&EPA
,' United States
Environmental Protection
Agency
Office of Water
Regulations and Standards
Criteria and Standards Division
Washington DC 20460
EPA 440/5-80-045
October 1980
Ambient
Water Quality
Criteria for
Dinitrotoluene
C.I
Do not weed. This document
should be retained in the EI*A
Region 5 Library Collection.
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AMBIENT WATER QUALITY CRITERIA FOR
2,4-DINITROTOLUENE
Prepared By,
U.S. ENVIRONMENTAL PROTECTION AGENCY
Office of Water Regulations and Standards
Criteria and Standards Division
Washington, D.C.
Office of Research and Development
Environmental Criteria and Assessment Office
Cincinnati, Ohio
Carcinogen Assessment Group
Washington, D.C.
Environmental Research Laboratories
Corvalis, Oregon
Duluth, Minnesota
Gulf Breeze, Florida
Narragansett, Rhode Island
rt * *
fd, 12th floor
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DISCLAIMER
This report has been reviewed by the Environmental Criteria and
Assessment Office, U.S. Environmental Protection Agency, and approved
for publication. Mention of trade names or commercial products does not
constitute endorsement or recommendation for use.
AVAILABILITY NOTICE
This document is available to the public through the National
Technical Information Service, (NTIS), Springfield, Virginia 22161.
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FOREWORD
Section 304 (a)(l) of the Clean Water Act of 1977 (P.L. 95-217),
requires the Administrator of the Environmental Protection Agency to
publish criteria for water quality accurately reflecting the latest
scientific knowledge on the kind and extent of all identifiable effects
on health and welfare which may be expected from the presence of
pollutants in any body of water, including ground water. Proposed water
quality criteria for the 65 toxic pollutants listed under section 307
(a)(l) of the Clean Water Act were developed and a notice of their
availability was published for public comment on March 15, 1979 (44 FR
15926), July 25, 1979 (44 FR 43660), and October 1, 1979 (44 FR 56628).
This document is a revision of those proposed- criteria based upon a
consideration of comments received from other Federal Agencies, State
agencies, special interest groups, and individual scientists. The
criteria contained in this document replace any previously published EPA
criteria for the 65 pollutants. This criterion document is also
published in satisifaction of paragraph 11 of the Settlement Agreement
in Natural Resources Defense Council, et. al. vs. Train, 8 ERC 2120
(O.D.C. 1976), modified, 12 ERC 1833 (D.D.C. 1979).
The term "water quality criteria" is used in two sections of the
Clean Water Act, section 304 (a)(l) and section 303 (c)(2). The term has
a different program impact in each section. In section 304, the term
represents a non-regulatory, scientific assessment of ecological ef-
fects. The criteria presented in this publication are such scientific
assessments. Such water quality criteria associated with specific
stream uses when adopted as State water quality standards under section
303 become enforceable maximum acceptable levels of a pollutant in
ambient waters. The water quality criteria adopted in the State water
quality standards could have the same numerical limits as the criteria
developed under section 304. However, in many situations States may want
to adjust water quality criteria developed under section 304 to reflect
local environmental conditions and human exposure patterns before
incorporation into water quality standards. It is not until their
adoption as part of the State water quality standards that the criteria
become regulatory.
Guidelines to assist the States in the modification of criteria
presented in this document, in the development of water quality
standards, and in other water-related programs of this Agency, are being
developed by EPA.
STEVEN SCHATZOW
Deputy Assistant Administrator
Office of Water Regulations and Standards
111
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ACKNOWLEDGEMENTS
Aquatic Life Toxicology:
William A. Brungs, ERL-Narragansett
U.S. Environmental Protection Agency
John H. Gentile, ERL-Narragansett
U.S. Environmental Protection Agency
Mammalian Toxicology and Human Health Effects*
V. M. Sadagopa Ramanujam (author)
University of Texas Medical Branch
Marvin S. Legator (author)
University of Texas Medical Branch
Christopher T. DeRosa (doc. mgr.)
ECAO-Cin
U.S. Environmental Protection Agency
Bonnie Smith (doc. mgr.) ECAO-Cin
U.S. Environmental Protection Agency
Myron Mehlman
Mobil"Oil Corporation
Debdas Mukerjee, ECAO-Cin
U.S. Environmental Protection Agency
Gordon W. Newell
National Academy of Science
Roy E. Albert, CAG*
U.S. Environmental Protection Agency
James Bruckner
University of Texas Medical School
Jacqueline V. Carr, OWP
U.S. Environmental Protection Agency
Herbert Cornish
University of Michigan
Patrick Durkin
Syracuse Research Corporation
Terri Laird, ECAO-Cin
U.S. Environmental Protection Agency
Si Duk Lee, ECAO-Cin
U.S. Environmental Protection Agency
Steven D. Lutkenhoff, ECAO-Cin
U.S. Environmental Protection Agency
Jerry F. Stara, ECAO-Cin
U.S. Environmental Protection Agency
F. W. Weir
University of Texas
Technical Support Services Staff: D.J. Reisman, M.A. Garlough, B.L. Zwayer,
P.A. Daunt, K.S. Edwards, T.A. Scan.dura, A.T. Pressley, C.A. Cooper,
M.M. Denessen.
Clerical Staff: C.A. Haynes, S.J. Faehr, L.A. Wade, D. Jones, B.J. Bordicks,
B.J. Quesnell , C. Russom, B. Gardiner.
*CAG Participating Members: Elizabeth L. Anderson, Larry Anderson, Ralph Arnicar,
Steven Bayard, David L. Bayliss, Chao W. Chen, John R. Fowle III, Bernard Haberman,
Charalingayya Hiremath, Chang S. Lao, Robert McGaughy, Jeffrey Rosenblatt,
Dharm V. Singh, and Todd W. Thorslund.
iv
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TABLE OF CONTENTS
Criteria Summary
Introduction A-l
Aquatic Life Toxicology B-l
Introduction B-l
Effects B-l
Acute Toxicity B-l
Chronic Toxicity B-l
Plant Effects B-2
Residues B-2
Summary B-2
Criteria B-3
References B-7
Mammalian Toxicology and Human Health Effects C-l
Introduction C-l
Exposure C-4
Ingestion from Water C-4
Ingestion from Food C-5
Inhalation C-7
Dermal C-7
Pharmacokinetics C-8
Absorption, Distribution, and Excretion C-8
Metabolism C-12
Effects C-16
Acute, Subacute, and Chronic Toxicity C-16
Synergism and/or Antagonism C-29
Teratogenicity C-30
Mutagenicity C-30
Carcinogenicity C-32
Criterion Formulation C-42
Existing Guidelines and Standards C-42
Current Levels of Exposure C-43
Special Groups at Risk C-43
Basis and Derivation of Criterion C-43
References C-48
Appendix I C-67
Appendix II C-69
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CRITERIA DOCUMENT
DINITROTOLUENE
CRITERIA
Aquatic Li£e
The available data for dinitrotoluenes indicate that acute and
chronic toxicity to freshwater aquatic life occur at concentrations
as low as 330 and 230 pg/1, respectively, and would occur at lower
concentrations among species that are more sensitive than those
tested.
The available data for dinitrotoluenes indicate that acute
toxicity to saltwater aquatic life occurs at concentrations as low
as 590 pg/1 and would occur at lower concentrations among species
that are more sensitive than those tested. No data are available
concerning the chronic toxicity of dinitrotoluenes to sensitive
saltwater aquatic life but a decrease in algal cell numbers occurs
at concentrations as low as 370 pg/1.
Human Health
For the maximum protection of human health from the potential
carcinogenic effects due to exposure of 2,4-dinitrotoluene through
ingestion of contaminated water and contaminated aquatic organisms,
the ambient water concentration should be zero based on the non-
threshold assumption for this chemical. However, zero level may
not be attainable at the present time. Therefore, the levels which
may result in incremental increase of cancer risk over the lifetime
are estimated at 10~ , 10~ and 10~ . The corresponding recommend-
ed criteria are 1.1 pg/1, 0.11 pg/1, and 0.011 pg/1, respectively.
vi
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If the above estimates are made for consumption of aquatic organ-
isms only, excluding consumption of water, the levels are 91 jug/1,
91.1 jag/1, and 0.91 ug/1, respectively.
VII
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INTRODUCTION
Dinitrotoluene (DNT) is an ingredient of explosives for com-
mercial and military use because of its waterproofing action and
explosive potential. Use is also made of DNT as a chemical stabi-
lizer in the manufacture of smokeless powder.
DNT is produced by nitration of toluene to nitrotoluene to
dinitrotoluene in a nitric and sulfuric acid solution (Lopez,
1977). The production of DNT is expected to increase yearly at a
rate of 20 to 25 percent (Sittig, 1974). There are six isomers of
dinitrotoluene, with the 2,4-isomer being the most important (Snell
and Ettre, 1971). Often this isomer alone is referred to as DNT
(Manufacturing Chemists Assoc., 1966) or dinitrotoluol (Sax, 1963).
Nitration of o-nitrotoluene yields mostly_.2,4- and 2,6-dini-
trotoluene, CH3CgH3 (NO.,^ in the ratio of about 65:35 (Wiseman,
1972).
2,6-DNT has a melting point of 66°C, a density of 1.2833 at
111°C, and is soluble in alcohol {Weast, 1975). Additional chemi-
cal and physical properties of this compound are: a boiling point
of 285°C (Maksimov, 1968); a molecular weight of 182.14 (Weast,
1977); and a log octanol/water partition coefficient of 2.05 (Tute,
1971). Table 1 lists some physiochemical constants for 2,4-dini-
trotoluene.
Except for their tendency to decompose at elevated tempera-
tures, dinitrotoluenes are relatively stable. At 250°C, commercial
grades of dinitrotoluene decompose at non-sustaining rates. How-
ever, at approximately 280°C rapid self-sustaining decomposition
A-l
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occurs. Dinitrotoluenes may burn safely if unconfined, but if con-
fined may result in an explosion. Decomposition may occur at lower
temperatures in the presence of impurities (Manufacturing Chemists
Assoc., 1966). Because of the deactivating effect of the two nitro
groups in dinitrotoluenes, the synthesis of trinitrotoluene (TNT)
does not occur readily (Wiseman, 1972).
A-2
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TABLE 1
Some Physicocheraical Constants
of 2,4-Dinitrotoluene*
Property
Value
Molecular weight
Melting point
Boiling point
Density
*r
*;
Vapor density (air=l)
J4
'I1
Vapor pressure at 25+2°C
Refractive index (n_)
Solubility, grams/liter
Water, at 22°C
Ethanol, at 15°C
Diethyl ether, at 22°C
Carbon disulfide, at 17°C
Heat of fusion (H~)
Log octanol/water partition
coefficient
(Calc. by method of Tute, 1971)
182.14
69.5-70.5°C
300°C (dec.)
1.521
1.321
6.27
1.4 x 10"4torr
1.442
0.27
30.46
94
21.9
26.4 cal/gram
2.01
*Source: Kirk and Othmer, 1967; St. John, et al. 1975;
Weast, 1978
A-3
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REFERENCES
Kirk, R.E. and D.F. Othmer. 1967. Encyclopedia of Chemical Tech-
nology. Vol. 13. John Wiley and Sons, New York.
