see Table B.2). MetHb was significantly increased in the low and
high-dose females and in the high-dose males (see Table B.2). The study authors noted the
following gross pathological observations in high-dose females and in low- and high-dose males:
discoloration (yellow), mottled appearance, and/or rough or granular surface of the liver.
Discoloration (green) of the kidneys was observed in high-dose males and females. Dark yellow
vaginal stains were also noted in two high-dose females, and an ovarian cyst was found in one
mid-dose female. Table B.3 presents the gross pathology results.
Based on the significant hematological changes in male rats, a LOAEL of
31.9 mg/kg-day is identified. Data preclude identification of a NOAEL.
Chronic Studies
CUT (1982a)
CUT (1982a) is selected as the principal study for deriving the screening subchronic
and chronic p-RfDs. CUT (1982a) administered tgDNT (consisting of 76.5% 2,4-DNT,
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18.8% 2,6-DNT, 2.4% 3,4-DNT, and <2.3% 2,3-, 2,5-, and 3,5-DNT) to groups of 130 F344
rats/sex/group (Charles River Breeding Laboratories, Wilmington, MA) via diet at target dose
levels of 0, 3.5, or 14.0 mg/kg-day for 2 years, or 35 mg/kg-day for 55 weeks (sacrificed early
due to treatment-related incidence of liver tumors). The control group rats received a basal diet
only. The study authors conducted interim sacrifices at 26 weeks, 52 weeks, 55 weeks (for
high-dose group only), 78 weeks, and 104 weeks at termination. For each dose group, a ADD
was calculated based on tgDNT concentration in food, food consumption, and time periods
(calculated based on TWA) provided by the study authors. The calculated ADDs are shown in
each data summary table (see Tables B.4 to B.15). The test diets were prepared weekly based on
measured body weight and food consumption data to ensure constant intake of tgDNT on a
mg/kg body-weight basis. The study authors did not provide a statement confirming GLP status.
Animals were examined twice daily for general physical appearance, mortality, and
morbidity. The study authors recorded food consumption in each cage and individual body
weights every week for the first 14 weeks, biweekly for the following 12 weeks, and every fourth
week for the remainder of the test period. Mean daily tgDNT consumption was calculated for
each dose level weekly from Weeks 1 through 14, biweekly through Week 26, and monthly for
the remainder of the treatment period.
Hematology, clinical chemistry, and urinalyses were performed on 10 animals/sex/group
at Weeks 26 and 52, and on 20 animals/sex/group at Weeks 78 and 104. An additional
20 animals from the high-dose group were tested for hematology and clinical chemistry at
Week 55. The measured hematological parameters included hematocrit, hemoglobin (Hb), and
MetHb; red blood cell (RBC), reticulocyte, Heinz body, total white blood cell (WBC), and
differential leukocyte counts; and mean corpuscular hemoglobin (MCH), mean corpuscular
volume (MCV), and mean corpuscular hemoglobin concentration (MCHC). Serum samples
were also analyzed for clinical chemistry, including alkaline phosphatase (ALP), BUN, and
SGPT. Urinalysis measured appearance, pH, specific gravity, glucose (GLU), ketone, total
protein (TPR), occult blood, and sediment. Ophthalmologic examinations were performed on
both eyes of each animal using an indirect ophthalmoscope 1 or 2 days prior to sacrifice at 26,
52, 78, and 104 weeks.
Ten rats/sex/group were sacrificed at Weeks 26 and 52. At Week 55, all surviving
high-dose rats were sacrificed due to severe toxicity (high incidence of tumors). At Week 78,
20 rats/sex/ group were sacrificed, and at Week 104, all surviving rats were sacrificed. The
study authors performed examinations for gross signs of toxicity and incidence and recorded the
location of tumors at identical intervals. At necropsy, the following organs were excised and
weighed: brain, heart, liver, kidneys, lungs, and testes with epididymides. Ovaries were not
weighed until after fixation. The following tissues were examined histopathologically from
animals in the control and high-dose groups at Weeks 26 and 52, in the control and mid-dose
groups at Weeks 78 and 104, and in the high-dose groups at Week 55: brain (cerebellum,
cerebrum, brain stem), eyes, testes with epididymides, thoracic spinal cord, pituitary, thyroid,
parathyroid, adrenal, heart, aorta, lungs, spleen, liver, kidneys, stomach, small intestine
(duodenum, jejunum, ileum), large intestine (upper and lower colon, rectum), pancreas, ovary
with oviduct, uterus, prostate, thymus, esophagus, trachea, nasal turbinate, adipose tissue,
submaxillary salivary gland, lymph nodes (mesenteric and thoracic), urinary bladder, thigh
skeletal muscle with sciatic nerve, bone marrow (sternum), skin (flank), mammary gland, and
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unusual lesions. Histopathological examination was also performed on the livers from the
low-dose groups at Weeks 26, 52, 78, and 104 and from the mid-dose group at Weeks 26 and 52.
The study authors reported a decrease in survival in male rats in the mid-dose group at
termination and in the high-dose group at Week 55. All surviving rats in the high dose group
were sacrificed at Week 55 due to severe liver toxicity and tumor formation noted at the
Week 52 interim sacrifice. Survival in the remaining dose groups was similar to controls. The
study authors reported treatment-related hunched, thin, and/or bloated appearance in all animals.
The study summaries are presented below in the order of the sacrifice. The study authors
reported that the results of urinalysis were unremarkable throughout the 2-year period.
26-week study
The ADDs at this interim sacrifice were calculated to be as follows: 0, 3.47, 13.6, or
34.6 mg/kg-day for males, and 0, 3.22, 13.9, or 34.9 mg/kg-day for females. At Week 26,
10 animals/sex/group were sacrificed. Hematology, clinical chemistry, urinalyses, gross
pathology, organ/body-weight ratios, and histopathology evaluations were conducted. Selected
hematology results are provided in Table B.5. There was a 31% increase in WBC count in the
mid-dose males and dose-related increases in MetHb levels (by up to 400%), reticulocyte count
(by up to 83%), as well as a decrease in RBC count in high-dose males (8%). The hematology
results for females are not summarized in this document because no biological significance was
found. RBC and reticulocyte counts were not significantly changed, while MetHb was
significantly decreased in all treatment groups. Clinical chemistry results indicated that BUN
levels in high-dose males and females were significantly increased by 17% and 27%,
respectively (see Table B.6). Both males and females experienced dose-related decreases in
body weight compared to control animals (see Tables B.4 and B.7). However, the decreased
body weight may be partially due to the reduction of food intake (see Table B.4).
The study authors reported significant dose-dependent increases in both absolute (12%
and 50%>) and relative (25% and 95%) liver weight in the mid- and high-dose males, respectively
(see Table B.8). A similar liver weight increase was also reported in tgDNT-treated females (see
Table B.9). In addition, there were dose-dependent increases in relative kidney weights with
significant changes observed in the high dose group (38% in males and 24% in females; see
Tables B.8 and B.9). Other observed significant organ-weight changes included increased
relative heart, relative lung weight in high-dose males and females, and increased relative testis
weight in high-dose males. Gross pathology findings revealed tgDNT-treatment-related gross
alterations in the livers. Histopathology findings indicated hepatotoxicity in the mid- and
high-dose males and females (see Table B. 10). Indicators of hep atotoxi city consisted of
minimally to moderately severe nonsuppurative pericholangitis in high-dose males and in mid-
and high-dose females; necrosis of hepatocytes in mid- and high-dose males and females;
vacuolated hepatocytes in high-dose males and females; and slightly to moderately severe biliary
hyperplasia, periportal fibrosis, and necrosis of bile duct epithelium in high-dose males. Other
signs of histopathology were found in the heart, spleen, and kidney (incidence data are not
displayed in this document). These findings included an increase in the incidence and severity of
chronic myocarditis in high-dose males. Considering 4/10 control males also had minimal
chronic myocarditis, the study authors concluded that this probably represents a treatment-related
exacerbation of spontaneous disease. Also, an increase in spleen hemosiderin and
extramedullary hematopoiesis was observed in the high-dose males and females. In the kidney,
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slightly increased incidence of chronic interstitial nephritis was noted in the high-dose males. In
addition, there was increased incidence and amount of tubular pigment in the mid- and high-dose
males and females. Although moderately severe testicular degeneration was observed in
high-dose males, the study authors suggested that the unilateral change may represent a
spontaneous lesion.
Two high-dose males had hepatocellular carcinomas, which were not observed in the
control group (see Table B.14), suggesting these tumors may have been induced by tgDNT.
Based on increased absolute and relative liver weight and hepatotoxicity in mid-dose
males, a LOAEL of 13.6 mg/kg-day and aNOAEL of 3.47 mg/kg-day are identified.
52-week study
The ADDs at this interim sacrifice were calculated as follows: 0, 3.47, 13.9, or
34.9 mg/kg-day for males, and 0, 3.46, 13.9, or 35.1 mg/kg-day for females. At Week 52,
10 animals/sex/group were sacrificed. Hematology, clinical chemistry, urinalyses, gross
pathology, organ/body-weight ratio, and histopathology evaluations were conducted. Compared
to controls, there were dose-related increases in MetHb levels (not significant) and reticulocyte
count (significant in high-dose group), and decreases in RBC count (significant in the mid-and
high-dose groups) and Hb (significant in the high-dose group; see Table B.5). Also, WBC
counts in the high-dose group were increased. In female rats, no treatment-related hematological
alterations were observed.
Body-weight changes were similar to the findings at Week 26 (see Table B.7).
Significant increases in both absolute and relative liver weights were reported in the low-dose
males and mid- and high-dose males and females. There were dose dependent increases in male
(significant in the mid- and high-dose groups) and female relative kidney weight (significant in
the high-dose group) and in male relative heart weight (significant in the high-dose group) (see
Tables B.8 and B.9). Gross pathology findings revealed distinct gross alterations of the livers,
namely, focal discolorations in the mid- and high-dose males and females. Also, liver nodular
lesions were noted in 8/10 males and 4/10 females in the high-dose group. The liver
histopathology findings included hyperbasophilia, megalocytosis of hepatocytes, and vacuolation
and necrosis of individual hepatocytes (see Table B. 11). Other treatment-related lesions in the
high-dose males consisted of exacerbation of chronic interstitial nephritis and renal tubular
pigment, increased incidence and severity of testicular degeneration, and increased proliferation
of hematopoietic cells in the splenic red pulp and sternal marrow, suggesting an increased RBC
turnover rate. Also, the study authors noted that the cardiomyopathy observed in the high-dose
males at 26 weeks was not obvious in this group at 52 weeks.
A dose-dependent increase in hepatocellular carcinomas (3/10 in mid-dose and 10/10 in
high-dose) was observed in males (see Table B. 14). Hepatocellular carcinomas were observed in
4/10 females in the high-dose group. Neoplastic nodules were noted in the livers of
4/10 mid-dose and 3/10 high-dose males, as well as 8/10 high-dose females.
Cholangiocarcinomas were observed in 2/10 high-dose males and 2/10 high-dose females. One
of 10 mid-dose males had biliary hyperplasia with atypia of the bile duct epithelium, which the
study authors believed to be a precursor lesion of cholangiocarcinoma.
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Based on increased absolute and relative liver weight and hepatotoxicity in low-dose
males, a LOAEL of 3.47 mg/kg-day is identified. Data preclude identification of aNOAEL.
55-week study
The ADDs at this interim sacrifice were calculated as follows: 34.9 mg/kg-day for males
and 35.1 mg/kg-day for females (only high-dose group). At Week 55, all surviving high-dose
animals were sacrificed. Hematology, clinical chemistry, urinalyses, and histopathology
evaluations were conducted on 20 rats/sex, but the results on hematology and clinical chemistry
were only reported on 10 rats/sex. Because there were no significant hematological findings in
the treated females, Table B.5 displays male results only. Tables B.6, B.8, and B.9 present
clinical chemistry and organ/body-weight ratios. Histopathological findings (not displayed in
tables) in high-dose animals indicated that hepatocellular carcinomas occurred in 20/20 males
and in 11/20 females. Two male rats had hepatocellular-cholangiocarcinomas and 3/20 males
had cholangiocarcinomas. Biliary hyperplasia with atypia of bile duct epithelium was also
observed in 2/20 males. Due to lack of a control group, data preclude identification of a NOAEL
or a LOAEL.
78-week study
The ADDs at this interim sacrifice were calculated as follows: 0, 3.49, or 14.0 mg/kg-day
for males, and 0, 3.45, or 14.0 mg/kg-day for females. At Week 78, 20 animals/sex from each
dose level were sacrificed (only control, low-dose, and mid-dose groups were available; the
high-dose males and females were all sacrificed at Week 55). Hematology, clinical chemistry,
urinalyses, gross pathology, organ/body-weight ratio, and histopathology evaluations were
conducted. Among the hematology findings were decreased hematocrit and RBC counts in the
mid-dose males and dose-dependent increases of 13% and 112% in reticulocyte counts in the
low-and mid-dose males, respectively (see Table B.5). In treated female rats, significantly
increased WBC counts were observed in mid- and high-dose groups, and no other significant
hematological changes were observed. Clinical chemistry indicated significantly higher mean
SGPT values in mid-dose males (93% higher than controls) (see Table B.6). Body-weight
changes in the mid-dose males and females were similar to those observed in the 26-week
treatment. Absolute and relative liver weights were significantly increased in the low- and
mid-dose males and in the mid-dose females (all the high-dose rats were sacrificed at Week 55;
see Tables B.8 and B.9). In low- and mid-dose males, the study authors also reported
significantly dose-dependent increases in the relative weights of testes (27% and 37%,
respectively), lungs (14% and 30%, respectively), and kidneys (12% and 55%, respectively; see
Table B.8). Also, the relative kidney weight was increased by 32% and the absolute brain
weight was decreased by 11% in the mid-dose females see Table B.9). Hepatotoxicity including
cystic degeneration, necrosis of individual hepatocytes and fatty metamorphosis were observed
in low- and mid-dose males and females (see Table B. 12). An increase in the severity of chronic
interstitial nephritis was observed in mid-dose males and females. Treatment-related testicular
pathology observed at the Week 52 interim sacrifice could not be determined because all control
and mid-dose males had interstitial cell testicular tumors. Hepatocellular carcinomas were
observed in 19/20 mid-dose males (see Table B.14); neoplastic nodules were observed as
follows: 1/20 in low-dose males, 11/20 in mid-dose males, 2/20 in low-dose females, and
10/20 in mid-dose females. Four cholangiocarcinomas were recognized in mid-dose males in
addition to 2/20 incidence of biliary hyperplasia with atypia of bile ductal epithelium in the same
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group. In this interim sacrifice, the study authors observed increases in relatively common
benign neoplasms, such as mammary fibroadenomas, subcutaneous fibromas, pituitary
chromophobe adenomas, and pulmonary carcinomas.
Based on increased relative liver weight and hepatotoxicity in low-dose females, a
LOAEL of 3.45 mg/kg-day is identified. Data preclude identification of a NOAEL.
104-week study
The ADDs doses at this terminal sacrifice were calculated as follows: 0, 3.51, or
14 mg/kg-day for males, and 0, 3.46, or 14 mg/kg-day for females. At Week 104, all surviving
animals were sacrificed. Hematological results indicated that in males there were
dose-dependent increases in MetHb (not significant); a significant increase in reticulocyte counts
in the mid-dose group; and significant decreases in hematocrit, Hb, and RBC counts in the
mid-dose group (see Table B.5). No remarkable hematological changes were observed in treated
female rats (data not shown). Evaluation of clinical chemistry revealed significantly increased
SGPT and BUN in the mid-dose males (see Table B.6). Similar body weight changes were
observed in mid-dose males and females as those observed in the 52 or 78-week treatments (see
Table B.7). Dose-dependent, significantly increased organ weights in the low- and mid-dose
groups included relative heart weights in males and females, both absolute and relative liver
weights in males and females, relative testis weight in males, and relative kidney and ovary
weights in females (see Table B.8 and Table B.9). Gross pathology findings revealed apparent
treatment-related liver lesions and nodular and/or mass formation in low- and mid-dose males
and females. Histopathology indicated that hepatotoxicity occurred in the livers of treated
animals and consisted of fatty metamorphosis, necrosis, cystic degeneration, and megalocytosis
in low- and mid-dose males and females (see Table B. 13). An increase in the severity of chronic
interstitial nephritis occurred in the kidneys of mid-dose males and females. The study authors
did not observe treatment-related histopathological changes in the ovaries or in the hearts from
either sexes; therefore, the biological significance of the increased relative heart and ovary
weights in the low- and mid-dose groups is unclear. Testicular pathological changes were not
determined as observed in Week 78. Therefore, the biological significance of the increased
relative testes weight is also unclear.
There is a clear dose-dependent increased incidence of hepatocellular carcinomas and
neoplastic nodules in both males and females (see Table B. 15). "Neoplastic nodule" is a term
used in rodent liver pathology. Because neoplastic nodules are believed to progress to
hepatocellular carcinomas (Bannasch et al.. 1982; Bannasch.. 1976). this endpoint is included in
the dose-response analysis. Hepatocholangiocarcinomas were observed in 1/68 mid-dose
female, and cholangiocarcinomas were observed in 2/23 mid-dose males. In addition, 2/23
mid-dose males had biliary hyperplasia with atypia of bile duct epithelium. Parathyroid
hyperplasia was also observed in mid-dose males and females, and 2/23 mid-dose males had
parathyroid adenomas. Increased relatively common benign neoplasms were observed in both
males and females (see Table B.15).
Based on increased absolute and relative liver weight and hepatotoxicity in low-dose
females, a LOAEL of 3.46 mg/kg-day was identified. Data preclude identification of a NOAEL.
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Leonard et al. (1987)
Leonard et al. (1987) administered 0 or 35 mg/kg-day tgDNT (final composition of
76.5% 2,4-DNT, 18.8% 2,6-DNT, 2.43% 3,4-DNT, 1.54% 2,3-DNT, 0.69% 2,5-DNT, and
0.04%) 3,5-DNT) via diet to groups of 28 male F344/CrlBR rats (Charles River Breeding
Laboratories, Kingston, NY) for 1 year. New food batches were prepared monthly and the
tgDNT concentration was adjusted in each batch based on food consumption and average body
weight in order to maintain target dose levels. It is unclear if this study was conducted under
GLP.
Rats were housed four per cage, and average dietary consumption for each cage was
determined weekly. Body weights were measured every 2 weeks throughout the study.
Four animals in each group were sacrificed after 6 and 26 weeks of feeding. At the end of the
52-week treatment period, all surviving animals were sacrificed and necropsied, the liver and
lungs were weighed, histopathological examination was performed, and hepatic microsomal
epoxide hydrolase (EH) and cytosolic DT-diaphorase (DTD) activities were measured. Serum
enzyme activities (SGPT) and glutamyl transferase (GGT) were also determined. Other clinical
chemistry, hematology, and pathology examinations besides liver and lung were not conducted.
The study authors reported that body weight was significantly reduced 11% and 26% in
rats treated with tgDNT compared with controls at 26 and 52 weeks, respectively (see
Table B. 16). Relative liver weight was significantly increased at both time periods (see
Table B. 16) as well. At the end of 52 weeks, absolute liver weight was 89% more and relative
liver weight was 155% more than that of controls (see Table B.16). After 52 weeks of treatment,
the study authors noted nonneoplastic lesions consisting of hepatocytic degeneration and
vacuolation in the majority of animals, as well as acidophilic and basophilic cell foci in over
90% of the animals. Bile duct hyperplasia and a highly variable incidence of cholangiofibrosis
were also noted.
Based on significant decreases in body weight, and increases in absolute liver and relative
liver weight accompanied by pathological findings in the liver and bile duct, a LOAEL of
35 mg/kg-day is identified from this study. Data preclude identification of a NOAEL.
Table B. 17 summarizes neoplastic lesions of the liver. Neoplastic nodules were found in
the livers in 53% of animals treated with tgDNT. Hepatocellular carcinomas were seen in 47%
of the tgDNT-treated animals; all lesions had a typical trabecular pattern. Cholangiocarcinomas
were also reported in 11% of the treated animals. The study authors concluded that tgDNT is a
potent, complete hepatocarcinogen in male F344 rats. The study is limited by the use of only
one dose of tgDNT, which precludes examining dose-response relationships. In addition, the
study authors did not provide quantitative data for nonneoplastic lesions and only reported
general findings.
Developmental Studies
Price et al. (1985) and CUT (1982b)
Research Triangle Institute (RTI) performed a study investigating the potential
developmental toxicity of tgDNT following maternal gestational exposure in F344 rats. The
study was performed in 1980, and a Final Report was submitted under the Toxic Substances
Control Act (TSCA) to EPA's Office of Toxic Substances by CUT (1982b). Additionally, Price
and colleagues, the study authors from RTI, reported on the maternal and fetal toxicity in a
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peer-reviewed, published study (Price et ai. 1985). The tgDNT used in the study contained the
following composition of isomers: 76% 2,4-DNT; 19% 2,6-DNT; 2.4% 3,4-DNT;
1.5% 2,3-DNT; <1%; 2,5-DNT; and <1%; 3,5-DNT. The rats were administered the tgDNT by
gavage, with laboratory-grade corn oil used as the vehicle. The study authors administered
tgDNT at doses of 0 (vehicle control), 35, 75, or 150 mg/kg-day (first breeding round) to groups
of 13 pregnant F344 rats on gestation days (GDs) 7 through 20. Due to high mortality rates
observed in the 150 mg/kg-day dose group, the doses used in the second and third breeding
rounds were reduced to 14, 37.5, and 100 mg/kg-day. Table B.18 shows the numbers of females
in each dose group.
The study authors examined dams daily for clinical signs of toxicity and recorded body
weights on GDs 0 and 7-20. On GD 20, 13, 7, 13, 7, 13, and 6 dams from each dose group (14,
35, 37.5, 75, 100, and 150 mg/kg-day, respectively) and 22 dams from the control group were
sacrificed and evaluated for implantations, resorptions, and dead or live fetuses. In addition,
body weight, liver weight, spleen weight, number of corpora lutea, and gravid uterine weight for
each dam were recorded. The pregnancy rates in mated dams from the control and low- through
high-dose groups were 20 (91%), 10 (77%), 7 (100%), 12 (92%), 6 (86%), 12 (92%), and
5 (83%>), respectively. No histopathological examinations were conducted on the dams. Live
fetuses were examined for uterine position, body weight, crown-rump length, placenta weight,
sex, and gross morphological abnormalities. Maternal and fetal blood samples from the
100-mg/kg-day treatment group were analyzed for MetHb content. In addition, blood samples
from dams and one male and one female fetus per litter from the 100-mg/kg-day treatment group
were evaluated for RBC count, WBC count, hematocrit, MCV, RBC distribution width (RDW),
and platelet count. The study authors also examined 50% of the fetuses in each litter for visceral
and skeletal malformations, malformations of the head, and liver and spleen weights.
A high mortality rate was observed in dams from the first breeding date exposed to
150 mg/kg-day tgDNT, as 46.2% of rats (6/13) in this treatment group died between GDs 11 and
18. Therefore, in the second and third breedings, the study authors reduced the tgDNT doses.
Treatment with tgDNT at 14, 35, and 100 mg/kg-day also resulted in mortality rates of
4.5%) (1/22), 7.7%) (1/13), and 4.3% (1/23), respectively, through GD 20 (see Table B.18). No
deaths occurred in females treated with the vehicle control (corn oil). The study authors stated
that the cause of death of one rat/group from the 14-, 35-, and 100-mg/kg/day-DNT groups was
initially suspected to be related to gavage error. Gavage error was also suspected to be the cause
of death in 2/6 rats from the highest dose group (150 mg/kg); however, the study authors
concluded that the cause of death in the remaining 4 rats appeared to be treatment related as
death was preceded by clinical signs of toxicity. Clinical signs related to tgDNT treatment
included rough coat, lethargy, and hind-limb weakness, and were observed in 7/13 females in the
150-mg/kg-day dose group beginning on the fifth to eighth day of dosing (GDs 11-14) and
continuing until death (GDs 12-18) or scheduled sacrifice (GD 20).
On GD 20, significant increases in MetHb, reticulocyte count, MCV (RBC size), RDW,
and platelet count were observed in dams treated with 100-mg/kg-day tgDNT. Significant
decreases in RBC count and hematocrit were also observed in this group (see Table B.19).
Because the study authors did not analyze low-dose blood samples, it is not clear if
hematological parameters were adversely affected at lower doses.
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A significant dose-related decrease in absolute maternal weight gain (maternal weight
gain during treatment minus gravid uterine weight) was observed in the 14, 100, and
150-mg/kg-day tgDNT dose groups (see Table B.20). However, no decreases in absolute
maternal weight gain was observed at 35, 37.5, and 75 mg/kg-day, suggesting the reduction of
weight gain at 14 mg/kg-day was not treatment related, at least at dose levels <100 mg/kg-day.
Significant dose-related increases in relative maternal liver weight were observed in the
75- (11%), 100- (12%), and 150-mg/kg-day (17%) treatment groups (see Table B.20). A
significant increase in maternal relative spleen weight was observed at all doses of tgDNT
>35 mg/kg-day (see Table B.20). Due to lack of histopathological data, the biological
significance of the increased spleen weight is not clear. There was an increased resorption rate
(46.0%) in the high-dose group compared to the control (16.8%); however, this change was not
significant. No other significant effects on measures related to reproduction (incidence of live or
dead fetuses per dam, see Table B.20) or fetal morphology (see Table B.21) were observed. The
study authors, therefore, concluded that tgDNT was not observed to be teratogenic in F344 rats
even at dose levels that produced significant maternal toxicity.
In litters with live fetuses, no significant difference was observed in the proportion of
male fetuses per litter, average fetal body weight per litter, average fetal crown-rump length per
litter, or average placental weight per litter (see Table B.22). Changes in liver/body and
spleen/body-weight ratios were observed in some treatment groups, but no dose-response
relationship was apparent (see Table B.22). Fetuses from the 100-mg/kg-day group exposed to
tgDNT exhibited decreased reticulocyte count, decreased RBC count, and increased MCV (see
Table B. 19). Although the decreased RBC count and increased MCV were significant, the
P/o RBC count decrease (2.15 x io6 compared to 2.17 x io6) and 2% RBC size increase
(160.61 |im3 compared to 156.54 |im3) were not considered biologically significant.
The Final Report submissions (CUT. 1982b) include the teratological study [the same
information included in Price et al. (1985)1 as well as a postnatal developmental evaluation. In
the postnatal developmental evaluation, the remaining female rats that were not sacrificed on
GD 20, including 8, 5, 15, 6, 9, and 1 pregnant females from the low- through high-dose groups,
respectively, and 15 controls were observed through parturition, death, or GD 24, whichever
came first. One female in the 14-mg/kg-day group died on GD 22, and 1 female in the
150-mg/kg-day group died on GD 23 (the females from this group were not available for
postnatal developmental evaluation). Twelve females failed to deliver by GD 24 and were
sacrificed; 11 of these females were determined not to be pregnant. In total, pups were observed
from 5-14 litters per treatment group from birth (postnatal day [PND] 0) to PND 60.
Body weight and crown-rump length of each live pup were recorded on PND 0, then
litters were culled to no more than eight live pups (four male and four female, as possible). The
study authors recorded body weight daily and noted age of appearance of physical landmarks
(pinna detachment, pilation, incisor eruption, eye opening, testes descent, vaginal opening),
neurobehavioral landmarks (surface righting, cliff avoidance, auditory startle, wire grasping, and
mid-air righting), and open field behavior on PND 30. A limited number of pups were sacrificed
from each treatment group on PNDs 0, 10, 25, and 50; the remaining pups were sacrificed on
PND 60. Body weight, liver weight, and spleen weight were recorded at each sacrifice date;
testis weights were recorded from the sacrifice at PND 60. All dams were sacrificed on PND 30,
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and maternal body weight, liver weight, and spleen weight at sacrifice were recorded. Blood
samples were collected from dams at PND 30 and pups on PNDs 0, 10, 25, and 50 from the 75-
and 100-mg/kg-day groups and the corresponding controls.
The study author (CUT. 1982b) reported that dams in the 100-mg/kg-day group exhibited
decreased body weight on PND 15, and dams in the 75-mg/kg-day group had reduced
reticulocyte counts on PND 30; no other signs of maternal toxicity were observed. The study
authors state that significant differences in some observations from vehicle controls were
observed in the litters, but these differences were not dose-related, including elevated litter size
on PND 0 in the 75-mg/kg-day group; elevated female crown-rump length on PND 0 in the 14-
and 37.5-mg/kg-day groups; increased male body weight on PND 0 in the 14-mg/kg-day group;
increased reticulocyte count in the 75-mg/kg-day group or decreased reticulocyte count in the
100-mg/kg-day group on PND 50; and either early or delayed appearance of eye opening in
the 14-mg/kg-day group or the 35- and 75-mg/kg-day groups, respectively. There was a
dose-dependent increase in relative liver weight for pups in all treatment groups on PND 0, but
no difference was observed on PND 60 in any group, indicating that tgDNT toxicity was
reversed by PND 60. The study authors stated that a dose-related decrease in rearing behavior in
the open field was observed at 100 mg/kg-day in female pups, suggestive of sex-specific
neuromotor deficits (see Table B.23).
The Price et al. (1985) study stated that tgDNT was not found to be teratogenic following
oral administration and concluded that there was no evidence for selective sensitivity of the
developing conceptus to tgDNT because prenatal viability was reduced only at the dose near the
maternal LD50. The CUT (1982b) studies concluded that while various dosages of tgDNT could
produce facilitation or retardation of growth or development, dose-response relationships for
these changes do not exist.
Based on significantly increased relative liver weight in pregnant F344 dams at GD 20, a
LOAEL of 75 mg/kg-day and a NOAEL of 37.5 mg/kg-day are identified for maternal toxicity.
Considering a 29.2% increase in resorption rate accompanied by an increase in dead fetuses and
a decrease in live fetuses in the high-dose group, a LOAEL of 150 mg/kg-day and a NOAEL of
100 mg/kg-day are established for developmental toxicity. Further, a LOAEL of 100 mg/kg-day
and a NOAEL of 75 mg/kg-day were identified for postnatal toxicity based on decreased rearing
behavior.
Reproductive Studies
No studies were identified.
Carcinogenicity Studies
Carcinogenicity studies by CUT (1982a) and Leonard et al. (1987) were summarized in
the Chronic Studies section. The carcinogenicity study by CUT (1982a) is selected as the
principal study for deriving the screening provisional oral slope factor (p-OSF).
Inhalation Exposures
No studies were identified.
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Other Data (Other Examinations)
Mutagenicity or genotoxicity of tgDNT has been evaluated in in vitro and in vivo test
systems, and Table 4 summarizes the study results. Mixed results were reported from both in
vitro and in vivo test systems. Mutagenicity tests were positive in Salmonella typhimurium
Ames assays, with and without metabolic activation, while they were negative in mammalian
cell systems (e.g., HGPRT gene mutations in Chinese hamster ovary [CHO] cells and TK
mutations in mouse lymphoma cells). Similar to the in vitro test systems, mixed results were
also seen in in vivo studies. While unscheduled DNA synthesis showed a positive response in
most rat hepatocytes (Mirsalis et at.. 1989; Hamilton and Mirsalis. 1987; Ashbv et at.. 1985;
Mirsalis and Butterworth. 1982; Mirsalis et at.. 1982) and in rat lymphocytes (Ktigerman et at..
1982). a mouse bone marrow micronucleus test (Ashbv et at.. 1985) and dominant lethal assay
(Spares and Lock, 1980) indicated negative responses.
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Table 4. Summary of tgDNT Genotoxicity
Endpoint
Test System
Dose
Concentration"
Resultsb
Comments
References
Without
Activation
With
Activation
Genotoxicity studies in prokaryotic organisms
Reverse
mutation
S. typhimurium
strains TA98, TA100,
TA1535, TA1537,
TA1538
1000 |ig/platc
+ (TA98,
TA1538)
+ (TA98,
TA1538)
NA
Couch et al.
(1981)
Forward
mutation
S. typhimurium strain
TM 677
500 (ig/mL
+
+
NA
Couch et al.
(19811
Genotoxicity studies in mammalian cells—in vitro
HGPRT
Mutation
Chinese hamster
ovary (CHO)
<2mM


