United
Environmental Protection
Agency
Drinking Water Health Advisory
for 2,4-Dinitrotoluene and
2,6-Dinitrotoluene
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Drinking Water Health Advisory
for 2,4-Dinitrotoluene and 2,6-Dinitrotoluene
Prepared by:
Health and Ecological Criteria Division
Office of Science and Technology
Office of Water
U.S. Environmental Protection Agency
Washington, DC 20460
www.epa.gov/safewatr/ccl/pdf/DNTs.pdf/
EPA Document Number: 822-R-08-010
January, 2008
2,4- and 2,6-Dinitrotoluene - January 2008
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2,4- and 2,6-Dinitrotoluene - January 2008
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CONTENTS
Page
ACKNOWLEDGMENTS vii
LIST OF ABBREVIATIONS AND ACRONYMS ix
EXECUTIVE SUMMARY 1
1.0 INTRODUCTION 5
2.0 GENERAL INFORMATION AND PROPERTIES 7
2.1 Chemical Identity 7
2.2 Physical and Chemical Properties 7
3.0 OCCURRENCE/EXPOSURE 9
3.1 Production and Use 10
3.2 Air 10
3.3 Food 11
3.4 Water 11
3.4.1 Drinking Water Occurrence 11
3.4.2 Bed Sediment 12
3.5 Soil 13
4.0 ENVIRONMENTAL FATE 15
4.1 Environmental Media Transport 15
4.2 Environmental Degradation 16
4.3 Bioaccumulation 19
5.0 TOXICOKTNETICS 21
5.1 Absorption 21
5.2 Distribution 22
5.3 Metabolism 23
5.4 Excretion 26
6.0 HEALTH EFFECTS DATA 29
6.1 Human Studies 29
6.1.1 Short-term Exposure 29
6.1.2 Long-term Exposure 29
6.1.3 Reproductive and Developmental Effects 29
6.1.4 Carcinogenicity 29
6.2 Animal Studies 30
6.2.2 Short-term Exposure 30
6.2.1 Dermal/Ocular Effects 34
6.2.3 Long-term Exposure 34
6.2.4 Reproductive and Developmental Effects 39
6.2.5 Mutagenicity 40
6.2.6 Carcinogenicity 41
6.3 Sensitive Populations 43
6.4 Proposed Mode of Action 43
7.0 QUANTIFICATION OF TOXICOLOGICAL EFFECTS 45
7.1 One-day Health Advisory 45
7.2 Ten-day Health Advisory 46
v
2,4- and 2,6-Dinitrotoluene - January 2008
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7.3 Longer-term Health Advisory 46
7.4 Lifetime Health Advisory 49
7.5 Evaluation of Carcinogenic Potential 51
8.0 OTHER CRITERIA, GUIDANCE, AND STANDARDS 55
9.0 ANALYTICAL METHODS 57
10.0 TREATMENT TECHNOLOGIES 59
11.0 REFERENCES 61
APPENDIX A. CALCULATION OF DINITROTOLUENE INGESTION BY RATS IN
MCGOWN ET AL. (1983) A-l
APPENDIX B. BENCHMARK DOSE MODELING RESULTS FOR 2,4-
DINITROTOLUENE (2,4-DNT) B-l
APPENDIX C. BENCHMARK DOSE (BMD) MODELING OUTPUT C-2
2,4- and 2,6-Dinitrotoluene - January 2008
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ACKNOWLEDGMENTS
This document was prepared under the U.S. EPA Contract Number 68-C-02-009, Work
Assignment Nos. 2-13 and 3-13, with ICF Consulting, Fairfax, VA. The Lead Scientist is
Octavia Conerly, Health and Ecological Criteria Division, Office of Science and Technology,
Office of Water, U.S. Environmental Protection Agency.
2,4- and 2,6-Dinitrotoluene - January 2008
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2,4- and 2,6-Dinitrotoluene - January 2008
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LIST OF ABBREVIATIONS AND ACRONYMS
4Ac2NBacid
4A2NBacid
3,4-DNT
3,5-DNT
2,4-DNBacid
2,4-DNBalc
2,4-DNBalcG
2,4-DNT
2,6-DAT
2,6-DNBacid
2,6-DNBalc
2,6-DNBalcG
2,6-DNT
2,5-DNT
2,4-DAT
2,3-DNT
2Ac6NT
2A4NBacid
2A6NBacid
2A6NT
AK
AR
ATSDR
BCF
BMD
BMDL
BMR
BW
CA
CAS
CAS No. 121-14-2
CAS No. 606-20-2
CUT
CNS
CO2
cws
DNA
DNT
DWEL
DWI
BCD
EPA, U.S. EPA
FL
4-(N-Acetyl)amino-2-nitrobenzoic acid
4-amino-2-nitrobenzoic acid
3,4-dinitrotoluene
3,5-dinitrotoluene
2,4-dinitrobenzoic acid
2,4-dinitrobenzyl alcohol
2,4-dinitrobenzyl alcohol glucuronide
2,4-dinitrotoluene
2,6-diaminotoluene
2,6-dinitrobenzoic acid
2,6-dinitrobenzyl alcohol
2,6-dinitrobenzyl alcohol glucuronide
2,6-dinitrotoluene
2,5-dinitrotoluene
2,4-diaminotoulene
2,3-dinitrotoluene
2-acetylamino-6-nitrotoluene
2-amino-4-nitrobenzoic acid
2-amino-6-nitrobenzoic acid
2-amino-6-nitrotoluene
Alaska
Arkansas
Agency for Toxic Substances and Disease Registry
bioconcentration factor
benchmark dose
benchmark dose level
benchmark risk
body weight
California
Chemical Abstracts Service
2,4-dinitrotoluene
2,6-dinitrotoluene
Chemical Industry Institute of Toxicology
central nervous system
carbon dioxide
community water system
deoxyribonucleic acid
dinitrotoluene
drinking water equivalent level
drinking water ingestion
electron capture detection
U.S. Environmental Protection Agency
Florida
2,4- and 2,6-Dinitrotoluene - January 2008
IX
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GC
GDR
g/L
G6PD
HA
HPLC
HSDB
IA
IARC
IL
IN
ip
log Koc
log Kow
KY
LA
Ibs/yr
LD50
Lifetime HA
LOAEL
log Koc
log Kow
mg/kg/day
mg/L
mg/m3
mL
MI
mL
mm Hg
MO
MRL
MS
MTD
NAWQA
NCEA
NCI
NE
NJ
NOAEL
NPL
NTNCWS
NV
OH
OW
p value
gas chromatography
German Democratic Republic (formerly East Germany)
grams per liter
glucose-6-phosphate dehydrogenase
health advisory
high-performance liquid chromatography
Hazardous Substances Data Bank
Iowa
International Agency for Research on Cancer
Illinois
Indiana
intraperitoneal
organic carbon soil partition coefficient
octanol/water partition coefficient
Kentucky
Louisiana
pounds per year
50% of the lethal dose
Lifetime health advisory
lowest observed adverse effect level
organic carbon soil partition coefficient
octanol/water partition coefficient
milligrams per kilograms (of body weight) per day
milligrams per liter
milligrams per cubic meter
milliliter
Michigan
milliliter
millimeters of mercury
Missouri
minimum reporting level
Mississippi, mass spectrometry
maximum tolerated dose
National Water-Quality Assessment (Program) (USGS)
National Center for Environmental Assessment (U.S. EPA)
National Cancer Institute
Nebraska
New Jersey
no observed adverse effect level
National Priorities List
non-transient non-community water system
Nevada
Ohio
Office of Water (U.S. EPA)
probability value
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POD
ppm
ppt
PWS
RfD
RL
RSC
SC
SVOC
Tg-DNT
TN
TNT
TRI
TWA
TX
UF
ug/L
U.S. EPA
USGS
UT
UV
VA
vs.
WV
point of departure
parts per million
parts per trillion
public water system
reference dose
reporting level
relative source contribution
South Carolina
semivolatile organic compound
technical grade DNT
Tennessee
trinitrotoluene
Toxics Release Inventory, the TRI Program
time-weighted average
Texas
uncertainty factor
micrograms per liter
Environmental Protection Agency
U.S. Geological Survey
Utah
ultraviolet
Virginia
not v, vs, or versus
West Virginia
2,4- and 2,6-Dinitrotoluene - January 2008
XI
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X11
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EXECUTIVE SUMMARY
The 2,4-dinitrotoluene (2,4-DNT) and 2,6-dinitrotoluene (2,6-DNT) isomers, Chemical
Abstracts Service Nos. 121-14-2 and 606-20-2, respectively, are yellow to reddish crystal solids
at room temperature. Both isomers are moderately soluble in water (0.30 g/L and 0.18 g/L,
respectively) and have a low affinity for organic paniculate matter (Koc 1.65 and Koc 1.96,
respectively). A mixture of DNTs is sold as an explosive and is a starting material for the
production of 2,4,6-trinitrotoluene (TNT). The mixture is also used as a modifier for smokeless
powders in the munitions industry, in airbags of automobiles, and as an intermediate in the
production of urethane foams.
2,4-DNT and 2,6-DNT are released into the environment primarily from facilities that
manufacture or process DNT. 2,4-DNT and 2,6-DNT may subsequently contaminant surface and
groundwaters during runoff events. However, monitoring data from sampling conducted under
the U.S. Environmental Protection Agency's (U.S. EPA) first Unregulated Contaminant
Monitoring Regulation (UCMR1) program indicate that the frequency of detection of 2,4-DNT
and 2,6-DNT in public water systems is low.
Human exposure to 2,4-DNT and 2,6-DNT occurs through inhalation, dermal contact, and
incidental ingestion, usually in occupational settings. 2,4-DNT and 2,6-DNT are readily and
rapidly absorbed following oral or inhalation exposure and are eliminated through urinary and
fecal excretion.
Human toxicity has been evaluated in DNT factory workers, munitions handlers, and
underground mining workers. DNT-related effects have been noted in the central nervous
system, heart, and circulatory system. Other effects that are possibly due to 2,4-DNT and 2,6-
DNT exposure include increased mortality from ischemic heart disease, hepatobiliary cancer,
and urothelial and renal cell cancers. No apparent reproductive or developmental effects have
been evaluated in humans exposed to DNT.
In animal studies with rats, mice, and dogs, 2,4-DNT and 2,6-DNT isomers have similar
effects and have been shown to cause adverse neurological, hematological, reproductive, hepatic,
and renal effects. Dogs generally are the most sensitive of the three species. Oral studies of 50%
of the lethal dose in rats and mice indicate that both 2,4-DNT and 2,6-DNT are moderately to
highly toxic.
Short-term (5 days to 4 weeks) oral exposures of 2,4-DNT were both lethal and toxic to
experimental animals. Chemical-related mortality at doses as low as 145 milligrams per kilogram
of body weight (BW) per day (mg/kg/day) was observed in rats and mice at doses > 1,250
mg/kg/day. At sublethal doses in rats (>45 mg/kg/day), toxicity was characterized by decreased
food consumption and decreased BW gain, BW loss, cyanosis, changes in serum chemistry
levels, increased absolute and relative liver weights, splenic hemosiderosis, testicular lesions and
atrophy, and aspermatogenesis. Treatment-related toxicity was observed in dogs at 25 mg/kg/day
and manifested as decreased food consumption, BW loss, neurological effects,
aspermatogenesis, and histopathology of the liver, brain, and spinal cord. Short-term (4 weeks)
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oral exposures to 2,6-DNT were toxic, but not lethal, to experimental animals. The Lowest
Observed Adverse Effect Level (LOAEL) was 35 mg/kg/day for rats, 55 mg/kg/day for mice and
20 mg/kg/day for dogs. Toxic effects were similar to those found for 2,4-DNT. Rats fed diets
with technical grade-DNT (Tg-DNT) for 4 weeks exhibited toxicity at doses >75 mg/kg/day,
experiencing similar symptoms to that listed above for isomeric components.
Subchronic (13 weeks) oral exposures to 2,4-DNT were both lethal and toxic to all species
tested. In rats there was chemical-related mortality at doses as low as 145 mg/kg/day, in mice at
doses >413 mg/kg/day and in dogs at 25 mg/kg/day. Toxic effects observed in rats included
decreased BW gain, urine-stained fur, neurological effects, anemia, increased liver and kidney
weights, and decreased spermatogenesis. Subchronic oral exposures to 2,6-DNT were lethal only
to dogs at doses >20 mg/kg/day; however, the toxic effects observed in rats, mice, and dogs were
very similar to the effects noted above for 2,4-DNT. There were no Subchronic animal exposures
to Tg-DNT found in the available literature.
In chronic studies, oral doses of 2,4-DNT given to rats, mice, and dogs for 1-2 years were
lethal and toxic. 2,4-DNT was lethal to rats at doses >34 mg/kg/day and to mice at 898
mg/kg/day; dogs given 10 mg/kg/day 2,4-DNT were sacrificed after 19 weeks due to their poor
condition. Toxic effects in rats included reduced BW gain, liver histopathology, bile duct
hyperplasia, cholangiofibrosis, seminiferous tubule atrophy, almost complete aspermatogenesis,
pigmentation of the spleen, anemia, and reticulocytosis. Toxicity in mice was reported to include
testicular atrophy, decreased food consumption, decreased BW, hemosiderosis of many organs
(primarily the liver and spleen), and an elevated incidence of malignant renal tumors. Toxicity in
dogs was reported as neuropathology, methemoglobinemia with associated reticulocytosis and
Heinz body formation, biliary tract hyperplasia, and pigmentation of the gallbladder, kidneys,
and spleen. When 2,6-DNT was administered in the diet of rats for 12 months, the observed
effects of hepatic histopathology included acidophilic and basophilic foci, elevated serum
alanine aminotransferase and gamma-glutamyl transferase, and bile duct hyperplasia. Tg-DNT
administered in the diets of rats for 6 months to 2 years did not cause any mortality, but toxic
effects were notable.
Studies in rats demonstrate that oral exposure to 2,4-DNT causes severe reproductive effects.
Some of the toxic effects observed in parental animals include reduced BW, cyanosis, decreased
mating index, reduced fertility, testicular atrophy and degeneration, reduced spermatogenesis
and sperm count, increases in serum follicle stimulating hormone and luteinizing hormone,
cessation of follicular function, reduced number of corpora lutea, and histopathology of Sertoli
cells, spermatocytes, and spermatids. Effects observed in offspring were lower mean litter size,
reduced viability, decreases in BW at birth and at weaning, changes in relative organ weights
and hematologic parameters, and reduced fertility. Limited available data suggest that orally
administered 2,4-DNT is not teratogenic in mice. Data on the reproductive or developmental
effects of 2,6-DNT were not found in the current literature. Tg-DNT was not teratogenic to rats
administered oral doses up to 150 mg/kg/day; however, embryotoxicity was observed at
maternally toxic levels (> 14mg/kg/day). Developmental effects noted in the fetuses were
reduced liver weight and increased spleen weight.
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Both 2,4-DNT and 2,6-DNT are weak mutagens in Salmonella test systems. Tg-DNT is
negative for unscheduled DNA synthesis except when an in vivo/in vitro testing system was
used.
Experimental studies with 2,4-DNT administered in the diet demonstrate that it is
tumorigenic in rats and mice but not in dogs. In a 1-year feeding study, 2,6-DNT administered in
the diet to mice induced hepatocellular carcinomas and cholangiocarcinomas (a type of liver
cancer involving the bile diet). Males fed Tg-DNT in the diet for 1 year and males and females
fed Tg-DNT in the diet for up to 2 years developed hepatocellular carcinomas and/or
cholangiocarcinomas.
2,4,6-TNT can cause hemolysis in humans with a genetic defect in the enzyme glucose-6-
phosphate dehydrogenase (G6PD). There is a lack of experimental data that indicate that the
DNTs have a similar effect but based on animal data showing hemolytic anemia, GSPD deficient
individuals may be a sensitive population for DNT exposure as well.
Health advisories (HAs) were determined for 1-day, 10-day, and longer term (up to 7 years)
exposures. The 1-day HA for 2,4-DNT is 0.5 mg/L, and the 10-day HA is 1.0 mg/L. The longer
term HA for 2,4-DNT for the 10-kg child is 0.3 mg/L; for the 70-kg adult, it is 1.0 mg/L. The
reference dose (RfD) for 2,4-DNT is 0.002 mg/kg/day, and the drinking water equivalent level
(DWEL)isO.l mg/L.
The 1-day HA for 2,6-DNT is 0.4 mg/L, and the 10-day HA is 0.4 mg/L. The longer term
2,6-DNT HA for the 10-kg child is 0.4 mg/L; for the 70-kg adult, it is 1.0 mg/L. The RfD for
2,6-DNT is 0.001 mg/kg/day, and the DWEL is 0.04 mg/L.
A mixture of 2,4-DNT and 2,6-DNT is classified as "likely to be carcinogenic to humans";
thus, Lifetime HAs for 2,4-DNT and 2,6-DNT are not recommended. The cancer risk estimate
for the 2,4-DNT/2,6-DNT mixture is derived from a feeding study where female rats were the
sensitive species/sex and mammary gland tumors were the critical endpoint. The point of
departure selected for the quantification of cancer risk from DNT is the lower 95% confidence
bound on the benchmark dose level of 0.15 mg/kg/day, derived the cancer incidence data in
female rats. The oral slope factor is 6.67 x 10"1 mg/kg/day, and the drinking water unit risk is
-v-5
1.90 x 10" ug/L. The drinking water concentrations at specific risk levels are 5 ug/L for a risk of
10'4(linlO
1,000,000).
10'4 (1 in 10,000); 0.5 ug/L for a risk of 10'5 (1 in 100,000); and 0.05 ug/L for a risk of 10'6 (1 in
The only other criteria, guidance, or standard found for any of the DNT isomers was a U.S.
EPA ambient water quality criteria to protect human health for 2,4-DNT at a 10"6 risk level. The
criteria are 0.11 ug/L for ingestion of water and organisms and 9.1 ug/L for ingestion of
organisms only.
Analytical methods for DNT isomers most frequently rely on gas chromatography and high-
performance liquid chromatography. Other methods include electron spin resonance
2,4- and 2,6-Dinitrotoluene - January 2008
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spectrometry, tandem mass spectrometry, and cluster analysis. Treatment technologies include
adsorption, chlorination, ozonation, and ultraviolet radiation.
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1.0 INTRODUCTION
The Health Advisory (HA) Program, sponsored by the Office of Water (OW), provides
information on the health effects, analytical methodology and treatment technology that are
useful in dealing with the contamination of drinking water. Health Advisories describe non-
regulatory concentrations of drinking water contaminants at which adverse health effects are not
anticipated to occur over specific exposure durations. Health Advisories contain a margin of
safety to protect sensitive members of the population.