Lopez, A.W. 1977. Toluene diisocynate. A paper presented at the
Am. Ind. Chem. Eng. Conf., Houston, Tex., March 23.
Maksimov, Y.Y. 1968. Vapor pressures of aromatic nitro compounds
at various temperatures. Zh. Fiz. Khim. 42: 2921; CA. 1969:
70: 61315y. (Abst.)
Manufacturing Chemists Association. 1966. Chemical safety data
sheet Sd-93, Dinitrotoluenes. Washington, D.C.
Sax, N.I. 1963. Dangerous properties of industrial materials.
Reinhold Publishing Corp., New York.
Sittig, M. 1974. Pollution control in the organic chemical indus-
try. Noyes Data Corp., Park Ridge, New Jersey.
Snell, F.D. and L.S. Ettre (eds.) 1971. Encyclopedia of Indus-
trial Chemical Analysis. Interscience Publishers, John Wiley and
Sons, Inc., New York.
A-4
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St. John, G.A., et al. 1975. Determination of the concentration
of explosives in air by isotope dilution analysis. Forensic Sci.
6: 53.
Tute, M.S. 1971. Principles and practice of Hansch analysis: A
guide to structure-activity correlation for the medicinal chemist.
Adv. Drug Res. 6: 1.
Weast, R.C. (ed.) 1975. CRC Handbook of Chemistry and Physics.
CRC Press Inc., Cleveland, Ohio.
Weast, R.E. (ed.) 1977. CRC Handbook of Chemistry and Physics.
58th ed. CRC Press, Inc., Cleveland, Ohio.
Weast, R.C. (ed.) 1978. CRC Handbook of Chemistry and Physics.
CRC Press, Cleveland, Ohio.
Wiseman, P. 1972. An Introduction to Industrial Organic Chemis-
try. Interscience Publishers, John Wiley and Sons, Inc., New York.
A-5
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Aquatic Life Toxicology*
INTRODUCTION
The data base for dinitrotoluenes is limited but 2,3-dinitrotoluene ap-
pears to be up to two orders of magnitude more acutely toxic to freshwater
fish and invertebrate species than 2,4-dinitrotoluene. The tested fish and
invertebrate species are similarly sensitive to these two dinitrotoluenes.
Acute toxicity tests using static conditions have been conducted with
2,3-dinitrotoluene and the sheepshead minnow and the mysid shrimp. The
LC-- and EC50 values range from 370 ug/1 for algal cell numbers to 2,280
ug/1 for the sheepshead minnow.
EFFECTS
Acute Toxicity
Forty-eight-hour EC,_n values are available for Daphnia magna for both
2,3- and 2,4-dinitrotoluene and are 660 and 35,000 ug/1, respectively (Table
1). The 96-hour LC5Q for the fathead minnow and 2,4-dinitrotoluene is
31,000 ug/1 (Table 1), and the 96-hour LC™ for the more toxic 2,3-dini-
trotoluene and the bluegill is 330 ug/1.
The 96-hour LC,-g values for the saltwater mysid shrimp and sheepshead
minnow and 2,3-dinitrotoluene are 590 and 2,280 ug/1, respectively.
Chronic Toxicity
The chronic value for 2,3-dinitrotoluene, derived from an embryo-
larval test with the fathead minnow, is 230 ug/1 (Table 2) and is based on
survival of these life stages (U.S. EPA, 1978). No acute-chronic ratio is
calculable in the absence of a 96-hour LC for this fish species.
*The reader is referred to the Guidelines for Deriving Water Quality Cri-
teria for the Protection of Aquatic Life and Its Uses in order to better un-
derstand the following discussion and recommendation. The following tables
contain the appropriate data that were found in the literature, and at the
bottom of each table are calculations for deriving various measures of tox-
icity as described in the Guidelines.
B-l
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No chronic toxicity data are available for any dinitrotoluene and salt-
water organisms.
Plant Effects
Cell numbers of the freshwater alga, Selenastrum capricornutum. were
reduced by 50 percent at a concentration of 2,3-dinitrotoluene of 1,370 ug/1
(Table 3). A comparable inhibition in chlorophyll a_ occurred at a concen-
tration of 1,620 ug/1.
A 50 percent reduction in cell numbers of the saltwater alga, Skele-
tonema costatum, occurred at a concentration of 370 ug/l 2,3-dinitrotoluene
(Table 3). There was a 50 percent inhibition of chlorophyll a production at
400 ug/1.
Residues
No bioconcentration data are available for dinitrotoluenes and any
aauatic organisms.
Summary
Few data are available for freshwater organisms but these data indicate
that 2,3-dinitrotoluene is two orders of magnitude more toxic to fish and
invertebrate species than is 2,4-dinitrotoluene. Also, the tested fish and
invertebrate species appear to be of similar sensitivity. The 50 percent
effect levels for 2,3-dinitrotoluene were within the range of 330 to 660
ug/1, and for 2,4-dinitrotoluene the range was 31,000 to 35,000 ug/1. A
chronic value of 230 ug/1 was calculated for the fathead minnow and 2,3-
dinitrotoluene. The results of an algal assay with Selenastrum capri-
cornutum and 2,3-dinitrotoluene were 96-hour EC5Q values of 1,370 and
1,620 ug/1 for cell number and chlorophyll ^ reduction.
Saltwater species have only been tested with 2,3-dinitrotoluene; the
96-hour IC<-0 values for the mysid shrimp and the sheepshead minnow were
8-2
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590 and 2,280 ug/1, respectively. The saltwater alga, Skeletonema costatum,
was of similar sensitivity as the mysid shrimp, with 96-hour EC5Q values
of 370 and 400 yg/1.
CRITERIA
The available data for dinitrotoluenes indicate that acute and chronic
toxicity to freshwater aauatic life occur at concentrations as low as 330
and 230 ug/^> respectively, and would occur at lower concentrations among
species that are more sensitive than those tested.
The available data for dinitrotoluenes indicate that acute toxicity to
saltwater aquatic life occurs at concentrations as low as 590 ug/1 and would
occur at lower concentrations among species that are more sensitive than
those tested. No data are available concerning the chronic toxicity of
dinitrotoluenes to sensitive saltwater aquatic life but a decrease in algal
cell numbers occurs at concentrations as low as 370 ug/1.
B-3
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Table I. Acute values for dinltrotoluenes
Species
Method*
Chemical
LC50/EC50
Species Acute
Value (ug/l) Reference
Cladoceran,
Daphnla magna
Cladoceran,
Daphnia magna
Fathead minnow,
Plmephales promelas
Bluegl 1 1,
Lepomls macrochlrus
CO
1
Mysid shrimp,
Hysldopsis bah la
Sheepshead minnow,
Cyprlnodon variegatus
FRESHWATER
S, U 2,3-dlnitro-
to 1 uene
S, U 2,4-dinitro-
toluene
S, U 2.4-dlnltro-
toluene
S, U 2,3-dlnitro-
toluene
SALTWATER
S, U 2,3-dlnltro-
toluene
S, U 2,3-dinitro-
toluene
SPECIES
660
35,000
31,000
330
SPECIES
590
2,280
660 U.S. EPA, 1978
35,000 U.S. Army, 1976
31,000 U.S. Army, 1976
330 U.S. EPA, 1978
590 U.S. EPA, 1978
2,280 U.S. EPA, 1978
* S = static, U = unmeasured
No Final Acute Values are calculable since the minimum data base requirements are not met.
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Table 2. Chronic values for dlnltrotoluenes (U.S. EPA, 1978)
Species
Fathead minnow,
Picnephales promelas
Method*
FRESHWATER
E-L
Chemlcal
SPECIES
2,3-dlnltro-
toluene
Limits
(ug/D
200-270
Value
(ug/l)
230
* E-L = embryo-larva I
No acute-chronic ratio can ba estimated since no acute test data are
available for this species.
Cd
I
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Table 3. Plant values for dlnltrotoluenes (U.S. EPA, 1978)
tx)
I
CTi
Species
Alga,
Selenastrum capricornutum
Alga,
Selenastrum capricornutum
Alga,
Skeletonema costatum
Alga,
Skeletonema costatum
Chemical
FRESHWATER SPECIES
2,3-dinitro-
tol uene
2,3-dinitro-
toluene
SALTWATER SPECIES
2,3-dinitro-
toluene
2,3-dinitro-
toluene
Effect
Cel 1 numbers
96-hr EC50
Ch lorophy 1 1 a
96- hr EC50
Cel 1 numbers
96-hr EC50
Ch lorophy 1 1 a
96-hr EC50
Result
(U9/I)
1,370
1,620
370
400
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REFERENCES
U.S. Army Research and Development Command. 1976. Toxicity of TNT waste-
water (pink water) to aquatic organisms. U.S. Army Res. Dev. Comm., Wash-
ington, O.C. Final Report, Contract DAMD17-75-C-5056.
U.S. EPA. 1978. In-depth studies on health and environmental impacts of
selected water pollutants. U.S. Environ. Prot. Agency, Contract No. 68-01-
4646.
B-7
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Mammalian Toxicology and Human Health Effects
INTRODUCTION
2,4-Dinitrotoluene (2,4-DNT) is a pale yellow crystalline
solid that is widely used as a raw material for dyestuffs and for
urethane polymers through a conversion to the corresponding diamine
and then to diisocyanate (Kirk and Othmer, 1967). Some of its
physical properties are presented in Table 1. It is commercially
prepared in the United States by the direct dinitration of toluene.
The process produces an 80:20 ratio of 2,4-:2,6-isomers, which on
fractionation gives pure 2,4-DNT (Kirk and Othmer, 1967). Precise
production figures for 2,4-DNT are not available; however, the U.S.
International Trade Commission (1975) reported a combined produc-
tion of 272,610,000 pounds for the 2,4- and 2,6-DNT isomers in 1975.
The name given by the Chemical Abstracts Service (1977) for
this compound is l-methyl-2,4-dinitrobenzene (CAS registry number
121-14-2). Other synonyms for 2,4-DNT include 2,4-dinitrotoluol
and toluene-2,4-dinitro. 2,4-DNT has a moderate fire and explosion
risk and it can be detonated only by a very strong initiator.
Aside from its use by the dye and polyurethane manufacturing
industries, 2,4-DNT is used by the munition industry as a modifier
for smokeless powders and, to a limited extent, as a gelatinizing
and waterproofing agent in military and commercial explosive com-
positions (Hamilton and Hardy, 1974). 2,4-DNT is also used as a
chemical intermediate in the production of toluene diisocyanate
(TDI) which, in turn, is consumed in the production of flexible and
C-l
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TABLE 1
Some Physicochemical Constants
of 2,4-Dinitrotoluene*
Property
Value
Molecular weight
Melting point
Boiling point
Density
*r
*;
Vapor density (air=l)
'4
•I1
Vapor pressure at 25+2°C
Refractive index (n_)
Solubility, grams/liter
Water, at 22°C
Ethanol, at 15°C
Diethyl ether, at 22°C
Carbon disulfide, at 17°C
Heat of fusion (Hf)
Log octanol/water partition
coefficient
(Calc. by method of Tute, 1971)
182.14
69.5-70.5°C
300°C (dec.)
