DINITROTOLUENE
Permanent Collection
Ambient Water Quality Criteria
Criteria and Standards Division
Office of Water Planning and Standards
U.S. Environmental Protection Agency
Washington, D.C.
-------
CRITERION DOCUMENT
DINITROTOLUENES
CRITERIA
Aquatic Life
2,3-d initrotoluene
For 2,3-dinitrotoluene the criterion to protect fresh-
water aquatic life as derived using the Guidelines is 12 ug/1 as a
24-hour average and the concentration should not exceed 27 ug/1 at
any time.
For 2,3-dinitrotoluene the criterion to protect salt-
water aquatic life as derived using procedures other than the
Guidelines is 4.4 ug/1 as a 24-hour average and the concentration
should not exceed 10 ug/1 at any time.
2,4-d initrotoluene
For 2,4-dinitrotoluene the criterion to protect fresh-
water aquatic life as derived using procedures other than the
Guidelines is 620 ug/1 as a 24-hour average and the concentration
should not exceed 1,400 ug/1 at any time.
For saltwater aquatic life, no criterion for 2,4-di-
nitrotoluene can be derived using the Guidelines, and there are
insufficient data to estimate a criterion using other procedures.
Human Health
For the maximum protection of human health from the potential
carcinogenic effects of exposure to 2,4-dinitrotoluene through
ingestion of water and contaminated aquatic organisms, the ambient
water concentration is zero. Concentrations of 2,4-dinitrotoluene
estimated to result in additional lifetime cancer risks ranging
Erom no additional risk to an additional risk of 1 in 100,000 are
-------
presented in the Criterion Formulation section of this document.
The Agency is considering setting criteria at an interim target
risk level in the range of 10~5, 10~6f or 10~7 with corresponding
criteria of 740 ng/1, 74.0 ng/lf and 7.4 ng/1, respectively.
-------
DINITROTOLUENES
Introduction
Dinitrotoluene (DNT) is an ingredient of explosives
for commercial and military use because of its waterproofing
action and explosive potential. Use is also made of DNT
as a chemical stabilizer in the manufacture of smokeless
powder.
DNT has been shown to enter the body through inhalation
of vapors or dust particles, ingestion of contaminated food,
and absorption through the skin. As a result of exposure
to DNT, workers have experienced muscular weakness, headaches,
and dizziness; this exposure also has been suspected of
causing pallor, cyanosis,.and anemia.
DNT is produced by nitration of toluene to nitrotoluene
to dinitrotoluene in a nitric and sulfuric acid solution
(Lopez, 1977). In 1975, the production of 2,4- and 2,6-
DNT in the United States was 264,030 metric tons (U.S. Int.
Trade Comm., 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 Chem-
ists Acsoc., 1966) or dinitrotoluol (Sax, 1963).
Nitration of o-nitrotoluene yields mostly 2,4- and
2 ,6-dini trotoluene, CH-^CgE^ (NO2) 2' i° tne ratio of about
65.35 (Wiseman, 1972).
A-l
-------
2,4-DNT has a molecular weight of 182.14, a melting
point of 71°C, a boiling point of 300°C with decomposition,
and a density of 1.3208 at 71°C (Weast, 1975). Its solubility
in water is 270 mg/1 water at 22°C? 94 g/1 ether at 22°C
and 21.9 g/1 carbon disulfide at 17°C (Kirk and Othmer,
1967). It is also readily soluble in ethanol at 15°C (30.5
g/1) (Kirk and Othmer, 1967).
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).
Except for their tendency to decompose at elevated
temperatures, dinitrotoluenes are relatively stable. At
250°C, commercial grades of dinitrotoluene decompose at
non-sustaining rates. However, at approximately 280 C rapid
self-sustaining decomposition occurs. Dinitrotoluenes may
burn safely if unconfined, but if confined may result in
an explosion. Decomposition may occur at lower temperatures
in the presence of impurities (Manufacturing Chemists Assoc.,
1966) . Mixtures of dinitrotoluene isomers are intermediates
in the manufacture of toluene diisocyanates (Wiseman, 1972}.
Because of the deactivating effect of the two nitro groups
in dinitrotoluenes, the synthesis of trinitrotoluene (TNT)
does not occur as readily (Wiseman, 1972).
A-2
-------
REFERENCES
Kirk, R.E., and D.F. Othmer. 1967. Kirk-Othmer Encyclopedia
of Chemical Technology. 2nd ed. John Wiley and Sons, Inc.,
New York.
Lopez, A.W. 1977. Toluene diisocynate. A paper presented
at the Am. Ind. Chem. Eng. Conference, Houston, Tex., March 23
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
industry- Noyes Data Corp., Park Ridge, N.J.
Snell, F.D., and L.S. Ettre, eds. 1971. Encyclopedia of
Industrial Chemical Analysis. Interscience Publishers, John
Wiley and Sons, Inc., New York.
U.S. International Trade Commission. 1977. Synthetic organic
chemicals, United States production and sales, 1975. U.S
Government Printing Office, Washington, D.C.
A-3
-------
Weast, R.C., ed. 1975. Handbook of chemistry and physics.
CRC Press, Cleveland, Ohio.
Wiseman, P- 1972. An introduction to industrial organic
chemistry. Interscience Publishers, John Wiley and Sons/
Inc., New York.
A-4
-------
AQUATIC LIFE TOXICOLOGY*
FRESHWATER ORGANISMS
Introduction
The data base for dinitrotoluenes is limited but 2,3-dinitro-
toluene appears to be up to two orders of magnitude more adutely
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
The unadjusted 96-hour LC50 for the fathead minnow and
2,4-dinitrotoluene is 31,000 ug/1 (Table 1). After adjustment for
test methods and species sensitivity, the Final Fish Acute Value
for this compound is 4,300 ug/1. The unadjusted 96-hour LC50 for
the more toxic 2,3-dinitrotoluene and the bluegill is 330 ug/1,
and this datum results in a Final Fish Acute Value of 46 ug/1
(Table 1).
*The reader is referred to the Guidelines for Deriving Water
Quality Criteria for the Protection of Aquatic Life [43 FR 21506
(May 18, 1978) and 43 FR 29028 (July 5, 1978)] in order to better
understand 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 the calcula-
tions for deriving various measures of toxicity as described in
the Guidelines.
B-l
-------
Forty-eight-hour EC50 values are available for Daphnia magna
for both 2,3- and 2,4-dinitrotoluene and are 660 and 35,000 ug/1,
respectively (Table 2). The Final Invertebrate Acute Values are
27 and 1,400 ug/1 for 2,3- and 2,4-dinitrotoluene, respectively,
and since these concentrations are lower than the comparable
concentrations for fish, they also become the Final Acute Values.
Chronic Toxicity
The chronic value for 2,3-dinitrotoluene, derived from a
embryo-larval test with the fathead minnow, is 116 ug/1 (Table 3)
and is based on survival of these life stages (U.S. EPA, 1978).
The Final Fish Chronic Value is 17 ug/1/ and this concentration
also becomes the Final Chronic Value for 2,3-dinitrotoluene in the
absence of data on any invertebrate species.
Plant Effects
Cell numbers of the alga, Selenastrum capricornutum, were
reduced by 50 percent at a concentration of 2,3-dinitrotoluene of
1,370 ug/1 (Table 4). A comparable inhibition in chlorophyll a_
occurred at a concentration of 1,620 ug/1. No other data on
dinitrotoluenes and freshwater plants are available.
Residues
No measured steady-state bioconcentration factor (BCF) is
available for 2,4-dinitrotoluene. A BCF can be estimated using
the octanol-water partition coefficient of 100. This coefficient
is used! to derive an estimated BCF of 19 for aquatic organisms
that cohtain about 8 percent lipids. If it is known that the diet
ot~ the consuming species of concern contains a significantly dif-
terent lipid content, an appropriate adjustment in the estimated
BCF should be made.
B-2 / .
-------
CRITERION FORMULATION
Freshwater-Aquatic Life
Summary of Available Data
The concentrations below have been rounded to two significant
figures.
2,3-d initrotoluene
Final Fish Acute Value = 46 ug/1
Final Invertebrate Acute Value = 27 ug/1
Final Acute Value = 27 ug/1
Final Fish Chronic Value = 17 ug/1
Final Invertebrate Chronic Value = not available
Final Plant Value = 1,400 ug/1
Residue Limited Toxicant Concentration = not available
Final Chronic Value = 17 ug/1
0.44 x Final Acute Value = 12 ug/1
2,4-dinitrotoluene
Final Fish Acute Value = 4,300 ug/1
Final Invertebrate Acute Value = 1,400 ug/1
Final Acute Value = 1,400 ug/1
Final Fish Chronic Value = not available
Final Invertebrate Chronic Value = not available
Final Plant Value = not available
Residue Limited Toxicant Concentration = not available
Final Chronic Value = not available
0.44 x Final Acute Value = 620 ug/1
2,3-dinitrotoluene
The maximum concentration of 2,3-dinitrotoluene is the Final
Acute Value of 27 ug/1 and the 24-hour average concentration is
B-3
-------
0.44 times the Final Acute Value. No important adverse effects on
freshwater aquatic organisms have been reported to be caused by
concentrations lower than the 24-hour average concentration.
CRITERION: For 2,3-dinitrotoluene the criterion to protect
freshwater aquatic life as derived using the Guidelines is 12 ug/1
as a 24-hour average and the concentration should not exceed 27
u.g/1 at any time.
2,4-dinitrotoluene
No freshwater criterion can be derived for 2,4-dinitrotoluene
using the Guidelines because no Final Chronic Value for either
fish or invertebrate species or a good substitute for either value
is available.
Results obtained with 2,3-dinitrotoluene and freshwater or-
ganisms indicate how a criterion may be estimated for 2,4-dinitro-
toluene and freshwater organisms.
For 2,3-dinitrotoluene and freshwater organisms 0.44 times
the Final Acute Value is less than the Final Chronic Value based
on an embryo-larval test with the fathead minnow. Therefore/ a
reasonable estimate of a criterion for 2,4-dinitrotoluene and
freshwater organisms would be 0.44 times the Final Acute Value.
The maximum concentration of 2,4-dinitrotoluene is the Final
Acute Value of 1,400 ug/1 and the estimated 24-hour average con-
centration is 0.44 times the Final Acute Value. No important ad-
verso effects on freshwater aquatic organisms have been reported
to be caused by concentrations lower than the 24-hour average con-
centration.
B-4
-------
CRITERION: For 2,4-dinitrotoluene the criterion to protect
freshwater aquatic life as derived using procedures other than the
Guidelines is 620 ug/1 as a 24-hour average and the concentration
should not exceed 1,400 ug/1 at any time.