Health Advisories serve as informal technical guidance to assist Federal, State and local
officials responsible for protecting public health when emergency spills or contamination
situations occur. They are not to be construed as legally enforceable Federal standards. The HAs
are subject to change as new information becomes available. HAs usually are based on adverse
health effects but HA documents may also provide information on the organoleptic or aesthetic
properties (color, taste, odor) of contaminants in drinking water.
Health Advisories are developed for both short-term and long-term (Lifetime) exposure
periods based on data describing non-carcinogenic end points of toxicity. Short-term exposures
can include one-day to ten-day exposure periods. In many cases a longer-term value is included
covering approximately 7 years, or 10 percent of an individual's lifetime. For those substances
that are "known" or "likely to be carcinogenic to humans," Lifetime HAs are not recommended.
The Health Advisory evaluation of carcinogenic potential includes the U.S. EPA
classification for the weight of evidence of the likelihood that the agent is a human carcinogen
and the conditions under which the carcinogenic effects may be expressed as well as quantitative
estimates of cancer potency (slope factor) where available. The cancer slope factor is the result
of the application of a low-dose extrapolation procedure and is presented as the risk per
mg/kg/day. The Health Advisory includes the drinking water concentration equivalent to cancer
risks of one-in-ten-thousand (10"4), one-in-one-hundred-thousand (10"5), to one-in-one-million
Cancer assessments conducted before 1996 used the five-category, alphanumeric system for
classifying carcinogens established by the Guidelines for Carcinogen Risk Assessment (U.S.
EPA, 1986). After 1999, assessments were conducted using Guidelines for Carcinogen Risk
Assessment (U.S. EPA, 1996, 1999). Between 1996 and 2001, assessments were conducted using
both the 1986 and 1996 guidelines. Currently, the Agency Administrator has issued a directive
that all new cancer assessments should be in accordance to the 1999 Draft Guidelines for
Carcinogen Risk Assessment (U.S. EPA, 2001). This has been superseded by the final guidelines,
Guidelines for Carcinogen Risk Assessment (U.S. EPA 2005).
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2,4- and 2,6-Dinitrotoluene - January 2008
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2.0 GENERAL INFORMATION AND PROPERTIES
2.1 Chemical Identity
The 2,4-dinitrotoluene (2,4-DNT) and 2,6-dinitrotoluene (2,6-DNT) isomers, Chemical
Abstracts Service (CAS) Nos. 121-14-2 and 606-20-2, respectively, are the major components of
technical grade DNT (Tg-DNT). Other DNT isomers make up 5% of Tg-DNT (ATSDR, 1998).
Analysis of Tg-DNT reveals the following composition: 76.49% 2,4-DNT, 18.83% 2,6-DNT,
0.65% 2,5-DNT, 2.43% 3,4-DNT, 1.54% 2,3-DNT, 0.040% 3,5-DNT, 0.050% trinitrotoluene
(TNT), 0.005% cresols, 0.003% mononitrobenzene, and 0.003%, 0.0005%, and 0.006%, for
ortho-, meta-, and para-, mononitrotoluenes, respectively (HSDB, 2004a,b,c).1994. The chemical
structures of 2,4-DNT and 2,6-DNT are displayed in Figure 2-1.
-o
0
-o-
(a)
(b)
Figure 2-1. Chemical Structures of (a) 2,4-Dinitrotoluene and (b) 2,6-Dinitrotoluene
(ChemFinder.com, 2004)
2.2 Physical and Chemical Properties
DNT is a white- to buff-colored solid at room temperature and exists as a mixture of two or
more of its six isomers: 2,3-DNT, 2,4-DNT, 2,5-DNT, 2,6-DNT, 3,4-DNT, and 3,5-DNT. Upon
heating, DNT forms an oily liquid that turns yellow when exposed to sunlight (Hartley et al.,
1994). The chemical and physical properties of 2,4-DNT and 2,6-DNT are listed in Table 2-1.
The 2,4 and 2,6-DNT isomers are combustible, nitroaromatic compounds that are soluble in
water (Hartley et al., 1994). At room temperature, 2,4-DNT appears as yellow or orange needles
or monoclinic prisms (HSDB, 2004a). At room temperature, 2,6-DNT exists as yellow to red
rhombic crystals (HSDB, 2004b).
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Table 2-1. Chemical and Physical Properties of 2,4-Dinitrotoluene and 2,6-Dinitrotoluene
Property
CAS No.
U.S. EPA Pesticide Chemical
Code
Synonyms
Registered Trade Name(s)
Chemical Formula
Molecular Weight
Physical State
Boiling Point
Melting Point
Density (at 71 °C)
Vapor Pressure:
At 20 °C
At 25 °C
Partition Coefficients:
Log Kow (octanol/water
partition coefficient)
Log Koc (organic carbon
soil partition coefficient)
Solubility in:
Water at 22 °C
Other Solvents
Conversion Factors
(at 25 °C, 1 atm)
2,4-Dinitrotoluene
121-14-2
NA
l-methyl-2,4-
dinitrobenzene, 2,4-
dinitrotoluol
2,4-DNT
No data
C7H6N204
182.14
Yellow solid
300 °C (slight
decomposition)
71 °C
1.3208
0.0051 mmHg
1.4xlO'4mmHg
1.98
1.65
300 mg/L
Acetone, alcohol, benzene,
ethanol, diethyl ether,
pyridine, CS2
1 ppm = 7.40 mg/m3
1 mg/m3 = 0.13 ppm
2,6-Dinitrotoluene
606-20-2
NA
1 -methyl-2,6-dinitrobenzene,
2,6-DNT
NA
C7H6N204
182.14
Yellow to red solid
285 °C
66 °C
1.2833
0.0 18 mmHg
5.67xlO'4mmHg
1.72 or 2. 10
1.96
180 mg/L
Ethanol, chloroform
1 ppm = 7.40 mg/m3
1 mg/m3 = 0.13 ppm
Sources: ATSDR, 1998; HSDB, 2004a,b; Hartley et al., 1994
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3.0 Occurrence/Exposure
DNTs are not known to occur naturally in the environment but have been detected in the soil,
surface water, and groundwater of hazardous waste sites that contain buried ammunition wastes
and wastes from manufacturing facilities that release DNT (ATSDR, 1998). No recent
quantitative estimates of DNT production or use are available. Combined 2,4- and 2,6-DNT
production was -1.24 x 106 kg in 1975 (U.S. EPA, 1980). Tg-DNT produced in the United States
was reported to be 3.27 x 109 g in 1982 (HSDB, 2004a,b,c).
Sources of exposure to 2,4-DNT and 2,6-DNT include facilities that manufacture or process
DNT, as well as hazardous waste sites (ATSDR, 1998). 2,4-DNT and 2,6-DNT have been
detected in soil, sediment, water, or air at 69 out of 1,467 and 53 out of 1,467, respectively,
current or former National Priorities List (NPL) hazardous waste sites (HazDat, 1998). The
general population may be exposed via inhalation, dermal contact, and incidental ingestion.
Occupational exposure to DNTs, which is much more likely than general exposure, can occur by
inhalation, skin absorption, or inadvertent ingestion during DNT production and DNT use as
intermediates (ATSDR, 1998; International Agency for Research on Cancer [IARC], 1996;
Tchounwou et al., 2003).
Both 2,4-DNT and 2,6-DNT are listed as Toxics Release Inventory (TRI) chemicals. TRI
data for both isomers are reported for the years 1988-2002 (U.S. EPA 2004).
2,4-Dinitrotoluene
TRI releases for 2,4-DNT were reported from 21 States (AK, CA, FL, IA, IL, IN, KY, LA,
MI, MO, MS, NE, NJ, NV, OH, SC, TN, TX, UT, VA, WV). Surface water discharges in 1988
and 1989 were slightly greater than 12,000 Ibs/year. They declined significantly in the!990s,
ranging from 3,735 Ibs/yr to as low as 90 Ibs/yr. Surface water discharges were even lower
during the years 2000, 2001, and 2002, at levels of 177, 10, and 6 Ibs/yr, respectively. Combined
releases of all kinds (i.e., onsite [air, surface water discharge, underground injection, releases to
land] and offsite) declined in the early 1990s and then peaked again around 1999-2001 to a high
of almost 700,000 Ibs/yr (U.S. EPA, 2004).
2,6-Dinitrotoluene
TRI releases for 2,6-DNT were reported from 10 States (AR, CA, IN, KY, LA, MI, NV, OH,
TX, WV), with no more than 9 States reporting in any one year. Surface water discharges in
1988 and 1989 were approximately 1,000 Ibs/yr. They declined in the!990s, ranging from 702
Ibs/yr to as low as 24 Ibs/yr. Surface water discharges remained low during the years 2000, 2001,
and 2002, at levels of 32, 0, and 1 Ibs/yr, respectively. Combined releases of all kinds (i.e., onsite
[air, surface water discharge, underground injection, releases to land] and offsite) declined in the
early 1990s and then peaked again around 1999-2001 to more than 1 million Ibs/yr (U.S. EPA,
2004).
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3.1 Production and Use
DNT is made by reacting toluene with a mixture of nitric and sulfuric acids (ATSDR, 1998).
Typically, the process yields 75% 2,4-DNT, 19% 2,6-DNT, 2.5% 3,4-DNT, 1.0% 2,3-DNT, and
0.5% 2,5-DNT by weight (HSDB, 2004a). TNT and mononitrotoluenes account for the
remaining percentage (ATSDR, 1998). This mixture typically is Tg-DNT. An alternative method
for the production of DNT is the nitration of mononitrotoluene with mixed acid (HSDB, 2004c).
Small concentrations of DNT isomers also occur as byproducts in the production of TNT
(Hartley et al., 1994). The military requirement for DNT specifies a minimal melting point of
65.5 °C, which corresponds to a 2,4-DNT purity of 92%. Another specification for the
production of military munitions requires the use of DNT mixture composed of at least 98.5% of
the 2,4-isomer (Hartley et al., 1994).
Most DNT is produced for production of toluene diisocyanate, a precursor to polyurethane
polymers. 2,4-DNT also is used in the production of TNT as a modifier for smokeless powders
in the munitions industry, in the production of waterproofing for explosives, as a dye
intermediate, as a plasticizer in propellants (HSDB, 2004a). The 2,4-DNT isomer is used in
airbags of automobiles (ATSDR, 1998). Similar to 2,4-DNT, 2,6-DNT is used in the production
of waterproofing for explosives, and as a plasticizer in propellants. It also is an intermediate in
the production of TNT, urethane polymers, flexible and rigid foams, surface coatings, and dyes
(HSDB, 2004b).
Currently, there are a small number of companies manufacturing DNT in the United States.
They include U.S. Bayer Corporation, Pittsburgh, PA (production site: Baytown, TX) and
Rubicon LLC, Ascension Parish, LA (production site: Geismar, LA) (HSDB, 2004c).
Information from the late 1990s (SRI Consulting, 1999 and ). Hartley et al., 1994) identified
other companies that produced 2,4-DNT and/or 2,6-DNT [Air Products and Chemicals, Inc.,
Allentown, PA; Allied Chemical Corporation, Moundsville, WV, and E.I. Du Pont de Nemours
and Company, Deepwater, NJ].
3.2 Air
Most measurements of 2,4-DNT and 2,6-DNT in air are from occupational environments
where explosives or explosive devices are manufactured or processed (ATSDR, 1998). The TRI
Program records air releases to the ambient environment. These releases for 2,4-DNT and 2,6-
DNT were discussed in Section 3.0 of this document.
2,4-Dinitrotoluene
Ambient air concentrations of 2,4-DNT in the work environment have been reported to range
from less than detectable levels up to 2,680 mg/m3 (Letzel et al., 2003; Woollen et al., 1985;
Ahrenholz, 1980).
2,4- and 2,6-Dinitrotoluene - January 2008
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Personal (breathing zone) samples taken from workers at these same workplaces also ranged
from less than detectable levels but only up to air concentrations of
440 mg/m3. In other airborne samples collected for the detection of nitroaromatics (from
unspecified sources), Matsushita and lida (1986) reported a concentration of 0.024 ng/m3 for 2,4-
DNT.
2,6-Dinitrotoluene
Studies primarily measured DNT mixtures or 2,4-DNT in air samples. Levine et al. (1985a)
detected personal breathing zone samples of 2,6-DNT at 50 mg/m3 to 590 mg/m3 in the
workplace.
Dinitrotoluene Mixture
In biological monitoring studies among workers exposed to Tg-DNT in an explosives
factory, routine personal air sampling revealed levels ranging from undetectable to 100 mg/m3.
The detection limit was not reported by the ATSDR (1998); however, air monitoring analysis
has a detection limit of 20 parts per trillion (ppt) for 2,4-DNT (Nacson et al., 1994). In the same
study, static samples positioned near potentially dusty areas revealed atmospheric concentrations
ranging from 20 mg/m3 to 2,680 mg/m3 (mean of 400 mg/m3) (Woollen et al., 1985). In another
occupational environment, breathing zone concentrations of Tg-DNT ranged from undetectable
to 23 mg/m3 (time-weighted average [TWA]) (Ahrenholz, 1980). Concentrations of Tg-DNT in
area air samples ranged from undetectable to 420 mg/m3 (TWA). Ahrenholz and Meyer (1982)
reported that area air samples in a manufacturing facility contained TWA concentrations of Tg-
DNT that ranged from undetectable to 890 mg/m3.
3.3 Food
2,4-DNT or 2,6-DNT were not detected in fish samples from Lake Michigan tributaries,
Grand Traverse Bay, and other Great Lakes harbors and tributaries in Ohio and Wisconsin
(Camanzo et al., 1987; De Vault, 1985). Reports of other food sources that contained levels of
2,4-DNT were not found in the available literature.
3.4 Water
3.4.1 Drinking Water Occurrence
The first Unregulated Contaminant Monitoring Regulation (UCMR1) was established to
satisfy the requirements of the 1996 Safe Drinking Water Act amendments. It was designed to
collect information on the national occurrence of select emerging contaminants in drinking
water. 2,4-DNT and 2,6-DNT were scheduled to be monitored by all large community water
systems (CWSs) and non-transient non-community water systems (NTNCWSs) and a
statistically representative sample of qualifying small CWSs and NTNCWSs. Although
monitoring was conducted primarily between 2001 and 2003, some results were not collected
and reported until as late as 2006. Because the health reference level (0.05 |ig/L) for each
2,4- and 2,6-Dinitrotoluene - January 2008
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contaminant (2,4-DNT and 2,6-DNT) is less than the minimum reporting level (MRL) of 2 |ig/L,
the data are analyzed only as detections (>MRL).
Among small systems, there were no detections of 2,4-DNT. There was only one detection in
large systems that reported results of 2,4-DNT; this surface water system represented 0.03% of
reporting large systems and 0.02% of the population served by them. The analysis of samples
from large and small systems did not detect any 2,6-DNT.
The state of Illinois submitted data to EPA indicating that significant levels of DNT (2,4-
DNT and 2,6-DNT; other isomers not specified) had been detected in groundwater at a number
of sites within the state (Illinois EPA, 2007). DNT in potable groundwater at the Joliet Army
Ammunition Plant and the Savanna Army Depot was detected at levels of 10,000 |ig/L and 58.8
|ig/L, respectively. Data were also obtained from the state of Wisconsin (2007) where the 2,3-,
2,5-, 3,4-, and 3,5-NDT isomers have been identified in both monitoring well and a few private
water supply wells near the Badger Army Ammunition Plant site. These data demonstrate the
risk for DNT contamination of groundwater at military bases where DNT munitions have been
used or stored.
3.4.2 Bed Sediment
SVOCs such as 2,4-DNT and 2,6-DNT have a slight tendency to sorb to sediment and
particles because of their low to moderate log octanol/water coefficients (K0ws) (1.98 and 1.72,
respectively). The NAWQA Pesticide National Synthesis Project includes an analysis of SVOC
monitoring in bed sediment from representative watersheds across the country between 1992 and
2001. Sampling was conducted at 1,029 sites. The RL for all SVOCs was 50 |ig/L (Nowell and
Capel, 2003).
2,4-Dinitrotoluene
NAWQA data indicate that 2,4-DNT was not detected in bed sediment in agricultural, urban,
or undeveloped settings. In mixed land use settings, 2,4-DNT was detected in 1.3% of samples,
with a maximum concentration of 173 |ig/kg dry weight (Nowell and Capel, 2003).
2,6-Dinitrotoluene
2,6-DNT was detected in bed sediment at frequencies ranging from 1.6% in urban settings to
4.4% in agricultural settings, 6.6% in mixed land use settings, and 6.9% in undeveloped settings.
The 95th percentile concentrations were less than the RL in all settings. The highest
concentration, 291 |ig/kg dry weight, was found in an undeveloped setting (Nowell and Capel,
2003).
12
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3.5 Soil
2,4-Dinitrotoluene
Concentrations of 2,4-DNT in soil ranged from <0.1 mg/kg to 117 mg/kg at the Joliet Army
Ammunition Plant in Joliet, IL, an NPL site (Simini et al., 1995). The 2,4-DNT isomer was
detected in the soil at 2.2% of hazardous waste sites, with a geometric mean concentration of 1.0
mg/kg (ATSDR, 1989). The concentration of 2,4-DNT in the soil in a waste lagoon abandoned
for 20 years at the Iowa Army Ammunition Plant was 3.0 mg/kg (Ryon et al., 1984).
2,6-Dinitrotoluene
Concentrations of 2,6-DNT in soil ranged from <0.1 mg/kg to 8 mg/kg at the Joliet Army
Ammunition Plant, an NPL site (Simini et al., 1995). The 2,6-DNT isomer was detected in the
soil at 1.3% of hazardous waste sites, with a geometric mean concentration of 0.140 mg/kg
(ATSDR, 1989).
2,4- and 2,6-Dinitrotoluene - January 2008
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14
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4.0 ENVIRONMENTAL FATE
DNT has been found in the soil, surface water, and groundwater of hazardous waste sites that
contain buried ammunition wastes and wastes from manufacturing facilities that release DNT
(ATSDR, 1998). The water solubilities of 2,4-DNT and 2,6-DNT are moderate, and the log Kow
and log Koc are low for both isomers (Table 2-1). Since the partitioning of organics to the
sediment from the aqueous phase does not become a major loss until the log Koc values exceed
3.5, the relatively low log Koc values for 2,4-DNT and 2,6-DNT indicate that these compounds
would have only a slight tendency to sorb to sediments, suspended solids, and biota. Therefore,
there is potential for transport via surface water or groundwater. The low lipophilicity of this
compound predicts it is not expected to bioaccumulate in animal tissues (ATSDR, 1998).
4.1 Environmental Media Transport
2,4-Dinitrotoluene
2,4-DNT adsorption to sediments, suspended solids, and biota is not a significant
environmental fate due to its relatively low log Koc (Spanggord et al., 1980). Additionally, the
low vapor pressure (1.4 x 10"4 mm Hg at 25 °C) and Henry's law constant (solubilities) (8.79 x
10"8 atm»m3»mol) of 2,4-DNT indicate that 2,4-DNT is not expected to volatilize from water or
soil (ATSDR, 1998; HSDB, 2004a,b,c).