1.521
1.321
6.27
1.4 x 10"4torr
1.442
0.27
30.46
94
21.9
26.4 cal/gram
2.01
*Source: Kirk and Othmer, 1967; St. John, et al. 1975;
Weast, 1978
C-2
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rigid polyurethane foams and elastomers. Most TDI producers, how-
ever, use toluene as the starting material, generating 2,4-DNT as a
captive intermediate (Kirk and Othmer, 1967).
The potential risk of exposure to 2,4-DNT is greatest for
workers in the dye and explosives industries and at chemical plants
producing TDI. 2,4-DNT encountered chiefly as a major component in
the wastewater from munitions industries. The general population
may experience exposure as a result of this discharge of 2,4-DNT
into rivers and streams from munition plants (National Cancer
Institute (NCI), 1978). Aromatic nitro compounds are one of several
classes of chemicals thought to contribute to the increased cancer
risk in dye and explosive manufacturing industries (Wynder, et al.
1963). The structural relationship of 2,4-DNT to the known car-
cinogen, 2,4-toluenediamine (2,4-TDA), is also a factor in its
selection for testing as a possible carcinogen (NCI, 1978).
The usual methods of identification and quantitative determi-
nation of 2,4-DNT include spot tests (Ames and Yallop, 1966),
colorimetry (Goldman and Jacobs, 1953), chromatographic methods
such as thin layer chromatography (Yoshida, et al. 1967), gas
chromatography (Krzymien and Elias, 1975; Pella, 1976; Fukuda, et
al. 1977), and high pressure liquid chromatography (HPLC) (Walsch,
et al. 1973; Doali and Juhasz, 1974; Stanford, 1977? National
Institute for Occupational Safety and Health (NIOSH), 1978), and
spectroscopic methods such as infrared (Priestera, et al. 1960) or
ultraviolet spectrophotometry (Conduit, 1959), nuclear magnetic
resonance spectrometry (Gehring and Reddy, 1968), mass spectrometry
(Murrmann, et al. 1971; Plimmer and Klingebiel, 1974; Zitrin and
C-3
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Yinon, 1976) and isotope dilution analysis (St. John, et al. 1975,
1976). In many other instances where the residues of explosives
needed to be identified after an explosion, special wet chemical
separation techniques were used (Hoffman and Byall, 1974; Jenkins
and Yallop, 1970; Fukuda, et al. 1977).
EXPOSURE
Ingestion from Water
2,4-DNT has limited solubility (270 mg/1 at 22°C) in water.
Possible sources of 2,4-DNT in the aqueous environment, either
surface water, ground water or drinking water, include the dumping
of chemical wastes and accidental loss during transfer and
transport.
Dinitrotoluene waste products are dumped into surface water or
sewage by manufacturing industries that make— dyes, isocyanates,
polyurethanes, and munitions. The occurrence of organic micropol-
lutants due to the dumping of aromatic nitro and amino compounds in
river water has been reported by Meijers and Van der Leer (1976).
The pollution of the Rhine and Maas Rivers in the Netherlands by
these aromatics and oils was examined by extracting water samples
in hexane followed by analysis of the extracts by gas chromato-
graph/mass spectrometry (GC/MS). The results showed that the Rhine
is heavily polluted by oil, a number of aromatic hydrocarbons,
aromatic amines and aromatic nitro compounds including 2,4-DNT.
The Maas River, however, is much less polluted by these substances
with the exception of oil.
The second source of water contamination by 2,, 4-DNT develops
when the chemical is accidentally spilled during the process of
C-4
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transfer and/or transportation. No specific incident of this type
has been reported in the literature, however.
The ability of microorganisms to degrade 2,4-DNT and related
compounds has been studied by a number of investigators (Schott, et
al. 1943? Ruchhoft, et al. 1945; Ruchhoft and Norris, 1946; Rogov-
skaya, 1951; Nason, 1956; U.S. Army, 1970,1971; Osinon and Klaus-
mier, 1972; Walsh, et al. 1973; Nay, 1974; Traxler, et al. 1974;
Won, et al. 1974; McCormick, et al. 1976; Parrish, 1977). Bio-
transformation of 2,4-DNT does occur but its frequency is much
lower than the equivalent activity for 2,4,6-trinitrotoluene (2,4,6-
TNT). The influence of aromatic nitrated hydrocarbons including
2,4-DNT, on the activated sludge process has been extensively stud-
ied (Bogatyrev, 1973; Matsui, et al. 1975; Roth and Murphy, 1978).
At concentrations of 50 mg/1 of nitro-aromatics, there was no
effect on the activated sludge process.
Ingestion from Food
The likelihood of 2,4-DNT existing in food is minimal, since
it is not used as a pesticide or herbicide. There is no report in
the literature, however, on the toxic effect of 2,4-DNT in humans
due to ingestion from food.
A bioconcentration factor (BCF) relates the concentration of a
chemical in aquatic animals to the concentration in the water in
which they live. The steady state BCFs for a lipid-soluble com-
pound in the tissues of various aquatic animals seem to be propor-
tional to the percent lipid in the tissue. Thus the per capita
ingestion of a lipid-soluble chemical can be estimated from the per
C-5
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capita consumption of fish and shellfish, the weighted average per-
cent lipids of consumed fish and shellfish, and a steady-state BCF
for the chemical.
Data from a recent survey on fish and shellfish consumption in
the United States were analyzed by SRI International (U.S. EPA,
1980). These data were used to estimate that the capita consump-
tion of freshwater and estuarine fish and shellfish in the United
States is 6.5 g/day (Stephan, 1980). In addition, these data were
used with data on the fat content of the edible portion of the same
species to estimate that the weighted average percent lipids for
consumed freshwater and estuarine fish and shellfish is 3.0
percent.
No measured steady-state bioconcentration factor (BCF) is
available for 2,4-dinitrotoluene, but the equation "Log BCF =
(0.85 Log P) - 0.70" can be used (Veith, et al. 1979) to estimate
the BCF for aquatic organisms that contain about 7.6 percent lipids
(Veith, 1980) from the octanol/water partition coefficient (P).
Based on a measured log P value of 1.98 (Hansch and Leo,, 1979), the
steady-state bioconcentration factor for 2,4-dinitrotoluene is
estimated to be 9.62. An adjustment factor of 3.0/7.6 = 0.395 can
be used to adjust the estimated BCF from the 7.6 percent lipids, on
which the equation is based, to the 3,0 percent lipids that is the
weighted average for consumed fish and shellfish. Thus, the
weighted average bioconcentration factor for 2,4-dinitrotoluene
and the edible portion of all aquatic organisms consumed by Ameri-
cans is calculated to be 9.62 x 0.395 = 3.8.
C-6
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Inhalation
An estimate of the number of individuals involved in the man-
ufacture of 2,4-DNT in the United States is not available at pre-
sent. But the U.S. International Trade Commission (1975) reports a
combined production of 272,610,000 pounds for the 2,4-and 2,6-DNT
isomers in 1975. Since DNT is produced in such large quantities, a
considerable proportion of the population may be at risk.
Inhalation has been reported to be one of the major routes of
exposure to 2,4-DNT in either the particulate or vapor state. The
effects of inhalation exposure to 2,4-DNT are a consequence of its
capacity to produce anoxia due to the formation of methemoglobin
(see Effects Section).
There are no data in the literature on the ambient atmospheric
concentration of 2,4-DNT. Thus, it is not possible to estimate the
extent of possible human exposure.
Dermal
Since 2,4-DNT is readily soluble in organic solvents such
as alcohol, ether, etc., it penetrates the intact skin readily
(Patty, 1958; Hamblin, 1963). From a survey of the literature
(Toxic and Hazardous Industrial Chemicals Safety Manual, 1976; Key,
et al. 1977; Proctor and Hughes, 1978), it is obvious that skin
contact is another important route for 2,4-DNT absorption in plant
workers. The quantitative data on the threshold doses for dermal
absorption of 2,4-DNT are unavailable in the literature. However,
the Occupational Safety and Health Administration (OSHA) recommends
a threshold limit value (TLV) of 1.5 mg/m of air including dermal
exposure (American Conference of Governmental Industrial Hygienists
C-7
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(ACGIH), 1978). This TLV was set by analogy to chemically
similar nitro-aromatic compounds (ACGIH, 1974).
Because of the limited availability of data on the human expo-
sure to 2,4-DNT, it is difficult to assess quantitatively the con-
tribution of each route of exposure to the total dose; it is likely
that the greatest contribution comes via inhalation, particularly
in an occupational setting. The next most likely route is dermal
and the least likely is ingestion.
PHARMACOKINETICS
Absorption, Distribution, and Excretion
2,4-DNT is absorbed mainly by inhalation of its vapor or by
percutaneous aborption of its solution in organic solvents. Hodg-
son, et al. (1977) recently reported a study on the comparative
absorption, distribution, and excretion of 2,4,6-TNT and isomers of
DNT in rats. It was noticed that the 14C ring-labeled nitrotoluenes
were well absorbed after oral administration in the rat. The
absorption was essentially complete in 24 hours with (50 to 90 per-
cent of the dose being absorbed. The extent of absorption occurred
in the following order: 2,4-DNT = 3,4-DNT > 3,5-DNT = 2,4,6-TNT =
2,5-DNT > 2,3-DNT = 2,6-DNT. The liver, kidneys and blood con-
tained small amounts of radioactivity. The ratios of radioactivity
(tissue:plasma) indicated a retention of 14C in both the liver and
kidneys, while negligible amounts of 14C were found in the other
14
tissues. No C was recovered in the expired air; most of the
absorbed radioactivity was eliminated in the urine. When 14C-
labeled nitrotoluenes were administered to bile duct-cannulated
rats, 10.3 to 27.3 percent of the C was recovered in the bile,
C-8
-------
suggesting that biliary excretion is also an important elimination
pathway. Thin layer chromatographic analysis of the urine from
rats treated with 2,4,6-TNT or dinitrotoluene indicated extensive
metabolism of the parent compounds. However, this study does not
report the characterization of the metabolic products from dinitro-
toluenes and 2,4,6-TNT.
In another study the distribution and excretion of tritium-
labeled 2,4-dinitrotoluene (3H-2,4-DNT) in the rat was examined
(Mori, et al. 1977). Approximately 21.3 percent of the radio-
activity was excreted in the feces on the first day after a single
oral administration of 3H-2,4-DNT. The amount of radioactivity
excreted in the feces on the second and third days were 4.1 and 1.25
percent of the administered dose, respectively. About 13.5 percent
of the radioactivity administered was excreted in the urine on the
first day, but after the second day the urinary excretion of radio-
activity occurred in only trace quantities. In all, about 47 per-
cent of the radioactivity administered was excreted in the feces
and urine during the first seven days following administration (see
Table 2).