NA
Abernethv
and Couch
(19821
TK+/-
P388 mouse
lymphoma cells
1.6-1000 (ig/mL
—
—
NA
Styles and
Cross (19831
Unscheduled
DNA synthesis
(UDS)
Primary hepatocyte
cultures from adult
male F344 rats
0, 1 x 1CT4, or
1 x l(T5 M

ND
Evaluated
Bermudez et
al. (19791
Genotoxicity studies in mammals—in vivo
Sister
chromatid
exchange
(SCE)
Rat lymphocyte
culture from male
F344 rats
0 or 100 mg/kg
(oral)
+
ND
NA
Kligerman et
al. (19821
UDS
Hepatocytes/Male
Alderley Park rats
0, 100, or 200
mg/kg (oral)
+
ND
NA
Aslibv et al.
(19851
UDS
Hepatocytes/Male
F344 rats
0, 25, 100, 150,
or 200 mg/kg
(oral)
+
ND
NA
Ashbv et al.
(1985)
UDS
Hepatocytes/Male
F344 rats
0 or 100 mg/kg
(oral)
+
ND
Mixture of DNTs,
with dissimilar
composition as
tgDNT
Hamilton
and Mirsalis
(1987)
UDS
Hepatocytes/Male
and female F344 rats
<200 mg/kg
(oral)
+
ND
NA
Mirsalis and
Buttcrworth
(1982)
UDS
Hepatocytes/Male
germ-free (axenic)
F344 rats or Charles
River Altered
Schaedler Flora rats
(CRASF; similar to
normal gut
microflora)
0 or 100 mg/kg
(oral)
Axenix -
CRASF +
ND
Results indicate
that gut flora is
necessary for
tgDNT to induce
UDS
Mirsalis et
al. (1982)
UDS
Hepatocytes/Male
F344 rats
0, 35, 125, or
250 mg/kg (oral)
+ (at doses
>125
mg/kg)
ND
NA
Mirsalis et
al. (19891
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Table 4. Summary of tgDNT Genotoxicity
Endpoint
Test System
Dose
Concentration3
Resultsb
Comments
References
Without
Activation
With
Activation
Mouse
biochemical or
visible specific
locus test
T stock male and
C57BL/6J female
mice;
C57BL/6 J male and
C57BLBL/6J female
mice
0 or 100 mg/kg

ND
Recessive spot
test; different
matings were
tested; treated on
a day designated
as 1/4 of
pregnancy
Soares and
Lock (1980)
Micronucleus
test
Bone marrow/Male
(CBA x BalbQFl
mice
0, 200, 400
mg/kg (IP)