2,6-Dinitrotoluene
Measurements in two types of soil indicated that 2,6-DNT's low Kocs were 1.86 and 1.28
(Kenaga, 1980). These values indicate that 2,6-DNT would have high mobility in soil (HSDB,
2004a,b,c). It is concluded that adsorption on sediments is not a significant environmental fate.
Additionally, the low vapor pressure (5.67 x 10"4 mm Hg at 25 °C) and Henry's law constant
(9.26 x 10"8 atm»m3»mol) of this DNT isomer indicate that it is not expected to volatilize from
water or soil (ATSDR, 1998; HSDB, 2004a,b,c).
Dinitrotoluene Mixture
DNT may be released and transported in the air in the form of dusts or aerosols from
manufacturing plants. It can enter surface water and groundwater by releases of wastewater from
TNT manufacturing facilities and from buried munition wastes (ATSDR, 1998). The relatively
low volatility and moderate solubility of DNT indicate that it will remain in water for long
periods of time. DNT is degraded by light, oxygen, and biota. As a result, it can be transported to
groundwater or surface water (ATSDR, 1998).
2,4- and 2,6-Dinitrotoluene - January 2008
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4.2 Environmental Degradation
2,4-Dinitrotoluene
Vapor-phase 2,4-DNT is degraded in the atmosphere by reaction with photochemically
produced hydroxyl radicals and has an estimated half-life of 75 days (HSDB, 2004a,b,c; Meylan
and Howard, 1993).
Ho (1986) found that intermediates formed during 2,4-DNT's degradation include
1,3-dinitrobenzene, hydroxynitrobenzene derivatives, and carboxylic acids. DNT's half-life
following photolysis from sunlight ranged from 2.7 hours to 9.6 hours in natural waters
(Spanggord et al., 1980). Dissolved humic substances in natural waters enhance the sunlight-
induced photodegradation rates (by 10-17 times) compared with rates observed in distilled water
(Simmons and Zepp, 1986). Chlorination and ozonation reduce 2,4-DNT 35% and 60%,
respectively, regardless of length of contact time.
Microbial biodegradation of DNT in water has been observed under both aerobic and
anaerobic conditions. Biotransformation occurs mainly through the reduction of the nitrogroup
(Spanggord et al., 1981). Microorganisms isolated from DNT-contaminated sites are capable of
growth on 2,4-DNT as their sole carbon and energy source (ATSDR, 1998; Lewis et al., 2004;
Spanggord et al., 1980). 2,4-DNT degradation occurred in waters taken downstream from the
Radford Army Ammunition Plant (Radford, VA) but not in those from a Maryland surface
freshwater source (Bausum et al., 1992). Biotransformation of DNT by Pseudomonas
aeruginosa isolated from a propellant wastewater treatment plant, was observed under both
aerobic and anoxic conditions (Noguera and Freedman, 1996).
In soil, microorganisms can degrade DNT. Jenkins et al. (2001) determined that 2,4-DNT's
half-life in soil was 25 days at 22 °C, from an initial concentration of 0.5 mg/kg, and concluded
that it was concentration dependent. Microorganisms indigenous to surface soils from munitions-
contaminated sites transformed 2,4-DNT to aminonitro intermediates within 70 days (Bradley
et al., 1994). Lower temperatures slow 2,4-DNT's breakdown (Grant et al., 1995).
Since microorganisms readily metabolize 2,4-DNT to CC>2 as the final product, DNT is not
expected to persist in the environment. However, studies show persistence is water-body
dependent, and the length of time any particular nitroaromatic compound resides in the
environment ultimately depends on the compound's unique interaction with the natural organics
and biota in its surroundings (Spanggord et al., 1980; Liu et al., 1984). Multiple studies show
that the breakdown/intermediate products of 2,4-DNT include 4-amino-2-nitrotoluene,
2-amino-4-nitrotoluene, and/or 2,4-diaminotoluene (Bradley et al., 1997; Cheng et al., 1996;
Freedman et al., 1996; Liu et al., 1984; Noguera and Freedman, 1996, 1997).
2,6-Dinitrotoluene
Vapor-phase 2,6-DNT is degraded in the atmosphere by reaction with photochemically
produced hydroxyl radicals (HSDB, 2004b). The half-life for this reaction in air is estimated to
2,4- and 2,6-Dinitrotoluene - January 2008
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be 75 days, as calculated from its rate constant of 2.2 x 10"13 cm3/molecule-sec at 25 °C, which
was determined using a structure estimation method (Meylan and Howard, 1993).
In a study of chemicals associated with TNT in the soil, the half-life of 2,6-DNT was
measured to be 20 days at 22 °C when the initial concentration of 2,6-DNT was 0.5 mg/kg. The
study also showed that the half-life of 2,6-DNT was concentration dependent (Jenkins et al.,
2001). In another study, microorganisms indigenous to surface soils collected at munitions-
contaminated sites were reported to transform 2,4-DNT and 2,6-DNT to aminonitro
intermediates within 70 days (Bradley et al., 1994). Degradation rates of 2,6-DNT measured in
Mississippi and Texas soils were 0.5 mg/kg/day and 0.7 mg/kg/day, respectively, which
correspond to half-lives of 92 and 73 days, respectively (Loehr, 1989).
In oxygenated waters, photolysis is probably the major route of degradation of DNT
(ATSDR, 1998). Dillert et al. (1995) reported that degradation rates followed first-order kinetics,
and were dependent on time, solution pH, and light intensity. Simmons and Zepp (1986) studied
the influence of various humic substances on the photoreactions of 19 nitroaromatic substances,
including 2,4-DNT and 2,6-DNT. The results observed indicate that dissolved humic substances
in natural waters enhance the sunlight-induced photodegradation rates (by 10-17 times)
compared with rates observed in distilled water. The half-life of 2,6-DNT in river water exposed
to sunlight was measured to be 12 minutes, and the degradation was determined to be from an
indirect photoreaction (Zepp et al., 1984).
Degradation of DNT by ozonation and chlorination was measured by Lee and Hunter (1985).
2,6-DNT was reduced by less than 17% with both chlorine and ozone, regardless of length of
contact time.
Microbial biodegradation of DNT in water has been observed under both aerobic and
anaerobic conditions. Biotransformation occurs mainly through the reduction of the nitrogroup
(Spanggord et al., 1981). Several studies have isolated microorganisms from DNT-contaminated
sites, which are capable of growth on 2,4-DNT and 2,6-DNT as their sole carbon and energy
source (ATSDR, 1998; Lewis et al., 2004; Spanggord et al., 1980). Data from studies cited in
Lewis et al. (2004) suggest that the initial biological breakdown of DNTs involves the activity of
dioxygenase enzymes, leading to electrophilic degradation by a monooxygenase enzyme on an
aromatic, nitro-substituted carbon atom.
Since microorganisms readily metabolize 2,6-DNT to CC>2 as the final product, it is not
expected to persist in the environment. Persistence is water-body dependent, and the half-life
ultimately depends on the compound's unique interaction with the natural organics and biota in
its surroundings. Spanggord et al. (1980) observed biodegradation in an aerobic environment
with a half-life of less than 1 hour. Complete degradation of 20 ppm of
2,6-DNT was observed in water samples taken downstream from the Radford Army Ammunition
Plant. Up to 60% of the substrate carbon appeared as CC>2 (Bausum et al., 1992). In another
study, 2,6-DNT was degraded to CO2 more slowly than 2,4-DNT . The conversion rate was
concentration dependent and increased with increasing concentration (Bausum et al., 1992). The
bacterium Burkholderia cepacia was isolated from the Radford Army Ammunition plant in West
17
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Virginia and can use 2,4-DNT as its sole source of carbon and nitrogen (Johnson et al., 2002). In
contrast, degradation was not observed in samples taken from Maryland surface freshwater
sources (Bausum et al., 1992).
Approximately 14% of 2,6-DNT was degraded by indigenous microorganisms in
microcosms (30 mL vials) prepared from contaminated aquifer material within 30 days (Bradley
et al., 1997). Following a 1-week acclimation period at a hydraulic residence time of 6 hours,
76% of the 2,6-DNT in the influent to a fluidized-bed biofilm reactor was degraded using a
mixed bacterial culture (Lendenmann et al., 1998).
Under anaerobic conditions, the half-life of 2,6-DNT in non-acclimated sewage was found to
be 28 days, with no loss of the compound under aerobic conditions during the same period
(Hallas and Alexander, 1983). In anaerobic conditions, three of six methanogens studied were
capable of degrading 55% to 95% of 2,6-DNT in an aqueous solution in 30 days; degradation
was not observed for the other three organisms (Boopathy, 1994). Biotransformation of DNT by
Pseudomonas aeruginosa isolated from a propellant wastewater treatment plant, was observed
under both aerobic and anoxic conditions. The primary products of the biotransformation, which
was mainly reductive, were 4-amino-2-nitrotoluene and 2-amino-4-nitrotoluene, with some 2,4-
diaminotoluene. DNT metabolites from acetylation of the arylaminos were identified, including
2-acetamide-2-nitrotoluene, 2-acetamide-4-nitrotoluene, 4-acetamide-2-aminotoluene, and 2,4-
diacetamidetoluene (Noguera and Freedman, 1996).
A study to identify and quantify the routes taken by 2,6-DNT in a model waste stabilization
pond (12-hour detention time) was conducted by Davis et al. (1981). The percentages of 2,6-
DNT lost by degradation, volatilization, sedimentation, water column residuals, and effluent
were 92.2, 0.3, 3.6, 1.2, and 2.7, respectively. The half-life was 8.3 days.
Dinitrotoluene Mixture
Studies indicate that DNT may be degraded through several mechanisms in the environment,
including photolysis, microbial biodegradation, ozonation, chlorination, and oxidation by strong
oxidants such as hydrogen peroxide, ozone, or oxone (potassium peroxomonosulfate) (ATSDR,
1998).
In the air, DNT is thought to break down by a variety of chemical reactions that take place
upon exposure to sunlight (ATSDR, 1998). In oxygenated waters, photolysis is probably the
major route of degradation of DNT (ATSDR, 1998). Based on its rapid photolysis in water, DNT
presumably is subject to oxidation of its methyl group, decarboxylation, ring oxidation, and/or
nitroreduction in air and sunlight (ATSDR, 1998). Degradation rates follow first-order kinetics
and are dependent on time, solution pH, and light intensity (Dillert et al., 1995).
18
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4.3 Bioaccumulation
2,4-Dinitrotoluene
2,4-DNT's relatively low log K0w (1-98) indicates that it is not expected to bioaccumulate in
animals (ATSDR, 1998; Callahan et al., 1979; Mabey et al., 1982). Calculated estimates of 2,4-
DNT's BCF were 7 (Meylan et al., 1999; HSDB, 2004a) and 204 calculated for guppy (Poecilia
reticulatd) (Deneer et al., 1987); the first estimate suggests a low potential for bioconcentration
in aquatic organisms while the second is an experimental value. Since DNT, however, is quite
soluble in water, it is expected to accumulate readily in plants via root uptake from soils
(ATSDR, 1998).
2,6-Dinitrotoluene
The relatively low log KOW of 2,6-DNT (1.72) indicates that 2,6-DNT in the environment
would not bioaccumulate in animals (ATSDR, 1998; Callahan et al., 1979; Mabey et al., 1982).
However, since DNT is quite soluble in water, it can be transferred to plants via root uptake from
soils and is expected to accumulate readily in plants (ATSDR, 1998). The structural analogy
with 1,3-dinitrobenzene and 4-nitrotoluene suggests that 2,6-DNT would be readily taken up by
plants (McFarlane et al., 1987; Nolt, 1988)., Davis et al. (1981) reported a bioconcentration
factor (BCF) of 5225 L/kg.
19
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20
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5.0 TOXICOKINETICS
5.1 Absorption
2,4-Dinitrotoluene
No experimental studies were identified that characterize how 2,4-DNT is absorbed in
humans and other animals. However, studies that demonstrate distribution, metabolism, and
excretion suggest that in both humans (Woollen et al., 1985) and animals (Medinsky and Dent,
1983; Lee et al., 1975, 1978; Mori et al., 1977, 1978), 2,4-DNT is absorbed rapidly, within 24
hours to 72 hours post-exposure or post-dosing.
2,6-Dinitrotoluene
No experimental studies were identified in the literature that characterize how 2,6-DNT is
absorbed in humans and animals. Studies suggest that humans absorb both DNT isomers
following dermal and inhalation exposure (Woollen et al., 1985; Medinsky and Dent, 1983; Lee
et al., 1975, 1978; Mori et al., 1977, 1978).
Dinitrotoluene Mixture
Absorption of Tg-DNT following oral exposure in humans has not been determined.
Occupational studies indicate that after inhalation or dermal exposure, Tg-DNT is absorbed by
humans and excreted in the urine. Absorption of DNT and excretion from the body occur rapidly
and are usually complete within 24 hours to 72 hours post-dosing. Percentage absorption of the
DNT isomers is difficult to estimate since biliary excretion is significant in most animals leading
to fecal excretion of a portion of the absorbed material. The amount of DNT reabsorbed from
bile also varies among species.
Two studies in which a total of 33 workers in an explosives factory were exposed to Tg-DNT
(20% 2,6-DNT) were published by Woollen et al. (1985). The authors suggest that the skin was
probably the primary route of exposure for these workers, and the lungs constituted the
secondary route because of low atmospheric levels of DNT (0.02 - 2.68 mg/m3). Blood levels of
DNT (2,4-DNT and 2,6-DNT combined) were low before the workday began (<10 ng/mL),
gradually increased during the exposure period (20-90 ng/mL), and peaked at the end of the
work shift (70-250 ng/mL). These data suggest that DNT is absorbed, is readily cleared from the
body, and does not tend to accumulate. However, the extent of DNT absorption by humans
cannot be determined from this study.
21
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5.2 Distribution
2,4-Dinitrotoluene
Oral administration of a single dose of 2,4-DNT in rats, mice, rabbits, and monkeys show
that both the unchanged compound and its metabolites distribute to the liver, kidneys, lung,
brain, skeletal muscle, blood, and adipose tissue (Lee et al., 1975; Rickert and Long, 1980; Mori
et al., 1977, 1978). Most tissues acquired between 0.36% and 1.6% of DNT and its metabolites
within 24 hours; the liver, kidneys, and lungs showed a preferential initial uptake. Very little 2,4-
DNT was retained in the single-dose studies after 24 hours (Lee et al., 1975, 1978). Results were
similar in dogs given multiple doses of 2,4-DNT for 5 days; however, tissue concentrations were
two to four times greater than those of single-dose studies, indicating that 2,4-DNT and/or its
metabolites can accumulate in the body (Lee et al., 1978). Schut et al. (1981, 1982) reported
similar tissue distribution in male mice given single intraperitoneal (ip) doses of 2,4-DNT,
except that blood and tissues reached peak levels quicker (30 minutes to 2 hours). There was no
indication in the data that any organ had preferential uptake.
Radioactivity was detected in the placenta and amniotic fluid of rats (strain not given)
administered a single oral dose of 14C-2,4-DNT on gestation day 20 (Rickert et al., 1980). The
investigators reported that approximately 10% to 50% of the 14C dose was recovered from these
two fractions; however, the time of sacrifice was not reported. The concentrations of 14C in fetal
tissues were reported to be similar to those in maternal tissues, but supporting data were not
provided.
2,6-Dinitrotoluene
Schut et al. (1983) examined the disposition of a single oral or ip dose of [3H]2,6-DNT in
male A/J mice. Animals were given 1-, 10-, or 100-mg/kg doses of the radiolabeled compound
(2.5 jiCi/mouse). Blood and liver levels of 3H were similar in orally dosed mice and remained
reasonably constant for the first 8 hours after dosing. In contrast, hepatic concentrations of
radioactivity in ip-dosed mice peaked during the first hour post-dosing and decreased steadily
thereafter. The amount of 3H in the blood was two to four times lower than that in the liver
through the first 2 hours after compound administration. Kidney 3H residue levels reportedly
were equivalent to those in the liver and showed no treatment-related differences (data not
provided). Uptake of 2,6-DNT in the small intestine peaked between 1 and 3 hours. The
maximum concentration was higher in orally dosed mice (9.5-16.9% of the administered 3H)
than in ip-dosed animals (5.2-8.9%). Lungs contained no more than 0.35% of the administered
dose for either group, and the levels of radioactivity in the brain, heart, and spleen remained low
throughout the experiment. Preferential tissue uptake was not apparent, but total recovery data
suggest that the 100-mg/kg dose may have been saturating for both routes of exposure.
22
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In a study by Ellis et al. (1980), only a small amount (<5%) of the radioactivity administered
to female CD rats was recovered from the carcass 24 hours after dosing. Each animal received,
by gavage, 10 |iCi of [ring-UL-14C]2,6-DNT at a dose equivalent to 1/10 of 50% of the lethal
dose (LD50) (~80 mg/kg).
5.3 Metabolism
Covalent binding of DNT to hepatic macromolecules may be particularly important due to
data showing a higher incidence of hepatic carcinomas and hepatic neoplastic nodules in DNT-
exposed male rats compared with treated female rats (Rickert et al., 1983; Swenberg et al.,
1983). Results of several studies indicate that conjugation, biliary excretion, microbial
metabolism in the gut, and intestinal reabsorption may be prerequisites to hepatic binding of
DNT. Hepatic binding may be greater for 2,6-DNT than for 2,4-DNT (Rickert et al., 1983), and
binding of DNT isomers appears to be lower in females than in males. Diet (i.e., as it affects
microbial activity and number) also may influence the degree to which binding of DNT
metabolites occurs.
Hartley et al., (1994) summarized DNT biotransformation from gastric absorption to urinary
excretion. After DNT is absorbed from the gastrointestinal tract, it is oxidized in the liver and
forms metabolites. Some metabolites are conjugated, which then allows them to be excreted in
urine or into bile. Metabolites that move from the bile to the gut are hydrolyzed and reduced by
intestinal microflora (e.g., Escherichia coli, from human intestines). Many of these compounds,
then, are reabsorbed from the gut into the systemic circulation and then oxidized in the liver.
Urinary elimination may occur next, but biliary excretion of these metabolites into the gut may
occur again, resulting in additional reduction by intestinal bacteria prior to elimination from the
body.
2,4-Dinitrotoluene
The effect on tissue uptake, following repeated administration of 2,4-DNT in the diet, was
examined in rats. Animals given both the treated and untreated (controls) diets were given a
single oral dose of 2,4-DNT after cessation of the feeding regimen. Ellis et al. (1979) found that
after 24 hours, tissue levels of 2,4-DNT were comparable in those that received the chemical in
feed for 20 months when compared with untreated controls. Mori et al. (1980) found slightly
lower levels of 2,4-DNT in the tissues of rats fed the chemical in the diet compared with those on
a standard diet. Both studies suggest that prior exposure to 2,4-DNT does not stimulate
metabolism.