B-5
-------
Table i. Freshwater fiah acute valueu for dinilrotoluenes
Bioacaay Ttbt Chemical Time
ftJ._fr*iSN/4" ^V-v. . .* W * F\A*3ffrmm.**mtr\ < Fl •-• O\
Adjusted
Ketc-t c-iiCfc
Kaihcad minnow,
Pituephales promelas
Bluegill.
lepomis macrochirus
S U 2,4-dinltro- 96
toluene
S U 2,3-dinitro- 96
toluene
31,000 17,000 U.S. Army. 1976
330 180 U.S. EPA, 1978
* S - static
** U - unmeasured
Geometric mean of adjusted values: 2,3-dinitrotoluene - 180
160
03
I
2,4-dinitrotoluene - 17,000 ng/1
-------
Table 2. Freshwater invertebrate acute values for diniLroLoluenea
Bioaesay Test Chemical Time
ftetf)cxl* cone ,** pescrijitfon
Adjusted
LC'jU l.C t>l<
(u l/i) (u.|/ 11 Heterence
Cladoceran,
Daphnia magna
Cladoceran,
Daphnia roagna
* S - static
** U - unmeasured
Geometric mean of adj
S U 2,3-dinltro- 48
toluene
S U 2,4-dinitro- 48
toluene
lusted values i 2 , 3-dinitrotoluene <* 560 \>
660 560 U.S. EPA, 1978
35,000 30,000 U.S. Army, 1976
g/1 J£ - 27 Mg/l
to
J
-J
2,4-dlnitrotoluene » 30,000
1.400 Mg/1
-------
B)
I
03
3. Freshwater fish chronic values for dinitrololuenea (U.S. EPA, 1978)
Chronic
Limits Value
Organism TfeBt* lu
-------
DO
I
IO
Table 4. Freshwater plant effects for dlnitrocoluonos (U.S. EPA, 1978)
Concentration
Organism Effect (ug/l>
2,3-dinlcrotoluene
Alga. ECSO 96-hr 1,370
Sfclenaatrum cell numbers
caprlcornutum
Alga, ECSO 96-hr 1,620
Selenastrum chlorophyll a
caprlcornutum ~
l-oweac plant value; 2,3-dlnlcrotoluene « 1,370 pg/1
-------
SALTWATER ORGANISMS
Introduction
Acute toxicity tests using static conditions have been con-
ducted with 2,3-dinitrotoluene and the sheepshead minnow, the
mysid shrimp Mysidopsis bahia, and an alga, Skeletonema costatum.
The LC50 and EC50 values range from 370 ug/1 for algal cell num-
bers to 2,280 ug/1 for the sheepshead minnow. No other data on
any dinitrotoluene are available.
Acute Toxicity
The Final Fish Acute Value 'for 2,3-dinitroto4uene is 340 ug/1
(Table 5) and is based on a single 96-hour static test with the
sheepshead minnow (U.S. EPA, 1978).
The unadjusted 96-hour LC50 for 2,3-dinitrotoluene and
Mysidopsis bahia, is 590 ug/1 (Table 6) and after adjustment for
test methods and species sensitivity, a Final Invertebrate Acute
Value of 10 ug/1 is obtained. This also becomes the Final Acute
Value for 2,3-dinitrotoluene and saltwater organisms since the
comparable acute value for fish is higher.
Chronic Toxicity
No chronic toxicity data are available for any dinitrotoluene
and saltwater organisms.
Plant Effects
A 50 percent reduction in cell numbers of the alga,
Skeletonema costatum, occurred at a concentration of 370 ug
B-10
-------
2,3-dinitrotoluene/l (Table 7). There was a 50 percent inhibition
of chlorophyll a_ production at 400 ug/l«
Residues
No measured steady-state bioconcentration factor (BCF) is
available for 2,4-dinitrotoluene. A BCF can be estimated using
the octanol-water partition coefficient of 100.. This coefficient
is used to derive an estimated BCF of 19 for aquatic organisms
that contain about 8 percent lipids. If it is known that the diet
of the consuming species of concern contains a significantly dif-
ferent lipid content, an appropriate adjustment in the estimated
BCF should be made.
B-ll
-------
CRITERION FORMULATION
Saltwater-Aquatic Life
Summary of Available Data
The concentrations below have been rounded to two significant
figures.
2,3-dinitrotoluene
Final Fish Acute Value = 340 ug/1
Final Invertebrate Acute Value = 10 ug/1
Final Acute Value = 10 ug/1
Final Fish Chronic Value = not available
Final Invertebrate Chronic Value = not available
Final Plant Value = 370 ug/1
Residue Limited Toxicant Concentration = not available
Final Chronic Value = 370 ug/1 d for any dinitrotoluene
0.44 x Final Acute Value = 4.4 ug/1 Value for either
No saltwater criterion can be derived for any dinitrotoluene
using the Guidelines because no Final Chronic Value for either
fish or invertebrate species or a good substitute for either value
is available.
Results obtained with 2,3-dinitrotoluene and freshwater
organisms indicate how a criterion may be estimated for 2,4-di-
nitrotoluene and saltwater organisms.
For 2,3-dinitrotoluene and freshwater organisms 0.44 times
the Final Acute Value is less than the Final Chronic Value based
on an embryo-larval test with the fathead minnow. Therefore, a
reasonable estimate of a criterion for 2,3-dinitrotoluene and
saltwater organisms would be 0.44 times the Final Acute Value.
B-12
-------
The maximum concentration of 2,3-dinitrotoluene is the Final
Acute Value of 10 ug/1 and the estimated 24-hour average concen-
tration is 0.44 times the Final Acute Value. No important adverse
effects on saltwater aquatic organisms have been reported to be
caused by concentrations lower than the 24-hour average concentra-
tion.
CRITERION: For 2,3-dinitrotoluene the criterion to protect
saltwater aquatic life as derived using procedures other than the
Guidelines is 4.4 ug/1 as a 24-hour average and the concentration
should not exceed 10 ug/1 at any time.
B-13
-------
D3
Tdble 5. Marine fish acute values for dinitrotoluenes (U.S. EPA, 1978)
Adjusted
BiOd&eay Test Chemical Time LCt>o LLt>u
i*_ £2fl£i.** Description ({Vra) lltliiL
Sheepshead minnow. S U 2,3- 96 2,280 1.246
Cyprlnodon varifegatua dinitrotoluene
* S - static
** U • unmeasured
1 *? A A
Geometric mean of adjusted values for 2,3-dinltrotoluene - 1,246
-------
CO
I
»—
tn
Table 6. Marine invertebrate acute values fur dinitrotoluencs (U.S. EPA, 1978)
Adjusted
BiOctssay Test Chemical Time LCbu l.CLiu
tt£lii2d*_ Cone,** Description (jug) (u'l/i^
Mysld shrimp, S U 2,3- 96 590 500
Mysidopsia bahla dinitrotoluene
* S - static
** U = unmeasured
Geometric mean of adjusted values for 2,3-dlnltrotoluene - 500 |ig/l —rs " 10 MB/I
-------
03
M
cn
Table 7. Marine plant effects for dlnitrotoluenes (U.S. EPA. 1978)
Concentration
organism Effect (ue/1)
2.3-Dinttrotoluene
Alga, ECSO 96-hr 400
Skeletonema costatum chlorophyll a
Alga. ECSO 96-hr 370
Skeleconema coatatum cell numbers
Lowest plant value: 2,3-dlnttrotoluene • 370 yg/l
-------
w
I
* S = static
** U = unmeasured
^ O / £
Geometric mean of adjusted values for 2,3-dinitrotoluene = 1,246 pg/1 —4—^— = 340 pg
-------
DINITROTOLUENE
REFERENCES
U.S. Army Research and Development Command. 1976. Toxicity
of TNT wastewater (pink water) to ^aquatic organisms. Final
Report, Contract DAMD17-75-C-5056. Washington, D.C.
U.S. EPA. 1978. In-depth studies on health and environmental
impacts of selected water pollutants. Contract No. 68-01-
4646.
B-18
-------
2,4-DINITROTOLUENE
Mammalian Toxicology and Human Health Effects
EXPOSURE
Introduction
2,4-Dinitrotoluene (2,4-DNT) is a pale yellow crystal-
line solid that is widely used as a raw material for dyestuffs
and for urethane polymers through a conversion to the corres-
ponding 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. This process produces
a 80/20 ratio of 2,4-/2,6-isomers, which on fractionatioit
i
gives pure 2,4-DNT (Kirk and Othmer, 1967). Precise produc-
tion figures for 2,4-DNT are not available; however, the
U.S. International Trade Commission (1977) reported a combin-
ed production 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 1-methyl 2, 4-dinitrobenzene (CAS regis-
try 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 manufactur-
ing industries, 2,4-DNT is used by the munition industry
as a modifier for smokeless powders and, to a limited extent,
C-l
-------
TABLE 1
Some Physical Constants of 2,4-Dinitrotoluene
(Data collected from Kirk and Othmer, 1967;
St. John, et al. 1975; Weast, 1978)
PROPERTY
VALUE
Molecular weight
Melting Point,
Boiling point
Density
15
d4
71
d4
Vapor density (air=l)
Vapor pressure at 25+2°C
Refractive index (nQ)
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)
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 caI/gram
C-2
-------
as a gelatinizing and waterproofing agent in military and
commercial explosive compositions (Hamilton and Hardy, 1974).
2,4-DNT is also used as a chemical intermediate in the pro-
duction of toluene diisocyanate (TDI) which, in turn, is
consumed in the production of flexible and rigid polyurethane
foams and elastomers. Most TDI producers, however, 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 is encountered chief-
ly 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 (NCI, 1978). Aromatic nitro
compounds are one of several classes of chemicals thought
to contribute to the increased cancer risk in dye and explo-
sive manufacturing industries (Wynder, et al. 1963). The
structural relationship of 2,4-DNT to the known carcinogen
2,4-toluenediamine (2,4-TDA) is also a factor in its selec-
tion for testing as a possible carcinogen (NCI, 1978).
The usual methods of identification and quantitative
determination 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 HPLC (Walsh, et al. 1973;
Doali and Juhasz, 1974, Stanford, 1977; Natl. Inst. Occup.
Safety Health Manual of Analytical Methods, 1978), and spec-
C-3
-------
troscopic methods such as infrared (Priestera, et al. 1960)
or ultraviolet (Conduit, 1959) spectrophotometry, nuclear
magnetic resonance spectrometry (Gehring and Reddy, 1968),
mass spectrometry (Murrmann, et al. 1971; Plimmer and Klingebiel,
1974; Zitrin and 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).