In the same experiment, relatively high amounts of radioactiv-
ity were found in adipose tissue, skin, and liver of the rats seven
days after administration; the relative amounts of radioactivity
remaining in other organs were not significant (Table 3). This
investigation, utilizing the single oral administration of H-2,4-
DNT, suggests that 2,4-DNT remains in the liver, skin, and adipose
tissue.
09
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TABLE 2
Urinary and Fecal Excretion of Radioactivity,
Expressed as Percentages of Administered Radioactivity*
Day
1st
2nd
3rd
4th
5th
6th
7th
Urine (%)
13.52+1.44
0.61+0.12
0.66+0.12
0.48+0.18
0.28+0.08
0.19+0.09
0.15+0.03
Feces (%)
21.34+3.10
4.11+0.53
1.25+0.41
0.78+0.12
0.77+0.14
0.84+0.21
1.23+0.02
Values are indicated as means and deviations of three rats
*Source: Mori, et al. 1977
C-10
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TABLE 3
Remaining Radioactivity in the Tissues of Rat
Seven Days after Administration of H-2,4-DNT*
Tissue
Brain
Heart
Lung
Liver
Spleen
Pancreas
Kidney
Adrenal
Stomach
Small intestine
Large intestine
Testis
Mesenter iolum
Adipose tissue
Skin
dpm per 100 mg
Tissue x 10
0.93
0.99
1.14
1.98
0.81
1.30
0.98
2.11
0.80
0.99
1.02
0.85
0.82
13.99
0.79
Radioactivity
Total dpm x 104
1.19
0.49
1.12
17.23
0.36
0.71
1.77
0.03
0.60
4.56
0.84
1.98
1.54
68.30
25.53
% of Dose
0.03
0.01
0.03
0.40
0.01
0.02
0.04
trace
0.01
0.10
0.02
0.04
0.04
1.60
0.60
Mean of three rats given 50 mg of H-2,4-DNT/kg p.o.
Weights of skin and adipose tissue were roughly calculated as:
skin = body weight x 1/25; adipose tissue = body weight x 1/40.
*Source: Mori, et al. 1977
C-ll
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Metabolism
No report has yet been published on the metabolic fate of 2,4-
DNT in humans. Even the two studies (Hodgson, et al. 1977; Mori, et
al. 1977) which describe the absorption, distribution, and excre-
tion of 2,4-DNT in rats do not give details on the characterization
of metabolites and metabolic pathways.
The isolation, identification and synthesis of biotransforma-
tion products derived from 2,4-DNT have been reported by McCormick,
et al. (1978) from a detailed study on the microbial transforma-
tion of 2,4-DNT by Mucrosporium sp. (Strain QM 9651). The bio-
transformation products were identified by thin layer chromato-
graphy (using silica gel plates with fluorescent indicator to
visualize the metabolites and developing in benzene-hexane 50:50
percent v/v solvent mixtures) and then were followed by GC/MS. The
metabolites identified were 2-amino-4-nitrotoluene, 4-amino-2-
nitrotoluene, 2,2'-dinitro-4,4'-azoxytoluene, 4,4'-dinitro-2,2'-
azoxytoluene, and 4-acetamido-2-nitrotoluene; a third azoxy com-
pound, believed to be a "mixed" type (i.e., 2,4'-azoxy or 4,2'-
azoxy), was also isolated, but not identified. These authors pre-
sent a scheme for the biotransformation of 2,4-DNT (Figure 1).
Although no 2,4-toluenediamine (2,4-TDA) was detected in the
present system, complete reduction of both nitro groups to amino
groups has been reported in the biotransformation of 2,4-DNT by
anaerobic bacterial systems (McCormick, et al. 1976); hence, 2,4-
TDA is also included in Figure 1.
In a study of the microbial transformation of 2,4-DNT, 2,4,6-
TNT and other nitroaromatic compounds by anaerobic bacterial
C-12
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NO: NO:
(0)
CH3
CH3
CH3
NHOH
NO2
NO2
\
CH3
CH3
NO2
NO2
FIGURE 1
Proposed Pathways for the Formation of Biotransformation
Products from 2,4-Dinitrotoluene (A)
Source: McCormick, et al. 1978
The hypothetical nitroso and hydroxylamino intermediates are
enclosed in brackets. The potential formation of 2,4-toluene-
diamine (L) is indicated by dashed arrows.
(B) 2-Nitroso-4-nitrotoluene; (C) 2-Hydroxylamino-4-nitrotoluene;
(D) 4,4'-Dinitro-2,2'-azoxytoluene; (E) 2-Amino-4-nitrotoluene;
(F) 4-Nitroso-2-nitrotoluene; (G) 4-Hydroxylamino-2-nitrotoluene;
(H) 4-Amino-2-nitrotoluene; (I) 2,2'-Dinitro-4,4'-azoxytoluene;
(J) 4,2'-Dinitro-2,4'-azoxytoluene; (K) 4-Acetamido-2-nitrotoluene
C-13
-------
systems (McCormick, et al. 1976), these compounds were reduced by
hydrogen in the presence of enzyme preparations from Veillonella
alkalescens. Consistent with the proposed reduction pathways,
R-N02 H2^R-NO "^R-NHOH H2^R-NH2, 3 moles of H2 were utilized per
mole of nitro group. From the rates of reduction of 40 mono-, di-,
and trinitroaromatic compounds by Veillonella alkalescens, it was
noticed that reactivity of the nitro group depended on other sub-
stituents and on the position of the nitro groups relative to these
substituents. The order of reduction rate of nitro compounds is
consistent with the "electronegativity rule" (Shikata. and Tachi,
1938):
-N02 > -COOH > -CH3 > -H > -OH > -NH2
In the case of nitrotoluenes, the para nitro group was the most
readily reduced, the 4-nitro position of 2,4-DNT being reduced
first. The "nitro-reductase" activity of Veillonella alkalescens
extracts was associated with protein fractions, one having some
ferredoxin-like properties and the other possessing hydrogenase
activity. The question of whether ferredoxin acts as a nonspecific
reductase for nitroaromatic compounds remains unresolved.
Since the microbial transformation pathway of 2,4-DNT (McCor-
mick, et al. 1978) is similar to that of 2,4,6-TNT (McCormick, et
al. 1976), it can be assumed that these two compounds may behave
similarly during biochemical transformation in animals and humans.
Hence, it is reasonable to discuss a few studies on the metabolism
of 2,4,6-TNT in animals and humans in this context.
The explosive 2,4,6-TNT has been extensively investigated
because of the toxic symptoms which it produces in people engaged
C-14
-------
in its manufacture (Palmer, et al. 1943; Schwartz, 1944; Crawford,
1954; Goodwin, 1972; Djerassi and Vitany, 1975; Morton, et al.
1976). It is generally agreed that its toxicity is due to its meta-
bolic products (Won, et al. 1974, 1976; Carpenter, et al. 1978).
Earlier studies (White and Hay, 1901; Moore, 1918; Schereschewsky,
1918; Voegtlin, et al. 1920) have shown that the urine of 2,4,6-TNT
workers and of experimental animals receiving 2,4,6-TNT orally or
by injection contained 2,2',6,6'-tetranitro-4,4'-azoxytoluene and
2- or 4-aminodinitrotoluene. The investigations of Channon, et al.
(1944) showed that rabbits, when given small oral doses of 2,4,6-
TNT, excreted 2- and 4-aminodinitrotoluenes and 4-hydroxylamino-
2,6-dinitrotoluene. Of the two amino compounds excreted, the
4-amino-2,6-dinitrotoluene was found in larger quantities and the
4-hydroxylamino-2,6-dinitrotoluene was obviously an intermediate
in the reduction of 2,4,6-TNT to the corresponding amino compound.
The 4-amino-2,6-dinitrotoluene was also formed when 2,4,6-TNT was
incubated with an acetone extract of pig liver (Bueding and
Jolliffe, 1946). When administered to pigs, some 24 to 30 percent
of the 2,4,6-TNT appears in the urine as compounds containing a
diazotizable amino group. In man, 2,4,6-TNT appears to be convert-
ed to the same metabolites as in the rabbit (Williams, 1959). Dale
(1921) showed that 2, 2' , 6,6'-tetranitro-4,4'-azoxytoluene could be
isolated from the urine of 2,4,6-TNT workers, a fact which indi-
cates that 2,4,6-TNT is reduced in man to 4-hydroxylamino-2,6-
dinitrotoluene. Lemberg and Callaghan (1944) also detected the
4-amino-2,6-dinitrotoluene and 2-amino-4,6-dinitrotoluene in human
urine. These authors stated that the qualitative and quantitative
C-15
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distribution of 2,4,6-TNT metabolites in human urine is similar to
that found in rabbit urine. A scheme for the biotransformation of
2,4,6-TNT is presented in Figure 2. It is interesting to note that
no study in the literature reports the formation of 2,4,6-triamino-
toluene as a metabolic product of 2,4,6-TNT, though such a possi-
bility cannot be ruled out.
Thus, by analogy of metabolism of 2,4,6-TNT to that of 2,4-DNT
(compare Figures 1 and 2), one might expect most of the products
presented in Figure 1 to be present in the urine of humans and ani-
mals exposed to 2,4-DNT. Most of these metabolites are either
toxic (Fairchild, et al. 1977) or suspected carcinogens
(Christensen, et al. 1976).
EFFECTS
Acute, Subacute, and Chronic Toxicity
Acute toxic effects of 2,4-DNT include methemoglobinemia fol-
lowed by cyanosis. The inhalation of the fumes or dust, the inges-
tion of the compound, or the absorption by the skin through contact
of 2,4-DNT all cause a chemical change of the blood oxyhemoglobin
into methemoglobin (via oxidation of Fe(II) to Fe(III)). The onset
of symptoms of methemoglobinemia due to the absorption of 2,4-DNT
is often insidious and may be delayed up to four hours; headache is
commonly the first symptom and may become quite intense as the
severity of methemoglobinemia progresses. The following symptoms
have been reported as a result of varying doses of 2,4-DNT:
vertigo, fatigue, dizziness, weakness, nausea, vomiting, dyspnea,
drowsiness, arthralgia, insomnia, tremor, paralysis, unconscious-
ness, chest pain, shortness of breath, palpitation (rapid throbbing
C-16
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07H g NO2
H3C -vO/~ N=N \O/~ CH3
02N _ NO2
N02 NO2
(H)
FIGURE 2
Proposed Pathways for the Formation of Biotransformation Products
from 2,4,6-Trinitrotoluene (A) The hypothetical nitroso inter-
mediates are enclosed in brackets. The potential formation of 2,4-
Diamino-6-nitrotoluene (J) is indicated by dashed arrows.