Humans and experimental laboratory animals (i.e., rats, mice, rabbits, dogs, monkeys)
transform 2,4-DNT to metabolites that are excreted in urine or into bile. The types of metabolites
formed vary among species and include reduction, oxidation, acetylation, and glucuronidation
products (Turner, 1986; Shoji et al., 1985; Turner et al., 1985; Levine et al., 1985a; Woollen et
al., 1985; Schut et al., 1985; Medinsky and Dent, 1983; Mori et al., 1981a; Rickert et al., 1981;
Rickert and Long, 1981; Ellis et al., 1979; Lee et al., 1978). Some studies show that the
microsomal metabolism of 2,4-DNT involves cytochrome monooxygenases (e.g., P-450 and P-
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448) to produce compounds such as 2,4-diaminotoulene (2,4-DAT) (Bond and Rickert, 1981;
Mori et al., 1981b) and dinitrobenzaldehyde (2,4-DNBal) (Sayama et al., 1989a). A more
detailed summary of 2,4-DNTs microbial metabolism can be found in Hartley et al. (1994).
2,6-Dinitrotoluene
The metabolism of 2,6-DNT has not been studied extensively. The primary urinary
metabolites of 2,6-DNT excreted by individuals (14 men, 3 women) exposed occupationally to
Tg-DNT (0.05-0.59 mg/m3) were the corresponding 2-6 dinitrobenzyl alcohols (2,6-DNBalc)
and their glucuronides (2,6-DNBalcG), the corresponding dinitrobenzoic acids (2,6-DNBacid),
and metabolite (2-amino-6-nitrobenzoic acid (2A4NBacid) (Levine et al., 1985a; Turner, 1986;
Turner et al., 1985). Males excreted more DNBacid (2,4- and 2,6- combined) than females
(52.5% vs. 28.8% of all urinary metabolites, respectively); most of the DNBacid eliminated was
2,4-DNBacid (50.5% for men and 28.9% for women). In contrast, men excreted less of the
combined DNBalcG isomers than females (9.5% vs. 33.3%, respectively). Approximately 73.9%
of the urinary DNBalcG metabolites in females and 35.6% in males were the 2,6- isomeric
forms. Urinary excretion of 2,6-DNBacid was slightly higher in males (2.2-14.3% of all
metabolites) than in females (2.5%), and levels of 2,6-DNBalc were comparable between the
sexes (4.8-6.6%). The unchanged parent compound (i.e., 2,6-DNT) also was recovered from the
urine, and the hydrolyzed urine of one individual contained trace amounts of 4-amino-2-
nitrobenzoic acid (4A2NBacid) and 4-(N-acetyl)amino-2-nitrobenzoic acid. Reduction of both
nitro groups was not evident. Wide variations in urinary metabolite profiles were attributed to
differences in exposure and in the pathways by which an individual may metabolize DNT.
Very limited data on the metabolism of 2,6-DNT in mice were available. In a study by Schut
et al. (1983), male A/J mice were given a single oral or ip dose (1, 10, or 100 mg/kg) of
3H-labeled compound. No unchanged 2,6-DNT was recovered from the blood, liver, lungs, or
small intestine of animals given a 1-mg/kg dose by either route, and less than 2% of the 3H
recovered from the urine of these animals during the 8-hour post-dosing period was unchanged
parent compound. In contrast, unchanged 2,6-DNT was isolated in the tissues of animals in all
other groups, with the highest levels in high-dose mice and the slowest rate of disappearance of
unchanged 2,6-DNT in orally dosed animals. The data indicate that 2,6-DNT is rapidly and
extensively metabolized by mice following oral or ip dosing. The liver and intestines appear to
be the primary sites for the metabolism of 2,6-DNT in mice.
Three metabolites accounted for about 95% of the urinary 14C excreted by male and female
Fischer 344 rats given a single oral dose of [ring-UL-14C]-2,6-DNT: 2,6-DNBacid, 2,6-
DNBalcG, and 2A6NBacid) (Long and Rickert, 1982). In males, these metabolites accounted for
about 21, 22, and 14% of the 14C dose, respectively; in females, the corresponding values were
20, 19, and 11%.
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The primary metabolite of 2,6-DNT produced by hepatocytes under aerobic conditions from
male A/J mice and male Fischer 344 rats was 2,6-DNBalc; most (61.3-70.9%) was conjugated
(Dixit et al., 1986). In another study, three metabolites of 2,6-DNT (2,6-DNBacid, 2,6-
DNBalcG, and 2A6NBacid) were recovered from the bile and liver perfusate of male and female
Fischer 344 rats (Long and Rickert, 1982).
The role of gut microflora in the metabolism of 2,6-DNT has been examined in vivo and
in vitro. In a study by Mori et al. (1984), Escherichia coli isolated from human intestines
converted 2,6-DNT to 2-amino-6-nitrotoluene (2A6NT) via the corresponding
hydroxylaminonitrotoluenes. Nitrosotoluenes, however, were not recovered in any human
samples (Mori et al., 1984). Microflora in the cecal contents of male A/J mice and male Fischer
344 rats anaerobically converted [3H]2,6-DNT to 2A6NT, 2-acetylamino-6-nitrotoluene
(2Ac6NT), and 2,6-diaminotoluene (2,6-DAT) (Dixit et al., 1986). Most of the parent compound
(82.1-88.7%) was recovered as unchanged 2,6-DNT at the end of the 30-minute incubation
period.
Dinitrotoluene Mixture
Following gastrointestinal absorption, DNT isomers undergo oxidation in the liver.
Metabolites generated from this reaction are often conjugated with sulfate or glucuronate and
subsequently excreted in urine or into bile. Metabolites that are transported from the bile to the
gut are hydrolyzed and reduced by intestinal microflora. Many of these compounds, in turn, are
reabsorbed from the gut into the systemic circulation and then oxidized in the liver. Urinary
elimination may occur next, but biliary excretion of these metabolites into the gut results in
additional reduction by intestinal bacteria prior to elimination from the body.
Several investigators conclude that, in humans exposed occupationally to Tg-DNT,
metabolism is similar to that in DNT-exposed Fischer rats (Levine et al., 1985a; Turner, 1986;
Turner et al., 1985). The primary urinary metabolites formed include dinitrobenzyl alcohols and
their glucuronides, the corresponding dinitrobenzoic acids, and 2-amino-4-nitrobenzoic acid
(2A4NBacid). Sex-related differences in the metabolism of 2,4-DNT have been observed only in
Fischer rats and humans (Levine et al., 1985a; Turner, 1986; Turner et al., 1985). Females
produce up to three times more 2,4-DNBalc and/or 2,4-DNBalcG than males, and in humans,
men excrete almost double the 2,4-DNBacid as women.
Woollen et al. (1985) found that the primary urinary metabolite of workers exposed to Tg-
DNT was 2,4-DNBacid. Other urinary metabolites isolated were 2A4NBacid, 4A2NBacid,
2A6NBacid, and 4Ac2NBacid. The urine of these workers contained no 2,4-DNBalc or 2,6-
DNBalc.
Mori et al. (1989) supported the finding of Sayama et al. (1989b) that dinitrobenzaldehyde is
a metabolite of DNT and that the metabolism of DNT isomers is strain dependent, which was
evident in Wistar and Sprague-Dawley rats.
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5.4 Excretion
Absorption of DNT and excretion from the body occur rapidly and are usually complete
within 24 hours to 72 hours post-dosing. Elimination via feces or lungs has not been examined in
humans. In explosives workers, urinary excretion of Tg-DNT's primary metabolite (2,4-
DNBacid) was highest at the end of the work shift and generally was much greater at the end of
each workweek than at the beginning. This suggests that DNT is cleared from the body readily
and does not accumulate (Woollen et al., 1985). Elimination half-lives for all five DNT
metabolites excreted in the urine of three workers exposed to 0.05-0.59 mg Tg-DNT/m3 were
between 0.88 hour and 2.76 hours (Turner, 1986; Turner et al., 1985). Values for individual
metabolites were between 0.80 and 4.26 hours. The urine is the primary route of elimination for
both DNT isomers in most animals, including rodents, rabbits, dogs, and monkeys.
2,4-Dinitrotoluene
2,4-DNT is eliminated rapidly in humans through urine and in laboratory animals through
feces and urine. Information concerning the elimination of 2,4-DNT from humans via feces or
lungs was not found in the literature. Woollen et al. (1985) estimated that the half-life for the
urinary elimination of 2,4-DNT was between 2 hours and 5 hours. Low, but detectable, levels of
2,4-DNBacid were found in the urine 3 days after exposure, suggesting that urinary elimination
of DNT is biphasic (Turner, 1986; Turner et al., 1985; Woollen et al., 1985).
In animals, the primary elimination route varies by species or strain. In Fischer 344 and CD
rats, rabbits, dogs, and monkeys, urine is the primary excretion route, and up to 90% is
eliminated within 24 hours to 72 hours (Rickert et al., 1981, 1984; Ellis et al., 1979, 1985; Lee et
al., 1975, 1978). In CD-I and female B6C3F1 mice (Lee et al., 1978) and in Wistar rats (Mori et
al., 1977), however, up to 84% of administered 2,4-DNT was eliminated in feces between 24
hours and 7 days. Similar results were observed in male AJ mice given ip doses of 2,4-DNT
(Schut et al., 1982, 1985). Rickert et al. (1981) observed that axenic (lacking intestinal bacteria)
male Fischer 344 rats eliminated significantly (p<.05) less 2,4-DNT in urine and feces,
suggesting that metabolism by gut flora may have a role in excretion. The only report of
elimination of 2,4-DNT by exhalation was noted in a study with A/J mice, where only 0.20%
was detected in the breath.
2,6-Dinitrotoluene
Orally administered 2,6-DNT was eliminated primarily via urine. Male AJ mice excreted
about 54, 54, and 49% of a single oral dose of 1, 10, or 100 mg [3H]2,6-DNT/kg, respectively, in
urine within 8 hours post-dosing (Schut et al., 1983). Feces contained no more than 2.1% of the
3H dose, and levels in the small intestine accounted for about 3.0% to 3.6%. Elimination via the
lungs was negligible (<0.35%).
Twenty-four hours following administration of a single oral dose of uniformly 14C labeled
ring [ring-UL-14C]2,6-DNT (80 mg/kg) to female CD rats, about 60% of the 14C was recovered
from urine, and 40% was recovered from feces and gastrointestinal contents (Ellis et al., 1980;
^f\
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Lee et al., 1975). Long and Rickert (1982) reported that male and female Fischer 344 rats
eliminated about 54% of a single oral dose of 14C-labeled 2,6-DNT (10 mg/kg, 2 mCi/mmol) in
the urine within 72 hours after dosing; feces contained about 20%. No sex-related differences
were noted. The authors reported that urinary excretion of 14C was complete within 24 hours, but
that fecal elimination was still evident at 72 hours.
A major route of elimination of 2,6-DNT is biliary excretion. For example, female CD rats
excreted about 25% of a single oral dose of [ring-UL-14C]2,6-DNT into bile 24 hours after
dosing (Ellis et al., 1980; Lee et al., 1978). The rate of biliary excretion of 14C peaked at 6 hours
after dosing compared with 2 hours in female CD rats administered 2,4-DNT (Lee et al., 1978).
Long and Rickert (1982) reported that total recovery of 14C-labeled 2,6-DNBalcG from liver
perfusate and bile was comparable for both male and female CD rats when livers were incubated
with 20 jiM [ring-UL-14C]2,6-DNT. At 70 jiM, however, total recovery of this metabolite in the
bile was significantly less (p<.05) in females than in males. These data indicate a possible
saturation point in the metabolism and excretion of 2,6-DNT in females. Biliary flow rates were
similar for both sexes, and disappearance of parent compound (20 jiM or 70 jiM) from the
perfusate was biphasic: half-times of elimination were 7.5 minutes and 8.4 minutes for males and
females, respectively, for the initial phase, and 53.9 minutes and 52.7 minutes, respectively, for
the second phase.
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6.0 HEALTH EFFECTS DATA
6.1 Human Studies
In humans, the toxic effects of 2,4-DNT or 2,6-DNT are on the central nervous system
(CNS) and also may be on the heart and circulatory system.
6.1.1 Short-Term Exposure
Reports of short-term exposure of 2,4-DNT or 2,6-DNT on humans were not located in the
available literature.
6.1.2 Long-Term Exposure
Chronic DNT exposure, primarily via the inhalation route, is characterized in munitions
workers by nausea, vertigo, methemoglobinemia, cyanosis, pain or paresthesia in extremities,
tremors, paralysis, chest pain, and unconsciousness (Etnier, 1987; Levine et al., 1985b; Ellis et
al., 1979). Following a latency period of 15 years, workers exposed to 2,4-DNT and Tg-DNT
exhibited excessive mortality from ischemic heart disease and residual diseases of the circulatory
system (Levine et al., 1986a,b).
Letzel et al. (2003) performed a cross-sectional study of 82 employees from a munitions
dismantling mechanical plant in the Free State of Saxony, Germany. The workers were exposed
to TNT and DNT regularly (51 persons) or occasionally (19) or were unexposed (12) for a
median period of 59 months. Air analyses yielded maximum concentrations of 20 ug/m3 for 2,4-
DNT and 3,250 ug/m3 for 2,4,6-TNT, respectively. In 63 workers where TNT, DNT, and/or their
metabolites were detected in their urine, workers frequently reported symptoms such as bitter
taste, burning eyes, and discoloration of skin and hair.
6.1.3 Reproductive and Developmental Effects
Limited evidence suggests that neither 2,4-DNT, 2,6-DNT, nor the DNT mixture causes
adverse effects on human reproductive performance (Hamill et al., 1982; Ahrenholz and Meyer,
1982).
6.1.4 Carcinogenicity
An epidemiology study was performed by Stayner et al. (1993) to evaluate the relationship
between DNT exposure and increased risk of cancers of the liver and biliary tract. The study
included a total of 4,989 white male workers exposed to DNT and 7,436 white male unexposed
workers who had worked for at least 5 months at the Radford Army Ammunition Plant between
January 1, 1949, and January 21, 1980. Women and non-Caucasian employees were a small
percentage of the worker population and therefore were excluded from the analysis. Workers
were considered exposed if they had worked at least 1 day on a job with probable exposure to
DNT; however, exposure data were not available. An excess of hepatobiliary cancer was
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observed among workers exposed to DNT, but the increase was not statistically significant
compared with the general population.
Briining et al. (1999, 2001, 2002) investigated the carcinogenicity of DNT on the urinary
tract of underground mining workers. Between 1984 and 1997, 6 cases of urothelial cancer and
14 cases of renal cell cancer were diagnosed in a group of 500 underground mining workers.
They worked in the copper mining industry of the former German Democratic Republic (GDR)
(East Germany) and had high exposures to explosives containing Tg-DNT. The incidences of
both urothelial and renal cell tumors in this group were 4.5 and 14.3 times higher, respectively,
than anticipated on the basis of the cancer registers of the GDR. A group of 161 miners highly
exposed to DNT was investigated for signs of subclinical renal damage. Biomarker excretion
(alphai-microglobulin, glutathione S-transferases a and TI) indicated that DNT-induced damage
was directed toward the tubular system. The authors indicate that their observations appear
consistent with the concept of cancer initiation by DNT isomers and the subsequent promotion of
renal carcinogenesis by selective damage to the proximal tubule. The differential pathways of
metabolic activation of DNT appear to apply to the proximal tubule of the kidney and to the
urothelium of the renal pelvis and lower urinary tract as target tissues of carcinogenicity.
These studies are limited by a variety of determinants including inadequate exposure
information (i.e. concentrations and duration). Confounding information on exposure to other
chemicals and lifestyles factors such a smoking behavior was also not reported.
6.2 Animal Studies
There are several short- and long-term animal studies with 2,4-DNT and 2,6-DNT that
demonstrate acute, subchronic, and chronic adverse effects. Both DNT isomers cause adverse
neurological, hematological, reproductive, hepatic, and renal effects in rats, mice, and dogs.
Dogs generally are the most sensitive of the three species.
6.2.1 Short-Term Exposure
studies indicate that both 2,4-DNT and 2,6-DNT are moderately to highly toxic to rats
and mice (Hartley et al., 1994). Times to death, recovery, and gross pathology were similar for
both isomers (Lee et al., 1975; Vernot et al., 1977).
2, 4-Dinitrotoluene
Acute oral toxicity studies indicate that rats are more susceptible to 2,4-DNT than mice. The
LDso values ranged from 1,340 mg/kg to 1,954 mg/kg in mice and from 270 to 650 mg/kg in rats
(Lee et al., 1975; Vernot et al., 1977). Both species exhibited ataxia and cyanosis.
Lane et al. (1985) orally administered 2,4-DNT (purity not specified) daily in corn oil to
groups of 10 male Sprague-Dawley rats by gavage as part of a study of reproductive toxicity.
The dose levels were 0, 60, 180, or 240 mg/kg/day for 5 consecutive days. High mortality was
observed in the high-dose group; therefore, another group of 15 rats was started at
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240 mg/kg/day on the same schedule; 8 of the 15 animals in this group died within 2 weeks after
receiving the first dose. No other deaths were seen. Rats at the mid- and high-dose levels
exhibited cyanosis, while BW loss was seen at the high dose.
Groups of 10 female CD-I mice were given oral doses of 2,4-DNT (purity not specified) by
gavage in corn oil daily for 8 consecutive days (Smith, 1983). Dose levels were 0, 310, 525,
1,250, 2,500, or 3,500 mg/kg/day BW. There was 100% mortality in all groups receiving > 1,250
mg/kg/day and 60% mortality in the 525-mg/kg/day group. No treatment-related mortality was
seen at the low dose. The survivors of the 525-mg/kg/day group had significantly (p<.007) lower
mean BW and exhibited toxicity characterized by lethargy, dyspnea, rough hair coat, hunched
posture, tilted head, tremors, ataxia, and prostration. The no observed adverse effect level
(NOAEL) was 310 mg/kg/day.