C-4
-------
Ingestion from Water
2,4-DNT has limited solubility (270 ing/liter at 22°C)
in water as noted in Table 1. Possible sources of 2,4-DNT
in the aqueous environment-either surface water, ground
water or drinking water-are from the dumping of chemical
wastes and from accidental loss during transfer and transport.
Dinitrotoluene waste products are dumped into surface
water or sewage by manufacturing industries that make dyes,
isocyanates, polyurethans, and munitions. The occurrence
of organic micropollutants due to the dumping of aromatic
nitro and amino compounds in the river water has been reported
by Meijers and Van der Leer (1976). The pollution of the
rivers Rhine and Maas in the Netherlands by these aromatics
and oils was examined by extracting water samples in hexane
followed by analyzing the extracts by gas chromatograph/mass
spectrometry (GC/MS). The results showed that the river
Rhine is heavily polluted by oil/ a number of aromatic hydro-
carbons, aromatic amines and aromatic nitro compounds includ-
ing 2,4-DNT. The river Maas, 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 accidently spilled during
the process of transfer and/or transportation. No specific
incidences of this type have 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 investi-
C-5
-------
gators (Schott, et al. 1943; Ruchhoft, et al. 1945; Ruchhoft
and Norris, 1946; Rogovskaya, 1951; Nason, 1956; Anon, 1970,
1971; Osmon and Klausmier, 1972; Walsh, et al. 1973; Nay,
1974; Traxler, et al. 1974; Won, et al. 1974; McCormick,
et al. 1976; Parrish, 1977.) Biotransformation of 2,4-DNT
does occur but its frequency is much lower than the equivalent
activity on 2,4,6-TNT. The influence of aromatic nitrated
hydrocarbons including 2,4-DNT, on the activated sludge
process has been extensively studied (Bogatyrev, 1973; Matsui,
et al. 1975; Roth and Murphy, 1978). At concentrations
of 50 mg/liter 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 water to the concentration in aquatic organ-
isms, but BCF's are not available for the edible portions
of all four major groups of aquatic organisms consumed in
the United States. Since data indicate that the BCF for
lipid-soluble compounds is proportional to percent lipids,
BCF's can be adjusted to edible portions using data on percent
lipids and the amounts of various species consumed by Ameri-
cans. A recent survey on fish and shellfish consumption
in the United States (Cordle, et al. 1978) found that the
per capita consumption is 18.7 g/day. From the data on
the nineteen major species identified in the survey and
C-6
-------
data on the fat content of the edible portion of these species
(Sidwell, et al. 1974), the relative consumption of the four
major groups and the weighted average percent lipids for each
group can be calculated:
Consumption Weighted Average
Group (Percent) Percent Lipids
Freshwater fishes 12 4.8
Saltwater fishes 61 2.3
Saltwater molluscs 9 1.2
Saltwater decapods 18 1.2
Using the percentages for consumption and lipids for each
of these groups, the weighted average percent lipids is
2.3 for consumed fish and shellfish.
No measured steady-state bioconcentration factor (BCF)
is available for 2,4-dinitrotoluene, but the equation "Log
BCF = 0.76 Log P - 0.23" can be used (Veith, et al. Manuscript)
to estimate the BCF for aquatic organisms that contain about.
eight percent lipids from the octanol-water partition coeffi-
cient (P). Based on an octanol-water partition coefficient
of 100, the steady-state bioconcentration factor for 2,4-
dinitrotoluene is estimated to be 19. An adjustment factor
of 2.3/8.0 = 0.2875 can be used to adjust the estimated
BCF from the 8.0 percent lipids on which the equation is
based to the 2.3 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 Americans is
calculated to be 19 x 0.2875 = 5.5.
C-7
-------
Inhalation
The current estimate in the United States for the number
of individuals involved in the manufacture of 2,4-DNT is
not available at present. But the U.S. International Trade
Commission (1977) 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
population may be at risk.
Inhalation has been reported to be one of the major
routes of exposure of 2,4-DNT either in its particulate
or vapor state. The effects from inhalation exposure to
2,4-DNT are caused by 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 possi-
ble to estimate the extent of possible human exposure.
Dermal
Since 2,4-DNT is readily soluble in organic solvents
such as alcohol, ether, etc., as noted in Table 1, it pene-
trates the intact skin readily (Patty, 1958; Hamblin, 1963).
From a survey of the literature (Toxic and Hazardous Indus-
trial 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 (Threshold Limit Values,
C-3
-------
1978). This TLV was set by analogy with chemically similar
nitro aromatic compounds (American Conference of Governmental
Industrial Hygienists, 1974).
Because of the availability of only limited data on
the human exposure to 2,4-DNT/ it is difficult to assess
quantitatively the contribution of each route of exposure
to the total dose; it is likely that the greatest contri-
bution 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 absorption of its solution in organic
solvents. Hodgson, 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
14
that the C-ring labeled nitrotoluenes were well absorbed
after oral administration in the rat. The absorption was
essentially complete in 24 hours with 60 to 90 percent 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 contained small amounts
of radioactivity. The ratio of radioactivity in tissue/plasma
14
indicated a retention of C in both the liver and kidneys,
14
while negligible amounts of C 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.
C-9
-------
14
When C-labeled nitrotoluenes were administered to bile
duct-cannulated rats, 10.3 to 27.3 percent of the 14C was
recovered in the bile, suggesting that biliary excretion
is also an important elimination pathway. Thin layer chroma-
tographic 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.
Another study examining the excretion and distribution
of tritium-labeled 2,4-dinitrotoluene ( H-2,4-DNT) in the
rat has been reported recently (Mori, et al. 1977). Approxi-
mately 21.3 percent of the radioactivity was excreted in
the feces on the first day after a single oral administration
of H-2,4-DNT. The amount of radioactivity excreted in
the feces on the second and third days were 4.1 and 1.1
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 radioactivity was found in only trace
quantities. In all, about 46 percent of the radioactivity
administered was excreted in the feces and urine during
the 7 days (see Table 2).
In the same experiment, relatively high amounts of
radioactivity 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 by the single
oral administration of H-2,4-DNT suggests that 2,4-DNT
remains in the liver, skin, and adipose tissue.
C-10
-------
TABLE 2
Urinary and Fecal Excretion of Radioactivity,
Expressed as Percentages of Administered
Radioactivity
(From Mori, et al. 1977)
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
C-ll
-------
TABLE 3
Remaining Radioactivity in the Tissues of Rat
Seven Days after Administration of 3H-2, 4-DNT
(From Mori, et al. 1977)
Tissue
Brain
Heart
Lung
Liver
Spleen
Pancreas
Kidney
Adrenal
S tomach
Small intestine
Large intestine
Testis
Mesenteriolum
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 10
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
C-12
-------
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 excretion of 2,4-DNT in rats do not give
details on the characterization of metabolites and metabolic
pathways.
The isolation, identification and synthesis of biotrans-
formation products from 2,4-DNT have been reported by McCormick,
et al. (1978) from a detailed study on the microbial trans-
formation of 2,4-DNT by Mucrosporium Sp. (Strain QM 9651).
The biotransformation products were identified by thin layer
chromatography (by 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-nitro-toluene; a third azoxy compound, believed
to be a "mixed" type (i.e. 2,4'-azoxy or 4,2'-azoxy), was
also isolated, but not identified. These authors present
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.
C-13
-------
CH3
NHOH
NO2
NH2
(I)
FIGURE 1.
Proposed Pathways for the formation of Biotransforraation
Products from 2,4-Dinitrotoluene (A)
(Taken from McCormick, et al. 1978)
The hypothetical nitroso and hydroxylamino intermediates are enclosed
brackets. The potential formation of 2,4-toluenediamine (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;
(P) 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-14
-------
In a study of the microbial transformation of 2,4-DNT,
2,4,6-TNT and other nitroaromatic compounds by anaerobic
bacterial systems (McCormick, et al. 1976), these compounds
were reduced by hydrogen in the presence of enzyme prepara-
tions from Veillonella alkalescens. Consistent with the
proposed reduction pathways, R-NO2 H2^R-NQ H2;R^-NHOH H2 ^
R-NH2, 3 moles of H~ were utilized per mole of nitro group.
From the rates of reduction of 40 mono-, di-, and trinitroaro-
matic compounds by Veillonella alkalescens, it was noticed
that reactivity of the nitro group depended on other substi-
tuents 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):
-NO2 > -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
(McCormick, 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 trans-
formation 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.
C-15
-------
The explosive 2,4,6-TNT has been extensively investi-
gated because of the toxic symptoms which it produces in
people engaged in its manufacture (Palmer, et al. 1943;
Schwartz, 1944; Dobbin Crawford, 1954; Goodwin, 1972; Djerassi
and Vitany, 1975; Morton, et al. 1976). It is generally
agreed that its toxicity is due to its metabolic 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-dinitro-
toluene was found in larger quantities and the 4-hydroxy-
lamino-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 converted 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
C-16
-------
fact which indicates that 2,4,6-TNT is reduced in man to
4-hydroxylaminc~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 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-triaminotoluene as a metabolic product of 2,4,6-
TNT, though such a possibility cannot be ruled out.
Thus, from an analogy of metabolism of 2,4,6-TNT with
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 animals exposed to 2,4-DNT.
Most of these metabolites are either toxic (Fairchild, et
i
al. 1977) or suspected carcinogens (Christensen, et al.
1976) .
EFFECTS
Acute, Sub-acute, and Chronic Toxicity
Acute toxic effects of 2,4-DNT include methemoglobinemia
followed by cyanosis. The inhalation of the fumes or dust,
the ingestion of the compound, or the absorption by the
skin through contact of 2,4-DNT bring about a chemical change
of the blood oxyhemoglobin into methemoglobin (basically,
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
-------
NO2
NH2
N02 N02
(H)
FIGURE 2
Proposed Pathways for the formation of Biotransformation
Products from 2,4, 6-Trinitrotoluene (A) (Taken from Williams,
1959; Won, et al. 1974). The hypothetical nitroso inter-
mediat^es are enclosed in brackets. The potential formation
of 2/4-Diamino-6-nitrotoluene (J) is indicated by dashed arrows.
4-Nitroso-2,6-dinitrotoluene; (C) 4-Hydroxylamino-2,6-
dinitrotoluene; (D) 2,2', 6,6 '-Tetranitro-4,4 '-azoxy toluene; (E)
4-Amino-2,6-dinitrotoluene; (F) 2-Nitroso-4,6-dinitrotoluene;
(G) 2-Hydroxylamino-4,6-dinitrotoluene; (H) 4r4', 6 ,6 '-Tetranitro-
2, 2 '-azoxy toluene; (I) 2-aminQ-4,6-dinitrotoluene
C-18
-------
is commonly the first symptom and may become quite intense
as the severity of methemoglobinemia progresses. The follow-
ing 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, unconsciousness, chest pain, shortness of breath,
palpitation (rapid throbbing 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 damage
of the hemoglobin molecules) in the cat (Bredow and Jung,
1942). Human subjects are similarly susceptible, and workers
handling such compounds as nitrobenzenes, nitrotoluenes
and phenylhydrazines occasionally exhibit Heinz bodies in
their blood (Hughes and Treon, 1954; De Bruin, 1976).