(B) 4-Nitroso-2,6-dinitrotoluene; (C) 4-Hydroxylamino-2,6-dinitro-
toluene; (D) 2 , 2',6,6'-Tetranitro-4,4'-azoxytoluene; (E) 4-Amino-
2,6-dinitrotoluene; (F) 2-Nitroso-4,6-dinitrotoluene; (G) 2-
Hydroxylamino-4,6-dinitrotoluene; (H) 4,4',6,6'-Tetranitro-2,2'-
azoxytoluene; (I) 2-Amino-4,6-dinitrotoluene
Source: Williams, 1959; Won, et al. 1974
C-17
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of heart), anorexia (lack of appetite), and loss of weight
(Koelsch, 1917; Von Oettingen, 1941; Mangelsdorff, 1952, 1956;
Hamblin, 1963; Toxic and Hazardous Industrial Chemicals Safety
Manual, 1976; Key, et al. 1977; Proctor and Hughes, 1978). 2,4-DNT
also produces Heinz bodies (granules in red blood cells due to dam-
age of the hemoglobin molecules) in the cat (Bredow and Jung,
1942). Human subjects are similarly susceptible, and workers
handling compounds such as nitrobenzenes, nitrotoluenes and phenyl-
hydrazines occasionally exhibit Heinz bodies in their blood (Hughes
and Treon, 1954; De Bruin, 1976).
Inactivation of hemoglobin due to 2,4-DNT and related com-
pounds has been noted by Vasilenko, et al. (1972). These authors
observed the transformation of hemoglobin into methemoglobin,
nitrosylhemoglobin, and sulfhemoglobin when rats received 0.1 to
0.2 LD5Q of 2,4-DNT orally for a period of 30 days. An increase in
the levels of methemoglobin and sulfhemoglobin was accompanied by a
decrease in oxyhemoglobin, but the total level of hemoglobin
remained unchanged.
Methemoglobin formation of nitrotoluenes in relation to the
number and positioning of nitro groups was studied by Kovalenko
(1973). When administered orally at doses corresponding to 0.1 to
0.2 LD5Q values to rats for one to three months, the hemotoxicity
of the nitrotoluenes decreased in the order: trinitrotoluene > di-
nitrotoluene > m-nitrotoluene, p-nitrotoluene > o-nitrotoluene.
Cyanosis due to the absorption of 2,4-DNT occurs when the
methemoglobin concentration of the blood is 15 percent or more. The
symptoms observed include blueness in the lips, the nose, and the
C-18
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earlobes. The individual usually feels well, has no complaints,
and insists that nothing is wrong until the methemoglobin concen-
tration approaches approximately 40 percent, when there usually is
weakness and dizziness; at levels of about 70 percent methemoglobin
there may be ataxia, dyspnea on mild exertion, tachycardia, nausea,
vomiting, and drowsiness (Hamblin, 1963). With an increase in
ambient temperature, and an associated increase in vapor pressure
there is an increased susceptibility to cyanosis due to higher
exposure levels of 2,4-DNT (Linch, 1974).
Some earlier studies provide useful information on the toxi-
city of 2,4-DNT. Animal experiments reported by White, et al.
(1902) indicate that 2,4-DNT is comparatively less toxic than 1,3-
dinitrobenzene. They found that cats may tolerate the repeated
oral administration of 2 or 4 ml of a 1 percent solution of 2,4 DNT
in cod liver oil, until a total of 24 ml has been given, without any
apparent toxic effect. Similarly, Zieger (1913) observed no toxic
effects due to the inhalation of 2,4 DNT vapors by cats, whereas
Kuhls (1908) found that the subcutaneous injection of cats with
0.05 to 0.5 g of 2,4-DNT dissolved in mineral oil resulted in death
within 2 to 23 days. Dambleff (1908) reported no indication of
percutaneous toxicity; similarly, Kuhls (1908) observed no toxic
effects in cats resulting from the cutaneous administration of 0.3
g/kg body weight, while Zieger (1913) found that two doses of 5 g
each were fatal to cats eight hours after administration.
A list of the toxic doses for a number of animal species is
presented in Table 4. The rat oral LD50 values listed in Table 4
are comparable to those of nitrobenzene and 2,6-DNT. The mouse
C-19
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TABLE 4
Acute Toxic Levels of 2,4-Dinitrotoluene
for Different Species*
Species
Rat
Mouse
Cat
Route
Oral
Oral
Oral
s.c.
Toxicity
LD50
LD50
MLD
LDLO
Dose
(mg/kg)
268
1,625
27
50-500
s.c. - subcutaneous; LDLo - lowest published lethal dose-
LD5Q - lethal dose 50 percent kill; MLD - minimum lethal
dose
*Source: Spector, 1956; Fairchild, et al. 1977; Vernot,
etal. 1977
C-20
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oral toxicity follows the order: aniline > 1,3,5-trinitrobenzene
>2,6-DNT > 3-nitrotoluene = 4-nitrotoluene = 2,5-DNT > 2,4-DNT
>2-nitrotoluene.
With regard to the human toxicity of 2,4-DNT, toxic effects
may only occasionally be observed from the handling of the pure
material. In addition to the complaints discussed above due to
methemoglobinemia, more severe cases involving dyspnea, dizziness,
sleepiness, and pain in the joints (especially in the knee) have
been reported (Perkins, 1919). Perkins (1919) also pointed out
that during the purification of the crude 2,4-DNT cakes, toxic
vapors may be inhaled and the material may be sufficiently absorbed
through the skin to cause toxic effects. Floret (1929) reported a
severe case of 2,4-DNT poisoning, in which the patient (a plant
worker) suffered from severe cyanosis and complained later of head-
ache, palpitation of heart, tightness in the chest, insomnia and
lack of appetite. Upon examination, medical findings indicated
tremors of varying intensity in the hands, arms, head, extended
fingers and tongue, nystagmus, and impaired reflexes. Lewin (1921)
stated that exposure to 2,4-DNT may result in temporary visual
disturbances.
The metabolic disturbances in workers exposed to 2,4-DNT were
extensively studied by McGee, et al. (1942). A number of signs and
symptoms of chemical intoxication appeared in a large group of
inexperienced workmen following their introduction into military
screening and coating houses which use 2,4-DNT. The chief symptoms
of a group of 154 workers so exposed were an unpleasant metallic
taste, weakness, headache, loss of appetite, and dizziness. Two-
thirds of the men in the group selected for study had these
C-21
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complaints at one time or another during the 12-month exposure
period. One-half of the group developed clinical signs of intoxi-
cation, chiefly pallor, cyanosis and low-grade anemia. Jaundice
was observed in two patients. No instances of permanent physical
impairment were found. The symptoms described by these workers are
presented in Table 5; Table 6 presents the chief findings from
clinical examinations of these workers.
There is no report in the literature that discusses the mecha-
nism of toxic action of 2,4-DNT per se. Usually its toxic action is
presented along with other structurally related aromatic nitro and
amino compounds. Most of the aromatic nitro and amino compounds
are not in themselves cyanogenie, but oxidation-reduction enzyme
systems promote biotransformation to active cyanogenic derivatives
that arise from either reduction of the nitro group or oxidation of
the amine. Most of the aromatic nitro and amino compounds that
have been investigated, regardless of species, including man, come
to a point of equilibrium,
Methemoglobin 7" ' ^ Hemoglobin,
beyond which, in spite of further dosage, no appreciable increase
in methemoglobin concentration can be obtained (Hamblin, 1963).
Bodansky (1951) also points out that there normally exists an equi-
librium in blood between hemoglobin and methemoglobin, which is
usually shifted far to the right. He believes that this shift is
regulated by various oxidizing and reducing substances produced
during _in vivo metabolism, and that such a concept helps to explain
the difference in degree of methemoglobin formation in various spe-
cies, as well as the differing rates of reduction of methemoglobin
C-22
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TABLE 5
Symptoms Presented by 154 2,4-Dinitrotoluene Workers*
Screening
House
Symptom Number of
Workmen
Unpleasant taste
in mouth
Weakness
Headache
Inappetence
Dizziness
Nausea
Insomnia
Pain in extremities
Vomiting
Numbness and tingling
Loss of weight
(5 pounds or more)
Diarrhea
62
51
48
42
43
39
37
26
22
18
7
3
Coating
House and
Air dry
Number of
Workmen
34
27
28
30
25
18
20
14
13
11
3
5
Total
Number
96
78
76
72
68
57
57
40
35
29
10
8
Percent
62
51
49
47
44
37
37
26
23
19
6.5
5.2
*Source: McGee, et al. 1942
C-23
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TABLE 6
Clinical Findings in 154 2,4-Dinitrotoluene Workers*
Finding
Pallor
Cyanosis
Anemia
Leucocy tosis
Hypotension
Skin rash
Leukopenia
Hepatitis and
Jaundice
Screening
House
(Number of
workmen)
40
38
28
12
8
2
2
1
Coating
House
(Number of
workmen)
15
14
8
7
1
4
3
1
Total
55
52
36
19
9
6
5
2
Percent
36
34
23
12
5.8
3.9
3.2
1 4
-L • T
*Source: McGee, et al. 1942
C-24
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to hemoglobin. Methemoglobdn-forming capacity in the cat of some
aromatic nitro and amino compounds including 2,4-DNT are presented
in Table 7.
From a ten-year study on the biological monitoring for indus-
trial exposure to cyanogenic aromatic nitro and amino compounds,
Linen (1974) establishes a reasonably good relationship between
causative agent structure and biochemical hazard in order to rank
the relative hazard of these chemicals. In this study, dinitro-
toluenes are ranked No. 12 (1 most potent, 13 least potent) indi-
cating that 2,4-DNT does not produce cyanosis as rapidly as other
cyanogenic aromatic nitro and amino compounds. From the similari-
ties of its toxic effects with other structurally related aromatic
nitro compounds, and also from the available information of its
metabolic pathway (as presented in Figure 1), a possible cyanosis
mechanism for 2,4-DNT is presented in Figure 3.
Subacute toxicity of 2,4-DNT in dogs, rats, and mice was stud-
ied by Ellis, et al. (1976). 2,4-DNT was given orally to dogs in
daily doses of 1, 5, or 25 mg/kg and to rats and mice in feed as
0.07, 0.2, or 0.7 percent of their diet for 13 weeks. Toxic effects
in the dogs and rats included inhibition of muscular coordination
in the hind legs, rigidity in extension of the hind legs, decreased
appetite, and weight loss. Only the appetite and weight effects
were observed in mice. The highest doses were lethal to some ani-
mals in all three species, while the lowest doses produced no toxic
effects. All species showed methemoglobinemia and anemia with
reticulocytosis. Characteristic tissue lesions were extramedul-
lary hematopoeisis in the spleen and liver, gliosis and demyelination
C-25
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TABLE 7
Methemoglobin-forming Capacity of Some Aromatic
Nitro and Amino Compounds in Cat*
Compound Molar ratio3
Nitrobenzene 0.86
1,3-Dinitrobenzene 7.1
1,3,5-Trinitrobenzene 4.3
2-Nitrotoluene 0.05
3-Nitrotoluene 0.04
4-Nitrotoluene Very slight
2,4-Dinitrotoluene 1.4
2,6-Dinitrotoluene 0.55
2,4,6-Trinitrotoluene 1.7
Aniline 2.5 (2.7)
Phenylhydroxylamine 34.0
3-Aminonitrobenzene 3.0
1,3-Diaminobenzene 1.4
Nitrosobenzene 8.6
*Source: Hamblin, 1963; De Bruin, 1976
Molar ratio of methemoglobin formed to dose of: test
compound
C-26
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Methemoglobm
reductase
Hemoglobin
NAD*
CH3
NO2
NHOH
4-HYDROXYLAMINO—2—^ITROTOLUE.NE
\
\
\
RAPID
Methemoglobm
NAOH *H*
CH3
N02
4—NITROSO—2—NITROTOLUENE
(0)
•••„ cvanooathic -,-'
intermediates
(H)
SLOW
CHS
CH3
n
• NO2
NH2
4—AMINO—2—NITHOTOLUSNH
NQ2
N02
2.4—OINITROTOI USNc
FIGURE 3
Suggested Metabolic Pathway for Cyanosis by
2,4-Dinitrotoluene and for 4-Amino-2-nitrotoluene
Based upon Data from Related Compounds.