As reported by Hartley et al. (1984), Sprague-Dawley rats (5/sex/dose) were fed diets
containing 0, 900, 1,200, 1,900, or 3,000 mg of 2,4-DNT kg/day for 14 days (McGown et al.,
1983). The chemical administered contained 97% 2,4-DNT, 2% 2,6-DNT, and 1% unspecified
contaminants. The intake was 0, 97, 126, 180, or 257 mg/kg/day 2,4-DNT for males and 0, 96,
121, 186, or 254 mg/kg/day for females. Body weight gain and food consumption were
decreased in a dose-related manner in males and females. A number of serum chemistry
parameters (cholesterol, glucose, alanine aminotransferase) were elevated significantly in dosed
males and/or females. Hyaline droplets were found histologically in the epithelium of the
proximal convoluted tubules of the kidneys of all dosed rats, but lacked a dose-response trend;
the males appeared to be more susceptible than females. Oligospermia was found in a dose-
related manner in males, with accompanying degenerative changes of the testes. Based on
decreased BW gain, decreased food consumption, and changes in serum chemistry levels in
females, the lowest observed adverse effect level (LOAEL) was 96 mg/kg/day.
Groups of 10 male Sprague-Dawley rats were fed a diet containing 0, 0.1, or 0.2% 2,4-DNT
(equivalent to 50 or 100 mg/kg/day, based on Lehman [1959]) for 3 weeks as part of a study of
reproductive toxicity (Bloch et al., 1988). The final BWs were significantly lower (p<.01) in
treated animals at both dietary levels compared with controls. No systemic toxicity was seen.
In a mouse study (8/sex/dose), groups of animals were fed diets containing 0, 0.07, 0.20, or
0.70% 2,4-DNT (98% pure) daily for 4 weeks (Lee et al., 1978). This represents daily intakes of
0, 47, 137, or 413 mg/kg/day for males and 0, 52, 147, or 468 mg/kg/day for females (U.S. EPA,
1986). No mortality occurred in the mice. The low- and mid-dose levels were nontoxic, and mice
at the high-dose level showed slight BW loss. No abnormalities were seen in blood parameters,
organ weights, or gross pathology. Histopathology revealed a mild depression of
spermatogenesis in two males at the high dose. After 4 weeks, the mice recovered. The NOAEL
was 137 mg/kg/day in males and 147 mg/kg/day in females. Based on BW loss in males and
females and depression of spermatogenesis in males, the LOAEL was 413 mg/kg/day in males
and 468 mg/kg/day in females.
In the companion study, Lee et al. (1978) fed the same DNT concentrations (and purity) in
feed to groups of rats (8/sex/dose), where the corresponding daily intakes were 0, 34, 93, or 266
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mg/kg/day for males and 0, 38, 108, or 145 mg/kg/day for females (U.S. EPA, 1986).
2,4-DNT was toxic to both sexes at all levels. At the high dose, two males and two females died;
the surviving rats showed BW loss and decreased food consumption. Rats dosed at the lower
levels exhibited a slight depression in BW gain and food consumption. No consistent changes
were observed in hematology or clinical chemistry parameters. A significant (p<.05) increase
was seen in both the absolute and relative liver weights of males fed 93 mg/kg/day and females
fed 38 or 145 mg/kg/day. Histopathology revealed mild to moderate hemosiderosis in the spleen
of males fed 93 or 266 mg/kg/day and females fed 108 or 145 mg/kg/day. Males at the high dose
(266 mg/kg/day) showed aspermatogenesis and testicular atrophy. After 4 weeks, there was only
partial recovery; rats regained the BW lost, but the hemosiderosis and testicular lesions were not
reversible. Based on BW loss and decreased food consumption in both sexes, the LOAEL was 34
mg/kg/day in males and 38 mg/kg/day in females; a NOAEL was not established.
In a dog study, Lee et al. (1978) gave groups of two males and two females 2,4-DNT in
capsules at doses of 0, 1, 5, or 25 mg/kg/day for 4 weeks. No treatment-related toxicity was
observed in dogs given 1 or 5 mg/kg/day. At the high dose (25 mg/kg/day), dogs showed signs of
toxicity, including decreased food consumption, BW loss, yellow stain on and near hind legs,
pale gums, neuromuscular incoordination, and paralysis. Histopathology of the high-dose dogs
revealed hemosiderosis in the liver, cloudy swelling and tubular degeneration of the kidneys, and
lesions of the brain and spinal cord in both sexes. Males exhibited aspermatogenesis. After 4
weeks, the animals partially recovered, and two kept for 8 months recovered completely. Based
on decreased BW gain, decreased food consumption, neurotoxic signs, and histopathology, the
LOAEL was 25 mg/kg/day, and the NOAEL was 5 mg/kg/day.
2,6-Dinitrotoluene
The oral LDso values for 2,6-DNT ranged from 621 to 1,000 mg/kg in mice and from 180
mg/kg to 795 mg/kg in rats. The acute toxicity of 2,6-DNT appears to be less species specific
than 2,4-DNT. Male rats were slightly less tolerant than females (Lee et al., 1975; Vernot et al.,
1977).
Lee et al. (1976) fed 2,6-DNT (>99% pure) in the diet to mice (8/sex/dose) for 4 weeks at
levels of 0.01, 0.05, or 0.25%, equivalent to a daily intake of 0, 11,51, or 289 mg/kg/day for
males and 0, 11, 55, or 299 mg/kg/day for females. No treatment-related effects were observed in
the 11-mg/kg/day dose group. No treatment-related deaths occurred in any dose group. The mid-
and high-dose levels caused decreased BW gain and decreased food consumption. The high-dose
males and females exhibited extramedullary hematopoiesis in the spleen and the liver. High-dose
males developed aspermatogenesis and testicular atrophy that reversed 4 weeks after treatment
was discontinued. The NOAEL was 11 mg/kg/day for males and females, based on decreased
BW gain and decreased food consumption.
In a similar study, Lee et al. (1976) fed 2,6-DNT (>99% pure) in the diet to rats (8/sex/dose)
for 4 weeks at levels of 0.01, 0.05, or 0.25%, equivalent to a daily intake of 0, 7, 35, or 145
mg/kg/day for males and 0, 7, 37, or 155 mg/kg/day for females. No treatment-related deaths
were observed in rats fed 2,6-DNT. The rate of BW gain was decreased in a dose-related manner
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in treated males and females but was not significantly different from controls. Food consumption
was also depressed in a dose-related manner. Histopathology revealed hematopoiesis in the
spleen and liver of both sexes and degeneration of spermatogenesis in males. The LOAEL was
the lowest dose tested, 7 mg/kg/day for both sexes, based on decreased BW gain and decreased
food consumption.
Dogs were given doses of 0, 4, 20, or 100 mg/kg/day 2,6-DNT in capsules for 4 weeks by
Lee et al. (1976). There were no signs of toxicity at 4 mg/kg/day, but the 20- and 100-mg/kg/day
groups had BW loss and reduced food consumption. The affected animals showed listlessness,
incoordination, lack of balance, pale gums, dark urine, and hind limb weakness. They also were
anemic, with decreased hematocrit, decreased hemoglobin concentration, and compensatory
reticulocytosis. Histopathology revealed extramedullary hematopoiesis in the liver and spleen
and bile duct hyperplasia in both sexes. There also was decreased spermatogenesis in males.
After 4 weeks of recovery, the dogs showed some improvement, with lesser amounts of
extramedullary hematopoiesis and testicular lesions, and two high-dose dogs that were allowed
to recover for 19 weeks showed complete recovery. The NOAEL was 4 mg/kg/day, based on
decreased BW gain and decreased food consumption in both sexes.
Dinitrotoluene Mixture
Reports of LDso studies with Tg-DNT or any other mixture in animals were not located in the
available literature.
The Chemical Industry Institute of Toxicology (CUT, 1977) conducted a 30-day toxicity
study in rats with Tg-DNT. Groups of Fischer 344 rats (10/sex/dose) were fed diets containing
0, 37.5, 75, or 150 mg/kg/day for 30 days. There were no deaths during the treatment period.
Urine stains on the fur were seen in six rats in the high-dose group. Two females in the mid-dose
group developed alopecia around the eyes; no clinical signs were observed at the low dose. The
mean BWs of females in the high-dose group and of males in all groups were significantly
(p<.05) lower than those of the controls and were most severe in rats fed the high-dose diet. A
significant (p<.05), dose-related increase in mean values was observed for methemoglobin,
reticulocyte counts, and Heinz body formation, with the exception of mean percentage
methemoglobin for females in the mid-dose group. Methemoglobin values for females in the
low-dose group and for males and females in the high-dose group were significantly
(p<.05) higher than the control values. Treatment-related gross pathological alterations seen in
rats in the high-dose group included discoloration, enlargement, and irregular surfaces of the
spleen in both sexes and discoloration of the kidneys in males. In addition, males at all dietary
levels had livers with discolorations and/or surface irregularities. Based on decreased BW gain
and decreased food consumption, blood effects, and gross pathological changes in males, the
LOAEL was 37.5 mg/kg/day, the lowest dose tested.
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6.2.2 Dermal/Ocular Effects
Lee et al. (1975) reported that neither 2,4-DNT nor 2,6-DNT produced ocular irritation when
instilled as a 50% paste in peanut oil into the eyes of groups of six New Zealand white rabbits.
As a result of primary skin irritation tests to New Zealand white rabbits (sex not reported),
2,4-DNT (98% pure) and 2,6-DNT (>99% pure), as a 50% paste with peanut oil, were classified
as very mild skin irritants (Lee et al., 1975).
6.2.3 Long-Term Exposure
2,4-Dinitrotoluene
Groups of CD rats (16/sex/dose) were fed diets of 2,4-DNT (98% pure) for up to 13 weeks,
at intake levels of 0, 34, 93, or 266 mg/kg/day for males and 0, 38, 108, or 145 mg/kg/day for
females (Lee et al., 1978; Ellis et al., 1985). Four animals/sex/group were sacrificed at 4 and 13
weeks after being returned to normal diets for 1 month. All high-dose females died within 3
weeks. One male in the mid-dose group and 6 in the high-dose group died between weeks 4 and
13. All surviving animals exhibited dose-dependent decreases in BW gain. Orange to yellowish
urine stains were observed on the fur of high-dose rats, and one male had widespread and stiff
hind legs. Mid- and high-dose animals of both sexes were anemic, characterized by decreases in
erythrocyte count, hematocrit, and hemoglobin and concurrent reticulocytosis. Absolute liver
and kidney weights were slightly increased in mid-dose males, and relative weights of these
organs were significantly increased. There was splenic hemosiderosis in mid- and high-dose
males and females. Spermatogenesis was decreased in mid-dose males and completely arrested
in high-dose males. One high-dose male showed some signs of neuromuscular effects with
demyelination in the cerebellum and brain stem. The LOAEL was 34 mg/kg/day based on
decreased BW gain and decreased food consumption in males. There was no NOAEL because
effects occurred at all doses tested.
In a longer, but limited (one-dose) study, Kozuka et al. (1979) reported similar effects
observed in a group of 20 male Wistar rats given 0.5% 2,4-DNT (purity not reported) in feed for
a period of 6 months. The estimated dose rate was 190 mg/kg/day for the first month and
214 mg/kg/day for the last 3 months. A total of 12 animals died before the end of treatment.
Adverse neurological effects included piloerection, whitened skin color, humpback, jerky
movements, decreased spontaneous movement, and general weakness. Toxic effects included
decreased BW (58%), decreased BW gain, and significantly (p<.01) increased relative weights
of the liver, spleen, and kidney.
Relative testicular weights were significantly lower. Methemoglobinemia was increased
significantly (p<.01) in treated animals compared with controls, and there were significant
(p<.01) differences in serum components (triglycerides, glucose, albumin, and albumin/globulin
ratios) and serum enzymes (aspartate aminotransaminase, alanine aminotransferase, alkaline
phosphatase, and acid-phosphatase). Gross pathology showed hepatic hypertrophy and testicular
atrophy. No histopathology was conducted.
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In a separate study (Lee et al., 1978; Ellis et al., 1985), CD-I mice (16/sex/dose) were fed
diets of 2,4-DNT (98% pure), with intakes equivalent to 0, 47, 137, or 413 mg/kg/day for males
and 0, 52, 147, or 468 mg/kg/day for females for 13 weeks. Five mice died during the study—
one low-dose male, two high-dose males, and two high-dose females. The males exhibited a
dose-dependent decrease in BW. The high-dose groups of both sexes were anemic (decreased
erythrocyte count, decreased hematocrit, and decreased hemoglobin) with concurrent
reticulocytosis, mild hepatocellular dysplasia, and Kupffer cell dysplasia. High- and mid-dose
males had mild degeneration of the seminiferous tubules or testicular degeneration. After
4 weeks off treatment, the mice recovered completely. The LOAEL was 47 mg/kg/day, based on
BW loss in males. There was no NOAEL because effects occurred at all doses tested.
Beagle dogs (2/sex/dose) were exposed to 2,4-DNT in capsules at doses of 0, 1, 5, or 25
mg/kg/day for 13 weeks (Lee et al., 1978; Ellis et al., 1985). No treatment-related findings were
observed in the mid- and low-dose groups. Mortality was observed after 22 days in the high-dose
group. There was great variation in individual susceptibility in the high-dose group. All affected
dogs exhibited decreased food consumption, BW loss, urine stains on the fur, pale gums,
neuromuscular incoordination, and paralysis. Hematological indices showed
methemoglobinemia, anemia, and Heinz bodies. The dogs were in fair to poor nutritional
condition, with little or no body fat. Histologically, there was hemosiderosis in the liver and
spleen, cloudy swelling of the kidneys in males and females, and aspermatogenesis in males.
Dogs sacrificed during weeks 6 and 7 had brain lesions characterized by gliosis, edema, and
demyelination of the cerebellum, spinal cord, and brain stem. Dogs were retained for 4 weeks
without exposure to 2,4-DNT, and partial recovery from the various effects was observed. The
LOAEL was 25 mg/kg/day, based on BW loss, hematological abnormalities, neurological signs,
and histopathology. The NOAEL was 5 mg/kg/day.
Leonard et al. (1987) gave 24 male CDF Fischer 344/CrlBR rats 27 mg/kg/day of 2,4-DNT
(>99.4% pure) in the diet for 12 months, with a 6-month interim sacrifice of four rats. BW gain
increased significantly (p<.05), and liver weight increased approximately 150% compared with
control animals. Most of the animals exhibited liver histopathology characterized by hepatocyte
degeneration and vacuolation and acidophilic and basophilic foci. Bile duct hyperplasia and a
highly variable incidence of cholangiofibrosis occurred in the majority of the treated animals.
The National Cancer Institute (NCI) (1978) conducted a study with Fischer 344 rats
(50/sex/dose) that were fed diets containing 0, 0.008% (80 ppm), or 0.02% (200 ppm) 2,4-DNT
(>95% pure) for 78 weeks. Applying estimates by Lehman (1959), which assume that daily food
consumption by rats is approximately 5% of their BW, the 2,4-DNT equivalent doses were 0, 4,
or 10 mg/kg/day, respectively. Upon termination of dosing, the animals were observed for
another 26 weeks. The only significant clinical observation was that high-dose males and
females had mean average BWs that were 25% and 18%, respectively, lower than those of
controls. The incidence and variety of nonneoplastic lesions in the major organs were similar in
control and treated rats.
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CD (Sprague-Dawley) rats (38/sex/dose) were fed 2,4-DNT (98% pure) in the diet for up to
2 years (Ellis et al., 1979; Lee et al., 1985). The intake of 2,4-DNT was 0, 0.57, 3.9, or 34
mg/kg/day for males and 0, 0.71, 5.1, or 45 mg/kg/day for females. There was an interim
sacrifice (8/sex/group) after!2 months. In both sexes of high-dose rats, lifespan was shortened
where there was 50% mortality by month 20; the same rate did not occur in controls until month
23. BW gains were reduced significantly (30-40%) in high-dose animals compared with controls.
After 12 months of exposure, severe atrophy of the seminiferous tubules occurred in a dose-
related manner in 16%, 26%, 33%, and 81% of the controls and low-, mid-, and high-dose
groups, respectively. In some high-dose males, the atrophy caused almost complete
aspermatogenesis. The spleen developed excessive pigmentation, and anemia and reticulocytosis
occurred in mid- and high-dose males and in high-dose females after 12 months. The LOAEL
was 34 mg/kg/day, based on seminiferous tubules effects in the males. The NOAEL was 3.9
mg/kg/day.
In a study of CD-I mice (38/sex/dose), animals were fed (98% pure) 2,4-DNT up to
24 months (Ellis et al., 1979; Hong et al., 1985). The dose levels were 0, 14, 95, or 898
mg/kg/day. All the animals fed 898 mg/kg/day died by month 18 (males) or month 21 (females).
Effects at 14 mg/kg/day, the lowest dose tested, included testicular atrophy, decreased BW in
males, and hemosiderosis of many organs, primarily the liver and spleen. The incidence of
malignant renal tumors was elevated in males fed 95 mg/kg/day (15/17 compared with
0/20 concurrent controls).
B6C3F1 mice (50/sex/dose) were given 2,4-DNT in feed at 0, 0.008, or 0.04% (0, 80, or
400 ppm, respectively) for 78 weeks, followed by 13 weeks without treatment (NCI, 1978).
Following consumption estimates similar to the rat study, where daily food consumption by mice
was approximately 15% of their BW (Lehman, 1959), the doses were 0, 11, or 57 mg/kg/day,
respectively. BW gain depression decreased significantly in all treatment groups. At the end of
the study, BW gain was depressed by 9% and 11% for low- and high-dose males, respectively;
for females BW gain depression was 18% and 24%, respectively.
Beagle dogs (6/sex/dose) were fed 2,4-DNT (98% pure) in gelatin capsules at 0, 0.2, 1.5, or
10 mg/kg/day up to 24 months (Ellis et al., 1979, 1985). In the 10-mg/kg/day group, four of the
six males were sacrificed due to moribund conditions by study week 19 after exhibiting
progressive paralysis. The high-dose animals displayed incoordination and paralysis within
6 months of study initiation and during month 16 in one dog receiving 1.5 mg/kg/day.
Histopathology of the CNS confirmed treatment-related lesions, which included vacuolization,
endothelial proliferation, and gliosis of the cerebellum. In dogs fed 1.5 and 10 mg/kg/day, there
was methemoglobinemia, with associated reticulocytosis and Heinz body formation. There also
was biliary tract hyperplasia and pigmentation of the gallbladder, kidneys, and spleen. The
hematologic effects were minimal during year 2, presumably due to an adaptive response. No
males had testicular effects. The LOAEL in this study was 1.5 mg/kg/day based on neurotoxicity
and the presence of Heinz bodies and biliary tract hyperplasia. The NOAEL was 0.2 mg/kg/day.
2,4- and 2,6-Dinitrotoluene - January 2008
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2,6-Dinitrotoluene
Lee et al. (1976) conducted subchronic toxicity studies of 2,6-DNT (>99% pure) in CD-I
mice, CD rats, and beagle dogs. The basic experimental design and procedures were similar to
those described above in studies conducted with 2,4-DNT.