Inactivation of the hemoglobin under the effect of
2,4-DNT and related compounds 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 LD50 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 hemo-
globin 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
C-19
-------
corresponding to 0.1 to 0.2 LD50 values to rats for one
to three months, the hemotoxicity of the nitrotoluenes de-
creased in the order: trinitrotoluene >dinitrotoluene >
Vy
m-nitrotoluener p-nitrotoluene> 0-nitrotoluene.
Cyanosis due to the absorption of 2,4-DNT occurs when
the methemoglobin concentration is 15 percent or more. The
symptoms observed include blueness in the lips, the nose,
and the ear lobes. The individual usually feels well, has
no complaints, and insists that nothing is wrong until the
methemoglobin concentration 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). Because of the increased
vapor pressure with higher ambient temperatures, there is,
in general, an increased susceptibility to cyanosis from
exposure to 2,4-DNT (Linen, 1974).
Some earlier studies provide useful information on
the toxicity 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 oraj. administration of 2 or 4 ml
of a 1 percent solution in cod liver oil, until a total
of 24 ml has been given, without any toxic effect. Similarly
Zieger (1913) observed no toxic effects from the inhalation
of vapors, whereas in the experience of Kuhls (1908), the
subcutaneous injection in cats of 0.05 to 0.5 g of 2,4-DNT
dissolved in mineral oil resulted in death after periods
of 2 to 23 days. Regarding the possibility of absorption
C-20
-------
through the skin, Dambleff (1908) found in rabbits no indica-
tion of a toxic action by this route, and similarly Kuhls (1908)
observed in cats no toxic effects from the cutaneous adminis-
tration of 0.3 g/kg body weight, while Zieger (1913) found
two doses of 5 g each were fatal to cats in eight hours.
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 oral toxicity follows the order: aniline >
1,3,5-trinitrobenzene >2,6-DNT >3-nitrotoluene = 4-nitrotol-
uene = 2,5-DNT >2,4-DNT ?2-nitrotoluene.
TABLE 4
Acute Toxic Levels of 2,4-Dinitrotoluene for
Different Species
(Data collected from Spector, 1956; Fairchild,
et al. 1977; Vernot, et al. 1977)
Species
Rat
Mouse
Cat
Route
Oral
Oral
Oral
S.C.
Toxicity
LD50
LD50
MLD
LDLo
Dose
(mg/kg)
268
1625
27
50-500
S.C. - subcutaneous; LDLo - lowest published lethal dose;
LD50 - lethal dose 50 percent kill; MLD - minimum lethal dose
C-21
-------
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 painrin"the joints (es-
pecially 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 headache, palpitation of-heart, oppression 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- coat ing
which use-2,4-DNT'., The chief symptomsnof a group of
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 complaints
C-22-
-------
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 mechanism 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 aminp compounds are not in themselves
cyanogenic, but oxidation-reduction enzyme systems promote
biotransformation to known active 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,
Hemoblobin^ ^Methemoglobin,
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 equilibrium 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;
C-23
-------
TABLE 5
Symptoms Presented by 154 2,4-Dinitrotoluene Workers
(From McGee, et al. 1942)
Screening
House
Symptom Number of
Wor kmen
Unpleasant taste
in mouth
Weakness
Headache
Inappetence
Dizziness
Nausea
Insomnia
Pain in extremities
Vomi t i ng
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 Percent
96
78
76
72
68
57
57
40
35
29
10
8
62
51
49
47
44
37
37
26
23
19
6.5
5.2
C-24
-------
TABLE 6
Clinical Findings in 154 2,4-Dinitrotoluene Workers
(From McGee, et al. 1942)
Screening
House
Finding (Number of
Workmen)
Pallor
Cyanosis
Anemia
Leucocytosis
Hypotension
Skin rash
Leukopenia
Hepatitis and
Jaundice
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.
3.
3.
1.
8
9
2
4
C-25
-------
he believes such a concept helps to explain the difference
in degree of methemoglobin formation in various species,
as well as the differing rates of reduction of methemoglobin
to hemoglobin. Methemoglobin-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 industrial exposure to cyanogenic aromatic nitro and
amino compounds, Linch (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, dinitrotoluenes are ranked No. 12 (1 most
potent, 13 least potent) indicating that 2,4-DNT does not
produce cyanosis as rapidly as other cyanogenic aromatic
nitro and amino compounds. From the similarities 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 studied 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 includ-
ed 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
animals in all three species, while the lowest doses produced
C-26
-------
TABLE 7
Methemoglobin-forming Capacity of Some Aromatic
Nitro and Amino Compounds in Cat
(Data collected from Hamblin, 1963; De Bruin, 1976)
Compound
Molecular ratio*
Nitrobenzene
1,3-Dinitrobenzene
1,3,5-Trinitrobenzene
2-Nitrotoluene
3-Nitrotoluene
4-Nitrotoluene
2,4-Dinitrotoluene
2,6-Dinitrotoluene
2,4,6-Trinitrotoluene
Aniline
Phenylhydroxylamine
3-Aminonitrobenzene
1,3-Diaminobenzene
Nitrosobenzene
0.86
7.1
4.8
0.05
0.04
Very slight
1.4
0.55
1.7
2.5 (2.7)
34.0
3.0
1.4
8.6 '
Molar ratio of methemoglobin formed to dose of test compound
C-27
-------
Methemoglobin
reductase
Hemoglobin Methemoglobin
CH3
NO2
NAD*
NHOH
4-HYDROXYUAMINO— 2— NITROTOLUENE
\
RAPID
NADH + H+
CH3
NO2
NO
4—NITROSO—2—NITROTOLUENE
/
/
(0)
cyanopathic
intermediates
(H)
SLOW
CHS
CH3
(Q
.NO2
NH2
4—AMINO—2—NITROTOLUENE
NO2
NO2
2.4—DINITROTOI UENE
FIGURE 3
Suggested Metabolic Pathway for Cyanosis by 2,4-Dinitrotoluene
based upon data from related compounds.
C-28
-------
no toxic effects. All species showed methemoglobinemia
and anemia with reticulocytosis. Characteristic tissue
lesions were extramedullary hematopoeisis in the spleen
and liver, gliosis and demy-elination 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 0.25 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,
jaundice 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 protein (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 sulfhydryl 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 procarcinogenic
diet were less resistant to toxicity from parenteral 2,4-
DNT than mice raised on the other diets.
C-29
-------
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 containing 5 or 30 percent
of added fat. Pat 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 Goldwater (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 tolerance for alcohol
C-30
-------
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 experienc-
ing reactions such as substernal pressure, precardial "palpi-
tation", fullness in the head, and severe acute illness.
The ingest ion of alcohol normally causes increased
susceptibility 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 alcoholic 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 dinitro-
toluene.
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) also studied somatic cell mutation
effects by cytogenetic analysis of lymphocyte and kidney
031
-------
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 observed in kidney cultures
after five weeks and in lymphocytes at 19 weeks. This would
suggest that 2,4-DNT has a potential for inducing damage
in somatic cells. In vitro studies using the CHO-K1 test
system were negative. On the other hand, microbial tests
using Salmonella typhimurium TA 1535 indicated that 2,4-
DNT is capable of producing base-pair mutations.
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 munitions plants were examined
before and after ozonation or chlorination to determine
whether such 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; pent-
aerythritol tetranitrate, and trinitroresorcinol. The ir\
vitro mutagenic assays used were the Salmonella/microsome
assay (Ames, et al. 1973) with strains TA 1535, TA 1537,
TA 1538, TA 98, and TA 100 and mitotic recombination in
the yeast, Saccharomyces cerevisiae D3. A metabolic acti-
vation system using the postmitochondrial supernatant fraction
C-32
-------
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 these nitro aromatic materials tested including
2,4-DNT.
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 elevated activity was indicated
in some of the experiments, it was not dose-related. At
the concentrations tested (0.08 jug/plate, highest dose),
2,4-DNT was not mutagenic in the Salmonella assay before
or after ozonation. The highest concentration tested in
the Saccharomyces 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
(a mixture of complex nitroaromatics containing primarily
2,4-and 2,6-DNT's) were also tested for mutagenicity. Two
new products (m/e 166 and 270) were found in the GC/MC profile.
The fragmentation pattern of the m/e 166 compound was found
to be consistent with a nitrosonitrotoluene but was not
confirmed. Prior to ozonation, the TNT condensate water
mixture was mutagenic in Salmonella assays but not in Saccha-
romyces. 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 afer ozonation. A duplicate experiment showed no
C-33
-------
activity. These mutagenicity results are presented in Table
8.
Carcinogencity
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) was conducted using Fisher 344
rats and B6C3F1 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 subcutaneous tissue occurred in the high
and low dose groups when compared to their respective controls
(Table 9). A statistically significant incidence of fibroade-
noma 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., hemangio-
sarcoma 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,
1973) considered that these tumors were not related to chemical
i
administration.
C-34
-------
TABLE 8
O
I
ui
Mutagenic Assay Results
of Munitions Compounds
(From Cotruvo, et al. 1977)
Munitions
Compounds
Initial
Concentration
(ppm)
Reaction
Time
(min)
Reacted pH
Salmonella
Activity
Saccha-
romyces
Activity
Comments
2,4-Dinitrotoluene 28.3
TNT condensate water 35.4
20
100
96
8.4/3.8
9.3 7.2/3.6
-/+ elevated activity
in high dose, not
dose related
-/- activity found in
one test, reduced
by ozonation
-------
o
TABLE 9
Summary of the Significant Primary Tumors at
Specific Sites in Male Rats Treated with 2,4-Dinitrotoluene
(From NCI, 1978)
TOPOGRAPHY: MORPHOLOGY
Subcutaneous Tissue or Skin: Fibroma
P Values0
Relative Risk (Control)
Lower Limit
Upper Limit
Weeks to First Observed Tumor
LOW DOSE
CONTROL
0/46(0.00)
HIGH DOSE
CONTROL LOW DOSE
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
Treated groups received time-weighted average concentrations of 17.6 and 44.0 mg/kg/day in feed.
Number of tumor-bearing animals/number of animals examined at site (proportion).