C-27
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in the brain, and atrophy with aspermatogenesis in the testes.
2,6-DNT tested similarly in dogs (Ellis, et al. 1976) at 4, 20, or
100 mg/kg/day and in rats and mice at 0.01, 0.05, and 0.2S percent
in their diet, produced similar effects. It was concluded that the
primary subacute toxic effects of 2,4- and 2,6-DNT are seen in the
red cells, nervous system, and testes.
Chronic exposure of 2,4-DNT may produce liver damage', jaun-
dice, and reversible anemia due to blood damage (Linch, 1974; Key,
et al. 1977; Proctor and Hughes, 1978). Liver injury may be more
common than cyanosis, especially if the diet is deficient in pro-
tein (von Oettingen, 1941; Gleason, et al. 1969). Kovalenko (1973)
reports that the chronic exposure of 2,4-DNT in rats caused anemia
accompanied by reticulocytosis, a decrease in the level of sulf-
hydryl groups, and an increase in that of fibrinogen in the blood.
Influence of diet on the chronic toxicity of 2,4-DNT in mice
was studied by Clayton and Baumann (1944). Mice fed with 2,4-DNT
grew better on diets high in fat than those fed on other diets.
Those animals maintained on diets low in fat and fed 2,4-DNT showed
a retardation in the rate of growth, and many died within five
weeks. Mice raised to maturity on the low fat diet or on a procar-
cinogenic diet were less resistant to toxicity from parenteral 2,4-
DNT than mice raised on the other diets.
From another study on the effect of fat and calories on the
resistance of mice to chronic toxicity of 2,4-DNT, Clayton and
Baumann (1948) observed that mice ingesting 2,4-DNT grew less and
died faster when fed a diet moderately low in fat (0.46 percent)
than when fed the same amount of 2,4-DNT per calorie in diets
C-28
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containing 5 or 30 percent added fat. Fat likewise appeared to
minimize the toxic effects of 2,4-DNT in rats. When the effects of
a low calorie intake are corrected for, 2,4-DNT per se retarded
growth only slightly. Clayton and Baumann (1948) noted that many
different fats and oils appeared equally active in minimizing the
toxic effects of 2,4-DNT.
The effect of diet on the susceptibility of the rat to chronic
poisoning by 2,4-DNT was also studied in detail by Shils and Gold-
water (1953). A high intake of fat, in the form of corn oil, was
found to be definitely beneficial with respect to the survival of
rats subsisting on a low-protein intake and receiving 2,4-DNT
parenterally. Increased amounts of protein with a low fat diet
prevented death, regardless of the mode of 2,4-DNT administration.
Synergism and/or Antagonism
Ingestion of alcohol has a synergistic effect on the toxicity
of 2,4-DNT. Friedlander (1900) discussed a patient who exhibited
acute confusion and retrograde amnesia after exposure to 2,4-DNT
and drinking a small amount of beer. This synergistic effect of
alcohol on the toxicity of 2,4-DNT was also noted by McGee, et al.
(1942). Of the group of 154 male workers exposed to 2,4-DNT in
military screening and coating houses, 23 showed a reduced toler-
ance for alcohol and 31 stated that their toxic symptoms had been
aggravated by ingesting alcohol. Some workers reported that they
had found it impossible to drink any alcoholic beverage within two
to three hours after finishing a shift without experiencing reac-
tions such as substernal pressure, precardial palpitation, fullness
in the head, and severe acute illness.
C-29
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The ingestion of alcohol normally causes increased suscepti-
bility to cyanosis; thus, alcohol in any form should never be
administered to a victim of 2,4-DNT poisoning. Furthermore, since
the body eliminates 2,4-DNT rather slowly, abstention from alcohol-
ic beverages should be practiced for several days after 2,4-DNT
exposure (Von Oettingen, 1941; Key, et al. 1977; Proctor and Hughes
1978).
Teratogenicity
No studies were found in the literature which addressed the
teratogenicity of 2,4-DNT or the other isomers of dinitrotoluene.
Mutagenicity
The data available in the literature on the mutagenicity of
2,4-DNT are limited and rather confusing. Studies by Hodgson, et
al. (1976) show some positive results. The mutagenic effect of
2,4-DNT on germinal cells was studied by these authors using the
dominant lethal assay on rats fed a diet containing 2,4-DNT for 13
weeks. Females mated to males treated with 0.2 percent 2,4-DNT
showed a significant increase in the number of dead implants/total
implants over control animals.
Hodgson, et al. (1976 abstract) also screened for somatic cell
mutation effects by cytogenetic analysis of lymphocyte and kidney
cultures derived from rats fed 0.2 percent of 2,4-DNT for 19 weeks.
No increase in the frequency of translocations or chromatid breaks
was observed in either the lymphocyte or kidney cultures. However,
significant increases in the frequency of chromatid gaps were ob-
served in kidney cultures after five weeks and in lymphocytes at 19
weeks. This would suggest that 2,4-DNT has a potential for inducing
C-30
-------
damage in somatic cells. _I_n vitro studies using the CHO-KI
test system were negative. On the other hand, microbial tests
using Salmonella typhimurium TA 1535 indicated that 2,4-DNT is cap-
able of producing base-pair mutations. Details of the methodology
used were not available.
There are two other reports in the literature (Simmon, et al.
1977; Cotruvo, et al. 1977) which discuss the mutagenic effects of
products from ozonation or chlorination reactions of 2,4-DNT and
other related di- and trinitrotoluenes. In the study by Simmon, et
al. (1977), a number of compounds present in waste water from muni-
tions plants were examined before and after ozonation or chlorina-
tion to determine whether mutagenic activity was affected by the
treatment. Test materials included 1,3-dinitrobenzene; 2,4-DNT;
3,5-DNT, 2,4,6-TNT; 2,4,6-TNT production waste water; hexahydro-
1,3,5-trinitro-s-triazine (RDX); octahydro-1,3,5,7-tetranitro-s-
tetrazine (HMX); components of photolyzed 2,4,6-TNT; penta-
erythritol tetranitrate, and trinitroresorcinol. The in vitro
mutagenic assays used were the Salmonella/microsome assay (Ames, et
al. 1973a,b) with strains TA 1535, TA 1537, TA 1538, TA 98, and TA
100 and mitotic recombination in the yeast, Saccharomyces cere-
vis iae D3. A metabolic activation system using the postmitochon-
drial supernatant fraction of liver from rats, pretreated with
Aroclor 1254, was included in each assay procedure. Under these
conditions, neither ozonation nor chlorination significantly
altered the mutagenic activity of the nitro aromatic materials
tested, including 2,4-DNT.
C-31
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In the investigation of mutagenicity of products of ozonation
in water by Cotruvo, et al. (1977), compounds such as 2,4-DNT,
phenol, hydroquinone and nitrilotriacetic acid were found to give
anomalous results in Saccharomyces after ozonation. Although ele-
vated activity was indicated in some of the experiments,, it was not
dose-related. At the concentrations tested (0.08 ug/plate, highest
dose), 2,4-DNT was not mutagenic in the Salmonella assay before or
after ozonation. The highest concentration tested in the Sacchar-
omyces assay, 0.004 percent was not mutagenic or toxic. There was
generally a higher number of mitotic recombinants after ozonation,
but the response was not dose-related. The products of ozonation
of TNT condensate-water mixture (complex nitroaromatics containing
primarily 2,4-and 2,6-DNTs) were also tested for mutagenicity. Two
new products (m/e 166 and 270) were found in the GC/MS profile. The
fragmentation pattern of the m/e 166 compound was found to be con-
sistent with a nitrosonitrotoluene but was not confirmed. Prior to
ozonation, the TNT condensate-water mixture was mutagenic in
Salmonella assays but not in Saccharomyces. After ozonation, the
mixture was weakly mutagenic in only one experiment with TA 1535
and TA 100 in the absence of metabolic activation; thus, activity
was considerably reduced after ozonation. A duplicate experiment
showed no activity. These mutagenicity results are presented in
Table 8.
Carcinogenicity
There are two reports in the literature (NCI, 1978; Lee, et
al. 1978) which address the Carcinogenicity of 2,4-DNT. A bioassay
of practical-grade 2,4-DNT for possible carcinogencity (NCI, 1978)
C-32
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TABLE 8
Mutagenic Assay Results of Munitions Compounds*
o
u>
U)
Munitions
Compounds
2, 4 -Dinitro toluene
TNT condensate water
Initial
Concentration
(ppm)
28.3
35.4
ReTime°n «"«*•«
(min) m
20 96
100 9.3
Salmonella
v Activity
8.4/3.8 -/-
7.2/3.6 +/-
Saccha-
romyces
Activity
-/±
V-
Comments
elevated activity
in high dose, not
dose related
activity found in
one test, reduced
by ozonation
*Source: Cotruvo, et al. 1977
-------
was conducted using Fisher 344 rats and B6C3F-L mice. 2,4-DNT was
administered in the feed to male and female rats; the low and high
time-weighted average doses were 17.6 and 44.0 mg/kg/day for male
rats and 25.3 and 63.4 mg/kg/day for female rats, respectively.
For male and female mice, the low and high time-weighted average
doses were 16.3 and 81.5 mg/kg/day, respectively. Both rats and
mice were treated with 2,4-DNT for 78 weeks. In the male rats, a
significantly higher incidence of fibroma of the skin and subcuta-
neous tissue occurred in the high and low dose groups when compared
to their respective controls (Table 9). A statistically signifi-
cant incidence of fibroadenoma of the mammary gland occurred in the
treated female rats of the high dose group (Table 10). It should be
noted that the above-mentioned tumors were benign.
There were certain unusual neoplasms (i.e., hemangiosarcoma in
the subcutis, hemangiosarcoma of the urinary bladder, and prostrate
gland adenocarcinoma) that occurred at low incidences in high dose
male rats but did not occur in either low dose or control male rats.
The authors (NCI, 1978) considered that these tumors were not re-
lated to chemical administration.
For the mice, there were no tumors in either sex having a sta-
tistically significant positive association between administration
of 2,4-DNT and incidence of tumor. As such there is no convincing
evidence of tumorigenicity in B6C3F, mice at the dose levels of
2,4-DNT used in these experiments.