CD rats were fed diets containing 0, 0.01, 0.05, or 0.25% 2,6-DNT for 13 weeks (Lee et al.,
1976). The dose of 2,6-DNT was equivalent to 0, 7, 35, or 145 mg/kg/day for males and 0, 7, 37,
or 155 mg/kg/day for females. No adverse effects were observed in the low-dose group. Both
mid-dose males and females had decreases in BW gain and food consumption. They also
exhibited extramedullary hematopoietic activity in the liver and spleen and bile duct hyperplasia.
The males also developed depressed spermatogenesis and testicular atrophy. The high-dose
animals developed a variety of adverse conditions, including decreases in BW, BW gain, and
food consumption. They exhibited various hematological conditions such as
methemoglobinemia, Heinz body formation, anemia, compensatory reticulocytosis, and severe
extramedullary hematopoiesis in the spleen and liver. There was bile duct hyperplasia and renal
degeneration in both sexes. By the end of 13 weeks, testicular lesions in males had progressed to
encompass virtually all connective tissues. After 4 weeks, only partial recovery was observed;
rats regained the BW lost, but lesions continued to occur in the spleen, liver, and testes of treated
rats. The LOAEL was 35 mg/kg/day in males and 37 mg/kg/day in females, based on BW loss,
hematological effects, and histopathology. The NOAEL was 7 mg/kg/day for both sexes.
Mice also were fed diets containing 0, 0.01, 0.05, or 0.25% 2,6-DNT for 13 weeks (Lee et
al., 1976). Daily intake was 0, 11,51, or 289 mg/kg/day for males and 0, 11, 55, or 299
mg/kg/day for females. The low-dose group did not show any treatment-related effects, and only
a few animals in the mid-dose group had BW loss. The higher doses of 2,6-DNT caused
decreases in BW gain and food consumption, hematopoiesis in the liver and/or spleen, and bile
duct hyperplasia in both sexes. Males developed testicular atrophy and depression of
spermatogenesis. There was partial recovery of some of the adverse effects after a 4-week
recovery after removal of 2,6-DNT from the diet; however, hematopoiesis continued in the liver
or spleen. The LOAEL was 51 mg/kg/day in males and 55 mg/kg/day in females, based on the
effects to BW and food consumption and histopathologic changes in the spleen, liver, bile duct,
and testes. The NOAEL was 11 mg/kg/day for both sexes.
Lee et al. (1976) also evaluated the effect of 2,6-DNT on dogs (4/sex/dose) that were given
2,6-DNT in capsules at doses of 0, 4, 20, or 100 mg/kg/day for 13 weeks. There were no adverse
effects observed in the low-dose animals. 2,6-DNT did, however, produce toxicity at higher dose
levels. All high-dose animals of both sexes and half of the females in the mid-dose group died
before the end of the study. The animals had BW loss due to decreased food consumption.
Adverse neurological effects observed were listlessness, incoordination leading to rigid
paralysis, and occasional tremors. Clinical chemistry effects included elevations in serum
alkaline phosphatase, alanine aminotransferase, and/or blood urea nitrogen. Adverse
hematological effects included methemoglobinemia leading to Heinz body formation, anemia
with compensatory reticulocytosis and extramedullary hematopoiesis, and lymphoid depression
leading to peripheral lymphocytopenia. Bile duct hyperplasia and degenerative and inflammatory
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2,4- and 2,6-Dinitrotoluene - January 2008
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changes in the liver and kidneys of both sexes were noted from histopathological examination.
Testicular changes observed were degeneration and atrophy of the spermatogenic cells. Effects
in the high-dose animals were more pronounced and appeared earlier than those in the mid-dose
animals. A great variation in the onset of symptoms was seen among dogs given the same dose.
The surviving animals completely recovered from the 2,6-DNT effects 19 weeks post-treatment.
The NOAEL was 4 mg/kg/day, based on mortality, BW loss, hematology, neurological effects,
and histopathology. The LOAEL was 20 mg/kg/day.
Leonard et al. (1987) studied the effects of purified 2,6-DNT (>99.4% pure) administered in
the diet to 24 male CDF Fischer 344/CrlBR rats for 12 months. The dose was either 7 mg/kg/day
or 14 mg/kg/day, with a 6-month interim sacrifice of four rats/group. After 1 year, serum alanine
aminotransferase was elevated at both dose levels. Treatment-related liver effects were
prominent at both treatment levels. Serum gamma-glutamyl transferase was increased in the
high-dose group after both the 6-month and 1-year exposure periods. Histopathological changes
in most animals included hepatocytic degeneration and vacuolization and acidophilic and
basophilic foci. Most rats also exhibited bile duct hyperplasia.
Dinitrotoluene Mixture
Leonard et al. (1987) administered Tg-DNT in the diet to 24 male CDF Fischer 344/CrlBR
rats for 12 months at concentrations that resulted in a dose of 35 mg/kg/day. There was a 6-
month interim sacrifice of four animals. Both the 6-month and 1-year groups had significantly
reduced BW gain. Liver weights were significantly increased at 1 year. There were no effects
observed on serum alanine aminotransferase or serum gamma-glutamyl transferase activities.
Hepatocyte histopathology (degeneration and vacuolization) was apparent in the majority of
treated animals but was not dose dependent. Acidophilic and basophilic foci and bile duct
hyperplasia were observed in most of the treated animals. There also was a highly variable
incidence of cholangiofibrosis.
The CUT (1982) studied the effects of Tg-DNT (composition not reported) in Charles River
CDF Fischer 344 rats (130/sex/group) that were given the chemical in feed at doses of 0, 3.5,
14.0, or 35.0 mg/kg/day. Animals were sacrificed and necropsied at weeks 26 and 52
(10 rats/sex/group) and at week 78 (20 rats/sex/group). All surviving high-dose rats were
sacrificed and necropsied at week 55, as were all the other surviving animals after 104 weeks.
Treatment-related effects varied by exposure duration, but overall they included decreased BW
gain, decreased mean BW, and anemia, characterized by increased hemosiderin and splenic
extramedullary hematopoiesis. There were increases in relative liver, brain, kidney, and ovary
weights and in absolute testicular weights in low-dose males. Adverse histopathological effects
were noted for the liver, kidney, pancreas, testes, spleen, adrenal and parathyroid glands, and
bone marrow. The LOAEL for Tg-DNT in this study was 3.5 mg/kg/day, the lowest dose tested,
based on histopathological changes in the liver, kidney, and parathyroid gland.
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6.2.4 Reproductive and Developmental Effects
2,4-Dinitrotoluene
Experimental studies with rats demonstrate that 2,4-DNT causes severe reproductive effects.
A 5-day oral reproduction study in male Sprague-Dawley rats resulted in the deaths of 8 of 15
rats dosed at 240 mg/kg/day, the highest dose tested (Lane et al., 1985). Survivors exhibited BW
loss and cyanosis; sharp decreases in the mating index and in the number of resulting sperm-
positive and pregnant females were observed at 240 mg/kg/day. Cyanosis also was exhibited at
180 mg/kg/day.
A three-generation study was conducted by Ellis et al. (1979), where Sprague-Dawley rats
(10-24/sex/dose) were fed approximately 0, 0.75, 5, or 35 mg/kg/day 2,4-DNT (98% pure) for up
to 6 months prior to mating. The highest dose was associated with reduced parental BW, reduced
pup survival, reduced fertility in FI animals, and slightly lower mean litter size and pup BW. At
mid- and low-dose levels, there were slight reductions in BW for first- and third-generation
pups; however, parental fertility and offspring viability were not affected. The LOAEL was 35
mg/kg/day, based on severe reductions in fertility. The NOAEL was 5 mg/kg/day.
Oral exposure to 2,4-DNT resulted in testicular atrophy and degeneration as well as
reductions in spermatogenesis in males. In females, oral exposure resulted in cessation of
follicular function and reduction in the number of corpora lutea. Consequently, fertility was
reduced in both sexes. Also, 2,4-DNT caused reduced viability as well as decreases in the BW of
offspring at birth and at weaning. Limited available data suggest that 2,4-DNT is not teratogenic
in mice following ingestion (Hardin et al., 1987).
Bloch et al. (1988) fed groups of 9-10 Sprague-Dawley rats 2,4-DNT (97% pure) 0, 100, or
200 mg/kg/day. Significant (p<.05) BW reduction was observed in the high-dose group. The
high-dose group also showed significant (p<.05) increases in serum follicle stimulating hormone
and luteinizing hormone, significantly (p<.01) reduced sperm count, disruption of
spermatogenesis, and histological alterations or degeneration in Sertoli cells, spermatocytes, and
spermatids. No significant effects were observed in the low-dose rats.
Studies of systemic toxicity reported in Sections 6.2.1 and 6.2.3 identified effects of 2,4-
DNT on the testes and spermatogeneisis following short and long term exposures (Elllis et al.,
1979; Hong set al., 1985; Kazuka et al., 1979; Lee et al., 1978, 1985 and McGown et al., 1983).
2,6-Dinitrotoluene
No data on the reproductive effects or developmental effects of 2,6-DNT were found in the
current literature. However, studies of systemic toxicity reported in Sections 6.2.1 and 6.2.3
identified effects of 2,4-DNT on the testes and spermatogeneisis following short and long term
exposures (Lee et al., 1976).
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Dinitrotoluene Mixture
Tg-DNT (76% 2,4-DNT, 19% 2,6-DNT, 5% other isomers) was not teratogenic to time-
mated female Fischer 344 rats administered gavage doses in corn oil (0, 14, 35, 37.5, 75, 100, or
150 mg/kg/day) (Price et al., 1985). Embryotoxicity, however, was observed at maternally toxic
levels. In the 150-mg/kg/day group, there was 46% mortality, and clinical signs of toxicity began
on gestation day 11. Corrected BW gain (minus gravid uterine weight) was significantly reduced
in dams receiving > 14 mg/kg/day. Relative liver weight increased significantly in the 75- and
100-mg/kg/day groups. Relative spleen weight was significantly increased at >35 mg/kg/day.
There was a statistically insignificant increase in percentage resorption in the 150-mg/kg/day
group, which was considered to be indicative of a compound-related effect. Developmental
effects noted in the fetuses were reduced liver weight at 14 mg/kg/day and increased spleen
weight at 35 and 75 mg/kg/day.
6.2.5 Mutagenicity
2,4-Dinitrotoluene
2,4-DNT is a weak mutagen in Salmonella test systems. A detailed summary of 2,4-DNT's
mutagenic potential can be found in Hartley et al. (1994).
Metabolites of 2,4-DNT are mutagenic without metabolic activation, particularly the 2,4-
nitrobenzyl alcohol and the 2-amino- and 2,nitroso-4-nitrotoluenes (Couch et al., 1987). It was
concluded that biliary excretion, metabolism by gut flora, and resorption from the intestine are
prerequisites for genotoxic activity (Mirsalis et al., 1982; Popp and Leonard, 1982). Metabolites
of 2,4-DNT can bind to liver DNA, and 2,4-DNT appears to act as a promoter, inducing gamma
glutamyl transferase-positive foci in the livers of rats initiated with dimethylnitrosamine
(Leonard et al., 1983, 1986).
2,6-Dinitrotoluene
2,6-DNT is a weak mutagen in Salmonella test systems. A detailed summary of 2,6-DNT's
mutagenic potential can be found in Hartley et al. (1994).
2,6-DNT appears to have both initiation and promoting activity (Popp and Leonard, 1982;
Mirsalis et al., 1982; Doolittle et al., 1983).
Dinitrotoluene Mixture
DNTs are negative genotoxins in mammalian cells in vitro., in the dominant lethal test in
mice and rats, and in Drosophila melanogaster systems (Abernethy and Couch, 1982; Styles and
Cross, 1983; Scares and Lock, 1980; Lane et al., 1985; Ellis et al., 1979; Woodruff et al., 1985).
Tg-DNT gave negative responses for unscheduled DNA synthesis except when an in vivo/in
vitro testing system was used (Bermudez et al., 1979; Mirsalis and Butterworth, 1982).
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6.2.6 Carcinogenicity
2,4-Dinitrotoluene
In a 2-year NCI study (1978), 2,4-DNT (>95% pure) was administered in the diet of Fischer
344 rats (50/sex/dose) at doses of 80 and 200 ppm. Controls consisted of 75 rats/sex. The
animals were tested for 78 weeks, followed by an additional observation period of 13-26 weeks.
Survival was adequate in all groups, and a reduced BW gain in high-dose groups showed that a
maximum tolerated dose (MTD) had been approached, indicating that study conditions were
valid. Only benign tumors were noted. 2,4-DNT induced a statistically significant increase in
fibromas of the skin and subcutaneous tissue in males (0/71, 7/49, 13/49) and fibroadenomas of
the mammary gland in high-dose females (13/71, 12/49, 23/50).
CD (Sprague-Dawley) rats (38/sex/dose) were fed 2,4-DNT (98% pure, with 2% 2,6-DNT)
in the diet, at concentrations of 0, 15, 100, or 700 ppm, for up to 2 years (Ellis et al., 1979; Lee et
al., 1985). The intake of 2,4-DNT was 0, 0.57, 3.9, or 34 mg/kg/day for males and 0, 0.71, 5.1, or
45 mg/kg/day for females. Mortality was high in all treatment groups; the control group survival
rate at 2 years was only 40-45%. The test chemical induced increased incidences of
hepatocellular carcinomas in high-dose males (1/25, 2/28, 2/19, and 6/30, respectively) and a
statistically significant increase in the same tumor type in high-dose females (0/23, 0/35, 1/27,
and 19/35, respectively). The incidence of hepatocellular neoplastic nodules was not considered
statistically significantly elevated in any of the treatment groups. A statistically significant
increase in the incidence of benign mammary gland tumors was observed in high-dose females
(8/23, 9/35, 16/27, and 33/35, respectively).
Leonard et al. (1987) treated 20 male Fischer 344 rats with 2,4-DNT (99.9% pure) in the diet
for 1 year and compared results with an untreated control group of 20 rats. No tumors were
found in controls or rats exposed to 2,4-DNT at 27 mg/kg/day.
In a 2-year NCI study (1978), 2,4-DNT (>95% pure) was administered in the diet of B6C3F1
mice (50/sex/dose) at concentrations of 80 ppm and 400 ppm. Controls consisted of 50 mice/sex.
The animals were on test for 78 weeks, followed by an additional observation period of 13-26
weeks. Survival was adequate in all groups, and a reduced BW gain in high-dose groups showed
that an MTD had been approached, indicating that the study conditions were valid. No
statistically significant increase in incidence of tumors was noted in males or females.
In a study of CD-I mice (38/sex/dose), the animals were fed 2,4-DNT (98% pure with 2%
2,6-DNT), at concentrations of 0, 100, 700, or 5,000 ppm, for up to 24 months (Ellis et al., 1979;
Hong et al., 1985). The dose levels were 0, 14, 95, or 898 mg/kg/day. All the animals fed 898
mg/kg/day died by month 18 (males) or month 21 (females), and mortality was high in all
treatment groups. The survival rate for the control group at 2 years was only 20% to 30%. All
animals that died before 12 months were not included in the tumor incidence. In males, the
incidence of kidney tumors (both benign and malignant) was 0/33, 8/33, and 19/28 for the
control and low- and mid-dose groups, respectively. This was a significant (p = .059) elevation
in the mid-dose group. No evidence of treatment-related increases in tumor frequency was noted
41
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in females.
Ellis et al. (1979, 1985) gave groups of six male and six female beagle dogs oral doses of
2,4-DNT at 0, 0.2, 1.5, or 10.0 mg/kg/day in gelatin capsules for 2 years. The high dose was
toxic to all dogs and lethal to five. The medium dose was toxic to some dogs, but the low dose
had no apparent adverse effects. Each animal received a thorough clinical and histopathological
examination following sacrifice. No evidence of carcinogenicity was seen in any of the dogs fed
2,4-DNT.
2,6-Dinitrotoluene
Leonard et al. (1987) treated 20 male Fischer 344 rats with 2,6-DNT (99.9% pure) in the diet
for 1 year and compared results with an untreated control group of 20 rats. 2,6-DNT induced
hepatocellular carcinomas in 100% (19/19) of the high-dose rats (14 mg/kg/day) and 85%
(17/20) of the low-dose rats (7 mg/kg/day). No tumors were found in controls or rats exposed to
2,4-DNT at 27 mg/kg/day. Two low-dose males receiving 2,6-DNT and two males receiving
Tg-DNT developed cholangiocarcinoma.
Dinitrotoluene Mixture
Leonard et al. (1987) treated 20 male Fischer 344 rats with 35 mg/kg/day Tg-DNT (76% 2,4-
DNT, 19% 2,6-DNT) in the diet for 1 year and compared results with an untreated control group
of 20 rats. Tg-DNT induced hepatocellular carcinomas in 47% (9/19) of the treated males. No
tumors were found in controls. Two males receiving Tg-DNT developed cholangiocarcinoma.
Although the duration of these studies was limited to 1 year and the number of animals tested
was small, these results and those from the 2,4-DNT and 2,6-DNT studies suggest that 2,6-DNT
accounts for much of the carcinogenic activity observed in mixed-isomer DNT bioassays.
Fischer 344 rats (130/sex/dose) were fed Tg-DNT (76% 2,4-DNT, 19% 2,6-DNT) at
concentrations of 0, 3.5, 10.0, or 35.0 mg/kg/day (CUT, 1982). All males and females in the
high-dose group were sacrificed at 55 weeks because of significantly reduced survival.
Histopathological studies were performed on sacrificed animals (20 rats/sex) with 100%
incidence of hepatocellular carcinoma in males (20/20) and 55% incidence in females (11/20).
Mid- and low-dose animals were kept on test for 104 weeks. The incidences of liver carcinoma
in males at 104 weeks were 1/61 for the control group, 9/70 for the low-dose group, 22/23 for the
mid-dose group, and 20/20 (at 55 weeks) for the high-dose group; the incidences in females at
104 weeks were 0/57 for the control group, 0/61 for the low-dose group, 40/68 for the mid-dose
group, and 11/20 (at 55 weeks) for the high-dose group. The incidence of neoplastic nodules in
males was 9/61, 11/70, 16/23, and 5/20, and the incidence in females was 5/57, 12/61, 53/68, and
12/20, at 104 weeks for the control and low- and mid-dose groups and (at 55 weeks) for the
high-dose groups, respectively. Cholangiocarcinomas, presumably derived from the bile duct
epithelium, also were observed in three high-dose males at 55 weeks and two mid-dose males at
104 weeks.
42
2,4- and 2,6-Dinitrotoluene - January 2008
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6.3 Sensitive Populations
Reports identifying populations or groups of people sensitive to 2,4-DNT or 2,6-DNT were
not located in the available literature.