Q
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; other-
wise, not significant (N.S.) is indicated. A negative designation (N) indicates a 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.
-------
For the mice, there were no tumors in either sex having
a statistically significant positive association between
administration of 2,4-DNT and incidence of tumor. As such
there is no convincing evidence of tumorigenicity in B6C3F1
mice at the dose levels of 2,4-DNT used in these experiments.
The possibility of a negative association between administra-
tion and incidence was observed for pituitary adenomas in
female mice and for alveolar/bronchiolar neoplasms in male
mice.
At this point, it is relevant to present some of the
comments made regarding this carcinogenesis study by the
Data Evaluation/Risk Assessment Subgroup of the Clearinghouse
on Envirnomental Carcinogens: (NCI, 1978)
1. The tumors in the treated rats must be viewed
with concern, especially since the maximum tolerat-
ed 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
increased 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
conversion 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 another species and
route of exposure, especially dermal.
-------
TABLE 10
Summary of the Significant Primary Tumors at
Specific Sites in Female Rats Treated with 2,4-Dinitrotoluene*
(From NCI, 1978)
LOW DOSE HIGH DOSE
TOPOGRAPHY: MORPHOLOGY CONTROL CONTROL
Mammary Gland:
P Values0
Relative Risk
Weeks to First
Fibroadenomab 9/48(0.19) 4/23(0.17)
(Control) d
Lower Limit
Upper Limit
Observed Tumor 92 109
LOW DOSE
12/49(0.24)
N.S.
1.306
0.559
3.183
83
HIGH DOSE
23/50(0.46)
P = 0.016
2.645
1.062
9.435
69
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).
GThe 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^O.05; other-
wise, 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.
-------
Another bioass'ay of practical grade 2,4-DNT for possible
carcinogenicity 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 mg/kg/day in female rats, was quite
toxic, caus.ing decreased weight gain and shortened life
span. Target organs included the blood (toxic anemia),
the liver (hep'atocellular carcinoma), the testis (aspermato-
genesis) , 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 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.
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 above study, it is reasonable to
discuss 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 carcinomas (Ito, et al. 1969). Similarly
Swiss-Webster mice fed 2,4-TDA showed a high incidence of
C-38
-------
TABLE 11
Summary of the Male Rats with Apparent Tumors
After being Fed 2,4-Dinitrotoluene for 18 months
(From Lee, et al. 1978)
Dose
(mg/kg/day)
0
0.57
3.90
34.0
Tumor/Total
1/37
0/37
0/29
17/23
Percent
3
0
0
74
TABLE 12
Summary of the Female Rats with Apparent Tumors.
After being Fed 2,4-Dinitrotoluene for 18 months
(From Lee, et al. 1978)
Dose
(mg/kg/day
0
0.71
5.10
45.0
Tumor/Total
8/29
11/40
10/27
28/32
Percent
28
28
37
88
C-39
-------
lung neoplasms (Stoats, 1972). In contrast, the recent
study by Giles, et al. (1976) indicates that the 2,4-TDA
and other hair dye ingredients did not augment the development
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 experimen-
tal conditions was found to be nontoxic and noncarcinogenic
to the skin of mice.
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 transformation 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 demon-
strated that 2,4-TDA is metabolized by the S9 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
Drosophilia melanogaster male germ cells (Blijleven, 1977;
_I^H^MMB»«.^A^M«^^H^MBW ~-^^^—^—~—^^^*-^——^~ ^
Fahmy and Fahmy, 1977; Venitt, 1978).
C-40
-------
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 Russian study (Korolev, et al. 1977) recommends
that a maximum permissible 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 (Am. Conf. Gov. Ind. Hyg.) 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 workday
of 40-hour workweek (Am. Conf. Gov. Ind. Hyg. 1978). This
value represents the highest level to which nearly all workers
may be repeatedly exposed, 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 (Am. Conf. Gov- Ind. Hyg.
1978). The TLV-STEL is defined as the maximal allowable
concentration to which workers can be exposed for a period
of up to 15 minutes continuously without suffering from
1) irritation, 2) chronic or irreversible tissue change,
or 3) narcosis of sufficient degree to increase accident
pront-ness, impair self-rescue, or materially reduce work
efficiency. No more than four exposures to the TLV-STEL
per day are permitted, with at least 60 minutes between
exposure periods, and the daily TLV-TWA must also not be
exceeded. . .
C-41
-------
Current Levels of Exposure
No data on the extent of human exposure to 2,4-DNT
are available in the literature. However, a study of the
concentration of explosives in air by isotope dilution analy-
sis (St. John, et al. 1975) reported a concentration of
184 ppb V/V (=1.384 mg/m3) 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 handling of 2,4-DNT in places such as ammunition, dye,
and polyurethane plants.
Basis and Derivation of Criterion
The data from the bioassay of 2,4-DNT for possible
carcinogenicity obtained by the National Cancer Institute
(1978) and Lee, et al. (1978) were used for the determina-
tion of a water quality criterion for the protection of
human health. It should be noted at this point, however,
that the Data Evaluation/Risk Assessment Subgroup of the
Clearinghouse on Environmental Carcinogens (NCI, 1978) expres-
sed reservations about the adequacy of this bioassay for
use in assessing human risk. Nevertheless, the criterion
was developed from the animal carcinogenicity data from
these two studies by utilizing the linear non-threshold
model (see Appendix I). The rat carcinogencity studies
t
with dietary administration 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.
C-42
-------
Under the Consent Decree in NRDC vs Train/ criteria
are to state "recommended maximum permissible concentrations
(including where appropriate, zero) consistent with the
protection of aquatic organisms, human health, and recreation-
al activities." 2,4-DNT is suspected of being a human carcino-
gen. Because there is no recognized 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 concentrations of 2,4-DNT corresponding
to several incremental lifetime 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, indi-
cates a probability of one additional case of cancer for
every 100,000 people exposed, a risk of 10" indicates one
additional case of cancer for every million people exposed,
and so forth.
In the Federal Register notice of availability of draft
ambient water quality criteria, EPA stated that it is consid-
ering setting criteria at an interim target risk level of
10~\ 10~ or 10~ as shown in the table below.
Exposure Assumptions Risk Levels and Corresponding Criteria (1)
(per day) £ 1£~7 10/~6 1CTD
2 liters of drinking water 7.4 ng/1 74.0 ng/1 740 ng/1
and consumption of 18.7
grams fish and shellfish. (2)
Consumption of fish and .156 jug/1 1.56 jug/1 15.6jug/l
shellfish only.
C-43
-------
(1) Calculated by applying a modified "one-hit" extrapolation
model described in the FR 15926, 1979 to the animal
bioassay data presented in Appendix I and in Table
9. Since the extrapolation model is linear at low
doses, the additional lifetime risk is directly propor-
tional 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, 1000 and so
forth.
(2) Approximately five percent of the DNT exposure results
from the consumption of aquatic organisms which exhibit
an average bioconcentration potential of 5.5 fold.
The remaining 95 percent of DNT exposure results from
drinking water.
Concentration levels were derived assuming a lifetime
exposure to various amounts of DNT, (1) occurring from the
consumption of both drinking water and aquatic life grown
in waters containing the corresponding DNT concentrations
and, (2) occurring solely from consumption of aquatic life
grown in the waters containing the corresponding DNT concen-
trations. Although total exposure information for chloroform
is discussed and an estimate of the contributions from other
sources of exposure can be made, this data will not be factor-
ed into ambient water quality criteria. The criteria present-
ed, therefore, assume an incremental risk from ambient water
exposure only.
C-44
-------
Results obtained from the linear non-threshold model
give a value of 740 ng/liter as the lowest value obtained,
the dose level which establishes a carcinogenicity risk
level in water for humans of 1 in 100,000. This was obtained
from the study of fibroadenomas of the mammary gland and
inanition of the female rats (Lee, et al., 1978). It should
be noted that this level is 1/500 the level of 0.5 mg/liter
for surface water recommended in the U.S.S.R. (Korolev,
et al. 1977).
Using the TLV-TWA value of 1.5 mg/m of air for 2,4-
DNT recommended by the Am. Conf. Gov- Ind. Hyg. (1978),
the daily occupational exposure gives a value of 5.4 mg
of 2,4-DNT per day (see Appendix II for calculation). At
an ambient water level of 740 ng/liter, assuming a daily
intake of 2 liters and a daily aquatic organism intake of
18.7 g with a bioaccumulation factor of 5.5, it can be
shown (see Appendix II for calculation) that the daily intake
of 2,4-DNT is 0.0016 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 (0.0016 mg/day) which would cause an
insignificant effect in terms of contribution to methemoglobi-
nemia (25 mg of 2,4-DNT/liter) (Cartwright, 1977; Proctor
and Hughes, 1978). It would thus appear that the linear
non-threshold model, using female rat data on fibroadenomas
of the mammary gland and inanition (Lee, et al. 1978) provides
a level of ambient water exposure which achieves a high
margin of safety.
C-45
-------
It should be noted that data are urgently needed in
the following areas to evaluate properly any hazard from
2,4-DNT:
1. Monitoring of workers exposed to 2,4-DNT in industries
manufacturing or using the chemical.
2. Monitoring of public water supplies and industrial
and municipal 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, occu-
pationally exposed humans.
4. Evaluation of chronic toxicity and teratogenicity using
currently acceptable techniques.
5. Detailed and definitive mutagenicity studies of 2,4-
DNT and its metabolites using several assay systems
such as:
a) Salmonella/microsomal, b) dominant lethal, c)
Drosophila, arid 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-46
-------
REFERENCES
American Conference of Governmental Industrial Hygienists.
1974. Dinitrotoluene. Documentation of the threshold limit
values for substances in workroom air. 3rd ed., 2nd printing,
Cincinnati.
American Conference of Governmental Industrial Hygienists.
P
1978. TLV's : Threshold limit values for chemical substances
and physical agents in the workroom environment with intended
changes for 1978.
Ames, B.N., et al. 1973a. An improved bacterial test system
for the detection and classification of mutagens and carcino-
gens. Proc. Natl. Acad. Sci. 70: 782.
Ames, B.N., et al. 1973b. Carcinogens are mutagens: a
simple test system combining liver homogenates for activation
and bacteria for detection. Proc. Natl. Acad. Sci. 70:
2281.
Ames, S.A.H., and H.J. Yallop. 1966. The identification
of industrial blasting explosives of the gelignite type.
Jour. Forensic Sci. Soc. 6: 185.
Blijleven, W.G.H. 1977. Mutagenicity of four hair dyes
in Drosophila melanogaster. Mutat. Res. 48: 181.