At this point, it is relevant to present some of the com-
ments made regarding this carcinogenesis study by the Data
C-34
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TABLE 9
Summary of the Significant Primary Tumors at Specific Sites in Male Rats Treated with 2,4-Dinitrotoluene
Low Dose
Topography: Morphology Control
Subcutaneous Tissue or Skin: Fibroma 0/46(0.00)
P Values0
Relative
Weeks to
Risk (Control)*1
Lower Limit
Upper Limit
First Observed Tumor
High Dose Low Dose
Control
0/25(0.00) 7/49(0.14)
P = 0.008
Infinite
1.827
Infinite
96
High Dose
13/49(0.27)
P = 0.003
Infinite
2.106
Infinite
85
o
Jj *Source: NCI, 1978
ui
aTreated groups received time-weighted average concentrations of 17.6 and 44.0 mg/kg/day in feed.
bNumber of tumor-bearing animals/number of animals examined at site (proportion).
cThe probability level for the Fisher exact test for the comparison of a treated group with the control group
is given beneath the incidence of tumors in the treated group when P<0.05? otherwise, not significant (N.S.)
is indicated. A negative designation (N) indicates a lower incidence in the treated group than in the control
group.
dThe 95* confidence interval of the relative risk of the treated group to the control group.
-------
TABLE 10
Summary of the Significant Primary Tumors at Specific Sites in Female Rats Treated with 2,4-Dinitrotoluene*'a
Topography:
Mammary Gland:
P Values0
Relative Risk
Weeks to First
Morphology
Fibroadenoma
(Control)d
Lower Limit
Upper Limit
Observed Tumor
Low Dose
Control
9/48(0.19)
92
High Dose
Control
4/23(0.17)
109
Low Dose
12/49(0.24)
N.S.
1.306
0.559
3.183
83
High Dose
23/50(0.46)
P = 6.016
2.645
1.062
9.435
69
*Source: NCI, 1978
u>
Treated groups received time-weighted average concentrations of 25.3 and 63.4 mg/kg/day in feed.
Number of tumor-bearing animals/number of animals examined at site (proportion).
£
The probability level for the Fisher exact test for the comparison of a treated group with the control group is
given beneath the incidence of tumors in the treated group when P<0.05; otherwise, not significant (N.S.) is
indicated. A negative designation (N) indicates lower incidence in the treated group than in the control group.
The 95% confidence interval of the relative risk of the treated group to the control group.
-------
Evaluation/Risk Assessment Subgroup of the Clearinghouse on Envi-
ronmental Carcinogens (NCI, 1978):
1. The tumors in the treated rats must be viewed with
concern, especially since the maximum tolerated
dose may not have been attained.
2. Since 2,4-DNT is an intermediate in the production
of dyes, there may be considerable human exposure
from its residues in dye products. Hence, there may
be a potential for human risk because of the in-
creased tumor incidence seen in the treated rats.
3. The biological activity of 2,4-DNT may ,be due to its
possible conversion to the diamine compound, 2,4-
toluenediamine. The rate of its enzymatic conver-
sion may limit its activity.
4. These data do not allow an assessment of human risk.
5. In view of the significant number of benign tumors
in the treated rats and widespread human exposure,
2,4-DNT should be considered for retest using an-
other species and route of exposure, especially
dermal.
Another bioassay of practical grade 2,4-DNT for possible car-
cinogenicity was conducted by Lee, et al. (1978) using CD rats
(Charles River Breeding Laboratory, Wilmington, Mass.) The high
dose, with 2,4-DNT intake of 34.0 mg/kg/day in male rats and 45.0
rog/kg/day in female rats, was quite toxic, causing decreased weight
gain and shortened lifespan. Target organs included the blood
(toxic anemia), the liver (hepatocellular carcinoma), the testis
(aspermatogenesis), and connective tissue in male rats (fibromas),
and the mammary tissue in female rats (fibroadenomas). No specific
effects were seen on the reproductive process, on chromosomes, or
on the metabolism of 2,4-DNT. The middle dose, with 2,4-DNT intake
of 3.90 mg/kg/day in male rats and 5.10 mg/kg/day in female rats,
was somewhat toxic. It caused similar effects in some, more
C-37
-------
susceptible, individual rats. The low dose, with 2,4-DNT intake of
0.57 and 0.71 mg/kg/day in male and female rats respectively, had
no apparent toxic effects. The carcinogenicity results for male
and female rats are summarized in Tables 11 and 12, respectively.
The interim results (weeks 52 and 55) of a feeding study in
rats given 2,4-DNT indicated a significant increase in the inci-
dence of hepatocellular carcinomas in both males and females (Chem-
ical Industry Institute of Toxicology, 1978). Although this study
has not yet been published or reviewed in detail, it supports the
results of Lee, et al. (1978).
Since 2,4-toluenediamine (2,4-TDA) is a possible metabolic
product of 2,4-DNT (as seen in Figure 1) and is mentioned in the
critique of the Lee, et al. (1978) study, it is reasonable to dis-
cuss briefly the carcinogenicity and mutagenicity of 2,4-TDA.
2,4-TDA is widely used in the production of human hair dyes.
Umeda (1955) reported that the repeated subcutaneous injections of
2,4-TDA induced rhabdomyosarcomas in 100 percent of rats treated.
Rats fed diets containing 2,4-TDA developed hepatocellular carcino-
mas (Ito, et al. 1969). Similarly Swiss-Webster mice fed 2,4-TDA
showed a high incidence of lung neoplasms (Stoats, 1972). In con-
trast, the recent study by Giles, et al. (1976) indicates that the
2,4-TDA and other hair dye ingredients did not augment the develop-
ment of primary lung neoplasms in mice. Skin neoplasms were seen
in most groups of Swiss-Webster mice, but the incidence of these
tumors in treated animals when compared to control mice, was not
significant. The 2,4-TDA under these experimental conditions was
found to be nontoxic and noncarcinogenic to the skin of mice.
C-38
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TABLE 11
Summary of the Male Rats with Tumors
After being Fed 2,4-Dinitrotoluene for 24 months*
Dose Mammary
(mg/kg/day) Tumor/Total Percent
0
0.57
3.90
34.0
1/37
0/37
0/29
17/23
3
0
0
74
*Source: Lee, et al. 1978
C-39
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TABLE 12
Separate Tumor Incidence for All Age Groups
Female Rats fed 2,4-DNT*
n
i
Liver
Mammary ,
gland tumor
Combined mammary
gland and liver
tumor
*Source: Lee, e
ci . r
Control
(0 ppm)
(0 mg/kg/day)
0/31
11/31
11/31
t al. 1978
0.0015%
(15 ppm)
(0.71 mg/kg/day)
3/43
(N.S.)
12/43
(N.S. )
13/43
(N.S. )
0.01%
(100 ppm)
(5.1 mg/kg/day)
3/35
(N.S. )
18/35
(N.S. )
18/35
(N.S. )
.
0.07%
(700 ppm)
(45 mg/kg/day)
30/42d
(p=3.96 x 10
34/43d
(p=1.75 x 10
35/43d
(p=7.01 x 10
.
4
T »
4
T Y
s
\
in
which livers were examined.
Number of animals with either adenoma, fibroadenoma, fibroma, or adenocarcinoma of the
mammary gland/no, of animals in which mammary gland tissues were examined.
°The number of animals which had either liver or mamary gland tumors or both/no, of ani-
mals in which the liver and mammary glands were examined.
The total number of animals examined microscopically for mammary gland tumors was 43.
One animal out of these 43 rats was missing liver tissue, i.e., livers examined were 42.
However, the animals which was missing liver tissue had a mammary gland tumor, so it was
counted as an animal having a tumor. Therefore, the total number of animals examined
0.07% dose was 43.
-------
On the other hand, it has been shown that 2,4-TDA is a mutagen
in several systems. A good correlation between mutagenicity of
2,4-TDA in the Salmonella/ microsome test and morphological trans-
formation in hamster embryo cell system was observed by Shah, et
al. (1977). 2,4-TDA usually requires metabolic activation by rat
liver microsomal enzymes (S9) for mutagenesis in tester strains TA
1538 and TA 98 (McCann, et al. 1975; Shah, et al. 1977; Dybing, et
al. 1977; Pienta, et al. 1977). In contrast, transformation of
hamster cells was induced without the addition of external enzymes
(Shah, et al. 1977), presumably because the cells can metabolize
2,4-TDA to its active derivatives. There was no mutagenic activity
in the strain TA 100, indicating that 2,4-TDA is not a base pair
mutagen. The dose-response curves obtained with tester strains TA
1538 and TA 98 demonstrated that 2,4-TDA is metabolized by the 39
activation mixture to a frameshift mutagen (Shah, et al. 1977).
2,4-TDA was also found to be mutagenic in the sex-linked recessive
lethal test in Drosophila melanogaster male germ cells (Blijleven,
1977; Fahmy and Fahmy, 1977; Venitt, 1978).
C-41
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CRITERION FORMULATION
Existing Guidelines and Standards
At present, no standard for exposure to 2,4-DNT in drinking or
ambient water has been set in the United States. However, a Rus-
sian study (Korolev, et al. 1977) recommends that a maximum permis-
sible concentration in the surface waters should be set at a level
of 0.5 mg/1 for each DNT isomer.
The American Conference of Governmental Industrial Hygienists
(ACGIH) recommends a threshold limit value-time weighted average
(TLV-TWA) concentration of 1.5 mg of 2,4-DNT per cubic meter of air
(1.5 mg/m ) including dermal exposure for a normal eight-hour work-
day of a 40-hour workweek (ACGIH, 1978). This value represents the
highest level to which nearly all workers may be repeatedly ex-
posed, day afer day, without adverse effect. This TLV-TWA was set
by analogy with chemically similar nitro aromatic compounds. A
threshold limit value short-term exposure level (TLV-STEL) of 5 mg
of 2,4-DNT/m of air was also set by the ACGIH (1978). The TLV-STEL
is defined as the maximal allowable concentration to which workers
can be exposed for a continuous period of up to 15 minutes without
suffering from 1) irritation, 2) chronic or irreversible tissue
change, or 3) narcosis of sufficient degree to increase accident
proneness, impair self-rescue, or materially reduce work effi-
ciency. No more than four exposures to the TLV-STEL per day are
permitted, with at least 60 minutes between exposure periods.
Additionally, the daily TLV-TWA must not be exceeded.
C-42
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Current Levels of Exposure
No data on the extent of human exposure to 2,4-DNT are avail-
able in the literature. However, a study of the concentration of
explosives in air by isotope dilution analysis (St. John, et al.
1975) reported a concentration of 184 ppb v/v (=1.384 mg/m ) of
2,4-DNT in air at 25°C, which is very close to the TLV-TWA value
noted above.
Special Groups at Risk
The main group expected to be at high risk for exposure to
2,4-DNT is industrial workers involved in the manufacturing or han-
dling of 2,4-DNT in places such as ammunition, dye, and polyure-
thane plants.
Basis and Derivation of Criteria
Although both bioassays for carcinogenicity were considered in
assessing the potential carcinogenic effect of dinitrotoluene (NCI,
1978; Lee, et al. 1978), the Data Evaluation/Risk Assessment Sub-
group of the Clearinghouse on Environmental Carcinogens (NCI, 1978)
expressed reservations about the adequacy of its bioassay for use
in assessing human risk. Therefore, the criterion was developed
from the Lee, et al. (1978) study.