However, 2,4-DNT and 2,6-DNT are metabolic products of 2,4,6-trinitrotoluene (2,4,6-
TNT). Therefore, it should be noted that TNT has been associated with the development of
hemolytic crisis in individuals deficient in the G6PD enzyme (ATSDR, 1995). African
Americans and people from Africa, the Middle East, and Southeast Asia exhibit higher
incidences of G6PD deficiencies. G6PD deficiency is a genetic disorder and therefore can be
passed on to offspring, who may display symptoms when stressed. Other populations that may
show increased sensitivity to 2,4,6-TNT include very young children, who have immature
hepatic detoxification systems; individuals with impaired liver function, including alcoholics, or
impaired kidney function; and those who are prone to anemia or who are anemic. Also at
increased risk may be individuals with sickle cell trait, genetically induced unstable hemoglobin
forms, or congenital hypercholesterolemia (ATSDR, 1995).
Letzel et al. (2003) did not find the DNT-induced G6PD deficiency as the result of 2,4,6-
TNT exposure that has been reported elsewhere.
6.4 Proposed Mode of Action
DNT and/or its metabolites oxidize the ferrous ion in hemoglobin and form methemoglobin
(Ellis et al., 1979). Methemoglobin can form aggregates of hemoglobin degradation products
called Heinz bodies, which is a sensitive indicator of hemoglobin destruction. High levels of
methemoglobin lead to the development of anemia, which is compensated by reticulocytosis.
When reticulocytosis cannot compensate adequately, then frank anemia develops.
The relationship between DNT metabolism and the formation of liver tumors is associated
with the formation of reactive intermediates (ATSDR, 1998; Kedderis et al., 1984; Sayama et al.,
1989b). When DNT is oxidized by cytochrome P450 and conjugated with glucuronic acid, it
forms DNBalcG, which is excreted in urine and into bile (Long and Rickert, 1982; Medinsky and
Dent, 1983). That in bile is metabolized further by intestinal microflora, is hydrolyzed, and then
is reduced to form an aminonitrobenzyl alcohol (Chadwick et al., 1993; Guest et al., 1982; Mori
et al., 1985). Nitroso and hydroxylamino derivatives probably are intermediates in the formation
of the alcohol (ATSDR, 1998). Enterohepatic circulation allows the metabolites in the bile,
which now are no longer conjugated, to transport back to the liver (Medinsky and Dent, 1983)
where the amine group is N-hydroxylated by cytochrome P450 to form an unstable sulfate
conjugate (Kedderis et al., 1984). The sulfate conjugate can decompose and form carbonium or
nitrenium ions, which then can bind to hepatic macromolecules, leading to mutations and
subsequently to liver tumors. This mechanism is thought to be applicable to the carcinogenicity
of 2,6-DNT (Long and Rickert, 1982; Mirsalis and Butterworth, 1982).
Based on hepatic tumor initiation-promotion experiments, Leonard et al. (1983, 1986) and
Mirsalis and Butterworth (1982) concluded that Tg-DNT has tumor-promoting and -initiating
43
2,4- and 2,6-Dinitrotoluene - January 2008
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activity. They further concluded that 2,6-DNT is a complete hepatocarcinogen and has the
primary role in Tg-DNT's carcinogenic activity.
44
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7.0 QUANTIFICATION OF TOXICOLOGICAL EFFECTS
Health advisories (HAs) generally are determined for 1-day, 10-day, longer term (up to 7
years), and lifetime exposures if adequate data are available that identify a sensitive
noncarcinogenic endpoint of toxicity. The HAs for noncarcinogenic toxicants are derived using
the following formula:
HA = (NOAEL or LOAEL) x (BW) = mg/L (ug/L)
(UF) (DWI)
where:
HA = Health advisory
NO AEL or LOAEL = No or lowest observed adverse effect level (in mg/kg BW/day)
BW = Assumed body weight of a 10-kg child or a 70-kg adult
UF = Uncertainty factor (10, 100, 1,000, or 10,000) in accordance with the
National Academy of Sciences (1983, 1994)
DWI = Drinking water ingestion; assumed daily water consumption of a 10-
kg child (1 L/day) or a 70-kg adult (2 L/day)
7.1 1-Day Health Advisory
2,4-Dinitrotoluene
No suitable information was found in the available literature for determining a 1-day HA for
2,4-DNT. Due to the acute toxicity of 2,4-DNT, no appropriate NO AEL or LOAEL values were
identified in the reviewed literature. The 5-day oral study in male Sprague-Dawley rats by Lane
et al. (1985) was considered. Since only one sex was tested, this study was considered to be
inappropriate for deriving a 1-day HA.
Since these data were not judged suitable for determining a 1-day HA value for 2,4-DNT, it
is recommended that the 10-day HA for a 10-kg child (0.5 mg/L) be used as a conservative
estimate of the 1-day HA value.
2,6-Dinitrotoluene
No suitable information was found in the available literature for determining the 1-day HA
for 2,6-DNT. Due to the acute toxicity of 2,6-DNT, no appropriate NO AEL or LOAEL values
were identified in the reviewed literature. It is recommended that the longer term HA for a 10-kg
child (0.4 mg/L) be used as a conservative estimate for the 1-day HA value.
45
2,4- and 2,6-Dinitrotoluene - January 2008
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7.2 10-Day Health Advisory
2,4-Dinitrotoluene
The 14-day study with 2,4-DNT in Sprague-Dawley rats (McGown et al., 1983) is acceptable
for derivation of the 10-day HA. Based on decreased BW gain, decreased food consumption,
changes in serum chemistry levels in males and females, and testicular lesions in males, the
LOAEL was 96 mg/kg/day, the lowest dose tested.
The 10-day HA for a 10-kg child is calculated as follows:
10-day HA = (96 mg/kg/day) (10 kg) = 0.96 mg/L (rounded to 1 mg/L or 1,000 ug/L)
(1,000) (1 L/day)
where:
96 mg/kg/day = LOAEL, based on decreased BW gain, decreased food consumption and
changes in serum chemistry levels in females following 14-day dietary
dosing
10 kg = Assumed BW of a child
1000 = UF, which includes a tenfold UF for intraspecies variability, another
tenfold UF to account for interspecies extrapolation, and another tenfold
UF for use of a LOAEL in the absence of a NOAEL
1 L/day = Assumed DWI of a 10-kg child
2,6-Dinitrotoluene
No suitable information was found in the available literature for determining the 10-day HA
for 2,6-DNT. Again, owing to the acute toxicity of 2,6-DNT, no appropriate NOAEL or LOAEL
values were identified in the reviewed literature. It is recommended that the longer term HA for
a 10-kg child (0.4 mg/L) be used as a conservative estimate for the 10-day HA value.
7.3 Longer Term Health Advisory
2,4-Dinitrotoluene
The 13-week feeding study in CD rats by Lee et al. (1978) (also reported by Ellis et al.,
1985) is used to derive the longer term HA. Based on dose-related decreases in BW gain and
food consumption, the LOAEL was 34 mg/kg/day for males and 38 mg/kg/day for females, the
lowest doses tested. The LOAEL for males, 34 mg/kg/day, is used as the most conservative
LOAEL for derivation of the longer term HA.
46
2,4- and 2,6-Dinitrotoluene - January 2008
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The longer term HA for the 10-kg child is calculated as follows:
Longer term HA = (34 mg/kg/day) (10 kg) =0.34 mg/L (rounded to 0.3 mg/L or 300 ug/L)
(1,000) (1 L/day)
where:
34 mg/kg/day = LOAEL, based on dose-related decreases in BW gain and food
consumption in males and females following 13-week dietary dosing
10 kg = Assumed BW of a child
1000 = UF, which includes a tenfold UF for intraspecies variability, another
tenfold UF to account for interspecies extrapolation, and another tenfold
UF for use of a LOAEL in the absence of a NOAEL
1 L/day = Assumed DWI of a 10-kg child
The longer term HA for a 70-kg adult is calculated as follows:
Longer term HA = (34 mg/kg/day) (70 kg) =1.19 mg/L (rounded to 1.0 mg/L or 1,000 ug/L)
(1,000) (2 L/day)
where:
34 mg/kg/day = LOAEL, based on dose-related decreases in BW gain and food
consumption in males and females following 13-week dietary dosing
70 kg = Assumed BW of an adult
1000 = UF, which includes a tenfold UF for intraspecies variability, another
tenfold UF to account for interspecies extrapolation, and another tenfold
UF for use of a LOAEL in the absence of a NOAEL
2 L/day = Assumed DWI of a 70-kg adult
2,6-Dinitrotoluene
The 13-week dog study by Lee et al. (1976) was used to derive the longer term HA for 2,6-
DNT. The animals (4/sex/dose) were administered 2,6-DNT in capsules at doses of 0, 4, 20, or
100 mg/kg/day for 13 weeks. All dogs in the high-dose group and two mid-dose females died
before study termination. Toxic effects observed in the study included decreased food
consumption leading to BW loss, adverse liver and kidney effects, bile duct hyperplasia, and
atrophy of spermatogenic cells in males. There also were neurological, clinical chemistry, and
hematological deficits. The LOAEL was 20 mg/kg/day based on BW loss, blood and
neurological effects, and histopathology. The NOAEL was 4 mg/kg/day.
47
2,4- and 2,6-Dinitrotoluene - January 2008
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The longer term HA for the 10-kg child is calculated as follows:
(4mg/kg/day)(10kg) = 0.4 mg/L (400 ug/L)
(100) (1 L/day)
where:
4 mg/kg/day = NOAEL, based on neurotoxicity, Heinz bodies, bile duct hyperplasia, liver
and kidney histopathology, and death
10 kg = Assumed BW of a child
100 = UF, which includes a tenfold UF for intraspecies variability and another
tenfold UF to account for interspecies extrapolation
1 L/day = Assumed DWI of a 10-kg child
The longer term HA for a 70-kg adult is calculated as follows:
(4 mg/kg/day) (70 kg) = 1.4 mg/L (rounded to 1.0 mg/L or 1,000 ug/L)
(100) (2 L/day)
where:
4 mg/kg/day = NOAEL, based on neurotoxicity, Heinz bodies, bile duct hyperplasia, liver
and kidney histopathology, and death
70 kg = Assumed BW of an adult
100 = UF, which includes a tenfold UF for intraspecies variability and another
tenfold UF to account for interspecies extrapolation
2 L/day = Assumed DWI of a 70-kg adult
48
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7.4 Lifetime Health Advisory
The Lifetime HA represents that portion of an individual's total exposure that is attributed to
drinking water and is considered protective of noncarcinogenic adverse health effects over a
lifetime exposure. The Lifetime HA is derived in a three-step process: Step 1 determines the
reference dose (RfD), formerly called the acceptable daily intake. The RfD is an estimate (with
uncertainty spanning perhaps an order of magnitude) of a daily exposure to the human
population (including sensitive subgroups) that is likely to be without appreciable risk of
deleterious health effects during a lifetime. The RfD is derived from the NOAEL (or LOAEL),
identified from a chronic (or subchronic) study, and divided by the UF(s). From the RfD, a
drinking water equivalent level (DWEL) can be determined (Step 2). A DWEL is a medium-
specific (i.e., drinking water) lifetime exposure level, assuming 100% exposure from that
medium, at which adverse, noncarcinogenic health effects would not be expected to occur. The
DWEL is derived from the multiplication of the RfD by the assumed BW of an adult and divided
by the assumed daily drinking water ingestion (DWI) of a 70-kg adult (2 L/day). The Lifetime
HA in drinking water alone is determined in Step 3 by factoring in other sources of exposure, the
relative source contribution (RSC). The RSC from drinking water is based on actual exposure
data or, if data are not available, a value of 20% is assumed.
For those substances that are "known or likely to be carcinogenic to humans" (U.S. EPA,
2005) or "carcinogenic to humans" or "probably carcinogenic to humans" (Group 1 and Group
2A, respectively, according to the IARC classification categories), the development of a Lifetime
HA is not recommended. The risk manager must balance this assessment of carcinogenic
potential and the quality of the data against the likelihood of occurrence and significance of
health effects related to noncarcinogenic toxicity. To assist the risk manager in this process,
drinking water concentrations associated with estimated excess lifetime cancer risks over the
range of 1 in 10,000 to 1 in 1,000,000 for the 70-kg adult drinking 2 L water/day are provided in
the Evaluation of Carcinogenic Potential, Section 7.5 below.
2,4-Dinitrotoluene
The 2-year chronic study with beagle dogs (Ellis et al., 1979, 1985) is used for derivation of
lifetime values. The dogs (6/sex/dose) were fed 2,4-DNT (98% pure) in gelatin capsules at 0,
0.2, 1.5, or 10 mg/kg/day. In the 10-mg/kg/day group, four of the six males were sacrificed due
to moribund conditions after exhibiting progressive paralysis early in the study. Typical effects
observed in the remaining high-dose and in the mid-dose animals were methemoglobinemia,
with associated reticulocytosis and Heinz body formation. There also was biliary tract
hyperplasia and pigmentation of the gallbladder, kidneys, and spleen. The LOAEL in this study
was 1.5 mg/kg/day based on neurotoxicity and the presence of Heinz bodies and biliary tract
hyperplasia. The NOAEL was 0.2 mg/kg/day.
49
2,4- and 2,6-Dinitrotoluene - January 2008
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Using this study, the DWEL is derived as follows:
Step 1. Determination of the RfD
RfD = (0.2 mg/kg/day) =0.002 mg/kg/day
100
where:
0.2 mg/kg/day = NOAEL, based on neurotoxicity, Heinz bodies, biliary tract hyperplasia,
and organ pigmentation
100 = UF, which includes a tenfold UF for intraspecies variability and another
tenfold UF to account for interspecies extrapolation
Step 2. Determination of the DWEL
DWEL = (0.02 mg/kg/day) (70 kg) =0.07 mg/L (rounded to 0.1 mg/L or 100 ug/L)
(2 L/day)
where:
0.002 mg/kg/day
70kg
2L
=
=
=
RfD
Assumed BW of an adult
Assumed DWI of a 70-kg adult
Step 3. Determination of Lifetime HA
The 2,4-DNT/2,6-DNT mixture is classified as "likely to be carcinogenic to humans" (U.S.
EPA, 2005); thus, the development of a Lifetime HA for 2,4-DNT is not recommended.
2,6-Dinitrotoluene
The 13-week study by Lee et al. (1976) of 2,6-DNT effects on beagle dogs is used for
derivation of lifetime values. The dogs (4/sex/dose) were given 2,6-DNT in capsules at doses of
0, 4, 20, or 100 mg/kg/day for 13 weeks. There were no adverse effects observed in the low-dose
animals. 2,6-DNT did, however, produce toxicity at higher dose levels. All high-dose animals of
both sexes and half of the females in the mid-dose group died before the end of the study. The
animals had BW loss due to decreased food consumption. Adverse effects in this study were
neurological and hematological, and there were altered clinical chemistry parameters. There also
were bile duct hyperplasia and histopathological effects to the liver and kidneys of both sexes
and to the testes in males. The LOAEL was 20 mg/kg/day, based on mortality, BW loss,
hematology, neurological effects, and histopathology. The NOAEL was 4 mg/kg/day.
2,4- and 2,6-Dinitrotoluene - January 2008
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Using this study, the DWEL is derived as follows:
Step 1. Determination of the RfD
RfD = (4 mg/kg/day) =0.001 mg/kg/day
3,000
where:
4 mg/kg/day = NOAEL, based on neurotoxicity, Heinz bodies, bile duct hyperplasia, liver
and kidney histopathology, and death
3000 = UF, which includes a tenfold UF for intraspecies variability, another tenfold
UF to account for interspecies extrapolation, and another tenfold UF for use
of a less-than-lifetime study. An additional factor of 3 is used to account for
the limited database.
Step 2. Determination of the DWEL
DWEL = (0.001 mg/kg/day) (70 kg) =0.035 mg/L (rounded to 0.04 mg/L or 40 ug/L)
(2 L/day)
where:
0.001 mg/kg/day = RfD
70 kg = Assumed BW of an adult
2L = Assumed DWI of a 70-kg adult
Step 3. Determination of Lifetime HA
A 2,4-DNT/2,6-DNT mixture is classified as "likely to be carcinogenic to humans" (U.S.
EPA, 2005); thus, the development of a Lifetime HA for 2,6-DNT is not recommended.
7.5 Evaluation of Carcinogenic Potential
The U.S. EPA reports the cancer classification for DNT as the 2,4-DNT/2,6-DNT mixture.
Although usually accompanied by 2,6-DNT, 2,4-DNT is the more significant component of the
mixture by volume in commercial formulations. For example, Tg-DNT is composed of
approximately 76.5% 2,4-DNT and 18.8% 2,6-DNT. The carcinogenic assessment for lifetime
exposure of DNT was determined using a chronic toxicity/oncogenicity study conducted with a
mixture consisting of 98% 2,4-DNT and 2% 2,6- DNT (Ellis et al., 1979; Lee et al. 1985).
2,4- and 2,6-Dinitrotoluene - January 2008
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Therefore, in this HA, the cancer risk potential and estimates for each of the isomers (i.e.,
2,4-DNT and 2,6-DNT) are the same as that of the mixture. The U.S. EPA classifies the 2,4-
DNT/2,6-DNT mixture as "likely to be carcinogenic to humans."
The cancer risk estimate for the 2,4-DNT/2,6-DNT mixture is derived from a study by Ellis
et al. (1979) (also reported by Lee et al., 1985) where female rats were the sensitive species and
mammary gland tumors were the critical endpoint. Selected information from the study is as
follows:
Tumor type: Liver: hepatocellular carcinomas, neoplastic nodules; mammary gland:
adenomas, fibroadenomas, fibromas, adenocarcinomas/carcinomas
Test animals: Female Sprague-Dawley rats
• Route: Oral (diet)
• References: Ellis et al., 1979; Lee et al., 1985
To estimate the potential cancer risk to exposed human populations from the combined
incidence of mammary gland tumors developed by female rats in the Ellis et al. (1979) study, the
doses administered to the animals are adjusted to a human equivalent exposure by using a
surface area correction factor. The concentration of DNT administered in food (in ppm) was
converted to dose (in mg/kg/day) using estimates from Lehman (1959), where 1 ppm = 0.05
mg/kg/day for the aging rat. The animal dose was divided by the ratio of the human BW to the
aging rat BW raised to the 1/3 power, which is the human equivalent dose, as shown below.