ND
NA
Ashbv et al.
(1985)
Dominant
lethal
Male DBA/2J mice
0 or 250 mg/kg
(IP or oral)
-
ND
Treated on two
consecutive days
Soares and
Lock (1980)
aLowest effective dose for positive results or highest dose tested for negative results.
b+ = positive; IP= intraperitoneal injection; NA = not applicable; ND = no data.
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DERIVATION OF PROVISIONAL REFERENCE DOSES
Tables 5 and 6 present a summary of noncancer reference values and cancer values, respectively.
Table 5. Summary of Noncancer Reference Values for Technical Grade Dinitrotoluene (CASRN 25321-14-6)
Toxicity Type (Units)
Species/Sex
Critical Effect
p-Reference
Value
POD
Method
PODhed
UFC
Principal
Study
Screening Subchronic p-RfD
(mg/kg-d)
F344 Rat/M
Increased hepatocyte necrosis at
week 26
5 x 1(T3
BMDLjo
0.52
100
CUT f1982a)
Screening Chronic p-RfD (mg/kg-d)
F344 Rat/M
Increased hepatocyte necrosis at
week 104
9 x 1(T4
BMDL10
0.087
100
CUT CI 982a)
Subchronic p-RfC (mg/m3)
NDr
Chronic p-RfC (mg/m3)
NDr
NDr = not determined.
Table 6. Summary of Cancer Values for Technical Grade Dinitrotoluene (CASRN 25321-14-6)
Toxicity Type
Species/Sex
Tumor Type
Cancer value
Principal
Study
Screening
p-OSF
F344 Rat/M
Combined tumor incidence for hepatocellular carcinomas, liver neoplastic nodules, mammary
fibroadenomas and subcutaneous fibromas
4.5 x 10"1
(mg/kg-d)-1
CUT f 1982a)
p-IUR
NDr
NDr = not determined.
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DERIVATION OF ORAL REFERENCE DOSES
Derivation of Subchronic Provisional RfD (Subchronic p-RfD)
A subchronic p-RfD cannot be derived for tgDNT because the only potential principal
study by CUT (CUT. 1983) is limited and is not suitable to derive a subchronic p-RfD (see
Appendix A for details). However, Appendix A provides a "screening level" value for
subchronic oral exposure based on a comprehensive unpublished study (CUT, 1982a).
Derivation of Chronic Provisional RfD (Chronic p-RfD)
A chronic p-RfD cannot be derived for tgDNT because no peer-reviewed studies are
suitable to derive a chronic p-RfD (see Appendix A for details). However, Appendix A provides
a "screening level" value for chronic oral exposure based on a comprehensive unpublished
chronic study (CUT. 1982a).
DERIVATION OF INHALATION REFERENCE CONCENTRATIONS
No studies were identified that could be used to derive provisional inhalation RfCs for
tgDNT. Available epidemiological studies consist primarily of occupational studies in which
workers were exposed to a tgDNT mixture, and/or other known and unknown chemicals. In
addition, none of the epidemiological studies provided quality exposure information. No animal
inhalation studies for tgDNT were identified.
CANCER WEIGHT-OF-EVIDENCE DESCRIPTOR
Table 7 identifies the cancer weight-of-evidence (WOE) descriptor for tgDNT.
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Table 7. Cancer WOE Descriptor for tgDNT
Possible WOE
Descriptor
Designation
Route of Entry
(Oral, Inhalation,
or Both)
Comments
"Carcinogenic to
Humans "
NA
Inhalation
Relevant human cancer studies on tgDNT are
available on the effects of human inhalation/dermal
exposure to mixed DNT.
"Likely to Be
Carcinogenic to
Humans"
Selected
Both3
In the cancer studv bv CUT (1982a). oral exposure
to tgDNT caused an increased incidence of
hepatocellular tumors, liver neoplastic nodules,
mammary fibroadenomas, and subcutaneous
fibromas in both male rats and an increased
incidence of hepatocellular tumors, liver neoplastic
nodules, and subcutaneous fibromas in female
F344 rats. In another cancer studv bv Leonard et
al. (1987), tsDNT caused an increased incidence of
hepatocellular carcinomas and liver neoplastic
nodules in F344 male rats after 1 year of oral
exposure.
"Suggestive Evidence
of Carcinogenic
Potential"
NA
NA
The evidence from animal and human data is more
than suggestive of carcinogenicity, which raises a
concern for carcinogenic effects and is judged
sufficient for a stronger conclusion.
"Inadequate
Information to Assess
Carcinogenic
Potential"
NA
NA
There is evidence to assess the carcinogenic potential
of tgDNT.
"Not Likely to Be
Carcinogenic to
Humans "
NA
NA
Evidence of the carcinogenic potential of tgDNT is
available in animals and humans.
atgDNT is considered "Likely to be Carcinogenic to Humans" by all routes of exposure based on Guidelines for
Carcinogen Risk Assessment (U.S. EPA. 2005). which indicates that for tumors occurring at a site other than the
initial point of contact, the cancer WOE descriptor may apply to all routes of exposure that have not been
adequately tested at sufficient doses.
NA = not applicable.
Under the 2005 Guidelines for Carcinogen Risk Assessment (U.S. EPA. 2005). the WOE
descriptor for tgDNT is "Likely to Be Carcinogenic to Humans'' by all routes of exposure (see
Table 7). This descriptor is based on (1) suggestive evidence of carcinogenicity in humans and
(2) strong evidence in animals by oral exposure.
There is some evidence for an increased risk of certain types of cancer in occupational
populations exposed to tgDNT. An association between tgDNT exposure and an increased risk
of hepatobiliary cancer was found in a retrospective mortality study involving workers at a
U.S. Army munitions facility (Stavner et al.. 1993). A study of underground mining workers
exposed to tgDNT as an explosive (Burning et ai. 1999) also indicated that tgDNT might be
associated with urothelial tumor formation. The workers in this study were believed to be
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exposed to tgDNT via dermal or inhalation exposure. However, these studies were limited by a
variety of factors, including inadequate exposure information (i.e., concentration and duration),
and therefore, do not permit a definitive conclusion on the carcinogenicity of tgDNT in humans.
Results from experimental animal studies showed that tgDNT increased the incidence of
multiple tumor types in F344 rats in two separate studies (Leonard et al .. 1987; CUT. 1982a).
Significant increases in hepatocellular neoplastic nodules and carcinomas (males and females),
subcutaneous fibromas (males and females), and mammary fibroadenomas (males only) were
observed in F344 rats in chronic-duration dietary exposure bioassays (CUT, 1982a), and
increases in hepatocellular carcinoma were also observed in male F344 rats in a 1-year dietary
study (Leonard et at., 1987). In addition, tgDNT caused hepatocellular tumors in rats as early as
26 weeks; therefore, the positive tumor results observed in these studies can be considered an
early onset of carcinogenicity. As stated in the Guidelines for Carcinogen Risk Assessment (U.S.
EPA, 2005), examples for a chemical to be considered "Likely to Be Carcinogenic to Humans"
are (1) "an agent that has tested positive in animal experiments in more than one species, sex,
strain, site, or exposure route, with or without evidence of carcinogenicity in humans;" (2) "a
positive tumor study that raises additional biological concerns beyond that of a statistically
significant result, for example, a high degree of malignancy, or an early age at onset." Based on
these examples from the cancer guidelines and the carcinogenicity data from available human
and animal studies, the WOE descriptor of "Likely to Be Carcinogenic to Humans'' is appropriate
for tgDNT.
The Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005) indicate that for
tumors occurring at a site other than the initial point of contact, the cancer WOE descriptor may
apply to all routes of exposure that have not been adequately tested at sufficient doses. An
exception occurs when there are convincing toxicokinetic data that absorption does not occur by
other routes. Information available on the carcinogenic effects of tgDNT demonstrates that
tumors occur in tissues remote from the site of absorption. tgDNT has been shown to be a
hepatocarcinogen in rats in two bioassays of various experimental designs by oral exposure.
Increased hepatocellular carcinoma in munition workers and urothelial cancer in mining workers
are presumed to have been exposed predominantly through the inhalation route with a
contribution from the dermal route. Information on the carcinogenic effects of tgDNT via the
dermal route in humans and animals is limited or absent. There are no toxicokinetic data
indicating absorption does not occur by other routes. Therefore, based on the observation of
liver tumors in animals following oral exposure and in humans following occupational inhalation
and dermal exposure, it is assumed that an internal effective dose will be achieved regardless of
the route of exposure. Thus, tgDNT is considered "Likely to be Carcinogenic to Humans" by all
routes of exposure.
MODE-OF-ACTION DISCUSSION
The Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005) define mode-of-action
"as a sequence of key events and processes, starting with the interaction of an agent with a cell,
proceeding through operational and anatomical changes, and resulting in cancer formation".
Examples of possible modes of carcinogenic action for any given chemical include
"mutagenicity, mitogenesis, inhibition of cell death, cytotoxicity with reparative cell
proliferation, and immune suppression".
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The potential mode of action for tgDNT is unclear. Table 4 summarizes the studies
examining genotoxicity (e.g., clastogenicity, mutagenicity) of tgDNT. tgDNT was shown to be
positive for mutagenicity in S. typhimurium strains (Couch et ai. 1981) but was not mutagenic in
mammalian cell systems [e.g., HGPRT mutation in CHO cells and TK mutation in mouse
lymphoma cells (Styles and Cross. 1983; Abernethv and Couch. 1982). While most assays of
unscheduled DNA synthesis in rat hepatocytes showed a positive response following oral dosing
(Mirsalis et at.. 1989; Hamilton and Mirsalis. 1987; Ashbv et ai. 1985; Mirsalis and Butterworth.
1982; Mirsalis et ai. 1982). mouse bone marrow micronuclei (Ashbv et ai. 1985) and dominant
lethal assays (Spares and Lock. 1980) were negative.
DERIVATION OF PROVISIONAL CANCER POTENCY VALUES
Derivation of Provisional Oral Slope Factor (p-OSF)
A p-OSF cannot be derived for tgDNT because no peer-reviewed studies are suitable to
derive a p-OSF. However, Appendix A provides a "screening level" value for a p-OSF based on
a comprehensive unpublished carcinogenicity study (CUT, 1982a).
Derivation of Provisional Inhalation Unit Risk (p-IUR)
No human or animal studies examining the carcinogenicity of tgDNT following
inhalation exposure were identified. Therefore, derivation of a p-IUR is precluded.
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APPENDIX A. PROVISIONAL SCREENING VALUES
For the reasons noted in the main document, subchronic and chronic p-RfDs for tgDNT
could not be derived. However, information is available for this chemical which, although
insufficient to support derivation of a provisional toxicity value, under current guidelines, may
be of limited use to risk assessors. In such cases, the Superfund Health Risk Technical Support
Center summarizes available information in an appendix and develops a "screening value".
Appendices receive the same level of internal and external scientific peer review as the main
documents to ensure their appropriateness within the limitations detailed in the document. Users
of screening toxicity values in an appendix to a PPRTV assessment should understand that there
is considerably more uncertainty associated with the derivation of an appendix screening toxicity
value than for a value presented in the body of the assessment. Questions or concerns about the
appropriate use of screening values should be directed to the Superfund Health Risk Technical
Support Center.
DERIVATION OF SCREENING SUBCHRONIC PROVISIONAL RfD (SCREENING
SUBCHRONIC p-RfD)
No human oral studies were identified. The database for oral tgDNT toxicity in animals
includes one subchronic-duration study (CUT. 1983). two chronic-duration studies (Leonard et
at., 1987; CUT, 1982a), and one developmental study (Price et ai, 1985; CUT, 1982b). The
chronic-duration F344 rat study by CUT (1982a) is composed of five interim evaluations at
Weeks 26, 52, 55, 78, and 104. C " ' : I ?83) (4 weeks exposure duration) reported significant
treatment-related hematological effects. As a result, a LOAEL of 31.9 mg/kg-day is identified
based on the significant hematological changes in male rats. However, this study was not
selected as the principal study because it only focused on hematological and gross pathological
examinations, and no clinical chemistry, organ weight, and histopathology endpoints were
examined. The 26-week interim study within the chronic-duration study (CUT, 1982a) provided
a comprehensive toxicity evaluation and is the closest exposure duration to the standard
13 weeks of a subchronic-duration study. Thus, in the absence of a comprehensive evaluation
following a shorter exposure duration, the 26-week study from CUT (1982a) is selected as the
principal study in lieu of the 4-week study by CUT (1983) and is protective of
subchronic-duration exposure. In this 26-week study, hematology, clinical chemistry, urinalyses,
gross pathology, and histopathology were all conducted to evaluate tgDNT toxicity. The study
authors reported toxicity of tgDNT on organ weight (e.g., liver and kidneys) and hematological
(e.g., MetHb levels, reticulocyte count, MCV, and RBC count), and histopathological end points
(e.g., liver and spleen). A NOAEL of 3.47 mg/kg-day and a LOAEL of 13.6 mg/kg-day were
identified based on increased absolute and relative liver weight and hepatotoxicity in male rats.
For comparison purposes, Table A.l summarizes all the potential critical effects from the
26-week study. All the endpoints shown in the table were modeled with benchmark dose
software (BMDS) (version 2.2.2), and the estimated BMDLi0s are also summarized in the table.
Among all the candidate endpoints for potential critical effect, the increased incidence of
hepatocyte necrosis in male rats resulted in the lowest BMDLio of 2.16 mg/kg-day, which is
followed by a BMDLio of 2.27 mg/kg-day for periportal hyperbasophilic hepatocytes in females.
Therefore, increased hepatocyte necrosis is considered the critical effect, and using the BMDLio
for this endpoint as the point of departure (POD) would protect all the sensitive effects observed
in rats after 26 weeks of oral exposure. In addition to the subchronic-duration studies mentioned
above, there is a developmental study (Price et at.. 1985) that is also considered as part of the
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database for derivation of a screening subchronic p-RfD. In this study, a NOAEL of
37.5 mg/kg-day and LOAEL of 75 mg/kg-day were established based on maternal toxicity (i.e.,
increased relative liver weight). A NOAEL of 100 mg/kg-day and a LOAEL of 150 mg/kg-day
were established for developmental toxicity based on increased resorption rate accompanied by
an increase in dead fetuses and a decrease in live fetuses. Among all available subchronic data
(including subchronic-duration and developmental studies), the BMDLio of 2.16 mg/kg-day for
increased incidence of hepatocyte necrosis in male rats is the most sensitive and is considered
protective for all potential tgDNT-induced effects including developmental toxicity. Therefore,
the BMDLio of 2.16 mg/kg-day is selected as the POD for derivation of the screening subchronic
p-RfD. The NOAEL of 3.47 mg/kg-day for increased absolute and relative liver weights in
males in the same 26-week interim sacrifice is considered supportive.
Table A.l. Potential Critical Effects in Male and Female F344 Rats After Dietary
Exposure to tgDNT for 26 Weeks



NOAEL
LOAEL
BMDL10
POD
End points
(mg/kg-d)
(mg/kg-d)
(mg/kg-d)
(mg/kg-d)
Males
Relative liver weight
3.47
13.6
No fit
3.47
Relative kidney weight
13.6
34.6
No fit
13.6
Hepatocyte necrosis
3.47
13.6
2.16
2.16
Females
Relative liver weight
3.22
13.9
5.60
5.60
Relative kidney weight
13.9
34.9
No fit
13.9
Periportal hyperbasophilic hepatocytes
3.22
13.9
2.27
2.27
In Recommended Use of Body Weight3/4 as the Default Method in Derivation of the Oral
Reference Dose (U.S. EPA. 2011b). the Agency endorses a hierarchy of approaches to derive
human equivalent oral exposures from data from laboratory animal species, with the preferred
approach being physiologically based toxicokinetic modeling. Other approaches may include
using some chemical-specific information, without a complete physiologically based
toxicokinetic model. In lieu of chemical-specific models or data to inform the derivation of
human equivalent oral exposures, EPA endorses body-weight scaling to the 3/4 power (i.e.,
BW3 4) as a default to extrapolate toxicologically equivalent doses of orally administered agents
from all laboratory animals to humans for the purpose of deriving a RfD under certain exposure
conditions. More specifically, the use of BW3 4 scaling for deriving a RfD is recommended
when the observed effects are associated with the parent compound or a stable metabolite but not
for portal-of-entry effects or developmental endpoints.
A validated human physiologically based pharmacokinetic (PBPK) model for tgDNT is
not available for use in extrapolating doses from animals to humans. In addition, the selected
POD of 2.16 mg/kg-day is based on increased incidence of hepatocyte necrosis, which is not a
portal-of-entry or developmental effect. Therefore, scaling by BW3 4 is relevant for deriving
human equivalent doses (HEDs) for this effect.
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Following U.S. EPA (2011b) guidance, the POD for the 26-week rat study is converted to
a HED through the application of a dosimetric adjustment factor (DAF1) derived as follows:
DAF = (BWa1/4 - BWh1/4)
where
DAF	=	dosimetric adjustment factor
BWa	=	animal body weight
BWh	=	human body weight
Using a BWa of 0.25 kg for rats and a default BWh of 70 kg for humans (U.S. EPA.
|), the resulting DAF is 0.24. Applying this DAF to the BMDLio identified in the 26-week
rat study yields a BMDLiqhed as follows:
PODhed = BMDLio (mg/kg-day) x DAF
= BMDLio (mg/kg-day) x 0.24
= 2.16 (mg/kg-day) x 0.24
= 0.52 mg/kg-day
Screening Subchronic p-RfD = PODhed ^ UFc
= 0.52 mg/kg-day -M00
= 5 x 10~3 mg/kg-day
Table A.2. Uncertainty Factors for the Screening Subchronic p-RfD for tgDNT
UF
Value
Justification
ufa
3
For the POD based on an increased incidence of heratocvte necrosis (CUT. 1982aN). an UF, of 3 dO0 5)
has been applied to account for uncertainty in characterizing the toxicodynamic differences between
rats and humans following oral tgDNT exposure. The toxicokinetic uncertainty has been accounted for
by calculation of a HED through application of a DAF as outlined in the Recommended Use of Body
Weisht3'4 as the Default Method in Derivation of the Oral Reference Dose ('U.S. EPA. 20 lib).
ufd
3
An UFn of 3 has been applied because there is one developmental toxicity studv (Price et al.. 1985^ in
addition to subchronic- and chronic-duration studies, but there are no two-generation reproductive
toxicity studies.
UFh
10
An UFh of 10 has been applied for inter-individual variability to account for human-to-human
variability in susceptibility in the absence of quantitative information to assess the toxicokinetics and
toxicodynamics of tgDNT in humans.
ufl
1
An UFl of 1 has been applied for LOAEL-to-NOAEL extrapolation because the POD is a BMDL10.
UFS
1
An UFS of 1 has been applied because a subchronic-duration study was selected as the principal study.
UFC
100

:As described in detail in Recommended Use of Body Weight3/4 as the Default Method in Derivation of the Oral
Reference Dose (U.S. EPA. 2011b'). rate-related processes scale across species in a manner related to both the direct
(BW11) and allometric scaling (BW3/4) aspects such that BW3 4 BW11 = BW ' converted to a
DAF = BWa1/4 - BWhI/4.
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DERIVATION OF SCREENING CHRONIC PROVISIONAL RfD (SCREENING
CHRONIC p-RfD)
The chronic-duration study database for tgDNT includes two studies (Leonard et al..
1987; CUT. 1982a). Leonard et al. (1987) conducted a 1-year study with two doses of tgDNT
(0 and 35 mg/kg-day) administered to male F344 rats. The study authors reported significantly
decreased body weight, increased absolute and relative liver weights, and liver pathological
changes. A LOAEL of 35 mg/kg-day is established based on the observed effects; no NOAEL
could be identified. The chronic-duration study by CUT (1982a) is a comprehensive study
composed of three interim chronic-duration evaluations (i.e., 52-,78-, and 104-week interim
sacrifices). Table A.3 summarizes all potential critical effects and corresponding NOAELs and
LOAELs for each interim study. All the endpoints shown in this table were modeled with
BMDS (version 2.2.2), and the estimated BMDLs are also summarized. As shown in Table A.3,
the interim sacrifices consistently indicated that the liver and kidneys are the target organs of
tgDNT, and the liver is a more sensitive target organ than the kidneys. During all three interim
sacrifices, the responses observed in male rats were consistently more sensitive than in the
female rats (e.g., relatively lower PODs for hepatocyte necrosis ranged from 0.059 to
0.5 mg/kg-day in males vs. 1.31 to 3.64 [LOAEL] in females). Among the responses observed
in male rats, hepatocyte necrosis is consistently shown as the most sensitive response.
Therefore, male rat hepatocyte necrosis data have been further evaluated (see Table A.4) to find
the most sensitive POD. Although, the necrosis incidences in male rats at 52 weeks appeared to
be more sensitive than the 78- and 104-week groups, the results might be questionable because
(1) the 52-week high-dose group showed a much lower response (50%) compared to 70% and
90% incidence at the low- and mid-doses, respectively; and (2) the 78- and 104-week studies,
which had a longer exposure duration and larger sample sizes, showed less response (35—60%) at
low- and mid-doses compared to those (70% and 90%, respectively) at 52-week sacrifice. At
104 weeks, male hepatocyte necrosis incidence at the low-dose (54%) was higher than that at the
mid dose (48%); therefore, BMD modeling cannot adequately model the full data set (see
Appendix C for details). In order to provide a best estimate for this data set, the mid-dose data
point (48%>) was dropped and two data points (control and low dose) were modeled with BMDS
(version 2.2.2), with an estimated BMDLio of 0.363 mg/kg-day (see Appendix C for details). As
part of the database to derive a chronic RfD, the developmental study by Price et al. (1985)
identified higher NOAELs for maternal toxicity (37.5 mg/kg-day) and developmental toxicity
(100 mg/kg-day). Therefore, the BMDLio of 0.363 mg/kg-day for hepatocyte necrosis at
104 weeks in the CUT (1982a) study is the most sensitive and is considered protective for all the
observed effects and is chosen as the POD for derivation of the screening chronic p-RfD.
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Table A.3. Potential Critical Effects in Male and Female F344 Rats After Dietary Exposure
to tgDNT for 52, 78, and 104 Weeks
Endpoints
NOAEL
(mg/kg-d)
LOAEL
(mg/kg-d)
BMDL10
(mg/kg-d)
POD
(mg/kg-d)
POD Selection
(mg/kg-d)
52-Week Males
Relative liver weight
NA
3.47
1.66
1.66

Relative kidney weight
3.47
13.9
No fit
3.47

Hepatocyte necrosis
NA
3.47
0.059
0.059
0.059
Hyperbasophilic hepatocyte
3.47
13.9
No fit
3.47

Vacuolation
NA
3.47
No fit
3.47 (LOAEL)

52-Week Females
Relative liver weight
3.46
13.9
No fit
3.46

Relative kidney weight
13.9
35.1
No fit
13.9

Hepatocyte necrosis
13.9
35.1
3.64
3.64

Hepatocyte megalocytosis
13.9
35.1
6.86
6.86

Hyperbasophilic hepatocyte
3.46
13.9
2.20
2.20
2.20
78-Week Males
Relative liver weight
NA
3.49
1.34
1.34

Relative kidney weight
NA
3.49
1.07
2.72

Hepatocyte necrosis
NA
3.49
0.50
0.5
0.5
Cystic degeneration
3.49
14
No fit
3.49

78-Week Females
Relative liver weight
NA
3.45
2.15a
2.15

Relative kidney weight
3.45
14
3.56
3.67

Hepatocyte necrosis
3.45
14
1.31
1.31
1.31
104-Week Males
Relative liver weight
NA
3.51
1.36a
1.36

Relative kidney weight
Original data illegible
Hepatocyte necrosis
NA
3.51
0.3633
0.363
0.363
Cystic degeneration
3.51
14.0
No fit
3.51

Hepatocyte megalocytosis
NA
3.51
No fit
3.51 (LOAEL)

104-Week Females
Relative liver weight
NA
3.46
Control SD from original
data is illegible
3.46 (LOAEL)
3.46 (LOAEL)
Relative kidney weight
3.46
14.0
No fit
3.46

Hepatocyte necrosis
NA
3.46
No fit
3.46 (LOAEL)

Hepatocyte megalocytosis
NA
3.46
2.09
2.09

aBMD modeling was performed with two data points (control and low dose).
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A.4. Hepatocyte Necrosis in Male F344 Rats After Dietary Exposure to
tgDNT for 52, 78 and 104 Weeks"
Week
Control
Low-dose
Mid-dose
High-dose
52 (n = 10)
0/10
7/10 (70°)
9/10 (90)
5/10 (50)
78 {n = 20)
0/20
7/20 (35)
12/20 (60)
NAb
104 (n = 61, 70, and 23 for control, low-, and
mid-dose groups, respectively)
0/61
38/70 (54)
11/23 (48)
NAb
"CUT (1982a).
bAll the high-dose rats were sacrificed at Week 55.
°Percent animals with necrosis
Following U.S. EPA (201 lb) guidance, the POD for the rat 104-week study is converted
to a HED through an application of a DAF derived as follows:
DAF	=	(BWa1/4 - BWh1/4)
where
DAF	=	dosimetric adjustment factor
BWa	=	animal body weight
BWh	=	human body weight
Using a BWa of 0.25 kg for rats and a default BWh of 70 kg for humans (U.S. EPA.
1988). the resulting DAF is 0.24. Applying this DAF to the BMDLm identified in the rat
104-week study yields a BMDLiohed as follows:
PODhed =	BMDLio (mg/kg-day) x DAF
=	BMDLio (mg/kg-day) x 0.24
=	0.363 (mg/kg-day) x 0.24
=	0.087 mg/kg-day
Screening Chronic p-RfD = PODhed ^ UFc
= 0.087 mg/kg-day -M00
= 9 x 10~4 mg/kg-day
2As described in detail in Recommended Use of Body Weight3/4 as the Default Method in Derivation of the Oral
Reference Dose (U.S. EPA. 2011b'). rate-related processes scale across species in a manner related to both the direct
(BW11) and allometric scaling (BW3/4) aspects such that BW3 4 BW11 = BW ' converted to a
DAF = BWa1/4 - BWhI/4.
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Table A.5. Uncertainty Factors for the Screening Chronic p-RfD for tgDNT
UF
Value
Justification
ufa
3
For the POD based on an increased incidence of heratocvte necrosis (CUT. 1982aN). a UF -, of 3 CIO05)
has been applied to account for uncertainty in characterizing the toxicodynamic differences between
rats and humans following oral tgDNT exposure. The toxicokinetic uncertainty has been accounted for
by calculation of a HED through application of a dosimetric adjustment factor (DAF) as outlined in
Recommended Use of Body Weight3/4 as the Default Method in Derivation of the Oral Reference Dose
(U.S. EPA. 2011W.
ufd
3
An UFn of 3 has been applied because there is one developmental toxicity studv (Price et al.. 1985s) in
addition to subchronic- and chronic-duration studies, but there are no two-generation reproductive
toxicity studies.
UFh
10
An UFh of 10 has been applied for interindividual variability to account for human-to-human variability
in susceptibility in the absence of quantitative information to assess the toxicokinetics and
toxicodynamics of tgDNT in humans.
ufl
1
An UFl of 1 has been applied for LOAEL-to-NOAEL extrapolation because the POD is a BMDLi0.
UFS
1
An UFS of 1 has been applied because a chronic-duration study was selected as the principal study.
UFC
100