C-47
-------
Bodansky, O. 1951. Methemoglobinemia and methemoglobin-
producing compounds. Pharm. Rev. 3: 144.
i
Bogatyrev, 0. 1973. Influence of aromatic nitrated hydro-
carbons on the activated sludge process. Acta Hydrochim.
Hydrobiology. 1: 455.
•t
Brandt, A.D. 1943. Engineering control of air contamination
of the working environment. Pages 198-266 _in W.M. Gafafer
ed. Manual of industrial hygiene and medical service in
war industries. W.B. Saunders Co., Philadelphia.
Bredow, M.V., and F. Jung. 1942. Methemoglobin formation.
XXV. Comparative toxicity of some aromatic nitro derivatives,
Arch. Exp. Pathol. Pharmakol. 200: 335.
Bueding, E., and N. Jolliffe. 1946. Metabolism of trinitro-
toluene (TNT) _in vitro. Jour. Pharmacol. Exp. Ther. 88:
300.
Burrows, D., and J.C. Dacre. 1975. Toxicity to aquatic
organisms and chemistry of nine selected waterborn pollutants
from munitions manufacture. Literature evaluation. Gov.
Rep. Announce Index 75: 45. A review. AD-A Rep. No. 010660.
Natl. Tech. Inf. Serv-, Springfield, Va.
C-48
-------
Carpenter, D.F., et al. 1978. Microbial transformation
14
of C-labeled 2,4,6-trinitrotoluene in an activated sludge
system. Appl. Environ. Microbiol. 35: 949.
Cartwright, G.E. 1977. Methemoglobinemia and sulfhemoglo-
binemia. Pages 1710-1713 _in G.W. Thorn, et al., eds. Harrison's
principles of internal medicine, 8th ed. McGraw-Hill, New
York.
Channon, H.J., et al. 1944. The metabolism of 2,4,6-trini-
trotoluene ( -TNT). Biochem. Jour. 38: 70.
Chemical Abstracts Service. 1977. The chemical abstracts
service (CAS) ninth collective index. Vol. 76-85. Am. Chem.
Soc., Washington, D.C.
Christensen, H.E., et al., eds. 1976. Suspected carcinogens.
2nd ed. A subfile of the NIOSH registry of toxic effects
of chemical substances. (USDHEW Publ. No. 77- 149). U.S.
Government Printing Office, Washington, D.C.
Clayton, C.C., and C.A. Baumann. 1944. Some effects of
diet on the resistance of mice toward 2,4-dinitrotoluene.
Arch. Biochem. 5: 115.
Clayton, C.C., and C.A. Baumann. 1948. Effect of fat and
calories on the resistance of mice to 2,4-dinitrotoluene.
Arch. Biochem. 16: 415.
C-49
-------
Conduit, C.P. 19.59- Ultraviolet-and infrared spectra of
some aromatic nitro compounds.. Jour. Chem. Soc. London
(P. 4): 3273.
Cordle, P., et al., 1978. Human exposure to polychlorinated
biphenyls and polybrominated biphenyls. Environ. Health
Perspect. 24: 157.
Cotruvo, J.A., et al. 1977. Investigation of mutagenic
effects of products of. ozonation reactions in water. Ann.
N.Y. Acad. Sci. 298: 124.
Dale, H.H. 1921. Characterization of a metabolite from
2,4,6-TNT. Spec. Rep. Sex. Med. Res. Council No. 58. London.
Dambleff, J. 1908. On the knowledge of the toxic action
of nitrated benzenes especially from the skin. Inaug. Diss.
Wurzburg.
DeBruin, A. 1976. Anomalies in hemoglobinT-methemoglobinemia.
Pages 1259-1282 Ln Biochemical, toxicology of environmental
agents. Elsevier/North Holland Biomedical Press, The Netherlands,
Djerassi, L.S., and L. Vitany. 1975.. Hemolytic episode
in G6 PD deficient workers exposed to TNT. Br. Jour. Ind.
Med. 32: 54,
C-50
-------
Doali, J.O, and A.A. Juhasz. 1974. Application of high
speed liquid chromatography to the qualitative analysis
of compounds of propellant and explosives interest. Jour.
Chromatogr. Sci. 12: 51.
Dobbin Crawford, M.A. 1954. Aplastic anemia due to trini-
trotoluene intoxication. Br. Med. Jour. 2: 430.
Dybing, E., et al. 1977. Use of the Salmonella mutagenicity
test in drug metabolism studies. Acta Pharmacol. Toxicol.
41(S4): 31.(Abstract.)
Ellis, H.V. Ill, et al. 1976. Subacute toxicity of 2,4-
dinitrotoluene and 2,6-dinitrotoluene. Toxicol. Appl. Pharma-
col. 37: 116. (Abstract from 15th Annu. Meet. Soc.Toxicol.
March 14-18.).
Fahmy, M.J., and O.G. Fahmy. 1977. Mutagenicity of hair
dye components relative to the carcinogen benzidine in Droso-
phila melanogaster. Mutat. Res. 56: 31.
Fairchild, E.J., et al. eds. 1977. Registry of toxic effects
of chemical substances. USDHEW Publ.No. (NIOSH) 78-104-B.
U.S. Government Printing Office, Washington, D.C.
C-51
-------
Floret, F. 1929. Medical opinions on industrial poisonings.
Centr. Gewerbehyg. Unfallverhut. 16: 280.
Friedlander, A. 1900. On the clinical picture of poisoning
with benzene and toluene derivatives with special reference
to the so-called anilinism. Neurol. Centrlbl. 19: 155.
Fukuda, H., et al. 1977. Detection of nitroglycerine and
nitro compounds in fragments after dynamite explosion.
Kagaku Keisatsu Kenkyusho Hokoku 30: 263.
Gehring, D.G., and G.S. Reddy. 1968. Nuclear magnetic
resonance examination and determination of the di- and trini-
trotoluene isomers in 2,4,6-trinitrotoluene. Anal. Chem.
40: 792.
Giles, A.L., et al. 1976. Dermal carcinogenicity study
by mouse-skin painting with 2,4-toluenediamine alone or
in representative hair dye formulations. Jour. Toxicol.
Environ. Health 1: 433.
Gleason, M.N., et al. 1969. In Clinical toxicology of commer-
cial products, 3rd ed. The Williams and Wilkins Co., Baltimore.
Goldman, F.H., and M.B. Jacobs. 1953. Chemical methods
in industrial hygiene. Interscience Publishers Inc., New
York.
C-52
-------
Goodwin, J.W. 1972. Twenty years handling TNT in a shell
loading plant. Am. Ind. Hyg. Assoc. Jour. 33: 41.
Hamblin, D.O. 1963. Aromatic nitro and amino compounds.
Pages 2105-2169 _in F.A. Patty, et al., eds. Industrial hygiene
and toxicology. Vol. II: Toxicology. John Wiley and Sons.,
New York.
Hamilton, A., and H.L. Hardy- 1974. In Industrial toxico-
logy. Publishing Sciences Group, Inc., Massachusetts.
Hodgson, J.R., et al. 1976. Mutation studies on 2,4-dinitro-
toluene. Mutat. Res. 38: 387- (Abstract from the 7th Annu.
Meet. Am. Environ. Mutagen Soc., Atlanta, March 12-15.).
Hodgson, J.R., et al. 1977. Comparative absorption, distri-
bution, excretion, and metabolism of 2,4,6-trinitroluene
(TNT) and isomers of dinitrotoluene (DNT) in rats. Fed.
Proc. 36: 996.
Hoffman, C.M., and E.B. Byall. 1974. Identification of
explosive residues in bomb scene investigations. Jour.
Forensic Sci. 19: 54.
Hughes, J.P., and J.F. Treon. 1954. Erythrocytic inclusion
bodies in the blood of chemical workers. Arch. Ind. Hyg.
Occup. Med. 10: 192.
C-53
-------
Ito, N., et al. 1969. The development of carcinoma in
liver of rats treated with toluenediamine and the antagonistic
effects with other chemicals. Cancer Res. 29: 1137.
Jenkins, R., and H.J. Yallop. 1970. The identification
of explosives in trace quantities on objects near an explosion,
Explosivstoffe 18: 139.
Key, M.M., et al. eds. 1977. Pages 278-279 in Occupational
diseases: a guide to their recognition.U.S.Dept.Health.Edu.
Welfare. U.S. Government Printing Office, Washington, D.C.
Kirk, R.E., and D.F. Othmer. 1967. Encyclopedia of chemical
technology- Vol. 13. John Wiley and Sons., New York.
Koelsch, F- 1917. Contributions to the toxicology of aro-
matic nitro compounds. Zentr, Gewerbehyg, Unfallverhut.
5: 60.
Korolev, A.A., et al. 1977. Experimental data for hygienic
standardization of dinitrotoluene and trinitrobenzene in
reservoir waters. Gig. Sanit. Iss. 10: 17-
Kovalenko, I.I. 1973. Hemotoxicity of nitrotoluenes in
relation to number and positioning of nitro groups. Farmakol.
Toxicol. (Kiev.) 8: 137.
C-54
-------
Krzymien, M., and L. Elias. 1975. A continuous flow trace
vapor source. Lab. Tech. Rep. LTR-UA-32. Natl. Res. Counc.
Can., Unsteady Aerodyn. Lab.
Kuhls, F. 1908. Quantitative experiments on the absorption
of poisons through the skin. Inaug. Dissert. Wurzburg.
Lee, C.C., et al. 1978. Mammalian toxicity of munition
compounds. Phase III: Effects of life-time exposure. Part
I: 2,4-Dinitrotoluene. U.S. Army Med. Res. Dev. Command.
Contract No. DAMD-17-74-C-4073. Rep. No. 7, September.
Lemberg, R., and J.P. Callaghan. 1944. Metabolism of symmet-
rical trinitrotoluene. Nature (London) 154: 768.
Lewin, L. 1921. The poisoning from trinitrotoluene. Arch.
Exp". Pathol. Pharmakol. 89: 340.
Linch, A.L. 1974. Biological monitoring for industrial
exposure to cyanogenic aromatic nitro and amino compounds.
Am. Ind. Hyg. Assoc. Jour. 35: 426.
Mangelsdorff, A.F. 1952. Methemoglobinemia - recognition,
treatment, and pevention. Ind. Med. Surg. 21: 395.
Mangelsdorff, A.F. 1956. Treatment of methemoglobinemia.
Am. Med. Assoc. Arch. Ind. Health 14: 148.
C-55
-------
Matsui, S., et al. 1975. Activated sludge degradability
of organic substances in the waste water of the Kashiraa
Pertroleum and Petrochemical Industrial Complex in Japan.
Prog. Water Technol. 7: 645.
McCann, J., et al. 1975. Detection of carcinogens as muta-
gens in the Salmonella/microsome test: assay of 300 chemicals,
Proc. Natl. Acad. Sci. 72: 5135.