Dose
Administered
(ppm)
0
15
100
700
Administered
(mg/kg/day)
0
0.71
5.10
45.00
Human Equivalent*
(mg/kg/day)
0
0.129
0.927
7.557
Tumor
Incidence
11/23
12/35
17/27
34/35
*Human equivalent dose = (administered dose)/(70 kg/0.425 kg)033
The dose-response data sets presented above were modeled using the Benchmark Dose
Software system (Version 1.3.2) developed by the U.S. EPA National Center for Environmental
Assessment (NCEA). The benchmark dose (BMD) was estimated using the numbers of female
rats with mammary gland tumors, as indicated previously. For a benchmark risk (BMR) level of
0.10, the estimated BMD value is 0.25 mg/kg/day with a lower bound (95%) (BMDL) of 0.15
mg/kg/day using the multistage model. The multistage model had a chi square p value of 0.39
and an Akaike Information Criterion value of 127. The BMDL was used as the point of departure
selected for the quantification of cancer risk from DNT. Additional information concerning
BMD modeling and model output is in Appendices B and C, respectively. These values result in
the following drinking water risk estimates:
2,4- and 2,6-Dinitrotoluene - January 2008
52
-------
Oral slope factor (mg/kg/day)"1—6.67 E-l
Drinking water unit (ug/L) risk—1.90 E-5
The concentrations of DNT in drinking water at the 10"4, 10"5, and 10"6 risk levels were
calculated using the following equation:
35,000 xR = C
ql*
where:
35,000 = Conversion factor for mg to ug and exposure assumption that a 70-kg adult
I ingests 2 L water/day
ql* = (mg/kg/day)"1, human oral slope factor
! R = RiskatlO'4, 10'5, 10'6, etc.
C = Concentration of chemical in ug/L -
The resultant risk levels are 5 ug/L for the E-4 (1 in 10,000) risk level, 0.5 ug/L for the E-5 (1 in
100,000) risk level, and 0.05 ug/L for the E-6 (1 in 1,000,000) risk level.
2,4- and 2,6-Dinitrotoluene - January 2008
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54
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8.0 OTHER CRITERIA, GUIDANCE, AND STANDARDS
Ambient Water Quality Criteria to Protect Human Health for 2,4-DNT at a 10E-6 risk
level (U.S. EPA, 1980):
- Ingestion of water and organisms: 0.11 ug/L
- Ingestion of organisms only: 9.10 ug/L
2,4- and 2,6-Dinitrotoluene - January 2008
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9.0 ANALYTICAL METHODS
Published analytical methods for DNT isomers for a variety of situations refer predominantly
to gas chromatography (GC) and high-performance liquid chromatography (HPLC); however,
other methods include electron spin resonance spectrometry, tandem mass spectrometry (MS),
and cluster analysis.
Gas Chromatography
GC has been studied by a number of scientists utilizing different detection methods for
various situations. Hartley et al. (1981) and Belkin et al. (1985) describe methods to detect and
quantitate DNT in water using GC with electron capture detection (BCD). Richard and Junk
(1986) describe a procedure for determining munitions in water utilizing a macroreticular resin
for extraction, elution with ethyl acetate, concentration of the eluate, separation by GC, and
detection by BCD. Lichtenberg et al. (1987) described a study that utilized GC in conjunction
with either BCD or flame ionization detection to identify and quantitate toxic organic substances
in complex matrices. Eichelberger et al. (1983) utilized both packed column GC (method 1) and
fused silica capillary GC (method 2) coupled with MS to determine the presence of a number of
compounds in water. Capillary GC or capillary GC/MS was used in conjunction with robotics to
analyze wastewater samples for a variety of DNT isomers (Hornbrook and Ode, 1987). A
method for determining DNT isomers in biosludge using GC and a thermal energy analyzer was
described by Phillips et al. (1983). Air samples were collected on a quartz filter and extracted
with a benzene/ethanol mixture by Matsushita and lida (1986), who subsequently detected DNTs
by analysis with GC using flame thermoionic detection.
High-Performance Liquid Chromatography
Krull et al. (1981) reported the use of HPLC with BCD and HPLC with GC/ECD for the
analysis of 2,4-DNT. Lloyd (1983) described a technique for screening trace amounts of
explosives that detected 2,4-DNT using a pendant mercury drop electrode in conjunction with
HPLC. Reverse phase HPLC has been used in the analysis of munitions wastewater samples
(Bauer et al., 1986; Jenkins et al., 1986). Preslan et al. (1991) modified a method for detecting
TNT by adding an intermediate derivatization that allows the separation of 2-amino-4,6-DNT, 4-
amino-2,6-DNT, and DNT. Bongiovanni et al. (1984) used a combination of HPLC and
ultraviolet (UV) light to detect and analyze explosives-bearing soils for trace amounts of DNT
isomers.
Other Methods
Yinon (1989) analyzed and identified a number of 2,4-DNT metabolites using electron
impact and chemical ionization MS. Hable et al. (1991) detected DNT isomers in drinking water
at levels below those measured by GC by using BCD together with a DB-1301 widebore-fused
silica capillary column. Burns et al. (1987) reported on the possibility of identification and
determination of 2,6-DNT by electron spin resonance spectrometry. McLuckey et al. (1985)
studied the use of tandem MS for the analysis of explosives, where the first stage serves as a
2,4- and 2,6-Dinitrotoluene - January 2008
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separator; negative chemical ionization was the most sensitive detector for nitroaromatic
compounds such as 2,4-DNT. Spanggord and Suta (1982) describe the use of cluster analysis to
characterize the distribution of waste components resulting from the production and purification
of TNT.
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10.0 TREATMENT TECHNOLOGIES
Treatment technologies found in the available literature include adsorption, chlorination,
ozonation, UV radiation, and several lesser used techniques.
The use of activated carbon for the adsorptive displacement of 2,4-DNT and 2,6-DNT has
been investigated. Activated carbon adsorption is the technique most frequently used to clean
nitroorganic-contaminated wastewater in military munitions plants. When the carbon becomes
exhausted, it must be disposed of at an approved hazardous waste disposal site. Ho and Daw
(1988) investigated the possibilities of regenerating spent carbons. Solvents tested for extracting
the adsorbed DNT were water, acetone, methanol, and mixtures of the solvents. Both acetone
and methanol were effective for the removal of DNT from activated carbon.
Thakkar and Manes (1987) also studied the adsorptive displacement of 2,4-DNT and 2,6-
DNT. After being preloaded onto activated carbon, the compounds were equilibrated with
benzo[a]anthracene-7,12-dione in methylene chloride/methanol. The 2,6-DNT isomer showed
essentially complete displacement, while 2,4-DNT exhibited nonlinear displacement.
The use of resin for the adsorption of munitions components in aqueous solutions followed
by desorption in acetone was studied by Maskarinec et al. (1984) and Richard and Junk (1986).
Resin adsorption techniques appear to offer several advantages: specific sorptivity toward
nitrogroups, increased stability, and field sorption to ensure sample integrity.
Lloyd (1985) determined the distribution coefficients of DNT for the adsorption of 10
representative adsorbents used in cleanup procedures. The adsorbents were placed in solutions of
methanol, and DNT was loaded into the solution. Two of the adsorbents gave nonsignificant
results, three showed negative selectivity, and the remaining five gave distribution coefficients of
0.129 to 0.356.
The effects of chlorination and ozonation on 2,4-DNT and 2,6-DNT were studied by Lee and
Hunter (1985). Concentrations of 21.3 mg/L (ozone) and 45.5 mg/L (chlorine) were added to
compound concentrations of 100 mg/L and observed. Reduction recoveries of 2,4-DNT by
chlorine and ozone at 1 hour were 35% and 60%, respectively. Corresponding values for 2,6-
DNT were 17% and 13%.
Ho (1986) studied the synergistic effects of hydrogen peroxide and UV radiation on the
decomposition of 2,4-DNT in water and found that at molar ratios of H2O2/DNT between 26
and 52, DNT disappeared very rapidly. The degradation rate of DNT in aqueous solution also
was found to be affected by the energy of the incident light.
Other treatment methods investigated include solvent and sediment extraction and partial
reduction. Hwang (1981) conducted a literature review to determine the feasibility of solvent
extraction as a treatment method for separating organic materials in wastewater effluent. He
found that solvent extraction can remove up to 99.9% of targeted materials. Lopez-Avila et al.
(1983) used an extraction technique involving the homogenization of a sediment sample with
59
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dichloromethane at dual pH and phase separation by centrifugation to determine priority
pollutants in a standard reference sediment sample. Total recoveries for 2,4-DNT and 2,6-DNT
were 95 and 93%, respectively. Ono and Kitazawa (1983) obtained a successful partial reduction
of 2,4-DNT by mild reduction under controlled conditions with a metal and organic acid system.
The reduction products obtained were 4-methyl-3-nitroaniline and 2,4-diaminotoluene. This
method is useful for the "recycling" of 2,4-DNT. In addition, biodegradation and photolytic
techniques may be considered as treatment technology alternatives because of the rapid
degradation of DNT by these two processes (Liu et al., 1984; Davis et al., 1981; Hallas and
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Greater than 99.9% of 2,4-DNT (present at 2.1 mmoles) was removed in 162-202 days in an
upflow anaerobic sludge bed reactor, using a granular sludge and glucose or a volatile fatty acid
mixture as cosubstrate (Razo-Flores et al., 1997).
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APPENDIX A. CALCULATION OF DINITROTOLUENE INGESTION
BY RATS IN MCGOWN ET AL. (1983)
The previous version of the Drinking Water Health Advisory (HA) for 2,4-dinitrotoluene
(2,4,-DNT) and 2,6-dinitrotoluene (2,6-DNT) (Hartley et al., 1994; ATSDR, 1998) describes the
14-day rat feeding study by McGown et al. (1983). The HA estimates DNT ingestion rates of 0,
45, 60, 94, or 143 mg/kg/day for both sexes. The ATSDR estimates the rates as 0, 78, 104, 165,
or 261 mg/kg/day for males and 0, 82, 109, 173, or 273 mg/kg/day for females. Neither reference
describes how the ingestion values were calculated.
The ingestion rates were recalculated from data derived from the report by McGown et al.
(1983). The authors did not report the animals' body weights (BWs) or DNT consumption rates;
however, these data were depicted in graphs. The values for the BW gain and food consumption
were estimated from the graphs and are shown in Table A-l.
Table A-l. Estimated Body Weight Gain and Food Consumption
Dose Group
(g/kg)a
Body Weight Gain (g)
Day of Study
2
4
6
8
10
12
14
16
Food Consumption (g/day)b
Day of Study
2
4
6
8
10
12
14
Males
0.0
0.9
1.2
1.9
3.0
165
164
164
166
164
177
172
172
168
164
198
190
190
187
175
215
204
204
194
182
225
212
214
193
190
240
226
222
206
195
257
238
235
217
201
242
224
215
196
183
21
20.5
20
21
20
21.8
20
20
15.8
13
24.5
22
22
20
15
24.5
21.8
20
17
15
22.5
22
21
15.2
15
23.5
21.5
20
17.5
15
25
22
22
18.5
15
Females
0.0
0.9
1.2
1.9
3.0
137
138
134
135
138
136
142
135
134
132
144
154
145
144
135
152
158
148
146
135
158
164
156
152
140
164
170
158
154
140
170
175
164
158
144
155
164
148
144
130
17.5
18
16
17.5
17.5
16
15
13
11.5
8.5
16.5
17
16
15
9.5
17
16
15
14
11
16
16.5
14.5
14.5
12
16
17.8
15
13
11.8
17
17
15
14
11.5
agrams of DNT per kilogram of feed
bgrams of feed per day
Table A-l data were used to calculate the daily and mean DNT ingestion rates (Table A-2)
with the formula:
Ingestion rate = (dose x food consumption) •*• body weight
2,4- and 2,6-Dinitrotoluene -January 2008
A-l
-------
Table A-2. Ingestion Rates of Dinitrotoluene (mg/kg/day)
Dose Group
(g/kg)a
Day of Study
2
4
6
8
10
12
14
Meanb
Males
.0
0.9
1.2
1.9
3.0
165
164
164
166
164
177
172
172
168
164
198
190
190
187
175
215
204
204
194
182
225
212
214
193
190
240
226
222
206
195
257
238
235
217
201
242
224
215
196
183
Females
0.0
0.9
1.2
1.9
3.0
137
138
134
135
138
136
142
135
134
132
144
154
145
144
135
152
158
148
146
135
158
164
156
152
140
164
170
158
154
140
170
175
164
158
144
155
164
148
144
130
agrams of DNT per kilogram of feed
bMean values are reported in the HA.
2,4- and 2,6-Dinitrotoluene - January 2008
A-2
-------
APPENDIX B. BENCHMARK DOSE MODELING RESULTS FOR 2,4-
DINITROTOLUENE (2,4-DNT)
Benchmark dose (BMD) modeling was performed to identify potential critical effect levels
for derivation of the reference dose (RfD) for 2,4-DNT. The modeling was conducted according
to draft U.S. Environmental Protection Agency guidelines (U.S. EPA, 2000c), using Benchmark
Dose Software (BMDS) system Version 1.3.2, which is available from the U.S. EPA (U.S. EPA,
2002). The BMD modeling results are summarized in Table B-l below, and selected output is
attached as Appendix C. A brief discussion of the modeling results is presented below. Because
the endpoint is a quantal tumor incidence, the multistage models available with BMDS were
used. For all of the modeling conducted, the benchmark risk was defined as an excess risk of
10% (U.S. EPA, 2000c).
The incidence of mammary gland tumors (including benign and malignant tumors from
epithelial or mesenchymal cells) in female CD (Sprague-Dawley) rats given 2,4-DNT in feed for
24 months (Ellis et al., 1979; Lee et al., 1985) was chosen as the endpoint to model. As
summarized in Table B-l, BMD and benchmark dose level (BMDL) estimates were identical
between the two-stage and three-stage multistage models. The goodness-of-fit p values
calculated for these two models were identical, as was the Akaike Information Criterion (AIC), a
measure of goodness of fit that takes into account the number of degrees of freedom. The two-
stage multistage model was chosen as the basis for the BMDL for this endpoint, based on its
simpler form.
Table B-l. Benchmark Dose Estimates of 2,4-DNT From Female Rat Mammary Gland
Tumors
Model
Multistage (2)
Multistage (3)
BMD
0.25
0.25
BMDL
0.15
0.15
Chi square
p value
0.39
0.39
AIC
127
127
2,4- and 2,6-Dinitrotoluene - January 2008
B-l
-------
2,4- and 2,6-Dinitrotoluene - January 2008
B-2
-------
APPENDIX C. BENCHMARK DOSE (BMD) MODELING OUTPUT
Multistage Model. SRevision: 2.1 $ $Date: 2000/08/21 03:38:21 $
Input Data File: E:\BMDS\DATA\DNT-CANCER.(d)
Gnuplot Plotting File: E:\BMDS\DATA\DNT-CANCER.plt
ThuJun09 13:35:51 2005
BMDS MODEL RUN 1
The form of the probability function is:
P[response] = background + (l-background)*[l-EXP(-betal*doseAl-beta2*doseA2)]
The parameter betas are restricted to be positive
Dependent variable = Incidence
Independent variable = HED
Total number of observations = 4
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 = 0.420273
Beta(l) = 0.399193
Beta(2) = 0
Asymptotic Correlation Matrix of Parameter Estimates
(***The model parameter(s) -Beta(2) have been estimated at a boundary point, or have
been specified by the user, and do not appear in the correlation matrix.)
Background Beta(l)
Background 1 -0.37
Beta(l) -0.37 1
2,4- and 2,6-Dinitrotoluene - January 2008
C-l
-------
Variable
Background
Beta(l)
Beta(2)
Parameter Estimates
Estimate
0.392887
0.420126
0
Standard Error
0.0966033
0.138015
NA*
*Indicates that this parameter has hit a bound implied by some inequality constraint and thus has
no standard error.
Model
Full model
Fitted model
Reduced model
Analysis of Deviance Table
Log(likelihood) Deviance Test DF p Value
-60.7606
-61.6992 1.87722 2 0.3912
-79.8807 38.2401 3 <.0001
AIC:
i: 1
127.398
Goodness of Fit
Dose Est. Prob. Expected Observed Size Chi Sq. Res.
0.0000 0.3929 9.036
11
23
0.358
0.1290 0.4249
0.9270 0.5887
7.5570 0.9746
14.872
15.896
34.112
Chi square =
1.87 DF = 2
BMD Computation
Specified effect = 0.1
Risk type = Extra risk
Confidence level = 0.95
BMD = 0.250783
BMDL = 0.154257
12
17
34
35
27
35
p value = 0.3929
-0.336
0.169
-0.129
2,4- and 2,6-Dinitrotoluene - January 2008
C-2
-------
with 0.95
1
I
I
IS
eg
u.
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
" BMD Lower Bound _— - — —
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L BMD , , , , , , , 1
01234i878
dose
13:3508/092005
Multistage Model. SRevision: 2.1 $ $Date: 2000/08/21 03:38:21 $
Input Data File: E:\BMDS\DATA\DNT-CANCER.(d)
Gnuplot Plotting File: E:\BMDS\DATA\DNT-CANCER.plt
ThuJun09 13:37:222005
BMDS MODEL RUN 2
The form of the probability function is:
P[response] = background + (l-background)*[l-EXP(-betal*doseAl-beta2*doseA2-
beta3*doseA3)]
The parameter betas are restricted to be positive
Dependent variable = Incidence
Independent variable = HED
2,4- and 2,6-Dinitrotoluene - January 2008
C-3
-------
Total number of observations = 4
Total number of records with missing values = 0
Total number of parameters in model = 4
Total number of specified parameters = 0
Degree of polynomial = 3
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 = 0.420273
Beta(l) = 0.399193
Beta(2) = 0
Beta(3) = 0
Asymptotic Correlation Matrix of Parameter Estimates
(***The model parameter(s) -Beta(2) -Beta(3) have been estimated at a boundary point, or have
been specified by the user, and do not appear in the correlation matrix.)
Background
Background
Beta(l)
1
-0.37
Beta(l)
-0.37
1
Parameter Estimates
Variable
Background
Beta(l)
Beta(2)
Beta(3)
Estimate
0.392887
0.420126
0
0
Std. Err.
0.0966033
0.138015
NA*
NA*
*Indicates that this parameter has hit a bound implied by some inequality constraint and thus has
no standard error.
Model
Full model
Fitted model
Reduced model
Analysis of Deviance Table
Log(likelihood) Deviance
-60.7606
-61.6992 1.87722 2
-79.8807 38.2401 3
Test DF p Value
0.3912
<0001
AIC:
127.398
2,4- and 2,6-Dinitrotoluene - January 2008
C-4
-------
Goodness of Fit
Dose Est. Prob. Expected Observed Size Chi Sq. Res.
i: 1
0.0000 0.3929 9.036
0.1290 0.4249 14.872
0.9270 0.5887 15.896
7.5570 0.9746 34.112
Chi square = 1.87 DF = 2
Benchmark Dose Computation
Specified effect
Risk Type
Confidence level
BMD
BMDL
0.1
Extra risk
0.95
0.250783
0.154257
11
12
17
34
23
35
27
35
p value = 0.3929
0.358
-0.336
0.169
-0.129
0,95 Confidence
"O
0)
1
03
u_
1
0.9
0,8
0.7
0,6
0.5
0,4
0,3
0,2
Multistage
Lower
/
BMD
0
4
2,4- and 2,6-Dinitrotoluene - January 2008
C-5
------- |