DERIVATION OF PROVISIONAL CANCER POTENCY VALUES
Derivation of Screening Provisional Oral Slope Factor (p-OSF)
There are two oral carcinogenicity studies in rats (Leonard et ai. 1987; CUT. 1982a).
Leonard et al. (1987) indicated that exposure to tgDNT (35 mg/kg-day) for 1 year caused a
47% increase in the incidence of hepatocellular tumors in male rats compared to the control
(only one treatment dose). In the study conducted by CUT (1982a). the incidence of
hepatocellular carcinoma was observed beginning at Week 26, and the highest incidence
occurred at Week 104. In addition, increased liver neoplastic nodules, mammary fibroadenomas,
and subcutaneous fibromas were observed in males at Week 104. Appendix B summarizes these
carcinogenicity results. The overall tumor response is relatively higher in male rats than in
female rats at 104 weeks from the CUT (1982a) study, and, therefore. Table A.6 summarizes
only the male incidence data.
Table A.6 shows BMD dose-response modeling was conducted for various tumor types.
All these tumor incidence data successfully fit to the Multistage-Cancer model in BMDS
(version 2.2.21 see Appendix D for details), and Table A.7 summarizes the modeling results.
Based on the BMD modeling results, the calculated cancer slope factors (oral slope factor or
OSF) for hepatocellular carcinomas, liver neoplastic nodules, combined hepatocellular
carcinoma and/or neoplastic nodules, mammary fibroadenomas, and subcutaneous fibromas are
0.047, 0.037, 0.060, 0.027, and 0.072 (mg/kg-day)-1, respectively. Because treatment with
tgDNT produced multiple types of tumors in male rats in three different tissues in the CUT
bioassay (CUT. 1982a). the overall oral cancer slope factor for tgDNT exposure was derived
based on the male incidence data for combined hepatocellular carcinoma and/or neoplastic
nodules, mammary fibroadenomas, and subcutaneous fibromas by assuming that different tumor
types are independent from each other. The overall tumor incidence was fit with the MSCombo
multiple tumor model (BMDS version 2.2.2; see Appendix D for details), and the estimated
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BMDLio is 0.852 mg/kg-day. Similar to the screening subchronic and chronic p-RfDs, this
BMDLio was further converted from an animal dose to an HED, and then used as the PODHed to
derive the p-OSF for tgDNT.
Table A.6. Incidences of Hepatocellular Carcinomas, Liver Neoplastic Nodules, Mammary
Fibroadenomas, and Subcutaneous Fibromas in Male Rats at Week 104a
Parameter
Exposure Group,
ADD mg/kg-db
Control
(0)
Low-dose
(3.51)
High-dose
(14.0)
Number examined
61
70
23
Hepatocellular carcinoma
1(2)
9(13)*
21 (91)*
Neoplastic nodules
9(15)
11(16)
15 (65)*
Hepatocellular carcinomas and/or neoplastic nodules
10(16)
19 (27)
23 (100)*
Mammary fibroadenomas
3(5)
7(10)
5 (22)*
Subcutaneous fibromas
5(8)
14 (20)
14 (61)*
"CUT (1982a).
bValues expressed as number of animals (% of animals with lesion/effect); % calculated by EPA.
* Statistically different from controls, p < 0.05.
Table A.7. Goodness-of-Fit Statistics and BMDio and BMDLio Values for
Dichotomous Model for Four Types of Tumors and Combined Tumors in Male F344
Rats Exposed to tgDNT Orally for 104 Weeks"
Multistage-Cancer Model
Goodness-of-fit
p-V alueb
BMD10
(mg/kg-d)
BMDL10
(mg/kg-d)
Cancer Slope
Factor
(mg/kg-d)1
(Animal Dose)
Hepatocellular carcinoma
0.6332
3.04
2.15
0.047
Liver neoplastic nodules
0.5454
4.86
2.69
0.037
Hepatocellular carcinoma and/or
neoplastic nodule(s)
0.2389
2.42
1.68
0.060
Mammary fibroadenomas
0.9081
7.37
3.73
0.027
Subcutaneous fibromas
0.4138
2.01
1.38
0.072
Combined tumors
NA
1.20
0.852

aCIIT (1982a1.
bValues >0.1 meet conventional goodness-of-fit criteria.
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PODhed = BMDLio(mg/kg-day) x DAF
= BMDLio (mg/kg-day) x (BWa1/4 - BWh1/4)
= BMDLio (mg/kg-day) x (0.3331/4 - 701/4)
= 0.852 (mg/kg-day) x 0.263
= 0.224 mg/kg-day
Note: The BWa of 0.333 kg is the mean body weight from the low-dose male group at Week 104
(see Table B.7).
Screening p-OSFHuman = BMR h- BMDLiohed (mg/kg-day)
= 0.1-0.224
= 4.5 x 10-1 (mg/kg-day)-1
The p-OSF is 4.5 x 10_1 (mg/kg-day)-1 based on combined tumor incidences in male rats
from CUT (1982a).
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APPENDIX B. DATA TABLES
Table B.l. Average Body Weights (g) of Male and Female F344 Rats After Dietary
Exposure to tgDNT for 4 Weeks"
Number of animals/group
10
10
10
10
Males
Time period
Exposure Group
(ADD, mg/kg-d;

0 (Control)
31.9
61.9
134
Initial
176.5 ± 11.0
177.9 ± 11.1 (101)
177.1 ± 10.8(100)
177.9 ± 11.7(101)
Week 1
207.1 ±7.4
205.9 ± 10.4(99)
201.8 ±9.4 (97)
186.6 ± 10.5* (90)
Week 2
228.0 ±8.4
221.4 ± 11.8(97)
207.8 ±9.8* (91)
167.6 ± 10.7* (74)
Week 3
236.7 ±9.6
231.4 ± 13.0(98)
209.6 ± 10.1* (89)
152.2 ± 10.2* (64)
Week 4
251.8 ± 13.2
236.9 ± 13.2* (94)
208.8 ± 11.9* (83)
155.8 ± 10.9* (62)
Females
Time period
Exposure Group
(ADD, mg/kg-d)b

0 (Control)
32.0
63.6
120
Initial
125.8 ±3.2
127.7 ±3.4 (102)
128.4 ±3.7 (102)
126.3 ±6.7 (100)
Week 1
136.2 ±2.7
138.0 ±5.5 (101)
137.2 ±4.3 (101)
139.1 ±7.5 (102)
Week 2
146.0 ±3.2
147.3 ±5.8 (101)
142.3 ±4.8 (97)
135.6 ±9.5* (93)
Week 3
153.8 ±6.0
151.9 ±7.7 (99)
149.4 ± 6.6 (97)
127.7 ± 11.0* (83)
Week 4
158.4 ±4.3
155.1 ±7.5 (98)
149.4 ±7.4* (94)
124.5 ± 10.1* (79)
"CUT fl.983).
bValues expressed as mean ± SD (% of control); % calculated by EPA.
* Statistically different from controls, p < 0,05.
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Table B.2. Hematology Results for Male and Female F344 Rats After Dietary Exposure to
tgDNT for 4 Weeks"
Number of animals/group
10
10
10
10
Males
Parameter
Exposure Group
(ADD, mg/kg-d)b

0 (Control)
31.9
61.9
134
MetHb (%)
1.12 ± 0.61
1.20 ±0.40 (107)
1.38 ±0.71 (123)
2.63 ± 1.03* (235)
Reticulocytes (%)
1.11 ± 0.31
1.99 ±0.82* (179)
2.81 ± 1.55* (253)
6.84 ±3.05* (616)
Heinz bodies (%)
0.00 ±0.00
0.04 ±0.05*
0.06 ±0.07*
5.25 ±3.28*
Females