McCormick, N.G., et al. 1976. Microbial transformation
of 2,4,6-trinitrotoluene and other nitroaromatic compounds.
Appl. Environ. Microbiol. 31: 949.
McCormick, N.G., et al. 1978. Identification of biotrans-
formation products from 2,4-dinitrotoluene. Appl. Environ.
Microbiol. 35: 945.
flcGee, L.C., et al. 1942. Metabolic disturbances in workers
exposed to dinitrotoluene. Am. Jour. Dig. Dis. 9: 329.
Meijers, A.P., and R.C. Van der Leer. 1976. The occurrence
of organic micropollutants in the river Rhine and the river
Maas in 1974. Water Res. 10: 597.
Moore, B. 1918. Causation and prevention of trinitrotoluene
poisoning. Jour. Am. Med. Assoc. 70: 412.
C-56
-------
Mori, M., et al. 1977. Studies on the metabolism and toxi-
city of dinitrotoluenes — on excretion and distribution
of tritium-labelled 2,4-dinitrotoluene (3H-2,4-DNT) in the
rat. Radioisotopes 26: 780.
Morton, A.R., et al. 1976. Biological effects of trinitro-
toluene from exposure below the threshold limit value.
Am. Ind. Hyg. Assoc. Jour. 37: 56.
Nason, A. 1956. Enzymatic steps in the assimilation of
nitrate and nitrite in fungi and green plants. Pages 109-
136 in Inorganic nitrogen metabolism. W.D. McElroy, and
B. Glass, eds. John Hopkins Press., Baltimore.
National Cancer Institute. 1978. Bioassay of 2,4-dinitrotoluene
for possible carcinogenicity. Carcinogenesis Tech. Rep.
Ser. No. 54. USDHEW (NIH) Publ. No. 78-1360. U.S. Government
Printing Office, Washington, B.C.
National Institute for Occupational Safety and Health. 1977.
Information profiles on potential occupational hazards.
Nitrotoluenes. Contract No. 210-77-0120.
National Institute for Occupational Safety and Health. 1978.
DinLtrotoluene. Ln NIOSH manual of analytical methods.
Vol. 4. U.S. Government Printing Office, Washington, D.C.
Nay, M.W., Jr., 1974. Biological treatability of trinitro-
toluene manufacturing waste water. Jour. Water Pollut.
Control Fed. 46: 485.
C-57
-------
Osmon, J.L., and R.E. Klausmier. 1972. The microbial degra-
dation of explosives. Dev. Ind. Microbiol. 14: 247.
Palmer, W.L., et al. 1943. Toxic necrosis of the liver
from trinitrotoluene. Jour. Am. Med. Assoc. 123: 1025.
Parrish, P.w. 1977. Fungal transformation of 2,4-dinitro-
toluene and 2,4,6-trinitrotoluene. Appl. Environ. Microbiol,
34: 232.
Patty, F.A. 1958. The mode of entry and action of toxic
materials. Page 162 in F.A. Patty, ed. Industrial hygiene
and toxicology. Vol. I. General principles. Interscience
Publishers, Inc., New York.
Pella, P.A. 1976. Generator for producing trace vapor
concentrations of 2,4,6-trinitrotoluene, 2,4-dinitrotoluene,
and ethylene glycol dinitrate for calibrating explosives
vapor detectors. Anal. Chem. 48: 1632.
Perkins, R.G. 1919. A study of the munitions intoxications
in France. U.S. Pub. Health Rep. 34: 2335.
Pienta, R.J., et al. 1977. Correlation of bacterial muta-
genicity and hamster cell transformation with tumorigenicity
induced by 2,4-toluenediamine. Cancer Lett. 3: 45.
C-58
-------
Plimmer, J.R., and U.I. Klingebiel. 1974. The application
of mass spectrometry to the study of nitroaniline-derived
herbicides. Pages 99-112 ir\ Mass spectrometry and NMR spectro-
scopy in pesticide chemistry- Plenum Press., New York.
Priestera, F., et al. 1960. Analysis of explosives using
infrared spectroscopy. Anal. Chem. 32: 495.
Proctor, N.H., and J.P. Hughes. 1978. Chemical hazards
of the workplace. J.B. Lippincott Co., Philadelphia/Toronto.
Rogovskaya, T.I. 1951. The effect of trinitrotoluene on
the microorganisms and biochemical processes of self purifi-
cation in waters. Mikrobiologiya 20: 480.
Roth, M., and J.M. Murphy, Jr. 1978. Correlation of oxygen
demand and total organic carbon tests on waste waters from
ammunitions plants. Proc. Ind. Waste Conf. 32: 674.
Ruchhoft, C.C,, et al. 1945. TNT wastes from shell-loading
plants. Ind. Eng. Chem. 37: 937.
Ruchhoft, C.C., and F.I. Norris. 1946. Estimation of ammoni-
um picrate in wastes from bomb and shell-loading plants.
Ind. Eng. Chem. 38: 480.
Schott, S., et al. 1943. TNT wastes. Ind. Eng. Chem.
35: 1122.
C-59
-------
Schereschewsky, J.W. 1918. Trinitrotoluene: practical
points in its safe handling. U..S. Pub. Health Serv. Rep.
434.
Schwartz, L. 1944. Dermatitis from explosives. Jour.
Am. Med. Assoc. 125: 186.
Shah, M.J., et al. 1977. Comparative studies of bacterial
mutation and hamster cell transformation induced by 2,4-
toluenediamine. Proc. Am. Assoc. Cancer Res. 18: 23.
(Abstract.)
Shikata, M., and I. Tachi. 1938. Polarographic studies
with the dropping mercury cathode. LXXIX. The electronega-
tivity rules of the reduction of organic compounds. Collect.
Czech. Chem. Conunun. 10: 368.
Shils, M.E., and L.J. Goldwater. 1953. Effect of diet
on the susceptibility of the rat to poisoning by 2,4-dini-
trotoluene. Am. Med. Assoc. Arch. Ind. Hyg. Occup. Med.
8: 262.
Sidwell, V.D., et al. 1974. Composition of the edible portion
of raw (fresh or frozen) crustaceans, finfish, and mollusks.
I. Protein, fat, moisture, ash, carbohydrate, energy value,
and cholesterol. Mar. Fish. Rev. 36: 21.
C-60
-------
Simmon, V.F., et al. 1977. Munitions wastewater treatments:
does chlorination or ozonation of individual components
produce microbial mutagens? Toxicol. Appl. Pharmacol.
41: 197. (Abstract from the 16th Annu. Meet. Soc. Toxicol.,
Toronto, Can., March 27-30.)
Spector, W.S. ed. 1956. Handbook of toxicology. Vol. I.
Acute toxicities of solids, liquids, and gases to labora-
tory animals. W.B. Saunders Co., Philadelphia/London.
St. John, G.A., et al. 1975. Determination of the concen-
tration of explosives in air by isotope dilution analysis.
Forensic Sci. 6: 53.
St. John, G.A., et al. 1976. Determination of the concen-
tration of explosives in air by isotope dilution. Edgewood
Arsenal Spec. Publ. EO-SO-76001. U.S. Dep. Army (Proc.
6th Annu. Symp. Trace Anal. Detect. Environ., 1975.
Stanford, T.B., Jr. 1977. The determination of tetryl
and 2,3-, 2,4-, 2,5-, 2,6-, 3,4-, and 3,5-dinitrotoluenes
using high performance liquid chromatography. AD Rep. AO42598.Natl<
Tech. Inf. Serv-
Stoats, J. 1972. Standardized nomenclature for inbred
strains of mice: fifth listing. Cancer Res. 32: 1609.
C-61
-------
Toxic and Hazardous Industrial Chemicals Safety Manual.
1976. Toxic and hazardous industrial chemicals safety manual
for handling and disposal with toxicity and hazard data.
The Inter. Tech. Inst., Japan.
Traxler, R.W., et al. 1974. Bacterial degradation of
TNT. Dev. Ind. Microbiol. 16: 71.
Umeda, M. 1955. Production of rat sarcoma by injections
of propylene glycol solution of m-toluenediamine. Gann.
46: 597.
U.S. Army. 1970. Special study of the effect of alpha TNT
on microbial systems and the determination of the biodegrad-
ability of alpha TNT. U.S. Army Environ. Hyg. Agency San.
Eng. Spec. Study No. 24-017-70/71. Edgewood Arsenal, Md.
U.S. Army. 1971. Evaluation of toxicity of selected TNT
wastes of fish. Phase I. Acute toxicity of alpha TNT to
bluegills. U.S. Army Environ. Hyg. Agency San. Eng. Spec.
Study No. 24-007-70/71. Edgewood Arsenal. Md.
U.S. International Trade Commission. 1975. Synthetic organic
chemicals: United States production and sales. USITC Publ.
804 Washington, D.C.
C-62
-------
Vasilenko, N.M., et al. 1972. Inactivation of the blood
respiratory pigment under the effect of aromatic nitro and
amino compounds from the benzene series. Sovrem. Probl.
Biokhim. Dykhaniya Klin., 2nd Mater. Vses. Konf., 1971.
1: 411.
Veith, G.D., et al. An evaluation of using partition coeffi-
cients and water solubility to estimate bioconcentration
factors for oganic chemicals in fish. (Manuscript).
Venitt, S. 1978. Mutagenicity of hair dyes: some more
evidence and the problems of its interpretation. Mutat.
Res. 53: 214. (Abstract from 2nd Int. Conf. Envrion. Mutagens,
July 11-15, 1977, Edinburgh, Great Br,)
Vernot, E.H., et al. 1977. Acute toxicity and skin corro-
sion data for some organic and inorganic compounds and aqueous
solutions. Toxicol. Appl. Pharmacol. 42: 417.
Voegtlin, C., et al. 1920. Trinitrotoluene poisoning -
its nature, poisoning, and prevention. U.S. Pub. Health
Serv. Hyg. Bull. 126: 7.
Von Oettingen, W.F. 1941. The aromatic amino and nitro
compounds, their toxicity and potential dangers. U.S. Pub.
Health Serv. Bull. No. 271. U.S. Government Printing Office,
Washington, D.C.
C-63
-------
Walsh, J..T., et al. 1973. Application of liquid chromato-
graphy to pollution abatement studies of munition wastes.
Anal. Chem. 45: 1215.
Weast. R.C., ed, 1978. CRC handbook of chemistry and physics.
A ready-reference book of chemical and physical data. CRC
Press, Inc., Cleveland, Ohio.
White, R.P., and J. Hay. 1901. Some recent inquires and
researches into the poisonous properties of naphthalene
and the aromatic compounds. Lancet 2: 582.