Both of these carcinogencity studies with dietary administra-
tion of 2,4-DNT showed increased incidences of fibroadenomas of the
subcutaneous tissue and inanition in male rats and fibroadenomas of
the mammary gland and inanition in female rats. In addition, the
Lee, et al. study showed a significant increase in liver tumors in
female rats. It should be noted that both of these bioassays
used technical grade 2,4-DNT which contained other DNT isomers as
C-43
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impurities. The influence of the other isomers and impurities on
the carcinogenic activity of technical grade 2,4-DNT cannot be
properly assessed at this time.
Under the Consent Decree in NRDC v. Train, criteria are to
state "recommended maximum permissible concentrations (including
where appropriate, zero) consistent with the protection of aquatic
organisms, human health, and recreational activities." 2,4-DNT is
suspected of being a human carcinogen. Because there is no recog-
nized safe concentration for a human carcinogen, the recommended
concentration of 2,4-DNT in water for maximum protection of human
health is zero.
Because attaining a zero concentration level may be infeasible
in some cases and in order to assist the Agency and states in the
possible future development of water quality regulations, the con-
centrations of 2,4-DNT corresponding to several incremental life-
time cancer risk levels have been estimated. A cancer risk level
provides an estimate of the additional incidence of cancer that may
be expected in an exposed population. A risk of 10 for example,
indicates a probability of one additional case of cancer for every
100,000 people exposed, a risk of 10"6 indicates one additional
case of cancer for every million people exposed, and so forth.
In the Federal Register notice of availability of draft ambi-
ent water quality criteria, EPA stated that it is considering set-
ting criteria at an interim target risk level of 10~5, 10"6, or
10 as shown in the following table.
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Exposure Assumptions Risk Levels and Corresponding Criteria (I]
(per day) _7 _6 _5
10 7 10 b 10 a
2 liters of drinking 0.011 jjg/1 0.11 jug/1 1.1 jug/1
water and consumption
of 6.5 grams fish and
shellfish. (2)
Consumption of fish 0.91 jug/1 9.1 ug/1 91 jug/1
and shellfish only.
(1) Calculated by applying a linearized multistage model, as dis-
cussed in the Human Health Methodology Appendices to the
October 1980 Federal Register notice which announced the
availability of this document, to the animal bioassay data
presented in Appendix II and in Table 12. Since the extrapola-
tion model is linear at low doses, the additional lifetime
risk is directly proportional to the water concentration.
Therefore, water concentrations corresponding to other risk
levels can be derived by multiplying or dividing one of the
risk levels and corresponding water concentrations shown in
the table by factors such as 10, 100, 1,000 and so forth.
(2) Approximately 1.2 percent of the 2,4-DNT exposure results from
the consumption of aquatic organisms which exhibit an average
bioconcentration potential of 3.8-fold. The remaining 98.8
percent of 2,4-DNT exposure results from drinking water.
Concentration levels were derived assuming a lifetime exposure
to various amounts of 2,4-DNT, (1) occurring from the consumption
of both drinking water and aquatic life grown in waters containing
the corresponding 2,4-DNT concentrations and, (2) occurring solely
from consumption of aquatic life grown in the waters containing the
corresponding 2,4-DNT concentrations. Although total exposure
C-45
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information for 2,4-DNT is discussed and an estimate of the contri-
butions from other sources of exposure can be made, this data will
not be factored into ambient water quality criteria. The criteria
presented, therefore, assume an incremental risk from ambient water
exposure only.
Results obtained from the linearized multistage model give 1.1
jug/1 as the dose level which establishes a carcinogenicity risk
level in water for humans of 1 in 100,000. It should be noted that
this level is one five-hundredth the level of 0.5 mg/1 for surface
water recommended in the USSR (Korolev, et al. 1977).
Using the TLV-TWA value for 2,4-DNT of 1.5 mg/m3 recommended
by the ACGIH (1978), the daily occupational exposure gives a value
of 5.4 mg of 2,4-DNT per day (see Appendix I for calculation). At
an ambient water level of 1.1 jjg/1, assuming a daily intake of 2
liters and a daily aquatic organism intake of 6.5 g with a bio-
accumulation factor of 3.8, it can be shown (see Appendix I for
calculation) that the daily intake of 2,4-DNT is 0.0015 mg/day
which is substantially below the occupational exposure level and
hence, will not pose a significant additional burden of exposure by
those at risk occupationally. This proposed level in ambient water
leads to an intake (0.0015 mg/day) which would cause an insignifi-
cant effect in terms of contribution to methemoglobinemia (25 mg of
2,4-DNT/l) (Cartwright, 1977; Proctor and Hughes, 1978). It would
thus appear that this extrapolation, using female rat data (Lee, et
al. 1978) provides a level of ambient water exposure which achieves
a high margin of safety.
C-46
-------
It should be noted that data are urgently needed in the fol-
lowing areas to evaluate properly any hazard from 2,4-DNT:
1. Monitoring of workers exposed to 2,4-DNT in industries manu-
facturing or using the chemical.
2. Monitoring of public water supplies and industrial and munici-
pal effluents to determine an expected range of concentrations
under differing environmental conditions.
3. More detailed studies on the pharmacokinetics of 2,4-DNT using
several animal species and if possible, occupationally exposed
humans.
4. Evaluation of chronic toxicity and teratogenicity using cur-
rently acceptable techniques.
5. Detailed and definitive mutagenicity studies of 2,4-DNT and
its metabolites using several assay systems such as: a) Sal-
monella/microsomal, b) dominant lethal, c) Drosophila, and
d) host mediated assay.
6. More definitive studies on the carcinogenicity of 2,4-DNT and
its metabolites using several animal species (and if possible,
occupationally exposed humans) using oral and dermal routes.
C-47
-------
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APPENDIX I
1. Calculation of Daily Occupational Exposure level of 2,4-Dini-
trotoluene based on its Threshold Limit Value-Time Weighted
Average (TLV-TWA) concentration (ACGIH, 1978):
TLV-TWA for 2,4-DNT =1.5 mg/m° of air for a normal 8-hour
workday or 40-hour workweek
= 1.5 x 10~3mq
liter of air
= 1.5 jig.
liter of air
Therefore, the daily occupational level for
2 4-nMT = 1.5 jug 7.5 liter of air . 60 minute 8 hour
' ~ liter — minute hour x day
= 5,400 ug
= 5.4 mg
where 7.5 liter of air is the ventilation rate for an average 70 kg
man doing moderately hard work (Kamon, 1979).
2. Calculation of Daily Intake Level of 2,4-DNT:
The assumptions used for this calculation are:
a) Bioaccumulation factor of 3.8 as determined for the blue-
gill sunfish (U.S. EPA report, Duluth, Minnesota),
b) Average weight of aquatic organisms consumed per day is
6.5 g, and
c) Consumption of water per person per day is 2 liters over
a period of 70 years.
d) A concentration of 2,4-DNT in water of 740 ng/1.
C-67
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The concentration of 2,4-DNT in fish = 740 x 3.8 x 0.0065 =
18 ng from aquatic organisms
Daily intake of 2,4-DNT from 2 liters of drinking water =
740 ng/1 x 2 = 1,480 ng
Total intake/day = 1,480 + 18 ng or 1,498 ng (1.50 jag or
.00150 mg)
C-68
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APPENDIX II
Summary and Conclusions Regarding the Carcinogenicity
of 2,4-Dinitrotoluene*
2,4-Dinitrotoluene (2,4-DNT) is a pale yellow crystalline
solid with a melting point of 70 C and has a moderate fire explo-
sion risk. A combined U.S. production of approximately 272 billion
pounds of 2,4- and 2,6-dinitrotoluene isomers was reported in 1975.
2,4-DNT is widely used as a raw material for dyestuffs and for ure-
thane polymers, as a modifier for smokeless powders, and as a gela-
tinizing and waterproofing agent in military and commercial explo-
sives.
The reports concerning the mutagenicity of 2,4-DNT are limited
and their results conflicting. However, this compound was found to
be mutagenic in the dominant lethal assay in rats and in microbial
tests using Salmonella typhimurium TA1535 indicating base-pair sub-
stitution.
Two reports concerning the Carcinogenicity of 2,4-DNT are in
the literature. The first is a National Cancer Institute (NCI)
two-year bioassay in male and female Fisher 344 rats and B6C3F,
mice fed 2,4-DNT (1978). The major pathologic findings were pre-
sent in the rats. These included fibromas of the skin and subcu-
taneous tissues in males and fibroadenomas of the mammary gland in
the females. These tumors are benign and were dose-related. The
mice had no statistically significant carcinogenic response to the
administration of 2,4-dinitrotoluene.
The second study relating oral administration of 2,4-DNT to
Carcinogenicity was a bioassay in male and female Charles River CD
rats and CD-I mice fed 2,4-DNT for two years (Lee, et al. 1978).
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The major pathologic findings in the rats included a significant
increase of hepatocellular carcinomas (p = 7.1 x 10~6) and neo-
plastic nodules (p = 0.01) in the liver of females, mammary gland
tumors of the female (p = 8.3 x 10 ) and the suspicious increase
of hepatocellular carcinomas of the liver in males. All of these
rat tumors were in high dose animals. The pathologic finding in
the mice was the highly significant (p = 1.5 x 10 ) increase of
kidney tumors in the males of the middle dose group.
The induction of hepatocellular carcinomas, hepatocellular
neoplastic nodules and mammary tumors in female rats and kidney
tumors in male mice from the administration of 2,4-dinitrotoluene
indicates that it is likely to be a human carcinogen.
The water quality criterion for 2,4-dinitrotoluene is based on
the induction of mammary tumors, hepatocellular carcinomas, and
hepatocellular neoplastic nodules in female Charles River CD rats
fed various doses of 2,4-DNT for 24 months (Lee, et al. 1978). It
is concluded that the water concentration of 2,4-dinitrotoluene
should be less than 1.1 ug/1 in order to keep the lifetime cancer
-5
risk below 10 .
*This summary has been prepared and approved by the Carcinogens
Assessment Group of EPA on June 19, 1979.
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Summary of Pertinent Data
The water quality criterion for 2,4-dinitrotoluene is derived
from the oncogenic effects observed in the mammary gland and liver
of female Charles River CD rats fed various doses of 2,4-DNT for 24
months, with the surviving animals sacrificed one month later. The
incidence of mammary and/or liver tumors is listed below for the
various doses, along with other parameters used in the extrapo-
lation:
Dose Incidence
(mg/kg/day) (no. responding/no, tested)
0.0 11/31
0.75 13/43
5.0 18/35
35.0 35/43
le = 720 days w = 0.464 kg
Le = 750 days R = 3.8 I/kg
L = 750 days
With these paramethers the carcinogenic potency factor for
-2 -1
humans, <3i*> is 3.6965 x 10 (mg/kg/day) . The resulting water
concentration of 2,4-dinitrotoluene calculated to keep the indivi-
dual lifetime cancer risk below 10" is 1.1 ug/1.
C-71 * U S. GOVERNMENT PRINTING OFFICE 1980 720-016/4378
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