Exposure Group
(ADD, mg/kg-d)b

0 (Control)
32.0
63.6
120
MetHb (%)
0.57 ±0.48
1.46 ±0.76* (256)
1.04 ±0.77 (182)
1.99 ±0.88* (349)
Reticulocytes (%)
1.45 ±0.27
3.27 ±0.94* (226)
2.95 ±0.81* (203)
4.05 ±0.91* (279)
Heinz bodies (%)°
0.00 ±0.00
0.07 ±0.07*
0.09 ±0.06*
0.14 ± 0.11*
aCIIT fl.983).
bValues expressed as mean ± SD (% of control); % calculated by EPA.
* Statistically different from controls, p < 0,05.
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Table B.3. Gross Pathology for Male and Female F344 Rats After Dietary Exposure to
tgDNT for 4 Weeks"
Number of animals/group
10
10
10
10
Parameter
Exposure Group
(ADD, mg/kg-d)b
0 (Control)
31.9
61.9
134
Males
Mottled lungs (dark-red or brown areas)
0
6 (60)
10 (100)
3 (30)
Liver
Surface rough or granular
0
5 (50)
10 (100)
0(0)
Mottled appearance
0
5 (50)
0(0)
3 (30)
Yellowish tinge
0
3 (30)
0(0)
6(60)
Spleen
Dark or black
0
1(10)
0(0)
10 (100)
Surface rough or granular
0
1(10)
0(0)
0(0)
Thickened
0
0(0)
0(0)
3 (30)
Enlarged
0
0(0)
0(0)
1(10)
Kidneys
Greenish tinge
0
0(0)
0(0)
3 (30)
Dark zone at cortico-medullary junction
0
1(10)
0(0)
1(10)
Small white raised nodule on ventral
surface
0
0(0)
0(0)
1(10)
Parameter
Exposure Group
(ADD, mg/kg-d)b
0 (Control)
32.0
63.6
120
Females
Mottled lungs
0
0(0)
0(0)
1(10)
Liver
Yellowish tinge
0
0(0)
0(0)
4 (40)
Spleen
Dark or black
0
0(0)
0(0)
5 (50)
Thickened
0
0(0)
0(0)
7 (70)
Enlarged
0
0(0)
0(0)
2 (20)
Kidneys
Greenish tinge
0
0(0)
0(0)
1(10)
Water cyst in ovary
0
0(0)
1(10)
0(0)
Dark yellow stains around vagina
0
0(0)
0(0)
2 (20)
"CUT (19831.
bValues expressed as number of animals affected (% affected); % calculated by EPA.
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Table B.4. Selected Average Body Weights (g) and Food Consumption (g/week/rat) of Male
and Female F344 Rats After Dietary Exposure to tgDNT for up to 2 Years"
Time Period
Exposure Group
(ADD, mg/kg-d)
Control (0)
Low-dose (3.51)
Mid-dose (14.0)
High-dose (34.9)
Average body weightb (Males)
Week 0(« = 130)
154 ± 14.5
153 ± 14.2 (99)
151 ± 14.2 (98)
153 ± 14.6 (99)
Week 26 (« = 130)
351 ±22
338 ±21.6 (96)
301 ± 19.2 (86)
265 ± 17.6 (75)
Week 50 (« = 113-119)c
387 ±23.9
370 ± 20.3 (96)
316 ± 18.3 (82)
270 ± 18 (70)
Week 78 (n = 95-106)
396 ±22.3
373 ±21.7 (94)
309± 25.8 (78)
NA
Week 102 (n = 25-75)
370 ±31
351 ±30.8 (95)
293 ± 39.3 (80)
NA
Average food consumption (Males) (g/week/rat)
Week 0(n = 130)
0
0
0
0
Week 26 (n = 130)
108
111(103)
101 (94)
101 (94)
Week 50 (n = 113-119)c
112
109 (97)
110(98)
114(102)
Week 78 (n = 95-106)
107
107 (100)
106 (99)
NA
Week 102 (n = 25-75)
105
104 (99)
92 (88)
NA
Time Period
Exposure Group
(ADD, mg/kg-d)
Control (0)
Low-dose (3.46)
Mid-dose (14.0)
High-dose (35.1)
Average body weightb (Females)
Week 0(n = 130)
118 ±8.4
121 ±8.2 (103)
121 ±7.7 (103)
118 ±8.4 (100)
Week 26 (n = 130)
198 ± 10.7
193 ±11.6 (97)
187 ± 11.2 (94)
172 ± 11.2 (87)
Week 50 (n = 120)c
226 ± 14.5
220 ± 14.9 (97)
197 ± 13.5 (90)
180 ± 11.1 (80)
Week 78 (n = 104-107)
272 ± 20
251 ±24.1 (92)
213 ± 12.8 (78)
NA
Week 104 (n = 61-69)
288 ± 29
267 ±25.1 (93)
213 ± 18.8 (74)
NA
Average food consumption (Females) (g/week/rat)
Week 0(« = 130)
0
0
0
0
Week 26 (n = 130)
79
78 (99)
73 (92)
73 (92)
Week 50 (n = 120)c
81
79 (98)
72 (89)
71 (88)
Week 78 (n = 104-107)
87
85 (98)
80 (92)
NA
Week 104 (n = 61-69)
88
89 (101)
91(103)
NA
TUT fl.982a).
''Body-weight values expressed as mean ± SD (% of control); % calculated by EPA.
°Body weight and food consumption data were collected on Week 50, not Week 52.
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Table B.5. Selected Hematology Results for Male F344 Rats After Dietary Exposure to
tgDNT for up to 2 Years3
Parameter
Exposure Group
(ADD, mg/kg-df
Control (0)
Low-dose (3.47)
Mid-dose (13.6)
High-dose (34.6)
Week 26 (n = 10)
Hb (g/dL)
17.12 ±0.42
17.36 ±0.48 (101)
17.26 ± 1.12(101)
16.48 ± 0.44 (96)
Hematocrit (100%)
48.70 ±2.21
50.00 ±2.71 (103)
52.05 ±4.37 (107)
49.30 ±2.21 (101)
RBC (x 106/mm3)
8.967 ±0.554
9.209 ±0.428 (103)
8.921 ±0.525 (99)
8.272 ± 0.392* (92)
Reticulocyte (%)
1.97 ±0.80
2.23 ±0.70 (113)
2.83 ±0.85 (144)
3.60 ±0.84* (183)
MetHb (%)
0.36 ±0.36
0.97 ±0.71 (269)
0.74 ± 0.55 (205)
1.80 ± 1.18* (500)
WBC (x 103/mm3)
12.31 ± 1.89
14.60 ±3.15 (119)
16.13 ±2.11* (131)
15.04 ±2.73 (122)
Parameter
Exposure Group
(ADD, mg/kg-dr
Control (0)
Low-dose (3.47)
Mid-dose (13.9)
High-dose (34.9)
Week 52 (n = 10)
Hb (g/dL)
16.05 ±0.59
16.11 ±0.61 (100)
16.29 ±0.66 (101)
14.07 ± 1.16* (88)
Hematocrit (100%)
46.70 ± 1.70
46.45 ± 1.57 (99)
46.90 ± 1.91 (100)
47.30 ±2.89 (101)
RBC (x 106/mm3)
8.507 ±0.778
8.572 ±0.570 (101)
7.585 ±0.647* (89)
5.546 ± 0.728* (65)
Reticulocyte (%)
2.08 ±0.73
2.42 ±0.79 (116)
2.01 ±0.74 (97)
3.87 ± 1.90* (186)
MetHb (%)
0.87 ±0.56
1.37 ±0.61 (157)
1.49 ±0.96 (171)
1.55 ±0.92 (178)
WBC (x 103/mm3)
13.29 ±3.23
11.60 ±4.13 (87)
14.45 ±3.60 (109)
19.30 ± 3.47* (145)
Parameter
Exposure Group
(ADD, mg/kg-dr
NA
NA
NA
High-dose (34.9)
Week 55 (n = 10)
Hb (g/dL)
NA
NA
NA
12.82 ±0.78
Hematocrit (100%)
NA
NA
NA
38.37 ±2.08
RBC (x 106/mm3)
NA
NA
NA
4.129 ± 1.245
Reticulocyte (%)
NA
NA
NA
1.61 ±0.77
MetHb (%)
NA
NA
NA
1.42 ± 1.33
WBC (x 103/mm3)
NA
NA
NA
4.33 ±0.69
55
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4-4-2013
Table B.5. Selected Hematology Results for Male F344 Rats After Dietary Exposure to
tgDNT for up to 2 Years3
Parameter
Exposure Group
(ADD, mg/kg-df
Control (0)
Low-dose (3.49)
Mid-dose (14.0)
NA
Week 78 (n = 20)
Hb (g/dL)
16.81 ± 1.60
18.25 ±2.16 (109)
15.34 ±2.79 (91)
NA
Hematocrit (100%)
51.37 ± 3.55
55.60 ±5.58* (108)
44.80 ±7.11* (87)
NA
RBC (x 106/mm3)
9.303 ±0.897
9.608 ±0.859 (103)
7.998 ± 1.328* (86)
NA
Reticulocyte (%)
2.02 ± 1.04
2.28 ± 1.25* (113)
4.29 ±2.64* (212)
NA
MetHb (%)
1.77 ± 1.16
1.56 ±0.46 (88)
1.31 ± 1.01(74)
NA
WBC (x 103/mm3)
10.36 ±2.26
9.71 ±2.86 (94)
10.76 ± 3.89 ( 104)
NA
Parameter
Exposure Group
(ADD, mg/kg-dr
Control (0)
Low-dose (3.51)
Mid-dose (14.0)
NA
Week 104 (n = 19-20)
Hb (g/dL)
18.48 ±3.13
19.40 ±2.86 (105)
12.08 ±2.72* (65)
NA
Hematocrit (100%)
54.60 ±9.38
56.42 ±8.09 (103)
36.95 ±7.21* (68)
NA
RBC (x 106/mm3)
9.227 ± 1.602
9.543 ± 1.112 (103)
6.819 ± 1.807* (74)
NA
Reticulocyte (%)
3.53 ±3.53
2.99 ± 1.89 (85)
6.72 ±2.93* (190)
NA
MetHb (%)
1.16 ± 1.01
1.32 ±0.99 (114)
1.46 ±0.86 (126)
NA
WBC (x 103/mm3)
11.22 ±4.06
12.85 ±3.62 (115)
9.35 ± 10.16(83)
NA
"CUT (1982a).
bValues expressed as mean ± SD (% of control); % calculated by EPA.
* Statistically different from controls, p < 0.05.
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Table B.6. Selected Clinical Chemistry Results for Male and Female F344 Rats After
Dietary Exposure to tgDNT for up to 2 Years"
Males
Parameter
Exposure Group
(ADD, mg/kg-d)b
Week 26 (n = 10)
Control (0)
Low-dose (3.47)
Mid-dose (13.6)
High-dose (34.6)
SGPT (IU/L)
43.0 ± 13.6
24.7 ±9.3* (57)
25.8 ±4.9* (60)
39.1 ±8.3 (91)
ALP (IU/L)
77.60 ± 7.90
67.70 ± 7.24* (87)
63.10 ±3.90* (81)
78.56 ±6.17 (101)
BUN (mg/dL)
21.03 ±2.09
17.98 ± 1.37* (85)
18.66 ± 1.55 (89)
24.56 ±2.63* (117)
Week 52 (n = 10)
0 (Control)
Low-dose (3.47)
Mid-dose (13.9)
High-dose (34.9)
SGPT (IU/L)
41.5 ±6.5
22.2 ±5.8 (53)
25.1 ±5.7 (60)
138.7 ±72.4* (334)
ALP (IU/L)
68.70 ± 10.95
53.50 ±4.25 (78)
57.60 ± 8.67 (84)
134.20 ±65.45*
(195)
BUN (mg/dL)
18.00 ± 1.08
17.30 ±0.96 (96)
16.77 ± 1.87 (93)
29.91 ±5.83* (166)
Week 55 (n = 10)
NA
NA
NA
High-dose (34.9)
SGPT (IU/L)
NA
NA
NA
136.8 ±96.1
ALP (IU/L)
NA
NA
NA
161.55 ±66.89
BUN (mg/dL)
NA
NA
NA
28.92 ±3.37
Week 78 (n = 20)
Control (0)
Low-dose (3.49)
Mid-dose (14.0)
NA
SGPT (IU/L)
28.7 ±6.5
24.1 ±16.2 (84)
55.3 ± 56.3* (193)
NA
ALP (IU/L)
74.80 ± 10.35
64.95 ± 29.20 (87)
79.95 ±47.52 (107)
NA
BUN (mg/dL)
21.55 ± 1.73
17.09 ±2.22* (79)
25.14 ±6.52 (117)
NA
Week 104 (n = 19-20)
0
Low-dose (3.51)
Mid-dose (14.0)
NA
SGPT (IU/L)
22.5 ±4.6
21.7 ±5.9 (96)
78.7 ±42.7* (350)
NA
ALP (IU/L)
65.10 ± 11.54
45.05 ± 9.59* (69)
89.58 ±51.09 (138)
NA
BUN (mg/dL)
27.54 ±7.53
19.33 ±3.08* (70)
77.49 ±47.03*
(281)
NA
Females
Parameter
Exposure Group
(ADD, mg/kg-d)b
Week 26 (n = 10)
Control (0)
Low-dose (3.22)
Mid-dose (13.9)
High-dose (34.9)
BUN (mg/dL)
18.82 ± 1.43
19.20 ±3.04 (102)
18.90 ± 1.49(100)
23.88 ±4.31*( 127)
aCIIT (1982a).
bValues expressed as mean ± SD (% of control); % calculated by EPA.
* Statistically different from controls, p < 0.05.
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Table B.7. Terminal Body Weights (g) of Male and Female F344 Rats After Dietary
Exposure to tgDNT for 2 Years"
Average body weight—interim and terminal sacrifices (males)
Time Period
Exposure Group
(ADD, mg/kg-d)b
Week 26 (n = 10)
Control (0)
Low-dose (3.47)
Mid-dose (13.6)
High-dose (34.6)
328 ± 23
311 ±14* (95)
297 ±31* (91)
253 ± 13* (77)
Week 52 (n = 10)
Control (0)
Low-dose (3.47)
Mid-dose (13.9)
High-dose (34.9)
363 ± 22
335 ±21 (92)
287 ±21* (79)
245 ± 14* (67)
Week 55 (n = 101)
NA
NA
NA
High-dose (34.9)
NA
NA
NA
259 ± 18
Week 78 (« = 20)
Control (0)
Low-dose (3.49)
Mid-dose (14.0)
NA
374.30 ± 16.936
358.20 ± 15.531*
(96)
286.00 ±20.261*
(76)
NA
Week 104 (n = 58, 68, and 19
for 0, 3.5, and 14 mg/kg-d,
respectively)
0
Low-dose (3.51)
Mid-dose (14.0)
NA
352.03 ±28.836
333.19 ±20.806*
(95)
257.79 ±30.608*
(73)
NA
Average body weight—interim and terminal sacrifices (females)
Time Period
Exposure Group
(ADD, mg/kg-d)b
Week 26 (n = 10)
Control (0)
Low-dose (3.22)
Mid-dose (14.0)
High-dose (34.9)
184 ±8
182 ± 12* (99)
182 ± 13* (99)
158 ± 6* (86)
Week 52 (n = 10)
Control (0)
Low-dose (3.46)
Mid-dose (13.9)
High-dose (35.1)
214 ± 14
207 ± 15 (97)
190 ± 12* (89)
169 ±11* (79)
Week 55 (n = 109)
NA
NA
NA
High-dose (35.1)
NA
NA
NA
170 ± 13
Week 78 (n = 20)
Control (0)
Low-dose (3.45)
Mid-dose (14.0)
NA
259.65 ± 14.651
238.80 ± 11.551*
(92)
201.35 ±11.815*
(78)
NA
Week 104 (n = 55, 59, and 59
for 0, 3.5, and 14 mg/kg-d,
respectively)
Control (0)
Low-dose (3.46)
Mid-dose (14.0)
NA
271.88 ±28.15
256.22 ± 22.627*
(94)
198.84 ± 19.27*
(73)
NA
"CUT Q982a).
bValues expressed as mean ± SD (% of control); % calculated by EPA.
* Statistically different from controls, p < 0.05.
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Table B.8. Selected Organ Weights (g) of Male F344 Rats After Dietary Exposure to
tgDNT for 2 Years"
Time Period
Exposure Group
(ADD, mg/kg-d)b
Week 26 (n = 10)
Control (0)
Low-dose (3.47)
Mid-dose (13.6)
High-dose (34.6)
Absolute liver weight
8.78 ±0.87
8.80 ±0.56 (100)
9.87 ±0.80* (112)
13.19 ± 1.01* (150)
Relative liver weight
2.68 ±0.23
2.83 ±0.07 (105)
3.35 ±0.36* (125)
5.23 ±0.48* (195)
Absolute brain weight
1.97 ±0.05
1.96 ±0.06 (99)
1.95 ±0.05 (99)
1.95 ±0.04 (99)
Relative heart weight
0.32 ±0.03
0.32 ±0.03 (100)
0.30 ±0.03 (94)
0.37 ±0.05* (116)
Relative kidney weight
0.685 ± 0.04
0.687 ±0.025 (100)
0.736 ± 0.066 (107)
0.942 ±0.044* (138)
Relative lung weight
0.415 ±0.049
0.407 ± 0.022 (98)
0.416 ±0.045 (100)
0.496 ±0.031* (120)
Relative testis weight
1.32 ± 0.11
1. 35 ±0.07 (102)
1.34 ±0.08 (102)
1.67 ±0.18* (127)
Week 52 (n = 10)
Control (0)
Low-dose (3.47)
Mid-dose (13.9)
High-dose (34.9)
Absolute liver weight
9.11 ±0.66
10.15 ±0.54 (111)
15.46 ± 1.23* (170)
28.73 ±7.86* (315)
Relative liver weight
2.51 ±0.12
3.00 ±0.20 (120)
5.41 ±0.54* (216)
11.81 ± 3.54* (471)
Absolute brain weight
2.13 ±0.16
2.01 ±0.08 (94)
1.99 ±0.07 (93)
2.00 ±0.13 (94)
Relative heart weight
0.296 ±0.02
0.335 ±0.05 (113)
0.355 ±0.035 (120)
0.455 ±0.25* (154)
Relative kidney weight
0.704 ±0.083
0.718 ±0.066 (102)
0.909 ±0.035*
(129)
1.160 ±0.190* (165)
Relative lung weight
0.417 ±0.055
0.400 ± 0.054 (96)
0.476 ±0.060 (114)
0.591 ±0.130* (142)
Relative testis weight
1.42 ±0.21
1.46 ±0.19 (103)
1.71 ±0.10 (120)
1.59 ±0.40 (112)
Week 55 (n = 101)
NA
NA
NA
High-dose (34.9)
Absolute liver weight
NA
NA
NA
29.88 ±5.77
Relative liver weight
NA
NA
NA
11.65 ±2.45
Absolute brain weight
NA
NA
NA
1.97 ±0.08
Relative heart weight
NA
NA
NA
0.39 ±0.04
Relative kidney weight
NA
NA
NA
1.15 ±0.28
Relative lung weight
NA
NA
NA
0.620 ±0.471
Relative testis weight
NA
NA
NA
1.48 ±0.45
Week 78 (n = 20)
Control (0)
Low-dose (3.49)
Mid-dose (14.0)
NA
Absolute liver weight
9.422 ±0.6001
11.363 ±0.9594*
(121)
18.580 ±3.8101*°
(197)
NA
Relative liver weight
2.518 ±0.1279
3.175 ±0.2682*
(126)
6.259 ± 1.0671*
(249)
NA
Absolute brain weight
2.1010 ±0.11026
2.1180 ±0.06661
(101)
2.0150 ±0.12089
(96)
NA
Relative heart weight
0.290 ±0.03
0.302 ±0.02 (104)
0.400 ±0.60* (138)
NA
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4-4-2013
Table B.8. Selected Organ Weights (g) of Male F344 Rats After Dietary Exposure to
tgDNT for 2 Years"
Relative kidney weight
0.665 ± 0.0282
0.744 ±0.0419*
(112)
1.029 ±0.1003*
(155)
NA
Relative lung weight
0.365 ±0.262
0.416 ±0.819*
(114)
0.476 ± 0.0372*
(130)
NA
Relative testis weight
1.450 ±0.3114
1.845 ±0.2662*
(127)
1.981 ±0.3586*
(137)
NA
Week 104 (n = various)
Control (0)
Low-dose (3.51)
Mid-dose (14.0)
NA
Absolute liver weight
10.615 ± 1.7036
12.126 ± 1.5875*
(114)
17.349 ±2.112*
(163)
NA
Relative liver weight
3.024 ±0.4842
3.644 ±0.4394*
(121)
6.901 ± 1.1686*
(228)
NA
Absolute brain weight
2.0791 ±0.08113
2.0926± 0.10566
(101)
2.0542 ±0.10089
(99)
NA
Relative heart weight
0.321 ±0.31
0.337 ±0.38* (105)
0.389 ±0.08* (121)
NA
Relative kidney weight
c
0.642 ±0.0661
1.227 ±0.2115*
NA
Relative lung weight
0.490 ±0.116
0.519 ±0.112 (106)
0.588 ±0.170*
(120)
NA
Relative testis weight
2.150 ±0.4924
2.581 ±0.5747*
(120)
2.978 ± 1.316*
(139)
NA
aCIIT (1982a).
bValues expressed as mean ± SD (% of control); % calculated by EPA.
Data illegible.
* Statistically different from controls, p < 0.05.
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4-4-2013
Table B.9. Selected Organ Weights (g) of Female F344 Rats After Dietary Exposure to
tgDNT for up to 2 Years"
Parameter
Exposure Group
(ADD, mg/kg-d)b
Week 26 (n = 10)
Control (0)
Low-dose (3.22)
Mid-dose (13.9)
High-dose (34.9)
Absolute liver weight
4.81 ±0.17
4.98 ± 0.35 (103)
5.89 ±0.44* (118)
7.26 ±0.56* (151)
Relative liver weight
2.62 ±0.10
2.74 ±0.08 (105)
3.24 ±0.11* (124)
4.60 ±0.34* (176)
Absolute brain weight
1.85 ±0.07
1.84 ±0.06 (99)
1.80 ±0.03 (97)
1.80 ±0.04 (97)
Relative heart weight
0.36 ±0.02
0.36 ±0.03 (100)
0.37 ±0.03 (103)
0.41 ±0.04* (114)
Relative kidney weight
0.689 ±0.017
0.725 ± 0.027 (105)
0.738 ±0.027* (107)
0.853 ±0.051* (124)
Relative lung weight
0.53 ±0.04
0.50 ±0.01 (94)
0.53 ±0.05 (100)
0.59 ±0.05* (111)
Relative ovary weight
0.040 ± 0.008
0.041 ±0.007 (103)
0.043 ±0.007 (108)
0.048 ± 0.007 (120)
Week 52 (n = 10)
Control (0)
Low-dose (3.46)
Mid-dose (13.9)
High-dose (35.1)
Absolute liver weight
5.74 ±0.30
5.80 ±0.50 (101)
7.06 ±0.41* (123)
8.90 ±0.61* (155)
Relative liver weight
2.69 ±0.18
2.82 ±0.27 (105)
3.73 ±0.14* (139)
5.28 ±0.48* (196)
Absolute brain weight
1.94 ±0.16
1.85 ±0.06 (95)
1.83 ±0.08 (94)
1.85 ±0.07 (95)
Relative heart weight
0.43 ±0.10
0.34 ±0.03* (79)
0.38 ±0.03 (88)
0.46 ±0.03 (107)
Relative kidney weight
0.727 ± 0.074
0.720 ± 0.047 (99)
0.762 ±0.105 (105)
0.949 ±0.044* (131)
Relative lung weight
0.538 ±0.113
0.480 ± 0.050 (89)
0.500 ±0.040 (93)
0.570 ± 0.020 (106)
Relative ovary weight
0.045 ± 0.008
0.051 ±0.0125
(113)
0.0740 ± 0.0699
(164)
0.057 ±0.015 (127)
Week 55 (n = various)
NA
NA
NA
High-dose (35.1)
Absolute liver weight
NA
NA
NA
9.92 ± 1.21
Relative liver weight
NA
NA
NA
5.87 ±0.93
Absolute brain weight
NA
NA
NA
1.84 ± 0.11
Relative heart weight
NA
NA
NA
0.44 ±0.04
Relative kidney weight
NA
NA
NA
0.964 ± 0.080
Relative lung weight
NA
NA
NA
0.616 ±0.114
Relative ovary weight
NA
NA
NA
0.058 ±0.032
Week 78 (n = 20 for control
and low-dose, n = 19 for
mid-dose)
Control (0)
Low-dose (3.45)
Mid-dose (14.0)
NA
Absolute liver weight
6.639 ± 0.4462
6.731 ±0.7549
(101)
9.074 ± 0.4790*
(137)
NA
Relative liver weight
2.560 ±0.155
2.826 ±0.334*
(110)
4.543 ± 0.2875*
(177)
NA
Absolute brain weight
2.1035 ±0.15936
2.0290 ±0.18547
(96)
1.8730 ±0.13483*
(89)
NA
Relative heart weight
0.378 ± 0.0626
0.374 ± 0.0803 (99)
0.430 ±0.0619 (114)
NA
Relative kidney weight
0.718 ±0.0741
0.731 ±0.0906
(102)
0.951 ±0.2626*
(132)
NA
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Table B.9. Selected Organ Weights (g) of Female F344 Rats After Dietary Exposure to
tgDNT for up to 2 Years"
Parameter
Exposure Group
(ADD, mg/kg-d)b
Relative lung weight
0.475 ± 0.0598
0.491 ±0.0798
(103)
0.508 ±0.826 (107)
NA
Relative ovary weight
0.0852 ±0.17849
0.1293 ±0.17126
(152)
0.0573 ±0.01170
(67)
NA
Week 104 (n = various)
Control (0)
Low-dose (3.46)
Mid-dose (14.0)
NA
Absolute liver weight
7.462 ± 1.0733
8.702 ± 1.1791*
(117)
12.309 ± 1.7879*
(165)
NA
Relative liver weight
2.783 ±c
3.486 ±0.4438 *
(125)
6.159 ±0.8252*
(221)
NA
Absolute brain weight
1.9211 ±0.18071
1.9129 ±0.08483
(100)
1.9085 ±0.11577
(99)
NA
Relative heart weight
0.342 ±0.0512
0.373 ± 0.0456*
(109)
0.449 ±0.0501*
(131)
NA
Relative kidney weight
0.732 ±0.0971
0.793 ± 0.0732*
(108)
1.157 ±0.1445*
(128)
NA
Relative lung weight
0.496 ±0.1963
0.488 ± 0.0934 (98)
0.647 ±0.3783*c
(130)
NA
Relative ovary weight
0.0392 ±0.01892c
0.0460 ±0.01491*
(117)
0.0823 ±0.1136*
(210)
NA
TUT f 1982a).
Values expressed as mean ± SD (% of control); % calculated by EPA.
Data were illegible.
* Statistically different from controls, p < 0.05.
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Table B.10. Selected Hepatotoxicity Incidences for Male and Female F344 Rats After
Dietary Exposure to tgDNT for 26 Weeks"
Parameter
Exposure Group
(ADD, mg/kg-d)b
Males (10/group)
Control (0)
Low-dose (3.47)
Mid-dose (13.6)
High-dose (34.6)
Fatty metamorphosis
0
4 (40)
4(40)
0
Hepatocyte necrosis
0
0
7 (70)*
9 (90)*
Megalocytosis
0
0
0
4 (40)
Periportal hyperbasophilic
hepatocytes
0
0
8(80)*
0
Vacuolation
0
1(10)
0
4(40)
Parameter
Exposure Group
(ADD, mg/kg-d)b
Females (10/group)
Control (0)
Low-dose (3.22)
Mid-dose (13.9)
High-dose (34.9)
Fatty metamorphosis
0
0
0
0
Hepatocyte necrosis
0
0
2 (20)
4 (40)
Megalocytosis
0
0
0
0
Periportal hyperbasophilic
hepatocytes
1(10)
2 (20)
6(60)
10 (100)*
Vacuolation
0