White, R.P., et al. 1902. Some notes from an inquiry into
the action of dinitrobenzene upon the urine of man. Lancet
160: 1393.
Williams, R.T. 1959. Metabolism of aromatic nitro compounds.
Pages 410-427 in Detoxication mechanisms. John Wiley and
Sons, Inc., New York.
Won, W.D., et al. 1974. Metabolic disposition of 2,4,6-
trinitrotoluene. Appl. Microblol. 27: 513.
Won, W.D., et al. 1976. Toxicity and mutagenicity of 2,4,6-
trinitrotoluene and its microbial metabolites. Appl. Environ,
Microbiol. 31: 576.
C-64
-------
Wynder, E.L., et al. 1963. An epidemiological investiga-
tion of cancer of the bladder. Cancer 16: 1388.
Zieger, J. 1913. Studies on the effect of nitrobenzene,
dinitrobenzene, nitrotoleuene and dinitrotoluene with absorp-
tion through the lungs and the skin. Inaug. Dissert. Wurzburg
Zitrin, S., and J. Yinon. 1976. Chemical ionization mass
spectrometry of explosives. Pages 369-381 in Advances in
mass spectrometry in biochemistry and medicine. Vol. 1.
C-65
-------
APPENDIX I
Carcinogenicity Risk Assessment by Extrapolation
from Laboratory Animal Toxicity Tests
An assessment of health risks associated with exposures
of a general environmental nature requires prediction of
effects from low level exposures of lifetime duration.
Carcinogenic risks effects from environmental exposures
must normally be estimated from animal data obtained at
much higher levels because of the difficulty in detecting
a small increase in tumor induction resulting from long-
term low level exposure. Because the carcinogenic process
is generally believed to be irreversible, self-replicating,
and often originating from a single somatic cell mutation,
assumptions of threshold levels of effect are believed to be
invalid for many, if not all, cancer-causative compounds.
Although many models have been proposed for extrapolation
from animal data to human risk assessment, the one utilized
here was chosen to facilitate uniform treatment of the variety
of chemical compounds that are discussed in the development
of those water criterion documents that deal with animal
carcinogens.
It is recognized that the process of evaluating existing
studies and resultant data in preparation for application
of mathematical methods involves a high level of professional
judgment. Many questions will necessarily arise due to
the unique characteristics of the specific compounds under
C-66
-------
discussion and the tremendous variability in completeness
and comparability among the available studies.
A general explanation of the evaluation and extrapolation
procedures to be used are as follows:
1. Since the compounds discussed are known, or suspect
carcinogens, emphasis was placed on those studies
with carcinogenic or mutagenic endpoints. In
particular, those studies dealing with" mammalian
species.
2. The extrapolation method employed is a mathematical
procedure that uses a single dose and observed
response of a toxicologic experiment to estimate
a dose level for humans that will not increase
the risk of tumors by more than a specified level
(1 in 100,000) (Personal communication, Dr. Todd
Thorsland, CAG, U.S.EPA, Washington, D.C.). Clearly
this method is predicated on sound toxicologic
test procedures. Hence, each included study was
evaluated for adherence to sound toxicological
and statistical principles.
3. Judgment was exercised in prioritizing the signifi-
cance of toxicologic studies that use different
routes of administration. In general, the preferred
route of exposure is oral (food, water, or gavage)
followed by intraperitoneal, intravenous, inhalation,
or dermal routes of administration for the same
species. However, in some instances, consideration
of absorption rates required that other routes
be evaluated.
C-67
-------
The NCI's Ad Hoc committee on the Evaluation of Low Levels
of Environmental Chemical Carcinogens outlined two conditions
that would render the extrapolations of animal carcinogenesis
to man inappropriate. This committee reported to the Surgeon
General as follows:
Any substance which is shown conclusively to cause
tumors in animals should be considered carcinogenic
and therefore a potential hazard for man. Exceptions
should be considered only where the carcinogenic effect
is clearly shown the results from physical rather than
chemical induction or where the route of administration
is shown to be grossly inappropriate in terms of conceiv-
able human exposure.
4. After selection of the sound toxicologic studies that
form the basis for development of a recommended criteria,
a, single dose and observed response were selected for
the most "sensitive" sex (if both males and females
were tested) according to the following method: Select
the lowest dose that yields a tumor response rate
that is greater than the control rate. If the standard
controls and media control response rates are not signif-
icantly different (d<0.05), a combined rate was calcul-
ated from controls.
5. The extrapolation methods were applied independently
to each selected dose and response pair. The lowest
projected dose was selected as the "safe level" based
on the available toxicologic studies, if judgment indica-
ted equal confidence in the various dose-response pairs.
6. The calculated safe dose was evaluated along with the
results from human studies to develop a recommended
criteria.
C-68
-------
Calculation of Estimated Safe Levels for Humans;
The specific data analyses performed along with required
input data are described in Mathematical Description of
Extrapolation Method. This model provides the additional
risk associated with ingestion of 2 liters of water per
day and contaminated aquatic foods. Any other risks associated
with air, food, or other exposure are not addressed by this
model. A copy of the working data sheet is also included.
Mathematical Description of Extrapolation Method
A. Necessary information:
nt = No. of animals (males or females) exposed to selected
dose that developed tumors (all sites combined
unless tumors appear to be related to route of
administration, e.g., peritoneal tumors would
not be included if intraperitoneal injection method
is used).
Nt = Total number of animals (male or females) exposed
to selected dose level.
nc = Number of control animals (males or females) with
tumors.
NC = Total number of control animals (males or females).
C-69
-------
Le = Actual maximum lifespan for test animals.
le = Length of exposure (no. of hours, days, weeks,
etc.).
d = Average dose per unit of time (mg/kg).
w = Average weight of test animals (kg).
B. Necessary information from general literature:
70 kg = Average weight of man.
L = Theoretical average length of life for test species,
unless specified in articles. (See attached table
for appropriate values.)
p = Average weight of fish consumed per day, assumed
18.7 grams.
C. Necessary ecological information.
R = Bioaccumulation factor for edible portions of
fish (supplied by Environmental Research Laboratory,
Duluth)
(Note: If a bioaccumulation factor is provided
for the total fish or for some part other than
the total edible portion (such as the fat) an
attempt should be made to estimate factor for
edible portion.)
C-70
-------
D. Mathematical Model:
Pt = PC + (1-Pc) 1 - e"
Where:
Pt = nt - NT = Proportion of test animals with tumors.
PC = nc - NC = Proportion of control animals with tumors
D = = Lifespan weighted average dose level
Le (mg/kg)/(unit of Time)
I 1 - Pt
B = V-ln
-\ FD x t3 "I where t = li£esPan £or test animals = Le
1 - PC I I *— -1 length of life for species L
3 | — (Note: It is assumed that average weight of man = 70 kg
B1 = B
If and only if B1 0.1, then
«• • 1°;' *7° = Safe level (mg/1) for
~"O \t* T r\xr ^
If B'20.1, then
SL = ^B'^ti +°RxF) x 70 = Safe level (mg/1) for man
(Note: It is assumed average daily consumption of water is
2 liters/day)
071
-------
APPENDIX II
1. Calculation of Daily Occupational Exposure level of
2,4-Dinitrotoluene based on its Threshold Limit Value-
Time Weighted Average (TLV-TWA) concentration (Am. Conf. Gov,
Ind. Hyg. 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 10~3rng
liter of air
= 1-5
liter of air
Therefore, the daily occupational level for
2,4-DNT = 1.5 jag x 7.5 liter of air x 60 minute x 8 hour
liter minute hour day
= 5,400
= 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 5.5 as determined for the
blue-gill sunfish (U.S. EPA report, Duluth, Minnesota),
b) Average weight of aquatic organisms consumed per day
is 18.7 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-72
-------
Bioaccumulation factor of 2,4-DNT = 5.5
The concentration of 2,4-DNT in fish =
740 x 5.5 x 0.0187 = 76 ng from aquatic organisms
Daily intake of
2,4-DNT from 2 liters
of drinking water = 740 ng/1 x 2 = 1480 ng
Total intake/day = 1480 + 76 mg
or 1556 ng
(1.55 /ug or .00155 mg)
C-73
-------
APPENDIX III
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
explosion 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 ma-
terial for dyestuffs and for urethane polymers, as a modifier
for smokeless powders, and as a gelatinizing and waterproofing
agent in military and commercial explosives.
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 typhimu-
rium TA1535 indicating base-pair substitution.
Two reports concerning the carcinogenicity of 2,4-DNT
are in the literature. The first is a National Cancer Insti-
tute (NCI) two-year bioassay in male and female Fisher 344
rats and B6C3F1 mice fed 2,4-DNT (1978). The major pathologic
findings were present in the rats. These included fibromas
of the skin and subcutaneous 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 signifi-
cant carcinogenic response to the administration of 2,4-
dinitrotoluene.
C-74
-------
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) . The major pathologic findings
in the rats included a significant increase of hepatocellular
carcinomas (p = 7.1 x 10~ ) and neoplastic nodules (p = .01)
in the liver of females, mammary gland tumors of the female
_c
(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, hepatocellu-
lar 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
V
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 200 ppm 2,4-DNT for 24 months
(Lee, et al. 1978). It is concluded that the water concentra-
tion of 2,4-dinitrotoluene should be less than 740 ng/1
in order to keep the lifetime cancer risk below 10
*This summary has been prepared and approved by the Carcino-
gens Assessment Group of EPA on June 19, 1979.
C-75
-------
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 200
ppm in the diet. The time-weighted average dose of 45 mg/kg/day
was given in the feed for 24 months, with the surviving
animals sacrificed one month later. The mammary tumor inci-
dence was 11/23 and 33/35 in the control and treated groups,
respectively. The incidence of hepatocellular carcinomas
and neoplastic nodules was 0/23 and 24/34 in the control
and treated groups, respectively. Assuming a fish bioconcen-
tration factor of 5.5, the criterion is calculated from
the following parameters:
nt mammary =33 d - 45 mg/kg/day
t mammary =35 R = 5.5
nc mammary =11 L = 25 months
Nc mammary =23 W = 0.464 .kg
nt liver =24 F = 0.0187 kg/day
Nt liver = 34
nc liver = 0
Nc liver = 23
le = 24 months
Le = 25 months
g
Based on these parameters, the one-hit slope, H, is
2.95 x 10 for mammary tumors and 1.53 x 10" for hepatocell-
ular carcinomas and hepatocellular neoplastic nodules.
The resulting water concentration of 2,4-dinitrotoluene
calculated to keep the individual lifetime cancer risk below
10~5 is 740 ng/1.
C-76
------- |