0
2(20)
aCIIT (1982a).
bValues expressed as number of animals with lesions (% of animals with lesion/effect); % calculated by EPA.
* Statistically different from controls, p < 0,05.
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Table B.ll. Selected Hepatotoxicity Incidences for Male and Female F344 Rats After
Dietary Exposure to tgDNT for 52 Weeks"
Parameter
Exposure Group
(ADD, mg/kg-d)b
Males (10/group)
Control (0)
Low-dose (3.47)
Mid-dose (13.9)
High-dose (34.9)
Hepatocyte necrosis
0
7(70)*
9(90)*
5 (50)*
Megalocytosis
0
1(10)
7 (70)*
3 (30)
Periportal hyperbasophilic
hepatocytes
0
2 (20)
9(90)*
6 (60)*
Vacuolation
0
7 (70)*
7 (70)*
6 (60)*
Parameter
Exposure Group
(ADD, mg/kg-d)b
Females (10/group)
Control (0)
Low-dose (3.46)
Mid-dose (13.9)
High-dose (35.1)
Hepatocyte necrosis
0
0
2 (20)*
7 (70)*
Megalocytosis
0
0
2 (20)
4 (40)
Periportal hyperbasophilic
hepatocytes
0
1(10)
6 (60)*
5 (50)*
Vacuolation
0
0
0
1(10)
"CUT f1982a).
bValues expressed as number of animals with lesions (% of animals with lesion/effect); % calculated by EPA.
* Statistically different from controls, p < 0,05.
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Table B.12. Selected Hepatotoxicity Incidences at Week 78a
Fatty Metamorphosis Incidence
Males (20/group)
Exposure Group
(ADD, mg/kg-d)b
Grading of Finding
Control (0)
Low-dose (3.49)
Mid-dose (14.0)
Minimal
0
7(35)
1(5)
Slight
0
6(30)
13 (65)
Moderate
0
0
3(15)
Moderately severe
0
0
0
Females (20/group)
Exposure Group
(ADD, mg/kg-d)b
Grading of Finding
Control (0)
Low-dose (3.45)
Mid-dose (14.0)
Minimal
0
0
5(25)
Slight
0
1(5)
11 (55)
Moderate
0
0
0
Moderately severe
0
0
1(5)
Other Hepatotoxicity Incidence
Males (20/group)
Exposure Group
(ADD, mg/kg-d)b
Grading of Finding
Control (0)
Low-dose (3.49)
Mid-dose (14.0)
Cystic degeneration
0
0
8 (40)*
Necrosis of individual hepatocytes
0
7 (35)*
12 (60)*
Females (20/group)
Exposure Group
(ADD, mg/kg-d)b
Grading of Finding
Control (0)
Low-dose (3.45)
Mid-dose (14.0)
Necrosis of individual hepatocytes
0
3(15)
20 (100)*
Megalocytosis of hepatocytes
0
0
2(10)
aCIIT (1982a).
bValues expressed as number of animals with lesions, (% of animals with lesion/effect), % calculated by EPA.
* Statistically different from controls, p < 0,05.
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Table B.13. Selected Hepatotoxicity Incidences at Week 104a
Fatty Metamorphosis Incidence
Males
Exposure Group
(ADD, mg/kg-d)b
Grading of Finding
Control (0) (n = 61)
Low-dose (3.51) (n = 70)
Mid-dose (14.0) (n = 23)
Minimal
8(13)
17 (24)
3(13)
Slight
1(2)
33 (47)
5(22)
Moderate
0(0)
1(1)
5(22)
Moderately severe
0(0)
0(0)
0(0)
Females
Exposure Group
(ADD, mg/kg-d)b
Grading of Finding
Control (0) (n = 57)
Low-dose (3.46) (n = 61)
Mid-dose (14.0) (n = 68)
Minimal
2(4)
6(10)
1(1)
Slight
3(5)
6(10)
24 (35)
Moderate
2(4)
1(2)
38 (56)
Moderately severe
0(0)
0(0)
2(3)
Other Hepatotoxicity Incidence
Males
Exposure Group
(ADD, mg/kg-d)b
Grading of Finding
Control (0) (n = 61)
Low-dose (3.51) (n = 70)
Mid-dose (14.0) (n = 23)
Cystic degeneration
2(3)
2(3)
16 (70)*
Necrosis of individual hepatocytes
0(0)
38 (54)*
11 (48)*
Megalocytosis of hepatocytes
0(0)
34 (49)*
8 (35)*
Females
Exposure Group
(ADD, mg/kg-d)b
Grading of Finding
Control (0) (n = 57)
Low-dose (3.46) (n = 61)
Mid-dose (14.0) (n = 68)
Number examined
57
61
68
Cystic degeneration
3(5)
0(0)
6(9)
Necrosis of individual hepatocytes
1(2)
18 (30)*
22 (32)*
Megalocytosis of hepatocytes
1(2)
22 (36)*
37 (54)*
"CUT (1982a).
bValues expressed as number of animals with lesions, (% of animals with lesion/effect), % calculated by EPA.
* Statistically different from controls, p < 0,05.
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Table B.14. Incidences of Hepatocellular Carcinomas in Male F344/CrlBR Rats at
Weeks 26, 52, and 78a
Parameter
Exposure Group
(ADD, mg/kg-d)b
26 Week (n = 10)
Control
(0)
Low-dose
(3.47 mg/kg-d)
Mid-dose
(13.6 mg/kg-d)
High-dose
(34.6 mg/kg-d)
Hepatocellular carcinoma
0
0
0
2 (20)
52 Week (n = 10)
Control
(0)
Low-dose
(3.47 mg/kg-d)
Mid-dose
(13.9 mg/kg-d)
High-dose
(34.9 mg/kg-d
Hepatocellular carcinoma
0
0
3 (30)
10 (100)*
78 Week (n = 20)
Control
(0)
Low-dose
(3.49 mg/kg-d)
Mid-dose
(14.0 mg/kg-d)
NA
Hepatocellular carcinoma
0
0
19 (95)*
NA
aCIIT (1982a).
bValues expressed as number of animals (% of animals with lesion/effect); % calculated by EPA.
* Statistically different from controls, p < 0,05.
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Table B.15. Incidences of Hepatocellular Carcinomas, Liver Neoplastic Nodules, and
Other Tumors in Male and Female F344 Rats at Week-104a
Parameter
Exposure Group
(ADD, mg/kg-d)b
Males
Control
(0)
Low-dose
(3.51 mg/kg-d)
Mid-dose
(14.0 mg/kg-d)
Number examined
61
70
23
Hepatocellular carcinoma
1(2)
9 (13)*
21 (91) *
Neoplastic nodule(s)
9(15)
11(16)
15 (65)*
Hepatocellular carcinoma and /or neoplastic nodule(s)
10 (16)
19 (27)
23 (100)*
Mammary fibroadenomas
3(5)
7(10)
5 (22)*
Subcutaneous fibromas
5(8)
14 (20)
14(61)*
Parameter
Exposure Group,
(ADD, mg/kg-d)b
Females
Control
(0)
Low-dose
(3.46 mg/kg-d)
Mid-dose
(14.0 mg/kg-d)
Number examined
57
61
68
Hepatocellular carcinoma
0
0
40 (59)*
Neoplastic nodule(s)
5(9)
12 (20)
53 (78)*
Hepatocellular carcinoma and /or neoplastic nodule(s)
5(9)
12 (20)
66 (97)*
Mammary fibroadenomas
15 (26)
12 (20)
24 (35)
Subcutaneous fibromas
0
2(3)
7(10)*
TUT (1982a).
Values expressed as number of animals (% of animals with lesion/effect); % calculated by EPA.
* Statistically different from controls, p < 0.05.
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Table B.16. Body and Liver Weights of Male F344 Rats After Dietary Exposure to tgDNT
for 1 Year3
Parameter
Exposure Group
(Adjusted Daily Dose, mg/kg-d)b
0 (Control)
35
26 Week
Number of animals
4
4
Body weight (g)
395 ±2
353 ± 8 (89)*
Liver/body weight
2.43 ±0.07
2.82 ±0.07 (116)*
52 Weeks
Number of animals
20
19
Terminal body weight (g)
434 ±3
321 ±4 (74)*
Liver weight (g)
10.30 ±0.16
19.49 ±0.35 (189)*
Liver/body weight (relative liver weight)
2.38 ±0.04
6.08 ±0.10 (255)*
"Leonard et al. (1987).
Values expressed as mean ± SEM (% of control); % calculated by EPA.
* Statistically different from controls, p < 0.05.
Table B.17. Incidence of Hepatic Neoplastic Lesions and Hepatic Metastases in Male F344
Rats After Dietary Exposure to tgDNT for 1 Year"

Exposure Group
(Adjusted Daily Dose, mg/kg-d)b
Parameter
0 (Control)
35
Total number of animals
20
19
Neoplastic nodules
0
10 (53)*
Hepatocellular carcinoma
Trabecular
0
9 (47)*
Adenocarcinoma
0
0
Cholangiocarcinoma
0
2(11)
"Leonard et al. (1987).
bValues expressed as number of animals (% with lesion).
* Statistically different from controls, p < 0.05.
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Table B.18. Distribution of Experimental Subjects Across Dose Groups and Breeding
Dates in the Developmental Study of tgDNT in F344 Rats3
Parameter
Exposure Group
(Adjusted Daily Dose, mg/kg-d)b
Vehicle Control
Technical DNT
14
35
37.5
75
100
150
Mortality for all treated females
Total females treated
37
22
13
22
13
23
13
No. deaths (GDs 0-20) (%)
0(0)
1 (4.5)
1 (7.7)
0(0)
0(0)
1 (4.3)
6 (46.2)
Assignment of surviving females for maternal and developmental evaluation
First breeding
9
0
7
0
7
0
6
Second breeding
7
6
0
6
0
6
0
Third breeding
6
7
0
7
0
7
0
Total No. assigned
22 (91)
13
7
13
7
13
6
No. of Pregnancy (% pregnantd)
20 (91)
10 (77)
7 (100)
12 (92)
6 (86)
12 (92)
5(83)
"Price et al. (1985).
bValues expressed as number of animals (% of animals death).
°Number of dams scarified at GD 20.
dPercent of pregnant dams.
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Table B.19. Hematology Parameters in F344 Dams and Fetuses on GD 20 Following
Maternal Exposure to tgDNT from GDs 7-20a
Parameter
Exposure Group
(Adjusted Daily Dose, mg/kg-d)b
Dams
Fetuses
Vehicle Control
tgDNT
(100)
Vehicle Control
tgDNT
(100)
% MetHb
3.8 ±0.5 (16)
7.7 ±0.6** (11)
13.4 ± 1.6(14)
8.9 ± 1.4(10)
% Reticulocytes
2.32 ±0.57 (12)
6.31 ±1.51* (11)
99.67 ± 0.06 (23)
98.98 ±0.22* (21)
WBC (x 103/mm3)
5.34 ±0.32 (5)
6.06 ± 0.64 (5)
0.93 ±0.19 (8)
0.83 ±0.12 (8)
RBC (x 1 oW)
6.24 ± 0.20 (5)
4.88 ±0.18* (5)
2.17 ±0.08 (10)
2.15 ±0.06** (9)
Hematocrit (%)
32.5 ± 1.00 (5)
26.0 ±0.98* (5)
34.08 ± 1.24(10)
32.17 ± 1.97 (9)
MCV (nm3)
52.14 ±0.29 (5)
54.54 ±0.55* (5)
156.54 ±0.88 (10)
160.61 ±1.17* (9)
RDW
7.96 ± 0.24 (5)
9.68 ±0.41* (5)
14.71 ±0.12 (10)
14.71 ±0.19 (9)
Platelets (x 103/mm3)
1063.00 ±20.92 (4)
1625.00 ± 97.50* (5)
406.86 ± 27.90 (7)
412.2 ±35.76 (5)
"Price et al. (1985).
bData are presented as x ± SE. Number of individual maternal or fetal blood samples evaluated is shown in
parentheses, except for fetal MetHb, which represents the number of litters evaluated after pooling individual fetal
blood within each litter.
*p < 0.05 t-test (two-tailed).
**p < 0.01 /-test (two-tailed).
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Table B.20. Summary of Select Organ Weights and Production Endpoints in
Pregnant F344 Dams Exposed to tgDNT from GDs 7-20a
Parameter
Exposure Group
(Adjusted Daily Dose, mg/kg-d)b
Vehicle Control
tgDNT
14
35
37.5
75
100
150
Number sacrificed
20
10
7
12
6
12
5
Weight gain, GDs 0-20
(g)c
61.83 ±3.61f
64.94 ±4.63 (105)
66.09 ±6.01 (107)
55.81 ±5.76 (90)
64.75 ± 6.78 (105)
52.67 ± 4.54 (85)
8.08 ±20.13
(13)**
Gravid uterine weight
(g)
37.70 ±3.22
47.75 ±2.39 (127)
39.39 ±3.25(104)
33.64 ±5.16 (89)
41.69 ±5.90 (111)
37.43 ± 3.84 (99)
22.09 ± 9.82 (59)
Absolute weight gain
(g)d
24.14 ±2.09§§ff
17.19 ±3.88 (71)*
26.70 ±4.31 (111)
22.17 ±2.10 (92)
23.06 ±2.31 (95)
15.24 ± 1.94 (63)**
14.01 ± 13.38
(58)**
Liver weight
(% body weight)
4.09±0.08§§ff
3.91 ±0.10 (96)*
4.12 ±0.09 (101)
3.96 ±0.09 (97)
4.55 ±0.10 (111)**
4.58 ±0.08 (112)**
4.79 ±0.36 (117)
Spleen weight
(% body weight)
0.197 ±0.003ff
0.185 ±0.007 (94)*
0.223 ±0.011
(113)*
0.215 ±0.006
(109)*
0.246 ±0.010
(125)**
0.320 ± 0.027
(162)**
0.284 ±0.059
(144)*
% Resorptions6
16.8 ±5.4
2.3 ± 1.5
4.1 ± 4.1
14.6 ±5.2
11.0 ±9.3
12.7 ±5.4
46.0 ±22.3
% Dead fetuses6
0.0
2.4 ± 1.6
0.0
0.0
1.3 ± 1.3
0.0
3.6 ±3.6
% Live fetuses6
83.2 ±5.4
95.4 ± 1.9
95.9 ±4.1
85 ±5.2
87.7 ±9.1
87.3 ± 5.4
50.4 ±20.6
"Price et al. (1985).
bData are presented as x ± SE (% compared to control). Number using dam or average litter value as the experimental unit.
Includes gravid uterine weight.
dWeight gain during gestation minus gravid uterine weight.
"Expressed as the percentage of total implants per dam (compared to controls).
*p < 0.05 Mann-Whitney U test (two-tailed).
**p < 0.01 Mann-Whitney U test (two-tailed),
tp < 0.05 Kruskal-Wallis one-way ANOVA.
< 0.01 Kruskal-Wallis one-way ANOVA.
§§p < 0.01 Jonckheere's test.
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Table B.21. Teratological Defects in F344 Rat Fetuses Following Maternal Exposure
to tgDNT or Vehicle Control on GDs 7-20a

Exposure Group
(Adjusted Daily Dose, mg/kg-d^
b



tgDNT
Parameter
Vehicle Control
14
35
37.5
75
100
150
Number of live litters examined
20
10
7
12
6
12
3
No. of live fetuses examined0
146
92
63
77
50
88
22
Gross malformations
Anophthalmia (bilateral or right side)
0
1
2
0
0
0
0
Agnathia
0
1
0
0
0
0
0
Umbilical hernia
0
1
0
0
0
0
0
Visceral malformations
Hydronephrosis (bilateral)
0
1
0
0
0
0
0
Skeletal malformations
Abnormal skull fusion
4
2
0
1
0
3
0
Fused thoracic arches
0
1
0
0
0
1
0
Thoracic centra off center
0
0
0
0
0
1
0
Lumbar centra off center
0
0
0
0
0
1
0
Ribs fused to each other
0
1
0
0
0
0
0
Short rib
0
1
0
0
0
1
0
Variations
0
0
0
0
0
0
0
Hematoma (back)
0
0
0
2
0
0
0
Misaligned sternebrae
3
4
0
0
0
6
0
Doubled centra
2
1
8
1
3
1
0
Clubbed limb without bone change
2
0
0
0
0
0
0
"Price et al. (1985).
bData are expressed as the number of fetuses exhibiting each type of defect. Thus, a single fetus may be represented
more than once in this table.
°Only 50% of the fetuses were examined for visceral malformations and internal malformations of the head.
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Table B.22. Status of Live Fetuses from F344 Rats Following Maternal Exposure
to tgDNT or Vehicle Control on GDs 7-20a
Parameter
Exposure Group
(Adjusted Daily Dose, mg/kg-d)b
Vehicle
Control
tgDNT
14
35
37.5
75
100
150
No. litters with
live fetuses
20
10
7
12
6
12
3
Live litter size
7.3 ±0.7
9.2 ±0.7
9.0 ±0.9
6.4 ± 1.1
8.3 ± 1.3
7.3 ±0.9
7.3 ± 1.7
Male/live x 100
(%)
48.8 ±5.0
53.0 ±6.3
46.4 ±4.5
44.4 ±8.0
52.7 ±4.9
43.0 ±5.9
57.4 ±22.8
Body weight (g)
3.21 ±0.05
3.39 ±0.07
(106)
3.29 ±0.07
(100)
3.34 ±0.06
(104)
3.29 ±0.13
(102)
3.17 ±0.08
(98)
3.14 ± 0.18
(98)
Crown-rump
length (cm)
3.55 ±0.03
3.51 ±0.07
(99)
3.57 ±0.07
(101)
3.58 ±0.07
(101)
3.53 ±0.05
(99)
3.46 ±0.07
(97)
3.53 ±0.15
(99)
Liver weight
(% body weight)
8.09 ± O.lltt
7.38 ±0.12
(91)**
8.35 ±0.14
(103)*
7.82 ±0.10
(96)
8.44 ±0.29
(104)
8.12 ±0.08
(100)
8.50 ±0.30
(105)
Spleen weight
(% body weight)
0.097 ±
0.0005ff
0.081 ±
0.008 (83)
0.131 ±0.006
(135)**
0.084 ±
0.004 (87)
0.119 ±
0.003 (123)*
0.085 ±
0.004 (88)
0.128 ±
0.012(132)
Placental weight
(g)
0.494 ± 0.022
0.539 ±
0.054 (109)
0.440 ± 0.009
(89)
0.536 ±
0.046 (108)
0.453 ±
0.018 (92)
0.51 ±
0.028 (103)
0.458 ±
0.057 (93)
"Price et al. (1985).
bData are presented as x ± SE. Number using dam or average litter value as the experimental unit.
*p < 0.05 Mann-Whitney U test (two-tailed).
**p < 0.01 Mann-Whitney U test (two-tailed),
"ft/? < 0.01 Kruskal-Wallis one-way ANOVA.
Table B.23. Rearing Behavior in Postnatal F344 Rat Female Pups when Dams were
Exposed to tgDNT from GDs 7-20a
End point
Exposure Group
(Adjusted Daily Dose, mg/kg-d)b
tgDNT
0 (control)
14
35
37.5
75
100
PND 30
Rearing behavior
20.2 ±2.2
14.5 ±2.4
24.6 ±2.2
17.0 ±0.8
16.7 ± 1.5
12.7 ± 1.8*
aCIIT ("1982b1.
bData are presented as x ± SE.
* Statistically different from controls, p < 0.05.
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APPENDIX C. BMD MODELING RESULTS
MODELING PROCEDURE FOR DICHOTOMOUS DATA
The BMD modeling of dichotomous data was conducted with EPA's 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) available within the software were fit
using a default benchmark response (BMR) of 10% extra risk. Adequacy of model fit was
judged based on the % goodness-of-fitp-value (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 BMDL was selected if the BMDLs estimated from different models
varied greater than 3-fold; otherwise, the BMDL from the model with the lowest Akaike
Information Criterion (AIC) was selected as a potential POD from which to derive the RfD.
In addition, in the absence of a mechanistic understanding of the biological response to a
toxic agent, data from exposures much higher than the study LOAEL do not provide reliable
information regarding the shape of the response at low doses. Such exposures, however, can
have a strong effect on the shape of the fitted model in the low-dose region of the dose-response
curve. Thus, if lack of fit is due to characteristics of the dose-response data for high doses, then
the Benchmark Dose Technical Guidance document allows for data to be adjusted by eliminating
the high-dose group (U.S. EPA. 2012). Because the focus of BMD analysis is on the low-dose
region of the response curve, elimination of the high-dose group is deemed reasonable.
MODELING PROCEDURE FOR CONTINUOUS DATA
The BMD modeling of continuous data was conducted with 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 relative risk. For liver-, body-, and kidney-weight
changes, a BMR of 10% relative risk was also used. An adequate fit was judged based on the
X goodness-of-fit p-v alue (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 homogeneous. If a homogeneous variance model was deemed appropriate based on the
statistical test provided by BMDS (i.e., Test 2), the final BMD results were estimated from a
homogeneous 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 nonhomogeneous variance. If this nonhomogeneous variance model did not adequately fit
the data (i.e., Test 3; p-v alue < 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 3-fold; otherwise, the BMDL from the model
with the lowest AIC was selected as a potential POD from which to derive the RfD.
INCREASED INCIDENCE OF HEPATOCYTE NECROSIS IN MALE RATS
TREATED WITH tgDNT FOR 26 WEEKS
The procedure outlined above was applied to the data for increased hepatocyte necrosis
(see Table C. 1) in male rats exposed to tgDNT via diet (CUT, 1982a) for 26 weeks. Table C.2
summarizes the BMD modeling results. All the models fit except the Logistic and Probit
models. Among the fitting models, the LogLogistic model has the lowest AIC. Thus, the
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BMDLio of 2.16 mg/kg-day from this model is selected for this end point (see Figure C. 1 and the
BMD text output for details).
Table C.l. Hepatocyte Necrosis in Male F344 Rats After Dietary Exposure

to tgDNT for 26 Weeks"



Low-dose
Mid-dose
High-dose

Control
(3.47 mg/kg-d)
(13.6 mg/kg-d)
(34.6 mg/kg-d)
Sample size
10
10
10
10
Incidence
0
0
7
9
"CUT (1982a).
Table C.l. BMD Modeling Results on Hepatocyte Necrosis in Male F344 Rats After
Dietary Exposure to tgDNT for 26 Weeks
Model Name
AIC
/>-value
BMD
BMDL10
Scaled Residual of Interest
Gamma
25.6203
0.2905
4.54802
1.36
-0.794
Logistic
30.1827
0.0174
5.63874
3.47
-1.058
LogLogistic
24.262
0.5178
5.17793
2.16
-0.61
LogProbit
24.2957
0.5089
5.07807
2.32
-0.569
Multistage
27.0948
0.1987
2.96349
1.14
-1.159
Probit
30.5661
0.029
5.67662
3.59
-1.058
Weibull
26.3218
0.2588
3.60128
1.23
-1.024
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Log-Logistic Model with 0.95 Confidence Level
Log-Logistic
1
0.8
0.6
0.4
0.2
0
BMDL
BMD
0
5
10
15
20
25
30
35
dose
16:25 07/24 2012
Figure C.l. LogLogistic Model for Increased Hepatocyte Necrosis in Male F344 Rats After
Dietary Exposure to tgDNT for 26 Weeks
Text Output for LogLogistic Model for Increased Hepatocyte Necrosis in Male Rats After
Dietary Exposure to tgDNT for 26 weeks (CUT, 1982a)
Logistic Model
BMDS Model Run
The form of the probability function is:
P[response] = background+(1-background)/[1+EXP(-intercept-siope*Log(dose))]
Dependent variable = Effect
Independent variable = Dose
Slope parameter is restricted as slope >= 1
Total number of observations = 4
Total number of records with missing values = 0
Maximum number of iterations = 250
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 =	-5.72644
slope =	2.32112
Asymptotic Correlation Matrix of Parameter Estimates
( *** The model parameter(s) -background
have been estimated at a boundary point, or have been specified by
the user,
and do not appear in the correlation matrix )
intercept	slope
intercept 1	-0.98
slope -0.98	1
Interval
Variable
Limit
background
intercept
slope
Parameter Estimates
Estimate
-6.69767
2.73682
Std. Err.
Indicates that this value is not calculated.
95.0% Wald Confidence
Lower Conf. Limit Upper Conf.
Analysis of Deviance Table
Model	Log(likelihood) # Param's Deviance Test d.f.
Full model	-9.35947	4
Fitted model
Reduced model
P-value
-10.131
-26.9205
1.54303
35.122
0.4623
<.0001
AIC:
24 .262
Dose
Goodness of Fit
Est._Prob. Expected Observed	Size
Scaled
Residual
0.0000
3.4700
13.6400
34.6100
0.0000
0.0358
0.6115
0.9527
0.000
0.358
6.115
9.527
0.000
0.000
7.000
9.000
10
10
10
10
0. 000
-0.610
0.574
-0.784
Chi^2 =1.32
d.f. = 2
P-value = 0.5178
Benchmark Dose Computation
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Specified effect
Risk Type
Confidence level
BMD
BMDL
0.1
Extra risk
0. 95
5.17793
2.16115
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INCREASED INCIDENCE OF HEPATOCYTE NECROSIS IN MALES TREATED
WITH tgDNT FOR 104 WEEKS (( 11 1 . 1982a)
The procedure outlined above was applied to the data for increased hepatocyte necrosis
(see Table C.3) in male rats exposed to tgDNT via diet (CUT. 1982a) for 104 weeks. Table C.4
summarizes the BMD modeling results. As assessed by the % goodness-of-fit^-values, all the
models failed to model this data set (see Table C.4).
To further attempt to calculate a BMDL based on this data set, BMD models were run
with the control and low-dose data points after dropping the mid-dose data point. Table C.5
presents the BMD modeling results. All the models available from the BMDS except the
Weibull model successfully fit the data set. The estimated BMDLios from these models differed
by more than 3-fold, so the lowest BMDL of 0.363 mg/kg-day (from the Multistage 2, 3) is
selected for this endpoint (see Figure C.2 and the BMD text output for Multistage 2 model).
Table C.3. Hepatocyte Necrosis in Male F344 Rats After Dietary Exposure to

tgDNT for 104 Weeks3



Low-dose
Mid-dose

Control
(3.51mg/kg-d)
(14 mg/kg-d)
Sample size
61
70
23
Incidence
0
38
11
aCIIT 0 982a).
Table C.4. BMD Results for Hepatocyte Necrosis in Male F344 Rats After Dietary
Exposure to tgDNT for 104 Weeks
Model Name
AIC
/j-valuc
BMD10
BMDL10
Scaled Residual of Interest
Gamma
154.943
0
0.856072
0.677433
0
Logistic
181.412
0
2.98469
2.21859
4.388
LogLogistic
141.63
0.0015
0.479011
0.335441
0
LogProbit
156.474
0
1.27286
1.02557
0
Multistage 2
154.943
0
0.856072
0.677433
0
Multistage 3
154.943
0
0.856072
0.677433
0
Probit
180.738
0
2.85092
2.17342
4.457
Weibull
154.943
0
0.85607
0.677433
0
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Table C.5. BMD Results After Dropping Mid-dose Point for Hepatocyte Necrosis in Male
F344 Rats After Dietary Exposure to tgDNT for 104 Weeks
Model Name
AIC
/j-valuc
BMD10
BMDL10
Scaled Residual of Interest
Logistic
100.526
NA
3.07144
1.576
0
Multistage 2
100.526
NA
0.774156
0.363194
0
Multistage 3
102.526
NA
1.02805
0.363194
0
Probit
100.526
NA
2.66223
1.36408
0
0	0.5	1	1.5	2	2.5	3	3.5
dose
10:38 07/23 2012
Figure C.2. Multistage 2 BMD Model for Increased Hepatocyte Necrosis in Male Rats
After Dietary Exposure to tgDNT for 104 Weeks (CUT, 1982a)
Multistage Model with 0.95 Confidence Level
I	I
Multistage
BMDL
BMD
Text Output for Multistage 2 BMD Model for Increased Hepatocyte Necrosis in Male Rats
After Dietary Exposure to tgDNT at 104 weeks (CUT, 1982a)
Multistage Model. (Version: 3.2; Date: 05/26/2010)
BMDS Model Run
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Observation # < parameter # for Multistage model.
The form of the probability function is:
P[response] = background + (1-background)*[1-EXP(
-betal*dose/sl-beta2*dose/s2) ]
The parameter betas are restricted to be positive
Dependent variable = Effect
Independent variable = Dose
Total number of observations = 2
Total number of records with missing values = 0
Total number of parameters in model = 3
Total number of specified parameters = 0
Degree of polynomial = 2
Maximum number of iterations = 250
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
Default Initial Parameter Values
Background =	0.542857
Beta(l) =	0.223008
Beta(2) = 0.0635351
Asymptotic Correlation Matrix of Parameter Estimates
( *** The model parameter(s) -Background
have been estimated at a boundary point, or have been specified by
the user,
and do not appear in the correlation matrix )
Beta(1)	Beta (2)
Beta (1)	NA	NA
Beta (2)	NA	NA
NA - This parameter's variance has been estimated as zero or less.
THE MODEL HAS PROBABLY NOT CONVERGED!!!
Parameter Estimates
95.0% Wald Confidence
Interval
Variable	Estimate	Std. Err. Lower Conf. Limit Upper Conf.
Limit
Background	0	* * *
Beta(1)	0.111504	* * *
Beta(2)	0.0317676	* * *
* - Indicates that this value is not calculated.
At least some variance estimates are negative.
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THIS USUALLY MEANS THE MODEL HAS NOT CONVERGED!
Try again from another starting point.
Error in computing chi-sguare; returning 2
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Model
Full model
Fitted model
Reduced model
AIC:
Analysis of Deviance Table
Log(likelihood) # Param's Deviance Test d.f.
-48.2628
-48.2628
-78.8908
100.526
0
61.256
P-value
NA
<.0001
Dose
Est. Prob.
Goodness of Fit
Expected Observed	Size
Scaled
Residual
0.0000
3.5100
Chi^2 = 0.00
0.0000
0.5429
0.000
38.000
d.f. = 0
0.000
38.000
P-value =
NA
61
70
0. 000
0. 000
Benchmark Dose Computation
Specified effect
Risk Type
Confidence level
BMD
BMDL
BMDU
0.1
Extra risk
0. 95
0.774156
0.363194
1.48533
Taken together, (0.363194, 1.48533) is a 90
interval for the BMD
two-sided confidence
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APPENDIX D. BENCHMARK DOSE CALCULATIONS FOR THE SCREENING p-OSF
MODEL-FITTING PROCEDURE FOR CANCER INCIDENCE DATA
The model-fitting procedure for dichotomous cancer incidence data is as follows. The
Multistage-Cancer model in the EPA's BMDS (version 2.2.2) is fit to the incidence data using
the extra risk option. The Multistage-Cancer model is run for all polynomial degrees up to n - 1
(where n is the number of dose groups including control). An adequate model fit is judged by
three criteria: (1) goodness-of-fit/>-value (p > 0.1), (2) visual inspection of the dose-response
curve, and (3) scaled residual at the data point (except the control) closest to the predefined
benchmark response (BMR). Among all the models providing adequate fit to the data, the
BMDL from the best fitting Multistage-Cancer model as judged by the goodness-of-fit />value,
is selected as the POD. In accordance with U.S. EPA (2012) guidance, BMDs and BMDLs
associated with an extra risk of 10% are calculated.
HEPATOCELLULAR CARCINOMAS IN MALE F344 RATS AFTER DIETARY
EXPOSURE TO tgDNT FOR 104 WEEKS (CUT. 1982a)
Table A.6 shows the dose-response data on hepatocellular carcinomas, liver neoplastic
nodules, hepatocellular carcinomas and/or neoplastic nodules, mammary fibroadenomas, and
subcutaneous fibromas in male F344 rats administered tgDNT via the diet for 104 weeks (CUT.
1982a). Modeling was performed according to the procedure outlined above using BMDS for
each individual tumor type based on the ADDs, and Table D.l summarizes the results. For
incidence of hepatocellular carcinomas in male rats, the 2-degree Multistage-Cancer model
provided an adequate fit (goodness-of-fitp-wdXut >0.1; see Table D.l and Figure D.l). The
estimated BMDio value is 3.04 mg/kg-day with a BMDLio of 2.15 mg/kg-day.
Table D.l. BMD Results for Hepatocyte Carcinomas in Male F344 Rats After Dietary

Exposure to tgDNT for 104 Weeks


Degree of




Scaled Residual of
Model Name
polynomial
AIC
/>-value
BMD
BMDL10
Interest
Multistage-Cancer
1
95.5511
0.0006
1.3531
0.998
0.35
Multistage-Cancer
2
81.7442
0.6332
3.03847
2.15
-0.366
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Multistage Cancer Model with 0.95 Confidence Level
Multistage Cancer
Linear extrapolation
1
0.8
0.6
0.4
0.2
0
BMDL
BMD
0	2	4	6	8	10	12	14
dose
09:50 07/18 2012
Figure D.l. Multistage-Cancer BMD Model for Increased Hepatocellular Carcinomas in
Male Rats at 104 Weeks (CUT. 1982a)
Text Output for Multistage-Cancer BMD Model for Increased Hepatocellular Carcinomas
in Male Rats (CUT. 1982a)
Multistage Cancer Model
BMDS_Model_Run
The form of the probability function is:
P[response] = background + (1-background)*[1-EXP(
-betal*dose/sl-beta2*dose/s2) ]
The parameter betas are restricted to be positive
Dependent variable = Effect
Independent variable = Dose
Total number of observations = 3
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Total number of records with missing values = 0
Total number of parameters in model = 3
Total number of specified parameters = 0
Degree of polynomial = 2
Maximum number of iterations = 250
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
Default Initial Parameter Values
Background = 0.000900222
Beta(l) =	0
Beta(2) =	0.012451
Asymptotic Correlation Matrix of Parameter Estimates
( *** The model parameter(s) -Beta(l)
have been estimated at a boundary point, or have been specified by
the user,
and do not appear in the correlation matrix )
Background	Beta (2)
Background	1	-0.32
Beta (2)	-0.32	1
Parameter Estimates
95.0% Wald Confidence
Interval
Variable	Estimate	Std. Err. Lower Conf. Limit Upper Conf.
Limit
Background	0.0147051	* * *
Beta(1)	0	* * *
Beta(2)	0.0114121	* * *
* - Indicates that this value is not calculated.
Analysis of Deviance Table
Model
Full model
Fitted model
Reduced model
Log(likelihood)
-38.7541
-38.8721
-77.3384
# Param's
3
2
1
Deviance Test d.f.
0.236087
77.1687
P-value
0. 627
<.0001
AIC:
81.7442
Goodness of Fit
Scaled
Dose	Est._Prob. Expected Observed	Size	Residual
0.0000	0.0147	0.897	1.000	61	0.110
3.5100	0.1439	10.076	9.000	70	-0.366
14.0000	0.8948	20.580 21.000	23	0.286
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Chi^2 =0.23	d.f. = 1	P-value = 0.6332
Benchmark Dose Computation
Specified effect =	0.1
Risk Type =	Extra risk
Confidence level =	0.95
BMD =	3.03847
BMDL =	2.14507
BMDU =	3.67911
Taken together, (2.14507, 3.67911) is a 90	% two-sided confidence
interval for the BMD
Multistage Cancer Slope Factor =	0.0466186
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LIVER NEOPLASTIC NODULES IN MALE F344 RATS AFTER DIETARY
EXPOSURE TO tgDNT FOR 104 WEEKS (CUT. 1982a)
Modeling was performed according to the procedure outlined above using BMDS based
on the ADDs, and Table D.2 summarizes the results. For incidence of liver neoplastic nodules in
male rats, the 2-degree Multistage-Cancer model provided an adequate fit (goodness-of-fit
p-wdXut >0.1; see Table D.2 and Figure D.2). The estimated BMDio value is 4.86 mg/kg-day
with a BMDLio of 2.69 mg/kg-day.
Table D.2. BMD Results for Liver Neoplastic Nodules in Male F344 Rats After Dietary

Exposure to tgDNT for 104 Weeks


Degree of




Scaled Residual of
Model Name
polynomial
AIC
/j-valuc
BMD
BMDL10
Interest
Multistage-Cancer
1
150.829
0.0262
2.43085
1.58
-1.606
Multistage-Cancer
2
146.021
0.5454
4.85647
2.69
-0.45
Multistage Cancer Model with 0.95 Confidence Level
0.9
Multistage Cancer
Linear extrapolation
0.8
0.7
0.6
0.5
0.4
0.3
0.2
1
BMDL
BMD
0
0
2
4
6
8
10
12
14
dose
11:30 07/25 2012
Figure D.2. Multistage-Cancer BMD Model for Increased Neoplastic Nodules in Male Rats
at 104 Weeks (("11 1 . 1982a)
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Text Output for Multistage-Cancer BMD Model for Increased Neoplastic Nodules in Male
Rats (('11 1 . 1982a)
Multistage Cancer Model. (Version: 1.9; Date: 05/26/2010)
Input Data File: C:/USEPA/BMDS220/Data/104 week/msc_104 week male neoplastic
nodules_Opt.(d)
Gnuplot Plotting File: C:/USEPA/BMDS220/Data/104 week/msc_104 week male
neoplastic nodules_Opt.pit
Wed Jul 25 11:30:27 2012
BMDS Model Run
The form of the probability function is:
P[response] = background + (1-background)*[1-EXP(
-betal*dose/sl-beta2*dose/s2) ]
The parameter betas are restricted to be positive
Dependent variable = Effect
Independent variable = Dose
Total number of observations = 3
Total number of records with missing values = 0
Total number of parameters in model = 3
Total number of specified parameters = 0
Degree of polynomial = 2
Maximum number of iterations = 250
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
Default Initial Parameter Values
Background =	0.128217
Beta(l) =	0
Beta(2) = 0.00468028
Asymptotic Correlation Matrix of Parameter Estimates
( *** The model parameter(s) -Beta(l)
have been estimated at a boundary point, or have been specified by
the user,
and do not appear in the correlation matrix )
Background	Beta(2)
Background	1	-0.3 6
Beta (2)	-0.36	1
Parameter Estimates
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95.0% Wald Confidence
Interval
Variable	Estimate	Std. Err. Lower Conf. Limit Upper Conf.
Limit
Background	0.131201	* * *
Beta(1)	0	* * *
Beta(2)	0.00446721	* * *
* - Indicates that this value is not calculated.
Analysis of Deviance Table
Model
Full model
Fitted model
Reduced model
Log(likelihood)
-70.8268
-71.0106
-82.5378
# Param's	Deviance	Test d.f.	P-value
3
2	0.367614	1	0.5443
1	23.422	2	<.0001
AIC:
146.021
Dose
Goodness of Fit
Est._Prob. Expected Observed	Size
Scaled
Residual
0.0000
3.5100
14.0000
0.1312
0.1777
0.6380
8.003
12.441
14.675
9.000
11.000
15.000
61
70
23
0.378
-0.450
0.141
Chi^2 =0.37
d.f. = 1
P-value = 0.5454
Benchmark Dose Computation
Specified effect =	0.1
Risk Type	=	Extra risk
Confidence level =	0.95
BMD =	4.85647
BMDL =	2.68723
BMDU =	6.53922
Taken together, (2.68723, 6.53922) is a 90	% two-sided confidence
interval for the BMD
Multistage Cancer Slope Factor =	0.037213
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COMBINED HEPATOCELLULAR CARCINOMAS AND LIVER NEOPLASTIC
NODULES IN MALE F344 RATS AFTER DIETARY EXPOSURE TO tgDNT FOR
104 WEEKS (( 11 1 . 1982a)
Modeling was performed according to the procedure outlined above using BMDS based
on the ADDs, and Table D.3 summarizes the results. For incidence of combined hepatocellular
carcinomas and liver neoplastic nodules in male rats, the 2-degree Multistage-Cancer model
provided an adequate fit (goodness-of-fitp-value >0.1; see Table D.3 and Figure D.3). The
estimated BMDio value is 2.42 mg/kg-day with a BMDLio of 1.68 mg/kg-day.
Table D.3. BMD Results for Combined Hepatocellular Carcinomas and Liver Neoplastic
Nodules in Male F344 Rats After Dietary Exposure to tgDNT for 104 Weeks
Model Name
Degree of
polynomial
AIC
/>-value
BMD
BMDL
Scaled
Residual of
Interest
Multistage-Cancer
1
156.06
0.0008
0.991314
0.721042
0.828
Multistage-Cancer
2
142.255
0.2389
2.41883
1.67831
-0.762
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Multistage Cancer Model with 0.95 Confidence Level
Multistage Cancer
Linear extrapolation
1
0.8
0.6
0.4
0.2
BMDL
BMD
0
0
2
4
6
8
10
12
14
dose
11:41 08/01 2012
Figure D.3. Multistage-Cancer BMD Model for Increased Combined Hepatocellular
Carcinomas and Neoplastic Nodule in Male Rats at 104 Weeks (('11 1 . 1982a)
Text Output for Multistage-Cancer BMD Model for Increased Combined Hepatocellular
Carcinomas and Neoplastic Nodule in Male Rats (CUT, 1982a)
Multistage Cancer Model. (Version: 1.9; Date: 05/26/2010)
Input Data File: C:/USEPA/BMDS220/Data/msc_livercombined_Msc2-BMR10.(d)
Gnuplot Plotting File: C:/USEPA/BMDS220/Data/msc_livercombined_Msc2-
BMR10.pit
Wed Aug 01 11:41:18 2012
BMDS Model Run
The form of the probability function is:
P[response] = background + (1-background)*[1-EXP(
-betal*dose/sl-beta2*dose/s2) ]
The parameter betas are restricted to be positive
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Dependent variable = Response
Independent variable = Dose
Total number of observations = 3
Total number of records with missing values = 0
Total number of parameters in model = 3
Total number of specified parameters = 0
Degree of polynomial = 2
Maximum number of iterations = 250
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
Default Initial Parameter Values
Background =	0
Beta(l) =	0
Beta(2) = 5.25101e+017
Asymptotic Correlation Matrix of Parameter Estimates
( *** The model parameter(s) -Beta(l)
have been estimated at a boundary point, or have been specified by
the user,
and do not appear in the correlation matrix )
Background	Beta (2)
Background	1	-0.3 6
Beta (2)	-0.36	1
Parameter Estimates
Interval
Variable
Limit
Background
Beta(1)
Beta(2)
Estimate
0.143194
0
0.0180081
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)
-68.1416
-69.1276
-98.4788
# Param's
3
2
1
Deviance Test d.f.
P-value
1.97201
60.6745
0.1602
<.0001
AIC:
142 .255
Goodness of Fit
Scaled
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Dose
Est. Prob.
Expected
Observed
Size
Residual
14.0000
3.5100
0.0000
0.9749
0.3137
0.1432
22.422
21.957
8 .735
23.000
19.000
10.000
23
70
61
0.770
-0.762
0. 462
Chi^2 = 1.39
d.f. = 1
P-value = 0.2389
Benchmark Dose Computation
Specified effect =	0.1
Risk Type	=	Extra risk
Confidence level =	0.95
BMD =	2.418 83
BMDL =	1.67831
BMDU =	3.09631
Taken together, (1.67831, 3.09631) is a 90	% two-sided confidence
interval for the BMD
Multistage Cancer Slope Factor =	0.0595837
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MAMMARY FIBROADENOMAS IN MALE F344 RATS AFTER DIETARY
EXPOSURE TO tgDNT FOR 104 WEEKS (CUT. 1982a)
Modeling was performed according to the procedure outlined above using BMDS based
on the ADDs, and Table D.4 summarizes the results. For incidence of mammary fibroadenomas
in male rats, both 1- and 2-degree Multistage-Cancer models provided an identical fit
(goodness-of-fit /rvalue >0.1; see Table D.4 and Figure D.4). The estimated BMDio value is
7.37 mg/kg-day with aBMDLio of 3.73 mg/kg-day.
Table D.4. BMD Results for Mammary Fibroadenomas in Male F344 Rats After Dietary

Exposure to tgDNT for 104 Weeks



Degree of




Scaled Residual
Model Name
polynomial
AIC
/j-valuc
BMD
BMDL10
of Interest
Multistage-Cancer
1
97.5334
0.9081
7.37334
3.73
0.085
Multistage-Cancer
2
97.5334
0.9081
7.37334
3.73
0.085
Multistage Cancer Model with 0.95 Confidence Level
Multistage Cancer
Linear extrapolation
0.4
0.3
0.2
0.1
0
BMDL
BMD
0
2
4
6
8
10
12
14
dose
10:28 07/25 2012
Figure D.4. Multistage-Cancer BMD Model for Increased Mammary Fibroadenomas in
Male Rats at 104 Weeks (CUT. 1982a)
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Text Output for Multistage-Cancer BMD Model for Increased Mammary Fibroadenomas
in Male Rats (("11 1 . 1982a)
Multistage Cancer Model. (Version: 1.9; Date: 05/26/2010)
Input Data File: C :/USEPA/BMDS220/Data/msc_Maittmary_Mscl-BMR10 . (d)
Gnuplot Plotting File: C:/USEPA/BMDS220/Data/msc_Mammary_Mscl-BMR10.pit
Wed Aug 01 15:18:00 2012
BMDS Model Run
The form of the probability function is:
P[response] = background + (1-background)*[1-EXP(
-betal*doseAl) ]
The parameter betas are restricted to be positive
Dependent variable = Response
Independent variable = Dose
Total number of observations = 3
Total number of records with missing values = 0
Total number of parameters in model = 2
Total number of specified parameters = 0
Degree of polynomial = 1
Maximum number of iterations = 250
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
Default Initial Parameter Values
Background = 0.0518599
Beta(1) = 0.0137724
Asymptotic Correlation Matrix of Parameter Estimates
Background	Beta(l)
Background	1	-0.62
Beta (1)	-0.62	1
Parameter Estimates
95.0% Wald Confidence
Interval
Variable	Estimate	Std. Err.	Lower Conf. Limit Upper Conf.
Limit
Background	0.0505334	*	*	*
Beta(1)	0.0142894	*	*	*
* - Indicates that this value is not calculated.
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Analysis of Deviance Table
Model
Full model
Fitted model
Reduced model
Log(likelihood)
-46.7601
-46.7667
-49.1781
Param's
3
2
1
Deviance Test d.f.
0.0133121
4.83601
P-value
0.9081
0.0891
AIC:
97.5334
Goodness of Fit
Scaled
Dose	Est._Prob. Expected Observed	Size	Residual
0.0000
0.0505
3.083
3.000
61
-0.048
3.5100
0.0970
6.789
7.000
70
0. 085
14.0000
0.2227
5.122
5.000
23
-0.061
Chi^2 = 0.01	d.f. = 1	P-value = 0.9081
Benchmark Dose Computation
Specified effect =	0.1
Risk Type	=	Extra risk
Confidence level =	0.95
BMD =	7.37334
BMDL =	3.72 678
BMDU =	33.6009
Taken together, (3.72678, 33.6009) is a 90	% two-sided confidence
interval for the BMD
Multistage Cancer Slope Factor =	0.0268328
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SUBCUTANEOUS FIBROMAS IN MALE F344 RATS AFTER DIETARY EXPOSURE
TO tgDNT FOR 104 WEEKS (CUT. 1982a)
Modeling was performed according to the procedure outlined above using BMDS based
on theADDs, and Table D.5 summarizes the results. For incidence of subcutaneous fibromas in
male rats, the 1-degree Multistage-Cancer model provided an adequate fit (goodness-of-fit
/rvalue >0.1; see Table D.5 and Figure D.5). The estimated BMDio value is 2.01 mg/kg-day
with a BMDLio of 1.38 mg/kg-day.
Table D.5. BMD Results for Subcutaneous Fibromas in Male F344 Rats After Dietary

Exposure to tgDNT for 104 Weeks



Degree of




Scaled Residual of
Model Name
polynomial
AIC
/>-value
BMD
BMDL10
Interest
Multistage-Cancer
1
140.118
0.4138
2.01413
1.38
-0.582
Multistage-Cancer
2
141.438
NA
2.79287
1.45
0
Multistage Cancer Model with 0.95 Confidence Level
Multistage Cancer
Linear extrapolation
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
BMDL
BMD
0
2
4
6
8
10
12
14
dose
11:36 07/25 2012
Figure D.5. Multistage-Cancer BMD Model for Increased Subcutaneous Fibromas in Male
Rats at 104 Weeks (CUT. 1982a)
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Text Output for Multistage-Cancer BMD Model for Increased Subcutaneous Fibromas in
Male Rats (('II I . 1982a)
Multistage Cancer Model. (Version: 1.9; Date: 05/26/2010)
Input Data File: C:/USEPA/BMDS220/Data/104 week/msc_Subcutaneous
fibromas_Opt.(d)
Gnuplot Plotting File: C:/USEPA/BMDS220/Data/104 week/msc_Subcutaneous
fibromas_Opt.pit
Wed Jul 25 10:38:29 2012
BMDS Model Run
The form of the probability function is:
P[response] = background + (1-background)*[1-EXP(
-betal*doseAl) ]
The parameter betas are restricted to be positive
Dependent variable = Effect
Independent variable = Dose
Total number of observations = 3
Total number of records with missing values = 0
Total number of parameters in model = 2
Total number of specified parameters = 0
Degree of polynomial = 1
Maximum number of iterations = 250
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
Default Initial Parameter Values
Background = 0.0491346
Beta(1) = 0.0625806
Asymptotic Correlation Matrix of Parameter Estimates
Background	Beta(l)
Background	1	-0.5 6
Beta (1)	-0.56	1
Parameter Estimates
95.0% Wald Confidence
Std. Err.	Lower Conf. Limit Upper Conf.
k
k	k	-k
Interval
Variable	Estimate
Limit
Background	0.0738705
Beta(1)	0.0523107
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* - Indicates that this value is not calculated.
Analysis of Deviance Table
Model
Full model
Fitted model
Reduced model
Log(likelihood)
-67.7191
-68.0589
-80. 0153
# Param's
3
2
1
Deviance Test d.f.
0.679587
24.5923
P-value
0.4097
<.0001
AIC:
140.118
Dose
Goodness of Fit
Est._Prob. Expected Observed	Size
Scaled
Residual
0.0000
3.5100
14.0000
0.0739
0.2292
0.5547
4.506
16.045
12 .759
5.000
14.000
14.000
61
70
23
0.242
-0.582
0.521
Chi^2 =0.67
d.f. = 1
P-value = 0.4138
Benchmark Dose Computation
Specified effect =	0.1
Risk Type =	Extra risk
Confidence level =	0.95
BMD =	2.01413
BMDL =	1.37981
BMDU =	3.2 9703
Taken together, (1.37981, 3.29703) is a 90	% two-sided confidence
interval for the BMD
Multistage Cancer Slope Factor =	0.0724738
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MSCOMBO-MULTIPLE TUMOR BMD MODEL FOR ALL TUMOR TYPES IN
MALE F344 RATS AFTER DIETARY EXPOSURE TO tgDNT FOR 104 WEEKS (CUT.
1982a)
MSCombo-multiple tumor BMD modeling was used to combine tumor incidence data
for combined hepatocellular carcinomas and/or neoplastic nodules, mammary fibroadenomas,
and subcutaneous fibromas in male rats. For each tumor type, the best-fitting multistage model
(i.e., the degree of Poly setting) was maintained in the MSCombo model run. The calculated
combined tumor BMDLio based on the MS Combo model is 0.852 mg/kg-day (see MS Combo
text output for details). This BMDLio is used as the POD to derive the p-OSF.
Text Output for MS COMBO Multiple Tumor Model for Combined Tumors in Male Rats
MS_COMBO. (Version: 1.5 Beta; Date: 01/25/2011)
Input Data File: C:\USEPA\BMDS220\Data\New.(d)
Gnuplot Plotting File: C:\USEPA\BMDS22 0\Data\New.plt
Wed Aug 01 15:35:40 2012
BMDS Model Run
The form of the probability function is:
P[response] = background + (1-background)*[1-EXP(
-betal*dose/sl-beta2*dose/s2) ]
The parameter betas are restricted to be positive
Dependent variable = Response
Independent variable = Dose
Total number of observations = 3
Total number of records with missing values = 0
Total number of parameters in model = 3
Total number of specified parameters = 0
Degree of polynomial = 2
Maximum number of iterations = 250
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
Default Initial Parameter Values
Background =	0
Beta(l) =	0
Beta(2) = 5.25101e+017
Asymptotic Correlation Matrix of Parameter Estimates
( *** The model parameter(s) -Beta(l)
have been estimated at a boundary point, or have been specified by
the user,
and do not appear in the correlation matrix )
Background	Beta (2)
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Background
Beta(2)
1
-0.36
-0.36
1
Parameter Estimates
Interval
Variable
Limit
Background
Beta(1)
Beta(2)
Estimate
0.143194
0
0.0180081
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)
-68.1416
-69.1276
-98.4788
# Param's	Deviance	Test d.f.	P-value
3
2	1.97201	1	0.1602
1	60.6745	2	<.0001
AIC:
142 .255
Log-likelihood Constant
63.914608924352244
Dose
Goodness of Fit
Est._Prob. Expected Observed	Size
Scaled
Residual
14.0000
3.5100
0.0000
Chi^2 = 1.39
0.9749
0.3137
0.1432
d.f. = 1
22.422 23.000	23
21.957 19.000	70
8.735 10.000	61
P-value = 0.2389
0.770
-0.762
0. 462
Benchmark Dose Computation
Specified effect =	0.1
Risk Type =	Extra risk
Confidence level =	0.95
BMD =	2.418 83
BMDL =	1.67831
BMDU =	3.09631
Taken together, (1.67831, 3.09631) is a 90	% two-sided confidence
interval for the BMD
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MS_COMBO. (Version: 1.5 Beta; Date: 01/25/2011)
Input Data File: C:\USEPA\BMDS220\Data\New.(d)
Gnuplot Plotting File: C:\USEPA\BMDS22 0\Data\New.plt
Wed Aug 01 15:35:40 2012
BMDS Model Run
The form of the probability function is:
P[response] = background + (1-background)*[1-EXP(
-betal*doseAl) ]
The parameter betas are restricted to be positive
Dependent variable = Response
Independent variable = Dose
Total number of observations = 3
Total number of records with missing values = 0
Total number of parameters in model = 2
Total number of specified parameters = 0
Degree of polynomial = 1
Maximum number of iterations = 250
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
Default Initial Parameter Values
Background = 0.0518599
Beta(1) = 0.0137724
Asymptotic Correlation Matrix of Parameter Estimates
Background	Beta(l)
Background	1	-0.62
Beta (1)	-0.62	1
Parameter Estimates
95.0% Wald Confidence
Interval
Variable	Estimate	Std. Err.	Lower Conf. Limit Upper Conf.
Limit
Background	0.0505334	*	*	*
Beta(1)	0.0142894	*	*	*
* - Indicates that this value is not calculated.
Analysis of Deviance Table
Model	Log(likelihood) # Param's Deviance Test d.f. P-value
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Full model
Fitted model
Reduced model
-46.7601
-46.7667
-49.1781
0.0133121
4.83601
0.9081
0.0891
AIC:
97.5334
Log-likelihood Constant
41.819300823452593


Goodness of Fit







Scaled
Dose
Est. Prob.
Expected
Observed
Size
Residual
0.0000
0.0505
3.083
3.000
61
-0.048
3.5100
0.0970
6.789
7.000
70
0. 085
14.0000
0.2227
5.122
5.000
23
-0.061
Chi^2 = 0.01
d.f. = 1
P-value = 0.9081
Benchmark Dose Computation
Specified effect =	0.1
Risk Type =	Extra risk
Confidence level =	0.95
BMD =	7.37334
BMDL =	3.72 678
BMDU =	33.6009
Taken together, (3.72678, 33.6009) is a 90	% two-sided confidence
interval for the BMD
MS_COMBO. (Version: 1.5 Beta; Date: 01/25/2011)
Input Data File: C:\USEPA\BMDS220\Data\New.(d)
Gnuplot Plotting File: C:\USEPA\BMDS22 0\Data\New.plt
Wed Aug 01 15:35:40 2012
BMDS Model Run
The form of the probability function is:
P[response] = background + (1-background)*[1-EXP(
-betal*doseAl) ]
The parameter betas are restricted to be positive
Dependent variable = Response
Independent variable = Dose
Total number of observations = 3
Total number of records with missing values = 0
Total number of parameters in model = 2
Total number of specified parameters = 0
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Degree of polynomial
Maximum number of iterations = 250
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
Default Initial Parameter Values
Background = 0.0491346
Beta(1) = 0.0625806
Asymptotic Correlation Matrix of Parameter Estimates
Background	Beta(l)
Background	1	-0.5 6
Beta (1)	-0.56	1
Parameter Estimates
95.0% Wald Confidence
Interval
Variable	Estimate	Std. Err.	Lower Conf. Limit Upper Conf.
Limit
Background	0.0738705	*	*	*
Beta(1)	0.0523107	*	*	*
* - Indicates that this value is not calculated.
Analysis of Deviance Table
Model
Full model
Fitted model
Reduced model
Log(likelihood)
-67.7191
-68.0589
-80. 0153
# Param's	Deviance	Test d.f.	P-value
3
2	0.679587	1	0.4097
1	24.5923	2	<.0001
AIC:
140.118
Log-likelihood Constant
62.107410554719884
Dose
Goodness of Fit
Est._Prob. Expected Observed	Size
Scaled
Residual
0.0000
3.5100
14.0000
Chi^2 =0.67
0.0739
0.2292
0.5547
d.f. = 1
4.506	5.000	61
16.045 14.000	70
12.759 14.000	23
P-value = 0.4138
0.242
-0.582
0.521
Benchmark Dose Computation
Specified effect =	0.1
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Risk Type =	Extra risk
Confidence level =	0.95
BMD =	2.01413
BMDL =	1.37981
BMDU =	3.2 9703
Taken together, (1.37981, 3.29703) is a 90	% two-sided confidence
interval for the BMD
**** Start of combined BMD and BMDL Calculations.****
Combined Log-Likelihood	-183.95322559798808
Combined Log-likelihood Constant	167.84132030252471
Benchmark Dose Computation
Specified effect =	0.1
Risk Type	=	Extra risk
Confidence level =	0.95
BMD =	1.19552
BMDL =	0.852494
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APPENDIX E. REFERENCES
Abernethv. D; Couch. D. (1982). Cytotoxicity and mutagenicity of dinitrotoluenes in Chinese
hamster ovary cells. Mutat Res 103: 53-59. http://dx.doi.org/10.1016/Q165-
7992(82)90087-2
Ahrenholz. S. (1980). Health hazard evaluation determination, report no. HE-79-1 13-728, 01 in
Chemical Company, Brandenburg, Kentucky [TSCA Submission], (HE-79-113-728).
Cincinnati, OH: National Institute of Occupational Safety and Health.
Ahrenholz. SH; Mever. CR. (1982). Health hazard evaluation report no HETA 81-295-1 155,
Olin (formerly Allied) Chemical Co, Moundsville, West Virginia. Cincinnati, OH:
National Institute for Occupational Safety and Health.
http://www2a.cdc. gov/hhe/select.asp?PitName=5509&bFlag=0&IP=3049
Ashbv. J; Burlinson. B; Lefevre. PA; Topham. J. (1985). Non-genotoxicity of 2,4,6-
trinitrotoluene (TNT) to the mouse bone marrow and the rat liver: implications for its
carcinogenicity. Arch Toxicol 58: 14-19.
AT SDR (Agency for Toxic Substances and Disease Registry). (1998). Toxicological profile for
2,4-dinitrotoluene and 2,6-dinitrotoluene (Update) [ATSDR Tox Profile], Atlanta, GA:
Agency for Toxic Substances & Disease Registry.
Bannasch. P. (1976). Cytology and cytogenesis of neoplastic (hyperplastic) hepatic nodules.
Cancer Res 36: 2555-2562.
Bannasch. P; Moore. \1A: Klimek. F; Zerban. H. (1982). Biological markers of preneoplastic
foci and neoplastic nodules in rodent liver. Toxicol Pathol 10: 19-34.
http://dx.doi. org/10.1177/019262338201000204
Bermudez. E; Tillerv. D; Butterworth. BE. (1979). The effect of 2,4-diaminotoluene and isomers
of dinitrotoluene on unscheduled DNA synthesis in primary rat hepatocytes. Environ Mol
Mutagen 1: 391-398. http://dx.doi.Org/l0.1002/ern.2860010412
Bruning. T; Chronz. C; Thier. R; Havelka. J; Ko. Y; Bolt. HM. (1999). Occurrence of urinary
tract tumors in miners highly exposed to dinitrotoluene. J Occup Environ Med 41: 144-
149.
Bruning. T; Thier. R; Mann. H; Melzer. H; Brode. P; Dallner. G; Bolt. HM. (2001). Pathological
excretion patterns of urinary proteins in miners highly exposed to dinitrotoluene. J Occup
Environ Med 43:610-615.
Cat EPA (California Environmental Protection Agency). (2009). Appendix A: Hot spots unit risk
and cancer potency values. Sacramento, CA: Office of Environmental Health Hazard
Assessment, http://www.oehha.ca.gov/air/hot spots/2009/AppendixA.pdf
Cat EPA (California Environmental Protection Agency). (2012a). All OEHHA acute, 8-hour and
chronic reference exposure levels (chRELs) as on February 2012. Sacramento, CA:
Office of Environmental Health Hazard Assessment.
http://www.oehha.ca.gov/air/allrels.html
Cat EPA (California Environmental Protection Agency). (2012b). OEHHA toxicity criteria
database. Sacramento, CA: Office of Environmental Health Hazard Assessment.
http ://www. oehha.ca. gov/tcdb/
CUT (Chemical Industry Institute of Toxicology). (1982a). 104-week chronic toxicity study in
rats: Dinitrotoluene, final report, volume I of II. (86940000342). Research Triangle Park,
NC.
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CUT (Chemical Industry Institute of Toxicology). (1982b). Teratological and postnatal
evaluation of dinitrotoluene in Fischer 344 rats [TSCA Submission], (FYI-OTS-1282-
0221). Research Triangle Park, NC.
CUT (Chemical Industry Institute of Toxicology). (1983). A thirty day toxicology study in
Fischer-344 rats given dinitrotoluene, technical grade [TSCA Submission], (878212040).
Research Triangle Park, NC.
http://www.ntis. gov/search/product.aspx?ABBR=QTS0205947
Couch. D; Allen. P; Abernethv. P. (1981). The mutagenicity of dinitrotoluenes in Salmonella
typhimurium. MutatRes 90: 373-383. http://dx.doi.ore/10.1016/0165-1218(81)90060-4
Hamill. PVV; Steinberaer. E; Levine. RJ; Rodriauez-Riaau. LJ; Lemeshow. S; Avrunin. JS.
(1982). The epidemiologic assessment of male reproductive hazard from occupational
exposure to TDA and DNT. J Occup Environ Med 24: 985-993.
Hamilton. CM; Mirsalis. JC. (1987). Factors that affect the sensitivity of the in vivo-in vitro
hepatocyte DNA repair assay in the male rat. Mutat Res 189: 341-347.
Harth. ¥; Bolt. HM; Brunina. T. (2005). Cancer of the urinary bladder in highly exposed
workers in the production of dinitrotoluenes: a case report. Int Arch Occup Environ
Health 78: 677-680. http://dx.doi.org/10.1007/s00420-005-0012-
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